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. Author manuscript; available in PMC: 2024 Apr 12.
Published in final edited form as: Sci Transl Med. 2023 Sep 20;15(714):eadi7244. doi: 10.1126/scitranslmed.adi7244

TP63 fusions drive multicomplex enhancer rewiring, lymphomagenesis, and EZH2 dependence

Gongwei Wu 1,2,*, Noriaki Yoshida 1,18,#, Jihe Liu 3,#, Xiaoyang Zhang 1,4,5,#, Yuan Xiong 6,7,#, Tayla B Heavican-Foral 1, Elisa Mandato 1, Huiyun Liu 1, Geoffrey M Nelson 7,8, Lu Yang 9, Renee Chen 9, Katherine A Donovan 6,7, Marcus K Jones 1, Mikhail Roshal 10, Yanming Zhang 10, Ran Xu 1, Ajit J Nirmal 1, Salvia Jain 11, Catharine Leahy 1, Kristen L Jones 1, Kristen E Stevenson 1, Natasha Galasso 12, Nivetha Ganesan 12, Tiffany Chang 12, Wen-Chao Wu 1, Abner Louissaint 1,13, Lydie Debaize 1, Hojong Yoon 6,7, Paola Dal Cin 14, Wing C Chan 15, Shannan J Ho Sui 3, Samuel Y Ng 1,16, Andrew L Feldman 17, Steven M Horwitz 12, Karen Adelman 4,7, Eric S Fischer 6,7, Chun-Wei Chen 9, David M Weinstock 1,4,18,*, Myles Brown 1,2,*
PMCID: PMC11014717  NIHMSID: NIHMS1979348  PMID: 37729434

Abstract

Gene fusions involving tumor protein p63 gene (TP63) occur in multiple T- and B-cell lymphomas and portend a dismal prognosis for patients. The function and mechanisms of TP63 fusions remain unclear, and there is no target therapy for patients with lymphoma harboring TP63 fusions. Here we show that TP63 fusions act as bona fide oncogenes and are essential for fusion-positive lymphomas. Transgenic mice expressing TBL1XR1::TP63, the most common TP63 fusion, develop diverse lymphomas that recapitulate multiple human T- and B-cell lymphomas. Here, we identify that TP63 fusions coordinate the recruitment of two epigenetic modifying complexes, the nuclear receptor co-repressor (NCoR) - histone deacetylase 3 (HDAC3) by the N-terminal TP63 fusion partner and the lysine methyltransferase 2D (KMT2D) by the C-terminal TP63 component, which are both required for fusion-dependent survival. TBL1XR1::TP63 localization at enhancers drives a unique cell state that involves upregulation of MYC and the polycomb repressor complex 2 (PRC2) components EED and EZH2. Inhibiting EZH2 with the therapeutic agent Valemetostat is highly effective at treating transgenic lymphoma murine models, xenografts and patient-derived xenografts (PDXs) harboring TP63 fusions. One patient with TP63-rearranged lymphoma showed a rapid response to Valemetostat treatment. In summary, TP63 fusions link partner components that together coordinate multiple epigenetic complexes, resulting in therapeutic vulnerability to EZH2 inhibition.

One-Sentence Summary

TP63 fusions coordinate multiple epigenetic complexes, drive lymphomagenesis, and create a therapeutic vulnerability to EZH2 inhibition.

Introduction

Recurrent chromosomal rearrangements causing gene fusions are common across cancers (1, 2). Investigations of gene fusions have provided fundamental insights into mechanisms of tumorigenesis and risk stratification but only a small subset of these, such as BCR::ABL, PML::RARα, can be selectively targeted (3). The locus encoding the tumor protein p53 gene (TP53) analog tumor protein p63 gene (TP63) is rearranged in a diverse set of lymphomas, including 5-10% of ALK-negative anaplastic large cell lymphoma (ALK- ALCL), peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) and primary cutaneous ALCL, 7% of mycosis fungoides with large cell transformation (MF-LCT), and 1% of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL)(4–9). Patients with TP63-rearranged lymphomas have dismal outcomes, with 5-year overall survival rates between 0-17%, depending on cohorts (4, 5). TP63 fusions are also present in other cancer types (0.02% incidence overall), including lung adenocarcinoma, adenocarcinoma of unknown primary, glioblastoma multiforme, invasive breast carcinoma, and mucosal melanoma of the head and neck (10).

TP63 proteins are TP53 family members that have essential roles in the development and homeostasis of multiple different cell types (11). All TP63 fusion proteins reported to date lack the N-terminus of TP63 encoded by the three 5’ coding exons, which results in the same TP63 isoform ΔNp63 that has previously been shown to exert dominant negative activity on TP53 family members (6, 11). Transducin β-like 1-X-linked receptor 1 (TBL1XR1) is the fusion partner in most TP63 rearrangements (6, 8). TBL1XR1 is a component of both nuclear receptor corepressor (NCoR) and histone deacetylase 3 (HDAC3) complexes, which contribute to transcriptional regulation (12, 13). However, the mechanisms through which TBL1XR1::TP63 acts in tumorigenesis and the associated therapeutic vulnerabilities remain unknown. Here, we investigate the molecular mechanisms by which TP63 fusions drive lymphomagenesis and develop a therapeutic strategy for directly countering the oncogenic effects of TP63 fusions.

Results

Multiple TP63 fusions link interaction domains with ΔNp63 and promote lymphoma growth

We recently identified TP63 rearrangements in four human T-cell lymphoma (TCL) cell lines (14). SMZ1, DL-40, and MTA cell lines harbor the TBL1XR1::TP63 rearrangement and the HH cell line harbors the FOXK2::TP63 rearrangement (fig. S1A). The TBL1XR1::TP63 fusion constitutes the N-terminal portion of TBL1XR1 (LisH and F-box-like domains) and the C-terminal portion of TP63. The latter, also known as ΔNp63, includes the DNA-binding (DB) domain and an oligomerization (OD) domain but not the short N-terminal transactivation (TA) domain (Fig. 1A). We confirmed these genomic arrangements by both whole genome (table S1) and Sanger sequencing (fig. S1B). RNA sequencing (RNA-seq) of additional peripheral T-cell lymphoma (PTCL) patient samples identified a BCL6::TP63 fusion that fuses the N-terminal Broad-complex, Tramtrack, and Bric-à-brac/poxvirus and zinc finger (BTB/POZ) and proline, glutamic acid, serine, threonine-rich (PEST) domains of the oncogenic transcription factor B cell lymphoma 6 (BCL6) to ΔNp63 (Fig. 1A; fig. S1C and S1D).

