Myelodysplastic Syndromes Hematopoietic Stem Cells Research Articles

Hematopoietic stem cells with granulo-monocytic differentiation state overcome venetoclax sensitivity in patients with myelodysplastic syndromes

The molecular mechanisms of venetoclax-based therapy failure in patients with acute myeloid leukemia were recently clarified, but the mechanisms by which patients with myelodysplastic syndromes (MDS) acquire secondary resistance to venetoclax after an initial response remain to be elucidated. Here, we show an expansion of MDS hematopoietic stem cells (HSCs) with a granulo-monocytic-biased transcriptional differentiation state in MDS patients who initially responded to venetoclax but eventually relapsed. While MDS HSCs in an undifferentiated cellular state are sensitive to venetoclax treatment, differentiation towards a granulo-monocytic-biased transcriptional state, through the acquisition or expansion of clones with STAG2 or RUNX1 mutations, affects HSCs’ survival dependence from BCL2-mediated anti-apoptotic pathways to TNFα-induced pro-survival NF-κB signaling and drives resistance to venetoclax-mediated cytotoxicity. Our findings reveal how hematopoietic stem and progenitor cell (HSPC) can eventually overcome therapy-induced depletion and underscore the importance of using close molecular monitoring to prevent HSPC hierarchical change in MDS patients enrolled in clinical trials of venetoclax.

Myelodysplastic Syndrome Hematopoietic Stem Cells Can Undergo Differentiation Trajectory Rewiring to Escape Venetoclax Vulnerability

Myelodysplastic syndromes (MDS) are incurable neoplasms that arise from early hematopoietic stem cells (HSCs). The standard of care for MDS is hypomethylating agent (HMA) therapy. However, the disease invariably fails these agents, resulting in fatal cytopenias or progression to acute myeloid leukemia (AML) with a dismal prognosis. We previously showed that the cellular hierarchy of MDS HSCs predicts the mechanisms of progression after HMA failure and can guide the design or choice of specific therapeutic approaches targeting these cells (Ganan-Gomez et al., Nature Medicine 2022). MDS HSCs in one of two differentiation states, long-term HSCs or lymphoid-primed multipotent progenitors, have a “common myeloid progenitor (CMP) pattern” or “granulocytic-monocytic progenitor (GMP) pattern” of differentiation. HSCs in these 2 differentiation states persist throughout the disease course and, driven by the recurrent activation of BCL2- or NF-κB-mediated survival pathways, respectively, expand at disease progression. In our preclinical studies, pharmacologically inhibiting these pathways depleted MDS HSCs and reduced tumor burden, which suggests that only patients with “CMP pattern” MDS may benefit from venetoclax-based therapies. Accordingly, patients with “CMP pattern” MDS, whose disease progressed after frontline HMA failure, enrolled in clinical trials of venetoclax-based therapy (NCT04160052, NCT04550442, or NCT04655755; n=28: 12 “CMP pattern” MDS and 16 “GMP pattern” MDS) had a shorter cumulative time to complete remission (P=0.012) and a longer relapse-free survival duration (14.0 months vs 5.2 months) than those with “GMP pattern” MDS did. Consistent with the hypothesis that venetoclax-based therapy selectively targets different HSC populations after HMA therapy failure, bone marrow (BM) HSC frequencies were significantly decreased at the time of remission after venetoclax-based therapy in “CMP pattern” MDS patients, but not in those with “GMP pattern” MDS. These results explain why “GMP pattern” MDS are resistant to venetoclax therapy. However, 8 of the 12 patients with “CMP pattern” MDS relapsed during venetoclax treatment and/or progressed to AML after an initial remission. In 2 patients with “CMP pattern” MDS, sequential flow cytometry and next generation sequencing analyses showed that the HSC hierarchy switched to “GMP pattern” MDS before venetoclax failure. This immunophenotypic hierarchical change was associated with the detection or expansion of STAG2- or RUNX1-mutant clones with isolated trisomy 8. To evaluate whether the immunophenotypic switch resulted from the distinct differentiation potential of early HSCs or from the aberrant expression of cell surface markers, we performed sequential single-cell RNA sequencing analyses of BM mononuclear cells isolated from the 2 “CMP pattern” MDS patients whose disease changed hierarchy before venetoclax failure. Our analyses revealed that after undergoing significant depletion during disease remission, HSCs expanded at therapy failure. Differential expression analysis showed that the expansion of STAG2- or RUNX1-mutant clones in the HSC compartment not only rewired MDS HSCs' differentiation state towards a granulo-monocytic-biased trajectory but also changed HSCs' survival dependency from BCL2-mediated anti-apoptotic pathways to TNFa-induced pro-survival NF-κB signaling, thus enabling HSCs to evade the cytotoxic effects of venetoclax (Fig. 1). The anti-apoptotic gene MCL1, a direct transcriptional target of NF-κB, was also significantly upregulated in HSCs at the time of progression, which suggests that the combination of venetoclax with an MCL1 inhibitor can overcome secondary failure to venetoclax. Together, these results reveal a novel mechanism of venetoclax resistance. HSCs under venetoclax therapy undergo survival pressure, which results in the expansion or acquisition of clones carrying specific mutations that change HSCs' survival dependencies (Fig. 2). Our study suggests that MDS patients receiving venetoclax-based therapy should be monitored at the molecular level and enrolled in clinical trials of other targeted agents before their disease undergoes clonal evolution and HSC hierarchical rewiring.

Mutant Natural Killer Cell Dysfunction Enables the Immune Escape of Premalignant MDS Cell Clones

