Single-cell insights into acute myeloid leukemia treated with venetoclax-based therapy

Abstract Introduction Acute myeloid leukemia(AML) is a heterogeneous malignancy marked by impaired myeloid differentiation and resistance to apoptosis. Venetoclax, a selective BCL-2 inhibitor that restores apoptotic signaling, has shown improved outcomes when combined with hypomethylating agents in older or unfit patients; however, many remain refractory or experience relapse. We used single-cell RNA sequencing(scRNA-seq) to compare bone marrow samples from patients with AML before and during venetoclax-based therapy with those from healthy controls, focusing on cellular type diversity and gene expression dynamics. Methods We analyzed 10 bone marrow samples from patients with AML treated with venetoclax plus azacitidine or decitabine, as well as from healthy donors at the University of Iowa. These included samples from 3 healthy controls, 4 responders (1 diagnostic[RD1] and 4 during treatment[RT1-RT4]), and 2 non-responders(1 diagnostic[NRD1] and 1 during treatment[NRT1]). scRNA-seq was performed using 10X Genomics 3' v3 chemistry, sequenced on an Illumina NovaSeq 6000, and processed with Cell Ranger v8.0.0. We excluded cells with <100 or >7,500 genes or >75% mitochondrial reads. Seurat (v5.0.1) was used for normalization, scaling, integration, clustering(resolution = 0.1), and UMAP reduction using the top 30 principal components. Cell type annotation used SCINA with references from Human Cell Atlas ((https://doi.org/10.1016/j.exphem.2018.09.004)). We focused on CD34+ cells to study stem cells and early hematopoietic progenitors. Results We identified various hematopoietic populations, including hematopoietic stem cells(HSCs), multilineage-primed(Multi-Lin) cells, monocyte-dendritic progenitors(MDP-1 and MDP-2), megakaryocyte-erythroid progenitors(MEPs), and other early progenitors, as well as mature blood cells. Control samples showed consistent distributions with expected proportions of early progenitors, while AML samples displayed distinct cellular profiles. RD1 sample was dominated by a high percentage of blasts(47%) and extensive expansion of MDP-2 cells(~90%). Refractory samples(NRD1 and NRT1) exhibited elevated blasts(60% and 12%) and significant expansion of immature T progenitors(pre-T cells, 33–49%). RT1 had residual blasts(7%) and showed increased numbers of Multi-Lin cells(34%) and MEPs(7%), which may indicate the persistence of immature leukemic clones. RT2, RT3, and RT4 were negative for blasts, with recovery of diverse hematopoietic populations, notably an expansion of MDP-2(13–70%) and lymphoid cells, consistent with marrow recovery. In differential gene expression analysis, a consistent finding across multiple cell types was the downregulation of FKBP5, a glucocorticoid-responsive gene involved in stress signaling and resistance to apoptosis. In HSCs, we observed BCL2 downregulation along with upregulation of pro-apoptotic genes(BAX, BAK, CASP7, CASP9). There was suppression of STAT5(growth-promoting) and SMAD3(growth-inhibitory) signals in HSCs, which may indicate disrupted stem cell signaling and early commitment or exhaustion. Downregulation of PTGER4, IL3RA, and IL12RB2 suggested decreased signaling through pro-survival and proliferative cytokine pathways. Additionally, ribosomal protein downregulation was observed, a phenomenon commonly associated with apoptosis, quiescence, or differentiation, where protein synthesis was actively suppressed. CD14 was upregulated in HSCs and MDP cell types, indicating monocytic skewing. In MDP-1 cells, heat shock proteins(HSPA1A and HSPA1B) were downregulated, and DUSP7(a MAPK phosphatase) was suppressed, pointing to impaired stress-response signaling. Changes in the expression of RASA1, RASA2, and MAP3K family genes suggest disrupted Ras-MAPK pathway activity. In MDP-2 cells, decreased CD1B coupled with increased CD14 expression marked a shift away from dendritic differentiation toward an aberrant monocytic identity. Conclusion Venetoclax-based therapy is associated with transcriptional remodeling across different cell types in bone marrow. Downregulation of pro-survival and stress-related genes and upregulation of differentiation and apoptotic programs were observed. Distinct transcriptional programs in MDP-1 vs. MDP-2 suggest functional remodeling in different lineages during therapy. These data provide insight into how venetoclax can reshape the bone marrow microenvironment in AML and may help identify early markers of treatment response.