Venkataswamy Sorna

Reversible LSD1 Inhibition Interferes with Global EWS/ETS Transcriptional Activity and Impedes Ewing Sarcoma Tumor Growth

Purpose: Ewing sarcoma is a pediatric bone tumor that absolutely relies on the transcriptional activity of the EWS/ETS family of fusion oncoproteins. While the most common fusion, EWS/FLI, utilizes lysine-specific demethylase 1 (LSD1) to repress critical tumor suppressors, small-molecule blockade of LSD1 has not yet been thoroughly explored as a therapeutic approach for Ewing sarcoma. We therefore evaluated the translational potential of potent and specific LSD1 inhibition with HCI2509 on the transcriptional program of both EWS/FLI and EWS/ERG as well as the downstream oncogenic phenotypes driven by EWS/ETS fusions in both in vitro and in vivo models of Ewing sarcoma. Experimental Design: RNA-seq was used to compare the transcriptional profiles of EWS/FLI, EWS/ERG, and treatment with HCI2509 in both EWS/FLI- and EWS/ERG-containing cell lines. We then evaluated morphologic phenotypes of treated cells with immunofluorescence. The induction of apoptosis was evaluated using caspase-3/7 activation and TUNEL staining. Colony forming assays were used to test oncogenic transformation and xenograft studies with patient-derived cell lines were used to evaluate the effects of HCI2509 on tumorigenesis. Results: HCI2509 caused a dramatic reversal of both the up- and downregulated transcriptional profiles of EWS/FLI and EWS/ERG accompanied by the induction of apoptosis and disruption of morphologic and oncogenic phenotypes modulated by EWS/FLI. Importantly, HCI2509 displayed single-agent efficacy in multiple xenograft models. Conclusions: These data support epigenetic modulation with HCI2509 as a therapeutic strategy for Ewing sarcoma, and highlight a critical dual role for LSD1 in the oncogenic transcriptional activity of EWS/ETS proteins. Clin Cancer Res; 20(17); 4584–97. ©2014 AACR.

Reversible inhibition of lysine specific demethylase 1 is a novel anti-tumor strategy for poorly differentiated endometrial carcinoma

BackgroundEndometrial cancer is the most common gynecologic malignancy. Type II endometrial carcinoma is often poorly differentiated and patients diagnosed with Type II disease (~11%) are disproportionately represented in annual endometrial cancer deaths (48%). Recent genomic studies highlight mutations in chromatin regulators as drivers in Type II endometrial carcinoma tumorigenesis, suggesting the use of epigenetic targeted therapies could provide clinical benefit to these patients. We investigated the anti-tumor efficacy of the LSD1 inhibitor HCI2509 in two poorly differentiated Type II endometrial cancer cell lines AN3CA and KLE.MethodsThe effects of HCI2509 on viability, proliferation, anchorage-independent growth, global histone methylation, LSD1 target gene induction, cell cycle, caspase activation and TUNEL were assayed. KLE cells were used in an orthotopic xenograft model to assess the anti-tumor activity of HCI2509.ResultsBoth AN3CA and KLE cells were sensitive to HCI2509 treatment with IC50s near 500 nM for cell viability. Inhibition of LSD1 with HCI2509 caused decreased proliferation and anchorage independent growth in soft agar, elevated global histone methylation, and perturbed the cell cycle in both cell lines. These effects were largely dose-dependent. HCI2509 treatment also caused apoptotic cell death. Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen. Tumor burden was distributed log-normally. Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p = 0.034).ConclusionsOur findings demonstrate the anti-cancer properties of the LSD1 inhibitor HCI2509 on poorly differentiated endometrial carcinoma cell lines, AN3CA and KLE. HCI2509 showed single-agent efficacy in orthotopic xenograft studies. Continued studies are needed to preclinically validate LSD1 inhibition as a therapeutic strategy for endometrial carcinoma.

