DF/HCC Breast Cancer Specialized Program of Research Excellence (SPORE)

Inhibitors of cyclin-dependent kinases CDK4 and CDK6 have been approved for treatment of luminal-type estrogen receptor (ER)-positive breast cancers. Unfortunately, a large proportion of patients with breast cancer develops resistance to CDK4/6 inhibition.

In Aim 1, we will test our hypothesis that CDK4/6 resistant breast cancer cells become dependent on hyperactivation of the cyclin-dependent kinase CDK2 for proliferation. Consequently, we hypothesize that inhibition of CDK2 in CDK4/6 inhibitor-resistant cancer cells would block their proliferation. We further hypothesize that combined inhibition of CDK4/6 and CDK2 would have a synergistic effect and might prevent the development of resistant disease. Currently, a major limitation in studying the role of CDK2 is the absence of CDK2-specific inhibitors. To overcome this limitation, our laboratory has applied the ‘analog-sensitive’ kinase approach, which allows us to specifically, potently and reversibly inhibit CDK2 using a compound that does not inhibit any other kinases. In Aim 1, we will use the analog-sensitive approach to test the impact of CDK2 inhibition on proliferation of CDK4/6-inhibitor resistant tumors in vivo. We will also compare side-by-side the effects of potent CDK2 inhibition using our system, versus CDK2 inhibitors that are currently in clinical trials. In an effort to assess the role of CDK2 in the development of CDK4/6 resistance in the clinic, we will obtain 120 baseline biopsies from patients starting CDK4/6 inhibitor treatment to obtain 60 paired biopsies at baseline and when resistance develops. These biopsies will be interrogated for CDK2 activation status, and we will develop new approaches to gauge CDK2 activity in the clinical setting. 

In Aim 2, we will extend our investigations to triple negative breast cancer (TNBC). In contrast to luminal-type breast cancers, TNBC is intrinsically resistant to CDK4/6 inhibition. Nonetheless, we have observed that a significant fraction of human TNBC cell lines critically requires CDK4/6 for proliferation. Our preliminary data indicate that CDK4/6 inhibitors become sequestered into TNBC cell lysosomes, thereby blocking the inhibitors’ therapeutic effect. Importantly, we found that treatment of TNBC cells with compounds that inhibit lysosomal acidification, such as chloroquine, reverses the sequestration and renders TNBC cells sensitive to CDK4/6 inhibitor treatment. We also identified a new CDK4/6 inhibitor compound that on its own inhibits proliferation of TNBC cells. We will test the utility of combining CDK4/6 inhibitors with chloroquine for treatment of TNBC, using patient-derived xenografts, as well as short-term cultures of cells isolated directly from human tumors. We will also use these systems to evaluate the efficacy of the novel CDK4/6 inhibitor described above. We will conduct a phase I/II study of palbociclib and chloroquine to test the hypothesis that the addition of chloroquine can circumvent lysosomal sequestration, and patients in this trial will undergo paired biopsies to assess CDK4/6 inhibitor sequestration. The expected overall impact of this proposal is that it may provide a highly effective therapeutic strategy for overcoming acquired resistance to CDK4/6 inhibitors and may extend the benefits of anti-CDK4/6 therapy to patients with TNBC.

Despite significant advances in the management of metastatic HER2+ breast cancer (BC), it remains incurable. The reason for this is that cancers invariably develop resistance to standard therapies – both cytotoxic chemotherapies and those that specifically target HER2. Modern attempts to improve upon standard therapies have largely focused on agents that inhibit HER2 downstream signaling more potently, but these have yielded only incremental benefits. Therefore, new treatment approaches are urgently needed. Recently it has become clear that HER2+ BCs are immunogenic. HER2 is a strong tumor antigen, and a proportion of HER2+ BCs harbor a lymphocytic infiltrate, which predicts for improved outcomes. In addition, anti-HER2 antibodies exert their effects in part by stimulating immune effector cells. Collectively, these facts provide rationale for testing immunotherapy in HER2+ metastatic BC. Our co-investigator Dr. Loi recently conducted a phase II study of trastuzumab and pembrolizumab (a PD-1 inhibitor) in patients with HER2+ metastatic BC (PANACEA). The study met its primary endpoint and thus provides proof-of-principle for the use of immunotherapy in HER2+ disease – however, only a small minority of patients benefited.

In Project 2, we will therefore study two novel therapeutic approaches designed to boost the anti-tumor immune response against HER2+ BC further: 

1. the use of CDK4/6 inhibitors, given together with trastuzumab and PD-1 blockade
2. the addition of PDL1blockade and a 4-1BB agonist to chemotherapy and trastuzumab.

Both regimens are rationally designed, based on our convincing preclinical data showing that these approaches markedly amplify anti-tumor immunity. In each Aim, we will perform a randomized, multicenter phase II clinical trial to determine the efficacy of these novel approaches. Each Aim will also employ a “co-clinical trial” model, with mouse studies running in parallel to human trials. The animal experiments will be performed using our state-of-the-art transgenic model of human HER2- driven mammary carcinoma (MMTV-rtTA/tetO-HER2). Our mouse studies incorporate cutting edge technologies to understand the mechanisms of activity for these novel immunotherapy approaches, as well as detailed studies of resistance mechanisms (including next-generation sequencing and multiplexed immunofluorescent profiling of tumor tissue). Meanwhile, the trials involve serial collection of tumor biopsies and blood samples. Biospecimens from mice and patients will be analyzed in parallel, with each informing the other. Ultimately, these studies will:

1. characterize the immune landscape of advanced HER2-positive BC in unprecedented detail
2. determine whether either of the two novel approaches is an effective clinical strategy
3. establish cellular mechanisms of activity for each regimen
4. explore mechanisms of therapeutic resistance

This work has the potential to uncover new therapies that enhance immune responses against HER2-positive BC, and thus significantly improve patient outcomes.

