Eric Gourley

In vitro and in vivo characterization of SGI-1252, a small molecule inhibitor of JAK2

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 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 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.

Abstract B263: Mechanistic, functional, and in vivo efficacy of inhibiting ETK/BMX in cancer models

Epithelial and endothelial tyrosine kinase (ETK), also known as bone marrow tyrosine kinase gene in chromosome X (BMX), belongs to the Burton9s 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 SuperGen9s 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 SuperGen9s 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, SuperGen9s lead ETK compounds also yielded concentration‐dependent responses with EC50 values in the nanomolar region. Given ETK9s 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 SuperGen9s 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.