Fig. 1. TP63 fusions promote lymphoma growth in vitro and in vivo.

Fig. 1.

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(A) Schematic of fusions TBL1XR1, FOXK2, BCL6, TP63, ΔNp63, TBL1XR1::TP63, FOXK2::TP63 and BCL6::TP63. Different colors show different domains. (B) Cell viability of TCL cell lines treated with shTP63 or shSeed. (C) Western blot analysis for TBL1XR1::TP63 in Cas9-SMZ1-sgwtTP63 cells expressing Dox-inducible shSeed or shTP63 treated with or without Dox for four days. (D) Cell proliferation assay of cells from (C) using CellTiter-Glo (CTG). (E) Tumor volume at the indicated timepoint after subcutaneous implantation of Cas9-SMZ1-sgwtTP63 cells with Dox-inducible shTP63 into mice (n=5 per group). The red arrow indicates the beginning of treatment with Dox or vehicle in vivo. (F) Rescue experiment of SMZ1 cells expressing shSeed or shTP63 with empty vector (EV) or TBL1XR1::TP63 expressing vector. (G) The proliferation of Ba/F3 cells expressing EV or TBL1XR1::TP63 cultured in the presence of the indicated concentrations of mIL-3 measured using CTG. Data are normalized to the empty vector (EV) group. (H) Western blot analysis using an anti-TP63 antibody in Cas9-SMZ1-sgwtTP63-shTP63 cells expressing Dox-inducible BCL6::TP63 fusion. Cells were treated with or without Dox for four days. (I) Cell proliferation assay of cells in (H) using CTG. (J, K) Normalized CRISPR score (NCS) of each sgRNA (dots) and the smoothed score (line) of the pooled TBL1XR1 and TP63 (J) and TBL1XR1::TP63 (K) survival screen on day 30 in SMZ1-Cas9+ cells.

All data unless specified are presented as mean ± SD or ±SEM, n=3 biological replicates. * P<0.05, *** P<0.001 or **** P<0.0001 as compared between indicated groups (Student’s t-test).

Next, we sought to verify lymphoma dependence on TP63 fusions. An shRNA selectively targeting the fusion junction sequence had little knockdown effect on the fusion gene (fig. S2A). Thus, we transduced eight different TCL cell lines with doxycycline (Dox)-inducible shRNA targeting the 3’UTR of TP63 (shTP63). We failed to transduce any plasmids or oligos into DL-40 cell line. TP63 knockdown inhibited the growth of TCL lines with TP63 fusions (SMZ1, MTA and HH), but had no effect on non-TP63 fusion lines (Fig. 1B). We confirmed this result with additional shRNAs (fig. S2A and S2B). Next, we performed genome-wide clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout of the wild-type (WT) TP63 allele in SMZ1 cells (Cas9-SMZ1-sgwtTP63) with single guide RNAs (sgRNAs) targeting exon 3 (fig. S2C), which specifically targets the full-length WT TP63 and not the fusion. As expected, knockout of WT TP63 did not affect cell growth (fig. S2D). Knockdown of the TP63 fusion in Cas9-SMZ1-sgwtTP63 cells inhibited cell growth (Fig. 1C and 1D) and induced apoptosis (fig. S2E). Knockdown of TP63 in MTA cells led to a similar extent of growth inhibition (fig. S2F). In vivo knockdown of the TP63 fusion after xenografting of Cas9-SMZ1-sgwtTP63 cells also suppressed tumor growth (Fig. 1E). Overexpression of an shRNA-resistant TBL1XR1::TP63 cDNA rescued the growth-inhibitory effects from TP63 knockdown (Fig. 1F). Expression of TBL1XR1::TP63 in pro-B Ba/F3 cells, which are rendered IL-3 independent when transformed by a variety of oncogenes (15), conferred IL-3 independent growth (Fig. 1G), consistent with a mitogenic, pro-survival effect from the fusion.

To determine whether different TP63 fusions provide redundant functions, we expressed BCL6::TP63 in Cas9-SMZ1-sgwtTP63 cells and knocked down TBL1XR1::TP63 (Fig. 1H). BCL6::TP63 successfully rescued the effect of shRNA (Fig. 1I; fig. S2G). The small molecule BI-3802 can trigger degradation of BCL6 by binding the Broad-complex, Tramtrack, and Bric-à-brac (BTB) domain of BCL6 (16), which is present in the BCL6::TP63 fusion. Treatment of BCL6::TP63-expessing SMZ1 cells with BI-3802 dose-dependently degraded BCL6::TP63 (fig. S2H) and reduced cell growth (fig. S2I). Together, these data demonstrate that TP63 fusions act as oncogenes in lymphoma cells.

To clarify essential oncogenic domains within TBL1XR1::TP63, we performed a domain-focused CRISPR-Cas9 knockout screen (17, 18) using 452 sgRNAs tiling the entire coding regions of TBL1XR1 and TP63 in SMZ1 cells (fig. S2J). Only sgRNA targeting domains included in the TBL1XR1::TP63 fusion were selectively depleted at day 30 after transduction, while none of sgRNAs targeting the remaining domains of either TBL1XR1 or TP63 were depleted (Fig. 1J). In particular, sgRNA targeting LisH and F-box like domains of TBL1XR1 and DB and OD domains of TP63 were preferentially depleted at day 30 (Fig. 1K). Taken together, our data show that domains within both the N-terminal TBL1XR1 and C-terminal TP63 portions contribute to the function of this fusion.

TBL1XR1::TP63 drives the development of diverse T- and B-cell lymphomas in mice

TP63 fusions are present in multiple subtypes of both T- and B-cell lymphomas, which is distinct from other transcription factor fusions (6, 7, 9). The diversity of lymphomas suggests that TP63 translocations could originate in both precursor B- and T-cells or even in a common lymphoid progenitor due to off-target activity of the V(D)J recombinase recombination activating gene (RAG). We analyzed translocation breakpoints from reported cases of TBL1XR1::TP63 lymphomas but were unable to identify convincing target sequences for RAG.

To investigate the role of TP63 fusions in T- and B-cell development and lymphomagenesis, we engineered a CAG-Loxp-Stop-Loxp-TBL1XR1::TP63-IRES-GFP conditional knock-in mouse model (transgenic TBL1XR1::TP63) (Fig. 2A). We crossed these mice to CD2-Cre mice (hereafter Cre/tgTT), which results in transgene expression after Cre-mediated recombination in T- and B-cell progenitors (19).

