Namita Kumar

Selective Inhibition of EZH2 by EPZ-6438 Leads to Potent Antitumor Activity in EZH2-Mutant Non-Hodgkin Lymphoma

Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers. Mol Cancer Ther; 13(4); 842–54. ©2014 AACR.

Evasion of immunosurveillance by genomic alterations of PPARγ/RXRα in bladder cancer

Muscle-invasive bladder cancer (MIBC) is an aggressive disease with limited therapeutic options. Although immunotherapies are approved for MIBC, the majority of patients fail to respond, suggesting existence of complementary immune evasion mechanisms. Here, we report that the PPARγ/RXRα pathway constitutes a tumor-intrinsic mechanism underlying immune evasion in MIBC. Recurrent mutations in RXRα at serine 427 (S427F/Y), through conformational activation of the PPARγ/RXRα heterodimer, and focal amplification/overexpression of PPARγ converge to modulate PPARγ/RXRα-dependent transcription programs. Immune cell-infiltration is controlled by activated PPARγ/RXRα that inhibits expression/secretion of inflammatory cytokines. Clinical data sets and an in vivo tumor model indicate that PPARγHigh/RXRαS427F/Y impairs CD8+ T-cell infiltration and confers partial resistance to immunotherapies. Knockdown of PPARγ or RXRα and pharmacological inhibition of PPARγ significantly increase cytokine expression suggesting therapeutic approaches to reviving immunosurveillance and sensitivity to immunotherapies. Our study reveals a class of tumor cell-intrinsic “immuno-oncogenes” that modulate the immune microenvironment of cancer.Muscle-invasive bladder cancer (MIBC) is a potentially lethal disease. Here the authors characterize diverse genetic alterations in MIBC that convergently lead to constitutive activation of PPARgamma/RXRalpha and result in immunosurveillance escape by inhibiting CD8+ T-cell recruitment.

EP4 Antagonism by E7046 diminishes Myeloid immunosuppression and synergizes with Treg-reducing IL-2-Diphtheria toxin fusion protein in restoring anti-tumor immunity

ABSTRACT Reprogramming of immunosuppressive tumor microenvironment (TME) by targeting alternatively activated tumor associated macrophages (M2TAM), myeloid-derived suppressor cells (MDSC), and regulatory T cells (Tregs), represents a promising strategy for developing novel cancer immunotherapy. Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite and mediator of chronic inflammation, has emerged as a powerful immunosuppressor in the TME through engagement with one or more of its 4 receptors (EP1-EP4). We have developed E7046, an orally bioavailable EP4-specific antagonist and show here that E7046 has specific and potent inhibitory activity on PGE2-mediated pro-tumor myeloid cell differentiation and activation. E7046 treatment reduced the growth or even rejected established tumors in vivo in a manner dependent on both myeloid and CD8+ T cells. Furthermore, co-administration of E7046 and E7777, an IL-2-diphtheria toxin fusion protein that preferentially kills Tregs, synergistically disrupted the myeloid and Treg immunosuppressive networks, resulting in effective and durable anti-tumor immune responses in mouse tumor models. In the TME, E7046 and E7777 markedly increased ratios of CD8+granzymeB+ cytotoxic T cells (CTLs)/live Tregs and of M1-like/M2TAM, and converted a chronic inflammation phenotype into acute inflammation, shown by substantial induction of STAT1/IRF-1 and IFNγ-controlled genes. Notably, E7046 also showed synergistic anti-tumor activity when combined with anti-CTLA-4 antibodies, which have been reported to diminish intratumoral Tregs. Our studies thus reveal a specific myeloid cell differentiation-modifying activity by EP4 blockade and a novel combination of E7046 and E7777 as a means to synergistically mitigate both myeloid and Treg-derived immunosuppression for cancer treatment in preclinical models.

Discovery of Selective Estrogen Receptor Covalent Antagonists (SERCAs) for the treatment of ERa(WT) and ERa(MUT) breast cancer.

Mutations in estrogen receptor alpha (ERα) that confer resistance to existing classes of endocrine therapies are detected in up to 30% of patients who have relapsed during endocrine treatments. Because a significant proportion of therapy-resistant breast cancer metastases continue to be dependent on ERα signaling, there remains a critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Through our drug-discovery efforts, we identified H3B-5942, which covalently inactivates both wild-type and mutant ERα by targeting Cys530 and enforcing a unique antagonist conformation. H3B-5942 belongs to a class of ERα antagonists referred to as selective estrogen receptor covalent antagonists (SERCA). In vitro comparisons of H3B-5942 with standard-of-care (SoC) and experimental agents confirmed increased antagonist activity across a panel of ERαWT and ERαMUT cell lines. In vivo, H3B-5942 demonstrated significant single-agent antitumor activity in xenograft models representing ERαWT and ERαY537S breast cancer that was superior to fulvestrant. Lastly, H3B-5942 potency can be further improved in combination with CDK4/6 or mTOR inhibitors in both ERαWT and ERαMUT cell lines and/or tumor models. In summary, H3B-5942 belongs to a class of orally available ERα covalent antagonists with an improved profile over SoCs.Significance: Nearly 30% of endocrine therapy-resistant breast cancer metastases harbor constitutively activating mutations in ERα. SERCA H3B-5942 engages C530 of both ERαWT and ERαMUT, promotes a unique antagonist conformation, and demonstrates improved in vitro and in vivo activity over SoC agents. Importantly, single-agent efficacy can be further enhanced by combining with CDK4/6 or mTOR inhibitors. Cancer Discov; 8(9); 1176-93. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.

