Hariprasad Vankayalapati

A novel EPAS1/HIF2A germline mutation in a congenital polycythemia with paraganglioma

Congenital polycythemias have diverse etiologies, including mutations in the hypoxia sensing pathway. These include HIF2A at exon 12, VHL gene (Chuvash polycythemia), and PHD2 mutations, which in one family was also associated with recurrent pheochromocytoma/paraganglioma (PHEO/PGL). Over the past two decades, we have studied seven unrelated patients with sporadic congenital polycythemia who subsequently developed PHEO/PGL with, until now, no discernible molecular basis. We now report a polycythemic patient with a novel germline HIF2AF374Y (exon 9) mutation, inherited from his mother, who developed PHEO/PGL. We show that this is a gain-of-function mutation and demonstrate no loss-of-heterozygosity or additional somatic mutation of HIF2A in the tumor, indicating HIF2AF374Y may be predisposing rather than causative of PHEO/PGL. This report, in view of two other concomitantly reported PHEO/PGL patients with somatic mutations of HIF2A and polycythemia, underscores the PHEO/PGL-promoting potential of mutations of HIF2A that alone are not sufficient for PHEO/PGL development.

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.

Repurposing of Proton Pump Inhibitors as first identified small molecule inhibitors of endo-β-N-acetylglucosaminidase (ENGase) for the treatment of NGLY1 deficiency, a rare genetic disease

N-Glycanase deficiency, or NGLY1 deficiency, is an extremely rare human genetic disease. N-Glycanase, encoded by the gene NGLY1, is an important enzyme involved in protein deglycosylation of misfolded proteins. Deglycosylation of misfolded proteins precedes the endoplasmic reticulum (ER)-associated degradation (ERAD) process. NGLY1 patients produce little or no N-glycanase (Ngly1), and the symptoms include global developmental delay, frequent seizures, complex hyperkinetic movement disorder, difficulty in swallowing/aspiration, liver dysfunction, and a lack of tears. Unfortunately, there has not been any therapeutic option available for this rare disease so far. Recently, a proposed molecular mechanism for NGLY1 deficiency suggested that endo-β-N-acetylglucosaminidase (ENGase) inhibitors may be promising therapeutics for NGLY1 patients. Herein, we performed structure-based virtual screening utilizing FDA-approved drug database on this ENGase target to enable repurposing of existing drugs. Several Proton Pump Inhibitors (PPIs), a series of substituted 1H-benzo [d] imidazole, and 1H-imidazo [4,5-b] pyridines, among other scaffolds, have been identified as potent ENGase inhibitors. An electrophoretic mobility shift assay was employed to assess the inhibition of ENGase activity by these PPIs. Our efforts led to the discovery of Rabeprazole Sodium as the most promising hit with an IC50 of 4.47±0.44μM. This is the first report that describes the discovery of small molecule ENGase inhibitors, which can potentially be used for the treatment of human NGLY1 deficiency.

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.

Ruthenium(II)- and copper(I)-catalyzed synthesis of click-xylosides and assessment of their glycosaminoglycan priming activity

Xylosides are small molecules that serve as primers of glycosaminoglycan biosynthesis. Xyloside mediated modulation of biological functions depends on the extent of priming activity and fine structures of primed GAG chains. In earlier studies, copper (Cu) catalyzed synthesis of click-xylosides and their priming activity were extensively documented. In the current study, ruthenium (Ru) mediated catalysis was employed to synthesize xylosides with a 1,5-linkage between the xylose and the triazole ring instead of a 1,4-linkage as found in Cu-catalyzed click-xyloside synthesis. Mono- and bis-click-xylosides were synthesized using each catalytic method and their glycosaminoglycan priming activity was assessed in vitro using a cellular system. Ru-catalyzed click-xylosides showed a higher priming activity as measured by incorporation of radioactive sulfate into primed glycosaminoglycan chains. This study demonstrates that altering the linkage of the aglycone to the triazole ring changes the priming activity. Computational modeling provides a molecular rationale for higher priming ability of Ru-mediated click-xylosides. Higher GAG priming activity is attributed to the formation of more stable interactions between the 1,5-linked xylosides and β-1,4-galactosyltransferase 7 (β4GalT7).

Abstract 5543: Inhibition of the tyrosine kinase receptor Axl blocks cell invasion and promotes apoptosis in pancreatic cancer cells.

