Citations

Abstract Next-generation sequencing is now commonly applied to measure genomic modifications, providing the opportunity for a more accurate view of cancer progression. That being said, several challenges including tumor purity, heterogeneity and the broad set of cancer associated cellular functions, increase the difficulty of interpreting cancer genomes and make analyzing them distinct from analysis of germline diseases. The ability to detect low allelic mutations in the broad set of cancer-associated genes is critically important. To address these issues, we developed an extended cancer panel, a targeted enrichment sequencing platform that includes over 1,300 cancer genes and 200 miRNAs that improves coverage in traditionally difficult to sequence NGS regions. In this study, we apply an integrated RNA/DNA analysis approach as well as our extended cancer panel to explore mutations in metastatic renal cell carcinoma (RCC) tissue samples before and after treatment. Our goal with this study was to test the importance of integrating DNA and RNA analysis, which allows for detection of mutational events that cannot be observed by either method alone. With RNA analysis it is possible to detect gene fusion events, expression of small variants (SNVs/indels), and gene expression levels while DNA analysis allows for detection of loss of heterozygosity (LOH), copy number variants (CNVs), and small variants. By integrating both the RNA and DNA analysis, it is possible to identify unique variant classes, such as unexpressed variants, allele specific expression (ASE), effects of CNVs on gene expression levels, and how fusion gene integration sites affect CNVs. In our study, each variant class was interpreted pre- and post-treatment, allowing for optimal analysis of treatment effectiveness. By applying both our RNA/DNA integration approach and our extended cancer panel to investigate metastatic RCC samples, we were able to classify many genomic alterations that drive cancer progression. For example, we found that anywhere from 5% to 30% of small variant mutations in driver genes called in the DNA reside in genes that are unexpressed in the RNA. Likewise, we noted multiple cases of ASE, where high profile variants observed in DNA were entirely absent in the RNA despite high expression levels. Strong correlations between CNVs observed in DNA and gene expression changes found in RNA were also detected. We also observed that about half of the RCC samples we tested were biallelic for VHL mutations, suggesting inherited predisposition combined with second hit somatic mutations, facilitating cancer progression. This analysis demonstrates how combining an integrated RNA/DNA approach with a cancer focused augmented enrichment panel allows for detection of both low allelic representation variants and unique variant classes, both of which are critical for accurate interpretation of cancer samples. Citation Format: Sean M. Boyle, Michael J. Clark, Elena Helman, Alexander S. Parker, Thai Ho, Shujun Luo, Scott Kirk, Parin Sripakdeevong, Mirian Karbelashvili, Deanna M. Church, Michael Snyder, John West, Rich Chen. Integrating RNA/DNA analysis with a comprehensive cancer panel to improve interpretations of stage four metastatic renal cell carcinoma. [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 3899. doi:10.1158/1538-7445.AM2015-3899

Abstract Introduction: Pulmonary carcinoid tumors account for up to 5% of all lung malignancies in adults, and comprise 30% of all carcinoid malignancies. They are defined histologically as typical carcinoid (TC) and atypical carcinoid (AC) tumors, and are characterized by neuroendocrine differentiation and the potential to metastasize. Method: We characterized genomic alterations in pulmonary carcinoid tumors using whole genome and exome sequencing in addition to mRNA expression and SNP genotyping from specimens of normal lung, typical and atypical carcinoid, and SCLC. Results: Analysis of the sequencing