Abstract 4502: Eribulin alters vascular function in human triple-negative (TN) breast MX-1 and MDA-MB-231 tumor xenograft models as measured by DCE-MRI.

Objective: Eribulin mesylate (ERI) is a simplified synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B and an inhibitor of microtubule dynamics. Literature reports have increasingly shown tubulin binding agents have vascular disruptive activity. In this study, we evaluated the effect of eribulin on tumor vasculature using DCE-MRI of TN human breast MX-1 xenografts in nude rats. Methods: The effects of ERI on human TN breast MX-1 and MDA-MB-231 tumor are examined in nude rats and nude mice. ERI was administered at MTD dose (0.3 mg/kg at Q4D4 in nude rats and 3.0 mg/kg at D1 in nude mice). In nude rats, DCE-MRI was conducted on a Bruker 4.7 T scanner with Magnevist contrast. Imaging was conducted on day -1, 6 hrs, day 2, day 5-6 and day 10. In nude mice, morphology of tumor vasculature was analyzed with immunohistochemistry (IHC) staining with anti-mouse endothelial marker CD31 antibody. Microvessel density (MVD) and vessel perimeter were analyzed using Aperio Image Scope. Results and discussion: ERI inhibited tumor growth of MX-1 (day 5; 290 +/- 169 vs 1195 +/- 36 mm3, N=5, p Conclusions: ERI altered tumor vasculature acutely in the tumor rim and increase vascular perfusion in the tumor core 5 days after treatment. In addition to regulation of mitosis, ERI may have induced normalization of tumor vasculature in preclinical TN breast cancer cell models. Further analysis of the mechanism of ERI on vascular normalization will be warranted. Citation Format: Paul J. McCracken, Ken Ito, Mamoru Yanagimachi, Xavier Tizon, Peggy Provent, Shanqin Xu, Namita Kumar, Denice Welsh, Tyler J. Teceno, Galina Kuznetsov, Yasuhiro Funahashi. Eribulin alters vascular function in human triple-negative (TN) breast MX-1 and MDA-MB-231 tumor xenograft models as measured by DCE-MRI. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4502. doi:10.1158/1538-7445.AM2013-4502

Abstract B6: Advanced image analysis of H3K27 trimethylation in skin from subjects dosed with the EZH2 inhibitor tazemetostat

Tazemetostat (EPZ-6438 or E7438) is a selective small molecule inhibitor of the enhancer of zeste homolog 2 (EZH2) histone methyltransferase (HMT). EZH2 is the catalytic subunit of the multi-protein HMT complex known as polycomb repressive complex 2 (PRC2), which is responsible for mono-, di-, and trimethylation (me3) of histone H3 lysine 27 (H3K27). An exploratory objective for the phase 1 study of tazemetostat in subjects with advanced solid tumors or B-cell lymphomas was to explore the pharmacokinetic: pharmacodynamic (PK: PD) relationships such as correlation of H3K27me3 inhibition in patient tissue with systemic drug exposure. Pre-clinical xenograft models of EZH2 inhibition have previously shown tumor regression correlating with dose-dependent H3K27me3 decreases in both tumor tissue and skin epidermis (Knutson 2014). H3K27me3 reduction in the epidermis was calculated as a change in percentage of H3K27me3 positive cell employing an immunohistochemistry (IHC) assay with subsequent image analysis. These studies and the low feasibility of obtaining matched pre- and post-treatment tumor biopsies, led to the selection of skin as a surrogate tissue for evaluation in the phase 1 study at screening and 28 days post dose and the use of IHC as a PD biomarker of target inhibition. Examination of phase 1 skin biopsies exhibited dose and exposure-dependent decreases in the percentage of H3K27me3 positive cells, however, the magnitude of reduction (60%) was less than observed in pre-clinical models (80%). Further investigation of the IHC images revealed a difference of H3K27me3 levels in distinct layers or strata of the skin. The stratum spinosum exhibited a distinct dose and exposure-dependent loss of H3K27me3 staining while the stratum basale showed a concomitant minimal loss of H3K27me3. These observations gave rise to the possibility of a stratum-specific PD response in skin to tazemetostat exposure. To fully evaluate this notion, a more sophisticated, unbiased, image analysis algorithm was employed to measure H3K27me3 levels across the strata of the epidermis. The image analysis algorithm was designed to identify three distinct regions: the full thickness epidermis, stratum basale to stratum corneum; the stratum basale alone; and the stratum spinosum. Iterative rounds of optimization were performed to identify the basale layer while excluding follicles and glands. Because skin has inherent challenges related to the chromogenic similarity between DAB and melanin, careful attention was paid to the negative control to ensure that endogenous melanin was not included in the analysis. Segregation of the stratum basale layer from the remainder of the epidermis led to a refined H3K27me3 analysis and resulted in an improved correlation of H3K27me3 inhibition with drug exposure. This analysis highlights that the zonal layers of the epidermis may require separate evaluation when analyzing skin as a surrogate tissue for proof of mechanism studies. Although more accessible for repeat biopsy, the use of skin as a surrogate tissue does not preclude the need to understand the PD effects in the target tumor tissue. To that end, optional paired tumor biopsies are being collected in the Phase 2 tazemetostat trial and will be evaluated for PD endpoints including H3K27me3 in addition to other biomarkers. Citation Format: Alice A. McDonald, Galina Kuznetsov, Namita Kumar, Amy Weaver, Trevor Do, Jeff Dzubay, Heike Keilhack, Nigel waters, Nathalie Rioux, Stephen J. Blakemore. Advanced image analysis of H3K27 trimethylation in skin from subjects dosed with the EZH2 inhibitor tazemetostat. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B6.

