bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1536: CRISPR/Cas9 biased mutagenesis in the mouse genome: Is it useful or harmful for cancer therapeutics
Authors: Shin Ha Youn
Published: July 2017

Advances in CRISPR/Cas9 genome editing have provided unprecedented opportunities to interrogate the functional significance of any given genomic site. While this technology is rapidly spreading and extended to with almost 4,000 PubMed entries as of today, there is a paucity of data on the extent of unexpected and undesired molecular consequences established upon targeting the mouse genome. We have introduced deletions at more than 20 genomic sites including CTCF and STAT5 transcription factor binding sites, screened over 500 founder mice and established and analyzed more than 50 lines. These data sets revealed hitherto unknown deletion patterns, unexpected large deletions using single sgRNAs and the consistent deletion of sequences bridging distant loci that were targeted simultaneously. Our findings provide guidelines for the application of CRISPR/Cas9 in the mouse germline and provide the insight into designing CRISPR-based cancer therapeutics.

Note: This abstract was not presented at the meeting.

Citation Format: Ha Youn Shin. CRISPR/Cas9 biased mutagenesis in the mouse genome: Is it useful or harmful for cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1536. doi:10.1158/1538-7445.AM2017-1536
Abstract 1537: 3p13-14 FOXP1-SHQ1 deletion spanning multiple potential tumor suppressor genes cooperates with PTEN loss in cancer
Authors: Hieronymus Haley , Iaquinta Philip J. , Wongvipat John , Gopalan Anuradha , Murali Rajmohan , Mao Ninghui , Carver Brett S. , Sawyers Charles L.
Published: July 2017

The recurrent 3p13-14 deletion spanning from FOXP1 to SHQ1 occurs frequently in prostate cancer and is broadly lost in a range of cancer types, but this deletion has unknown tumor suppressive potential. FOXP1-SHQ1 deletion significantly co-occurs with PTEN loss in prostate cancer and other cancers. We find that FOXP1-SHQ1 deletion cooperates with PTEN loss to accelerate prostate cancer development in mice, resulting in tumors with increased proliferation and highly anaplastic dedifferentiation. FOXP1-SHQ1 deletion in these PTEN null tumors results in selective mTORC1 pathway hyperactivation beyond that mediated by PTEN loss alone. FOXP1-SHQ1 deletion also partially rescues AR target gene inhibition caused by PTEN loss, circumventing the repression of the androgen axis seen upon PI3K pathway activation. Clinically, combined FOXP1 and PTEN loss is associated with increased prostate cancer recurrence, and this finding extends to other cancer types, most notably breast cancer.

Citation Format: Haley Hieronymus, Philip J. Iaquinta, John Wongvipat, Anuradha Gopalan, Rajmohan Murali, Ninghui Mao, Brett S. Carver, Charles L. Sawyers. 3p13-14 FOXP1-SHQ1 deletion spanning multiple potential tumor suppressor genes cooperates with PTEN loss in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1537. doi:10.1158/1538-7445.AM2017-1537
Abstract 1543: Caspase-10 suppresses tumorigenesis by targeting ATP-citrate lyase
Authors: Kumari Rajni , Das Sanjeev
Published: July 2017

p53 fosters metabolic reprogramming, which restricts metabolic adaptation of tumor cells under energy stress conditions. However, modulation and directionality of underlying cascades to induce or restrict metabolism are determined by the downstream target of p53. To explore the role of p53 in tumor metabolism upon metabolic stress, we performed a microarray screen in the presence or absence of p53 under glucose starvation. We observed caspase-10 to be upregulated by p53 upon metabolic stress. Caspase-10 belongs to the class of initiator caspases and is poorly understood in terms of substrate specificities. Emerging evidences also suggest intrinsic activation of caspase-10, distinct from its homologue caspase-8. Moreover, inactivating mutations as well as downregulation of caspase-10 in several carcinomas are suggestive of its role in tumorigenesis. Thus, we investigated the function of caspase-10 as a tumor suppressor. Our results indicated that caspase-10, upregulated in a p53-dependent manner in response to metabolic stress, was activated by homodimerization-induced intrinsic activation mechanism involving Adenylate Kinase 2 and FADD. We further performed a proteomic screen to identify novel caspase-10 substrates. We observed that several anabolic enzymes are caspase-10 interacting partners. However, in vitro and in vivo assays confirmed only ATP-Citrate Lyase (ACLY) as a novel substrate of caspase-10. Cleavage of ACLY by caspase-10 abrogates enzymatic conversion of cytosolic citrate to acetyl-CoA, leading to overall decrease in nucleocytosolic acetyl-CoA pool. Decrease in cytosolic acetyl-CoA levels suppress lipogenesis upon metabolic stress. Moreover, decreased levels of nuclear acetyl-CoA alter global histone acetylation. Further studies using RNAi established that alteration in histone acetylation is GCN5-dependent. Caspase-10-dependent abrogation of histone acetylation reprograms the epigenetic profile of metastatic and proliferative genes, leading to their diminished expression under metabolic stress. Concomitant suppression of both cytosolic lipogenesis and nuclear histone acetylation by caspase-10-mediated ACLY cleavage decreases proliferation capacity, migration potential and invasiveness of tumor cells. Further, studies in mouse tumor models confirmed that caspase-10 restricts tumorigenesis and metastases under metabolic stress via ACLY and GCN5. Taken together, our findings establish caspase-10 as a novel integrator of metabolism and epigenetics, by regulating acetyl-CoA levels. Hence, it functions as a tumor suppressor by disrupting lipogenesis-mediated metabolic reprogramming and GCN5-mediated epigenetic reprogramming.

