Dinesh Rakheja Research Articles

Abstract B005: Metabolic liabilities in high L-2HG kidney cancer

Abstract Renal cell carcinoma (RCC) is among the top 10 cancers in the USA. Despite several approved therapies, patients with the advanced disease rarely have durable responses and therefore, face a poor prognosis (median survival 2-3 years). This underscores the need for new strategies. Alterations in metabolism are well-established in cancers including RCC. The oncometabolite, L-2-hydroxyglutarate (L-2HG) is elevated in the most common form of RCC (clear cell histology) and promotes tumor progression. However, L-2HG’s roles in RCC progression and its mediated therapeutic vulnerability are yet to be explored. RCC cell lines lack the L-2HG dehydrogenase enzyme (L2HGDH) which results in their high L-2HG level. RNA-seq of control (high L-2GH) and an L2HGDH reconstituted (low L-2HG) RCC cell line reveals that L-2HG suppresses the expression of serine biosynthesis genes, PHGDH and PSAT1. In agreement, high L-2HG renal tumors demonstrate lower levels of serine biosynthesis enzymes compared to their matched normal kidneys. Mechanistic studies reveal L-2HG-mediated remodeling of both the epigenome and epitranscriptome suppress serine biosynthesis genes. Consistently, 13C-metabolomics labeling studies demonstrate that raised L-2HG suppresses de novo serine biosynthesis. Moreover, LC-MS analysis of the metabolites isolated from the kidneys of L2hgdh KO and wild-type (WT) mice revealed lower serine levels in L2hgdh KO kidneys. In accordance with these data, found that high L-2HG RCC cells require exogenous serine for in vitro proliferation and in vivo tumor growth. Likewise, the pharmacologic blockade of serine uptake decreases the proliferation of high L-2HG RCC cells. Furthermore, this serine liability can be rescued upon lowering cellular L-2HG levels. Untargeted metabolomics analyses demonstrate that exogenous serine is required to maintain cellular pools of glutathione (GSH+GSSG) in high L-2HG RCC. This is particularly relevant as glutathione is among the most highly enriched metabolites in RCC compared to normal kidneys. Our metabolomics data also suggest that serine might be essential for the transsulfuration process of glutathione biosynthesis in RCC that lacks the xCT system required to uptake cysteine for transsulfuration. In vivo, we find that intratumoral levels of glutathione are reduced in mice fed a chow diet lacking serine compared to regular chow diet-fed mice. Pharmacologic inhibition of glutathione synthesis ablates the growth of high L-2HG RCC cells even in the presence of serine, suggesting the importance of redox homeostasis for RCC proliferation. The data indicate that the L-2HG elevation in RCC reconfigures tumor metabolism, resulting in serine liability. Collectively, our data unmask a metabolic vulnerability that can be harnessed for precision-based approaches to kidney cancer. Citation Format: Anirban Kundu, Garrett J. Brinkley, Hyeyoung Nam, Suman Karki, Devin Absher, William J. Placzek, Jason Locasale, Dinesh Rakheja, Victor Darley-Usmarc, Jason Tennessen, Sunil Sudarshan. Metabolic liabilities in high L-2HG kidney cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B005.

Abstract PR004: Comparative genomic analysis of novel translocation renal cell carcinoma model reveals molecular mechanisms of disease progression and therapeutic opportunities

