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XLS file - 167K, Table 1: Cell line drug sensitivity data for SB2343 and mutations in cancer genes. Table 2: Results from MANOVA analysis of cancer genes as modifiers of drug sensitivity

PDF file - 2.9MB, In-vitro kinase assay, Histology, Cell proliferation, MANOVA analysis for genomic correlates of drug sensitivity

PDF file - 663K, Ambit kinome tree, Cell proliferation data, Pharmacokinetics, Body weight changes in PC3 efficacy study, Active vessel stain in COLO-205 efficacy study, MV4-11 efficacy model, Drug sensitivity profiling and correlation to mutation status

Dysregulation of the PI3K/mTOR pathway, either through amplifications, deletions, or as a direct result of mutations, has been closely linked to the development and progression of a wide range of cancers. Moreover, this pathway activation is a poor prognostic marker for many tumor types and confers resistance to various cancer therapies. Here, we describe VS-5584, a novel, low-molecular weight compound with equivalent potent activity against mTOR (IC50 = 37 nmol/L) and all class I phosphoinositide 3-kinase (PI3K) isoforms IC50: PI3Kα = 16 nmol/L; PI3Kβ = 68 nmol/L; PI3Kγ = 25 nmol/L; PI3Kδ = 42 nmol/L, without relevant activity on 400 lipid and protein kinases. VS-5584 shows robust modulation of cellular PI3K/mTOR pathways, inhibiting phosphorylation of substrates downstream of PI3K and mTORC1/2. A large human cancer cell line panel screen (436 lines) revealed broad antiproliferative sensitivity and that cells harboring mutations in PI3KCA are generally more sensitive toward VS-5584 treatment. VS-5584 exhibits favorable pharmacokinetic properties after oral dosing in mice and is well tolerated. VS-5584 induces long-lasting and dose-dependent inhibition of PI3K/mTOR signaling in tumor tissue, leading to tumor growth inhibition in various rapalog-sensitive and -resistant human xenograft models. Furthermore, VS-5584 is synergistic with an EGF receptor inhibitor in a gastric tumor model. The unique selectivity profile and favorable pharmacologic and pharmaceutical properties of VS-5584 and its efficacy in a wide range of human tumor models supports further investigations of VS-5584 in clinical trials. Mol Cancer Ther; 12(2); 151–61. ©2012 AACR.

SB939 is an oral histone deacetylase (HDAC) inhibitor currently in phase II clinical trials potently inhibiting class I, II, and IV HDACs with favorable pharmacokinetic properties, resulting in tumor tissue accumulation. To show target efficacy, a Western blot assay measuring histone H3 acetylation (acH3) relative to a loading control was developed, validated on cancer cell lines, peripheral blood mononuclear cells (PBMC), and in animal tumor models. Exposure of cells to 60 nmol/L (22 ng/mL) SB939 for 24 hours was sufficient to detect an acH3 signal in 25 μg of protein lysate. AcH3 levels of liver, spleen, PBMCs, bone marrow and tumor were measured in BALB/c mice, HCT-116 xenografted BALB/c nude mice, or in SCID mice orthotopically engrafted with AML (HL-60) after oral treatment with SB939. AcH3 could only be detected after treatment. In all tissues, the highest signal detected was at the 3-hour time point on day 1. On day 15, the signal decreased in normal tissues but increased in cancerous tissues and became detectable in the bone marrow of leukemic mice. In all tissues, acH3 correlated with SB939 dose levels (r2 = 0.76–0.94). When applied to PBMCs from 30 patients with advanced solid malignancies in a phase I clinical trial, a dose-dependent (10–80 mg) increase in relative acH3 was observed 3-hour postdose on day 1, correlating with Cmax and AUC of SB939 concentrations in plasma (r = 0.97, P = 0.014). Our data show that the favorable pharmacokinetic and pharmacodynamic properties of SB939 are translated from preclinical models to patients. Mol Cancer Ther; 10(7); 1207–17. ©2011 AACR.

