Myelodysplastic Syndromes CD34 Research Articles

Novel Clinically Useful Inhibitor of Mediator Complex, RVU120, Relives Differentiation Block in MDS/AML

Background: Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemias (AML) are characterized by a block in hematopoietic differentiation that can often be traced to aberrant stage-specific transcription. The kinase module of the mediator complex, consisting of CDK8, its paralog CDK19, Cyclin C, MED12, and MED13, have been identified as a molecular switch that regulates gene expression. A variety of cancers have exploited this mechanism to maintain stemness and an undifferentiated state. RVU120 is a first-in-class, specific, and selective inhibitor of CDK8/19, currently in Phase Ib clinical trial in patients with AML or High-Risk MDS (HR-MDS) (NCT04021368). RVU120 has been shown to stimulate erythroid differentiation in transformed CD34+ cord blood (CB) cells and in CD34+ cells from Diamond-Blackfan Anemia (DBA). To evaluate the further therapeutic potential of CDK8/19 inhibition, we confirmed aberrant expression of mediator complex components and erythroid-stimulating activity in a collection of CD34+ primary samples of AML and MDS. Results: We investigated the expression of mediator complex components in a large database of MDS CD34+ marrow samples and age-matched controls. Our findings revealed that MED12, a critical component of the mediator complex, was significantly overexpressed in MDS samples from refractory anemia with excess blasts (RAEB), a higher risk subset of MDS(p=0.018) (Fig A). Furthermore, we observed that this overexpression of MED12 was associated with a higher rate of transformation to AML. To delve deeper, we examined the expression of MED12 in sorted leukemic stem cells from primary AML samples and found significantly elevated levels in Long Term Hematopoietic Stem Cells from AML samples with both normal karyotype and complex karyotypes compared to healthy controls. Next, we conducted an evaluation of the efficacy of the mediator kinase module inhibition in MDS-derived cell line (MDS-L). Inhibition of CDK8/19 with RVU120 led to increased erythroid differentiation in this model which confirmed our observations in transformed CD34+ CB cells in CB cells, these phenotypic changes were correlated with the inhibition of CDK8-dependent phosphorylation marker S726 on STAT5 and a profound increase in the expression of genes involved in erythroid commitment and hemoglobin metabolism. In order to confirm therapeutic potential of CDK8/CDK19 inhibition in MDS and AML, we performed colony-forming assay on primary samples of MDS and AML (n= 15). The treatment with RVU120 resulted in increased erythroid differentiation in a majority of samples, evident from an augmented number of colonies. Detailed analysis indicated changes in the expression of erythroid differentiation markers, including increased CD71 and Glycophorin A expression on colonies, as assessed by FACS analysis (Figure B, shows representative sample). Conclusion Our data highlight that inhibition of the overexpressed mediator complex proteins can alleviate the differentiation blocks seen in MDS/AML. These results provide a compelling rationale for further developing the CDK8/19 inhibitor RVU120 for transfusion- dependent MDS/AML patients.

Inhibition of Toll-like Receptor 4 (TLR4) Promotes Erythroid and Myeloid Differentiation in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML)

Introduction: The hallmark of Myelodysplastic Syndrome (MDS) is a block in differentiation of hematopoietic precursors resulting in single or multilineage cytopenias. Evaluation of regulatory pathways that are inappropriately overactive and lead to a blockade in erythroid and myeloid differentiation is an important process in determining future therapeutic targets for the treatment of MDS. The Toll-like receptor (TLR) pathways play an important role in the innate immune response and are also highly expressed on hematopoietic stem cells. Aberrant overactivation of these pathways can stimulate inflammation and cytokine production in the marrow microenvironment. Also, while the overactivation of interleukin-1 receptor-associated kinase 4 (IRAK4), tumor necrosis factor receptor associated factor 6 (TRAF6) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways has been seen in Myelodysplastic Syndrome and Acute Myeloid Leukemia (AML), the upstream signals that drive their activation are not fully identified. Results: We investigated the expression of all TLRs in a large database of MDS CD34+ marrow samples and age-matched controls. Our findings revealed that TLR6 and TLR4 were the most significantly overexpressed receptors in MDS samples (FDR<0.001). Furthermore, we observed that the TLR4 cofactor lymphocyte antigen 96 (LY96), also known as myeloid differentiation factor 2 (MD-2), was also overexpressed in MDS samples when compared to controls. Furthermore, the S100A8 and S100A9 proteins are ligands for TLR4 and were found to be positively associated with severity of anemia in the cohort of MDS samples. These data provided the rationale for determining the functional role of the TLR4 pathway in MDS/AML models. To obtain a potent and specific antibody against TLR4, we inoculated rabbits with recombinant human TLR4 and obtained high affinity antibodies that neutralized TLR4 function at low nano-molar range of doses. The TLR4 polyclonal antibody was able to inhibit lipopolysaccharide (LPS)-induced NF-κb activation in leukemic THP-1 cells. The TLR4 antibody also led to a decrease in proliferative phosphorylated extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase in leukemic cells. We then treated samples from MDS/AML patients (n=14) with the TLR4 antibody (1 nM dosing) and assessed for erythroid and myeloid growth and differentiation. Fluorescence activated cell sorting (FACS) analysis for erythroid differentiation showed increased expression of either CD71, CD235a or both after treatment with the TLR4 antibody. FACS analysis for myeloid differentiation showed increased expression of either CD11b, CD14 or both after treatment with the TLR4 antibody. This effect was more pronounced in the intermediate/high risk group than in the low-risk group of patients. Conclusion: Inhibition of the TLR4 pathway with a potent TLR4 antibody promotes erythroid and myeloid maturation and relieves the differentiation block on these lineages in MDS patient samples in the in vitro setting. Our preclinical data supports further evaluation of TLR4 inhibition as a therapeutic target in patients with MDS, as anemia and neutropenia are hallmarks of the disease.

RVU120 (SEL120) CDK8/19 Inhibitor - a Drug Candidate for the Treatment of MDS Can Induce Erythroid Differentiation

