Mark L. Wade

Conditional Expression of 15-Lipoxygenase-1 Inhibits the Selenoenzyme Thioredoxin Reductase MODULATION OF SELENOPROTEINS BY LIPOXYGENASE ENZYMES

The selenoenzyme thioredoxin reductase regulates redox-sensitive proteins involved in inflammation and carcinogenesis, including ribonucleotide reductase, p53, NFκB, and others. Little is known about endogenous cellular factors that modulate thioredoxin reductase activity. Here we report that several metabolites of 15-lipoxygenase-1 inhibit purified thioredoxin reductase in vitro. 15(S)-Hydroperoxy-5,8,11-cis-13-trans-eicosatetraenoic acid, a metastable hydroperoxide generated by 15-lipoxygenase-1, and 4-hydroxy-2-nonenal, its non-enzymatic rearrangement product inhibit thioredoxin reductase with IC50 = 13 ± 1.5 μm and 1 ± 0.2 μm, respectively. Endogenously generated metabolites of 15-lipoxygenase-1 also inhibit thioredoxin reductase in HEK-293 cells that harbor a 15-LOX-1 gene under the control of an inducible promoter complex. Conditional, highly selective induction of 15-lipoxygenase-1 caused an inhibition of ribonucleotide reductase activity, cell cycle arrest in G1, impairment of anchorage-independent growth, and accumulation of the pro-apoptotic protein BAX. All of these responses are consistent with inhibition of thioredoxin reductase via 15-lipoxygenase-1 overexpression. In contrast, metabolites of 5-lipoxygenase were poor inhibitors of isolated thioredoxin reductase, and the overexpression of 5-lipoxygenase did not inhibit thioredoxin reductase or cause a G cell cycle arrest. The influences of 15-lipoxygenase-1 on 1inflammation, cell growth, and survival may be attributable, in part, to inhibition of thioredoxin reductase and several redox-sensitive processes subordinate to thioredoxin reductase.

Abstract 1172: Phase I study of histone deacetylase inhibitor belinostat in combination with warfarin in patients with solid tumors or hematological malignancies.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: The family of Histone Deacetylase (HDAC) enzymes serves as important epigenetic regulators of gene expression through modulation of acetylation on important histone and non-histone proteins. Aberrant acetylation of histones can alter gene expression believed to be important in the tumorigenic process. Belinostat, a potent inhibitor of HDAC proteins has demonstrated anti tumor activity in animal models and in humans. The purpose of this study was to examine the pharmacokinetic and pharmacodynamic properties of warfarin in combination with belinostat and to evaluate the safety profile of belinostat with concomitant warfarin. Methods: Eligible patients enrolled on the study received 5 mg PO warfarin 14 days prior to administration of belinostat. Belinostat was administered as an iv infusion, 1000mg/m2 over 30 minutes for 5 consecutive days every 21 days. On day 3, cycle 1 of belinostat treatment, a second dose of 5mg PO warfarin was administered 2 hours prior to belinostat. Pharmacokinetic blood samples were obtained during cycle 1 of the study to measure warfarin and belinostat metabolism. Toxicities were monitored regularly throughout treatment and response was monitored according to standard of care guidelines. Results: 18 patients, with solid tumors or hematologic malignancies, treated with belinostat and warfarin were included in this analysis. Median age was 55 years (31-77). 11 (61%) patients were male and 7(39%) patients were female. The most common Grade 1 or 2, toxicities observed during the study were anemia (78%), fatigue (72%) and nausea (61%). The most frequent Grade 3 or 4 toxicities were nausea (11%) and hyperuricemia (11%). No Grade 3 or 4 thrombocytopenia or neutropenia were reported. No treatment related Grade 5 toxicities were reported. During cycle 1 no patient experienced treatment delays or discontinued study as result of treatment related toxicity. Conclusion: Belinostat was generally well tolerated in patients with solid tumors or hematologic malignancies with the major toxicity being anemia, fatigue or nausea. Pharmacokinetic results will be presented at the conference. Citation Format: Neeraj Agarwal, Mark L. Wade, Julia Batten, Cynthia Davidson, Show-Li Sun, Sunil Sharma. Phase I study of histone deacetylase inhibitor belinostat in combination with warfarin in patients with solid tumors or hematological malignancies. [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 1172. doi:10.1158/1538-7445.AM2013-1172

