C-MYC Targeting By Degradation: Novel Dual c-Myc/GSPT1 Degrader GT19715 Induces TP53-Independent Cell Death in Acute Myeloid Leukemia and Lymphomas

The oncoprotein c-Myc, a major regulator of the epigenome and transcriptome is dysregulated in 70% of all human cancers. MYC is highly expressed in Burkitt's leukemia/lymphomas and also in TP53 mutant and venetoclax (ven) resistant AML (Sallman, Blood 2021, Nishida, ASH 2021). However, targeting c-Myc or the MYC pathway has not been successful and remains a major unmet clinical need. We here report the first c-Myc degrading agent in pre-clinical development. Cerebron E3 ligase modulators (CELMoDs) is an emerging treatment modality to target hitherto undruggable oncoproteins. We developed the first c-Myc degrader GT19630 (GT19715 is the salt form of GT19630). C-Myc was pulled down by biotinylated GT19630 in a cell-free, in vitro affinity purification assay and was degraded withIC₅₀ of 1.5 nM in HL-60 cells. A proteasome inhibitor ixazomib completely blocked c-Myc degradation, confirming CRL4^CRBN-dependent c-Myc degradation as mechanism. IC₅₀ of GT19715 in HL-60 cells was 0.33 nM, considerably lower than the IC₅₀ of 44.9 nM and 42.9 nM in normal erythroid and myeloid progenitors CFU assays, suggesting a therapeutic window. In agreement with other CELMoDs, proteomic analyses revealed degradation of translation termination factor GSPT1 (G1 to S phase transition proteins 1), an important factor in LSC survival (Surka et al. Blood 2021), suggesting a dual activity of GT19630 against c-Myc and GSPT1. Whereas a selective GSPT1 degrader CC-90009 reduced GSPT1 protein levels but not c-Myc, GT19715 reduced both c-Myc and GSPT1 (Fig. A) and exerted a 20x higher cytoreductive potency compared to CC-90009 (IC₅₀ of 1.8 nM vs 40.4 nM for GT19715 and CC-90009, respectively) in HL-60 cells. GT19630 degraded c-Myc and GSPT1 in a xenograft model with HL-60 cells, and inhibited tumor growth at a dose as low as 0.3 mg/kg/bid. GT19630 had no effect on normal myeloid lineages in rats at 6 mg/kg. As expected, GT19715 eliminated circulating blasts and prolonged survival in the c-Myc-driven systemic Burkitt's leukemia/lymphoma model (Daudi) (Fig. B). GT19715 significantly reduced human CD45+ AML blasts in peripheral blood, bone marrow and spleens compared to vehicle controls in vivo in a chemotherapy-resistant AML PDX model . Importantly, GT19715 induced cell death independent of TP53 status. Baseline c-Myc protein levels significantly correlated with sensitivity to GT19715 in MOLM-13 cells with CRISPR engineered knockout/mutations of TP53 (R² = 0.86, P = 0.02). Indeed, GT19715 induced comparable cell death in primary AML samples with wild-type or mutant TP53 (62.6% and 61.6% annexin V/DAPI positivity, P = 0.65 for wild-type and mutant TP53 samples at 64 nM of GT19715, respectively). CD34+ AML cells were more susceptible to GT19715 than CD34- AML cells, suggesting a greater efficacy in AML stem/progenitor than in more mature AML cells. Chromatin-enriched proteomics confirmed c-Myc as one of the most significantly downregulated proteins; and identified ERF1, a binding partner of GSPT1 that promotes translation termination, as one of the top upregulated proteins, implicating a compensatory response against GSPT1 degradation. Finally, we found that MV4;11 ven resistant (VR) cells generated through long-term ven exposure demonstrated elevated protein levels of c-Myc and GSPT1 and exhibited greater sensitivity to GT19715 compared to ven-sensitive parental cells. CK1α, another target protein of thalidomide derivatives, was not degraded by GT19715 in MV4;11 parental and VR cells with no induction of p53, supporting p53-independent cell death. Conclusions: The novel dual c-Myc/GSPT1 degrader GT19715 exerts TP53 independent preclinical anti-lymphoma and -leukemia efficacy, providing rationale for its clinical development.

Nishida:Daiichi-Sankyo Inc.: Research Funding; Kintor Pharmaceutical: Research Funding. Carter:PinotBio: Research Funding; Syndax: Research Funding; Revolution Medicines: Research Funding. Zhou:Kintor Pharmaceutical: Current Employment. Yang:Kintor Pharmaceutical: Current Employment. Yan:Kintor Pharmaceutical: Current Employment. Ma:Kintor Pharmaceutical: Current Employment. Andreeff:Syndax: Consultancy, Research Funding; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Aptose: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Reata: Current holder of stock options in a privately-held company; Brooklyn ITX: Research Funding; Glycomimetics: Consultancy; Chimerix: Current holder of stock options in a privately-held company; Senti Bio: Consultancy, Research Funding; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo Inc.: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; Medicxi: Consultancy; Oxford Biomedical UK: Research Funding; Pinot Bio: Research Funding; Oncolyze: Current holder of stock options in a privately-held company; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI: Membership on an entity's Board of Directors or advisory committees; Kintor Pharmaceutical: Research Funding.