Jeenah Park

Epigenetic modulation with histone deacetylase inhibitors in combination with immunotherapy

Understanding the contribution of dysregulated gene silencing to epigenomic alterations in cancer development provides the rationale for the use of epigenetic modulators, such as histone deacetylase (HDAC) inhibitors, in cancer therapy. HDAC inhibitors have been approved as single agents for cutaneous and peripheral T-cell lymphoma and have shown promising activity in reversing therapy resistance in other tumor types. The effects of HDAC inhibitors on immune modulation have created a recent interest in their potential role in immunotherapy. This review describes the current understanding on integrating HDAC inhibitors into various immunotherapeutic approaches, such as cancer vaccines, adoptive T-cell transfer and immune checkpoint inhibitors. Furthermore, it summarizes promising treatment strategies in epigenetic immune priming from clinical trials that are currently underway.

HDAC inhibition potentiates immunotherapy in triple negative breast cancer

Triple-negative breast cancer (TNBC) represents a more aggressive and difficult subtype of breast cancer where responses to chemotherapy occur, but toxicity is significant and resistance often follows. Immunotherapy has shown promising results in various types of cancer, including breast cancer. Here, we investigated a new combination strategy where histone deacetylase inhibitors (HDACi) are applied with immune checkpoint inhibitors to improve immunotherapy responses in TNBC. Testing different epigenetic modifiers, we focused on the mechanisms underlying HDACi as priming modulators of immunotherapy. Tumor cells were co-cultured with human peripheral blood mononuclear cells (PBMCs) and flow cytometric immunophenotyping was performed to define the role of epigenetic priming in promoting tumor antigen presentation and immune cell activation. We found that HDACi up-regulate PD-L1 mRNA and protein expression in a time-dependent manner in TNBC cells, but not in hormone responsive cells. Focusing on TNBC, HDACi up-regulated PD-L1 and HLA-DR on tumor cells when co-cultured with PBMCs and down-regulated CD4+ Foxp3+ Treg in vitro. HDACi significantly enhanced the in vivo response to PD-1/CTLA-4 blockade in the triple-negative 4T1 breast cancer mouse model, the only currently available experimental system with functional resemblance to human TNBC. This resulted in a significant decrease in tumor growth and increased survival, associated with increased T cell tumor infiltration and a reduction in CD4+ Foxp3+ T cells in the tumor microenvironment. Overall, our results suggest a novel role for HDAC inhibition in combination with immune checkpoint inhibitors and identify a promising therapeutic strategy, supporting its further clinical evaluation for TNBC treatment.

A phase I trial of panobinostat and epirubicin in solid tumors with a dose expansion in patients with sarcoma

BACKGROUND Treatment options for sarcoma are limited. Histone deacetylase inhibitors increase the efficacy of topoisomerase II inhibitors by promoting access to chromatin and by down-regulating DNA repair. Thus, combined panobinostat and epirubicin therapy was evaluated to treat refractory sarcoma. PATIENTS AND METHODS Patients with advanced solid tumors were enrolled in a 3 + 3 dose-escalation phase I trial of panobinostat given on days 1, 3, and 5 followed by 75 mg/m(2) of epirubicin on day 5 in 21-day cycles, with a dose expansion at maximum tolerated dose (MTD) in 20 sarcoma patients. Peripheral blood mononucleocyte histone acetylation was also evaluated. RESULTS Forty patients received 20-60 mg panobinostat. Dose-limiting toxicities included thrombocytopenia, febrile neutropenia, and fatigue at 60 mg, defining a panobinostat MTD at 50 mg. Four responses were seen in 37 assessable patients, all after progression on prior topoisomerase II inhibitors. For those with sarcoma, 12 of 20 derived clinical benefit (1 partial response and 11 stable disease, median overall survival 8.3 months), including 8 of 14 previously progressed on topoisomerase II therapy. Treatment benefits correlated with increased histone acetylation and decreased neutrophil count on day 5. CONCLUSIONS Panobinostat and epirubicin treatment is well tolerated and may reverse anthracycline resistance. Changes in histone acetylation and associated decrease in neutrophil count correlated with clinical benefit and warrant investigation as predictive biomarkers. CLINICAL TRIAL This trial is registered at www.Clinicaltrials.gov, Identifier: NCT00878904.

