Treatment of acute myeloid leukemia models by targeting a cell-surface RNA-binding protein

Abstract

Immunotherapies for acute myeloid leukemia (AML) and other cancers are limited by a lack of tumor-specific targets. Here, we discover that RNA-binding proteins and glycoRNAs form precisely organized nanodomains on cancer cell surfaces. We characterize nucleophosmin (csNPM1) as an abundant cell surface protein on a variety of tumor types. With a focus on AML, we observe csNPM1 on blasts and leukemic stem cells, but not on normal hematopoietic stem cells. We develop a monoclonal antibody to target csNPM1, which exhibits robust anti-tumor activity in multiple syngeneic and xenograft models of AML, including patient-derived xenografts, without observable toxicity. We find that csNPM1 is expressed in a mutation-agnostic manner on primary AML cells and may therefore offer a general strategy for detecting and treating AML. Surface profiling and in vivo work also demonstrates csNPM1 as a target on solid tumors. Our data suggest that csNPM1 and its neighboring glycoRNA-csRBP clusters may serve as an alternative antigen class for therapeutic targeting or cell identification.

In recent years there has been a surge in new therapies that target cell surface molecules, including monoclonal antibodies, antibody drug-conjugates, bispecific antibodies, and chimeric antigen receptor T-cells^1–4. These approaches have improved overall survival in a variety of cancers, including non-Hodgkin lymphoma, breast cancer, and acute leukemia. While the molecular toolkit for targeting cell surface molecules has rapidly expanded, there remain few clinically actionable targets to leverage these approaches. There have been efforts to better understand and characterize cell surface proteins that are differentially expressed between healthy and cancer states using techniques like proteomics, transcriptomics, and phage display^5–7. Despite the emergence of these unbiased techniques, acute myeloid leukemia (AML) has remained challenging to target with surface-directed therapies as many highly-expressed antigens on AML cells are also present on critical healthy tissue, including hematopoietic stem cells.

Anti-CD33 molecules, like the antibody-drug conjugate gemtuzumab ozogamicin⁸, have seen some success; however, high expression of CD33 on healthy myeloid cells and hepatic sinusoidal endothelial cells has been associated with severe hematological and hepatic toxicity, respectively⁹. Newer approaches studying both the expression and the physical conformation of proteins on the cell surface have demonstrated promising results, such as an antibody fragment targeting an AML-specific conformation of integrin-β2¹⁰. Another approach that has gained traction has been targeting HLA-restricted peptides, such as WT1¹¹; however, this approach is limited to a fraction of patients that express specific HLA subtypes. Now there are methods to genetically delete surface proteins from healthy hematopoietic tissue to allow AML cytotoxicity with limited off-target effects¹²; however, this may be technically cumbersome and costly. Though the majority of clinical efforts have focused on canonical cell surface proteins¹³,such as those containing a transmembrane domain, there appears to be a need to expand our scope.

There are now three examples of RBPs as surface proteins in preclinical cancer models: PABP1¹⁴, nucleolin^15–18, and U5-snRNP200¹⁹. There are multiple examples of mutated and dysregulated RBPs in AML, such as SF3B1, U2AF1, SRSF2 and nucleophosmin 1 (NPM1). In adult AML, nearly 30% of all cases and approximately 60% of those with a normal karyotype are driven by mutations in NPM1^20,21. In these cases, C-terminal mutations of NPM1 cause the replacement of a nucleolar localization signal with a novel nuclear export sequence, leading to cytoplasmic localization of the mutant protein (NPM1c)²². However, it has been unclear whether NPM1 (either wildtype or mutated) is present on the cell surface. Outside of leukemia, autoantibodies in the sera from prostate cancer patients were shown to react with NPM1, while healthy controls did not²³, indicating potential aberrant cell-surface localization leading to immune recognition.

Here, we provide evidence that NPM1 is expressed on the cell-surface in a tumor-selective manner. Cell surface NPM1 (csNPM1) is present on a diverse set of clinically relevant human and mouse leukemia models and forms nanoscale clusters in physical proximity to other csRBPs and glycoRNAs^24,25. We demonstrate that csNPM1 is expressed at increased levels in primary AML in comparison to healthy hematopoietic stem and progenitor cells (HSPCs). We also optimize a novel mouse IgG2a antibody (mAb2) targeting NPM1, which does not cause toxicity in healthy mice in vivo. mAb2 shows preferential targeting of murine leukemia stem cells (LSCs), and increases survival in murine models of AML while restoring healthy hematopoiesis. csNPM1 is also present on solid cancers cell lines and using in vivo models, we find that targeting csNPM1 has anti-tumor activity in murine models of prostate and colorectal (csNPM1 high) cancer but not melanoma (csNPM1 absent). Collectively, we provide evidence of an RBP that can localize selectively to the surface of cancer cells, which could have significant therapeutic implications.

Results

Full length NPM1 is presented on the surface of living cells

Our examination of cell surface proteomes for csRBPs²⁶ identified NPM1 as a putative cell surface protein, with at least 7 datasets showing NPM1 enrichment on the cell surface (Extended Data Figure 1A). Since NPM1 mislocalization is a driver of leukemia, we first examined the subcellular profile of NPM1 across a panel of 9 human myeloid and lymphoid leukemia cell lines (Extended Data Figure 1B, 1C), one of which harbors the NPM1c mutation (OCI-AML3). We isolated soluble cytosol and crude membrane fractions and assayed for cytosolic β-actin and endoplasmic reticulum-membrane tethered RPN1, confirming efficient separation of these pools (Extended Data Figure 1B, 1C). Blotting with an anti-NPM1 (FC8791) antibody produced a robust band corresponding to NPM1 from the cytosolic and membrane-enriched fraction of most cell lines tested (Extended Data Figure 1B, 1C). Because OCI-AML3 harbors the C-terminal mutated form of NPM1 (NPM1c), we examined its cellular distribution using a commercially available anti-NPM1c antibody. We found that the NPM1c signal was detected in both the cytosol and the membrane fraction of OCI-AML3 cells (Extended Data Figure 1B, 1C). We next tested if the wild-type antibody could bind to the surface of live cells. To directly visualize the cell surface localization of NPM1, we performed confocal imaging of OCI-AML3 and found that when applied to live cells, anti-NPM1 (FC8791) binds the cell surface in distinct puncta (Figure 1A, Extended Data Figure 1D). To demonstrate the specificity of the antibody, we performed intracellular immunofluorescence on fixed cells, resulting in the expected nucleolar predominance of NPM1 (Figure 1A, Extended Data Figure 1D). To confirm the cell surface pattern seen when staining live cells, we repeated the experiment with WGA, which is a lectin that binds cell surface glycans. We observed robust WGA cell surface staining with colocalized anti-NPM1 (FC8791) signal (Figure 1B, Extended Data Figure 1E), further confirming NPM1 cell surface presentation.

Figure 1. Full-length NPM1 forms nanoclusters on the surface of cells with other RBPs.

A. Confocal microscopy images of OCI-AML3 cells presented as one z-slice (all images are z-slices unless otherwise specified). Cells in the top row were stained with the anti-NPM1 (FC8791) antibody after fixation and permeabilization while the bottom row of cells were stained while alive. 4′,6-diamidino-2-phenylindole (DAPI) in shown in blue. Scale bars are 10 μm. Nucleoli are indicted with white lines. Representative of three individual replicates.

B. Confocal microscopy images of OCI-AML3 stained alive with anti-NPM1 (FC8791) and biotinylated WGA. DAPI shown in blue. Scale bars are 10 μm. Representative of three individual replicates.

C. Western blot of K562 cells first labeled with sNHS-SS-biotin. NPM1 immunoprecipitation (IP, left) or streptavidin IP (right) were performed and stained with anti-NPM1 or streptavidin. Molecular weights are shown in kilodaltons (kDa). Red asterisks mark the expected weight of full-length NPM1. Representative of three individual replicates.

D. HL-60 cells stained live with anti-NPM1 antibody and subsequently fixed. Shown are diffraction-limited (DL) widefield microscopy images (left) or super-resolution reconstructions (SR, right), presented as 2D histograms (bin size: 17.7 nm). Scale bar 10 μm. For SR images, the calibration bar indicates the number of localizations per histogram bin.

E. A zoomed view of the indicated region in (D). Scale bar 1.42 μm.

F. OCI-AML3 cells stained and analyzed as in (D).

G. A zoomed view of the indicated region in (F). Scale bar 1.42 μm.

H. Quantification of the cluster size in nanometers (nm), cluster to cluster distance (nm), and localizations per cell, for each cell. Median value is shown as a horizontal bar. Unpaired, two-sided t-tests were performed to evaluate the significance; ns = not significant.

I. Schematic of HRP-based cell surface proximity labeling.

J. Gene ontology (GO) cellular compartment (CC) analysis of csNPM1 proximity labeling MS data. The top 4 terms across the three cell lines were intersected and the union shown, significance (y-axis) and set size (circle size) plotted.

K. Intersection analysis of the proximity labeling MS data. For each bar, the number of RBPs is overlaid in orange.

To assess whether full-length NPM1 or a peptide fragment was presented on the cell surface, we used a sequential biochemical enrichment strategy. Live cell labeling with sulfo-NHS-SS-biotin, followed by membrane isolation, anti-NPM1 (FC8791) immunoprecipitation, and finally western blotting resulted in robust enrichment of NPM1 from K562 membrane fractions (Figure 1C, left). Using a streptavidin detection reagent on the same material resulted in a specific band migrating at the same molecular weight of NPM1 (Figure 1C, right), supporting the presence of full-length cell-surface NPM1 (csNPM1) rather than MHC-presentation, as shown in other cases²⁷. Since many cell surface proteins are glycosylated, we next investigated whether NPM1 was similarly modified. While NPM1 has no N-glycan sequons it does have many predicted sites for O-linked N-acetylgalactosamine (Extended Data Figure 1F). Further we selectively enriched NPM1 from membrane lysates with a sialic acid binding lectin (SNA) (Extended Data Figure 1G), suggesting that NPM1 can become glycosylated, consistent with a previous report²⁸.

Super-resolution localization of csNPM1

The presence of full-length csNPM1²⁷ and the punctate pattern observed with confocal microscopy in Figure 1A motivated a more detailed investigation of its cell surface organization. We performed super-resolution (“SR”) single-molecule localization microscopy (Extended Data Figure 1H) using a primary-conjugated anti-NPM1 (FC8791) antibody for staining HL-60 (NPM1-WT) and OCI-AML3 (NPM1c) cells (Figure 1D-H, Extended Data Figure 1H). Staining live cells as in Figure 1A and imaging with diffraction-limited (“DL”) widefield epifluorescent microscopy yielded an expected punctate pattern of csNPM1 (Figure 1D and 1F). We obtained SR reconstructions for a total of 5 OCI-AML3 cells and 3 HL-60 cells (Table S1, Extended Data Figure 1H, S1I). In both AML models, the SR reconstructions showed csNPM1 forming distinct clusters of apparently uniform size and regular patterning (Figure 1D-G). We implemented a semi-automated cluster analysis workflow (Methods and Extended Data Figure 1I). The diameter of csNPM1 clusters was on average 119 nm and 165 nm in HL-60 and OCI-AML3 cells, respectively (Figure 1H). We then determined the number of localizations observed per cluster and the cluster-to-cluster distance. Across both cell lines, clusters were regularly spaced at approximately 300-330 nm apart (Figure 1H), while the number of localizations was less uniform with clusters of two types: one with 50-80 and a second with 120-140 localizations per cluster (Figure 1H). Using the SR data, we calculated the approximate number of antibodies bound per cell, finding ~1,000-2,400 and ~3,000-24,000 per cell on HL-60 and OCI-AML3, respectively (Table S1). A similar number was obtained by an orthogonal method using quantitative flow cytometry (Extended Data Figure 1J). Together, the overall distribution of csNPM1 is reminiscent of a tessellated pattern on the cell surface.

Molecular neighborhoods of csNPM1

Aside from rare examples, RBPs like NPM1 are not commonly thought to occupy the surface of living cells. Our recent work has begun to explore this concept more generally²⁶, however, there remains an overall lack of molecular understanding of what factors may associate with cell surface RBPs. To address this, we adapted a strategy previously used to label glycoRNAs²⁴, csRBPs²⁶, and other proteins²⁹ in proximity to glycan ligands. Using a primary antibody against csNPM1, a secondary antibody conjugated to horseradish peroxidase was applied, which activates biotin-phenol (protein labeling) or biotin-aniline (RNA labeling) to label cell surface molecules in the presence of hydrogen peroxide (Figure 1I, Table S2). To ensure robust recovery of proximal proteins, we selected the three AML models with the highest csNPM1 mean fluorescence intensity (MFI) fold change over isotype: MOLM-13, OCI-AML3, and Kasumi-1 cells (Extended Data Figure 2A). After capture of the biotinylated proteins and analysis of those hits enriched over the isotype labeling, we examined the enriched gene ontology (GO) cellular compartment (CC) terms. Membrane terms were highly enriched suggesting our approach was overall successful for assaying components of the cell surface (Figure 1J). In addition, other GO terms like cytosol, ribonucleoprotein (RNP) complex, and nucleoplasm were also enriched (Figure 1J). At the individual gene level, we found that MOLM-13, OCI-AML3, and Kasumi-1 cells had many unique hits (Figure 1K, left three bars), and RBPs comprised 61.1%, 60.5%, and 71% of the proximal proteins to csNPM1 (Figure 1K). Notably, NPM1 was found in all three proximity labeling datasets as anticipated from our data above (Table S2). Hits found in common between the AML models tested were more likely to be RBPs as 75.9% of hits found proximal to csNPM1 in at least two cell lines were annotated RBPs (Table S2). Together these data suggest that csNPM1 clusters into distinct RBP-enriched nanodomains on the surface of leukemia cells.