Abstract Introduction. Translocation Renal Cell Carcinoma (tRCC) is a distinct and aggressive molecular subtype of kidney cancer unusually prevalent in children and adolescents. tRCC has a poor prognosis with no specific therapies and is incurable in the metastatic setting. tRCC is characterized by chromosomal translocations involving three out of the four Microphthalmia (MiT) family transcription factors, TFE3, TFE3, and MITF. The most commonly involved gene is TFE3, which most often fuses with ASPSCR1. However, the molecular mechanisms by which ASPSCR1-TFE3 (and more broadly MiT/TFE fusion proteins) dictates tRCC tumorigenesis remain unclear and advances are hampered by the paucity of animal models. Herein, we sought to model tRCC and performed integrative genomic analyses to dissect molecular mechanisms underpinning tRCC tumorigenesis with the goal of translating this knowledge into therapeutic opportunities. Methods. We conditionally expressed a human ASPSCR1-TFE3 fusion in the Pax8 and Sglt2 lineages and performed phenotypic analyses as well as WES and RNAseq of emerging tRCC tumors. Similar genomic analyses of a human tRCC cohort were performed. The mutational landscape, cooperating pathways, and potential therapeutic targets were identified through comparative genomic and transcriptomic analyses. Emerging hypotheses were tested through in vitro and in vivo assays. Results. We show that human ASPSCR1-TFE3 driven by Pax8-Cre, a credentialled RCC driver, disrupted nephron development causing neonatal death, whilst Sglt2-Cre resulted in tRCC with complete penetrance and short latency. In both models, ASPSCR1-TFE3 induced characteristic morphological changes, loss of epithelial markers, and an EMT program, but only in the Sglt2-Cre model was proliferation observed. Given that Pax8-Cre, but not Sglt2-Cre¸ induce clear cell RCC (ccRCC), these data show that tRCC and ccRCC originate from different cell types. Comparative murine/human genomic analyses show that tumorigenesis by MiT/TFE fusion proteins is broadly cooperative (i.e. p53, ATM/ATR, Fanconi anemia, SWI/SNF, Hippo, and Wnt). Gene expression and functional studies implicate EPHA5 as well as Met/Ret in tRCC tumorigenesis. Antitumor/antiproliferative activity was observed with the therapeutic targeting of these pathways. Conclusion. This study provides insight into the tRCC mutational landscape, the cell of origin, and MiT/TFE-driven transcriptional programs offering several avenues for therapeutic development. Citation Format: Gopinath Prakasam, Akhilesh Mishra, Alana Christie, Jeffrey Miyata, Vanina T. Tcheuyap, Hui Ye, Quyen N. Do, Kevin B. Jones, Mai-Carmen Requena-Komuro, Ivan Pedrosa, Dinesh Rakheja, Payal Kapur, James Brugarolas. Comparative genomic analysis of novel translocation renal cell carcinoma model reveals molecular mechanisms of disease progression and therapeutic opportunities [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR004.

Abstract 3705: L-2HG, oncometabolite-driven epigenetic and epitranscriptomic reprogramming creates metabolic vulnerability in renal cancer

Abstract The oncometabolite, L-2-hydroxyglutarate (L-2HG) is elevated in the most common form of renal cell carcinoma-RCC (clear cell histology) and promotes tumor progression. L-2HG is structurally similar to α-ketoglutarate (α-KG). Therefore, L-2HG can competitively inhibit enzymes that utilize α-KG as a cofactor including α-KG-dependent dioxygenases that can profoundly impact gene expression via effects on the epigenome and epitranscriptome. RCC cell lines lack the L-2HG dehydrogenase enzyme (L2HGDH), resulting in their high L-2HG level. RNA-seq of control (high L-2GH) and an L2HGDH reconstituted (low L-2HG) RCC cell line has revealed that L-2HG suppresses the expression of serine biosynthesis genes, PHGDH and PSAT1. The findings were consistent in the patient samples where high L-2HG renal tumors had lower levels of PHGDH and PSAT1 expressions than that of the low L-2HG renal tumors and the patient-matched normal kidneys. Consistently, 13C-metabolomics labeling studies demonstrate that raised L-2HG suppresses de novo serine biosynthesis. Moreover, LC-MS analysis of the metabolites isolated from the kidneys of L2HGDH KO and wild-type (WT) mice revealed less serine content in the absence of L2HGDH, further confirming that high L-2HG suppresses serine biosynthesis in vivo. We found that L-2HG-mediated inhibition of the α-KG-dependent histone demethylase KDM4C silences ATF4 transcription. ATF4 is a master regulator of amino acid biosynthetic genes including PHGDH and PSAT1. Using ATF4 gain of function analysis, we confirmed that high L-2HG causes the suppression of PHGDH and PSAT1 in an ATF4-dependent manner. In addition, we demonstrate that L-2HG promotes the accumulation of the epitranscriptomic mark N⁶-methyladenosine (m6A) via inhibiting α-KG-dependent RNA demethylases ALKBH5 and FTO. In the setting of high L-2HG, m6A is enriched in the 3’-UTR region of transcripts including PSAT1. Using mutational analysis, we demonstrate that L-2HG promotes m6A accumulation at a specific site within the 3’UTR of PSAT1 that silences its translation. In accord with these data, found that high L-2HG RCC cells require exogenous serine for in vitro proliferation and in vivo tumor growth. Furthermore, this serine liability can be rescued upon lowering cellular L-2HG levels. Metabolomics analyses demonstrate that exogenous serine is required to maintain cellular pools of glutathione in high L-2HG RCC which supports both proliferation and resistance to oxidative stress. The data indicate that the L-2HG elevation in RCC reconfigures tumor metabolism through a bimodal mechanism via remodeling of both the epigenome and epitranscriptome. This results in a serine liability in the setting of raised L-2HG. Collectively, our data unmask a metabolic vulnerability that can be harnessed for precision-based approaches to kidney cancer. Citation Format: Anirban Kundu, Garrett J. Brinkley, Hyeyoung Nam, Suman Karki, Richard Kirkman, Hayley Widden, Michelle Johnson, Juan Liu, Yasaman Heidarian, Nader Mahmoudzadeh, Devin Absher, Han-Fei Ding, David Crosman, William J. Placzek, Jason Locasale, Dinesh Rakheja, Victor Darley-Usmar, Jason Tennessen, Sunil Sudarshan. L-2HG, oncometabolite-driven epigenetic and epitranscriptomic reprogramming creates metabolic vulnerability in renal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3705.

