ALBERT

Description: CMML is an aggressive adult myeloid neoplasm hallmarked by peripheral monocytosis and a propensity for acute myeloid leukemia (AML) transformation. Although 5AZA and decitabine are FDA approved for use in CMML, no pharmacologic treatment has been shown to impact the natural history of this disease. We have previously demonstrated that the majority of CMML cases display GM-CSF hypersensitivity and that this pathway is a therapeutic target via GM-CSF neutralization or pharmacologic JAK2 inhibition. We have clinically tested the latter using the FDA approved JAK1/2 inhibitor ruxolitinib (RUX) with favorable results. Our data suggests that JAK inhibitors may be clinically effective and may serve as a foundation for future combination therapy in CMML. However, both RUX and 5AZA are associated with severe myelosuppression and therefore have overlapping toxicities that would make combination therapy clinically problematic. PAC is a JAK2 inhibitor that also inhibits CSF1 and IRAK1, also activated in CMML, and does not cause clinical myelosuppression. We therefore hypothesized that the combination of PAC and 5AZA may be an active and clinically feasible therapeutic combination in CMML. We first explored the preclinical activity of PAC in the monocytic and/or GM-CSF hypersensitive cell lines THP-1, U937, and MO7e (HL-60 negative control). These cell lines were cultured in the presence of GM-CSF (except for THP-1), and six increasing doses of PAC (0-10uM). After 48 hours, these cultures were analyzed for proliferation (MTT) and apoptosis (Annexin V/DAPI incorporation). In contrast to HL-60s, all monocytic cell lines tested had a dose dependent decrease in proliferation and increase in apoptosis with an IC50 approximating 0.5uM (Fig 1a). We additionally measured the capacity of PAC to augment GM-CSF dependent STAT5 phosphorylation in these cell lines by phos-flow. All cell lines had a dose-dependent decrease in pSTAT5 suggesting appropriate target inhibition. We next explored the activity of PAC in primary CMML BMNCs by measuring apoptosis and 14-day colony forming capacity (n=10). Using the exact dosing schema as above, we confirmed a dose dependent increase in apoptosis and decrease in colony forming capacity with an IC50 that approximated 0.5 uM (Fig 1b-c). When testing inhibition of basal and GM-CSF dependent STAT5 phosphorylation, PAC was able to inhibit pSTAT5 more potently than seen in cell lines. We next sought to determine whether PAC was synergistic in model cell lines by generating a synergy topology map using a proliferation assay that determines if there are synergistic doses of two drugs in an unbiased manner. We used the Bliss method to determine if formal synergy was present and identified profound synergy between PAC and 5AZA at a dose of approximately 0.5 and 2.5 uM respectively (fig 1d,e). Interestingly, the synergy topology of the PAC and 5AZA combination was distinct from that of RUX combination therapy suggesting a PAC-specific effect. We next confirmed our synergy results in primary CMML BMNCs by measuring the change in colony formation assay in control, PAC (0.5uM), 5AZA (2.5uM), or combination treated cells (n=5). As shown in Figure 1e, a decrease in colony forming capacity with combination therapy compared to other conditions was identified (p