Supplementary Methods, Figures 1-2, Tables 1-2 from Pharmacodynamic Evaluation of the Target Efficacy of SB939, an Oral HDAC Inhibitor with Selectivity for Tumor Tissue

Supplementary Table from Comprehensive Analysis of R-Spondin Fusions and RNF43 Mutations Implicate Novel Therapeutic Options in Colorectal Cancer

Supplementary Figures and Materials and Metholds from SB939, a Novel Potent and Orally Active Histone Deacetylase Inhibitor with High Tumor Exposure and Efficacy in Mouse Models of Colorectal Cancer

Supplementary Table 2 from SB939, a Novel Potent and Orally Active Histone Deacetylase Inhibitor with High Tumor Exposure and Efficacy in Mouse Models of Colorectal Cancer

Purpose:Gene fusions involving R-spondin (RSPOfp) and RNF43 mutations have been shown to drive Wnt-dependent tumor initiation in colorectal cancer. Herein, we aimed to characterize the molecular features of RSPOfp/RNF43 mutated (mut) compared with wild-type (WT) colorectal cancers to gain insights into potential rationales for therapeutic strategies.Experimental Design:A discovery cohort was classified for RSPOfp/RNF43 status using DNA/RNA sequencing and IHC. An independent cohort was used to validate our findings.Results:The discovery cohort consisted of 7,245 colorectal cancer samples. RSPOfp and RNF43 mutations were detected in 1.3% (n = 94) and 6.1% (n = 443) of cases. We found 5 RSPO fusion events that had not previously been reported (e.g., IFNGR1–RSPO3). RNF43-mut tumors were associated with right-sided primary tumors. No RSPOfp tumors had RNF43 mutations. In comparison with WT colorectal cancers, RSPOfp tumors were characterized by a higher frequency of BRAF, BMPR1A, and SMAD4 mutations. APC mutations were observed in only a minority of RSPOfp-positive compared with WT cases (4.4% vs. 81.4%). Regarding RNF43 mutations, a higher rate of KMT2D and BRAF mutations were detectable compared with WT samples. Although RNF43 mutations were associated with a microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) phenotype (64.3%), and a tumor mutation burden ≥10 mt/Mb (65.8%), RSPOfp was not associated with MSI-H/dMMR. The validation cohort replicated our genetic findings.Conclusions:This is the largest series of RSPOfp/RNF43-mut colorectal cancers reported to date. Comprehensive molecular analyses asserted the unique molecular landscape associated with RSPO/RNF43 and suggested potential alternative strategies to overcome the low clinical impact of Wnt-targeted agents and immunotherapy.

Supplementary Table 1 from SB939, a Novel Potent and Orally Active Histone Deacetylase Inhibitor with High Tumor Exposure and Efficacy in Mouse Models of Colorectal Cancer

Supplementary Table 3 from SB939, a Novel Potent and Orally Active Histone Deacetylase Inhibitor with High Tumor Exposure and Efficacy in Mouse Models of Colorectal Cancer

Supplementary Figure from Comprehensive Analysis of R-Spondin Fusions and RNF43 Mutations Implicate Novel Therapeutic Options in Colorectal Cancer

Although clinical responses in liquid tumors and certain lymphomas have been reported, the clinical efficacy of histone deacetylase inhibitors in solid tumors has been limited. This may be in part due to the poor pharmacokinetic of these drugs, resulting in inadequate tumor concentrations of the drug. SB939 is a new hydroxamic acid based histone deacetylase inhibitor with improved physicochemical, pharmaceutical, and pharmacokinetic properties. In vitro, SB939 inhibits class I, II, and IV HDACs, with no effects on other zinc binding enzymes, and shows significant antiproliferative activity against a wide variety of tumor cell lines. It has very favorable pharmacokinetic properties after oral dosing in mice, with >4-fold increased bioavailability and 3.3-fold increased half-life over suberoylanilide hydroxamic acid (SAHA). In contrast to SAHA, SB939 accumulates in tumor tissue and induces a sustained inhibition of histone acetylation in tumor tissue. These excellent pharmacokinetic properties translated into a dose-dependent antitumor efficacy in a xenograft model of human colorectal cancer (HCT-116), with a tumor growth inhibition of 94% versus 48% for SAHA (both at maximum tolerated dose), and was also effective when given in different intermittent schedules. Furthermore, in APCmin mice, a genetic mouse model of early-stage colon cancer, SB939 inhibited adenoma formation, hemocult scores, and increased hematocrit values more effectively than 5-fluorouracil. Emerging clinical data from phase I trials in cancer patients indicate that the pharmacokinetic and pharmacologic advantages of SB939 are translated to the clinic. The efficacy of SB939 reported here in two very different models of colorectal cancer warrants further investigation in patients. Mol Cancer Ther; 9(3); 642–52

