David J. Bearss

Chemical genetic screen reveals a role for desmosomal adhesion in mammary branching morphogenesis.

Background: Mammary gland branching morphogenesis is a highly regulated developmental process often disrupted in breast cancer. Results: A chemical genetic screen in primary three-dimensional culture revealed that activation of the aryl hydrocarbon receptor promotes desmosomes to block branching. Conclusion: Down-regulation of desmosomes is required for proper mammary branching morphogenesis. Significance: Desmosomes are a novel mechanism through which exposure to environmental pollutants may affect mammary development. During the process of branching morphogenesis, the mammary gland undergoes distinct phases of remodeling to form an elaborate ductal network that ultimately produces and delivers milk to newborn animals. These developmental events rely on tight regulation of critical cellular pathways, many of which are probably disrupted during initiation and progression of breast cancer. Transgenic mouse and in vitro organoid models previously identified growth factor signaling as a key regulator of mammary branching, but the functional downstream targets of these pathways remain unclear. Here, we used purified primary mammary epithelial cells stimulated with fibroblast growth factor-2 (FGF2) to model mammary branching morphogenesis in vitro. We employed a forward chemical genetic approach to identify modulators of this process and describe a potent compound, 1023, that blocks FGF2-induced branching. In primary mammary epithelial cells, we used lentivirus-mediated knockdown of the aryl hydrocarbon receptor (AHR) to demonstrate that 1023 acts through AHR to block branching. Using 1023 as a tool, we identified desmosomal adhesion as a novel target of AHR signaling and show that desmosomes are critical for AHR agonists to block branching. Our findings support a functional role for desmosomes during mammary morphogenesis and also in blocking FGF-induced invasion.

Competitive enhancement of HGF-induced epithelial scattering by accessory growth factors.

HGF signaling induces epithelial cells to disassemble cadherin-based adhesion and increase cell motility and invasion, a process termed epithelial-mesenchymal transition (EMT). EMT plays a major role in cancer metastasis, allowing individual cells to detach from the primary tumor, invade local tissue, and colonize distant tissues with new tumors. While invasion of vascular and lymphatic networks is the predominant route of metastasis, nerves also can act as networks for dissemination of cancer cell to distant sites in a process termed perineual invasion (PNI). Signaling between nerves and invasive cancer cells remains poorly understood, as does cellular decision making that selects the specific route of invasion. Here we examine how HGF signaling contributes to PNI using reductionist culture model systems. We find that TGFβ, produced by PC12 cells, enhances scattering in response to HGF stimulation, increasing both cell-cell junction disassembly and cell migration. Further, gradients of TGFβ induce migratory mesenchymal cells to undergo chemotaxis towards the source of TGFβ. Interestingly, VEGF suppresses TGFβ-induced enhancement of scattering. These results have broad implications for how combinatorial growth factor signaling contributes to cancer metastasis, suggesting that VEGF and TGFβ might modulate HGF signaling to influence route selection during cancer progression.

Discovery of biologically active oncologic and immunologic small molecule therapies using zebrafish: Overview and example of modulation of T Cell activation

Zebrafish models continue to gain popularity as in vivo models for drug discovery. Described in this overview are advantages and challenges of zebrafish drug screening, as well as a novel in vivo screen for immunomodulatory compounds using transgenic, T cell reporting zebrafish larvae designed for discovery of compounds targeting T cell leukemia. This assay system allows rapid screening of large numbers of compounds while avoiding the pitfalls of assays based on cell cultures, which lack biologic context and are afflicted by genomic instability. The rationale for this approach is based on similarities of immature normal T cells and developmentally arrested, malignant lymphoblasts in mammalian species. The screening algorithm has been used to identify a nontoxic compound with activity in both acute leukemia models and models of multiple sclerosis, demonstrating the utility of this screening procedure. Curr. Protoc. Pharmacol. 60:14.24.1‐14.24.13.