Background: Myelodysplastic syndromes (MDS) are a group of malignant blood disorders characterized by ineffective hematopoiesis and cytopenias and frequent evolution to acute myeloid leukemia (AML). MDS results from the expansion of genetically and epigenetically changed clones with impaired differentiation and maturation. Primary clinical goals in MDS are to achieve remissions, alleviate symptoms associated with cytopenias, and minimize the transfusion burden. While supportive red blood cell transfusions, erythropoiesis-stimulating agents and novel targeted agents may lead to clinical improvement, an allogeneic bone marrow transplant (BMT) remains the only potential curative option for patients with MDS. RVU120 (SEL120) is a specific, selective inhibitor of CDK8 and its paralog CDK19. A first-in-human Phase Ib clinical trial with RVU120 in patients with AML or high risk (HR)-MDS is currently ongoing. Preclinical studies indicated the strong antileukemic potential of RVU120 that was often associated with the multilineage commitment of CD34+ AML cells. Moreover, RVU120 could improve proliferation and induce erythroid differentiation of CD34+ cells derived from Diamond-Blackfan anemia (DBA) patients.Aims: Primary aim was to evaluate the erythroid differentiation potential of RVU120 in primary MDS and transformed cord blood CD34+ blood cells characterized with an early block in erythroid differentiation.Methods: Efficacy and mechanism of action of RVU120 and other CDK8 inhibitors were investigated in cord blood (CB) cells transduced with TLS-ERG, a fusion gene generated from t(16;21)(p11;q22). Transformed cells displayed an increased capacity for self-renewal, proliferation, and altered erythroid differentiation. Efficacy was further tested in CD34+ cells isolated from BM of MDS patients and established MDS cell lines. Cells were treated with RVU120 and global transcriptional changes and chromatin status were analyzed by RNA-seq, ATAC-seq, and ChIP-seq. Cell cycle, proliferation, and lineage-specific markers were studied by flow cytometry in liquid and semi-solid methylcellulose-based media.Results: RVU120 treatment leads to transcriptional reprogramming of transformed CD34+ CB cells. The most profound changes included decreased CDK8 occupancy followed by increased STAT5 and RNA Polymerase II loading at transcription start site and gene bodies. RVU120 treatment transcriptionally represses multiple genes associated with hematopoietic and leukemia stem cells such as CD34, FLI1, ENG and RGS18 and importantly induce the expression of genes involved in erythroid commitment, including regulators of erythroid/megakaryocytic fate, such as RGS18, KLF1, FLI1, INHBA, GATA1/2 and hemoglobin genes. Detailed analysis of transformed CB and MDS CD34+ cells by flow cytometry at early and late time points reflected sequential changes in the expression of lineage-specific surface markers, leading to erythroid differentiation.Conclusions: Presented results indicate strong erythroid differentiation potential of RVU120 in CD34+ cells that acquired genetic abnormalities leading to arrested erythroid commitment, characteristics of many MDS and AML subtypes. Observed differentiation phenotype strikingly resembles effects of RVU120 in DBA cells caused by disruption of genes encoding ribosomal proteins. Detailed transcriptomic profiling strongly links the differentiation with enrichment of genes representing regulators of erythroid commitment and hemoglobin metabolism. Further studies are warranted to investigate the efficacy of RVU120 in chronic anemias associated with bone marrow failures in AML and MDS patients. [Display omitted] DisclosuresPakulska: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Obacz: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Goller: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Combik: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Keska: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mazan: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Juszczynski: Ryvu Therapeutics: Current equity holder in publicly-traded company. Brzozka: Ardigen: Current Employment, Membership on an entity's Board of Directors or advisory committees; Selvita SA: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Dziedzic: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Angelosanto: Ryvu Therapeutics: Current Employment. Shamsili: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rzymski: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Selective Targeting of Splicing Factor Mutant Hematopoietic Stem and Progenitor Cells Via STAT3 Inhibition

Myelodysplastic syndrome (MDS) is a bone marrow failure disorder driven by dysfunction of hematopoietic stem and progenitor cells (HSPCs). Patient sequencing studies over the last decade have revealed that mutations in splicing machinery predominate in MDS, thus selective targeting of these cells is therapeutically attractive. STAT3 inhibition has been explored previously as a means to eradicate HSPCs in MDS. While efficacy was demonstrated in a subset of samples, the underlying mechanism for this selectivity remains unknown. We examined RNAseq of MDS CD34+ HSPCs with splicing factor mutations versus wildtype, finding alternative splicing and differential expression of STAT3 pathway components. Functionally, we explored if STAT3 signaling represents a novel vulnerability in SF3B1 mutant HSPCs using a multi-model approach of in vivo zebrafish and mouse systems, and in vitro assays of CRISPR-engineered human leukemia K562 cells and primary MDS samples. Utilizing the small molecule STAT3 inhibitor STATTIC, we found that human cells carrying MDS-associated SF3B1 point mutations had heightened sensitivity to STAT3 inhibition compared to wildtype controls. To evaluate the activity of STAT3 inhibition in vivo, we utilized an Mx1-cre conditional knock-in mouse model of mutant SF3B1 (Sf3b1+/K700E). We demonstrated that in vivo STATTIC treatment selectively depleted Sf3b1 mutant cells over wildtype in vivo. RNAseq of sf3b1 homozygous mutantzebrafish cells revealed conserved dysregulation of STAT3 pathway splicing and target expression. Diminishing Stat3 (via morpholino knockdown, stable mutants, or STATTIC treatment) decreased HSPCs in sf3b1 heterozygotes but not wildtype embryos, demonstrating synthetic lethality between Sf3b1 and Stat3. Our data indicate that SF3B1 heterozygosity, regardless of the type of mutation, confers a heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Critically, our data indicate that SF3B1-mutant cells can be selectively killed in vivo while sparing wildtype cells. We sought to rescue HSPCs in sf3b1 homozygous mutant zebrafish, however overexpression of ligands Osm and Il6 or wildtype Stat3 was insufficient. Instead, overexpression of constitutively-active Stat3 partially restored HSPCs, indicating that functional Stat3 signaling downstream of Sf3b1 is critical for HSPC formation. To investigate the specificity of the synthetic lethality for SF3B1, we assessed the STAT3 synthetic lethal interaction with other mutated splicing factors in MDS. Similar to SF3B1, we demonstrated STAT3 synthetic lethality with U2AF1 and SRSF2 heterozygosity in zebrafish and human cells. RNA-sequencing analysis of STATTIC-treated K562 cells revealed an exacerbation of splicing alterations upon STAT3 inhibition that was more pronounced in SF3B1+/K666N cells compared to wildtype. Even more strikingly, we demonstrated that constitutive activation of STAT3 could partially reverse defective splicing in zebrafish sf3b1 homozygous mutant cells. Mechanistically, these data strongly support coordinated splicing dysfunction as the underlying cause for STAT3-SF3B1 synthetic lethality. Together, we demonstrated a conserved and selective synthetic lethal interaction between STAT3 function and splicing factor defects that represents a novel liability for mutant HSPCs with important implications for MDS treatment. DisclosuresShastri: Guidepoint: Consultancy; Kymera Therapeutics: Research Funding; Onclive: Honoraria; GLC: Consultancy. Verma: Curis: Research Funding; BMS: Research Funding; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; GSK: Research Funding; Novartis: Consultancy; Acceleron: Consultancy; Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company.

Activation of SIRT1 Deacetylase As a Therapeutic Approach for Myelodysplastic Syndromes By Restoring TET2 Function