Purpose: Gene fusions involving R-spondin (RSPOfp) and RNF43 mutations have been shown to drive Wnt-dependent tumor initiation in colorectal cancer. Herein, we aimed to characterize the molecular features of RSPOfp/RNF43 mutated (mut) compared with wild-type (WT) colorectal cancers to gain insights into potential rationales for therapeutic strategies. Experimental Design: A discovery cohort was classified for RSPOfp/RNF43 status using DNA/RNA sequencing and IHC. An independent cohort was used to validate our findings. Results: The discovery cohort consisted of 7,245 colorectal cancer samples. RSPOfp and RNF43 mutations were detected in 1.3% (n = 94) and 6.1% (n = 443) of cases. We found 5 RSPO fusion events that had not previously been reported (e.g., IFNGR1–RSPO3). RNF43-mut tumors were associated with right-sided primary tumors. No RSPOfp tumors had RNF43 mutations. In comparison with WT colorectal cancers, RSPOfp tumors were characterized by a higher frequency of BRAF, BMPR1A, and SMAD4 mutations. APC mutations were observed in only a minority of RSPOfp-positive compared with WT cases (4.4% vs. 81.4%). Regarding RNF43 mutations, a higher rate of KMT2D and BRAF mutations were detectable compared with WT samples. Although RNF43 mutations were associated with a microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) phenotype (64.3%), and a tumor mutation burden ≥10 mt/Mb (65.8%), RSPOfp was not associated with MSI-H/dMMR. The validation cohort replicated our genetic findings. Conclusions: This is the largest series of RSPOfp/RNF43-mut colorectal cancers reported to date. Comprehensive molecular analyses asserted the unique molecular landscape associated with RSPO/RNF43 and suggested potential alternative strategies to overcome the low clinical impact of Wnt-targeted agents and immunotherapy.

SUMMARY While immune checkpoint blockade is associated with prolonged responses in multiple cancers, most patients still do not benefit from this therapeutic strategy. The Wnt-β-catenin pathway is associated with diminished T cell infiltration; however, activating mutations are rare, implicating a role for autocrine/paracrine Wnt ligand-driven signaling in immune evasion. In this study, we show that proximal mediators of the Wnt signaling pathway are associated with anti-PD-1 resistance, and pharmacologic inhibition of Wnt ligand signaling supports anti-PD-1 efficacy by reversing dendritic cell tolerization and the recruitment of granulocytic myeloid-derived suppressor cells in autochthonous tumor models. We further demonstrate that the inhibition of Wnt signaling promotes the development of a tumor microenvironment that is more conducive to favorable responses to checkpoint blockade in cancer patients. These findings support a rationale for Wnt ligand-focused treatment approaches in future immunotherapy clinical trials and suggest a strategy for selecting those tumors more responsive to Wnt inhibition., Graphical abstract, In brief Anti-PD-1-refractory melanoma exhibits elevated Wnt ligand signaling activity. DeVito et al. demonstrate that pharmacologic inhibition of proximal Wnt ligand signaling sensitizes transgenic models of melanoma and lung cancer to anti-PD-1 checkpoint inhibitor immunotherapy by reversing dendritic cell tolerization and suppressing recruitment of granulocytic myeloid-derived suppressor cells to the tumor bed.

