Citations

Abstract Recent studies suggest that progression to castrate-resistant prostate cancer (CRPC) is due to generation of androgen receptor (AR) splice variants, lacking the ligand binding domain. These AR variants originally identified in cell-based models of CRPC, have also been detected in benign and malignant human prostate tissue, with the highest levels observed in late state CRPC. The diversity and complexity of AR variants is well appreciated but not fully understood. Using RT-PCR we assessed the expression of a number of AR variants in cell-based models of both castrate-resistant and androgen-dependent prostate cancer. Several AR variants were expressed in both 22Rv1 and VCaP cells, but not in LNCaP cells. Coding sequences for AR variants were cloned into expression vectors and constitutive transcriptional activity was observed using a luciferase reporter assay. Augmented transcriptional activity of AR variants was observed in cells that express full-length AR, when full-length AR was silenced using shRNA targeting exons 7/8. This observation suggests the constitutive activity of AR variants may be independent of full-length AR. We profiled the AR variant transcriptome in 22Rv1 cells, using the Illumina HumanHT12 v4 whole-genome expression array platform. shRNA targeting exon 1 was used to silence all AR isoforms and shRNA targeting exons 7/8 was used to silence full-length AR. 5,256 genes were modulated by knocking down all AR isoforms and 5,086 genes were modulated by knocking down full-length AR. A total of 3,714 genes were common to both groups, leaving 1542 genes modulated by AR variants and 1372 modulated by full-length AR (p<0.05). The represents a subset of genes differentially regulated by AR variants independently of full length AR. Aggregate function of multiple AR variants may confer a CRPC phenotype independent of full-length AR. Pathway analysis of these genes differentially regulated by AR variants may reveal potential targets for therapeutic intervention in patients who progress despite androgen deprivation or anti-androgen therapy. (Supported by NIH grants CA125747, CA091956 and CA121277, T.J. Martell Foundation and the Mayo Clinic Prostate Cancer SPORE). 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 2924. doi:1538-7445.AM2012-2924

Background Clinical classification of rare sequence changes identified in the breast cancer susceptibility genes BRCA1 and BRCA2 is essential for appropriate genetic counselling of individuals carrying these variants. We previously showed that variant BRCA1 c.5096G>A p.Arg1699Gln in the BRCA1 transcriptional transactivation domain demonstrated equivocal results from a series of functional assays, and proposed that this variant may confer low to moderate risk of cancer. Methods Measures of genetic risk (report of family history, segregation) were assessed for 68 BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) families recruited through family cancer clinics, comparing results with 34 families carrying the previously classified pathogenic BRCA1 c.5095C>T p.Arg1699Trp (R1699W) mutation at the same residue, and to 243 breast cancer families with no BRCA1 pathogenic mutation (BRCA-X). Results Comparison of BRCA1 carrier prediction scores of probands using the BOADICEA risk prediction tool revealed that BRCA1 c.5096G>A p.Arg1699Gln variant carriers had family histories that were less ‘ BRCA1 -like’ than BRCA1 c.5095C>T p.Arg1699Trp mutation carriers (p<0.00001), but more ‘ BRCA1 -like’ than BRCA-X families (p=0.0004). Further, modified segregation analysis of the subset of 30 families with additional genotyping showed that BRCA1 c.5096G >A p.Arg1699Gln had reduced penetrance compared with the average truncating BRCA1 mutation penetrance (p=0.0002), with estimated cumulative risks to age 70 of breast or ovarian cancer of 24%. Conclusions Our results provide substantial evidence that the BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) variant, demonstrating ambiguous functional deficiency across multiple assays, is associated with intermediate risk of breast and ovarian cancer, highlighting challenges for risk modelling and clinical management of patients of this and other potential moderate-risk variants.