csNPM1 is expressed on human and murine models of AML

We next assessed the levels of csNPM1 on the 9 human cell lines tested in Extended Data Figure 1B and in all cases, live cell staining with the anti-NPM1 (FC8791) antibody demonstrated staining above the isotype levels, albeit to different degrees (Figure 2A, Extended Data Figure 2A). While cancer cell lines are efficient at growing in vitro, over time, cultures adapt to these conditions and may select for states that deviate from their in vivo state, where niche environments are important^30–32. Here, we harvested leukemic marrow from four primary murine leukemia models driven by MLL-AF4, MLL-AF9, MLL-ENL, and NPM1c, all in a Flt3^ITD/+ background^33,34. In all four genotypes, we observed cell surface binding with anti-NPM1 (B0556) (Figure 2B, top row), demonstrating that primary murine AML also presents csNPM1. To understand if this effect was stable ex vivo, we evaluated how csNPM1 changed upon in vitro culture of Npm1^cA/+/Flt3^ITD/+ AML cells. After 7 days in culture, these cells still robustly expressed csNPM1 (Extended Data Figure 2B), although direct comparison to the in vivo data is not possible because these samples were analyzed on different days. As compared to the AML cell lines, we saw a larger distribution in the fraction and intensity of csNPM1-positive cells in primary murine AMLs. Whole-cell levels of NPM1 assessed by flow cytometry after fixation and permeabilization demonstrated similar levels and profiles across all four genotypes (Figure 2B, bottom row), suggesting csNPM1 levels are not purely a product of total cellular abundance. The broad expression of csNPM1 on models with or without NPM1c and the higher expression on models with NPM1c (i.e. OCI-AML3 and Npm1c/Flt3^ITD/+ murine AML) raises the question of whether both the wild-type and mutant forms of NPM1 can be presented on the cell surface, especially considering NPM1c can form heterodimers with wild-type NPM1³⁵. To explore this, we designed over-expression vectors encoding NPM1-WT or NPM1c with Ty1 N-terminal tags. Using an anti-NPM1 antibody, we found the overexpression of either NPM1-WT-Ty1 or NPM1c-Ty1 further increased the levels of csNPM1 as compared to K562 cells bearing an empty vector (Figure 2C). Using the anti-Ty1 antibody, we also found that both the NPM1-WT-Ty1- and NPM1c-Ty1-expressing cells were detectable by flow cytometry (Figure 2C), suggesting that both the WT and NPM1c forms are capable of reaching the cell surface. Consistently, cells expressing an empty vector had little binding when stained with the anti-Ty1 antibody (Figure 2C). Taken together, these data suggest that csNPM1 is presented on many in vitro leukemia models and in vivo primary murine AML models, regardless of NPM1 mutation status.

Figure 2. Commercial and novel anti-NPM1 antibodies detect csNPM1 across many human and murine AML models.

A. Histograms of live cell flow cytometry of nine human leukemia cell lines stained with an isotype (gray) or the commercially available anti-NPM1 FC8791 (red) antibody.

B. Histograms of flow cytometry of four primary murine leukemias isolated from bone marrow-derived cells. AML-driving mutations are noted, all models are in a Flt3^ITD/+ background. The top row shows flow cytometry on the surface s while the bottom shows intracellular staining of cells that were first fixed and permeabilized before adding the anti-NPM1 (B0556) or isotype antibodies.

C. Histograms of live cell flow cytometry on K562 cells that were transduced to express a plasmid containing no cDNA (empty vector) or cDNAs encoding Ty1-tagged NPM1-WT (Ty1-NPM1-WT) or the NPM1c mutant (Ty1-NPM1c). Cells were stained either with anti-NPM1 (B0556, left) or anti-Ty1 (right).

D. Contour plot of OCI-AML3 cells live staining with the mAb2 (red) or isotype (gray) antibody. The cells shifted above the isotype are boxed and the total percent of this population is noted.

E. Confocal microscopy images of OCI-AML3 cells. Cells in the top row were stained with antibodies while they were alive, while the bottom row was first fixed with formaldehyde, permeabilized, then stained with antibodies; anti-NPM1 FC8791 (left) and mAb2 (right). All cells are counterstained with (DAPI) in blue. Scale bars are 10 μm. Representative of three individual replicates.

F. Cell surface proximity labeling assisted by Protein A-HRP and biotin-aniline (bio-Aniline) to label cell surface RNAs. Isotype or mAb2 antibodies were used as anchors and the resulting total RNA (SybrGold signal, Sybr) was analyzed on a northern blot, detecting biotinylated species (Streptavidin IR800, Strep). A high molecular weight (MW) smear is highlighted (orange) that is sensitive to RNase and sialidase treatment. Representative of three individual replicates.

Development of a monoclonal antibody to target csNPM1

The FC8791 monoclonal antibody was raised against the C-terminal region of human NPM1. This domain is highly conserved between mice, monkeys, and humans (Extended Data Figure 2C), suggesting that there is a possibility of developing a cross-species reactive antibody, which would be desirable for clinical translation. We sought to develop an antibody derived from a human immune system, thus minimizing the potential risk of anti-drug antibodies in human subjects^36,37. Previously, an NPM1c mutant-specific binder was selected from a human scFv library³⁸. Using this as a baseline, we constructed a mouse IgG2a antibody (mAb2) targeting NPM1 (Extended Data Figure 2D) and evaluated its binding features. Mouse IgG2a was selected for its capacity to induce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)³⁹, which would enable anti-cancer immunity. First, we analyzed the signal of FC8791 and mAb2 from whole cell lysate as well as extracted membranes which demonstrated nearly identical banding patterns (Extended Data Figure 2E). Next, we used mAb2 for live cell flow cytometry where mAb2 was able to fully shift OCI-AML3 cells above the isotype control (Figure 2D). Evaluation of the binding pattern by confocal microscopy of OCI-AML3 cells showed that both FC8791 and mAb2 formed puncta on the cell periphery (Figure 2E, Extended Data Figure 2F, top row). The distribution of signal was different upon fixation and permeabilization. Specifically, FC8791, which was raised against a 100% NPM1-WT peptide, displayed a highly nucleolar pattern with limited signal from the cytosol (Figure 2E, bottom row). In contrast, mAb2 had a more prominent cytosolic signal, while still retaining nucleolar staining (Figure 2E, bottom row), consistent with mAb2 having some specificity for NPM1c, which is mislocalized to the cytoplasm. Co-staining live cells with mAb2 and WGA demonstrated that the mAb2 pattern was restricted to the cell surface (Extended Data Figure 2G) and co-staining of cells with mAb2 and FC8791 demonstrated overlapping signals (Extended Data Figure 2H) suggesting a common cell surface target. We also examined the cell surface binding of mAb2 on the primary murine MLL-ENL/Flt3^ITD/+ bone marrow cells which showed poor and non-specific staining with an isotype control antibody, while mAb2 staining produced punctate, cell surface specific clusters (Extended Data Figure 2I) as seen on the human AML cell lines.

To validate the specificity of mAb2 using functional genetics, we generated CRISPR/Cas9 NPM1 knockout cells using the OCI-AML2 cell line. Upon genetic ablation of NPM1 we lost cell surface mAb2 binding, while surface expression of CD47 remained unchanged (Extended Data Figure 2J, 2K), suggesting that mAb2 loss was not a non-specific finding. Finally, to further explore the csRBP clustering phenotype we observe with csNPM1 and given that we have also found glycoRNAs being in proximity to other csRBP clusters²⁶, we performed an RNA proximity labeling experiment on OCI-AML3 cells with mAb2. RNA from cells stained with mAb2 and then labeled with biotin-aniline revealed a high molecular weight biotin smear that was RNase-sensitive and sialidase-sensitive (Figure 2F), which is consistent with cell surface glycoRNA labeling²⁴. Together, these data suggest that mAb2 targets csNPM1 and that this RBP is in proximity to glycoRNAs.

csNPM1 is present on primary AML

Given the presence of csNPM1 on primary murine models of AML and human cell lines, we next assessed its presence on primary human AML. We obtained cryopreserved bone marrow aspirates (BM) from healthy donors and AML patients and performed flow cytometry using a myeloid leukemia panel (CD45/CD34/CD117/CD33/CD13/HLA-DR). Malignant blasts were coarsely identified as SSC low (SSC-lo) and CD45dim (Figure 3A, middle, Extended Data Figure 3A). Across 12 bone marrow aspirates (Set A), including the gating example from Patient #3 (Figure 3A), the blast population generally showed a robust shift above isotype control with mAb2 (Figure 3B). We next expanded our primary sample cohort to 31 patient samples encompassing various genotypes and immunophenotypes, including primary and secondary AMLs (i.e. progressing from myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN)) (Figure 3C, Table S3). AML blasts consistently showed a significantly enriched csNPM1 population as compared to isotype control (Figure 3D). Despite the Set A patients having a wide range of identified mutations through genetic analysis (Figure 3C), all except Patient #8 demonstrated clear mAb2 binding (Figure 3B). Most profiles were unimodal with between 21-54% of cells being mAb2+ (Figure 3B, e.g. AML BM #3, #5, #9, #10, #11, and #12). Next we compared the relative binding efficacy of mAb2 compared to FC8791, which is a well-studied anti-NPM1 antibody (Extended Data Figure 3B). On OCI-AML3 and two primary patient samples, we see that both antibodies bind the malignant SSClo CD45dim populations, while mAb2 consistently shows higher MFI. We then examined whether mAb2 can also target csNPM1 in primary murine AML models by performing live staining followed by flow cytometry analysis), finding mAb2 can bind these cells as well driven by Npm1^A/+/Flt3^ITD/+, MLL-AF9/Flt3^ITD/+, MLL-ENL/Flt3^ITD/+, and Npm1c/Nras^G12D/+ (Extended Data Figure 3C).Overall, the broadly observed and high levels of mAb2 binding on AML bone marrow suggests that csNPM1 could serve as a genotype-agnostic marker of AML.

Figure 3. mAb2 stains primary human AML bone marrow in a genotype-agnostic manner.

A. Flow cytometry analysis of an AML patient bone marrow (BM) sample (Set A, #3). Gates first identify lymphocytes, then CD45 dim vs. bright populations, and finally the mAb2 vs. isotype staining of the CD45dim population in a histogram. The percent positive of each population is noted in the panels.

B. Flow cytometry analysis of the OCI-AML3 cell line and 11 AML patient BM samples as analyzed in (A).

C. Pathogenic mutations identified by clinical pathology for each of the 31 AML patient BM samples analyzed in this study (across three Sets; A, B, C). A green cell denotes a mutation of that gene was identified, a gray cell denotes no testing of mutations for this gene, and blue highlights mutations in the NPM1 gene.

D. Dot plot of the percent NPM1+ cells identified in each patient’s BM CD45dim population.

csNPM1 is a myeloid-biased marker that spares healthy HSPCs

Though mAb2 strongly stains cancer cells, avoiding healthy hematopoietic stem and progenitor cells (HSPCs) will be critical to avoid the associated toxicities seen with other AML targets (i.e. CD33 and CD123)⁴⁰. To understand in detail where, and to what degree csNPM1 is presented on healthy cells, we studied this in both human and primary mouse models. We examined this by measuring mAb2 binding on B cells (CD19), T cells (CD3), myeloid cells (CD33), and HSPCs (CD34). In both human PB and BM from healthy donors, there was minimal binding in T lymphocytes, stronger binding in CD33+ myeloid cells, while B lymphocytes showed higher binding on BM as compared to PB (Extended Data Figure 4A, 4B). Both CD33+ SCC-hi (neutrophils) and CD33+ SSC-lo (monocytes) had ~70% mAb2 binding. To understand how this degree of positivity compares to a malignant state, we assessed the MFI for mAb2 across bone marrow cell populations and the OCI-AML3 human AML cell line (Extended Data Figure 4C). On average, lineage-differentiated bone marrow populations showed 10-fold less binding intensity than OCI-AML3. Next, examining the CD45dim/CD34+ cells (HSPCs), we found an approximate 10-fold reduction in binding compared to lineage cells, and 100-fold lower binding as compared to AML cells (Extended Data Figure 4C). Thus, while a subpopulation of healthy hematopoietic cells are stained by mAb2 (percent positive), the total amount of antibody binding (MFI) is significantly less on healthy human cells as compared to a human AML model.

We next assessed mAb2 binding on healthy mouse hematopoietic cells. Similar to our findings with human samples, mAb2 binding was preferentially seen in bone marrow neutrophils (Mac1+, Gr1+) and monocytes (Mac1+, Gr1-), ranging between 20 and 30% of each population (Figure 4D). There was ~10% binding in bone marrow T and B cells. Again, we found low level binding on mouse hematopoietic progenitors (Lineage-, Kit+) and stem cells (Lineage-, Kit+, Sca1+) (Extended Data Figure 4D, 4E). Due to limited binding on HSPCs, we predicted that mAb2 would have minimal impact on normal hematopoiesis.

Figure 4. In vivo anti-AML efficacy and mechanism of killing.

A. Real-time PCR quantification of Cas9 gene in PB of Npm1^cA/+/Flt3^ITD/+/Rosa^Cas9/+-driven AML treated with either isotype control (Iso., n=4) or mAb2 (n=5) (mean ± SD). Statistical significance was determined by two-tailed Mann–Whitney U test.

B. Spleen weight of Npm1^cA/+/Flt3^ITD/+/Rosa^Cas9/+ murine AML models following treatment with either Iso. (n=4) or mAb2 (n=5) (mean ± SD). Statistical significance as in A.

C. Kaplan-Meier (KM) survival after transplantation of Npm1^cA/+/Flt3^ITD/+/Rosa^Cas9/+ cells treated with either Iso. (5 mg/kg, n=4) or mAb2 (5 mg/kg, n=5). Log-rank (Mantel–Cox) test was used for survival comparisons. Dashed vertical line denotes end of study sacrifice, deaths not due to illness.

D. Percentage of YFP+ MLL-AF9/Flt3^ITD/+ in peripheral blood (PB) 72 hours post-treatment with either Iso. (5 mg/kg) or mAb2 (5 mg/kg) (n=5, mean ± SD). Statistical significance as in A.

E. Spleen weight MLL-AF9/Flt3^ITD/+ in PB 72 hours post-treatment with either Iso. (5 mg/kg) or mAb2 (5 mg/kg) (n=5, mean ± SD). Statistical significance as in A.

F. KM survival after transplantation of MLL-AF9/Flt3^ITD/+ in PB 72 hours post-treatment with either Iso. (5 mg/kg, n=5) or mAb2 (5 mg/kg, n=5). Survival comparisons as in C.