Description: SRSF2 is mutated at proline 95 (P95) in approximately 45% of patients with CMML. The consequence of SRSF2 P95H mutations on splicing has been associated with change in target RNA motif preference compared to wild type that favors CCNG over GGNG resulting in aberrant splicing abnormalities. Although this has led to several downstream mis-spliced candidates that may contribute to SRSF2 leukemic pathogenesis, the biologic consequences of SRSF2 mutation have not been fully elucidated and its effect on non-splicing pathways has yet to be explored. SRSF2 has two major functions within the nucleus: (1) bind to cis elements on pre-mRNA transcripts that functionally redefines putative exon-intron boundaries and (2) interact with other members of the spliceosome complex at nuclear suborganelles known as speckles. To explore the effects of SRSF2 P95 mutation on nuclear speckle dynamics, we transfected HeLa cells with GFP-SRSF2 wildtype (WT) and mutant (MT) constructs and performed Fluorescent Recovery After Photo-bleaching (FRAP) to determine the mutant specific kinetics of SRSF2 molecules localized to the nuclear speckle. Using this approach, we confirmed that SRSF2 WTs are characterized by complete photo-recovery with a half-life of approximately 35 (30-42) seconds as previously reported. However, when we performed FRAP of GFP-SRSF2 MTs a statistically significant difference in photo-recovery was observed compared to WT cells (Fig. 1a). The difference between baseline fluorescence and maximal recovered fluorescence in SRSF2 MT cells, or the immobile fraction, suggests that trapped bleached SRSF2 molecules are sterically inhibiting the diffusion of unbleached SRSF2 molecules. We reasoned that the observed differences in RNA binding capacity and nuclear speckle dynamics could be related. This rationale identified MALAT1 as a link connecting both increased RNA binding and nuclear speckle trafficking abnormalities because it is a Long Non-Coding (lnc) RNA that localizes to and has a critical role in SR protein recruitment and retention to the nuclear speckle. MALAT1, which is highly enriched for CCNG motifs, has also been demonstrated to bind to SRSF2 directly and has been implicated in a wide spectrum of carcinomas making it an attractive intermediary to study in our system. To determine whether SRSF2 MTs have increased MALAT1 binding capacity compared to SRSF2 WT, we performed RNA IP of myc-his-SRSF2 WT and MT transfected HeLa cells. Using this approach, we demonstrated a 60% enrichment of MALAT1 in SRSF2 WT transfected cells and a 120-150% enrichment of MALAT1 in SRSF2 MT cells compared to the empty vector control (Fig. 1b-c). We next performed the above FRAP experiment in the context of MALAT1 depletion and demonstrated that observed immobile fraction seen in SRSF2 MT cells is rescued (Fig. 1d). To determine the clinical relevance of MALAT1 in CMML we profiled a cohort of 54 CMML cases for MALAT1 expression by PCR. As shown in Figure 2a-b and d-e, MALAT1 expression is statistically elevated in CMML BMNCs and is among the highest differentially expressed transcripts in CMML monocytes (CD14+). We additionally characterized SRSF2 expression in our CMML BMNCs and CD14+ cohort and identified a linear correlation (Pearson R=0.7 p