Myelodysplastic syndromes (MDS) which arise from a primitive CD34+ hematopoitic stem/progenitor cell (HSPC), are incurable by non-transplant therapy. Better understanding of MDS HSPC regulation is critical for developing targeted therapies against the fatal disease. Tet methylcytosine dioxygenase 2 (TET2), initiating DNA demethylation through converting 5-methylcytosine to 5-hydromethylcytosine (5hmC), is the most frequently mutated gene in MDS. TET2 alterations lead to DNA hypermethylation and dysregulated gene expression in HSPCs, resulting in enhanced self-renewal and skewed myeloid specific expansion. While TET2's biological function has been well studied, the knowledge of its regulation is limited. We had demonstrated the TET2 c-terminus acetylation was reversed by SIRT1 deacetylase which maintains normal HSPCs activity under stress. We also observed that SIRT1 protein in MDS CD34+CD38- was expressed below normal levels, and that knockdown (KD) of SIRT1 enhanced in vivo growth of MDS-L cells, a patient derived MDS cell line, is associated with TET2 acetylation (Blood. 2016;128:1053). These results indicate an important role for SIRT1 in MDS HSPCs maintenance.To further assess SIRT1 function in MDS, we ectopically expressed SIRT1 in primary MDS CD34+ cells enriched for HSPCs and MDS-L cells, respectively. Overexpression (OE) of SIRT1 resulted in inhibition of colony formation of MDS CD34+ cells (p<0.01, n=3) but not normal counterpart (p>0.05, n=3). SIRT1 OE also suppressed MDSL colony formation (p<0.01) and cell growth (p<0.01). SIRT1 OE significantly reduced MDS-L cell BM engraftment in NSGs mice relative to controls (human CD45+ cells: 5.7±0.8% of BM in SIRT1 OE group vs 31.4 ± 5.8% in control, p<0.01, n=7), resulting in delayed mortality of recipient mice (median survival: 52 days vs 35 days; p<0.05, n=5). Meanwhile, treatment of SIRT1 agonist SRT1720, reduced colony formation of primary MDS CD34+ cells in a dose dependent manner (p<0.01, n=9). Similar results were also achieved in SRT1720 treated MDS-L cells. Furthermore, TET2 was hyperacetylated in primary MDS cells (n=3) compared to normal counterpart (n=3). Next, we tested whether inhibition of MDS HSPCs following SIRT1 activation was related to TET2 activation via protein deacetylation. Indeed, enhanced TET2 activity marked by 5hmC was observed in MDS CD34+ cells upon SRT1720 treatment (P<0.05, n=3). TET2 KD diminished growth inhibition induced by SIRT1 OE or SRT1720, indicating a requirement of TET2 for SIRT1 function. Through mass spectrometry analysis, three conserved acetylated lysine residuals (K1472, K1473, K1478) were identified in TET2 catalytic domain (CD). Engineered lysines to glutamines (TET2 3KQ) mutant, mimicking acetylated TET2, produced lower 5hmC, and led to increased colony growth compared to MDS-L expressing TET2 WT (p<0.01). We further evaluated the effects of SRT1720 using NUP98-HOXD13 (NHD13)MDS mouse. This murine MDS is transferable and secondary recipients receiving primary NHD13 MDS cells succumb to MDS at 4 months, allowing generation of a cohort of MDS mice for preclinical studies of therapeutics against MDS. CD45.2+ BM cells from donor NHD13+ mice showed evidence of dysplasia were harvested, and transplanted into preconditioned congenic recipients expressing CD45.1. SRT1720 treatment (100mg/kg/day for 12 weeks, p.o.) resulted in partial reversal of MDS-like disease developed in recipients receiving MDS cells, including an increase in HGB, PLT, RBC and WBC (p<0.01,n=6). Importantly, significant reduction of NHD13+ CD45.2+ chimerism in LSK and other progenitors from BM were also observed (p<0.01, n=6). As expected, 5hmC level in lineage depleted cells from SRT1720 treated mice was increased (p<0.01). Secondary transplantation was performed to evaluate the effects of SRT1720 on MDS initiating cells. We observed that the mice receiving cells from SRT1720 treated donors showed reduced NHD13+ CD45.2+ cell long-term engraftment (p<0.01, n=5).In summary, we demonstrated that genetic overexpression or pharmacological activation of SIRT1 drastically inhibited MDS maintenance, and that TET2 function was required for the inhibitory effect of SIRT1. These results support the hypothesis that SIRT1 deficiency promotes MDS cell expansion through TET2 inactivation and support the rationale for exploring SIRT1 activation as a promising approach to selectively target MDS stem cells. DisclosuresNo relevant conflicts of interest to declare.

Overexpression of Histone Demethylase KDM6B in the Hematopoietic Compartment Promotes the Effect of Innate Immune Activation and Results in Myelodysplastic Syndrome (MDS)-like Phenotype in Mice

The histone demethylase KDM6B regulates transcriptional activity of genes involved in innate immune signals and controls differentiation of hematopoietic cells. Our laboratory previously reported that KDM6B is overexpressed in bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) of over 50% MDS pts (Leukemia 2013). Pharmacologic targeting of KDM6B has shown pre-clinical activity in several neoplasms such as glioma and ALL. It still remains to be elucidated whether KDM6B overexpression directly alters hematopoiesis, in order to test the hypothesis that overexpression of KDM6B contributes to aberrant epigenetic and innate immune signal in MDS and thus forms an important pathological mechanism and potential therapeutic target.Here we report an animal model of KDM6B overexpression in mouse hematopoietic compartment (Vav-KDM6B). In 20-wk old mice, KDM6B overexpression led to mild phenotype including reduced white cell count (WBC) in peripheral blood (PB) (15% p<0.05 N=47) and decreased LSK in BM (20% p<0.05 N=12). When exposed to chronic low dose Toll-like receptor ligand lipopolysaccharide (LPS) (6mg every other day, i.p. 6 wks), phenotype severity was more profound in Vav-KDM6B mice than in wildtype (WT) mice. First, hematopoietic cell numbers in both PB and BM were significantly reduced: LPS led to a 40% decrease of PB WBC (p<0.01 N=17) and a 38% decrease of BM cell (p<0.01 N=13 Fig 1A) in KDM6B mice, compared to 20% and 28% reduction respectively in WT mice treated by LPS. Second, both number and repopulating function of BM long term (LT) HSC were severely compromised: LPS led to decrease of BM LT-HSC by 52% (p<0.01 N=13) in KDM6B mice compared to a 35% decrease in WT mice. Functionally, colonies formed in methylcellulose by LSKs of LPS treated KDM6B mice decreased by 75% (p<0.01 N=13) at 2nd plating, whereas LPS led to a reduction of 60% for WT-LSK colony formation at 2nd plating. Similar effect was also observed when BM repopulating capacity was evaluated by chimerism (CD45.2:CD45.1) in competitive transplantation assay (p<0.05 N=11). Third, increase of megakaryocytic dysplasia was documented in BM of LPS treated KDM6B mice compared to WT counterparts (Fig 1B).To identify KDM6B overexpression mediated molecular signals under innate immune stimuli, we performed genome-wide transcriptome study using BM LSK of treated mice. Results reveal that over 130 genes were more profoundly activated by LPS in LSK of Vav-KDM6B than in WT LSK. Gene set enrichment assays (GSEA) indicated that major molecular pathways related to these genes include MDS-relevant signals, such as inflammatory and complement innate immune signals, MAPK pathway, as well as activated gene signature of RPS14 knockout in human BM CD34+ cell. We also performed RNA-Seq analysis in BM CD34+ HSPCs of MDS (N=26) and healthy controls (N=10). Results confirmed that a significant proportion of these genes (N>20) are overexpressed in MDS patients. Furthermore, we demonstrated in mouse BM cells that KDM6B overexpression mediates histone H3K4 and H3K27 methylation at promoters of several key innate immune genes towards transcription activation.We next evaluated effect of pharmacological KDM6B targeting using a highly specific KDM6B inhibitor (GSK-J4). At relatively low-dose (0.5mM), GSK-J4 could increase in vitro colony formation of LSKs of LPS treated KDM6B mice by 80% (p<0.05) at 2nd plating. Injection of GSK-J4 (160mg once a week, 5 weeks) resulted in 50% increase of WBM cells in LPS treated Vav-KDM6B mice (p<0.05). These results suggest that GSK-J4 can improve compromised hematopoiesis of Vav-KDM6B mice. We next treated primary BM HSPCs isolated from patients with low risk MDS (N=11) with GSK-J4. Results indicated that at 0.5mM, GSK-J4 could improve colony formation of MDS CD34+ cells (p=0.05). Flow cytometry analysis using cells from colonies indicated that GSK-J4 led to increased myeloid mark CD11b (p=0.08) and reduced apoptotic mark annexin-V (p<0.05). The same concentration of GSK-J4 did not affect colony formation of normal BM CD34+ HSPCs isolated from healthy donors (N=4).Taken together, our results indicate that overexpression of KDM6B mediates the hyperactivation of innate immune signals and has a functional impact in BM HSPCs, supporting role for KDM6B overexpression in MDS pathogenesis. Furthermore, our study also indicates that pharmacological inhibition of KDM6B may have therapeutic potential in MDS. [Display omitted] DisclosuresKhoury:Kiromics: Research Funding; Angle: Research Funding; Stemline Therapeutics: Research Funding; Pfizer: Research Funding.

