Deregulation of the cell cycle machinery is a hallmark of cancer. While CDK4/6 inhibitors are FDA approved (palbociclib) for treating advanced estrogen receptor-positive breast cancer, two major clinical challenges remain: (i) adverse events leading to therapy discontinuation and (ii) lack of reliable biomarkers. Here we report 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 tumours in vivo. Furthermore, intact G1/S transition (Rb-positive and low-molecular-weight isoform of cyclin E (cytoplasmic)-negative) is a reliable prognostic biomarker in ER positive breast cancer patients, and predictive of preclinical sensitivity to this drug combination. Inhibition of CDK4/6 and autophagy is 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 tumours.

Cyclin E (CCNE) is altered in nearly a third of invasive breast cancers where it is a powerful independent predictor of survival in women with stage I-III disease. Full-length cyclin E is post-translationally cleaved into low molecular weight (LMW-E) isoforms, which are tumor-specific and accumulate in the cytoplasm because they lack a nuclear localization sequence (NLS). We hypothesized that aberrant localization of cytosolic LMW-E isoforms alters target binding and activation ultimately contributing to LMW-E-induced tumorigenicity. To address this hypothesis, we used a retrovirus-based protein complementation assay to find LMW-E binding proteins in breast cancer, identifying ATP-citrate lyase (ACLY), an enzyme in the de novo lipogenesis pathway, as a novel LMW-E-interacting protein in the cytoplasm. LMW-E upregulated ACLY enzymatic activity, subsequently increasing lipid droplet formation, thereby providing cells with essential building blocks to support growth. ACLY was also required for LMW-E-mediated transformation, migration and invasion of breast cancer cells in vitro along with tumor growth in vivo. In clinical specimens of breast cancer, the absence of LMW-E and low expression of adipophilin (PLIN2), a marker of lipid droplet formation, associated with favorable prognosis, whereas overexpression of both proteins correlated with a markedly worse prognosis. Taken together, our findings establish a novel relationship between LMW-E isoforms of cyclin E and aberrant lipid metabolism pathways in breast cancer tumorigenesis, warranting further investigation in additional malignancies exhibiting their expression.

Purpose Preoperative aromatase inhibitor (AI) therapy has demonstrated efficacy in hormone receptor (HR)-positive postmenopausal breast cancer. However, many patients have disease that is either intrinsically resistant to AIs or that responds initially but develops resistance after prolonged exposure. We have shown that patients with breast tumors expressing the deregulated forms of cyclin E (low molecular weight forms [LMW-E]) have poor overall survival. Herein, we hypothesize that LMW-E expression can identify HR-positive tumors that are unresponsive to neoadjuvant AI therapy due to the inability of AIs to induce a cytostatic effect. Experimental Design LMW-E was examined in breast cancer specimen from 58 patients enrolled in the American College of Surgeons Oncology Group Z1031, a neoadjuvant AI clinical trial. The mechanisms of LMW-E mediated resistance to AI were evaluated in vitro and in vivo using an inducible model system of cyclin E (full-length and LMW-E) in aromatase-overexpressing MCF7 cells. Results Breast cancer recurrence-free interval was significantly worst in LMW-E positive patients who received AI neoadjuvant therapy. Upon LMW-E induction, MCF7 xenografts were unresponsive to letrozole in vivo, resulting in increased tumor volume after treatment with AIs. LMW-E expression overcame cell cycle inhibition by AIs in a CDK2/Rb-dependent manner and inhibition of CDK2 by dinaciclib reversed LMW-E-mediated resistance, while treatment with palbociclib, a CDK4/6 inhibitor, did not. Conclusion Collectively, these findings suggest that cell cycle deregulation by LMW-E mediates resistance to AIs and a combination of CDK2 inhibitors and AIs may be an effective treatment in patients with HR-positive tumors that express LMW-E.

Background: CDK 4 and 6 inhibitors (CDK4/6i), which arrest unregulated cancer cell proliferation, show clinical efficacy in breast cancer. Unexpectedly, a patient treated on a CDK4/6i-based trial, as first-line therapy in metastatic breast cancer, developed rapid disease progression following discontinuation of study drug while receiving standard second-line therapy off trial. We thus sought to expand this observation within a population of patients treated similarly at The University of Texas MD Anderson Cancer Center. Methods: Using an IRB-approved protocol, 4 patients previously enrolled on CDK4/6i trials were analyzed for outcomes after discontinuing study drug. These patients were treated on a randomized trial of first-line endocrine therapy +/-a CDK4/6i. Rapid disease progression was defined as progression occurring within 4 months of CDK4/6i discontinuation. Results: In total, 4 patients developed rapid disease progression and died; 2 of whom died within 6 months of CDK4/6i discontinuation. Conclusion: This case series suggests a potential for rapid disease progression following CDK4/6i discontinuation. However, the clinical course following progression must be validated in large CDK4/6i clinical trials and standard-of-care cohorts. If confirmed, such observations may alter the algorithm for subsequent therapy in patients with disease progression on CDK4/6i. Nevertheless, the need remains to define a mechanistic basis for this rapid progression and formulate alternative therapeutic strategies.