data identified novel mutations in the pulmonary neuroendocrine tumor spectrum, including KRAS, ABCA9, GALNT10, HSFX1, PTHLH, RANBP2, BRIP1, FXR1, BACE2, NCOR2, DGKI, HERC1, HERC5 and GLI3. Pathway analysis found 45% of the cases to harbor a mutation in genes that are involved in protein-protein interaction with ubiquitin c (UBC) and may therefore play a role in ubiquitin protein degradation pathway. We also identified genes differentially expressed in corresponding regions of copy number variation, 52 of which were linked to ubiquitination pathways in 88% of the samples genotyped. Genes amplified in 15 to 23% of the cases included ITM2A, MAOA, EGR2, HAGH, TSC22D3, NBEA, TCEAL2, and TSPYL2 whereas deleted regions in 15% of the cases implicated CFD, GDI1, IFITM1 and IFITM2. Conclusion: These results suggest that in addition to previously identified pathways, dysregulation of the ubiquitin pathway may contribute to lung carcinoid tumorigenesis and could be exploited for therapeutic purposes. Citation Format: Michael K. Asiedu, Charles L. Thomas, Jie Dong, Prasidda Khadka, Zhifu Sun, Farhad Kosari, Jin Jen, Julian Molina, George Vasmatzis, Marie Christine Aubry, Ping Yang, Dennis Wigle. Genomic alterations modify ubiquitination pathways in pulmonary carcinoid tumors. [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 3895. doi:10.1158/1538-7445.AM2015-3895

Abstract Background: Hepatocellular carcinomas (HCCs) are heterogenous primary liver cancers with pleomorphic histologic features. We present a comprehensive genetic, genomic, transcriptomic and proteomic analysis of 203 HCCs in The Cancer Genome Atlas Program (TCGA) on behalf of the TCGA Liver analysis working group to provide a deeper understanding of the molecular features of HCCs, to classify them in a clinically-relevant manner, and to provide a public resource that identifies potential targets for emerging therapies.Results: Mutation analyses confirmed the presence of TERT promoter mutations in 40% of HCCs. The analysis also revealed the first reported germline mutation in the TERT promoter in a 30 year old male, possibly reflecting an inherited predisposition to HCC. TERT promoter mutations were enriched in hepatitis C virus infected subjects. Analyses of hepatitis B virus (HBV)-induced HCCs demonstrated recurrent integrations of HBV in the TERT locus. Thus, HBV integration may substitute for TERT promoter mutations as an alternate mechanism of telomerase activation. Other tumor suppressor genes inactivated by mutation included the cell cycle genes TP53 (32% of cases), RB1 (8%), and PTEN (3%), the Wnt/beta catenin pathway gene AXIN1 (4%), and the chromatin remodeling genes ARID1A (9%), BAP1 (6%), ARID1B (4%), ARID2 (4%), and PBRM1 (3%). The oncogene CTNNB1 was mutated in 27% of HCCs. Mutations were also found in the MTOR pathway genes, MTOR (4%), TSC2 (4%) and TSC1 (3%). The patterns of mutational signatures observed included signatures characteristic of the known carcinogens aristolochic acid and aflatoxin B1. Clustering of mRNA, miRNA, or protein expression, DNA hypermethylation and DNA copy number profiles identified five, four, two, five and three clusters, respectively. Analysis of mRNA expression data identified five groups. In particular, Group 1 was associated with Asian race, higher histologic grade, and micro- and macrovascular invasion. Integrated clustering showed a grouping of HCCs into three superclusters: iCluster 1 had a low frequency of CDKN2A silencing, lack of CTNNB1 mutation, a normal-like methylation pattern, and overexpression of proliferation marker genes; iClusters 2 and 3 had a high frequency of CDKN2A silencing, CTNNB1 mutation, and overexpression of oxidative response genes; iCluster 3 tumors had a higher degree of chromosomal instability, high frequency of 17p loss, and a highly altered methylation profile. Copy number aberrations included gains in the MYC, VEGFA, CCND1, TERT, and MET loci and losses in the TP53, RB1, ERFFI1, CDKN2A, ARID1A