Molecular-Based Score inspired on metabolic signature improves prognostic stratification for myelodysplastic syndrome

Deregulated cellular energetics is formally incorporated as an emerging hallmark of cancer, however little is known about its processes in myelodysplastic syndromes (MDS). Using transcriptomic data of CD34+ cells from 159 MDS patients and 17 healthy donors, we selected 37 genes involved in cellular energetics and interrogated about its clinical and prognostic functions. Based on the low expression of ACLY, ANPEP, and PANK1, as well as high expression of PKM and SLC25A5, we constructed our Molecular-Based Score (MBS), that efficiently discriminated patients at three risks groups: favourable risk (n = 28; 3-year overall survival (OS): 100%); intermediate (n = 60; 76% [62–93%]) and adverse (n = 71; 35% [17–61%]). Adverse MBS risk was independently associated with inferior OS (HR = 10.1 [95% CI 1.26–81]; P = 0.029) in multivariable analysis using age, gender and the revised international prognostic score system as confounders. Transcriptional signature revealed that Favourable- and intermediate-risk patients presented enriched molecular programs related to mature myeloid progenitors, cell cycle progression, and oxidative phosphorylation, indicating that this cells differs in their origin, metabolic state, and cell cycle regulation, in comparison to the adverse-risk. Our study provides the first evidence that cellular energetics is transcriptionally deregulated in MDS CD34+ cells and establishes a new useful prognostic score based on the expression of five genes.

Replication stress signaling is a therapeutic target in myelodysplastic syndromes with splicing factor mutations.

Somatic mutations in genes coding for splicing factors, e.g., SF3B1, U2AF1, SRSF2, and others are found in approximately 50% of patients with myelodysplastic syndromes (MDS). These mutations have been predicted to frequently occur early in the mutational hierarchy of the disease, therefore, making them particularly attractive potential therapeutic targets. Recent studies in cell lines engineered to carry splicing factor mutations have revealed a strong association with elevated levels of DNA:RNA intermediates (R-loops) and a dependency on proper ATR function. However, data confirming this hypothesis in a representative cohort of primary MDS patient samples have so far been missing. Using CD34+ cells isolated from MDS patients with and without splicing factor mutations as well as healthy controls we show that splicing factor mutation- associated R-loops lead to elevated levels of replication stress and ATR pathway activation. Moreover, splicing factor mutated CD34+ cells are more susceptible to pharmacological inhibition of ATR resulting in elevated levels of DNA damage, cell cycle blockade, and cell death. This can be enhanced by combination treatment with the low-dose splicing modulatory compound Pladienolide B. We further confirm the direct association between R-loops and ATR sensitivity and the presence of a splicing factor mutation using lentiviral overexpression of wild-type and mutant SRSF2 P95H in cord blood CD34+ cells. Collectively, our results from n=53 MDS patients identify replication stress and associated ATR signaling to be critical pathophysiological mechanisms in primary MDS CD34+ cells carrying splicing factor mutations, and provide a preclinical rationale for targeting ATR signaling in these patients.

Inhibition of LSD1 in Myelodysplastic Syndrome Progenitors Restores Differentiation of CD141Hi Conventional Dendritic Cells

Background: Immunotherapeutic approaches for myelodysplastic syndrome (MDS) show promise, but progress is limited by our incomplete understanding of the immunologic milieu. In a recent Phase I trial, we found that MDS patients with higher numbers of CD141Hi conventional dendritic cells (cDCs) were more likely to respond to NY-ESO-1 vaccination. In solid tumor models, the CD141Hi cDC is critical for initiating anti-tumor immune responses but its impact in myeloid malignancies is unknown. In studies of primary human specimens and mouse models, we tested the hypothesis that MDS patients exhibit decreased quantity and quality of CD141Hi cDCs due to impaired myeloid differentiation. Methods: Bone marrow (BM) cells were collected from MDS patients (pre-treatment) and age matched healthy donors (HD; defined as absence of hematologic malignancy). We quantified DC populations, stem, progenitor cells and interferon regulatory factor-8 (IRF-8) expression using flow cytometry and RT-qPCR. Histone modifications were assessed by chromatin immunoprecipitation. To assess DC differentiation of progenitors, human CD34+ and mouse c-kit+ cells were expanded and differentiated in vitro. Results: We found fewer CD141Hi cDCs (p&lt;0.0001), CD1c+ cDCs (p&lt;0.005) and plasmacytoid DCs (CD123+ pDCs; p&lt;0.005) overall in BM samples from MDS patients (n=71) compared to HD (n=17). We stratified MDS patients based on the relative number of DCs and found that only those patients with highest number of CD141Hi cDCs had superior survival (p &lt; 0.05). No differences in survival were seen in patients stratified by the CD1c+ cDCs (p = 0.96) and CD123+ pDCs (p = 0.32) populations. We hypothesized that decreased numbers of CD141Hi cDCs and adverse survival in MDS patients resulted from impaired differentiation of DC progenitors. We showed that MDS patients (n=19) have fewer monocyte-DC progenitors (MDP) and common DC progenitors (CDP) compared to HD (n=11; p&lt;0.01). We then hypothesized that MDS progenitors express lower levels of IRF8, a master regulator of CD141Hi cDC differentiation. IRF8 expression was significantly lower in CDPs from MDS patients compared to HD (p&lt;0.05). Furthermore, MDS patients with lower levels of IRF8 (n=8) in their MDPs showed a trend towards production of fewer CDPs and significantly fewer CD141Hi cDCs compared to those with higher levels of IRF8 (n=10; p&lt;0.005). These results suggest that approaches to increase IRF8 expression could enhance CD141Hi cDC differentiation. We hypothesized that inhibition of lysine-specific histone demethylase 1A (LSD1), which increases IRF8 expression in myeloid leukemia cells, would induce CD141Hi cDC differentiation. Pharmacologic inhibition of LSD1 increased IRF8 expression (both mRNA and protein) in KG-1 cells, a model of human CD34+ cells, and in HD and MDS CD34+ progenitors (p&lt;0.05). LSD1 inhibition in KG-1 cells resulted in increased H3K27 acetylation (3861-fold change) and H3K4 dimethylation (922.4-fold change) compared to PBS (p&lt;0.05) at a region -70 kb to the IRF8 transcriptional start site, a putative regulatory element that demonstrated the highest level of LSD1 binding. These data indicate that LSD1 inhibition alters histone modifications at the IRF8 locus, resulting in increased expression of IRF8. Pharmacologic inhibition of LSD1 in HD CD34+ cells increased the number of mature CD141Hi cDCs in 92% of specimens (n = 12; 3.4 fold-change) compared to PBS. Similarly, LSD1 inhibition in MDS CD34+ cells increased the number of CD141Hi cDCs (n = 12) in 75% of patient specimens (16.8 fold-change) compared to PBS. IRF8 function is conserved between mice and humans. To test whether the effect of LSD1 inhibition on cDC differentiation was dependent on IRF8, we compared the effect of LSD1 inhibition on BM c-kit+ cells from Irf8 knock-out mice (Irf8-KO) and littermate controls (WT). LSD1 inhibition in WT c-kit+ cells resulted in increased numbers of CD141Hi cDCs in vitro (p&lt;0.05). By contrast, LSD1 inhibition of Irf8-KO c-kit+ cells did not result in differentiation of CD141Hi cDCs. These data suggest that LSD1 inhibition drives CD141Hi cDCs differentiation through IRF8. Conclusion: These data reveal a previously unrecognized determinant of the immune microenvironment in MDS. The opportunity for epigenetic regulation of CD141Hi cDC differentiation in MDS offers an opportunity for intervention and a potential adjunct to immunotherapy for patients. Disclosures Sait: Celgene: Consultancy. Griffiths:Boston Scientific: Consultancy; Boston Scientific: Consultancy; Genentech, Inc.: Research Funding; Persimmune: Consultancy; Persimmune: Consultancy; Abbvie, Inc.: Consultancy; Genentech, Inc.: Research Funding; Novartis Inc.: Consultancy; Celgene, Inc: Consultancy, Research Funding; New Link Genetics: Consultancy; New Link Genetics: Consultancy; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Novartis Inc.: Consultancy; Partner Therapeutics: Consultancy; Partner Therapeutics: Consultancy; Appelis Pharmaceuticals: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Appelis Pharmaceuticals: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Celgene, Inc: Consultancy, Research Funding; Abbvie, Inc.: Consultancy, PI on a clinical trial.

