RAF-independent MEK mutations drive refractory histiocytic neoplasms but respond to ERK inhibition

SUMMARY

Histiocytic neoplasms are clonal disorders of the monocyte/macrophage lineage defined by mutations activating MAPK signaling. Recently, the MEK1/2 inhibitor cobimetinib was FDA-approved for patients with adult histiocytoses. Here, aided by a prospective registry of patients with histiocytoses (NCT03329274), we identify that MEK1/2 mutations which constitutively activate MEK independently of RAF are associated with worse progression-free survival with MEK1/2 inhibition as compared to patients with other MEK1/2 mutational classes. The most common RAF-independent MEK1 mutation (MEK1^E102_I103del) drove a lethal histiocytic-like neoplasm in mice which was sensitive to the ERK1/2 inhibitor ulixertinib. We subsequently treated five MEK1^E102_I103del-mutant patients with ulixertinib on prospective protocols, four of whom were refractory to MEK inhibition. Four of five patients experienced objective responses to ulixertinib. These data reveal the impact of oncogenic MEK mutations in vivo, identify patients with likelihood of resistance to MEK inhibition, and nominate ERK inhibition to overcome resistance to MEK inhibition in histiocytoses.

Graphical Abstract

eTOC blurb

Diamond et al. find RAF-independent (“class III”) mutations in MEK1/2 kinases are common in patients with systemic histiocytic neoplasms, drive disease in a conditional knock-in mouse model, and associate with progression on FDA-approved MEK inhibitors. Patients with these mutations who progress on MEK inhibitors respond to the ERK inhibitor ulixertinib.

INTRODUCTION

Histiocytic neoplasms (histiocytoses) are a diverse group of clonal hematopoietic disorders defined by the accumulation of macrophages, dendritic cells, or monocyte-derived cells in various tissues¹. Histiocytoses subtypes include Langerhans Cell Histiocytosis (LCH), Erdheim-Chester Disease (ECD), Juvenile Xanthogranuloma (JXG), Rosai-Dorfman-Destombes Disease (RDD), and histiocytic sarcoma (HS).

The etiology of histiocytoses was long obscure, and these disorders were previously considered inflammatory, non-malignant conditions. However, over the last decade, each of the histiocytoses have been defined by recurrent, mutually exclusive alterations activating mitogen-activated protein kinase (MAPK) and receptor tyrosine kinase (RTK) signaling. This unified etiological understanding of histiocytoses began with the discovery of somatic BRAF^V600E in ~50% of patients with LCH and ECD^2,3. Since then, patients lacking BRAF^V600E have been found to have activating mutations in MAP2K1 (encoding MEK1), MAP2K2 (encoding MEK2), ARAF, CSF1R, N/KRAS, or PIK3CA; in-frame fusions activating BRAF, ALK, RET, and NTRK1; or in-frame BRAF deletions^4–11. Therefore, most patients with histiocytic neoplasms have a mutation activating mitogenic kinases, and molecular genotype (i.e. presence of BRAF or MAP2K1 mutations) has superseded morphological classification of histiocytoses subtypes (i.e., ECD, RDD) in the clinical management of these disorders.

The discovery of BRAF^V600-mutations in patients with histiocytoses initiated clinical trials targeting BRAF in adults with histiocytic neoplasms. In the initial trial of the BRAF inhibitor vemurafenib for adults with BRAF^V600-mutant ECD, an overall response rate (ORR) of 100% was observed by modified PERCIST criteria (and 61.5% by RECIST criteria) leading the USA Food and Drug Administration (FDA) to approve vemurafenib for BRAF^V600E-mutant ECD in 2017. To date, >100 patients with BRAF^V600E-mutant histiocytoses have been treated with BRAF inhibitor therapy^12–22, with few cases of acquired resistance documented²¹.

Given the excellent efficacy of vemurafenib for BRAF^V600-mutant histiocytoses and the near-universal presence of mutations activating ERK in patients with histiocytoses, we investigated MEK1/2 inhibitor therapy in BRAF^V600-wild type (WT) histiocytoses. Based on our phase 2 clinical trial results²³, the FDA approved the MEK1/2 inhibitor cobimetinib for the treatment of adults with histiocytic neoplasms in 2022.

Although responses to cobimetinib among patients with histiocytoses have been favorable, they have not recapitulated the consistently durable responses to BRAF inhibition. For example, in the phase II cobimetinib trial, ORR was 79% (modified PERCIST criteria) and 53% (RECIST criteria) while for vemurafenib, ORR was 100% and 61.5%, respectively. Additionally, clinical experience, as well as a modest but growing literature, reflects variable responsiveness and even instances of resistance to MEK1/2 inhibition^24–26, the basis of which is currently unknown. Importantly, there are no FDA-approved therapies beyond vemurafenib or cobimetinib (for BRAF^V600-mutant or WT patients) if patients progress on one of these agents.

We performed genetic analysis of a large patient cohort with histiocytic neoplasms which revealed that a specific MEK1 kinase mutation, resulting in an in-frame deletion of two amino acids within the kinase catalytic domain (MEK1^E102_I103del), is the most recurrent mutation following BRAF^V600E. Aided by use of an international collaboration and a prospective clinical registry of patients with histiocytic neoplasms (clinicaltrial.gov identifier NCT03329274), this cohort included the largest group of patients treated with MEK1/2 inhibitors to date. Analyses of these patients revealed an enrichment of MEK1^E102_I103del with progressive disease on cobimetinib.

Since the in vivo biological effect of cancer-associated MEK1 mutations is unknown, we generated a conditional knock-in mouse of MEK1^E102_I103del and found that expression of this mutation in the hematopoietic compartment results in a lethal, fully penetrant, aggressive, mature, myelomonocytic neoplasm reminiscent of human histiocytic neoplasms. Importantly, MEK1^E102_I103del-mutant mice were sensitive to pharmacological inhibition of ERK1/2, the kinases downstream of MEK1/2. This led us to evaluate the orally bioavailable ERK1/2 inhibitor ulixertinib in patients with MEK1^E102_I103del-mutant histiocytoses within prospective, IRB-approved, single-patient Investigational New Drug (IND) trials, which revealed promising and durable clinical activity.

RESULTS

MEK1^E102_I103del mutations are common and adverse in histiocytoses

Prior genomic landscape studies of histiocytoses utilized cohort sizes of a maximum of 270 patients²⁷. Since these studies, we instituted a prospective registry to collect clinical and genomic data from patients with histiocytoses across multiple institutions internationally. The analyzed cohort included 498 patients: 189 (38%) LCH, 122 (25%) ECD, 75 (15%) RDD, 69 (14%) juvenile or adult xanthogranuloma (JXG/AXG), and 43 (8%) rare histiocytoses (ALK⁺ histiocytosis, indeterminate cell histiocytosis, or malignant histiocytoses) (Fig. 1A, Table S1).

Fig. 1. Genomic analysis of 498 adult and pediatric patients with histiocytoses identifies class 3 MEK1 mutations as common drivers of histiocytoses associated with progression of disease with MEK1/2 inhibitor treatment.

(A) Alluvial plots of kinase mutations identified in patients (n=498; patient number on y-axis) with diverse histiocytoses. The mutant gene/protein (left column) and the histiocytoses subtypes (right column) are noted. Bands between the two columns indicate the patient fractions with each mutation. (B) Protein diagram of MEK1 mutations (n=108 patients) across the histiocytosis patient cohort. The circle diameter correlates with the patient number with the specified mutation while the color-coding documents the MEK1 mutational class (class 1, green; class 2, orange; class 3, red; class unknown = gray). (C) Kaplan-Meier curve of progression-free survival of patients with histiocytoses undergoing MEK1/2 inhibitor therapy, comparing class 1/2 MEK1 mutations (blue) to class 3 (red). (D) Two patients with MEK1^E102_I103del histiocytoses experiencing disease progression on MEK1/2 inhibition. One ECD patient has increased fibrothorax (top panels), and another with ECD has increased peri-ocular xanthelasmas (bottom panels) despite MEK1/2 inhibition. (E) Swimmer plot of class 3 MEK1-mutant patients showing histiocytoses subtype, treatment modality (conventional therapy, MEK1/2 inhibition, ERK1/2 inhibition), treatment responses, and subsequent progression events. Abbreviations: CR = complete response, PR = partial response, SD = stable disease, POD = progression of disease. See also Tables S1, S2.

As in prior histiocytoses genomic studies, BRAF^V600 mutations were the most common mutation (27%;136/498) of patients with histiocytoses overall, nearly all of which (134/136) were BRAF^V600E substitutions. Other recurrent BRAF mutations in our cohort included in-frame deletions (2.6%; 13/498) near the αC-helix region (most commonly BRAF^N486_P490del), which are notable since BRAF indels are insensitive to vemurafenib or dabrafenib but sensitive to RAF dimer inhibitors in preclinical studies²⁸. In-frame BRAF fusions were also seen in our cohort (2.8%; 14/498) (Fig. 1A, Table S1).

Following BRAF mutations, MEK1/MEK2 mutations were the second largest group of alterations in our cohort (22%; 111/498) (Fig. 1A, Table S1). Prior in vitro studies by our group identified that mutations in MEK1/2 occur in distinct biochemical classes: those depending on RAF kinase to activate MAPK signaling (class 1 MEK1), those activating ERK1/2 by both RAF-dependent and independent mechanisms (class 2 MEK1), and those activating ERK1/2 independently of RAF (class 3 MEK1)²⁹. This patient cohort’s unprecedented size allowed evaluation of the frequency of patients with each biochemical class of MEK1 mutations. Class 3 was the most common MEK1 mutational class (47%; 52/111) of MEK1/2 mutations overall (Figs. 1A, 1B, Table S1). Nearly all (88%; 46/52) class 3 MEK1 mutations consisted of an in-frame deletion of two amino acids at E102 and I103 (MEK1^E102_I103del). These data indicate that MEK1^E102_I103del is the most common mutation among patients with histiocytoses after BRAF^V600E.

Given the distinct MEK1 mutational classes and the patient cohort numbers with each mutational type, we evaluated the clinical characteristics of the MEK1-mutant patient subsets where we had detailed clinical annotation (n=73; Table S2). Disease sites and treatment data were collected from patients with histiocytic neoplasms harboring MEK1^E102_I103del or other class 3 mutations (n=40) and from patients with class 1/2 mutations (n=33). MEK1-mutant patients did not differ by sex; the class 3 mutant patient median age at diagnosis was lower (42.1; IQR 32–51) than class 1/2 mutant patients (51.6; IQR 41–61; p=0.004). “Risk-organ” involvement as conceived in the pediatric LCH context, defined as involvement of hepatobiliary or splenic structures by PET/CT, or biopsy-proven bone marrow (BM) involvement, was present in only one patient with class 3 MEK1 mutations. One well-established prognostic variable among patients with histiocytoses is the anatomic disease site location with central nervous system (CNS) involvement being known to confer the worst outcome³⁰. Importantly, comparison of disease sites among patients with MEK1 class 3 versus class 1/2 mutations revealed higher frequency of bone (98% versus 73%, P=0.004), CNS (45% versus 21%, P=0.03), and lymph node (48% versus 21%, P=0.02) disease in class 3 MEK1-mutant patients, and less frequently retroperitoneal (20% versus 42%, P=0.04) and abdominal disease (10% versus 30%, P=0.03) (Table S2).

