Search

DNA damage resistance is a major barrier to effective DNA-damaging anticancer therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense therapy targeting ILF2, an important DNA damage regulator in MM (Marchesini et. al., Cancer Cell 2017). We continuously treated JJN3 and KMS11 cells with an ILF2-targeting antisense oligonucleotide (ILF2 ASOs) or control non-targeting antisense oligonucleotide (NT ASOs). Whereas KMS11 cells maintained a high level of DNA damage activation and a significantly increased rate of apoptosis after 3 weeks of ILF2 ASOs treatment, JJN3 cells overcame ILF2 ASO-induced DNA damage activation and became resistant to ILF2 ASOs treatment. To evaluate whether continuous ILF2 ASOs exposure could lead to the selection of MM clones intrinsically resistant to ILF2 ASO-induced DNA damage, we performed single-cell RNA seq (scRNA-seq) analysis of JJN3 cells treated with NT or ILF2 ASOs for 3 weeks. Our analysis divided JJN3 cells into 2 main clusters that were independent of treatment (Fig. 1A), suggesting that persistent exposure to ILF2 ASOs did not induce clonal selection. Differential gene expression analysis of NT ASO- and ILF2 ASO-treated cells in each of these clusters revealed that DNA damage resistant ILF2 ASO-treated cells had significantly upregulated oxidative phosphorylation (OXPHOS), DNA repair signaling, and reactive oxidative species (ROS). Consistent with these results, metabolomic analysis of JJN3 cells after long-term exposure to ILF2-ASOs showed a significant enrichment of tricarboxylic acid cycle (TCA) intermediates (Fig. 1B). ILF2-ASO-resistant MM cells were significantly more sensitive to the OXPHOS inhibitor IACS-010759 than ILF2-ASO-sensitive cells were. These data suggest that MM cells can undergo an adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage. We then hypothesized that ILF2-ASO-resistant cells' metabolic reprogramming relies on the repair of DNA damage induced by ILF2 depletion or by the generation of ROS from activated mitochondrial metabolism and that targeting DNA repair proteins involved in these processes overcomes DNA damage resistance. We used a CRISPR/Cas9 library screening strategy to identify DNA repair genes whose loss of function suppresses MM cells' ability to overcome ILF2-ASO-induced DNA damage. Compared with those in NT-ASO-treated cells, DNA2-targeting sgRNAs were significantly depleted after 3 weeks of treatment in ILF2-ASO-treated JJN3 cells but not in ILF2-ASO-treated KMS11 cells. These data suggest that DNA2 is needed to promote resistance to ILF2 depletion. Accordingly, the DNA2 inhibitor NSC105808 (NSC) significantly enhanced ILF2-ASO-induced apoptosis in JJN3 cells. These data gain added significance in light of previous findings that DNA2 is a nuclear and mitochondrial DNA nuclease/helicase that enables cancer cells to counteract the DNA replication stress and mitochondrial oxidative DNA damage induced by DNA-damaging agents. Accordingly, we observed that DNA2 was mainly localized into the mitochondria of MM cells. To dissect the mechanisms of DNA2 inhibition-induced synthetic lethality, we evaluated whether DNA2 activity is essential to maintain activated OXPHOS, which ILF2-ASO-resistant cells require to survive. The quantification of mitochondrial respiratory activity in NT-ASO-and ILF2-ASO-treated MM cells exposed to NSC for 72 hours showed that DNA2 activity inhibition significantly decreased the oxygen consumption rate while increasing ROS production in only ILF2-depleted cells. Transmission electron microscopy analysis showed that NSC-treated ILF2-depleted cells had fragmented mitochondrial cristae structures, whose perturbations affect the OXPHOS system structure and impair cell metabolism. These data suggest that DNA2 is essential to counteract oxidative DNA damage and maintain mitochondrial respiration after MM cells' metabolic reprogramming. In conclusion, our study has revealed a novel mechanism through which MM cells can overcome DNA damage activation. Further studies will clarify whether targeting DNA2 is synthetically lethal in tumors with increased demand of mitochondrial metabolism. Figure 1 Figure 1. Disclosures Konopleva: Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Sanofi: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Agios: Other: grant support, Research Funding; Cellectis: Other: grant support; Rafael Pharmaceuticals: Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Stemline Therapeutics: Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; KisoJi: Research Funding.