and PTEN loci. Discussion: Based on integrated analysis of genome, transcriptome, methylome and proteome we showed that HCCs naturally separate into three to five distinct groups with associated molecular and clinical characteristics. Importantly, we identified a novel germline mutation in the TERT promoter that may be associated with an inherited risk for HCC. Note: This abstract was not presented at the meeting. Citation Format: Lewis R. Roberts, David A. Wheeler. Comprehensive integrative characterization of hepatocellular carcinoma: The TCGA HCC project. [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 3745. doi:10.1158/1538-7445.AM2015-3745

Abstract Immunomodulatory drugs (IMiDs) are therapeutically active compounds that bind to the E3 ubiquitin ligase substrate recruiter cereblon (CRBN) and induce many cellular responses, including cytotoxicity. We had found that the expression of CRBN in multiple myeloma (MM) cells provided the basis for their responses to IMiDs. In this report, we have identified argonaute 2 (AGO2) as a new member of CRBN-downstream binding protein and found that: 1) the steady-state levels of CRBN in IMiD-sensitive cells are significantly higher than in IMiD-resistant cells; 2) the steady-state levels of AGO2 in IMiD-sensitive cells are significantly less compared to IMiD-resistant cells, suggesting that CRBN plays a role in regulating AGO2. AGO2 is a component of microRNA (miRNA)-induced silencing complexes and plays an important role in miRNA maturation and function. The matured miRNAs are considered as key regulators of almost all cellular pathways. Treatment of IMiD-sensitive MM cells with lenalidomide, an IMiD, significantly increased CRBN, decreased both AGO2 and miRNAs and inhibited cell growth. In contrast, although treatment of IMiD-resistant MM cells with lenalidomide moderately increased CRBN and decreased both AGO2 and miRNAs, this treatment did not significantly inhibit cell growth. However, silencing of AGO2 with small hairpin RNA significantly decreased both AGO2 and miRNAs and inhibited cell growth regardless of whether the cells are sensitive or resistant to IMiDs. Therefore, AGO2 could be considered as a new drug target for overcoming IMiDs resistance in MM cells. Citation Format: Qinqin Xu, Yuexian Hou, Paul Langlais, Patrick Erickson, James Zhu, Changxin Shi, Moulun Luo, Yuanxiao Zhu, Lawrence Mandarino, Keith Stewart, Xiu-Bao Chang. Expression of CRBN binding protein AGO2 plays important roles for myeloma cell growth. [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 3625. doi:10.1158/1538-7445.AM2015-3625

Abstract Glioblastoma multiforme (GBM) is an aggressive and fatal disease commonly treated with radiation and temozolomide (TMZ), but emergence of therapy resistance invariably limits treatment efficacy. Inhibition of Poly-ADP ribose polymerases 1 and 2 (PARP1/2) sensitizes GBM cells, and inhibitors of PARP are emerging as combination partners with TMZ. However, limited brain penetration precludes use of several promising PARP inhibitors in GBM. BGB-290 is a relatively new and potent PARP inhibitor that crosses the blood brain barrier with a brain to plasma ratio of ∼0.2. The focus of the present study was to evaluate antitumor effects of BGB-290, as single agent or in combination with TMZ, in patient derived GBM xenografts and glioma cell lines. In a biochemical assay, low doses (30-100 nM) of BGB-290 suppressed PARP activity, while single agent anti-tumor activity of BGB-290 was modest and restricted to concentrations above 3 μM. Combinations of 0.1 μM BGB-290 with 10 μM TMZ significantly diminished primary neurosphere formation in a patient-derived xenograft line, GBM12, decreasing relative neurosphere count to 3.1 ± 1.3% as compared to 9.2 ± 3.1% with TMZ alone, p = 0.03. Consistent with a mechanism involving disruption of DNA repair, treatment of GBM12 cells with TMZ and BGB-290 combination resulted in robust γH2AX foci formation and a synergistic increase