Search

Background: Preclinical studies in myeloid neoplasms have demonstrated efficacy of Bromodomain and Extra-Terminal protein inhibitors (BETi). However, BETi demonstrate poor single agent activity in clinical trials. Several studies suggest that combination with other anti-cancer inhibitors may enhance the efficacy of BETi. Experimental Design: To nominate BETi combination therapies for myeloid neoplasms, we used a chemical screen with therapies currently in clinical cancer development and validated this screen using a panel of myeloid cell line, heterotopic cell line models, and PDX models of disease. We utilized standard protein and RNA assays to determine the mechanism responsible for synergy in our disease models. Results: We identified PIM inhibitors (PIMi) as therapeutically synergistic with BETi in myeloid leukemia models. Mechanistically, we show that PIM kinase is increased after BETi treatment, and that PIM kinase upregulation is sufficient to induce persistence to BETi and sensitize cells to PIMi. Further, we demonstrate that miR-33a downregulation is the underlying mechanism driving PIM1 upregulation. We also show that GM-CSF hypersensitivity, a hallmark of chronic myelomonocytic leukemia (CMML), represents a molecular signature for sensitivity to combination therapy. Conclusion: Inhibition of PIM kinases is a potential novel strategy for overcoming BETi persistence in myeloid neoplasms. Our data supports further clinical investigation of this combination.

Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ∼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients who had shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1−/− accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+;Asxl1−/− (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or short hairpin RNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8 T-cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations.

SummaryPreclinical studies in myeloid neoplasms have demonstrated efficacy of Bromodomain and Extra-Terminal protein inhibitors (BETi). However, BETi demonstrate poor single agent activity in clinical trials. Several studies suggest that combination with other anti-cancer inhibitors may enhance the efficacy of BETi. To nominate BETi combination therapies for myeloid neoplasms, we used a chemical screen with therapies currently in clinical cancer development. We identified PIM inhibitors (PIMi) as therapeutically synergistic with BETi in myeloid leukemia models. Mechanistically, we show that PIM kinase is increased after BETi treatment, and that PIM kinase upregulation is sufficient to induce resistance to BETi and sensitize cells to PIMi. Further, we demonstrate that miR-33a downregulation is the underlying mechanism driving PIM1 upregulation. We also show that GM-CSF hypersensitivity, a hallmark of chronic myelomonocytic leukemia (CMML), represents a molecular signature for sensitivity to combination therapy and credential this using patient-derived xenografts supporting the clinical investigation of this combination.

Casitas B-lineage lymphoma (CBL) encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and nonreceptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10% to 20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein’s E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) recruitment, and downstream phosphatidylinositol 3-kinase (PI3K)/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine kinase–binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.

Modeling chronic myelomonocytic leukemia (CMML) in immunodeficient NSGS mice relies on unique human CMML specimens and consistent murine engraftment. Only anecdotal comments have thus far supported the notion that research data may be altered by Corynebacterium bovis, an opportunistic cutaneous pathogen of immunodeficient mice. C. bovis disseminated by asymptomatic and clinically affected mice with hyperkeratotic dermatitis, resulting in resilient facility contamination and infectious recurrence. Herein we report that, compared with C. bovis PCR-negative counterparts, C. bovis PCR-positive NSGS mice developed periocular and facial hyperkeratosis and alopecia and had reduced metrics indicative of ineffective human CMML engraftment, including less thrombocytopenia, less splenomegaly, fewer CMML infiltrates in histopathologic sections of murine organs, and fewer human CD45(+) cells in samples from murine spleen, bone marrow, and peripheral blood that were analyzed by flow cytometry. All CMML model metrics of engraftment were significantly reduced in the C. bovis PCR-positive cohort compared with the -negative cohort. In addition, a survey of comprehensive cancer center practices revealed that most murine facilities do not routinely test for C. bovis or broadly decontaminate the facility or its equipment after a C. bovis outbreak, thus increasing the likelihood of recurrence of invalidated studies. Our findings document that CMML engraftment of NSGS mice is diminished—and the integrity of murine research data jeopardized—by C. bovis infection of immunodeficient mice. In addition, our results indicate that C. bovis should be excluded from and not tolerated in murine facilities housing immunodeficient strains.