Local delivery of hormonal therapy with silastic tubing for prevention and treatment of breast cancer

Broad use of germline testing has identified an increasing number of women at risk for breast cancer with a need for effective chemoprevention. We report a novel method to selectively deliver various anti-estrogens at high drug levels to the breast tissue by implanting a device comprised of silastic tubing. Optimized tubing properties allow elution of otherwise poorly bioavailable anti-estrogens, such as fulvestrant, into mammary tissue in vitro and in vivo with levels sufficient to inhibit estrogen receptor activation and tumor cell proliferation. Implantable silastic tubing delivers fulvestrant selectively to mouse mammary fat tissue for one year with anti-tumor effects similar to those achieved with systemic fulvestrant exposure. Furthermore, local delivery of fulvestrant significantly decreases cell proliferation, as assessed by Ki67 expression, most effectively in tumor sections adjacent to tubing. This approach may thereby introduce a potential paradigm shift and offer a promising alternative to systemic therapy for prevention and early interception of breast cancer.

Abstract B10: HDAC inhibition modulates immune checkpoint pathway in triple-negative breast cancer

Background: Breast cancer remains one of the most serious and common diseases, second only to lung cancer as a leading cause of cancer death in women, accounting for 30% of new diagnoses. In particular, hormone-sensitive metastatic breast cancer and triple-negative breast cancer (TNBC) remains a therapeutic challenge. Despite aggressive therapeutic options, resistance develops in almost all patients and survival remains poor, prompting the need for novel approaches. This project focuses on the characterization of the molecular mechanisms underlying epigenetic modifiers and in particular Histone deacetylase inhibitors (HDACi) as priming modulators of immunotherapy, mainly focusing on the diversity between TNBC and hormone responsive breast cancer. HDACi represent a new class of anticancer agents that can affect multiple cancer pathways, including reverse hormone therapy resistance, resulting in prolonged antitumor responses in patients. In addition to their effects on tumor signaling, HDACi have immune modulatory functions, including modulation of regulatory T-cells, Foxp3 expression, changes in tumor-infiltrating lymphocyte composition and induction of PD-L1 expression. The PD-1/PD-L1 pathway is an immune checkpoint inhibitor pathway that under normal conditions down-regulates cytotoxic T-cell activity to maintain immune homeostasis. Cancer cells take advantage of this pathway to suppress immune control and inhibit the anti-tumor immune response by deactivating cytotoxic T-cells in the tumor microenvironment. Breast cancer is considered a less immunogenic cancer compared to melanoma and lung cancer. PD-L1 expression is mainly found in TNBC, HER2+, ER- and PR- tumors. Increased PD-L1 expression correlates with higher tumor infiltrating lymphocytes and those data together correlate with better response in breast cancer patients. PD-1/PD-L1 pathway represents one of the primary immunosuppressive drivers in multiple types of cancer. Thus, inhibiting PD-1/PD-L1 interactions may prevent T-cell suppression and reactivate immune-surveillance. Methods and Results: Evaluating PD-L1 basal expression in a different set of human and mouse breast cancer cell lines by Western blotting and real-time PCR, we found TNBC and HER2+ cells express the highest PD-L1 levels. Testing different epigenetic modifiers, we found that HDACi were able to up-regulate PD-L1 mRNA and protein expression in a time-dependent manner up to 72 hours in the TNBC MDA-MB231 cells. Co-culturing tumor cells with human peripheral blood mononuclear cell (PBMCs), we performed comprehensive flow cytometric immunophenotyping to define the role of epigenetic priming in promoting immune cell activation. We found that HDACi upregulate PD-L1 and HLA-DR expression on MDA-MB231 tumor cells independently from PBMCs presence. Opposite effects were observed with hormone responsive ER+ MCF-7 cells, where PD-L1 was not significantly increased and HLA-DR was down-regulated upon HDACi treatment. These effects were associated with reduced Foxp3 expression on CD4+ Tregs in vitro after HDACi treatment. Importantly, HDACi significantly enhanced the in vivo response to PD-1 and CTLA-4 blockade in the triple-negative 4T1 breast cancer mouse model. This was associated with increased tumor infiltrating T cells, increased survival, and tumor growth inhibition in vivo. Conclusion: Overall, this study suggests that the combination of HDACi with immune checkpoint inhibitors identify a novel therapeutic strategy and warrants further clinical evaluation for the treatment of TNBC. Citation Format: Manuela Terranova Barberio, Scott Thomas, Niwa Ali, Nela Pawlowska, Jeenah Park, Michael D. Rosenblum, Alfredo Budillon, Pamela N. Munster. HDAC inhibition modulates immune checkpoint pathway in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B10.