Transcriptomic Analysis Implicates Necroptosis in Disease Progression and Prognosis in Myelodysplastic Syndromes

INTRODUCTION: Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis and cytopenias due to uncontrolled programmed cell death. The presence of pro-inflammatory cytokines and constitutive activation of innate immunity signals in MDS cells suggest inflammatory cell death, such as necroptosis, may be responsible for disease phenotype. There is no data evaluating the association of RIPK1, RIPK3 and MLKL with response and prognosis in MDS. METHODS: We evaluated 64 bone marrow samples from 55 patients with MDS or chronic myelomonocytic leukemia (CMML) obtained prior to (n=46) or after (n=18) therapy with hypomethylating agents (HMAs). RNA from sorted bone marrow CD34+ cells was isolated and subject to amplification and RNA-seq. Gene co-expression was evaluated using Spearman correlation. Pathway enrichment analysis was performed using gene set enrichment analysis, with the fgsea library in R. Sequencing data was obtained by use of a 81-gene targeted PCR-based next generation sequencing (NGS) platform. Previously described somatic mutations registered at the Catalogue of Somatic Mutations in Cancer (COSMIC: http://cancer.sanger.ac.uk/cosmic) were considered as potential driver mutations. RESULTS: Compared to healthy controls, MLKL (CMML vs controls: 2.09 log2FC, p=0.0013; MDS vs control: 1.89 log2FC, p=0.003), but not RIPK1 or RIPK3, were significantly upregulated in patients with MDS and CMML (Figure 1A-C). No differences in the level of expression of RIPK1, MLKL or RIPK3 were observed based on the mutation context or burden. No significant difference in RIPK1, RIPK3 or MLKL expression levels was observed based on presence of cytogenetic abnormalities (RIPK1: 0.10 log2FC, p=0.6; RIPK3: -0.39 log2FC, p=0.40; MLKL: 0.34 log2FC, p=0.30). Higher expression levels of MLKL were associated with lower hemoglobin levels at the time of diagnosis (-0.19 log2FC per 1g/dL increase of Hgb, p=0.03) (Figure 1D). Exposure to HMA therapy was associated with a trend to decreased expression of MLKL (-0.52 log2FC, p=0.08) when all post-HMA therapy samples were evaluated. Among patient matched samples, significant reduction in MLKL levels was observed after HMA therapy (-1.06 log2FC, p=0.05). The degree of reduction in expression levels was greater among non-responders (-2.89 log2FC, p=0.06) compared to responders (-0.78, log2FC, p=0.06). Expression levels of RIPK1 at the time of diagnosis predicted for shorter survival for patients with high RIPK1 levels, defined as a log expression higher than median mRNA expression values, (median OS 10.7 vs 24.2 months, HR 1.92, 95% CI 1.00-3.67, p=0.049 by Cox proportional hazards) (Figure 1E). A multivariate analysis for overall survival using both IPSS-R risk and RIPK1 expression levels demonstrated that high RIPK1 expression was an independent adverse prognostic factor in MDS patients (HR 6.83, 95% CI 1.74-26.8, p=0.006). A total of 359 genes were significantly correlated with RIPK1 levels in MDS CD34+ cells (Spearman's method q&lt;0.2). Among these, 21 genes were positively correlated with RIPK1 expression, while 17 genes were negative correlated (Spearman's correlation, q&lt;0.1). Gene set enrichment analysis identified 373 gene sets that significantly correlated with RIPK1 expression. Upregulated genes were associated with molecular signals associated with innate immunity and inflammatory signaling including TNF-α signaling via NFkB, NFkB signaling, the IFN-g pathway, and RIG-I-like receptor (RLRs) signaling genes (Figure 1F). CONCLUSIONS: This data provides further support for a role of necroptosis in MDS, and potentially response to HMAs and prognosis. This data also indicates that RIPK1/RIPK3/MLKL are potential therapeutic targets in MDS. Figure 1 Disclosures Sasaki: Otsuka: Honoraria; Pfizer: Consultancy. Bueso-Ramos:Incyte: Consultancy. Kantarjian:Cyclacel: Research Funding; Jazz Pharma: Research Funding; AbbVie: Honoraria, Research Funding; Astex: Research Funding; Amgen: Honoraria, Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Research Funding; Ariad: Research Funding; Agios: Honoraria, Research Funding; Takeda: Honoraria. Garcia-Manero:Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding.

Oxidized Mitochondrial DNA Is a Catalyst and Biomarker of Pyroptotic Cell Death in Myelodysplastic Syndromes