Citation Format: Rajni Kumari, Sanjeev Das. Caspase-10 suppresses tumorigenesis by targeting ATP-citrate lyase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1543. doi:10.1158/1538-7445.AM2017-1543
Abstract 1545: Loss of glycogen debranching enzyme (AGL) promotes rapid growth of non-small cell lung cancer cells
Authors: Meier David R. , Weinhaus Benjamin , Oldenburg Darby , Guin Sunny
Published: July 2017

Glycogen Debranching Enzyme (AGL) is involved in glycogen breakdown. Compromise in glycogen breakdown due to loss of function mutations in the AGL gene lead to metabolic disease - Glycogen Storage Disease Type III. We have previously shown that loss of AGL results in rapid growth of bladder cancer cells using extensive in vitro and in vivo experiments. Here we test whether AGL regulate growth of non-small cell lung cancer (NSCLC) cells. We show that loss of AGL promotes rapid growth of NSCLC cells using anchorage independent growth assay. Loss of AGL also resulted in rapid xenograft growth of NSCLC cells when injected subcutaneously in immunocompromised mice. Whereas overexpression of AGL reduce the growth of these NSCLC cells. Further loss of glycogen phosphorylase the other enzyme involved in glycogen breakdown does not promote aggressive growth of these cancer cells. This observation is similar to our findings in bladder cancer which confirms that AGL regulates tumor growth independent of its role in glycogen metabolism. Thus using in vitro and in vivo experiments we show that AGL can regulate the growth of multiple tumor types such as bladder cancer and NSCLC. The exact mechanism on how AGL regulates NSCLC growth needs further investigation.

Citation Format: David R. Meier, Benjamin Weinhaus, Darby Oldenburg, Sunny Guin. Loss of glycogen debranching enzyme (AGL) promotes rapid growth of non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1545. doi:10.1158/1538-7445.AM2017-1545
Abstract 1551: GULP1 is an epigenetically altered and functional tumor suppressor in urothelial carcinoma through regulation of Nrf2-Keap1 signaling axis
Authors: Hayashi Masamichi , Guida Elisa , Goldberg Rachel , Inokawa Yoshikuni , Reis Leonardo , Oki Akira , Izumchenko Evgeny , Maldonado Leonel , Marchionni Luigi , Brait Mariana , Bivalacqua Trinity , Baras Alexander , Netto George J. , Koch Wayne , Sidransky David , Hoque Mohammad O.
Published: July 2017

We identified GULP1 as a novel tumor suppressor gene (TSG) selectively silenced during urothelial cancer (UC) progression through promoter hypermethylation (PH) analyzed by quantitative methylation specific PCR (QMSP) and novel methylation specific digital droplet PCR (ddPCR) assay. Numerous cell based assays revealed that GULP1 silencing confers growth advantage to tumor cells. Correspondently, in vivo tumorigenicity after xenotransplantation of GULP1 knockdown T24 cells was significantly higher than control cells. Further mechanistic analysis revealed that GULP1 has a crucial role in the regulation of Nrf2-Keap1 axis, maintaining actin cytoskeleton architecture and helping Keap1 to scaffold Nrf2 in the cytoplasm. Moreover, GULP1 silencing induces constitutive activation of Nrf2 target signature, responsible for chemoresistance of UCs. Additionally, we analyzed GULP1 PH and expression in cisplatin-based therapy responsive and resistant primary UC samples, and in isogenic cisplatin sensitive and resistant T24 cell lines. Interestingly, GULP1 expression at transcription level was lower in both resistant primary UC samples and resistant T24 cell line. Cell lines with lower expressions of GULP1 (SW780 and UM-UC-3) also showed higher resistance to cisplatin than those with higher expression (T24 and BFTC905). Altogether, our findings determined that GULP1 is an epigenetically silenced potential TSG in UC and GULP1 expression and/or PH may guide in selecting candidate patients for cisplatin based neo-adjuvant therapy.

Citation Format: Masamichi Hayashi, Elisa Guida, Rachel Goldberg, Yoshikuni Inokawa, Leonardo Reis, Akira Oki, Evgeny Izumchenko, Leonel Maldonado, Luigi Marchionni, Mariana Brait, Trinity Bivalacqua, Alexander Baras, George J. Netto, Wayne Koch, David Sidransky, Mohammad O. Hoque. GULP1 is an epigenetically altered and functional tumor suppressor in urothelial carcinoma through regulation of Nrf2-Keap1 signaling axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1551. doi:10.1158/1538-7445.AM2017-1551
Abstract 1556: Algorithms for discovery of somatic single nucleotide mutation display specific artifacts and different detection capabilities under the effect of read coverage and sample heterogeneity
Authors: Xiao Wenming , Wu Leihong , Yavas Gokhan , Hong Huixiao , Ning Baitang , Tong Weida , Donaldson Eric F. , Tezak Zivana , Philip Reena , Jacob Howard , Staudt Louis M.
Published: July 2017

