Chronic Myelomonocytic Leukemia Cells Research Articles

Cellular Interactions within Clonal Dendritic Cell Aggregates Drive Immune Tolerance in Chronic Myelomonocytic Leukemia Bone Marrow Microenvironment

Introduction: Clonal dendritic cell (DC) aggregates in chronic myelomonocytic leukemia (CMML) are seen in approximately 20-30% patients at diagnosis. Previously, these were thought to be comprised of CD123+ plasmacytoid DCs but our data has suggested that these are heterogenous and comprise of myeloid DCs, monocytes, and myeloid-derived suppressor cells. Furthermore, these aggregates uniformly express immune checkpoints such as indoleamine 2,3-dioxygenase-1 (IDO), associate with an IDO-specific systemic impact (increased trytophan catabolism), and expanded regulatory T cell population in CMML, suggesting a role in inducing immune tolerance. DC aggregates are also associated with increased risk of acute myeloid leukemia (AML) transformation and resistance to hypomethylating agents, highlighting that they are important for disease progression in CMML (Mangaonkar A et al. Blood Adv 2020). We performed this study to decipher the architecture of a CMML DC aggregate to understand the complex inter-cellular interactions, and assess their impact on the adaptive immune system. Methods: In this study, we used archival fresh-frozen paraffin-embedded (FFPE) bone marrow biopsy specimens obtained from CMML patients, and isolated monocytes (CD14+ magnetic bead sorting) and lymphocytes (CD14-ve fraction) from untreated CMML and age-matched normal control (NC) peripheral blood mononuclear cells (PBMCs). DCs were cultured from monocytes using a commercial protocol. Spatial architecture of the CMML BM microenvironment was interrogated through multiplex imaging of FFPE samples by co-detection by indexing (CODEX) using 30 markers (CCR6, CD107a, CD11c, CD20, CD21, CD31, CD3e, CD4, CD44, CD45, CD45RO CD56, CD68, CD8, E-Cadherin, FoxP3, HLA-DR, CD14, CD16, CD11b, CD33, CD15, CD66b, CD123, CD303, CD34, CD163 and IDO). Single-cell transcriptomic signatures were analyzed using the 10X single cell Fixed RNA profiling platform, and validated using bulk RNA seq (Illumina stranded mRNA prep) from isolated and cultured cells. Impact of DCs on lymphocytes was assessed via autologous mixed DC-T culture experiments. Results: BM FFPE samples from 5 CMML cases (3 with DC aggregates) were included for multiplex imaging via CODEX. Results confirm the significant interactions of CD8+ and CD4+ T cells with CD123+/HLA-DR+ DCs within a CMML DC aggregate (Fig. 1A). Upon quantitative assessment of cell-cell interactions, IDO+/CD8+T ( P=0.006), IDO+/MDSC ( P=0.005), and IDO+/macrophage ( P=0.001) interactions were significantly increased in CMML samples with versus without DC aggregates . Specific findings were validated using single-stain immunohistochemistry and multiplex immunoflourescence staining. Next, in order to assess transcriptomic signatures within BM at the single-cell level, we chose two cases; CMML (BM aspirate blast% 1, WBC 49.6 x 10 9/L, ASXL1/GATA2/SETBP1/SRSF2 mutations) and AML transformed from CMML (BM aspirate blast% 81, WBC 152 x 10 9/L, NPM1/DNMT3A/TET2 mutations). Approximately, 10,000 cells were obtained and analyzed with cell type annotation through human PBMC reference (Azimuth). Results show an expanded regulatory T cell population in AML compared to CMML (UMAP plot, Fig. 1B), suggestive of immune tolerance. In order to validate findings and perform functional experiments, we used sorted monocytes and cutured DCs from CMML and NC. DC phenotype was confirmed both through visual inspection and flow cytometry, and clonality was confirmed through Sanger sequencing (for previously identified mutations). Differential gene expression analysis through bulk RNAseq showed significant differences between CMML and NC lymphocytes with immunoinhibitory receptor, LILRB2 (log2fold change=7.05, adjusted P=0.0002) being one of the top five significantly upregulated genes, further confirming immune tolerance. Finally, mixed autologous DC-lymphocyte reaction (1:4 ratio) experiments demonstrated that DCs induce T cell tolerance in CMML (expansion of exhausted PD1/TIM3/LAG3+ CD8 T cells) when compared to NC. Conclusion: Clonal dendritic cell aggregates within the CMML bone marrow microenviroment interact with T cells and induce immune tolerance. This represents a novel mechanism of disease progression in CMML with potential therapeutic targets (IDO, LILRB2, and/or CD123).

Development of Novel Protein-Drug Conjugates for the Treatment of Chronic Myelomonocytic Leukemia

Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid stem cell neoplasm with few effective therapies and poor prognosis. We have developed CCL2-drug conjugates that specifically bind, internalize and eliminate cells of the disease clone via inhibition of the key metabolic enzyme NAMPT. CCL2 is a chemokine which binds CCR2, a receptor expressed predominantly on the classical monocytes canonically expanded in CMML, while CMML stem and progenitors display ~100-fold higher CCR2 expression compared with healthy equivalents ( Ferrall-Fairbanks et al, Blood Cancer Discov 2022 3(6):536-553). CCL2-NAMPTi conjugates thus represent an attractive and novel strategy, co-opting both lineage specificity and therapeutic window to deliver payload drugs for efficient and targeted cell killing. Methods: Four NAMPTi warheads were made in adaptable seven-step synthetic route and covalently conjugated to human CCL2 though TGase. We screened the range of CCL2-NAMPTi conjugates on CCR2-expressing cell lines (THP-1; MV411) for their ability to reduce NAD/NADH and induce cytotoxicity. The lead CCL2-NAMPTi conjugate was identified based on its ability to reduce NAD/NADH levels, induce cell death, and show impermeability when using the unconjugated warhead alone. Plasma stability studies confirmed conjugate stability in mouse and human plasma with no release of unconjugated active warhead. The lead compound was then tested on CD14+ cells isolated from CMML patient peripheral blood (PB); examining NAD/NADH levels, cell viability and induction of caspase 3/8. To better understand its specificity and target cell population, we conducted in vivo studies using fluorescently labelled CCL2 in wild type and CCR2 KO mice. Therapeutic effectiveness of the lead conjugate was evaluated by means of NAD/NADH reduction in THP1 subcutaneous models. Results: All CCL2-NAMPTi tested induced cell death in THP-1 cells (IC50 1.1-7.2 nM) following 72 hours incubation. The lead compound, MM015, effectively reduced NAD/NADH levels in CCR2+ cell lines (THP-1; MV411) 24-48 hours following treatment (IC 50 0.1-0.8 nM), with a corresponding decrease in cell viability at 72 and 96 hours (IC 50 1.1- 2.1 nM). No effect was observed on CCR2- cell lines (Jurkat; PC9). Specificity of the lead conjugate was confirmed by measuring NAD/NADH levels and cytotoxicity in presence of 100-fold excess of unconjugated CCL2. Efficacy studies on primary CMML PB samples confirmed that MM015 exposure specifically reduced NAD/NADH levels (n=6, IC 50 0.5-15 nM) and cell viability (n=6, IC 50 1.9-250 nM), and induced caspase 3/8 activation. Flow cytometry analysis of CMML PB confirmed CCR2 expression was largely restricted to monocytes and precursors, as compared with other immune cell subsets (n=6). Accordingly, efficacy measures in treated whole PB were exclusively seen in the CD14+ population, sparing lymphoid and other lineage cells, demonstrating ability of CCL2-NAMPTi to selectively target the CMML disease clone. In vivo experiments using fluorescently labelled CCL2 confirmed selective uptake of intravenously administered human CCL2 by CCR2+ cells within mouse blood, bone marrow and in THP-1 subcutaneous tumour cells. No uptake of fluorescently labelled CCL2 was observed in CCR2 KO mice, further confirming CCR2 specific uptake. Additionally, measurement of human CCL2 in CCR2 KO mouse peripheral blood by ELISA confirmed CCR2 dependent CCL2 uptake, 1 hour following intravenous administration. In vivo efficacy studies showed consistent reduction of NAD/NADH levels in xenografted tumor cells, and monocytic cells in the murine BM compartment, 24 hours following IV injection of MM015. Conclusion: We have developed a novel drug delivery strategy utilizing CCL2-NAMPTi conjugates to selectively target and eliminate key CMML cell populations. CCL2-NAMPTi can effectively reduce NAD levels and induce subsequent cell death in CCR2-expressing cell lines, primary CMML cells and in vivo models. This innovative approach holds therapeutic promise to address this major unmet clinical need.