in DNA damage signaling as compared to TMZ or BGB-290 alone. TMZ-sensitizing effects of BGB-290 were heterogeneous in TMZ-resistant (GBM12TMZ) sublines that were developed previously by in vivo selection of parental GBM12 with cyclical TMZ therapy. The average neurosphere formation across 4 resistant sublines lacking MGMT expression after 100 μM TMZ only was 75.3 ± 9.0% versus 14.9 ± 7.4% with TMZ and 0.1 μM BGB-290, p<0.01. In contrast, 2 MGMT overexpressing sublines were much less sensitive to the combination with relative neurospheres 91.9 ± 5.2% versus 73.2 ± 9.2% respectively, p< 0.01. Next Gen Sequencing revealed discrete non-synonymous somatic mutations in the mismatch repair pathway of GBM12TMZ sublines lacking MGMT, which led us to examine the relationship between mismatch repair and sensitizing effects of PARP inhibition. Silencing Msh2 resulted in marked TMZ resistance in U251 cells (94.7± 5.4% relative growth with 100 μM TMZ), while co-treatment with 0.1 μM BGB-290 and 100 μM TMZ decreased growth to 23.5 ± 9.6% (p<0.01), which is similar sensitivity to TMZ alone in U251 parental cells. In an initial in vivo study with orthotopic GBM12 xenografts, administration of BGB-290 at 3 mg/kg twice per day combined with 25 mg/kg TMZ extended survival of animals beyond 300 days (80% survival at day 320), while in the group treated with TMZ alone 80% animals were moribund before day 150. In conclusion, BGB-290 is a promising PARP inhibitor with high potency, cytotoxicity and favorable in vivo efficacy in TMZ sensitive and possibly a subset of recurrent TMZ resistant GBM. Citation Format: Shiv K. Gupta, Brett L. Carlson, Mark A. Schroeder, Katrina K. Bakken, Ann C. Tuma, Jann N. Sarkaria. Inhibition of PARP activity by BGB-290 potentiates efficacy of temozolomide in patient derived xenografts of glioblastoma multiforme. [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 3505. doi:10.1158/1538-7445.AM2015-3505

Abstract The success of clinical trials for patients with malignant melanoma (MM) designed from the “promising” results using pre-clinical models is minimal. With its increase in incidence, there is a dire need to develop both pre-clinical models comparable to clinical practice that have the capability to translate from bench-to-bedside and novel therapeutic agents for this life-threatening form of skin cancer. To address this concern, we have developed a panel of pre-clinical MM models including patient matched cell lines, patient cell-line derived xenograft models and patient tumor tissue-derived xenograft models. Patient derived xenograft (PDX) models are purported to most closely replicate a patient's response to therapy. Since the development of patient-derived cell lines and tumors grown in mice were more efficacious, their genetic, phenotypic, and therapeutic responses were compared to those of their corresponding patient-matched PDX model. Each model was molecularly characterized to confirm a match to that of the originating patient tumor tissue via protein immunohistochemistry (IHC), mRNA quantitative polymerase chain reaction (qPCR), DNA short tandem repeat (STR) analysis, and Sanger sequencing. Each pre-clinical model provided a genetic match to their respective patient tumor sample. All pre-clinical models were positive for the melanoma-specific markers (S-100 beta, HMB-45, and Melan-A) confirming a melanoma phenotype. Soft agar studies and tumor growth of our PDX and patient cell-line derived xenograft models confirmed tumorigenicity within our models providing the foundation to perform therapeutic studies. Mutation status data (i.e. BRAF, RAS), patient therapeutic clinical history and oncogene overexpression analysis (pERK, pAKT, PI3K, pmTOR, etc.) provide the platform and rationale behind the use of specific therapeutic agents per model. BRAF, NRAS and KRAS mutations were identified along with elevated pERK, pAKT and pmTOR in the models. Targeted therapies (trametinib/MEK inhibitor; vemurafenib/BRAF