OBJECTIVE:Constitutive activation of the Janus kinase 2 (JAK2) due to a somatic mutation (JAK2(V617F)) arising in hematopoietic stem cells plays a central role in the pathophysiology of myeloproliferative neoplasms (MPNs). To investigate the hypothesis that drugs that inhibit JAK2 have therapeutic potential, we developed a small molecule inhibitor, SGI-1252, that targets the adenosine triphosphate-binding and solvent pocket of the protein. MATERIALS AND METHODS:Established cells lines each expressing different JAK2(V617F) copy numbers, a cell line transfected with wild-type and mutant JAK2, ex vivo expanded erythroid progenitor cells from patients with MPNs, and a murine xenograft model were used to characterize the activity of SGI-1252. RESULTS:In vitro studies showed that SGI-1252 potently inhibits the kinase activity of wild-type JAK2, JAK2(V617F) and JAK1, but not JAK3. SGI-1252 blocked phosphorylation of signal transducers and activators of transcription 5, a downstream target of JAK2 and inhibited expression of the JAK2-dependent antiapoptotic gene BCL-X(L). Additional studies confirmed induction of apoptosis in JAK2(V617F)-positive cell lines by SGI-1252. Moreover, cell lines transfected with either wild-type JAK2 or JAK2(V617F) were equally susceptible to the antiproliferative effects of SGI-1252 and the antiproliferative activity of SGI-1252 toward ex vivo--expanded erythroid progenitors from patients with polycythemia vera and primary myelofibrosis appeared independent of the JAK2(V617F) allele burden. Pharmacodynamic studies in a murine xenograft model demonstrated both anti-tumor activity and inhibition of signal transducers and activators of transcription 5 phosphorylation by SGI-1252, and the drug was active and well-tolerated whether delivered intraperitoneally or orally. CONCLUSIONS:Together, these studies support further development of SGI-1252 for clinical use.

Abstract Epithelial and endothelial tyrosine kinase (ETK), also known as bone marrow tyrosine kinase gene in chromosome X (BMX), belongs to the Burton's Tyrosine Kinase (BTK) family. ETK is a nonreceptor tyrosine kinases involved in tumorigenicity, adhesion, motility, angiogenesis, proliferation, and differentiation. It is overexpressed in metastatic breast and prostate cancers and is implicated in the neuroendocrine transformation of prostate cells. Significant oncogenes such as Src, focal adhesion kinase (FAK), and phosphatidyl-inositol (PI)-3 kinase are upstream regulators of ETK while several hub proteins, also critical for cancer development and progression, such as AKT, STAT3, and p21 activated kinase 1 (PAK1), are downstream of ETK. Cell-based validation studies also demonstrate that inhibition of ETK diminishes cellular transformation, proliferation, and migration. Such evidence provides substantial reasoning for developing new small molecule inhibitors against ETK. Applying SuperGen's proprietary small molecule drug discovery process, CLIMB™ a series of lead compounds were effectively developed to potently and selectively inhibit ETK. Biochemical data from kinase screens against ETK demonstrated nanomolar potency of SuperGen's inhibitors and good selectivity toward ETK from a panel of 75 kinases. To mechanistically demonstrate ETK inhibition in cell-based models, immunoprecipitations were carried out and phospho-ETK levels were detected. These compounds exhibited a dose-dependent response against phospho-ETK. Functionally, the inhibition of ETK will produce changes in phospho-AKT (S473) levels as well as phospho-STAT3 (Y705) levels downstream of EGF stimulation. Using Luminex bead-based technology and western blot techniques respectively, SuperGen's lead ETK compounds also yielded concentration-dependent responses with EC50 values in the nanomolar region. Given ETK's reported angiogenic role in endothelial cell migration and proliferation, these compounds were evaluated in an in vitro tube formation assay. Likewise, these ETK inhibitors demonstrated potent nanomolar inhibition of endothelial tube formation. Finally, having shown mechanistically and functionally the efficacy of ETK inhibitors through in vitro assays, the compounds were tested with in vivo solid tumor efficacy models in nude mice. All five lead inhibitors effectively decreased tumor burden volume relative to the vehicle treatment group in an AN3CA xenograft model. Taken together, these data suggest SuperGen's small molecule ETK inhibitors represent a potentially new therapeutic avenue for treating patients with solid tumor malignancies in the future. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B263.