MYC oncoproteins regulate transcription of genes directing cell proliferation, metabolism, and tumorigenesis. A variety of alterations drive MYC expression in acute myeloid leukemia (AML), and enforced MYC expression in hematopoietic progenitors is sufficient to induce AML. Here we report that AML and myeloid progenitor cell growth and survival rely on MYC-directed suppression of Transcription Factor EB (TFEB), a master regulator of the autophagy–lysosome pathway. Notably, although originally identified as an oncogene, TFEB functions as a tumor suppressor in AML, where it provokes AML cell differentiation and death. These responses reflect TFEB control of myeloid epigenetic programs by inducing expression of isocitrate dehydrogenase-1 (IDH1) and IDH2, resulting in global hydroxylation of 5-methycytosine. Finally, activating the TFEB–IDH1/IDH2–TET2 axis is revealed as a targetable vulnerability in AML. Thus, epigenetic control by an MYC–TFEB circuit dictates myeloid cell fate and is essential for maintenance of AML. Significance: Alterations in epigenetic control are a hallmark of AML. This study establishes that a MYC–TFEB circuit controls AML differentiation and epigenetic programs by inducing IDH1/IDH2 and hydroxylation of 5-methylcytosine, that TFEB functions as a tumor suppressor in AML, and that this circuit is a targetable vulnerability in AML. See related commentary by Wu and Eisenman, p. 116.

CBL encodes an E3 ubiquitin ligase and signaling adaptor that acts downstream of cytokine receptors. Recurrent CBL mutations occur in myeloid malignancies, but the mechanism by which these mutations drive oncogenesis remains incompletely understood. Here we performed a series of studies to define the phosphoproteome, CBL interactome and molecular mechanisms of signaling activation in cells expressing an allelic series of CBL mutants. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced PIK3R1 recruitment and downstream PI3K/AKT signaling in CBL-mutant cells. Furthermore, we demonstrated in vitro and in vivo efficacy of LYN inhibition by dasatinib in CBL-mutant cell lines and primary chronic myelomonocytic leukemia cells. Overall, our data provide rationale for exploring the therapeutic potential of LYN inhibition in patients with CBL-mutated myeloid malignancies.Statement of SignificanceWe investigated the oncogenic mechanisms of myeloid malignancy-associated CBL mutations by mass spectrometry-based proteomics and interactomics. Our findings indicate that increased LYN kinase activity in CBL-mutant cells stimulates PI3K/AKT signaling, revealing opportunities for the use of targeted inhibitors in CBL-mutated myeloid malignancies.

Chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML) are myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) overlap disorders characterized by monocytosis, myelodysplasia, and a characteristic hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Currently, there are no available disease-modifying therapies for CMML, nor are there preclinical models that fully recapitulate the unique features of CMML. Through use of immunocompromised mice with transgenic expression of human GM-CSF, interleukin-3, and stem cell factor in a NOD/SCID-IL2Rγnull background (NSGS mice), we demonstrate remarkable engraftment of CMML and JMML providing the first examples of serially transplantable and genetically accurate models of CMML. Xenotransplantation of CD34+ cells (n = 8 patients) or unfractionated bone marrow (BM) or peripheral blood mononuclear cells (n = 10) resulted in robust engraftment of CMML in BM, spleen, liver, and lung of recipients (n = 82 total mice). Engrafted cells were myeloid-restricted and matched the immunophenotype, morphology, and genetic mutations of the corresponding patient. Similar levels of engraftment were seen upon serial transplantation of human CD34+ cells in secondary NSGS recipients (2/5 patients, 6/11 mice), demonstrating the durability of CMML grafts and functionally validating CD34+ cells as harboring the disease-initiating compartment in vivo. Successful engraftments of JMML primary samples were also achieved in all NSGS recipients (n = 4 patients, n = 12 mice). Engraftment of CMML and JMML resulted in overt phenotypic abnormalities and lethality in recipients, which facilitated evaluation of the JAK2/FLT3 inhibitor pacritinib in vivo. These data reveal that NSGS mice support the development of CMML and JMML disease-initiating and mature leukemic cells in vivo, allowing creation of genetically accurate preclinical models of these disorders.