inhibitor; MK-2206/pAKT inhibitor; and everolimus/mTOR inhibitor) demonstrated dose dependent inhibition of cell proliferation in each of the models based upon oncogenic expression or mutations. In vivo experiments are in progress, which will allow comparison of the three models. The next steps include combination therapy to identify antitumor synergy targeting oncogenic signaling pathways. Citation Format: Antoneicka L. Harris, Laura Marlow, Adam Mathias, Louis Dawson, William Durham, Kenneth Meshaw, Robert Mullin, Daniel Small, Aidan Synnott, Kevin Wu, Dragana Milosevic, Brian Netzel, Stefan Grebe, Svetomir Markovic, John Copland. Characterization of novel patient derived melanoma xenografts and cell lines in response to targeted therapies. [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 2417. doi:10.1158/1538-7445.AM2015-2417

Abstract Introduction: The role of macrophages in the tumor microenvironment is of increasing interest for tumor progression and invasion. Here we investigated the difference in CD68+ cell density in tissue samples from women with Benign Breast Disease (BBD) and women with no clinical breast disease from the Komen Tissue Bank (KTB) at Indiana University. Method: Archived breast tissue samples from women with BBD and no clinical breast disease were obtained for this age-matched case-control study. BBD samples included cases [later got Breast Cancer (BC)] and controls (did not get BC). Samples were characterized via H&E stain for epithelial proliferation [normal (N), fibrocystic non-proliferative (NP), proliferative (P), or atypia (A)]. CD68+ cell density was obtained by digital image quantitation on a per lobule basis (Total CD68+ cells/mm2). Statistical analysis was performed with mixed effects regression. Results: Among 276 women (median age 53, range 35-79) comprising 92 age-matched triplets, 2508 lobules were evaluated [789 KTB, 839 BBD case, 880 BBD control]. 1407 (56%) lobules were normal and 1101 (44%) were fibrocystic {NP [613 (56%)], P [448 (41%)] and A [40 (4%)]}. Out of 2508 lobules analyzed, 2362 (94%) had CD68+ cells, while 146 (6%) showed zero CD68+ cells [19 (2%) BBD cases, 20 (2%) BBD controls, 107 (14%) KTB, p<0.0001]. KTB samples showed significantly lower (p<0.0001) CD68+ density (median 136 cells/mm2) as compared to BBD cases (median 313 cells/mm2) and BBD controls (median 299 cells/mm2). (See Table) For each lobule type, KTB samples had lower CD68+ density compared with the same lobule type in BBD samples (p<0.0001). Conclusion: CD68+ cells are more frequent in breast lobules from women with BBD compared to normal donors. This finding in premalignant breast tissues is consistent with inflammation as a driver of tumorigenesis. BBD case (n = 92 patients)BBD control (n = 92 patients)KTB(n = 92 patients)n lobulesMedian (IQR)n lobulesMedian (IQR)n lobulesMedian (IQR)Overall839313 (165 - 538)880299 (164 - 527)789136 (55 - 272)By Lobule TypeNormal344310 (177 - 557)434331 (177 - 567)629128 (55 - 255)All non-normal/fibrocystic lobules combined495314 (157 - 527)446274 (152 - 478)160159 (58 - 393)Fibrocystic non-proliferative231319 (153 - 543)263274 (140 - 496)119163 (71 - 359)Fibrocystic proliferative246295 (153 - 496)169277 (153 - 478)33128 (30 - 385)Atypia18484 (313 - 739)14260 (223 - 375)8216 (0 - 493)IQR - Interquartile range (25th percentile - 75th percentile) Citation Format: Muhammad A. Arshad, Daniel W. Visscher, Tanya L. Hoskin, Rushin D. Brahmbhatt, Alvaro Pena Jimenez, Melody L. Stallings Mann, Erin E. Miller, Linda M. Murphy, Jodi M. Carter, Stacey J. Winham, Keith L. Knutson, Derek C. Radisky, Amy C. Degnim. CD68+ immune cells show different infiltration patterns in tissue samples from women with no clinical breast disease and those who have benign breast disease. [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 2364. doi:10.1158/1538-7445.AM2015-2364

