Chemotherapy-Free Treatment with Venetoclax, Azacitidine and Flumatinib Induces an Early and Deep Molecular Response in Patients with Newly Diagnosed Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia：What Is the Underlying Mechanism?
Authors: Liu Lingling , Li Mengyun , Cui Qingya , Li Yangzi , Yao Li , Fu Jianxin , Wu Depei , Tang Xiaowen 
Published: December 2024

Background:
Anti-apoptotic BCL-2 family proteins, including BCL-2, MCL-1, and BCL-XL, are recognized as crucial pro-survival factors in various subtypes of acute lymphoblastic leukemia (ALL), particularly in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) (Felix Seyfried, Leukemia. 2022 Apr;36(4):901-912). Previous research has demonstrated that the combination of venetoclax, a selective Bcl-2 inhibitor, with tyrosine kinase inhibitors (TKIs)-based regimens holds promise as a therapeutic strategy in Ph+ALL (Huafeng Wang, Blood Cancer J. 2022 Jan 28;12(1):20.). Our prior studies have shown that the combination of venetoclax, azacitidine, and flumatinib (VAF regimen) induces early and deep molecular responses in patients with de novo Ph+ALL (2023 EHA.P378). The rate of complete molecular remission (CMR) after two cycles of VAF was 78.9%, with a median time to CMR of 14 days (update data).The mechanism underlying the significant synergistic effect of the VAF regimen remains unclear.
Methods:
We initially evaluated the impact of single-agent and combined treatments with venetoclax, azacitidine, and flumatinib on cell viability and apoptosis in vitro, utilizing the Ph+ALL cell line (SUP-B15) and primary patient samples (n=3). To elucidate differential gene expression following VAF treatment, we conducted pathway enrichment analysis and key gene identification through RNA-seq, subsequently analyzed using Gene Ontology Enrichment Analysis (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). We then assessed the mRNA and protein expression of potential key genes via quantitative real-time PCR (qRT-PCR) and western blotting (WB) to validate our findings. To substantiate the role of the identified key gene in the VAF treatment efficacy against Ph+ALL, we employed CRISPR-Cas9 to knock out the gene. Furthermore, we utilized WB to examine alterations in pro/anti-apoptotic protein expression post-VAF treatment, with a particular focus on BCL-2 family members that facilitate cell survival in a BCR-ABL1-dependent manner, including BCL-2, BCL-XL, and MCL-1.
Results:
The monotherapeutic administration of venetoclax, azacitidine, or flumatinib exhibits limited efficacy against Ph+ALL cells, with inhibition rates of cell viability ranging from 5% to 45%. However, VAF regimen markedly amplifies their anti-leukemic efficacy, achieving a 95% inhibition rate. A synergistic interaction was evident in both SUP-B15 cell lines and primary patient samples, as indicated by a combination index less than 1. Comparative analyses using Annexin-V staining and caspase-3 cleavage assays demonstrated that VAF induces a more pronounced pro-apoptotic effect in Ph+ALL cells than any single agent alone (p&lt;0.05).
GO and KEGG pathway analyses highlighted that the key differentially expressed genes were predominantly involved in apoptosis and autophagy pathways. qRT-PCR and WB analysis were employed to assess the expression levels of selected genes, including BBC3 and PMAIP1. PMAIP1, which encodes NOXA, showed significant upregulation at both mRNA and protein levels in VAF-treated samples compared to controls (p&lt;0.05). Furthermore, PMAIP1 knockout attenuated the synergistic suppression of cell proliferation and the pro-apoptotic effects of VAF in SUP-B15 cells, with viability rates of 82.26±1.33% and 19.29±0.65%, respectively (p&lt;0.0001).
The pro-apoptotic protein BIM was notably upregulated by azacitidine or flumatinib monotherapy, as well as by their combination. Conversely, the expression of anti-apoptotic proteins such as BCL-2, BCL-XL, and MCL-1 remained stable under monotherapy conditions, with MCL-1 being the only anti-apoptotic protein downregulated following combination treatment. These findings underscore the contribution of azacitidine and flumatinib to the enhanced pro-apoptotic activity of venetoclax in Ph+ALL.
Conclusion:
Our study demonstrates that VAF regimen exhibits synergistic cytotoxic effects, promoting apoptosis in both a Ph+ALL cell line and primary patient samples. Azacitidine and flumatinib enhance venetoclax-induced apoptosis in Ph+ALL cells by upregulating pro-apoptotic proteins NOXA and BIM, and downregulating the anti-apoptotic protein MCL-1. Our study provided a theoretical foundation for the clinical efficacy of VAF regimen.