Chapter 40 – Clinical Applications of Histone Deacetylase Inhibitors

Abstract Aberrations in histone acetyltransferases and histone deacetylases (HDAC) have been associated with tumor development and tumor progression. Pharmacological interference with histone acetylation and methylation has shown to be promising for the treatment and prevention of cancer. Several structurally and functionally different HDAC inhibitors have been developed as a novel modality to reverse aberrant epigenetic changes in cancer cells. With a growing body of literature, there is ample preclinical rationale to test these drugs in various stages and settings of cancer, either as single agents or in rationally designed combinations as a therapeutic strategy in hematological as well as solid tumor malignancies. The first HDAC inhibitor vorinostat (Zolinza) was approved for the treatment of patients with cutaneous T-cell lymphoma. HDAC inhibitors have also demonstrated great promise in treating a variety of noncancer diseases, including epilepsy, cystic fibrosis, spinal muscular atrophy, and human immunodeficiency virus infection.

Abstract B111: HDAC inhibitors modulate immune checkpoint blockade in breast cancer

Background: Breast cancer is one of the most common diseases, second only to lung cancer as the leading cause of cancer death in women. In particular hormone sensitive metastatic breast cancer (MBC) and triple-negative breast cancer (TNBC) remains a therapeutic challenge as resistance develops in almost all patients. Despite aggressive treatment strategies, survival is poor due to the development of resistance, prompting the need for novel approaches. Here, we characterize the molecular mechanisms underlying epigenetic modifiers and in particular histone deacetylase inhibitors (HDACi) as priming modulators of immunotherapy, with a specific focus on TNBC and hormone resistant breast cancers. HDACi represent a new class of anticancer agents that can reverse hormone therapy resistance, resulting in prolonged anti-tumor responses in patients. In addition to their effects on estrogen receptor (ER) signaling, HDACi can also influence immune cell function, including but not limited to modulation of Foxp3+ regulatory T-cells (Tregs), tumor-infiltrating lymphocyte (TILs) composition, as well as induction of the co-inhibitory receptors PD-L1 and PD-1. Under normal conditions the PD-1/PD-L1 pathway down-regulates cytotoxic T-cell activity to maintain immune homeostasis. Cancer cells exploit this pathway in the tumor microenvironment to suppress cytotoxic T-cell activation, significantly diminishing the anti-tumor immune response. In breast cancer, PD-L1 expression is less frequent and mainly found in TNBC, HER2+, ER- and PR- tumors. Increased PD-L1 expression correlates with increased TILs and these criteria together are indicative of improved response rates in breast cancer patients. The PD-1/PD-L1 pathway represents one of the primary immunosuppressive drivers in multiple types of cancer. Thus, inhibiting PD-1/PD-L1 interactions may prevent T-cell suppression and reactivate immunosurveillance mechanisms necessary for tumor cell eradication. Methods and Results: Evaluation of basal PD-L1 expression in a range of human and mouse breast cancer cell lines by western blotting and real-time PCR identified TNBC and HER2+ cells as the highest expressing cells. Testing different epigenetic modifiers, we found that HDACi were able to up-regulate PD-L1 mRNA and protein in a time-dependent manner up to 72 hours. This was a direct transcriptional effect induced by HDACi and was confirmed even in tamoxifen resistant breast cancer cells, characterized by increased basal expression of PD-L1 as compared to the parental cells. To define the role of epigenetic priming in promoting immune cell activation, we co-cultured tumor cells and human peripheral blood mononuclear cell (PBMCs) and performed comprehensive immunophenotyping by flow cytometry. HDACi were able to up-regulate PD-L1 on tumor cells independent of PBMCs, while exhibiting a selective decrease in the frequency of immunosuppressive Tregs. Conclusion: Our data demonstrate that the combination of HDACi with immune checkpoint inhibitors represents a novel therapeutic anti-tumor strategy and warrants further clinical evaluation for the treatment of TNBC and hormone resistant breast cancer. Citation Format: Manuela Terranova Barberio, Scott Thomas, Niwa H. Ali, Jeenah Park, Michael D. Rosenblum, Alfredo Budillon, Pamela N. Munster. HDAC inhibitors modulate immune checkpoint blockade in breast cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B111.