Abstract 1045: Activity of the LSD1 inhibitor HCI-2509 in ER-negative breast cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnLysine-specific demethylase 1 (LSD1/AOF2/KDM1A) is a flavin-dependent histone demethylase that catalyzes the post-translational oxidative demethylation of mono- and di-methylated lysines on histones. 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. High LSD1 protein levels have been reported in tissue specimens from estrogen receptor (ER)-negative breast cancers and high expression of LSD1 in these tumors correlates with aggressive biology. Thus, LSD1 is emerging as an important target for the development of specific inhibitors as a new class of antitumor drugs. Only a few existing compounds, most of which are monoamine oxidase inhibitors, have been shown to act as inhibitors of LSD1. More potent and specific inhibitors of LSD1 lacking the monoamine oxidase activity are needed to advance LSD1 biology and test for potential efficacy in tumor models. We have previously reported using a structure-based drug discovery approach using solved 3-D crystal structures of LSD1 to computationally dock more than 10 million virtual compounds into the active site of the protein. From the docking experiments, we physically screened a selected group of compounds in an LSD1 biochemical assay. Initial hits from the screen were optimized utilizing a structure-based synthetic design strategy and a lead was identified as a potent inhibitor of LSD1 enzymatic activity, with an IC50 of 13 nM. To evaluate inhibitor specificity, our lead compounds were tested against 5 closely related flavin-dependent enzymes and showed minimal inhibition in these assays. Additionally, cell lines treated with our lead, HCI-2509, show increased levels of H3K4 methylation. To examine the cell-based activity of various cancer cell lines to our lead, a large panel of cancer cell lines was tested for sensitivity to HCI-2509 using a cell viability assay. Among the most sensitive cell lines are several derived from ER-negative breast cancers. We further demonstrated potent activity of HCI-2509 in cells taken directly from ER-negative breast cancer patients. Using gene expression analysis after treatment of highly sensitive and less sensitive breast cancer cell lines we have developed a gene expression signature associated with sensitivity to HCI-2509. Our efforts are now focused on examining HCI-2509 potential as novel therapeutic targeting LSD1 for treatment of ER-negative breast cancers using mouse models of breast cancer.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1045. doi:1538-7445.AM2012-1045

Abstract 2776: Inhibition of Nek2 by novel small molecules affects proteasome activity

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnNek2 is a serine/threonine kinase that has been associated with centrosome function and cell cycle progression. Nek2 overexpression has been reported in several tumor types including breast and lung cancers. We have previously shown that elevated expression of Nek2 in clinical samples of multiple myeloma (MM) correlates with bortezomib resistance. Furthermore, the overexpression of Nek2 in MM cell lines decreases sensitivity to bortezomib and knockdown with RNAi sensitizes bortezomib resistant cells. Due to the proteaseome inhibitory activity of bortezomib, we hypothesized that Nek2 overexpression may increase proteasome activity. 26S proteasomes were isolated by ultracentrifugation from stably transfected Hela cells (+Nek2 or +GFP) and Nek2 was shown to be involved in the 26S proteasome complex by western blot. Proteasome activity assays were performed both biochemically and in cell culture demonstrating that proteasome activity in Hela+Nek2 is significantly higher than in Hela+GFP. Furthermore, the isolated 26S proteasomes were incubated with novel Nek2 inhibitors (HCI-2184 and HCI-2389) resulting in a significant reduction in proteasome function when used as single agents or in combination with bortezomib. HCI-2184, a reversible inhibitor of Nek2, demonstrated an IC50 value in the range of 13-38 nM, and HCI-2389, an irreversible inhibitor of Nek2, showed an IC50 value between 8-26 nM. Similarly, both compounds significantly increased the efficacy of bortezomib in inhibiting the proteasome activity. These results were confirmed in multiple cancer cell lines, including ARP1 (human MM cell line), H299 (human lung cancer cell line) and K28 (mouse Leydig tumor cell line). The effect of inhibiting Nek2 on cell cycle was also investigated. The expression levels of cyclinB1 and cdc2, proteins degraded by the proteasome for cells to exit mitosis, were increased in HCI-2389 treated Hela+Nek2 and to a lesser extent Hela+GFP treated cells. While Nek2 overexpression leads to the down-regulation of cyclinB1 and cdc2, HCI-2389 treatment successfully rescued this effect. Flow cytometry data showed that both treatments of HCI-2184 and HCI-2389 significantly arrested Hela+Nek2 and Hela+GFP in G2/M phase after 24 hours at concentration as low as 10nM. In conclusion, we have discovered a novel biological function of Nek2 related to elevated proteasome activity. We also demonstrated that our novel Nek2 inhibitors efficiently inhibit Nek2 function, resulting in reduced proteasome activity. These Nek2 inhibitors have the potential to be applied clinically for treatment of the MM patients resistant to bortezomib.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2776. doi:1538-7445.AM2012-2776