Patients (pts) with myelodysplastic syndromes (MDS) have few therapy options. Interventions to improve outcomes should consider strategies that arrest MDS in its early phases, when symptoms are minimal and prolonged survival is expected. To develop prevention strategies that arrest MDS before the disease outcomes become irreversibly dismal, we dissected the molecular and biological mechanisms that maintain MDS in one of its premalignant phases, clonal cytopenia of undetermined significance (CCUS). Recognizing that CCUS is an aging-related disease, we first sought to determine, at the single-cell level, how CCUS affects the transcriptional and epigenetic profile of the aging hematopoietic stem and progenitor cell (HSPC) compartment and overcomes aging-induced degenerative phenotypes. We performed single-cell RNA sequencing (scRNA-seq) analysis of Lin -CD34 + HSPCs isolated from the bone marrow (BM) of 3 young healthy donors (yHDs), 4 elderly HDs (eHDs), and 3 elderly pts with CCUS carrying mutations in common MDS driver genes. We found that the frequencies of hematopoietic stem cells (HSCs) and megakaryocytic (Mk)/erythroid (Er) progenitors were increased at the expense of myeloid (My) progenitors in eHDs as compared with yHDs (Fig. a). In contrast, CCUS pts had a predominant My-biased HSPC distribution (Fig. a). However, immunophenotypic quantification in large cohorts of eHDs and CCUS pts revealed that CCUS pts' BM had significantly fewer CD34 +CD38 - HSC populations and CD34 +CD38 + My progenitors, suggesting that My bias in CCUS results from the aberrant My differentiation of HSCs rather than My cell expansion. Further differential expression analysis among the scRNA-seq clusters showed that, compared with yHD HSCs, eHD HSCs were characterized by a significant upregulation of genes involved in the TNFα-induced activation of NF-κB (e.g., MCL1; Fig. b), which is consistent with previous findings that aged HSCs undergo transcriptional reprogramming to maintain their survival in response to changes in the systemic environment (He et al. Blood 2020). In contrast, CCUS HSCs, compared with eHD HSCs, overexpressed regulators of translation, respiratory electron transport, and mitochondrial translation initiation (Fig. c), which underscores these cells' state of proliferation and metabolic activation and their ability to overcome aging-induced phenotypic alterations. To evaluate whether the aberrant lineage differentiation in eHD and CCUS HSPCs arose from the altered fate determination of HSCs, we performed single-cell assays for transposase-accessible chromatin sequencing to profile chromatin accessibility in sorted HSCs or Lin -CD34 + HSPCs from yHDs, eHDs, and CCUS pts. Consistent with our transcriptomic data, compared with yHD HSCs, eHD HSCs were mostly poised for Mk differentiation, whereas CCUS HSPCs were poised for lymphoid/My differentiation. Indeed, eHD HSCs had an increased activity of transcriptional factors belonging to the NF-κB family and open peaks at the distal elements of genes involved in hemostasis (Fig. d). In contrast, CCUS HSCs were poised to downregulate the expression of genes involved in NF-κB signaling and Mk/Er differentiation (Fig. e). These results suggested that CCUS HSCs are highly metabolically active to maintain My differentiation. Indeed, metabolomic analyses confirmed that intermediates of oxidative phosphorylation were significantly upregulated in CCUS CD34 + cells as compared with eHD CD34 + cells (Fig. f). Further, scRNA-seq analysis of mononuclear cells isolated from the BM of 3 CCUS and 3 eHD samples revealed the widespread upregulation of genes involved in protein processing and mitochondrial metabolism. This analysis also revealed impaired terminal My differentiation despite the HSPC My bias, with decreased frequencies of monocytic cells, and an intriguing expansion of cytotoxic cell subsets in the BM of CCUS pts. In conclusion, our results demonstrate that CCUS HSCs carrying MDS driver mutations evade aging-induced phenotypic degeneration, become metabolically active, and have aberrant My skewing. Our study clarifies the molecular mechanisms underlying MDS initiation and offers an opportunity for early therapeutic intervention. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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