Fig. 2. TBL1XR1::TP63 regulates the T- and B-cells in mice.

Fig. 2.

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(A) Strategy for insertion of the CAG-loxP-Stop-LoxP-TBL1XR1::TP63-IRES-EGFP cassette into the mouse Rosa26 locus. (B, C) Representative flow activated cell sorting (FACS) plot (B) and quantification (C) showing TCRb staining of viable cells. (D, E) Representative FACS plot (D) and quantification (E) showing CD4/CD8 staining of TCRb+ T cells; (F, G) Representative FACS plot (F) and quantification (G) showing Treg (Foxp3 staining) of TCRb+ T cells; (H, I) Representative FACS plot (H) and quantification (I) showing B cells (CD19/B220 staining) of viable cells; (J, K) Representative FACS plot (J) and quantification (K) showing FoB and MZB (CD21/CD23 staining) of B220+ B cells; (L, M) Representative FACS plot (L) and quantification (M) showing GCB (CD38/CD95 staining) of B220+ B cells; (N, O) Representative FACS plot (N) and quantification (O) showing naïve B cell (CD27/IgD staining) of B220+ B cells; (P, Q) Representative FACS plot (P) and quantification (Q) showing PC (CD138/B220 staining) of viable cells. All spleen samples were from 11-week-old Cre and Cre/tgTT mice. Data are presented as mean ± SD, *P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 as compared between indicated groups (Student’s t-test). n=4 mice/arm except for B cells (n=6 mice for Cre and n=7 mice for Cre/tgTT). (R) Quantification of cytokines in peripheral blood serum from 11-week-old Cre and Cre/tgTT mice. Cytokine quantification is presented as the ratio in Cre/tgTT versus Cre mice (n=3 mice/arm).

We characterized the effects of TBL1XR1::TP63 expression on splenocytes from 11-week-old Cre mice and 11-week-old Cre/tgTT mice by flow cytometry. We confirmed TP63 fusion expression in 95% of TCRβ+ T cells and 90% of B220+ B cells based on GFP expression (fig. S3A–D). Among the T cells, we observed a decrease of TCRβ+ percentage (Fig. 2B and 2C; P<0.0001). In the TCRβ+ T-cell population, we observed an increase in the CD4:CD8 ratio (Fig. 2D and 2E; P<0.01). Fractions of regulatory T (Treg) cells, CD4+ and CD8+ activated T cells, and CD4+ and CD8+ effector memory (TEM) cells were increased whereas CD4+ naïve cells and CD8+ central memory (TCM) cells were decreased in Cre/tgTT mice compared to control Cre mice (Fig. 2F and 2G; fig. S3E–N; P<0.05). There were no significant changes in splenic CD8+ naïve cells, CD4+ follicular helper (Tfh) and CD4+ TCM cells between Cre and Cre/tgTT mice (fig. S3I–N).

As to B cells, we observed a decrease of the overall B cell population in Cre/tgTT mice compared to Cre mice (Fig. 2H and 2I; P<0.05). Further analyses showed that the fractions of naïve B cells, follicular B (FoB) cells and germinal center B (GCB) cells were decreased, whereas marginal zone B (MZB) cells and plasma cells (PCs) were increased in the Cre/tgTT mice (Fig. 2J-Q; P<0.001). There were no significant changes in transitional B (TrB) cells between Cre and Cre/tgTT mice (Fig. S3O–Q). These data demonstrated that TBL1XR1::TP63 regulates immune homeostasis. Consistent with systemic immune stimulation, serum from Cre/tgTT had increased relative amount of multiple inflammatory cytokines, including IL-2, IL-6, IL-17, TNF-a and IL-1β (Fig. 2R; P<0.05).

We also performed single cell RNA sequencing (scRNA-seq) on whole spleen cells from 11-week-old WT mice (23,619 cells, n = 3 mice) and 11-week-old Cre/tgTT mice (22,178 cells, n = 3 mice). Transcriptome-based cluster annotation based on gene expression programs selectively associated with sorted mouse immune cells (20) confirmed TP63 fusion expression in Cre/tgTT specimens (fig. S3R) as well as immune response-related genes (fig. S3S and S3T; table S2) that were differentially enriched compared to WT spleens. Based on single-cell T cell receptor sequencing (scTCR-seq), T cells from Cre/tgTT mice had reduced TCR diversity with enriched clonotypes compared to normal spleens (fig. S3U and S3V), suggesting that TBL1XR1::TP63 induces clonal expansion that results in reduced TCR diversity.

To directly assess the role of TBL1XR1::TP63 in T- and B-cell transformation, we monitored Cre/tgTT mice for disease symptoms. In this cohort, 60% (12/20) of mice developed lymphomas within 210 days, with a median latency of 172.1 days (Fig. 3A; fig. S4A). All mice with lymphoma developed splenomegaly with involvement of cells expressing TBL1XR1::TP63 (Fig. 3B and 3C; fig. S4B and S4C). Histopathologic examination of lymphoid tissues from diseased mice showed an infiltrative process that disrupted typical splenic architecture (Fig. 3D). Immunohistochemical (IHC) analyses confirmed the atypical lymphoid cells to be CD3+CD4+ in TCL cases (Fig. 3E and 3F). In these TCLs, expression of T-cell differentiation markers differed across lymphomas (Fig. 3F), with some capturing ALCL-like phenotypes (CD30+CD4+) and others T-cell lymphoma phenotypes (CD30-CD4+). Six mice developed B-cell lymphoma (BCL) (B220+), with some capturing germinal-center B-cell-like (GCB) DLBCL-like phenotypes and activated B-cell-like (ABC) DLBCL-like phenotypes (Fig. 3F). RNAseq analysis of these representative transgenic tumors showed that tumor transcriptomes displayed enrichment of gene signatures derived from human ALCL, PTCL-NOS, GCB-DLBCL or ABC-DLBCL diseases (Fig. 3G) that were consistent with pathological characterizations. Clonotype analysis showed that these tumors had enriched clonal T- and B-cell populations in T- and B-cell lymphoma samples, respectively (Fig. 3H). Thus, as observed in patients, TBL1XR1::TP63 drove the development of diverse T- and B-cell lymphomas that recapitulated aspects of human lymphomas (Fig. 3I). Tumors readily engrafted into wild-type, irradiated, or immunodeficient recipients after intravenous transplantation (Fig. 3J and 3K; fig. S4D–F), confirming their malignant phenotype and competitive advantage.

