Somatic estrogen receptor α mutations that induce dimerization promote receptor activity and breast cancer proliferation.

Physiologic activation of estrogen receptor α (ERα) is mediated by estradiol (E2) binding in the ligand-binding pocket of the receptor, repositioning helix 12 (H12) to facilitate binding of coactivator proteins in the unoccupied coactivator binding groove. In breast cancer, activation of ERα is often observed through point mutations that lead to the same H12 repositioning in the absence of E2. Through expanded genetic sequencing of breast cancer patients, we identified a collection of mutations located far from H12 but nonetheless capable of promoting E2-independent transcription and breast cancer cell growth. Using machine learning and computational structure analyses, this set of mutants was inferred to act distinctly from the H12-repositioning mutants and instead was associated with conformational changes across the ERα dimer interface. Through both in vitro and in-cell assays of full-length ERα protein and isolated ligand-binding domain, we found that these mutants promoted ERα dimerization, stability, and nuclear localization. Point mutations that selectively disrupted dimerization abrogated E2-independent transcriptional activity of these dimer-promoting mutants. The results reveal a distinct mechanism for activation of ERα function through enforced receptor dimerization and suggest dimer disruption as a potential therapeutic strategy to treat ER-dependent cancers.

A Druggable FOXA1-Glucocorticoid Receptor Transcriptional Axis Drives Tumor Growth in a Subset of Non-Small Cell Lung Cancer.

The FOXA1 pioneer factor is an essential mediator of steroid receptor function in multiple hormone-dependent cancers, including breast and prostate cancers, enabling nuclear receptors such as estrogen receptor (ER) and androgen receptor (AR) to activate lineage-specific growth programs. FOXA1 is also highly expressed in non-small cell lung cancer (NSCLC), but whether and how it regulates tumor growth in this context is not known. Analyzing data from loss-of-function screens, we identified a subset of NSCLC tumor lines where proliferation is FOXA1 dependent. Using rapid immunoprecipitation and mass spectrometry of endogenous protein, we identified chromatin-localized interactions between FOXA1 and glucocorticoid receptor (GR) in these tumor cells. Knockdown of GR inhibited proliferation of FOXA1-dependent, but not FOXA1-independent NSCLC cells. In these FOXA1-dependent models, FOXA1 and GR cooperate to regulate gene targets involved in EGF signaling and G1-S cell-cycle progression. To investigate the therapeutic potential for targeting this complex, we examined the effects of highly selective inhibitors of the GR ligand-binding pocket and found that GR antagonism with ORIC-101 suppressed FOXA1/GR target expression, activation of EGF signaling, entry into the S-phase, and attendant proliferation in vitro and in vivo. Taken together, our findings point to a subset of NSCLCs harboring a dependence on the FOXA1/GR growth program and provide rationale for its therapeutic targeting. Significance: NSCLC is the leading cause of cancer deaths worldwide. There is a need to identify novel druggable dependencies. We identify a subset of NSCLCs dependent on FOXA1-GR and sensitive to GR antagonism.

INK4 Tumor Suppressor Proteins Mediate Resistance to CDK4/6 Kinase Inhibitors.

Cyclin-dependent kinases 4 and 6 (CDK4/6) represent a major therapeutic vulnerability for breast cancer. The kinases are clinically targeted via ATP competitive inhibitors (CDK4/6i); however, drug resistance commonly emerges over time. To understand CDK4/6i resistance, we surveyed over 1,300 breast cancers and identified several genetic alterations (e.g., FAT1, PTEN, or ARID1A loss) converging on upregulation of CDK6. Mechanistically, we demonstrate CDK6 causes resistance by inducing and binding CDK inhibitor INK4 proteins (e.g., p18INK4C). In vitro binding and kinase assays together with physical modeling reveal that the p18INK4C-cyclin D-CDK6 complex occludes CDK4/6i binding while only weakly suppressing ATP binding. Suppression of INK4 expression or its binding to CDK6 restores CDK4/6i sensitivity. To overcome this constraint, we developed bifunctional degraders conjugating palbociclib with E3 ligands. Two resulting lead compounds potently degraded CDK4/6, leading to substantial antitumor effects in vivo, demonstrating the promising therapeutic potential for retargeting CDK4/6 despite CDK4/6i resistance. SIGNIFICANCE: CDK4/6 kinase activation represents a common mechanism by which oncogenic signaling induces proliferation and is potentially targetable by ATP competitive inhibitors. We identify a CDK6-INK4 complex that is resilient to current-generation inhibitors and develop a new strategy for more effective inhibition of CDK4/6 kinases.This article is highlighted in the In This Issue feature, p. 275.