The biological mechanisms of abnormal terminal erythroid differentiation (TED) in MDS-RS and SF3B1 mutations (SF3B1MT) are largely unknown. This gap in understanding, which has dramatically delayed the design of second-line approaches for SF3B1MT patients whose disease has failed hypomethylating agent (HMA) therapy, is primarily due to a lack of studies molecularly characterizing how SF3B1MTaffect distinct stages of erythropoiesis. Here, we dissected at the single-cell level the cellular and transcriptomic changes induced by SF3B1MT in cells undergoing erythroid differentiation and elucidated how HMA therapy can overcome SF3B1MT-defective erythropoiesis. We first analyzed the expression profile of the lineage-negative CD34+ stem and progenitor (HSPC) compartment. Single-cell RNA sequencing (scRNA-seq) analysis of HSPCs isolated from 2 healthy donors (HDs) and 5 untreated MDS-RS patients with SF3B1MT revealed cell clusters driven by the cells' differentiation potential that we defined based on the differential expression of validated lineage-specific transcriptional factors and cellular markers. Whereas the HD HSPCs had equally distributed erythroid/megakaryocyte and myeloid/lymphoid differentiation trajectories, the SF3B1MT HSPCs had a predominant erythroid differentiation route (Fig. 1a). Differential expression analysis revealed that the SF3B1MT HSPCs undergoing erythroid differentiation were characterized by the expression of genes involved in translation, oxidative phosphorylation, and cell cycle progression, which underlines these cells' metabolic and proliferative activation. Consistent with these findings, scRNA-seq of bone marrow (BM) mononuclear cells (MNCs) from the same samples showed that SF3B1MT MNCs had a predominant population of erythroid cells at the expense of B lymphocytes and myeloid cells (Fig. 1b). An analysis of the erythroid cluster distribution inside the erythroid compartment showed that the frequency of early-stage maturation (BFU-E [cluster #18], CFU-E [#9]) was lower in the SF3B1MT erythroblasts than in the HD erythroblasts, owing to a significantly increased frequency of SF3B1MT cells in the latest stages of TED (proerythroblasts [#4]), basophilic normoblasts [#12], polychromatophilic normoblasts [#7], orthochromatic normoblasts [ONs; #1/11], pre-reticulocytes [#13]). These data suggest that SF3B1MT enhance HSPC differentiation towards the erythroid lineage but arrest erythroblasts at the last step of their maturation by inhibiting the transition of ONs to pre-reticulocytes, resulting in the accumulation of TED cells in the BM. Transcriptomic analysis of SF3B1MT ONs revealed a significant upregulation of genes regulating heme metabolism, including those involved in EIF2AK1's response to impaired heme production, and of major effectors of cell cycle checkpoint activation (e.g., CHK1, CDKN1A, GADD45). These data are consistent with previous studies showing that the SF3B1MT-induced defective accumulation of iron in erythroblasts' mitochondria leads to activated cell stress response, cell cycle arrest, and apoptosis. Of note, the growth differentiation factor 11 receptors ACVR1B, TGFBR1, and ACVR1C were not expressed at any step of erythroid differentiation, which challenges this factor's role as a target of luspatercept-induced differentiation of late-stage erythroblasts. To evaluate how HMA therapy can overcome inefficient erythropoiesis, we performed scRNA-seq of HSPCs and BM MNCs isolated from SF3B1MT MDS-RS patients before any therapy and at the time of hematological response to HMA therapy. HMA therapy did not reduce the aberrant differentiation of SF3B1MT HSPCs towards the erythroid lineage but temporarily induced the TED of SF3B1MT ONs into reticulocytes and these cells' release into peripheral blood. Accordingly, blast progression following the initial HMA therapy-induced hematological response coincided with the further expansion of the erythroid-primed cells arising from the earliest stem cell population (Fig. 1c), which suggests that failure to eradicate or genetically correct SF3B1MT stem cells leads to disease relapse. In conclusion, our results elucidate how SF3B1MT molecularly affect distinct stages of erythropoiesis and have implications for developing approaches that achieve lasting hematological remission in patients with MDS-RS. Disclosures Garcia-Manero: Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Consultancy; Merck: Research Funding; AbbVie: Honoraria, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Novartis: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amphivena Therapeutics: Research Funding; Acceleron Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Research Funding. Colla:Amgen: Other: Unspecified.