Myelodysplastic syndromes (MDS) cannot be cured by standard pharmacological therapies owing to their inability to eradicate aberrant hematopoietic stem cells (HSCs). Given that developing early intervention strategies for MDS patients requires an improved understanding of the mechanisms underlying aberrant HSC expansion, we sought to elucidate how MDS HSCs evade immune surveillance and expand in patients with clonal cytopenias of undetermined significance (CCUS), the pre-malignant stage of MDS. To characterize transcriptomic changes induced by CCUS in the aged immune microenvironment, we used single-cell RNA sequencing (scRNA-seq) of bone marrow (BM) mononuclear cells (MNCs) isolated from 2 young healthy donors (yHDs), 2 elderly healthy donors (eHDs), and 3 CCUS patients harboring mutations in MDS driver genes such as DNMT3A or TET2. CCUS immune cells were enriched in CD8 + T effector and NK cells, which were in activated states, characterized by the significant (P adj<0.05) upregulation of genes involved in protein translation, mitochondrial metabolism, and response to interferon signaling. A computational interactome analysis of ligand-receptor pair expression predicted gained intercellular communications among myeloid cells and CD8 + T and NK cells in CCUS patients compared with yHDs and eHDs, consistent with a tumor recognition process. However, CCUS CD8 + T and NK cells had downregulated NF-kB-mediated inflammatory signaling genes, which suggested a loss of proinflammatory capacity. Because an efficient antitumor CD8 + T-cell response requires functional NK cells, we further dissected their functional state by performing scRNA-seq of CD3 + and/or CD56 + BM cells isolated from 2 yHDs, 2 eHDs, and 3 CCUS patients. A trajectory reconstruction analysis showed that CCUS NK cells were in more advanced differentiation states than HD NK cells were, which was confirmed by the significant downregulation of the transcription factor FOXO1 (P adj=1.17 x 10 -81). Differential gene expression analyses revealed that CCUS NK cells had significantly (P adj<0.05) upregulated MYC and mTOR target genes and genes involved in oxidative metabolism, protein synthesis, and immune signaling (consistent with an NK cell proliferative burst) compared with eHD NK cells, which suggests that CCUS NK cells are hyperactivated. However, cytokine secretion assays with K562 cells revealed that NK cells from 5 CCUS patients secreted significantly (P=0.002) less interferon gamma than those from 6 eHDs did. CCUS NK cells and eHD NK cells produced similar levels of granzyme and perforin, and CCUS NK cells could degranulate (based on CD107a expression); however, CCUS NK cells had significantly lower CD16 expression (P=0.008), which suggests an impaired capacity to form stable immune synapses and degranulate in a polarized manner. Indeed, compared with those from 3 yHDs and 4 eHDs, NK cells from 3 CCUS patients had a significantly (P<0.0001) impaired cytolytic capacity against leukemic cells that could not be rescued by prolonged culture with feeder cells. Moreover, NK cells from 4 CCUS patients failed to kill both heterotypic and isotypic CCUS CD34 + cells ex vivo (Fig. 1). To investigate the molecular mechanisms of NK cell dysfunction, we performed joint targeted single-cell DNA sequencing and immunophenotypic analyses of MNCs from 5 CCUS patients with DNMT3A or TET2 mutations, which revealed that NK cells were enriched in aberrant clones and had mutational burdens similar to those of myelomonocytes (Fig. 2). Deletion of DNMT3A or TET2 by CRISPR/Cas9 in cord blood-derived NK cells and subsequent killing assays showed a significant (P<0.0001) progressive loss of cytolytic capacity with tumor re-challenging in knock-out NK cells, which demonstrated that loss of function mutations in those genes are a direct cause of NK cell dysfunction in the context of sustained immune activation. This study demonstrates that MDS driver mutations in CCUS NK cells induce immune dysfunction, thus enabling the expansion of malignant cells and thereby contributing to MDS onset. Our results suggest that immunotherapy with adoptive NK cell transfer could restore the anti-tumor immune response in patients with CCUS and may be an effective approach to arrest disease progression and prevent MDS onset.

Abstract A23: Less is more: Ultra-low input sequencing allows for evaluation of stem cells as a predictor of post-transplant relapse in the myelodysplastic syndromes

Abstract Malignant hematopoietic stem cells (HSCs) initiate the myelodysplastic syndromes (MDS) and drive treatment failure through their resistance to all non-transplant therapies. As a result, allogeneic stem cell transplantation (allo-SCT) is the only curative therapy for MDS. However, relapse remains the leading cause of treatment failure after allo-SCT. Improved tools to predict relapse in this setting are necessary to allow for pre-emptive therapeutic interventions, which may be more effective when the burden of disease is still low. Given prior evidence that HSCs serve as the functional unit of resistance to non-transplant therapies, we asked whether relapse after transplant originates from MDS HSCs, and whether their persistence can predict relapse. To overcome the limiting number of HSCs available in clinical bone marrow (BM) samples, we adapted a workflow developed for assessment of cell-free DNA and utilized a hybridization capture-based targeted sequencing panel covering 260 myeloid malignancy-associated genes. Unique dual index primers (UDIs) were utilized to tag input DNA, improving PCR duplicate identification, which overcome issues with amplification bias typically faced by low-input sequencing. This ultimately allowed us to consistently capture evaluable read depths from as few as 10 sorted cells. We assembled an initial cohort of 21 patients who underwent allo-SCT for MDS or secondary acute myeloid leukemia. Inclusion criteria were follow-up time greater than 1.5 years (or relapse in this timeframe), BM sampling prior to Day+120 post-transplant and relapse, and biospecimens allowing for sorting of greater than 10 HSCs. We sequenced sorted CD34+CD38-CD45RA- HSCs and multipotent progenitors (MPPs), CD34+CD38- CD45RA+ lymphoid-primed multipotent progenitors (LMPPs), and CD34+CD38+ progenitors, as well as bulk BM. With a median follow-up of two years, 9 of the 21 patients relapsed, with an average time to relapse of 362 days. Detection of disease mutations in HSCs/MPPs in any BM at D+30-D+120 post-transplant was 100% specific and 88% sensitive for relapse, showing improvements in both metrics over bulk BM assessment (22% sensitive and 66% specific), despite far lower cell input and read depth. Average time from detection to relapse was 6.9 months. In patients destined to relapse, disease mutations are often present in HSCs at a variant allele frequency of greater than 45%, while simultaneously undetectable on high-depth sequencing of bulk cells (>400x coverage). While even higher depth error-corrected sequencing bulk BM could likely detect such mutations, it would require >20x the sequencing depth, and could not identify the involved cell populations. In conclusion, we show for the first time that relapse of MDS after allo-SCT is driven by failure to eradicate MDS HSCs. Detection of MDS HSCs early post-transplant is highly predictive for relapse and opens new doors for pre-emptive interventions to prevent relapse. These findings are being validated in an independent cohort of 46 patients and updated data will be presented at the meeting. Citation Format: Benjamin Kroger, Yi Huang, Prapti Patel, Aimaz Afrough, Gurbakhash Kaur, Larry Anderson, Farrukh Awan, Praveen Ramakrishnan, Kunal Verma, Russell Vittrup, Robert H Collins, Madhuri Vusirikala, Yazan F Madanat, David Sallman, Carmelo Gurnari, Jaroslaw Maciejewski, Roni Tamari, Stephen Chung. Less is more: Ultra-low input sequencing allows for evaluation of stem cells as a predictor of post-transplant relapse in the myelodysplastic syndromes [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A23.

Inactivation of p53 provides a competitive advantage to del(5q) myelodysplastic syndrome hematopoietic stem cells during inflammation.

Inflammation is associated with the pathogenesis of myelodysplastic syndromes (MDS) and emerging evidence suggests that MDS hematopoietic stem and progenitor cells (HSPC) exhibit an altered response to inflammation. Deletion of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. Although this MDS subtype contains several haploinsufficient genes that impact innate immune signaling, the effects of inflammation on del(5q) MDS HSPC remains undefined. Utilizing a model of del(5q)-like MDS, inhibiting the IRAK1/4-TRAF6 axis improved cytopenias, suggesting that activation of innate immune pathways contributes to certain clinical features underlying the pathogenesis of low-risk MDS. However, low-grade inflammation in the del(5q)-like MDS model did not contribute to more severe disease but instead impaired the del(5q)-like HSPC as indicated by their diminished numbers, premature attrition and increased p53 expression. Del(5q)-like HSPC exposed to inflammation became less quiescent, but without affecting cell viability. Unexpectedly, the reduced cellular quiescence of del(5q) HSPC exposed to inflammation was restored by p53 deletion. These findings uncovered that inflammation confers a competitive advantage of functionally defective del(5q) HSPC upon loss of p53. Since TP53 mutations are enriched in del(5q) AML following an MDS diagnosis, increased p53 activation in del(5q) MDS HSPC due to inflammation may create a selective pressure for genetic inactivation of p53 or expansion of a pre-existing TP53-mutant clone.

PI3-kinase deletion promotes myelodysplasia by dysregulating autophagy in hematopoietic stem cells.

