Finding synergies for cancer treatment : new ways to modulate DNA damage repair by CX3CR1 and PFKFB3 inhibition

The goal of targeted cancer therapy is to selectively kill cancer cells based on their molecular survival mechanisms. DNA repair is as a promising cancer target as many cancers have chronic replication stress and deficiencies in the DNA damage response. Moreover, combining DNA damaging chemo- and radiotherapy with inhibitors of DNA repair can lead to improved treatment responses, reduced resistance to treatments, as well as lowering of effective doses and thereby reduced toxicity to healthy tissues. In this thesis, two cancer targets, CX3CR1 and PFKFB3, were investigated for their emerging roles in DNA repair. Furthermore, small molecule inhibitors KAN0438757, developed in Paper I to target PFKFB3, and KAND567 targeting CX3CR1, were evaluated in combination treatments with ionizing radiation (IR) and platinum drugs in vitro.

In Paper II and III we characterize the role of CX3CR1 in the DNA damage response. We reveal that CX3CR1 inhibition by KAND567 reduces cancer cell survival and impairs DNA replication, reducing RPA and ATR activation (Paper II). CX3CR1 inhibition increases DNA damage levels and S phase arrest when combined with platinum drugs, resulting in reduced cancer cell survival at doses not affecting non-transformed cells (Paper II and III). Mechanistically, we reveal that upon DNA damage induction CX3CR1 is relocated to the nucleus and regulates interstrand crosslink (ICL) repair by facilitating the recruitment of the key repair proteins in the Fanconi Anemia (FA) repair pathway, FANCD2 and FANCI, to the chromatin (Paper III). Notably, CX3CR1 inhibition sensitizes cancer cells to platinum treatment and especially platinum resistant cancer cell lines demonstrate good synergy for this combination treatment (Paper III).

In Paper I and IV, we identify novel roles for PFKFB3 in regulating DNA repair. We show that PFKFB3 locates to DNA damage sites upon IR and PFKFB3 inhibition results in impairment of DNA double-strand break repair by homologous recombination (HR). Mechanistically, PFKFB3 triggers recruitment of RRM2, responsible of local nucleotide supply, and the HR factors, RPA and RAD51, to DNA damage sites, to allow for DNA repair (Paper I). Moreover, we develop a selective small molecule inhibitor, KAN0438757, that targets PFKFB3 and selectively radiosensitizes transformed cells (Paper I). In Paper IV, we discover a role for PFKFB3 in FA repair upon ICL induction in cancer cells. We demonstrate that PFKFB3 associates to the chromatin following treatment with ICL-inducing agents and regulates establishment of the FA repair pathway, needed for initiation of ICL repair. Importantly, we demonstrate that PFKFB3 inhibition synergizes with platinum treatments in blocking proliferation of transformed cells.

In summary, our work identifies novel roles of CX3CR1 and PFKFB3 in DNA repair processes critical for cancer cell survival following treatment with DNA damaging agents.

List of scientific papers

I. Targeting PFKFB3 radiosensitizes cancer cells and suppresses homologous recombination. Nina M.S. Gustafsson, Katarina Färnegårdh, Nadilly Bonagas, Anna Huguet Ninou, Petra Groth, Elisee Wiita, Mattias Jönsson, Kenth Hallberg, Jemina Lehto, Rosa Pennisi, Jessica Martinsson, Carina Norström, Jessica Hollers, Johan Schultz, Martin Andersson, Natalia Markova, Petra Marttila, Baek Kim, Martin Norin, Thomas Olin, Thomas Helleday. Nature Communications. 2018, 9(1):3872.
https://doi.org/10.1038/s41467-018-06287-x

II. Blocking the fractalkine receptor disrupts replication and ovarian cancer cell proliferation. Jemina Lehto, Anna Huguet Ninou, Petra Marttila, Emma Åkerlund, Greta Gudoityte, Ulrika Joneborg, Joseph Carlson, Brinton Seashore-Ludlow, Nina M.S. Gustafsson. [Manuscript]

III. Targeting CX3CR1 suppresses the Fanconi Anemia DNA repair pathway and synergizes with platinum. Jemina Lehto, Anna Huguet Ninou, Dimitrios Chioureas, Jos Jonkers, Nina M.S. Gustafsson. Cancers. 2021, 13(6):1442.
https://doi.org/10.3390/cancers13061442

IV. PFKFB3 regulates repair of DNA interstrand crosslinks via modulation of the Fanconi Anemia repair pathway. Anna Huguet Ninou, Jemina Lehto, Dimitrios Chioureas, Hannah Stigsdotter, Korbinian Schelzig, Emma Åkerlund, Greta Gudoityte, Ulrika Joneborg, Joseph Carlson, Jos Jonkers, Brinton Seashore-Ludlow, Nina M.S. Gustafsson. [Manuscript]