Breast cancer brain metastases (BCBM) affect up to half of patients with advanced HER2+ breast cancer and 10-15% of patients with advanced ER+/HER2- breast cancer. Standard of care includes surgery and/or radiotherapy; however, these approaches can be associated with substantial toxicities, do not address systemic disease, and leave patients at risk for future central nervous system progression (CNS) and death. Despite their clinical impact, existing preclinical models of BCBM have been limited, and the factors which influence BCBM growth are not well elucidated. To date, no systemic therapy has gained regulatory approval for the treatment of BCBM—hence this represents an area of major, persistent, and unmet medical need. Preclinical investigations, including our own, have suggested a role for at least two key pathways—PI3K/PTEN/mTOR and cyclin D1/CDK4—in the growth and maintenance of BCBM. The overarching goals of this project are to elucidate the roles of the PI3K/PTEN/mTOR pathway and the Cyclin D1/CDK4 pathway in the growth and development of BCBM, to dissect the basis of site-specific response/resistance to inhibitors of these pathways, to test the clinical utility of targeting the pathways in patients with BCBM, and to identify ways to predict and overcome therapeutic resistance, with the long-term goal of identifying more effective treatment and prevention strategies. To accomplish our aims, we have assembled a multidisciplinary team enabling close bi-directional collaboration between the laboratory and clinic. We will leverage our unique collection of patient-derived xenograft (PDX) models generated from human BCBM specimens, and genetically-engineered mouse models (GEMMs), married with state-of-the art molecular pathology techniques.

In Aim 1, we will:

1. test whether PTEN loss promotes the growth and maintenance of BCBMs, and evaluate the effects of genetic or pharmacologic restoration of PTEN expression
2. evaluate brain-penetrant PI3K/mTOR inhibitors in preclinical models of BCBM and uncover potential mechanisms of site-specific resistance
3. test the efficacy of combined PI3K/mTOR blockade in patients with HER2+ BCBM.

In Aim 2, we will: 

1. evaluate the efficacy of CDK4/6 inhibition, alone and in rational combinations
2. evaluate the efficacy and immuno-modulatory effects of CDK4/6 inhibitors, alone and in combination with immune checkpoint blockade and in varying genetic backgrounds
3. explore the clinical efficacy of combined HER2 and CDK4/6 inhibition in patients with HER2+ BCBM.

Together, these studies will further our understanding of the pathophysiology of BCBM, strengthen our ability to overcome therapeutic resistance, and improve outcomes for patients with this disease.

Although several lines of evidence support the use of immunotherapy in triple-negative breast cancer (TNBC), the modest clinical efficacy achieved in current clinical trials suggests that the immunosuppressive microenvironment cannot be overcome by PD-1/PD-L1 blockade alone. In order to improve outcomes, this project will investigate the immunologic effects of two emerging classes of targeted breast cancer therapies, poly (ADP-ribose) polymerase (PARP) and BET bromodomain (BBD) inhibitors, and will test the hypothesis that combinations of these agents with immunotherapies will be effective therapeutic strategies for BRCA-mutated and sporadic TNBC. The rationale for this work is based on our preliminary data indicating that PARP inhibition can activate the STING pathway, alter tryptophan metabolism and stimulate the infiltration and activation of cytotoxic T cells, and that BBD inhibitors synergize with paclitaxel and PD-L1 blockade in preclinical models.

Two specific aims are proposed. In Aim 1, the effects of PARP inhibition alone and in combination with PD-1blockade on the immune microenvironment and on tumor growth will be assessed in an animal model of BRCA associated TNBC and in a clinical trial. Experiments will be conducted in mice bearing TNBCs derived from the K14Cre;BRCA1f/f;p53f/f genetically-engineered mouse model, and will translate to a phase 2 trial in the neoadjuvant setting using niraparib or combined niraparib/PD-1 therapy, in which changes in T cell infiltrate and pathologic complete response (pCR) rate will be defined. Preclinically, combined PARP inhibition and PD-1 blockade will also be investigated in BRCA wild-type syngeneic TNBC models, including EMT-6 and JC.

In Aim 2, the effects of the BBD inhibitor JQ1 alone and in combination with PD-L1 blockade will be evaluated in the same syngeneic and genetically-engineered mouse models of TNBC used in Aim 1, as well as in a clinical trial. Changes in the composition and activation of components of the immune microenvironment will be assessed following JQ1 or JQ1 and PD-L1 treatment, with or without paclitaxel. Finally, a Phase 1 dose-escalation trial combining the JQ1 derivative RO6870810 and atezolizumab as a doublet, or with paclitaxel as a triplet, will be performed using concomitant and sequential schedules, in which tumor biopsies will be studied to document changes in the immune microenvironment and in copy number and expression of CD274, encoding PD-L1. The successful completion of this project will improve our understanding of the immune effects of these targeted therapies and may identify biomarkers to aid the selection of patients most likely to benefit.