Comparing treatment outcomes between both mutational cohort subsets treated with MEK1/2 inhibitors by Kaplan-Meier methodology, progression-free survival was significantly worse in the 28 patients with MEK1^E102_I103del or similar in-class mutation as compared to 20 with class 1/2 MEK1 mutations (P=0.02) (Fig. 1C). On a patient level, those with class 3 mutations (36%; 10/28) experienced progression during MEK1/2 inhibitor treatment versus those with class 1/2 MEK1 mutations (5%; 1/20) (Fig. 1C). Progression during MEK1/2 inhibitor therapy was observed across histiocytoses subtypes in patients with and without prior chemotherapy (Fig. 1D, 1E). Altogether, these results indicate class 3 MEK1 mutations represent common drivers of histiocytoses associated with more aggressive disease presentation, relapse following chemotherapy, and higher likelihood of disease progression on cobimetinib (only FDA-approved therapy for this patient molecular subgroup).

Physiological expression of MEK1^E102_I103del drives aggressive multi-system histiocytosis-like, mature myelomonocytic neoplasm in vivo

Prior studies have evaluated the BRAF^V600E contribution to histiocytosis initiation in vivo using mice.^11,31,32 These studies utilized a variety of promoters to conditionally express the heterozygous Braf^V600E mutation from the endogenous Braf locus, contributed to our understanding of the potential histiocytoses cellular origins, and resulted in several histiocytoses animal models for preclinical therapeutic studies. In contrast to the extensive prior mouse studies of Braf^V600E, no cancer-associated MEK1/2 mutational animal models have been created. Given this fact, combined with the high frequency and clinical importance of MEK1^E102_I103del mutations among patients with histiocytoses, we generated animals for conditional expression of the MEK1^E102_I103del mutation from the endogenous Map2k1 locus. We inserted a mutated exon 3 containing deletion of six nucleotides and resulting in an in-frame deletion of amino acids E102 and I103 from MEK1, mirroring the histiocytoses patient mutation (Fig. 2A, Fig. S1A–B). This mutated exon was inserted in the fourth intron of Map2k1 along with a Neomycin cassette and flanked by mutant LoxP sites. An additional LoxP site was inserted upstream of endogenous WT exon 3 such that in the presence of Cre recombinase, endogenous WT exon 3 would be excised and replaced by mutated exon 3 in the correct orientation to produce a MEK1^E102_I103del encoding transcript (Fig. 2A).

Fig. 2. Hematopoietic-specific expression of the MEK1^E102_I103del mutation drives lethally aggressive histiocytoses-like mature myelomonocytic neoplasms in vivo.

(A) Schema of conditional MEK1^E102_I103del knock-in allele in the endogenous Map2k1 gene. An inverted synthetic Map2k1 exon 3 with a six-nucleotide deletion to create the MEK1^E102_I103del mutation was inserted within an intron downstream of wild-type exon 3 and flanked by two mutant LoxP sites. In addition, a 5’ LoxP cassette was inserted 250 bp upstream of wild-type exon 3. An initial recombination event between the two mutant LoxP sites flips the mutant exon 3 into the correct orientation and allows a second recombination event to excise the wild-type exon 3. (B) RNA sequencing reads from Mx1-Cre MEK1^{E102_I103del/WT} mice demonstrating deletion of six nucleotides resulting in in-frame deletion of amino acids E102 and I103 in MEK1 in bone marrow (BM) but not tail tip. (C) Kaplan-Meier survival curves of Mx1-Cre (n=45) and Vav-Cre (n=9) MEK1^{E102_I103del/WT} mice and littermate controls (n=59). Log-rank (Mantel-Cox) test. (D-E) Hematoxylin and eosin (H&E) staining and Iba1 and B220 immunohistochemistry (IHC) of BM (D) and spleen (E) from Mx1-Cre MEK1^{E102_I103del/WT} mice and littermate controls (400x magnification; 50 µm scale bars). (F) Spleen photos (top) and weights (bottom). Unpaired two-tailed t test. (G) H&E staining and Iba1 IHC of liver (400x magnification; 50 µm scale bars). Liver photos (top) are also shown. (H) Photographs of skin lesions (left) and corresponding histology (right) in Mx1-Cre MEK1^{E102_I103del/WT} mice (200x and 400x magnifications; 100 µm and 50 µm scale bars). (I) Western blot of BM cells from mice in (D). (J) IHC for phosphorylated ERK1/2 (pERK1/2) in tissue sections from Mx1-Cre MEK1^{E102_I103del/WT} mice and littermate controls (400x magnification; 50 µm scale bars). Box-and-whisker plots, bar indicates median; box edges, first and third quartile values; and whisker edges, minimum and maximum values. *P<0.05; **P<0.01; ***P<0.001; ****P< 0.0001. See also Fig. S1, S2.

Mice with a heterozygous MEK1^{E102_I103del/WT} allele were crossed to Mx1-Cre transgenic mice to allow for conditional expression of the MEK1 mutation upon administration of polyinosinic:polycytidylic acid (pIpC). Mx1-Cre MEK1^{E102_I103del/WT} were born at normal Mendelian ratios, and RNA-seq analyses of blood at 6-weeks, two weeks after pIpC administration, demonstrated clear expression of the MEK1^E102_I103/WT mutation at ~50% allele frequency in the blood indicative of MEK1 mutational expression in this model (Fig. 2B). Across a cohort of 45 Mx1-Cre MEK1^E102_I103/WT-mutant mice, median survival was 59 days (Fig. 2C). Necropsy of Cre-positive MEK1^E102_I103/WT mice revealed massive expansion of mature myeloid cells with an appearance consistent with monocytes and Iba1⁺ macrophages in the BM, spleen, and liver, as well as splenomegaly (Fig. 2D–G). There were also greatly diminished B220⁺ B-cells in spleen and BM (Fig. S2A–C). Additionally, granulomas, a histopathological feature commonly seen in human histiocytoses were evident in these tissues, especially within the liver (Fig. 2G). Beyond the hematopoietic abnormalities noted above, extensive necropsy evaluations of Cre+ MEK1-mutant mice revealed no histological abnormalities of the epithelial, mesenchymal, or endothelial tissue components.

One nearly universal phenotypic feature of the Cre-positive MEK1^E102_I103/WT mice was the development of red cutaneous lesions (Fig. 2H). Histological evaluation of these lesions revealed massive dermal accumulation of Iba1⁺ macrophages, consistent with histiocytosis skin involvement.

Consistent with the gain of function activity of the MEK1^E102_I103/WT, Western blot analysis of BM mononuclear cells (BMMNCs) revealed clear upregulation of phosphorylated MEK1/2 and ERK1/2 in tissue from the Mx1-Cre MEK1^E102_I103/WT mice compared to Cre-negative littermates (Fig. 2I). Similar findings were noted by phospho-ERK1/2 immunohistochemical (IHC) analysis of the neoplastic cells within the BM, spleen, liver, and skin of these animals (Fig. 2J).

We next evaluated the composition of hematopoiesis in Mx1-Cre MEK1^E102_I103/WT mice and Cre-negative MEK1^E102_I103/WT-mutant controls. The 5-to-6-week-old Cre-positive animals developed leukopenia, macrocytic anemia, and thrombocytopenia indicative of BM involvement (Fig. 3A–C, Fig. S1C–D). The leukopenia was largely attributable to a reduction in blood B-lymphocytes while there was an expansion of monocytes in the blood of Mx1-Cre MEK1^E102_I103/WT mice relative to controls (Fig. 3D, Fig. S1E–G). Reduction of B220⁺ cells was also apparent in BM and spleen of mutant mice by IHC analyses (Fig. S2A–C).

Fig. 3. Development of anemia, thrombocytopenia and expansion of monocytes and macrophages in MEK1^E102_I103del mice.

(A-D) Box-and-whisker plots of (A) white blood cell (WBC) and platelet counts, (B) hemoglobin (Hgb), (C) mean corpuscular volume (MCV), and (D) lymphocytes and monocytes in Mx1-Cre MEK1^E102_I103del mice and Cre-negative mutant control mice at six-weeks. n=10–14 mice/group. (E) Representative FACS plots of gating of live, myeloid (CD45⁺ CD11b⁺) cells, neutrophils (Ly6C^high Ly6G^high), classical monocytes (Ly6C^high Ly6G⁻ CD115⁺), non-classical monocytes (Ly6C^low Ly6G⁻ CX3CR1⁺ CD115⁺), and macrophages (Ly6C^low Ly6G⁻ F4/80⁺) in the mice blood from (A). (F) Box-and-whisker plots of data from (E). n=10. Box-and-whisker plots, bar indicates median; box edges, first and third quartile values; and whisker edges, minimum and maximum values. *P<0.05; **P<0.01; ***P<0.001; ****P< 0.0001. Unpaired two-tailed t test (A-D). One-way ANOVA (F). See also Fig. S1, S2

Given the expansion of mature myeloid cells in tissues from histopathological analysis, we performed multi-parameter flow cytometric analyses of monocyte and macrophage subsets in each tissue compartment. This revealed clear expansions of neutrophils (CD45⁺ CD11b⁺ Ly6C^high Ly6G^high), classical monocytes (CD45⁺ CD11b⁺ Ly6C^high Ly6G⁻ CD115⁺), non-classical monocytes (CD45⁺ CD11b⁺ Ly6C^low Ly6G⁻ CX3CR1⁺ CD115⁺), and macrophages (CD45⁺ CD11b⁺ Ly6C^low Ly6G⁻ F4/80⁺) among hematopoietic (CD45⁺) cells in blood, BM, spleen, and liver (Fig. 3E–F, Fig. S2D). Finally, given prior data identifying expansion of dendritic cell (DC) subsets in CD11c-Cre Braf^V600E-mutant mice, we examined DC populations in our model. Mx1-Cre MEK1^E102_I103/WT mice developed clear expansion of the Cd11b⁺ classical dendritic cells type 2 (DC2) in BM, spleen, and liver without clear expansion of classical dendritic cells type 1 (DC1) (CD24⁺ classical DC1 in hematopoietic tissues and CD103⁺ DC1 in non-hematopoietic tissues (e.g. liver and skin)) (Fig. S2E–F).