Myelodysplastic syndrome (MDS) is a clonal malignancy arising in hematopoietic stem cells (HSCs). The mechanisms of MDS initiation in HSCs are still poorly understood. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is frequently activated in acute myeloid leukemia, but in MDS, PI3K/AKT is often down-regulated. To determine whether PI3K down-regulation can perturb HSC function, we generated a triple knockout (TKO) mouse model with Pik3ca, Pik3cb, and Pik3cd deletion in hematopoietic cells. Unexpectedly, PI3K deficiency caused cytopenias, decreased survival, and multilineage dysplasia with chromosomal abnormalities, consistent with MDS initiation. TKO HSCs exhibit impaired autophagy, and pharmacologic autophagy induction improved HSC differentiation. Using intracellular LC3 and P62 flow cytometry and transmission electron microscopy, we also observed abnormal autophagic degradation in patient MDS HSCs. Therefore, we have uncovered an important protective role for PI3K in maintaining autophagic flux in HSCs to preserve the balance between self-renewal and differentiation and to prevent MDS initiation.

Evaluating Stem Cells to Predict Post-Transplant Relapse in the Myelodysplastic Syndromes

Malignant hematopoietic stem cells (HSCs) both initiate the myelodysplastic syndromes (MDS) and drive treatment failure through their resistance to frontline non-transplant therapies. Consequently, allogeneic transplant is the only curative therapy, but relapse can still occur. While post-transplant relapse is hypothesized to be driven by persistent MDS HSCs, their detection has proven challenging due to their limiting frequency in biospecimens. The accuracy of sequencing low-input specimens is limited by allelic drop-out and amplification bias. We adapted a hybridization capture-based targeted sequencing panel covering 260 myeloid malignancy associated genes that incorporates unique molecular identifiers capable of retaining read depths of >12x from as few as 19 sorted HSCs. We aimed to characterize residual disease after transplant with greater resolution than previously possible by sequencing sorted CD34+CD38-CD45RA- HSCs and multipotent progenitors (MPPs), CD34+CD38-CD45RA+ lymphoid-primed multipotent progenitors (LMPPs), and CD34+CD38+ progenitors. We applied our technique to an MDS case characterized by bi-allelic TP53 mutations (Y220C and P75fs), assessing a bone marrow (BM) specimen D+168 after transplant, which was five months prior to clinical relapse. Both mutations were present at variant allelic frequencies (VAFs) of 8% and 4% in 258 sorted CD34+CD38- cells with 105x coverage (xc), but simultaneously undetectable in bulk BM (481xc) and CD34+CD38+ progenitor cells (239xc) despite greater cell number and read depth. We next evaluated a D+179 post-transplant BM from a case of MDS harboring ASXL1 (R713G), DNMT3A (R882H), and IDH2 (R140Q) mutations. All three mutations were detectable in 102 sorted HSCs/MPPs at VAFs of 20% (92xc), 13% (68xc), and 35% (89xc), respectively, while absent in bulk BM (369xc) and CD34+CD38+ progenitors (506xc). These findings preceded clinical relapse by nearly two years. We additionally investigated pre- and post-transplant BM samples of a patient with a germline DDX41 M1I mutation treated for MDS. In such cases, a second somatic DDX41 mutation is often acquired, impairing ribosomal biogenesis. This mutation is always present at a low VAF, and it is unclear how it promotes disease. Prior to transplant, we identified a somatic DDX41 R525H mutation at a VAF of 2.6% in bulk BM. However, this mutation was present at a VAF of 45% in sorted HSCs/MPPs, revealing the pervasiveness of this "minor clone” at the apex of the hematopoietic hierarchy (Fig. 1). Additionally, two previously unknown TP53 clones were discovered at VAFs of 22% (K132R) and 1.8% (C275F) in HSCs/MPPs, and these were undetectable in bulk BM. At D+102 post-transplant, we detected persistence of both M1I (VAF 43%) and R52H (VAF 27%) DDX41 mutations in 19 sorted HSCs/MPPs (12xc) 78 days prior to relapse. These data suggest that while impaired ribosomal biogenesis in HSCs with bi-allelic DDX41 mutations prevents effective hematopoiesis, it also increases self-renewal and accumulation of HSCs. Next, we sought to test if early identification of residual MDS HSCs post-transplant is predictive of disease relapse. We evaluated a cohort of patients who underwent myeloablative allogeneic transplant for MDS at UTSW and MSKCC (n=21). We evaluated BMs available from post-transplant dates ranging from D+30 to D+120, including any specimen from which we were able to sort at least 15 HSCs/MPPs and achieve an effective read depth of >10. We censored specimens with follow-up of less than one year. With a median follow-up of two years, detection of disease mutations in HSCs/MPPs was 100% specific and 88% sensitive for relapse (Fig. 2). The average time from detection of disease in HSCs to clinical relapse was 6.9 months. Detection in CD38+CD34+ progenitors was also 100% specific but only 67% sensitive for relapse. Assessment of mutations in bulk BM (mean 529xc) was only 22% sensitive and 50% specific for relapse. In conclusion, our research shows for the first time that relapse of MDS after allogeneic transplant is driven by failure to eradicate MDS HSCs, and that detection of MDS HSCs early after transplant is highly predictive for relapse. Validation of this assay in larger cohorts promises to yield a tool to identify patients who may benefit from early post-transplant interventions to forestall relapse. We also demonstrate how this assay can reveal novel insights into human disease HSC biology. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High Throughput Multi‐omic Characterization of Clonal Hematopoietic Stem Cells During Progression of Myeloid Malignancies

Hematopoietic stem cells (HSCs) are responsible for giving rise to all other lineages of blood cells in the body. Over time, mutations in HSCs can promote the outgrowth of clonal populations that outcompete other HSCs, resulting in a phenomenon called Clonal Hematopoiesis of Indeterminate Potential (CHIP). Though not cancerous in and of itself, CHIP can progress to more serious hematologic disorders, such as the Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). The mechanisms of clonal expansion, by which certain mutant HSCs acquire a competitive advantage over other HSCs, currently remain largely undeciphered, as are the mechanisms by which clonal HSCs drive the initiation of MDS and contribute to the development of AML. Moreover, previous studies have shown that such mutant HSCs are resistant to conventional therapies and may act as reservoirs for disease relapse and progression. In the past, researchers were hindered by bulk cell analyses and the relative rarity of HSCs, but advances in single‐cell omics have now enabled us to explore the molecular heterogeneity of clonal HSCs and identify distinct clonal populations based on genotype and cell surface phenotype. We used single cell RNA sequencing to examine the transcriptional dynamics of purified HSCs from MDS and AML patients before and after treatment, as well as from age matched elderly controls. Interestingly, dimensionality reduction methods such as UMAP and tSNE revealed a reservoir of control HSCs that clustered with MDS HSCs. Upon comparison with other normal control HSCs, we found that genes associated with aging, mitochondrial function, and particular ion channels were strongly upregulated in these “MDS‐like” control HSCs, while genes involved in ribosomal and translation activity, along with certain surface markers, were substantially downregulated. Additionally, ribosomal transcripts were depleted in MDS HSCs from patients who did not respond to treatment. These results support the notion that the most immature HSCs, which impose the strictest constraints on translation, might clonally expand and initiate CHIP and/or MDS upon acquiring driver mutations, also serving as treatment resistant populations that underlie disease relapse. We also subjected purified MDS HSCs to simultaneous single cell targeted DNA sequencing and cell surface phenotyping, which allowed us to correlate the cell surface phenotype and genotype for specific clonal populations and identify cell surface markers that can be used to isolate HSCs with enhanced engraftment ability. Taken together, these results indicate that combined single cell genotyping and phenotyping can be used to track clonal populations across different stages of pathogenesis, providing further insight about the development of CHIP and its progression to MDS and AML.