G. KM survival after transplantation of MLL-AF9/Flt3^ITD/+ in NSG mice treated with either Iso. (5 mg/kg, n=7) or mAb2 (5 mg/kg, n=5). Survival comparisons as in C.

H. Spleen weight of NSG mice after transplantation of MLL-AF9/Flt3^ITD/+ treated with either Iso. (5 mg/kg) or mAb2 (5 mg/kg) (n=5, mean ± SD). Significance as in A.

I. KM survival after transplantation of OCI-AML3 in CB17-SCID mice treated with either Iso. (10 mg/kg, n=4) or mAb2 (10 mg/kg, n=5). Survival comparisons as in C.

J. KM survival after transplantation of AML PDX (MLL-R) in CB17-SCID mice treated with either Iso. (10 mg/kg, n=5) or mAb2 (10 mg/kg, n=5). Survival comparisons as in C.

K. Percentage of human myeloid cells in the PB after transplantation of AML PDX (MLL-R) in CB17-SCID mice on day 24 post-transplantation and after two treatments of either Iso. (10 mg/kg) or mAb2 (10 mg/kg) (n=5, mean ± SD). Significance as in A.

To examine the potential toxicity that mAb2 treatment might have in vivo, we used WT (C57BL/6J) mice and injected them intraperitoneally (IP) with 3 different doses (2.5, 5, and 10 mg/kg) of mAb2 or an isotype control (5 mg/kg). No significant differences were observed in the PB counts or the total body weight of mice after 4 weekly treatments at any given dose (Extended Data Figure 5A). At the end of treatment, no significant weight differences were observed in the spleens, livers, or kidneys (Extended Data Figure 5B). We next examined the impact of mAb2 treatment in sub-lethally irradiated mice. After weekly injections of 10 mg/kg of mAb2 or 5 mg/kg of isotype control, there were no differences in blood counts or total body weight between treatment arms after 4 weeks (Extended Data Figure 5C). These mice were kept for 5 additional weeks after final treatment and there were still no detectable health issues or tissue weight changes observed (Extended Data Figure 5D). Overall, these data suggest that the mAb2 antibody is well-tolerated and shows no transient or lasting toxicity on normal hematopoiesis in vivo.

In vivo efficacy of mAb2 in mouse AML models

To establish if mAb2 has anti-leukemic efficacy we first used a primary mouse AML model driven by Npm1^cA/+/Flt3^ITD/+/Rosa^Cas9/+. This model possesses the highest levels of csNPM1 among the primary murine models tested (Figure 2B, Extended Data Figure 3C). We used sub-lethally irradiated, immunocompetent mice which were injected with primary murine Npm1^cA/+/Flt3^ITD/+/Rosa^Cas9/+ AML cells. After AML engraftment, mice were treated weekly with either isotype control or mAb2 at 5 mg/kg. Examining the PB 4 weeks post- treatment, mice treated with mAb2 had significantly lower levels of Cas9 transgene compared with those given isotype control (Figure 4A). Moreover, we observed significantly lower white blood cell (WBC) counts in mAb2 compared to isotype control, indicating decreased leukemic burden. With improved leukemia control, there was hematopoietic recovery as shown by WBC counts, red blood cells (RBC) and hemoglobin (HGB) returning to irradiation-only control levels (Extended Data Figure 6A, 6B). Because splenomegaly is one of the key indicators of AML engraftment and expansion in vivo, we examined spleen sizes and found that the mAb2-treated cohort had significantly smaller spleens as compared to the isotype-treated cohort (Figure 4B). Finally, examination of the overall survival demonstrated that weekly IP treatment with mAb2 significantly prolonged survival when compared to the isotype-treated cohort (Figure 4C). Overall, our results show that mAb2 treatment impaired the leukemic engraftment and expansion in a primary murine Npm1^cA/+/Flt3^ITD/+/Rosa^Cas9/+ AML model while significantly prolonging survival.

Based on mAb2’s efficacy in high-csNPM1 models, we next assessed its in vivo efficacy in AML models with lower levels of csNPM1. We used the primary murine MLL-AF9/Flt3^ITD/+ AML model which showed an intermediate amount of mAb2 binding (Extended Data Figure 3C4). These cells, which contain a YFP marker, were transplanted and treated as above. We observed that the AML burden (YFP+) in PB was significantly lower in the mAb2-treated cohort compared to isotype control after just 72 hours of treatment (Figure 4D). Total blood counts further showed that markers of leukemia were reduced in the treated cohort while again RBCs, HGB, and PLT were all increased, suggesting improved hematopoiesis (Extended Data Figure 6C). Notably, the leukemia-infiltrated spleen, liver, and lungs were significantly smaller in the mAb2-treated cohort than the isotype control suggesting strong anti-leukemic efficacy in those tissues (Figure 4E, Extended Data Figure 6D). Treatment with mAb2 significantly prolonged survival (Figure 4F), indicating that mAb2 can also effectively target AML with intermediate csNPM1 levels.

To establish the killing mechanism of mAb2, we transplanted MLL-AF9/Flt3^ITD/+ AML into immunocompromised mice (NSG) which lack natural killer (NK) cells to participate in an Fc-mediated ADCC and also have an impaired complement system. In NSG mice, we did not observe any differences in the overall survival or the spleen size of mice treated with mAb2 compared to isotype control (Figure 4G, 4H). These findings suggest that mAb2’s mechanism of action is dependent on ADCC and/or CDC. To further investigate our findings we used CB17-SCID mice, which are deficient in B and T cells, but have intact innate immunity, including complement⁴¹. CB17-SCID mice harboring human OCI-AML3 cells had significantly improved survival in the mAb2-treated cohort (Figure 4I) solidifying that the anti-leukemic effect we observe is immune-mediated.

Finally, to assess whether mAb2 has the potential to target human AML patient cells in vivo, we performed flow cytometry analysis to examine the csNPM1 levels in a patient-derived xenograft (PDX) model driven by an MLL-rearrangment (MLL-R), which are seen in 40% of pediatric leukemia and 5-12% of adult AML⁴². We found high csNPM1 levels on the engrafted PDX model whereas the host mouse BM had significantly lower csNPM1 levels (Extended Data Figure 6E, 6F). Finally, mAb2 treatment of CB17-SCID mice bearing this PDX led to significant survival prolongation (Figure 4J) and reduced PDX engraftment (Figure 4K). These results suggest that mAb2 is highly efficacious in vivo and in the context of clinically relevant human AML.

mAb2 targets leukemic stem cells

It has been reported that L-GMP and CD93+ subpopulations are linked to the generation and maintenance of AMLs driven by MLL^43,44. Using the primary murine MLL-AF9/Flt3^ITD/+ AML model, we observed considerable csNPM1 levels detected on the surface of L-GMP+ and CD93+ populations compared to the isotype control (Figure 5A). Additionally, there was a significant reduction of L-GMP+ and CD93+ subpopulations in the mAb2-treated cohort when compared to the isotype control mice (Figure 5B). To show that mAb2 treatment leads to functional impairment of leukemic stem cells, we performed secondary transplantations using the engrafted BM from leukemic mice treated with either mAb2 or isotype control. Following secondary transplantation, mice were left untreated and we observed that secondary recipients from the mAb2 cohort showed an improved overall survival as compared to isotype control (Figure 5C). These results indicate that mAb2 treatment targets AML stem cell- or leukemia-propagating compartments.

Figure 5. mAb2 treatment is targeting key LSCs.

A. Histogram of live cell flow cytometry from primary murine MLL-AF9/Flt3^ITD/+ L-GMP+ (left) and CD93+ (right) LSC populations stained with an Iso. (gray) or mAb2 (red) antibody.

B. Percentage of L-GMP+ (left) or CD93+ (right) cells in the bone marrow of MLL-AF9/Flt3^ITD/+ model treated with either Iso. (n=5) or mAb2 (n=5). Statistical significance was determined by two-tailed Mann–Whitney U test and bars showing the median ± SD.

C. Kaplan-Meier survival after re-transplantation of cells isolated from primary recipients transplanted with MLL-AF9/Flt3^ITD/+ and treated with either Iso. (n=5) or mAb2 (n=5). Log-rank (Mantel–Cox) test was used for survival comparisons.

D. Histograms of live cell flow cytometry using WT, NPM1^cA/+ and NPM1^cA/+/Flt3^ITD/+ primary murine bone marrow HSPCs (Lineage negative) stained with an Iso. (gray) or mAb2 (red) antibody.

E. Flow cytometry analysis of a healthy donor BM sample (Set B, #3). Gates first identify CD45dim, then lineage negative, then CD34+, and finally displays mAb2 or Iso. antibody binding. The percent positive of each population is noted in the panels.

F. Flow cytometry analysis as in (A) here of an AML patient BM (Set B, #2).

G. Dot plot of the percent NPM1+ cells from OCI-AML3 cells and Set B donor and patient BM samples. Whole CD45+ vs. CD45dim, CD3-, CD19-, CD34+ cells are analyzed separately. Statistical significance was determined by an unpaired t-test, p-value noted. Bars displayed are median values.

H. MFI analysis of mAb2 stained cells from (C) Unpaired t-tests was used to calculate significance between the healthy and AML BM samples. Statistical significance was determined by an unpaired t-test, p-values shown. Bars displayed are median values.

I. Bar plot of the percent NPM1+ cells from OCI-AML3 cells, human CD34+ enriched healthy cells, or 13 BM samples from AML patients (Set C). Data are stratified by CD34 positive (red) or CD34 negative (blue) and the NPM1c status is noted below each sample.

Furthermore, to assess the potential of csNPM1 as a marker of leukemia initiation and progression, we examined mAb2 surface staining of isogenic primary murine BM HSPCs (lineage negative) from WT, Npm1^cA/+ or Npm1^cA/+/Flt3^ITD/+ mice. In this model, Npm1^cA/+ has a pre-leukemic phenotype, while Npm1^cA/+/Flt3^ITD/+ mice have overt leukemia. There was a clear ‘stage-dependent’ increase in mAb2 surface binding where Npm1^cA/+/Flt3^ITD/+ cells had evidently more csNPM1 than the Npm1^cA/+ pre-leukemic cells, or the WT bone marrow, which had little to no surface binding (Figure 5D). Taken together, these findings suggest that csNPM1 may be preferentially expressed in the leukemia initiating cell fraction in murine models of leukemia.

We coarsely assessed NPM1 binding in the HSPC population from primary patient leukemia samples. We defined HSPCs as CD45dim/CD3-/CD19-/CD34+ cells⁴⁵. As expected, this immature population was in low abundance in healthy bone marrow (Figure 5E, Set B) and poorly stained with mAb2 over the isotype signal (13.4% positive, Figure 5E). Examining a bone marrow aspirate from an AML patient demonstrated a markedly expanded CD45dim population, representing malignant blasts. This patient’s blasts had aberrant CD19 expression, which is a relatively common phenomenon in AML⁴⁶. Within this sample, there was a minor population of CD19-/CD3-/CD34+ HSPCs which were demonstrably better bound by mAb2, both in percent positive (63%) and fluorescent intensity (Figure 5F).

To understand how this pattern looks across AML patient sample Set B (3 healthy and 6 AML bone marrow aspirates), we first looked at all CD45+ cells, where 34-54% of healthy and 14-61% of AMLs were mAb2+, with the reference OCI-AML3 as 100% positive (Figure 5G). However, when next considering the CD45dim, CD34+, CD3-, and CD19- stem-like population, only 8-13% of healthy but 25-75% of AML were mAb2+ (Figure 5G). Motivated by the intensity difference seen between Figure 3A and 3B, we next quantified the MFI of mAb2 binding on CD45dim/CD34+/CD3-/CD19- HSPCs. HSPC populations from AML samples had significantly higher MFI values of mAb2 binding as compared to the homogeneously low MFI values observed on healthy HSPCs (Figure 5H). This set of healthy and patient bone marrow samples suggests that, by both fraction-bound and intensity-bound, mAb2 selectively binds csNPM1 on the surface of primitive malignant cells. We expanded this type of analysis to examine the 13 patient samples in Set C (Figure 5I, Extended Data Figure 7A-7C). NPM1c leukemic blasts are generally CD34- negative, unlike most other AML subtypes. However, studies characterizing the minor fraction of CD34+ cells from NPM1c samples show that mutated NPM1 protein can also be found in this subset. Additionally, when CD34+ cells from NPM1c samples are transplanted, they can reconstitute a CD34- leukemia, thus suggesting CD34+ cells from NPM1c are leukemia initiating cells⁴⁷. First looking at one AML patient (Set C, AML BM #12) we observed that csNPM1 expression among CD45dim cells is predominantly within the CD34+ fraction (Extended Data Figure 7A). Analyzing the other 12 patients as well as CD34+ enriched healthy donor cells showed that csNPM1 was upregulated in the CD34 subset of all AML samples (Extended Data Figure 7B, 7C, 7E). The CD45dim/CD34+ fraction from NPM1c samples had the most csNPM1 signal as compared to CD34- blasts. Overall, in human samples there is a clear preferential binding for csNPM1 in the HSPC population of malignant marrows that is not present in the healthy marrows. Additional studies will be needed to assess whether this binding is preferential to bona fide human leukemic stem cells.

mAb2 enables in vivo targeting of solid tumors

Given the genotype-agnostic binding of mAb2 across a diverse set of AML models and primary samples, we hypothesized that csNPM1 could also mark solid tumors. To assess this, we measured mAb2 binding across 47 human and mouse solid tumor models from various tissue origins using flow cytometry (Figure 6A, Table S4). We saw varying degrees of mAb2 binding in multiple tissue types. There was no binding in B16F10 murine melanoma and little binding in multiple models of human pancreatic cancer. Given the binding on solid tumors, we next sought to assess whether we could achieve in vivo efficacy with mAb2. We turned to a syngeneic model of prostate cancer, where primary normal murine prostate epithelial cells had little to no mAb2 binding while primary murine prostate carcinoma cells (Pten^-/-Tp53^-/-) had clear staining above isotype (Figure 6B). After engrafting syngeneic tumors in C57BL/6 mice, animals were treated on days 5, 7, and 9 with 10 mg/kg of mAb2 or isotype control. We found that mAb2 treatment reduced the tumor volume while isotype treated animals had significantly larger tumors at 14 and 17 days post engraftment (Figure 6C, 6D). To extend this observation beyond prostate carcinoma, we also evaluated the activity of mAb2 against a murine colorectal cancer model (MC38) which is also bound by mAb2 (Figure 6E). In this syngeneic model, we also observed significant reduction in tumor volume at 10 and 13 days post transplantation in C57BL/6 mice (Figure 6F, 6G). Finally, to establish the specificity of this effect, we examined the activity of mAb2 on the murine melanoma line B16F10, which has no significant mAb2 binding (Figure 6A, 6H). As expected, treatment of B16F10-transplanted syngeneic mice with mAb2 has no impact on the tumor volume when compared to isotype treated controls (Figure 6I). These data suggest that mAb2 induces anti-tumor activity in vivo in csNPM1-positive solid tumors.

