Welcome to the MDS 2023 Congress Calendar

Abstract Body

Myelodysplastic syndromes (MDS) originate with the acquisition of genetic alterations by multipotent hematopoietic stem cells (HSC), which confers them selective advantage over their normal counterparts. This advantage enables aberrant clones to overpopulate the bone marrow (BM) of the patients in a process that may start as an indolent condition called clonal hematopoiesis (CH) and evolves over time in the face of diverse forms of stress. The genetic alterations that lead to the development of MDS are well known; however, the mechanisms enabling aberrant cells to take over the BM niche and leading to inefficient hematopoiesis are still poorly understood. To dissect these mechanisms at the onset of MDS, we employed single-cell transcriptomics and chromatin accessibility analyses in BM lineage-negative (Lin^-) CD34⁺ hematopoietic stem and progenitor cells (HSPCs) from patients with clonal cytopenias of undetermined significance (CCUS), a premalignant stage of MDS that presents with CH and cytopenias. Acknowledging that CCUS and MDS are disorders of aging, we used young and elderly healthy donor (HD) BM cells as controls. Our single-cell RNA sequencing (scRNA-seq) analysis showed that, in normal aging, primitive HSCs significantly downregulated genes enriched in proliferation and oxidative metabolism pathways, consistent with increased quiescence in old HSCs, whereas downstream HSPC differentiation was skewed toward the megakaryocytic (Mk) and erythroid (Er) lineages. Single-cell Assay for Transposase-Accessible Chromatin (scATAC-seq) analyses demonstrated that this bias was the consequence of increased Mk priming in the aged primitive HSC compartment. In contrast, similar to our observations in MDS patients (Ganan-Gomez et al. 2022), scRNA-seq and flow cytometry analyses indicated that the HSPC compartment was contracted and myeloid-skewed in patients with CCUS. ScATAC-seq analyses showed that HSCs from CCUS patients lost Mk/Er priming, indicating that differentiation defects in CCUS and MDS are not a consequence of the expansion of myeloid-biased cells but, rather, the result of predominant myeloid differentiation of surviving (advantageous) cells in the context of HSPC loss. To dissect the mechanisms eliciting this advantage and myeloid bias, we performed differential gene expression analyses of our scRNA-seq data. Compared with those of age-matched HDs, HSCs from patients with CCUS significantly upregulated genes enriched in protein translation and mitochondrial metabolism, suggesting a state of cellular activation and growth. Remarkably, CCUS HSCs also upregulated genes involved in the endoplasmic reticulum-phagosome pathway, including the pro-inflammatory cytokine IL-1β, which induces myeloid differentiation in HSCs at the expense of self-renewal. Thus, we hypothesized that proinflammatory signaling induces cell growth and increased myeloid differentiation in CCUS HSCs, generating higher metabolic needs that the cells cover by enhancing their oxidative metabolism. In line with this, downstream lineage-primed progenitor cells also upregulated genes involved in protein synthesis, oxidative metabolism and myeloid fate. Metabolomic analyses in Lin^-CD34⁺ HSPCs by ion chromatography-mass spectrometry confirmed that HSPCs from patients with CCUS had significantly higher levels of intermediates of the tricarboxylic acid cycle and lower levels of glycolysis metabolites than those from elderly HDs. Moreover, a highly oxidative transcriptional profile was also observed in a scRNA-seq analysis of Lin^-CD34⁺ HSPCs from a CCUS patient with high clonal burden whose disease progressed, and was exacerbated at the time of MDS diagnosis, particularly in committed progenitors. This indicated that the metabolic activation was primarily driven by aberrant cells and was an early manifestation of MDS. Collectively, our results suggest that CCUS HSPCs escape aging-related quiescence and epigenetic reprogramming toward emergency hematopoiesis and become metabolically rewired in order to meet higher energy demands and sustain myeloid-skewed differentiation. This activation state may confer them survival advantage over normal HSPCs in the presence of extracellular and cell-intrinsic stressors and aggravates during progression to MDS, potentially contributing to ineffective hematopoiesis.