Cell mediated immunity plays a vital role in defense against influenza infection in humans. Less is known about the role of vaccine-induced cell mediated immunity and the cytokine responses elicited. We measured CD4+ and CD8+ T-cell reactivity in human subjects following vaccination with licensed trivalent influenza vaccine and a novel virus-like particle based vaccine. We detected influenza-specific CD4+ T-cell responses following vaccination with the licensed trivalent influenza vaccine and found that these correlated with antibody measurements. Administration of the novel virus-like particle based vaccine elicited influenza-specific CD4+ and CD8+ T-cell responses and the induction of the cytokines IFN-γ, IL-17A, IL17F, IL-5, IL-13, IL-9, IL-10 and IL-21. Pre-existing cytokine responses influenced the profile of the cytokine response elicited by vaccination. In a subset of individuals the VLP vaccine changed pre-vaccination production of type 2 cytokines such as IL-5 and IL-13 to a post-vaccination type 1 cytokine signature characterized by IFN-γ. A transcriptional signature to vaccination was found to correlate with antibody titer, IFN-γ production by T-cells and expression of a putative RNA helicase, DDX17, on the surface of immune cells.

The interleukin (IL)-6-type cytokines play major roles in a variety of biological processes by signaling through a common receptor subunit, glycoprotein (gp) 130. We performed yeast two-hybrid screening to identify new binding partners of the activated gp130 and the associated Janus kinases. LMO4, a LIM domain-containing protein that belongs to a family of oncogenes, was identified in this assay. Further studies show that LMO4 associates with gp130 and Janus kinase1 in several mammalian cell types. It also interacts with protein-tyrosine phosphatase 2 (SHP2) and suppressor of cytokine signaling 3 (SOCS3). The binding domains involved in these interactions were mapped, and the interactions were shown to be in a direct manner by in vitro binding assays. It is likely that LMO4 exists in the gp130 complex. The cellular localization of LMO4 was detected primarily in the nucleus with a substantial amount also detected in the cytoplasm in several cell types. The effect of LMO4 in IL-6 signaling was subsequently examined. Overexpression of LMO4 enhanced the transcriptional activity and target gene expression of Stat 3 (signal transducers and activators of transcription 3). Consistent with this, silencing LMO4 expression in stable cell lines expressing the small interfering RNA of LMO4 decreased Stat3 activity. Furthermore, the half-life of gp130 was shortened, and the production of acute phase proteins induced by IL-6 was reduced. Together, our data reveal a positive regulatory role of LMO4 in IL-6 signaling, possibly by acting as a scaffold for stabilization of the gp130 complex. These studies may open up a link between the oncogenic effect of LMO proteins and their regulatory role in cytokine signaling in general.

Signal transducer and activator of transcription 3 (Stat3) is a latent cytoplasmic transcription factor that can be activated by cytokines and growth factors. Stat3 plays important roles in cell growth, anti-apoptosis and cell transformation, and is constitutively active in various cancers. We examined its potential regulators by yeast two-hybrid screening. GRIM-19, a gene product related to interferon-beta- and retinoic acid-induced cancer cell death, was identified and demonstrated to interact with Stat3 in various cell types. The interaction is specific for Stat3, but not for Stat1 and Stat5a. The interaction regions in both proteins were mapped, and the cellular localization of the interaction was examined. GRIM-19 itself co-localizes with mitochondrial markers, and forms aggregates at the perinulear region with co-expressed Stat3, which inhibits Stat3 nuclear translocation stimulated by epidermal growth factor (EGF). GRIM-19 represses Stat3 transcriptional activity and its target gene expression, and also suppresses cell growth in Src-transformed cells and a Stat3-expressing cell line. Our data suggest that GRIM-19 is a novel negative regulator of Stat3.