The interplay between tumor heterogeneity and microenvironmental factors is a critical mechanism for clonal selection in leukemia. Evidence of unique clonal capacities to engraft within patient-derived xenograft (PDX) models suggests that intrapatient genetic architecture may be defined by functional differences at the clonal level. However, methods to detect functional differences assigned to genetically defined clones remain limited. Here, we describe a scalable method to directly measure the functional properties of clones within the same leukemia patient by coupling intracellular flow cytometry and next-generation sequencing (NGS). We provide proof of concept utilizing primary chronic myelmonocytic leukemia (CMML) samples and granulocyte-macrophage colony stimulating factor (GM-CSF) to elucidate the interaction between tumor heterogeneity and microenvironmental factors. Mixtures of human leukemia cell lines, with known response to GM-CSF, were used to validate the accuracy of our methodology. Using this approach, we confirm that our method is capable of discriminating GM-CSF sensitive cell lines, identifies somatic variants in primary leukemia samples, and resolves functional clonal architecture in an illustrative patient. Taken together, our data describes a novel method to determine intrapatient functional clonal heterogeneity and provides proof-of-concept for future investigation aimed at elucidating the clinical relevance of functional clonal differences.

MYC gene amplification and somatic mutations are frequent in both adult and pediatric AML although how MYC drives and contributes to the development and maintenance of AML has not been resolved. Transcription factor EB (TFEB) is a master regulator of genes that control autophagy and lysosome biogenesis, a central catabolic recycling pathway that regulates cell survival. Given the oncogenic effects of MYC in AML and that the induction of autophagy compromises AML cell growth and survival, we tested if the oncogenic effect of MYC depends on its suppression of TFEB transcription programs in AML. In support of this hypothesis, inducible MYC expression in K562 and THP-1 leukemia cells was sufficient to suppress expression of TFEB and its target genes. Further and conversely, MYC knockdown in NB4 AML cells provoked increased expression of TFEB mRNA and protein, as well as increased expression of TFEB target genes. Notably, dose response studies demonstrated that expression of TFEBS211A, constitutively nuclear form of TFEB that is refractory to control by mTORC1 signaling, dramatically impairs proliferation of HL60, OCI-AML2 and OCI-AML3 AML cells. In addition, induction of TFEBS211A provoked rampant apoptosis. Of important, overexpression of TFEBS211A in HL-60 and OCI-AML3 cells was also sufficient to promote monocytic and granulocytic differentiation, as judged by morphological changes and the acquisition of mature monocytic and granulocytic markers including CD11b, Gr1, and CD15. To identify TFEB targets that might contribute to myeloid/granulocytic differentiation, we performed RNA-seq analysis of HL60 leukemia cells engineered to inducibly express the TFEBS211A transgene. Using a cut-off of fold change>4 with q Surprisingly, among genes induced by TFEB in HL60 cells is IDH1, which catalyzes the production of α-ketoglutarate (α-KG), a required substrate of the TET family of dioxygenases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). In particular, TFEBS211A expression provoked increases in levels of α-KG and significant increases in global levels of 5hmC in genomic DNA of HL60 leukemia cells both ex vivo and in vivo. Furthermore, TFEB-mediated induction of IDH1/2 mRNA and protein, and of IDH1 promoter activity, was antagonized by inducible expression of MYC. To assess the global effects of TFEB on 5mC/5hmC landscapes, we performed paired reduced representation bisulfite (BS)- and oxidation bisulfite (oxBS)-sequencing. As predicted, TFEBS211A induced both loss and gains of 5mC, but there were more losses (n = 722) than gains (n = 459) across all 22 chromosomes. Quite remarkably, and consistent with TFEB-provoked increases of 5hmC signals, TFEBS211A exclusively induced 5hmC gains (in a total of 863 genes), and 37% and 36% of these 5hmC gains occurred in promoter regions and CpG islands, and across all 22 chromosomes. Comparison of BS- and oxBS-seq versus RNA-seq analyses of HL60 cells expressing TFEB revealed significant changes in mRNA levels and concomitant differential changes in 5mC and 5hmC marks in KLF4, KLF6, STAT3, TP73, andFOXO1 that have pivotal roles in controlling myeloid cell differentiation and death. Collectively, these findings demonstrate that MYC suppresses TFEB expression and function in AML cells, and that TFEB functions as a tumor suppressor that provokes AML cell differentiation and death. Strikingly, these responses rely on epigenetic control, where TFEB directly induces the transcription of IDH1 and IDH2 to provoke global hydroxylation of 5-methylcytosine and the expression of genes that drive terminal differentiation and apoptosis. Thus, a MYC-TFEB-IDH1/2-TET2 circuit controls AML cell fate. Disclosures Murphy: Merck: Research Funding; Puma Biotech: Research Funding. Ballabio:CASMA Therapeutics: Other: Co-Founder. Kaufmann:Takeda Pharmaceuticals: Research Funding.

