Citations

AbstractMutations in the all-trans retinoic acid (ATRA)–targeted ligand binding domain of PML-RARα (PRα/LBD+) have been implicated in the passive selection of ATRA-resistant acute promyelocytic leukemia clones leading to disease relapse. Among 45 relapse patients from the ATRA/chemotherapy arm of intergroup protocol C9710, 18 patients harbored PRα/LBD+ (40%), 7 of whom (39%) relapsed Off-ATRA selection pressure, suggesting a possible active role of PRα/LBD+. Of 41 relapse patients coanalyzed, 15 (37%) had FMS-related tyrosine kinase 3 internal tandem duplication mutations (FLT3-ITD+), which were differentially associated with PRα/LBD+ depending on ATRA treatment status at relapse: positively, On-ATRA; negatively, Off-ATRA. Thirteen of 21 patients (62%) had additional chromosome abnormalities (ACAs); all coanalyzed PRα/LBD mutant patients who relapsed off-ATRA (n = 5) had associated ACA. After relapse Off-ATRA, ACA and FLT3-ITD+ were negatively associated and were oppositely associated with presenting white blood count and PML-RARα type: ACA, low, L-isoform; FLT3-ITD+, high, S-isoform. These exploratory results suggest that differing PRα/LBD+ activities may interact with FLT3-ITD+ or ACA, that FLT3-ITD+ and ACA are associated with different intrinsic disease progression pathways manifest at relapse Off-ATRA, and that these different pathways may be short-circuited by ATRA-selectable defects at relapse On-ATRA. ACA and certain PRα/LBD+ were also associated with reduced postrelapse survival.

AbstractThe biological role of vitamin D receptors (VDR), which are abundantly expressed in developing zebrafish (Danio rerio) as early as 48 h after fertilization, and before the development of a mineralized skeleton and mature intestine and kidney, is unknown. We probed the role of VDR in developing zebrafish biology by examining changes in expression of RNA by whole transcriptome shotgun sequencing (RNA-seq) in fish treated with picomolar concentrations of the VDR ligand and hormonal form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3)].We observed significant changes in RNAs of transcription factors, leptin, peptide hormones, and RNAs encoding proteins of fatty acid, amino acid, xenobiotic metabolism, receptor-activator of NFκB ligand (RANKL), and calcitonin-like ligand receptor pathways. Early highly restricted, and subsequent massive changes in more than 10% of expressed cellular RNA were observed. At days post fertilization (dpf) 2 [24 h 1α,25(OH)2D3-treatment], only four RNAs were differentially expressed (hormone vs. vehicle). On dpf 4 (72 h treatment), 77 RNAs; on dpf 6 (120 h treatment) 1039 RNAs; and on dpf 7 (144 h treatment), 2407 RNAs were differentially expressed in response to 1α,25(OH)2D3. Fewer RNAs (n = 481) were altered in dpf 7 larvae treated for 24 h with 1α,25(OH)2D3vs. those treated with hormone for 144 h. At dpf 7, in 1α,25(OH)2D3-treated larvae, pharyngeal cartilage was larger and mineralization was greater. Changes in expression of RNAs for transcription factors, peptide hormones, and RNAs encoding proteins integral to fatty acid, amino acid, leptin, calcitonin-like ligand receptor, RANKL, and xenobiotic metabolism pathways, demonstrate heretofore unrecognized mechanisms by which 1α,25(OH)2D3 functions in vivo in developing eukaryotes.

