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RAS mutations are highly prevalent in tumors, especially those of the lung, colon, pancreas and cutaneous melanoma. The tissues in which these tumors arise are enriched for specific RAS isoform and codon preferences. Mutations in KRAS are prevalent in lung, colon and pancreas. While NRAS mutations are predominately found in cutaneous melanoma. Generally, mutations in KRAS occur at codon 12 and NRAS mutations at codon 61. Independent of the RAS isoform, the mutated codon contributes to distinct biochemical differences. Codon 61 mutations are largely viewed to be more biochemically active than codon 12 mutations. Previous RAS-mutant genetically engineered mouse models (GEMMs) resembled the tissue-specificity observed in patients. In the colon, expression of mutant Kras (G12D, G12C, G12R, G13D, A146T) but not Nras (G12D) induced colonic hyperplasia. The degree of colonic hyperproliferation was dependent on the specific Kras mutation. In terms of Nras, melanocytes expressing NrasQ61R, but not NrasG12D, led to the development of melanoma. These data suggest the specific Ras mutation contributes to the strength in signaling in both isoforms. Given these data, we aimed to understand the preference for codon 61 mutations in Nras and to determine if increased biochemical activity from a codon 61 mutation led to accelerated tumorigenesis in Kras. To achieve this, we generated four conditional knock-in GEMMs to directly compare Kras and Nras mutations at codons 12 and 61; G12D and Q61R. To robustly determine the effects of RAS-mutant expression across a wide-range of tissues, we utilized the ubiquitous and temporally expressed Cre, Rosa26-CreERT2. Induction of mutant Kras resulted in rapid tumorigenesis compared to Nras. Moreover, KrasQ61R-mutant mice had reduced survival compared to KrasG12D. Expression of KrasG12D and KrasQ61R drove hyperproliferation in both lung and colon tissues, but to varying degrees. While we observed colonic hyperplasia in both Kras-mutant mice, KrasQ61R expression dramatically reduced the number of goblet cells in the colonic epithelium. This phenotype is distinct from the published Kras mouse models and highlights an unmet understanding of KRASQ61-mutant colorectal cancers (CRC); contributing to roughly 5% of all KRAS-mutant CRCs. In the lung, both Kras-mutant mice developed lung adenomas but KrasQ61Rexpression increased the size and number of the adenomas. Neither Nras-mutant models had an effect on the colon or lung tissues. However, all Ras-mutant GEMMs developed myeloproliferative disorders (MPD). Notably, leukemias and MPDs frequently have mutations in KRAS and NRAS at either codon 12 or 61. Our GEMMs recapitulate the tissue-specificity of RAS mutations observed in patients and highlight the unique oncogenic activity of KrasQ61R. Citation Format: Amanda R. Moore, Jason Liang, Robert Piskol, Lisa McGinnis, Donald S. Kirkpatrick, Shiva Malek. RAS-mutant mouse models confirm tissue-specificity and reveal unique oncogenic activity of KrasQ61R [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2923.

Background: Seviteronel (Sevi), a CYP17-lyase (L) inhibitor (reduces testosterone (T) and estradiol (E2) biosynthesis) and a competitive AR antagonist, has activity in castration resistant prostate cancer at a dose of 600mg nightly. Sevi potently inhibits the growth of ER(+)/AR(+) MCF7, tamoxifen-resistant (TAMR) MCF7, and ER(-)/AR(+) MDA-MB-453 cells. In a TAMR xenograft BC model, Sevi decreases tumor growth greater than enzalutamide (Enza), an AR antagonist (Ellison et al, SABCS 2015). Nearly all subtypes of BC, including AR(+) TNBC, are potential targets for Sevi based on its mechanism of action (MOA). Phase (Ph) 1 of this study established the recommended Ph 2 dose (RP2D) of Sevi in women with BC as 450mg once nightly, based upon preliminary tolerability and pharmacokinetics (PK) (Bardia et al, ASCO 2016). The primary objective of Ph 2 is to estimate the activity of Sevi, as measured by clinical benefit rate (CBR) at 16 and 24 weeks (wks) for AR(+) TNBC and ER(+) BC, respectively. The secondary objectives include an estimation of Sevi tolerability and pharmacodynamics (PD) (NCT02580448). Methods: Women with advanced AR(+) TNBC (stratified by prior Enza use) or ER(+) BC were enrolled using 3 parallel Simon's 2-stage designs powered to evaluate CBR. ER(+) BC patients must have had ≥1 prior line of endocrine therapy; no limit to prior treatment for TNBC. AR(+) status was confirmed using central IHC analysis in all patients, with a ≥10% tumor cell nuclear staining cutoff for evaluable TNBC patients. Sevi was administered once nightly with dinner at 450mg (28d cycle). Tumor and blood samples were collected for PK and PD analysis (circulating tumor cells, ctDNA, sex steroids). Response was assessed every 8 wks for 52 wks, then every 12 wks thereafter. Current tolerability and PD results are presented herein for this ongoing Ph 2 study. Results: As of June 7, 2016, 17 patients received Sevi at 450mg nightly between Ph1 and Ph2 with 10 in screening. 14 patients are currently on study in Cycles 1-6. The most common adverse events (AEs > 10% regardless of causality or grade) were tremor (24%), pain (18%), fatigue (18%) and dyspnea (18%), nausea (12%), AST increase (12%), ALT increase (12%) and abdominal pain (12%), all of which were Grade 1 or 2 except for Grade 3 dyspnea (n=1; unrelated). No dose reductions were reported and there were no drug-related discontinuations. Nine patients underwent central AR testing (4 AR(+) of 6 TNBC; 3 AR(+) of 3 ER(+) BC). Median AR tumor cell nuclear staining was 90% (15-100%). Preliminary sex steroid analyses from 6 Ph 1 patients receiving Sevi at 450, 600, or 750mg nightly (n=2 at each dose) for 1 cycle showed a median decline in E2 concentration of 52% (-29 to -87%) to 12.4pmol/L (4 to 33pmol/L) from baseline. There was a similar magnitude of decline for T. Conclusions: Sevi was well-tolerated at 450mg nightly with exposures similar to the RP2D in men (600mg nightly). The CYP17-L inhibition activity of Sevi was demonstrated with an early and potent reduction in E2 and T. Sevi's unique CYP17-L and AR antagonist MOA may provide a new novel treatment option for AR(+) TNBC or ER(+) BC. Citation Format: Gucalp A, Bardia A, Gabrail N, DaCosta N, Danso M, Elias AD, Ali H, Lemon SJ, Riley EC, Eisner JR, Fleming RA, Kurman MR, Moore WR, Traina TA. Phase 1/2 study of oral seviteronel (VT-464), a dual CYP17-lyase inhibitor and androgen receptor (AR) antagonist, in patients with advanced AR positive triple negative (TNBC) or estrogen receptor (ER) positive breast cancer (BC) [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 P2-08-04.

