Mechanisms of Resistance to Apto-253

Abstract

APTO-253 is a small molecule indolyl-phenanthroline-imidazole that exerts potent cytotoxicity against human acute myeloid leukemia and lymphoma cells but not normal PBMC or bone marrow cells. The goal of this project was to provide fundamental information on the mechanism of resistance of APTO-253 so as to identify synthetic lethal interactions that can guide combination drug studies.The human Burkitt’s lymphoma cell line Raji was exposed to progressively increasing concentrations of APTO-253 over a period of 6 months to generate the resistant subline Raji/253R. Raji/253R cells were 16.7 ± 3.9-fold resistant to a 120 h exposure to APTO-253 (IC50: Raji IC50 91.9 ± 22.3 nm; Raji /253R IC50 1387.7 ± 98.5 nm). The level of resistance remained stable for at least 3 month during culture in drug-free media. Exposure of the parental Raji cells to 500 nM APTO-253 for 24 h produced: a) G1 arrest accompanied by up-regulation of proapoptotic proteins BIK and BAD and down-regulation of anti-apoptotic protein MCL-1; b) cleavage of PARP; and, c) time-dependent up-regulation of γH2AX. None of these changes were detected in the Raji/253R cells subjected to the same exposure. RNA-seq analysis of the parental and resistant cells in the absence of drug exposure demonstrated marked up-regulation of ATP-binding cassette sub-family G member 2 (ABCG2) in the Raji/253R cells that was confirmed by qRT-PCR and Western blot analysis. Ko143 is a specific inhibitor of ABCG2 that was not toxic to either Raji or Raji/253R when used as a single agent. When Raji/253R cells were concurrently treated with either 5 nM or 50 nM Ko143 and APTO-253, resistance to APTO-253 was reversed by 1.6- and 6.8-fold, respectively, confirming that overexpression of ABCG2 is one mechanism of resistance to APTO-253. The overexpression of ABCG2 in Raji/253R resulted in 2.2-fold less intracellular APTO-253 in Raji/253R cells relative to the Raji cells after a 6 h exposure to 500 nM APTO-253. Raji/253R was found to be cross-resistant to topotecan, a known ABCG2 substrate, and Ko143 also reverses topotecan resistance in Raji/253R. Etoposide is also an ABCG2 substrate but, surprisingly, Raji/253R cells were found to be hypersensitive to etoposide. Etoposide is a non-intercalating topoisomerase II inhibitor and a potent inducer double strand breaks. The hypersensitivity to etoposide in Raji/253R suggests that DNA repair pathways are aberrant in the Raji/253R cells. The second major finding in the Raji/253R cells is that they express only a truncated 45 kDa form of c-Myc in which 159 nucleotides (53 amino acids) are missing from exon 2. Sanger sequencing disclosed microhomologies at each end of the deletion suggesting that microhomology mediated end-joining (MMEJ) repair of a double strand break produced as a result of APTO-253 exposure was responsible for the deletion. BRCA1 plays crucial roles in both homologous recombination (HR) and non-homologous end joining (NHEJ) repair. The RNA-seq RPKM (reads per kilobase exon per million) data revealed that there was less BRCA1 in Raji/253R suggesting that the two classic DNA repair mechanisms (HR and NHEJ) may be impaired in the Raji/253R cells. We conclude that high level resistance to APTO-253 is mediated in part by ABCG2, and that adaption to the drug has resulted in expression of an altered form of c-Myc whose link to the resistant phenotype is not yet established. DisclosuresNo relevant conflicts of interest to declare.