Pancreatic cancer is virtually a uniformly lethal disease and a better understanding of the molecular basis of this malignancy is needed to discover new ways to prevent or treat this deadly disease. A central feature of malignant cells is their ability to disseminate from the primary tumor and establish local and distant metastases. In most cases, cancer patients with localized disease have significantly better prognosis than those with metastatic tumors and the majority of cancer mortality is associated with metastatic disease rather than the primary tumor. The receptor tyrosine kinase Axl is overexpressed in over 50% of pancreatic cancers and expression of Axl in these cancers is highly associated with a poor prognostic outcome for patients. 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 known ligand for Axl is the Growth Arrest Specific Gene-6 (Gas6) protein and it9s binding to Axl leads to Axl autophosphorylation and activation of downstream signaling pathways including MAPK and PI3K/Akt pathways. We discovered and developed a small molecule Axl kinase inhibitor, HCI-2084, and explored it for the effectiveness of targeting the Axl kinase in cell-based models of pancreatic cancer. HCI-2084 is a 2-((2,5-substitutedpyrimidin-4-yl)amino)-N,N-dimethyl benzene sulfonamide that has low nanomolar (IC50 = 12 nM) activity against the Axl kinase in a biochemical assays. In further biochemical evaluation, HCI-2084 was shown to inhibit the entire TAM family of kinases (IC50 Axl = 12 nM; IC50 Mer = 60 nM; Tyro3 = 71% inhibition at 200 nM). HCI-2084 inhibits a small number of additional kinases when screened in a kinase panel of over 500 kinases and has demonstrated an ADMET profile suggesting it may be a potential clinical candidate. In cell proliferation assays, HCI-2084 significantly inhibited pancreatic cancer cell growth at concentrations as low as 30 nM. In pharmacodynamic assays, HCI-2084 dramatically inhibited Akt signaling (pAKT S473) downstream of GAS6 stimulation in pancreatic cancer cell lines. Consistent with the known function of Axl, HCI-2084 inhibited Gas6-induced migration and invasion of pancreatic cancer cells in vitro and potently induces apoptosis. Mechanistically, HCI-2084 decreases the expression of genes involved in Epithelial-Mesenchymal Transition (EMT) and induces cells to take on more epithelial phenotypes. HCI-2084 also significantly inhibited the growth of pancreatic cancer cell lines grown in xenograft tumor mouse model and taken together, these results suggest Axl is a potential therapeutic target in pancreatic cancer and that HCI-2084 is a potential agent treat this disease. Citation Format: Malia Anderson, Alex Ober, Alexis Mollard, Lee Call, Jared J. Bearss, Hariprasad Vankayalapati, Sunil Sharma, Steven Warner, David J. Bearss. Inhibition of the tyrosine kinase receptor Axl blocks cell invasion and promotes apoptosis in pancreatic cancer cells. [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 5543. doi:10.1158/1538-7445.AM2013-5543

Abstract C92: HCI-2577 inhibits LSD1 and modulates histone marks in Ewing's sarcoma models

Lysine-specific demethylase 1 (LSD1/AOF2/KDM1A) is a flavin-dependent histone demethylase that catalyzes the posttranslational oxidative demethylation of mono- and dimethylated 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 mark, and demethylation of this mark by LSD1 prevents expression of tumor suppressor genes important in human cancer. Whereas, H3K9 methylation is a repressive mark and LSD1 activity has been shown to upregulate tumor promoting pathways. This makes LSD1 emerge as an important target for the development of novel antitumor inhibitors. The compound HCI-2577 was identified as a potent reversible inhibitor of LSD1 enzymatic activity, with an IC50 of 7nM. In a diverse cell screen panel for cellular viability, Ewing9s sarcoma was identified as being sensitive to HCI-2577. Here we show that HCI-2577 is efficacious in in vivo models of Ewing9s sarcoma as a single agent with a favorable drug profile. In conclusion, HCI-2577 is a novel LSD-1 inhibitor with therapeutic potential in Ewing9s sarcoma demonstrating promising activity in biochemical, cell-based and in vivo assays. Citation Format: Jared J. Bearss, Adrianne Neiss, Xiao-Hui Liu, Hariprasad Vankayalapati, Sunil Sharma. HCI-2577 inhibits LSD1 and modulates histone marks in Ewing9s sarcoma models. [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 C92.