VT-464 is a lyase-selective inhibitor of the dual-activity CYP17A1 enzyme that is required for the synthesis of androgens and estrogens in the gonads, adrenals, and tumors. In addition to its role as a CYP17A1 lyase inhibitor, we previously showed that VT-464 also functions as a direct and effective AR antagonist in prostate cancer models. In our current study, we evaluated the therapeutic potential of VT-464 in breast cancer by analyzing its effectiveness in several AR-positive breast cancer cell lines, including those that are ER-positive and ER-negative. Through in vitro assays and xenograft analysis, we compared the activity of VT-464 to enzalutamide, a second generation AR antagonist that is approved for the treatment of castration resistant prostate cancer and is in multiple Phase 2 breast cancer studies. Our results showed that VT-464 was highly effective in preventing proliferation of both ER-positive and ER-negative breast cancer cell lines in vitro. Importantly, significant inhibition was also observed in soft agar assays that assesses anchorage-independent growth. In mechanistic studies, VT-464 and enzalutamide both inhibited induction of AR target genes and recruitment of AR to target promoters, verifying direct AR antagonistic activity observed previously in prostate cancer cells. Furthermore, we evaluated the ability of VT-464 and enzalutamide to inhibit tumor formation in a tamoxifen-resistant model of breast cancer. Oral administration of either enzalutamide or VT-464 significantly decreased tumor growth in mice, with VT-464 achieving greater growth inhibition than enzalutamide. Together, these data provide further rationale for the future study of the AR as a viable therapeutic target in breast cancer, and importantly, suggest that AR inhibition can impact tamoxifen-resistant tumor growth. The dual effects of CYP17A1 lyase inhibition and AR antagonism that are achieved with VT-464 further supports its development as an effective oral therapy option for AR-positive breast cancer. A phase 1 / 2 clinical study of oral VT-464 in women with AR+ triple-negative breast cancer or ER+ cancer resistant to aromatase inhibitors will commence in 2015. Citation Format: Ellison SJ, Norris JD, Wardell S, Eisner JR, Hoekstra WJ, Stagg DB, Alley HM, Moore WR, McDonnell DP. Effects of the dual selective CYP17 lyase inhibitor and androgen receptor (AR) antagonist, VT-464, on AR+ and ER+ tumor models in vitro and in vivo. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-04.

The purpose of this study was to determine the role of BAP1 levels in cutaneous melanoma (CM). BAP1 is a tumor suppressor in which loss of heterozygosity (LOH) from mutation and copy number alteration is well described in germline and somatic cancers. Although BAP1 genomic alterations in CM are extremely rare (2% of 665 samples from 5 datasets), marked variability in BAP1 expression is observed in CM. We show that low nuclear BAP1 levels portend a significantly worse clinical outcome in stage III CM (n=37, log rank p ≤0.01 for both overall survival and progression-free survival). Gene Set Enrichment Analysis (GSEA) revealed low BAP1 expression to be most highly ranked with an increased epithelial–mesenchymal transition (EMT) gene expression profile in CM tumors (n=379, FDR q = 1.34E-26) and cell lines (n=53, FDR q = 2.86E-116). We identify the expression of ZEB1, a master regulator of EMT, to be significantly associated with low BAP1 expression in CM tumors and cell lines (p= 1.5E-04 and 3.3E-05, respectively). Analysis of the BAP1 promoter indicates three canonical ZEB1 binding sites. Functional experiments show ZEB1 to bind to the BAP1 promoter, and luciferase activity assays indicate that ZEB1 acts as a transcriptional suppressor of BAP1 expression with differential utilization of the promoter binding sites. Targeted reduction of endogenous ZEB1 caused increased BAP1 levels, while targeted reduction of BAP1 did not modulate ZEB1 levels, consistent with ZEB1 having a suppressive effect on BAP1. Phenotypically, targeted reduction of BAP1 in CM cells resulted in a switch from a more differentiated, melanocytic state, to a less differentiated, more migratory and invasive phenotype. Extinguishing melanocyte-specific BAP1 in mice with a BRAF V600E mutant genetic background resulted in the emergence of primary melanoma tumors, with a marked EMT gene expression profile, and resultant metastases. Given the phenotypic changes associated with BAP1 levels in our mouse and human studies, we then tested the effect of modulating BAP1 on BRAF targeted therapy. Exogenous expression of BAP1 sensitized BRAF inhibitor (vemurafenib)-resistant melanoma cells, while the targeted reduction of BAP1 desensitized BRAF inhibitor-sensitive melanoma cells. BRAF mutant/BAP1 loss mice failed to exhibit a marked response to vemurafenib treatment compared to control mice. These data implicate regulation of BAP1 to be a major mechanism that characterizes a highly malignant and treatment-resistant subset of tumors. Our study indicates that nongenomic reduction in BAP1 through ZEB1 transcriptional modulation may be a key factor in aggressive CM. This abstract is also being presented as Poster A30. Citation Format: Haifeng Zhu, Junna Oba, Xiaoxing Yu, Caitlin A. Creasy, Marie-Andrée Forget, Fernando Carapeto, Cara L. Haymaker, Chang-Jiun Wu, Tatiana V. Karpinets, Wei-Lien Wang, Michael T. Tetzlaff, Alexander J. Lazar, Gordon B. Mills, Amanda R. Moore, Yu Chen, Jianhua Zhang, Jeffrey E. Gershenwald, Jennifer A. Wargo, Chantale Bernatchez, Patrick Hwu, P. Andrew Futreal, Scott E. Woodman. Nongenomic BAP1 aberrancy drives highly aggressive cutaneous melanoma phenotype [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR03.

Uveal melanoma (UVM) is the most common intraocular malignancy, which represents about 5% of all melanoma cases per year in the United States. Half of all patients eventually develop metastatic disease, resulting in an average survival rate of six months and a five-year survival rate of ~15%. Recent studies have revealed the underlying genetic landscape of uveal melanoma but have not resulted in any therapeutic options to effectively treat UVM. UVMs harbor mutually exclusive activating mutations in GPCR signaling: ~90% in G-protein alpha q (Gq) subunits GNAQ/GNA11, ~5% in the GPCR CYSLTR2, and ~4% in a downstream effector. These mutations aberrantly activate canonical PLCβ signaling: cleavage of PIP2 into DAG and IP3, both of which lead to PKC activation. In addition to uveal melanoma, recent studies have shown this exact pathway to be activated in a subset of cutaneous and mucosal melanomas as well as other diseases like blue nevi, leptomeningeal melanocytic neoplasms (LMNs), and Sturge-Weber syndrome. My project focuses on understanding how Gq signals in UVM tumorigenesis and if Gq inhibition has therapeutic potential in UVM by addressing the following aims: 1) Using a highly specific Gq inhibitor, I will ask how Gq inhibition effects different activating mutations in UVM using representative in vitro models. I have generated melanocytes dependent on different mutations in the PLCβ pathway to study pathway inhibition and therapeutic efficacy. Based on my preliminary data I will also interrogate the mechanistic basis for Gq inhibition sensitivity in GNAQQ209L driven melanocytes. 2) Determining clinical potential of this drug by testing its pharmacologic properties in vivo. Using the melanocytes I have generated along with UVM cell lines and our GEMM, we will be able to thoroughly test the drug’s efficacy against a variety of xenografts. 3) Taking advantage of the ability to inhibit active Gq, I will aim to identify other pathways activated downstream of Gq using proteomic and transcriptomic approaches. This may provide alternative therapeutic strategies to target this recalcitrant disease. The outcome of this research will indicate the effectiveness of Gq inhibition in UVM as well as elucidate currently unknown signaling pathways essential for UVM tumorigenesis. Citation Format: Tyler D. Hitchman, Emilie Ceraudo, Amanda R. Moore, Alexander N. Shoushtari, Jasmine H. Francis, Youxin Guan, Juliet Chen, Matthew T. Chang, Barry S. Taylor, Thomas P. Sakmar, Thomas Huber, Ping Chi, Yu Chen. Activating mutations in uveal melanoma convey sensitivity to g-alpha q inhibition [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A07.