Stem cell architecture drives myelodysplastic syndrome progression and predicts response to venetoclax-based therapy

Myelodysplastic syndromes (MDS) are heterogeneous neoplastic disorders of hematopoietic stem cells (HSCs). The current standard of care for patients with MDS is hypomethylating agent (HMA)-based therapy; however, almost 50% of MDS patients fail HMA therapy and progress to acute myeloid leukemia, facing a dismal prognosis due to lack of approved second-line treatment options. As cancer stem cells are the seeds of disease progression, we investigated the biological properties of the MDS HSCs that drive disease evolution, seeking to uncover vulnerabilities that could be therapeutically exploited. Through integrative molecular profiling of HSCs and progenitor cells in large patient cohorts, we found that MDS HSCs in two distinct differentiation states are maintained throughout the clinical course of the disease, and expand at progression, depending on recurrent activation of the anti-apoptotic regulator BCL-2 or nuclear factor-kappa B-mediated survival pathways. Pharmacologically inhibiting these pathways depleted MDS HSCs and reduced tumor burden in experimental systems. Further, patients with MDS who progressed after failure to frontline HMA therapy and whose HSCs upregulated BCL-2 achieved improved clinical responses to venetoclax-based therapy in the clinical setting. Overall, our study uncovers that HSC architectures in MDS are potential predictive biomarkers to guide second-line treatments after HMA failure. These findings warrant further investigation of HSC-specific survival pathways to identify new therapeutic targets of clinical potential in MDS.

Hematopoietic Stem Cells (HSC) and Granulocyte Macrophage Progenitors (GMP) are the Oxidative Stress Targets in Low/Intermediate-1 Risk Myelodysplastic Syndromes.

Myelodysplastic Syndromes (MDS) are a heterogenous group of clonal hematopoietic stem cell malignancies. Previous studies showed that Reactive Oxygen Species (ROS) play a role in the pathogenesis and clinical evolution of MDS, contributing to Hematopoietic Stem and Progenitor Cells (HSPC) genetic instability. Less is known about ROS levels in the various sub-populations of MDS HSPC and how they correlate with clinical data in MDS patients. Our study aims to analyze ROS levels in MDS Hematopoietic Stem Cells (HSC), common myeloid progenitors (CMP), Granulocyte Macrophages Progenitors (GMP) and megakaryocyte-erythrocyte progenitors (MEP); afterwards, we looked at the relationship between ROS levels and clinical data. thirty-eight MDS and 27 Normal Bone Marrow (NBM) samples were collected; ROS levels were analyzed via multicolor flow cytometry. In both NBM and MDS, HSC showed much higher ROS levels than progenitors (3 to 4 folds, p < 0.0001); HSC ROS were significantly more elevated in MDS-no excess blasts versus MDS with excess blasts and versus NBM. GMP from MDS-no excess blasts showed higher ROS compared to NBM GMP. The 3 MDS with Ringed Sideroblasts (RS) showed more elevated ROS in HSC and GMP compared to the not RS low/intermediate-1 MDS; the 2 monosomy 7 patients displayed higher ROS levels in each subpopulation compared to the normal karyotype MDS; the only del(5q) patient did not show relevant differences in ROS levels compared to the median of the normal karyotype MDS ROS. The 9 high transfusion burden patients exhibited higher ROS in HSC and GMP compared to NBM HSC and GMP. These data were not confirmed in low transfusion burden (n:2) and non-transfused patients (n:26). In low/intermediate-1 MDS, a direct correlation between ferritin values and ROS levels in progenitors, but not in HSC, was detected. Interestingly, low/intermediate-1 risk patients that are no longer responding to recombinant human erythropoietin (rh-EPO) showed higher ROS levels in GMP and HSC. Our data showed that ROS can play a role in the pathogenesis and maintenance of low and intermediate-1 risk MDS clone; ROS status can be influenced by several clinical factors as ferritin levels and rh-EPO treatment. In this scenario, high ROS levels can contribute to genetic instability and influence progression to AML. Further biological studies are needed to elucidate ROS role in MDS pathogenesis and analyze the possible benefit of antioxidant drugs added to the standard MDS treatments.

Acquired Gene Mutation of MAP4K2 Identified from Myelodysplastic Syndromes Patients Confers Azacitidine Resistance

Myelodysplastic syndromes (MDS) is clinically characterized by ineffective hematopoiesis and frequent development of acute myeloid leukemia (AML). Accumulating literature has addressed a variety of abnormalities in MDS hematopoietic stem cells, including chromosomal abnormalities, genetic mutations and epigenetic changes. At present, however, these recent discoveries have hardly led to improvement in patient outcomes. Except for allogeneic stem cell transplantation as a potentially curative treatment, few treatment options are available. Among them, DNA methyltransferases inhibitor azacitidine has been widely used against MDS and AML with proven efficacy in overall survival improvement, but subsequent recurrence is inevitable even after marked response. In this study, we searched genomic mutations associated with azacitidine resistance in high risk MDS patients by next generation sequencing, using longitudinally collected samples from patients undergoing azacitidine treatment in our institution. Mutational analysis was performed by whole exome sequencing in 4 consecutive cases, with patients' BM samples taken at the point of diagnosis, during treatment, after relapse. Control sequencing was performed using T cell fraction from during-treatment samples. We verified variant allele frequency of the detected mutations by targeted deep sequencing, to identify gene mutations strongly related to clinical azacitidine resistance.We identified an acquired gene mutation of MAP4K2, which is a member of the serine/threonine protein kinase family and activated by TNF-α/TRAF2 signal. MAP4K2 is required for the efficient activation of JNKs by stress-induced pathway. The mutation was exclusively found in the region encoding the protein-binding domain, which is required for signal transduction of JNK and p38 pathway by binding MAP3Ks such as MEKK1. We introduced the mutated form of MAP4K2 retrovirally into the human AML cell line Kasumi-1, and assessed its impact on azacitidine resistance. We found that the MAP4K2 mutation recaptured azacitidine resistance without affecting proliferation in the absence of azacitidine. In addition, another AML cell line HL60 with MAP4K2 mutation demonstrated higher growth ratio compared with control vector or MAP4K2 wild type after azacitidine treatment. Moreover, Kasumi-1 cells harboring MAP4K2 mutation showed low apoptosis rate and increased colony forming capacity after azacitidine treatment. We knocked in the mutations into MAP4K2 alleles Kasumi-1 cells using CRISPR/Cas9 system and found that Kasumi-1 cells with heterozygous MAP4K2 mutation similarly showed azacitidine resistance compared with control cells.We examined whether associated with stress-induced cytokines are associated with MAP4K2 mutation-induced azacitidine resistance. We revealed that MAP4K2 mutation increased TNF-α expression after azacitidine treatment. In addition, the mutant-form of MAP4K2 exhibited enhanced stability compared with the wild type. These results imply that MAP4K2 mutation we detected in this study is gain of function mutation that confers altered cytokine signaling and growth advantage on MDS cells by stabilizing MAP4K2 protein.In summary, we found that MAP4K2 mutation in MDS patients who have lost azacitidine responsiveness. Mutant MAP4K2 played a causative role in azacitidine resistance. Targeting MAP4K2 has become an attractive therapeutic strategy to conquer azacitidine resistance in MDS. DisclosuresTsukamoto:Pfizer Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Research Funding; Astellas Pharma Inc.: Research Funding. Masamoto:Pfizer Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory; Astellas Pharma Inc.: Research Funding. Arai:Bristol-Myers Squibb: Research Funding. Kurokawa:Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Celgene Corporation: Speakers Bureau; Astellas Pharma Inc.: Research Funding; Pfizer Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau.

