Highlights
- •RANK signaling contributes to resistance to CDK4/6 inhibitors in ER+ breast cancer
- •RANK overexpression predicts resistance to CDK4/6 inhibitors in ER+ breast cancer
- •RANK pathway activates an interferon (IFN) response program
- •RANKL inhibitors treat and prevent/delay resistance to CDK4/6 inhibitors
Summary
The combination of endocrine therapy (ET) and cyclin-dependent kinase 4/6 (CDK4/6) inhibitors (CDK4/6i) was a hallmark in metastatic luminal breast cancer (BC). However, intrinsic and acquired resistance affects long-term efficacy. Here, we study the role of the receptor activator of nuclear factor-κB (RANK) pathway in CDK4/6i resistance. We find that RANK overexpression in luminal BC is associated with intrinsic resistance to CDK4/6i, both in vitro and in mouse xenografts, and decreased proliferation rate and chronic interferon (IFN) γ response are highlighted as resistance drivers. Gene expression data from the NeoPalAna CDK4/6i clinical trial, and studies with palbociclib-resistant cell lines, show that RANK is upregulated after treatment with CDK4/6i, supporting a role in acquired resistance. Our study shows that RANK ligand (RANKL) inhibitors can restore sensitivity to CDK4/6i and prevent acquired resistance. On the basis of these findings, we conclude that pharmacological inhibition of the RANK pathway through RANKL blocking could represent an add-on to ET + CDK4/6i, warranting further clinical studies.
Introduction
Breast cancer (BC) is a heterogeneous disease with multiple histological and molecular presentations, in which about 70% of all cases belong to the luminal sub-type, characterized by the expression of estrogen receptor (ER) and/or progesterone receptor (PR). Although these patients have the better prognosis at early stages, the prognosis is dismal in advanced disease (4-year survival rate 35.9%), accounting for 10% of all deaths of cancer among women in the United States.
Currently, the first-line standard-of-care treatment for advanced ER+HER2− BC is the combination of endocrine therapy (ET) with cyclin-dependent kinase 4/6 inhibitors (CDK4/6i), which remarkably improved outcomes in this setting.
Unfortunately, up to 20% of patients are unresponsive to therapy, and the majority develop clinical resistance within two years of starting treatment. So far, there are no validated biomarkers to predict response to CDK4/6i, although there are evidences of mechanisms of resistance, such as amplification or deletion of cell cycle proteins, stabilizing cyclin D1 mutations, increased transcription of cyclin genes via mTORC1 or androgen receptor (AR), or increased upstream signaling, such as PI3K-AKT pathway activation. Therefore, strategies to predict and overcome intrinsic resistance to CDK4/6i, or to extend benefit by delaying acquired resistance, are a clear unmet need.
The receptor activator of nuclear factor-κB (RANK) is a transmembrane receptor of the tumor necrosis factor receptor (TNFR) family, activated by RANK ligand (RANKL), a homotrimeric transmembrane or soluble protein.
Activated RANK recruits TNFR-associated cytoplasmic factors (TRAFs), and subsequently activates the NF-κB, c-Jun N-terminal kinase (JNK), p38, extracellular signal-regulated kinase (ERK), and Src pathways, modulating cellular responses. In the clinical setting, pharmacological inhibition of RANKL with denosumab, a fully human anti-RANKL monoclonal antibody, is approved for prevention of skeletal-related events in patients with multiple myeloma and in patients with cancer-induced bone metastases, as well as for the treatment of osteoporosis.
Over the past decade, RANK pathway has emerged as an important mediator of breast morphogenesis and carcinogenesis, mostly associated with triple-negative BC aggressiveness.
However, the relevance of RANK pathway in luminal BC has been recently acknowledged. We have found that RANK overexpression (OE) mediates an aggressive luminal BC phenotype, with decreased proliferation rate and susceptibility to chemotherapy and ET.
Moreover, it was demonstrated that ectopic RANK expression in non-transformed mammary epithelia induces senescence, leading to delayed onset of subsequent aggressive luminal-like tumors.
Stemming from these findings, we decided to investigate whether RANK pathway could be involved in luminal BC resistance to CDK4/6i. Here, we report that RANK OE drives intrinsic resistance to CDK4/6i, associated with decreased proliferation rate and aberrant interferon (IFN) response in tumor cells. Moreover, we have found not only that elevated RANK metagene expression in the tumor prior to CDK4/6i therapy could predict resistance to treatment in patients enrolled in the NeoPalAna neoadjuvant clinical trial (NCT01723774) but also that it increases after treatment in therapy-responding patients, a feature we also observed in cells with acquired resistance to CDK4/6i. Focusing on the therapeutic potential of RANK pathway inhibition via RANKL targeting, we demonstrate that it not only sensitized RANK OE luminal BC cells to CDK4/6i in vitro and in vivo but also effectively restored sensitivity of cells that have acquired resistance as well as prevented its onset when used in combination with CDK4/6i. Therefore, we propose that RANK pathway status might represent a predictive biomarker for CDK4/6i therapy, and our results unveil the clinical relevance of the combination of CDK4/6i + ET with RANKL inhibition.
Results
RANK OE in luminal BC cells induces intrinsic resistance to CDK4/6i reversible by RANK pathway inhibition
Standard-of-care first-line therapy for patients with metastatic luminal BC is the combination of ET and CDK4/6i. Our previous studies demonstrate that RANK expression has effects in the phenotype of luminal BC, both in vitro and in vivo. To investigate if RANK expression would influence the response to CDK4/6i we assessed the sensitivity of two luminal BC cell lines, MCF-7 and T47, to the three currently approved CDK4/6i, palbociclib (Palbo), ribociclib (Ribo), and abemaciclib (Abema), in comparison with their RANK OE derivatives (MCF-7OE and T47DOE). These RANK OE cell lines express high levels of RANK as confirmed by qRT-PCR (Figure S1A) and western blot (Figure S1B) and, without exception, were more resistant to CDK4/6i compared with their parental (PAR) counterparts (Figure 1A )….
