Jared Bearss

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.

LSD1 activates a lethal prostate cancer gene network independently of its demethylase function

Significance Medical castration or interference with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, progression is universal, and therapies following the emergence of castration resistance do not offer durable control of the disease. Lysine-specific demethylase 1 (LSD1) is an important regulator of gene expression, including in cancer. Here, we show that LSD1 is highly expressed in tumors of patients with lethal castration-resistant prostate cancer (CRPC) and that LSD1 promotes AR-independent survival in CRPC cells in a noncanonical, demethylase-independent manner. We determined that the drug SP-2509 acts as an allosteric inhibitor of LSD1–blocking demethylase-independent functions. Our demonstration of tumor suppression with this inhibitor in CRPC preclinical models provides the rationale for clinical trials with LSD1 inhibitors. Medical castration that interferes with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, clinical progression is universal, and tumors with AR-independent resistance mechanisms appear to be increasing in frequency. Consequently, there is an urgent need to develop new treatments targeting molecular pathways enriched in lethal prostate cancer. Lysine-specific demethylase 1 (LSD1) is a histone demethylase and an important regulator of gene expression. Here, we show that LSD1 promotes the survival of prostate cancer cells, including those that are castration-resistant, independently of its demethylase function and of the AR. Importantly, this effect is explained in part by activation of a lethal prostate cancer gene network in collaboration with LSD1’s binding protein, ZNF217. Finally, that a small-molecule LSD1 inhibitor―SP-2509―blocks important demethylase-independent functions and suppresses castration-resistant prostate cancer cell viability demonstrates the potential of LSD1 inhibition in this disease.

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 C206: In vivo activity of SGI‐1252, a potent, small‐molecule dual inhibitor of JAK2 and ALK2

Activation of JAK2, an intracellular protein tyrosine kinase, is known to induce cell proliferation and apoptosis and it has been shown to be mediated by pro‐inflammatory cytokines. JAK2 dysregulation is implicated in myeloproliferative disorders, solid tumor malignancies, and autoimmune diseases. Utilizing our proprietary CLIMB™ technology, a computationally driven drug discovery process, we have rapidly designed and developed, SGI‐1252, a potent oral inhibitor which exhibits low nanomolar in vitro activity against all of the JAK kinase family, except JAK3. Additionally, it has low nanomolar activity against ALK2, a high‐affinity binding receptor for bone morphogenic protein. As predicted from computational models, SGI‐1252 is well tolerated across several rodent species and demonstrates a wide safety margin in both short‐ and long‐term toxicity studies. Acute maximum tolerated doses of greater than 900 mg/kg are observed in mice and rats dosed orally with SGI‐1252. Over a 5‐week period, daily oral dosing of up to 200 mg/kg SGI‐1252 shows no hematological toxicity and no body weight loss in mice. Pharmacokinetic studies of SGI‐1252 in rodents also demonstrate excellent systemic exposure, with oral bioavailabilities of greater than 50% in mice and rats. Because inflammatory cytokines are known to play a role in the pathogenesis of pancreatic and lung cancers, tumor xenograft models of those cell lines were evaluated. In BxPC3 and A549 xenografts, three weeks of daily oral dosing of 200 mg/kg SGI‐1252 demonstrate exceptional activity, with tumor inhibitions of greater than 50% in both models. Pharmacodynamic studies were also performed to further elucidate the effect of SGI‐1252 on pro‐inflammatory cytokines related to these models. In several acute‐phase mouse models, where turpentine‐oil injection are utilized for cytokine stimulation, SGI‐1252 is observed to down‐regulate IL‐6 and VEGF expression 16–24 hours after dosing. In summary, SGI‐1252 is a potent oral inhibitor that is well tolerated in rodent models, provides excellent PK exposure, and demonstrates tumor growth inhibition as well as modulation of pro‐inflammatory cytokines. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C206.

Abstract 2174: Small molecule iInhibitors of PIM kinases as potential treatments for urothelial carcinomas.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The proto-oncogene PIM kinases (PIM-1, PIM-2, PIM-3) are serine/threonine kinases that have been shown to be involved in a number of signaling pathways important to cancer cells. PIM kinases act as downstream effectors as inhibitors of apoptosis and as positive regulators of G1-S phase progression through the cell cycle. PIM kinases are upregulated in multiple cancer indications, including lymphoma, leukemia, multiple myeloma, prostate, gastric, and head & neck cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The aim of this investigation was to evaluate PIM expression in low- and high-grade urothelial carcinoma, and to assess for expression that may contribute to disease progression and serve as a potential site for targeted therapy. Seventy-two cases of urothelial carcinoma were included in this retrospective study of surgical biopsy and resection specimens from the University of Utah Department of Pathology (retrieved from 2008-2011). Tissue was stained with commercially available antibodies against PIM-1, PIM-2, and PIM-3. Cases were divided into three groups (invasive high grade urothelial carcinoma (n=49), non-invasive urothelial carcinoma/carcinoma in situ (n=16), and non-invasive low grade urothelial carcinoma (n=7)). Individual cases were then given a score (0-4) based upon a percentage of cells staining positive for each antibody ( 75%=4). A score of 2 or greater was considered expressed. PIM-1, PIM-2 and PIM-3 expression was noted in 29% (2/7), 43% (3/7) and 86% (6/7) cases of non-invasive low-grade urothelial carcinoma; 44% (7/16), 50% (8/16), 44% (7/16) cases of non-invasive high-grade urothelial carcinoma; 10% (5/49), 27% (13/49), and 18% (9/49) cases of invasive high-grade urothelial carcinoma, respectively. These results suggest that expression of PIM-1, PIM-2 and PIM-3 is present in a significant percentage of urothelial carcinomas and may serve as a source for targeted PIM-kinase inhibition. We have developed PIM inhibitors exhibiting 4-10 fold improved potency against the PIM kinase family compared to our original PIM inhibitor SGI-1776. Our PIM inhibitors display sub-μM activity in pharmacodynamic marker modulation, proliferation and 2D colony formation assays using the UM-UC-3 bladder cancer cell line. These PIM kinase inhibitors also are potent inducers of apoptosis in T24, RT4, and UM-UC-3 bladder cancer cell lines. These compounds have favorable hERG and CYP inhibition profiles compared with SGI-1776, and demonstrate excellent oral bioavailability. In vivo xenograft studies using bladder cancer cell line models show that PIM kinase inhibition can reduce the tumor growth of these tumor models suggesting that PIM kinase inhibitors may be active in human urothelial carcinomas. Citation Format: Kent J. Carpenter, Rachel Brog, Christopher Moreno, Daniel J. Albertson, Jared J. Bearss, Ting Liu, Steven Warner, David J. Bearss. Small molecule iInhibitors of PIM kinases as potential treatments for urothelial carcinomas. [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 2174. doi:10.1158/1538-7445.AM2013-2174

