Benjamin Boyerinas

MICRORNAS: KEY PLAYERS IN THE IMMUNE SYSTEM, DIFFERENTIATION, TUMORIGENESIS AND CELL DEATH

Micro (mi)RNAs are small, highly conserved noncoding RNAs that control gene expression post-transcriptionally either via the degradation of target mRNAs or the inhibition of protein translation. Each miRNA is believed to regulate the expression of multiple mRNA targets, and many miRNAs have been linked to the initiation and progression of human cancer. miRNAs control various activities of the immune system and different stages of hematopoietic development, and their misexpression is the cause of various blood malignancies. Certain miRNAs have oncogenic activities, whereas others have the potential to act as tumor suppressors. Because they control fundamental processes such as differentiation, cell growth and cell death, the study of the role of miRNAs in human neoplasms holds great promise for novel forms of therapy. Here, we summarize the role of miRNAs and their targets in contributing to human cancers and their function as regulators of apoptotic pathways and the immune system.

The role of let-7 in cell differentiation and cancer

MicroRNAs (miRNAs or miRs) are small noncoding RNAs capable of regulating gene expression at the translational level. Current evidence suggests that a significant portion of the human genome is regulated by microRNAs, and many reports have demonstrated that microRNA expression is deregulated in human cancer. The let-7 family of microRNAs, first discovered in Caenorhabditis elegans, is functionally conserved from worms to humans. The human let-7 family contains 13 members located on nine different chromosomes, and many human cancers have deregulated let-7 expression. A growing body of evidence suggests that restoration of let-7 expression may be a useful therapeutic option in cancers, where its expression has been lost. In this review, we discuss the role of let-7 in normal development and differentiation, and provide an overview of the relationship between deregulated let-7 expression and tumorigenesis. The regulation of let-7 expression, cancer-relevant let-7 targets, and the relationship between let-7 and drug sensitivity are highlighted.

Identification of Let-7–Regulated Oncofetal Genes

MicroRNAs (miRNA) are small RNA molecules of approximately 20 to 22 nucleotides that reduce expression of proteins through mRNA degradation and/or translational silencing. Each known miRNA has a large number of predicted targets. Members of the let-7/miR-98 family of miRNAs are up-regulated at the end of embryonic development. Let-7 is often down-regulated early during cancer development, suggesting that let-7-regulated oncofetal genes (LOG) may become reexpressed in cancer cells. Using comparative bioinformatics, we have identified 12 conserved LOGs that include HMGA2 and IMP-1/CRD-BP. IMP-1 has growth-promoting activities through stabilization of c-myc mRNA. We experimentally confirmed that IMP-1 is a direct let-7 target that promotes cell growth and motility of tumor cells, and we confirmed by proteomics analysis that IMP-1 and HMGA2 are major miRNA targets. Our data suggest that a substantial part of the growth inhibitory activities of let-7 comes from suppressing the expression of IMP-1. LOGs could be novel therapeutic targets and potential biomarkers for cancer treatment.

Antibody-Dependent Cellular Cytotoxicity Activity of a Novel Anti–PD-L1 Antibody Avelumab (MSB0010718C) on Human Tumor Cells