Phase 1/2 Study of Pacritinib in Combination with Azacitidine in Chronic Myelomonocytic Leukemia

Introduction and preclinical rationale Treatment options for chronic myelomonocytic leukemia (CMML) are limited. Hypomethylating agents (HMAs) are the only approved therapies but are associated with unsatisfactory response rates and do not impact disease evolution (Merlevede Nat Comm 2016). JAK inhibition can reduce GM-CSF signaling, which contributes to CMML pathobiology; ruxolitinib has been clinically evaluated in CMML demonstrating promising, but insufficient clinical activity. Pacritinib is a JAK inhibitor that has a unique profile which suggests it may be uniquely well suited for the treatment of CMML, including targeting of CSF1R and IRAK1, both of which are upregulated in CMML and related diseases (Obba Autophagy 2015, Rhyasen Cancer Cell 2013). Additionally, pacritinib was recently shown to be a potent inhibitor of ACVR1 (Oh AJH 2023), potentially allowing for increased iron availability and improvement in anemia. Pacritinib has been evaluated preclinically in CMML primary samples and mouse models where it demonstrated significant, albeit heterogenous activity (Yoshimi Blood 2017). To enhance the activity of pacritinib in CMML, it was also evaluated in combination with azacitidine in GM-CSF hypersensitive cell lines and primary CMML cells where profound synergy was noted (Ma ASH 2015). Based on encouraging preclinical data, we hypothesize that the combination of pacritinib with azacitidine will be an effective treatment strategy in CMML patients. Methods In this phase 1/2 trial, we will evaluate the safety and efficacy of pacritinib and azacitidine in newly diagnosed or previously treated CMML. Patients must be JAK inhibitor and HMA naïve (≤1 cycle of HMA allowed). Phase 1 will follow a ‘3+3‘ dose de-escalation design and will enroll 6-12 patients. The primary objective of this phase is to determine the recommended phase 2 dose (RP2D) of pacritinib in combination with a fixed dose of azacitidine. Pacritinib will be administered at 200mg twice daily (with a lower dose of 100mg twice daily being explored in the case of dose limiting toxicity) in combination with azacitidine 75mg/m 2 for 7 days. Phase 2 will utilize a Simon's two stage design to assess the preliminary efficacy of pacritinib in combination with azacitidine at the RP2D. In the first stage, after treating 6 patients at the RP2D, there will be an interim response assessment as part of the phase 2 portion. If at least 4 among these 6 patients show a response, the trial will proceed to a second stage and enroll an additional 12 patients. Patients will be treated for 6 cycles of 28 days each with an interim bone marrow biopsy after cycle 3. The primary endpoint of the phase 1 portion is the recommended RP2D through the assessment of dose limiting toxicities and of the phase 2 portion is the overall response rate (ORR) by the end of cycle 6 as defined by a clinical benefit or greater by myelodysplastic syndrome/myeloproliferative neoplasm International Working Group response criteria (Savona 2015). Secondary endpoints include duration of response, disease free survival, overall survival, changes in MPN-SAF total symptom scores, palpable splenomegaly, and adverse events. Several correlative studies are planned as part of this clinical trial. Modulation of biomarkers of disease activity with treatment including GM-CSF dependent STAT5 phosphorylation, MAPK, and PI3K signaling will be analyzed. We will also evaluate changes in mutational and cytogenetic burden with treatment as well as at baseline to identify predictive biomarkers of response.

Suppression of KRAS and CBL Mutations and Hematological Improvement By Lenzilumab and Azacitidine Treatment in Proliferative Chronic Myelomonocytic Leukemia

Introduction: Mutations in RAS-pathway genes ( NRAS, KRAS, CBL) are amongst the most common somatic mutations in cancer and historically resistant to most therapies. Targeted therapies that impact the RAS-pathway constitute a major unmet need in oncology. The proliferative form of chronic myelomonocytic leukemia (CMML) is commonly associated with RAS-pathway mutations and has a high propensity to develop into acute myeloid leukemia. In pre-clinical models, Lenzilumab (LENZ; Humanigen, Inc., Short Hills, NJ), a proprietary Humaneered ® first-in-class monoclonal antibody with best-in-class off-rate and affinity that neutralizes GM-CSF, resulted in a reduction of colony numbers and viability of CMML cells, with the greatest sensitivity in cells possessing RAS-pathway ( NRAS/ KRAS/ CBL) mutations, suggesting benefit in the targeted treatment of CMML. The PREcision Approach to CHronic Myelomonocytic Leukemia (PREACH-M) trial assesses the efficacy of LENZ, in addition to azacytidine (AZA), in CMML subjects with RAS-pathway mutations and high dose sodium ascorbate (ASC) and AZA in CMML subjects without RAS-pathway mutations. Interim data from 11 subjects with RAS-pathway ( NRAS/ KRAS/ CBL) mutations in PREACH-M receiving LENZ/AZA demonstrate reductions in circulating GM-CSF and CRP with 8 subjects achieving complete response or optimal marrow response. This report describes improvements in variant allele frequencies (VAF) for RAS-pathway mutations and hematologic improvements associated with LENZ/AZA treatment in CMML. Methods: PREACH-M is a Phase 2/3 non-randomized, open-label trial in 72 subjects, aged at least 18 years, with newly diagnosed CMML based on the WHO 2016 criteria; and RAS-pathway mutations at a variant allele frequency ≥3%. Subjects received 24 cycles (every 28 days) of AZA (SC; 75 mg/m 2 for 7 days) and LENZ (IV; 552 mg; d1 & d15 of cycle 1 and d1 only for all subsequent cycles). Subjects without RAS-pathway mutations received the same AZA regimen and sodium ascorbate (IV; 30g for 7 days (15g for 1st dose only; 30g thereafter if no evidence of tumor lysis syndrome); PO; 1.1g on all other days). VAF was determined from bone marrow mononuclear cells using a 41-myeloid panel using Illumina Hi-Seq with a depth of 1000x performed on bone marrow aspirates obtained at baseline and Day 1 of treatment cycles 4, 7, and 12. Results: As of July 2023, 11 subjects completed at least 3 months of LENZ/AZA treatments and follow-up sequencing data were available for 10 subjects. Five of 10 subjects showed >10% decrease in VAF for at least one mutation detected at baseline, including 5 decreased alleles in the RAS-pathway ( KRAS, CBL) out of 22 total VAF mutation responses. CBL mutations showed the largest change with a decrease in >50% VAF in three subjects. Decreases in RAS-pathway clones by LENZ/AZA were durable at 6 months. No decreases in VAF for any mutations were observed in 2 evaluable subjects following ASC/AZA treatment. Following 3 months LENZ/AZA treatment, blood monocyte count improved from a baseline of 11.0 ± 6.0 x 10 9/L to 2.0 ± 1.0 x 10 9/L (p=0.030). Blast differential improved from 10.5 ± 5.0% to 4.0 ± 3.5% (p=0.038). Platelet count increased from 90.0 ± 50.0 x 10 9/L to 150.0 ± 60.0 x 10 9/L (p=0.010). Statistical improvements remained durable for at least 6 (n=6) and 12 (n=3) months. Hemoglobin concentration increased 100.0 ± 22.0 g/L to 115.0 ± 10.0 g/L at 3 months (p=NS, n=11) and progressively increased to 122.0 ± 10.0 g/L at 12 months (p=0.024, n=3). Spleen length decreased from 15.0 ± 4.0 cm to 13.0 ± 4.0 cm (p=0.03) and decreased further to 11.0 ± 2.0 cm in subjects who received 12 months of treatment (n=3). No evidence of disease progression or relapse was observed in any subject. Conclusion: In 11 evaluable subjects with proliferative CMML and RAS-pathway mutations, GM-CSF neutralization with LENZ in addition to AZA standard of care, resulted in significant decreases in the proportion of KRAS and CBL mutant leukemic cells, accompanied by clinically significant hematologic improvements and a reduction in splenomegaly. Lenzilumab may have efficacy in preventing outgrowth of RAS-pathway mutations, specifically KRAS and CBL, in the context of CMML and other myeloid malignancies.