Abstract 3525: VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis

Abstract Clinical sequencing efforts are rapidly identifying sarcoma gene fusions that have not been functionally validated for their transformation capacity and biological activity. An example is the new fusion of transcriptional coactivators, VGLL2-NCOA2, found in infantile rhabdomyosarcoma. To delineate VGLL2-NCOA2 tumorigenic mechanisms and identify therapeutic vulnerabilities, we implemented a cross-species comparative oncology approach with zebrafish, mouse allograft, and patient samples. We found that in our transgenic zebrafish and mouse allograft models, VGLL2-NCOA2 is sufficient to generate mesenchymal tumors that display features of immature skeletal muscle. Zebrafish and mouse VGLL2-NCOA2 tumors are consistent with the human disease histologically and transcriptionally, and express diagnostic rhabdomyosarcoma markers. We found a shared molecular feature of these tumors was a suppression of myogenic differentiation pathways and reactivation of developmental programs. To identify impacted developmental programs, we transcriptionally clustered zebrafish VGLL2-NCOA2 tumors with the trajectory of early zebrafish development. A subset of VGLL2-NCOA2 zebrafish tumors cluster with embryonic somitogenesis and pinpoint VGLL2-NCOA2 developmental targets, including a RAS family GTPase, ARF6. In VGLL2-NCOA2 zebrafish, mouse allograft, and patient tumors, ARF6 is highly expressed and is absent from mature skeletal muscle. ARF6 knockout in our C2C12-VGLL2-NCOA2 cell culture model suppresses VGLL2-NCOA2 soft agar colony formation capacity, suggesting a genetic cooperating event in the disease. Our data indicate that VGLL2-NCOA2 is an oncogene which leverages developmental programs for tumorigenesis, and that the reactivation or persistence of ARF6 could represent a therapeutic opportunity. Citation Format: Sarah Watson, Collette A. LaVigne, Lin Xu, Didier Surdez, Joanna Cyrta, Delia Calderon, Matthew V. Cannon, Matthew R. Kent, Katherine M. Silvius, Jack P. Kucinski, Emma N. Harrison, Whitney Murchison, Dinesh Rakheja, Franck Tirode, Olivier Delattre, James F. Amatruda, Genevieve C. Kendall. VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3525.