RAS mutations are highly prevalent in tumors, especially those of the lung, colon, skin, and pancreas. The tissues in which these tumors arise are enriched for specific RAS isoform and codon preferences. For example, mutations in KRAS are prevalent in lung, colon, and pancreas, while NRAS mutations are predominantly found in melanoma. Additionally, mutations in KRAS predominantly occur at codons 12 and NRAS mutations at codon 61. Independent of the RAS isoform, the mutated codon contributes to distinct biochemical differences, with codon 61 mutations largely viewed as more biochemically active than codon 12 mutations. Previous RAS-mutant genetically engineered mouse (GEM) models resembled the tissue specificity observed in patients. In the colon, expression of KrasG12D but not NrasG12D induced colonic hyperplasia. While in melanocytes, the expression of NrasQ61R, but not NrasG12D, led to melanoma development. Given these data, we developed RAS-mutant GEM models to identify the tissues susceptible to RAS mutations and the differences among these animal models. To achieve this, we generated four conditional knockin GEM models to directly compare Kras and Nras mutations in codons 12 and 61; G12D and Q61R. To robustly determine the effects of mutant expression across a wide range of tissues, we utilized the ubiquitous and temporally expressed Cre, Rosa26-CreERT2. Following the induction of recombination by tamoxifen treatment, we observed rapid mortality in Kras-mutant animals. KrasQ61R mice survived to a median of 13 days while KrasG12D mice survived to a median of 22 days post-tamoxifen. However, the Nras-mutant mice have extended survival. While ongoing, these survival data suggest tissues acutely sensitive to Kras-mutant expression can tolerate or are resistant to Nras-mutant expression. When comparing the two Kras conditional knockin GEM models, we found expression of KrasG12D and KrasQ61R drove hyperproliferation in both lung and colon tissues to varying degrees and different tissue types in the colon. For instance, we observe colonic hyperplasia in KrasG12D mice and polypoid hyperplasia in KrasQ61R mice. Interestingly, the hematopoietic cells were exquisitely sensitive to expression of KrasQ61R, and these animals rapidly developed acute myeloid leukemia leading to death. These data illustrate how the strength of the constitutive activation of RAS proteins drives differential tumorigenesis. We plan to further our understanding of the tissue-specific tumorigenesis of mutant-RAS proteins by comparing the Kras- and Nras-mutant GEM models. This abstract is also being presented as Poster A01. Citation Format: Amanda R. Moore, Lisa McGinnis, Shiva Malek. Modeling the tissue-specific oncogenesis of mutant RAS [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PHA01.

Background: Castration-resistant prostate cancers that express the constitutively active androgen receptor (AR) variant AR-V7 exhibit significantly reduced progression-free and overall survival in response to the AR targeting agents, enzalutamide (ENZ) and abiraterone. Inhibition of Phosphoinositide-3-Kinase (PIK3CA) or CDK4/6 has been proposed as alternative therapies that directly target the cell cycle pathway in these tumors. However, the combined role of AR plus cell cycle inhibition in this treatment setting has not been established. G1T38 is a potent, selective clinical stage CDK 4/6 inhibitor. Seviteronel (SEVI) is a selective CYP17-lyase and AR inhibitor that blocks the growth of CRPC tumors with clinically relevant AR mutations, including T878A and AR-F876L. The activity of SEVI, ENZ and G1T38, alone or in combination, was evaluated using the 22Rv1 (AR-V7+) CRPC model in vitro and in vivo. Methods: Cellular proliferation (7 days) of 22Rv1 prostate cancer cells treated with SEVI, ENZ, and G1T38 alone or in combination with SEVI or ENZA was assessed by measuring DNA content. Orchiectomized male nu/nu mice bearing 22Rv1 were randomized to receive vehicle, SEVI (150mg/kg/day p.o.), ENZ (30 mg/kg/day p.o.), G1T38 (25-100 mg/kg/day p.o.), or SEVI or ENZ in combination with G1T38. A docetaxel (20 mg/kg weekly i.p.) group was included as a standard of care comparison. Time to progression (X-fold increase in tumor volume) and tumor volume changes were assessed over a 3-7-week dosing period. Blood and tissues were collected for analysis of exposure and pharmacodynamics markers of response. Results: SEVI and G1T38 alone or in combination, significantly reduced the proliferation of 22Rv1 cells in vitro (p Conclusions: SEVI and G1T38 mono- or combination therapy significantly decreased the growth of the AR-V7+ 22Rv1 CPRC model in vitro and in vivo, an activity that distinguishes SEVI from ENZ. Therefore, combining SEVI with a CDK4/6 inhibitor such as G1T38 may be an effective therapy for the treatment of CRPC that is resistant to current standards of care. Citation Format: Suzanne E. Wardell, Alexander P. Yllanes, John D. Norris, James P. Stice, Hannah White, Ronald A. Fleming, Jay C. Strum, William R. Moore, Donald P. McDonnell. Effects of the selective CYP17-lyase and androgen receptor (AR) inhibitor, seviteronel, and the cyclin-dependent kinase (CDK) 4/6 inhibitor, G1T38, on tumor growth in an AR-V7+ castration-resistant prostate cancer (CRPC) xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1588. doi:10.1158/1538-7445.AM2017-1588

Pancreatic ductal adenocarcinoma (PDA) is a highly recalcitrant disease. One factor contributing to chemoresistance is thought to be the desmoplastic response seen in these tumors. Fibronectin (FN) is a major constituent of the desmoplastic reaction of PDA. It has been shown that integrin activation by fibronectin can induce reactive oxygen species (ROS) production by stromal cells. Increased ROS has been observed in many cancers, including PDA. Therefore, we propose that oxidation therapy is an attractive approach to increase chemoresponse in PDA as this strategy takes advantage of the sensitivity of these tumors to further ROS insults. We discovered that the matricellular protein Fibulin5 (Fbln5) is upregulated in PDA and reduces FN-mediated integrin-induced ROS production by competing with FN for binding to α5β1 integrin and blocking integrin-induced ROS production. Mutation of the RGD-integrin binding domain in Fbln5 to RGE abolishes the binding of Fbln5 to the integrin and increases integrin-induced ROS production. Fbln5RGE/RGE (RGE) mice, display reduced tumor growth in implant and genetic models of PDA (KIC and KPC) as a direct consequence of elevated ROS levels in the tumor microenvironment. Furthermore, ROS induction correlates with reduced angiogenesis in tumors from RGE animals and had an additive therapeutic effect when combined with standard therapy. We have identified multiple oxidative stress-responsive genes that are elevated in stromal fibroblasts isolated from tumors from RGE animals. One of the most promising targets is NAD(P)H dehydrogenase (quinone 1) (Nqo1). In support of this observation we found that the level of Nqo1 is significantly higher in RGE fibroblasts compared with WT fibroblasts when plated on FN but not collagen or plastic. Furthermore we have demonstrated that FN-mediated induction of ROS and Nqo1 is dependent on β1 integrin and 5-lipooxygenase (5-LOX). Our studies reveal a unique integrin-based mechanism to enhance ROS production to a level that is specifically toxic to the tumor by exploiting the tumor-specific expression of Fbln5. Current studies are focused on understanding the mechanism by which Fbln5 is induced in PDA. Citation Format: Mary Topalovski, Miao Wang, Zachary Moore, David Boothman, Rolf Brekken. Fibulin-5 supports pancreatic tumor growth through inhibition of integrin-induced ROS. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5093.