Abstract Purpose. Prostate cancer (PCa) continues to be a leading cause of cancer death in males worldwide. In the prostate, androgens play a crucial role in both normal and cancerous growth by acting through the androgen receptor (AR), a member of the nuclear receptor super-family; hence, the AR has become a target of therapeutic intervention. The standard treatment for advanced PCa is androgen ablation therapy, initially leading to tumor regression through inhibition of AR activity. However, the success of androgen ablation therapy is often terminated by the emergence of castration- recurrent PCa (CRPC), for which there is still no cure. CRPC remains dependent on a functional AR-axis for proliferation and drugs targeting this network are continually being developed. We have examined the effects of Millennium-Takeda investigational compound T-1759243 on both androgen-responsive and androgen-refractory PCa cells in order to assess its therapeutic potential. Methods. We used PCa cell lines LNCaP, LNCaP-C42 and 22RV1 and assessed effects on cell viability and proliferation using MTS and BrdU assays. AR activity was measured using PSA-luciferase reporter constructs and protein levels by western blot. Microarray analysis with the Illumina HT-12 platform was used to identify genes affected by treatment with T-1759243, followed by real-time PCR confirmation. Pathway analysis was performed using MetaCoreTM software. Results. Our studies have revealed that T-1759243 reduces viability in all cell lines examined, regardless of androgen responsiveness. Treatment resulted in decreased proliferation in androgen-responsive cells and increased apoptosis in refractory cells. AR activity, as measured by PSA-luciferase assay, was greatly decreased in all cell lines by 10uM T-1759243 and T-1759243 abrogated the ability of synthetic androgen R1881 to increase AR activity by inhibiting nuclear translocation of the AR in refractory cells. Pathway analysis revealed changes in genes involved in cell-cycle, cell adhesion and cytoskeletal remodeling in addition to genes in androgen target pathways. Conclusions. T-1759243 is a novel AR antagonist that inhibits AR activity in both androgen-dependent and androgen-refractory prostate cancer cells. Additionally, it inhibits the ability of androgen to promote nuclear translocation of the AR and thus stimulate AR activity in castrate-resistant prostate cancer cells. Pathways affected by treatment with T-1759243 are cell-specific and dependent on AR status and may reveal novel gene targets in PCa treatment. This work was supported in part by Grant #35 from Millennium Pharmaceuticals, Inc the Takeda Oncology Company. 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 940. doi:1538-7445.AM2012-940

Abstract Bladder cancer is the 4th most commonly diagnosed cancer in the United States. Because superficial bladder cancer (SBC) recurs in about 60% of cases, standard treatment involves tumor resection followed by intravesical therapies to prevent recurrence, as well as lifelong periodic cystoscopy. There is an urgent need for better intravesical therapies to prevent both the recurrence and progression of SBC. The Mammalian Target Of Rapamycin (mTOR) complex is upregulated in many bladder cancer tumors. We have targeted both the mTORc1 and mTORc2 complexes with drugs that inhibit both complexes. We used PP242 and a second inhibitor, OSI-027, which is currently in a phase I clinical trial for solid tumors (NCT00698243). Our in vitro model targets cell proliferation in three bladder cancer cell line (HT1376, T24 and UM-UC-3). Both drugs are effective in inhibiting the growth of bladder cancer cell lines with IC50 values between 0.6 and 18µM. In comparison with rapamycin, both drugs demonstrate effects on downstream targets of both the mTORc1 and mTORc2 pathways including 4EBP1 and pAkt. Receptor tyrosine kinases are another therapeutic target for bladder cancer. Lapatinib which targets EGFR and Her3, in combination with OSI-027 forms a highly synergistic treatment that inhibits bladder cancer cell line growth. Downstream molecular targets of the combination therapy, including the apoptosis markers PARP and caspase 3, were evaluated by Western blots. Our in vitro data suggests that a combination of dual mTOR inhibitors with RTK inhibitors may provide a novel effective therapeutic treatment for bladder cancer. Future studies of the in vivo efficacy of these drug combinations in a mouse model are planned. 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 896. doi:1538-7445.AM2012-896

Abstract Recent RNA-sequencing studies have identified variant transcripts, fusion transcripts and novel transcripts in cancer. However, little is known about the role of these variant transcripts in tumor development. To identify a specific role of these variant transcripts in tumor development toward chemotherapy resistance, we generated a custom retroviral cDNA expression library from 10 ovarian tumors with resistance to chemotherapy. Retrovirues were generated from the custom library, and they were used to transduced exogenous genes into ovarian cancer cell line A1847, a cell line chosen for its sensitivity to paclitaxel. A1847 cells transduced with retroviruses carrying alkaline phosphatase was used as a control. Transuced cells were selected with 25 nM paclitaxel. 87 clones survived the selection in library-tranduced cells whereas none survived the selection in alkaline phosphatase-tranduced control cells. Forty seven clones survive the secondary screens. Sequence analysis of transduced genes in these surviving clones indicate that 4 paclitaxel-resistant clones contain RABL3 splice variant. Although 195 accessions corresponding to wildtype transcripts are reported in AceView, there is a single accession corresponding to RABL splice variant that we identified in this screen. RNAi-mediated downregulation of RABL3 resulted in enhance sensitivity toward paclitaxel in these resistant clones. Ectopic expression of RABL3 splice variant resulted in increased resistant to paclitaxel. Collectively, these results demonstrate the feasibility of performing functional genetic screens to identify novel splice variants involved in tumor development. 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 5120. doi:1538-7445.AM2012-5120