Abstract Background: The transcription factor Krüppel-like factor 5 (KLF5) is essential for normal homeostasis, stemness, and tumorigenesis. Typically, KLF5 functions as a transcriptional activator, including in esophageal epithelia where KLF5 promotes keratinocyte proliferation and migration by activating EGFR and ILK, respectively, and prevents transformation by activating Notch1. Yet, the function of KLF5 is context dependent, and mutation of the tumor suppressor p53 switches KLF5 from a transcriptional activator to a repressor of p21Waf1/Cip1 and from pro-proliferative to anti-proliferative. Interestingly, KLF5 also inhibits p53 itself, though the mechanism of this inhibition is unknown. We hypothesized that the functional switch of KLF5 from transcriptional activator to repressor involves the recruitment of a co-repressor. Methods: Primary human esophageal keratinocytes immortalized with hTERT were stably infected with inducible lentivirus containing shRNA against KLF5 or Sin3A. p53 mRNA and protein levels were assessed by real-time quantitative PCR and Western blotting, respectively, and p53 protein stability was evaluated by pulse-chase experiments. We employed ChIP across a 4 kb region on the p53 gene, from 2 kb upstream to 2 kb downstream of the transcription start site, to identify the region of KLF5 binding on p53. Promoter activity was assessed with a series of luciferase reporters containing varying lengths of p53 and its upstream regulatory region. Protein-protein interaction was examined by co-IP. Results: KLF5 knockdown significantly increased p53 both at the mRNA and protein levels. In unstressed cells p53 protein undergoes rapid degradation such that p53 levels are typically low. Yet, with KLF5 knockdown, despite increased p53 protein levels, we saw no changes in p53 protein stability. By ChIP, we identified a region of KLF5 binding to p53 between -600 to -356 bp upstream of the transcription start site. Several other KLF family members can interact with the co-repressor Sin3A, and we found that Sin3A also complexed with KLF5 in human primary esophageal keratinocytes. Knockdown of Sin3A abolished the inhibitory effect of KLF5 on p53, resulting in elevated p53 expression, growth inhibition, and increased apoptosis. Conclusion: KLF5 transcriptionally represses p53 via the co-repressor Sin3A. Thus, KLF5 promotes cell proliferation in normal keratinocytes by both activating pro-growth signals and inhibiting the brake machinery of the cell cycle. Citation Format: Yizeng Yang, Taicheng Zhou, Zhen Zong, Gwen Lomberk, Raul Urrutia, Jonathan Katz. Regulation of p53 function by KLF5. [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 2087. doi:10.1158/1538-7445.AM2015-2087

Abstract Background: Cytarabine and an anthracycline, the mainstay of AML therapy, affords a five-year survival below 30%. Accordingly, there is a continuing need for improved AML therapies. CPX-351 encapsulates within liposomes a fixed 5:1 molar ratio of cytarabine and daunorubicin that is synergistic (in vitro) and markedly improved in anti-leukemic activity (in vivo). Promising evidence of improved remission and survival rates with CPX-351 compared to standard of care treatments has also been observed clinically and a pivotal Phase 3 trial is currently ongoing. Because Chk1 inhibition has been shown to enhance the actions of both cytarabine and anthracyclines, there has been interest in assessing the effect of CPX-351 when combined with Chk1 inhibitors. Here we describe the evaluation of CPX-351 alone and in combination with the Chk1 inhibitors MK-8776 and LY2603618 in AML cell lines and in clinical AML samples ex vivo. Methods: Effects of CPX-351 on Chk1 phosphorylation and cell cycle distribution in the human AML lines U937 and HL-60 were analyzed by immunoblotting and flow cytometry. Effects of Chk1 inhibitors on CPX-351-induced apoptosis were assessed by flow cytometry after propidium iodide staining (DNA cleavage), Hoechst staining (nuclear fragmentation) and colony formation in soft agar (long-term survival). Effects CPX-351 ± MK-8776 on clinical