Design, Synthesis, and Biological Evaluation of a Series of Anthracene-9,10-dione Dioxime β-Catenin Pathway Inhibitors

The Wnt/β-catenin signaling pathway plays a vital role in cell growth, the regulation, cell development, and the differentiation of normal stem cells. Constitutive activation of the Wnt/β-catenin signaling pathway is found in many human cancers, and thus, it is an attractive target for anticancer therapy. Specific inhibitors of this pathway have been keenly researched and developed. Cell based screening of compounds library, hit-to-lead optimization, computational and structure-based design strategies resulted in the design and synthesis of a series of anthracene-9,10-dione dioxime series of compounds demonstrated potent inhibition of β-catenin in vitro (IC50 < 10 nM, 14) and the growth of several cancer cell lines. This article discusses the potential of inhibiting the Wnt/β-catenin signaling pathway as a therapeutic approach for cancer along with an overview of the development of specific inhibitors.

Fragment-based design, synthesis, biological evaluation, and SAR of 1H-benzo[d]imidazol-2-yl)-1H-indazol derivatives as potent PDK1 inhibitors

In this work, we describe the use of the rule of 3 fragment-based strategies from biochemical screening data of 1100 in-house, small, low molecular weight fragments. The sequential combination of in silico fragment hopping and fragment linking based on S160/Y161/A162 hinge residues hydrogen bonding interactions leads to the identification of novel 1H-benzo[d]imidazol-2-yl)-1H-indazol class of Phosphoinositide-Dependent Kinase-1 (PDK1) inhibitors. Consequent SAR and follow-up screening data led to the discovery of two potent PDK1 inhibitors: compound 32 and 35, with an IC50 of 80 nM and 94 nM, respectively. Further biological evaluation showed that, at the low nanomolar concentration, the drug had potent ability to inhibit phosphorylation of AKT and p70S6, and selectively kill the cancer cells with mutations in both PTEN and PI3K. The microarray data showed that DUSP6, DUSP4, and FOSL1 were down-regulated in the sensitive cell lines with the compound treatment. The in vivo test showed that 35 can significantly inhibit tumor growth without influencing body weight growth. Our results suggest that these compounds, especially 35, merit further pre-clinical evaluation.

Abstract 3: Targeted inhibition of LSD1 in castration-resistant prostate cancer.

Epigenetic dysfunction is recognized as a driver in the pathology of various cancers. Promoter DNA hypermethylation, genomic hypomethylation, and aberrant histone acetylation and methylation contribute to epigenetic lesions. Further, genetic mutations in important epigenetic regulators have been identified across various solid and hematological malignancies. These aberrant marks are reversible due to the dynamic nature of epigenetic regulation and present attractive therapeutic targets. While epigenetic therapies, like DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, have shown survival benefit in the clinic there are several potential epigenetic targets whose therapeutic potential remains undefined. The histone lysine specific demethylase 1 (LSD1) is such a target. LSD1 is a FAD-dependent amine oxidase which catalyzes the oxidative demethylation of mono- and dimethyl marks from both lysines 4 and 9 on histone H3 (H3K4 and H3K9). Overexpression of LSD1 correlates with aggressive tumor biology, dedifferentiation, and poor prognosis in various solid malignancies, including prostate, breast, bladder, lung, liver and colorectal tumors. In prostate cancer, upregulation of LSD1 is sufficient to promote ligand-independent androgen-receptor-dependent transcription through demethylation of the repressive histone 3 lysine 9 dimethyl mark (H3K9me2). Currently reported inhibitors of LSD1 exhibit poor potency and specificity, solubility issues, and irreversibility and preclinical studies using these compounds have offered little in the way of single agent efficacy in solid tumors. Furthermore, inducible LSD1 knockdown experiments in vivo raise significant concerns about the potential for LSD1 inhibitors to block terminal hematopoetic differentiation. More potent and reversible inhibitors are required to discern which solid malignancies present an opportunity for LSD1 enzymatic inhibition as a single-agent and if there is a therapeutic window between single-agent efficacy and hematopoetic toxicity. HCI-2509 was previously reported as a potent and specific inhibitor for LSD1 with in vitro efficacy in breast cancer and in vivo efficacy in acute myeloid leukemia (AML). Here we report single-agent efficacy of HCI-2509 dosed both orally and intraperitoneally over the course of 21 days using the castration-resistant prostate cancer (CRPC) PC-3 xenografts in vivo. We observed target modulation with increased levels of H3K9me2 in both treatment groups. Additionally, the literature-predicted hematopoetic toxicity was not observed. This suggests that reversible and potent LSD1 inhibition provides a viable therapeutic strategy for CRPC and that dosing strategies can be optimized to avoid serious hematological toxicity. Our efforts are now focused on building a pharmacokinetic/pharmacodynamic model to better understand the clinical potential of LSD1 inhibition in CRPC. Citation Format: Emily Theisen, Jared Bearss, Venkataswamy Sorna, David Bearss, Sunil Sharma. Targeted inhibition of LSD1 in castration-resistant prostate cancer. [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 1003. doi:10.1158/1538-7445.AM2013-1003