Understanding the performance and capability of different bioinformatics algorithms used for the discovery of somatic variants is very important for helping scientists choose an appropriate tool for cancer research. In this study, we developed a comparison approach and created a series of data sets that could be used to provide such guidance. We mixed reads from two well characterized individuals, NA12878 and NA24385, and generated a series of data sets with different coverages and sample heterogeneity. We then used these data sets to evaluate five commonly utilized somatic mutation detection tools. Our results indicate that read coverage has a significant impact on the accuracy and capability of mutation calling by individual bioinformatics algorithms. The mutation caller that performs well with high read coverage may perform poorly with low read coverage. On the other hand, the tool that performed well in calling variants in a relatively higher homogeneity sample may not have the same power to detect rare variants with low mutation allele frequency. In addition, we demonstrated that different mutation calling algorithms are associated with specific artifacts that were sensitive to read coverage. Furthermore, there were large numbers of false positives and false negatives shared by five callers, indicating that other factors, such as read alignment, library preparation, and even the properties of the sequencing platform could be the sources of false discovery for somatic variants. We observed similar behavior of the five variant calling algorithms using the sequencing data of a pair of matched tumor/normal cell lines, confirming the findings from the comparative analyses on the mixture of reads from the two normal individuals. Our findings are expected to facilitate selection of bioinformatics pipelines that fit for specific purposes in cancer research based on sequencing data.

Citation Format: Wenming Xiao, Leihong Wu, Gokhan Yavas, Huixiao Hong, Baitang Ning, Weida Tong, Eric F. Donaldson, Zivana Tezak, Reena Philip, Howard Jacob, Louis M. Staudt. Algorithms for discovery of somatic single nucleotide mutation display specific artifacts and different detection capabilities under the effect of read coverage and sample heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1556. doi:10.1158/1538-7445.AM2017-1556
Abstract 1558: Data-driven approach to detecting novel gene interactions in cancer with applications to drug response prediction and cancer stratification
Authors: Magen Assaf , Das Avinash , Lee Joo , Hannenhalli Sridhar , Ruppin Eytan
Published: July 2017

The effect of a gene’s activity on a phenotype depends on the context of a complex network of functionally interacting genes. Several genetic interactions (GIs), such as Synthetic lethality and Synthetic Rescues, have been reported to have a significant functional role in cancer progression and provide potential candidates for selective cancer treatments. However, numerous other types of GIs with potential clinical significance are yet to be explored. In this work we generalize the concept of GI and detect ~70,000 GIs of different types with both molecular and clinical signature. We demonstrate their clinical predictive value as well as their ability to stratify breast cancer patients into refined clinical subtypes that might allow for better diagnosis and modified course of treatment. These results compare favorably with previous sequence based approaches and provide evidence for the importance of context specific genomic events and their effect on tumor progression. Additionally, the GI network accurately predicts patients’ drug response, where difference GI types are found to be predictive of distinct drugs in a complementary manner. This work provides the basis for future exploration of novel GI types as well as individual interactions with major impact on cancer progression.

Citation Format: Assaf Magen, Avinash Das, Joo Lee, Sridhar Hannenhalli, Eytan Ruppin. Data-driven approach to detecting novel gene interactions in cancer with applications to drug response prediction and cancer stratification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1558. doi:10.1158/1538-7445.AM2017-1558
Abstract 1571: Decrypting the transcriptome profile of Pak4 using next generation sequencing
Authors: Rane Chetan , Patel Misaal , Cai Li , Minden Audrey
Published: July 2017

The PAK4 (p-21 Activated Kinase 4) protein kinase has long been associated with cancer because of its key roles in regulating cell proliferation, cell cycle progression and cell morphology. The PAK4 gene is amplified in different types of cancers, including breast cancer. PAK4 overexpression is associated with oncogenic transformation in several breast cancer cell lines, while PAK4 inhibition has been shown to significantly reduce their tumorigenic potential. However, there is limited information available on PAK4’s mechanism of action in promoting tumorigenesis. To gain an insight into PAK4 downstream signaling pathways, we performed Next Generation Sequencing (NGS) on RNA samples collected from non-transformed immortalized mouse mammary epithelial cells (WT iMMECs) and iMMECs overexpressing PAK4. Research from our lab has shown that unlike WT iMMECs, iMMECs overexpressing PAK4 formed tumors when injected into mammary fat pads of mice, suggesting a central role for PAK4 in tumor formation. Previous studies to delineate PAK4 signaling pathways have mostly focused on substrates phosphorylated by PAK4. This study, however, takes into account genes which may act downstream of PAK4 and play an important role in mediating long term PAK4 function. RNA-seq analysis offers the ability to discover new genes and splice variants and measure transcript expression. mRNA was isolated in triplicates from WT iMMECs and iMMECs overexpressing PAK4; mRNA from each condition was then sequenced using Illumina NextSeq platform. The RNA-seq data generated was analyzed using Top Hat and Cufflinks software, which created a list of genes whose expression was significantly different in these two cell types, and previously unknown to be regulated by PAK4 (26 genes were up regulated and 51 genes were down regulated by more than a log2 fold change of 3). A qPCR analysis of 8 of these genes validated the sequencing data. Among the different genes we identified, we chose to focus on ParvB, which is consistently down regulated by almost seven fold in the iMMECs overexpressing PAK4. Previous studies have suggested a tumor suppressor role for ParvB in regulating breast tumorigenicity. We hypothesize that PAK4 mediates mammary tumor formation through ParvB downregulation. Additional studies investigating the role of ParvB in the PAK4 signaling pathway are under investigation. Future studies will also involve studying the other genes we found to be regulated by PAK4 overexpression and determining their role in tumorigenesis.