Figure 6. mAb2 stains multiple solid cancer models and impairs in vivo tumor growth.

A. Percent NPM1+ cells from flow cytometric analysis of 47 human and mouse solid cancer models from various tissue origins (Table S4).

B. Histogram of live cell flow cytometry from primary, healthy murine prostate epithelial cells (left) or primary murine Pten^-/-/Tp53-/- prostate carcinoma cells (right) stained with an Iso. (gray) or mAb2 (red) antibody.

C. Tumor volume of the primary murine Pten^-/-/Tp53^-/- prostate carcinoma model following treatment with either Iso. (n=3) or mAb2 (n=3) (mean ± SD). Syngeneic tumors were engrafted subcutaneously and treatment began after development of palpable tumors. Red arrows indicate the relevant timepoints of treatment. Statistical significance was determined by two-tailed Mann–Whitney U test.

D. Primary murine Pten^-/-/Tp53^-/- prostate carcinoma tumors from the animal cohorts shown in (C), treated with either the Iso. (top) or mAb2 (bottom) and dissected on day 17 post-transplantation.

E. Histogram of live cell flow cytometry from the mouse colorectal cancer model MC38 stained with an Iso. (gray) or mAb2 (red) antibody.

F. Tumor volume of the mouse colorectal cancer model MC38 following treatment with either Iso. (n=4) or mAb2 (n=4) (mean ± SD). Syngeneic tumors were engrafted subcutaneously and treatment began after development of palpable tumors. Red arrows indicate the relevant timepoints of treatment. Statistical significance was determined by two-tailed Mann–Whitney U test.

G. MC38 tumors from the animal cohorts shown in (F), treated with either the Iso. (top) or mAb2 (bottom) and dissected on day 13 post-transplantation.

H. Histogram of live cell flow cytometry from the mouse melanoma cancer model B16F10 stained with an Iso. (gray) or mAb2 (red) antibody.

I. Tumor volume of the mouse melanoma cancer model B16F10 following treatment with either Iso. (n=4) or mAb2 (n=5) (mean ± SD). Syngeneic tumors were engrafted subcutaneously and treatment began after development of palpable tumors. Red arrows indicate the relevant timepoints of treatment. Statistical significance was determined by two-tailed Mann–Whitney U test.

Discussion

Here we show that NPM1, a frequently mutated protein in AML, and often dysregulated protein in malignancy, can be found on the cell surface. Since csRBPs like NPM1 lack a transmembrane and GPI-linked anchors, we focused on the molecular organization of csNPM1 by combining mass spectrometry and super-resolution microscopy methods. Cell surface proximity labeling defined many other csRBPs and glycoRNAs within the molecular neighborhoods of csNPM1, consistent with our recent observation of csRBPs clusters (e.g. DDX21, hnRNP-U etc.) on other cell types²⁶. Similarly, the SR reconstructions of csNPM1 on AML cells are highly similar to those of DDX21 and hnRNP-U on adherent cell lines²⁶; such a non-random, structured organization suggests the existence of an underlying regulatory mechanism guiding the nanoscale arrangement of these clusters.

Although this study does not fully elucidate the mechanism that drives the surface expression of csNPM1, we do identify that csNPM1 from membrane fractions is bound by sialic acid specific lectins. Given the predicted O-linked glycosylation sites in NPM1, these findings would suggest that NPM1 can be glycosylated. Protein glycosylation is not only critical to appropriate folding, but it can dictate trafficking, which may in turn be indispensable for NPM1’s cell surface localization. Interestingly, others have identified that cell surface nucleolin is glycosylated, and this modification is important for its localization⁴⁸. There is much to be learned about the mechanisms that govern how csRBPs are glycosylated, and how this might relate to malignancy.

Aberrant protein expression and localization is a common feature of malignancies. Recent work from our groups has shown the glycoRNA-csRBP clusters can serve as regulators of cell penetrating peptide entry²⁶, while others have shown csRBPs can bind viruses and mediate ligand internalization⁴⁹. Given these findings and the commonality of csNPM1 in multiple models of malignancy shown here, it suggests that csNPM1 and its molecular neighborhood on the cell surface may play a functional role in tumorigenesis. Future work enabled by new tools to manipulate csRBPs directly could allow for a direct understanding of csNPM1’s (and other csRBPs) impact on cellular state.

In this study, we focused on csNPM1 as a target for opsonization and immune mediated destruction of target cells. This approach, if successfully translated to humans, offers significant advancements on existing anti-cancer therapies. Firstly, csNPM1 expression was mutation agnostic, which enables its potential use in a multitude of patients. Current advancements in AML therapy have used small molecules that work on a subset of patients based on their driver mutations^50,51. Secondly, other approved anti-AML therapies that are not mutation specific work by inducing DNA damage, which can cause off-target toxicities⁵². Within the healthy hematopoietic system, csNPM1 has limited expression, and importantly is not present on healthy HSCs. Whereas most approved therapies drive life-threatening cytopenias by also affecting normal hematopoiesis⁵³, we do not see this in mouse models. Upon mAb2 treatment, we saw improvement in blood counts, which indicate simultaneous eradication of leukemia and unperturbed normal hematopoiesis.

Further, there were no observable weight changes in mice to suggest broader toxicity, which is reassuring as NPM1 is well-conserved between human and mouse. Ultimately, non-human primate and human studies will be needed to assess whether csNPM1 is expressed on vital non-hematopoietic tissue. If it were seen, one interesting prospect is to develop antibodies against mutant NPM1, as we observed both wildtype and mutant forms on NPM1-mutant models. By targeting a mutant form with an antibody, one could limit off-target effects further. Interestingly, given that there are other RBPs mutated in AML⁵⁴, if they are also found to be on the cell surface, they could represent novel targets.

Finally, an important goal in AML therapy development has been to target leukemic stem cells⁵⁵. In our mouse models, we indeed see that csNPM1 is a marker of LSCs, which may in part explain the significant survival benefit offered by anti-csNPM1 therapy. Though our study is not powered to assess human LSCs, we do see increased anti-NPM1 expression in the CD34+ fraction of NPM1c AML marrows, which are notable for having CD34- blasts. This would suggest that there is a primitive population that has csNPM1 in these samples, which we do not observe in healthy bone marrow tissue. Phenotypically, LSCs are defined by their ability to initiate leukemia in limiting dilution transplantation experiments⁵⁵. Therefore, a focus of future efforts will be to characterize csNPM1+ leukemic cells in transplantation models that have been created to foster human leukemogenesis. LSCs are better characterized in mouse leukemia models^43,44, and as we illustrated in this study, csNPM1 is upregulated in well-defined and clinically relevant murine LSCs.

The finding of csNPM1 prompts a need to reevaluate cell surface proteomics to ensure that non-canonical findings are studied, rather than discarded as noise. With multiple examples now of cancer specific csRBPs (csNPM1, csSNRNP200¹⁹, etc.), there is a clear need to better investigate their biology in other normal and abnormal contexts. Beyond monoclonal antibodies, these proteins could serve as optimal targets for ADCs and chimeric antigen receptor cell products, thus bringing novel therapies to the patients who need them most.

Methods

Cell culture

All cells were cultured at 5% CO₂ and 37°C. Suspension cell lines including OCI-AML2, OCI-AML3, Nalm6, MOLM-13, Jurkat, Jeko1, SupT1, K562, HL-60, and Kasumi1 were maintained by spinning cells down, removing the medium, and resuspending cells in fresh complete media. Suspension cell cultures were split when cell density reached 2 million cells per mL. Culture media used was 1x RPMI-1640 base media (ThermoFisher Scientific) with 1% Pen/Strep and 10% Heat Inactivated Fetal Bovine Serum (FBS, ThermoFisher Scientific). OCI-AML2 and OCI-AML3 were cultured in MEM-alpha (Fisher Scientific) supplemented with 20% FBS (Fisher Scientific) and 1% Pen/Strep. A549, MC38, A673, SH-SY5Y, H4, U251, MDA-MB-231, B16F10 and primary mouse PDAC1-10¹ were cultured in DMEM (Fisher Scientific) supplemented with 10% FBS (Fisher Scientific) and 1% Pen/Strep. LA-N-5 was cultured in DMEM (Fisher Scientific) supplemented with 20% FBS (Fisher Scientific) and 1% Pen/Strep. KNS-42 was cultured in DMEM (Fisher Scientific) supplemented with 10% FBS (Fisher Scientific), 1% NaPy and 1% Pen/Strep. UT-SSC-42B was cultured in DMEM (Fisher Scientific) supplemented with 10% FBS (Fisher Scientific), 1% NEAA and 1% Pen/Strep. Calu-1, HCT-116, MHH-ES-1 and U2OS were cultured in McCoy’s 5A (Fisher Scientific) supplemented with 10% FBS (Fisher Scientific) and 1% Pen/Strep. SaOS were cultured in McCoy’s 5A (Fisher Scientific) supplemented with 15% FBS (Fisher Scientific) and 1% Pen/Strep. HOS, MG-G3, FADU, DETROIT, HT1080, HPAF-II were cultured in MEM (Fisher Scientific) supplemented with 10% FBS (Fisher Scientific) and 1% Pen/Strep. NCI-H520, KYSE-30, KYSE-140, SiMa, PFSK1, PC3, 22Rv1, ASPC1, BXPC3, SU86.86 and YAPC were cultured in RPMI-1640 (ThermoFisher Scientific) supplemented with 10% FBS (Fisher Scientific) and 1% Pen/Strep. OE21 and OE33 were cultured in RPMI-1640 (ThermoFisher Scientific) supplemented with 10% FBS (Fisher Scientific), 2% L-Glutamine (ThermoFisher Scientific) and 1% Pen/Strep. All the primary murine AML cells were cultured in X-VIVO 20 media with Gentamicin and PR1 (Lonza) supplemented with 5% FBS, recombinant murine IL3 (10 ng/mL; Peprotech) IL6 (10 ng/mL; Peprotech) and SCF (50 ng/mL; Peprotech), and 1% penicillin/streptomycin/glutamine (Gibco). Cell lines were acquired from ATCC and Sanger Institute Cancer Cell Collection unless otherwise noted. Full list of the cell lines source is provided in the reporting summary. Cell cultures were periodically checked for and maintained as mycoplasma negative. Human cell lines employed were either not listed in the cross-contaminated or misidentified cell lines database curated by the International Cell Line Authentication Committee (ICLAC) or were previously verified by karyotyping.

Ex vivo culture of murine primary leukemia

Flt3^ITD/+ (Flt3 internal tandem duplication) mice were kindly provided by Gary Gilliland and crossed with Rosa26^Cas9/+ mice. Freshly isolated bone marrow from 6- to 10-week-old female Flt3^ITD/+; Rosa26^Cas9/+ or moribund Npm1^{flox− cA/+}; Flt3^ITD/+; Rosa26^Cas9/+, Npm1^{flox− cA/+}; Nras^G12D/+ mice were used. Bone marrow cells were exposed to erythrocyte lysis (BD PharmLyse, BD Bioscience), followed by magnetic bead selection of Lin^- cells using the Lineage Cell Depletion Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Lin^- were cultured in X-VIVO 20 (Lonza) supplemented with 5% FBS (Life Technologies) 10 ng/mL IL3 (Peprotech), 10 ng/mL IL6 (Peprotech) and 50 ng/mL of SCF (Peprotech) and 1% penicillin/streptomycin/glutamine. Retroviral constructs pMSCV-MLL-AF9-IRES-YFP and pMSCV-MLL-ENL-IRES-Neo were used with package plasmid psi-Eco to produce retrovirus. 293T cells (Life Technologies) were cultured and prepared for transduction in 10 cm plates. For virus production, 5 μg of the above plasmids and 5 μg psi-Eco packaging vector were transfected drop wise into the 293T cells using 47.5 μL TransIT LT1 (Mirus) and 600 μL Opti-MEM (Invitrogen). The resulting viral supernatant was harvested as previously described. Transduction of primary Flt3^ITD/+; Rosa26^Cas9/+ mouse cells was performed in 6-well plates as mentioned above. After transduction, transduced cells were sorted for YFP (for MLL-AF9) or selected with neomycin (for MLL-ENL).

Dissection and culture of primary mouse prostate tissue

Normal prostate tissue derived from Rosa26-LSL-Cas9 knock-in mice on B6J (strain #26175, Jackson Laboratories) and Pten^-/-/Tp53^-/- prostate tumors derived from a mouse model of prostate cancer of the same genetic background akin to Feng et al². Tissues were minced into small pieces and transferred into a gentleMACS C tube (Miltenyi Biotec) for enzymatic digestion using the Multi Tissue Dissociation Kit 1 (catalog #130-110-201, Miltenyi Biotec). Digestion was performed in a gentleMACS Dissociator utilizing program 37C_Multi_A. Following enzymatic dissociation, the cell suspension was pelleted, resuspended, and filtered through a 70- μm cell strainer. Normal and tumoral dissociated prostate cells were cultured as 3D organoids using the protocol and medium composition described by Drost et al³. Cultures were maintained under these conditions for 6-7 days to enrich for epithelial cells, after which cells were maintained as monolayer cultures on plates coated with collagen I (#354236, Corning) using the same medium composition.