Chronic myelomonocytic leukemia (CMML) is an aggressive hematological malignancy with limited treatment options. Whole exome (WES) and targeted sequencing of several independent cohorts of CMML patients, comparing dysplastic (dCMML) to proliferative (pCMML) CMML, as well as paired chronic phase disease and acute leukemic transformation (LT), associate acquisition of oncogenic RAS pathway mutations, the most common being NRASG12D, with aggressive disease and with disease progression. Using patient derived progenitor colony assays and a NRASG12D-Vav-Cre mouse model, we further demonstrate the role of mutant RAS signaling in driving and maintaining pCMML phenotype. RNA-sequencing links RAS pathway mutations with an increased expression of genes encoding the mitotic checkpoint kinases PLK1 and WEE1. Further, we dmeoinstrated that non-mutated lysine methyltransferase KMT2A (MLL1) acts as mediator of NRAS-induced PLK1 and WEE1 expression. Finally, we demonstrate the translational value of our findings by showing that pharmacological PLK1 inhibition decreases monocytosis and hepatosplenomegaly while improving hematopoiesis in RAS mutant patient-derived xenografts. Hence, we define severe CMML as oncogenic RAS pathway-enriched malignancies, with a unique gene expression profile regulated by KMT2A, amenable to therapeutic intervention.

