Emerging roles of PFKFB3 and CX3CR1 in the DNA damage response and their potential as therapeutic targets in cancer

Abstract

Genomic instability in cancer is exploited therapeutically using DNA damaging therapies that cause irreparable lesions above the threshold of tolerable DNA damage levels. Nevertheless, toxic side effects on healthy tissues limit the therapeutic potential of such therapies. Through the concept of synthetic lethality, therapeutic targeting of factors involved in the DNA damage response (DDR) appears as an attractive strategy to increase the efficacy and improve the therapeutic window of traditional DNA damaging therapies. The aim of this thesis was to study how the metabolic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), and the fractalkine membrane receptor (CX3CR1) contribute to the maintenance of genome stability and whether they can constitute therapeutic targets in cancer.

In paper I, we reveal a hitherto unknown role for PFKFB3 in repair of DNA double-strand breaks (DSBs) upon ionizing radiation (IR). PFKFB3 relocates to IR-induced foci (IRIF) in the nucleus where it colocalizes with homologous recombination (HR) factors. Assembly of PFKFB3 IRIF is dependent on ATM activity and is essential for the recruitment of RAD51 and RPA recombinant mediators, HR activity, and survival upon IR. To interrogate PFKFB3 molecular function, we develop and validate the small molecule inhibitor KAN0438757. Pharmaceutical inhibition of PFKFB3 results in defective HR repair of IR-induced DSBs and impairment of nucleotide incorporation for DNA synthesis by direct interaction with RRM2 at DNA damage sites. Furthermore, targeting PFKFB3 with KAN0438757 sensitizes transformed cells to IR while sparing non-transformed cells.

In paper II, we describe a novel role for PFKFB3 in supporting replication-coupled Fanconi anemia (FA) repair of DNA crosslinks, which is essential to resolve stalled replication forks, resume replication, and cell survival. PFKFB3 inhibition provides cancer-selective sensitization to the DNA crosslinkers cisplatin and carboplatin (platinum drugs). This sensitization is not due to a general distortion of glycolysis but appears associated to enhanced PFKFB3 chromatin loading in transformed cells, which is further enriched in platinum resistant cells. Activation of FA pathway upon replication stress results in PFKFB3 recruitment into nuclear foci that depends on ATR activity and FANCM assembly at stalled forks, where PFKFB3 directly interacts with FANCD2 and BLM to ensure FA-mediated repair. Moreover, targeting PFKFB3 increases replication fork stalling and limits fork restart, which results in inability to progress through S phase and fork collapse.

In paper III, we introduce an unknown function of CX3CR1 in promoting FA pathway repair of DNA crosslinks that has functional consequences for the resolution of DNA adducts, DNA replication and survival upon treatment with platinum drugs. Targeting CX3CR1 by siRNA transfections, lentiviral delivery of shRNA, or with the non-competitive inhibitor KAND567, renders resistant cells hypersensitive to platinum treatments. FA pathway activation under replication stress conditions triggers CX3CR1 nuclear localization, which is vital for the assembly of FANCD2 into foci. Suggesting a potential role in fork stabilization, CX3CR1 inhibition hampers chromatin loading of FANCD2, and its partners FANCI, RAD51 and γH2AX.

Lastly, in paper IV we provide an initial characterization of CX3CR1 molecular function in survival and proliferation of ovarian cancer cells. KAND567 treatment is cytotoxic in a panel of ovarian cancer cells and in cells derived from an ovarian cancer patient, whereas viability of non-transformed cells is not affected at the same range of concentrations. CX3CR1 pharmacological inhibition slows down G1 to S phase transition and results in defective DNA replication, accumulation of DNA damage and apoptotic cell death.

Altogether, the present thesis provides evidence for emerging roles of PFKFB3 and CX3CR1 in the DDR that are critical for cancer cells to maintain genomic stability, and approaches the feasibility of a rational design for a future therapeutic intervention in cancer.