Abstract 1043: Combined targeting of chromatin modifying enzymes LSD1, histone deacetylases (HDACs) and EZH2 has superior efficacy against human acute myeloid leukemia cells.

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnLSD1 or KDM1A is a FAD-dependent demethylase, with homology to amine oxidases. LSD1 demethylates di- and mono-methylated lysine (K)4 on histone H3, reducing the permissive H3K4Me3 chromatin mark. LSD1 inhibition attenuates growth of pluripotent cancer cells with OCT4 and SOX2 expression. LSD1 complexes with HDAC1/2 and Co-REST, and high LSD1 expression confers poor prognosis in cancers. Previous studies have shown that HDAC inhibitors downregulate LSD1 thru Sp1 inhibition. Inhibition of LSD1 leads to increase in H3K4Me3-a permissive mark for gene expression. HCI2509 is an FAD-binding pocket, non-MAOA and MAOB LSD1 inhibitor. In the present studies, we determined the chromatin-modifying and cytotoxic effects of HCI2509 alone and in combination with the pan-histone deacetylase inhibitor, panobinostat (PS) in cultured (HL-60 and OCI-AML3) and primary human acute myeloid leukemia (AML) cells. Treatment with HCI2509 (100 to 500 nM), dose-dependently increased the levels of H3K4Me2 & Me3, p16 and p27, which was associated with inhibition of cell proliferation as measured by a decrease in Ki-67 expression. Chromatin immunoprecipitation followed by PCR demonstrated that treatment with HCI2509 increased the H3K4Me3 mark on the promoters of KLF4, HMOX1, and CDH1 in AML cells. Treatment with HCI2509 also induced C/EBPα expression and features of morphologic differentiation of cultured and primary AML cells. Treatment with HCI2509 (25 mg/kg B.I.W. via IP injection) significantly improved the survival of NOD/SCID mice bearing OCI-AML3- AML xenografts. We have previously reported that treatment with PS (Novartis Pharma) depleted PRC2 complex proteins EZH2, and SUZ12 but also modestly depleted LSD1 expression in AML cells. Co-treatment with PS enhanced the chromatin modifying effects of HCI2509 on K4 of Histone H3 in AML cells. Co-treatment with HCI2509 and PS synergistically induced apoptosis of the cultured AML cells (combination indices, CI <1.0). This was associated with greater induction of p16, and p27. Co-treatment with HCI2509 and PS also induced significantly greater loss of viability of primary AML cells but not of normal CD34+ cells. We have previously reported that treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep, dose-dependently depleted EZH2, and SUZ12 levels in cultured and primary AML cells. Combined treatment with HCI2509 enhanced the apoptosis of AML cells induced by DZNep. Taken together, these findings indicate that combined targeted depletion of the level and activity of LSD1 and HDACs by PS and HCI2509, along with PS-mediated depletion of PRC2 proteins, exerts superior and selective cytotoxic activity against AML cells. These findings also support the in vivo testing of combined epigenetic therapies in the treatment of AML.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1043. doi:1538-7445.AM2012-1043

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.