Contribution of mutant HSC clones to immature and mature cells in MDS and CMML, and variations with AZA therapy

AbstractMyelodysplastic neoplasms (MDSs) and chronic myelomonocytic leukemia (CMML) are clonal disorders driven by progressively acquired somatic mutations in hematopoietic stem cells (HSCs). Hypomethylating agents (HMAs) can modify the clinical course of MDS and CMML. Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated HSCs. However, in patients with established disease it is unclear whether (1) HSCs with multiple mutations progress through differentiation with comparable frequency to their less mutated counterparts or (2) improvements in peripheral blood counts following HMA therapy are driven by residual wild-type HSCs or by clones with particular combinations of mutations. To address these questions, the somatic mutations of individual stem cells, progenitors (common myeloid progenitors, granulocyte monocyte progenitors, and megakaryocyte erythroid progenitors), and matched circulating hematopoietic cells (monocytes, neutrophils, and naïve B cells) in MDS and CMML were characterized via high-throughput single-cell genotyping, followed by bulk analysis in immature and mature cells before and after AZA treatment. The mutational burden was similar throughout differentiation, with even the most mutated stem and progenitor clones maintaining their capacity to differentiate to mature cell types in vivo. Increased contributions from productive mutant progenitors appear to underlie improved hematopoiesis in MDS following HMA therapy.

The Functional and Molecular Alterations in Human Bone Marrow Mesenchymal Stem Cells in Chronic Myelomonocytic Leukemia

Background: Crosstalk between the bone marrow microenvironment and leukemia cells plays a pivotal role in pathogenesis. Mesenchymal stem cells (MSCs) acquire defects during natural ageing, with reduced regenerative potential and functional impairment. Since chronic myelomonocytic leukemia (CMML) mainly affects older adults, defective MSCs could play a critical and targetable role in disease initiation and progression. Identifying molecular abnormalities associated with CMML MSCs would reveal novel therapeutic targets for patient benefit. Methods: Phenotypes of age-matched healthy and CMML MSCs were compared by a range of functional approaches, including senescence, multilineage differentiation and hematopoietic stem cell maintenance assays. To identify altered epigenetic and transcriptional networks we performed RNA-Seq (n=18 CMML/10 healthy) and ATAC-Seq (n=12 CMML/5 healthy) on cultured MSCs (CD44+73+90+105+146+), integrating datasets to explore gene regulatory networks operational within CMML MSCs. To investigate direct crosstalk between MSCs and malignant cells we performed RNA-Seq on healthy MSCs following 7 days co-culture with CMML blood mononuclear cells (MNCs). Functional validation was performed through inhibitor studies using apoptosis, senescence, osteogenesis, and stem cell maintenance as readouts. Results: CMML MSCs displayed consistent and profound intrinsic growth defects, with higher senescence and apoptosis, and lower proliferation and clonogenic capacity. CMML MSCs also displayed a marked differentiation bias, with increased osteogenic lineage potential but reduced adipogenicity and chondrogenicity. Importantly, CMML MSCs demonstrated reduced stem cell maintenance capability in co-culture experiments, confirming a functional consequence on hematopoiesis. Transcriptomic analysis showed activation of novel pathways including mTOR, PI3K-Akt signaling, and oxidative phosphorylation in CMML MSCs, in addition to Wnt pathway upregulation as shown previously (Figure 1A). Consistent with functional readouts, CMML MSCs were transcriptionally primed towards the osteo-lineage, with high expression of osteogenic promoting factors (CORIN, LTF, TREM2, SMAD5, ALX5) and downregulation of osteogenic inhibitors (RBPJ, CLU). In line with transcriptomic analysis, ATAC-Seq of CMML MSCs highlighted aberrantly accessible chromatin regions linked to genes involved in mTOR, PI3K-Akt and Wnt signaling as well as promotion of osteogenesis (Figure 1A). Integration of datasets identified downregulated expression and activity of the transcription factor EBF1, where reduced expression is associated with self-renewal defects, decreased adipogenesis, cellular senescence and defective stem cell maintenance ability, compatible with our observations in CMML MSCs. Similarly, we identified downregulated activity of the transcription factor RBPJ, potentially mediating the preferential enhancement of osteogenesis observed in CMML MSCs. Following co-culture with CMML MNCs, healthy MSCs showed activation of mTOR, PI3K-Akt and osteogenesis, verifying a direct influence of leukemic cells on MSCs. Remarkably, integration of all datasets identified consistently upregulated genes (RGS4, TPI1) which positively regulate mTOR and PI3K-Akt signaling. Inhibiting these aberrantly active pathways in CMML MSCs using small molecule modulators significantly reversed the phenotypic abnormalities with a reduction in apoptosis, senescence and osteogenesis and restoration of their ability to support and maintain stem cells (Figure 1B). Discussion: Our integrated analysis identified altered gene regulatory networks that are linked to abnormal pathways active in CMML MSCs, which can be targeted to restore their normal function. These results highlight novel pathogenic roles for non-clonal MSCs in the CMML microenvironment, and potential adjunctive strategies for targeting microenvironmental alongside cell intrinsic vulnerabilities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P748: SINGLE CELL GENOTYPING OF MATCHED BONE MARROW AND PERIPHERAL BLOOD CELLS IN TREATMENT NAIVE AND AZA-TREATED MDS AND CMML