Abstract P6-13-13: Developing silastic tubing for local delivery of hormonal therapy: A novel approach to breast cancer prevention

Background: For women at high risk for breast cancer, preventative interventions are limited to bilateral mastectomy with or without oophorectomy or prolonged anti-estrogen therapy. For many, these two options are unacceptable due to drug related toxicity or the irreversible consequences of prophylactic surgery. As such, many women will choose neither preventive measures. This highlights the need for prevention alternatives that are less invasive, less toxic, and less permanent. Ideally, only the tissue at risk should be treated. Therefore, we have developed a novel approach to release an anti-estrogen to the breast tissue only, with the goal to reach high breast tissue concentrations with minimal systemic exposure. Method: Cultured ER-positive breast cancer cells were used to validate the activity of fulvestrant released from the silastic tubing. CD-1 mice were used to demonstrate accumulation of fulvestrant released from the silastic tubing in the target breast tissue. LC-MS/MS was used to quantify released fulvestrant in both in vitro and in vivo experiments. Results: After 10 cm of silastic tubing was loaded with dry fulvestrant (0.076 mg/cm), it was placed in tissue culture media. Media was collected and replaced with fresh media every 3.5 days for 30+ weeks. It was then used to treat MCF7 and T47D cancer cells for 3 days. Within 7 days, fulvestrant released from the tubing was sufficient to modulate the ER signaling pathway of both cell lines and inhibit cell growth comparable to cells directly treated with a clinically feasible concentration of fulvestrant (100 nM). LC-MS/MS analysis demonstrated a fulvestrant release rate of rate of 10.7 ng fulvestrant per cm of tubing per day in culture media. To date, sustained release at this rate has been confirmed for 7+ months. To ascertain differential uptake of fulvestrant in the mammary tissue, we implanted fulvestrant-loaded tubing proximal to the inguinal mammary fat pad of CD-1 female mice and characterized biodistribution of fulvestrant. Various organs (blood, heart, lung, liver, kidney, and mammary fat pad) were harvested over time post-implantation. Using LC-MS/MS, we determined that fulvestrant preferentially accumulates in the mammary fat pad (175 nM), with minimal to no detection in other organs. Summary: Our data from in vitro and in vivo breast cancer models suggest that implantable silastic tubing has the capacity for long-term release of the anti-estrogen fulvestrant at high local concentrations that are sufficient to inhibit ER signaling and tumor cell proliferation with minimal systemic exposure. Further work is underway to design the optimal design for delivery. If successful, this option will provide a more acceptable alternative for breast cancer prevention and allow women at high risk to delay or forgo bilateral mastectomies. Citation Format: Park J, Thomas S, Zhong A, Piper M, Terranova Barberio M, Munster PN. Developing silastic tubing for local delivery of hormonal therapy: A novel approach to breast cancer prevention. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-13-13.