Abstract 2577: Targeting the Axl tyrosine kinase receptor in pancreatic cancer

With the worst five-year survival rate of any cancer, pancreatic adenocarcinomas now rank as the fourth leading cause of cancer death in adults in the United States. This necessitates increased efforts to identify new leads that will serve as therapeutic targets in our fight against this aggressive cancer. Axl is a TAM family receptor tyrosine kinase involved in multiple aspects of tumorigenesis. Increased expression of Axl is associated with increased oncogenic transformation, cell survival, proliferation, migration, angiogenesis, and cellular adhesion. The oncogenic potential of Axl was first discovered in chronic myelogenous leukemia (CML), but it has been demonstrated to play a role in the progression and metastasis of other cancer types. The known ligand for Axl is the Growth Arrest Specific Gene-6 (Gas6) protein and its binding to Axl leads to Axl autophosphorylation and activation of downstream signaling pathways including MAPK and PI3K/Akt pathways. Furthermore, target validation studies of in vivo cancer models show that inhibition of Axl expression by RNAi blocked tumor growth in those models. Taken together, this information makes Axl kinase an exciting target for small molecule drug discovery. Using HCI-2084, a small molecule Axl kinase inhibitor, we explored the effectiveness of targeting the Axl kinase in pancreatic cancer. HCI-2084 demonstrates low nanomolar (IC50 = 12 nM) activity against the Axl kinase in a biochemical assay with good selectivity for Axl when screened in a kinase panel. In 2D and 3D cell proliferation assays, HCI-2084 significantly inhibited pancreatic cancer cell growth at concentrations as low as 30 nM. In pharmacodynamic endpoint assays, HCI-2084 dramatically inhibited Akt signaling (pAKT S473) downstream of GAS6 stimulation in pancreatic cancer cell lines. Inhibition of Axl autophosphorylation by HCI-2084 was also observed in an Axl-transfected cell line system. Consistent with the known function of Axl, HCI-2084 inhibited Gas6-induced migration and invasion of pancreatic cancer cells in vitro. The proteolytic processing of the extracellular domain of the Axl receptor is a known event downstream of Axl activation and results in the release of soluble Axl (sAxl) into the cell culture media (in vitro) or into the blood stream (in vivo). We hypothesized that sAxl levels could function as a biomarker for target inhibition. Indeed, conditioned media from pancreatic cancer cell lines treated with HCI-2084 showed significant dose-dependent reductions in sAxl levels compared to the vehicle treated controls. Taken together, these results suggest Axl is a potential therapeutic target in pancreatic cancer and that HCI-2084 is an exciting agent to potentially treat this disease. HCI-2084 is currently undergoing evaluation in animal efficacy and pharmacodynamic endpoint studies. 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 2577. doi:10.1158/1538-7445.AM2011-2577

Abstract 1368: Fragment-based design, synthesis and biological evaluation of a series of novel PDK1 inhibitors

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnThe PDK1 is a monomeric serine/threonine kinase and a promising oncology target of significant current interest for drug development due to its central role in the PI3K/AKT/mTOR pathway. It phosphorylates highly conserved Ser or Thr residues in the activation loop of several AGC super family kinases including PKC, SGK, PKB/Akt, p70S6K, and PDK-1 itself. Approximately, 40-50% of all tumors involve mutations in the PTEN protein, which results in elevated levels of PIP3 and enhanced activation of PKB/AKT, p70S6K, and SGK. It has been proposed that inhibitors of PDK-1 could provide a valuable therapeutic approach to targeting cancer, particularly those with PTEN deficiencies. Using a fragment-based design strategy, we screened a collection of 1100 low molecular weight (< 250 MW) fragments against the PDK1 kinase and identified 9 fragments with moderate inhibitory activity against PDK1 (IC50 values from 45-82 μM).nnOf these fragments, those based off of a 5-Br,4-I-1H-indazol-3-amine scaffold seemed the most promising based on initial activity and in keeping with the “rule of 3” for fragment-based drug design. Subsequent molecular docking studies using a crystal structure of PDK1 allowed for the structural rationalization of how these fragments bound in the ATP-binding pocket (hydrogen bonding to S160/A162 hinge residues) and provided insight for further optimization. Concurrently, we carried out scaffold-hopping searches at 2-site points for hydrophobic and solvent pocket fragments. With the addition of one fused heterocyclic ring, the potency increased to 8.8 and 10.9 μM. Our systematic fragment based workflow led to the preparation of target molecules in 4 steps beginning with the condensation, cyclization, and reduction and finally installing the hydrophobic binding site fragments under normal amide coupling. Subsequent SAR and follow-up screening led to the discovery of HCI-1680, a potent PDK1 inhibitor with an IC50 of 97 nM. Additional productive interactions sites with PDK1 were introduced to further improve both biochemical and cellular activities in panel of cancer cells. In cell-based assays HCI-1680 demonstrated enhanced activity in PTEN-deficient cell lines compared to PTEN-wild type lines. HCI-1680 and additional compounds from this series were also shown to inhibit the activation of AKT and other downstream signaling molecules. Kinase selectivity profiling, additional cell-based assays, and animal studies are ongoing. The details of these studies will be presented.nnCitation 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 1368. doi:10.1158/1538-7445.AM2011-1368