Background: Somatic mutations in hematopoietic stem cells (HSCs) are a central pathogenic event in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) where they induce proliferative advantages and impaired differentiation with subsequent cytopenias in peripheral blood (PB). Patients with high-risk disease who are ineligible for allogenic stem cell transplantation are treated with hypomethylating agents, including 5-azacytidine (AZA). AZA treatment can improve PB counts and delay progression to Akute Myeloid Leukemia. However, AZA response does not require eradication of mutated HSCs and may be related to improved differentiation capacity of mutated hematopoietic stem and progenitor cells (HSPCs). However, the contribution of mutated HSPCs to steadystate hematopoiesis in MDS and CMML is unclear. Aims: In this project, we aimed to determine the distribution of individual subclones and even wild-type cells within the HSPC-compartment and the contribution of these subclones and wild-type cells to effective hematopoiesis in patients with MDS or CMML. Methods: We used a combination of index sorting and single cell genotyping to characterize the somatic mutations of individual stem- and progenitor cells and matched high-turnover circulating cells in three patients, one treatment naïve and two AZA-treated. Using this approach, myeloid driver mutations previously detected in a traditional approach were tracked in HSC/multipotent progenitors (MPP), MDS stem cells MDS-SC, progenitor cells (common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP), megakaryocyte erythroid progenitor (MEP)), as well as in monocytes, neutrophils and naïve B cells (nBC). Results: Patient H198302 and Patient H198303 both had CMML and had been treated with AZA for ̴10 years with complete response. Patient H198304 had MDS-EB1 and has never been treated with hypomethylating agents. For each patient, the figure shows the proportions of cells across the haematopoietic hierarchy carrying zero, one, two, three, or four mutations in the specified alleles. Only very small numbers of HSPCs carrying no mutation could be detected. In all three patients, most stem and progenitor cells carried a majority of tracked mutations, with no major differences between stem cells, progenitor cells and MDS-stem cells. In one patient, H198304, MDS-stem cells had a higher proportion of cells with 3 or 4 mutations detected. Overall, in all three patients, a similar frequency of mutations was observed in differentiated monocytes and neutrophils with a substantial proportion of the circulating cells derived from highly mutated progenitors. One notable exception were nBCs in Patient H198304, which were mostly wild-type, suggesting that the small wildtype HSC population is the predominant origin of nBCs in this individual. Analysis of additional lymphoid populations in PB from this patient revealed that naïve T, but not NK cells were also predominantly wild-type, suggesting specific impairment of B- and T-lineage maturation in the mutated cells. Image:Summary/Conclusion: In conclusion, attrition of highly mutated cells during myeloid maturation was not observed in any of the three patients, irrespective of HMA therapy. This suggests that in vivo, highly mutated stem and progenitor cells retain the capacity to differentiate to mature myeloid and in some patients mature lymphoid cells and contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment.

Single-Cell RNA Sequencing Analysis Reveals Mechanisms of Initiation and Progression in Chronic Myelomonocytic Leukemia

Despite advances in the genetic characterization of chronic myelomonocytic leukemia (CMML), the molecular mechanisms that drive the disease during its distinct phases remain unclear. To uncover vulnerabilities in CMML that could be therapeutically targeted to halt its evolution, we sought to dissect at the single-cell level the cellular and transcriptomic changes that occur in the hematopoietic system at the time of CMML's initiation and its progression after hypomethylating agent (HMA) therapy.To evaluate the molecular mechanisms underlying CMML maintenance, we performed single-cell RNA sequencing (scRNA-seq) analysis of lineage-negative (Lin -)CD34 + hematopoietic stem and progenitor cells (HSPCs) and bone marrow (BM) mononuclear cells (MNCs) isolated from untreated CMML patients (n=5 and 6, respectively) and age-matched healthy donors (HDs; n=2 and 3, respectively). Our integrated analysis revealed that CMML Lin -CD34 + HSPCs had a predominant granulomonocytic differentiation route with an increased frequency of early and committed myeloid-monocytic progenitors at the expense of HSCs and megakaryocyte/erythroid progenitors (Fig. 1a). Differential expression analysis among the clusters revealed that most transcriptomic differences occurred in CMML HSCs, which were characterized by the upregulation of genes involved in oxidative phosphorylation, type I interferon (IFN) and IFNγ response, myeloid development, and inflammatory signaling and had downregulated expression of genes involved in TNFα-mediated NF-κB activation (Fig. 1b). These data suggest that CMML HSCs undergo metabolic reprogramming and demand a higher level of mitochondrial activity to maintain activated monocytic differentiation in response to inflammatory signaling.Consistent with these results, scRNA-seq analysis of MNCs isolated from the same HD and CMML BM samples showed that monocytes were significantly increased at the expense of erythroid precursors and B cells in CMML (Fig. 1c). CMML monocytes had upregulated genes involved in IFNγ response, oxidative phosphorylation, MYC targets, NF-κB activation, and inflammation (e.g., S100A9, CCL3, IL1B). Interestingly, among the anti-apoptotic BCL2 family members, only the NF-κB transcriptional target BCL2A1 was significantly overexpressed.To investigate the mechanisms of resistance to HMA therapy, we performed integrated scRNA-seq analysis of sequential Lin -CD34 + cells and BM MNCs isolated from CMML patients at the time of disease initiation and progression after HMA therapy failure. CMML progression was driven by a significant expansion of lympho-myeloid progenitors (LMPPs) at the expense of earlier HSCs , which exacerbated myelomonocytic differentiation in the HSPC compartment (Fig. 1d). Expanded LMPPs were characterized by higher levels of IFNγ response, NF-κB survival signaling, and cell cycle regulators. Accordingly, scRNA-seq analysis of MNCs cells from the same patients showed significantly increased frequencies of monocytes and a reduction of naïve CD4 +/CD8 + T cells and effector memory CD8 + T cells. Differential expression analysis of the 2 sample groups in the monocyte population identified five different cellular clusters, one of which emerged only at progression (Fig. 1e). This population was characterized by high expression levels of inflammatory cytokines and the anti-apoptotic modulators MCL1 and BCL2A1. Together, these data suggest that CMML progression arises from immature myeloid progenitors at the stem cell level and that downstream monocytes undergo transcriptomic rewiring and acquire survival mechanisms that induce therapy resistance and further accelerate disease progression.In conclusion, our results elucidate the differentiation hierarchies and transcriptional programs associated with CMML's initiation and its progression after HMA therapy. Our data suggest that therapies targeting downstream effectors of NF-kB-mediated survival signaling could overcome treatment failure. [Display omitted] DisclosuresWei: Daiichi Sanko: Research Funding. Kantarjian: AbbVie: Honoraria, Research Funding; Immunogen: Research Funding; KAHR Medical Ltd: Honoraria; Jazz: Research Funding; Ipsen Pharmaceuticals: Honoraria; Astellas Health: Honoraria; NOVA Research: Honoraria; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Astra Zeneca: Honoraria; Ascentage: Research Funding; Aptitude Health: Honoraria; Daiichi-Sankyo: Research Funding; Amgen: Honoraria, Research Funding; BMS: Research Funding; Precision Biosciences: Honoraria; Taiho Pharmaceutical Canada: Honoraria.

Serially Transplantable Patient-Derived Xenograft Model of Chronic Myelomonocytic Leukemia (CMML)

