Venetoclax Enhances the Efficacy of the DA Treatment Regimen By Targeting Quiescent Leukemia Stem Cells
Authors: Zhuo Nanfang , Liu Yongjing , Yao Yiyi , Sun Jie , Jin Jie , Huang Jinyan , Wang Huafeng 
Published: December 2024

Background
Our previous research introduced an effective induction treatment of venetoclax plus daunorubicin and cytarabine (DAV regimen), which showed high CR rates of 91% after one cycle of induction therapy in newly diagnosed young adult patients with AML. Of note, adding venetoclax to the conventional DA induction regimen resulted in a higher complete remission rate than the DA regimen, the underlying mechanisms are still under investigation. We aimed to observe differences in the effects of two therapeutic strategies (DA and DAV) at the transcriptome and genome levels and explore factors contributing to variations in treatment response.
Methods
Acute myeloid leukemia (AML) bone marrow samples from the First Affiliated Hospital of Zhejiang University School of Medicine were analyzed pre- and post-treatment using targeted panel sequencing and whole transcriptome RNA sequencing. Of the 86 RNA-seq samples collected, 84 were quality-filtered and categorized into four groups based on sampling time points (before/after therapy) and treatment regimens (DA/DAV). Panel sequencing included patient-matched normal controls. We performed differential expression and gene set enrichment analyses (GSEA) to identify biologically significant gene sets. Single-cell RNA-seq data from Van Galen et al. were utilized for deconvolution analysis on bulk samples, with gene set-based scoring was performed by mean expression levels. Finally, we verified our findings using cell viability assay to assess the proliferation of cells, as well as using flow cytometry to analyze apoptosis and current state of cell cycle between DA and DAV regimens in vitro.
Results
To clarify disparities in treatment responses, we conducted pre-treatment GSEA analysis on CR and non-CR samples, categorized by treatment strategies. Focusing on pre-treatment differences due to post-treatment tumor cell scarcity and expression convergence, we found non-CR samples under DA therapy showed reduced OXPHOS, contradicting with existing literature. This, along with decreased mitochondrial activity, hinted at LSC characteristics, including a quiescent state and enhanced mitochondrial autophagy, evidenced by reduced proteasome gene activity and upregulation of PINK1 and ULK1. The need for additional evidence is highlighted due to transcriptional variations and intervening factors.
Gene sets reflecting LSC traits were analyzed, revealing higher scores in non-CR DA samples (5 in top 6 of the LSC4 gene set). Deconvolution of RNA-seq data using a leukemia single-cell dataset as a reference showed significant HSC-like score differences between CR and non-CR groups pre-DA treatment, not observed in the DAV group. This suggests LSC traits significantly affect DA therapy efficacy, an effect DAV may mitigate.
DAV therapy showed stronger inhibition of the HOXA gene family which is linked to LSCs, and a more pronounced promotion of the cell cycle in CR samples compared to DA therapy. In both non-CR samples from the two groups, the DA therapy resulted in a non-significant increase in cell cycle activity, while the DAV non-CR samples initially exhibited high cell cycle activity, leading to a slight decrease after treatment. This suggests a potential promoting effect of venetoclax on the cell cycle, but further evidence is required for confirmation.
LSCs treated with NC, VEN, DA, DAV showed reduced viability and increased apoptosis in DAV groups, indicating higher efficacy of the combination of daunorubicin, cytarabine, and venetoclax in LSCs. Apoptosis rates were significantly higher post-DAV treatment, and venetoclax-induced LSCs from quiescence to increased cell cycle entry, apoptosis, and growth in vitro.
Conclusion
We show evidence that venetoclax mediates a functional interplay between stem cell quiescence and activity. Non-CR samples from DA treatment group exhibited more HSC-like features and might reduce treatment efficacy, while DAV treatment could counteract this influence. In comparison to the DA regimen, the DAV therapy exhibited more potent inhibition of the HOXA gene family, whose high expression is known to be linked to LSCs. Venetoclax inhibits cell viability and promotes cell apoptosis as well as cell cycle entry in LSCs, providing new insight in eradication of quiescent leukemia stem cells.