Window-of-Opportunity Study of Valproic Acid in Breast Cancer Testing a Gene Expression Biomarker

Purpose The anticancer activity of valproic acid (VPA) is attributed to the inhibition of histone deacetylase. We previously published the genomically derived sensitivity signature for VPA (GDSS-VPA), a gene expression biomarker that predicts breast cancer sensitivity to VPA in vitro and in vivo. We conducted a window-of-opportunity study that examined the tolerability of VPA and the ability of the GDSS-VPA to predict biologic changes in breast tumors after treatment with VPA. Patients and Methods Eligible women had untreated breast cancer with breast tumors larger than 1.5 cm. After a biopsy, women were given VPA for 7 to 12 days, increasing from 30 mg/kg/d orally divided into two doses per day to a maximum of 50 mg/kg/d. After VPA treatment, serum VPA level was measured and then breast surgery or biopsy was performed. Tumor proliferation was assessed by using Ki-67 immunohistochemistry. Histone acetylation of peripheral blood mononuclear cells was assessed by Western blot. Dynamic contrast-enhanced magnetic resonance imaging scans were performed before and after VPA treatment. Results Thirty women were evaluable. The median age was 54 years (range, 31-73 years). Fifty-two percent of women tolerated VPA at 50 mg/kg/d, but 10% missed more than two doses as a result of adverse events. Grade 3 adverse events included vomiting and diarrhea (one patient) and fatigue (one patient). The end serum VPA level correlated with a change in histone acetylation of peripheral blood mononuclear cells (ρ = 0.451; P = .024). Fifty percent of women (three of six) with triple-negative breast cancer had a Ki-67 reduction of at least 10% compared with 17% of other women. Women whose tumors had higher GDSS-VPA were more likely to have a Ki-67 decrease of at least 10% (area under the curve, 0.66). Conclusion VPA was well tolerated and there was a significant correlation between serum VPA levels and histone acetylation. VPA treatment caused a decrease in proliferation of breast tumors. The genomic biomarker correlated with decreased proliferation. Inhibition of histone deacetylase is a valid strategy for drug development in triple-negative breast cancer using gene expression biomarkers.

Abstract P2-10-05: PowerPIINC trial: Changes in tumor proliferation index and quality of life with 7 days of preoperative tamoxifen

BACKGROUND: A decrease in Ki67 has been shown to be a predictor of response to tamoxifen. Previous trials have shown a decreased Ki67 proliferation index in breast tumors with as little as 2 weeks of preoperative tamoxifen. However, shortening the preoperative treatment time in window of opportunity studies increases patient acceptance for trial participation. The POWERPIINC trial examined the effect of 7 days of preoperative tamoxifen on breast tumor proliferation and patient symptoms. METHODS: Adult women with untreated stage I or II invasive breast cancer that was ER positive (>1%) planning on breast surgery with no contraindications to tamoxifen were enrolled. Women received 20mg of tamoxifen for 7 days up to the day of surgery and for 14 days afterwards. Proliferation was assessed by Ki67 immunohistochemistry before and after 7 days of tamoxifen. The proliferation genes from the PAM50 were also assessed by RT-PCR. Symptoms and QOL were assessed by the FACT-ES, MENQoL, and BMQ. RESULTS: 52 women were enrolled, and 44 were evaluable for Ki67. The median age was 58.5 years, and the median tumor diameter was 1.2cm. Most women (73%) were post-menopausal. Most tumors were PR positive (88%). Only 8% of tumors were HER2-positive. The Ki67 decreased by a geometric mean of 40% (95% CI 29%-63%), and 73% (95% CI 57%-85%) of women had tumors with decreased proliferation after 7 days of tamoxifen (p=0.0001 by paired t-test). No correlation was seen between the change in Ki-67 and change in FACT-ES or MENQoL scores. Women reported minimal to no bother from psychosocial or physical symptoms at baseline or on the day of surgery. Expression level of individual proliferation genes did not change after 7 days of tamoxifen. CONCLUSION: Seven days of tamoxifen showed a similar relative decrease in the Ki67 proliferation index as that reported for longer courses. Therefore, short window of opportunity trials can be informative. Citation Format: Cohen AL, Factor RE, Mooney K, Wade M, Serpico V, Salama M, Nelson E, Porretta J, Matsen C, Ostrander E, Bernard P, Boucher K, Neumayer L. PowerPIINC trial: Changes in tumor proliferation index and quality of life with 7 days of preoperative tamoxifen [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-10-05.