Introduction: The establishment of preclinical mouse models for MDS or MDS/MPN overlap syndromes from patient cells often fails or results in low engraftment. If successful, engrafting cells in primary mouse recipients usually do not engraft secondary or later recipients, thus precluding genetic manipulation of the cells for functional studies. We hypothesized that oncogenic complementation of patient cells would a) allow engraftment and serial transplantation, b) enable genetic modification of the cells for functional evaluation and c) preserve the disease-associated biology of the cell. To study these hypotheses we chose the MN1 oncogene for complementation of cells from a patient with chronic myelomonocytic leukemia (CMML). Importantly, healthy cord blood cells, when transduced with MN1, induce myeloproliferation in vivo, but cannot be serially transplanted and do not induce acute myeloid leukemia (Imren et al. Blood 2014).Aim: To establish a serially transplantable CMML patient-derived xenograft (PDX) model and perform a targeted in vivo shRNA screening in this model.Patient and Methods: Bone marrow (BM) cells were harvested from a CMML patient with 15% blasts in BM (CMML-2). BM cells were depleted for T cells and transduced with a lentiviral vector encoding MN1 and GFP. One million MN1-transduced or untransduced cells were injected intravenously in sublethally irradiated NOD/SCID IL-2Rγ-/- mice transgenic for SCF, IL3 and GM-CSF (NSGS). BM and spleen cells from engrafted mice were immunophenotyped and retransplanted in NSGS recipients up to 6 times. Mutations were evaluated by whole exome sequencing and confirmed by Sanger sequencing. A targeted shRNA library was generated and lentivirally transduced in serially transplantable CMML cells. Representation of transduced cells was monitored by next-generation sequencing of individual shRNAs.Results: MN1 transduction of human CMML cells resulted in mean engraftment of human CD45+ cells of 9% in peripheral blood (PB) and 41% in BM at week 12 after first transplantation. In contrast, untransduced CMML cells were hardly detected in PB and BM even 22 weeks after transplantation (mean engraftment 1.1% and 1.7%, respectively). Transplantation of BM cells from primary into secondary recipients led to stable engraftment of MN1-transduced CMML cells in PB (11%), spleen (4%) and BM (26%). In contrast, transplantation from primary to secondary recipients of untransduced CMML cells showed no engraftment in PB over a period of 40 weeks. Importantly, MN1+CMML cells have been serially transplanted up to the sixth recipient with good engraftment levels in PB, BM and spleen and could be re-derived from frozen cells. Overall, MN1+CMML cells could be expanded in vivo to allow engraftment in 33 mice so far.MN1+CMML cells in spleen and BM showed a myeloid phenotype expressing CD33, CD38 and CD14, but lacked expression of lymphoid markers CD3 and CD19. The stem and progenitor phenotype CD34+CD38- was found in 2.5% of these cells. By morphologic evaluation, monocytic cells prevailed in BM up to the third transplantation, while blast cells increased in later transplantations. Molecular analysis of primary patient cells identified mutations in BCOR, DNMT3A, U2AF1, NOTCH1 and NRAS . All mutations were still present in human cells isolated from recipient mice after the 4th and 6th transplantation, confirming the genetic stability of the model.A shRNA pool directed against the above mentioned mutated genes, MN1, and luciferase was lentivirally transduced into cells from the fifth recipient and transplanted into the sixth recipient. After 8 weeks, shRNAs against luciferase and BCOR were well preserved in vivo in MN1+CMML cells, while shRNAs against U2AF1 and MN1 were depleted. Thus, MN1+CMML cells remained dependent on at least one additional gene besides MN1, thus validating our hypothesis that the disease biology is preserved in our induced PDX model.Conclusion: We present a novel approach to establish serially transplantable MDS/MPN-PDX models by complementation with the MN1 oncogene. Our CMML model preserves the genetic and morphologic characteristics of the disease and allows dissection of the functional architecture of CMML and preclinical drug studies in this underserved disease. DisclosuresNo relevant conflicts of interest to declare.

Phenotypic characterization of leukemia-initiating stem cells in chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a stem cell-derived neoplasm characterized by dysplasia, uncontrolled expansion of monocytes and a substantial risk to transform to secondary acute myeloid leukemia (sAML). So far, little is known about CMML-initiating cells. We found that leukemic stem cells (LSC) in CMML reside in a CD34+/CD38– fraction of the malignant clone. Whereas CD34+/CD38– cells engrafted NSGS mice with overt CMML, no CMML was produced by CD34+/CD38+ progenitors or the bulk of CD34– monocytes. CMML LSC invariably expressed CD33, CD117, CD123 and CD133. In a subset of patients, CMML LSC also displayed CD52, IL-1RAP and/or CLL-1. CMML LSC did not express CD25 or CD26. However, in sAML following CMML, the LSC also expressed CD25 and high levels of CD114, CD123 and IL-1RAP. No correlations between LSC phenotypes, CMML-variant, mutation-profiles, or clinical course were identified. Pre-incubation of CMML LSC with gemtuzumab-ozogamicin or venetoclax resulted in decreased growth and impaired engraftment in NSGS mice. Together, CMML LSC are CD34+/CD38– cells that express a distinct profile of surface markers and target-antigens. During progression to sAML, LSC acquire or upregulate certain cytokine receptors, including CD25, CD114 and CD123. Characterization of CMML LSC should facilitate their enrichment and the development of LSC-eradicating therapies.

Curcumol and FTY720 synergistically induce apoptosis and differentiation in chronic myelomonocytic leukemia via multiple signaling pathways.

Chronic myelomonocytic leukemia (CML) is a myeloid tumor characterized by MDS (myelodysplastic syndrome) and MPN (myeloproliferative neoplasms). Allogeneic hematopoietic stem cell transplantation, chemotherapy, interferon, and targeted therapy are the main treatment methods for CML. Tyrosine kinase inhibitors (TKIs) are also a treatment option, and patients are currently recommended to take these drugs throughout their lives to prevent CML recurrence. Therefore, there is a need to investigate and identify other potential chemotherapy drugs. Currently, research on CML treatment with a single drug has shown little progress. Fingolimod (FTY720), an FDA-approved drug used to treat relapsing multiple sclerosis, has also shown great potential in the treatment of lymphocytic leukemia. In our study, we find that FTY720 and curcumol have a significant inhibitory effect on K562 cells, K562/ADR cells, and CD34+ cells from CML patients. RNAseq data analysis shows that regulation of apoptosis and differentiation pathways are key pathways in this process. Besides, BCR/ABL-Jak2/STAT3 signaling, PI3K/Akt-Jnk signaling, and activation of BH3-only genes are involved in CML inhibition. In a K562 xenograft mouse model, therapy with curcumol and FTY720 led to significant inhibition of tumor growth and induction of apoptosis. To summarize, curcumol and FTY720 synergistically inhibit proliferation involved in differentiation and induce apoptosis in CML cells. Therefore, synergistic treatment with two drugs could be the next choice of treatment for CML.

Effective drug treatment identified by in vivo screening in a transplantable patient-derived xenograft model of chronic myelomonocytic leukemia

To establish novel and effective treatment combinations for chronic myelomonocytic leukemia (CMML) preclinically, we hypothesized that supplementation of CMML cells with the human oncogene Meningioma 1 (MN1) promotes expansion and serial transplantability in mice, while maintaining the functional dependencies of these cells on their original genetic profile. Using lentiviral expression of MN1 for oncogenic supplementation and transplanting transduced primary mononuclear CMML cells into immunocompromised mice, we established three serially transplantable CMML-PDX models with disease-related gene mutations that recapitulate the disease in vivo. Ectopic MN1 expression was confirmed to enhance the proliferation of CMML cells, which otherwise did not engraft upon secondary transplantation. Furthermore, MN1-supplemented CMML cells were serially transplantable into recipient mice up to 5 generations. This robust engraftment enabled an in vivo RNA interference screening targeting CMML-related mutated genes including NRAS, confirming that their functional relevance is preserved in the presence of MN1. The novel combination treatment with azacitidine and the MEK-inhibitor trametinib additively inhibited ERK-phosphorylation and thus depleted the signal from mutated NRAS. The combination treatment significantly prolonged survival of CMML mice compared to single-agent treatment. Thus, we identified the combination of azacitidine and trametinib as an effective treatment in NRAS-mutated CMML and propose its clinical development.