Description: BACKGROUND: CMML is a myelodysplastic/myeloproliferative neoplasm with a median survival of 32 months and no therapies that improve its natural history. We have previously demonstrated that CMML bone marrow mononuclear cells (BMNCs) are hypersensitive to GM-CSF and that the GM-CSF axis is a viable therapeutic target (Padron et al., Blood 2013). Lenzilumab is a novel, humaneered IgG1κ monoclonal antibody, with high affinity for human GM-CSF that has activity in preclinical models of CMML. We report a Phase 1 clinical trial testing the safety and preliminary efficacy of this agent in CMML. METHODS: The study was approved by scientific and ethical review boards. This was a multicenter Phase 1 study designed to evaluate the safety and determine the recommended phase 2 dose of lenzilumab in subjects with CMML. Dose escalation proceeded using a standard 3+3 study design to determine the maximum tolerated dose (MTD). Three dose cohorts included 200 mg, 400 mg, and 600 mg, were given IV on day 1 and 15 of cycle 1 and then only on day 1 of subsequent 28-day cycles. Key inclusion criteria included a WHO-defined diagnosis of CMML and a platelet count greater than 20 x103 cells/dL. Response was evaluated utilizing the MDS/MPN International Working Group Criteria (Savona Blood 2015). Pharmacokinetic analysis and pharmacodynamics were evaluated by pSTAT5 by flow cytometry. RESULTS: Between July 2016 and June 2018, a total of 15 patients were enrolled. The median age at study entry was 74 years (range 52-85) and 80% were male. Nine patients were classified as CMML-0, 3 as CMML-1, and 3 as CMML-2. Seventy three percent of patients had normal cytogenetics or -Y. The most commonly mutated genes at screening included TET2 60%, ASXL1 53%, SRSF2 47%, and RAS pathway (i.e. NRAS or CBL) mutations 40%. Nine patients were previously treated with hypomethylating agents and/or experimental therapies, 3 were treated with hydroxyurea only, and 3 were untreated. The mean Hgb was 9.7g/dL (7.6-14g/dL), the mean platelet count was 147 x103 cells/dL (16-942 x103 cells/dL), and 66% of cases were MPN-CMML by the French-American-British classification at study entry. Three patients were enrolled at each dose level and an additional 6 patients were enrolled at 600mg as planned. Consistent with prior studies of lenzilumab, no dose limiting toxicities were identified and no treatment emergent grade 3 or 4 toxicities were reported. The mean duration on therapy was 221.8 days (14-787 days) and the majority of patients discontinued study drug because of disease progression or lack of response (69%). Five of 15 (33%) patients enrolled achieved clinical benefit by MDS/MPN IWG criteria with 3 platelet responses, 1 neutrophil response, and 1 spleen response. An additional patient had bone marrow myeloblast reduction from 6% to 1% which allowed that patient to undergo allogeneic stem cell transplantation. Clinical response was not statistically associated with somatic mutations or changes in pSTAT5 between screening and cycle 3. However, 3 of 4 patients with NRAS mutation achieved clinical benefit or had clinical meaningful bone marrow myeloblast reductions. CONCLUSION: Lenzilumab is well tolerated in patients with CMML, with no grade 3 or 4 treatment emergent adverse events or DLTs reported. Durable clinical benefit was achieved in 33% of patients and one patient was bridged to allogenic transplant, providing proof of concept that GM-CSF inhibition has activity in CMML. The favorable safety and activity profile of lenzilumab warrants future evaluation as part of a combination regimen targeted to specific subtypes more likely to respond, including patients with NRAS mutations. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Sallman:Celgene: Research Funding, Speakers Bureau; Celyad: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Abbvie: Speakers Bureau; Novartis: Speakers Bureau; Jazz: Research Funding. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding. Komrokji:celgene: Consultancy; Agios: Consultancy; JAZZ: Consultancy; Novartis: Speakers Bureau; JAZZ: Speakers Bureau; pfizer: Consultancy; DSI: Consultancy; Incyte: Consultancy. Lo:Humanigen: Employment. Durrant:Humanigen: Employment. Chappell:Humanigen: Employment. Ahmed:Humanigen: Employment. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: In this phase 1 trial, inhibition of granulocyte-macrophage colony-stimulating factor (GM-CSF) was associated with clinically meaningful responses in 5 of 15 patients with relapsed or refractory chronic myelomonocytic leukemia (CMML). Preliminary data suggest that this approach may be tractable in CMML bearing activating NRAS mutations.

Description: Key Points Genetically accurate xenografts of CMML are achievable with near 100% frequency in NSGS mice. Robust human engraftment and overt phenotypes of CMML and JMML xenografts here facilitate preclinical therapeutic evaluation in vivo.

