Apcin-derived CDC20 inhibitors synergize with microtubule disruptors to induce mitotic catastrophe in acute leukemias Free

CDC20, a coactivator of the Anaphase-Promoting Complex/Cyclosome (APC/C), is a key regulator of chromosome segregation and mitotic exit (Bruno et al. J Exp Clin Cancer Res 2022). Aberrant CDC20 overexpression has been associated with increased proliferation, poor prognosis and treatment resistance in multiple solid tumors. CDC20 is frequently upregulated in acute lymphoblastic (ALL) and acute myeloid (AML) leukemia, especially high-risk cases with complex karyotype (Simonetti et al. Cancer 2019). Despite its biological relevance, pharmacological targeting of CDC20 has remained largely underexplored. Apcin, the first CDC20-specific inhibitor (Sackton et al. Nature 2014), prevents substrate interaction, but demonstrates limited activity in preclinical tumor models due to the high concentrations required for efficacy (Lubet al. Oncotarget 2016).

To overcome these limitations, we designed, synthesized by a multistep approach, and screened five novel Apcin-based analogs for improved potency and cellular activity. Among them, M3 and M4 emerged as the most effective compounds in terms of anti-leukemic activity. We evaluated their therapeutic potential in a panel of AML (KG-1, OCI-AML3, MOLM-13, KASUMI-1) and Philadelphia-negative B-ALL (NALM-6, HAL-01) cell lines, as well as in primary leukemic blasts obtained from patients. Apcin exhibited modest activity, with IC₅₀ values between 30–100 μM at 48h. In contrast, M3 and M4 demonstrated 25- to 100-fold lower IC₅₀ values, indicating a significant increase in cytotoxic efficacy across leukemia models. Mechanistically, treatment with M3 and M4 led to robust G2/M cell cycle arrest, severe DNA damage, as shown by increased phosphorylation of H2AX^Ser139 even at sub-toxic concentrations, induction of apoptosis (40-60% at 24h, 2.5 μm) and inhibition of long-term clonogenic potential. Flow cytometry and immunoblotting revealed a consistent response pattern characterized by accumulation of cells in G2/M and sub-G1 phases, increased phosphorylation of CDK1^Tyr15, elevated cyclin B1 and cleavage of PARP1. Notably, an inverse relationship was observed between G2/M accumulation and apoptosis levels, suggesting that more sensitive cell lines were prone to rapid mitotic failure and death, whereas resistant lines exhibited prolonged arrest. Importantly, while CDC20 expression remained stable, its proteolytic activity was functionally inhibited, as proved by securin stabilization. Overall, these findings confirm checkpoint activation and effective disruption of APC/C^CDC20 function. The therapeutic potential of M3 and M4 was further supported by the response achieved on primary leukemic cells isolated from AML and ALL patients. Indeed, both compounds induced a dose- and time-dependent reduction of viability (˜50% at 48h, 5-10 μM) and clonogenic capacity. Strikingly, no cytotoxicity was observed in peripheral blood mononuclear cells from healthy subjects, even at concentrations exceeding therapeutic thresholds, indicating a favorable safety profile and selectivity for leukemic cells. Beyond CDC20 inhibition, confocal microscopy and HRP-based immunoassays demonstrated that M3 and M4 also disrupted tubulin polymerization, mimicking the mechanism of the mitotic poison vincristine. This dual mechanism prompted us to explore novel combination strategies. Combined treatment of acute leukemia cells with M3 and vincristine resulted in strong synergistic effects as quantified by both the Combination Index and ZIP synergy models, with enhanced apoptosis induction across nearly all cell lines. These results suggest that M3 amplifies the mitotic disruption mediated by vincristine, supporting the rationale for combination regimens targeting both microtubule dynamics and CDC20 activity.

In conclusion, our study identifies M3 and M4 as novel potent, dual-acting anti-leukemic agents that effectively target CDC20 function and microtubule integrity. Their activity against both cell lines and primary leukemic samples, combined with minimal toxicity in normal hematopoietic cells, highlights their therapeutic promise. These findings provide compelling preclinical evidence supporting the development of M3 and M4 for clinical application in novel combination strategies, including the one with established mitotic agents as vincristine. Future studies are needed to characterize the in vivo pharmacokinetics and efficacy of the compounds and to elucidate the molecular predictors of response in patient-derived models.