Lentiviral vector production and infection

For virus production, 293FT cells were transfected with the lentiviral vector (LentiCRISPR-v2) containing either an empty vector or the NPM1 guide RNA together with the packaging plasmids psPAX2 (Addgene:12260) and pMD2.G (Addgene: 12259). The viral supernatant was harvested 48 and 72 hour after transfection and concentrated overnight at 6000g at 4°C. 1x10⁶ cells and viral supernatant were mixed in 2 ml culture medium supplemented with 8 μg/mL polybrene (Millipore) followed by spinfection (60 min, 900g, 32°C) and were further incubated overnight at 37°C. The medium was refreshed on the following day and the transduced cells were cultured further. Pellets for protein were collected 7- and 9-days post-transduction.

Protein extraction and western blot for knockout experiment

OCI-AML2 cells were transduced with either NPM1 gRNAs or control gRNAs. Cell pellets collected on day 7 post-transduction and lysed with whole-cell lysis buffer (0.2% Nonidet NP-40, 50 mM Tris-HCl pH 8.0, 450 mM NaCl, 1 mM EDTA), 1x protease inhibitor cocktail 1 (Merck), 1x phosphatase inhibitor 2 (Merck), 1x phosphatase inhibitor 3 (Merck), and 1 mM DTT (Epigentek)) and incubated on ice for 10 minutes. The lysates were then centrifuged at 20,000 x g and 4°C for 10 minutes. The supernatant was transferred to a fresh tube and quantified using Bradford Assay (BioRad). Following quantification, the samples were supplemented with 1x LDS sample buffer (ThermoFisher Scientific) and 1x Sample Reducing agent (ThermoFisher Scientific) and they were then incubated at 70°C for 10 minutes. 10 μg of protein was loaded. Western blot was performed using SDS-PAGE gels and blotted onto a PVDF membrane. It was performed using the following antibodies NPM1 (FC8791) H3 (Abcam, ab1220) as a loading control in 1:1000 dilution and goat anti-mouse IgG H&L HRP- conjugated (Abcam, ab205719) in 1:10000 dilution.

Blood Counting

For blood counts, 20 μL of blood was collected from the tail-vein of the mice using a capillary pipette containing anticoagulants (EDTA). The EDTA anticoagulated blood samples were used to obtain a complete blood count with a VetABC analyzer (Horiba ABX). Samples were counted no longer than 20 minutes after blood was drawn.

Real-time PCR

For Figure 4A, genomic DNA was extracted from murine peripheral blood using the DNeasy Blood & Tissue Kit (Qiagen). Genomic DNA (10 ng) was used and the levels of Cas9 and Gapdh were analyzed on a QuantStudio™ 5 real-time PCR instrument (Applied Biosystems) using PowerUp™ SYBR™ Green Master Mix (Applied Biosciences). The relative quantification of Cas9 was performed using the comparative cycle threshold (Ct) method against the housekeeping gene Gapdh. The primer sequences are listed in Table S3.

PCR

For Extended Data Figure 6B, genomic DNA was extracted from murine peripheral blood two weeks post-injection using the DNeasy Blood and Tissue kit (Qiagen). Primers for Flt3-ITD were used for the PCR amplification of the genomic DNA (20ng). The PCR product was analyzed by an agarose gel electrophoresis stained with GelRed. 1kb plus DNA ladder (NEB) was used.

Antibody staining and flow cytometry analysis of AML cells

For primary murine AML experiments related to Figures 2B, Extended Data Figure 2B and Extended Data Figure3B, 6- to 10-week-old C57BL/6 male mice were injected with 10⁶ primary murine AML cells by intravenous injection from the indicated animal AML models, as described above. Upon animal sickness, bone marrow (BM) was isolated and lysed in 0.85% NH₄Cl for 5 minutes. Primary antibodies, in a concentration of 0.5 μg per reaction, either anti-NPM1 (mAb2) or IgG2a-isotype control (BioXCell) were pre-complexed with 1:1000 dilution of the secondary antibody Goat Anti-Mouse IgG, Alexa Fluor 488 (Abcam) for 30 minutes. For the intracellular staining cells were first permeabilized with 0.1% Triton X100 (Sigma) for 10 minutes at room temperature and rinsed with 2% FBS in 1x PBS. For cell surface staining (“Live cell”) cells were processed directly. Bone marrow cells were blocked in 2% FBS in 1x PBS for 30 min on ice and then stained with the pre-complexed mix of antibodies as stated above. Cells were washed once with 150 µL of 2% FBS in 1x PBS, resuspended in 2% FBS in 1x PBS containing 0.1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI, Sigma). For intracellular staining cells were fixed with 4% formaldehyde followed by a wash with 2% FBS in 1x PBS. Flow cytometry analysis was performed using Cytoflex (Beckman Coulter) and analyzed using FlowJo (v10, BD).

For primary murine AML experiments related to Figures 4D, 6- to 10-week-old C57BL/6 male mice were sub-lethally irradiated with a whole body dose of 5.5 Gy and then injected with 10⁶ primary murine AML cells by intravenous injection from the indicated animal AML model, as described above. On day 15 post-transplantation, mice were treated intraperitoneally (IP) with a single dose of either 5 mg/kg of mouse IgG2a isotype control antibody or 5 mg/kg of anti-NPM1 (mAb2) antibody. On day 18 post-transplantation, peripheral blood was isolated and lysed in 0.85% NH₄Cl for 5 minutes. Flow cytometric analysis of YFP+ cells was performed as above.

For Figure 2C, K562 cells were transduced with the lentiviral cDNA constructs pKLV-TY1-NPM1-PURO, pKLV-TY1-NPM1c-PURO (mutant NPM1) or an empty pKLV-TY1-PURO vector control. Transduced cells were selected for puro and then live cells were stained with either a mouse anti-B23 NPM1 antibody (Merck) or a mouse anti-TY1 antibody (Diagenode) for 45 minutes followed by a staining with 1:1000 dilution of the secondary antibody Goat Anti-Mouse IgG Alexa Fluor 488 (Abcam) for 45 minutes. Cells were washed once with 2% FBS in 1x PBS and finally resuspended in 2% FBS in 1x PBS containing 0.1 µg/mL DAPI. Flow cytometry analysis was performed as above.

For Extended Data Figure 2G, K562 and OCI-AML3 cells were incubated with human Fc block (Biolegend, cat. No. 101319). Afterward, they were incubated with WGA conjugated to fluorescein (Vector Laboratories, cat. No. FL-1021-5) at a concentration of 1:1000. When co-labeled with Fc8791 or mAb2, antibodies were precomplexed with anti-mouse IgG Alexafluor 647 in a ratio of 2:1 prior to being incubated with cells. Final concentration of Fc8791 and mAb2 were 5ug/mL and secondary was used at 2.5ug/mL. Cells were washed after binding, subsequently stained with DAPI, and then applied to slides using CytoSpin. Images were obtained on a Leica TCS SP8.

For AML PDX experiments related to Extended Data Figures 6E-F, 6- to 10-week-old SCID-CB17 female mice (Charles River, Strain: 236) were injected with 10⁶ patient-derived AML cells by intravenous injection. Bone marrow (BM) was isolated and lysed in 0.85% NH₄Cl for 5 minutes. Primary antibodies, in a concentration of 0.5 μg per reaction, either anti-NPM1 (mAb2) or IgG2a-isotype control (BioXCell) were pre-complexed with 1:1000 dilution of the secondary antibody Goat Anti-Mouse IgG Alexa Fluor 488 (Abcam) for 30 minutes. Bone marrow cells were blocked in 2% FBS in 1x PBS for 30 min on ice and then stained with the pre-complexed mix of antibodies as stated above. Cells were washed once with 150 µL of 2% FBS in 1x PBS, resuspended in 2% FBS in 1x PBS containing 0.1 µg/mL DAPI. Flow cytometry analysis was performed as above.

For primary murine experiments related to Figure 5D, freshly isolated bone marrow from male 20-week-old WT and pre-leukemic Npm1^{flox− cA/+} or moribund Npm1^{flox− cA/+}; Flt3^ITD/+ mice were used. Bone marrow cells were exposed to erythrocyte lysis (BD PharmLyse, BD Bioscience), followed by magnetic bead selection of Lin^- cells using the Lineage Cell Depletion Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Primary antibodies, in a concentration of 0.5 μg per reaction, either anti-NPM1 (mAb2) or IgG2a-isotype control (BioXCell) were pre-complexed with 1:1000 dilution of the secondary antibody Goat Anti-Mouse IgG Alexa Fluor 488 (Abcam) for 30 minutes. Bone marrow cells were blocked in 2% FBS in 1x PBS for 30 min on ice and then stained with the pre-complexed mix of antibodies as stated above. Cells were washed once with 150 µL of 2% FBS in 1x PBS, resuspended in 2% FBS in 1x PBS containing 0.1 µg/mL DAPI. Flow cytometry analysis was performed as above.

For primary murine AML experiments related to Figure 5A-B, 6- to 10-week-old C57BL/6 male mice were sub-lethally irradiated with a whole body dose of 5.5 Gy and then injected with 10⁶ primary murine AML cells by intravenous injection from the indicated animal AML model, as described above. Upon animal sickness on day 18 post-transplantation, bone marrow (BM) was isolated and lysed in 0.85% NH₄Cl for 5 minutes. BM cells were resuspended in 10% DMSO in FBS and stored in -80°C for further applications. BM cells were thawed suspended in PBS supplemented with 2% FBS and stained with Biotin anti-mouse Ly-6A/E (Sca-1) (Biolegend), biotin anti-mouse CD127 (Biolegend, cat. no. 135005), biotin anti-mouse CD3 (Biolegend, cat. no. 100201), biotin anti-mouse TER-119/Erythroid cells (Biolegend, cat. no. 116203), biotin anti-mouse/human CD45R/B220 (Biolegend, cat. no. 103203), BV605 anti-mouse Gr-1 (Biolegend, cat. no. 108439), BV650 anti-mouse CD11b (Biolegend, cat. no. 101239), PerCP/Cy5.5 anti-mouse CD16/32 (Biolegend, cat. no. 101323), PE anti-mouse CD93 (Biolegend, cat. no. 136503), APC anti-mouse CD48 (Biolegend, cat. no. 103411), APC/Fire 750 anti-mouse CD117 (c-kit) (Biolegend, cat. no. 135139), Zombie aqua viability dye (Biolegend, cat. no. 423101), and BV421 streptavidin (Biolegend, cat. no. 405226). All the antibodies were used in a 1:400 dilution apart from the viability dye which was used in a 1:1000 dilution. The samples were then stained for either anti-NPM1 (mAb2) antibody or IgG2a-isotype control (BioXCell, BE0085), pre-complexed with an anti-mouse IgG Alexa Fluor 594 (Abcam, cat.no. ab150108) as a secondary antibody. FMO controls were included in the experiments to provide a measure of spillover in each channel. This allows for correct gating in each experimental sample. Flow cytometry analysis was performed using Cytek Aurora Spectra Analyser and analyzed using FlowJo (v10, BD). Data in this section were plotted using GraphPad Prism (version 9).

Antibody staining and flow cytometry analysis of solid cancer models

Primary antibodies, in a concentration of 0.5 μg per reaction, either anti-NPM1 (mAb2) or IgG2a-isotype control (BioXCell) were pre-complexed with 1:1000 dilution of the secondary antibody Goat Anti-Mouse IgG, Alexa Fluor 488 (Abcam) for 30 minutes. 5 x 10⁴ of cells for each model used in Figure 6 (Figures 6A, B, E and H) was blocked in 2% FBS in 1x PBS for 30 min on ice and then stained with the pre-complexed mix of antibodies as stated above. Cells were washed once with 150 µL of 2% FBS in 1x PBS, resuspended in 2% FBS in 1x PBS containing 0.1 µg/mL DAPI (Sigma). Flow cytometry analysis was performed using Cytoflex (Beckman Coulter) and analyzed using FlowJo (v10, BD).

In vivo treatment of normal, primary murine AML and PDX models

For experiments related to Extended Data Figures 4D-E, 16- to 20-week-old C57BL/6 male mice were used that were housed at Boston Children’s Hospital. All mouse procedures and protocols were approved by the Animal Care and Use Committee of Boston Children’s Hospital and followed all relevant guidelines and regulations. After euthanization, peripheral blood, liver, spleen, and bone marrows were harvested.

For experiments related to Extended Data Figures 5A-B, 6- to 10-week-old C57BL/6 male mice were given intraperitoneal injections of either anti-NPM1 (mAb2) or IgG2a-isotype control (BioXCell, BE0085) antibody at the indicated doses, once per week for a total of four weeks tenure (total 4 treatments). Weights and peripheral blood from the tail vein were collected at the indicated time points. Weight measurements of the indicated mouse organs were taken from all treated-cohorts at the end of the study, on the 28^th day post-initiation of the relevant treatments.

For experiments related to Figure 4D-E and Extended Data Figures 5C-D, 6- to 10-week-old C57BL/6 male mice were sublethally irradiated with a whole body dose of 5.5 Gy. On day 12 post-irradiation mice were given intraperitoneal injections of either anti-NPM1 (mAb2) or IgG2a-isotype control (BioXCell, BE0085) antibody at the indicated doses, once per week for a total of four weeks tenure (total 4 treatments). Weights and peripheral blood from the tail vein were collected at the indicated time points. Weight measurements of the indicated mouse organs were taken from all treated-cohorts at the end of the study, on the 66^th day post-irradiation.

For primary murine NPM1c/Flt3^ITD/+/Cas9 AML experiments related to Figures 4A-C and Extended Data Figures 6A, 6- to 10-week-old C57BL/6 male mice were sub-lethally irradiated with a whole body dose of 5.5 Gy and then injected with 10⁶ primary murine AML cells by intravenous injection from the indicated animal AML model, as described above. On day 14 post-transplantation, mice were given intraperitoneal injections of either anti-NPM1 (mAb2) or IgG2a-isotype control (BioXCell, BE0085) antibody at the indicated doses, once per week for a total of 4 weeks tenure (total 4 treatments). Weight measurements of leukemic spleens were taken from each animal, after humane endpoints had reached.

For primary murine MLL-AF9/Flt3^ITD/+/Cas9 AML experiments related to Figure 4G-H, 6- to 10-week-old NSG male mice were injected with 10⁶ primary murine AML cells by intravenous injection from the indicated animal AML model, as described above. On day 14 post-transplantation, mice were given intraperitoneal injections of either anti-NPM1 (mAb2) or IgG2a-isotype control (BioXCell, BE0085) antibody at the indicated doses, once per week for a total of 3 weeks tenure (total 3 treatments). Weight measurements of leukemic spleens were taken from each animal, after humane endpoints had reached.