Inhibition of PD-L1 interferes with an immunosuppressive signal, thereby prolonging antitumor responses. A novel monoclonal antibody to PD-L1 also mediated antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells, an additional mode of action for checkpoint inhibitors. Several anti–PD-1/PD-L1 monoclonal antibodies (mAb) are currently providing evidence of clinical benefit in subsets of cancer patients. The mode of action of these mAbs is to inhibit PD-1 on immune cells interacting with PD-L1 on tumor cells. These mAbs are either designed or engineered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), which, however, has been implicated as an important mechanism in several highly effective mAb-mediated cancer therapies. A fully human anti–PD-L1 mAb would potentially be able to block PD-1/PD-L1 interactions and also mediate the ADCC lysis of tumor cells. MSB0010718C (designated avelumab) is a fully human IgG1 anti–PD-L1 mAb. The studies reported here demonstrate (i) the ability of avelumab to lyse a range of human tumor cells in the presence of PBMC or NK effectors; (ii) IFNγ can enhance tumor cell PD-L1 expression and, in some cases, enhance ADCC tumor cell lysis; (iii) purified NK cells are potent effectors for avelumab; (iv) similar levels of avelumab-mediated ADCC lysis of tumor cells are seen using purified NK as effectors from either healthy donors or cancer patients; (v) very low levels of avelumab-mediated lysis are seen using whole PBMCs as targets; this finding complements results seen in analyses of PBMC subsets of patients receiving avelumab; and (vi) the addition of IL12 to NK cells greatly enhances avelumab-mediated ADCC. These studies thus provide an additional mode of action for an anti–PD-L1 mAb and support the rationale for further studies to enhance avelumab-mediated ADCC activity. Cancer Immunol Res; 3(10); 1148–57. ©2015 AACR.

Let-7 modulates acquired resistance of ovarian cancer to Taxanes via IMP-1-mediated stabilization of multidrug resistance 1.

Ovarian cancer patients frequently develop resistance to chemotherapy regiments using Taxol and carboplatin. One of the resistance factors that protects cancer cells from Taxol‐based therapy is multidrug resistance 1 (MDR1). micro(mi)RNAs are small noncoding RNAs that negatively regulate protein expression. Members of the let‐7 family of miRNAs are downregulated in many human cancers, and low let‐7 expression has been correlated with resistance to microtubule targeting drugs (Taxanes), although little is known that would explain this activity. We now provide evidence that, although let‐7 is not a universal sensitizer of cancer cells to Taxanes, it affects acquired resistance of cells to this class of drugs by targeting IMP‐1, resulting in destabilization of the mRNA of MDR1. Introducing let‐7g into ADR‐RES cells expressing both IMP‐1 and MDR1 reduced expression of both proteins rendering the cells more sensitive to treatment with either Taxol or vinblastine without affecting the sensitivity of the cells to carboplatin, a non‐MDR1 substrate. This effect could be reversed by reintroducing IMP‐1 into let‐7g high/MDR1 low cells causing MDR1 to again become stabilized. Consistently, many relapsed ovarian cancer patients tested before and after chemotherapy were found to downregulate let‐7 and to co‐upregulate IMP‐1 and MDR1, and the increase in the expression levels of both proteins after chemotherapy negatively correlated with disease‐free time before recurrence. Our data point at IMP‐1 and MDR1 as indicators for response to therapy, and at IMP‐1 as a novel therapeutic target for overcoming multidrug resistance of ovarian cancer.

Adhesion to osteopontin in the bone marrow niche regulates lymphoblastic leukemia cell dormancy

Malignant cells may evade death from cytotoxic agents if they are in a dormant state. The host microenvironment plays important roles in cancer progression, but how niches might control cancer cell dormancy is little understood. Here we show that osteopontin (OPN), an extracellular matrix molecule secreted by osteoblasts, can function to anchor leukemic blasts in anatomic locations supporting tumor dormancy. We demonstrate that acute lymphoblastic leukemia (ALL) cells specifically adhere to OPN in vitro and secrete OPN when localized to the endosteal niche in vivo. Using intravital microscopy to perform imaging studies of the calvarial bone marrow (BM) of xenografted mice, we show that OPN is highly expressed adjacent to dormant tumor cells within the marrow. Inhibition of the OPN-signaling axis significantly increases the leukemic cell Ki-67 proliferative index and leads to a twofold increase in tumor burden in treated mice. Moreover, using cell-cycle-dependent Ara-C chemotherapy to produce minimal residual disease (MRD) in leukemic mice, we show that OPN neutralization synergizes with Ara-C to reduce detectable BM MRD. Taken together, these data suggest that ALL interacts with extracellular OPN within the malignant BM, and that this interaction induces cell cycle exit in leukemic blasts, protecting them from cytotoxic chemotherapy.