Atypical Teratoid/ Rhabdoid Tumor (ATRT) is an aggressive cancer that accounts for up to 20% of infant brain tumors. Even with intense chemotherapy and radiation, approximately 75% of children die from their disease at a median of 10 months after diagnosis. We found that the pathognomonic genetic lesion of ATRTs and related malignant rhabdoid tumors (MRTs), loss of the SWI/SNF chromatin remodeling complex member INI1/hSNF5/SMARCB1, is associated with high expression of NAD(P)H:quinone oxidoreductase 1 (NQO1), a two-electron oxidoreductase. Thus, we hypothesized that ATRTs will be vulnerable to drugs such as ß-lapachone (ß-lap, ARQ761 in clinical form) that selectively kill NQO1-expressing cells via mu-calpain mediated cell necrosis. A panel of ATRT cell lines exhibited overall high NQO1 expression and sensitivity to ß-lap (IC50 1.6-4 uM). A two-hour pulse with ß-lap caused a burst of ROS-mediated DNA damage and PARP1 hyperactivation, resulting in NAD+ and ATP depletion. Lack of NQO1 expression or inhibition of NQO1 with dicoumarol abrogated sensitivity to ß-lap. Low expression of NQO1 in normal brain and other pediatric tissues suggests a wide therapeutic window for ß-lap, and a survival advantage was observed after treatment in a heterotopic mouse xenograft model with several mice achieving apparent cures. Furthermore, the mechanism of ß-lap results in synergy with standard of care agents such as ionizing radiation, increasing its potential for clinical applications. Citation Format: Julia C. Meade, Zachary R. Moore, Sarai H. Stuart, Agnieszka Cholka, Richard L. Boriack, Jingying Xu, Anat Erdreich-Epstein, Dinesh Rakheja, David A. Boothman, James F. Amatruda, Theodore W. Laetsch. NQO1 as a therapeutic target for atypical teratoid/ rhabdoid tumor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B39.

VT-464, an oral dual lyase-selective CYP17 inhibitor and AR antagonist (wild-type and mutated forms [e.g., F876L and T877A]), is in multiple Phase (Ph) 2 studies as treatment for men with castration-resistant prostate cancer (CRPC). VT-464 inhibits the growth of multiple BC cell lines in vitro including MCF7 (ER(+)/AR low), tamoxifen-resistant MCF7, and MDA-MB-453 (ER(-)/AR(+)) in a dose-dependent manner and with greater potency/efficacy than enzalutamide (submitted, Ellison et al., 2015). A subset of TNBC and most ER(+) BC express AR, making them potential targets for VT-464 since it directly inhibits both androgen/estrogen synthesis and AR transcriptional activity. Objectives: The primary objective of Ph 1, now enrolling, is to establish the once-daily dose of VT-464 in women. Secondary objectives include safety, PK and efficacy endpoints, including determination of clinical benefit rate (CBR) which is the primary objective of Ph 2. Exploratory objectives include the determination of the extent of AR expression and signaling in breast tissue and to evaluate the relationship of expression with VT-464 effects on circulating tumor biomarkers, circulating hormones and clinical outcomes. Study Design: This study is an open-label, single arm, Ph 1/2 study of VT-464 in women with AR(+) TNBC or ER(+)/HER2 normal unresectable locally advanced or metastatic BC. Ph 1 will follow a modified 3+3 Fibonacci design with cohort expansion to 6 patients following a single DLT in the first 28-days of treatment. Approximately 2-3 dose-levels will be explored in Ph 1. Ph 1 start dose will be the MTD for men with CRPC. AR(+) TNBC and ER(+)/HER2 BC cohorts will be expanded in Ph 2 using the MTD from Ph 1. Ph 2 will follow a Simon's two-stage design with pre-determined futility parameters. Eligible patients will have ER≥1% BC or AR≥1% (as determined by central IHC testing using the Dako antibody) TNBC. ER(+) patients must be postmenopausal and must have received at least 1 prior line of endocrine therapy. Additional eligibility criteria include: ≥ 18 years of age, ECOG PS ≤ 1, unresectable locally advanced or metastatic BC, available representative tumor specimen to enable correlative science. Treatment Plan: Eligible patients will receive VT-464 once-nightly with dinner in a continuous dosing schedule. Adverse events and concomitant medications will be collected from the time of signing of informed consent until 30 days after end of study visit (EOS). Safety labs will be monitored monthly through EOS. Dense PK will be collected after the first dose of study drug in Ph 1 and single morning samples collected approximately every two cycles thereafter in Ph 1 and Ph 2 until EOS. Blood samples for steroids, circulating tumor DNA and circulating tumor cells will be collected through Cycle 2 and then at EOS. Tumor biopsy will be collected at baseline and at disease progression. Radiographic response will be assessed every 8 weeks and EOS. Patient Accrual: Accrual is ongoing with 12-18 patients expected to be enrolled in Ph 1. Citation Format: Gucalp A, Hudis C, Norton L, Patil S, Kurman MR, Eisner JR, Moore WR, Traina TA. A phase 1/2 study of once-daily oral VT-464 in patients with advanced androgen receptor (AR) positive triple negative (TNBC) or estrogen receptor (ER) positive breast cancer (BC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT2-01-03.

Macrophage-induced bystander effects have been implicated as an important mediator of chromosomal instability and colon cancer triggered by Enterococcus faecalis, a human intestinal commensal bacteria. There is little understanding about how inflammatory cytokines mediate bystander effects, but questions in this area are important because of the pivotal contributions made by inflammatory processes to cancer initiation and progression. Here, we report that the central proinflammatory cytokine TNF-α acts as a diffusible mediator of the bystander effects induced by macrophages, an effect caused by a proliferation of macrophages that trigger epithelial cell production of Netrin-1, a neuronal guidance molecule. TNF-α-mediated bystander assays used a murine coculture system of primary colonic epithelial cells and E. faecalis-infected macrophages (in vitro), with an interleukin 10 (IL-10)-deficient mouse model of colon cancer that involves long-term colonization with E. faecalis (in vivo). In cell cocultures, we observed increased expression of the TNF-α receptor Tnfrsf1b and Netrin-1. These effects were blocked by anti-TNF-α antibody or by pretreatment with an inhibitor of NF-κB signaling. RNAi-mediated attenuation of Tnfrsf1b decreased TNF-α-induced netrin-1 production and augmented epithelial cell apoptosis in culture. Extending these observations, colon biopsies from E. faecalis-colonized IL-10−/− mice exhibited crypt hyperplasia and increased staining for macrophages, TNF-α, netrin-1, NF-κB, Tnfrsf1b, and the proliferation marker proliferating cell nuclear antigen while also displaying a reduction in epithelial cell apoptosis. Together, our results define a pathway for macrophage-induced bystander effects in which TNF-α triggers TNFRSF1b receptor signaling leading to increased production of Netrin-1, crypt hyperplasia, and decreased epithelial cell apoptosis. In elucidating an important commensal-associated proinflammatory mechanism in the intestinal microenvironment, our work highlights the role of Netrin-1 and a specific TNF-α receptor as candidate targets to prevent or treat colorectal cancer. Cancer Res; 72(20); 5219–29. ©2012 AACR.