Abstract B90: Eribulin mesylate alters immune homeostasis in mice bearing syngeneic tumors.

Numerous studies indicate that certain tumor-infiltrating macrophage subsets can support neoplastic growth. Recent reports showed that spleen can serve as an important reservoir for macrophages and neutrophils that replicate in situ, then migrate to support tumor growth. Eribulin mesylate (Halaven®) is a microtubule-dynamics inhibitor approved for third line clinical use in patients with heavily pretreated metastatic breast cancer based upon statistically significant increase in median overall survival (OS) compared to treatment of physician9s choice. In an effort to understand whether eribulin has additional effects beyond its anti-mitotic activity, the current study addressed the effects of eribulin mesylate on immune cells. Using the subcutaneous syngeneic murine tumor model Lewis lung carcinoma (LLC), we found that the tumor shrinking effect of eribulin correlated with increased extramedullary hematopoiesis observed in the spleens of treated mice. Eribulin was also found to inhibit the proliferation of M2 tumor-associated macrophages (TAMs) in the spleens of LLC-bearing mice. Early effects of eribulin were studied using the CT-26 colon tumor isograft model and included increased differentiation and death of CD4 + and CD8 + effector T cells. Furthermore, eribulin caused increased CD25 expression on CD4 + Foxp3 - T cells but not on CD4 + Foxp3 + T regulatory cells. Finally, frequencies of exhausted CD8 + T cells were reduced following eribulin treatment. Taken together, these results suggest that eribulin-mediated modulation of immune homeostasis especially in the spleen, may contribute to its anti-tumor effect. Citation Format: Diana I. Albu, Namita Kumar, Galina Kusnetzov, Shanqin Xu, Bruce Littlefield, Mary Woodall-Jappe. Eribulin mesylate alters immune homeostasis in mice bearing syngeneic tumors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B90.

Abstract 4126: Optimization of clinical sample collection for detection of c-Met phosphorylation

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The receptor tyrosine kinase c-Met is being investigated as a target for cancer chemotherapy. c-Met and its ligand HGF (hepatocyte growth factor) play important roles in cell growth, survival and migration. Dysregulation of the HGF-c-Met pathway (overexpression, constitutive activation, gene amplification, ligand-dependent activation, mutation or epigenetic mechanisms) can lead to oncogenic changes, and has been described in multiple types of cancer including lung, gastric, and colorectal cancer. For cancer therapies targeting the HGF-c-Met signaling pathway, elevated levels of c-Met expression and increased phosphorylation of c-Met have been explored as both the predictive and PD biomarkers. However, detection of c-Met phosphorylation by IHC in human clinical cancer samples has proven to be extremely challenging. We have conducted a study aimed at finding a way to detect phosphorylation of c-Met in clinical samples. In the first attempt, we have acquired a set of 30 gastric cancer samples which were fixed in formalin in the presence of phosphatase inhibitors (PhosphoGuardTM fixation kit). While this approach has helped us to improve detection of several other phospho-proteins in tissues, it did not help with the detection of phospho-c-Met. We then hypothesized that this is a very labile phosphorylation which is rapidly lost during the cold ischemia time occurring between the surgical excision of the tissue and its placement in formalin for fixation (which could last 30-60 minutes or longer in the operating room). Studies with xenograft tissues showed that a 15 minute delay in fixation resulted in approximately 50% decrease in c-Met phosphorylation, while a 30 minute delay resulted in almost complete disappearance of phospho-c-Met. We therefore acquired a set of colorectal cancer samples which were placed into formalin within 5 minutes of surgical excision. Using these rapidly fixed samples, we were able to detect strong phosphorylation of c-Met in a small number of samples. The staining pattern for phospho-c-Met correlated with that of phospho-ERK (a downstream signaling molecule in the c-Met pathway) suggesting that the relatively low occurrence of c-Met phosphorylation may reflect the status of activation of this pathway in vivo at a given time point. This work suggests that rapid fixation of human tissues facilitates detection of phospho-c-met and its use as a PD and predictive biomarker in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4126. doi:10.1158/1538-7445.AM2011-4126