Fig. 3. TBL1XR1::TP63 drives the development of diverse T- and B-cell lymphomas in mice.

Fig. 3.

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(A) Kaplan-Meier curve of lymphoma-specific survival in Cre and Cre/tgTT mice within 210 days. n=20 mice/arm. P value by Log-rank test. (B) Representative image of the spleen from moribund Cre/tgTT mice and age-matched control wildtype (WT) and Cre mice. (C) Western blot analysis for indicated proteins in spleen samples from (B). (D) H&E staining of spleen tissues in (B) showing representative splenic architecture of each group of mice. Scale bar: 1 mm. (E) Immunohistochemical (IHC) analysis showing the expression of CD3 and CD4 in representative spleen tissues in (B). Scale bars are as indicated. (F) H&E and IHC analysis for the indicated markers in representative spleen tissues harvested from four lymphoma-bearing Cre/tgTT mice. Scale bar: 100 mm. (G) RNAseq analysis of transgenic tumors. Enrichment of individual human lymphoma subtype signatures was assessed. (H) Contribution of CDR3b and IgH sequences to the T-cell receptor repertoire and B-cell receptor repertoire in transgenic tumor samples, respectively. The colors between samples do not indicate the same clone. The top 5 clones are shown. (I) Bar graph showing the cumulative incidence of transgenic tumors. 11 of 12 tumors were characterized. One mouse having an enlarged spleen (tumor) was found dead, and the tumor was not further analyzed. (J, K) Kaplan-Meyer survival curve for C57BL/6 mice after being irradiated with 5.5 Gy and intravenously transplanted with 0.5 million spleen cells from diseased Cre/tgTT mice (Tumor 1 (J) or Tumor 3 (K)) or from Cre mice. n=5 mice/arm. P value by Log-rank test.

TBL1XR1::TP63 rewires enhancers to regulate gene expression

To define the effects of TBL1XR1::TP63 on gene expression, we performed RNA-seq on SMZ1 cells after knockdown of the fusion; this identified 3,155 genes regulated by TBL1XR1::TP63 (1,619 up and 1,539 down with fusion expression) (fig. S5A). TCR signaling and T-cell differentiation-related genes were significantly enriched among the TBL1XR1::TP63-upregulated genes (Fig. 4A; FDR<0.01). Chromatin immunoprecipitation-sequencing (ChIP-seq) using an antibody that bound TBL1XR1::TP63 in Cas9-SMZ1-sgwtTP63 cells identified the localization at the transcriptional start sites (TSS) of only 142 (4.5%) of these 3,155 genes (Fig. 4B). In contrast, we found that TBL1XR1::TP63 binding sites were primarily located in introns (48-49%) and in intergenic regions (41-43%) (Fig. 4C), suggesting that the fusion can act at distal regulatory elements.

Fig. 4. TBL1XR1::TP63 fusion rewires enhancers to regulate gene expression.

Fig. 4.

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(A) GSEA plots of indicated gene sets in SMZ1 cells expressing shTP63 versus shSeed control. Normalized enrichment score (NES); FDR: false discovery rate (FDR). (B) Venn diagram showing genes regulated by TBL1XR1::TP63 from RNA-seq in SMZ1 and genes bound by TBL1XR1::TP63 from ChIP-seq in SMZ1. (C) Pie graph showing the locations of TBL1XR1::TP63 binding peaks based on ChIP-seq of SMZ1 and DL40 cells. (D) Schematic of IP-MS of flag-tagged TP63, ΔNp63, TBL1XR1::TP63, TBL1XR1, and the truncated TBL1XR1 (ΔTBL1XR1). (E-G) IP using anti-TP63 antibody that binds TBL1XR1::TP63 in Cas-SMZ1-sgwtTP63 cells (E), anti-KMT2D antibody in SMZ1 cells and Cas9-SMZ1-sgwtTP63 cells (F), and anti-TP63 in SMZ1 cells and OCI-Ly12 cells (G). (H) IP of KMT2D in SMZ1 cells with 150 mM NaCl (low-salt) IP buffer or 300 mM NaCl (high-salt) IP buffer then immunoblotted for TP63. (I) Analysis of TBL1XR1::TP63-bound enhancers from HiChIP and ChIP-seq. (J) Analysis of PRO-seq signal at the genes associated with TBL1XR1::TP63-bound enhancers in Cas9-SMZ1-sgwtTP63 cells with or without TP63 fusion knockdown using Dox-inducible shRNA. PRO-seq signal is shown as violin plots of summed reads from the TSS to +75nt. P-value is from Wilcoxon test. (K) Motif analysis at TBL1XR1::TP63-bound enhancers by using HOMER.

The highly conserved oligomerization domain of TP63 allows homotetramerization, which is required for high DNA affinity (21), and heterotetramerization with tumor protein p73 gene (TP73) (22). Both TP63 and ΔNp63 are found in a tetrameric form. Under non-reducing conditions, TBL1XR1::TP63 existed at high molecular mass consistent with a large multimeric assembly (fig. S5B). Immunoprecipitation-mass spectrometry (IP-MS) identified more than 300 unique proteins that interact with TBL1XR1::TP63 (Fig. 4D; fig. S5C; table S3). Among these interacting partners, nuclear receptor corepressor 1 (NCOR1) and nuclear receptor corepressor 2 (NCOR2) (also known as SMRT) were the top ranked, which is consistent with the known interaction between the N-terminal region (LisH and F-box-like domains) of TBL1XR1 and the NCoR complex (23). Additional components of NCoR complex (transducin β-like protein 1 (TBL1X), G-protein suppressor 2 (GPS2), HDAC3) were also recovered by IP-MS of TBL1XR1::TP63 (fig. S5C; table S3). We confirmed the interactions between TBL1XR1::TP63 and NCOR1, NCOR2 and HDAC3 by IP assay (Fig. 4E).

To address the targetability of HDAC3 in these cells, we developed and tested multiple heterobifunctional degraders of HDAC3 (fig. S5D–F). Treatment of SMZ1 cells with either of these molecules led to highly selective degradation of HDAC3 (fig. S5G) and reduced cell growth (fig. S5H and S5I).