Atypical teratoid rhabdoid tumors (ATRTs) are aggressive malignant neoplasms that occur in the CNS, most commonly in children 15 uM). A two-hour pulse with ß-lap caused a burst of ROS-mediated DNA damage and PARP1 hyperactivation, resulting in NAD+ and ATP depletion and caspase-independent, mu-calpain-mediated apoptosis. Co-treatment with dicoumarol, a specific inhibitor of NQO1, prevented these effects by blocking the NQO1-dependent bioactivation of ß-lap. Low expression of NQO1 in normal brain and other pediatric tissues suggests a wide therapeutic window for ß-lap. These findings encourage the continued study of ß-lap and other NQO1 bioactivated therapeutics for the treatment of this recalcitrant disease. This work was supported by NIH/NCI R01 CA102972 to DAB and a REACH award from the Alex's Lemonade Stand Foundation to JFA and TWL. Citation Format: Zachary R. Moore, Sarai Stuart, Agnieszka Cholka, Ashwin Venkataraman, Jingying Xu, Anat Erdreich-Epstein, Dinesh Rakheja, David A. Boothman, James F. Amatruda, Theodore W. Laetsch. β-lapachone: a novel targeted therapy for atypical teratoid rhabdoid tumors (ATRTs). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1613. doi:10.1158/1538-7445.AM2015-1613

Metabolic adaptations in tumors are necessary to balance the need for cellular energy supply, macromoleclar biosynthesis and maintenance of redox balance. A critical molecule produced as a result of this altered metabolism is nicotinamide adenine dinucleotide phosphate (NADPH). NADPH is a critical co-factor that provides reducing power to maintain cytoprotective concentrations of reduced glutathione against reactive oxygen species (ROS) that are produced during rapid growth and proliferation. In pancreatic cancers, NADPH can be formed by the NAD+ salvage pathway through the enzyme, Nicotinamide phosphoribosyltransferase (NamPT), as well as through the non-canonical metabolism of glutamine through glutaminase-1 (GLS1). While NamPT and GLS1 inhibitors have shown some anti-tumor activity in vitro and in vivo, these inhibitors lack tumor specificity and can have an extensive toxicity profile due to the requirement of NADPH synthesis in proliferating normal cells/tissue. We recently discovered that NAD(P)H:quinone oxidoreductase 1 (NQO1) levels were elevated 5- to 40-fold in >80% of pancreatic tumors vs associated normal tissue. As important, we noted that catalase levels were inversely expressed comparatively, elevated in normal and suppressed in tumor tissue. Thus, NQO1 represents a unique target to exploit for the therapeutic elimination of pancreatic cancers. While NQO1 is normally a detoxifying (Phase II) enzyme, we have discovered two unique classes of compounds that are ‘bioactivated’ in a futile redox cycle by the enzyme. NQO1 bioactivatable drugs, such as β-lapachone (β-lap) and deoxynyboquinone (DNQ), generate hydrogen peroxide as a mechanism to hyperactivate poly(ADP-ribose) polymerase 1 (PARP1), and to rapidly deplete NAD+ and ATP in a selective manner to kill tumors. Normal cells are protected due to low NQO1 and relatively high Catalase levels. We found that the tumor specificity and efficacy of NamPT or GLS1 inhibition can be greatly increased when small molecule NamPT inhibitors (e.g., FK866) or GLS1 inhibitors (e.g., BPTES) are used in combination with NQO1-bioactivatable drugs. With combination treatment, cells are primed for ROS-mediated damage due to reduced glutathione and NADPH levels; they are unable to recover ATP, NAD+, and NADPH synthesis even after only a 2 h exposure to NQO1 bioactivatable drugs, and they rapidly die through a caspase-independent, parthanatos (programmed necrosis) mechanism. Our findings indicate that NQO1-bioactivatable drugs may improve the sensitivities of clinical grade GLS1 and NamPT inhibitors in pancreatic cancer patients. This work was supported by an AACR/Pancreatic Cancer Action Network - George and June Block Foundation - Innovator Award and an NIH/NCI R01 CA102971 grant to DAB. Citation Format: Zachary Moore, Gaurab Chakrabarti, Costas Lyssiotis, Ralph Deberardinis, Boothman A. David. Modulating the NQO1-dependent ‘kiss of death’ mechanism of action of NQO1 bioactivatable drugs. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B47.

The major problem plaguing DNA repair and metabolic inhibitors is their lack of tumor-selectivity, resulting in unwanted toxicities. In contrast, NAD(P)H:quinone oxidoreductase 1 (NQO1) bioactivatable drugs (e.g., ß-lapachone (ß-lap) and deoxynyboquinine (DNQ) and their derivatives) offer exquisite tumor-selectivity, with one form of the drug, ARQ761, currently in Phase I clinical trials against solid cancers, most of which have elevated levels of NQO1; pancreatic and nonsmall cell lung cancers (NSCLC) have the highest levels of this two-electron oxidoreductase. NQO1 levels are elevated during early steps of carcinogenesis in most solid cancers in response to activated K-Ras, c-Myc or other oncogenic drivers, as well as re-expression of human telomerase (hTERT). NQO1 catalyzes an unique futile redox cycle using either of these two unique classes of NQO1 bioactivatable drugs, resulting in super-lethal doses of hydrogen peroxide; in fact one mole of ß-lap used by NQO1 produces >60 moles H2O2 in 2 mins. The elevated levels of H2O2 generates tremendous DNA lesions (e.g., 8-oxoguanine formation) that ultimately results in massive levels of DNA single strand breaks. The H2O2 generated also results in release of ERCa2+ and culminates in the hyperactivation of Poly(ADP-ribose) polymerase (PARP1). GAPDH is then inhibited initially by direct suppression by H2O2 and then by posttranslational modification, resulting in accumulation of glyceraldehyde-3phosphate (GA3P). This unique, NQO1-dependent tumor-selective hyperactivation of PARP1 results in simultaneous losses of NAD+ (generated by the NQO1 futile redox cycle) and ATP, with suppressed recovery by inhibition of glycolysis (by inhibition of GAPDH) and the Krebs cycle through suppression of pyruvate production. Understanding the mechanism of action of these two classes of unique drugs has allowed tumor-selective combination therapies with DNA damaging agents, DNA repair inhibitors, and more recently metabolic inhibitors, such as agents that block NaMPt and GLS1. Addition of these inhibitors, in turn, results in a dramatic lowering of the required doses of NQO1 bioactivatable drugs-a true synergy is established with nontoxic levels of drugs combined to achieve one log or greater loss of colony forming ability. This work is supported by grants from Texas CPRIT, AACR/PanCan Action Network and NIH R01 CA102792-11 to DAB. Citation Format: Xiumei Huang, Zachary Moore, Xiuquan Luo, Gaurab Chakrabarti, Mariya Ilcheva, Rolf Brekken, Sandeep Burma, Jinming Gao, Ralph Deberardinis, William Bornmann, Paul Hergenrother, David A. Boothman. DNA repair and metabolism inhibitors are tumor-selective when combined with NQO1 bioactivatable drugs for therapy against pancreatic and nonsmall cell lung cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 787. doi:10.1158/1538-7445.AM2014-787