Abstract A190: In vivo activity of SGI‐110, a novel hypomethylating agent for treatment in hematology and solid malignancies

SGI‐110 is a second generation hypomethylating agent being developed for treatment in myelodysplastic syndrome (MDS) and solid tumor malignancies. In previous work, SGI‐110 has demonstrated potent in vivo activity in a number of tumor types, including non‐hematological cell lines. Current efforts are underway to optimize formulation and delivery of SGI‐110 for first‐in‐human (FIH) studies. In animals, SGI‐110 is well‐tolerated across multiple species utilizing multiple routes of drug delivery. Tolerability studies have been performed in mouse, rat, and rabbit models with multiple dose routes and schedules. Myelosuppression is an observed toxicity endpoint for hypomethylating agents. Hence, myelotoxic effects were investigated by comparing RBCs and bone marrow cellularity of mice treated with and without SGI‐110. Mice dosed with SGI‐110 for five consecutive days showed a significant decrease in RBCs at the end of the dosing period and a continued decrease one week after dosing. Bone marrow cellularity also showed a decrease at the end of dosing, but recovered to near normal levels one week later. Interestingly, RBCs from SGI‐110 treated mice were elevated in the bone marrow after the dosing period. Pyrosequencing methylation analysis of colon samples was also evaluated in this study. A significant decrease in B1 methylation was observed in colon samples of treated mice, indicating global DNA methylation is being inhibited. Decreased levels in several hematology parameters and decreases in bone marrow cellularity were also observed in rat and rabbit studies after five consecutive days of SGI‐110 dosing. Increased dosing frequency, while maintaining the same total dose per week, appears to result in increased toxicity. Previous pharmacokinetic studies have shown that SGI‐110 rapidly metabolizes to decitabine, an FDA‐approved drug for MDS. Multiple formulations and different routes of delivery were examined to determine the optimal dose form to be used in FIH studies. Subcutaneous dosing results in bioavailability that is similar to that of intravenous dosing. Subcutaneous dosing appears to reduce the Cmax while maintaining similar AUC values when compared to intravenous dosing. Similar results in pharmacokinetic parameters are observed when the delivery vehicle is changed from an aqueous to non‐aqueous formulation. SGI‐110 is a novel hypomethylating agent that is well‐tolerated in rodent models, provides excellent PK exposure, and demonstrates inhibition of DNA methylation in a mouse model. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A190.

Abstract C199: Development of potent, small‐molecule inhibitors of ETK

Epithelial and endothelial tyrosine kinase (Etk) is a nonreceptor tyrosine kinase that plays a central role in the proliferation, differentiation, apoptosis, and tumorigenicity of epithelial cells. Inhibition of Etk signaling can result in impaired cellular transformation, down‐regulation of angiogenesis, and increased apoptosis. Employing our proprietary CLIMB™ technology, a computationally driven drug discovery process, we designed and synthesized approximately 35 small molecules for ETK‐inhibition testing in biochemical and cellular assays. Most of these compounds exhibited low nanomolar activity and selectivity across a wide panel of kinases. Five compounds were subsequently chosen for further evaluation in in vivo studies. As predicted from CLIMB™, all of the compounds showed sufficient tolerability and pharmacokinetics in mice to advance into tumor efficacy studies. Endometrial and hepatocellular cancers were selected for these studies based on previous in vitro results indicating high ETK expression and potent compound activity. All five compounds demonstrated marked activity in these models; in one cell line, two of the compounds inhibited tumor growth by more than 50% after less than two weeks of dosing. Using these same tumor lines in pharmacodynamic studies, the compounds also showed significant modulation of cellular transformation and anti‐apoptotic markers consistent with ETK inhibition. Moreover, quantitative analysis of microvessel density, a key indicator of angiogenesis, demonstrated clear inhibition of blood vessel formation from tumors excised after treatment with the five compounds. Utilizing our CLIMB™ technology, we have rapidly developed a new class of potent inhibitors that consistently demonstrate in vivo activity against ETK‐relevant tumor cell lines. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C199.