A novel CDK4 inhibitor for myeloid protection in chemotherapy-treated triple-negative breast Cancer

Abstract

Background: Despite advances in cancer treatment, chemotherapy remains a cornerstone of clinical practice. However, its efficacy is often compromised by dose-limiting haematologic toxicities. Recent strategies aim to enhance chemotherapy tolerability while preserving its effectiveness. One emerging approach involves selective CDK4 inhibitors to serve as myeloid-protective agents in retinoblastoma (RB)-negative tumours, such as triple-negative breast cancer (TNBC). Because bone marrow (BM) cells rely on RB for proliferation, CDK4 inhibitors may protect these cells while sparing RB-deficient tumour cells. The present study investigated the potential of AU2-94, a first-in-class CDK4 inhibitor, to protect BM cells during myelosuppressive chemotherapy in TNBC, beyond its established application in RB-positive cancers.

Methods: This study employed in vitro, ex vivo, and in vivo experiments to evaluate the myeloid-protective effects of AU2-94 against chemotherapy-induced damage.

Results: AU2-94 induced a transient G1 arrest that protects BM cells from chemotherapy-induced apoptosis by preventing DNA double-strand breaks. Pre-treatment with AU2-94 prior to 5-fluorouracil (5-FU) administration reduced BM cells apoptosis, preserved Ki67-positive cells, and mitigated declines in red blood cells and neutrophils. Similarly, AU2-94 pre-treatment before cisplatin administration reduced cisplatin-induced haematologic toxicity in RB-deficient TNBC bearing mice without compromising the efficacy of chemotherapy.

Conclusion: These findings support the repurposing of AU2-94 as a myeloprotective agent, highlighting its therapeutic potential in RB-deficient tumours. With AU2-94 advancing to clinical trials, these results underscore its broader therapeutic promise, extending to both RB-positive and RB-negative cancer treatment.

Keywords: CDK4 inhibitor; Chemotherapy-induced myelosuppression; RB-negative tumour; Triple negative breast cancer.

Fig. 1

AU2–94 induced G1 arrest in RB-competent cells but not RB-deficient cells. A Representative western blots showing basal expression of cell cycle-related proteins in some breast cancer cell lines. Scatter plot illustrates the inverse correlation between RB expression and GI₅₀ values in a panel of breast cancer cell lines. GI₅₀ values were calculated as the average from three independent MTT proliferation assays conducted over 144 hours for each cell line. B Treatment of RB-positive cells (MCF-7 and MDA-MB-231) with AU2–94 for 24 hours resulted in G1 arrest. In contrast, AU2–94 treatment had no effect on the cell cycle distribution of RB-deficient MDA-MB-468 cells at the same time point. C AU2–94 induced transient G1 arrest in murine BM cells after 24 hours of treatment. D BM cells were exposed to increasing concentrations of AU2–94 for 24 hours, and the percentage inhibition of p-RB (S780) was assessed by alpha assay. Data are representative of three independent experiments, except for panel A, which was performed twice. Statistical significance of differences between sample groups was assessed using a t-test for panel A, and one-way ANOVA for the others. Error bars indicate SEM. ns, non-significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 compared to control

Fig. 2

AU2–94 pre-treatment reduced the sensitivity of BM cells to chemotherapy. A and B BM cells were treated with AU2–94 or 0.1% DMSO for 24 hours and subsequently exposed to various DNA damaging chemotherapeutic drugs for another 24 hours. Cell viability was assessed using MTT assays, and the relative activity of caspase-3/7 was measured to evaluate apoptotic cell death. C and D AU2–94 pre-exposure prevented cisplatin-induced sub-G1 arrest, thereby inhibiting apoptotic cell death in BM cells. E Western blot analysis depicting the effect of AU2–94 pre-treatment on reducing cisplatin-induced DNA damage, as indicated by the level of γH2AX in BM cells. The fold-change in γH2AX expression was normalized to the loading control (GAPDH). Results shown are representative of three independent experiments, except for panel E, which was performed twice. Statistical significance of differences between sample groups was determined using one-way ANOVA analysis. Error bars represent SEM. ns, non-significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 compared to chemotherapy-treated cells