Abstract 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.

Pim-1 is a highly conserved serine-threonine kinase that is a key regulator in many cytokine signaling pathways. Activated Pim-1 kinase can induce progression of the cell cycle, inhibition of apoptosis, and modulation of other signal transduction pathways, including its own. Transformation of Pim-1 kinase in conjunction with other oncogenes such as c-myc, N-Myc, and others such as gfi-1, runx-2 leads to malignancies including Burkitt\#8217;s lymphoma, prostate cancer, diffuse large cell lymphoma several other types of human leukemias. These findings suggest that Pim-1 may be a promising drug target for development of anticancer agents. Through the use of Pim-1kinase crystal structure we began our discovery process by performing large-scale virtual screening composed of focused and or diverse virtual libraries. With the application of consensus scoring, binding energies and several drug-like filters we have selected limited set of 72 compounds. Five compounds belongs to imidazo[1,2-b]pyridzaine series were found to inhibit the Pim-1 kinase enzyme with an IC50 from 2 to 10 \#956;M. These active compounds and their binding modes lead to the lead optimization, synthesis and for further design of potent Pim1 kinase inhibitors. The strategies for optimizing the potency and selectivity against the Pim-1 target were undertaken with the focus on the modifications of R1 and R2 groups. A novel synthetic route was developed starting from 1,4-dibromo-trans-2-butene. We prepared 3-bromo-6-chloro-imidazo[1,2-b]pyridazine scaffold in three steps. Suzuki coupling with various substituted aryl boronic acids and followed by introduction of aliphatic amines lead to the synthesis of more than 30 novel chemical entities as Pim-1 kinase inhibitors. Initial SAR studies revealed that the introduction of hydrophobic moiety at R1 and small ring substituent groups with 1 or 2 -CH2 spacer at the 6-amine position are vital for the Pim-1 kinase inhibition. The lead compound SGI-1776 showed very potent activity against Pim-1 kinase with an IC50 of 7 nM and kinases selectivity. We recently filed an IND and anticipate enrollment into the First-in-Human Phase I study in the near future. Design, synthesis and SAR studies will be presented. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2013.

Abstract Abstract 2758 Poster Board II-734 Axl kinase, a member of the TAM family and also known as UFO, ARK, and Tyro7, is a receptor tyrosine kinase implicated in tumorigenesis. Overexpression of Axl is associated with increased cellular transformation, cell survival, proliferation, migration, angiogenesis, and adhesion. The oncogenic potential of Axl was first discovered in chronic myelogenous leukemia (CML) and has been shown to play a role in the development of acute myelogenous leukemia (AML) and myelodysplasia. Binding of Growth Arrest Specific Gene-6 (GAS6), a vitamin K-dependent protein and a known ligand for Axl, leads to subsequent phosphorylation events of downstream effecter molecules such as mitogen-activated protein (MAP) kinase and phosphatidyl-inositol (PI)-3 kinase/AKT pathways which are critical in oncogenic transformation. Furthermore, target validation studies of in vivo cancer models show that inhibition of Axl expression by RNA interference blocked tumor growth in those models. Taken together, this information makes Axl kinase an exciting target for small molecule drug discovery. Effectively utilizing SuperGen's proprietary CLIMB“technology, a series of small molecule inhibitors were rapidly discovered and developed for potency and selectivity against Axl. Lead candidate compounds demonstrate low nano-molar IC50 values in an Axl kinase biochemical assay, and focused selectivity for Axl when screened in kinase panels. Likewise, these compounds showed low and sub micro-molar activity from an anti-proliferative leukemia/lymphoma cell-based panel. Similarly, treatment of leukemia cells with these compounds in combination with known chemotherapeutic agents produced results that implicate combinatorial therapies as potentially beneficial treatments for patients suffering from hematological malignancies. In mechanistic, target validation assays, EC50 data from immunoprecipitated western blots of transiently transfected liquid tumor cells showed nano-molar inhibition of phospho-Axl when detecting with an anti-phospho-tyrosine antibody. A western blot panel of leukemia and lymphoma cell lines showed overexpression of Axl in several of these cell lines, further validating Axl as a potential therapeutic target in hematological cancers. Functional cell-based assays using SuperGen's lead compounds also potently inhibited phospho-AKT (S473). We propose that SuperGen's small molecule inhibitors against Axl kinase represent a new class of compounds with potent and selective activity in hematological malignancies. Disclosures: No relevant conflicts of interest to declare.

