Abstract
Thrombocytopenia affects a substantial proportion of patients with myelodysplastic syndrome (MDS). Mechanisms for aberrant thrombopoiesis in MDS are not fully understood and treatment options remain limited. It is well documented that hypomethylating agents (HMAs), the primary frontline therapy for MDS, can significantly improve platelet counts during early treatment cycles in MDS. Notably, this early platelet response to HMA is significantly associated with favorable patient outcomes.
To investigate altered thrombopoiesis in MDS and the impacts of therapy, we analyzed longitudinal platelet count data from baseline to 100 days following single-agent HMA therapy in a cohort of MDS patients (n=319). Our results indicate that, among patients with severe thrombocytopenia (baseline platelet count <30 × 10⁹/L), HMA induced significantly greater platelet recovery at the end of C2 in patients harboring TET2 mutations (median increase=105 × 10⁹/L) compared to those without mutant TET2 (median increase=31 × 10⁹/L, P<0.01). This observation was not made for other common somatic mutations of MDS, including TP53, ASXL1, DNMT3A, or RUNX1, in the same cohort. In contrast, among patients with higher baseline platelet counts (>30 × 10⁹/L), enhanced platelet recovery was not observed in TET2-mutant patients. These findings suggest that TET2 mutation is associated with improved thrombopoiesis in MDS with severe thrombocytopenia.
To explore related biological mechanism, we utilized a Tet2 knockout (Tet2-KO) MDS mouse model and administered azacitidine (AZA) (10 mg/kg, intraperitoneally). Complete blood count (CBC) revealed that one treatment cycle of AZA (daily for five days) resulted in a significantly greater increase in platelet count in Tet2-KO mice compared to control Cre mice. However, this early platelet response was no longer observed after the 2nd treatment cycle. These results are consistent with the clinical observations in MDS and support the use of Tet2-KO mice as a model for further investigation of HMA-induced platelet responses.
Flow cytometry analysis of bone marrow (BM) samples isolated from mice showed that, although AZA induced a stronger peripheral platelet response in Tet2-KO mice, the increase in BM CD41⁺ megakaryocytes (Mk) was similar between Tet2-KO and Cre mice. Additionally, the frequencies of Mk progenitors (Lin-/cKit+/Sca1-/CD41+) and Mk-biased hematopoietic stem cells (CD41+/Lin-/cKit+/Sca1+/CD48-/CD150+/Flt3-) in BM were not significantly affected by AZA treatment. In contrast, AZA significantly increased the proportion of BM CD41⁺ Mk cells with high polyploidy in Tet2-KO mice. Together, these findings suggest that the HMA-induced platelet response in MDS is primarily driven by enhanced Mk maturation, rather than early Mk lineage determination.
To confirm these findings, we performed single-cell RNA sequencing (scRNA-seq) in CD41⁺ BM Mk cells isolated from Tet2-KO and Cre mice after one cycle of AZA or vehicle control treatment. This analysis revealed a marked increase for the frequencies of mature Mk cells, particularly the most mature platelet-producing megakaryocytes (Plt-Mk), in the BM of AZA-treated Tet2-KO mice. Furthermore, differentially expressed genes (DEGs) in Plt-Mk cells were significantly enriched in pathways known to promote Mk maturation and platelet production, including G-protein and vesicle trafficking signaling. In contrast, early-stage Mk populations and their gene expression were largely unaffected by AZA treatment in Tet2-KO mice. Bulk RNA-seq in CD41⁺ Mk made similar findings and demonstrated that the Mk gene activation induced by AZA occurred only in early treatment, explaining why platelet recovery in HMA therapy was specifically observed in early cycles and lost later. Moreover, whole-genome bisulfite and ATAC-seq identified chromatin regions specifically activated by AZA in the BM Mk of Tet2-KO mice.
In summary, this study represents the first systematic biological investigation of HMA-induced platelet responses in MDS. We identify a potential association between the TET2 mutation and early platelet recovery in HMA therapy. Moreover, we demonstrate that this response is primarily driven by the promotion of late-stage BM Mk maturation. The identification of relevant molecular pathways through Mk-specific scRNA and bulk-seq provides valuable insights for future research and highlights potential therapeutic targets to enhance HMA efficacy and alleviate thrombocytopenia in MDS.