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Clonal hematopoiesis of indeterminate potential (CHIP) is a common, age-associated condition in individuals who do not have a hematologic malignancy or altered blood counts. CHIP is defined by the presence of clonal, somatic mutations that are found in myeloid malignancies. These mutations are commonly the initiating events for myeloid malignancies, and CHIP is associated with a striking increased risk of hematologic malignancy. Analysis of large cohort studies has revealed that CHIP is also associated with a range of inflammatory disorders as well as solid tumor development. In murine models, Tet2 inactivation in blood cells leads to amplified responses to inflammatory stimuli, acceleration of atherosclerosis, and exacerbation of other disease processes. Clonal hematopoiesis is revealing insights into the initiation of hematologic malignancies, the biology of clonal selection, and the broad consequences of somatic mutations on the function of terminally differentiated blood cells.

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While significant progress has been made to understand the genetic landscape of MDS, less is known about the epigenetic makeup of this disease and how this may impact biology and response to azacitidine (AZA). To address this, we performed genomic, epigenomic and transcriptomic analysis on CD34+ cells from a multicenter cohort of 94 intermediate or higher risk MDS patients treated with AZA who had documented responses. DNA methylation (DNAme) by ERRBS, gene expression (GE) by RNA-seq, mutational profiling, and detailed clinical, cytogenetic and laboratory data were documented. Supervised and unsupervised analyses revealed that aberrant DNAme in MDS is not distributed randomly but rather is highly correlated with disease phenotypes, capturing clinically relevant heterogeneity, beyond what is identified by methodologies used in the past that focused solely on promoter regions. Moreover, this epigenetic information can be harnessed for the development of robust biomarkers predictive of AZA response and integrative approaches combining GE and DNAme data can further improve the predictive performance of these biomarkers, with an AUC score=0.92.

Abstract Body

The term of Clonal Cytopenia of Undetermined Significance (CCUS) has been introduced for describing a condition characterized by persistent cytopenia not caused by another comorbid condition and a somatic mutation in a myeloid neoplasm (MN) driver gene [Steensma et al. Blood 2015]. CCUS has been recognized as pre-malignant disorder by the recent proposals for the revision of the classification of MNs [Arber et al. Blood 2022; Khoury et al. Leukemia 2022].

Prospective studies showed that the risk of progression into MN is affected by clone features, including the number and type of driver mutations and the clone size [Malcovati et al. Blood 2017]. Unsupervised analyses based on mutation profiles identified distinct clusters of patients with CCUS, showing different survival and risk of progression into MN. Notably, subjects with selected clone metrics showed survival and risk of disease progression not dissimilar to those with MN, supporting the notion that a fraction of CCUS may represent an early manifestation of a MN rather than a pre-malignant condition [Gallì et al. Blood 2021]. Recently, genetic mutations and laboratory values have been combined to define a clonal hematopoiesis risk score (CHRS), which provides simple prognostic framework to distinguish subjects at high risk for progression to MN [Weeks et al. Blood 2022].

The clinical context appears to be relevant in shaping the evolutionary trajectories of these disorders. Patients developing CCUS after exposure to chemoradiotherapy for non-myeloid tumors are at higher risk of developing a therapy-related MN, while the significance of clonal cytopenia in patients treated for acute myeloid leukemia warrants interpretation with respect to minimal/measurable residual disease. A recent study in chronic idiopathic neutropenia reported a biased distribution of CH, associated with high relative risk for transformation into MN [Tsaknakis et al. Blood. 2021].

In cases with persistent mild monocytosis, evidence of CH but indeterminate bone marrow features, the definition of clonal monocytosis of undetermined significance (CMUS) has been proposed. Consistently, monocytosis (≥10% and ≥0.5x10⁹/L of the WBC) almost invariably segregated precursor conditions with potential to progress to MDS/MPN [Cargo et al. Blood 2019; Gallì et al. Blood 2021].

The association of cytopenia and CH can be observed in other disorders that are to be distinguished from CCUS. These include aplastic anemia with CH, paroxysmal nocturnal hemoglobinuria, or VEXAS syndrome. In addition, CH can be an incidental finding in well-established cytopenias of other origin, with uncertain clinical implications.

References

Arber et al. International Consensus Classification of Myeloid Neoplasms and Acute Leukemias: integrating morphologic, clinical, and genomic data. Blood. 2022 Sep 15;140(11):1200-1228.

Cargo et al. The use of targeted sequencing and flow cytometry to identify patients with a clinically significant monocytosis. Blood. 2019 Mar 21;133(12):1325-1334.