Overall, these data indicate pan-hematopoietic expression of MEK1^E102_I103/WT gives rise to a 100% penetrant, aggressive, mature myelomonocytic neoplasm reminiscent of human multi-system histiocytosis involving mature myeloid cell infiltration of hematopoietic organs, liver, and skin.

MEK1^E102_I103del promotes myeloid skewing, expansion of mature monocyte and macrophage subsets, and activation of inflammation in mutant myeloid cells

To perform an unbiased broader assessment of hematopoietic cell types and gain insights into molecular effects of MEK1^E102_I103del on distinct cell types, we performed single-cell RNA sequencing (scRNA-seq) of BM and splenic MNCs from three six-week-old Mx1-Cre MEK1^E102_I103/WT mice and three littermate Cre-negative MEK1-mutant control mice (two weeks after pIpC administration). Data integration and clustering identified a total of 61,022 cells across 15 transcriptional clusters (Fig. 4A, Fig. S3A–B). This notably included a broad range of mature myeloid cells (classical, intermediate, non−classical, and neutrophil-like monocytes as well as macrophages, neutrophils, and DCs) in addition to progenitor cells and B, T, and natural killer (NK) cells.

Fig. 4. Myeloid skewing, expansion of mature myeloid cells, and inflammation in the bone marrow and spleen of MEK1^E102_I103del mice.

(A) Uniform manifold approximation and projection (UMAP) dimensionality reduction of cell types from single-cell RNA sequencing of 61,022 bone marrow (BM) and spleen cells from three six-week-old Mx1-Cre MEK1^E102_I103del mice and three littermate Cre-negative MEK1-mutant control mice. (B) As in (A) but cell types colored by animal genotype (mutant or wild-type (WT)) and tissue source (BM or spleen (SP)). (C) Fraction of each cell type from all cells in (A) in BM (left) or spleen (right) from MEK1 WT or mutant mice. (D) Chord diagram showing the proportion of each cell type from (C) arising from MEK1 WT or mutant BM or spleen cells. (E) Heatmaps showing log-normalized expression of the top 20 differentially expressed genes in MEK1-mutant versus WT neutrophil-like, classical, and non-classical monocytes as determined by the absolute value of log₂FC multiplied by the - log₁₀ (Bonferroni-adjusted P value). (F) Gene Ontology (GO) analysis of differentially expressed genes in the monocyte subtypes analyzed in (E) showing select terms with Benjamin-Hochberg-corrected P values<0.05. See also Fig. S3, S5.

Evaluation of the cell type distribution based on MEK1-mutant genotype revealed a clear preponderance of mature monocyte and macrophage cell subsets (classical, intermediate, non−classical, and neutrophil-like monocytes, as well as macrophages) among MEK1-mutant cells with a striking depletion of B, T, and NK cells in MEK1-mutant mice (Fig. 4B–D). The myeloid skewing of the MEK1-mutant cells was evident in both BM and spleen (Fig. 4C). There was also an expansion of erythroid lineage cells among MEK1-mutant splenic cells (Fig. 4A–D), which in combination with the anemia in MEK1-mutant mice suggests a potential impairment of erythroid differentiation.

Given the clear monocyte expansion in the MEK1-mutant mice, we performed differential gene expression analyses of MEK1-mutant versus WT neutrophil-like, classical, and non-classical monocytes. This revealed a strikingly increased expression of the alarmins S100a8 and S100a9 as the top differentially expressed genes in MEK1-mutant versus WT classical, and non-classical monocytes (Fig. 4E). S100A8 and S100A9 are secreted by myeloid cells in inflammatory states and bind pattern recognition receptors, such as Toll-like receptor 4 on target cells to recruit neutrophils and activate inflammatory signaling³³. Consistent with this, numerous genes involved in regulation of monocyte proliferation (Mif, Cd38, Bst1), chemotaxis (Fpr2, Ctsg, Pla2g7, Ccl6), response to type I and II interferons (Ifitm1, Ifitm2, Ifitm6, Nos2), and interleukin signaling (Ilr2, Havcr2, App, Pycard) were upregulated in each of these monocyte subsets in MEK1-mutant mice (Fig. 4F).

We next evaluated these gene expression data using protein-level assays. Analysis of live, classical (CD45⁺ CD11b⁺ Ly-6C^high) and non-classical (CD45⁺ CD11b⁺ Ly-6C^low) monocytes and macrophages in the spleen and BM of 6-week-old Mx1-Cre MEK1^E102_I103del mice and littermate controls revealed an increased frequency of classical and non-classical monocytes and macrophages in MEK1-mutant mice (Fig. 5A–B). Splenic MEK1-mutant monocytes had increased expression of inflammatory proteins including IL-1β, TNFα, and phosphorylated Serine 536 NF-κB as determined by intracellular flow cytometry (Fig. 5C–D). These data were corroborated by Luminex-based assessment of serum cytokines in the same mice (Fig. 5E). Moreover, there was increased activation and proliferation of MEK1-mutant monocytes as evidenced by increased surface PD-L1 and Ki-67 staining by flow cytometry and IHC (Fig. 5D and Fig. 5F–H). Altogether, these immunophenotypic and scRNA-seq analyses of MEK1-mutant mice reveal clear expansions of mature monocyte and macrophage subsets with transcriptional profiles consistent with a hyperinflammatory state and a relative depletion of lymphoid cell types.

Fig. 5. Increased frequency and expression of protein indicators of inflammation, activation, and proliferation in MEK1^E102_I103del-mutant monocytes.

(A) Gating strategy used to identify live, classical (CD45⁺ CD11b⁺ Ly6C^high) and non-classical (CD45⁺ CD11b⁺ Ly6C^low) monocytes in the spleen and bone marrow (BM). (B) Percentage of classical and non-classical monocytes in the spleen and BM among live hematopoietic cells. (C) MFI (median fluorescence intensity) of IL-1β, TNFα, pS536 NF-κB, and PD-L1 in splenic classical and non-classical monocytes (normalized to Cre-negative control mice). (D) Representative FACS plots of IL-1β, TNFα, pS536 NF-κB, PD-L1, and Ki-67 expression in splenic CD45⁺ CD11b⁺ Ly6C^low cells. (E) Heatmap of serum cytokine concentration Z-score obtained from blood of MEK1-mutant and control mice. n=9 mice/group. (F) As in (C) but for Ki-67⁺ cells. (G, H) Representative IHC analysis of myeloid cells in (G) BM and (H) spleen of MEK1-mutant and control mice (400x magnification; 50 µm scale bars). Box-and-whisker plots, bar indicates median; box edges, first and third quartile values; and whisker edges, minimum and maximum values. Horizontal bar represents mean value. n = 5 mice/group. Statistical significance was assessed by two tailed Student’s t-test. *P<0.05; **P<0.01; ***P <0.001; ****P <0.0001.

Hematopoietic-cell autonomous effects of MEK1^E102_I103del

To ascertain if the phenotypes seen in Mx1-Cre MEK1^{E102_I103del/WT} mice were hematopoietic cell autonomous, we evaluated if hematopoietic cells from Mx1-Cre MEK1^E102_I103/WT mice were transplantable in recipient mice. Transplantation of 1.0 × 10⁶ BMMNCs from CD45.2⁺ Mx1-Cre MEK1^E102_I103/WT mice into lethally irradiated CD45.1⁺ 8-week-old C57BL/6 recipient mice resulted in death of recipient mice at a median of 53 days post-transplant (Fig. 6A). In contrast, transplantation of similar numbers of BMMNCs from Cre-negative MEK1-mutant mice had no impact on recipient mice. Recipients of Cre⁺ MEK1-mutant hematopoietic cells experienced similar expansions of Iba1⁺ myeloid cells and granulomata in BM, spleen, and liver with development of hepatosplenomegaly as seen in primary Mx1-Cre MEK1^E102_I103/WT mice (Fig. 6B–F). Blood analysis also revealed similar effects of the MEK1^E102_I103/WT mutation, including macrocytic anemia, thrombocytopenia, and decrement in B lymphocytes (Fig. S4A–D). Flow cytometric and IHC analysis of BM, spleen, and liver revealed similar expansions of macrophages and classical and non-classical monocytes in each of these organs, as well as similar alterations in DC populations as in primary mutant mice (Fig. 6G, Fig. S4E–G).

Fig. 6. Hematopoietic-cell autonomous development of aggressive mature myelomonocytic neoplasms reminiscent of histiocytosis in MEK1^E102_I103del-mutant mice.

(A) Kaplan-Meier curve of lethally irradiated CD45.1⁺ recipient mice transplanted with 1.0 × 10⁶ CD45.2⁺ bone marrow (BM) cells from Mx1-Cre MEK1^E102_I103del mice (n=10) or littermate Cre-negative MEK1-mutant controls (n=10). P-value indicated in figure. Log-rank (Mantel-Cox) test. (B-D) Hematoxylin and eosin (H&E) staining and Iba1 and B220 immunohistochemistry (IHC) of (B) BM, (C) spleen, and (D) liver from Mx1-Cre MEK1^{E102_I103del/WT} mice and littermate Cre-negative MEK1-mutant controls (400x magnification; 50 µm scale bars). (E,F) Photos and weights of (E) spleen and (F) liver. n=4 (transplanted Cre-negative MEK1^E102_I103del control); n = 7 (transplanted Mx1-Cre MEK1^E102_I103del). (G) Percent of donor (CD45.2⁺) cells in recipients’ blood that are classical monocytes (CD45⁺ CD11b⁺ Ly6C^high Ly6G⁻ CD115⁺), non-classical monocytes (CD45⁺ CD11b⁺ Ly6C^low Ly6G⁻ CX3CR1⁺ CD115⁺), and macrophages (CD45⁺ CD11b⁺ Ly6C^low Ly6G⁻ F4/80⁺). Box-and-whisker plots, bar indicates median; box edges, first and third quartile values; and whisker edges, minimum and maximum values. n=5 (transplanted Cre-negative MEK1^E102_I103del control); n=5 (transplanted Mx1-Cre MEK1^E102_I103del). *P<0.05; **P<0.01; ***P <0.001; ****P <0.0001. Unpaired two-tailed t test (E-F). One-way ANOVA. (G). See also Fig. S4.

Finally, we crossed MEK1^{E102_I103del/WT} floxed mice to Vav-Cre (pan-hematopoietic expression) mice and Vav-Cre MEK1^{E102_I103del/WT} mice died at a similar median timepoint as Mx1-Cre MEK1^E102_I103/WT mice (Fig. 2C). These studies indicate MEK1^E102_I103/WT expression drives histiocytosis development in a hematopoietic cell autonomous manner and that transplantation of these cells into recipient mice can propagate the disease model.

