TARGETING AUTOPHAGY TO IMPROVE EFFICACY OF CDK4/6 INHIBITION IN BREAST CANCER

Deregulation of the cell cycle machinery is a hallmark of cancer, leading to aberrant proliferation and tumorigenesis. The crucial role of the CDK4/6-Cyclin D pathway has led to the development and FDA approval (palbociclib, ribociclib) of CDK4/6 inhibitors for the treatment of advanced estrogen receptor positive breast cancer. However, three major clinical challenges remain: i) adverse events leading to discontinuation of therapy and ii) lack of reliable biomarkers to identify responsive patients and iii) acquired resistance to CDK4/6 inhibitors. Previous in vitro studies have shown that palbociclib mediated CDK4/6 inhibition induces G1 arrest and senescence in ER+ breast cancer cells, and a recent study in fibroblasts implicated a role for palbociclib in inducing autophagy, a catabolic process that facilitates survival of the cells under stress. Thus, we hypothesize that in the presence of an intact G1/S checkpoint, autophagy protects ER positive breast cancer cells from palbociclib induced senescence. Further, based on our preliminary results, we hypothesize that cancer stem cells and EMT mediates acquired resistance to palbociclib. Results from this study show that breast cancer cells activate autophagy in response to palbociclib, and that the combination of autophagy and CDK4/6 inhibitors induces irreversible growth inhibition and senescence in vitro, and diminishes growth of cell line and patient-derived xenograft tumors in vivo. Furthermore, intact G1/S transition is necessary and predictive of preclinical sensitivity to this drug combination, and Rb positive and low-molecular-weight isoform of cyclin E negative status are reliable prognostic biomarkers in advanced estrogen receptor positive breast cancer patients. Inhibition of CDK4/6 and autophagy was also synergistic in other solid cancers with an intact G1/S checkpoint, providing a novel and promising biomarker-driven combination therapeutic strategy to treat breast and other solid tumors. Lastly, combined targeting with STAT-3 and PARP inhibitors can effectively target acquired resistant to palbociclib. Collectively, results from this study can help improve the efficacy, selectivity and treat acquired resistance to CDK4/6 inhibition in breast and other solid tumors

Cyclin E overexpression sensitizes triple negative breast cancer to Wee1 kinase Inhibition

Purpose: Poor prognosis in triple-negative breast cancer (TNBC) is due to an aggressive phenotype and lack of biomarker-driven targeted therapies. Overexpression of cyclin E and phosphorylated-CDK2 are correlated with poor survival in TNBC patients, and the absence of CDK2 desensitizes cells to inhibition of Wee1 kinase, a key cell cycle regulator. We hypothesize that cyclin E expression can predict response to therapies, which include the Wee1 kinase inhibitor, AZD1775. Experimental Design: Mono and combination therapies with AZD1775 were evaluated in TNBC cell lines and multiple patient derived xenograft (PDX) models with different cyclin E expression profiles. The mechanism(s) of cyclin E-mediated replicative stress were investigated following cyclin E induction or CRISPR/Cas9 knockout by a number of assays in multiple cells lines. Results: Cyclin E overexpression (1) is enriched in TNBCs with high recurrence rates, (2) sensitizes TNBC cell lines and PDX models to AZD1775, (3) leads to CDK2-dependent activation of DNA replication stress pathways and (4) increases Wee1 kinase activity. Moreover, treatment of cells with either CDK2 inhibitors or carboplatin leads to transient transcriptional induction of cyclin E (in cyclin E-low tumors) and result in DNA replicative stress. Such drug mediated cyclin E induction in TNBC cells and PDX models sensitizes them to AZD1775 in a sequential treatment combination strategy. Conclusions: Cyclin E is a potential biomarker of response (1) for AZD1775 as monotherapy in cyclin E high TNBC tumors and (2) for sequential combination therapy with CDK2 inhibitor or carboplatin followed by AZD1775 in cyclin E low TNBC tumors

Combined Inhibition of STAT3 and DNA Repair in Palbociclib-Resistant ER-Positive Breast Cancer.

Purpose: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are currently used in combination with endocrine therapy to treat advanced hormone receptor-positive, HER2-negative breast cancer. Although this treatment doubles time to progression compared with endocrine therapy alone, about 25%-35% of patients do not respond, and almost all patients eventually acquire resistance. Discerning the mechanisms of resistance to CDK4/6 inhibition is crucial in devising alternative treatment strategies