NVX-412, a new oncology drug candidate, induces S-phase arrest and DNA damage in cancer cells in a p53-independent manner

Abstract

The new molecular entity quinoxalinhydrazide derivative NVX-412 was identified as a promising drug candidate for the treatment of various cancer types due to its strong cytotoxic activity and relative specificity. Here, we provide first data about the mechanisms of action of NVX-412. We show that NVX-412 exerts its anti-neoplastic activity in a p53-independent manner and induces S-phase arrest and DNA damage as assessed by γH2AX staining. We suggest a bi-modal (dose-dependent) mode of action of NVX-412, being primarily cytostatic at lower and predominantly cytotoxic at higher concentrations. Based on the broad and consistent anti-neoplastic activity observed, NVX-412 holds promise as an effective drug candidate for the treatment of various cancer types, especially for hematological malignancies with highly unmet medical need.

Figure 1. Chemical structure of NVX-412.

A: Pyrazine-2-carboxylic acid N’-(7-fluoro-pyrrolo[1,2-α]quinoxalin-4-yl)-hydrazide-oxalic acid co-crystal; Molecular Formula: C₁₈H₁₃FN₆O₅, Molecular Weight: 412 g/mol B: Solvent accessible mesh model. White: carbon; yellow: fluorine; blue: nitrogen; red: oxygen. Both structures were generated with ChemBio 3D Ultra 12.0 (CambridgeSoft, MA, USA).

Figure 2. NVX-412 exerts strong anti-neoplastic and dose-dependent bi-modal activity in various tumor cell lines. A:

Dose-response curves of HT-29, HepG2, HeLa and HCT116 cancer cells. Cells were incubated with the indicated concentrations of NVX-412 for 72 hours and counted with a Beckman Coulter ViCell XR. B: Clonogenic survival of HepG2 and HT-29 cells. HepG2 and HT-29 cells were incubated with the indicated concentrations of NVX-412 for 12 or 14 days, respectively. Clonogenic survival was significantly reduced in a dose-dependent manner. C: Proliferation kinetics over three days treatment with different concentrations of NVX-412 in HT-29 cells showing significantly reduced proliferation at 300 nM and a decline in cell numbers at 1 µM NVX-412 treatment. Data represent mean values (± SD) of at least 2 independent experiments. Statistical analysis was performed with GraphPad Prism 5.0. * indicates p value <0.05, **** indicates p value <0.0001 (Two-way ANOVA).

Figure 3. NVX-412 induces significant increase in cell sizes at IC₅₀ and higher concentrations in HCT116, HeLa and HT-29 cells.

HCT116, HeLa and HT-29 cells were treated for up to 72 hours as indicated. A DMSO control was performed but did not show any differences compared to UTC. After 24, 48 and 72 hours pictures were taken (A, C, E). Panels B, D and F show quantification of cell sizes after 48 hours treatment with indicated concentrations of NVX-412. Box plots represent data of cells counted within 5 different fields of view. Statistical analysis was performed with GraphPad Prism 5.0. **** indicates p value <0.0001 (Mann-Whitney U Test). A, B: HCT116. C, D: HeLa. E, F: HT-29. UTC, Untreated Control; CPT, Camptothecin.

Figure 4. NVX-412 induces p53 phosphorylation only at higher concentrations and acts p53 status independent. A:

Western Blot analysis for p-p53 (Ser15) in HCT116 cells after 24 and 48 hours incubation with 0, 0.15, 0.5 and 1 µM NVX-412 and 1 µM CPT as positive control. B: Corresponding immunofluorescence staining for p-p53 (Ser15) (green) of HCT116 cells after 24 hours of incubation with 0, 0.15, 0.5 and 1 µM NVX-412 and 1 µM CPT as positive control. Nuclei are stained with Hoechst 33342 (blue). C: Confirmation of p53 status in HCT116 p53+/+ and p53−/− cells by immunoblotting. Cells were cultured in the presence and absence of 375 µM 5-FU for 24 hours. D, E: Cell Numbers of HCT116 p53+/+ or p53−/− (D), RKO p53+/+ or p53−/− (E) cells after 72 hours incubation with NVX-412 or Nutlin-3. Cells were cultured for 72 hours with different concentrations of NVX-412 or Nutlin-3, respectively. Viable cell numbers were determined using a Beckman Coulter ViCell XR. Data represent mean values (± SD) of two independent experiments.

Figure 5. NVX-412 induces S-phase arrest and DNA damage and reduces DNA replication rate.

A: Cell cycle analyses by flow cytometry for HT-29, HeLa and HCT116 cells treated for 24 hours as indicated with NVX-412 or CPT. Percentages of cells in G1, S and G2/M phase of the cell cycle are shown. Cells were analyzed using a BD FACScan. B: NVX-412 reduces DNA replication in a reversible manner in HeLa and HCT116 cells. HeLa and HCT116 cells were treated for 24 hours as indicated. Additionally, after 24 hours of treatment cells were allowed to recover for 24 hours in normal growth medium without NVX-412 or CPT. DNA replication rate was analyzed by BrdU incorporation. C: Western Blot for pChk1(Ser296) in HCT116 cells after 0–48 hours incubation with 150 nM NVX-412. D: NVX-412 induces γH2AX in HeLa cells in a reversible manner. Cells were treated for 3 and 24 hours as indicated. Additionally, after 24 hours of treatment cells were allowed to recover for 24 hours in normal growth medium without NVX-412 or CPT. Data represent fold change in average γH2AX fluorescence intensity per nucleus (± SD) as quantified from immunofluorescence stainings. UTC, Untreated Control; CPT, Camptothecin. Mean values of at least 2 independent experiments are shown. Statistical analysis was performed with GraphPad Prism 5.0. **** indicates p value <0.0001 (Two-way ANOVA).