Imlunestrant, elacestrant, and fulvestrant mediated ER degradation, PGR gene inhibition and cell proliferation inhibition in T-47D parental and T-47D ESR1-Y537N cells.

In vivo single agent imlunestrant efficacy in ESR1-mutant and wild type xenograft and PDX breast cancer models.

Statistical analysis of imlunestrant and fulvestrant efficacy alone and in combination with abemaciclib, alpelisib, and everolimus in ESR1-wild type and mutant xenograft and PDX breast cancer models

Imlunestrant PK/PD and uterotrophic activity.

Statistical survival analysis and body weight change in MCF-7 intracranial model treated with imlunestrant or other SERDs.

The impact of first-generation covalent KRASG12C inhibitors has been reduced due to the development of drug resistance, tolerability and challenges combining with immunotherapy. We designed olomorasib, a next-generation GDP-binding KRASG12C inhibitor, for nanomolar potency as well as selectivity over wild-type inhibition. In both in vitro and in vivo models of KRASG12C -mutant cancers, olomorasib reduces RAS activity and pERK levels, leading to substantial and significant tumor growth inhibition. Additionally, olomorasib combined with immune checkpoint inhibitors demonstrates greater anti-tumor activity compared to monotherapy. Furthermore, we demonstrate that olomorasib binds tightly to KRASG12C even in the presence of clinically relevant second site mutations, a known mechanism of resistance and limitation to currently approved KRASG12C inhibitors. These findings suggest that olomorasib could be effective for patients with KRASG12C mutant cancers either as monotherapy or in combination with immunotherapy. Olomorasib monotherapy and combination treatments are currently being investigated clinically.

In vivo imlunestrant efficacy alone and in combination with abemaciclib, alpelisib, and everolimus in CDX breast cancer models.

Biochemical and in vitro characterization of imlunestrant.

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).

Abstract Targeting of the estrogen receptor (ER) by antiestrogens is the standard of care for patients with ER+ HER2− advanced/metastatic breast cancer. Although antiestrogens that degrade ERα (fulvestrant) or block estrogen production (aromatase inhibitors) have improved patient outcomes, clinically important challenges remain related to drug administration, limited bioavailability, lack of brain exposure, and acquired resistance due to ESR1 mutations. These limitations indicate a need for more robust ER-targeted therapies. Here, we discovered and characterized imlunestrant, a next-generation potent, brain-penetrant oral selective ER degrader. Imlunestrant degraded ERα and decreased ERα-mediated gene expression both in vitro and in vivo. Cell proliferation and tumor growth in ESR1 wild-type (WT) and mutant models were significantly inhibited by imlunestrant. Combining imlunestrant with abemaciclib (CDK4/6 inhibitor), alpelisib (PI3K inhibitor), or everolimus (mTOR inhibitor) further enhanced tumor growth inhibition, regardless of ESR1 mutational status. In an ER+ breast cancer intracranial tumor model, imlunestrant prolonged survival compared with vehicle or alternative selective ER degrader therapies. Together, these findings support the potential of imlunestrant to degrade ERα and suppress the growth of ESR1-WT and mutant breast cancer, including brain metastatic tumors. Significance: Imlunestrant, a next-generation, brain-penetrant oral ERα degrader, displays potent activity in ESR1 wild-type and mutant breast cancer, enhances combination activity with standard-of-care agents, and inhibits growth of ER+ intracranial tumors.

Despite the biological and therapeutic relevance of CDK4/6 for the treatment of HR+, HER2- advanced breast cancer, the detailed mode of action of CDK4/6 inhibitors is not completely understood. Of particular interest, phosphorylation of CDK4 at T172 (pT172) is critical for generating the active conformation, yet no such crystal structure has been reported to date. We describe here the x-ray structure of active CDK4-cyclin D3 bound to the CDK4/6 inhibitor abemaciclib and discuss the key aspects of the catalytically-competent complex. Furthermore, the effect of CDK4/6 inhibitors on CDK4 T172 phosphorylation has not been explored, despite its role as a potential biomarker of CDK4/6 inhibitor response. We show mechanistically that CDK4/6i stabilize primed (pT172) CDK4-cyclin D complex and selectively displace p21 in responsive tumor cells. Stabilization of active CDK4-cyclin D1 complex can lead to pathway reactivation following alternate dosing regimen. Consequently, sustained binding of abemaciclib to CDK4 leads to potent cell cycle inhibition in breast cancer cell lines and prevents rebound activation of downstream signaling. Overall, our study provides key insights demonstrating that prolonged treatment with CDK4/6 inhibitors and composition of the CDK4/6-cyclin D complex are both critical determinants of abemaciclib efficacy, with implications for this class of anticancer therapy.