Myelodysplastic Syndromes (MDS) are a group of heterogeneous stem cell disorders that result in inefficient hematopoiesis. Although the genetic and cytogenetic landscapes of MDS have been well characterized (Papaemmanuil 2013, Sperling 2017), little is known about the differentiation abnormalities that underlie the MDS phenotype. Gaining insights on how different hematopoietic stem and progenitor cell (HSPC) types contribute to MDS is essential for the design of new targeted therapies to supplement the currently limited effective therapeutic options. To understand the contribution of different cell types to the pathogenesis of MDS, we analyzed the expression profile of the Lin-CD34+ HSPC compartment at the single-cell level. Single-cell RNA-sequencing (scRNA-seq) analysis of HSPCs isolated from 2 MDS patients and 2 age-matched healthy donor samples revealed distinct cell clusters driven by the sample type and the differentiation potential of the cells. To annotate the specific subsets of HSPCs in each cluster, we scored them on the basis of previously reported population-specific gene signatures (Laurenti 2013, Psaila 2016, Van Galen 2019). Whereas CD34+ cells from the 2 healthy donor bone marrow (BM) samples largely overlapped with each other and displayed 2 distinct erythroid/megakaryocytic (Er/Mk; cluster 3) and lympho/myeloid (clusters 2, 5) differentiation trajectories in line with the current view of hematopoiesis, CD34+ cells from the 2 MDS BM samples clustered separately and showed predominantly myeloid differentiation routes (Fig a). Importantly, differential expression analysis of the HSPCs from the 2 MDS samples (Fig b) showed that cells residing atop of the HSPC hierarchy retained the transcriptional profile of immature HSCs in one of the samples (clusters 2, 4), while they were characterized by the expression of genes involved in the differentiation of myelo/lympho multipotent progenitor cells (clusters 0, 1) in the other. However, pseudotime analysis of the HSPCs' transcriptional dynamics showed that, despite the distinct differentiation state of the early hematopoietic cells in each group, the differentiation trajectories of those cells converged at the late myeloid progenitor state (clusters 3, 5, 6). These results suggest that, although the earlier HSC architecture is heterogeneous across MDS patients, the more differentiated myeloid progenitor compartment is similarly compromised and is responsible for the clinical phenotypes of MDS. To confirm differential cell-type contributions to the MDS hierarchy, we immunophenotyped BM samples from 123 untreated patients using multicolor flow cytometry. We applied principal component analysis and logistic regression to group samples based on their cellular compositions. Our mathematical classifier stratified patients in 2 groups, which had markedly different cellular repertoires consistent with our scRNA-seq results (Fig c). Patients with different MDS stem cell hierarchies did not present with significantly different clinical characteristics at diagnosis. These data confirm that different abnormal hematopoietic trajectories converge in the myeloid bias typically observed in MDS hematopoiesis. Next, we exome-sequenced mononuclear cells and T-cells from 45 untreated MDS patients and identified high-confidence somatic mutations in known oncogenes and/or leukemia driver genes. The median number of mutations (n=3) was not significantly different between MDS groups 1 and 2. We identified 4 genes that were differentially mutated in the 2 MDS architectures (Fig d), which suggested that certain mutations may predispose for a specific HSPC phenotype. However, mutation specificity could not fully account for the origin of the 2 differentiation architectures, which were independent on the genetic background in most patients. In conclusion, we demonstrated that MDS are sustained by distinct and recurrent abnormal HSPC differentiation hierarchies. Diverse cellular compositions suggest that different cell-type specific signaling pathways maintain the disease in each group of patients. Our work shows that the characterization of the cellular diversity in the hematopoietic compartment can be used as a biomarker to stratify MDS patients, and warrants further studies to predict the intrinsic vulnerabilities of the cells involved in the pathogenesis and maintenance of MDS in a patient-specific manner. Figure Disclosures 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. Colla:IONIS: Other: Intellectual property and research material IONIS); Amgen: Research Funding; Abbvie: Research Funding.