Abstract 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.

Discovery of somatic mutation of JAK-2 (G1849T that produces JAK-2V617F) in the hematopoietic cells of patients with Philadelphia chromosome negative myeloproliferative disorders (Ph−MPDs) was a watershed event that not only provided new insights into the pathobiology of polycythemia vera, essential thrombocytosis and primary myelofibrosis but also identified a potential target for therapy. Herein we report the results of preclinical studies designed to characterize the activity of a novel inhibitor of JAK-2. The compound, SGI-1252, developed by SuperGen (Dublin, CA) incorporates with high affinity into the ATP-binding site of JAK-2. SGI-1252 was tested against a panel of 75 kinases and was found to have significant activity against only FLT-3, TYK-2 and the SRC family members, ABL, LCK, YES, in addition to JAK-2 and JAK-1. SGI-1252 has an IC50 for JAK-2 of 5.4 nM with an IC50 for JAK-2V617F of 19.7 nM. The inhibitor also effectively blocks the activity of JAK-1 (IC50 14.8 nM) but has little JAK-3 inhibitory activity (IC50 1,700 nM). SGI-1252 is a potent inhibitor of STAT-5 phosphorylation (EC50 76.2 nM) and was also found to block the JAK-2 dependent expression of the anti-apoptotic protein, BCL-XL (EC50 778 nM). Drug treatment of a murine cell line (FDCP) transfected with either human wild-type JAK-2 or JAK-2G1849Tgenerated IC50 values of 83 nM and 108 nM, respectively, and SGI-1252 treatment of human cell lines, HEL, UKE-1 and SET-2, that express mutant JAK2 in different copy numbers, gave IC50 values of 472 nM, 83 nM and 63 nM, repectively. When tested in ex-vivo expanded native human erythroid progenitor cells from 17 patients with Ph−MPDs (10 PV and 7 MF), SGI-1252 showed an IC50 of ~100 nM, regardless of the JAK-2V617F allele burden. Using a flow cytometric assay, SGI-1252 was shown to induce apoptotic cell death in a concentration dependent manner. Luminex technology allows for concurrent quantitative analysis of multiple proteins from the same tissue source, and this technology was used to investigate simultaneously the effects of SGI-1252 on total and phospho ERK1/2, total and phospho STAT3, phospho STAT5, caspase 3, cleaved PARP and GAPDH (control) in untreated and drug treated cells at IC50 and IC80 concentrations. Significant in vivo efficacy of SGI-1252 was also observed using HEL and MV-4-11 xenograft models when compared to treatment with vehicle or daunorubicin. Using a murine model, we found that SGI-1252 has high oral bioavailability and is well tolerated with a five-day repeat maximum dose of at least 900 mg/kg. Together, these studies demonstrate that SGI-1252 is a potent inhibitor of JAK-2 dependent proliferation in both JAK-2V617F positive cell lines and in ex vivo expanded erythroid progenitors derived from patients with JAK-2V617F positive Ph−MPDs. Moreover, our studies show that the effects of SGI-1252 are mediated by blocking both JAK-2 dependent anti-apoptoic pathways and JAK-2 dependent proliferative pathways. Using the orally available form of the compound, pharmacokinetic, pharmacodynamic and toxicity studies in mice suggest that serum concentration of the drug well above the predicted therapeutic range can be achieved without significant hematological toxicity. Based on these preclinical experiments, SGI-1252 appears to be an excellent candidate for phase I/II studies in patients with Ph−MPDs.