Abstract Purpose: The study has two related purposes. To: 1. describe breast cancer risk factors and screening patterns in a sample of American Indian and Alaska Native (AI/AN) women from four tribes in the Northern Plains region; and 2. ascertain associations between women's risk factors and their adherence to mammographic screening guidelines with the intention of using the results to develop future interventions to increase screening, especially in higher risk non-adherent women. Background: The Indian Health Service (IHS) has noted that challenging economic factors make increases in AI/AN women's screening participation difficult. In this light, understanding the relationship between risk factors and screening adherence is an important first step in developing low-cost interventions to improve screening participation, especially for women at higher risk of breast cancer. Lending importance to the role of screening in reducing breast cancer deaths in Northern Plains AI/AN women are high rates of later stage (regional or distant disease) diagnoses that are equal to the Non-Hispanic White (NHW) population (95.8 and 95.4/100,000 respectively). Later stage at diagnosis is associated with worse survival profiles. We hypothesized that women at higher risk as compared to women at lower risk on the factors analyzed would be more likely to adhere to annual screening guidelines promoted by their clinics. Procedures/Methods: Criteria for inclusion in this study were: women with no history of breast cancer who were ≥ 40 years of age, and had at least one mammogram on file. We reviewed the charts of a representative sample of ∼20% of eligible women in four tribes (1250 records, 1088 of of which met inclusion criteria). The study collected Gail Model risk factor data and also assigned BIRADS scores to an early and late mammogram where they were available (888 pairs) for each woman. In addition, we collected age at menopause as an additional risk factor. For each Gail Model risk factor (age, age at menarche, age at first live birth, number of first degree relatives with a breast cancer history, breast biopsy—number of biopsies, history of atypical hyperplasia), women were assigned to higher or lower risk categories by comparison with calculated scores for women of the same age. Overall five-year and lifetime Gail Model risk scores, using standard criteria, also were calculated for each woman and compared with predicted risk for women of equivalent ages. For breast density, women were determined to be at lower risk if their BIRADS score was 1 or 2, and at higher risk if they were scored 3 or 4. To ascertain levels of screening adherence, we followed American Cancer Society (ACS) guidelines which recommend an annual mammogram beginning at age 40. We used this standard because it is the recommendation given to women in the clinics where the study was conducted. All women who received screening exams within 1.5 years (18 months) were considered adherent. Results are age-adjusted for each tribal site and across sites. Summary of Findings: Only P-values for older age at menopause (0.02), older chronological age (0.005), and 5-year Gail Model risk scores (0.02) were consistent with our hypotheses that women at higher risk would be more adherent with screening guidelines. There was no significant association between screening adherence and life-time risk (0.12), family history (0.28), age at menarche (0.99), age at first live birth (0.68), biopsy history (0.99), or BIRADS density scores (0.16). Conclusions: The minimal relationship between risk factors and adherence suggests that generally neither individual risk factors nor overall risk predict women's screening decisions. This suggests that personal and clinic-centered mediators should be identified to guide future interventions to increase the proportion of women who adhere to screening guidelines, especially women who are at greater risk. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B95.

Abstract Introduction: The mammalian target of rapamycin (mTOR), a downstream kinase of the PI3K/Akt signaling pathway, is a critical regulator of basic cellular functions, especially in tumor progression, and therefore has emerged as a key target for cancer chemotherapy. Rapamycin and its derivatives (e.g. Everolimus, Temsirolimus and Deferolimus) are mTOR inhibitors originally approved as immunosuppressants to prevent renal and cardiac allograft rejection. These mTOR inhibitors have recently been used in a large number of cancer clinical trials, including renal cell carcinoma, breast cancer, endometrial carcinoma, glioblastoma and mantle cell lymphoma. However, severe side effects such as toxicity and immune suppression limit the escalation of mTOR inhibitors' dosage, resulting in a low responsive rate. To maximize the efficacy and minimize side effects of mTOR inhibitors, there is a critical need to identify the genetic biomarkers for response to mTOR inhibitors and the mechanisms by which they manifest their therapeutic benefits in cancer. Purpose: This study aims to identify and validate novel genetic biomarkers that might influence the therapeutic responses of mTOR inhibitors. Methods: We used 273 human lymphoblastoid cell lines (LCLs), for which we have obtained genome-wide SNP data, Affymetric U133 plus 2 mRNA expression array data, and Illumina microRNA array data, to perform cytotoxicity assays with two mTOR inhibitors, Rapamycin and Everolimus. We then performed genome-wide association (GWA) analyses using SNPs, mRNA gene expression and microRNA data with cytotoxicity data (AUC values) collected on both mTOR inhibitors. Integrated analysis was then performed to identify the SNPs, and microRNAs that might be associated with genes, whose expressions in turn were significantly associated with the cytotoxicity. We then selected candidate genes to functionally validate their effects on sensitivity of mTOR inhibitors using an siRNA screening approach with 3 cell lines, a renal carcinoma (Caki2), a glioblastoma (U87) and a fibroblast (IMR90) cell line, followed by MTS cell proliferation assay and colony formation assay. Results: mRNA expression levels of 48 and 55 genes were associated with Rapamycin and Everolimus cytotoxicity (p<10−4) respectively, with 16 common genes between two drugs. One hundred and twenty seven and 100 SNPs were associated with Rapamycin and Everolimus cytotoxicity (p<10−4), respectively. We selected 23 candidate genes based on the GWA analyses (mRNA expression vs. cytotoxicity, SNPs vs. cytotoxicity and integrated analysis), and verified 13 genes that significantly altered cells' sensitivity to either one or both mTOR inhibitors in at least one cell line based on siRNA screening study. In addition, one microRNA, miR10a, was shown to be most significantly associated with cytotoxicity for both Rapamycin and Everolimus (p=3.3×10−4; p =1.4×10−4). Interestingly, this particular microRNA was also found to be strongly associated with gene expression levels of 9 out 23 selected candidate genes with p<10−4. Conclusions: This study identified a series of novel candidate genes and microRNA (miR10a) that might contribute to the response of mTOR inhibitors. These findings will enhance our understanding of the regulation of mTOR pathway and the mechanisms underlying the variation in response to mTOR inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-11.

Abstract Mortality from prostate cancer in 2011 is estimated at 33,720 deaths in United States which occurs primarily in advanced disease stages. The cornerstone treatment of advanced prostate cancer is androgen deprivation, first established by Huggins and Hodges in 1941. Since then, recognition for the central role of the androgen axis and the androgen receptor (AR) during prostate tumorigenesis and progression, has been accepted. The arrest of the androgen-dependent growth stimulus by surgical or medical castration (with LHRH analogues or antagonists) is the result of reduced circulating androgens including dihydro-testosterone (DHT), which induces apoptotic regression of androgen-dependent cancer cells. This control however is short lived with the emergence of castration-resistant prostate cancer (CRPC). Despite achieving sub-castrate levels of circulating androgens as a result of castration, the androgen-AR axis continues to play an active role in tumor progression. A functional AR, despite the paucity of circulating androgens, is evidenced by the elevation of AR mRNA and re-expression of some androgen-regulated genes during clinical castration, indicative of a persistently activated AR cell signaling pathway. Furthermore, although testosterone and DHT are depleted in serum following traditional androgen deprivation therapy, significant levels of androgens have been measured in locally CRPC tissue1 as well as in metastatic CRPC tissue2. These androgens continue to regulate AR transcriptional activity and stimulate expression of androgen-regulated genes thereby re-invigorating tumor progression. Indeed, a number of molecular and cellular changes have been shown to facilitate activation of the AR under these conditions: amplification of the AR gene allow the receptor to respond to lower levels of androgens; splice variants of the AR exhibit constitutive activity; increased expression of several co-activator proteins enhance activation of the AR; other signaling pathways, including Akt-PI3 Kinase and MAP Kinase, activate the AR; and gain-of-function mutations in the AR allow the receptor to become “promiscuous” and then bind to ligands other than DHT, including anti-androgens3 and weaker sex steroids such as androstenedione. Abiraterone acetate, a selective steroidal irreversible inhibitor of CYP17 (17 hydroxylase/C17,20-lyase) blocks two important enzymatic activities in the synthesis of testosterone4. In a phase III trial in patients progressing on docetaxel chemotherapy, the combination of abiraterone plus prednisone resulted in an improvement in overall survival compared to patients receiving prednisone plus placebo (14.8 months vs 10.9 months, HR=0.646;P<0.001)5. The PSA response to the active drug combination was 38% vs 10% for the placebo group. A second phase III trial testing abiraterone with prednisone vs placebo and prednisone in the castration resistant stage in patients who are chemotherapy naïve has completed accrual (COU-AA-302; NCT00887198). These results re-enforce the rationale behind previous similar clinical attempts in which non-selective and reversible inhibitors of sex steroid biosynthesis such as ketoconazole in combination with hydrocortisone were used6 to target androgen biosynthesis after failure of androgen deprivation therapy. Ketoconazole inhibits the cholesterol side chain cleavage enzyme 11 beta hydroxylase as well as CYP 17, albeit reversibly7. While no overall survival difference for the study group was demonstrated in this study, an increased survival was observed in patients treated with ketoconazole experiencing PSA responses (35% with a 50% decline in PSA). Interestingly, CYP 17 inhibition with abiraterone in CRPC patients may retain activity even in patients previously treated with ketoconazole8. Adverse events with abiraterone use include hypokalemia, hypertension, peripheral edema and headaches all sequelae of secondary mineralocorticoid excess which can be minimized with the use of combination prednisone. Additional drugs in development include Ortrenol (previously named TAK-700; Millenium Pharmaceuticals, Cambridge, MA/Takeda, Osaka, Japan), which inhibits CYP 17, 20 lyase more potently than 17-hydroxylase. Two on-going large phase III studies with ortrenol in combination with prednisone in chemotherapy naïve (ClinicalTrials.gov Identifier: NCT 01193244) and in the post docetaxel chemotherapy (ClinicalTrials.gov Identifier: NCT01193257) CRPC populations will evaluate response of the study combination vs prednisone plus placebo with overall survival as the primary endpoint. Another novel drug is ToK-001 (Tokai Pharmaceuticals, Cambridge, MA) which is a combined inhibitor of CYP 17 and AR currently in early clinical phase II trials. Resistance to abiraterone has been observed in patients initially responding to the drug. Xenograft models suggest that the resistance may occur through upregulation of CYP17A1 and/or induction of AR and AR splice variants that confer ligand independent AR transactivation9. Targeting the AR with next generation AR inhibitors has also emerged as a potential therapeutic maneuver in CRPC. MDV310010 (Medivation, San Francisco, CA), a second generation anti-androgen, is now in phase III clinical trials. This new diarylthiohydantoin compound targets AR by binding over-expressed AR with an affinity that is several fold greater than previously obtained with anti-androgens (bicalutamide and flutamide). Another drug, ARN-509 (Aragon, San Diego, CA) is currently in phase I/II clinical trials (NCT01171898) and further development of this drug is to be determined based on the completion of the on-going dose establishing trial. Both these drugs disrupt the nuclear translocation of AR and impair DNA binding to androgen response elements and recruitment of co-activators and thus have multi-functional anti-tumor capabilities10. Results of a phase III study with the primary endpoint of overall survival evaluating MDV3100 vs placebo in the post chemotherapy, castration resistant stage which has completed accrual is now awaited (NCT00974311). Another phase III study is currently accruing patients after androgen deprivation failure who are chemotherapy naïve evaluating MDV3100 vs placebo also with the primary endpoint of overall survival (NCT01212991). In conclusion, the AR signaling axis remains active during the progression to CRPC and should be considered to be a viable target for therapy. References: 1. Mohler JL. Castration-recurrent prostate cancer is not androgen-independent. Adv Exp Med Biol. 2008;617:223–234. 2. Montgomery RB, Mostaghel EA, Vessella R, et al. Maintenance of intratumoral androgens in metastatic prostate cancer: a mechanism for castration-resistant tumor growth. Cancer Res. 2008;68:4447–4454. 3. Dehm SM, Tindall DJ. Molecular regulation of androgen action in prostate cancer. J Cell Biochem. 2006;99:333–344. 4. Reid AH, Attard G, Barrie E, de Bono JS. CYP17 inhibition as a hormonal strategy for prostate cancer. Nat Clin Pract Urol. 2008;5:610–620. 5. de Bono JS, Logothetis CJ, Molina A, et al. Abiraterone and increased survival in metastatic prostate cancer. N Engl J Med. 2011;364:1995–2005. 6. Small EJ, Halabi S, Dawson NA, et al. Antiandrogen withdrawal alone or in combination with ketoconazole in androgen-independent prostate cancer patients: a phase III trial (CALGB 9583). Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2004;22:1025–1033. 7. Haidar S, Ehmer PB, Barassin S, Batzl-Hartmann C, Hartmann RW. Effects of novel 17alpha-hydroxylase/C17, 20-lyase (P450 17, CYP 17) inhibitors on androgen biosynthesis in vitro and in vivo. J Steroid Biochem Mol Biol. 2003;84:555–562. 8. Ryan CJ, Smith MR, Fong L, et al. Phase I clinical trial of the CYP17 inhibitor abiraterone acetate demonstrating clinical activity in patients with castration-resistant prostate cancer who received prior ketoconazole therapy. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2010;28:1481–1488. 9. Mostaghel EA, Marck BT, Plymate SR, et al. Resistance to CYP17A1 Inhibition with Abiraterone in Castration-Resistant Prostate Cancer: Induction of Steroidogenesis and Androgen Receptor Splice Variants. Clinical cancer research: an official journal of the American Association for Cancer Research. 2011;17:5913–5925. 10. Tran C, Ouk S, Clegg NJ, et al. Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science. 2009;324:787–790. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr CN08-01.

Abstract In early clinical trials, vaccination with allogeneic cancer cells enhanced the overall survival in some patients with malignancies. Because the administered vaccine cells were cultured at pO2=20 kPa, it is unclear whether they provided an adequate antigen match to tumor cells in situ where pO2 is generally much lower. Thus, we postulate that hypoxically grown vaccine cells will provide a better antigen match to tumors in situ. We are testing this hypothesis by studying the effects of hypoxia on prostate cancer (CaP) cells. LnCaP and VCaP cells were cultured at pO2=2 kPa or 20 kPa and analyzed by 2-D gel electrophoresis. The difference in pO2 affected the proteome mostly quantitatively (i.e., by change in spot intensity). Using a threshold of fivefold change, we found that hypoxia decreased the levels of thirteen proteins and increased of four in VCaP cells. These results are in line with the reports showing that hypoxia affects expression only of a small fraction of total cellular protein and that the content of total protein is not altered significantly. To determine if the hypoxia-dependent changes affected immune reactivity of the cells, we took advantage of the spontaneous autoantibodies against tumor-associated antigens (TAAs). Hence, we compared the binding to lysates of LnCaP and VCaP cells of antibodies pooled from CaP patients (n=25), healthy controls (n=25), rheumatoid arthritis (n=17), colorectal cancer (n=10) and lung cancer (n=10). CaP patient sera plasma reacted with numerous spots, some even in control groups and thus considered nonspecific. CaP sera plasma specifically bound to seven spots; four were hypoxia-specific. All selected spots were excised from the gel, trypsin-digested, and identified by MALDI-TOF mass spectrometry as heat shock 70 kDa protein 4; protein disulfide isomerase A3; heterogeneous nuclear ribonucleoprotein L and leucine-rich repeat-containing protein 47. With the exception of the latter, the proteins have been identified or validated as TAAs before, but apparently none in CaP. CaP patient antibodies bound to lysates of LnCaP cells grown at pO2=2 kPa, but less than to such VCaP cell lysates. Our results confirmed the findings that hypoxia affects the proteome mostly quantitatively. Lysates of hypoxic CaP cells revealed novel potential TAAs suggesting the potential immunologic relevance of antigens in the hypoxic proteome. Thus, pO2 control can provide a tool for the development of more effective immunotherapy for CaP and possibly other cancers. Support: DOD PC094680 (CRG), PCF Creativity Award (CRG), Mayo Clinic Prostate SPORE 5P50CA091956 (SV-P); Mrs. Adelyn L. Luther, Singer Island, Florida and Mayo Clinic Cancer Center (SV-P). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C223.

Abstract We have recently showed that the immunophilin FKBP5 (also known as FKBP5) as a scaffolding protein that can enhance PHLPP-AKT interaction and facilitate PHLPP-mediated dephosphorylation of AKT-Ser473, negatively regulating AKT activation in vitro. Therefore, FKBP5 might function as a tumor suppressor and levels of FKBP5 would affect cell response to chemotherapy. In the current study, we took a step forward by using pancreatic caner xenograft mice model to show that FKBP5−/− promotes tumor growth and renders resistance to gemcitabine and addition of AKT inhibitors have more significant sensitization effect on FKBP5−/− mice to gemcitabine treatment. The combination of triciribine and gemcitabine resulted in cooperative inhibition of pancreatic cancer cell proliferation and the growth of xenografts in vivo without increasing toxicity, as well as cooperative inhibition of AKT signaling pathways at concentrations that were ineffective as individual agents. These results provide evidence for future clinical studies designed to tailor the therapy based on the biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C15.

Abstract Introduction: Androstenedione is a precursor for estrogen biosynthesis catalyzed by aromatase. Plasma androstenedione concentrations in postmenopausal women with early stage breast cancer display large interindividual variation (Ingle et al., Cancer Res. 70:3278–86, 2010). We performed a GWAS to identify single nucleotide polymorphisms (SNPs) associated with plasma levels of androstenedione in 776 postmenopausal women with resected early stage breast cancer. Methods: Plasma androstenedione concentrations were measured by GC-MS/MS. GWAS was performed using Illumina Human610-Quad BeadChips. The GWAS results were controlled for population stratification (PS) by Eigenstrat analyses. Plasma androstenedione concentrations were associated with genotype by using a quasi-likelihood model with adjustment for PS, BMI, age and other clinical variables associated with androstenedione concentrations (p<0.01). Imputation using HapMap2 data was performed within 200 kb on either side of regions containing SNPs with p<E-05. Results: 563,945 SNPs were analyzed, followed by imputation. The genotyped SNP with the lowest p value, rs4736317 (p=4.61E-06), was on chromosome 8 near the CYP11B1 and CYP11B2 genes. This SNP had a multiplicative effect of 1.128 (95% Cl: 1.07, 1.19) and a MAF of 0.40. After imputation, 20 SNPs with apparent p<E-06 were observed in this same region, with the lowest p value for rs9773022 (p=8.1E-08). We then performed functional genomic studies, and observed that two of the SNPs with low p values (rs4736312, p=1.49E-05 and rs4736350, p=6.34E-06) created apparent Oct-1 transcription factor binding sites (TFBS). Differential nuclear protein binding of Oct-1 to probes for these SNPs was verified with EMS and supershift assays using nuclear extract from A549 and NCI-H295R cells. Treatment of these two cell lines with 10 nM androstenedione led to the disruption of Oct-1 binding with the A (wild type) allele on rs4736312. rs4736312 is located in the 3'-UTR of CYP11B1 15 bp distant from response elements for the progesterone receptor and the glucocorticoid receptor. We also demonstrated that CYP11B1 and CYP11B2 were induced by estradiol in U2OS cell lines that express ER receptor and by androstenedione in H295R cells that express the androgen receptor. Transfection of a reporter gene construct with the rs4736312 SNP sequences into COS-1 and H295R cells suppressed reporter gene activity for the variant SNP sequence as compared to wild type, an observation that may correlate with the higher plasma levels of androstenedione in women (549× 328 vs 410×208 mean×SD, M/ml) with the variant allele. Conclusion: We have identified a series of SNPs in a region of chromosome 8 in and around CYP11B1 and CYP11B2 that are associated with elevated plasma androstenedione concentrations in postmenopausal women with resected early stage breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C114.

Abstract Purpose/Background: The first-line chemotherapeutic temozolomide (TMZ) does not offer any therapeutic benefit in some cases of glioblastoma (GBM) and emergence of resistance leads to tumor progression in almost all patients. While the expression of O-6-methylguanine-DNA-methyltransferase (MGMT) correlates well with TMZ resistance, its expression varies among GBM tumors. The goal of this study is to identify additional genes with potential roles in TMZ resistance. Materials and Methods: Five primary GBM xenografts were generated from patient samples, and subsequent secondary models for resistance were developed by subjecting tumor-bearing mice to escalating doses of TMZ. Triplicate samples from parental and TMZ-resistant xenografts were analyzed for gene expression using the Illumina BeadChip microarray platform. The top 100 differentially expressed genes for each line were considered candidates. Genes were selected for qRT-PCR validation after ontological and pathway analysis. A lentivirus-mediated shRNA knockdown was performed to validate the role of several candidate genes in TMZ resistance in U87 and primary GBM12 cell lines. Knockdown was confirmed with qRT-PCR and western blots. TMZ responsiveness was assessed using CyQUANT and Neurosphere assays. Results: Upregulated MGMT expression was found in 3 of the 5 xenografts (GBM12TMZ, GBM14TMZ and GBM28TMZ) and was mechanistically linked with TMZ resistance in these lines using O6-benzylguanine to reverse the resistance phenotype. Differential expression of other DNA damage response genes included CCND2, CDKN1A, GADD45B, UBB as well as apoptosis regulators NFKB2 and BIRC3. In lines with unchanged MGMT expression (GBM22TMZ and GBM39TMZ), changes in anti-apoptosis genes NFKB2 and MAP3K1, transcription factor SRF and signal transduction gene PRKCDBP were validated by qRT-PCR. In GBM12TMZ, in addition to MGMT upregulation, expression of Ubiquitin B (UBB) was found to be the most significantly down-regulated gene both on microarray (Log2FC = −4.55, Fold change = 0.043) and qRT-PCR (Log2RQ =-4.04, RQ = 0.061). To test a possible role of UBB in TMZ resistance, knockdown was achieved in U87 (knockdown efficiency=91.2%) and in primary xenograft line GBM12 (knockdown efficiency=84.4%). The CyQUANT assay for U87UBB-KD and U87NT treated with escalating doses of TMZ revealed no significant difference in cell survival (10 M TMZ relative survival= 89.7% for U87UBB-KD and 79.2% for U87NT; p-value=0.53). Similarly, a neurosphere assay for GBM12UBB-KD and GBM12NT also yielded no significant difference in cell survival (10 M TMZ relative survival= 25.13% for GBM12UBB-KD and 23.35% for GBM12NT; p-value=0.83). Conclusions: We have used a TMZ resistance xenograft model to identify candidate TMZ resistance genes. The current data provides evidence on the role of MGMT in the development of acquired TMZ resistance while ruling out UBB as potential TMZ resistance gene in GBM. Additional studies are underway to assess the functional significance of other candidate genes, particularly those with differential expression in the setting of constant MGMT expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B68.

Abstract Heat shock protein (HSP) 90 is a molecular chaperone that modulates stability of several client proteins, many of which are key regulators of signaling pathways in glioblastoma multiforme (GBM). The potential for targeting multiple effectors makes HSP90 an appealing therapeutic target for GBM. The biological activity of HSP990, a novel HSP90 inhibitor (Novartis Pharmaceuticals) as single agent was evaluated in a panel of short-term explant cultures derived from primary GBM xenografts (n=10). HSP990 treatment resulted in a dose-dependent growth inhibition in the all xenograft lines, with an IC50 ranging from 5 to 30 nM. A 72 h exposure to HSP990 (10–30 nM) resulted in a dose-dependent decrease in the HSP90 client proteins Akt, Cdc2 and Cdc25C, a decrease in associated post-translational modifications p-Akt (S473), p-CDC2 (Y15), p-CDC25 (S216), and increased expression of HSP70. Additionally, a dose-dependent increase in HSP990-induced apoptosis (Annexin-V positivity), was observed within 24–72 hours of drug treatment. GBM tumors harbor a subpopulation of glioma stem cells that grow as neurospheres in neurobasal media and are considered important for tumor maintenance and progression. The effects of HSP990 were tested in neurospheres derived from 4 of the xenografts tested above (GBM12, GBM39, GB59, GBM12TMZ). These cultures demonstrated potential for tri-lineage differentiation upon serum challenge and had robust secondary neurosphere formation, demonstrating two hallmark features of cancer stem cells. In a bulk-culture neurosphere formation assay, co-incubation with 5 to 10 nM HSP990 was highly effective at suppressing neurosphere formation in all 4 xenograft lines. Notably, in GBM12 neurosphere cultures maintained in either serum-free media or challenged with serum, 30 nM HSP990 was equally effective at promoting up-regulation of HSP70, but exhibited less effect on Akt or Cdc2 in serum-free cultures as compared to cultures supplemented with serum. Incubation of serum-free neurosphere cultures with 5 nM HSP990 (5 days) resulted in increased glioneuronal differentiation exhibiting elevated GFAP, Oct-4 and beta-tubulin expression compared to untreated controls. To test ‘self renewal’ GBM12 neurospheres were treated with HSP990 (10, 30 nM) for 7 days followed by dissociation and plating equal numbers of viable cells at low density in fresh drug-free neurobasal media. Interestingly, HSP990 treatment did not affect the number of secondary neurospheres (surviving fraction: control = 1.00, 10 nM = 1.30, 30 nM = 1.05). Treatment of GBM12TMZ flank tumor xenografts with weekly HSP990 (13 mg/kg P.O.) resulted in a modest 9 day delay in tumor re-growth (40.3 and 49.3 respectively) compared to placebo treatment. In conclusion, HSP990 (a) had anti-proliferative, pro-apoptotic effect on differentiated serum-supplemented xenograft cultures (b) a pro-differentiation effect on serum-free GBM stem cell cultures (c) markedly suppressed neurosphere formation with prolonged drug exposure but not with more limited drug exposure (d) inhibited GBM growth in vivo. Thus, HSP990 may inhibit GBM growth both through direct growth inhibition and through pro-differentiation effects on GBM stem-like cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B100.