AML samples were assayed using clonogenic assays. Results: Treatment of U937 cells with CPX-351 resulted in phosphorylation of Chk1 on Ser317 and Ser296 (sites of ATR-mediated phosphorylation and autophosphorylation, respectively) as well as S phase slowing consistent with Chk1 activation. Co-treatment of AML cell lines with Chk1 inhibitors + CPX-351 resulted in increased apoptotic morphological changes and DNA fragmentation as well as diminished clonogenic survival compared to CPX-351 alone. The CPX-351/MK-8776 combination displayed enhanced antiproliferative effects against some clinical AML isolates ex vivo compared to CPX-351 alone, with additional assays ongoing. Conclusions: Collectively, these results show that Chk1 inhibition enhances the cytotoxicity of CPX-351 in AML cell lines and some clinical AML samples, offering the possibility that CPX-351 may be well suited for combining with such molecularly targeted agents. Citation Format: Nicole D. Vincelette, Larry M. Karnitz, Judith E. Karp, Douglas B. Smith, Allan D. Hess, Lawrence D. Mayer, Scott H. Kaufmann. CPX-351 (cytarabine:daunorubicin liposome for injection) anti-leukemia activity is potentiated by Chk1 inhibition. [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 3479. doi:10.1158/1538-7445.AM2015-3479

Abstract Radiotherapy is an important treatment regimen for prostate cancer. We have shown that the lymphangiogenic growth factor, Vascular Endothelial Growth Factor C (VEGF-C), induces docetaxel resistance and autophagy in prostate cancer (Muders et al., Cancer Res 2009 and Stanton et al., Cancer Res 2013). In this study we have evaluated the role of VEGF-C and VEGF-C induced autophagy in radioresistance of prostate cancer using cell culture experiments and human tissue samples. The human prostate carcinoma cell lines PC-3, LNCaP and DU145 were used in these cell culture experiments. Clonogenic survival of prostate cancer cells, after RNA interference (RNAi) of VEGF-C, autophagy related gene 5 (ATG5), and after addition of recombinant human VEGF-C following irradiation therapy, was evaluated. Autophagic flux was tested after ionizing irradiation using a Light-Chain-3 II immunoblot. To confirm our in vitro findings, human tissues of patients who underwent radical prostatectomy and adjuvant radiotherapy at Mayo Clinic were evaluated for VEGF-C expression. The quantity and intensity of VEGF-C staining was evaluated by two pathologists independently and correlated with biochemical relapse free survival (BRFS). The follow-up time is up to 23 years after radiotherapy. Multivariate Cox analysis was performed. VEGF-C levels correlated with a significantly higher radioresistance in colony formation assays and human tissue samples by multivariate analysis. The risk of biochemical recurrence increased 2.8 fold (95% confidence interval: 1.109 to 7.237; p = 0.03) when VEGF-C was highly expressed in prostatectomy patients with adjuvant radiotherapy. In line with our studies on autophagy in VEGF-C mediated docetaxel resistance of prostate cancer, autophagic flux was reduced after VEGF-C depletion during radiation. Interestingly, RNAi for ATG5 showed no effect on radiosensitivity which suggests that the decrease in autophagic flux after VEGF-C depletion is not responsible for the increased radiosensitivity. We conclude that VEGF-C is an important mediator of radiotherapy resistance in prostate cancer. In contrast to our published results on chemotherapy resistance, VEGF-C induced autophagy appears to be less important for resistance against ionizing radiation. Other VEGF-C dependent mechanisms for therapy resistance are under investigation. Citation Format: Steffi Haberlau, Pia Hönscheid, Rafael E. Jimenez, Gustavo B. Baretton, Donald J. Tindall, Mechthild Krause, Kaustubh Datta, Michael H. Muders. Role of Vascular Endothelial Growth Factor C in promoting radioresistance of 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 3304. doi:10.1158/1538-7445.AM2015-3304