C200 The Janus Kinases (JAKs) are a family of intracellular protein tyrosine kinases that are expressed in a wide variety of cell types including mammary gland, hematopoeitic cells, and prostate. Activation of JAK family members initiates signaling of in a number of intracellular signaling pathways important in cell differentiation, proliferation, migration, and apoptosis. JAK activation leads to the tyrosine phosphorylation and activation of the Signal Transducers and Activators of Transcription (STATs) proteins. The activated STAT proteins form dimers that translocate to the nucleus, bind to DNA, and activate transcription of target genes such as, SPI2.1, PIM-1, SOCS, and others. Dysregulation of JAK2 signaling, caused by point mutation of the JH2 autoinhibitory region or formation of JAK2 fusion proteins, has been implicated in myeloproliferative disorders (MPD), leukemia, lymphoma, and various solid tumors. We have identified a series of selective JAK2 inhibitors, based on a novel pharmacophore, using our proprietary CLIMBTM drug discovery process. Through the use of CLIMBTM, the published JAK2 crystal structure was used to build several models that were then used as a substrate for in silico docking of 2.3 million virtual small molecule compounds to generate a subset of leads based on calculated binding energies. These leads were then screened using a number of in silico physicochemical and ADMET prediction algorithms to determine “druggable” leads which were most likely to be successful in a biological context. Lead JAK2 inhibitor candidates, JAK2-1, JAK2-2, and JAK2-3, exhibit low nanomolar IC50 activity against the JAK2 and JAK2 V617F mutant enzymes. Cancer cell lines expressing either the wild-type or mutant JAK2 enzyme demonstrate sensitivity to these inhibitors resulting in IC50 values in low micromolar to nanomolar range. Consistent with the inhibition of the JAK2 enzyme, activity of downstream signaling partners are severely decreased. Activity of STAT5, a downstream modulator of JAK2 signaling, was determined by probing the phosphorylation state of the STAT5 protein from treated HEL cell lysates. Western blot analysis showed an inhibition of STAT5 phosphorylation at an EC50 of

The three Pim kinases represent a small subfamily of serine/threonine kinases known to be involved in a number of signaling pathways as downstream effectors and potent inhibitors of apoptosis. Unlike most other kinases, Pim kinases lack a regulatory domain which means they are controlled largely at the transcriptional level and that the mRNA expression levels of Pim kinases in cells correlate with their activity. While normal expression of Pim-1 kinase is seen in cells of hematopoietic origin, examination of gene expression of Pim-1 in different malignancies using cDNA microarray analysis suggests that Pim-1 is overexpressed in a large percentage of ALL, AML, CML, DLBCL, Prostatic Adenocarcinoma, Bladder, and Oral cancers. Pim-2 is also largely expressed in ALL, AML, Squamous Cell Lung and Adenocarcinoma of the Lung while Pim-3 is restricted to Melanoma, Pancreatic Adenocarcinoma, Gastric cancers. This implicates Pim-1 and Pim-2 in the onset and progression in several of hematological malignancies and therefore they represent interesting potential targets for drug development. To evaluate the potential of Pim-1 and -2 as a drug targets we have identified and synthesized a series of Pim kinase inhibitors using our proprietary CLIMB™ drug discovery process. Through the use of CLIMB™, the published Pim-1 kinase crystal structure was used to build several models that were then used to predict potential small molecule inhibitors of Pim-1 and Pim-2 from a large virtual library. A subset of leads, based on calculated binding energies as well as additional physical chemical properties, were screened using a number of in silico physicochemical and ADMET prediction algorithms to determine which compounds were most likely to be successful in a biological context. Lead candidates were initially screened using biochemical enzyme-based and cell-based assays. Cell-based activity was determined in HEL (acute megakaryocytic leukemia), K562 (chronic myeloid leukemia), MO7e (myeloid leukemia), and other human leukemia and lymphoma cell lines. From several lead candidates a series of analogs were produced with improved inhibitory activity and pharmacokinetic characteristics. In the Pim-1 and Pim-2 in vitro kinase assay and in the cell-based assay a number of leads exhibited inhibitory activity with IC50 concentrations in the low nanomolar range. Here we present the details of the biochemical and cell-based assay results as well as the activity in tumor xenograft models of our Pim kinase inhibitors.