Citation Format: Chetan Rane, Misaal Patel, Li Cai, Audrey Minden. Decrypting the transcriptome profile of Pak4 using next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1571. doi:10.1158/1538-7445.AM2017-1571
Abstract 1573: Detection of late-arising driver mutations using multi-sample tumor sequencing data
Authors: Gomez Karen , Miura Sayaka , Kumar Sudhir
Published: July 2017

Whole-genome sequencing of tumors has identified genetic mutations that drive the origination, growth, and spread of cancer. Most driver mutations detected from sequencing tumors have been categorized as early-occurring and are believed to play a role in initial tumor formation. However, single-region primary tumor samples represent just one snapshot of the continuously changing mutational landscape of a tumor. Drivers of initial tumor formation, occurring early on in tumor development, are present in more tumor cells and are more likely to be identified from a single tumor sample than drivers of later events like metastasis, which are likely to be found in only one or a few tumor samples. We analyzed 101 multi-region and/or multi-tumor sequencing data sets using driver mutation annotation and phylogenetic inference to determine whether driver mutations occur preferentially early on. We found driver mutations frequently arise late in the course of tumor evolution, even when statistically accounting for small amounts of total drivers and many early total mutations. In addition, the timing of driver mutation occurrence varies widely depending on cancer type as well as among different individuals with a single cancer subtype. Finally, we identify known cancer genes that are enriched in either early or late driver mutations, reflecting different selective pressures at various stages of cancer development. These results illustrate how drivers of primary tumorigenesis must be distinguished from drivers of treatment resistance, metastasis, or recurrence in order to better understand the genetic changes behind cancer progression.

Citation Format: Karen Gomez, Sayaka Miura, Sudhir Kumar. Detection of late-arising driver mutations using multi-sample tumor sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1573. doi:10.1158/1538-7445.AM2017-1573
Abstract 1577: GRK3 is a direct target of ADT-induced CREB1 activation and it promotes neuroendocrine differentiation of prostate cancer cells
Authors: Zheng Dayong , Hulsurkar Mohit , Sang Meixiang , Zhang Songlin , Xu Jianming , Gleave Martin , Ittmann Michael , Li Wenliang
Published: July 2017

Treatment-related neuroendocrine prostate cancer (t-NEPC) is an aggressive subtype of prostate cancer that is believed to arise through neuroendocrine differentiation (NED) from prostate adenocarcinoma (PAC) upon castration resistance to androgen deprivation treatment (ADT). t-NEPC is highly metastatic with poor prognosis. With the recent introduction of potent ADT drugs in clinic, the incidence of t-NEPC is expected to increase dramatically. No effective therapeutic is available for t-NEPC and its molecular mechanisms remain poorly understood. We reported that GRK3 (G protein-coupled receptor kinase 3) promotes prostate cancer progression. We further demonstrated that the ADT activates CREB1 (cAMP response element binding protein 1), which directly targets and induces GRK3. GRK3 expression is higher in t-NEPC than in PAC cells and mouse models, and it positively correlates with the expression and activity of CREB1 in human prostate cancers. Notably, overexpression of GRK3 in PAC cells increased the expression of NE markers. Conversely, silencing GRK3 blocked CREB1-induced NED in PAC cells, reversed NE phenotypes and inhibited proliferation of t-NEPC cells in culture and in xenograft. Currently, we are investigating the mechanisms underlying NEPC progression promoted by the CREB1/GRK3 axis. Taken together, these results indicate that GRK3 is a new critical activator of NE phenotypes and mediator of CREB1 activation in promoting NED of prostate cancer cells.

Citation Format: Dayong Zheng, Mohit Hulsurkar, Meixiang Sang, Songlin Zhang, Jianming Xu, Martin Gleave, Michael Ittmann, Wenliang Li. GRK3 is a direct target of ADT-induced CREB1 activation and it promotes neuroendocrine differentiation of prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1577. doi:10.1158/1538-7445.AM2017-1577
Abstract 1579: Bicalutamide-induced expression of T-type Ca2+ channels in prostate cancer cells undergoing neuroendocrine differentiation in vitro
Authors: Martin-Caraballo Miguel , Hall Megan , Todd Bryan , Kwon Yoon Jung , Nguyen Vu , Hearne Jennifer L.
Published: July 2017

The growth of prostate cancer cells depends on the activation of androgen receptors. Anti-androgen therapies that inhibit androgen synthesis or receptor activation are effective in limiting tumor growth. However, prolonged treatment with anti-androgen therapies result in the transition of prostate cancers into an androgen refractory state. Neuroendocrine differentiation (NED) has been associated with the progression of prostate cancers to an androgen resistant phenotype. In this work we investigated the effect of the androgen receptor blocker bicalutamide in promoting NED of LNCaP cells and whether it is accompanied by increased T-type Ca2+ channel expression. The ability of bicalutamide to evoke morphological and biochemical changes associated with NED was assessed by PCR, western blot and immuno-histochemical analysis. Changes in the Cav3.2 T-type Ca2+ channel subunit expression was studied using PCR analysis, western blot and whole cell recordings. The role of T-type Ca2+ channels in promoting the differentiation of LNCaP cells was assessed by a cell viability assay and changes in cell morphology, specifically the development of neurite-like processes. PCR analysis of bicalutamide-stimulated cells indicates no significant changes in Cav3.2 mRNA, yet results in Cav3.2 protein expression and functional channels. Western blot and immuno-histochemical analysis of LNCaP cells simulated with bicalutamide for 4-10 day reveals biochemical changes consistent with NED including the expression of tubulin IIIbeta and neuron-specific enolase. Pharmacological inhibition of T-type Ca2+ channel function with nickel ions and NNC 33-0936 disrupts the morphological differentiation and cell viability of LNCaP cells treated with bicalutamide. These results suggest that bicalutamide treatment of LNCaP cells evokes significant morphological and biochemical changes associated with NED and results in increased expression of T-type Ca2+ channels, which can significantly alter Ca2+ homeostasis.

