Satbir Thakur

ING1 and 5-Azacytidine Act Synergistically to Block Breast Cancer Cell Growth

Background Inhibitor of Growth (ING) proteins are epigenetic “readers” that recognize trimethylated lysine 4 of histone H3 (H3K4Me3) and target histone acetyl transferase (HAT) and histone deacetylase (HDAC) complexes to chromatin. Methods and Principal Findings Here we asked whether dysregulating two epigenetic pathways with chemical inhibitors showed synergistic effects on breast cancer cell line killing. We also tested whether ING1 could synergize better with chemotherapeutics that target the same epigenetic mechanism such as the HDAC inhibitor LBH589 (Panobinostat) or a different epigenetic mechanism such as 5-azacytidine (5azaC), which inhibits DNA methyl transferases. Simultaneous treatment of breast cancer cell lines with LBH589 and 5azaC did not show significant synergy in killing cells. However, combination treatment of ING1 with either LBH589 or 5azaC did show synergy. The combination of ING1b with 5azaC, which targets two distinct epigenetic mechanisms, was more effective at lower doses and enhanced apoptosis as determined by Annexin V staining and cleavage of caspase 3 and poly-ADP-ribose polymerase (PARP). ING1b plus 5azaC also acted synergistically to increase γH2AX staining indicating significant levels of DNA damage were induced. Adenoviral delivery of ING1b with 5azaC also inhibited cancer cell growth in a murine xenograft model and led to tumor regression when viral concentration was optimized in vivo. Conclusions These data show that targeting distinct epigenetic pathways can be more effective in blocking cancer cell line growth than targeting the same pathway with multiple agents, and that using viral delivery of epigenetic regulators can be more effective in synergizing with a chemical agent than using two chemotherapeutic agents. This study also indicates that the ING1 epigenetic regulator may have additional activities in the cell when expressed at high levels.

Src Regulates the Activity of the ING1 Tumor Suppressor

The INhibitor of Growth 1 (ING1) is stoichiometric member of histone deacetylase (HDAC) complexes and functions as an epigenetic regulator and a type II tumor suppressor. It impacts cell growth, aging, apoptosis, and DNA repair, by affecting chromatin conformation and gene expression. Down regulation and mislocalization of ING1 have been reported in diverse tumor types and Ser/Thr phosphorylation has been implicated in both of these processes. Here we demonstrate that both in vitro and in vivo, the tyrosine kinase Src is able to physically associate with, and phosphorylate ING1, which results in a nuclear to cytoplasmic relocalization of ING1 in cells and a decrease of ING1 stability. Functionally, Src antagonizes the ability of ING1 to induce apoptosis, most likely through relocalization of ING1 and down regulation of ING1 levels. These effects were due to both kinase-dependent and kinase-independent properties of Src, and were most apparent at elevated levels of Src expression. These findings suggest that Src may play a major role in regulating ING1 levels during tumorigenesis in those cancers in which high levels of Src expression or activity are present. These data represent the first report of tyrosine kinase-mediated regulation of ING1 levels and suggest that kinase activation can impact chromatin structure through the ING1 epigenetic regulator.

ING3 protein expression profiling in normal human tissues suggest its role in cellular growth and self-renewal.

Members of the INhibitor of Growth (ING) family of proteins act as readers of the epigenetic code through specific recognition of the trimethylated form of lysine 4 of histone H3 (H3K4Me3) by their plant homeodomains. The founding member of the family, ING1, was initially identified as a tumor suppressor with altered regulation in a variety of cancer types. While alterations in ING1 and ING4 levels have been reported in a variety of cancer types, little is known regarding ING3 protein levels in normal or transformed cells due to a lack of reliable immunological tools. In this study we present the characterization of a new monoclonal antibody we have developed against ING3 that specifically recognizes human and mouse ING3. The antibody works in western blots, immunofluorescence, immunoprecipitation and immunohistochemistry. Using this antibody we show that ING3 is most highly expressed in small intestine, bone marrow and epidermis, tissues in which cells undergo rapid proliferation and renewal. Consistent with this observation, we show that ING3 is expressed at significantly higher levels in proliferating versus quiescent epithelial cells. These data suggest that ING3 levels may serve as a surrogate for growth rate, and suggest possible roles for ING3 in growth and self renewal and related diseases such as cancer.

The use of automated Ki67 analysis to predict Oncotype DX risk-of-recurrence categories in early-stage breast cancer.

