Protein degradation of MYC/GSPT1 is a novel treatment modality for aggressive T-cell acute lymphoblastic leukemias

Abstract

Abstract Background: T-cell Acute Lymphoblastic Leukemia (T-ALL) is characterized by the aggressive proliferation of immature T-lymphoblasts. T-ALLs rely on the NOTCH1-MYC signaling pathway for proliferation and resistance, as NOTCH1 mutations are present in the majority of T-ALL subtypes. The NOTCH1 oncogene is essential to T-ALL leukemogenesis, while the MYC oncogene, a direct target of NOTCH1, encodes a transcription factor, executing transcriptomic control for proliferation and maintenance, providing rationale to target MYC in T-ALL. We recently discovered a feedforward loop between MYC and GSPT1, the key translation termination factor, where MYC regulates transcription of GSPT1 while GSPT1 recognizes the stop codon of MYC, promoting its translation. GT19715, the first-in-class MYC/GSPT1 degrader, induces integrated stress response and inhibition of the TCA cycle, resulting in profound cell death and promising activities in MYC-driven cancers (Nishida et al biorxiv 650490, in revision). T-ALLs specifically depend on BCL-XL except early T-cell precursor (ETP) subtype, where BCL-2 plays essential roles for its survival, providing opportunities for potential combinatorial approaches with targeting MYC/GSPT1 with BH3 mimetics. Methods: We treated T-ALL cell lines (CCRF-CEM, P12-ICHIKAWA, Loucy, DND41, KOPTK1, Jurkat and HPB-ALL.) with GT19715 and determined apoptosis induction and absolute live cell numbers by annexin V/DAPI assay with counting beads. We used O-propargyl-puromycin (OPP) flow cytometry to determine nascent protein synthesis in T-ALL cells. Single-cell mass cytometry (CyTOF) analysis in patient-derived xenograft (PDX) CU76 T-ALL cells was performed to characterize CU76 cells and investigate the impact of MYC/GSPT1 degradation. NSG mice were injected with CU76 cells and treated with vehicle or GT19715 (N = 5 per group) to determine the activities of GT19715 in vivo. Peripheral blood samples were collected to determine leukemia burden and survival analysis using the Kaplan-Meier method with a log-rank test. Lastly, T-ALL cells were treated with GT19715 in combination with BCL-2, BCL-XL and MCL-1 inhibitors to explore potential promising combination strategies. Bliss synergy analyses were performed to determine synergistic anti-T-ALL activities. Results: All but HPB-ALL cells were sensitive to GT19715 with IC50 values of less than 5 nM; HPB-ALL cells were relatively resistant to GT19715 with IC50 values > 80 nM. Primary CD1a+ T-ALL cells exhibited significantly increased nascent protein translation levels compared to normal CD3+ T-cells (median fluorescence intensity: 9,961 ± 625 vs 1,316 ± 198 for T-ALL vs CD3+ T-cells, respectively, P < 0.0001). GT19715 substantially reduced nascent protein synthesis determined by OPP incorporation in DND and P12 ICHIKAWA cells. CyTOF analysis identified subclassification of CU76 to be non-ETP, TAL1 double positive-like subtype based on high CD4, CD8a, CD2, CD5 and CD7 levels, and low CD33, CD34, HOXA9 and STAT5 levels (Pölönen et al Nature 2024). In an in vivo CU76 PDX experiment, circulating human CD45+ cells increased rapidly at week 1 in the vehicle group, and were significantly reduced in GT19715-treated group compared to the vehicle-treated group (12.6% vs 0.009%, P = 0.014). The reduction of circulating human CD45+ cells was sustained until week 14, and GT19715 significantly prolonged survival of mice compared to vehicle (median OS 14 vs 95 days [680% survival prolongation], P = 0.0021). Intriguingly, GT19715 reduced BCL-2 and MCL-1 but not BCL-XL levels in CU76 cells, suggesting persistent T-ALL cells with sustained BCL-XL after MYC/GSPT1 degradation. Indeed, ABT-263, a BCL-2/BCL-XL inhibitor, in combination with GT19715 induced synergistic cell death. Conclusion: MYC/GSPT1 protein degradation is highly active in T-ALL cells in vitro, with substantial reduction of MYC and GSPT1, leading to disruption of nascent protein synthesis. GT19715 profoundly extended survival of mice carrying PDX T-ALL cells by 680%. Persistent T-ALL cells showed sustained BCL-XL levels and combinatorial inhibition of BCL-XL with MYC/GSPT1 protein degradation synergistically induced cell death. Data suggests that targeting MYC/GSPT1 by protein degradation is a promising treatment approach and poses a potential combinatorial strategy with BCL-XL inhibition for aggressive T-ALLs.