Cancer cells require increased NAD+ synthesis to support anabolic metabolism, to sustain signaling processes such as sirtuin activity and ADP-ribosylation, and to maintain redox balance. Inhibitors of Nicotinamide phosphoribosyltransferase (NamPT), the rate-limiting step in NAD+ biosynthesis, have modest anti-tumor activity in monotherapy in vitro and in vivo, but failed clinical trials due to a lack of tumor specificity attributed to the necessity of NAD+ synthesis in normal proliferating cells. We found that the tumor specificity and efficacy of NamPT inhibition can be greatly increased when small molecule NamPT inhibitors, such as FK866, are used in combination with the NQO1 bioactivated therapeutic ß-lapachone (ß-lap). Most solid tumor cells overexpress NQO1, which catalyzes a futile redox cycle with ß-lap, resulting in the formation of a burst of reactive oxygen species (ROS). This results in substantial DNA single strand breaks and base damage in a tumor-specific manner, which hyperactivates poly(ADP-ribose) polymerase-1 (PARP-1), a DNA repair enzyme that utilizes NAD+ to generate poly(ADP-ribose) (PAR)-protein moieties. Rapid NAD+ and ATP loss occurs and tumor cells die through programmed necrosis. PARP-1-mediated NAD+ depletion caused by ß-lap treatment synergizes with reduced NAD+ synthesis as a result of FK866-mediated NamPT inhibition. Enhanced cancer cell death in terms of loss of viability and clonogenicity at lower ß-lap doses was noted, while tumor specificity for NQO1 overexpressing cells was maintained. With combination treatment, cells are primed for ROS-mediated damage due to reduced glutathione and NAD(P)+ levels, they are unable to recover ATP, NAD+, and NADP+ synthesis even after only a 2 hour treatment, and they rapidly die through a caspase-independent mechanism. This therapeutic approach is highly effective against a variety of tumor cell lines from pancreatic, breast, and non-small cell lung, and is expected to improve the efficacy and tumor specificity of both of these drugs by allowing their use at lower doses in vivo. These studies were supported by an AACR/PanCan Innovator Award and NIH R01 CA102972 to DAB. Citation Format: Zachary Moore, David A. Boothman. Tumor-specific targeting of the NAD metabolome with β-lapachone and NamPT inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1760. doi:10.1158/1538-7445.AM2014-1760

Alteration of DNA demethylation in five CpG sites (-547, -533, -475, -464, and -454) immediately upstream from the estrogen response element of lactoferrin promoter was determined in the uteri of immature (17-day-old) and mature (21- and 30-day-old) mice treated neonatally with DES. Only the CpG/-464 was found to be abnormally demethylated by diethylstilbestrol (DES) treatment in the mature uteri. This abnormal demethylation occurred in specific response to DES in neonatal mice, because DES injected into the 30-day-old mature mice did not demethylate CpG/-464. This site, however, remained methylated in the neonatally DES-treated/ovariectomized mice, indicating that this DES-elicited demethylation is under hormonal control. Thus, neonatal DES treatment appeared to imprint an abnormal, site-specific demethylation of CpG/-464, which requires ovarian hormones to occur in adult mice. Moreover, the demethylation was maintained in uterine tumors of the neonatally DES-treated mice. This mode of demethylation is reminiscent of uterine tumor formation, which also depends on both neonatal DES exposure and ovarian hormone stimulation in adulthood. Thus, neonatal DES treatment may induce tumor formation as well as demethylation through a common cellular process.

Antibodies directed to specific disease-related antigens have proven to be very successful therapeutics for a variety of disease indications. In spite of their high affinity and specificity for target antigen, target-mediated toxicity constitutes a major limitation for the development of antibodies to certain targets. We have addressed this type of on-target toxicity by developing a new class of targeting antibodies (Probody™ therapeutics) that remain in an inert, masked form until proteolytically activated at the site of disease. As a proof-of-concept for the construction of a Probody, we used cetuximab as a starting point. Cetuximab is an EGFR-targeted antibody approved for the treatment of colorectal and head-and-neck cancers that produces an on-target toxicity in the form of a skin rash that afflicts 88% of patients treated with the antibody. We engineered an EGFR Probody by incorporating an inhibitory masking peptide fused to the antibody light chain. Masking of the Probody is achieved through a linker that also incorporates a substrate that is cleaved by one or more proteases up-regulated in cancer. In vitro, EGFR binding and cell-based activities of the masked Probody were diminished compared to those of cetuximab, but treatment with exogenous target proteases activated the Probody and restored activity comparable to cetuximab. Using tumor xenograft models in mice, we demonstrated that the Probody remained masked in systemic circulation but was activated and accumulated in the tumor microenvironment. Tumor activation of the Probody translated to efficacy similar to that seen with cetuximab. Consistent with our results in mice, the Probody remained efficiently masked in non-human primates and did not cause skin toxicity such as that observed in animals treated with cetuximab. Together, these results demonstrate that antibody activity can be specifically targeted to diseased tissue by utilizing locally overexpressed proteases as activating agents, suggesting that a variety of antigens not previously amenable to an antibody therapeutic approach may be successfully addressed with Probodies. Citation Format: Luc R. Desnoyers, Annie Yang, Tony W. Liang, Stephen Moore, Jason Sagert, Daniel R. Hostetter, Elizabeth Menendez, Fei Han, Michael Krimm, Ken Wong, Jennifer Richardson, Jim W. West, Shouchun Liu, Olga Vasiljeva, Henry B. Lowman. Development of a proteolytically activatable EGFR Probody for cancer therapy. [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 4570. doi:10.1158/1538-7445.AM2013-4570

We have previously shown that selected commensal bacteria contribute to colorectal carcinogenesis through bystander effects. Specifically, Enterococcus faecalis, a human intestinal commensal, induces colitis and colon cancer in Il10−/- mice. In this study, we hypothesized that tissue macrophages triggered by E. faecalis were exclusively responsible for epithelial cell transformation. We evaluated the role of macrophages by depleting them from the colon in these mice and observing their loss on colorectal carcinogenesis. To deplete colon macrophages in Il10−/− and control mice, we administered encapsulated clodronate liposomes (ECL) via weekly rectal enemas. Mice were colonized with E. faecalis strain OG1RFSS. Controls consisted of: 1) Il10−/- mice given ECL enemas but not colonized with enterococci; 2) Il10−/− mice colonized with E. faecalis but given sham liposome enemas containing no clodronate (SL); and 3) Il10+/+ mice colonized with E. faecalis and given ECL enemas. Mice were treated for 9 months and colon biopsies evaluated by immunohistochemical staining for numbers of macrophages; inflammation; cancer; presence of 4-hydroxy-2-nonenal protein adducts; and expression of cyclooxygenase (COX)-2, TNF-α, netrin-1, and WNT/β-catenin signaling. Staining of colon biopsies using F4/80 (a murine macrophage marker) showed significant decreases in numbers of macrophages for mice administered ECL enemas compared to mice given SL enemas (P These findings strongly suggest that the activation of colon macrophages by E. faecalis is necessary for inflammation and cancer development in the IL-10 knockout model. The reduction or elimination of expression of COX-2, TNF-α, netrin-1, and 4-hydroxy-2-nonenal protein adducts further supports a key role for these innate immune cells in generating bystander effects. Our results provide potential new strategies for preventing colorectal cancer. Citation Format: Yonghong Yang, Xingmin Wang, Thomas Huycke, Danny Moore, Stanley Lightfoot, Mark Huycke, Department of Veterans Affairs Medical Center. Depletion of colon macrophages prevents colitis and colon cancer triggered by commensal bacteria. [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 2868. doi:10.1158/1538-7445.AM2013-2868