Peripheral Blood Cell Sorting Strategies for Transcriptomic Analysis in Chronic Myelomonocytic Leukemia

Introduction: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic neoplasm characterized by sustained peripheral blood (PB) monocytosis and an inherent risk for leukemic transformation. Clonal origins of the disease can be detected in hematopoietic progenitor cells (CD34+/CD38-), while the complete spectrum of mutational evolution can be seen in circulating monocytes (CD14+). Cell sorting strategies have been employed to select cells in CMML, and while there are adequate monocyte numbers in the PB, there are very few circulating progenitor cells. In addition, attrition related to the selection process significantly depletes primary cells available for biological experiments and multiomics studies such as RNA-seq, ChIP-seq, ATAC-seq, and DIP-seq. While single-cell methods may be able to overcome this challenge, bulk sequencing methods remain a robust and cost-effective approach. We hypothesized that, secondary to the stem cell origin of this disease and significant myeloproliferation, PB mononuclear cells (MNC) would provide comparable results with regards to transcriptomic analysis, in comparison to cell selection procedures. Methods: Peripheral blood obtained from 15 molecularly annotated patients with WHO-defined CMML was ACK-lysed and subjected to a Ficoll procedure for collection of MNC. MNC were left unsorted (n=5) or further selected for CD34+/CD38- (n=5) and CD14+ (n=5) using a fully automated RoboSep-S (StemCell Technologies) protocol. All samples were then subjected to bulk whole transcriptome shotgun sequencing (using Illumina TruSeq and an Illumina HiSeq 4000). After data quality control, counts of detectable transcripts were log2-normalized and Pearson's product-moment correlation coefficients were calculated to evaluate the correlation between the two cell-sorting strategies and unsorted cells in terms of detectable transcripts. To visualize sample differences log2-normalized transcripts counts were centered and scaled per gene for a select number of genes relevant to myeloid biology as well as a number of housekeeping genes. Results: Fifteen patients with WHO-defined CMML, median age 69 years (55-73 years), 66% male, were included. Next generation sequencing for somatic mutations was performed on PB MNC obtained at CMML diagnosis (Figure 1, top heatmap). Considering the small sample size, mutations were evenly distributed among groups with the exception of ASXL1 (higher frequency in CD14+ and CD34+/CD38- cells), ZRSR2 (higher frequency in unsorted cells), and TET2 (lower frequency in CD14+ cells). The three groups were also well matched with regards to other CMML-related variables such as WHO and FAB morphological subtypes, cytogenetic abnormalities, and risk stratification by the Mayo Molecular Model. Transcriptomic analysis revealed a strong positive correlation between the median number of log2-normalized detectable transcripts in unsorted cells and CD34+/CD38- cells (ρ = 0.96, p < 0.001, top scatterplot). Likewise, there was a strong positive correlation between the median number of log2-normalized detectable transcripts in unsorted cells and CD14+ cells (ρ = 0.91, p < 0.001, bottom scatterplot). The latter correlation was marginally lower, which was explained by increased global gene expression in 3 of the 5 CD14+ samples (bottom heatmap). Increased gene expression in these 3 samples involved key myeloid genes and housekeeping genes known to have stable expression across human tissues alike. In comparison to PB MNC, both cell sorting strategies resulted in significant depletion of primary cells required for other experiments, and for procedures such as ChIP-seq, DIP-seq and ATAC-seq (CD34+/CD38- had greater depletion than CD14+). Additional experiments to assess this strategy for the above mentioned epigenetic studies are currently being planned. Conclusions: Accounting for sample differences, different cell sorting strategies (unsorted, CD34+/CD38- selection, and CD14+ selection) yielded similar results when performing bulk transcriptomic assessments on PB MNC from patients with CMML. For the purpose of gene expression profiling there was no clear advantage with CD34+/CD38- or CD14+ selection. These results support the use of unsorted cells for bulk transcriptomic analysis in CMML. Figure 1 Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Phenotypic Characterization of Leukemia-Initiating Stem Cells in Chronic Myelomonocytic Leukemia (CMML)

Chronic myelomonocytic leukemia (CMML) is a stem cell-derived hematopoietic neoplasm characterized by dysplasia, uncontrolled expansion of monocytic (progenitor) cells in the bone marrow (BM) and in the peripheral blood (PB), and an increased risk of progression to secondary acute myeloid leukemia (sAML). Patients with advanced CMML and sAML are often highly resistant to therapy and their prognosis is dismal. It is thought that drug resistance in myeloid malignancies is a quality of leukemia stem cells (LSC), but little is known about CMML-initiating and propagating LSC. We investigated the phenotype and functional behavior of putative CMML-initiating cells in 15 patients with CMML (7 females, 8 males; median age 73 years; range 45-82 years) and 6 with sAML following CMML (1 female, 5 males; median age 67.5 years; range 66-76 years). BM and/or PB samples were examined by multicolor flow cytometry using antibodies against CD34, CD38 and various additional surface markers and target antigens. In a subset of patients, putative stem and progenitor cells (CD34+ cells, CD34+/CD38─ cells and CD34+/CD38+ cells) were FACS-sorted to high purity (>95%) and were employed in xenotransplantation experiments or in drug testing experiments. We found that CMML-initiating and propagating LSC reside within the CD34+/CD38─ fraction of the malignant clone. Whereas highly purified CD34+ cells engrafted NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG) 1Eav/MloySzJ (NSGS) mice with full-blown CMML (engraftment rate 44.8±26.0%), no CMML was produced by the bulk of CD34- monocytic cells (engraftment rate 0.8±0.5%; p=0.002). CMML engraftment was also detectable when transplanting unselected mononuclear cells (engraftment rate 19.7±10.9%). By contrast, no leukemic engraftment was produced by CD38+ CMML fractions (engraftment rate 0.1±0.1%; p=0.003), indicating that the NSGS-repopulating CMML LSC reside specifically in a CD34+/CD38- fraction of the clone. In sAML, both the CD34+/CD38- cell fraction (engraftment rate 92.2±6.2%) and the CD34+/CD38+ fraction (engraftment rate 80.5±7.2%) produced engraftment with AML blasts in NSGS mice. In a next step, we established the cell surface phenotype of CD34+/CD38- LSC in CMML and sAML. As assessed by multicolor flow cytometry, CD34+/CD38- CMML cells invariably expressed CD33/Siglec-3, CD117/KIT, CD123/IL3RA, CD133/AC133, CD135/FLT3, and IL-1RAP. In a subset of patients, CMML LSC also expressed CD52 (9/11 patients; 81%), CD114 (3/7 patients; 43%), CD184 (9/12 patients; 75%), CD221 (8/11 patients; 73%) and/or CLL-1 (7/13 patients; 54%). CMML LSC did not express CD25 or CD26. However, in patients with sAML, LSC also displayed CD25 (median fluorescence intensity, MFI: CMML: 0.9 vs. sAML: 23.0; p<0.001). Compared to hematopoietic stem cells in normal BM (NBM), CMML LSC displayed slightly increased levels of CD117/KIT (MFI CMML: 32.5 vs. MFI NBM: 15.0; p=0.019), CD135/FLT3 (MFI CMML: 1.9 vs. MFI NBM: 0.8; p=0.001), CD184/CXCR4 (MFI CMML: 1.6 vs. MFI NBM 0.9; p=0.027), and IL-1RAP (MFI CMML: 1.6 vs. MFI NBM: 0.8; p=0.004). No correlations between surface-marker expression on LSC and the type of CMML (CMML-0/1/2 or dysplastic vs. proliferative CMML) or the clinical course were found. To confirm the clinical relevance of expression of surface target antigens on CMML LSC, we applied the CD33-targeted drug gemtuzumab-ozogamicin (GO). As assessed by combined staining for LSC (CD34+/CD38-) and AnnexinV/DAPI, incubation of CMML LSC with GO (0.001-1 µg/ml) resulted in dose-dependent apoptosis in all donors tested, and the same result was obtained in the monoblastic cell lines THP-1 (GO at 1 µg/ml: 94.2±1.5% vs. control: 12.7±2.2%, p<0.05) and Mono-Mac-1 (GO at 1 µg/ml: 56.4±12.1% vs. control: 10.1±0.6% p<0.05). In conclusion, LSC in CMML and sAML reside within CD34+/CD38─ cell populations that express distinct profiles of surface markers and target antigens. During progression of CMML into sAML, LSC apparently acquire CD25. Characterization of CMML LSC and LSC in sAML should facilitate their enrichment and the development of LSC-eradicating therapies. Disclosures Hoermann: Novartis: Honoraria; Roche: Honoraria. Sperr:Celgene: Consultancy, Honoraria; Novartis: Honoraria. Sill:Astex: Other: Advisory board; Novartis: Other: Advisory board; AbbVie: Other: Advisory board; Astellas: Other: Advisory board. Geissler:Novartis: Honoraria; AOP: Honoraria; Roche: Honoraria; Amgen: Honoraria; AstraZeneca: Honoraria; Ratiopharm: Honoraria; Celgene: Honoraria; Abbvie: Honoraria; Pfizer: Honoraria. Deininger:Blueprint: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Research Funding; Ascentage Pharma: Consultancy, Honoraria; Fusion Pharma: Consultancy; TRM: Consultancy; Sangoma: Consultancy; Adelphi: Consultancy; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Humana: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Sangamo: Consultancy. Valent:Pfizer: Honoraria; Blueprint: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria; Deciphera: Honoraria, Research Funding.

