Harnessing redox homeostasis and cell death pathways for targeting pediatric T-cell acute lymphoblastic leukemia (T-ALL).

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of ALL. Despite significant advances in treatment, approximately one out of five patients develop primary or secondary resistance to current therapies, which include glucocorticoids as a key component; indeed, the clinical outcome depends on the initial response to glucocorticoids. To provide novel therapeutic options to these patients, our laboratory is exploring strategies to sensitize T-ALL cells to Venetoclax (ABT-199), a specific BCL-2 inhibitor employed in other hematological malignancies such as chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML), but ineffective in T-ALL cells. We recently showed that inhibition of mTOR, an oncogenic pathway activated in most T-ALL cases, increases Reactive Oxygen Species (ROS) levels in T-ALL cells, leading to apoptotic cell death. Consequently, we tested whether mTOR inhibition synergizes with Venetoclax in inducing T-ALL cell death. In vitro experiments showed that the combination of the mTOR inhibitor Everolimus with Venetoclax caused a synergistic increase in cell death in T-ALL cell lines, patient-derived xenograft (PDX) cells, and primary samples from T-ALL patients compared to the drugs used as single agents. Next Generation Sequencing (NGS) experiments revealed significant enrichment in genes belonging to the integrated stress response (ISR) pathway in Everolimus-responsive samples. These data were confirmed at the protein level by analyzing the induction of eIF2α phosphorylation and the increase in the expression of the pro-apoptotic transcription factor CHOP, both markers of ISR activation. Pre-treatment of T-ALL cell lines and PDX cells with ISRIB (an inhibitor of ISR) significantly blunted cell death induced by Everolimus alone or in association with Venetoclax, demonstrating that the ISR has a pro-apoptotic role in this system. The effect of ISRIB was phenocopied by silencing ATF4, the master transcriptional activator of the ISR. Moreover, Everolimus plus Venetoclax significantly reduced the leukemic burden in mice inoculated with T-ALL PDX cells compared to vehicle or the drugs used as single agents. Taken together, our findings demonstrate that Everolimus plus Venetoclax treatment unleashes a robust apoptotic response in T-ALL cells through the activation of the ISR, providing proof of principle for new pharmacological approaches to target refractory T-ALL.