We noted that lysine methyltransferase 2D (KMT2D) and multiple components of the KMT2D complex (ASH2L, RbBP5, WDR5, NCOA6, KDM6A) were also identified as interacting partners of TBL1XR1::TP63 by IP-MS (fig. S5C; table S3), which is consistent with a previous study demonstrating that KMT2D interacts with ΔNp63 in epithelial cells (24). KMT2D is a mono-methyltransferase for Lysine 4 of Histone H3 (H3K4me1), a marker of enhancers that remotely activates gene transcription (25). We confirmed that TBL1XR1::TP63 interacts with KMT2D in SMZ1 cells by pulling down either TBL1XR1::TP63 or KMT2D under different IP conditions (Fig. 4F–H). IP in high-salt buffer also detected the interaction between KMT2D and TBL1XR1::TP63 (but not WT TP63), suggesting the interaction is independent of their binding to DNA (Fig. 4H; fig. S5J).

To identify potential target genes of TBL1XR1::TP63-bound enhancers, we performed HiChIP, which globally maps enhancer-promoter interactions (26, 27). We selected H3K27ac as the bait for the HiChIP assay, as it distinguishes active enhancers from poised enhancers that are methylated at H3K27 (28). We integrated H3K27ac HiChIP and TBL1XR1::TP63 ChIP-seq datasets and found that TBL1XR1::TP63 bound 2,892 active enhancers looping to 2,010 genes (Fig. 4I). Next, we utilized Precision nuclear Run-On sequencing (PRO-seq), which quantifies nascent transcripts, to assess whether TBL1XR1::TP63 binding at enhancers drives transcription of the looped genes. Knockdown of TBL1XR1::TP63 significantly decreased overall promoter-proximal PRO-seq reads of these 2,010 enhancer-looped genes (Fig. 4J; P=0.00059), demonstrating that binding of the TBL1XR1::TP63 fusion at enhancers broadly serves to activate the associated promoter.

A motif analysis of the TBL1XR1::TP63 binding peaks within the 2,892 enhancers identified the known p63 binding sequence as the top motif (Fig. 4K). Other enriched motifs were primarily transcription factors involved in guiding the functional diversification of mature T cells (29), including BATF, JunB, RUNX1, IRF4, Tbet, Tbx21, RORγ and RORα (Fig. 4K). Of note, none of these transcription factors were identified as interacting partners with TBL1XR1::TP63 by IP-MS.

TBL1XR1::TP63 fusion activates enhancers to confer EZH2 dependence

Next, we investigated the therapeutic vulnerability of TP63-rearranged TCLs. CRISPR-Cas9 screening identified TP63 as a top dependency in SMZ1 (Fig. 5A), representing a selective dependency as compared to screening results from other cancers lines (Fig. 5B)(14, 30), consistent with the gain-of-function of the TBL1XR1::TP63 fusion. SMZ1 cells also had the highest dependency score among 391 cell lines analyzed for Enhancer of zeste homologue 2 (EZH2), the Histone H3 Lysine 27 (H3K27) methyltransferase (Fig. 5C). We confirmed that knockdown of EZH2 in TP63-rearranged lines impaired cell growth (Fig. 5D), as did treatment with the EZH2/ Enhancer of zeste homologue 1 (EZH1) dual inhibitor Valemetostat (31) (Fig. 5E and 5F; fig. S6A) or the EZH2-selective inhibitor GSK-126 (fig. S6B). The combination of targeting HDAC3 and EZH2/EZH1 had a profound impact on cell growth (fig. S6C and S6D; P<0.0001).

Fig. 5. TBL1XR1::TP63 activates enhancers to confer PRC2 dependence.

Fig. 5.

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(A) Ranked dependency score of genome-wide CRISPR screen in SMZ1 cells with TP63 and EZH2 highlighted. (B, C) Z scores for TP63 (B) and EZH2 (C) dependency across a pan-cancer set of 391 cell lines (14,30). (D) Cell proliferation assay of SMZ1 cells transduced with shRNAs targeting EZH2 using CTG. (E) IC50 of Valemetostat (VAL) for TCL cell lines. The B-cell lymphoma cell line Karpas-422 (blue), which harbors an EZH2 Y646F gain-of-function mutation serves as a positive control. TCL cell lines with TP63 rearrangements are in red and those with WT TP63 are in black. (F) Cell proliferation assay of SMZ1, DL40, and OCI-Ly12 cell lines treated with VAL using CTG. (G) Venn diagram showing genes regulated by TBL1XR1::TP63 from RNA-seq and genes bound by TBL1XR1::TP63 from ChIP-seq in SMZ1. (H) Possible mechanisms of regulating EED and EZH2 by TBL1XR1::TP63. (I, J) RT-qPCR analysis for EED, MYC, and EZH2 after TP63 knockdown with Dox treatment in SMZ1 cells (I) and MTA cells (J). *** P<0.001 or ****P<0.0001 for shTP63+Dox compared to shSeed+Dox for the same gene. (K, L) Western blot of EED, MYC, EZH2 and H3K27me3 after Dox-induced knockdown in Cas9-SMZ1-sgwtTP63 cells (K) and MTA cells (L). (M) The TBL1XR1-TP63-activated gene MYC is connected to a high-confidence distal TBL1XR1::TP63 binding site through chromatin loops identified from H3K27Ac HiChIP assays in Cas9-SMZ1-sgwtTP63 cells. HiChIP anchors that are looped to promoters of the target genes are presented. ChIP-seq shows the TBL1XR1::TP63 binding site as well as enhancer-associated chromatin marks. (N) ChIP-qPCR of TP63 fusion at the MYC enhancer in Cas9-SMZ1-sgwtTP63 cells. Control (CTR) primer sets are also included. (O) Luciferase reporter assay measuring the MYC enhancer activity in 293T cells expressing indicated proteins. The pGL3 plasmid without the MYC enhancer region (Empty vector, EV) is used as a negative control. (Y-axis) Relative (Rel.) Luciferase units are normalized to Renilla signal ± SD. (P) RT-qPCR analysis of MYC expression in SMZ1 cells with (+DOX) and without (−DOX) KRAB-dCas9 mediated repression of the MYC enhancer. (Q) PRO-seq reads at the MYC locus in Cas9-SMZ1-sgwtTP63 cells with (+DOX) and without (−DOX) TBL1XR1::TP63 knockdown using Dox-inducible shRNA.

Data are presented as mean ± SD, n=3 biological replicates. *** P<0.001 or ****P<0.0001 as compared between indicated groups (Student’s t-test).