Abstract 872: Genetic models reveal that the novel VGLL2-NCOA2 fusion oncogene leverages embryonic programs for sarcomagenesis

Abstract Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer characterized by a misregulation of skeletal muscle developmental pathways. To date, identified oncogenic drivers predominantly include RAS mutations or chromosomal translocations and gene fusions between PAX3 or PAX7 and FOXO1. RNAseq analysis of sarcomas with non-canonical gene fusions has identified new potential genetic drivers of tumorigenesis that have not been rigorously functionally validated for their transformation capacity and biological activity. One new fusion is a chromosomal translocation and inversion between chromosomes 6 and 8, which acts to juxtapose two transcriptional co-activators, VGLL2 and NCOA2. This VGLL2-NCOA2 fusion was identified in congenital rhabdomyosarcoma clinical cohorts by us and others, and characterizes aggressive RMS that express MYOD and MYOG histological markers. However, evidence of VGLL2-NCOA2 transformation capacity has not been verified, hindering insights into its functional contributions to tumorigenesis. Here, we interrogate the function ofVGLL2-NCOA2 using complementary genomic patient data and zebrafish model systems. We utilized the Tol2 transposon system to express mosaic human VGLL2-NCOA2 during early development, and found that VGLL2-NCOA2 is sufficient for tumorigenesis, and results in aggressive tumors with high penetrance by 75 days of age in zebrafish. Further, the histology of zebrafish tumors resembles the human disease, and tumors express markers indicative of RMS such as myog and desma. A cross-species RNAseq of patient and zebrafish VGLL2-NCOA2 RMS tumors highlights a significant enrichment and overlap between gene expression signatures. Finally, mapping the gene expression signatures of VGLL2-NCOA2 zebrafish RMS tumors along the spectrum of zebrafish embryogenesis indicates a clustering with developmental stages corresponding to early somitogenesis, highlighting their arrested developmental nature. Hence, we have generated the first animal model of human VGLL2-NCOA2 tumorigenesis, and have applied this model to understand the biology and identify potential therapeutic targets for this newly identified disease. Citation Format: Genevieve C. Kendall, Sarah Watson, Lin Xu, Collette LaVigne, Whitney Murchison, Dinesh Rakheja, Franck Tirode, Olivier Delattre, James Amatruda. Genetic models reveal that the novel VGLL2-NCOA2 fusion oncogene leverages embryonic programs for sarcomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 872.

Abstract B14: Establishment and characterization of in vitro models of alternative lengthening of telomeres (ALT) in pediatric high-grade glioma

Abstract Telomeres consist of many kilobases of repeated TTAGGG sequences at the ends of chromosomes, protected by a sequence-specific protein cap. Telomeres progressively shorten with each cell division and ultimately become critically short; due to their extensive proliferation, cancer cells must find a way to counteract this telomere loss. While most cancers utilize telomerase to maintain their telomere length, about 5% of cancers use a telomerase-independent telomere maintenance strategy, termed alternative lengthening of telomeres (ALT). While, overall, ALT is rare in cancer (~5-10% of cases), this telomere maintenance mechanism is enriched in pediatric high-grade glioma (pHGG). Previous work in our laboratory suggests that nearly half of pHGG utilize ALT. To date, therapeutic options are largely ineffective for children with HGG, reflected in the five-year survival rate, which is less than 33%. Our goal is to better harness ALT as a clinical marker in pHGG, specifically by the identification of drugs that target ALT-positive cancers. In order to study ALT in this context, we obtained and characterized a panel of six pHGG cell lines. Two of these six pHGG cell lines displayed features of ALT, including the presence of ALT-associated PML bodies and extrachromosomal telomeric DNA in the form of c-circles. Furthermore, these lines lacked measurable telomerase activity. It is well established that ATRX is commonly mutated in ALT-positive cancers, including pHGG. Interestingly, only one of these two ALT-positive pHGG cell lines displayed total loss of ATRX; the second cell line has an in-frame deletion in the ATRX gene, which may provide insight into the mechanism of how ATRX acts to suppress ALT. In addition, we have generated ATRX knockout cell lines from the four ALT-negative pHGG cells identified in this panel. Despite the strong link between ATRX loss and ALT in clinical samples, only one cell line displayed ALT characteristics after ATRX knockout. Comparison of the ALT-competent cell line to the ALT-resistant cell lines will yield important information about additional genetic or epigenetic events that allow ALT to occur. In conclusion, we have established in vitro models of ALT in pHGG cell lines based on endogenous ALT positivity and induction of ALT-like features based on ATRX modulation. These models will be invaluable resources as we strive to understand the molecular characteristics of ALT and translate these findings to better therapies for children with pHGG. Citation Format: Jacqueline A. Brosnan-Cashman, Mindy K. Graham, Anthony J. Rizzo, Kaylar Myers, Rebecca Zhang, Ezgi Göger, Reza Zarinshenas, Christine Davis, Ming Yuan, Dinesh Rakheja, Eric H. Raabe, Charles G. Eberhart, Christopher M. Heaphy, Alan K. Meeker. Establishment and characterization of in vitro models of alternative lengthening of telomeres (ALT) in pediatric high-grade glioma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B14.