The transcriptional regulation of Stat proteins is controlled through their C-terminal domains, which harbor both a tyrosine phosphorylation site, required for dimerization and subsequent nuclear translocation, and a serine phosphorylation site, required for maximum transcriptional activity. Previously, we reported that protein kinase Cdelta (PKCdelta) phosphorylates and interacts with Stat3 in an interleukin (IL)-6-dependent manner. In this study, we further characterized this interaction, and investigated the potential role of such an interaction. We show here that the catalytic domain of PKCdelta interacts with the Src homology 2 domain and part of the adjacent C-terminal transactivation domain of Stat3. This interaction, which does not seem to involve a classical phosphotyrosine SH2-mediated binding, however, significantly enhances the interaction of Stat3 and the IL-6 receptor subunit glycoprotein (gp) 130, which is the initial step for Stat3 activation by IL-6. Expression of a dominant negative PKCdelta or depletion of the endogenous PKCdelta by phorbol 12-myristate 3-acetate treatment abrogates the association of Stat3 with gp130. At the same time, PKCdelta is recruited to gp130 via association with Stat3, which may facilitate its phosphorylation on the gp130 receptor. Finally, we identified Thr-890, a putative PKC phosphorylation site on gp130, to be critical for the effect of PKCdelta. Our data indicate that PKCdelta plays important regulatory roles in IL-6 signaling.

Dysregulation of the PI3K/mTOR pathway, either through amplifications, deletions, or as a direct result of mutations, has been closely linked to the development and progression of a wide range of cancers. Moreover, this pathway activation is a poor prognostic marker for many tumor types and confers resistance to various cancer therapies. Here, we describe VS-5584, a novel, low-molecular weight compound with equivalent potent activity against mTOR (IC50 = 37 nmol/L) and all class I phosphoinositide 3-kinase (PI3K) isoforms IC50: PI3Kα = 16 nmol/L; PI3Kβ = 68 nmol/L; PI3Kγ = 25 nmol/L; PI3Kδ = 42 nmol/L, without relevant activity on 400 lipid and protein kinases. VS-5584 shows robust modulation of cellular PI3K/mTOR pathways, inhibiting phosphorylation of substrates downstream of PI3K and mTORC1/2. A large human cancer cell line panel screen (436 lines) revealed broad antiproliferative sensitivity and that cells harboring mutations in PI3KCA are generally more sensitive toward VS-5584 treatment. VS-5584 exhibits favorable pharmacokinetic properties after oral dosing in mice and is well tolerated. VS-5584 induces long-lasting and dose-dependent inhibition of PI3K/mTOR signaling in tumor tissue, leading to tumor growth inhibition in various rapalog-sensitive and -resistant human xenograft models. Furthermore, VS-5584 is synergistic with an EGF receptor inhibitor in a gastric tumor model. The unique selectivity profile and favorable pharmacologic and pharmaceutical properties of VS-5584 and its efficacy in a wide range of human tumor models supports further investigations of VS-5584 in clinical trials. Mol Cancer Ther; 12(2); 151–61. ©2012 AACR.

Islet1 (Isl1) belongs to the LIM homeodomain transcription factor family. Its roles in differentiation of motor neurons and organogenesis of pancreas and heart have been revealed. However, less is known about its regulatory mechanism and the target genes. In this study, we identified interactions between Isl1 and Janus tyrosine kinase (JAK), as well as signal transducer and activator of transcription (Stat)3, but not Stat1 and Stat5, in mammalian cells. We found that Isl1 not only forms a complex with Jak1 and Stat3 but also triggers the tyrosine phosphorylation of Jak1 and its kinase activity, thereby elevating the tyrosine phosphorylation, DNA binding activity, and target gene expression of Stat3. In vivo, the tyrosine-phosphorylated Stat3 was colocalized with Isl1 in the nucleus of the mouse motor neurons in spinal cord after nerve injury. Correspondingly, electroporation of Isl1 and Stat3 into the neural tube of chick embryos resulted in the activation of a reporter gene expression controlled by a Stat3 regulatory sequence, and cotransfection of Isl1 and Stat3 promoted the proliferation of the mouse motor neuron cells. Our data suggest a novel role of Isl1 as an adaptor for Jak1 and Stat3 and reveal a possible functional link between LIM homeodomain transcription factors and the Jak-Stat pathway.