CBL encodes an E3 ubiquitin ligase and signaling adaptor that acts downstream of cytokine receptors. Recurrent CBL mutations are found in a variety of myeloid disorders, including 10-15% of chronic myelomonocytic leukemia (CMML) cases, and specifically disrupt the protein's RING domain, which is responsible for E3 ligase activity; adaptor domains of CBL, including the tyrosine kinase-binding domain (TKB), proline-rich region (PRR) and C-terminal phosphotyrosine (pY) residues, remain intact in the context of RING mutations. In prior studies, CBL RING mutations were associated with hyperactivation of signaling pathways that drive cell proliferation. However, the precise mechanism by which CBL mutants act remains incompletely understood. Here we combined functional assays and mass spectrometry (MS) to comprehensively define the phosphoproteome, CBL interactome and molecular mechanism of signaling hyperactivation in a panel of cell lines expressing an allelic series of CBL RING mutants. We identified the SRC family kinase LYN as a key driver of signaling by CBL RING mutants; furthermore, we demonstrated in vitro and in vivo efficacy of LYN inhibition by dasatinib in CBL-mutant cell lines and primary CMML patient samples. We generated cell lines expressing wild-type (WT) or RING-mutant CBL using IL3-dependent mouse 32D cells and GM-CSF-dependent human TF1 cells. Cells expressing CBL RING mutants Y371H, C384Y or R420Q had a proliferative advantage over CBL WT or CBL knockout cells. To determine the role of CBL's adaptor domains in the proliferative advantage conferred by CBL RING mutants, we generated double mutants comprising the C384Y RING mutation in cis with mutations in the TKB domain (G306E), PRR (Δ477-688) or pY residues (Y700/731/774F). The proliferative advantage of cells expressing CBL C384Y was significantly reduced with mutation of the TKB domain, PRR or pY residues, indicating that CBL's adaptor domains are critical for the proliferative advantage of cells expressing RING-mutant CBL. To assess the effects of CBL RING mutation on signaling, we used MS to measure global protein phosphorylation in 32D cells expressing CBL WT or CBL C384Y. Activation of LYN and the PI3 kinase (PI3K) pathway were most significantly increased in cells expressing CBL C384Y compared to CBL WT; western blot confirmed increased phosphorylation of LYN, the PI3K p85 subunit and AKT in cells expressing CBL Y371H, C384Y or R420Q. We next employed immunoprecipitation (IP) followed by MS to characterize the global CBL interactome in 32D cells expressing CBL WT or RING mutants Y371H, C384Y or R420Q. In line with the phosphoproteomic analysis, LYN showed significantly increased binding to CBL RING mutants; the PI3K p85 subunit also showed increased binding to CBL RING mutants. Thus, global proteomic analyses revealed that increased binding of LYN and p85 to CBL RING mutants was directly associated with hyperactivation of LYN and PI3K-AKT signaling pathways. Deletion of CBL's PRR reduced interactions with both LYN and p85, and the CBL-p85 interaction required CBL Y731. Genetic ablation or inhibition of LYN by dasatinib decreased binding of p85 to CBL, suggesting that increased CBL Y731 phosphorylation by LYN enabled the CBL-p85 interaction. Indeed, CBL Y731 phosphorylation and AKT activation were diminished by deletion of CBL's PRR, LYN knockout or LYN inhibition by dasatinib. Altogether, these data demonstrated that enhanced LYN activation in cells expressing RING-mutant CBL drives increased CBL phosphorylation, p85 recruitment and downstream AKT signaling. Given the central role of LYN in signaling by CBL RING mutants, we hypothesized that LYN inhibition by dasatinib would abrogate the hyperproliferation of cells expressing CBL RING mutants. Dasatinib blocked the proliferative advantage of 32D and TF1 cells expressing CBL RING mutants. In addition, dasatinib significantly reduced the number of methylcellulose colonies formed by bone marrow mononuclear cells from 2 patients with CBL-mutated CMML; dasatinib treatment of mice xenografted with the same CMML cells resulted in a substantial decrease in leukemia burden compared to vehicle-treated mice. In summary, we have defined a mechanism by which LYN promotes PI3K-AKT signaling through CBL RING mutants. Our data provide rationale for exploring the therapeutic potential of LYN and/or PI3K-AKT inhibition in patients with CBL-mutated myeloid malignancies. Disclosures Ebert: Celgene: Research Funding; Deerfield: Research Funding.