Citation Format: Miguel Martin-Caraballo, Megan Hall, Bryan Todd, Yoon Jung Kwon, Vu Nguyen, Jennifer L. Hearne. Bicalutamide-induced expression of T-type Ca2+ channels in prostate cancer cells undergoing neuroendocrine differentiation in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1579. doi:10.1158/1538-7445.AM2017-1579
Abstract 1585: Endostatin inhibits androgen-independent prostate cancer growth by suppressing nuclear receptor-mediated oxidative stress
Authors: Lee Joo Hyoung , Kang Minsung , Naik Gurudatta , Mobley James A. , Sonpavde Guru , Garvey W. Timothy , Darley-Usmar Victor M. , Ponnazhagan Selvarangan
Published: July 2017

Androgen-deprivation therapy has been identified to induce oxidative stress in prostate cancer (PCa), leading to re-activation of androgen receptor (AR) signaling in a hormone-refractory manner. Thus, antioxidant therapies have gained attention as adjuvants for castration-resistant PCa (CRPC). Here, we report for the first time that human endostatin (ES) prevents androgen-independent growth phenotype in PCa cells through its molecular targeting of AR and glucocorticoid receptor (GR), and downstream pro-oxidant signaling. This reversal following ES treatment significantly decreased PCa cell proliferation through downregulation of GR, and upregulation of manganese superoxide dismutase and reduced glutathione levels. Proteome and biochemical analyses of ES-treated PCa cells further indicated a significant upregulation of enzymes in major ROS scavenging machinery, including catalase, glutathione synthetase, glutathione reductase, NADPH-cytochrome P450 reductase, biliverdin reductase and thioredoxin reductase, resulting in a concomitant reduction of intracellular ROS. ES further augmented antioxidant system through upregulation of glucose influx, pentose phosphate pathway (PPP) and NAD salvaging pathway. This shift in cancer cell redox homeostasis by ES significantly decreased the effect of protumorigenic oxidative machinery on androgen-independent PCa growth, suggesting that ES can suppress GR-induced CRPC phenotype upon AR antagonism, and dual targeting action of ES on AR and GR can be further translated to PCa therapy.

Citation Format: Joo Hyoung Lee, Minsung Kang, Gurudatta Naik, James A. Mobley, Guru Sonpavde, W. Timothy Garvey, Victor M. Darley-Usmar, Selvarangan Ponnazhagan. Endostatin inhibits androgen-independent prostate cancer growth by suppressing nuclear receptor-mediated oxidative stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1585. doi:10.1158/1538-7445.AM2017-1585
Abstract 16: Evaluation of the efficacy of clotrimazole treatment in human melanoma cell lines that overexpress the multidrug resistance-associated protein (MRP) and the lung resistance-related protein (LRP)
Authors: Blake Steven D. , McKamey Shelby G. , Tweed Christopher M. , Koh David W.
Published: July 2017

Drug resistance to chemotherapy, mediated in part by overexpression of P-glycoprotein (MDR-1), multidrug resistance-associated protein (MRP), or lung resistance-realted protein (LRP), continues to be a major problem in the treatment of melanoma. Because we previously demonstrated the ability of clotrimazole to enhance cytotoxicity in human metastatic melanoma cells, here we evaluated the ability of clotrimazole to treat drug-resistant human melanoma cells that overexpress MRP and/or LRP. We utilized human melanoma cell lines that overexpress either MDR or LRP, and human melanoma cell lines that overexpress both. Cell growth and cell death were analyzed by flow cytometry, proliferation assays, and immunoblotting after drug treatments and RNA interference. Clotrimazole caused significant decreases in proliferation and increases in cell death in both drug-sensitive melanoma lines and those overexpressing MRP and LRP. No extensive cell death was induced by clotrimazole treatment in noncancerous human skin cell lines. These results indicate that clotrimazole selectively induces cytotoxicity in both drug-sensitive and drug-resistant melanoma lines. As clotrimazole is known to inhibit various transient receptor potential (TRP) ion channels, we pretreated these melanoma lines with TRP inhibitors or RNAi. Inhibitors of the TRP melastatin-8 channel (TRPM8) or TRP vanilloid-1 (TRPV1) failed to produce comparable levels of cell death caused by clotrimazole. However, RNAi silencing of the TRPM2 cation channel caused significant levels of cell death in both drug-sensitive and drug-resistant melanoma lines. These results indicated that inhibition of TRPM2 channels may have a primary role in the ability of clotrimazole to treat drug-sensitive and drug-resistant melanoma lines.