Abstract 2016: Combined targeting of epigenetic mechanisms has superior efficacy against human mantle cell lymphoma cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnGene silencing is mediated by multi-protein complexes PRC (polycomb repressive complexes) 1 and 2. Of the three core protein components of PRC2, i.e., EZH2, SUZ12 and EED, EZH2 has the SET domain with its intrinsic histone methyltransferase activity. This induces the trimethylation (Me3) of lysine (K) 27 on histone (H) 3-a repressive chromatin mark for gene repression. The PRC1 components include BMI1, RING1 and RING2, which are responsible for the ubiquitylation (Ub) of K119 on H2A and compaction of the chromatin at PRC2 target genes. We have previously reported that treatment with the pan-histone deacetylase inhibitor panobinostat (PS, Novartis Pharma) depletes EZH2, SUZ12 and the DNA methyltransferase (DNMT) 1. We also showed that co-treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep, further depleted PRC2 complex proteins and, in combination with PS, induced synergistic apoptosis of cultured and primary AML cells. In the present studies we determined that DZNep dose-dependently depleted EZH2, SUZ12 and BMI1 expression as well as inhibited K27Me3 on H3. DZNep treatment also induced p21, p27 and FBXO32, while depleting the levels of cyclin D1 in the cultured MCL JeKo-1 and MO2058 cells. Similar induction of p21, p27 and FBXO32 were also observed, following siRNA knockdown of EZH2. Notably, DZNep also induced similar perturbations in primary, patient-derived MCL cells. Treatment with PS alone attenuated EZH2, SUZ12 and DNMT1, as well as depleted the protein expression of BMI1, RING2 and MEL18 in the cultured MCL cells. This was associated with attenuation of H3K27Me3 and H2K119Ub, and augmentation of H3K4Me3 chromatin marks. Depletion of BMI with shRNA also reduced the levels of H2K119 Ub, which was associated with growth inhibition of MCL cells. PS treatment also decreased the levels of LSD1, a demethylase of H3K4Me2, which led to increase in H3K4Me3. Treatment with the LSD1 inhibitor CIT0665, or knockdown of LSD1 by shRNA, increased H3K4Me2 &Me3 levels, as well as enhanced PS mediated growth inhibition and apoptosis of MCL cells. PS treatment also induced heat shock protein (hsp) 90 acetylation, and depleted the levels of hsp90 client proteins in JeKo-1, including CDK4, c-RAF and AKT. As compared to treatment with each agent alone, co-treatment with DZNep and PS caused greater depletion of EZH2, SUZ12 and BMI1, accompanied with greater induction of p21 and p27 but attenuation of cyclin D1 expression. Co-treatment with DZNep and PS also induced cell cycle growth arrest and synergistically induced apoptosis of JeKo-1, MO2058, and primary MCL cells derived from 3 patients with MCL (combination indices <1.0). Taken together these findings indicate that combined targeted depletion of the levels and activities of EZH2, HDACs, LSD1 and BM1 exerts superior activity against MCL cells. These studies also support the in vivo testing of combined epigenetic therapies in the therapy of MCL.nnCitation 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 2016. doi:10.1158/1538-7445.AM2011-2016