Pancreatic cancer will be the second leading cause of cancer-related deaths in the US by 2020, where 5-year survival is To improve its efficacy, we examined the synergistic effects of adding the AP site-modifying drug and base excision repair (BER) inhibitor, methoxyamine (MeOX), with beta-lap against NQO1 over-expressing pancreatic cancer cells. MeOX + beta-lap synergy resulted in: a, enhanced lethality of sublethal doses of beta-lap, reducing the shoulder (Dq), increasing the lethality rate (Do), and inducing apoptosis (TUNEL+) in NQO1+, but not in NQO1-, MIA PaCa-2 cells; b, increased DNA lesion formation; c, dramatic losses in ATP levels, with little recovery; and d, dramatic suppression of glycolysis. These data strongly suggests that MeOX enhances PARP1 hyperactivation and synergistic cell killing of beta-lap. Similar results were noted in shRNA-XRCC1 knockdown cells. Mechanistically, our data suggests that PARP1 detects MeOX-AP modified sites or SSBs, allowing PARP1 hyperactivation and synergistic cell death. Since MeOX is a nontoxic agent, and both agents are currently in clinical trials (i.e., beta-lap as Arq761, Arqule, Boston, MA), combination therapies for the treatment of pancreatic, as well as other NQO1 over-expressing solid cancers could be rapidly developed. An AACR Innovator Award from the George and June Block Foundation to DAB supported this work. Citation Format: Xiuquan Luo, Longshan Li, Xiumei Huang, Lifen Cao, Zachary Moore, Ralph Deberardinis, Rolf Brekken, Stanton Gerson, Lili Liu, David A. Boothman. Inhibiting base excision repair synergistically enhances beta-lapachone-mediated ‘kiss of death’ for tumor-selective therapy of pancreatic cancer. [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 3344. doi:10.1158/1538-7445.AM2013-3344

Cytochrome P450 17α-hydroxylase /17,20-lyase (CYP17) is a validated treatment target for metastatic castrate-resistant prostate cancer (mCRPC). Abiraterone acetate (AA; Zytiga) inhibits 17α-hydroxylase and 17,20-lyase reactions catalyzed by CYP17 and thus reduces androgen biosynthesis. However, co-administration of prednisone is required to suppress the mineralocorticoid excess from 17α-hydroxylase inhibitions. VT-464, a non-steroidal small molecule inhibits androgen production without mineralocorticoid excess or cortisol depletion by selective inhibition of CYP17 17,20-lyase. We determined the impact of VT-464 on tumor growth of a mCRPC xenograft, MDA-PCa-133, in vivo, and on androgen signaling in C4-2B prostate cancer cells in vitro. The MDA-PCa-133 xenograft is derived from a clinical CRPC bone metastasis. Subcutaneous MDA-PCa-133 tumor expresses PSA, full-length androgen receptor (AR) and AR-V7 isoform. We determined the effect of VT464 and AA on MDA-PCa-133 growing in tumor-bearing castrated male mice: randomization into three groups; oral treatment with vehicle only, VT-464, (100 mg/kg bid), or AA (100 mg/kg bid) for 25 days. Both VT-464 and AA reduced tumor volume (>two fold compared to vehicle; p We determined the effects of VT-464 on AR-dependent luciferase reporter activity and expression of AR signaling genes, PSA and NKX3.1, in cultured C4-2B prostate cancer cells with or without VT-464 or AA treatment. VT-464 or AA highly reduced AR-dependent reporter activity and specific expression of PSA and NKX3.1. AR-dependent signaling in the presence of VT-464 or AA was restored however by 10 nM DHT, consistent with VT-464- or AA-mediated depletion of androgen in C4-2B cells. The AR-dependent reporter activity was also measured following overexpression of full-length AR or AR-V7 isoform in C4-2B cells. Full-length AR-dependent reporter activity was (>80%) inhibited; AR-V7-dependent reporter activity was inhibited by 50% by 1 μM VT-464 or AA. We conclude that VT-464 inhibited AR signaling as effectively as AA in C4-2B prostate cancer cells. The findings suggest that AR signaling in cells, which express high levels of AR isoform (AR-V7) are more resistant to androgen biosynthesis inhibitors than those with wild type AR. Preliminary observations suggest C4-2B cells with AR-V7 are less sensitive to AA than VT- 464. Ongoing clinical and co-clinical studies will determine if the observed differences in steroid profile and relative efficacy of VT-464 compared to AA are clinically and biologically meaningful. Citation Format: Sankar N. Maity, Guanglin Wu, Jing-fang Lu, Joel R. Eisner, Stephen W. Rafferty, Robert J. Schotzinger, William R. Moore, Christopher J. Logothetis, John C. Araujo, Eleni Efstathiou. Efficacy of VT-464, a novel selective inhibitor of cytochrome P450 17,20-lyase, in castrate-resistant prostate cancer models. [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 4772. doi:10.1158/1538-7445.AM2013-4772

Introduction. Circulating tumor cells (CTCs) have emerged as a predictive biomarker in the treatment of metastatic breast, colorectal and prostate cancers. Non-epithelial tumors such as melanoma also present evidence of circulating cells that have been difficult to detect due to technological limitations. Previous clinical studies have shown presence of melanoma specific marker mRNAs circulating in the blood, used as a surrogate of the circulating melanoma cell (CMC). This study was designed to use a negative cell selection platform to enrich CMCs from blood. The approach involved depletion of red blood cells by centrifugation and lysis, and white blood cells by immunomagnetic tagging with pan-leukocyte (CD45) antibody and magnetic separation. The enriched non-hematopoietic cell analysis was optimized for high sensitivity and specificity of CMC detection using a panel of antibodies against known melanoma markers. The secondary goal was to estimate the number of CMCs per milliliter of blood based on immunophenotype and morphology criteria. Methods. The study was reviewed and approved by the IRB. The leukocyte depletion was accomplished using a fluid dynamics-based quadrupole magnetic separating device (1) which, in principle, enriches CMCs without affecting them by immunomagnetic reagents. CMC cytospin smears were immunostained against known melanoma markers (Melan-A and S100). A high magnification microscopy imaging and a computer-aided image analysis were developed for efficient CMC enumeration. Results. Based on preliminary studies with SKMEL-28 melanoma cell line, normal adult blood controls and blood samples from advanced melanoma patients, the CMC was defined as an intact, nucleated cell, positive for Melan A or S100 and negative for CD45. Separations of SKMEL-28 cells spiked at approximately one SKMEL-28 cell per million leukocytes resulted in high efficiency separation with a mean recovery of (93.0 ±5.4 S.E.M.)% of SKMEL-28 cells. Samples obtained from apparently healthy donor blood (n =5) had a baseline of 0-3 positive cells per mL blood, whereas samples from stage IV melanoma patients (n =8) exhibit CMC counts ranging from 78 to 28,500 cells per mL of blood. Conclusions. Our study combines negative selection platform with immunophenotypic and morphological criteria for CMC enrichment, identification and enumeration. Interestingly, our preliminary results indicate the presence of high numbers of CMCs in malignant melanoma patients, providing basis for larger studies on correlations with clinical outcomes of the disease. Moreover, negative selection method lends itself to in vitro or in vivo culture bioassays, and molecular marker assays such as qRT-PCR, genomic PCRs, FISH or microarrays. Citation Format: Powrnima Joshi, Barbara Jacobs, Adeeb Derakhshan, Lee R. Moore, Pierre Triozzi, Ernest C. Borden, Maciej Zborowski. Evaluation of negative selection strategy for enrichment of circulating melanoma cells (CMCs) from patients with metastatic melanoma. [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 1454. doi:10.1158/1538-7445.AM2013-1454

