Unveiling ZMAT2 and SMARCD3 As Therapeutic Targets to Mitigate Aberrant Hematopoiesis in MDS

Introduction

The molecular mechanisms underlying the ineffective hematopoiesis that characterizes patients with myelodysplastic syndromes (MDS) remain largely unknown, and effective treatments remain elusive. To deepen our understanding of the biological mechanisms driving aberrant hematopoiesis in these patients, we have undertaken a comprehensive study to characterize the transcriptional alterations that occur during the early stages of myeloid differentiation.

Methods

In this study, we performed RNA sequencing on HSCs, CMPs, GMPs, and MEPs, which were isolated via fluorescence-activated cell sorting from elderly healthy donors (n=12, median age ~67) and untreated MDS patients diagnosed with either unilineage or multilineage dysplasia (n=18, median age ~71, normal karyotype, 0-4% blasts in the bone marrow, IPSS-R: [very] low). To identify alterations linked to aberrant hematopoiesis, we employed an extensive computational analysis encompassing differential expression (DE), trajectory analysis, and gene regulatory network (GRN) analysis. These computational findings were subsequently validated in vitro through liquid differentiation and flow cytometry assays, where CRISPR-Cas9 inhibition (CRISPRi) and shRNA knock-down systems were used in both cell lines and primary MDS cells.

Results

DE analysis between HSPCs from MDS patients and healthy cells demonstrated that the genes showing altered expression in MDS were cell-type specific, highlighting an enrichment in apoptosis and cell cycle, as well as reduced oxidative stress response in all MDS subpopulations. To gain deeper insights into the transcriptional dynamics taking place across early hematopoiesis, we developed a statistical model that identified 579 and 711 genes with altered expression trajectories in MDS during early myeloid (HSC-CMP-GMP) and erythroid (HSC-CMP-MEP) differentiation, respectively. Importantly, the majority of these genes were not detected through conventional DE analysis, underscoring the value of sophisticated algorithms in uncovering potential contributors to the disease.

Functional ontology analysis suggested that genes exhibiting progressively lower expression levels in MDS were associated with the activation and functionality of myeloid cells. For instance, negatively altered genes across early granulocyte-monocyte differentiation, including MIF, CEACAM6 and XRCC6, were enriched in neutrophil activation and degranulation, whereas negatively altered genes in early erythropoiesis, including HBA1, HBA2 and HBQ1, were involved in gas transport. Additionally, GRN analysis identified transcription factors (TFs) that could be driving transcriptomic dysregulation in MDS by aberrantly regulating genes with altered trajectories. Among these TFs, ZMAT2 and SMARCD3 emerged as key regulators, as they could be potentially altering the expression of critical genes: ZMAT2 by downregulating key genes for myeloid differentiation (HBA1, HBA2, and MIF), and SMARCD3 by upregulating genes involved in drug resistance (MEG3, FKBP10, and ARAP3). Functional validation through CRISPRi of ZMAT2 in cell lines resulted in enhanced myeloid differentiation capacity, a finding that was corroborated by preliminary results in CD34⁺ cells from MDS patients. Similar results were observed upon knockdown of SMARCD3 in primary CD34⁺ cells from MDS patients. These results suggest that ZMAT2/SMARCD3 could serve as promising therapeutic targets in MDS.

Conclusion

Our study provides a novel and comprehensive approach to identifying transcriptional alterations associated with MDS pathogenesis. By highlighting specific master regulators as potential therapeutic targets, we open new avenues for the development of targeted treatments aimed at ameliorating ineffective hematopoiesis in MDS patients.

Pierola:Abbvie, BMS, Jazz Pharma, Novartis, Syros: Speakers Bureau; Astellas, BMS, Jazz Pharma, Syros: Consultancy; AstraZeneca: Research Funding. Diez-Campelo:BLUEPRINT MEDICINES: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; KEROS: Honoraria, Membership on an entity's Board of Directors or advisory committees; ASTEX/OTSUKA: Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL TO MEETINGS; CURIS: Membership on an entity's Board of Directors or advisory committees; SYROS: Membership on an entity's Board of Directors or advisory committees; HEMAVAN: Membership on an entity's Board of Directors or advisory committees; AGIOS: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board fees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Other: Travel reimbursement. Valcarcel:AbbVie: Consultancy, Other: Meeting and travel accommodation; Agios: Honoraria, Other: Meeting and travel accommodation, Speakers Bureau; Gebro: Honoraria, Speakers Bureau; Grifols: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Meeting and travel accommodation, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Meeting and travel accommodation, Speakers Bureau; Bristol Myers Squibb/Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Meeting and travel accommodation, Research Funding, Speakers Bureau; Astellas: Consultancy, Honoraria; Janssen: Honoraria, Speakers Bureau; Jazz Pharmaceuticials: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Meeting and travel accommodation, Speakers Bureau; Kite/Gilead: Consultancy, Honoraria, Speakers Bureau; MSD: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Meeting and travel accommodation, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria, Other: Meeting and travel accommodation, Speakers Bureau; Servier: Membership on an entity's Board of Directors or advisory committees; SOBI: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Meeting and travel accommodation, Speakers Bureau; TAKEDA: Consultancy, Honoraria, Speakers Bureau.