Abstract 3679: Inhibition of LSD1 disrupts global EWS/ETS transcriptional function in Ewing sarcoma

Ewing sarcoma is an aggressive pediatric bone tumor characterized by an absolute reliance on the transcriptional activity of the EWS/ETS family of transcription factor fusion oncoproteins. The most common fusion is EWS/FLI, present in 85-95% of cases, though less common fusions include EWS/ERG, EWS/ETV1, EWS/ETV4 and EWS/FEV. EWS/FLI utilizes lysine-specific demethylase 1 (LSD1) as member of the NuRD complex to mediate transcriptional repression of critical tumor suppressors in Ewing sarcoma. LSD1 overexpression has been observed in clinical samples from Ewing sarcoma patients and small molecule blockade of LSD1 using tranylcypromine has been suggested as a therapeutic approach for Ewing sarcoma. We therefore evaluated the effects of LSD1 inhibition with the small molecule inhibitor, HCI-2509, in both in vitro and in vivo models of Ewing sarcoma. HCI-2509 is an LSD1 inhibitor with a Ki of ∼30 nM and multiple Ewing sarcoma cell lines show IC50s in cell viability assays from 500 nM-1 μM. Using RNA-seq, we show that HCI-2509 dramatically reverses both the up- and downregulated transcriptional profiles of both EWS/FLI and EWS/ERG accompanied by the induction of apoptosis and disruption of oncogenic phenotypes modulated by EWS/FLI. We further developed a 9-gene panel based on the RNA-seq data to assess the transcriptional phenotype of HCI-2509 in additional cell lines and showed that it HCI-2509 disrupted both EWS/FLI-activated and -repressed genes similarly across the tested Ewing sarcoma cell lines. HCI-2509 impaired transformation of Ewing sarcoma cell lines in colony forming assays with IC50s from from 25 nM-1 μM. Notably, HCI-2509 displayed single-agent efficacy in multiple xenograft models. We investigated the PK/PD relationship using tumor histone H3K4 and H3K9 methylation. Taken together, these data suggest that epigenetic modulation through LSD1 inhibition may be a therapeutic strategy for Ewing sarcoma, and highlight a critical dual role for LSD1 in the oncogenic transcriptional activity of EWS/ETS proteins. Citation Format: Emily Rose Theisen, Savita Sankar, Jared Bearss, Timothy Mulvihill, Venkataswamy Sorna, Sunil Sharma, Stephen L. Lessnick. Inhibition of LSD1 disrupts global EWS/ETS transcriptional function in Ewing sarcoma. [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 3679. doi:10.1158/1538-7445.AM2014-3679

Abstract 2161: Targeting Bruton's tyrosine kinase (BTK) in multiple myeloma with novel BTK inhibitors.