For xenotransplantation AML experiments related to Figure 4I, 6- to 10-week-old SCID-CB17 (Charles River, Strain: 236) female mice were injected with 2x10⁶ OCI-AML3 human AML cells by intravenous injection. On day 14 post-transplantation, mice were given intraperitoneal injections of either anti-NPM1 (mAb2) or IgG2a-isotype control (BioXCell, BE0085) antibody at the indicated doses, once per week for a total of 3 weeks tenure (total 3 treatments).

For AML PDX experiments related to Figure 4J-K, 6- to 10-week-old SCID-CB17 (Charles River, Strain: 236) female mice were injected with 10⁶ patient-derived AML cells by intravenous injection. On day 14 post-transplantation, mice were given intraperitoneal injections of either anti-NPM1 (mAb2) or IgG2a-isotype control (BioXCell, BE0085) antibody at the indicated doses, once per week for a total of 3 weeks tenure (total 3 treatments). Peripheral blood from the tail vein was collected on day 24 post-transplantation.

For primary murine MLL-AF9/Flt3^ITD/+/Cas9 AML experiments related to Figure 5C, 6- to 10-week-old C57BL/6 male mice were sublethally irradiated with a whole body dose of 5.5 Gy and then injected with 10⁶ primary murine AML cells from primary recipients. For the primary recipients (related to Figure 4D-E and Extended Data Figures 6C-D), 6- to 10-week-old C57BL/6 male mice were sublethally irradiated with a whole body dose of 5.5 Gy and then injected with 10⁶ primary murine AML cells by intravenous injection from the indicated animal AML model, as described above. On day 15 post-transplantation, mice were treated intraperitoneally (IP) with a single dose of either 5 mg/kg of mouse IgG2a isotype control antibody or 5 mg/kg of anti-NPM1 (mAb2) antibody. On day 18 post-transplantation, bone marrow (BM) was isolated and processed accordingly for secondary transplantation as indicated above. Moreover, on day 18 post-transplantation, peripheral blood from the tail vein as well as weight measurements of spleens, lung and liver were taken from each animal, after humane endpoints had reached.

All mice used in the study were housed in specific pathogen-free conditions in the UBS animal facilities of the University of Cambridge. All cages were on a 12:12-hour light:dark cycle (lights on, 07:30) in a temperature-controlled and humidity-controlled room. Room temperature was maintained at 72 ± 2°F (22.2 ± 1.1°C), and room humidity was maintained at 30–70%. The animals were culled when leukemia-associated symptoms or humane endpoints reached. All animal studies were carried out in accordance with the Animals (Scientific Procedures) Act 1986, UK and approved by the Ethics Committee at the University of Cambridge. Randomization and blinding were not applied. All data in this section were plotted using GraphPad Prism (Version 9).

In vivo treatment of mouse solid tumor models

For primary prostate carcinoma experiments related to Figure 6C-D, 8- to 10-week-old C57BL/6 male mice were injected with 10⁶ primary murine prostate carcinoma cells by subcutaneous injection (1:1 ratio of Matrigel and cancer cells) from the indicated animal model, as described above. For colorectal carcinoma experiments related to Figure 6F-G, 8- to 10-week-old C57BL/6 male mice were injected with 5 x 10⁵ MC38 cells by subcutaneous injection (1:1 ratio of Matrigel and cancer cells). For melanoma experiments related to Figure 6H-I, 8- to 10-week-old C57BL/6 male mice were injected with 5 x 10⁵ B16F10 cells by subcutaneous injection (1:1 ratio of Matrigel and cancer cells). On days 5, 7 and 9 post-transplantation, mice were given intraperitoneal injections of either anti-NPM1 (mAb2) or IgG2a-isotype control (BioXCell, BE0085) antibody at the indicated doses (total 3 treatments). Tumors were dissected when sizes were approaching or reached the humane end point limit (1.2cm²) as per animal license; on day 17 post-transplantation for prostate tumors, on day 13 post-transplantation for colorectal carcinoma tumors, and on day 11 post-transplantation for melanoma. All tumor sizes were measured using a digital caliper (Jodsen).. All solid tumor models used in the study were housed in specific pathogen-free conditions in the UBS animal facilities of the University of Cambridge,. All cages were on a 12:12-hour light:dark cycle (lights on, 07:30) in a temperature-controlled and humidity-controlled room. Room temperature was maintained at 72 ± 2°F (22.2 ± 1.1°C), and room humidity was maintained at 30–70%. The animals were culled when humane endpoints reached. All animal studies were carried out in accordance with the Animals (Scientific Procedures) Act 1986, UK and approved by the Ethics Committee at the University of Cambridge. Randomization and blinding were not applied. All data in this section were plotted using GraphPad Prism (Version 9).

Cell surface biotinylation, immunoprecipitation, and western blotting

K562 cells were cultured as above. Cell surface protein labeling was accomplished using sulfo-NHS-SS-biotin (APExBio) as described above after which crude membrane fractions were isolated. To obtain cytosolic and membrane fractions⁴, suspension cells were directly resuspended in Membrane Isolation Buffer (10mM HEPES (ThermoFisher Scientific), 250mM Sucrose (Sigma), 1mM EDTA) at 5 million cells per 1 mL. Adherent cells were collected off the plate by scraping in the ice cold PBS, pelleted, and then similarly resuspended at 5M cells / 1 mL of Membrane Isolation Buffer. Cells were rested on ice for up to 5 minutes, moved to a glass dounce homogenizer (Sigma), and then homogenized using 40-80 strokes to obtain a resuspension of approximately 50% released nuclei. Over douncing can cause nuclei rupture and contamination of the cytosolic fraction. After douncing, unbroken cells and nuclei were pelleted by spinning at 4°C for 10 minutes at 700x g. Supernatants (cytosol and membranes) were carefully transferred to a new tube and pellets discarded. The supernatants were again centrifuged at 4°C for 30 minutes at 10,000x g. Most (90%) of the supernatant was removed and saved as cytosolic fractions. The remaining supernatant was discarded as it was near to the membrane pellet. The membrane pellet was gently washed with 500 µL of ice cold 1x PBS (the pellet was not resuspended here), the tube spun down briefly, and all supernatant was discarded to leave a cleaned membrane pellet. Finally the membrane pellet was resuspended in 500 µL of CLIP lysis buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl (Sigma), 1 mM EDTA, 10% glycerol (ThermoFisher Scientific), 0.1% NP-40, 0.2% Triton-X100, 0.5% N-lauroylsarcosine). Both the cytosolic and membrane lysates were stored at -80°C for later processing. After isolation and solubilization with CLIP lysis buffer, total protein quantification occurred using the BCA assay. For each sample 5 μg of protein was used for input and 20 μg used for either anti-NPM1 (FC8791) coated bead Protein A Dynabeads (ThermoFisher Scientific) or streptavidin coated bead (MyOne C1 beads, ThermoFisher Scientific) enrichments. In both cases, 10 μL bead surrey was added to the membrane lysates in 100 μL of CLIP lysis buffer and binding occurred at 4°C for 16 hours. After binding, the beads were washed three times with High Stringency buffer and then twice with 1x PBS. Proteins were released from the beads by heating at 85°C for 10 minutes in 20 µL 1x LDS (ThermoFisher Scientific) and 1 mM free biotin (ThermoFisher Scientific). Input and enriched proteins were analyzed by Western blot, staining with anti-NPM1 (Santa Cruz Biotechnology, sc-32256; FC8791) and streptavidin-IR800 (Li-Cor Biotechnology) and finally scanning on Li-Cor Odyssey CLx scanner. Fractionation as above was performed without cell surface biotinylation for data in Figure 1A and the western blots were developing using anti-NPM1 (Santa Cruz Biotechnology, sc-32256; FC8791), anti-NPM1c (ThermoFisher Scientific, 32-5200), anti-b-Actin (Santa Cruz Biotechnology, sc-47778), and anti-RPN1 (Santa Cruz Biotechnology, sc-48367).

mAb2 design and synthesis

VH and VL sequences were grafted to the constant region of the mouse IgG2a heavy chain and the mouse lambda light chain, respectively to generate a new mAb. These sequences were given to Curia Global. At Curia the gene synthesis process involves overlapping oligonucleotide synthesis and assembly, followed by cloning into Curia’s proprietary high expression mammalian vector. Production and mAb quality control was performed by Curia to produce a Protein A purified IgG fraction with low endo-toxin (< 1 EU / mg IgG) in 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4

Live and fixed cell staining (human samples), flow cytometry, and data analysis

Cells were cultured as above and directly counted. Typically, 50,000 cells were used and blocked with Human TruStain FcX (Fc block, BioLegend) or Mouse TruStain FcX (Fc block, BioLegend) in FACS buffer (0.5% BSA (Sigma) in 1x PBS) for at least 15 minutes on ice, cells were kept on ice from this point forward. For intracellular staining (“Fix/Perm”) cells were first fixed with 3.7% formaldehyde for 10 minutes at 25°C, rinsed once with 1x PBS, then permeabilized with 0.1% Triton X100 (Sigma) for 10 minutes at 25°C and finally rinsed once with 1x PBS. For cell surface staining (“Live cell”) cells were processed directly after the live Fc blocking on ice. To live or fixed cells, precomplexed antibodies were added. To precomplex, primary unconjugated antibodies including Mouse isotype (Santa Cruz Biotechnology, sc-2025), and anti-NPM1 (Santa Cruz Biotechnology, sc-32256; FC8791), and anti-Ty1 tag (Diagenode) were bound in solution (precomplexed) to a Goat anti-Mouse AF647 secondary antibody (ThermoFisher Scientific, A32728) or a Goat anti-Mouse AF488 secondary antibody (ThermoFisher Scientific, A28175) for at least 30 minutes on ice before using. The molar ratio was 2:1, primary:secondary. To the blocked cells, precomplexed antibody was added to a final concentration of 1 µg/mL (primary antibody) and allowed to bind to cells for 60 minutes on ice. After staining cells were spun at 4°C for 3 minutes at 400x g and the supernatant discarded. All cell spins took place using these conditions. Cells were washed once with 150 µL of FACS buffer, spun under the same conditions, and finally resuspended in FACS buffer containing 0.1 µg/mL DAPI. Data collection occurred on a BD Biosciences LSRFortessa 3 and a gating strategy was used to isolate live, single cells, to examine antibody binding using FlowJo Software (v10, BD).

For the frozen peripheral blood (PB) and bone marrow (BM) samples obtained from healthy donors as well as AML patients were processed with care to ensure as little cell lysis and high viability after thawing. Vials were warmed in a 37°C water bath for 2-3 minutes and then completely thawed in 5 mL of ice cold FACS buffer. Cells were pelleted from this initial resuspension at 400 x g for 5 minutes at 4°C. The supernatant was discarded, cells were resuspended in 1 mL of fresh ice cold FACS buffer and counted. For each sample 1M cells per mL were taken for Fc blocking and staining as per the above protocol. Next we stained the cells using the live protocol with the precomplexed isotype or anti-NPM1 (mAb2) antibodies for 30 minutes on ice in 100 µL FACS buffer. After which we spun the cells down and discarded the supernatant as before. The cells were then stained again with 200ng of dye-conjugated cell-type specific antibodies (anti-human CD45 (HI30), 304024; anti-human CD34 (581), 343516; anti-human CD117 (104D2), 313204; anti-human CD33 (WM53), 303416; anti-human CD13 (WM15), 301710; anti-human HLA-DR (L243), 307604; anti-human CD3 (OKT3), 317308; and/or anti-human CD19 (HIB19), 302206; all BioLegend) on ice for 30 minutes in 100 µL FACS buffer. Finally the cells were pelleted again, supernatant discarded, once 200 µL FACS buffer wash was performed, and the cells were finally resuspended in 200 µL of FACS buffer with 0.1 µg/mL DAPI for FACS analysis. Healthy donor and AML patient samples were obtained with informed consent under: 1) UK ethical approval (IRAS ref: 340167, previously 149581, REC 07-MRE05-44). Additionally, healthy donor and AML patient samples were collected from patients located at the Dana Farber Cancer Institute (DFCI) or Brigham and Women’s Hospital (USA). Samples are then processed and banked in the Pasquerello Tissue Bank in accordance with IRB-22-160 at DFCI, we obtained assistance from Hematologic Malignancies Data Repository to identify patient samples from the tissue bank that were bona fide AML. HMDR also provided relevant information regarding disease characteristics, such as cytogenetics, mutations and immunophenotype. Samples were banked in accordance with the DFCI Protocol 01-206: tissue and data collection for research studies in patients with hematologic malignancies, bone marrow disorders and normal donors. Sample characteristics were obtained using DFCI IRB Protocol 22-160. This is a DFCI specific tissue banking protocol, which is not available publicly for review but it can be shared upon request.

Confocal microscopy sample preparation, data acquisition, and analysis

For suspension cells, culturing, counting, and Fc blocking were carried out as above. Samples for “Live Cell” imaging were processed as per the live cell flow cytometry noted above; however after staining and washing, cells were fixed with 3.7% formaldehyde (37% stock, Sigma) for 30 minutes at 25°C. Primary and secondary antibodies noted above were used but sequentially, rather than precomplexed: Primary antibody was added at a final concentration of 2.5 µg/mL for 45 minutes on ice in FACS buffer. After staining, cells were washed twice with 1x PBS and then stained with a secondary antibody at a final concentration of 2.5 µg/mL. Secondary stains occurred for 30 minutes on ice and in the dark, after which cells were washed once with 1x PBS. A final fixation for the “Fix/Perm” samples was performed in parallel with the “Live Cell” samples with 3.7% formaldehyde in 1x PBS for 30 minutes at 25°C in the dark. Nuclei were stained with 0.1 µg/mL DAPI in FACS buffer for 15 minutes at 25°C. Suspension cells were applied to glass slides using a CytoSpin centrifuge (Fisher Scientific): this was accomplished by centrifugation at 500x g for 5 minutes on a CytoSpin 1867. Finally, cells were mounted in ProLong Diamond Antifade Mountant (ThermoFisher Scientific) and a coverglass was sealed over the sample with nail polish. All samples were then imaged on a Leica TCS SP8 STED ONE microscope. For all experiments, at least three regions of interest (ROIs) were acquired using a 63x oil immersion objective across one or more z-slices. Leica’s line-sequential scanning method was used and images were acquired at 1024 by 1024 resolution with a pinhole size of 1 AU. The DAPI channel was acquired with a PMT detector while all other channels were imaged using Hybrid detectors. For Extended Data Figure 2I DAPI channel was imaged using Hybrid detector and images were analyzed using Image J (v1.54f). Then, using Imaris Microscopy Image Analysis software (Oxford Instruments), single slices of the confocal stack were analyzed.

