FANCA KO Breast Cancer Cells are Sensitized to PARPi and ATRi by Homologous Recombination-Independent Mechanisms (2023)

Undergraduate: Simon Ellington

Faculty Advisor: Gaorav Gupta
Department: Biochemistry & Biophysics, Radiation Oncology

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PARP inhibitors (PARPi) are a class of targeted drugs used in the clinic to treat cancers with mutations in BRCA1/2, which are highly sensitive to PARPi. This PARPi sensitivity is traditionally thought to be mediated by deficiency in homologous recombination (HR), an important DNA repair process. As such, HR-deficiency, primarily identified as mutated BRCA1/2, is currently the sole criterion for selecting patients for PARPi treatment. However, the true mechanism underlying PARPi sensitivity remains poorly understood. Our lab recently conducted an in-vivo CRISPR-Cas9 screen of DNA damage response genes that identified loss of FANCA as a mutation which sensitizes cancer cells to PARPi or ATR inhibitor (ATRi), drugs that target DNA repair processes. Using CellTiter-Glo viability assays, we compared sensitivity to PARPi and ATRi in wild-type (WT), FANCA knockout (KO), and BRCA1 KO cancer cells, showing that FANCA KO cells display heightened sensitivity to PARPi, ATRi, or combination treatment. We then performed immunofluorescence-based assays to investigate whether HR deficiency plays a role in the PARPi sensitivity observed in FANCA KO cells. By detecting nuclear foci of Rad51 and 𝛾-H2AX — markers of HR repair and DNA damage, respectively — we showed that following treatment with PARPi, FANCA KO cells display no HR deficiency while accumulating greater DNA damage compared to WT cells. These findings challenge the traditional hypothesis that PARPi sensitivity is caused by impairment of HR and provide a rationale for investigating PARPi and ATRi activity in patients with FANCA-mutant cancers, which make up 2-3% of human metastatic cancer cases.

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