Deficiencies in the Fanconi Anemia or the Homologous Recombination Pathway enhance the antitumor effects of the novel hypoxia-activated prodrug CP-506

Schuitmaker, L.; van der Wiel, A. M. A.; Lieuwes, N. G.; Biemans, R.; Mutsters, N. A. M.; Jung, J.; Claudino Bastos, V.; Prades Sagarra, E.; Kuang, S.; Langie, S. A. S.; Setton, J.; Theys, J.; Yaromina, A.; Dubois, L. J.; Lambin, P.

doi:10.1101/2025.05.21.655302

bioRxiv · 10.1101/2025.05.21.655302

Deficiencies in the Fanconi Anemia or the Homologous Recombination Pathway enhance the antitumor effects of the novel hypoxia-activated prodrug CP-506

Schuitmaker, L.·van der Wiel, A. M. A.·Lieuwes, N. G.·Biemans, R.·Mutsters, N. A. M.·Jung, J.·Claudino Bastos, V.·Prades Sagarra, E.·Kuang, S.·Langie, S. A. S.·Setton, J.·Theys, J.·Yaromina, A.·Dubois, L. J.·Lambin, P.

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Abstract

The novel hypoxia-activated prodrug CP-506 has been shown to selectively target hypoxic tumor cells, which are associated with disease progression and resistance to conventional anti-cancer therapies. Given the alkylating effector metabolites, we hypothesize that defects in interstrand crosslink (ICL) and double strand break (DSB) DNA repair may serve as a predictive biomarker of sensitivity to CP-506. Here, we evaluated the role of DNA damage repair pathways in the antitumor response to CP-506. Isogenic cancer cell lines proficient or deficient in the Fanconi Anemia (FA), homologous recombination (HR), or non-homologous end joining (NHEJ) pathway were cultured as 2D monolayers and 3D spheroids. Cell viability, clonogenic cell survival, and spheroid growth inhibition were assessed following CP-506 exposure. Mice bearing subcutaneous isogenic xenografts received CP-506 (600 mg/kg; QD5) or vehicle treatment upon reaching a tumor starting volume (SV) of 244.9 {+/-} 72.0 mm3. Treatment response was quantified as time to reach 4xSV (T4xSV) and respective enhancement ratios (ER), defined as T4xSVCP-506/T4xSVvehicle. DNA damage and repair capacity were evaluated by {gamma}H2AX and alkaline comet assays. In vitro, cell lines deficient in FA or HR, but not NHEJ, showed enhanced sensitivity to CP-506 compared to parental cells in viability and clonogenic assays. This was confirmed in spheroid growth inhibition studies. In vivo, the antitumor response to CP-506 was more pronounced (P<0.0001) in LNCaP AR FANCA-/- (ER 4.0{+/-}1.1) and LNCaP AR FANCD2-/- (ER 3.4{+/-}0.8) xenografts compared to parental LNCaP AR (ER 1.5{+/-}0.5) xenografts. CP-506 treatment of DLD-1 BRCA2-/- (2.9{+/-}0.7; P<0.0001) xenografts resulted in significantly enhanced ER compared to parental (1.3{+/-}0.2) xenografts. Similar results were obtained in HCT116 BRCA2-/- (ER 4.0{+/-}0.6; P<0.0001) versus parental (ER 1.7{+/-}0.6) xenografts. In contrast, the ER of HCT116 DNA-PKcs-/- (1.4{+/-}0.3; P=0.18) xenografts was not different from HCT116 parental xenografts. Under anoxic conditions, CP-506 caused elevated {gamma}H2AX foci counts in FANCA-(2.0-fold increase at 48 hours) and FANCD2-deficient cells (1.4-fold increase at 72 hours) compared to LNCaP AR parental cells (P<0.0001). Similarly, {gamma}H2AX expression was increased in DLD-1 BRCA2-/- cells, but not in HCT116 BRCA2-/-cells, compared to their respective parental cells. In xenografts, FA- and HR-deficiency caused elevated {gamma}H2AX expression compared to respective parental tumors (1.6-9.3-fold increase). HCT116 DNA-PKcs-/- cells and xenografts displayed reduced {gamma}H2AX expression compared to their parental counterpart, with an 0.5-fold reduction. The comet assay confirmed CP-506-induced ICLs and DNA strand breaks but was unable to explain the differential therapeutic responses of CP-506 among isogenic tumor cells. Deficiencies within FA or HR, but not NHEJ, enhanced the antitumor effects of CP-506 through a mechanism consistent with the concept of synthetic lethality. Therefore, both DNA repair status and the presence of tumor hypoxia represent key biomarkers for patient stratification in clinical trials of CP-506.

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Schuitmaker, L., van der Wiel, A. M. A., Lieuwes, N. G., Biemans, R., Mutsters, N. A. M., Jung, J., Claudino Bastos, V., Prades Sagarra, E., Kuang, S., Langie, S. A. S., Setton, J., Theys, J., Yaromina, A., Dubois, L. J., Lambin, P.. 2025-05-24. Deficiencies in the Fanconi Anemia or the Homologous Recombination Pathway enhance the antitumor effects of the novel hypoxia-activated prodrug CP-506. https://doi.org/10.1101/2025.05.21.655302

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