Combined Inhibition of CXCR4 Signaling and System x_c^- Transporter Activity Induces Synthetic Lethality in T-ALL Cells By Suppressing Gsh and Inducing Ferroptosis

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. Prior studies have established that most cases pf T-ALL are addicted to CXCR4 signaling. Indeed, strong preclinical data demonstrating therapeutic activity of BL-8040, a potent CXCR4 antagonist, have led to a clinical trial of BL-8040 in combination with nelarabine for patients with relapsed/refractory T-ALL (NCT02763384). However, the molecular mechanisms by which CXCR4 blockade induces T-ALL cell death are unknown. Using a human T-ALL xenotransplantation model, we previously reported that treatment with BL-8040 killed T-ALL cells through a non-apoptotic mechanism. Transcriptome sequencing revealed that BL-8040 induced alterations in genes involved in oxidative phosphorylation and carbohydrate metabolism. Indeed, seahorse experiments show that BL-8040 markedly reduced both oxidative phosphorylation and glycolysis. However, metabolic tracing studies using ¹³C-labeled glucose show that BL-8040 treatment does not have a major effect on the contribution of glucose to either glycolysis or the citric acid cycle. Instead, the major alteration observed is the reduced entry of glucose into the pentose phosphate pathway (PPP). A major function of the PPP pathway is to generate NADPH, which regulates reactive oxygen species (ROS) by producing reduced glutathione (GSH). Indeed, BL-8040 treatment resulted in a significant decrease in the ratio of reduced glutathione to oxidized glutathione. Together, these data suggest that BL-8040 induces oxidative stress by inhibiting GSH production.

One mechanism utilized by cancer cells to regulate GSH levels and oxidative stress is the system x_c^- amino acid antiporter that mediates the exchange of extracellular L-cystine and intracellular l-glutamate across the plasma membrane, resulting in the production of GSH and oxidative protection. We measured L-cystine levels in the media of T-ALL cells cultured for 24 hours with or without BL-8040. A significant decrease in L-cystine in the media was observed. These data, along with increased expression of the x_c^- transporter (SLC7A11), suggested that increased system x_c^- activity was compensating for the loss of GSH induced by BL-8040. To test this possibility, we cultured T-ALL cells in L-cystine deficient media. Loss of L-cystine in the media resulted in a modest decrease in T-ALL cell viability that was markedly increased, in a synergistic fashion, upon treatment with BL-8040. Interestingly, caspase 3 was not activated, suggesting that, similar to in vivo results, BL-8040 induces a non-apoptotic cell death. This observation, coupled with the reduction in GSH, suggested the hypothesis that BL-8040 induces ferroptosis. Consistent with the hypothesis, treatment of T-ALL cells with ACXT-3102, a novel system x_c^- inhibitor, significantly enhanced BL-8040 killing of T-ALL cells in vitro. Collectively, these data suggest that T-ALL cells are sensitive to perturbations of the glutathione axis. Combined inhibition of CXCR4 signaling and system x_c^- activity exploits this vulnerability and presents a promising new therapeutic approach for T-ALL.