Ki67 is a commonly used marker of cancer cell proliferation, and has significant prognostic value in breast cancer. In spite of its clinical importance, assessment of Ki67 remains a challenge, as current manual scoring methods have high inter- and intra-user variability. A major reason for this variability is selection bias, in that different observers will score different regions of the same tumor. Here, we developed an automated Ki67 scoring method that eliminates selection bias, by using whole-slide analysis to identify and score the tumor regions with the highest proliferative rates. The Ki67 indices calculated using this method were highly concordant with manual scoring by a pathologist (Pearson’s r = 0.909) and between users (Pearson’s r = 0.984). We assessed the clinical validity of this method by scoring Ki67 from 328 whole-slide sections of resected early-stage, hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer. All patients had Oncotype DX testing performed (Genomic Health) and available Recurrence Scores. High Ki67 indices correlated significantly with several clinico-pathological correlates, including higher tumor grade (1 versus 3, P<0.001), higher mitotic score (1 versus 3, P<0.001), and lower Allred scores for estrogen and progesterone receptors (P = 0.002, 0.008). High Ki67 indices were also significantly correlated with higher Oncotype DX risk-of-recurrence group (low versus high, P<0.001). Ki67 index was the major contributor to a machine learning model which, when trained solely on clinico-pathological data and Ki67 scores, identified Oncotype DX high- and low-risk patients with 97% accuracy, 98% sensitivity and 80% specificity. Automated scoring of Ki67 can thus successfully address issues of consistency, reproducibility and accuracy, in a manner that integrates readily into the workflow of a pathology laboratory. Furthermore, automated Ki67 scores contribute significantly to models that predict risk of recurrence in breast cancer.

Oncolytic virotherapy as an immunotherapeutic strategy for multiple myeloma

Multiple Myeloma (MM), a clonal malignancy of antibody-producing plasma cells, is the second most common hematologic malignancy and results in significant patient morbidity and mortality. The high degree of immune dysregulation in MM, including T cell imbalances and up-regulation of immunosuppressive checkpoint proteins and myeloid derived suppressor cells, allows this malignancy to escape from host immune control. Despite advances in the therapeutic landscape of MM over the last decade, including the introduction of immunomodulatory drugs, the prognosis for this disease is poor, with less than 50% of patients surviving 5 years. Thus, novel treatment strategies are required. Oncolytic viruses (OV) are a promising new class of therapeutics that rely on tumour specific oncolysis and the generation of a potent adaptive anti-tumour immune response for efficacy. To date, a number of OV have shown efficacy in pre-clinical studies of MM with three reaching early phase clinical trials. OVs represent a rational therapeutic strategy for MM based on (1) their tumour tropism, (2) their ability to potentiate anti-tumour immunity and (3) their ability to be rationally combined with other immunotherapeutic agents to achieve a more robust clinical response.

Abstract 541: Successful oncolytic virotherapy in a bortezomib resistant syngeneic mouse model of multiple myeloma: Implications for translational significance

Introduction: Multiple myeloma (MM) is a malignancy of plasma cells that is still considered incurable due to the 90% relapse rate in patients. Despite the advent of new agents the majority of MM patients relapse secondary to therapy resistance. The potential of reovirus (RV) as a novel therapeutic agent for MM under in vitro, in vivo and ex vivo conditions has been demonstrated by us and a phase I clinical trial of MM with RV therapy has shown indications of efficacy. Recently we have shown that RV could synergize with several standard of care MM drugs such as bortezomib (BTZ), carfilzomib (CFZ) and dexamethasone and importantly, enhance its therapeutic potential in therapy resistant human MM cell lines in vitro. Utilizing the syngeneic Vk*MYC BTZ resistant transplantable MM mouse model, we demonstrate that mice harbouring BTZ insensitive MM tumors significantly respond to RV treatment. Our data indicate that this RV treatment sensitivity is manifested via, direct oncolysis in conjunction with immune modulatory effects. Methods: Vk*MYC myeloma cells (Vk12598) were injected via tail vein (IV) into 3 groups of C57BL/6 wt recipient mice. Seven days post tumor injection, mice were treated with PBS (vehicle) or live (LV) or dead reovirus (DV) (5×10⁁8 PFU) administered IV every 6 days in a total of 5 doses. Serum gamma globulins (M-spike) were assessed weekly by serum protein electrophoresis. Mice were sacrificed on day 35 post tumour injection and their spleens and bone marrow (BM) were harvested. Splenic and BM cells were stained for CD45+/CD45R-/CD138+ (MM tumors), CD4+/CD8+ (T cells), CD3+/NKG2D+ (NKT cells), CD11b+Ly6C+ (monocytic myeloid derived suppressor cells (Mo-MDSC) and CD11b+Ly6G+ (polymorphonuclear (PMN) - MDSCs) and analyzed by flow cytometry. Results: Mice treated with RV demonstrated highly significant (P Conclusions: Our results indicate reovirotherapy is successful in a validated syngeneic BTZ resistant MM model. These results have important implications for future clinical trials. Currently we are conducting further experiments to assess the immune modulatory (such as T - memory and T - regulatory cells) contributions of RV in this model. Citation Format: Chandini M. Thirukkumaran, Zhong Qiao Shi, Joanne Luider, Karen Kopciuk, Marta Chesi, Leif Bergsagel, Yuan Dong, Chunfen Zhang, Ahmed Mostafa, Kathy Gratton, Satbir Thakur, Don Morris. Successful oncolytic virotherapy in a bortezomib resistant syngeneic mouse model of multiple myeloma: Implications for translational significance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 541.