Stat3 is activated by cytokines and growth factors via specific tyrosine phosphorylation, dimerization, and nuclear translocation. However, the mechanism involved in its nuclear translocation is unclear. In this study, by systematic deletion and site-directed mutagenesis we identified Arg-214/215 in the alpha-helix 2 region of the coiled-coil domain of Stat3 as a novel sequence element essential for its nuclear translocation, stimulated by epidermal growth factor as well as by interleukin-6. Furthermore, we identified Arg-414/417 in the DNA binding domain as also required for the nuclear localization of Stat3. This sequence element corresponds to Lys-410/413 of Stat1, a reported sequence for Stat1 nuclear translocation. On the other hand, Leu-411 of Stat3, corresponding to Leu-407 of Stat1, a necessary residue for Stat1 nuclear transport, is not essential for Stat3 nuclear import. The mutant of Arg-214/215 or Arg-414/417 was shown to be tyrosyl-phosphorylated normally but failed to enter the nucleus in response to epidermal growth factor or interleukin-6. The defect, however, can be rescued by the wild-type Stat3 but cannot be compensated by these two mutants. Mutations on Arg-414/417, but not Arg-214/215, destroy the DNA binding activity of Stat3. Our data for the first time identified a sequence element located in the coiled-coil domain that is involved in the ligand-induced nuclear translocation of Stat3. This novel sequence together with a conserved sequence element in the DNA binding domain coordinates to mediate the nuclear translocation of Stat3.

Introduction: Janus kinase 2 (JAK2) and fms-related tyrosine kinase 3 (FLT3) are important tyrosine kinases in hematopoietic progenitor cell differentiation and proliferation. Aberrant signaling of JAK2 or FLT3 is implicated in various myeloid malignancies. SB1518, a new chemical entity, is an inhibitor of both JAK2 (IC50 = 22 nM) and its JAK2V617F mutant (IC50 = 19 nM) as well as FLT3 (IC50 = 22 nM) and its mutant FLT3D835Y (IC50 = 6 nM). It is currently being evaluated in Phase I and II clinical trials for advanced hematologic malignancies and is showing promising therapeutic effects. Here we present a pharmacological evaluation of SB1518 in in vitro and in vivo models of myeloid disease that provide a rationale for its selection for clinical development. Methods: The inhibitory effect of SB1518 on JAK2, FLT3 and STAT signaling was evaluated by Western-blot analysis in cell lines with different JAK2/FLT3 mutation status. Cell proliferation assays using the CellTiterGlo® assay were performed on a panel of different cancer cell lines and primary cells from patients. Selected cells were treated with SB1518 to determine effects on apoptosis (Caspase 3/7 activation) and cell cycle (propidium iodide stain). SET-2 cells (a megakaryoblastic cell line derived from an ET-patient bearing JAK2V617F) and MOLM-13 cells (an AML cell line bearing FLT3-ITD) were used to establish subcutaneous tumors in nude mice to test the anti-tumor efficacy of SB1518 after oral daily dosing. Results: SB1518 inhibits phosphorylation of JAK2 at Y221, FLT3 and of STAT proteins and shows anti-proliferative activity in cell lines driven by mutant or wt JAK2 or FLT3-ITD (IC50 range of 47-348 nM), leading to cell cycle arrest (in G0/1) and induction of apoptosis. Cells bearing JAK2 or FLT-3 mutations or genetic alterations leading to high expression of JAK2, are the most sensitive to the anti-proliferative effects of SB1518, as are primary cells isolated from PV or AML patients. In these cells SB1518 induces cell cycle arrest and apoptosis. SB1518 has favorable physicochemical properties and pharmacokinetics allowing oral dosing. It significantly blocks tumor growth in a subcutaneous model of megakaryoblastic AML (SET-2 cells, TGI = 61% at 150 mg/kg b.i.d.). In a model of late stage, aggressive AML (MOLM-13), SB1518 not only blocked tumor growth (TGI = 81%, at 150 mg/kg b.i.d.), but also significantly reduced lung metastasis. In both models SB1518 was very well tolerated and did not induce cytopenia. Conclusions: SB1518, a novel, orally bio-available small molecule inhibitor of JAK2/FLT3, shows promising activities in various hematological cancer cell lines as well as primary cells from patients. It is efficacious in mouse xenograft models of FLT-3 dependent MOLM-13 cells and JAK2-dependent SET-2 cells. SB1518 is currently undergoing evaluation in Phase 2 clinical trials in myelofibrosis (post-PV, post-ET and primary) and has shown encouraging clinical activity. 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 3570. doi:10.1158/1538-7445.AM2011-3570