EZH2 is the catalytic component of the polycomb repressor complex 2 (PRC2). Knockdown of the TP63 fusion reduced the expression of both EZH2 and the noncatalytic PRC2 complex subunit embryonic ectoderm development (EED) (Fig. 5G). ChIP-seq data showed that the TP63 fusion bound the TSS of EED but not the TSS of EZH2 (fig. S6E and S6F), suggesting two separate regulatory mechanisms. We noted that proto-oncogene MYC was among the 1,619 up-regulated genes by the TP63 fusion (Fig. 5G). MYC regulates EZH2 in several cancers, including lymphoma (32). We hypothesized that TBL1XR1::TP63 regulates EZH2 by MYC (Fig. 5H). Knockdown of TBL1XR1::TP63 in SMZ1 and MTA cells decreased the expression of MYC, EZH2, EED, and H3K27me3 (Fig. 5I–L). Additionally, knockdown of MYC decreased EZH2 mRNA and protein abundance (fig. S6G and S6H) and reduced cell growth (fig. S6I). Of note, Cre/tgTT tumors also had up-regulated MYC, EZH2 and EED (fig. S6J). As expected, knockdown of KMT2D decreased both mRNA and protein abundance of MYC, EZH2 and EED (fig. S6K and S6L) and impaired cell growth (fig. S6M).

ChIP-seq analysis did not identify binding of TBL1XR1::TP63 at the TSS of MYC (fig. S6E), suggesting that the fusion acted through a MYC enhancer. HiChIP identified multiple enhancers looped to the MYC promoter in SMZ1 cells. When analyzed together with the TBL1XR1::TP63 ChIP-seq data, there was only one TBL1XR1::TP63-bound enhancer looped to the MYC promoter (Fig. 5M). We validated the binding of TBL1XR1::TP63 at this putative enhancer by ChIP-qPCR in both SMZ1 and MTA cells (Fig. 5N; fig. S7A). We also confirmed TBL1XR1::TP63-dependent activity of this enhancer using a luciferase reporter assay (Fig. 5O; fig. S7B). MYC enhancer-dependent transcription was selectively driven by TBL1XR1::TP63 but not TP63, ΔNp63 or TBL1XR1 (Fig. 5O). CRISPR-mediated interference of the single TBL1XR1::TP63 motif in this MYC enhancer reduced MYC expression by approximately 50% in both SMZ1 and MTA cells (Fig. 5P; fig. S7C). PRO-seq showed an inhibition of nascent MYC transcription after knockdown of the TP63 fusion (Fig. 5Q). Together, our data demonstrate that TBL1XR1::TP63 activates a distal MYC enhancer to up-regulate MYC expression. We also found two TBL1XR1::TP63 specific enhancer-promoter loops for EED (fig. S7D) and confirmed that these two sites acted as TBL1XR1::TP63-dependent EED enhancers (fig. S7E–J).

EZH2 inhibition as a promising therapeutic strategy for TP63-rearranged TCLs

Although Valemetostat has shown promising activity in patients with PTCL (33), its activity in the poor-prognosis subset with TP63 rearrangements has not been described. Treating Nod.CgPrkdcscid.IL2rgtm1Wjl/SzJ (NSG) mice transplanted with SMZ1 cells with Valemetostat by oral daily administration significantly reduced H3K27me3 in tumors and decreased tumor growth, resulting in prolonged survival of mice (Fig. 6A–C; P<0.01). We also transplanted TBL1XR1::TP63-induced transgenic tumor cells into irradiated WT C57BL/6 mice (Fig. 6D). Our pharmacodynamics (PD) result showed that Valemetostat inhibited the tumor growth and H3K27me3 abundance after 10 days treatment (Fig. 6E–G). Survival results showed that Valemetostat treatment significantly improved the mouse survival and inhibited H3K27me3 abundance after systemic engraftment of Cre/tgTT lymphomas into C57BL/6 mice (Fig. 6H and 6I; P<0.01). As a third model, we tested the efficacy of Valemetostat in a patient-derived xenograft (PDX) of ALK- ALCL (COH1) that harbors the same TBL1XR1::TP63 coding sequence as the MTA cell line (fig. S8A and S8B). COH1 was transplanted into NSG mice and treated with Valemetostat by orally daily. Again, Valemetostat reduced H3K27me3, impaired the tumor growth and prolonged mouse survival (Fig. 6J–O; P<0.01). Tumors taken from mice that progressed on Valemetostat continued to have complete loss of H3K27me3 (Fig. 6I and 6N), strongly suggesting that the mechanism of acquired resistance is not through reactivation of PRC2 function.

Fig. 6. EZH2 inhibition as a promising therapeutic strategy for TP63-rearranged TCLs.

Fig. 6.

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(A) Tumor growth curve of NSG mice engrafted with SMZ1 cells injected subcutaneously (n=5/arm). Mice were treated with vehicle (Veh) or Valemetostat (VAL) when tumor size reached to 200 mm3 and sacrificed when subcutaneous tumors reached 2 cm in the longest dimension. (B) Kaplan-Meyer survival curve for mice in A. (C) Mean fluorescence intensity (MFI) of the H3K27me3 abundance in tumor samples from B determined by FACS. (D) Schematic outline of transplantation of Cre/tgTT lymphoma-infiltrated spleen cells (Tumor 1) and treating C57BL/6 recipients with Veh or VAL. C57BL/6 mice are irradiated with 5.5 Gy before receiving 0.5 million cells intravenously and treatments are initiated two days after transplantation. A cohort of mice are sacrificed after 10 days for pharmacodynamic (PD) analysis, and another cohort are observed for survival analysis. (E, F, G) Photo of spleens (E), spleen weight versus body weight ratio (F) and western blot analysis of H3K27me3 in spleen cells (G) from mice treated with VAL or Veh (n=3 mice/arm) for 10 days. (H) Kaplan-Meyer survival curve of C57BL/6 mice transplanted with Cre/tgTT lymphoma-infiltrated spleen cells and treated with Veh or VAL (n=6 mice/arm). (I) Western blot analysis for indicated proteins in spleen cells harvested from H. (J) Schematic of subcutaneous engraftment of ALK- ALCL PDX (COH1) harboring a TBL1XR1-TP63 rearrangement in NSG mice and treating recipients with VAL or Veh when tumor size reached to 200 mm3. (K) FACS analysis of H3K27me3 in tumor cells harvested from mice treated with VAL or Veh (n=3 mice/arm) for 10 days. (L, M) Tumor growth curve (L) and Kaplan-Meyer survival curve (M) of the COH1 PDX model. Mice were treated with Veh or VAL (n=6 mice/arm) by p.o. and sacrificed when the subcutaneous tumor reached 2 cm in the longest dimension. (N, O) Western blot analysis for indicated proteins in tumor cells harvested from M (N) and quantification of relative H3K27me3 abundance normalized to H3 (O). (P) Fluorescence in situ hybridization using a 5’ TP63 probe (green, 314 kb centromeric to TP63) and a 3’ TP63 probe (red, 310 kb telomeric to TP63). The translocated chromosomes are indicated with arrows. (Q, R) FACS showing abnormal T cells (purple) (Q) and associated quantifications (R) from pre-treatment and day 28 of VAL treatment in whole blood cells of Patient 2. Other cells shown are NK cells (blue), CD8+ T cells (green), and CD4+ T cells (red).