MEK1^E102_I103del-mutant mice are sensitive to S100A8/9 inhibition with Tasquinimod

As above, class 3 MEK1 mutations are clearly associated with progression of disease on cobimetinib. This motivated us to test different therapeutic approaches for patients with histiocytoses patients with these mutations using our MEK1-mutant mouse model. Given the increase in inflammatory cell signaling and expression of S100A8 and S100A9 in the MEK1-mutant mice, we tested the impact of tasquinimod, a small-molecule inhibitor of S100A8/S100A9 recently shown to ameliorate disease phenotypes in other myeloid neoplasia disease models associated with increased S100A8/S100A9 production^34,35. Six-week-old CD45.1⁺ recipient mice were lethally irradiated and engrafted with donor BM cells from CD45.2⁺ Mx1-Cre MEK1^E102_I103del mice or littermate Cre-negative MEK1-mutant controls. Two weeks later, these mice were treated with tasquinimod. Mice were then sacrificed following 3 weeks of oral gavage treatment and assessed for therapeutic impact on BM cellular populations. Tasquinimod robustly reduced the myeloid-biased hematopoiesis induced by the MEK1^E102_I103del mutation as evidenced by reduction in mature (CD11b⁺) myeloid cells in recipient mice engrafted with MEK1^E102_I103del cells with a concomitant restoration in B-lymphopoiesis to levels seen in control mice (Fig. S5A). Moreover, tasquinimod treatment reduced the number and proliferation of MEK1-mutant BM classical (CD45⁺ CD11b⁺ Ly-6C^high) monocytes (Fig. S5B–D). Although tasquinimod treatment for three weeks did not substantially reduce the frequency of MEK1-mutant hematopoietic stem or progenitor cells, tasquinimod reduced the increased proliferation of MEK1-mutant LSK and myeloid progenitor cells (Fig. S5E–F).

MEK1^E102_I103del-mutant mice are sensitive to ERK inhibition

Since class 3 mutants are dependent on ERK1/2 for signaling and several ERK1/2 inhibitors are in clinical development, we evaluated the activity of ulixertinib (ERK1/2 inhibitor) in Mx1-Cre MEK1^E102_I103/WT mice. We initially assessed cytotoxicity of ulixertinib against BMMNCs from primary Mx1-Cre MEK1^E102_I103/WT mice and Cre-negative control mice. This revealed enhanced sensitivity of MEK1-mutant BMMNCs to ulixertinib (Fig. S6A). For in vivo ulixertinib studies, we transplanted 1.0 × 10⁶ BMMNCs from CD45.2⁺ Mx1-Cre MEK1^E102_I103/WT mice into lethally irradiated CD45.1⁺ 8-week-old C57BL/6 recipients. Recipient mice were then randomized to receive vehicle or ulixertinib (100mg/kg BID) 15 days later (then continuously for 75 days). Ulixertinib treatment resulted in a clear survival benefit in this model (Fig. 7A). Moreover, timed-sacrifice of a second transplanted Mx1-Cre MEK1^E102_I103/WT CD45.1 recipient mice cohort and randomized to treatment with vehicle or ulixertinib for 15 days revealed significant reduction in splenomegaly and hepatomegaly with ERK1/2 inhibition (Fig. 7B–C). Given the anemia and expansion of early erythroid lineage cells in MEK1-mutant mice (Fig. 3B, 4C), we evaluated the impact of ulixertinib therapy in vivo on erythropoiesis. Analysis of BM erythroid cells revealed that the MEK1 mutation promotes expansion of erythroid precursors while reducing the frequency of terminally differentiated erythroid cells. However, ulixertinib treatment promoted terminal erythroid differentiation of MEK1-mutant cells (Fig. S6B–C). Furthermore, two weeks of ulixertinib treatment significantly reduced the frequency, proliferation (noted by Ki-67 staining), and activation state of MEK1-mutant monocytes (noted by surface PD-L1 staining), as well as inflammatory serum cytokines in MEK1-mutant animals (Fig. S6D–G). These data indicate that histiocytoses driven by MEK1^E102_I103/WT are responsive to pharmacological ERK1/2 inhibition, and ERK1/2 inhibition with ulixertinib reduces the proliferation and activation state of MEK1-mutant monocytes.

Figure 7. Patients and mice with histiocytosis driven by MEK1^E102_I103del respond to single-agent ERK1/2 inhibition with ulixertinib.

(A) CD45.1⁺ recipient mice engrafted with CD45.2⁺ cells from Mx1-Cre MEK1^E102_I103del knock-in mice and then orally treated with either ulixertinib (100 mg/kg BID) or vehicle (carboxymethylcellulose daily) starting from 15 days post-transplant to 90 days post-transplant continuously for 5 of 7 days each week. n=5 mice/group. Log-rank (Mantel-Cox) test. (B) Representative photos of spleen (top) and box-and-whisker plots (bottom) of spleen weights following 15 days of ulixertinib treatment of CD45.1⁺ recipient mice (n=10 mice/group) engrafted with CD45.2⁺ cells from Mx1-Cre MEK1^E102_I103del mice and treated with ulixertinib as in (A). (C) As in (B) but for liver. n=10 mice/group. Unpaired two-tailed t test. (D) Patient characteristics of five patients with histiocytoses treated with single-agent ulixertinib on single-patient IRB protocols. (E) Patient 2 with ECD involving the periarticular tissues (blue arrows, upper left panel) previously progressed on MEK inhibition, with partial response to ulixertinib. Top: axial fused FDG-PET/CT. Bottom: MEK1^E102_I103del-mutant allele frequency in plasma cell-free DNA prior to and during treatment with ulixertinib. (F) Patient 3 with LCH of the dura and brain (red arrow, upper panels, coronal post-gadolinium T1-weighted MRI) and bone with extraosseous extension (lower panels, axial fused FDG-PET/CT) with dramatic response to ulixertinib. Box-and-whisker plots, bar indicates median; box edges, first and third quartile values; and whisker edges, minimum and maximum values. *P<0.05; **P<0.01; ***P <0.001; ****P <0.0001. See also Figs. S5, S6; Tables S3, S4.

Safety and efficacy of ERK1/2 inhibition for MEK1^E102_I103del-mutant histiocytoses

Based on our promising preclinical findings, we next evaluated the safety and efficacy of ulixertinib in patients with treatment-refractory histiocytoses driven by MEK1^E102_I103del (or related class 3 MEK1 mutations). Five patients (four of whom were MEK1^E102_I103del and one with the related MEK1^{L101_I103delinsF}) were treated with ulixertinib, within prospective, IRB-approved, single-patient therapeutic trials (Fig. 7D; see STAR Methods). All patients had histiocytic neoplasms refractory to prior therapies (all clinical characteristics in Table S3). Three of these five patients treated had ECD, and two had LCH. All had two or more prior therapies, and 4/5 were treated with MEK1/2 inhibition with disease progression.

Of four patients radiologically evaluable for response with PET/CT, three had complete or partial metabolic responses, and one had progressive metabolic disease as best response. Of the three with complete or partial metabolic responses, one had subsequent progressive metabolic disease on reduced dose ulixertinib, and the other two have sustained responses with ongoing treatment of 17 and 38 cycles. Responses were observed across numerous disease sites, including the brain, bone, soft tissues, joint structures, and pituitary infundibulum (Fig. 7D–F). Interestingly, in one patient (Patient 2 in Table S3), we evaluated the MEK1^E102_I103del mutational allele burden in plasma cell-free DNA prior to and during therapy revealing a marked suppression of this mutation by ulixertinib treatment (Fig. 7E). One patient was non-evaluable for radiological response owing to withdrawal from study related to the COVID-19 pandemic; she had a marked clinical response (resolution of pleural effusions and removal of pleural drains) and remained alive and off treatment as of the data cutoff.

Per CTCAE 5.0 evaluation, three ulixertinib-related grade 3 adverse events were observed: ejection fraction decrease, hypertension, and urticaria. Two grade 3 events (hypoxia; vulvar erosion) were disease related. The remainder of treatment-adverse events were grade 1 or 2 (all patient adverse events are in Figure 7 and Table S4). Overall, these data, across genetically engineered animal models and patients indicate promising potential clinical efficacy of single-agent ERK1/2 inhibition with ulixertinib in patients with histiocytoses refractory to currently utilized therapies.

DISCUSSION

In this study, we identify MEK1 mutations as frequent drivers of histiocytoses based on both the high frequency of these mutations in patients and analyses of a MEK1-mutant animal model generated here. Furthermore, we identify RAF-independent MEK1 mutations associated with clinical progression on treatment with current FDA-approved allosteric MEK1/2 inhibitors and propose ERK1/2 inhibition with ulixertinib as a promising strategy to overcome MEK1/2 inhibitor resistance in patients with histiocytoses.

Outside of histiocytoses, mutations in MEK1/2 are infrequent in individual cancers (occurring in ~3–8% of melanoma and 1–2% of non-small cell lung cancer)³⁶, and their biological impact on cancer development have not been well-studied. However, despite their rarity in any single form of cancer, recent comprehensive genomic analyses have revealed that mutations in MEK1/2 are recurrent across a wide variety of human cancers and represent an important molecular cancer subtype in aggregate.³⁶ Moreover, MEK1/2 mutations acquired during treatment are increasingly recognized during resistance to therapies targeting KRAS, RAF, and MEK kinases³⁷. Thus, the high frequency of MEK1 mutations among patients with histiocytoses provides a unique setting to study these alterations clinically and to make observations that may have broader applications in cancer.

Prior preclinical in vitro studies from our group identified that different mutations in MEK1 kinases vary in their degree of dependence on RAF-mediated phosphorylation to activate MEK1 catalytic activity. Specifically, we found that constitutively activating MEK1 mutations which render MEK1 totally independent of upstream RAF signaling potently transform cells and confer resistance to allosteric MEK1/2 inhibitors, findings not seen with RAF-dependent and RAF-regulated MEK1 mutations²⁹. This occurs because current MEK1/2 inhibitors bind to the inactive conformation of the enzyme, and class 3 mutations in MEK1 create a constitutively active form of MEK1 enzyme. This latter point has potential clinical implications as all currently FDA-approved MEK1/2 inhibitors (cobimetinib, trametinib, and binimetinib) are allosteric inhibitors. Despite the potential importance of these findings, their clinical relevance was uncertain since previous results were based on preclinical studies. However, the present study verifies that RAF-independent (Class 3) MEK1 mutations are associated with increased risk of clinical progression in response to cobimetinib in patients compared to patients with RAF-dependent or RAF-regulated MEK1 mutations.

One study limitation is that we did not have an opportunity to evaluate for potential acquired genetic mechanisms of resistance to cobimetinib in any patients progressing on this therapy. Currently, there are very few examples of acquired MEK1 inhibitor resistance in patients³⁷. Nonetheless, since patients with progression on cobimetinib were responsive to ERK1/2 inhibition, this indicates the disease is still clearly ERK1/2 dependent. Importantly, our prior work demonstrated that despite the diverse impact of MEK1/2 mutations on MEK1/2 catalytic activity and ERK1/2 output, all MEK1/2 mutants appear to be similarly sensitive to ERK inhibition preclinically³⁶.