Oncolytic Reovirus and Immune Checkpoint Inhibition as a Novel Immunotherapeutic Strategy for Breast Cancer

As the current efficacy of oncolytic viruses (OVs) as monotherapy is limited, exploration of OVs as part of a broader immunotherapeutic treatment strategy for cancer is necessary. Here, we investigated the ability for immune checkpoint blockade to enhance the efficacy of oncolytic reovirus (RV) for the treatment of breast cancer (BrCa). In vitro, oncolysis and cytokine production were assessed in human and murine BrCa cell lines following RV exposure. Furthermore, RV-induced upregulation of tumor cell PD-L1 was evaluated. In vivo, the immunocompetent, syngeneic EMT6 murine model of BrCa was employed to determine therapeutic and tumor-specific immune responses following treatment with RV, anti-PD-1 antibodies or in combination. RV-mediated oncolysis and cytokine production were observed following BrCa cell infection and RV upregulated tumor cell expression of PD-L1. In vivo, RV monotherapy significantly reduced disease burden and enhanced survival in treated mice, and was further enhanced by PD-1 blockade. RV therapy increased the number of intratumoral regulatory T cells, which was reversed by the addition of PD-1 blockade. Finally, dual treatment led to the generation of a systemic adaptive anti-tumor immune response evidenced by an increase in tumor-specific IFN-γ producing CD8+ T cells, and immunity from tumor re-challenge. The combination of PD-1 blockade and RV appears to be an efficacious immunotherapeutic strategy for the treatment of BrCa, and warrants further investigation in early-phase clinical trials.

Stromal ING1 expression induces a secretory phenotype and correlates with breast cancer patient survival

BackgroundPrevious studies have established that levels of the Inhibitor of Growth 1(ING1) tumor suppressor are reduced in a significant proportion of different cancer types. Here we analyzed levels of ING1 in breast cancer patients to determine its prognostic significance as a biomarker for breast cancer prognosis.MethodsWe used automated quantitative analysis (AQUA) to determine the levels of ING1 in the tumor associated stromal cells of 462 breast cancer samples. To better understand how high ING1 levels affect nearby epithelium, we measured the levels of cytokines and secreted matrix metalloproteases (MMPs), using an ELISA based assay in mammary fibroblasts overexpressing ING1. These cells were also used in a 3-dimensional co-culture with MCF7 cells to determine the effect of released MMPs and other cytokines on growing colonies.ResultsWe find that high levels of ING1 in stroma are associated with tumor grade (p = 0.001) and size (p = 0.02), and inversely associated with patient survival (p = 0.0001) in luminal, but not in non-luminal cancers, suggesting that high stromal ING1 promotes cancer development. In this group of patients ING1 could also predict patient survival and act as a biomarker (HR = 2.125). While ING1 increased or decreased the expression of different cytokines, ING1 also increased the levels of MMP1, MMP3 and MMP10 by 5–8 fold, and concomitantly decreased levels of the tissue inhibitors of metalloproteases TIMP2, TIMP3 and TIMP4 by 1.5–3.3 fold, resulting in significant increases in MMP activity as determined by zymography. Co-culturing of MCF7 cells with stromal cells expressing ING1 in 3-dimensional organoid cultures suggested that MCF7 colonies were less well defined, suggesting that secreted MMPs might promote migration.ConclusionThese data indicate that stromal ING1 expression can predict the survival of patients with luminal breast cancer. High levels of ING1 in stromal cells can promote the development of breast cancer through increased expression and release of MMPs and down regulation of TIMPs, which may be an underlying mechanism of reduced patient survival.