Super-resolution imaging and reconstruction

Cells were prepared as described above for live cell imaging. Here, primary conjugated antibodies (AF647) were used directly for cell labeling and included Mouse isotype (Santa Cruz Biotechnology, sc-24636-AF647) and anti-NPM1 (Santa Cruz Biotechnology, sc-32256-AF647; FC8791). To avoid cell movement during the SR acquisition, cells were immobilized on glass bottom well plate pre-coated with Poly-L-lysine (Sigma, P4707) and Cell-Tak (Corning, 354240). An overview of the method and processing can be found in Extended Data Figure 3A. For single-molecule super-resolution microscopy we used direct stochastic optical reconstruction microscopy (dSTORM). To perform this, the PBS in which the cells were stored was replaced by a reducing oxygen scavenging buffer to induce blinking of fluorophores as described in literature⁵. The blinking buffer consisted of 2 μL/mL catalase (Sigma), 10% (w/v) glucose (BD Biosciences), 100 mM Tris-HCl (ThermoFisher Scientific), 560 μg/mL glucose oxidase (Sigma), and 20 mM cysteamine (Sigma). First, diffraction-limited images were obtained with low-intensity illumination of few W/cm². Then, the laser power was increased to approximately 3 kW/cm². Image acquisition was started after a short delay in order to ensure that most fluorophores were shelved into a dark state. The exposure time was 50 milliseconds (ms), and approximately 40000 frames were recorded.

As the data was obtained with a sCMOS camera, which typically exhibit few pixels with deviating sensitivity (“hot” and “cold” pixels), the obtained single-molecule data was corrected for individual pixels with abnormally high or low sensitivity first⁶. 4000 frames of a raw data stack were averaged. Hot and cold pixels, which are a systematic deviation, persist, in contrast to the random single-molecule signals. Each pixel was compared to its neighbors, using 8-connectivity. If a deviation of more than 3% from the median of the neighboring pixel was observed, a correction factor that set the pixel to the median of its neighbors was recorded as previously described⁷. Otherwise, the pixel was not considered to be significantly brighter or darker. This yielded a correction mask which was applied to all frames of the raw data. Finally, the no-light counts were subtracted from the pixel-corrected data.

For localization of single molecules, the Fiji plugin ThunderSTORM was used⁸. Each camera frame was filtered with a B-spline filter of order 3 and scale 2. Local maxima, corresponding to single-molecule signals, were detected with 8-neighborhood connectivity and a threshold of 1.1 or 1.2 times the standard deviation of the first wavelet level. Detected local maxima were fitted with a 2D-Gaussian using least squares, and the position was recorded. Next, to account for single-molecule signals being active in multiple frames, merging of localizations was performed, using a maximum distance of 30 nm and maximum of 5 off frames with no limit regarding on frames. Cross correlation-based drift correction (magnification 5, bin size 5) was performed, followed by filtering of the localizations (sigma of the point spread function (PSF) between 60 and 270 nm, intensity below 37800 photons, localization uncertainty smaller than 30 nm). For visualization the final localizations were reconstructed as 2D histograms with magnification of 5 (corresponding to a pixel size of 17.7 nm).

For cluster analysis, an automated pipeline was established, using the raw list of localizations. Frist, Ripley’s H-function was calculated on three areas with a large number of well-separated clusters. The resulting preferential cluster size from the three areas (which was, notably, always very similar) was averaged, multiplied by a correction factor of 0.45, and used as the seed radius for the following DBscan analysis. This DBScan script yielded all individual clusters, the total number of clusters, the average number of points per cluster and the spatial relation between Clusters. The identified final clusters were then analyzed with respect to their spatial relation, size, and number of localizations. This unbiased approach follows recommended analysis procedures recently described⁹. Crucially, each dataset was subjected to identical postprocessing and cluster analysis steps with no manual intervention, thus avoiding any biases arising from different parameter settings. Custom scripts used for this analysis can be shared upon request.

Cell surface proximity labeling of proteins, peptide generation, and mass spec data analysis

Samples were prepared similarly to the flow cytometry workflow as described above. However, rather than dye-conjugated secondaries, here a horseradish peroxidase (HRP) conjugated secondary (ThermoFisher Scientific, 31430) was used. The isotype (control) or anti-NPM1 (FC8791) (target) primary unconjugated antibody was precomplexed with an appropriate secondary-HRP antibody at 2:1, primary:secondary for at least 30 minutes on ice. Cells were grown as biological triplicate cultures and typically 2.5 million cells were used per replicate per labeling experiment. Cells were harvested from culture, washed of culture media, and resuspended on ice cold FACS buffer to which the Fc blocker was added for at least 15 minutes. After blocking cells were adjusted to 1 million cells per mL of FACS buffer and then the precomplexed antibodies were added for staining to a final concentration of 2.5 µg/mL. Staining occurred for 60 minutes at 4°C on rotation, after which cells were pelleted, supernatants discarded and cells washed once in ice cold 1x PBS. This wash is important to remove excess BSA in the FACS buffer. Next, cells were gently but quickly resuspended in 980 µL of 100 µM biotin-phenol (Iris Biotech) in 1x PBS at 25°C. To this, 10 µL of 100 Mm H₂O₂ (insert manufacturer) was quickly added, tubes capped and inverted, and the reaction allowed to proceed for 2 minutes at 25°C. Precisely after 2 minutes, the samples were quenched by adding sodium azide and sodium ascorbate to a final concentration of 5 mM and 10 mM, respectively. Samples were inverted and cells pelleted at 4°C. Samples were then washed sequentially once with ice cold FACS buffer and then twice ice cold 1x PBS, after which cell pellets were lysed in 500 µL of CLIP lysis buffer, briefly sonicated to solubilize chromatin, and frozen at -80°C for later processing.

Once all the proximity labeling was complete, lysates were thawed in batches to perform the following steps in parallel. Total protein amounts were quantified using the BCA assay and labeling efficiency and consistent was checked using Western blotting. For biotin enrichment, we used the streptavidin western QC to determine the biotin signal across all samples first, then calculated the total µg of lysate that was needed from that sample to generate 5,000,000 units of streptavidin-IR800 signal on the LiCor. That µg value was then used as input mass for each of the replicates across all of the proximity labeling samples for the biotin capture and MS prep. Samples were normalized to a final volume of 500 µL with CLIP lysis buffer and to each sample 100 µL of Pierce NeutrAvidin Agarose (ThermoFisher Scientific) was added and incubated at 4°C for 4 hours on rotation. Beads were then washed twice with 1 mL of High Stringency, twice with 1 mL of 4 M NaCl in 100 mM HEPES all at 37°C. Salts and detergents were then rinsed from the beads by sequentially washing twice with 1 mL 1x PBS, twice with 1 mL of LC-MS grade water (Fisher Scientific) and finally with 1 mL of 50 mM ammonium bicarbonate. An on-bead trypsin digestion was then set up by adding 200 µL of 50 mM ammonium bicarbonate and 1 µg MS-grade Trypsin to each sample and incubating for 16 hours at 37°C with occasional shaking. After digestion, the samples were acidified by adding formic acid to a final concentration of 0.5%. The solution containing released peptides was moved to a new tube and the beads were rinsed twice with 300 µL LC-MS grade water to capture any remaining peptides. All elution and wash samples for a given replicate were combined and SpeedVac’ed to a volume of < 200 µL. Samples were then desalted using a C18 spin column (ThermoFisher Scientific): preconditioned with 50% methanol in water, washed twice with 5% acetonitrile, 0.5% formic acid in water, the sample bound twice to the column, then the columns washed four times with 0.5% formic acid in water. Finally the peptides were eluted into Protein LoBind tubes (Eppendorf) with two applications of 40 µL of 70% acetonitrile, 0.5% formic acid in water. Organic solvents were removed with a SpeedVac and samples were fully dried with a lyophilizer. Resulting peptides were analyzed on the timsTOF Pro as described above.

For all peptides generated, we followed the same procedure for mass spectrometry analysis and peptide database searching. Specifically, a nanoElute was attached in line to a timsTOF Pro equipped with a CaptiveSpray Source (Bruker, Hamburg, Germany). Chromatography was conducted at 40°C through a 25 cm reversed-phase C18 column (PepSep) at a constant flow rate of 0.5 µL/minute. Mobile phase A was 98/2/0.1% water/acetonitrile/formic acid (v/v/v) and phase B was acetonitrile with 0.1% formic acid (v/v). During a 108 minute method, peptides were separated by a 3-step linear gradient (5% to 30% B over 90 minutes, 30% to 35% B over 10 minutes, 35% to 95% B over 4 minutes) followed by a 4 minute isocratic flush at 95% for 4 minutes before washing and a return to low organic conditions. Experiments were run as data-dependent acquisitions with ion mobility activated in PASEF mode. MS and MS/MS spectra were collected with m/z 100 to 1700 and ions with z = +1 were excluded. Raw data files were searched using PEAKS Online Xpro 1.7 (Bioinformatics Solutions Inc., Waterloo, Ontario, Canada). The precursor mass error tolerance and fragment mass error tolerance were set to 20 ppm and 0.03, respectively. The trypsin digest mode was set to semi-specific and missed cleavages was set to 3. The human Swiss-Prot reviewed (canonical) database version 2020_05 (downloaded from UniProt) and the common repository of adventitious proteins (cRAP v1.0, downloaded from The Global Proteome Machine Organization) totaling 20,487 entries were used. Carbamidomethylation was selected as a fixed modification. Oxidation (M), Deamidation (NQ) and Acetylation (N-term) were selected as variable modifications. Raw data files and searched datasets are available on the Mass Spectrometry Interactive Virtual Environment (MassIVE), a full member of the ProteomeXchange consortium under the identifier: MSV000092211. The complete searched datasets are also available in our supplementary files.

To identify enriched proteins from these datasets we took an approach that compared enrichment in the NPM1 to that of isotype antibodies. Results of the database search were first purged of non-human proteins and keratins as previously described. All proteins with fewer than two unique peptides were also filtered out of the list. Next, Excel was used to calculate the mean spectral count for each of the remaining proteins across triplicates. For each protein, the mean spectral counts associated with each antibody probe were divided by the mean spectral counts of the corresponding isotype, creating an enrichment factor of each protein in the proximity labeling over the isotype. Python (v3.13) was then used to calculate the ratio of total protein isolated from proximity labeling with protein-targeting antibodies divided by that collected with isotype antibodies. Proteins from each set of proximity labeling data with enrichments either less than two or the previously described ratio were filtered from the dataset. The resultant lists comprise the hits associated with each round of proximity labeling. To perform GO-term analysis, all proteins observed across all four fractions from each cell line were concatenated, creating a list of background proteins from each of the four cell lines tested. Lists of hits from the membrane hits pulldown from each cell line were submitted to DAVID (https://david.ncifcrf.gov/) and ran against their respective backgrounds. Then, for each category of GO-term (BP, CC, and MF), the union of the top four terms across all cell lines and their associated sizes and Benjamini values was plotted for comparison of enrichment across cell lines.

Anti-NPM1 Molecular Counting

OCI-AML3 cells were harvested, washed in FACS buffer and resuspended at 10e6 cells/mL. They were then blocked with Human TruStain FcX for 15 minutes with 5uL of Fc block per million cells. They were washed again afterward. The cells were then partitioned for live cell and fixed cell staining. Cells planned for fixation were stained with 1uL of fixable violet live/dead per 1e6 cells/mL for 30 minutes. Afterward, cells were washed with FACS buffer and resuspended in 4% PFA for 10 minutes at room temperature. They were washed with FACS buffer then resuspended in 0.1% Triton-X for 5 minutes at room temperature. Again the cells were washed in FACS buffer. Live cells and fixed cells were then incubated with 500ng of Fc8791-AlexaFluor 647 in 100uL for 30 minutes. Cells were washed twice afterwards, and live cells were finally resuspended in DAPI. For molecular counting, Quantum Alexa Fluor 647 molecules of equivalent soluble fluorochrome (MESF) beads were used from Bangs Labs. All samples were analyzed on BD LSRFortessa. Standard curves and fluorescence quantitation was performed as per Bang Labs’ QuickCal analysis tool.