Description: 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

Description: Leukemic clones within the same patient have unique sensitivities to drugs in vivo and may have unique capacities to engraft within xenograft models suggesting that there exist intra-patient functional clonal differences. However, methods to detect the functional differences assigned to genetically defined clones remain limited. Here, we describe a method to measure functional differences of clones within the same leukemia patient by coupling intracellular flow cytometry and next generation sequencing (NGS). We hypothesize that functionally distinct clones can be inferred by stimulating cells with cytokines, fluorescently staining with a relevant downstream marker of activation, and then isolating functionally distinct fractions by flow sorting. NGS can then digitally annotate somatic variants in sorted, functionally distinct samples that can be compared to the bulk leukemic cell population. To achieve this, we optimized a DNA isolation method that results in high-quality DNA despite originating from fixed/permeabilized cells. In this way, we annotate enrichment and de-enrichment of variants upon stimulation and infer the functional clonality of any given sample. To provide proof-of-concept, we explored the well-established relationship between Chronic Myelmonocytic Leukemia (CMML) and GMCSF. CMML cells are uniquely hypersensitive to GMCSF and simultaneously have a well-established mutational composition that can be annotated by sequencing only 49 genes. This allowed us to leverage an amplicon-based enrichment panel that employs emulsion PCR to achieve reproducible library preparation with 5 ng of input DNA hence bypassing the need for whole genome amplification. We tested our methodology by mixing two cell lines whose response to GMCSF was known and somatic variants had been annotated a priori. K562 (non-GMCSF responsive) and THP-1 (GMCSF responsive) leukemic cells were mixed at 1:1 and then stimulated with GMCSF. The resultant cells were stained with anti-pSTAT5 and cell sorting isolated positive and negative fractions. Unstimulated mixed cells and each positive and negative fraction was sequenced and variant allele fractions (VAF) was compared to unstimulated bulk (Figure 1A). To quantitate the change in VAF, we reasoned that summing the difference in VAF of unsorted/pSTAT5+ and unsorted/pSTAT5- populations would yield the most informative composite value as it would contain information about VAF difference for each mutation in both the GMCSF stimulated and GMCSF unstimulated fractions. Using this method, we found that 100% of THP1 and K562 variants were enriched in the pSTAT5+ and pSTAT5- fractions, respectively (Figure 1B). Receiver Operator Curves (ROC) demonstrated that the data functionally discriminated mixed THP-1 and K-562 cells with an AUC of 0.9868 (p

Description: Introduction: Proinflammatory cytokines/chemokines have been implicated in the pathogenesis and prognosis of myeloproliferative neoplasms (MPNs) and myelodysplastic syndromes (MDS). Augmenting cytokine levels by JAK2 inhibition or cytokine neutralization are promising therapeutic approaches in these diseases. Chronic Myelomonocytic Leukemia (CMML) is a myeloid neoplasm that has both features of MDS and MPNs for which JAK2 inhibition has yielded reductions in inflammatory cytokines and promising clinical activity. Therefore, we hypothesize that annotation of inflammatory cytokines may uncover cytokine subsets associated with CMML-specific clinical features and identify novel therapeutic targets. To address this, we perform the first comprehensive annotation of inflammatory cytokines in CMML. Methods: A custom 46-plex Luminex based inflammatory cytokine profiling assay was developed. Cryopreserved peripheral blood plasma was collected from three academic centers after institutional review board approval. Center specific, age-matched plasma controls processed in a similar manner to CMML plasma samples were collected for analytical comparison. All samples were profiled in duplicate and only cytokines above the range of negative controls were reported. CMML cases were also clinically annotated by retrospective chart review and genotyping performed to annotate the frequency of TET2, IDH1/2, DNMT3A, ASXL1, EZH2, SRSF2, SF3B1, U2AF1, ZRSR2, N/K-RAS, CBL, and JAK2 somatic mutations. Descriptive statistics were used to summarize data and the Mann-Whitney test was used to compare cytokine levels among clinically and genetically relevant groups. A Cox proportional hazards model was used to derive likelihood ratios for overall survival using JMP®, Version 12. SAS Institute Inc., Cary, NC. Results: A total of 219 CMML and 35 control plasma samples were profiled. The median age was 73.4 years and 67.1 years in CMML and healthy controls, respectively. Eighty seven percent were CMML-1 by the WHO criteria and 54% were MPN-CMML by FAB criteria. Significantly different levels of cytokines between CMML and controls are summarized in Figure 1 and included TNFa (P=0.0404), IL-8 (P=0.0271), MCP-1 (P

Description: 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.

Description: 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.