Oncogenic Mechanisms of CBL E3 Ubiquitin Ligase Mutations in Myeloid Malignancies

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.

LILRB4 expression in CMML and MDS based on response to hypomethylating agents.

e18550 Background: Leukocyte immunoglobulin-like receptor B4 (LILRB4) is expressed on M4 and M5 monocytic acute myeloid leukemia (AML) cells and leads to negative regulation of immune cell activation via T cell suppression. Its expression and role in chronic myelomonocytic leukemia (CMML) and myelodysplastic syndrome (MDS) are unknown. We investigated LILRB4 expression in patients with CMML and MDS and correlated it with response to hypomethylating agent (HMA) therapy. Methods: We evaluated bone marrow samples from 19 patients with CMML and 27 patients with MDS from 2008-2014. Transcriptomic and splicing analysis by RNA sequencing were performed on CD34-positive cells in CMML and MDS patient samples in addition to AML or MDS cell lines (n = 6). Gene set enrichment analysis was used to identify pathways associated with increased LILRB4 expression. Results: By the WHO 2016 classification for CMML, there were 8 CMML-0, 5 CMML-1, and 6 CMML-2 patients, and there were 2 low-risk, 8 intermediate-1-risk, 12 intermediate-2-risk, and 5 high-risk MDS patients by IPSS. A total of 12 CMML patients and 27 MDS patients received HMA, with 6 and 12 showing response, respectively. RNA expression of LILRB4 was increased in CMML patients when compared to MDS patients and healthy controls (q < 0.1) with no difference in expression based on risk groups. Pathway analysis revealed upregulation of PD-1 signaling (p = 0.004), CTLA-4 signaling (p = 0.004), and inflammatory response (p = 0.004), and gene correlates revealed a positive association with CTLA-4 expression (p = 0.05). LILRB4 expression was slightly increased in CMML patients who responded to HMAs (q > 0.1). In cell lines, LILRB4 was expressed in MOLM13 (AML M5), SKM1 (AML M5), and U937 (histiocytic lymphoma), with an increase in HMA-sensitive MOLM13 cells compared to HMA-resistant MOLM13 cells (q < 0.01) and no difference based on HMAs seen in other cell lines. Conclusions: LILRB4 expression is increased in CMML, with further increase in those exhibiting response to HMA therapy. It is positively correlated with immune checkpoint signaling pathways, particularly CTLA-4 expression. This may set the basis for exploring the possibility of anti-LILRB4 and anti-CTLA-4 combinations.

Biology and prognostic impact of clonal plasmacytoid dendritic cells in chronic myelomonocytic leukemia.

Islands of CD123high cells have been commonly described in the bone marrow of patients with chronic myelomonocytic leukemia (CMML). Using a multiparameter flow cytometry assay, we detected an excess of CD123+ mononucleated cells that are lineage-negative, CD45+, CD11c-, CD33-, HLA-DR+, BDCA-2+, BDCA-4+ in the bone marrow of 32/159 (20%) patients. Conventional and electron microscopy, flow cytometry detection of cell surface markers, gene expression analyses, and the ability to synthesize interferon alpha in response to Toll-like receptor agonists identified these cells as bona fide plasmacytoid dendritic cells (pDCs). Whole-exome sequencing of sorted monocytes and pDCs identified somatic mutations in genes of the oncogenic RAS pathway in the two cell types of every patient. CD34+ cells could generate high amount of pDCs in the absence of FMS-like tyrosine kinase 3-ligand(FLT3L). Finally, an excess of pDCs correlates with regulatory T cell accumulation and an increased risk of acute leukemia transformation. These results demonstrate the FLT3L-independent accumulation of clonal pDCs in the bone marrow of CMML patients with mutations affecting the RAS pathway, which is associated with a higher risk of disease progression.

Genotypic Representation of Myelodysplastic/Myeloproliferative Neoplasms in Nrg, Nrg-3GS and Srg-W41 Mice with Transgenic Expression of Human Cytokines

Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are complex clonal hematopoietic stem cell malignancies with overlapping dysplastic and proliferative features. Genomic analyses have charted the somatic mutation spectrum of MDS/MPN and revealed a major role for epigenetic dysregulation in their pathogenesis. No disease-modifying therapies are currently available, as progress has been hampered by a lack of genetically faithful in vivo model systems suitable for the preclinical development of new strategies. Yoshimi et al (Blood. 2017;130:397-407) recently showed that patients' chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML) cells transplanted into NOD/SCID-IL2Rγ-/-mice expressing human IL3, GM-CSF and SCF transgenes (NSG-3GS mice) produced xenografts that had mutations characteristic of the input cells. Since we had demonstrated a superior level of chimerism achieved from transplants of normal human CD34+cord blood cells in SirpaNOD/Rag1-/-/IL2rγc-/-/W41/41mice with c-KIT deficiency (with an otherwise mixed NOD-C57Bl/6 background - SRG-W41 mice) compared to conventional NSG or NRG hosts (Miller et al. Exp Hematol. 2017;48:41-49), it was of interest to explore their use as hosts of samples from patients with MDS/MPN: CMML, atypical chronic myeloid leukemia (aCML) and secondary acute myeloid leukemia (sAML) progressed from CMML or aCML.Heparinized blood or bone marrow samples were obtained from patients treated at Huntsman Cancer Institute after informed consent. Diagnoses included CMML (n=5), aCML (n=2), and sAML (n=2). Unseparated cells were shipped by overnight courier to Vancouver and CD34+cells isolated on the same day were injected intravenously into sub-lethally irradiated female NRG mice or SRG-W41 mice, or in some cases the same sex and strains also carrying the human 3GS transgenes (NRG-3GS or SRG-W41-3GS mice) in accordance with British Columbia Cancer Agency institutional guidelines. Occasionally when mice were not immediately available, or large numbers of cells were available, cells were viably cryopreserved and transplanted later after thawing. Mice were observed for up to 36 weeks after xenotransplantation with .05 to 1.1x106 human CD34+cells. Engraftment of human CD45+cells in xenografts was evaluated by immunophenotyping, and a median of 90% human chimerism (range: 1% - 95%) was achieved at the time of bone marrow harvest from xenografts.Variant allele frequencies (VAF) were determined in genomic DNA extracted from both the patient samples (CD34+cells) and matching fluorescence-activated cells (FACS)-sorted human CD45+cells (hCD45+cells) purified from xenografts (1-5 xenografts per patient sample). DNA samples were subjected to PCR amplification with extension primers and analyzed using a MALDI-TOF mass spectrometer (MassArray, Agena Bioscience, San Diego, CA). Each mutation call was assigned by the software based on the molecular weight of the extended primer. Analysis of hCD45+cells from eight xenograft samples so far demonstrated a strong correlation of VAF between the patient samples and hCD45+cells from xenografts, in both SRG-W41-3GS (R2=0.94, p<0.01) and NRG-3GS (R2=0.97, p<0.01) models (Figure 1). This tight correlation of VAF was illustrated in hCD45+cells from xenografts transplanted with CMML, aCML or sAML cells. The majority of mutations detected were those in epigenetic regulator genes, such as ASXL1, EZH2 and TET2. No significant difference in VAF was observed between CD34+and CD34- compartments within the hCD45+cells. Additional samples, including specimens from patients with the related myeloproliferative neoplasm, chronic neutrophilic leukemia (CNL) are being analyzed and will be presented.These findings demonstrate the utility of SRG-W41-3GS as well as NRG-3GS as receptive hosts of primary human MDS/MPN cells with genetic evidence of their growth in these mice closely recapitulating the mutational profiles of the transplanted cells. These new strains may facilitate the development of functional screening and pre-clinical testing of novel therapeutic strategies for a range of human MDS/MPN and related myeloid disorders. [Display omitted] DisclosuresDeininger:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