Introduction: SB939 is an hydroxamic acid based class I, II and IV HDAC inhibitor, with very favourable physiochemical, pharmaceutical and pharmacokinetic properties, resulting in an excellent bioavailability and marked accumulation in tumor tissue. In clinical phase I studies with SB939 in solid tumors, drug exposure was demonstrated to be higher and side effects milder/fewer compared to first generation HDAC inhibitors such as SAHA. Only 6.7% Grade3/4 fatigue (2/30 patients) or 6.9% (2/29 patients) were observed in two independent Phase I clinical studies making SB939 ideally suited for combination therapies. The aim of the present studies was to explore the potential of SB939 for the treatment of hepatocellular carcinoma (HCC). Methods: The effects of SB939 as single agent and combination therapy on the proliferation of HCC cell lines were explored in vitro studies. Single agent and combination therapy together with Sorafenib (Nexavar), a multitargeted VEGFR-b-Raf kinase inhibitor, which is now standard therapy for HCC, was explored in an orthotopic xenograft model of HCC, using Hep3B cells in female SCID mice. Mice were dosed daily with either 10 or 45 mg/kg Sorafenib per oral (p.o.) or with 125 mg/kg SB939 p.o. every other day, alone or in combination with Sorafenib. Sorafenib was dosed first and SB939 dosed 6 h thereafter. Plasma and tumor concentrations of SB939 and Sorafenib were measured using HPLC/MS, analyzed with the MassLynx software and PK parameters were calculated by a non-compartmental method using WinNonlin 4.0 software. Pharmacodynamic (PD) parameters such as HDAC inhibition, inhibition of the Raf-ras MAPK pathway, as well as anti-angiogenic effects of the treatment were analyzed by Western blotting and immunohistochemistry of tumor tissue. Results: HCC cell lines did not show high sensitivity to the anti-proliferative effects of either Sorafanib or SB939 (IC50 values for SB939 ranging from 0.9 to 1.23 μM). In contrast, in the in vivo model of HCC, Sorafenib at a dose of 10 mg/kg had no significant effect on tumor growth (tumor growth inhibition [TGI] was −12.6%), but a dose of 45 mg/kg was very effective (82% TGI). SB939 at a maximum tolerated dose and schedule was well tolerated and moderately effective in arresting tumor grow (40% TGI). When combined with the ineffective lower dose of Sorafenib, the anti-tumor efficacy of SB939 was significantly enhanced (58% TGI), with an in vivo combinatorial index of 0.4, indicating synergy. Conclusion: Although the Hep3B cell line is quite resistant to SB939 in vitro, SB939 is moderately effective as single agent therapy. Anti-tumor efficacy of SB939 was synergistically enhanced in combination with a low and ineffective dose of the VEGFR-Raf tyrosine kinase inhibitor Sorafenib, and the combination was well tolerated, providing a rationale for a phase II trial in combination with Sorafenib in HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5436.