CMML is a lethal myeloid neoplasm with no therapies that improve its dismal prognosis. Inhibition of BET family members has been proposed as a therapeutic strategy based on preclinical data identifying BRD4 as a therapeutic target in acute myeloid leukemia. However, despite potent on-target transcriptional remodeling, early phase clinical trials have demonstrated only modest activity secondary to a variety of resistance mechanisms. In ovarian cancer BET inhibitor (BETi) treated cells, compensatory upregulation and addiction to pro-survival kinase networks have been observed. Given that over 50% of CMML cases have mutations upregulating kinase signaling, we hypothesized that BETi resistance is mediated by these networks in CMML and can be targeted therapeutically. We tested this hypothesis by performing a limited screen of kinase inhibitors alone and in combination with the IC20 of the BETi INCB54329 in 8 human leukemia cell lines. This screen revealed that the IC50 of the PIM inhibitor (PIMi) INCB53914 decreased after co-treatment with BETi in a majority of the leukemia cell lines tested. Synergy was validated chemically in U937, TF1 and SKM1 leukemia cells using other selective inhibitors of BET and PIM. We next assessed the activity of the BET-PIM combination in 14-day colony formation assays with 10 unique CMML bone marrow mononuclear cell (BM-MNCs) patient samples(Fig. 1A). These studies revealed that combination therapy significantly suppressed clonogenicity versus BMNCs treated with vehicle or single drug alone. Finally, this synergy was validated in vivo in 36 patient derived xenografts (PDX) from 3 CMML patients, as manifest by reduced leukemic burden/engraftment in CMML PDX treated with combination therapy(Fig. 1B). To explore the mechanism by which BETi and PIMi therapeutically synergize we treated U937 and SKM1 leukemia cells with INCB54329 and measured mRNA and protein levels for all PIM isoforms. Surprisingly, we identified that PIM1 was increased following treatment with INCB54329, other BETi, or a JQ1-derived PROTAC (Fig. 1C). PIM1 upregulation was also manifest in INCB54329 persistor U937 leukemia cells generated by daily BETi treatment for 6 weeks. Testing across a broader panel of leukemia cell lines revealed an inverse correlation between PIM1 induction and decrease in the IC50 of PIMi following BETi treatment, suggesting PIM1 upregulation confers sensitivity to combination therapy. Consistent with this, isogenic SKM1 leukemia cells engineered to overexpress PIM1 were resistant to INCB54329 and were more sensitive to INCB53914 versus controls cells. Recent studies have demonstrated that inhibitory miRNAs, especially those located near super-enhancers, are suppressed by BET inhibition. Given that several miRNAs are known to control PIM1 expression, we hypothesized that paradoxical PIM1 upregulation following BETi treatment was due to down-regulation of select miRNAs. To test this, we treated our leukemia cell models with broad inhibitors of miRNA activity (i.e., AGO and Dicer inhibitors) and observed a dose dependent increase in PIM1 levels similar to that seen with BET inhibition(Fig. 1Di). Further, integrating public H3K27 CHIP-seq and miRNA super enhancer datasets and using computational prediction algorithms, we identified 6 candidate miRNAs that could regulate PIM1 and were predicted to be controlled by BET inhibitors. Of these, only miR-33a levels were reduced in a dose dependent manner in SKM1 cells by BETi treatment(Fig. 1Dii). This was confirmed by genetically silencing all BET proteins, which suppressed miR-33a levels in SKM1 leukemia cells. Finally, miR-33a mimics (but not control miRNAs) abolished BETi-induced upregulation of PIM1(Fig. 1Diii). Collectively, these studies established BET and PIM inhibition as a novel and potent combination therapy for CMML that is mediated by miR-33a-dependent upregulation of PIM1(Fig. 1E). Disclosures Liu: Incyte Corporation: Employment. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Lancet:Daiichi Sankyo: Consultancy, Other: fees for non-CME/CE services ; Agios, Biopath, Biosight, Boehringer Inglheim, Celator, Celgene, Janssen, Jazz Pharmaceuticals, Karyopharm, Novartis: Consultancy; Pfizer: Consultancy, Research Funding. Komrokji:Novartis: Speakers Bureau; JAZZ: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy; DSI: Consultancy; pfizer: Consultancy; celgene: Consultancy. Epling-Burnette:Incyte Corporation: Research Funding. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Haura:Incyte Corporation: Research Funding. Reuther:Incyte Corporation: Research Funding. Koblish:Incyte Corporation: Employment.