Abstract 5482: L-2HG/ L2HGDH axis as therapeutic target for kidney cancer

Abstract Background: The D-enantiomer of 2-hydroxygultarate (D-2HG), along with fumarate and succinate, are considered oncometabolites that promote tumorigenesis. Our lab has reported elevated levels of the L-enantiomer of 2-hydroxyglutare (L-2HG) in clear cell renal cell carcinoma (ccRCC), in part due to reduced expression of the enzyme L-2HG dehydrogenase (L2HGDH). Objectives: Here we evaluated the underlying biochemical mechanisms of L-2HG accumulation and characterized the contribution of the L-2HG/L2HGDH axis to tumorigenesis. Experimental strategy: We assessed the role of raised L-2HG in renal carcinogenesis via both in vitro and in vivo means. Mutagenesis and knock down approaches were applied to study involvement of loss of L2HGDH in accumulation of high L-2HG levels and associated tumor phenotypes. Metabolomics coupled with 13C tracer labeling studies were utilized to dissect the biochemical axis that promotes L-2HG accumulation in RCC cells. Results: Translational based studies demonstrate that loss of L2HGDH expression is associated with both cancer progression and worsened outcomes. In connection with our previous findings, we demonstrate that raising cellular L-2HG levels by treatment with cell permeable octyl ester of L-2HG as well as by shRNA-mediated knock down of L2HGDH in renal epithelial cells (HK-2) promotes in vitro tumor phenotypes. Concurrently, the epigenetic mark 5hmc was significantly decreased under high L-2HG levels. Further investigation with restoration of L2HGDH in RCC cells (RXF393 and A498) shows decreased L-2HG levels and suppression of in vivo tumor growth in nude mice (NU/NU). Interestingly, expression of loss-of-function mutant of L2HGDH was unable to decrease L-2HG levels and failed to suppress in vitro and in vivo tumor phenotypes. In addition, high L-2HG levels were found to upregulate epithelial-mesenchymal-transition (EMT) marker SNAIL1 and correspondingly downregulate E-cadherin. Biochemical studies demonstrate that the predominant carbon source for L-2HG in RCC is glutamine through the activity of glutaminase and malate dehydrogenase (MDH). Pharmacological inhibition of the glutamine/MDH axis by treatment of glutaminase inhibitor (CB-839) and MDH inhibitor (4k) in RCC cells reduced L-2HG levels and mitigates in vitro tumor phenotypes. Furthermore, suppression of in vitro phenotypes by CB-839 and shRNA-mediated MDH2 knockdown was rescued by concurrent treatment with octyl ester of L-2HG. Finally, restoration of L2HGDH promoted the expression of genes targeted by the polycomb repressor complex 2 (PRC2), whereas inhibition of PRC2 in high L-2HG cells suppressed tumor phenotypes. Conclusion: Collectively, our data demonstrate the biologic relevance of high L-2HG to renal carcinogenesis and reveal novel therapeutic opportunities for L-2HG driven kidney tumors. Citation Format: Sandeep Balu Shelar, Eun-hee Shim, Garrett Brinkley, Anirban Kundu, Hyeyoung Nam, Francesca Carobbio, Tyler Poston, Jubilee Tan, Daniel Benson, Dinesh Rakheja, Richard Kirkman, Yusuke Sato, Seishi Ogawa, Shilpa Dutta, Sadanandan E. Velu, David Crossman, Anja Becker, Conrad Kunick, Sunil Sudarshan. L-2HG/ L2HGDH axis as therapeutic target for kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5482.

