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Background: CDK4/6 inhibitors (CDK4/6i) combined with hormone therapies have demonstrated clinical benefit in HR+, HER2- breast cancer patients. However, the onset of resistance remains a concern and highlights a need for therapeutic strategies to improve outcomes. The objective of this study was to develop an in vitro model to better understand the mechanisms of resistance to CDK4/6i + hormone therapies and identify therapeutic strategies with potential to overcome this resistance. Methods: The HR+, HER2− T47D breast cancer cell line genetically modified with a Geminin–Venus reporter construct was treated with CDK4/6i (abemaciclib or palbociclib) in combination with 4-hydroxytamoxifen (tamoxifen). Resistant cells were identified by cell sorting for Geminin (%GEM+), a marker of the S/G2/M phases of the cell cycle, and confirmed by treatment with tamoxifen plus the CDK4/6i used to drive resistance. In resistant cells, following treatment with CDK4/6i + ET (tamoxifen or fulvestrant), the effects on cell proliferation (%GEM+) and viability, gene expression, and protein analysis to evaluate CDK4/6–cyclin D complex composition were examined. Results: Palbociclib + tamoxifen-resistant (PTxR) cells treated with abemaciclib + ET showed decreased %GEM+, %Ki67, and colony formation ability, compared to abemaciclib + tamoxifen-resistant (ATxR) cells treated with palbociclib + ET. Additionally, PTxR cells showed increased CDK4-p21 interaction, compared to ATxR. The CDK6 levels were greater in ATxR cells compared to PTxR cells, associated with CDK4/6i resistance. Additionally, abemaciclib + fulvestrant continued to robustly decrease pRb levels in PTxR models compared to palbociclib + fulvestrant in ATxR models. Transcriptome analysis revealed a depression of the cell cycle and E2F- and Rb-related genes in PTxR cells following treatment with abemaciclib + ET, not present in ATxR cells treated with palbociclib + ET. Both resistant models showed increased EGFR-related gene expression. Conclusion: Taken together, we describe CDK4/6i-dependent mechanisms resulting in early-onset resistance to CDK4/6i + ET, using clinically relevant drug concentrations, in preclinical breast cancer cell models. The characterization of these preclinical models post progression on CDK4/6 inhibitor + ET treatment highlights the potential that the specific sequencing of CDK4/6 inhibitors could offer to overcome acquired resistance to CDK4/6i + ET. Abemaciclib + fulvestrant is currently under clinical investigation in patients with HR+, HER2− breast cancer and progression on prior CDK4/6i + ET (NCT05169567, postMONARCH).

PP2A is a major tumor suppressor whose inactivation is frequently found in a wide spectrum of human tumors. In particular, deletion or epigenetic silencing of genes encoding the B55 family of PP2A regulatory subunits is a common feature of breast cancer cells. A key player in the regulation of PP2A/B55 phosphatase complexes is the cell cycle kinase MASTL (also known as Greatwall). During cell division, inhibition of PP2A-B55 by MASTL is required to maintain the mitotic state, whereas inactivation of MASTL and PP2A reactivation is required for mitotic exit. Despite its critical role in cell cycle progression in multiple organisms, its relevance as a therapeutic target in human cancer and its dependence of PP2A activity is mostly unknown. Here we show that MASTL overexpression predicts poor survival and shows prognostic value in breast cancer patients. MASTL knockdown or knockout using RNA interference or CRISPR/Cas9 systems impairs proliferation of a subset of breast cancer cells. The proliferative function of MASTL in these tumor cells requires its kinase activity and the presence of PP2A-B55 complexes. By using a new inducible CRISPR/Cas9 system in breast cancer cells, we show that genetic ablation of MASTL displays a significant therapeutic effect in vivo. All together, these data suggest that the PP2A inhibitory kinase MASTL may have both prognostic and therapeutic value in human breast cancer.

Full differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) results in expanded differentiation potency as well as improved efficiency in stringent assays such as tetraploid complementation and human-mouse interspecies chimerism. Mechanistically, these effects are mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasing of DNA methylation. As a proof of concept, miR-203 improves differentiation and maturation of PSCs into cardiomyocytes in vitro as well as cardiac regeneration in vivo, after cardiac injury. These data support the use of transient exposure to miR-203 as a general and single method to reset the epigenetic memory in PSCs, and improve their use in regenerative medicine. Footnotes * Supplemental files added.