ZBTB7A modulates response to BCL-2 inhibition by venetoclax in acute myeloid leukemia (AML) Free

Venetoclax, a selective BCL-2 inhibitor, in combination with azacytidine has become a standard-of-care treatment for older, and/or patients with newly diagnosed acute myeloid leukemia (AML); however, the emergence of drug resistance remains a major clinical challenge, limiting the long-term efficacy of this drug. Here we identified ZBTB7A, a member of the POK family of transcription factors involved in cell proliferation, differentiation, and development, as a key regulator of venetoclax response in AML.

We established sgRNA lentivirus library targeting approximately 900 genes that were identified as being overexpressed in venetoclax-resistant primary AML samples in the BEAT AML study and performed a CRISPR-Cas9 knockout (KO) screen to identify the direct effect on venetoclax response. The screen identified ZBTB7A amongst the top 3 hits enhancing venetoclax sensitivity across three AML cell lines. Dose response analysis confirmed that CRISPR-Cas9 depletion of ZBTB7A significantly enhanced sensitivity to venetoclax in three AML cell lines (MOLM-13, MV4-11, and OCI-AML3), increasing sensitivity by 4- to 7-fold compared to wild-type controls. Conversely, overexpression of ZBTB7A conferred significant resistance to venetoclax. We further show that ZBTB7A deletion also significantly enhanced venetoclax sensitivity in vivo. NRGS immunodeficient mice engrafted with ZBTB7A KO MOLM-13 cells were administered venetoclax orally (50 mg/kg). The proportion of leukemia cells was significantly reduced in both bone marrow and peripheral blood following venetoclax treatment, and overall survival was markedly prolonged (P = 0.0049), compared to mice engrafted with wild-type cells.

To investigate mechanisms by which ZBTB7A depletion enhances sensitivity to BCL-2 inhibition, we examined the expression of BCL-2 family proteins. Notably, in ZBTB7A KO AML cells, venetoclax treatment led to enhanced suppression of anti-apoptotic proteins, including MCL-1, BCL-2, and BCL-xl, thereby shifting the balance toward pro-apoptotic signaling. Interestingly, we observed a more rapid degradation of MCL-1 protein through proteasome-mediated pathways following venetoclax treatment in ZBTB7A KO AML cells.

RNA-seq analysis revealed that mitochondrial function and oxidative phosphorylation (OXPHOS) were among the top upregulated pathways in ZBTB7A-deficient AML cells. Venetoclax has been reported to suppress oxidative phosphorylation (OXPHOS) in AML cells by inhibiting uptake and metabolism of amino acid. Seahorse metabolic analysis revealed a marked increase in OXPHOS in ZBTB7A KO cells relative to controls, which is consistent with our RNA-seq data. Importantly, the inhibition of OXPHOS by venetoclax treatment was significantly enhanced in ZBTB7A KO AML cells compared to control cells (P < 0.001). Seahorse substrate oxidation stress tests using selective inhibitors of glycolysis, glutaminolysis, and fatty acid oxidation indicated that glycolysis was the primary contributor to OXPHOS in control AML cells, but that ZBTB7A KO cells exhibited increased sensitivity to inhibition of glutaminolysis inhibition in addition to glycolysis. These results indicate an important role of ZBTB7A in regulating mitochondrial energy metabolism, and enhancing its sensitivity to inhibition by venetoclax, associated with a shift toward a dual dependency on glycolysis and glutaminolysis for energy production.In conclusion, our data demonstrate that deletion of ZBTB7A enhances sensitivity to the BCL-2 inhibitor venetoclax, potentially through downregulation of anti-apoptotic proteins, as well as through modulation of energy metabolism. Given the observed upregulation of ZBTB7A in venetoclax-resistant AML cells, ZBTB7A may represent a promising therapeutic target for overcoming venetoclax resistance.