Abstract 3465: Context-dependent effects of ATRX loss on telomere biology in glioma cells

Abstract Telomeres progressively shorten with each cell division, leading to critically short telomeres during tumorigenesis. While most cancers activate telomerase to maintain their telomeres, a subset of cancers utilize a recombination-based process known as alternative lengthening of telomeres, or ALT. ALT has been widely characterized mostly in the context of immortalized cells, and characteristic features include ultrabright telomeric foci, colocalization of telomere foci with PML (ALT-associated PML bodies, or APBs), extrachromosomal telomeric DNA (e.g. c-circles), and overall telomere length heterogeneity. While the overall rate of ALT in cancer is low (roughly 5%, overall), the prevalence of ALT is concentrated in certain cancer subtypes, including gliomas. Gliomas employing ALT have a common genomic signature: inactivating mutation of ATRX, a chromatin-modifying protein that functions at the telomeres. In order to study the role of ATRX loss in the biology of ALT in cancer, we have characterized a panel of six human cell lines derived from pediatric high grade glioma, two of which are ALT(+) and lack functional ATRX. Furthermore, in four pediatric and three adult high grade glioma cell lines that endogenously express functional ATRX, we knocked out ATRX using CRISPR/Cas9n and knocked down ATRX expression using a stable shRNA-mediated approach. Knockout or knockdown of ATRX was successful in these seven glioma cell lines. Overall, knockout and/or knockdown of ATRX induced the formation of ultrabright telomeric foci, as assessed by telomere FISH, in two out of seven glioma cell lines. However, the effect of ATRX loss on formation of c-circles and APBs was more variable: the presence of ultrabright telomeric foci was not predictive of the presence of c-circles or ALT-associated PML bodies. These results indicate that ATRX loss, alone, is not necessarily sufficient to activate the ALT pathway in gliomas. Further examination of ALT-competent and ALT-resistant cell lines from our cohort will yield clues as to additional ALT-associated gene expression changes. In addition, our results suggest that previously identified hallmarks of ALT (notably c-circles and APBs) may be more variable in cancer cells compared to immortalized normal cell lines. The ALT pathway is an intriguing target for synthetic lethality in the cancer subtypes that employ it; our hope is to better understand the ALT phenotype in cancer so that we may effectively translate ALT-specific therapeutics to the clinic. Citation Format: Jacqueline A. Brosnan-Cashman, Ming Yuan, Anthony J. Rizzo, Kaylar M. Myers, Walter T. Barry, Christine Davis, David M. Esopi, Dinesh Rakheja, Eric H. Raabe, Charles G. Eberhart, Christopher M. Heaphy, Alan K. Meeker. Context-dependent effects of ATRX loss on telomere biology in glioma 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 3465. doi:10.1158/1538-7445.AM2017-3465

Abstract 3466: ALT-positive pediatric high grade glioma cells display chemosensitivity to ATR pathway inhibition

Abstract Overcoming the end-replication problem is a major hurdle for cancer cells due to progressive telomere shortening that results from excessive cell division during tumorigenesis. While most cancers activate telomerase to maintain their telomere length, about five percent of cancers use a telomerase-independent telomere maintenance mechanism, termed alternative lengthening of telomeres (ALT). Despite its low overall frequency in cancer, ALT is enriched in several cancer subtypes, including pediatric high grade glioma (HGG). Previous work in our laboratory suggests that nearly half of pediatric HGG utilize the ALT mechanism. To date, therapeutic options are largely ineffective for children with HGG, reflected in the dismal five-year survival rate, which is less than 33 percent. Our goal is to better harness ALT as a clinical marker in pediatric HGG, specifically by identifying drugs that target ALT-positive cancers. In order to study ALT in this context, we obtained and characterized a panel of six pediatric HGG cell lines. Two of the six cell lines in this panel display hallmarks of the ALT pathway. To assess the potential of ALT as a therapeutic biomarker in pediatric HGG, we measured cell viability in the presence of inhibitors, stratifying our analysis by ALT status. ALT is predicted to occur via a homologous-recombination-based mechanism. As such, we focused our attention on inhibitors of the DNA damage response, as well as agents that induce DNA damage. Treatment with inhibitors of DNA-PK, RAD51, and MRE11 did not result in significant differences in cell viability when stratified by ALT status, nor did treatment with temozolomide or hydroxyurea. However, inhibitors of ATR (VE-821, VE-822/VX-970, and AZD-6738) and CHK1 (MK-8776) led to significantly greater reductions in cell viability in ALT-positive cells. In order to further our understanding of the mechanism through which ATR inhibition preferentially targets ALT-positive cell lines, we examined the effect of these inhibitors on ALT-specific properties, such as the presence of ultrabright telomeric foci and extrachromosomal telomeric DNA (c-circles). Preliminary results indicate that ATR inhibition does not diminish these ALT-associated properties and, therefore, the differential sensitivity to these agents is unlikely to be due to directly blocking the ALT telomere maintenance mechanism. Continued study of the effect of ATR/CHK1 inhibition on ALT-positive cells will yield further insight into the mechanism of ALT-specific toxicity. Overall, we have identified ATR/CHK1 as a promising pathway to target for ALT-positive pediatric HGG. Our goal is to better understand the relationship between ATR/CHK1 signaling and ALT in order to effectively translate this observation to the clinic, both for pediatric HGG and for other ALT-positive cancers. Citation Format: Jacqueline A. Brosnan-Cashman, Anthony J. Rizzo, Ezgi Goger, Kaylar M. Myers, Reza Zarinshenas, Christine Davis, Ming Yuan, Dinesh Rakheja, Eric H. Raabe, Charles G. Eberhart, Christopher M. Heaphy, Alan K. Meeker. ALT-positive pediatric high grade glioma cells display chemosensitivity to ATR pathway inhibition [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 3466. doi:10.1158/1538-7445.AM2017-3466

