CC-90009, a Novel Cereblon E3 Ligase Modulator, Targets GSPT1 for Degradation to Induce Potent Tumoricidal Activity Against Acute Myeloid Leukemia (AML)

Abstract

The cereblon E3 ligase modulator (CELMoD) CC-885 co-opts the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex to trigger the ubiquitination and proteasomal degradation of the translation termination factor G1 to S phase transition 1 (GSPT1), resulting in robust growth inhibition in AML cell lines and primary patient blasts (Matyskiela ME, et al. Nature. 2016;535:252-7). However, CC-885 also triggers off-target degradation of other cereblon (CRBN) neosubstrates, potentially leading to toxicities. Here, we present the identification of a novel GSPT1-selective CELMoD, CC-90009, and the mechanistic rationale for its clinical development in relapsed or refractory (R/R) AML (CC-90009-AML-001; NCT02848001). To identify new CELMoD agents, we performed cell-based phenotypic screening using a panel of 11 human AML cell lines harboring common oncogenic mutations. The screening identified CC-90009, which demonstrated potent antiproliferative and proapoptotic activity with 50% inhibitory concentration (IC50) values ranging from 3 to 75 nM in 10 out of 11 cell lines. Maximal apoptosis was reached between 16 and 48 hours post exposure to CC-90009 in 5 AML cell lines with cells being committed to apoptosis within 8-16 hours of treatment. CC-90009 was then tested in a panel of samples obtained from 30 patients with newly diagnosed or R/R AML. Bone marrow aspirates obtained during patient diagnosis were plated without separation of constituent cells and tested for sensitivity to CC-90009 using the PharmaFlow PM test which was reported to predict clinical response to standard-of-care chemotherapy with 81% accuracy in AML (Martínez-Cuadrón D, et al. Leuk Res. 2019;76:1-10). The efficacy of CC-90009 was concentration dependent in 26 of 30 patient samples, with an average half-maximal response concentration (EC50) of 21 nM. Leukemic cell killing was rapid and highly efficient: > 82% of leukemic cells were eliminated within 24 hours and nearly all were eliminated within 96 hours. In contrast, CC-90009 showed only modest activity against normal lymphocytes in the same patient samples. Knockout of CRBN via CRISPR/Cas9-mediated gene editing completely abrogated the activity of CC-90009 in sensitive AML cell lines, suggesting that the anti-AML activity of CC-90009 is mediated by the degradation of GSPT1. Tandem mass tag quantitative mass spectrometry analysis of AML cells treated with CC-90009 revealed that CC-90009 selectively reduced the abundance of GSPT1 with little to no effect on the rest of the proteome. The degradation of GSPT1 by CC-90009 was blocked by proteasomal inhibition or inactivation of the CRL4CRBN E3 ubiquitinligase complex. Overexpression of a GSPT1-degradation-resistant mutant, G575N, conferred complete resistance to CC-90009, while RNA interference (RNAi)-mediated partial knockdown of GSPT1 gene expression enhanced the response to CC-90009. Mechanistically, GSPT1 degradation induced by CC-90009 triggers the activation of the integrated stress response pathway, which is associated with the induction of apoptosis and inhibition of proliferation. CC-90009 is a CELMoD and first-in-class GSPT1 degrader entering clinical development. The degradation of GSPT1 was confirmed to be essential for CC-90009-induced apoptosis and antiproliferative activity. The profound antiproliferative activity of CC-90009 in > 80% of human AML cell lines and patient blasts strongly supports the ongoing phase 1 study in R/R AML. Disclosures Lopez-Girona: Celgene Corporation: Employment. Lu:Celgene Corporation: Employment, Equity Ownership. Rychak:Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene Corporation: Employment. Lu:Celgene Corporation: Employment, Equity Ownership. Rappley:Celgene Corporation: Employment. Fontanillo:Celgene Corporation: Employment. Cathers:Global Blood Therapeutics (GBT): Employment; Celgene Corporation: Equity Ownership. Daniel:Celgene Corporation: Employment. Hansen:Celgene Corporation: Employment.