Fig. 3

AU2–94 pre-treatment mitigated the myelosuppressive effect of 5-FU in murine BM cells. A Experimental procedure illustrating treatment of female BALB/c mice (n = 10 per group) with vehicle (distilled water, PO), a single dose of AU2–94 (200 mg/kg, PO), 5-FU (100 mg/kg, IP), or AU2–94 (200 mg/kg, PO) administered 2 hours prior to 5-FU injection. Spleens, BM cells, and blood samples were collected at 2- and 6-days post-5-FU administration. B Spleen weight differences among treatment groups post-5-FU treatment. C Comparison of apoptotic events in BM cells among treatment groups, assessed by Annexin-V/PI staining. D Percentage of Ki-67 positive cells in BM among treatment groups, determined by Ki-67 intracellular staining. E Blood samples collected from 3 mice per group at 2 and 6 days after treatment. Statistical significance of differences between sample groups was evaluated using two-way ANOVA in GraphPad Prism 10. Error bars represent SEM. ns, non-significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 compared to the 5-FU treated group. ^, p ≤ 0.05; ^^, p ≤ 0.01; ^^^, p ≤ 0.001; ^^^^, p ≤ 0.0001 compared to the vehicle-treated group

Fig. 3

AU2–94 pre-treatment mitigated the myelosuppressive effect of 5-FU in murine BM cells. A Experimental procedure illustrating treatment of female BALB/c mice (n = 10 per group) with vehicle (distilled water, PO), a single dose of AU2–94 (200 mg/kg, PO), 5-FU (100 mg/kg, IP), or AU2–94 (200 mg/kg, PO) administered 2 hours prior to 5-FU injection. Spleens, BM cells, and blood samples were collected at 2- and 6-days post-5-FU administration. B Spleen weight differences among treatment groups post-5-FU treatment. C Comparison of apoptotic events in BM cells among treatment groups, assessed by Annexin-V/PI staining. D Percentage of Ki-67 positive cells in BM among treatment groups, determined by Ki-67 intracellular staining. E Blood samples collected from 3 mice per group at 2 and 6 days after treatment. Statistical significance of differences between sample groups was evaluated using two-way ANOVA in GraphPad Prism 10. Error bars represent SEM. ns, non-significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 compared to the 5-FU treated group. ^, p ≤ 0.05; ^^, p ≤ 0.01; ^^^, p ≤ 0.001; ^^^^, p ≤ 0.0001 compared to the vehicle-treated group

Fig. 4

The impact of AU2–94 pre-treatment on cisplatin-induced anti-cancer effect in RB-deficient MDA-MB-468 cells. A Flow cytometric analysis of cell cycle distribution in MDA-MB-468 cells following 24-hour pre-exposure to AU2–94 (2 and 4 μM) followed by 48-hour incubation with cisplatin (2 μM). B Colony formation assay in MDA-MB-468 cells treated with AU2–94 for 7 days, followed by exposure to cisplatin for another 7 days. Colonies were quantified using ImageJ software. C Assessment of apoptosis induction in MDA-MB-468 cells treated with AU2–94, cisplatin, and AU2–94 pre-treatment. D Immunoblotting analysis of MDA-MB-468 cells exposed to indicated concentrations of AU2–94 and cisplatin, showing protein expression levels. Results are representative of three independent experiments. Statistical significance between treatment groups was determined using one-way ANOVA analysis. Error bars represent SEM. ns, non-significant compared to cisplatin-treated cells; **, p ≤ 0.01; ****, p ≤ 0.0001 compared to control. Cis: Cisplatin

Fig. 5

The effect of pre-administration of AU2–94 before cisplatin in the MDA-MB-468 xenograft model. A and B Treatment schedule and relative changes in tumour volume and weight over a 21-day treatment cycle in MDA-MB-468-bearing BALB/c nude mice. Treatments included vehicle (distilled water PO QW), AU2–94 (100 mg/kg PO QW), cisplatin (5 mg/kg IP QW), AU2–94 (75 mg/kg PO QW) administered 2 hours before cisplatin, and AU2–94 (100 mg/kg PO QW) administered 2 hours before cisplatin. C Evaluation of peripheral blood cell counts 21 days after treatment initiation, illustrating that pre-administration of AU2–94 at 75 mg/kg before each cisplatin injection protected against chemotherapy-induced haematologic toxicity. Statistical significance between treatment groups was assessed using two-way ANOVA analysis. Error bars represent SEM. ns, non-significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 compared to cisplatin-treated group. ^, p ≤ 0.05; ^^, p ≤ 0.01; ^^^^, p ≤ 0.0001 compared to vehicle group