In addition to the direct therapeutic relevance of these data, our study provides an animal model to study the biological function of constitutively activating cancer-associated MEK1/2 mutations in vivo. Interestingly, expression of MEK1^E102_I103del in hematopoietic stem cells or committed myeloid cells resulted in aggressive expansion of inflammatory myeloid cells in hematopoietic tissues, liver, and skin. These results verify our in vitro predictions that RAF-independent MEK1 mutations are transformative on their own ²⁹. Thus, this study provides an animal model of MEK1-activating mutations which can be used to study diverse cancers, in addition to BRAF^V600-WT histiocytoses. However, this animal model does not fully and accurately capture the diverse and heterogenous features of human histiocytic neoplasms as the animals uniformly develop lethal, inflammatory myelomonocytic neoplasms.

Overall, we hope these findings will motivate future ulixertinib clinical trials in patients with histiocytoses failing to benefit from BRAF or MEK inhibition. Importantly, the preclinical animal and pilot patient clinical studies performed here are now the basis for an ongoing focused phase II trial which we have initiated to study the role and safety of ulixertinib in patients with histiocytoses (clinicaltrials.gov identifier NCT06411821).

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Benjamin Durham (bd559@cinj.rutgers.edu)

Materials availability

All materials generated or analyzed during this study are included in this article and its supplementary information files. The Map2k1^E102_I103del-mutant conditional knock-in mice are being deposited to JAX as JAX Stock No. 040482 C57BL/6-Map2k1^tm1.1Oaw/J (synonym: MEK1^E102_I103del cKI) and are available in the meantime from O.A.-W. (abdelwao@mskcc.org). Please contact the lead contact for unique material requests. Any material that can be shared will be released via a material transfer agreement for non-commercial usage.

Data and code availability

• Data supporting the findings of this study are available from the corresponding authors upon reasonable request. Datasets generated and/or analyzed during this study, including patient-level clinical and sequencing data have been deposited and are publicly available in the cBioPortal for Cancer Genomics under the accession code (https://www.cbioportal.org/study/summary?id=hdcn_msk_2025). Single-cell RNA-seq data have been deposited to GEO under accession ID GSE268618.

• This paper does not report original code.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

STAR METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Patients and Clinical Data

The study was conducted according to the Declaration of Helsinki, and clinical data were obtained with patient-informed consent and regulatory approval by the Institutional Review Boards of Memorial Sloan Kettering Cancer Center (MSK), New York, NY, USA; Publique-Hôpitaux de Paris, Boulogne, France; Pitié-Salpȇtrière Hospital, Paris, France; University Hospital Zurich, Switzerland; Assuta Medical Centers, Tel-Aviv, Israel; Dalhousie University, Halifax, Nova Scotia, Canada; Aix-Marseille Université, Marseille, France; Hopital de la Conception, Marseille, France. Clinical data was prospectively collected under a parent registry protocol (NCT03329274) (Fig. 1, Table S1, S2). Patients treated with the ERK1/2 inhibitor ulixertinib provided informed consent to prospective, IRB-approved investigational treatment protocols (MSK IRB #061–19, #011–20, #068–20, #030–21, #018–22) (Fig. 7, Table S3, S4).

All patients in this study had biopsy-proven histiocytoses confirmed by expert pathology review at one or more of the participating institutions. Data exported from the registry included sociodemographic variables and histiocytic neoplasm subtype (i.e. ECD, LCH, etc) (Fig. 1, Tables S1, S2). Sites of disease involvement, for a subset of patients were gleaned from pre-treatment FDG-PET/CT and/or from medical notes, categorized as previously published ^38,39 as neurologic (i.e. involving any neurologic structure), brain parenchyma (i.e. cerebrum, brainstem/cerebellum, or leptomeninges), cardiovascular, pulmonary, retroperitoneum, abdomen, bone, skin or subcutis, lymph nodes, or others. Presence of disease within these organ systems was dichotomized as present or absent. Treatments received were categorized into conventional therapies (defined as corticosteroids, immunosuppressive agents, cytotoxic chemotherapy) and targeted therapies (e.g. BRAF, MEK1/2, or ERK1/2 inhibitors) (Figs. 1, 7 and Tables S3, S4).

Best radiological responses to treatment were categorized as published extensively in histiocytic neoplasms ^40–44: (1) complete metabolic response (CMR) if all target lesions’ FDG avidity was reduced to below liver background (2) partial metabolic response (PMR) if targeted lesions’ FDG avidity was reduced, however not fully to liver background (3) progressive metabolic disease if any target lesions’ FDG avidity increased 3 units or more from nadir, or (4) stable metabolic disease (SMD) if not meeting other criteria. For patients evaluated by CT/MRI rather than PET/CT, radiological responses to treatment were categorized as a complete response (CR; complete resolution of tumorous lesions by CT/MRI), partial response (PR; partial reduction in tumorous lesions by CT/MRI), stable disease (SD; no change in tumorous lesions), and progressive disease (PD; progression of lesional radiologic measurements). Whether or not disease progression by FDG-PET/CT or CT/MRI was observed following every instance of radiological CMR/CR, PMR/PR, or SMD/SD was dichotomously captured.

Mouse Studies

All animal experiments performed in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Memorial Sloan Kettering Cancer Center (MSK). MEK1 (Map2k1) E102_I103del mice were created by InGenious Targeting Laboratory, Inc. in collaboration with Omar Abdel-Wahab, M.D. and Benjamin H. Durham, M.D. The Mx1-Cre (B6.Cg-Tg(Mx1-Cre)1Cgn/J; RRID: IMSR_JAX:003556) and Vav1-Cre (B6.Cg-Tg(VAV1-Cre)1Graf/MdfJ; RRID: IMSR_JAX:035670) promoter mice utilized in breeding were purchased from Jackson Laboratories.

We generated animals for conditional expression of the MEK1^{EI102_I103del} mutation from the endogenous gene encoding MEK1 (Map2k1). We inserted a mutant exon 3 bearing deletion of six nucleotides to give rise to in-frame deletion of two amino acids at positions E102 and I103 in MEK1 kinase (c.303_308del; GGAGAT) (Fig. 2A, Fig. S1A–B). This mutant exon was inserted in the fourth intron of Map2k1 in an inverted manner along with a Neomycin cassette and flanked by mutant LoxP sites. An additional LoxP site was inserted upstream of endogenous wild-type (WT) exon 3 (SV40pA-FRT-Neo-FRTLoxP cassette) such that in the presence of Cre recombinase, an initial recombination event flips the mutant exon 3 into the correct orientation. This initial recombination event then allows a subsequent recombination event to excise WT exon 3. In this manner, endogenous WT exon 3 is excised and replaced by mutant exon 3 in the correct orientation to produce a mutant MEK1^E102_I103del encoding transcript (Figs. 2A; S1A).

Mice with a heterozygous MEK1^{E102_I103del/WT} allele were crossed to Mx1-Cre and Vav-Cre transgenic mice to allow for conditional expression of the MEK1 mutation at different stages of hematopoietic development. Primary Mx1-Cre mice were treated with three intraperitoneal injections of pIpC every other day at a dose of 20 mg/kg of body weight starting at 4–6 weeks after birth.

METHOD DETAILS

Protocol Ulixertinib Treatment

Patients were treated under INDs (161369, 148179, 156867, 146494, 154697) with ulixertinib starting at 150mg twice daily, and then the dose was increased to 300mg twice daily to augment clinical or radiologic response; at any given dose, the investigational brochure was followed for dose modification or reduction. Patients were evaluated every cycle by a clinical trials nurse and physician investigator for toxicity, graded per CTCAE 5.0, and adverse events were attributed to ulixertinib as related (i.e. definitely, probably, or possibly related) or unrelated (unlikely or unrelated). Response assessment was performed with 18F-flouro-deoxyglucose positron emission tomography (FDG-PET/CT) every 4 cycles of treatment with the response criteria above.

Mutational Analyses

Excised lesions were either flash-frozen for DNA/RNA extraction and/or fixed in 4% neutral-buffered formalin, embedded in paraffin, and processed by routine histological methods. For patients undergoing whole exome sequencing (WES) and targeted exon sequencing, DNA extracted from peripheral blood mononuclear cells (PB MNCs), or fingernail clippings was utilized as a paired normal germline control. In total, specimens from 498 patients with diverse histiocytoses subtypes were analyzed, and some of the clinical, histological, and genetic characteristics are summarized in Tables S1, S2.

Genomic analyses were performed on DNA extracted from histiocyte tissue biopsies or circulating cell-free DNA from blood plasma using a variety of assays--most commonly, targeted exon sequencing using MSK-IMPACT, MSK-IMPACT-Heme, HemePACT, or MSK-ACCESS using previously described methodology and analysis pipelines ^45–47. Prior to DNA extraction, FFPE samples from all cases were reviewed to confirm that the tissue was of sufficient size to generate a minimum of 50 ng of 20% histiocyte nucleic acid. DNA was isolated from 40-µm-thick sections of FFPE tissue. Targeted RNA sequencing was also used for the purpose of detecting gene fusions using the MSK-Fusion targeted RNA-seq assay ⁴⁸. WES was also performed, based on DNA adequacy from fresh-frozen tissue biopsies or targeted sequencing libraries, using DNA from PB MNCs or fingernails as a germline control as previously described^6,27.

Analysis of WES data, which includes mapping, coverage and quality assessment, single-nucleotide variant (SNV)/indel detection, tier annotation for sequence mutations, and prediction of deleterious effects of missense mutations, has been described previously^49,50. Approximately 250 ng of DNA from each sample was sheared to an average of 150 bp in a Covaris instrument for 360 seconds (duty cycle, 10%; intensity, 5; cycles/burst, 200). Bar-coded libraries were prepared using the Kapa Low-Throughput Library Preparation Kit Standard (Kapa Biosystems), amplified using the KAPA HiFi Library Amplification Kit (Kapa Biosystems; 8 cycles), and quantified using Qubit Fluorimetric Quantitation (Invitrogen) and Agilent Bioanalyzer. An equimolar pool of the 4 bar-coded libraries (300 ng each) was used as input to capture the exome using one reaction tube of the Nimblegen SeqCap EZ Human Exome Library v3.0 (Roche; cat no. 06465684001), according to the manufacturer’s protocol. The pooled capture library was quantified by Qubit (Invitrogen) and Bioanalyzer (Agilent) and sequenced on an Illumina HiSeq 2500 using a paired end, 100 nucleotide in length run mode, to achieve an average of 100x coverage.