Abstract Background: In tamoxifen (TAM) treated patients, both CYP2D6 metabolism and low steady state concentrations of endoxifen (END), the active metabolite of TAM, have been associated with a higher risk of recurrence (Madlensky L, Clin Pharmacol Ther. 2011; 89(5):718-25). END drug development is ongoing at Mayo and NCI, and our previous data demonstrated that 1) END had high oral bioavailability in mice and yielded plasma concentrations and total exposure 20-fold greater as compared to an equivalent oral dose of TAM (MM Ames et al, Cancer Res 2011;70(8 Suppl 1):Abstract nr 3603) and 2) END is superior to TAM in both HER2 (Reinicke AACR 2011) and aromatase expressing (Goetz, SABC 2011) MCF-7 xenograft models. While subcutaneous (SQ) TAM is used by most researchers to study and develop models of TAM resistance, little is known about 1) whether murine and human TAM pharmacokinetics are comparable and 2) whether the route of administration affects concentrations of the active metabolites. Therefore, we conducted murine pharmacokinetic studies evaluating both the oral and SQ administration of TAM and END. Results: Following administration of TAM, concentrations of the active metabolites (END and 4-hydroxy TAM [4HT]) were therapeutic (Cmax- END 35.9 nM, 4HT 62.8 nM) only after a 0.5 mg oral dose of TAM. In contrast, following all doses of SQ TAM, concentrations of the active metabolites (END and 4HT) were sub-therapeutic (Cmax- END 5.2 nM; 4HT 18.7 nM). In contrast to humans, the Cmax of N-desmethyl TAM, the most abundant human TAM metabolite, was &lt; 16.5 nM in mice after oral and SQ TAM. SQ administration of END and TAM resulted in substantially greater bioavailability of the parent drug compared with oral dosing. Following administration of 0.25-5 mg oral END and 0.0625-0.625 mg subcutaneous END, the increase in AUC was greater than dose proportional and active concentrations (&gt;80 nM) were detected in plasma at every dose and both dosing routes. Comparable END and TAM plasma concentrations were achieved with a 0.0625 mg SQ END and a 0.5 mg SQ TAM, respectively. Conclusions: Our data strongly suggest that SQ dosing of TAM results in non-therapeutic concentrations of the active hydroxylated metabolites, and may be a simple pharmacokinetic factor influencing response and resistance in a murine in vivo xenograft model. Given the nonlinear pharmacokinetics and differences in metabolism, the dose and route of END must be carefully selected for comparative studies with TAM. These data support the ongoing human studies designed to bypass the need for TAM metabolism by the direct administration of END. Supported by the Mayo Comprehensive Cancer Center Grant (CA15083) and the Mayo Clinic Breast Cancer SPORE (CA 116201; MMA, JMR, MPG). 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 3781. doi:1538-7445.AM2012-3781

Abstract Batracylin (NSC 320846, BAT) is a heterocyclic arylamine and topoisomerase II inhibitor shown to be active in murine tumor models, but with demonstrated species differences in toxicity associated with metabolism to N-acetylbatracylin (NBAT) by N-acetyltransferase (NAT). BAT is a preferential substrate of human NAT2. Accordingly, production of NBAT should be subject to population variability as a function of NAT2 polymorphisms. A phase I clinical trial of BAT in patients with advanced cancer and slow acetylator NAT2 phenotypes to reduce the risk of NBAT-related toxicity found a dose-limiting toxicity (DLT) of cystitis/hemorrhagic cystitis. In subsequent in vitro and in vivo preclinical studies to elucidate metabolic pathways that might contribute to BAT toxicity, we found oxidative metabolites and thiol conjugates of BAT and NBAT (MM Ames et al, Cancer Res 2011;71(8 Suppl):Abstract nr 1303). Since these data are consistent with metabolic activation of BAT to potentially toxic metabolites, we assessed formation of reactive intermediates as measured by covalent binding (CB) to protein and DNA or by formation of glutathione conjugates. Radiolabeled BAT and NBAT were incubated with 3-MC-induced rat (r), human (h) and dog (d) liver microsomes (LM), as well as with recombinant human cytochromes P450 (CYPs) with or without added DNA. NADPH-dependent BAT protein and DNA CB were detected in all microsomal preparations (see table below). NADPH-dependent NBAT protein CB was detected in all microsomal preparations except hCYP1B1. NADPH-dependent NBAT DNA CB was detected in all microsomal preparations except 3MC-rLM. We also detected a glutathione conjugate of mono-hydroxy BAT by lc/ms/ms analysis when BAT was incubated with hLM in the presence of glutathione and NADPH. Thus, CYP-catalyzed oxidation of BAT yields reactive metabolites that that bind protein and DNA and may contribute to drug-related DLT. Supported in part by NCI Contract N01-CM-52206. 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 3769. doi:1538-7445.AM2012-3769