Abstract 602: A novel TGF-β/IL-12R signal conversion platform that protects CAR T cells from TGF-β-mediated immune suppression and concurrently amplifies effector function

Numerous immune-suppressive mechanisms exist within the tumor microenvironment that may hinder chimeric antigen receptor (CAR) T cell efficacy. One such mechanism is mediated by TGF-β, a cytokine secreted by tumor cells and infiltrating suppressive immune cells that directly inhibits effector T cell activity. Effector T cells express the TGF-β receptors TGFBR1 and TGFBR2, and exposure of T cells to TGF-β induces phosphorylation of the major TGF-β signal mediators SMAD2 and SMAD3. Phosphorylated SMAD proteins (pSMADs) induce a suppressive transcriptional program that ultimately leads to reduced cytokine production, reduced cytotoxicity, and a failure to proliferate in response to antigen stimulation. A dominant negative receptor version (DNR) of TGFBR2 that does not contain signaling domains protects T cells from the impacts of TGF-β by blocking the ability of TGF-β to induce pSMADs. Here, we report the development of a novel TGF-β signal conversion platform that provides a T cell stimulatory signal upon exposure to TGF-β. This platform utilizes co-expression of chimeric variants of TGFBR2 and TGFBR1 where the TGF-β-binding domain of each receptor is fused to the transmembrane and intracellular signaling domains of the T cell simulating IL-12 receptors IL-12R-β2 and IL-12R-β1, respectively. Using a single lentiviral vector encoding both chimeric TGF-β receptors (CTBR) and a CAR, we demonstrated that CAR-CTBR T cells were completely protected from TGF-β-mediated SMAD phosphorylation. In addition, CAR-CTBR T cells generated significant amounts of pSTAT4 and pSTAT5 in response to TGF-β exposure, a response that mimics the T cell stimulation effects of IL-12. To further demonstrate successful signal conversion, we evaluated the impact of TGF-β exposure on the secretion of IFNγ, a major downstream target of IL-12 signaling. CAR-CTBR cells secreted significantly greater amounts of IFNγ than either control CAR T or CAR-DNR T cells following activation in the presence of TGF-β. Lastly, we utilized a serial restimulation assay to expand CAR, CAR-DNR, and CAR-CTBR T cells in the presence or absence of TGF-β. As expected, TGF-β exposure resulted in a significant inhibition of T cell proliferation in control CAR T cells. By contrast, both CAR-DNR and CAR-CTBR cells were protected from TGF-β-mediated inhibition of expansion. Gene expression analysis following 21 days of weekly antigen-driven expansion revealed specific TGF-β-mediated gene expression changes in CAR-CTBR cells consistent with increased T cell potency, including significant upregulation of IFNγ, IL10, IL18RAP, IL18R1, IL21R and CD62L transcripts. These data demonstrate the successful development of a TGF-β signal conversion platform that transforms the inhibitory effects of TGF-β exposure into an IL-12R-like T cell stimulatory signal that has the potential to produce superior CAR T cell responses in vivo. Note: This abstract was not presented at the meeting. Citation Format: Benjamin Boyerinas, Sara Miller, Ryan Murray, Stacie Seidel, Geoffrey Parsons, Kathy Seidl, Kevin Friedman, Richard Morgan. A novel TGF-β/IL-12R signal conversion platform that protects CAR T cells from TGF-β-mediated immune suppression and concurrently amplifies effector function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 602. doi:10.1158/1538-7445.AM2017-602

Abstract 1316: Evaluation of immune cell subsets of cancer patients treated with a fully human IgG1 anti-PD-L1 MAb (MSB0010718C) capable of mediating ADCC of human tumor cells