Histiocytic neoplasms are routinely characterized by low tumor cellularity, which often results in low variant-allele fractions for established driver mutations such as BRAF^{V600E 6}. As such, for samples in which no MAPK-pathway mutations were called using established pipelines developed and optimized for use in more-cellular solid tumors, sequences were manually curated, and mutations with lower read support were salvaged.

Mutations identified by WES were validated using a custom-designed, TruSeq Custom Amplicon probe. Design Studio (Illumina) was used to design amplicons covering the regions of interest. The regions were amplified using 250 ng of template genomic DNA, using the manufacturer’s instructions, with 25 cycles of amplification, and were run on an Illumina MiSeq 2 × 250 cartridge.

Map2k1^E102_I103del In-Frame Deletion Conditional Knock-in Vector Design Outline

An 8.4 kb genomic DNA used to construct the targeting vector was first subcloned from a positively identified C57BL/6 BAC clone (RP23–10C6). The region was designed such that the long homology arm (LA) extends 6.8 kb 5’ to the 5’ LoxP cassette, and the short homology arm (SA) extends 2.6 kb 3’ to the insertion of the inversion cassette. The 5’ LoxP cassette was inserted 250 bp upstream of WT exon 3. The inversion cassette is flanked by two mutant Lox sites (Lox66/71) and consists of inverted mutant exon 3* (c.303_308del; GGAGAT) and its flanking genomic sequences for correct splicing (Inv.saEx3*Sd). Base pair changes were made in the inversion cassette for splicing optimization. The inversion cassette was inserted downstream of WT exon 3. The FRT-flanked Neo cassette was inserted immediately upstream of the inversion cassette and is 465 bp downstream from wild-type exon 3. The targeting region is 863 bp containing exon 3.

The targeting vector was confirmed by restriction analysis and sequencing after each modification step. The boundaries of the 2 homology arms were confirmed by sequencing with BAC5 (5’- CTG CAC CAT GGC TGT CTA ACA G-3’) and T73 (5’- TAA TGC AGG TTA ACC TGG CTT ATC G-3’) primers. The 5’ LoxP site was confirmed by sequencing with LOX1 (5’- GTG CCC ACC TAG CCT GGC ATA TG-3’) primer. Primers read from the selection cassette into the 3’ end of the middle arm (iNeo N2: 5’- AGT ATG GCT TTC CTT CCC GAT GG-3’) and the 5’ of the inversion cassette (IVNeo N3: 5’- TCT AAG GCC GAG TCT TAT GAG CAG-3’). The entire inversion cassette sequence and flanking Lox66 and Lox71 were confirmed by IVNeo N3 (5’- TCT AAG GCC GAG TCT TAT GAG CAG-3’), SQ4 (5’- CTT CCC TGC CTC CCC ATT CCA C-3’), and NeoSQ2 (5’- TTA CTA TAT ATA TGC CCA ATA AGC ATG AGC C −3’) primers. (Figs. 2A, Fig. S1A, Table S5).

Backbone Vector Information

The BAC was sub cloned into a ~2.4kb pSP72 (Promega) backbone vector containing an ampicillin selection cassette for retransformation of the construct prior to electroporation. A hUBS-gb2 FRT-flanked Neomycin cassette was inserted into the gene as described in the project schematic. The targeting construct can be linearized using Not I prior to electroporation into ES cells. The total size of the targeting construct (including vector backbone and DT cassette) is ~ 18.0 kb.

PCR Screening and Reconfirmation of Recombinant Clones

Ten micrograms of the targeting vector were linearized and then transfected by electroporation of FLP C57Bl/6 (BF1) embryonic stem (ES) cells. After selection with G418 antibiotic, surviving clones were expanded for PCR analysis to identify recombinant ES clones. The Neo cassette in the targeting vector has been removed during ES clone expansion. Screening primer A1 (5’- CTT CCT GTT TCT GGC TTA TCA GCA TTG ACT C –3’) was designed downstream of the short homology arm (SA) outside the 3’ region used to generate the targeting construct. PCR reactions using A1 with the FN1 (5’- GTT CGT GGG ATT GTG TCC GTG TCG –3’) primer amplify a 3.64 kb fragment. Positive clones were identified and selected for further expansion. Positive clones were expanded and reconfirmed for SA integration. Wild-type DNA was used as a negative control. DNA from an individual clone (before reconfirmation) was used as a positive control. No DNA was used as a negative control. Confirmation of distal LoxP retention was performed by PCR using the LOX1 (5’- GTG CCC ACC TAG CCT GGC ATA TG–3’) and FN2A (5’- AAC TTC GCG ACA CGG ACA CAA TCC –3’) primers. This reaction produces a 1.36 kb-sized product.

Sequencing was performed on purified PCR DNA to confirm the 3’ junction of the Inv.saE3* cassette using the SC1 (5’- TCT GGG CTG TTT AGG ATG ACA CTT G–3’) primer. Sequencing was performed on purified PCR DNA to confirm 5’ junction of the Inv.saE3* cassette using the FN1 (5’- GTT CGT GGG ATT GTG TCC GTG TCG–3’) primer. Sequencing was performed on purified PCR DNA to confirm presence of the distal LoxP cassette using the SDL2 (5’- GGT GGA TCA GCT GAA GGG AAG ACG–3’) primer. Gene targeting analysis of positive clones by real-time PCR using a probe which anneals to wild type allele and corresponds to the target site was conducted. Analysis of positive clones for copy number using a probe annealing to the 3’ homology arm region was also conducted by real-time PCR (Table S5.

Identification of F1 and F2 Heterozygous Mice

Targeted iTL BF1 (C57BL/6 FLP) embryonic stem cells were microinjected into Balb/c blastocysts. Resulting chimeras with a high percentage black coat color were mated to C57BL/6N WT mice to generate Germline Neo Deleted mice. Tail DNA was analyzed as described below from pups with black coat color.

The presence of the distal LoxP site was detected with primers LOX1 (5’- GTG CCC ACC TAG CCT GGC ATA TG −3’) and RNEOGT (5’- GAA AGT ATA GGA ACT TCG CGA CAC GGA C −3’). Lox1 is located on the long homology arm upstream of the Distal LoxP site. RNEOGT is located inside the remaining Neo cassette. The amplified size for Lox1/RNEOGT is 1.38 kb. The EconoTaq Plus Green 2x Master Mix (Lucigen catalog# 30033–1) was utilized and consisted of the following: 11.00 µL ddH₂0; 12.50 µL EconoTaq Plus Green 2x Master Mix; 0.25µL 100 µM Each Primer; and 1.00 µL DNA. The PCR parameters for LOX1/ RNEOGT are the following: a 94°C hot start for 2 minutes; 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 minute and 15s; and then 4°C for infinity. The PCR product was run on a 2% agarose gel with a 100 bp ladder as reference. An expanded ES clone was used as a positive control. Bands were excised and sequenced from positive samples (Fig. S1B, Table S5).

Primer sets newFLP1 (5’- ACA GAG ACA AAG ACA AGC GTT AGT AGG −3’) and newFLP2 (5’- ATT TCC CAC AAC ATT AGT CAA CTC CGT TAG G-3’) were used to screen mice for the FLP transgene. The amplified product for primer set newFLP1 and newFLP2 is 330bp. The EconoTaq Plus Green 2x Master Mix (Lucigen catalog# 30033–1) was utilized and consisted of the following: 11.00 µL ddH₂0; 12.50 µL EconoTaq Plus Green 2x Master Mix; 0.25µL 100 µM each primer; and 1.00 µL DNA. The PCR parameters for newFLP1 / newFLP2 are the following: a 94°C hot start for 2 minutes; 30 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 1 minute; and then 4°C for infinity. The PCR product was run on a 2% agarose gel with a 100 bp ladder as reference. A tail DNA sample from an FLP mouse was used as the positive control (Table S5).

Tail DNA samples from positive mice were amplified with primers FN1 (5’- GTT CGT GGG ATT GTG TCC GTG TCG −3’) and A1 (5’- CTT CCT GTT TCT GGC TTA TCA GCA TTG ACT C −3’). FN1 is located inside the remaining Neo cassette, and A1 is located downstream of the short homology arm, outside the region used to create the targeting construct. FN1/A1 amplifies a fragment of 3.64 kb in length. When the Neo cassette is intact the PCR yields a 6.29 kb product. The Expand High Fidelity PCR System (Roche catalog # 04 738 276 001) was utilized and consisted of the following: 17.50 µL ddH₂0; 2.50 µL 200.0 µM dNTP; 2.50 µL PCR Buffer with 15mM MgCl₂; 1.00 µL DMSO; 0.25 µL 100 µM each primer; and 1.00 µL 1.5µL DNA. The PCR parameters for FN1/A1 are the following: a 99°C hot start for 10 minutes; 40 cycles of 95°C for 30 s, 62°C for 30 s, and 68°C for 4 minutes and 30s; and then 4°C for infinity. After a 10-minute hot start at 99°C, 0.125 µL of Taq polymerase was added to each PCR sample followed by a layer of 2 drops mineral oil. The PCR product was run on a 0.8% gel with a 1 kb ladder as reference. The expanded ES clone was used as a positive control (Fig. S1B, Table S5).

Bone Marrow Serial Transplantation Assays

Freshly dissected femora and tibiae were isolated from Mx1-Cre Map2k1^E102_I103del and Cre-negative Map2k1^E102_I103del CD45.2⁺ primary mice (which underwent pIpC administration at 4 weeks). Bone marrow was flushed with a 3-cm³ insulin syringe into cold PBS supplemented with 0.5% fetal calf serum (FCS; heat-inactivated). The bone marrow was spun by centrifugation at 1,500 rpm for 5 minutes, and red blood cells were lysed in ammonium chloride–potassium bicarbonate lysis (ACK) buffer for 3 minutes on ice. Cells were then resuspended in PBS + 0.5% FBS, passed through a 40-µm cell strainer, and counted. Serial transplantation was performed by transplanting 1 × 10⁶ total bone marrow cells from primary mice into lethally irradiated (900 cGy), eight-week-old CD45.1⁺ recipient mice. Peripheral blood chimerism was assessed every 4 weeks by flow cytometry.

Ulixertinib treatment in mice

We transplanted 1.0 × 10⁶ BM MNCs from CD45.2⁺ Mx1-Cre MEK1^E102_I103/WT mice into lethally irradiated CD45.1⁺ 8-week-old C57/B6 recipients (two weeks after pIpC administration of primary mice). For ERK1/2 inhibitor treatment, ulixertinib (Selleck Chemicals) was dissolved in 0.5% cCMC-NA (carboxymethylcellulose) (Selleck Chemicals, S6703) to obtain a final concentration of 4 mg/mL. Mice were orally administrated with either 100 mg/kg ulixertinib (twice daily) or vehicle (0.5% CMC-NA daily) for 5 days/week starting from 15 days post-transplant to 90 days post-transplant.