Improvements in antisense technology have now enabled clinically relevant therapeutic credentialing of the noncoding genome. MALAT1 is a long non-coding RNA that, among other functions, is thought to serve as a nuclear scaffold for splicing and transcription factors. MALAT1 expression is associated with inferior prognosis across solid tumors and its depletion impairs proliferation and metastasis in preclinical solid tumor models. We found that elevated MALAT1 levels are independently associated with inferior overall survival in patients with CMML. Further, RNA-sequencing of primary CMML monocytes identified MALAT1 as the fourth most over-expressed transcript compared to controls. Therefore, we explored the biologic relevance and therapeutic candidacy of MALAT1 across several human and murine models of CMML. First, we crossed NRASQ61R/+Mx1-cre driven mice, which display a CMML-like phenotype, to MALAT1KO/KOmice. Although MALAT1KO/KOmice did not have abnormalities in complete blood counts, immunophenotyping of the hematopoietic stem cell (HSC) compartment identified statistically significantly lower numbers of HSC compared to wild type (WT) controls and a non-significant decrease in NRASQ61R/+/MALAT1KO/KOcompared to NRASQ61R/+alone. This decrease in HSC was not a result of impaired self-renewal as no differences were observed in these models after in vivo competitive transplant experiments. Therefore, we reasoned that MALAT1 expression may be controlling HSC differentiation. To test this, we transformed bone marrow cells from these models with an estrogen-regulated (ER) Hoxb8 construct enabling cells to maintain an HSC state until ER is withdrawn and myeloid differentiation is induced. ER-Hoxb8NRASQ61R/+/MALAT1KO/KOcells had increased basal levels of Gr-1 compared to ER-Hoxb8 transformed NRASQ61R/+alone that was dramatically enhanced upon ER withdrawal suggesting that MALAT1 depletion regulates myeloid differentiation. These findings were validated by assessment of morphology, transcriptome, and in vivo immunophenotyping of bone marrow and spleen cells. Further, moribund NRASQ61R/+/MALAT1KO/KOmice displayed a reduction in organomegaly typically associated with leukemic burden. We validated this in human monocytic leukemia by generating MALAT1 depleted THP-1 isogeneic cell lines using the CRISPR/Cas9 system. MALAT1 depleted THP-1 cells (MKO) had greater terminal differentiation according to immunophenotypic markers and morphology that was greatly enhanced when treated with phorbol myristate acetate. Last, MKO orthotopic xenografts demonstrated inferior human leukemia engraftment and decreased spleen and liver weights, and heterotopic xenografts exhibited reduced tumor volume, collectively suggesting diminished leukemic burden. Because ATRA has been clinically tested in CMML with minimal effects, we next explored whether MALAT1 depletion could potentiate ATRA differentiation in CMML. First, we treated MKO cells with ATRA and observed a large induction of myeloid differentiation by marker expression and morphologic assessment compared to isogenic controls. This was validated by NRASQ61R/+/MALAT1KO/KOmice demonstrating that ATRA more robustly induced differentiation compared to vehicle which was not seen in NRASQ61R/+/MALAT1+/+mice. Next, we tested MALAT1 antisense oligonucleotides (ASOs) currently under clinical development in THP-1 cells +/- ATRA and demonstrated both an increase in myeloid differentiation and apoptosis compared to ATRA alone. To test this therapeutic strategy in primary CMML specimens, we generated CMML patient-derived xenografts (n=30 mice) and treated each with ASO, ATRA, the combination, or controls and identified a more robust reduction in human HSC engraftment with the combination. To explore the mechanistic basis for these findings, we performed RNA-sequencing of MALAT1-depleted or control cells and identified that CREB target genes were differentially expressed. Basal protein levels of p-CREB were also decreased in MKO cells and were further reduced in the nucleus of MKO by western and microscopy. Lastly, overexpression of WT or constitutively active CREB but not its dominant negative rescued the differentiation effect seen in ATRA treated MKO cells. Taken together, MALAT1 is a novel, CREB-dependent regulator of myeloid differentiation and its depletion potentiates ATRA therapy. Disclosures Cluzeau: Menarini: Consultancy; Jazz Pharma: Consultancy; Abbvie: Consultancy. Komrokji:celgene: Consultancy; pfizer: Consultancy; DSI: Consultancy; JAZZ: Speakers Bureau; Novartis: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy. MacLeod:Ionis Pharmaceuticals: Employment. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Epling-Burnette:Forma Therapeutics: Research Funding; Celgene Corporation: Patents & Royalties, Research Funding; Incyte Corporation: Research Funding.