Abstract Background: Esophageal adenocarcinoma (EAC) is a lethal cancer, and the incidence is increasing. Our group demonstrated that over-expression of the epidermal growth factor receptor (EGFR) correlated with reduced survival in EAC. Panitumumab (P) is a fully human IgG2 monoclonal antibody against human EGFR. Cetuximab combined with irinotecan (I) has shown activity for second-line treatment of advanced colorectal cancer (CRC). P alone is active in EGFR expressing CRC as well. Our group has empiric evidence for the activity of cetuximab + I as third-line treatment for advanced EAC. As such, a phase II study was designed to evaluate P + I as second-line therapy for advanced EAC. Trial Design: The primary endpoint is overall response (OR) as measured by RECIST (cross-sectional imaging every 2 cycles). Secondary endpoints include overall survival, safety/toxicity and correlative studies. Patients with confirmed EAC with history of one prior treatment are treated with P 9 mg/m2 on day 1 and I 125 mg/m2 on days 1 and 8 of each 21 day cycle to a maximum of 6 cycles. Inclusion criteria include confirmed EAC, measurable disease, no prior I or P, PS &lt;2 and normal organ function. A Simon two-stage design is used with power of 80% and a type I error of 0.05. 18 patients will be tested in the first stage and will terminate the trial if 2 or fewer respond. A total of 43 patients will be studied. Blood and tissue are obtained to analyse the role of FCα-R receptors as well EGFR pathway components and kras mutations. Enrollment: The 1st patient was enrolled in May 2009. Through August 2011, 7 patients received treatment. Preliminary results: Of the 7 patients treated, all are white males, median age 62. 2 patients completed 6 cycles. 1 patient received 3 cycles, 3 received 2 cycles and 1 patient completed 1 cycle. 3 patients are still alive. However, only 1 patient is still in the trial. 4 patients received dose reductions due to grade 3 or 4 side effects, including GI disturbances (5), fatigue(1), skin rash(1), electrolyte imbalances(2) and hematologic abnormalities (5). Reasons for discontinuation included progressive disease (PD, 5 patients) and patient refusal (2). The longest survival was 338 days–1 of the 2 patients who completed 6 cycles. The second patient who completed 6 cycles is alive at 307 days. 1 patient completed 3 cycles and survived 213 days after PD. 1 patient survived 174 days followed PD after 2 cycles. 2 patients completed 2 cycles and are still alive, surviving 151 (pt refusal) and 103 days respectively. The shortest survival was 85 days who completed only 1 cycle. Conclusion: Preliminary data from this Phase II trial suggests that patients with advanced EAC might benefit from P+I regimen as 2nd line therapy. 3 additional centers will open the trial. Accrual is ongoing, and correlative studies will follow. This project used the University of Pittsburgh Cancer Institute and was supported in part by award P30CA047904 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 3708. doi:1538-7445.AM2012-3708

Abstract Pancreatic ductal adenocarcinoma (PDA) affects over 44,000 people in the United States every year and carries a dismal prognosis, with a 5-year survival rate of just 5%. A subset of these patients (5-10%) report a familial history of the disease. The genetic etiology in these familial cases is poorly defined, with known susceptibility genes, such as: BRCA2, PALB2, CDKN2A, BRCA1, PRSS1 and STK11, accounting for only 10-15% of familial pancreatic cancer. In an effort to identify previously unappreciated pancreatic cancer susceptibility genes, we used next generation sequencing technology to evaluate the whole genome and whole exome sequences of 16 (6 families) and 22 individuals (10 families) respectively. All of these families enrolled into one of the familial pancreatic cancer registries participating in the Pancreatic Cancer Genetic Epidemiology (PacGene) Consortium, all had at least three members with PDA, and DNA was available from at least two affected members in each kindred. Using this approach, we identified heterozygous, inactivating, ATM mutations in two kindreds with familial pancreatic cancer (c.8266A&gt;AT; p.K2756X and c.170G&gt;GA; p.W57X). These mutations were previously reported as disease causing variants in patients with ataxia-telangiectasia, an autosomal recessive condition resulting from bi-allelic deleterious mutations of ATM. Interestingly, heterozygotes of deleterious ATM mutations have an increased risk of breast cancer. However, there have been no previous reports of deleterious ATM mutations in the germline of familial PDA patients. In our study, mutations segregated with disease in both kindreds and tumor analysis demonstrated Loss of heterozygosity (LOH) of the wild-type allele. Sequence analysis of the entire ATM gene in an additional 166 familial pancreatic cancer probands indentified four additional patients with deleterious mutations in the ATM gene (c.3214G&gt;GT; p.E1072X, c.6095G&gt;GA; p.R2032K, IVS41-1G&gt;GT and c.3801delG), while no deleterious mutations were identified in 190 spouse controls (p=0.046). When considering only the mostly severely affected families, those with three or more pancreatic cancer cases, four deleterious mutations were found in 87 families (P=0.009). These results indicate that ATM mutations play an important role in familial pancreatic cancer predisposition and have significant implications in the management of affected individuals and the risk assessment of family members. 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 2628. doi:1538-7445.AM2012-2628