Tasquinimod treatment in mice

Tasquinimod (MedChemExpress, HY-10528) was dissolved in 1% cCMC-NA (carboxymethylcellulose) (Selleck Chemicals, S6703) to obtain a final concentration of 6 mg/mL. Mice were orally administrated with either 30 mg/kg tasquinimod (once daily) or vehicle (0.5% CMC-NA daily) for 5 days/week starting from 14 days post-transplant to 35 days post-transplant.

Peripheral blood analysis

Blood was collected by submandibular bleeding using heparinized microhematocrit capillary tubes (Thermo Fisher Scientific). Automated peripheral blood counts were obtained using a ProCyte Dx Hematology Analyzer (IDEXX).

Flow cytometry analysis

Peripheral blood, bone marrow, spleen, and liver samples collected from transgenic mice and littermate controls, as well as serially transplanted mice were first lysed with ACK lysis buffer to remove red blood cells and washed with ice-cold phosphate-buffered saline. Cells were stained with monoclonal antibodies against cell surface markers in phosphate-buffered saline/2% bovine serum albumin for 30 minutes on ice. For intracellular staining, cells were further fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences; 554714) according to the manufacturer’s protocol and then stained with antibodies.

The following anti-mouse antibodies were used for flow cytometry: anti-CD45.2-FITC (clone 104; BioLegend; 109806; 1:100), anti-CD45.2-PE (clone QA18A15; BioLegend; 111104; 1:100), anti-CD45.1-A700 (clone A20; BioLegend; 110724), anti-CD11b -APC (clone M1/70; BioLegend; 101212; 1:100), anti-CD11b-APC-Cy7 (clone M1/70; BioLegend; 101226; 1:100), anti-Ly6C-APC-Cy7 (clone HK1.4; BioLegend; 128026; 1:100), anti-Ly6G-PerCP-Cy5.5 (clone 1A8; BioLegend; 127616; 1:100), anti-CD3e-PE-Cy7 (clone 500A2; BioLegend; 152314; 1:100), anti-B220 (CD45RO)-BV605 (clone RA3–6B2; BioLegend; 103244; 1:100), anti-CD115-PE (clone W19330E; BioLegend; 165203; 1:100), anti-CX3CR1-BV605 (clone SA011F11; BioLegend; 149027; 1:100), anti-F4/80-A700 (clone BM8; BioLegend; 123130; 1:100), anti-CD11c-PerCP-Cy5.5 (clone N418; BioLegend; 117328; 1:100), anti-1A/1E (MHCII)-APC (clone M5/114.15.2; BioLegend; 107614, 1:100), anti-CD24-BV605 (clone M1/69; BioLegend; 101827; 1:100), and anti-CD103-FITC (clone W19396D; BioLegend; 110908; 1:100), anti-B220-FITC (clone RA3–6B2; Thermo Fisher Scientific; 11-0452-85; 1:500), anti-CD45.2-PE (clone 104; BioLegend; 109808; 1:1000), anti-CD34-PE/Cy7 (clone HM34; BioLegend; 128618: 1;100), anti-CD117 (c-kit)-APC (clone ACK2; BioLegend; 135108; 1:500), anti-CD45.1-Alexa Fluor 700 (clone A20; BioLegend; 110724: 1:500), anti-CD3-APC/Cy7 (clone 17A2; BioLegend; 100222; 1:500), anti-Ly-6A/E (Sca-1)-BV605 (clone D2; BioLegend; 108134; 1:500), anti-CD11b-BV711 (clone M1/70; BioLegend; 101242; 1:2000), anti-Ki-67-BV786 (clone SolA15; Thermo Fisher Scientific; 417-5698-82; 1:50), anti-CD45.2-FITC (clone 104; BioLegend; 109805; 1:500), anti-Ly-6G- PerCP/Cy5.5 (clone 1A8; BioLegend; 127616; 1:500), anti-CD45.1-Alexa Fluor 700 (clone A20; BioLegend; 110724; 1:500), anti-Ly-6C-APC/Cy7 (clone HK1.4; BioLegend; 128026; 1:500), anti-CD11b-BV605 (clone M1/70; BioLegend; 101257; 1:2000), anti-CD274 (PD-L1)-BV711 (clone 10F.9G2; BioLegend; 124319; 1:200), anti-TNF alpha-PE (clone MP6-XT22; Thermo Fisher Scientific; 12-7321-82; 1:100), anti-IL-1 beta (Pro-form)-PE/Cy7 (clone NJTEN3; Thermo Fisher Scientific; 25-7114-82; 1:100), anti-Phospho-NF-κB p65 (Ser536)-Alexa Fluor 647 (clone 93H1; Cell Signaling Technology; 4887; 1:100), anti-CD71-PE (clone RI7217; BioLegend; 113807; 1:500), anti-TER119-BV785 (clone TER-119; BioLegend; 116245; 1:500) following the manufacturer’s protocol. DAPI (BioLegend, 422801, 1:1000) or Zombie Violet Fixable Viability Kit (BioLegend; 423113; 1:500) was used to exclude dead cells. Flow cytometry data acquisition or cell sorting was performed by BD LSRFortessa or BD FACSAria II (BD Biosciences). Flow cytometric data were analyzed by FlowJo 10 software.

Histology and immunohistochemistry (IHC) analysis

Mouse tissue was collected and fixed in 4% paraformaldehyde for at least 48 hours. Samples were then processed, embedded in paraffin, sectioned at 4 µm thickness, and stained with hematoxylin and eosin by the Laboratory of Comparative Pathology at MSKCC. For immunohistochemistry, Bond Dewax Solution (Leica, AR9222) was used. Antigen retrieval was performed by using the Leica Bond RX H1(10) protocol: HIER 10 minutes ER1 (Citrate pH6) for B220 and H2(20) protocol: HIER 20 minutes ER2 (EDTA pH9) at 100C. Slides were quenched using the Bond Polymer Refine Detection (Leica, DS9800). Slides were incubated with the primary antibodies B220 (BDBioscience, 550286, 1:200), Iba1 (Abcam, ab5076, 1:2000) for 15 minutes, followed by secondary antibodies Rabbit Anti-Rat IgG (Vector Laboratories, BA-4001, 1:100) for B220 and Rabbit Anti-Goat IgG (Vector Laboratories, BA-5000, 1:1000) for Iba1. Visualization was done using diaminobenzidine 10 minutes followed by hematoxylin counterstain 5 minutes from the Bond Polymer Refine Detection Kit (Leica, DS9800).

The percentage of tumor cells exhibiting staining was scored by an American Board of Pathology–certified hematopathologist with extensive expertise in both human and mouse hematopathological evaluations (B.H.D). Microscopic slides were evaluated using an OLYMPUS BX41 microscope (Olympus Scientific Solutions, Waltham, MA), and images were acquired using an OLYMPUS DP72 camera (Olympus Scientific Solutions).

Western blotting

Anti-phospho-MEK1/2 (Ser217/221) (no. 9121), anti-MEK1/2 (D1A5) (no. 8727), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (no. 9101), and anti-p44/42 MAPK (ERK1/2) (137F5) (no.4695), as well as the secondary antibodies anti-rabbit IgG-HRP (no. 7076) and anti-mouse IgG-HRP (no. 7074) were purchased from Cell Signaling Technology. Anti-β-Actin (A5441) was purchased from Sigma-Aldrich^®. Cell lysates were prepared in RIPA buffer supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific). Equal amounts of protein, as measured by the BRADFORD protein assay, were resolved in 4–12% Bis-Tris NuPage gradient gels (Life Technologies) and transferred electrophoretically on a polyvinylidene difluoride 0.45-m membrane. Membranes were blocked for 30 minutes at room temperature in 5% bovine serum albumin (BSA) in TBST before being incubated overnight at 4°C with the primary antibodies. All primary antibodies were diluted 1:1,000 in 5% BSA in TBST, except anti-β-actin, which was diluted 1:5,000 in 5% BSA in TBST. After three washes of 5 min in TBST, secondary antibodies were diluted 1:2,000 in 5% BSA in TBST and incubated for 1 h at room temperature. After another three washes in TBST, detection of the signal was achieved by incubating the membrane on an ECL solution from Millipore and exposure on autoradiography films from Denville Scientific (Metuchen, NJ, USA).

Serum cytokine analysis

The MILLIPLEX^® Humanized Mouse Panel (Cat. No. HUMU-210K) was used to evaluate serum cytokine levels according to the kit protocol. Samples were tested neat and analyzed on Luminex^® 200^™ systems, and data were acquired via xPONENT^® v. 4.3 software. Data analysis was performed using the Belysa^® Immunoassay Curve Fitting Software (Cat. No. 40–122).

Cell viability assay

20,000 bone marrow mononuclear cells per well were seeded into 96-well plate. 96 hours after treatment with ulixertinib, cell viability was assessed by CellTiter-Glo Luminescent Cell Viability Assay (Promega; G7572) according to the manufacturer’s protocol.

Single-cell RNA-seq analyses

3’ single-cell RNA sequencing FASTQs were processed using the nf-core scRNA-seq pipeline v2.6.0 (10.5281/zenodo.3568187) with 10x Genomics CellRanger v8.0.0 and aligned to GRCm39. Count matrices and relevant metadata were loaded into Seurat v5.1.0 objects ⁵¹. Individual samples were then filtered to only retain cells with (1) greater than 200 and less than 5,000 detected unique RNA features, (2) greater than 200 and less than 10,000 total RNA molecules, and (3) less than 5 percent mitochondrial RNA reads. After filtering, doublets were removed sample-wise using scDblFinder v1.19.0 ⁵² and refined data was merged into a single Seurat object.

Expression count data was log-normalized using Seurat’s NormalizeData function. Variable features were identified with FindVariableFeatures using variance-stabilizing transformation followed by data scaling with ScaleData. Principle-component analysis (PCA) was performed using Seurat’s RunPCA function. To mitigate the effect of sample-specific technical covariates/batch effects, HarmonyIntegration v1.2.0 ⁵³ was used as part of the IntegrateLayers function. Elbow plots were manually inspected to determine the number of principal components (PCs) to use downstream followed by K-nearest neighbor graph construction with the FindNeighbors function using the first 11 PCs. Louvain clusters were then generated with FindClusters using a resolution of 0.5 and a global UMAP was constructed with the RunUMAP function.

To assign cell types, FindAllMarkers was used to compute cluster-wise differential gene expression. These markers were then cross referenced with the Cellmarker 2.0 database ⁵⁴ to include only those expressed within mouse spleen and bone-marrow tissue. The top 10 genes from each cluster that passed filtering were used as input to ChatGPT4o ⁵⁵ for cell-type prediction. These predictions were then manually verified using established markers. Next, separate Seurat objects were made for the clusters containing monocytes, macrophages, and neutrophils. These were reclustered as above using a resolution of 0.1, and refined cell types were assigned manually based on expression of known cell markers (Fig. S3).