Gallì et al. Relationship between clone metrics and clinical outcome in clonal cytopenia. Blood. 2021 Sep 16;138(11):965-976.

Khoury et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Myeloid and Histiocytic/Dendritic Neoplasms. Leukemia. 2022 Jul;36(7):1703-1719.

Malcovati et al. Clinical significance of somatic mutation in unexplained blood cytopenia. Blood. 2017 Jun 22;129(25):3371-3378.

Steensma et al. Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes. Blood. 2015 Jul 2;126(1):9-16.

Tsaknakis et al. Incidence and prognosis of clonal hematopoiesis in patients with chronic idiopathic neutropenia. Blood. 2021 Oct 7;138(14):1249-1257.

Weeks et al. Prediction of risk for myeloid malignancy in clonal hematopoiesis. Blood 2022 140 (Supplement 1): 2229–2231.

Background And Aims

The potential of RNA epigenetics-based diagnostics and therapeutics remains largely unexplored. In a recent study (Cheng et al. Nat Commun 2018), we first demonstrated that specific RNA cytosine methyltransferases (RCMTs), namely NSUN1 and NSUN2, directly interact with elongating RNA polymerase II (eRNAPII) and the bromodomain-containing protein 4 (BRD4) to form distinct drug-resistant active chromatin structures (ACS) at nascent RNAs in leukemia cells. The goal of this study is to develop novel RNA epigenetics/NSUN1/2-targeting diagnostics and therapeutics for predicting and overcoming drug resistance in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML).

Methods

We developed new technologies, including 5-ethynyl uridine click chemistry (EC) and proximity ligation rolling cycle amplification (PL-RCA)-coupled confocal microscopy (CM), flow cytometry (FCM) and RNA sequencing (RNA-seq). We also used supercomputing and artificial intelligence (AI) to design NSUN1/2-targeting drug libraries.

Results

Our IHC, PL-RCA/CM and PL-RCA/FCM demonstrated a marked increase in the expression and interactions of the ASC components, which is associated with disease progression and drug resistance in MDS/AML (Figure 1).

Our data demonstrated that RCMTs/ACT-targeting PL-RCA-FCM has a very high sensitivity to detect venetoclax-resistant ACS in pre-treatment clinical MDS/AML specimens and can rapidly predict drug response (Figure 2).

Our supercomputing/AI-assisted NSUN1/2-targeting drug library design and functional screening have identified small-molecule compounds that can disrupt the interactions between NSUN1 and eRNAPII/C-MYC and inhibit the growth of venetoclax-resistant leukemia cells (Figure 3).

Conclusions

We have developed novel RCMTs/NSUN1/2-targeting diagnostic/prognostic tools and small-molecule compounds with a high-promise for predicting and overcoming drug resistance in MDS and AML.

Background And Aims

Despite the large impact of clonal hematopoiesis (CH) on leukemogenesis, the pathogenesis of CH is still not fully elucidated.

Methods

Using a novel single-cell sequencing platform that enables simultaneous detection of mutations and gene expression, we analyzed gene expression of hematopoietic stem/progenitors carrying CH-related mutations from CH(+) cases, which was compared to that of wild-type (WT) cells from both CH(+) and CH(−) cases.

Results

In 10 patients with CH, mutant cells showed an upregulation of genes implicated in cell proliferation and down-regulation of inflammatory responses-related genes. Importantly, WT cells from CH(+) patients showed an enhanced response to proinflammatory cytokines, compared with those from CH(−) donors.　Unexpectedly, ‘WT’ cells　from 3 TET2-CH(+) patients showed significantly upregulated genes related to interferon response and cell proliferation, compared with those from CH(−) donors, suggesting altered BM environments. Of interest, when competitively transplanted with Tet2-knockout (KO) cells, WT cells exhibited enhanced expression of genes related to cell proliferation and interferon signalling, compared with those transplanted with WT cells, implying the non-cell autonomous effects of mutant cells. We next examined single-cell gene expression of whole BM cells. Intriguingly, TET2-mutated cases showed decreased expression of genes involved in heme metabolism in mature erythroid cells, compared with CH(−) cases, suggesting defected erythroid maturation.

Conclusions

Our results suggest that mutant cells in CH(+) BM have non-cell autonomous effects on endogenous WT cells, which might favor positive selection of CH-clones, and also affect the phenotype of endogenous WT cells, contributing to the pathogenesis of CH.