APTO-253 is a clinical stage small molecule that acts in part by down-regulation of MYC at mRNA and protein levels. MYC onco-protein dysregulation, which leads to increased proliferation and survival, is implicated in the etiology of numerous cancer subtypes. In the current study, we explored sensitivity of B-cell cancer cells to APTO-253 and sought to understand how changes in MYC can yield resistance to APTO-253. APTO-253 showed potency against B-cell cancer cell lines (IC50 30-600 nM) and bone marrow samples from patients. Sensitivity of cell lines correlated with MYC over-expression, amplification or translocation. APTO-253 resistant Raji cells (Raji253R) are characterized by upregulation of the ABCG2 transporter. Treatment with ABCG2 inhibitors only partially reversed the resistant phenotype suggesting additional contributing mechanisms. RNA-seq analysis revealed that while levels of MYC mRNA were unchanged in Raji253R, a truncation of exon 2 was present. This internal deletion occurred at the DNA level, not by alternative splicing, and resulted in a truncated MYC protein (45 kDa) lacking the conserved core sequence of MYC box III (MBIII). At early stages in the evolution of resistance residual full length MYC mRNA and protein was detectable; however, this expression was eventually lost in Raji253R cells as selection continued. Deletion of MBIII has been implicated in MYC protein stability, in vitro and in vivo transformation, gene repression, and MYC dependent apoptosis. Cycloheximide treatment of Raji and Raji253R cells demonstrated a modest increase in stability of truncated over full length MYC protein. In addition, Raji253R cells exhibited hypersensitivity to etoposide. The internal truncation is flanked by sites of microhomology suggesting a method for deletion. We hypothesize that DNA damage in response to APTO-253 treatment led to error prone repair by microhomology end joining and resulted in a MYC internal deletion. Interestingly, transcription from the canonical P1/P2 promoter, as seen in WT Raji cells, was systematically lost and transcription initiation shifted to a downstream promoter, P3. APTO-253 inhibits MYC expression via binding and stabilizing the G-quadruplex (G4) sequence in the P1/P2 promoter therefore switching of transcriptional initiation to P3 should relieve APTO-253 mediated repression. Indeed, APTO-253 treatment of Raji253R cells did not repress MYC expression, even at concentrations 50-fold higher than required for MYC repression in Raji WT cells. Presence of the active intra-cellular form of APTO-253, Fe(253)3, was confirmed in Raji253R cells. In summary, MYC driven Raji cells become resistant to APTO-253 by acquisition of a more stable MYC protein and by utilization of an alternate promoter not inhibited by G4 binding and stabilization, thereby confirming the essential role of MYC repression in the mechanism of action of APTO-253.

Citation Format: Andrea Local, Cheng-Yu Tsai, Hongying Zhang, Susan Sheng, Khalid Benbatoul, Stephen B. Howell, William G. Rice. Resistance to APTO-253 caused by internal deletion and alternate promoter usage of the MYC gene in malignant B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2096.