Constitutive innate immune activation is a pathogenetic driver of Myelodysplastic Syndromes (MDS) that directs ineffective hematopoiesis by NLRP3 inflammasome (IFM) assembly and pyroptotic cell death. IFM activation involves recruitment of caspase-1 (casp1) through the adapter protein, ASC, to facilitate autocatalytic cleavage of the zymogen to its active form that is responsible for interleukin (IL)-1β maturation, membrane pore formation and pyroptosis. Oxidized mitochondrial DNA (ox-mtDNA) has been proposed to serve as an alarmin that can activate the IFM by interaction directly with NLRP3 or engagement by DNA sensors, Toll-like receptor 9 (TLR9) and Cyclic GMP-AMP synthase (cGAS). Upon cytolysis, ox-mtDNA is released, permitting interaction with pattern recognition receptors on neighboring cells (Grishman, Pediatric Research, 2012, Shimada, 2012, Immunity. Vollmer, 2004, Immunology). Here, we investigate ox-mtDNA as an IFM-activator and pyroptotic biomarker in MDS.Incubation of TLR9 expressing cell lines, SKM1 (high expresser) and U937 (moderate expresser) with 50ng/mL ox-mtDNA (ND1 gene, amplified with oxidized guanosine) induced IFM activation evidenced by increased p-NFkβ, casp1 and IL-1β cleavage, ASC oligomerization and liberation of ASC specks. Direct interaction of ox-mtDNA with NLRP3 was confirmed by NLRP3 immunoprecipitation followed by probing for mtDNA using ND1 and CYTB specific primers and GAPDH primers as negative genomic control; mtDNA oxidation status was confirmed by dot blot. Furthermore, significantly increased expression of interferon stimulated genes (ISG) was seen in MDS bone marrow (BM) specimens (p≤0.01) compared to normal donors indicating TLR9 and/or cGAS activation. Ox-mtDNA engagement of TLR9 and cGAS was confirmed in MDS specimens by IF colocalization with corresponding IFM activation, as well as in MDS somatic gene mutation murine models (Tet2, SRSF2, U2AF) vs. Wt controls. Evaluation of surface TLR9 by flow cytometry showed significantly increased membrane expression in MDS CD34+ BMMC (n=4) vs. healthy donors (n=13) (p<0.05), suggesting priming for enhanced sensitivity to ox-mtDNA. Concentrations of ox-mtDNA in supernatants from THP1 (N=5, p=0.0547), U937 (N=3, p=0.0637), and SKM1 (N=3, p=0.028) cells confirmed extracellular release after LPS/ATP/Nigericin induced pyroptosis. We then investigated ox-mtDNA concentrations in peripheral blood plasma from MDS patients by ELISA. Glucose adjusted, ox-mtDNA levels were significantly increased in 177 MDS cases compared to 29 healthy donors (p<0.0001), which was validated in an independent cohort of 154 MDS cases and 34 healthy donors (p<0.0001). Ox-mtDNA was higher in lower risk (LR) compared to higher risk MDS (p<0.0001) consistent with greater pyroptotic cell fraction in LR-MDS. Further, we found a direct correlation between ox-mtDNA (n=134) and known MDS IFM-activating alarmins S100A9 (r=0.395), S100A8 (r=0.618), and percentage of ASC specks (r=0.589) (p<0.0001 for all), demonstrating specificity of ox-mtDNA as a biomarker for the magnitude of medullary pyroptosis in MDS. Importantly, compared to other hematologic malignancies, we found that plasma ox-mtDNA was significantly increased in LR-MDS (n=162) compared to ALL (n=7), de novo (n=20) and secondary AML (n=18), CML (n=8), CMML (n=18), LGL (n=19), MF (n=12), ET (n=20), PV (n=20), MM (n=18), and type 2 diabetes (n=25) where IFM activation promotes insulin resistance (p ≤0.036). There was no significant difference in oxDNA concentrations between MDS and CLL (n=50), which is reported to have high ox-mtDNA levels associated with genomic abnormalities (Collado, 2014, Biomed Res Int.). ROC/AUC analysis demonstrated that ox-mtDNA is an MDS pyroptosis-specific biomarker when compared to healthy donors (AUC=0.929). Similarly, a 5-fold cross-validation (k=5) repeated 30 times to compare MDS cases to non-MDS hematological malignancies (excluding CLL), similarly confirmed biomarker specificity (AUC=0.877).Collectively, these data indicate that ox-mtDNA both directly engages NLRP3 and the DNA sensors TLR9/cGAS to induce IFM activation and pyroptosis, creating a feed forward inflammatory cascade that extends to neighboring cells. Ox-mtDNA may serve as a biomarker and companion diagnostic for pyroptosis execution in MDS. DisclosuresList:Celgene: Research Funding.

Antisense STAT3 inhibitor decreases viability of myelodysplastic and leukemic stem cells.

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Transcriptomic analysis of stem and progenitor populations in MDS and AML demonstrated overexpression of STAT3 that was validated in an independent cohort. STAT3 overexpression was predictive of a shorter survival and worse clinical features in a large MDS cohort. High STAT3 expression signature in MDS CD34+ cells was similar to known preleukemic gene signatures. Functionally, STAT3 inhibition by a clinical, antisense oligonucleotide, AZD9150, led to reduced viability and increased apoptosis in leukemic cell lines. AZD9150 was rapidly incorporated by primary MDS/AML stem and progenitor cells and led to increased hematopoietic differentiation. STAT3 knockdown also impaired leukemic growth in vivo and led to decreased expression of MCL1 and other oncogenic genes in malignant cells. These studies demonstrate that STAT3 is an adverse prognostic factor in MDS/AML and provide a preclinical rationale for studies using AZD9150 in these diseases.

A Phase II Study Evaluating the Combination of Nivolumab (Nivo) or Ipilimumab (Ipi) with Azacitidine in Pts with Previously Treated or Untreated Myelodysplastic Syndromes (MDS)

A Phase II Study Evaluating the Combination of Nivolumab (Nivo) or Ipilimumab (Ipi) with Azacitidine in Pts with Previously Treated or Untreated Myelodysplastic Syndromes (MDS)

Down-regulation of EZH2 expression in myelodysplastic syndromes

EZH2 genetic mutations are common in myelodysplastic syndrome (MDS), which implies that this gene has a pathophysiological role in the disease. To further characterize molecular alterations of EZH2, and their potential prognostic impact in MDS, we assessed EZH2 RNA expression in primary bone marrow CD34+ cells from 78 patients.We found that 47% of patients have reduced EZH2 expression compared to normal controls. Further analyses revealed that EZH2 is significantly underexpressed in patients bearing chromosome 7 or 7q deletions (7-alt) when compared to controls, diploid patients, and patients with other cytogenetic alterations (p<0.05). In survival analysis, we found a non-significant trend toward overall survival (OS) being better among patients with EZH2 underexpression (median OS 55 vs. 36 months; p=0.71). Importantly, this trend became significant when the analysis was restricted to the subset of cases without alterations in chromosome 7 (62 vs. 36 months; p=0.033).Furthermore, our previous work has identified a spectrum of innate immune genes in MDS CD34+ cells that are deregulated via abnormal promoter histone methylation. Because EZH2 is a key regulator of histone methylation, we assessed the relationship between deregulation of these genes and EZH2 underexpression. We observed that the mRNA levels of 11 immune genes were higher in the EZH2 underexpression group and that immune gene expression was significantly higher in patients with concomitant EZH2 underexpression and KDM6B (also known as JMJD3, an H3K27 demethylase) overexpression.Taken together, these data indicate that EZH2 underexpression may have unique impact on the molecular pathogenesis and prognosis in MDS and be an important marker for patients without chromosome 7 alteration.

Apoptosis in human myelodysplastic syndrome CD34+ cells is modulated by the upregulation of TLRs and histone H4 acetylation via a β-arrestin 1 dependent mechanism

Excessive apoptosis of hematopoietic precursors in the bone marrow underlies the ineffective hematopoiesis characteristic of myelodysplastic syndrome (MDS). Toll-like receptor (TLR) signaling is abnormally activated in MDS and may be involved in excessive programmed cell death in the pathogenesis of MDS. TLRs expression and global histone H3/H4 acetylation were analyzed in bone marrow (BM) CD34+ cells from 20 lower-risk and 20 higher-risk MDS patients and 10 healthy controls. Apoptosis of BM CD34+ cells was examined by flow cytometry, and its correlation to histone acetylation and the expression of TLR2 and β-arrestin1 (β-arr1), measured by enzyme-linkedimmunosorbent assay and qRT-PCR, was assessed. TLR1, TLR2 and TLR6 expression and H4 acetylation levels were higher in lower-risk MDS patients than in higher-risk MDS patients or controls, and TLR2 expression and H4 acetylation levels were positively correlated with an increased rate of apoptosis. Lower-risk MDS was associated with increased β-arr1 expression and histone acetyltransferase p300 activity. In in vitro-cultured primary normal and lower-risk MDS CD34+ cells, TLR2 activation-induced apoptosis was mediated by the upregulation of β-arr1 leading to the recruitment of p300 and increased histone H4 acetylation. The nuclear accumulation of βarr1 following TLR2 activation promote H4 acetylation at specific target gene promoters and may thus affect transcription of target genes in BM CD34+ cells. The mechanisms underlying the deregulation of TLR2 and increased apoptosis in MDS may involve the β-arr1 mediated recruitment of p300 leading to increased levels of histone H4 acetylation.