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

With the worst five-year survival rate of any cancer, pancreatic adenocarcinomas now rank as the fourth leading cause of cancer death in adults in the United States. This necessitates increased efforts to identify new leads that will serve as therapeutic targets in our fight against this aggressive cancer. Axl is a TAM family receptor tyrosine kinase involved in multiple aspects of tumorigenesis. Increased expression of Axl is associated with increased oncogenic transformation, cell survival, proliferation, migration, angiogenesis, and cellular adhesion. The oncogenic potential of Axl was first discovered in chronic myelogenous leukemia (CML), but it has been demonstrated to play a role in the progression and metastasis of other cancer types. The known ligand for Axl is the Growth Arrest Specific Gene-6 (Gas6) protein and its binding to Axl leads to Axl autophosphorylation and activation of downstream signaling pathways including MAPK and PI3K/Akt pathways. Furthermore, target validation studies of in vivo cancer models show that inhibition of Axl expression by RNAi blocked tumor growth in those models. Taken together, this information makes Axl kinase an exciting target for small molecule drug discovery. Using HCI-2084, a small molecule Axl kinase inhibitor, we explored the effectiveness of targeting the Axl kinase in pancreatic cancer. HCI-2084 demonstrates low nanomolar (IC50 = 12 nM) activity against the Axl kinase in a biochemical assay with good selectivity for Axl when screened in a kinase panel. In 2D and 3D cell proliferation assays, HCI-2084 significantly inhibited pancreatic cancer cell growth at concentrations as low as 30 nM. In pharmacodynamic endpoint assays, HCI-2084 dramatically inhibited Akt signaling (pAKT S473) downstream of GAS6 stimulation in pancreatic cancer cell lines. Inhibition of Axl autophosphorylation by HCI-2084 was also observed in an Axl-transfected cell line system. Consistent with the known function of Axl, HCI-2084 inhibited Gas6-induced migration and invasion of pancreatic cancer cells in vitro. The proteolytic processing of the extracellular domain of the Axl receptor is a known event downstream of Axl activation and results in the release of soluble Axl (sAxl) into the cell culture media (in vitro) or into the blood stream (in vivo). We hypothesized that sAxl levels could function as a biomarker for target inhibition. Indeed, conditioned media from pancreatic cancer cell lines treated with HCI-2084 showed significant dose-dependent reductions in sAxl levels compared to the vehicle treated controls. Taken together, these results suggest Axl is a potential therapeutic target in pancreatic cancer and that HCI-2084 is an exciting agent to potentially treat this disease. HCI-2084 is currently undergoing evaluation in animal efficacy and pharmacodynamic endpoint studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2577. doi:10.1158/1538-7445.AM2011-2577

Abstract 3592: Preclinical combination strategies for development of JI-101, a selective inhibitor of Ephrin B4, VEGF R2 and PDGF-Rβ

