Cyclin E is a regulatory subunit of CDK2 that mediates S phase entry and progression. The cleavage of full-length cyclin E (FL-cycE) to low–molecular weight isoforms (LMW-E) dramatically alters substrate specificity, promoting G1–S cell cycle transition and accelerating mitotic exit. Approximately 70% of triple-negative breast cancers (TNBC) express LMW-E, which correlates with poor prognosis. PKMYT1 also plays an important role in mitosis by inhibiting CDK1 to block premature mitotic entry, suggesting it could be a therapeutic target in TNBC expressing LMW-E. In this study, analysis of tumor samples of patients with TNBC revealed that coexpression of LMW-E and PKMYT1-catalyzed CDK1 phosphorylation predicted poor response to neoadjuvant chemotherapy. Compared with FL-cycE, LMW-E specifically upregulates PKMYT1 expression and protein stability, thereby increasing CDK1 phosphorylation. Inhibiting PKMYT1 with the selective inhibitor RP-6306 (lunresertib) elicited LMW-E–dependent antitumor effects, accelerating premature mitotic entry, inhibiting replication fork restart, and enhancing DNA damage, chromosomal breakage, apoptosis, and replication stress. Importantly, TNBC cell line xenografts expressing LMW-E showed greater sensitivity to RP-6306 than tumors with empty vector or FL-cycE. Furthermore, RP-6306 exerted tumor suppressive effects in LMW-E transgenic murine mammary tumors and patient-derived xenografts of LMW-E–high TNBC but not in the LMW-E null models examined in parallel. Lastly, transcriptomic and immune profiling demonstrated that RP-6306 treatment induced interferon responses and T-cell infiltration in the LMW-E–high tumor microenvironment, enhancing the antitumor immune response. These findings highlight the LMW-E/PKMYT1/CDK1 regulatory axis as a promising therapeutic target in TNBC, providing the rationale for further clinical development of PKMYT1 inhibitors in this aggressive breast cancer subtype. Significance: PKMYT1 upregulation and CDK1 phosphorylation in triple-negative breast cancer expressing low–molecular weight cyclin E leads to suboptimal responses to chemotherapy but sensitizes tumors to PKMYT1 inhibitors, proposing a personalized treatment strategy.

Poly (ADP-ribose) polymerase inhibitors (PARPi) can encounter resistance through various mechanisms, limiting their effectiveness. Our recent research showed that PARPi alone can induce drug resistance by promoting autophagy. Moreover, our studies have revealed that anaplastic lymphoma kinase (ALK) plays a role in regulating the survival of ovarian cancer cells undergoing autophagy. Here, we explored whether the ALK-inhibitor crizotinib could enhance the efficacy of PARPi by targeting drug-induced autophagic ovarian cancer cell and xenograft models. Our investigation demonstrates that crizotinib enhances the anti-tumor activity of PARPi across multiple ovarian cancer cells. Combination therapy with crizotinib and olaparib reduced cell viability and clonogenic growth in two-olaparib resistant cell lines. More importantly, this effect was consistently observed in patient-derived organoids. Furthermore, combined treatment with crizotinib and olaparib led to tumor regression in human ovarian xenograft models. Mechanistically, the combination resulted in increased levels of reactive oxygen species (ROS), induced DNA damage, and decreased the phosphorylation of AKT, mTOR, and ULK-1, contributing to increased olaparib-induced autophagy and apoptosis. Notably, pharmacologic, or genetic inhibition or autophagy reduced the sensitivity of ovarian cancer cell lines to olaparib and crizotinib treatment, underscoring the role of autophagy in cell death. Blocking ROS mitigated olaparib/crizotinib-induced autophagy and cell death while restoring levels of phosphorylated AKT, mTOR and ULK-1. These findings suggest that crizotinib can improve the therapeutic efficacy of olaparib by enhancing autophagy. Implications: The combination of crizotinib and PARPi presents a promising strategy, that could provide a novel approach to enhance outcomes for patients with ovarian cancer.

Treatment strategies with a strong scientific rationale based on specific biomarkers are needed to improve outcomes in patients with advanced sarcomas. Suppression of cell-cycle progression through reactivation of the tumor suppressor retinoblastoma (Rb) using CDK4/6 inhibitors is a potential avenue for novel targeted therapies in sarcomas that harbor intact Rb signaling. Here, we evaluated combination treatment strategies (sequential and concomitant) with the CDK4/6 inhibitor abemacicib to identify optimal combination strategies. Expression of Rb was examined in 1,043 sarcoma tumor specimens, and 50% were found to be Rb-positive. Using in vitro and in vivo models, an effective two-step sequential combination strategy was developed. Abemaciclib was used first to prime Rb-positive sarcoma cells to reversibly arrest in G1 phase. Upon drug removal, cells synchronously traversed to S phase, where a second treatment with S-phase targeted agents (gemcitabine or Wee1 kinase inhibitor) mediated a synergistic response by inducing DNA damage. The response to treatment could be noninvasively monitored using real-time positron emission tomography imaging and serum thymidine kinase activity. Collectively, these results show that a novel, sequential treatment strategy with a CDK4/6 inhibitor followed by a DNA-damaging agent was effective, resulting in synergistic tumor cell killing. This approach can be readily translated into a clinical trial with noninvasive functional imaging and serum biomarkers as indicators of response and cell cycling. Significance: An innovative sequential therapeutic strategy targeting Rb, followed by treatment with agents that perturb DNA synthesis pathways, results in synergistic killing of Rb-positive sarcomas that can be noninvasively monitored.

Background/Objectives. Breast cancer is the most frequently diagnosed cancer worldwide, with approximately 15% classified as Triple-Negative Breast Cancer (TNBC). TNBC is characterized by the absence of estrogen receptor (ER) and progesterone receptor (PR), and the lack of HER2 overexpression, limiting use of targeted therapies. Current TNBC treatment relies heavily on chemotherapy, most commonly taxanes including paclitaxel that stabilize microtubules, disrupt chromosome separation and induce apoptosis. TNBCs frequently develop chemoresistance after multiple treatment cycles, highlighting a critical unmet need for novel therapeutic strategies. This study addresses this challenge by targeting salt-inducible kinase 2 (SIK2), which is overexpressed in 85% of TNBCs compared to normal breast tissue. Methodes. In collaboration with Arrien Pharmaceuticals and Greenfire Biologics, we developed ARN-3261/GRN-300, a novel orally bioavailable SIK2 inhibitor and evaluated its ability to sensitize TNBC cells to paclitaxel in vitro and in vivo. Results. GRN-300 demonstrated strong synergy with paclitaxel in all eight TNBC cell lines tested, as indicated by favorable combination indices. In xenograft models, the combination therapy significantly enhanced tumor growth inhibition and prolonged survival compared to either agent alone. Mechanistic studies showed that GRN-300 disrupts the anaphase-promoting complex/cyclosome (APC/C) pathway by downregulating key mitotic regulators, including CDC27, CDK1, and PLK1, thereby potentiating G2/M cell cycle arrest and apoptosis. Conclusions. Together, these findings establish GRN-300 as a promising therapeutic agent that enhances paclitaxel efficacy through complementary disruption of mitotic regulatory pathways, providing strong preclinical rationale for clinical development in TNBC.

This file contains all the information pertaining to supplementary methods that were referred to in the supplementary data.

Clinical and pathological characteristics of the 53 TNBC