Phase 2 Study of Monotherapy Galunisertib (LY2157299 Monohydrate) in Very Low-, Low-, and Intermediate-Risk Patients with Myelodysplastic Syndromes

Phase 2 Study of Monotherapy Galunisertib (LY2157299 Monohydrate) in Very Low-, Low-, and Intermediate-Risk Patients with Myelodysplastic Syndromes

Mechanisms of Resistance to 5-Azacytidine/Decitabine in MDS-AML and Pre-Clinical In Vivo Proof of Principle of Rational Solutions to Extend Response

Only 5-azacytidine (5Aza) and decitabine (Dec), both DNA methyltransferase (DNMT1) depleting drugs, are FDA-approved to treat all myelodysplastic syndrome (MDS) subtypes. Neither agent is curative. What are the mechanisms of resistance? MDS is genetically heterogeneous, thus, this question has been approached by attempting to correlate MDS genetics with response, or by examining genetics at relapse. Unfortunately, results have been contradictory/inconclusive. Another approach is to consider that achievement of the intended molecular pharmacodynamic (PD) effect of DNMT1-depletion is a minimum requirement for response, superceding other biological considerations including genetics/susceptibility to apoptosis or differentiation. As such, a logical first-step is to examine whether relapse is driven by sub-clones thriving despite DNMT1-depletion, or from failure to deplete DNMT1 in the first place. In both murine and clinical studies, relapse was from failure to deplete DNMT1 (Leukemia 2011;25(11):1739-50; Oncotarget 2012;3(10):1137-45; J Clin Invest 2015;125(3):1043-55). Thus, the question can be reframed as: Why is molecular PD not achieved, and can mechanisms be identified to extend response?Achievement of molecular PD by 5Aza/Dec depends on intra-cellular exposure/half-life (t½). Intra-cellular t½ depends in turn on extra-cellular exposure time (plasma t½) and pyrimidine metabolism enzymes that phosphorylate 5Aza/Dec, trapping these drugs in cells. There is controversy regarding the identify of the enzymes that phosphorylate and trap 5Aza: UCK1, UCK2 or both. To answer this question, we correlated UCK1, UCK2 and DCK expression with sensitivity of NCI60 cancer cell lines (n=60) to 5Aza or Dec. 5Aza sensitivity correlated strongly with UCK2 expression but not UCK1 or DCK (corr. coeff.: 5AzaGI50 vs UCK2-0.28, p=0.03; vs UCK1 0.22, p=0.1; vs DCK -0.07, p=0.5). Dec sensitivity correlated with DCK expression but not UCK2 or UCK1 (corr. coeff.: DecGI50 versus DCK-0.27, p=0.04; vs UCK1-0.01, p=0.9; vs UCK2-0.19, p=0.2). There was greater than 2-fold variation of UCK2/DCK expression in primary MDS CD34+ bone marrow cells, not linked with WHO-subtypes or genetics, suggesting flexible use of one or the other enzyme for pyrimidine salvage, although a trend for higher DCK expression was noted in MDS containing mutated TP53 (GSE58831). Does treatment select for sub-clones utilizing salvage enzymes that avoid drug? DCK and UCK2 expression was serially measured by QRT-PCR in bone marrow from MDS patients treated with a Dec regimen rationalized for non-cytotoxic DNMT1-depletion (0.1-0.2 mg/kg SC 1-3X/week, n=12) or with conventional 5Aza (n=6). UCK2 consistently increased (up to 100-fold) and DCK consistently decreased at relapse on Dec, with vice-versa for 5Aza (Fig. 1).Mechanism-based solutions to such resistance were evaluated in xenotransplant models of human MDS-AML that phenocopy human disease with bone marrow infiltration and death by cytopenia. Candidate solutions investigated were (i) Dec + thymidine, or HU, or methotrexate, to increase DCK (by inhibition of ribonucleotide reductase [RNR]); (ii) 5Aza or Dec at lower dose to decrease Cmax and avoid cytotoxicity + tetrahydrouridine (THU) to increase plasma t½/Tmax for DNMT1-depletion (THU inhibits the enzyme cytidine deaminase that rapidly inactivates 5Aza and Dec in vivo); (iii) 5Aza alternating or combined with Dec, since 5Aza-mediated increases in DCK are expected to increase sensitivity to Dec, while Dec-mediated increases in UCK2 are expected to increase sensitivity to 5Aza; Of these interventions, reduced dosage 5Aza or Dec + THU, and alternating THU-5Aza with THU-Dec, but not simultaneous THU-5Aza+THU-Dec, nor the RNR inhibitors, increased response and survival to striking extents (approaching 1 year)(Fig. 2). Bone marrow gH2AX and DNMT1-expression by flow-cytometry and preservation of murine hematopoiesis confirmed the non-cytotoxic, epigenetic mechanism of action (Fig. 2).We show that straightforward shifts in pyrimidine metabolism demonstrably account for much of the resistance to 5Aza or Dec. Fortunately, such resistance is amenable to logical, non-toxic, clinically applicable solutions, validated in pre-clinical in vivo models. Clinical trials of non-cytotoxic THU-Dec (IND#112914) and alternating THU-Dec/THU-5Aza, incorporating companion PD/metabolism biomarkers, are planned. [Display omitted] DisclosuresSekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Saunthararajah:Not applicable: Other: patent applications around decitabine, 5-azacytidine and tetrahydrouridine. Off Label Use: decitabine used by route of administration and dosages other than FDA approved regimens.

IL8-CXCR2 pathway inhibition as a therapeutic strategy against MDS and AML stem cells

AbstractAcute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Novel therapeutic targets against preleukemic stem cells need to be identified for potentially curative strategies. We conducted parallel transcriptional analysis of highly fractionated stem and progenitor populations in MDS, AML, and control samples and found interleukin 8 (IL8) to be consistently overexpressed in patient samples. The receptor for IL8, CXCR2, was also significantly increased in MDS CD34+ cells from a large clinical cohort and was predictive of increased transfusion dependence. High CXCR2 expression was also an adverse prognostic factor in The Cancer Genome Atlas AML cohort, further pointing to the critical role of the IL8-CXCR2 axis in AML/MDS. Functionally, CXCR2 inhibition by knockdown and pharmacologic approaches led to a significant reduction in proliferation in several leukemic cell lines and primary MDS/AML samples via induction of G0/G1 cell cycle arrest. Importantly, inhibition of CXCR2 selectively inhibited immature hematopoietic stem cells from MDS/AML samples without an effect on healthy controls. CXCR2 knockdown also impaired leukemic growth in vivo. Together, these studies demonstrate that the IL8 receptor CXCR2 is an adverse prognostic factor in MDS/AML and is a potential therapeutic target against immature leukemic stem cell–enriched cell fractions in MDS and AML.