Kinase inhibitors have found applications in multiple oncology settings due to their ability to target key signaling pathways in many different cancers. In general, the broad-spectrum kinase inhibitors have yielded better clinical outcomes than more selective ones because they block more than one pathway critical for tumor growth. We describe herein the pharmacological profile of TG02, a multi-kinase inhibitor being developed by Tragara Pharmaceuticals, which combines cell cycle regulatory and transcriptional CDK inhibition with activity against kinase targets important in the etiology of various leukemias. In vitro potency against the kinases was evaluated using recombinant enzymes with synthetic substrates. Potency on intracellular signaling pathways was assessed by Western blot analyses of the phosphorylated substrates in cell lysates. Functional potency was assessed using proliferation assays performed on human tumor cell lines as well as hematopoietic progenitors expanded from patients' blood. Evaluation of pharmacokinetics, pharmacodynamic biomarkers and anti-tumor efficacy was performed in nude mouse xenograft models of leukemia. TG02 was shown to be a potent inhibitor of several key CDKs (IC50 = 19, 11, 37 & 10nM vs. CDK1, 2, 7 & 9) as well as two other kinases implicated in hematological malignancies, FLT3 and JAK2, with similar potency. The CDK spectrum includes both cell cycle regulatory and transcriptional CDKs. TG02 potently inhibits proliferation across a broad panel of human liquid tumor cell lines (IC50 from 68 to 230 nM). Comparison with reference inhibitors that block only one of the kinase targets of interest demonstrated the significant added benefit of combined CDK and JAK2 targeting. Inhibition of signaling by the CDK and FLT3-JAK2 pathways in leukemia cell lines was detected at the same range (0.10 to 0.35 μM). TG02 was generally cleared from the blood within 8 hours of oral administration but was retained in tumor masses at supratherapeutic levels for 24-48 hours, depending on dose. Accordingly, pathway-related biomarkers were markedly suppressed for 24-72 hours after dosing. In a murine model of FLT3-mutated leukemia (MV4-11), TG02 induced tumor regression after oral daily dosing (40 mg/kg for 21 days). Furthermore, in an orthotopic model of AML with WT FLT3 and JAK2 (HL60), TG02 significantly prolonged median survival time (59 days vs. 40 days) after treatment at 100 mg/kg p.o. 2d on/5d off. Proliferation assays using hematopoietic progenitors expanded from patients with JAK2 mutations or FLT3 wild-type or mutant acute myeloid leukemia revealed higher efficacy of TG02 compared to reference inhibitors targeting only one of the kinases of interest, again demonstrating the benefits of co-targeting CDKs and additional kinases. TG02 is a multi-kinase inhibitor with a previously unreported spectrum of targets that shows promising preclinical activity for the treatment of hematological malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2542.

METHODS: A novel, fully bacterially produced recombinant virus-like particle (VLP) based influenza vaccine (gH1-Qbeta) against A/California/07/2009(H1N1) was tested in a double-blind, randomized phase I clinical trial at two clinical sites in Singapore. The trial evaluated the immunogenicity and safety of gH1-Qbeta in the presence or absence of alhydrogel adjuvant. Healthy adult volunteers with no or low pre-existing immunity against A/California/07/2009 (H1N1) were randomized to receive two intramuscular injections 21 days apart, with 100μg vaccine, containing 42μg hemagglutinin antigen. Antibody responses were measured before and 21 days after each immunization by hemagglutination inhibition (HAI) assays. The primary endpoint was seroconversion on Day 42, defined as percentage of subjects which reach a HAI titer ≥40 or achieve an at least 4-fold rise in HAI titer (with pre-existing immunity). The co-secondary endpoints were safety and seroconversion on Day 21. RESULTS: A total of 84 Asian volunteers were enrolled in this study and randomized to receive the adjuvanted (n=43) or the non-adjuvanted (n=41) vaccine. Of those, 43 and 37 respectively (95%) completed the study. There were no deaths or serious adverse events reported during this trial. A total of 535 adverse events occurred during treatment with 49.5% local solicited symptoms, of mostly (76.4%) mild severity. The most common treatment-related systemic symptom was fatigue. The non-adjuvanted vaccine met all primary and secondary endpoints and showed seroconversion in 62.2% and 70.3% of participants respectively on Day 21 and Day 42. While the adjuvanted vaccine showed an increased seroconversion from 25.5% (Day 21) to 51.2% (Day 42), it did not meet the immunogenicity endpoint. CONCLUSION: In summary, non-adjuvanted gH1-Qbeta showed similar antibody mediated immunogenicity and a comparable safety profile in healthy humans to commercially available vaccines. These results warrant the consideration of this VLP vaccine platform for the vaccination against influenza infection (HSA CTC1300092).