TRAF3 is a novel tumor suppressor identified in human non-Hodgkin lymphoma and multiple myeloma. We recently reported that TRAF3 deletion causes vastly prolonged survival of mature B cells, which eventually leads to B lymphoma development in mice. The long latency of B lymphoma development observed in B-TRAF3−/− mice suggest that additional oncogenic pathways are required for B lymphomagenesis. To delineate such oncogenic pathways in TRAF3−/− B lymphomas, we performed microarray analyses and identified Sox5 as a gene recurrently up-regulated in B lymphomas spontaneously developed in different individual B-TRAF3−/− mice. We confirmed the striking up-regulation of Sox5 expression in TRAF3−/− B lymphomas at both the mRNA and protein levels by quantitative real time PCR and Western blot analyses, respectively. We further cloned the full-length cDNA of Sox5 from B lymphomas derived from 4 different individual B-TRAF3−/− mice. Surprisingly, we found that the sequence of Sox5 expressed in TRAF3−/− B lymphomas represents a novel isoform of Sox5, which has not been previously reported. This new isoform of Sox5 contains a 35 aa deletion in the N-terminal region in front of the leucine zipper domain. When transduced into human multiple myeloma cells, the cloned Sox5 cDNA was expressed into a protein of 80 kDa, a size identical to that detected in TRAF3−/− B lymphomas. Taken together, our findings identified a novel isoform of Sox5 as a candidate oncogene in B lymphoma. Our ongoing experiments will further elucidate the roles and mechanisms of Sox5 in TRAF3 deficiency-initiated B cell malignant transformation. This study is supported by a seed grant from the New Jersey Commission on Cancer Research (10-1066-CCR-EO, P. Xie), a Faculty Research Grant (P. Xie), and the Arthur Herrmann Endowed Cancer Research Fund (P. Xie); supported in part by a grant from NCI (R. Hart) and an Aresty Research Grant (A. Desai). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-272. doi:1538-7445.AM2012-LB-272

Tetraploidy can be an intermediate condition for cells undergoing tranformation from diploidy to aneuploidy. We previously showed that Enterococcus faecalis, a human intestinal commensal, generated tetraploidy and aneuploidy in colonic epithelial cells via a macrophage-induced bystander effect. To explore potential mediators for this effect, supernatants from E. faecalis-infected macrophages were analyzed by HPLC coupled to a coulometric detection system. Significantly increased concentrations of 4-hydroxy-2-nonenal (4-HNE), a breakdown product of polyunsaturated fatty acids, were detected in macrophage supernatants. This system was used to purify macrophage-generated 4-HNE for further experiments. To investigate aneugenic effects of 4-HNE, a near-diploid human colon cancer cell line (HCT116) and non-transformed diploid mouse colonic epithelial cell line (YAMC) were exposed to 4-HNE and ploidy analyzed by FACS. For HCT116 cells the percent tetraploidy significantly increased following exposure to 4-HNE at 2.5 µM compared to untreated controls (9.90% ± 0.09 vs. 0.21% ± 0.08, P < 0.001). In comparison, the percent tetraploidy was increased to 0.68% ± 0.10 for YAMC cells exposed to 1 µM 4-HNE compared to 0.12% ± 0.06 for untreated controls (P < 0.001). This showed that 4-HNE generated tetraploidy in both transformed and non-transformed cells. To address potential mechanisms by which 4-HNE produced tetraploidy, HCT116 cells exposed to 4-HNE were stained with anti-α/β tubulin antibody and microtubules analyzed by confocal microscopy. We found cells exposed to 4-HNE had degraded microtubules and abnormal mitotic patterns, implicating the induction of tetraploidy through inhibition of microtubule assembly. Moreover, for HCT116 and YAMC cells, 4-HNE caused a G2/M cell cycle arrest with increased γH2AX foci suggesting DNA double-strand breaks. Next, we silenced mGSTα4-4, a key enzyme in the detoxification of 4-HNE, by constitutively expressing RNAi in cells. FACS analysis showed that γH2AX positivity remarkably increased in YAMC cells with silenced mGSTα4-4 that were exposed to E. faecalis-infected macrophages compared to non-infected macrophages. No changes were seen in wildtype YAMC cells. Similarly, γH2AX foci increased in mGSTα4-4-silenced YAMC cells exposed to 4-HNE compared to the wildtype control (41.8% ± 1.3 vs. 24.6% ± 6.5, P = 0.01). Finally, percent tetraploidy also significantly increased for mGSTα4-4-silenced YAMC cells exposed to 4-HNE, supporting the hypothesis that 4-HNE can specifically generate tetraploidy. In sum, E. faecalis-infected macrophages produce 4-HNE that, in turn, induces tetraploidy, DNA double-strand breaks and G2/M arrest in colonic epithelial cells. Inactivation of mGSTα4-4, a detoxifying enzyme for 4-HNE, exacerbated these effects. We propose 4-HNE as a mediator for the E. faecalis-infected macrophage bystander effect. 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 4391.

The development of glucose-6-phosphatase (G-6-Pase-)-deficient hyperbasophilic foci was analyzed at 4-week intervals in the livers of CD-1 and C57BL/6J x C3H/HeJ F1 (hereafter called B6C3F1) mice given a single i.p. injection of diethylnitrosamine (DEN) (0.1, 0.2, or 0.4 mumol/g body weight) within 24 hr after birth. Transections of G-6-Pase-deficient foci of hepatocytes were readily discernible in liver sections of DEN-treated mice of either sex at 8 weeks of age. The size and number of these foci per liver increased with time. The occurrence of G-6-Pase-deficient focus transections with diameters as large as 1 mm coincided with the gross appearance of 1-mm gray-white nodules in the livers of male B6C3F1 mice at 16 weeks of age and in females at 32 weeks of age. Transections of all grossly visible hepatic nodules from male and female mice were G-6-Pase deficient and hyperbasophilic; the great majority were diagnosed as mouse hepatomas type A. After a single neonatal dose of DEN, the number and rate of growth of the G-6-Pase-deficient foci and the incidence and rate of appearance of gross hepatomas were greater in the liver of male than in those of female mice. In contrast, the average numbers of G-6-Pase-deficient foci in the livers of male and androgenized female B6C3F1 mice at 36 weeks of age were approximately equal and about twice that observed for the livers of DEN-treated female controls. Quantitation of carcinogen-induced histochemically detectable foci and hepatomas as a function of time provides a useful tool for the analysis of initiation and promotion in the mouse liver.

Human anti-mouse immunoglobulin immune responses were studied in ten patients, eight with ovarian cancer and two with grade IV gliomas, diagnosed and treated with radiolabeled (123I, 131I) murine monoclonal antibodies. It was found that serum from these patients before treatment and from 18 control healthy individuals contained detectable antibodies to antigenic determinants on the Fc but not the F(ab')2 portion of mouse immunoglobulin. No change in this reactivity occurred after the initial (imaging) dose of monoclonal antibodies. However, repeated administration of mouse immunoglobulins for therapy resulted in an elevated immune response directed against determinants on both Fc and F(ab')2 regions of mouse immunoglobulin. This response contained increased levels of immunoglobulin M as well as immunoglobulin G and showed a marked prozone effect in our enzyme linked immunosorbent assay system. None of the immunized patients developed a detectable antiidiotypic response.