Innate Immune Signal Pathway Activation Induced By Combined Loss of Del(5q) MDS Genes, Tifab and Mir-146a, Accelerates Hematopoietic Stem Cell Exhaustion Driven By Chronic Exposure to a TLR Ligands

Deletion of the long arm of chromosome 5 (del(5q)) is the most frequently observed chromosomal abnormality in patients with myelodysplastic syndrome (MDS). We previously reported that deletion of TRAF-interacting protein with forkhead-associated domain B (TIFAB) and miR-146a, two haploinsufficient genes in del(5q) MDS, activates the Toll-like receptor (TLR) signaling cascade in hematopoietic stem/progenitor cells (HSPC) by increasing TRAF6 protein stability and mRNA translation, respectively (Varney et al., JEM 2015 and Varney et al., Leukemia 2017). To investigate the epistasis of TIFAB and miR-146a, we generated a mouse model in which Tifab and miR-146a were simultaneously deleted (Tifab-/-; miR-146a-/-, herein dKO). Consistent with increased TRAF6 expression, dKO mice exhibit phosphorylation of IRAK1 and IRAK4 kinases, indicating activation of the TLR/innate immune pathway in bone marrow (BM) HSPC of dKO mice. To determine whether combined deletion of Tifab and miR-146a exaggerates the hematopoietic-specific defects observed in either Tifab- or miR-146a-deficient mice in vivo, BM cells from WT, Tifab-/-, miR-146a-/-, and dKO mice were transplanted into lethally-irradiated syngeneic recipient mice. As previously reported, a minor subset of Tifab-/- and ~ 50% of miR-146a-/- mice succumbed to a bone marrow failure (BMF)-like disease. For dKO mice, 80% of mice developed a more rapid and penetrant BMF-like disease as compared to loss of Tifab or miR-146a alone. Moribund dKO mice presented with leukopenia, severe anemia, and thrombocytopenia. Since TLR activation is a common feature of MDS due to overexpression and secretion of damage-associated molecular patterns (DAMPs) within the BM microenvironment (Monlish et al., Front Immunol 2016), we explored the biological effect of systemic TLR activation on hematopoietic stem cell (HSC) function of dKO mice following exposure to chronic low dose lipopolysaccharide (LPS), a TLR4 agonist. After 4 weeks, the proportion of myeloid cells in peripheral blood (PB) of LPS-treated dKO mice was significantly increased as compared to WT mice exposed to LPS. BM cellularity of dKO mice was dramatically decreased by LPS exposure while that of WT mice was only slightly affected by LPS. The proportion of CD48-CD150+Lineage-Sca1+c-Kit+ (LSK) of dKO mice was significantly reduced by exposure to LPS as compared to WT mice, which correlated with loss of HSC quiescence. We next performed competitive repopulation assays using WT and dKO BM cells exposed to LPS. As expected, the chimerism of donor-derived PB cells from LPS-treated WT mice was significantly lower than that from WT control mice. Intriguingly, LPS-treated dKO mice showed significantly reduced chimerism of donor-derived PB and CD48-CD150+ LSK BM cells as compared to vehicle-treated dKO mice or WT mice exposed to LPS. Importantly, the impaired hematopoietic reconstitution was more pronounced following serial BM transplantation. These results indicate that deletion of Tifab and miR-146a diminishes the long-term repopulating potential of HSC driven by in vivo TLR stimulation. To uncover the molecular consequences that explain the functional HSC defect of dKO mice exposed to TLR activation, RNA-sequencing was performed on LSK from dKO and WT mice treated with LPS. Following TLR activation, we observed differential expression of distinct genes in dKO LSK as compared to WT LSK. Pathway network analyses revealed changes in chromatin organization, epigenetic regulations, and cellular senescence in WT LSK exposed to LPS as compared to control WT cells. In contrast, dKO LSK exposed to LPS exhibited enrichment in pathways associated with axon guidance, carbohydrate metabolism, and megakaryocyte development and platelet production as compared to control dKO cells. These results suggest the differential impact of TLR stimulation on hematopoiesis in Tifab;miR-146a-deficient mice. Collectively, our findings indicate that innate immune signal pathway activation induced by combined loss of Tifab and miR-146a accelerates HSC exhaustion driven by chronic exposure to TLR ligands via the activation of distinct signal pathways. These findings have important implications in the role of TLR stimulation in the cellular consequence of MDS HSC. DisclosuresNo relevant conflicts of interest to declare.

Anti-CD117 antibody depletes normal and myelodysplastic syndrome human hematopoietic stem cells in xenografted mice

AbstractThe myelodysplastic syndromes (MDS) represent a group of clonal disorders that result in ineffective hematopoiesis and are associated with an increased risk of transformation into acute leukemia. MDS arises from hematopoietic stem cells (HSCs); therefore, successful elimination of MDS HSCs is an important part of any curative therapy. However, current treatment options, including allogeneic hematopoietic cell transplantation (HCT), often fail to ablate disease-initiating MDS HSCs, and thus have low curative potential and high relapse rates. Here, we demonstrate that human HSCs can be targeted and eliminated by monoclonal antibodies (mAbs) that bind cell-surface CD117 (c-Kit). We show that an anti-human CD117 mAb, SR-1, inhibits normal cord blood and bone marrow HSCs in vitro. Furthermore, SR-1 and clinical-grade humanized anti-human CD117 mAb, AMG 191, deplete normal and MDS HSCs in vivo in xenograft mouse models. Anti-CD117 mAbs also facilitate the engraftment of normal donor human HSCs in MDS xenograft mouse models, restoring normal human hematopoiesis and eradicating aggressive pathologic MDS cells. This study is the first to demonstrate that anti-human CD117 mAbs have potential as novel therapeutics to eradicate MDS HSCs and augment the curative effect of allogeneic HCT for this disease. Moreover, we establish the foundation for use of these antibody agents not only in the treatment of MDS but also for the multitude of other HSC-driven blood and immune disorders for which transplant can be disease-altering.