BACKGROUND: CMML is an aggressive myeloid neoplasm with no known disease modifying treatments. We have shown both that CMML PDXs recapitulate the genetic and pathologic features of this disease, and that the JAK1/2 inhibitor ruxolitinib is a promising therapeutic in a phase 1/2 clinical trial. However, whether CMML PDXs can recapitulate clinical responses is unknown. To explore this, we generated PDXs from bone marrow mononuclear cells (BMMCs) of ruxolitinib clinical trial CMML patients and treated the resultant mice with pharmacologically equivalent doses of ruxolitinib or vehicle. These models were then used to determine if clinical trial responses could be recapitulated and if molecular analysis could identify novel therapeutic strategies for CMML. METHODS: Two million BMMCs from patient samples harvested no later than 30 days before ruxolitinib initiation were transplanted into 3 vehicle and 3 drug-treated NSGS mice as described (Yoshima Blood 2017). Fourteen days post-transplant, mice received either 60 mg/kg ruxolitinib or vehicle via oral gavage twice daily. Treatment continued until the animals became moribund. The primary endpoint of the study was intrapatient PDX overall survival. Secondary PDX response assessments included: (1) reduction in splenomegaly as measured by spleen weight at necropsy, (2) Magnetic Resonance Imaging (MRI) of the spleen at baseline and at the time that the first mouse became moribund (3) and improvement in BM and spleen pathology to include decreases in human CMML engraftment. PDX responses were compared to those seen in the clinical trial per MDS/MPN IWG criteria. Human CD45+ cells from exemplary ruxolitinib- and vehicle-treated mice were sorted and subjected to targeted DNA sequencing and transcriptional and proteomic profiling using the Nanostring Hemeplatform. RESULTS: As we previously reported, the overall response rate of our single-arm ruxolitinib clinical trial (n=50) was 36% and 9 of 23 patients (40%) with splenomegaly had a > 50% reduction in spleen size by physical exam. PDX models were generated from 6 of the patients who had splenomegaly at baseline (3 responders, 3 nonresponders). A total of 34 CMML PDX models were generated and the mean duration on therapy was 63 days (range 7-199 days) for all models. The median overall survival of PDX mice was 59.5 days in the ruxolitinib cohort versus 52 days for the vehicle cohort (HR=1.062, p=0.88). However, ruxolitinib-treated xenografts derived from responders experienced a survival benefit (p=0.0002), a greater reduction in spleen volume when compared to corresponding vehicles (mean decrease in volume -43.9mm3 vs -16.6mm3), and a reduction in bone marrow leukemic engraftment compared to vehicle not observed in PDX mice from nonresponders (p CONCLUSION: Our CMML PDX platform confirmed the biologic impact of ruxolitinib in CMML, identified a novel combination therapy, and recapitulated clinical responses manifest in human patients. To our knowledge, this is the first report of PDX models recapitulating clinical trial responses in leukemia. A cohort of 6 additional patient samples are currently being assessed to validate these results. Disclosures DeZern: Celgene: Consultancy; Astex Pharmaceuticals, Inc.: Consultancy. Lancet:Daiichi Sankyo: Consultancy, Other: fees for non-CME/CE services ; Pfizer: Consultancy, Research Funding; Agios, Biopath, Biosight, Boehringer Inglheim, Celator, Celgene, Janssen, Jazz Pharmaceuticals, Karyopharm, Novartis: Consultancy. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Roboz:Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amphivena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trovagene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Steensma:H3 Biosciences: Other: Research funding to institution, not investigator.; Pfizer: Consultancy; Onconova: Consultancy; Summer Road: Consultancy; Aprea: Research Funding; Stemline: Consultancy; Astex: Consultancy; Arrowhead: Equity Ownership. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Komrokji:JAZZ: Speakers Bureau; DSI: Consultancy; Incyte: Consultancy; pfizer: Consultancy; celgene: Consultancy; Novartis: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy.