Citations

Abstract Nilotinib has been approved to treat newly diagnosed, imatinib-resistant or -intolerant leukemia, but the mechanisms underlying its antileukemia actions remain elusive. Here, we report that nilotinib blocks AML growth through its DNA hypomethylating activity. We showed that, in addition to inducing a reduction of KIT and FLT3 protein expression and phosphorylation, nilotinib downregulated DNMT1, 3a and 3b in Kasumi-1, MV4-11 and C1498 cells. Since DNMT1 is maintenance and de novo DNMT, we focused on DNMT1 and proved that nilotinib impaired DNMT1 expression via Sp1 dysfunction, since the luciferase activity driven by DNMT1 promoter containing Sp1 binding sites was decreased by 2.6 fold by nilotinib, while increased by 10.6 fold after Sp1 overexpression. Electrophoretic mobility shift assay disclosed that nilotinib abolished Sp1 binding on DNMT1 promoter. Dotblot using 5mC antibody revealed global DNA hypomethylation and qPCR showed p15INK4B upregulation in Kasumi-1 (3.4 fold) or MV4-11 (5.2 fold). Bisulfite sequencing found a decrease of DNA methylation on p15INK4B promoter from 100% to 89% in Kasumi-1 or 13% to 0% in MV4-11. The functional assays showed a decrease of colony formation in Kasumi-1 (60%) or MV4-11 (58%), and an increase of cell apoptosis in Kasumi-1 (6.9 fold) or MV4-11 (3.2 fold). We verified these findings in AML patient blasts (n = 3) by showing that nilotinib treatment led to the dephosphorylation of KIT, the downregulation of DNMT1, 3a, 3b and Sp1, the reduction of global DNA methylation and the re-expression of p15INK4B (4.4 fold) via promoter demethylation (from 14% to 6%), which was followed by suppressed colony formation (77%), promoted cell apoptosis (1.84 fold) and increased caspase-3 and caspase-8. To test if nilotinib induces AML regression via DNA hypomethylation in vivo, 0.1×106 C1498 cells were injected into C57BL/6 mice (n = 3 mice/group) via the tail vein. The leukemic mice were treated with 5, 10 or 15 mg/kg of nilotinib in PEG400 plus saline via intraperitoneal injection twice a week for a total of 6 doses. No toxicity was observed for the tested drug dosage and schedule, because of no evident change of mouse body weight. Compared to the control group, nilotinib-treated mice had a decrease in spleen size. H&E staining showed that the control mice exhibited increased infiltration of AML cells into the spleen, lung and liver of recipients. The bone marrow (BM) histopathology identified a higher degree of mature post-mitotic cells containing metamyelocytes, bands and segmented neutrophils in nilotinib-treated mice than control. DNMT1 was downregulated (39%), p15INK4B was upregulated and global DNA methylation was reduced (60%) in BM cells from nilotinib-treated mice. Thus, our findings unveil DNA methylation machinery as an innovative target behind nilotinib's anticancer activities, and provide a new therapeutic avenue for nilotinib in overcoming aberrant epigenetics in AML. Note: This abstract was not presented at the meeting. Citation Format: Na Shen, Fei Yan, Jiuxia Pang, Naseema Gangat, Mark R. Litzow, Aref Al-Kali, Shujun Liu. Nilotinib abrogates DNMT1-depenent DNA methylation: A novel mechanism for induction of AML leukemia regression. [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 2945. doi:10.1158/1538-7445.AM2015-2945

Abstract Background. Oncogenic KRAS mutations occur in nearly one-half of colorectal cancers (CRC) where they contribute to de novo apoptosis resistance and treatment failure. Inhibition of MEK/ERK is a promising strategy to sensitize KRAS mutant tumors to apoptosis that may involve upregulation of pro-apoptotic BH3-only proteins, including BIM. However, BH3-only proteins can be neutralized by anti-apoptotic Bcl-xL that we found was upregulated in isogenic KRAS mutant compared to wild-type HCT116 and DLD1 CRC cells. Aims. We determined the ability and mechanism by which a novel MEK/ERK inhibitor, GDC-0623, can induce BIM to promote apoptotic signaling. A strategy of concurrent induction of BH3-only proteins and antagonism of Bcl-xL was studied to overcome mutant KRAS-mediated apoptosis resistance. Results. Using isogenic KRAS HCT116 cells and KRAS mutant SW620 cells, we found that GDC-0623 can potently induce the BH3-only protein BIM, and to a lesser extent BIK, in both KRAS wild-type and mutant cells. Only KRAS mutant cells remained resistant to GDC-0623-induced apoptosis. Knockdown of Bcl-xL or use of a BH3 mimetic drug, ABT-263, sensitized KRAS mutant cells to GDC-0623-mediated apoptosis. GDC-0623 and ABT-263 were shown to interact synergistically to promote apoptosis in KRAS mutant cells. Dependence on BIM or BIK for synergy between GDC-0623 and ABT-263 was shown by shRNA knockdown that each attenuated drug-induced apoptosis. Mechanistically, ABT-263 treatment was shown to release BIM from its binding to Bcl-xL using immunoprecipitation. The ability of GDC-0623 to induce apoptosis occurred via BIM that was phosphorylated by MEK/ERK activation leading to BIM degradation. GDC-0623 was shown to dephosphorylate BIM at serine 69 in cells with wild-type BIM or a phospho-mimic mutation (S69E). However, GDC-0623's ability to dephosphorylate BIM was lost in cells containing a phosphorylation-defective BIM mutation (S69G) that showed delayed BIM degradation. Conclusion. GDC-0623 was shown to dephosphorylate BIM to enhance its accumulation and to promote apoptosis. Concurrent induction of BIM/BIK and antagonism of Bcl-xL can overcome mutant KRAS-mediated apoptosis resistance, suggesting a promising therapeutic strategy against KRAS mutant CRCs. Note: This abstract was not presented at the meeting. Citation Format: Aziz Zaanan, Koichi Okamoto, Hisato Kawakami, Shengbing Huang, Frank Sinicrope. MEK/ERK inhibitor GDC-0623 dephosphorylates and accumulates BIM that enables a synergistic apoptosis with the Bcl-xL antagonist, ABT-263, in mutant KRAS colorectal cancer cells. [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 2937. doi:10.1158/1538-7445.AM2015-2937

Abstract Introduction: Overexpression and amplification of PD-L1 has been reported in a subset of cancers, including lymphoma, and gastric cancer. We have recently described amplification of chromosome 9p24 targeting PD-L1 in a subset of triple negative breast cancers (TNBC), as well as 1-3% of colorectal carcinomas and glioblastomas. The clinical impact of these amplifications is not known. Methods: Tumor samples from 41 subjects with TNBC were evaluated for copy number aberrations. We performed DNA-based flow cytometry to sort nuclei from diploid, tetraploid, and aneuploid populations of tumor cells. The genomes of each sorted tumor cell population were then interrogated with oligonucleotide CGH arrays. Copy number intervals were identified and their genomic boundaries mapped using a step gram algorithm. Clinical data was available on 36 of 45 (80%) patients. Results: A recurring top ranked high level (log2ratio >1) amplicon that spanned 9p24.1 was detected in 12/41 (29.3%) of TNBCs. In contrast this amplicon was absent in ER+ (n = 8) and Her2+ (N = 15) breast tumors. The shortest region of overlap (SRO) spanned 777 kb and always included the PD-1 ligands PD-L1, PD-L2, and Janus kinase 2 (JAK2) loci, termed the “PDJ” amplicon. Additional genes in this amplicon include RCL1, MIR101-2, INSL6, INSL4, RLN2, RLN1, and C9orf46. The height of the PDJ amplicon included mean log2ratios of >4, consistent with genomic drivers such as Her2 and Myc. Patients with PD-L1 amplification (n = 8) were noted to have larger tumors (mean 3.9 vs. 1.9 cm, p = 0.04) and a higher incidence of lymph node metastases (75% vs. 26%, p = 0.01). Lymphocytic infiltration was only noted in 4 patients, all without PDJ amplification. Almost all patients received chemotherapy after definitive surgical therapy. The recurrence rate at 5 years was 62.5% in the PDJ amplified patients, and 25% in the unamplified patients (p = 0.02). Overall survival at 5 years was 37.5% in the PDJ amplified patients, compared with 78.2% in the unamplified patients (p = 0.02). Conclusion: We identified a focal amplification of chromosome 9p24.1 involving PD-L1, PD-L2, and JAK2 in a significant proportion of TNBCs. The high level of amplification and inverse correlation with survival suggests that the PDJ amplicon is a genetic driver event in these cancers, and warrants confirmation in larger studies. Citation Format: Karen S. Anderson, Christine L. Klassen, Amylou C. Dueck, Ann E. McCullough, Donald W. Northfelt, Mariacarla Andreozzi, Barbara A. Pockaj, Michael T. Barrett. High-level amplification of chromosome 9p24 targeting PD-L1 and JAK2 correlates with worse DFS and OS in triple negative breast cancer. [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 2861. doi:10.1158/1538-7445.AM2015-2861

Abstract Background/purpose: Despite recent advances in the development of androgen deprivation therapies (ADT), prostate cancer (PCa) remains the second leading cause of cancer death in American men. Thus, there is a critical need to improve treatment outcomes for men with aggressive PCa. The purpose of this study was to identify and functionally characterize kinases that can serve as both prognostic biomarkers and therapeutic targets in PCa. Methods: To identify kinases associated with metastatic progression of PCa, we utilized high-density oligonucleotide arrays to interrogate the expression of all kinases in 545 prostatectomy samples, obtained from high-risk patients with long-term clinical follow-up (>10 years). We ranked all kinases by their fold change in expression between PCas that subsequently metastasized versus those that did not. We then characterized the top ranked kinase in preclinical PCa models with in vitro mechanistic experiments and in vivo therapeutic studies. Results: We nominated DNA-dependent protein kinase (DNAPK) as the top kinase associated with metastatic PCa progression. Interrogation of pathways associated with DNAPK knockdown in PC3, DU145, VCaP and C4-2B, identified canonical Wnt signaling as the top DNAPK-activated pathway. In PCa cells (PC3, LNCaP C.S. {LNCaP grown under charcoal-stripped serum condition}, C4-2B and LNCaP-AR) DNAPK inhibition significantly abrogates Wnt signaling and abolishes cell proliferation, migration and invasion. Interestingly, we found that DNAPK directly modulates the Wnt signaling independent of the androgen receptor (AR), highlighting the therapeutic potential of DNAPK inhibition in cancers resistant to ADT. Indeed, in VCaP xenograft models, pharmacologic inhibition of DNAPK with NU7441 demonstrated 3.4 fold (p<0.01) reduction of tumor growth at non-toxic doses. Using an independent prostate cancer tissue cohort, we validate DNAPK expression as a biomarker for metastatic disease progression (p = 6.4 e−06, hazard ratio = 2). Conclusion: In summary, we nominate DNAPK as a potential biomarker of disease progression and as a novel therapeutic target in aggressive prostate cancer. Our data suggests that DNAPK mediates PCa progression by upregulating Wnt signaling, and demonstrates that DNAPK inhibition results in significant tumor responses in PCa xenografts. Citation Format: Vishal Kothari, Jonathan F. Goodwin, Shuang Zhao, Elai Davicioni, Jeffrey R. Karnes, Robert B. Den, Rohit Mehra, Karen E. Knudsen, Felix Y. Feng. Investigating DNAPK as a biomarker and a novel therapeutic target in aggressive prostate cancer. [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 2853. doi:10.1158/1538-7445.AM2015-2853

Abstract Genome-wide association studies (GWAS) have identified several susceptibility regions that confer risk of two or more cancers. A region at 19p13 is associated with both breast and ovarian cancer risk in the general population as well as in BRCA1/BRCA2 mutation carriers. Using the iCOGs custom genotyping array, we analyzed 438 SNPs in this region in 7,435 ER-negative breast cancer cases and 42,599 controls from BCAC, and 15,347 ovarian cancer cases and 30,845 controls from OCAC and 15,252 BRCA1 mutation carriers. We identified two significant peaks of association. Peak 1, consisting of 13 highly correlated SNPs (r2>0.95), spans a 19.4kb region harboring the BABAM1, ANKLE1 and ABHD8 genes. Peak 1 SNPs are associated with serous ovarian cancer (top hit rs4808075, P = 9.2×10-20), ER-negative breast cancer (rs61494113, P = 1.1×10-13) and breast cancer in BRCA1 mutation carriers (rs61494113, P = 7.7×10-16). Peak 2 spans over 100kb and contains 7 SNPs associated with breast cancer in BRCA1 mutation carriers only (rs4464206, P = 8.9×10-8). Multiple assays were used to establish the putative functional roles of risk SNPs and candidate susceptibility genes in this region. Expression quantitative trait locus (eQTL) analyses identified significant genotype-gene expression correlations between genotype at the Peak 1 SNP rs480816 and ABHD8 expression in ovarian tumors (P = 3.0×10-5) and normal breast tissues (P = 2.8×10-3). The Peak 2 SNP, rs7246243, was significantly associated with BABAM1 expression (P = 5×10-3) in normal breast tissue only. Overexpression of BABAM1 in normal ovarian and breast cells did not modulate neoplastic phenotypes, whereas overexpression of ABHD8 induced a significant reduction in migration in both cell types in vitro. Differentially expressed genes in ABHD8 models, identified by RNAseq, were associated with cellular movement (P<1.82×10-44). Chromosome conformation capture (3C) identified an interaction between the ABHD8 promoter and a region containing 4 risk SNPs (rs4808075, rs10419397, rs56069439 and rs4808076) in breast and ovarian normal and cancer cells but only rs56069439 coincides with epigenetic enhancer marks in breast and ovarian cells. CRISPR/Cas9 genome editing was used to delete a 57bp region containing rs56069439 and expression of BABAM1, ANKLE1 and ABHD8 were measured. Taken together, these data suggest a common functional mechanism underlying risk of breast and ovarian cancer at this locus, mediated by rs56069439 variants differentially regulating expression of ABHD8, a gene involved in cell motility. Citation Format: Kate Lawrenson, Siddhartha Kar, Karoline Kuckenbaeker, Stacey Edwards, Qiyuan Li, Jonathan Tyrer, Jonathan Beesley, Kyriaki Michailidou, Susan Ramus, Alison Dunning, Fergus Couch, Alvaro Monteiro, Jacques Simard, Matthew Freedman, Douglas Easton, Georgia Chenevix-Trench, Paul Pharoah, Antonis Antoniou, Simon Gayther, OCAC, BCAC and CIMBA. Common functional mechanisms underlying pleiotropy at the 19p13.1 breast and ovarian cancer cusceptibility locus. [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 2783. doi:10.1158/1538-7445.AM2015-2783

Abstract Purpose: Clustering of breast and colorectal cancer has been observed within families in population-based studies giving rise to speculation that there are “breast-colon” cancer susceptibility genes. We performed a genome-wide association study (GWAS) to identify genetic markers associated with a potential breast-colon cancer phenotype. Methods: Cases and controls were ascertained from the Breast and Colon Cancer Family Registries’ (CFR) GWAS subjects. “Breast-colon phenotype” definition was based on 2 or more breast and colon cancer cases diagnosed in first- or second-degree relatives within a family. Cases (n = 985) were women with a history of breast cancer and a family history of colorectal cancer, or persons with colorectal cancer with a family history of breast cancer. Unrelated controls (n = 1769) were frequency matched to cases for age and gender. Following standard quality control measures, 6,220,060 directly measured and imputed single nucleotide polymorphisms (SNPs) were included in the discovery set. SNPs associated at p<1×10−5 were analyzed in a replication dataset of cases (n = 293) and controls (n = 2103) from the Genetics and Epidemiology of Colorectal Cancer Consortium. Results: In a regression model that included gender, age, CFR center and 6 principal components, the top association in the discovery set was in the chromosome 8q22.3 region overlying the BAALC gene (top SNP rs12548629, P = 3.63×10−7). In the replication dataset, of 341 SNPs tested, the top signal was found in multiple correlated SNPs overlying the ROBO1 gene on chromosome 3p12 (rs7429100, P = 2.8×10−3), however, the signal on chromosome 8, the BAALC gene did not replicate. In the discovery set, P values for the SNPs overlaying ROBO1 gene ranged from 2.2×10−5 to 9.7×10−5 (rs7429100, P = 3.9×10−5). The combined meta-analysis showed strongest association at the ROBO1 gene (rs7429100, Pfixed effects 1.84×10−6, Pheterogeneity >0.05 for testing for heterogeneity between the overall discovery set and the replication set). ROBO1 (roundabout, axon guidance receptor, homolog 1) is a transmembrane receptor of the immunoglobulin family and is differentially expressed in human cancers, with a possible role as a tumor suppressor gene. Low ROBO1 expression has been shown to be an adverse prognostic factor for invasive ductal breast cancer and may also play a role in pathogenesis of colorectal cancer. BAALC (brain and acute leukemia, cytoplasmic) is predominantly expressed by neural and blood cells and is largely implicated in acute leukemia. Conclusion: In this exploratory analysis, to elucidate genes/regions associated with pleiotropic effect for breast and colorectal cancer risk we identified germline variation in the region of ROBO1 and BAALC. Validation in a larger dataset and functional characterization of the loci is warranted to elucidate mechanisms by which these genes/SNPs may contribute to the development of breast and colorectal cancer. Citation Format: Mala Pande, Aron Joon, Sanjay Shete, Abenaa M. Brewster, Cathy Eng, Wei V. Chen, Habibul Ahsan, Irene L. Andrulis, Esther M. John, Yi Lin, Polly A. Newcomb, Noralane M. Lindor, Christopher I. Amos, John Hopper, Patrick M. Lynch. Risk loci for a breast-colon cancer phenotype: results from a genome-wide association study. [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 2782. doi:10.1158/1538-7445.AM2015-2782

Abstract Introduction: Increased levels of circulating estrogens are associated with elevated risks of postmenopausal breast cancer. Previously, we showed that levels of sex steroid hormones in breast adipose tissue are positively correlated with circulating hormones. Further evaluation of hormone levels in breast tissues may help to explain the relationship between estrogens and breast cancer risk. Specifically, data suggest that focal breast lesions consisting of macrophages encircling dying adipocytes, referred to as “crown-like structures (CLS)”, are associated with increased aromatase activity, which could increase conversion of androgens to estrogens locally in the breast. Therefore, we assessed whether the presence of CLS affects the correlations between blood and breast adipose tissue hormone levels. Methods: Participants included 84 postmenopausal women with invasive breast cancer in the Polish Breast Cancer Study, a population based case-control study conducted from 2000 to 2003. We previously reported sex steroid hormone levels in cancer-free breast adipose tissue removed as part of breast excisions performed for cancer. Here, using immunohistochemical stains, we evaluated the number of CD68 positive macrophages and the presence of full CLS (i.e., complete adipocyte encirclement) in benign breast tissue sections prepared from surgical pathology specimens containing breast cancer. The relationship between the number of CD68 positive macrophages per unit area of fat and hormone levels was examined using Spearman rank correlations (rs). We assessed potential effect modification of the association between blood and tissue hormone levels by presence or absence of CLS by including an interaction term in linear regression models. Results: The number of CD68 positive macrophages per unit area of fat was not significantly correlated with levels of estrone, estradiol, androstenedione or testosterone measured in blood (rs = 0.001, -0.04, -0.07, and -0.09, respectively; p>0.05) or breast adipose tissue (rs = -0.15, 0.06, -0.06, and -0.08, respectively; p>0.05). Correlations between serum and adipose tissue hormones did not statistically significantly differ between women with CLS (n = 30) compared to those without CLS (n = 54). CLS were found more frequently in breast adipose tissue from overweight (i.e., women with a body mass index (BMI) between 25 and 29.9) (43%) and obese women (BMI >30) (30%) versus normal weight women, with (BMI between 18.5 and 24.9), (27%); (p = 0.04, χ2 test). The number of CD68 positive macrophages per unit area of fat was not significantly related to BMI (p = 0.31). Conclusion: Among postmenopausal women with invasive breast cancer, the presence of CLS was more common among overweight and obese women, but CLS did not influence the correlation between circulating and breast adipose tissue sex steroid hormone levels. Citation Format: Maeve Mullooly, Hannah P. Yang, Roni T. Falk, Sarah Nyante, Renata Cora, Ruth M. Pfeiffer, Derek C. Radisky, Daniel W. Visscher, Lynn C. Hartmann, Amy C. Degnim, Frank Z. Stanczyk, Jonine F. Figueroa, Montserrat Garcia-Closas, Jolanta Lissowska, Melissa T. Troester, Louise A. Brinton, Mark E. Sherman, Gretchen L. Gierach. Investigation of the relationship between crown-like structures and adipose tissue hormone levels among postmenopausal women with breast cancer. [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 2767. doi:10.1158/1538-7445.AM2015-2767

Abstract Introduction Chronic lymphocytic leukemia (CLL) is a B-cell malignancy that is known to have a familial component to disease risk. Although 31 loci have been found to be associated with CLL risk, the functional variant(s) driving these associations is mostly unknown. Here we set out to identify rare, highly-penetrant, cosegregating, susceptibility variants within the known GWAS discovered loci using whole exome sequencing (WES) data in CLL families from the Mayo Clinic family study of B-cell malignancies. Methods We performed WES on germ line DNA of 93 CLL families with two or more members with CLL, using Agilent capture kits and Illumina HiSeq2000. Bioinformatics analyses leveraged the following software packages: Novoalign, Picard, The Genome Analysis Toolkit (GATK), and the Biological Reference Repository (bioR). Quality control filters were implemented; subjects with mis-specified relationships were removed, as were variants with <75% call rate, <8X coverage, and those identified as sequencing artifacts. Each GWAS locus was defined by +/- 1Mb of the top GWAS hit within the locus. Linkage disequilibrium (LD) was calculated among the single nucleotide variants (SNVs) located within each locus. Potentially functional SNVs were identified based on: a) uncommon in public databases (< 5%), b) cosegregating in at least two CLL families, c) being highly conserved and in coding regions, and d) functional prediction status of deleterious (SIFT Score), damaging (PolyPhen Score), and a moderate, or high variant impact (SNP Effect). Results In our 93 CLL families, we sequenced 443 individuals: 160 with CLL, 73 with monoclonal B cell lymphocytosis (MBL), and 210 relatives that were not diagnosed with CLL or MBL at the time of sequencing. Median age of CLL diagnosis was 59 years (range 34-87), and 56% were male. Among the MBL individuals and relatives, the median age at recruitment was 55 years (range 18-93), and 40% were male. A total of 317,666 SNVs passed our sequencing quality control filters of which 10,731 were within +/- 1 Mb of known GWAS hits from 31 loci. Of these SNVs, 91% were in coding regions, 18% were reported to have high or moderate impact, 6% were estimated to be damaging and 6% were predicted to be deleterious. From these SNVs, we identified 76 putatively functional variants distributed across 25 GWAS loci that were cosegregating in the individuals with CLL or MBL in multiple CLL pedigrees. These SNVs were all located in coding regions with high or moderate impact and were predicted to be damaging and deleterious. Of these 76 variants, 56 had a frequency of <0.005 in 1000 Genomes’ European population while the remaining 20 had a frequency of 1%. Conclusions Through WES, we identified a number of rare, penetrant and potentially predisposing SNVs located within 25 of the 31 CLL GWAS-discovered loci. These segregating variants provide a list for future validation and functional studies. Citation Format: Sara Beiggi, Daniel R. O'Brien, Sara J. Achenbach, Kari G. Chaffee, Timothy G. Call, Neil E. Kay, Tait D. Shanafelt, Julie Cunningham, James R. Cerhan, Celine M. Vachon, Susan L. Slager. Cosegregating variants in chronic lymphocytic leukemia (CLL) families that are located in loci discovered by genome wide association studies (GWAS). [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 2764. doi:10.1158/1538-7445.AM2015-2764

Abstract Recently, Wilson et al. (doi:10.1038/onc.2013.396) found that EYA4 was frequently inactivated biallelically in sporadic lung cancer, displays tumor suppressor gene-like properties and affects DNA repair, and that 5 of 17 common SNPs in this 0.3Mb region showed nominal association to familial lung cancer (FLC, p<0.05) although these associations are not genome-wide significant after correction for multiple testing.. However, this is an interesting candidate gene for FLC risk because it is located on chromosome 6q in a region where we previously showed evidence of linkage to FLC (PMID: 15272417, 20215501). Here, we sequenced 75 individuals from our 9 most strongly 6q-linked families, using Illumina technology and a custom Agilent kit to capture 37Mb of chromosome 6 from 130Mb to 167Mb. We closely examined a region around EYA4, between 133Mb and 135Mb. This region contains a number of other genes in addition to EYA4, including the Vanin cluster; ribosomal protein S12; the tumor suppressor gene TCF21 and its antisense RNA TARID which directs methylation and activation of TCF21; solute carriers SLC18B1 and SLC2A12 and the serum/glucocorticoid regulated kinase SGK1. To detect sharing of variants among affecteds