Data Driven Computational Modeling of Hematopoiesis in Myelodysplastic Syndromes Unveils Differences in Hematopoietic Stem Cell Kinetics Compared to Age-Matched Healthy Controls

Background: Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis, peripheral cytopenias and risk of transformation to acute myeloid leukemia (AML). Recently, HSC in MDS have been shown to carry acquired distinct genetic and epigenetic mutations, most frequently in epigenetic regulators or splicing machinery factors, which are considered disease-initiating events and are likely responsible for the dysplastic features observed in this disease. Hierarchies of hematopoiesis have been established for healthy HSC and their progeny but it is unclear whether MDS HSC follow the same patterns. Clonal hierarchies in MDS have been inferred on the basis of mutational data, these only constitute a snapshot of hematopoiesis in the patient at a given time and may not accurately reflect the kinetics of the disease. To understand how clonal dominance of MDS over healthy HSC is achieved, we sought to identify differences in proliferation kinetics and lineage fate decisions of MDS HSC in comparison to healthy hematopoiesis and to fit this real data into a mathematical model of MDS hematopoiesis.Methods: Lower-risk MDS (according to IPSS-R) bone marrow (BM) samples from patients harboring an SF3B1 mutation (n = 5) or higher-risk MDS BM samples from patients with an ASXL1 mutation (n = 5) were chosen for analysis to limit heterogeneity and allow for better comparability. Healthy age-matched control BM samples (n = 5) were obtained from patients undergoing routine hip replacement surgery. Distribution of stem and progenitor cell compartments (HSC, MPP, MLP, CMP, GMP, MEP) in BM on day 0 were determined by multiparameter flow cytometry. Cells were labeled with violet cell trace and HSC (Lin-CD34+CD38-CD90+CD45RA-) were sorted by flow cytometry. A fixed number of HSC as well single HSC cell cultures were set up in vitro in serum-free medium with defined growth factors (FL, KL, TPO, IL-6, G-CSF, GM-CSF and EPO). Cell divisions, proliferation rates, cell death and lineage maturation were determined by flow cytometry on days 1, 3, 5, 7 and 9. Clonogenic capacity was probed by standard CFU assays in methylcellulose on day 0 and day 7. Additional integration of genomic sequencing analyses of bulk BM and HSC was used to assess the contribution of the malignant clone to the population of proliferating cells and to each hematopoietic lineage.Results: While CFU numbers at day 0 were significantly reduced for all MDS samples compared to healthy controls, ASXL1 mutated MDS BM samples contained significantly higher numbers of HSC than SF3B1 MDS or healthy control BM samples. SF3B1 MDS samples had significantly larger CMP and GMP compartments compared to healthy controls or ASXL1 mutated samples. In cultures started with a fixed number of HSC, no apparent differences in proliferation kinetics were observed between MDS and healthy samples over time. However, cell trace labeling and sorting of single HSC into 96 well plates revealed clear differences between MDS and healthy HSC. Overall, MDS HSC were less robust, with fewer wells of the 96 plate containing cells after sorting than for healthy HSC samples. In contrast, the remaining HSC from both both ASXL1 and SF3B1 mutated MDS showed a significantly higher cell division rate than healthy HSC in vitro, suggesting MDS HSC are more active than their normal counterparts. Genotyping analyses to assess single MDS HSC contributions to proliferation are ongoing and data are currently being fitted to mathematical models.Conclusion: Detailed analysis of hematopoietic stem and progenitor cell compartments as well as time-resolved tracking of HSC revealed significant differences between MDS and healthy HSC kinetics in vitro. Integration of this data into a comprehensive mathematical model of MDS hematopoiesis may aid in dissecting the mechanisms of progression of MDS towards acute myeloid leukaemia (AML). DisclosuresGötze:Takeda: Honoraria, Other: Travel aid ASH 2017; Celgene: Honoraria, Research Funding; Novartis: Honoraria; JAZZ Pharmaceuticals: Honoraria.

Cell-Type Specific Mechanisms of Hematopoietic Stem Cell (HSC) Expansion Underpin Progressive Disease in Myelodysplastic Syndromes (MDS) and Provide a Rationale for Targeted Therapies

The mechanisms of HMA failure in MDS remain unclear, as recent advances in sequencing approaches did not enable the molecular characterization of the cells that survive therapy and drive resistance and disease progression.Here, through combined functional and transcriptomic analyses of highly-purified hematopoietic populations isolated from the BM of 132 MDS patients enrolled in clinical trials of single drug HMA therapy, we show that 2 immunophenotypically and molecularly distinct cell types maintain the disease and expand at progression, and we propose therapeutic approaches to overcome MDS evolution.Unsupervised hierarchical clustering followed by principal component analysis of 101 untreated MDS samples based on the frequency of immunophenotypically defined stem and myeloid progenitor cell populations identified the frequencies of the lymphoid-primed multipotent progenitors (LMPPs) and granulo-monocytic progenitors (GMPs) as the main sources of variation across the samples. Further logistic regression analysis enabled the systematic stratification of the samples in 2 main groups. “CMP pattern” MDS was characterized by the prevalence of common myeloid progenitors (CMPs) in the progenitor compartment while “GMP pattern” MDS was characterized by the increased frequency of GMPs in the progenitors and by increased LMPPs and decreased long-term (LT)-HSC frequencies in the BM (Fig A, B). Functional analysis of the CMPs by immunophenotypic characterization of lineage-primed fractions and colony assays revealed that this population was myeloid-biased only in “CMP pattern” MDS but not in “GMP pattern” MDS, suggesting that 2 distinct hierarchical differentiation routes underlie disease manifestation (Fig C). Further logistic regression analysis of 31 MDS samples from patients with progressive disease showed that whereas “CMP pattern” MDS patients with blast expansion were characterized by a significant increase in the BM frequency of LT-HSCs, “GMP pattern” MDS patients were characterized by the expansion of the LMPPs (Fig D).To investigate the molecular pathways underlying the 2 different hierarchical patterns of progression, we analyzed the transcriptional profiling of the HSCs that selectively expanded in the 2 groups of MDS patients. RNA-Seq analysis revealed that, compared with those isolated from patients at baseline, LT-HSCs isolated from “CMP pattern” MDS patients with blast expansion had significantly upregulated genes involved in promoting cell proliferation and survival, including the anti-apoptotic regulator BCL2. In contrast, genes involved in the response to TNF-a were significantly upregulated in the LMPPs from “GMP pattern” MDS patients with progressive disease as compared with the LMPPs isolated at baseline (Fig E). Then, we hypothesized that, despite genetic dissimilarities, the HSCs that expanded at progression were addicted to the molecular pathways that were upregulated and that targeting these pathways represented a potential therapeutic strategy to overcome HMA resistance. As proof-of-principle, we treated CD34+ cells from “CMP pattern” MDS isolated after HMA failure, co-cultured over a layer of stromal cells, with the BCL2 inhibitor ABT-199 for 72 hours. ABT-199, in combination with 5-azacytidine (AZA), significantly decreased the number of LT-HSCs from MDS patients with progressive disease (Fig F), but did not affect those from MDS patients in whom HMA therapy failed because of persistent MDS. These data suggest that ABT-199 selectively targets the LT-HSCs from “CMP pattern” MDS with progressive disease, which depend on increased levels of BCL2 to support their expansion. Treatment of WT/vav-cre-TET2L/L BM chimeras with ABT-199 therapy showed selective apoptosis and depletion of the MDS-like LT-HSCs and the concomitant proliferation of the WT LT-HSCs. This suggests that ABT-199 allows selective therapeutic targeting that eliminates abnormal HSCs while restoring normal hematopoiesis (Fig G). Furthermore, ABT-199 treatment of xenografts generated by transplanting the MDS-L cell line inNSGS mice reduced tumoral burden by depleting the human blast population (Fig H).Taken together, these findings demonstrate that targeting commonly deregulated pathways in MDS HSCs is a feasible approach to tackling disease progression and provide a rationale for the selective inhibition of BCL2 in “CMP pattern” MDS with progressive disease. [Display omitted] DisclosuresGiuliani:Celgene Italy: Other: Avisory Board, Research Funding; Takeda Pharmaceutical Co: Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding. Konopleva:Stemline Therapeutics: Research Funding. Colla:Abbvie: Research Funding.

