Abstract LB-C02: In vitro on-target and selectivity profiling of small-molecule inhibitors of the Myc/Max heterodimeric complex

Abstract

The expression and activity of the Myc oncoprotein is frequently dysregulated in human cancers due to Myc amplification, translocation, protein stabilization and/or convergence of oncogenic signaling pathways on Myc. Therapeutic inhibition of Myc is highly desirable however, prior efforts to identify specific and direct pharmacological inhibitors of Myc function have generally failed to yield Myc-selective drug-like small molecules. Herein, we have comprehensively profiled previously reported small molecule inhibitors of the Myc-Max heterodimeric complex (10058-F4, 10074-G5, MYCMI-6, KI-MS2-001/-008 (KI-MS2), Mycro3 & JKY-2-169), using an in vitro biological screening cascade in an effort to identify and characterize bona fide tool compounds for our Myc drug discovery program. Initial cellular screening of compounds using a reporter construct containing Myc binding elements upstream of the luciferase gene and cellular viability assays using the P493-6 (Myc On / Off) system identified JKY-2-169 (JKY) and KI-MS2 as compounds with on-target activity and acceptable specificity profiles to warrant further characterization. Consistent with its reported mode of action, we confirmed JKY perturbs binding of the c-Myc/MAX heterodimer to its canonical E-box DNA sequence without causing protein-protein dissociation through biochemical and in vitro Myc:MAX interaction assays. In a cellular thermal shift assay (CETSA) using P493-6 B-cells, JKY induced thermal stabilization of c-Myc and not MAX, suggesting direct and specific cellular engagement of JKY to Myc. Consistent with this, JKY phenocopied the genetic de-induction of c-Myc in P493-6 cells, inducing G₀/G₁ cell-cycle arrest in a dose-dependent manner. Moreover, 3’RNA-Sequencing (3’RNA-Seq.) of MV4-11 AML cells and P493-6 cells treated with JKY demonstrated an altered transcriptional program consistent with Myc deregulation that was independent of transcriptional modulation of Myc expression. Further transcriptional profiling revealed JKY exhibits a profile with high correlation to that of the small molecule MAX inhibitor, KI-MS2; consistent with the obligate dependence of Myc on heterodimerization with MAX for transcriptional activation. Together, these results highlight JKY and KI-MS2 as suitable chemical tools for deciphering Myc biology. MecRx’s small molecule Myc inhibitor MRX-1669 was developed using several orthogonal biochemical/biophysical assays including those described above. Similar to JKY, MRX-1669 exhibits cellular Myc engagement (CETSA), and evokes a similar transcriptional profile to that of JKY and KI-MS2 (3’RNA-Seq.). MRX-1669 is a drug-like molecule that provided a benchmark for further development and medicinal chemistry optimization.

Citation Format: SANG-KYU KIM, RICHARD C FOITZIK, SONGHUI LI, ANDREW VINSON, ALEX SHMAYLOV, KAREN JARVIS, SUSAN JAMES, SCOTT R WALKER, MARK YORK, BENJAMIN CAO, SUSAN K NILSSON, RICKY W JOHNSTONE. In vitro on-target and selectivity profiling of small-molecule inhibitors of the Myc/Max heterodimeric complex [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C02. doi:10.1158/1535-7163.TARG-19-LB-C02