Background: Several monoclonal antibodies (MAbs) with demonstrated clinical anti-cancer activities have been engineered as fully human IgG1 entities to also encompass their potential to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of human tumor cells. MSB0010718C is a fully human IgG1 MAb targeting the co-regulatory protein Programmed Death-Ligand 1 (PD-L1), and is thus distinct from other MAbs targeting the PD-L1/PD-1 axis currently being evaluated in clinical trials. One possibility is that an anti-PD-L1 antibody capable of inducing ADCC may negatively affect PD-L1 expressing immune cell subtypes. This work is intended to determine if there is any validity to this concern. Methods: The clinical activity of MSB0010718C, observed in several tumor types in ongoing clinical studies such as NCT01772004, has been and will be reported elsewhere. In the studies reported here, MSB0010718C is shown to mediate ADCC of several types of human tumor cell lines (e.g., breast, lung, bladder carcinomas) in vitro, with tumor cell lysis mediated mainly by human CD16+ monocytes and natural killer (NK) cells. Since some human immune cell subsets express PD-L1 on their cell surface (albeit at relatively low levels compared to many tumor cells), studies were undertaken to evaluate changes in the frequency of immune cell subsets in peripheral blood mononuclear cells (PBMC) from cancer patients pre- vs post-treatment with MSB0010718C. Immune cells evaluated were PD-L1 positive and PD-L1 negative subsets of the following: CD4+ T cells, CD8+ T cells, NK cells, regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSC), natural killer T cells (NKT), plasmacytoid dendritic cells (DC), conventional DC, and B cells. Results: Forty-two post-treatment PBMC samples were evaluated as follows: pre vs 1 dose of MSB0010718C (day 15, n = 19); pre vs 3 doses of MSB0010718C (day 43, n = 14); and pre vs 9 doses of MSB0010718C (day 127, n = 9). In all cases there were no statistical differences pre- vs post-treatment in any immune cell subset, and at any time point analyzed, regardless of whether the immune subset expressed PD-L1 or not. In addition, no changes were observed in absolute lymphocyte counts at any time point analyzed. Conclusion: While immune cell subsets pre- vs post-treatment continue to be analyzed in various patient cohorts, these studies provide evidence that MSB0010718C, a fully human IgG1 MAb, capable of mediating ADCC, can be administered safely to cancer patients without altering the balance of numerous PBMC immune cell subsets. Citation Format: Lauren M. Lepone, Renee N. Donahue, Benedetto Farsaci, Italia Grenga, Benjamin Boyerinas, Caroline Jochems, Kwong-Yok Tsang, Christopher R. Heery, Ravi A. Madan, Geraldine O9Sullivan Coyne, Harpreet Singh, James L. Gulley, Jeffrey Schlom. Evaluation of immune cell subsets of cancer patients treated with a fully human IgG1 anti-PD-L1 MAb (MSB0010718C) capable of mediating ADCC of human tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1316. doi:10.1158/1538-7445.AM2015-1316

Abstract 2561: Yeast vector-encoding multiple MUC1 agonist epitopes (yeast-MUC1) can induce MUC1-specific T-cell immune responses