The interleukin-3 receptor CD123 targeted SL-401 mediates potent cytotoxic activity against CD34+CD123+ cells from acute myeloid leukemia/myelodysplastic syndrome patients and healthy donors.

Diseases with clonal hematopoiesis such as myelodysplastic syndrome and acute myeloid leukemia have high rates of relapse. Only a small subset of acute myeloid leukemia patients are cured with chemotherapy alone. Relapse in these diseases occurs at least in part due to the failure to eradicate leukemic stem cells or hematopoietic stem cells in myelodysplastic syndrome. CD123, the alpha chain of the interleukin-3 receptor heterodimer, is expressed on the majority of leukemic stem cells and myelodysplastic syndrome hematopoietic stem cells and in 80% of acute myeloid leukemia. Here, we report indiscriminate killing of CD123+ normal and acute myeloid leukemia / myelodysplastic syndrome cells by SL-401, a diphtheria toxin interleukin-3 fusion protein. SL-401 induced cytotoxicity of CD123+ primary cells/blasts from acute myeloid leukemia and myelodysplastic syndrome patients but not CD123− lymphoid cells. Importantly, SL-401 was highly active even in cells expressing low levels of CD123, with minimal effect on modulation of the CD123 target in acute myeloid leukemia. SL-401 significantly prolonged survival of leukemic mice in acute myeloid leukemia patient-derived xenograft mouse models. In addition to primary samples, studies on normal cord blood and healthy marrow show that SL-401 has activity against normal hematopoietic progenitors. These findings indicate potential use of SL-401 as a “bridge-to-transplant” before allogeneic hematopoietic cell transplantation in acute myeloid leukemia / myelodysplastic syndrome patients.

313 - Anti-Human CD117 Antibodies Mediate Clearance of Myelodysplastic Syndrome Hematopoietic Stem Cells and Facilitate Establishment of Normal Hematopoiesis in Transplantation

313 - Anti-Human CD117 Antibodies Mediate Clearance of Myelodysplastic Syndrome Hematopoietic Stem Cells and Facilitate Establishment of Normal Hematopoiesis in Transplantation

An Analysis of the Transcriptional Response of Myelodysplastic Syndrome Stem Cells to Therapy at Single Cell Resolution

The myelodysplastic syndromes (MDS) are initiated in hematopoietic stem cells (HSCs) that persist despite excellent clinical responses to DNA methyltransferase inhibitors (DNMTIs). Given the likely contributions of genetic and phenotypic heterogeneity to therapeutic resistance, we sought to characterize the response of MDS HSCs to DNMTIs at single cell resolution. We identified patients who received the DNMTI decitabine and either sustained hematologic improvement (n=2) or were non-responders (n=2). Bone marrow (BM) specimens were obtained at pre- and post-treatment time points, as well as from untreated MDS patients (n=2) and age matched controls (n=2). From these specimens, HSCs (Lin-CD34+CD38-CD90+CD45RA-) were FACS-purified and the Fluidigm C1 platform was used to capture 686 single cells, with subsequent RNA-sequencing (Illumina HiSeq2500, 100 bp paired-end). The data was of high quality with an average mapping rate of 80%.We used t-distributed Stochastic Neighbor Embedding (t-SNE) to visualize differences between cells from responders, non-responders, and age matched controls (Fig.1A). This revealed separation between responder and non-responder cells prior to treatment, with non-responders more distant from normal HSCs. Post-treatment samples revealed a shift in responder cells closer to normal HSCs, with non-responder cells remaining distant from normal HSCs. A small number of cells from responders and normal HSCs clustered away from their respective groups and closely with cells from non-responders, highlighting intratumoral transcriptional heterogeneity. We speculate that such cells represent a reservoir for eventual therapeutic resistance in responders. Comparing pre-treatment HSCs from responders and non-responders, we found 455 differentially expressed genes (DEGs, FDR<0.01). DEGs were enriched for gene ontology (GO) terms including mRNA catabolic processes, NMD, and translational elongation, with 12/13 DEGs in the top five GO terms being ribosomal proteins (RPs). Pre-treatment cells formed four clusters based on these genes (Fig.1B), with responders comprising the two highest expressing clusters and non-responders comprising the next to lowest expressing cluster. The lowest expressing cluster was a mixture of cells from responders and non-responders, again suggestive of a source for eventual therapeutic resistance in responders.To assess the effect of decitabine on MDS HSCs agnostic to treatment response, we compared all pre-treatment MDS HSCs with all post-treatment MDS HSCs. We identified 45 DEGs (FDR <0.01) after filtering for high expression (FPKM >4) and removing genes that might reflect disease progression by excluding DEGs between early and late time points of untreated patients. Using these DEGs to cluster all pre- and post-treatment cells, the highest level of clustering separated a pre- and post-treatment cluster (Fig.1C). The pre-treatment cluster was primarily composed of pre-treatment cells from responders, with nearly all pre-treatment cells from non-responders falling in the post-treatment cluster, which also included post-treatment cells from both responders and non-responders. Thus MDS HSCs in responders occupy a unique transcriptional landscape prior to therapy.As aberrant splicing may contribute to MDS pathogenesis, we developed an algorithm to evaluate differential isoform usage in single cells. Comparing MDS HSCs to normal HSCs, we identified 22 genes with differentially expressed isoforms, including seven RPs (Fig.1D). A comparison between SRSF2 mutant (n=3) and wild-type (n=4) patients identified 13 differentially expressed isoforms, including five RPs. This suggests that even MDS HSCs from patients lacking splicing factor mutations may exhibit aberrant splicing, and that these changes may differ from those found in patients with mutated splicing genes.In conclusion, our studies of the transcriptional landscape of single MDS HSCs reveal RP gene signatures that represent novel biomarkers that distinguish MDS from age-matched controls and predict response to DNMTIs. Our studies also demonstrate significant intratumoral heterogeneity in gene expression that may underlie the biology of inevitable therapeutic resistance. Such heterogeneity includes differential isoform usage in both splicing factor mutant and wild type patients, suggesting that aberrant splicing may be a general feature of MDS. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Role of LUC7L2 in Splicing and MDS