In conclusion, this study demonstrated that clotrimazole selectively increases cell death in drug-resistant melanoma lines, with minimal deleterious effects in normal skin cells. Taken together, we conclude that clotrimazole has in vitro efficacy toward the treatment of drug-resistant human melanoma cells. Thus, these preliminary studies indicate that clotrimazole has the potential to successfully treat drug resistant melanoma in the future.

Citation Format: Steven D. Blake, Shelby G. McKamey, Christopher M. Tweed, David W. Koh. Evaluation of the efficacy of clotrimazole treatment in human melanoma cell lines that overexpress the multidrug resistance-associated protein (MRP) and the lung resistance-related protein (LRP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 16. doi:10.1158/1538-7445.AM2017-16
Abstract 1602: Generation of anti-IL-17B antibodies neutralizing IL-17B-mediated alterations of the immune microenvironment, promotion of tumor cell initiating capacity and chemoresistance
Authors: Laprevotte Emilie , Docquier Aurélie , Bastid Jeremy , Déjou Cécile , Lapierre Marion , Alberici Gilles , Bensussan Armand , Eliaou Jean-François , Bonnefoy Nathalie
Published: July 2017

Interleukin 17B (IL-17B) is a pro-inflammatory cytokine that belongs to a family encompassing 6 interleukins (IL-17A to F) and binds to the IL-17 receptor B (IL-17RB). Recently, amplified IL-17B/IL-17RB signaling was found critical for breast and pancreatic tumorigenesis and elevated expression of IL-17RB has been associated with the shortest survival rates in patients with breast or pancreatic cancer. Using IL-17B knock-out (IL-17B KO) mice we demonstrate here that melanoma, fibrosarcoma and breast cancer cell tumorigenicity is strongly impaired in immunocompetent IL-17B KO mice compared to WT littermates, including a large number of tumor free mice. Reduced tumor incidence in IL-17B KO mice is associated with alterations of the immune tumor microenvironment especially within innate lymphocyte and myeloid sub-populations. We further demonstrate that IL-17B is a key cytokine shaping the tumor initiating cancer cell niche. Indeed, MDA-MB-468 human breast cancer cells overexpressing IL-17B exhibit 10 times higher frequency of tumor initiating cells when xenografted at a serial limiting dilution in nude mice. Tumor progression is, again, associated with alterations of NK cells within the tumor microenvironment and with increased percentages of CD44hi/CD24lo tumor cells, a phenotype associated with breast cancer stem cells (CSC). This is associated with resistance to conventional chemotherapeutic agents such as taxol, an effect that is totally abrogated by disrupting IL-17B-IL-17RB signaling with a neutralizing antibody. Altogether our results point out the key role of IL-17B in regulating the immune microenvironment as well as cardinal features of CSC, one of the alleged causes of resistance to therapy and tumor relapse. Thereby, IL-17B and its receptor appear as potential therapeutic targets for cancer immunotherapy. Collectively, these data support the ongoing development of IL-17B neutralizing antibodies.

Citation Format: Emilie Laprevotte, Aurélie Docquier, Jeremy Bastid, Cécile Déjou, Marion Lapierre, Gilles Alberici, Armand Bensussan, Jean-François Eliaou, Nathalie Bonnefoy. Generation of anti-IL-17B antibodies neutralizing IL-17B-mediated alterations of the immune microenvironment, promotion of tumor cell initiating capacity and chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1602. doi:10.1158/1538-7445.AM2017-1602
Abstract 1614: Intratumoral delivery of a P2A-linked bicistronic IL-12 construct leads to high intratumoral expression and systemic anti-tumor response
Authors: Canton David A. , Burkart Christoph , Shirley Shawna A. , Mukhopadhyay Anandaroop , Connolly Richard J. , Bahrami Arya , Campbell Jean S. , Pierce Robert H.
Published: July 2017

The use of immunomodulatory cytokines has been shown effective in regressing a wide range of tumors. However, systemic delivery of recombinant cytokines can result in serious adverse effects, often life-threatening. DNA transfer via electroporation (EP) is a safe and effective method of delivering cytokines to target tissues.

Intratumoral (IT) electroporation of Interleukin 12 (IL-12), a potent immunomodulatory cytokine, is well tolerated with an acceptable safety profile and objective response rates of 34-40% and 33% in Phase II clinical trials in advanced melanoma and Merkel cell carcinoma, respectively. We sought to improve the systemic anti-tumor response of IT pIL-12-EP by improving IL-12p70 expression and electroporation conditions, which were evaluated in vitro and in vivo with a two-tumor syngeneic mouse model of melanoma. As functional IL-12 p70 is a heterodimer, we compared different expression constructs to achieve high levels of IL-12 protein expression. IL-12p70 protein expression from a plasmid that incorporated a picornavirus-derived co-translational cleavage site (P2A) was higher than constructs with an internal ribosomal entry sequence (IRES) or a fusion of the p35 and p40 subunits. In functional in vitro assays, pIL-12(P2A) was superior to pIL-12(IRES) and the fusion protein. Using the murine B16.F10 tumor model, we show that IT EP of pIL-12 (P2A) plasmid regresses the treated lesions in a dose-dependent manner compared to control treatments. Systemic effects of IT-expressed IL-12 was assessed by monitoring generation of an antigen-specific CD8 T cell response and regression of B16.F10 contralateral (untreated) tumors following primary tumor electroporation. IT-pIL12-EP treatment with the P2A-linked construct resulted in a significant increase in antigen-specific CD8 T cells, as well as enhanced contralateral tumor growth inhibition suggesting the induction of strong systemic anti-tumor immune response.