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The tumor-associated antigen MUC1 is overexpressed on various human hematological and epithelial malignancies. The MUC1 molecule, which has an N-terminal (MUC1-N) and a C-terminal (MUC1-C, which has been shown to act as an oncogene), is an attractive target for cancer immunotherapy. We have identified and reported 10 agonist epitopes (7 in the C-terminus, 2 in the N-terminus VNTR region, and 1 in the non-VNTR region) that enhance production of CD8 cytotoxic T lymphocytes. These epitopes span HLA-A2, HLA-A3, and HLA-A24 MHC class I alleles, thus encompassing the majority of the North American population. Compared to native epitopes, these agonist epitopes more efficiently generate MUC1-specific T cells, induce production of IFN-gamma by MUC1-specific T cells, and lyse human tumor cells expressing MUC1 native epitopes in an MHC-restricted manner. We have previously shown that heat-killed recombinant Saccharomyces cerevisiae yeast genetically modified to express carcinoembryonic antigen can efficiently activate human dendritic cells (DCs) and stimulate CEA-specific CD8+ T cells. Here, we investigated the ability of a Saccharomyces cerevisiae vector containing the MUC1 transgene (yeast-MUC1) and encoding MUC1 with 8 agonist epitopes to activate human DCs, stimulate MUC1-N- and MUC1-C-specific T cells, and generate MUC1-specific T cells. We show here for the first time that human DCs treated with yeast-MUC1 vectors can activate MUC1-N and MUC1-C agonist-specific T-cell lines and can act as antigen-presenting cells to generate MUC1-N- and MUC1-C-specific T cells specific to each agonist epitopes, and that these T cells are capable of lysing MUC1-expressing tumor cells. Together, these findings provide a rationale for further clinical evaluation of yeast-MUC1 constructs encoding MUC1 agonist epitopes in cancer vaccine immunotherapy. Citation Format: Kwong Y. Tsang, Benjamin Boyerinas, Caroline Jochems, Tim Rodell, Thomas King, Jeffey Schlom. Yeast vector-encoding multiple MUC1 agonist epitopes (yeast-MUC1) can induce MUC1-specific T-cell immune responses. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2561. doi:10.1158/1538-7445.AM2014-2561

Abstract 2546: Analysis of immune cell subsets in a multidrug therapeutic regimen for patients with metastatic castration-resistant prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: To investigate the effects of docetaxel-based combination therapy with bevacizumab and dexamethasone premedication on the immune response in patients with metastatic castration-resistant prostate cancer (mCRPC). Experimental Design: We studied immune responses in 13 patients enrolled in a phase II trial at the National Cancer Institute (NCI). The study was designed as a 13 patient expansion of a previously reported phase II study to evaluate the immunologic response after 2 cycles of treatment with a docetaxel-based chemotherapy regimen including docetaxel (75 mg/m2 every 3 weeks) and bevacizumab (15 mg/kg every 3 weeks). Dexamethasone pre-medication (4 mg) was given 12 h and 1 h prior to chemotherapy, and again after 12 h. Patients were evaluated before treatment and on day 40, 3 weeks after the second cycle. We compared PBMC and serum samples collected at baseline and after 40 days of treatment. We investigated CD4+ and CD8+ T-cells and regulatory T-cells (CD4+ CD25hi CD127- FoxP3+) by flow cytometry. T-cell proliferation, as well as NK-cell functional activity, was evaluated. Serum samples were analyzed for levels of cytokines, chemokines, sCD27, sCD40L and vascular endothelial growth factor (VEGF). Results: The baseline characteristics were: median age 64 years, Gleason score 9, PSA 100 ng/ml, and Halabi Predicted Survival 10.6 months. Patients had a median PSA decline of 66% after 2 cycles. The median TTP was 14.1 months, and OS 18.7 months. At 3 weeks after the second cycle we found no significant changes in absolute lymphocyte count, CD4+ and CD8+ T-cell proliferation and NK-cell function. The number of CD4+ T-cells decreased. CTLA4+ regulatory T-cells did not change. There was no change in the serum levels of IL-6, IL-8, IL-10 and TNFα. As expected, the serum levels of VEGF decreased substantially after therapy. The serum levels of sCD40L did not change after treatment. Interestingly, an increase in the serum level of sCD27 correlated with longer OS (P= 0.037, R= 0.58). Conclusions: Treatment of mCRPC patients with docetaxel-based combination therapy with bevacizumab and dexamethasone premedication for 40 days did not alter the immune response in a way that would decrease the likelihood of successful immunotherapy, either before or after this treatment. Citation Format: Caroline Jochems, Benjamin Boyerinas, Ravi A. Madan, Diane J. Poole, Yang-Min Ning, William D. Figg, David J. Liewehr, Seth M. Steinberg, James L. Gulley, Kwong-Yok Tsang, Jeffrey Schlom. Analysis of immune cell subsets in a multidrug therapeutic regimen for patients with metastatic castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2546. doi:10.1158/1538-7445.AM2014-2546