Transcriptional differences between mutant and wild-type mice for each cell type were characterized using Seurat’s implementation of the Wilcoxon-Rank Sum Test using log-normalized counts between WT and mutant mice as input. The top 20 genes as determined by the absolute value of the log₂FC multiplied by the -log₁₀(Bonferroni-adjusted P value) were then plotted as heatmaps using ComplexHeatmap ⁵⁶. GO analysis was performed with clusterProfiler ⁵⁷ using enrichGO within the compareCluster function. Plots were created using ggplot2 ⁵⁸ and SCpubr v2.0.2 ⁵⁹.

Statistical analyses

Data were plotted using GraphPad Prism 9 software as mean values, with error bars representing standard deviation (SD). Data are shown as mean, with individual values per mouse represented as circles, unless stated otherwise. Statistical significance was analyzed with GraphPad Prism 9 software as mean values with error bars representing standard deviation (SD) by using Mann-Whitney tests, unpaired 2-tailed t tests, 1-way analysis of variance, and Log-rank (Mantel-Cox) test as indicated in the figure legends. The n value represents biological replicates. Significance was considered at *P < 0.05. Kaplan-Meier survival analysis was used to estimate overall survival. Experiments were repeated to ensure reproducibility of the observations. Equal variance was assumed for cell counting experiments. No statistical methods were used to predetermine sample size. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Supplementary Material

2

Table S1. Histiocytoses diagnosis and kinase driver alterations identified in the patient cohort (n=498), related to Figure 1.

3

Table S2. Distributions of sites of disease by MEK1 mutational class, related to Figure 1.

4

Table S3. Characteristics of patients treated with ulixertinib, related to Figure 7.

5

Table S4. Adverse events in patients treated with ulixertinib, related to Figure 7.

6

Table S5. Oligonucleotide primers utilized in generating and screening the Map2k1^E102_I103del conditional knock-in mice, related to the STAR Methods.

Key resources table

REAGENT or RESOURCE		SOURCE	IDENTIFIER
Antibodies
CD45R (B220) Monoclonal Antibody (RA3-6B2), FITC		Thermo Fisher Scientific	Cat#1-0452-85; RRID: AB_465055
CD45R (B220) Monoclonal Antibody (RA3-6B2), BV605		BioLegend	Cat#103244; RRID: AB_2563312
PE anti-mouse CD45.2 Antibody		BioLegend	Cat#109808; RRID: AB_313445
PE/Cyanine7 anti-mouse CD34 Antibody		BioLegend	Cat#128618; RRID: AB_2721678
APC anti-mouse CD117 (c-kit) Antibody		BioLegend	Cat#135108; RRID: AB_2028407
Alexa Fluor 700 anti-mouse CD45.1 Antibody		BioLegend	Cat#110724; RRID: AB_493733
APC/Cyanine7 anti-mouse CD3 Antibody		BioLegend	Cat#100222; RRID: AB_2242784
Brilliant Violet 605 anti-mouse Ly-6A/E (Sca-1) Antibody		BioLegend	Cat#108134; RRID: AB_2650926
Brilliant Violet 711 anti-mouse/human CD11b Antibody		BioLegend	Cat#101242; RRID: AB_2563310
Ki-67 Monoclonal Antibody (SolA15), Brilliant Violet™ 786		Thermo Fisher Scientific	Cat#417-5698-82; RRID: AB_2925745
FITC anti-mouse CD45.2 Antibody		BioLegend	Cat#109805; RRID: AB_313442
PerCP/Cyanine5.5 anti-mouse Ly-6G Antibody (clone 1A8)		BioLegend	Cat#127616; RRID: AB_1877271
Alexa Fluor® 700 anti-mouse CD45.1 Antibody		BioLegend	Cat#110724; RRID: AB_493733
APC/Cyanine7 anti-mouse Ly-6C Antibody (clone HK1.4)		BioLegend	Cat#128026: RRID: AB_10640120
Brilliant Violet 605 anti-mouse/human CD11b Antibody		BioLegend	Cat#101257: RRID: AB_2565431
anti-CD11b -APC (clone M1/70)		BioLegend	Cat#101212; RRID: AB_312795
Anti-CD11b-APC-Cy7 (clone M1/70)		BioLegend	Cat#101226; RRID: AB_830642
Anti-CD3e-PE-Cy7 (clone 500A2)		BioLegend	Cat#152314; RRID: AB_2629847
Anti-CD115-PE (clone W19330E)		BioLegend	Cat#165203; RRID: AB_2910352
Anti-CX3CR1-BV605 (clone SA011F11)		BioLegend	Cat#149027; RRID: AB_2565937
Anti-F4/80-A700 (clone BM8)		BioLegend	Cat#123130; RRID: AB_2293450
Anti-CD11c-PerCP-Cy5.5 (clone N418)		BioLegend	Cat#117328; RRID: AB_2129641
Anti-1A/1E (MHCII)-APC (clone M5/114.15.2)		BioLegend	Cat#107614; RRID: AB_313329
Anti-CD24-BV605 (clone M1/69)		BioLegend	Cat#101827; RRID: AB_2563464
FITC anti-mouse CD103 Antibody (clone W19396D)		BioLegend	Cat#110907; RRID: AB_2936716
Brilliant Violet 711 anti-mouse CD274 (B7-H1, PD-L1) Antibody		BioLegend	Cat#124319; RRID: AB_2563619
TNF alpha Monoclonal Antibody (MP6-XT22), PE		Thermo Fisher Scientific	Cat#12-7321-82; RRID: AB_466199
IL-1 beta (Pro-form) Monoclonal Antibody (NJTEN3), PE-Cyanine7		Thermo Fisher Scientific	Cat#25-7114-82; RRID: AB_2573526
Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (Alexa Fluor 647 Conjugate)		Cell Signaling Technology	Cat#4887; RRID: AB_561198
TruStain FcX (anti-mouse CD16/32) Antibody		BioLegend	Cat#101320; RRID: AB_1574975
Purified rat anti-mouse CD45R (B220) (clone RA3-6B2)		BD Bioscience	Cat#550286; RRID: AB_393581
Anti-Iba1 Antibody (polyclonal)		Abcam	Cat#ab5076; RRID: AB_2224402
Rabbit Anti-Rat IgG		Vector Laboratories	Cat#BA-4001; RRID: AB_10015300
Rabbit Anti-Goat IgG		Vector Laboratories	Cat#BA-5000; RRID: AB_2336126
Anti-phospho-MEK1/2 (Ser217/221)		Cell Signaling Technologies	Cat#9121; RRID: AB_331648
Anti-MEK1/2 (D1A5)		Cell Signaling Technologies	Cat#8727; RRID: AB_10829473
Anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204)		Cell Signaling Technologies	Cat#9101; RRID: AB_331646
Anti-p44/42 MAPK (ERK1/2) (137F5)		Cell Signaling Technologies	Cat#4695; RRID: AB_390779
Anti-rabbit IgG-HRP		Cell Signaling Technologies	Cat#7076; RRID: AB_330924
Anti-mouse IgG-HRP		Cell Signaling Technologies	Cat#7074; RRID: AB_2099233
Anti-β-Actin		Sigma-Aldrich	Cat#A5441; RRID: AB_476744
Bacterial and virus strains
Biological samples
Chemicals, peptides, and recombinant proteins
Ulixertinib		Selleck Chemicals	Cat#S7854
Tasquinimod		MedChemExp ress	Cat#HY-10528
cCMC-NA (carboxymethylcellulose)		Selleck Chemicals	Cat#S6703
Critical commercial assays
Nimblegen SeqCap EZ Human Exome Library v3.0		Roche	Cat# 06465684001
MILLIPLEX® Humanized Mouse Panel		MILLIPLEX	Cat# HUMU-210K
CellTiter-Glo Luminescent Cell Viability Assay		Promega	Cat# G7572
Zombie Violet Fixable Viability Kit		BioLegend	Cat# 423113
BD Cytofix/Cytoperm Fixation/Permeabilization Kit		BD Biosciences	Cat# 554714
Deposited data
Datasets generated during and/or analyzed during the current study, including patient-level clinical data, as well as DNA sequencing data		This paper	cBioPortal:: https://www.cbioportal.org/study/summary?id=hdcn_msk_2025
Single-cell RNA-seq data raw and analyzed.		This paper.	GEO: GSE268618
Experimental models: Cell lines
Experimental models: Organisms/strains
Mouse: C57BL/6-Map2k1E102_I103del		This paper.	N/A
Mouse: B6.Cg-Tg(Mx1-Cre)1Cgn/J		The Jackson Laboratory	RRID: IMSR_JAX:003556
Mouse: B6.Cg-Tg(VAV1-Cre)1Graf/MdfJ		The Jackson Laboratory	RRID: IMSR_JAX:035670
Oligonucleotides
Oligonucleotide primers utilized in generating and screening the Map2k1E102_I103del conditional knock-in mice		This paper.	Table S5
Recombinant DNA
Software and algorithms
FlowJo		BD Biosciences	V10
GraphPad Prism		GraphPad Software	V9
Belysa® Immunoassay Curve Fitting Software		Millipore Sigma	Cat# 40-122
nf-core scRNA-seq pipeline v2.6.0		Hao et al.51	https://nf-co.re/scrnaseq/2.6.0/
10x Genomics CellRanger v8.0.0		Hao et al.51	https://www.10xgenomics.com/support/software/cell-ranger/8.0/release-notes
Seurat v5.1.0 objects		Hao et al.51	https://satijalab.org/seurat/
scDblFinder v1.19.0		Germain et al.52	https://github.com/plger/scDblFinder
HarmonyIntegration v1.2.0		Korsunsky et al.53	https://github.com/immunogenomics/harmony
Cellmarker 2.0 database		Hu et al.54	http://117.50.127.228/CellMarker/CellMarker_help.html
ChatGPT4o		Hou et al.55	https://openai.com/index/hello-gpt-4o/
ComplexHeatmap		Gu et al.56	https://bioconductor.org/packages/release/bioc/html/ComplexHeatmap.htm
clusterProfiler		Wu et al.57	https://www.bioconductor.org/packages/release/bioc/html/clusterProfiler.html
ggplot2	Wickham and Pedersen58		https://ggplot2.tidyverse.org/articles/ggplot2.html
SCpubr v2.0.2	Blanco-Carmona59		https://www.rdocumentation.org/packages/SCpubr/versions/2.0.2
Other

Highlights.

• RAF-independent (class III) MEK1 mutations are common in histiocytoses patients.

• Class III MEK1/2 mutations are associated with MEK inhibitor disease progression.

• Class III MEK1^E102_I103del mutation drives aggressive myeloid neoplasms in mice.

• Class III MEK1-mutant histiocytoses patients and mice respond to ERK inhibition.