Transcriptomic Evaluation of CD34+ Marrow Cells from Myelodysplastic Syndrome (MDS) Patients

Background: Prior studies using microarray platforms have shown alterations of gene expression profiles (GEPs) in MDS CD34+ marrow cells related to clinical outcomes (Sridhar et al, Blood 2009, Pellagatti et al, JCO 2013). Given the increased sensitivity and accuracy of high-throughput RNA sequencing (RNA-Seq) (Mortazavi et al, Nat Meth 2008, Soon et al, Mol Syst Bio 2012) for detecting and quantifying mRNA transcripts, we applied this methodology for evaluating differential gene expression between MDS and normal CD34+ marrow cells.Methods:RNA was isolated from magnetic bead affinity-enriched CD34+ (>90%) marrow aspirate cells (Miltenyi Biotec, Auburn, CA) and amplified using the Smarter Kit (Clontech, Mt View, CA). The amplified product (ds DNA) was fragmented to a size distribution of ~200-300bp using the E220 Focused Ultrasonicator (Covaris Inc, Woburn, MA). End repair, adapter ligation and PCR amplification were performed using the NEBNext Ultra RNA library prep kit for Illumina (New England Biolabs, Ipswich, MA). The indexed cDNA libraries were sequenced (paired end, 100bp) on an Illumina HiSeq2000 platform with median read counts of 69 million. The sequences were aligned to Human Reference sequence hg19 using DNAnexus mapper with gene detection focused on known annotated genes. The differential expression was analyzed using edgeR. DAVID and Ingenuity IPA programs were used for pathway analyses. Gene Set Enrichment Analysis (GSEA) was used to identify biologic processes in our dataset present across phenotypes.Results: Correlations of RNA-Seq data from unamplified to amplified transcripts demonstrated high fidelity of transcripts obtained (Pearson and Spearman R2 = 0.80). After filtering samples for adequate read counts, 12,323 genes were evaluated. Differential expression analysis yielded 719 differentially expressed genes (DEGs) in MDS (n=30) vs normal (n=21) with FDR <.05. Among the DEGs, 548 and 171 were over- and under-expressed ≥2 fold in MDS vs Normal, respectively: 20% of the overexpressed genes were present in >50% of the patients. Hierarchical cluster analysis using these DEGs confirmed clear separation of MDS patients from normals, with 2 differential expression clusters—one region overexpressed and one underexpressed. A distinctive trend toward clustering of the patients was seen which related to their IPSS categories and marrow blast %. In functional pathway analysis of the 2 distinctive gene clusters which distinguished MDS from normal, the underexpressed MDS DEGs demonstrated enrichment of inflammatory cytokines, oxidative stress and interleukin signaling pathways, plus mitochondrial calcium transport; whereas the MDS overexpressed DEG cluster showed enrichment of adherens junction/cytokeletal remodeling, cell cycle control of chromosome replication and DNA damage response pathways. Using GSEA analysis, significantly increased numbers of genes in MDS vs normal, common to those in gene sets present within curated public databases, were involved with TP53 targets and mTOR signaling pathways.Conclusions: Our study demonstrated that RNA-Seq methodology, a high-throughput and more comprehensive technique than most gene expression microarrays, was capable of showing significant and distinctive differences in gene expression between MDS and normal marrow CD34+ cells. Specific clustering of the DEGs was demonstrated to distinguish patient subsets associated with their major clinical features. Further, the stringently identified DEGs shown to be engaged in functional pathways and biologic processes highly relevant for MDS were extant within the patients’ CD34+ cells. These transcriptomic data provide information complementary to exomic mutational findings contributing to improved understanding of biologic mechanisms underlying MDS. DisclosuresNo relevant conflicts of interest to declare.

CD99 Is a Therapeutic Target on Disease Initiating Stem Cells in Acute Myeloid Leukemia and the Myelodysplastic Syndromes

Acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS) are initiated and sustained by self-renewing malignant stem cells. Any curative therapy for AML or MDS must eliminate leukemic stem cells (LSCs) or MDS hematopoietic stem cells (HSCs), respectively, as these are likely the only self-renewing disease cells. Through transcriptional profiling and flow cytometric validation, we previously identified CD99 as a cell surface marker highly expressed on MDS HSCs (85%, n=63). Assessment of 78 paired diagnosis/relapse AML specimens revealed that CD99 is also frequently overexpressed in AML at diagnosis (86%) and relapse (92%). Consistent with CD99’s potential as an LSC marker, we showed that the LSC enriched CD34+CD38- fraction of AML consistently expressed higher levels of CD99 compared with more differentiated blasts (p&lt;0.0001). We also found that CD99 expression was higher on leukemic blasts at relapse as compared with diagnosis (p=0.007). To test whether CD99 expression enriches for functional LSCs, we transplanted the top and bottom 10% of CD99 expressing blasts within the LSC enriched CD34+CD38-CD90-CD45RA+ fraction of a CD34-positive primary AML specimen into sublethally irradiated NOD/SCID/IL-2Rgc-null (NSG) mice, observing that leukemia-initiating cell (L-IC) activity was restricted to the top 10% of CD99 expressors (L-IC frequency 1 in 24,401). Similar results were obtained utilizing a CD34-negative AML (L-IC frequency 1 in 105,688 in the top 10% of CD99 expressors), suggesting that CD99 can enrich for functional LSCs independent of CD34-expression status. CD99 is the first LSC marker reported to selectively enrich for LSCs, as we found other previously identified LSC markers (CD47, TIM3, CD44, and CD123) to be expressed at equal or decreased levels on LSC-enriched fractions compared to more differentiated blasts. Thus, high CD99 expression provides a novel method to purify LSCs, allowing for more refined studies of this critical cell type. As therapeutically targeting CD99 with mAbs has been proposed in Ewing’s sarcoma and B cell acute lymphoblastic leukemia, we tested the ability of anti-CD99 mAbs to induce cell death in AML and MDS. Anti-CD99 mAbs (clones H036-1.1 and 12E7) were cytotoxic to primary AML blasts (n=5) and MDS CD34+ cells (n=2), as well as 17 myeloid leukemia cell lines in vitro. Normal HSCs and human umbilical vein endothelial cells express low and intermediate levels of CD99, respectively, but anti-CD99 mAbs were minimally toxic to these normal cell types, indicating a potentially large therapeutic window. Ex vivo incubation of primary AML blasts previously demonstrated to engraft NSG mice with H036-1.1 for 45 minutes prior to xenotransplantation completely abolished their engraftment. To rule out an effect on initial homing and lodging of LSCs, and to determine whether anti-CD99 mAbs can eradicate LSCs in vivo, NSG mice were transplanted with primary AML cells, allowed to engraft for two weeks, and then treated with 15 μg H036-1.1 or isotype control. One treatment with H036-1.1 was sufficient to completely abolish engraftment as measured in the BM five months post-treatment. To determine the mechanism of anti-CD99 mAb induced cell death, we assessed the effect of anti-CD99 mAbs on Src-family kinase (SFK) activation. We demonstrated that CD99 negatively regulates SFK activation in AML, and that cytotoxic anti-CD99 mAbs induce rapid and robust SFK activation. To determine whether SFK activation is sufficient to induce cell death, we generated a constitutively active c-Src mutant (Y530F) and overexpressed it in anti-CD99 mAb “sensitive” and “resistant” cell lines (MOLM13 and K562, respectively). While MOLM13 cells showed a marked decrease in cell growth, increased apoptosis, and G0 cell cycle arrest, K562 cells demonstrated only a modest decrease in growth and S-phase entry, with no G0 arrest or increase in apoptosis. We thus propose that anti-CD99 mAbs induce apoptosis by inducing dysregulated SFK activation and oncogenic stress. Taken together, our studies establish CD99 as a novel stem cell marker in AML and MDS, and as a promising therapeutic target in two diseases in need of novel therapies with validated activity against disease-initiating cells. Cytotoxic anti-CD99 mAbs appear to exert their effects by activating SFKs, revealing a novel therapeutic vulnerability in AML and MDS that has potential to be exploited using other modulators of this pathway. Disclosures No relevant conflicts of interest to declare.