C208 The Pim-1 serine/threonine kinase is known to be involved in a number of signaling pathways as a downstream effector and a potent inhibitor of apoptosis. Unlike most other kinases, Pim-1 lacks a regulatory domain which means it is controlled largely at the transcriptional level and that the mRNA expression levels of Pim-1 in cells correlate with its activity. While normal expression of Pim-1 kinase is seen in cells of hematopoietic origin, examination of gene expression of Pim-1 in different malignancies using cDNA microarray analysis suggests that Pim-1 is overexpressed in a large percentage of ALL, AML, CML, DLBCL, Prostatic Adenocarcinoma, Bladder, and Oral cancers. Pim-1 has been implicated in the onset and progression in several of these malignancies and therefore represents an interesting potential target for drug development. To evaluate the potential of Pim-1 as a drug target we have identified and synthesized a series of Pim-1 kinase inhibitors using our proprietary CLIMBTM drug discovery process. Through the use of CLIMBTM, the published Pim-1 kinase crystal structure was used to build several models that were then used to predict potential small molecule inhibitors of Pim-1 from a large virtual library. A subset of leads, based on calculated binding energies as well as additional physical chemical properties, were screened using a number of in silico physicochemical and ADMET prediction algorithms to determine which compounds were most likely to be successful in a biological context. Lead candidates were initially screened using biochemical enzyme-based and cell-based assays. Cell-based activity was determined in HEL (acute megakaryocytic leukemia), K562 (chronic myeloid leukemia), MO7e (myeloid leukemia), PC3 (prostate adenocarcinoma) and other human tumor cell lines. From several lead candidates a series of analogs were produced with improved inhibitory activity and pharmacokinetic characteristics. In the Pim-1 in vitro kinase assay and in the cell-based assay a number of leads exhibited inhibitory activity with IC50 concentrations in the low nanomolar range. Here we present the details of the biochemical and cell-based assay results as well as the activity in tumor xenograft models of our Pim-1 kinase inhibitors.

JAK2 is an intracellular protein tyrosine kinase whose dysregulation has been implicated in leukemia, lymphoma, and myeloproliferative disorders (MPD). Increased kinase activity of JAK2, caused by point mutation of the JH2 autoinhibitory region or formation of JAK2 fusion proteins, causes increased activation of downstream signaling pathways affecting cell differentiation, proliferation, migration, and apoptosis. Through the use of CLIMB™, our proprietary drug discovery process, the published JAK2 crystal structure was used to build several models that were then used as a substrate for in silico docking of 2.3 million virtual small molecule compounds to generate a subset of leads based on calculated binding energies. These leads were further screened using a number of in silico physicochemical and ADMET prediction algorithms to determine “druggable” leads which were most likely to be successful in a biological context. Lead JAK2 inhibitor candidates exhibit low nanomolar IC50 activity against the JAK2 and JAK2 V617F mutant enzymes. Cancer cell lines expressing either the wild-type or mutant JAK2 enzyme demonstrate sensitivity to these inhibitors resulting in IC50 values in low micromolar to nanomolar range. Consistent with the inhibition of the JAK2 enzyme, activity of downstream signaling partners are severely decreased. The phosphorylation level of STAT5, a downstream modulator of JAK2 signaling, in treated HEL cell lysates was analyzed by western blot analysis. These results showed that lead JAK2 candidates caused an inhibition of STAT5 phosphorylation at a low nanomolar EC50. This series of compounds are currently being tested in in vivo xenograft models. Evaluation of lead candidates in biochemical assays against the hERG and CYP450 enzymes showed that these compounds have little inhibitory activity against these enzymes. SuperGen’s lead selective JAK2 inhibitors exhibit potent inhibition of wild-type and mutant JAK2 kinase activity translating into potent inhibition of cellular signaling pathways and cancer cell proliferation.