Data are presented as mean ± SEM. ** P<0.01 or **** P<0.0001 as compared between indicated groups (Student’s t-test). P value for survival by Log-rank test.

To determine whether PRC2 upregulation is unique to TP63-rearranged PTCLs, we assessed both EZH2 expression and H3K27me3 by IHC in two separate cohorts of PTCL (table S4). Both TP63-rearranged and -unrearranged cases had abundant EZH2 and H3K27me3 staining (fig. S8C and S8D), suggesting that multiple mechanisms can drive PRC2 activity in PTCLs (32).

Our findings suggest that patients with lymphoma with TP63 rearrangements might respond to Valemetostat, which encouraged us to enroll these patients into our phase I/II study of Valemetostat in patients with relapsed or refractory PTCL (NCT04703192). Of the patients enrolled, three harbored TP63 rearrangements (table S5). Two of these patients died shortly before the start of Valemetostat therapy. The only treated patient (Patient 2) had ALK- ALCL (Ann Arbor stage IV) and was heavily pre-treated, including allogeneic hematopoietic stem cell transplantation (table S5). We confirmed a TBL1XR1::TP63 rearrangement by fluorescence in situ hybridization and RNA sequencing (Fig. 6P; fig. S8E). The patient received a standard dose of Valemetostat 200 mg daily. Compared to pre-treatment, the fraction of malignant T cells in peripheral blood dropped >99% by day 28 (Fig. 6Q and 6R; fig. S8F). Unfortunately, the patient experienced a cytomegalovirus reactivation and was removed from the study.

Discussion

TBL1XR1 is the rearrangement partner in one-half to two-thirds of TP63-rearranged cases (6, 8). Alternative TP63 fusion partners have not been comprehensively studied but both FOXK2 and ATXN are known to interact with NCoR/SMRT complex members and drive transcriptional repression (34, 35). We also identified a BCL6::TP63 fusion in a patient with PTCL and showed that it can act as an oncogene and is sufficient to substitute TBL1XR1::TP63 in TCL cells. BCL6 is a major transcriptional repressor in lymphoid cells and can also interact with NCoR/SMRT (36, 37). The Mitelman database includes at least 4 other partners involved in TP63 fusions, among which LPP::TP63 was reported in a case of breast adenocarcinoma. LPP is involved in multiple chromosomal translocations and can act as a transcriptional coactivator in epithelial cells (38, 39). Further studies are needed to clarify the lineage-specific functions of TP63 fusions, but it appears that co-recruitment of both the KMT2D complex (by the C-terminal portion of each fusion) and additional epigenetic modifiers by the N-terminal partner is a common component of TP63 fusions. A model for the mechanisms of lymphomagenesis induced by TP63 fusions and their targeting with EZH2 inhibitors is shown in fig. S9.

The majority of mice expressing TBL1XR1::TP63 upon activation of CD2 ultimately succumbed to spontaneous lymphomas that spanned precursor T cell-like, mature PTCL-like, and aggressive B-cell lymphomas. Additional studies are needed to determine whether expression restricted to CD4 (only CD4+ T cells), VAV (all hematopoietic cells) or Ighg1 (Cγ1, germinal center B cells) promoters can be used to drive individual tumor phenotypes. In the transgenic mouse model, we observed that TBL1XR1::TP63 expression disrupted the normal distributions of both T and B cell subtypes within the spleen. This dysregulation was associated with increases of IL2, IL10, IL6, and other cytokines in the serum. The contribution of these findings to transformation is unclear but it seems plausible that remodeling of the immune microenvironment plays a pro-lymphomagenic role in transgenic mice.

Mechanistically, the ability of TBL1XR1::TP63 to recruit NCOR/SMRT, KMT2D or both of these complexes confers unique capabilities on the fusion and may explain why half of the TBL1XR1::TP63 target genes are up-regulated and the other half are down-regulated. In SMZ1 PTCL cells, TBL1XR1::TP63 binding motif analysis at enhancers shows that it binds to motifs for several transcription factors known to regulate the functions of both nonmalignant T cells and T-cell lymphomas. We speculate that binding by TBL1XR1::TP63 may either co-activate with or negatively compete with those canonical T-cell transcription factors, which would result in up- or down-regulation, respectively. Alternatively, some sites such as the MYC enhancer we identified may be selectively activated by the fusion and otherwise quiescent within T cells. A subset of aggressive B-cell lymphomas with extranodal distribution carry somatic mutations in TBL1XR1 but not fusions. A recent manuscript demonstrated that these TBL1XR1 mutants can co-opt SMRT/HDAC3 complexes toward binding of the memory B-cell transcription factor BACH2, rather than BCL6 (40). As a result, the TBL1XR1 mutants mimic the context-dependent, repressor function of TP63 fusions but lack the ability to co-recruit KMT2D complexes and drive enhancer activation. We observed that similar to the association between TBL1XR1 mutations and extranodal B-cell lymphoma in patients, mice expressing TBL1XR1::TP63 could also develop extranodal DLBCL.

Our study has some limitations. We investigated the mechanisms of TP63 fusions in TCL but not in BCL. We were unable to identify BCL cell lines or PDX models harboring TP63 fusions and have thus far failed to establish cell lines from mouse lymphomas arising from our transgenic mouse models using a variety of starting material and conditions, which limited our ability to interrogate mechanistic links between the fusion and B-cell lymphomas. The EZH2/EZHZ1 dual inhibitor Valemetostat was effective both preclinically and in a patient with TP63-rearranged TCL, inducing a near complete remission in the blood after only 28 days of therapy. The specific contribution of EZH1 inhibition remains unclear, although EZH1 was not a vulnerability in our previous CRISPR screens of TCL cell lines. We recognized that any conclusions based on “n of 1” clinical anecdotes must be interpreted with skepticism. Nevertheless, the combination of mechanistic studies and the promising clinical response supports further testing of both Valemetostat and selective EZH2 inhibitors in patients with TP63-rearranged lymphomas, and possibly other cancers.