Cell surface proximity labeling of RNAs and gel analysis

Samples were prepared here in a similar manner as described above for the proximity label of proteins with key differences. The HRP-conjugated secondary antibody was switched for Protein A-HRP (Cell Signaling) and used at the same molar ration; 2 primary antibodies per 1 Protein A-HRP molecules. The biotinylation reagent used here was biotin-aniline (bioAn, Iris Biotech), it was used at 200 µM final concentration in 1x PBS for the labeling reaction, and the labeling reaction was allowed to proceed for 3 minutes at 25°C. After pelleting the cells from the quenching reaction, cells were directly lysed and total RNA isolated as described before¹⁰. Briefly, RNAzol RT (Molecular Research Center, Inc.) was used to lyse cell pellets by placing the samples at 50°C and shaking for 5 min. To phase separate the RNA, 0.4X volumes of water was added, vortexed, let to stand for 5 minutes at 25°C and lastly spun at 12,000x g at 4°C for 15 min. The aqueous phase was transferred to clean tubes and 1.1X volumes of isopropanol was added. The RNA is then purified over a Zymo column (Zymo Research). For all column cleanups, we followed the following protocol. First, 350 μL of pure water was added to each column and spun at 10,000x g for 30 seconds, and the flowthrough was discarded. Next, precipitated RNA from the RNAzol RT extraction (or binding buffer precipitated RNA, below) is added to the columns, spun at 10,000x g for 10-20 seconds, and the flowthrough is discarded. This step is repeated until all the precipitated RNA is passed over the column once. Next, the column is washed three times total: once using 400 μL RNA Prep Buffer (3M GuHCl in 80% EtOH), twice with 400 μL 80% ethanol. The first two spins are at 10,000x g for 20 seconds, the last for 30 seconds. The RNA is then treated with Proteinase K (Ambion) on the column. Proteinase K is diluted 1:19 in water and added directly to the column matrix and then allowed to incubate on the column at 37°C for 45 min. The column top is sealed with either a cap or parafilm to avoid evaporation. After the digestion, the columns are brought to room temperature for 5 min; lowering the temperature is important before proceeding. Next, eluted RNA is spun out into fresh tubes and a second elution with water is performed. To the eluate, 1.5 μg of the mucinase StcE (Sigma-Aldrich) is added for every 50 μL of RNA, and placed at 37°C for 30 minutes to digest. The RNA is then cleaned up again using a Zymo column. Here, 2X RNA Binding Buffer (Zymo Research) was added and vortexed for 10 seconds, and then 2X (samples + buffer) of 100% ethanol was added and vortexed for 10 seconds. The final RNA is quantified using a Nanodrop. In vitro RNase or Sialidase digestions took place by digesting 50 μg total RNA with either, nothing, 4 μL RNase Cocktail (ThermoFisher Scientific), or 4 μL of α2-3,6,8,9 Neuraminidase A (NEB) in 1x NEB Glyco Buffer #1 (NEB) for 60 minutes at 37°C. After digestion, RNA was purified using a Zymo column as noted above and was then ready for gel analysis.

In order to visualize the labeled RNA, it is run on a denaturing agarose gel, transferred to a nitrocellulose (NC) membrane, and stained with streptavidin¹⁰. After elution from the column as described above, the RNA is combined with 12 μL of Gel Loading Buffer II (GLBII, 95% formamide (ThermoFisher Scientific), 18 mM EDTA (ThermoFisher Scientific), 0.025% SDS) with a final concentration of 1X SybrGold (ThermoFisher Scientific) and denatured at 55°C for 10 minutes. It is important to not use GLBII with dyes. Immediately after this incubation, the RNA is placed on ice for at least 2 minutes. The samples are then loaded into a 1% agarose, 0.75% formaldehyde, 1.5x MOPS Buffer (Lonza) denaturing gel. Precise and consistent pouring of these gels is critical to ensure a similar thickness of the gel for accurate transfer conditions; we aim for approximately 1 cm thick of solidified gel. RNA is electrophoresed in 1x MOPS at 115V for between 34 or 45 min, depending on the length of the gel. Subsequently, the RNA is visualized on a UV gel imager, and excess gel is cut away; leaving ~0.75 cm of gel around the outer edges of sample lanes will improve transfer accuracy. The RNA is transferred with 3M NaCl pH 1 (with HCl) to an NC membrane for 90 minutes at 25°C. Post transfer, the membrane is rinsed in 1x PBS and dried on Whatman Paper (GE Healthcare). Dried membranes are rehydrated in Intercept Protein-Free Blocking Buffer, TBS (Li-Cor Biosciences), for 30 minutes at 25°C. After the blocking, the membranes are stained using streptavidin-IR800 (Li-Cor Biosciences) diluted 1:5,000 in Intercept blocking buffer for 30 minutes at 25°C. Excess streptavidin-IR800 was washed from the membranes using three washes with 0.1% Tween-20 (Sigma) in 1x PBS for 3 minutes each at 25°C. The membranes were then briefly rinsed with PBS to remove the Tween-20 before scanning. Membranes were scanned on a Li-Cor Odyssey CLx scanner (Li-Cor Biosciences).

Extended Data

Extended Data Figure 1. Organization of csNPM1 by confocal and super-resolution microscopy.

A. Histogram plotting the number of each RBP (y-axis) that was found in a given set of cell surface proteomics experiments (surfaceomes, x-axis) as curated in²⁶.

B. Western blot analysis of cytosolic (C) or membrane (M) lysates from 9 human leukemia cell lines. Red asterisks denote the molecular weight of the target protein. The subtle difference in NPM1c’s molecular weight is highlighted by a blue dashed line and arrows.

C. Higher exposure western blot panels from (B). Breakout panels below highlight the specific migration of NPM1c at the blue arrow. This band is only present in the OCI-AML3 cell line.

D. Confocal microscopy images of OCI-AML3 cells as in Figure 1A, here stained with the mouse isotype antibody. Scale bars are 10 μm.

E. Confocal microscopy images of OCI-AML3 cells as in Figure 1B, here stained with the mouse isotype antibody and biotinylated WGA. Scale bars are 10 μm.

F. Histogram of each amino acid of human NPM1 scored by the NetOGlyc4.0 algorithm. Residues predicted to be a site of O-GalNAc modification are highlighted in red. Protein domains of NPM1 are annotated as defined by the Uniprot ID P06748.

G. Western blot analysis of Lectin IP (SNA) from membrane lysates of K562 cells treated with or without sialidase. Input and IP samples were blotted for NPM1. Expected molecular weights of NPM1 is indicated with a red asterisk.

H. Individual DL (left columns, grayscale) and SR (right columns, colored scales) images of each individual cell. Scale bars are noted on each image. For SR panels, individual color bars are provided to indicate the relative signal intensity. Two cells have an orange asterisk in the bottom left corners, indicating these are the cells presented in Figure 1.

I. Schematic of the experimental, data acquisition, and data analysis pipeline used to collect and analyze SR reconstructions.

J. Quantitative flow cytometry of OCI-AML3 cells to quantify the amount of intracellular and cell surface NPM1. MESF is a measure of fluorescence intensity on the flow cytometer. F:P is the ratio of AF647 dyes per antibody molecule of the FC8791 stock that was used.

Extended Data Figure 2. Characterization of the novel anti-NPM1 antibody mAb2.

A. Mean fluorescence intensity (MFI) analysis of 9x human leukemia cell lines.)

B. Histograms of flow cytometry of the Npm1c/Flt3^ITD/+ model isolated from bone marrow lysates and then grown ex vivo for 7 days. The top row is flow cytometry on live cells, the bottom cellswere permeabilized and fixed before adding the anti-NPM1 or isotype antibodies..

C. Amino acid sequence comparison of the C-terminal domain of NPM1-WT (43 amino acids shown) from Homo sapiens (human), Rhesus Macaque (macmu), and Mus musculus (mouse), as well as the NPM1c mutant. The NPM1c 45 amino acids were used to identify the scFv mAb2 is designed on.

D. Histogram of a denaturing (SDS) capillary electrophoresis run of the expressed mAb2.

E. Western blot analysis of whole cell extract (WCE) or crude membrane fractions (Mem) from K562 cells. Membranes were stained with either FC8791 or mAb2.

F. Confocal microscopy images of OCI-AML3 cells. Top row is live cell stained while the bottom row is stained on fixed and permeabilized cells with antibodies indicated. DAPI shown in blue. Scale bars are 10 μm.

G. Confocal microscopy images of OCI-AML3 and K562 cells. Cells were stained either with an isotype control or mAb2 (yellow) and a co-stain with biotinylated WGA (green). Brightfield images are also shown with a triple merge (last column). DAPI shown in blue. Scale bars are 10 μm.

H. Confocal microscopy images of OCI-AML3 cells. Cells were stained either with an isotype control or anti-NPM1 (FC8791, red) and a co-stain with mAb2 (red). The third column is a merged presentation to highlight overlapping signals. DAPI shown in blue. Scale bars are 10 μm.

I. Confocal microscopy images of primary MLL-ENL/Flt3^ITD/+ bone marrow cells. Cells were stained either with an isotype control or mAb2 (green). DAPI shown in blue. Scale bars are 10 μm.

J. Flow cytometry analysis of live OCI-AML2 cells that have been subjected to genetic ablation of NPM1 and stained with isotype (control, gray), mAb2 (red), or anti-CD47 (blue).

K. Western blot analysis of native and NPM1 knockout cells blotting for NPM1 or histone H3 (H3) as a loading control.

Extended Data Figure 3. mAb2 stains multiple primary murine AML models.

A. Flow cytometry analysis of an AML patient bone marrow (BM) sample (Set A, #3). The three gates here demonstrate isolation of single, live cells. This gating strategy is representative of all flow cytometry in this study of patient samples.

B. Flow cytometry analysis of OCI-AML3 cells and three AML patient bone marrow (BM) samples (Set D, #1-3). For each sample, cells were stained comparing either mouse isotype to FC8791 (left column) or mouse isotype to mAb2 (right column).

C. Histograms of live cell flow cytometry of NPM1^cA/+/Flt3^ITD/+, MLL-AF9/Flt3^ITD/+, MLL-ENL/Flt3^ITD/+ and Npm1c/Nras^G12D/+ primary murine cells stained with an isotype (gray) or anti-NPM1 (mAb2) (red) antibody.

Extended Data Figure 4. mAb2 poorly stains the healthy hematopoietic niche with no observed in vivo toxicity.

A. Bar plot of the percent NPM1+ cells from peripheral blood (PB) samples of three independent healthy human donors. Populations analyzed include CD45+, CD3+, CD19+, CD33+ SSC-hi, CD33+ SSC-low. Error bars plotted are standard deviation (SD).

B. Bar plot as in (A), analyzing cells from BM samples of three independent healthy human donors. The CD45dim CD34+ population is additionally analyzed in BM samples. Error bars plotted are SD.

C. MFI analysis of mAb2 stained cells from (B) plotted on a log₁₀ scale. OCI-AML3 cells are included as a positive control. Unpaired t-tests were used to calculate significance between the CD34+ cells and the other mature lineage cells, p values shown. Error bars plotted are SD.

D. Flow cytometry analysis of a healthy mouse BM sample. The gates presented sequentially show: lymphocytes, single cells, single cells, live cells, Lin-, Kit vs. Sca1, and finally mAb2 or isotype staining on the Kit+ or Kit+. Sca1+ cells. This gating strategy is representative of all flow cytometry in this study of murine samples.

E. Dot plot of the percent NPM1+ cells from BM samples of three independent healthy mouse donors. Populations analyzed include Mac1+, Gr1+; Mac1+, Gr1-; CD3+; CD19+; Lin-, Kit+; and Lin-, Kit+, Sca1+. Bars plotted are mean values.

Extended Data Figure 5. Poor presentation of csNPM1 on normal mouse bone marrow.

A. Blood count results and total body weight from an animal experiment with WT C57BL/6J mice following 4 weekly treatments with either 5mg/kg isotype control (Iso.) or 2.5mg/kg, 5 mg/kg, and 10 mg/kg of mAb2 (n=5 mice used in each group). Statistical significance was determined by two-tailed Mann–Whitney U test and box plots showing median, IQR, and extremes. Whiskers extend from minimum to maximum values in all the box plots presented.

B. Spleen, liver, and kidney weight from WT C57BL/6J mice following 4 weekly treatments with either Iso. or 2.5, 5, and 10 mg/kg of mAb2 (n=5 mice used in each group) (mean ± SD). Statistical significance was determined by two-tailed Mann–Whitney U test.

C. Blood count results and total body weight from an animal experiment with sub-lethally irradiated WT C57BL/6J mice following 4 weekly treatments with either 5mg/kg Iso. or 10 mg/kg of mAb2 (n=5 mice used in each group). Statistical significance was determined by two-tailed Mann–Whitney U test and box plots showing median, IQR, and extremes. Whiskers extend from minimum to maximum values in all the box plots presented.

D. Spleen, liver, and kidney weight from sub-lethally irradiated WT C57BL/6J mice following 4 weekly treatments with either 5mg/kg Iso. or 10 mg/kg of mAb2 (n=5 mice used in each group) (mean ± SD). Statistical significance was determined by two-tailed Mann–Whitney U test.

Extended Data Figure 6. mAb2 treatment reduces leukemic burden in mice.

A. Blood count results after transplantation of Npm1^cA/+/Flt3^ITD/+/Rosa^Cas9/+ in sub-lethally irradiated WT C57BL/6J mice treated with either Iso. (5 mg/kg) or mAb2 (5 mg/kg) (n=4, mean ± SD. Statistical significance was determined by a two-tailed Mann–Whitney U test.

B. DNA gel of genotyping PCR of the PB on day 14 post transplantation of Npm1^cA/+/Flt3^ITD/+/Rosa^Cas9/+ in sub-lethally irradiated WT C57BL/6J mice treated with either Iso. (5 mg/kg) or mAb2 (5 mg/kg).

C. Blood count results after transplantation of MLL-AF9/Flt3^ITD/+ in sub-lethally irradiated WT C57BL/6J mice treated with either Iso. (5 mg/kg) or mAb2 (5 mg/kg) (n=5, mean ± SD). Statistical significance was determined by two-tailed Mann–Whitney U.

D. Liver and lung weight of MLL-AF9/Flt3^ITD/+ murine AML mice treated with either Iso. (5 mg/kg) or mAb2 (5 mg/kg) (n=5, mean ± SD). Statistical significance was determined by Mann–Whitney two-tailed U test.

E. Histogram of live cell flow cytometry on BM-derived AML PDX (MLL-R) stained with Iso. (gray) and mAb2 (red).

F. Histogram of live cell flow cytometry on host normal BM (SCID) from the PDX shown in (D).

Extended Data Figure 7. mAb2 binding to primary AML bone marrow and leukemic stem cells.

A. Flow cytometric gating strategy for deriving CD45dim blast cells and CD45 bright mature cells, and NPM1 expression within these subsets.

B. Gating strategy showing cell purity from CD34 enriched healthy control.

C. Histograms of live flow cytometry from CD34 enriched healthy control, OCI-AML3, and 13 AML bone marrow aspirates. When measurable, NPM1 positivity is shown within the CD45dim CD34+ and CD45dim CD34- populations for each AML bone marrow aspirate.

Data Availability

All raw data files and searched datasets related to our proteomics experiments are available on the Mass Spectrometry Interactive Virtual Environment (MassIVE), a full member of the ProteomeXchange consortium under the identifier: MSV000092211. Other requests for materials should be directed to the corresponding authors.

Code Availability

No custom code was generated in this work. Adobe Illustrator CC (Adobe) was used for the generation of all the figures shown in this manuscript.