in each family while allowing for phenocopies, singlepoint affected-only linkage analysis was performed on lung cancer affection status for all sequence variants using the Elston-Stewart algorithm implemented in the R package paramlink assuming penetrance of 0.01, 0.1 and 0.1 for dd/Dd/DD and a disease allele (D) frequency of 0.01, at a recombination fraction (theta) of 0. The highest total LOD score (summed over all 9 families) in the entire 37Mb targeted region was not near EYA4. In only one family was there any variant in EYA4 that appeared on the linked haplotype with close to the highest family-specific LOD score: Family 30 had a LOD of 0.6514 at several EYA4 variants (max LOD across region 0.6519). However, many variants across the region had similar LODs. In the remaining families, all EYA4 variants had LODs much lower than the maximum family-specific LOD score in the region. Thus, EYA4 variants are unlikely to explain our 6q linkage peak in these 6q-linked families. The second highest total LOD score for the entire 37Mb targeted region was found near EYA4 at 133.9Mb (LOD = 3.82), in the middle of the TARID antisense RNA gene. This variant appears to be entirely novel, as it is not present in any of the online variant databases, such as NHLBI's ESP or 1000 Genomes. In two families, the family LOD score at this locus was at or close to the highest in the region: in Family 44 the LOD score was 1.206 (max LOD across entire region 1.207) and in Family 35 the LOD score was 0.3958 (max LOD across region 0.3959). Therefore this variant in the TARID antisense RNA is a candidate in some of our families most strongly linked to chromosome 6q. Citation Format: Joan E. Bailey-Wilson, Claire L. Simpson, Susan M. Pinney, Mariza de Andrade, Colette Gaba, Ping Yang, Ming You, Elena Y. Kupert, Ann G. Schwartz, Diptasri Mandal, Christopher I. Amos, Marshall W. Anderson. Evaluation of EYA4 as a candidate risk locus in familial lung cancer families linked to 6q. [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 2757. doi:10.1158/1538-7445.AM2015-2757

Abstract Metabolic alterations in cancer are increasingly being recognized as important novel targets. Among different regulators of altered metabolism, 6-PhosphoFructo-2-Kinase/Fructose-2,6-Bisphosphatases 3 (PFKFB3) a glycolytic enzyme required for tumorigenic growth, and produces fructose-2,6-bisphosphate (F2,6BP), is recognized as important regulator of glycolysis. We determined that the active form of PFKFB3ser471 was overexpressed in chemo-resistant ovarian cancer (OVCA) cell lines and resistant patient derived xenograft (PDX) models compared to their isogenic chemo-sensitive cell lines and sensitive PDX models. These results suggest that targeting PFKFB3 will have beneficial effects in patients with OVCA. PFK-158 is a potent and selective inhibitor of PFKFB3 that is currently being investigated in a Phase 1 study in patients with advanced solid malignancies. Here we report for the first time, PFK-158 dose-dependently inhibits cell proliferation and colony formation ability in both carboplatin and paclitaxel (PTX) resistant cell lines. PFK-158 markedly suppresses glycolysis by reducing glucose uptake, ATP production, lactate dehydrogenase activity and lactate secretion. Interestingly, both Chou-Talalay synergy analysis and apoptosis assay by annexin V-PI staining showed PFK-158 strongly synergies with both PTX and carboplatin in PTX and cisplatin resistant cell lines respectively. PFK-158 induced apoptosis through a significant increase in caspase-3/7 activation and PARP cleavage. Moreover, in a highly metastatic PTX-resistant in vivo model, combination of PFK-158 with carboplatin and PTX significantly reduced tumor weight, ascites and metastasis compared to untreated and PFK-158, carboplatin or PTX alone treated mice. Since the majority of OVCA patients recur with chemo-resistant disease, our results suggest that PFK-158 in combination with standard chemotherapy may have clinical utility in late stage ovarian cancer patients. Citation Format: Susmita Mondal, Debarshi Roy, Eleftheria Kalogera, Ashwani Khurana, Gilles H. Tapolsky, Sucheta Telang, Jason Chesney, Viji Shridhar. Inhibition of PFKFB3/glycolysis overcomes chemoresistance in ovarian cancers. [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 2706. doi:10.1158/1538-7445.AM2015-2706