Materials and Methods

Study Design

Outcomes among patients with TP63 fusions remain poor. The purpose of this study was to define the molecular mechanisms and induced vulnerabilities of TP63 fusions in lymphomas. TP63 fusions were analyzed at the mRNA and protein expressions in multiple T-cell lymphoma cell lines. Isogenic cell lines were generated to study if TP63 fusions are essential or not for the cell growth. A TP63 fusion knock-in transgenic mouse model was generated to study its oncogenic role in lymphomagenesis. Different preclinical models, including a cell line-driven xenograft model, a patient-derived xenograft model and transgenic mouse lymphoma models, were treated in vivo with EZH2 inhibitor Valemetostat to assess efficacy at controlling the tumor growth. Mice were randomized into treatment arms by tumor volume so that each arm contained mice with the same mean tumor burden. End points for each treatment group were defined as the time when tumor size reached at 2000 mm3 of volume or survival timepoint. In general, group sizes of five or more mice were used. Differences in tumor growth were tested using Log-rank test. All in vitro experiments were run at least in three biological triplicates and tested using Student’s t-test (two-tailed). For clinical trial, patients with relapsed PTCL harboring TP63 fusions were enrolled to clinical trial NCT04703192. Patients received oral administration of Valemetostat tosylate at a dose of 200 mg once daily starting at Cycle 1, Day 1 (continuous for 28-day cycles), treatment was continuous until intolerance, progression, or discontinuation by physician or patient choice.

Ethics statement

Written informed consent was obtained for participation in the clinical trial of valemetostat and approved by the MSKCC Institutional Review Board (MSKCC #: 14-254, 16-042, 16-1542, 18-147, 21-176). All animal studies conform to the standards of good research practice and approved as components of DFCI IACUC protocols #10-035 and #13-034.

Statistical analyses

Clinicopathological characteristics were analyzed by the X2 test. Survival curves were analyzed by the Log-rank (Mantel-Cox) test. P-values for PRO-seq were analyzed by Wilcoxon test. Statistical analysis was determined using the Student’s t-test for other experiments. Data were presented as the mean (± SD) or mean (± SEM) of at least three biological replicates. P<0.05 was considered significantly different.

Supplementary Material

Supplementary Materials
Data S1

Acknowledgements

We thank the members of the Weinstock and Brown laboratories for thoughtful comments. We thank Drs. M. Müschen, A. Melnick and L. Su for comments or critical reading of the manuscript, Dr. C. Wu and the Transgenic Core at Beth Israel Deaconess Medical Center for generation of the transgenic mouse model, the Flow Cytometry Core, Molecular Biology Core Facilities and Center of Functional Cancer Epigentics at DFCI, Brigham and Women’s Hospital Pathology Core, the Nascent Transcriptomics Core and Taplin Biological Mass Spectrometry Facility at Harvard Medical School, and the Single Cell Core at Harvard University for their help.

Funding

This work is supported by NCI R35 CA231958 and P01 CA233412 (both to D.M.W.), a Specialized Center of Research 7026-21 from the Leukemia and Lymphoma Society (to D.M.W., M.B. and S.M.H.), NCI R37 CA233691 (to C-W.C.), NCI P01CA229100 (to A.L.F.), NCI P30 CA008748 (S.M.H.), NCI K08CA230498 (to S.J.), NCI K00CA253754 (to H.Y.), the Lymphoma Research Foundation Postdoctoral Fellowship (816777) (to E.M.), the Lash Family Foundation and Nonna’s Garden Foundation. Gongwei Wu is a Fellow of The Leukemia & Lymphoma Society. This manuscript was edited at Life Science Editors.

Competing interests

D.M.W. received research support from Daiichi Sankyo and is now an employee of Merck/MSD. M.B. is a consultant to and receives sponsored research support from Novartis and serves on the scientific advisory boards of Kronos Bio, H3 Biomedicine and GV20 Oncotherapy. G.W. received research support from Daiichi Sankyo. E.S.F is a founder, scientific advisory board (SAB) member, and equity holder of Civetta Therapeutics, Proximity Therapeutics, and Neomorph, Inc. (also board of directors). He is an equity holder and/or SAB member for Avilar Therapeutics, Photys Therapeutics and Lighthorse Therapeutics, and a consultant to Novartis, Sanofi, EcoR1 Capital, and Deerfield. The Fischer lab receives or has received research funding from Deerfield, Novartis, Ajax, Interline and Astellas. K.A.D is a consultant to Kronos Bio and Neomorph Inc. S.M.H. consulted, received honorarium from, or participated in advisory boards for Affimed, Daiichi Sankyo, Kyowa Hakko Kirin, ONO Pharmaceuticals, SecuraBio, Shoreline Biosciences, Inc. Takeda, Yingli Pharma Limited, Abcuro, Inc. and Tubulis. He received research support for clinical trials from ADC Therapeutics, Affimed, Auxilius Pharma, Celgene, Crispr Therapeutics, Daiichi Sankyo, Kyowa Hakko Kirin, Millennium/Takeda, Seattle Genetics, C4, and Verastem/SecuraBio. A.L.F. has received research funding from Seattle Genetics. A.L.F. is an inventor of technology for which Mayo Clinic holds an unlicensed patent. H.L., G.M.N. and K.L.J., are now employees of AstraZeneca. K.A. is a consultant to Syros Pharmaceuticals and Odyssey Therapeutics, is on the scientific advisory boards of CAMP4 Therapeutics, and received research funding from Novartis not related to this work. All other authors declare no relevant competing interests.

Data and materials availability

All data associated with this study are in the paper or supplementary materials. The data and materials generated in this study are available upon request from the corresponding author. The reported data have been deposited in the NCBI GEO, under accession number BioProject: 821322; GSE240151; GSE240201.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Materials
NIHMS1979348-supplement-Supplementary_Materials.docx (3.7MB, docx)
Data S1
NIHMS1979348-supplement-Data_S1.xlsx (46.8MB, xlsx)

Data Availability Statement

All data associated with this study are in the paper or supplementary materials. The data and materials generated in this study are available upon request from the corresponding author. The reported data have been deposited in the NCBI GEO, under accession number BioProject: 821322; GSE240151; GSE240201.

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