PLK1 Acts in Homologous Recombinatorial Repair and in Mitosis As Synthetically Lethal with the Fanconi Anemia/BRCA Pathway

Introduction: Fanconi anemia (FA) is a bone marrow failure syndrome that confers increased risk of cancer in myeloid leukemia and solid tumors, especially head and neck squamous cell carcinoma (HNSCC). FA mutations engender an increase in hypersensitivity to DNA damaging agents that prevents use of many conventional chemotherapies in cancers. In addition, somatic FA gene mutations are found broadly in a variety of cancers. Thus, combination targeted therapy using reduced therapeutic doses may be beneficial to confer less toxicity to FA patients, FA carriers, and non-FA patients with somatic mutations by capitalizing on molecular vulnerabilities. Using an siRNA-based synthetic lethality screen, we have identified polo-like kinase (PLK1) as a candidate target in an FA-mutant background. PLK1 plays a role in several cellular processes, including homologous recombinatorial (HR), DNA damage repair and mitosis. In this study, we aim to investigate the mechanism of synthetic lethality of PLK1 in a mutant FA background.

Method:

• Cell models

  Mutant and corrected cells of the following FA lines: CRISPR KO FANCA HNSCC, Patient derived FA-D2 fibroblasts (PD20), FA-D1/BRCA2 fibroblasts (EUFA 423), and lymphoma cells derived from Fancd2 -/- KO mice.

• Drugs

  PLK1 inhibitor (volasertib), PARP1 inhibitor (olaparib), Mitomycin C (MMC)

• TCGA

  Analysis of PLK1 and FANCD2 expression levels to score HR repair efficiency, disease prognosis in HPV-neg HNSCC, PLK1 expression in all cancer types, co-mutation mutation rate of FA and PLK1 genes.

• Assay

  Survival curves using mono and combined therapy of volasertib, olaparib, and MMC. Immunofluorescence (IF) microscopy for phospho-RPA, phospho-histone 3, RAD51 foci, tubulin, phosphor-histone 3. Cell cycle analysis using DNA flow cytometry HR-GFP assay for HR deficiency

Results: TCGA analysis of cancers with mutations in FA genes correlated with high PLK1 mRNA expression in 21 cancers and reduced overall survival in all cancers. HR repair efficiency also correlated with PLK1 expression. To assess the sensitivity of PLK1 inhibition of FA mutations lines were treated with an addback wildtype for their respective mutation to test if PLK1 inhibition is specific to FA mutations. PLK1 inhibitor, volasertib, we found FA gene mutant cell types selectively reduced cell survival of all FA gene mutant cell types versus corrected cells. The observed sensitivity was synergistic in combination with sublethal doses of the DNA interstrand crosslinking agent, MMC. Since PLK1 is a known mitotic kinase, we used DNA flow to analyze cell cycle dynamics. Although cell cycle analysis revealed increased G2/M phase upon both MMC or volasertib treatment, only FA mutant cells demonstrated improper entry into mitosis as evidenced by condensed chromosomes and phospho-S10-histone 3 with volasertib. Western blot analysis showed increase H2AX and phospho-RPA in FA mutant cells when treated with volasertib, suggestive of an additional defect in HR. Since RAD51 is a substrate of PLK1, we wanted to explore the effects of volasertib treatment on HR in wild type and mutant FA cells. After treatment, we saw a marked increase in RAD51 foci in wild type cells only. Using a HR-GFP recombination assay in U2OS cells, cells knocked down for FANCD2 had significant increased reduction of HR activity beyond that seen in FANCD2 knockdown alone, similar to that seen in BRCA2 mutants, when treated with volasertib. Given the inducible and increased HR defect observed in non-BRCA FA mutant cells, we hypothesized that PLK1 could sensitize PARP1-resistant FA mutant cells to olaparib. Use of low dose volasertib conferred marked olaparib sensitivity in synergistic fashion, as predicted. Taken together, PLK1 is synthetic lethal in an FA-mutant background, the inhibition of which appears to act via its mitotic and HR function. Such a strategy can be combined with low dose treatment with crosslinkers and with the induction of PARP1 inhibitor sensitivity, achieving synergy and avoiding toxicity.

Conclusion: PLK1 is synthetic lethal in the FA pathway, the targeting of which is caused by disruption of its HR and mitotic function. Such a strategy can be combined with low dose treatment with crosslinkers or with of PARP1 inhibitors.

No relevant conflicts of interest to declare.