Chromosomal abnormalities are common in myelodysplastic syndrome (MDS) and other myeloid malignancies. -7/del7q is found in 15% of MDS patients, but the pathological consequences of this deletion are unknown. To identify genes in the -7/del7q region that may contribute to pathogenesis of MDS, whole exome sequencing of MDS patient samples was used to detect novel somatic nonsense/frameshift mutations. Novel somatic mutations in the gene LUC7L2 were found in 27 malignant cases.Cases of mutant LUC7L2 hemizygosity, heterozygosity and homozygosity were observed in this cohort. The mutations were frameshift or nonsense, resulting in a premature stop codon and decreased expression of the LUC7L2 mRNA. LUC7L2 is located in the most commonly deleted region of -7/del7q; 7q34, which is deleted in 85% of -7/del7q patients. Patients with LUC7L2 mutations and those harboring -7/del7q have similar and statistically shorter overall survival than those with normal LUC7L2 expression levels.LUC7L2 is a poorly characterized, splicing-related protein. The function of LUC7L2 is largely unknown, but it is the ortholog of yeast protein LUC7p, which is involved in recruitment of early splicing factors. Recent studies have shown that over 65% of MDS patients harbor mutations in one of several proteins involved in pre-mRNA splicing. Therefore, we hypothesize that LUC7L2 acts as a mammalian splicing factor and deficiency of LUC7L2 results in aberrant splicing of transcripts that contribute to the pathogenesis of MDS.We characterized the role LUC7L2 in splicing by showing that LUC7L2 interacts with splicing factors using immunoprecipitation followed by mass spectrometry. Immunoprecipitation of LUC7L2 and interacting proteins revealed that LUC7L2 interacts with many proteins in the SR and HnRNP families of splicing regulators suggesting that LUC7L2 may also play a role as a splicing regulator. Splicing regulatory proteins are a diverse group that can influence constitutive splicing as well as alternative splicing. Therefore, to understand the mRNA targets of LUC7L2, we performed RNA CLIP-Seq. This revealed that LUC7L2 binds at least 301 pre-mRNA transcripts as well as spliceosomal snRNAs. LUC7L2 binding is enriched near splice junctions, with 117 of binding sites lying within 100bp of a splice site.To understand how LUC7L2 regulates splicing, we knocked down LUC7L2 in HEK293 cells and subjected the cells to a qRT-PCR based intron splicing efficiency assay as well as a splicing sensitive microarray. The intron splicing efficiency assay was performed on a subset of LUC7L2 targets identified by CLIP-Seq and showed that LUC7L2-deficiency often promotes the splicing of introns, suggesting that LUC7L2 acts as a splicing repressor. To understand the effect of LUC7L2 deficiency on all LUC7L2-targets, the splicing sensitive microarray identified splicing changes near LUC7L2-binding sites. LUC7L2-deficiency significantly alters the splicing of 151 transcripts that contain LUC7L2 binding sites.Missplicing of transcripts can alter protein function, leading to profound repercussions in the cell. The microarray analysis of LUC7L2-deficient cells identified several misspliced transcripts that are involved in the processes of proliferation, differentiation, and apoptosis, which are disrupted in MDS hematopoietic stem cells. Exons 2, 7 and 8 of the RUNX1 transcript are frequently skipped in the LUC7L2 knockdown cells. Although the mRNA expression of RUNX1 remains the same, several downstream transcripts that are regulated by RUNX1 have significantly increased or decreased expression, suggesting altered function of the RUNX1 protein. RUNX1 is a transcription factor that is required for HSC differentiation into myeloid progenitor cells. Altered splicing of RUNX1 and changes in the RUNX1 pathway could cause defects in the HSC differentiation process.In conclusion, novel somatic mutations in have been observed in LUC7L2 in patients with MDS. LUC7L2 is a protein that acts as a splicing regulator and deficiency of this protein results in missplicing of transcripts, including factors involved in differentiation, which may contribute to the pathogenesis of MDS. DisclosuresMakishima:The Yasuda Medical Foundation: Research Funding.

Analysis of Myelodysplastic Syndrome Stem Cells at Single Cell Resolution during DNA Methyltransferase Inhibitor Therapy

Abstract The myelodysplastic syndromes (MDS) arise in and are maintained by hematopoietic stem cells (HSCs). Serial sampling of patients treated with DNA methyltransferase inhibitors (DNMTIs) and lenalidomide has demonstrated that disease HSCs (MDS HSCs) persist at significant levels even in patients achieving complete clinical and cytogenetic responses. As MDS HSCs are the functional unit of clonal selection both during therapy and subsequent disease progression, we hypothesized that the molecular heterogeneity of MDS HSCs may underlie therapeutic resistance. We therefore sought to perform single cell RNA-sequencing (RNA-seq) on MDS HSCs from patients with known responses to therapy, with the intention of identifying novel therapeutic vulnerabilities. To characterize MDS HSC heterogeneity, we FACS-purified HSCs (Lin-CD34+CD38-CD90+CD45RA-) from paired bone marrow (BM) specimens taken from four MDS patients before and after two to four 28-day cycles of the DNMTI decitabine, as well as two patients who were not treated due to stable disease, and two normal age matched controls. Specimens from both responding and non-responding patients were included. We captured and sequenced a total of 869 single cells from 14 samples, sequencing to an average depth of 4.8 million reads. In a subset of samples (n=7) we also performed bulk RNA-seq (average 1500 cells) for comparison. The sequencing data was of high quality, with an average of 80% mapped reads. We confirmed our ability to accurately quantify transcript levels using ERCC spike-in controls, observing a linear correlation between expected concentration and observed FPKM (fragments per kilobase per million). Single cell RNA-seq revealed vast intratumoral heterogeneity in MDS HSCs that was otherwise missed by bulk RNA-seq, as evidenced by the presence of transcripts variably expressed among cells from the same specimen (Fig. 1A). Despite this intratumoral heterogeneity, single cell transcriptomes were able to completely separate individual MDS patients using principal components analysis and hierarchical clustering, consistent with the known heterogeneity of MDS. MDS HSCs further clustered separately from normal age-matched HSCs, with the top 10% of genes contributing to this separation enriched for Gene Ontology (GO) categories including pathways implicated in MDS biology such as "mRNA splicing," "nonsense mediated decay," and "P53 mediated DNA Damage Response" (all P&lt;1e-9). Unsupervised hierarchical clustering of all pre-treatment MDS HSCs revealed clustering of cells from responders separately from non-responders (Fig. 1B). Differential gene expression analysis identified a cluster of genes (FDR&lt;0.01) enriched for GO categories including "translational termination," "SRP dependent co-translational protein targeting to membrane," and "nonsense mediated decay" (all P&lt;1e-9). Notably, this cluster included 60 ribosomal proteins, all of which were decreased in non-responders (t-test, P&lt;1e-16), with responders demonstrating levels of expression closer to but still lower than normal controls (t-test, P&lt;1e-3). Thus, defective ribosomal biogenesis, a hallmark of MDS pathogenesis, may also contribute to therapeutic resistance. Finally, within each sample we measured the spread of gene expression using dispersion (log[variance/mean]) within bins based on expression levels, defining variable genes as those with a dispersion &gt;1.75 at a mean FPKM &gt;2. The highest number of variable genes were in normal HSCs (mean=141), with the next highest in responders prior to treatment (mean=80), and the least number of variable genes in non-responders prior to treatment (mean=9.5). We speculate that the low number of variable genes in non-responders reflects a higher degree of clonal dominance. All post-treatment MDS HSCs demonstrated a relatively low number of variable genes (mean=25), suggesting that therapy induces clonal selection. In sum, our data illustrate the robustness of single cell RNA-seq to define the intrinsic variability of individual MDS HSCs, implicating perturbed ribosomal biogenesis and transcriptional variability as novel predictors of response to therapy. As we expand our data set with additional patients, we expect to identify additional pathways that mediate therapeutic response and resistance, as well as mutations and variably expressed genes that are selected for during therapy and drive disease progression. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.