Results of a Phase 2 Trial of the Monocyte-Targeted Histone Deacetylase Inhibitor Tefinostat (CHR-2845) in Chronic Myelomonocytic Leukemia (CMML) — the UK Monocle Study

BackgroundChronic myelomonocytic leukemia (CMML) is a highly heterogeneous myeloid neoplasm with poor prognosis and limited therapeutic options. Tefinostat (CHR-2845) is a cell permeant ester that is converted to an active acid histone deacetylase (HDAC) inhibitor (CHR-2847) by human carboxylesterase 1 (hCE-1), an enzyme predominantly found in cells of monocyte lineage. Pre-clinical studies confirmed selective activity of tefinostat, correlating with hCE-1 expression and acetylation induction, in monocyte derived cell lines, primary CMML and AML (M4/M5) cells (1). A phase 1 study in refractory haematological malignancies showed tefinostat to be well tolerated, with complete remission reported in 1/2 CMML patients (2). Given the compelling rationale for selective activity in monocytoid malignancies, we conducted a phase 2 study to assess safety and efficacy of tefinostat in CMML.MethodsMONOCLE was a single arm phase 2 trial conducted to a Bryant and Day 2-stage design with dual primary endpoints of safety and clinical efficacy. CMML-2 patients were included; additionally CMML-1 patients with symptomatic bone marrow failure or proliferative disease, symptomatic splenomegaly, extramedullary involvement, systemic symptoms or CMML-specific Prognostic Score (CPSS) int-2/high. Tefinostat was administered orally in continuous 28-day cycles starting at 360mg once daily, increasing to 480mg after 4 weeks if well tolerated. Concomitant hydroxycarbamide was permitted only with cycles 1-3. Clinical response was assessed according to International Consortium MDS/MPN Response Criteria (3); responding patients at cycle 6 were permitted to continue therapy. Toxicity was assessed according to CTCAE v4.0.ResultsIn stage 1, 21 patients were enrolled at 9 centers (Jan-Sep 2017). 20 patients received tefinostat (median age 75 years [64-88], M/F 14/6) including 16 with CMML-1 (80%) and 4 CMML-2 (20%), 8 (40%) with myelodysplastic and 12 (60%) myeloproliferative CMML; respective proportions in CPSS low/int-1/int-2/high risk groups were 5/50/40/5%. Myeloid NGS analysis confirmed a molecularly relatively high risk population: 70% ASXL1 mutation frequency and median 4 (2-7) mutations per patient; other most commonly observed mutations being TET2 (65%), SRSF2 (50%), EZH2 (35%) and NRAS (35%). Prior therapy included azacitidine (3 patients) and hydroxycarbamide (7). 17/20 patients had high hCE-1 levels, assessed flow cytometrically at trial entry in monocytoid cells (vs bulk myeloid population). Median number of cycles of tefinostat received was 4 (1-15). Of 13 patients completing ≥3 cycles of tefinostat, 1 patient achieved clinical benefit (partial bone marrow response at cycle 6 with red cell transfusion independence sustained over 15 cycles of treatment), 9 had stable disease (of whom 1 had transient clinical benefit [MPN-SAF symptom reduction] which was not sustained to cycle 6) and 3 had progressive disease. Most frequent non-hematologic adverse events of any grade were raised creatinine (55% patients), fatigue (40%) and nausea/vomiting (30%). Grade ≥3 AEs judged potentially related to tefinostat included thrombocytopenia (3 patients), fatigue (2), raised creatinine (2), anorexia, AV block, nausea and neutropenia (1 each). Creatinine rises were in all cases reversible following tefinostat dose reduction/cessation. Induction of intracellular lysine acetylation in monocytes (a marker of HDAC inhibition) was observed in 50% of patients (including clinical responders), peaking between days 15-28 of cycle 1. While no clear relationship between baseline hCE-1 expression and clinical response was evident, reductions in marrow monocyte and myeloid blast fractions were seen in both clinical responders.ConclusionFollowing failure to achieve a pre-defined minimum number of clinical responses to tefinostat, patient recruitment was not continued into stage 2 of this phase 2 study. Drug tolerability was encouraging although observed renal effects will likely preclude dose escalation in this challenging, often frail patient group. Despite compelling scientific rationale and pre-clinical data favoring this monocyte targeted treatment approach we were unable to demonstrate a clinically significant single agent disease modifying effect in CMML.(1) Zabkiewicz J. Oncotarget 2016; 7: 16650-62(2) Ossenkoppele G. Br J Haem 2013; 162: 191-201(3) Savona M. Blood 2015; 125: 1857-65 DisclosuresKnapper:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Other: Meeting and travel support; Jazz: Other: Meeting and travel support; Daiichi Sankyo: Other: Meeting and travel support; Chroma Therapeutics: Research Funding. Hills:Daiichi Sankyo: Consultancy, Honoraria. Dillon:Teva Pharmaceuticals UK: Consultancy, Honoraria; AbbVie UK: Consultancy, Honoraria. Pocock:Kent & Canterbury Hospital: Employment. Culligan:Pfizer: Honoraria; Takeda: Honoraria, Other: Support to attend conferences; JAZZ: Honoraria; Merck Sharp & Dohme (MSD): Honoraria; Abbvie: Other: Support to attend conferences; Celgene: Other: Support to attend conferences; Daiichi-Sankyo: Other: Support to attend conferences.

CBL mutation and MEFV single-nucleotide variant are important genetic predictors of tumor reduction in glucocorticoid-treated patients with chronic myelomonocytic leukemia.

Glucocorticoid (GC) therapy occasionally relieves tumor-related fever and promotes tumor reduction in patients with chronic myelomonocytic leukemia (CMML). A mutation analysis of 24 patients with CMML revealed the relationship of GC effectiveness, defined as a monocyte reduction of > 50% within 3days of methylprednisolone administration, with the MEFV single-nucleotide variant (SNV) and CBL mutation. Lipopolysaccharide-stimulated monocytes harboring MEFV E148Q produced greater amounts of IL-1β and TNF-α than did wild-type monocytes; this was effectively suppressed by GC. Primary CMML cells harboring the MEFV SNV and CBL mutation, and the myelomonocytic leukemia cell line GDM-1, harboring the CBL mutation, were both more significantly suppressed than non-mutated cells following GC treatment in the presence of GM-CSF. A loss-of-function CBL mutation prolonged STAT5 phosphorylation after GM-CSF stimulation, which was rapidly terminated in both patient samples and GDM-1 cells. In conclusion, GC therapy effectively treats CMML cells harboring the MEFV SNV and CBL mutation by reducing inflammatory cytokine production and terminating prolonged STAT5 phosphorylation in the GM-CSF signaling pathway.