* Contributed equally to this work

Citation Format: David A. Canton, Christoph Burkart, Shawna A. Shirley, Anandaroop Mukhopadhyay, Richard J. Connolly, Arya Bahrami, Jean S. Campbell, Robert H. Pierce. Intratumoral delivery of a P2A-linked bicistronic IL-12 construct leads to high intratumoral expression and systemic anti-tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1614. doi:10.1158/1538-7445.AM2017-1614
Abstract 1615: C-Reactive protein and radiotherapy-induced skin toxicity in breast cancer
Authors: Hu Jennifer J. , Case Doug , Lively Mark O. , Lee Eunkyung , Takita Cristiane , Urbanic James J. , Lesser Glenn J. , Shaw Edward G.
Published: July 2017

Background: Post-surgery adjuvant radiotherapy (RT) for breast cancer significantly reduced the local recurrence rate. However, many patients develop early adverse skin reactions (EASRs) that impact quality of life.

Methods: In a large prospective study of 1,000 breast cancer patients undergoing RT, we evaluated an inflammatory biomarker, C-reactive protein (CRP) in predicting RT-induced EASRs. In each patient, we measured pre- and post-RT plasma CRP levels using a highly-sensitive ELISA CRP assay. RT-induced EASRs were assessed using the Oncology Nursing Society Skin Toxicity Criteria. Association between EASRs and CRP were assessed using logistic regression models after adjusting for potential confounders.

Results: The study population includes 405 non-Hispanic White, 280 African Americans, 218 Hispanic Whites, 52 Asians, and 45 others. RT-induced grade 3+ and 4+ skin toxicity at the end of RT were observed in 42% and 15% patients, respectively. CRP levels differ significantly by race/ethnicity at baseline and at the end of RT. RT-induced grade 4+ skin toxicity was significantly associated with: obesity and pre-RT CRP > 2mg/L (OR=3.27; 95%CI=1.88, 5.68), obesity and post-RT CRP > 2mg/L (OR=4.42; 95%CI=2.38, 8.23), or obesity and change of CRP > 1mg/L (OR=3.58; 95%CI=2.00, 6.39).

Conclusion: The current data validate our previous findings that the inflammatory biomarker CRP is associated with RT-induced EASRs, particularly combined with obesity.

Impact: Our current findings support the discovery and development of anti-inflammatory agents to protect normal tissue from RT-induced EASRs and improve quality of life in breast cancer patients undergoing RT.

Citation Format: Jennifer J. Hu, Doug Case, Mark O. Lively, Eunkyung Lee, Cristiane Takita, James J. Urbanic, Glenn J. Lesser, Edward G. Shaw. C-Reactive protein and radiotherapy-induced skin toxicity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1615. doi:10.1158/1538-7445.AM2017-1615
Abstract 1622: The role of the CCL-2 on lymphopenia-induced myeloid derived suppressor cells
Authors: Innamarato Pasquale P. , Weber Amy , Pilon-Thomas Shari
Published: July 2017

The induction of lymphopenia prompts the expansion of CD11b+Ly6ChiLy6G- monocytic MDSCs (M-MDSCs) and CD11b+Ly6C+Ly6G+ polymorphonuclear MDSCs (PMN-MDSCs). Patients receiving adoptive cell therapy (ACT) require nonmyeloablative chemotherapy to induce lymphopenia and support anti-tumor immunity. However, the role of MDSCs in the setting of ACT is not fully understood. As high levels of CCL-2 can be measured in lymphopenic mice, in this study we investigate the role of CCL-2 in the expansion and function of lymphopenia-induced MDSCs using CCR2KO mouse models. Lymphopenia was induced in melanoma-bearing C57BL/6 (WT) and CCR2KO mice by 600rad of total body irradiation or combination therapy with cyclophosphamide and fludarabine. We first evaluated the percentages of splenic MDSCs in WT and CCR2KO mice after the induction of lymphopenia. At day 14, the expansion of total MDSCs were similar in WT (279% of normal) and CCR2KO (241% of normal). However, the mean percentages of M-MDSCs (3.5%) and PMN-MDSCs (30.3%) in CCR2KO mice were altered compared to M-MDSCs (22.2%) and PMN-MDSCs (21.7%) in WT mice. In addition, M-MDSCs in B16 tumors grown in CCR2KO mice were decreased compared to intratumoral M-MDSCs in WT mice (p<0.001). In contrast, the percentages of M-MDSCs and PMN-MDSCs were unchanged in the bone marrow (BM). To investigate the suppressive capacity of lymphopenia-induced MDSCs, OVA antigen-specific CD8+ T cells were co-cultured with OVA peptide in the presence of MDSCs purified from the spleens of CCR2KO and WT mice. Both CCR2KO and WT MDSCs potently suppressed T cell proliferation as measured by 3H thymidine incorporation. CCR2KO and WT MDSCs had significant arginase activity and production of nitrites. To investigate the efficacy of ACT, B16 tumor-bearing CCR2KO and WT mice were lymphodepleted and gp100 antigen-specific Thy1.2+CD8+ T cells were adoptively transferred followed by 3 days of IL-2 treatment. We found that donor T cell tumor infiltration was similar and no difference in tumor growth was observed between CCR2KO and WT mice. Collectively, these results suggest that the role of CCL-2 is important for the egress of lymphopenia-induced M-MDSCs from the BM, but has no effect on the expansion or function of PMN-MDSCs in the setting of lymphopenia. These data suggest that factors besides CCL-2 play an important role in the expansion and function of MDSCs after the induction of lymphopenia.