Abstract 4413: Mechanisms of sensitivity to treatment with the PDK1 inhibitors HCI-1680 and HCI-1708.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The phosphoinositide-dependent kinase-1 (PDK1) is a serine/threonine kinase that has been considered a promising potential oncology drug target because of its role as an important regulator in the PI3K/AKT/mTOR pathway. PDK1 phosphorylates highly conserved Ser or Thr residues in the activation loop of several AGC super family kinases including PKC, SGK, PKB/Akt, p70S6K, and PDK1 itself. Approximately, 40-50% of all tumors involve mutations in the phosphatase and tensin homolog (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 PDK1 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). 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. Synthesis and follow-up screening led to the discovery of HCI-1680 and a related compound SGI-1708, as potent PDK1 inhibitors with IC50 values of 80 and 94 nM, respectively. We used a large cell line panel of over 100 cancer cell lines to examine the ability of these compounds to kill cancer cells. Both HCI-1680 and SGI-1708 demonstrated remarkable selectivity for cell killing in several cell lines (KATO3, KG-1, MV4-11, Kasumi-1, MFE296 and AN3CA) in the low nanomolar range compared to all of the remaining cell lines in which the compounds showed low micromolar activity. Based on the known mutations in these cell lines we determined that HCI-1680 and SGI-1708 were more active in cells with PTEN deletion/silencing as well as activation of PIK3CA through activating mutations in PIK3CA or Ras proteins. Our compounds from this series were also shown to inhibit the activation of AKT and other downstream signaling molecules. We have explored the effects on gene expression Moreover, the lead compounds had high ligand efficiency with promising solubility and permeability parameters. Early animal studies examining pharmacokinetics and efficacy in xenograft models of human cancers have suggested that HCI-1680 and SGI-1708 have properties and activity to be developed as potential clinical candidates. We hypothesize that tumors, which have inactivated PTEN through mutations or silencing and also harbor mutations that activate PI3K define a population of cancer cells that are uniquely sensitive to PDK1 Inhibition. By inhibiting PDK1 activity we will block signaling from the PI3K pathway and lead to inhibition of cell proliferation and survival of these cancer cells. Citation Format: Brian Walker, Sorna Venkataswamy, Steven Warner, Lee Call, Hariprasad Vankayalapati, Sunil Sharma, David J. Bearss. Mechanisms of sensitivity to treatment with the PDK1 inhibitors HCI-1680 and HCI-1708. [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 4413. doi:10.1158/1538-7445.AM2013-4413

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 3391: Overexpression of Nek2 promotes bortezomib resistance in multiple myeloma cells.

Drug resistance is a major complication in cancer therapy but the molecular mechanisms driving both innate and acquired drug resistance still remain largely unknown for many anti-cancer agents. Microarray analyses on paired primary multiple myeloma samples at baseline and after therapy or at relapse showed that the mitotic kinase NEK2 was one of the most up-regulated genes in myeloma cells after high-dose chemotherapy or at relapse. Nek2 is a Serine/Threonine kinase that is activated during mitosis and is associated centrosome function and structure at the G2/M transition. A role for Nek2 has been suggested in the separation of centrosomes as well as promoting cell cycle from G2 phase to M phase but no role for Nek2 in mediating drug resistance has been described. By analyzing the published (> 2,500) microarrays and clinical datasets, we found that NEK2 expression is increased in many malignancies, and that high expression of NEK2 was associated with a shorter event-free and overall survival. Moreover, NEK2 expression was typically increased in tumors with aggressive subtype and advanced stage. Our studies in myeloma cells indicate that over-expressing NEK2 results in enhanced cell proliferation and drug resistance, whereas knockdown of NEK2 induced significant cancer cell death and growth inhibition. Nek2 transfected myeloma cell lines ARP1, H929 and KMS28PE were used to investigate Nek2’s function relative to bortezomib resistance. We found that Nek2 over-expression significantly elevated the proteasome activity of transfected cells leading to decreased activity of Bortezomib in these cells. Using novel small molecule Nek2 inhibitors developed in our lab, we demonstrated efficient inhibition of proteasome activity in Nek2 over-expressing cancer cell lines. The treatment of cell with our lead compounds significantly inhibited the degradation of some proteasome targeted cell cycle regulators. Further, the combinations of these Nek2 inhibitors and bortezomib, greatly elevated the efficiency of bortezomib in decreasing the proteasome activity in vitro. The treatment with these novel Nek2 inhibitors successfully rescued the drug resistance in Nek2 over-expressed cells, indicating a potential therapy for bortezomib resistant myeloma patients. We conclude that NEK2 represents a predictor for drug resistance and poor prognosis in cancers and could be a potential target for cancer therapy. Citation Format: Jessica Flory, Rachel Brog, Ling Yao Meng, Lee Call, Hariprasad Vankayalapati, Guido Tricot, Fenghuang Zhan, David J. Bearss. Overexpression of Nek2 promotes bortezomib resistance in multiple myeloma cells. [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 3391. doi:10.1158/1538-7445.AM2013-3391