Multiple myeloma (MM) is a malignant disease that is characterized by an excess of monotypic plasma cells in the bone marrow (BM). A key clinical characteristic of MM is the localization of the MM cells to the bone marrow where they promote osteolytic bone destruction and impaired hematopoietic function. As a consequence MM patients experience bone pain, hypocalcaemia, anemia. Although there has been some progress in recent years in the development of novel drugs, such as proteasome inhibitors and derivatives of thalidomide, MM remains incurable and the majority of patients eventually succumb to their cancer. We have recently identified BTK over-expression in cancer cells taken from patients with multiple myeloma. In addition we have observed that BTK is also expressed in the activated osteoclasts of MM patients indicating that targeted inhibition of BTK might not only effect the MM cancer cells but may also influence the activity of osteoclasts and the associated osteolytic lesions. Bruton9s tyrosine kinase (BTK) is a cytoplasmic nonreceptor tyrosine kinase belonging to the Tec family of kinases. BTK has been extensively studied for its role in B-cell maturation and activation of B-cells by various ligands is accompanied by the translocation of BTK to the cell membrane where it binds phosphatidylinositol-3,4,5-trisphosphate through its PH domain. Activation of BTK results in downstream signaling through the PI3K/AKT, PLCγ1/2, NFκB, and other signaling pathways important for B-cell development and function. We propose that BTK plays an important role in multiple myeloma pathophysiology and that therapeutically targeting BTK will inhibit the growth of cancer cells and alter the tumor microenvironment in the bone marrow of multiple myeloma cancer patients. Using a structure-based approach we have developed a series of irreversible BTK inhibitors with selective and potent low nanomolar activity. In preclinical studies to date, our compounds have demonstrated promising activity in biochemical and cell-based experiments. Our BTK-targeted agents have shown activity in MM cells and have good pharmacokinetics when delivered IV and oral. MM cells create an adverse microenvironment from a pathophysiologic and clinical perspective because of the disruption of bone remodeling and this disruption of normal bone function has been shown to inhibit the response of MM cell to drug treatment. Therefore, a strategy to develop new drugs for MM must take into account the ability of the new agents to partition and distribute to the bone and function in the bone microenviroment of MM. Our compounds are being optimized for their ability to partition to bone and remain active in the BM microenvironment. By inhibiting BTK we seek to block BTK-dependent growth and migration of multiple myeloma cells and inhibit of the production of differentiated activated osteoclasts thereby disrupting the bone marrow microenvironment in MM. Citation Format: Destinee Bushman, Jared J. Bearss, Venkataswamy Sorna, Hariprasad Vankayalapati, Sunil Sharma, Fenghuang Zhan, David Bearss. Targeting Bruton9s tyrosine kinase (BTK) in multiple myeloma with novel BTK inhibitors. [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 2161. doi:10.1158/1538-7445.AM2013-2161

Abstract B84: HCI-2528 is a potent inhibitor of LSD1 with improved druglike characteristics.

Lysine Specific Demethylase 1 (LSD1/KDM1A) plays an important role in the regulation of histone methylation at lysine residues, and is currently being validated as an attractive therapeutic target for many diseases, particularly for multiple forms of cancer. Methylation of lysine residues on histones can signal transcriptional activation or repression depending on the specific residue involved. H3K4me2 is a transcription-activating chromatin mark at gene promoters, and demethylation of this mark by LSD1 is thought to prevent expression of tumor suppressor genes important in human cancer. In contrast, methylation of H3K9 is a repressive mark and LSD1 activity has been shown to upregulate tumor promoting pathways. Thus, LSD1 is emerging as an important target for the development of specific inhibitors as a new class of antitumor drugs. Several LSD1 inhibitors have been reported, but they have shown poor selectivity and/or pharmacological properties, making further exploration of LSD1 biology difficult. We previously reported the identification of CIT-0665, an LSD1 inhibitor identified through a virtual screening effort in our lab. Evaluation of the structure activity relationships of multiple analogs of our LSD1 inhibitor led to the identification of HCI-2528, which is more potent and exhibits improved drug like properties. Using a cell viability assay, a large panel of cancer cell lines was tested for sensitivity to HCI-2528. In addition to the well-documented sensitivity of breast cancer cell lines, we also found that Ewing9s sarcoma cell lines are uniquely sensitive to LSD-1 inhibition. We are currently engaged in a bioinformatic effort to determine possible mechanisms of sensitivity from our studies, and performing xenograft studies in mice. In conclusion, HCI-2528 is a novel LSD1 inhibitor with activity in biochemical and cell-based assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B84.