Citation Format: Pasquale P. Innamarato, Amy Weber, Shari Pilon-Thomas. The role of the CCL-2 on lymphopenia-induced myeloid derived suppressor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1622. doi:10.1158/1538-7445.AM2017-1622
Abstract 1648: ImmunoGraft® platform for the evaluation of Immuno-Oncology agents in PDX tumors models
Authors: Cerna David , Ciznadija Daniel , Verma Bhavna , Sidransky David , Izumchenko Evgeny , Davies Angela , Mancini Maria
Published: July 2017

Recent breakthroughs in Immunotherapy have given new hope to treating previously untreatable tumor types and provide a better tolerated alternative to standard agents. There is an unmet need for a pre-clinical platform to test potential immune-oncology therapeutics that would also provide a tool to examine the mechanisms of response to better predict clinical outcomes. We have previously presented the Champions ImmunoGraft®, an innovative pre-clinical model enabling immunotherapeutic agents to be evaluated for efficacy in solid tumors. This platform is more reflective of the human tumor microenvironment (both immune and non-immune cell-based) and may be one of the most translationally-relevant models to date for screening therapies targeting the immune system. However, optimization of humanization and PDX implantation protocols that allow a broader reconstitution of cell linage and higher engraftment rate are necessary to further improve the pre-clinical evaluation of immune-oncology therapeutics and enhance the value of this modality for patient’s benefit.

To this end, immune-compromised NOG (PrkdcscidIl2rgtm1Sug) mice were reconstituted (humanized) with human CD34+ cells using optimized procedure and blood was collected at different time points post engraftment to check for the major leukocyte linages. At ten weeks after humanization, mature human CD45+ cells comprised close to 50% of the leukocytes detected in the circulation and secondary lymphoid tissues of the humanized animals. As a result of improved methodology of the reconstitution protocol we have achieved an 85% success rate of humanization with a shortened engraftment period utilizing fewer CD34+ cells that maintain humanization reconfirmed at 22 weeks post reconstitution.

Champions TumorGraft® database contains more than one-thousand clinically relevant well-annotated PDX models. We have complied data from extensive genetic and protein expression analyses to design applicable ImmunoGraft® studies for testing of Immuno-Oncology agents. We have identified tumors expressing key markers that may predict response to immunotherapy agents including PDL-1, CD40, IDO-1 and IDO-2 as well as tumors that harbor a high mutational load. Here we present the efficacy and clinically relevant endpoints of two well-studied checkpoint inhibitors Ipilimumab and Pembrolizumab in this highly translational PDX platform.

Citation Format: David Cerna, Daniel Ciznadija, Bhavna Verma, David Sidransky, Evgeny Izumchenko, Angela Davies, Maria Mancini. ImmunoGraft® platform for the evaluation of Immuno-Oncology agents in PDX tumors models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1648. doi:10.1158/1538-7445.AM2017-1648
Abstract 1649: In vivo CRISPR-Cas9 library screen for immune checkpoint regulators
Authors: Karassek Sascha , Long Min , Li Xi , Peng Zhengang , Ji Qunsheng , Cang Yong
Published: July 2017

Therapeutic antibodies blocking immune checkpoints such as programmed cell death protein 1 (PD1) have proven clinically beneficial in multiple cancer types. However, anti-PD1 drugs approved by FDA are neither effective in treating all cancer types nor in all patients in a responsive cancer type. Therefore, understanding the mechanism underlying the efficacy of immune checkpoint blockade is critical for our rational design of synergistic treatment combinations. Here we describe how we employ the latest CRISPR-Cas9 genome-wide knockout screening strategies to identify targets in cancer cells or in T lymphocytes that control the sensitivity or resistance to anti-PD1 treatment. We constructed a sgRNA library targeting the functional domains of 116 mouse methyltransferases and demethylases, which was infected into the mouse Cloudman S91 melanoma cell line. Syngeneic mouse xenograft models derived from these infected cells were treated with anti-mPD1 monoclonal antibody, and the xenograft tumors were harvest at different time points after antibody treatment. Deep sequencing of the integrated sgRNA sequences in the tumors has identified potential druggable targets to sensitize or antagonize the tumors resistance to anti-mPD1 immunotherapy. Alternatively, we introduced the same library to naïve T cells purified from the OT-1 transgenic mice. The T cell library was then injected to B16-OVA melanoma syngeneic mouse models, followed by anti-mPD1 treatment. Deep sequencing of the integrated sgRNA sequences in the tumor-infiltrating T cells would allow us to uncover novel signaling molecules in the immune system that can potentially be manipulated to enhance the anti-PD1 therapy. In summary, we provide in vivo knockout screen strategies that can be applied to identify and validate combination therapies with immune checkpoint antibodies.

Citation Format: Sascha Karassek, Min Long, Xi Li, Zhengang Peng, Qunsheng Ji, Yong Cang. In vivo CRISPR-Cas9 library screen for immune checkpoint regulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1649. doi:10.1158/1538-7445.AM2017-1649