Synthetic Lethal Interaction between Apto-253 and Impaired BRCA1/2 Function

APTO-253 is a novel indolyl-phenanthroline-imidazole small molecule with antiproliferative activity against cell lines derived from a wide range of human malignancies including leukemia, lymphoma, and colon, non-small cell lung, renal and prostate carcinomas. Of particular significance, APTO-253 did not elicit myelosuppression in its initial Phase 1 trial in patients with solid tumors even at the maximum tolerated dose. The goal of this project was to provide fundamental information on the mechanism of action and resistance to APTO-253 so as to identify synthetic lethal interactions that can guide combination drug studies.

The human Burkitt'slymphoma cell line Rajiwas exposed to progressively increasing concentrations of APTO-253 over a period of 6 months to generate a resistant subline Raji/253R with 16.7 ± 3.9-fold resistance (IC50: Raji IC₅₀ 91.9 ± 22.3 nM; Raji /253R IC₅₀ 1387.7 ± 98.5 nM). RNA-seq analysis of the parental and resistant cells 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. Raji/253R was found to be cross-resistant to topotecan, a known ABCG2 substrate, and Ko143 also reversed topotecan resistance in Raji/253R. Intriguingly, Raji/253R was also significantly cross-resistant to carboplatin even though carboplatin is not thought to be an ABCG2 substrate; treatment with Ko143 did not reduce the carboplatin IC₅₀ in the Raji/253R cells. The curious finding that Raji/243R cells were cross-resistant to carboplatin led to the discovery that APTO-253 causes DNA damage as documented by the neutral comet assay and gH2AX phosphorylation and foci formation assays. Surprisingly, Raji/253R cells were hypersensitive to etoposide, an ABCG2 substrate and a potent DNA double strand break inducer. GO and pathway analysis from the RNA-seq data revealed that DNA repair pathways were downregulated in Raji/253R, which partially explained the hypersensitivity to etoposide. We then asked whether cells deficient in homologous recombination repair pathways were hypersensitive to APTO-253 and if there was synthetic lethality between APTO-253 and BRCA1 deficiency (using isogenic pairs of BRCA1-proficient and -deficient human cell lines). BRCA1 deficiency was documented by treating the BRCA1-proficient and -deficient isogenic cell lines with the PARP inhibitor olaparib. BRCA1-deficient sublines of MCF10A, MCF7 and hTERT -IMEC were all hypersensitive to both olaparib and APTO-253. We also found that the BRCA2deficient ovarian cancer cell line PEO4 and two colorectal HCT116 BRCA2 ^-/- subclones were hypersensitive to both olaparib and APTO-253.

We conclude that AP-253 causes DNA damage and that it exhibits a degree of synthetic lethality comparable to olaparib in cells deficient in BRCA1 and BRCA2-function. The ability of APTO-253 to exploit homologous recombination deficiency is of particular interest because, unlike all the other drugs for which loss of this repair function results in hypersensitivity, APTO-253 does not produce myelosuppression even at the maximum tolerated dose. The observations reported here also identify gH2AX as a potential biomarker of clinical drug effect and open the window more detailed studies of how APTO-253 causes DNA damage.