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: JI-101 is a novel, highly selective and potent angiogenesis inhibitor with unique EphB4 activity along with VEGFR2 and PDGFRβ inhibitor activity. JI-101 shows a good pre-clinical profile and is well tolerated to date in phase 1 and early phase 2 clinical trials. In order to investigate combination partners, in vitro and in vivo studies were undertaken to combine JI-101 with chemotherapies and various targeted agents. Materials and Methods: Cancer cell lines from different tissue types were seeded on 96 well plates at a density of 2,000 cell per well and allowed to adhere overnight. To determine if JI101 could enhance the toxic effects of traditional chemotherapy, cells were incubated with 10-0.003 μM Gemcitabine, Oxaliplatin and 5-Fluorouricil in the presence or absence of 1 μM JI101. To determine the effect of JI101 with other targeted therapies, cells were incubated with 10-0.03 μM JI101, Axitininb, and Imantinib and 1-0.003 μM BEZ235. In addition, cells incubated with BEZ235 were combined with 1 μM of JI101, Axitininb or Imatinib. Cells were incubated with the drugs for 96 hours at 37oC in a cell culture incubator. After incubation cell viability was tested with the MTS Assay. Results: Toxicity profiles of traditional chemotherapies were not significantly altered when combined with JI101. Incubation of BEZ235 with multiple cancer cell lines resulted in dose dependent toxicity to the cells whereas JI101, Axitinib and Imatinib showed little to no toxicity in the cancer cell lines tested. Coincubation of the PI3 kinase/mTOR inhibitor BEZ235 with JI101, an EphB4, VEGFR2, and PDGF-Rβ or Axitinib a specific VEGF-R2 inhibitor enhanced the cellular toxicity of BEZ235 in a cell line specific manner. Conclusions: JI-101 is highly synergistic with mTOR inhibitors and PI 3 kinase inhibitors in vitro and in vivo. The molecular mechanism will be presented for this synergy. A combination trial of JI101 and PI3 kinase inhibitor or JI101 and mTOR inhibitor is indicated. 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 3592. doi:10.1158/1538-7445.AM2011-3592

Abstract 3609: Homology structure-based design, synthesis and biological evaluation of a series of novel Axl and Mer kinase inhibitors

Axl and Mer kinases belong to the TAM family that was first identified as a transforming gene in chronic myeloid leukemia. Its intracellular region has the typical receptor tyrosine kinase (RTK) structure and its extracellular domain is similar to cadherin-type adhesion molecules in that it is composed of fibronectin type II and immunoglobulin (Ig) motifs. Axl binds various growth factors, with vitamin K-dependent protein growth-arrest-specific gene 6 (GAS6) being the best studied. Axl is involved in mesenchymal and neuronal development as well as in cell survival, adhesion and blood vessel function. High levels of Axl are found in various tumors, including malignant glioma and metastatic colon, ovarian and breast cancers, and it plays a role in cancer invasion. Recent development of Axl inhibitors suggested that inhibition of Axl activity may be impact both tumor angiogenesis and tumor growth. We describe here the design, synthesis, molecular modeling, and biological evaluation of a series of small molecule, inhibitors of Axl and Mer kinases. Our initial lead compound was identified via cross-docking experiments utilizing the homology model of Axl kinase and screening of a diverse in-house chemical library. We subsequently carried out structure-activity relationship studies and optimized the lead structure which has 2 to 6.1 μM inhibition activity to 2-fold improvement in the Axl and Mer kinase activities to 730 nM. As a starting point for further optimization, it was considered that the modest of these series could be improved by introducing a spacer at the aryl piperazine moiety and focused on maximizing the in vitro potency, addressing the SAR and molecular properties. We further explored Leu620, hydrophobic, Gly543, Phe547 and DFG motif sites through scaffold hoping lead to the identification of an electron-withdrawing functional groups lead to the compound HCI-2084 and HCI-2091 which exhibited potent Axl kinase inhibition activity of 7 and 12 nM in Axl kinase assay and 30-50 nM is panel of cancer cell lines. These novel series of compounds were synthesized in two-step procedure via a standard cross-coupling reaction or with various substituted aryl amine derivatives under amination conditions. In a subsequent step we employed Buchwald-Hartwig amination reaction to prepare target molecules. Due to the ready synthesis accessibility, we extensively investigated the SAR improving the RO5, solubility and permeability parameters. The details of these results will be presented. 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 3609. doi:10.1158/1538-7445.AM2011-3609