Abstract 4880: MAGE gene family expression in pediatric medulloblastoma: frequency and possible therapeutic target

Abstract Background: The melanoma antigen gene family (MAGE) is a group of related genes that were first discovered in malignant melanoma. Among the approximately 40 MAGE genes in the human genome, two thirds are classified as Type I (MAGE-A, MAGE-B, and MAGE-C), referred to also as cancer testis antigens (CTAs), proteins that are restricted to testis but often aberrantly expressed in many adult cancers. Despite their common expression in adult cancers and their oncogenic potential, little is known about their expression and role in pediatric cancers. To determine the frequency of MAGE expression in pediatric medulloblastoma, we measured expression levels of Type I MAGEs in patient tumors. In addition, we examined how MAGE expression in medulloblastoma cells affects their growth and oncogenic potential. Methods: We searched the Children’s Health pathology database for pediatric medulloblastoma between 2008 - 2015 and were able to recover an adequate amount of RNA from formalin-fixed paraffin-embedded tissue samples of 34 patients. The RNA was converted to cDNA using reverse transcriptase, and the relative expression of 23 Type I MAGE genes was measured by qRT-PCR. To determine if MAGE expression is critical for medulloblastoma cell survival we knocked down several MAGEs using siRNAs in two MAGE-expressing medulloblastoma cell lines (D283 and DAOY) and evaluated the effect by cell viability (Alamar Blue and Cell titer Glo), apoptosis assays (Annexin V), and colony formation in soft agar. Results: In 34 medulloblastoma patient samples we found that 10 (29.4%) expressed at least one MAGE gene, and one case had high levels of expression of 10 of the 23 Type I MAGEs. MAGE expression was associated with the non-WNT/SHH (G3/G4) molecular subgroup of tumors in our study. There was no correlation between MAGE expression and histologic subtype. Furthermore, using MAGE-A/-B2 positive medulloblastoma cell lines, we showed that siRNA knock down of multiple MAGE-As and -B2 decreased cell viability by 15 - 80%, increased apoptosis (-B2), and decreased growth of cells in soft agar. Conclusion: Type I MAGE genes are expressed in 30% of pediatric medulloblastoma in our study. In MAGE positive medulloblastoma cells, MAGE genes are important for cell survival and clonogenic growth. The decreased cell survival after MAGE knockdown appears to be related, at least in part, to increased apoptosis. Our results indicate that targeting of MAGEs in medulloblastoma may be a potential therapeutic option. Citation Format: Rebecca R. Collins, Klementina Fon Tacer, Dinesh Rakheja, Patrick Ryan Potts. MAGE gene family expression in pediatric medulloblastoma: frequency and possible therapeutic target [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 4880. doi:10.1158/1538-7445.AM2017-4880

Abstract 1004: GLS inhibitor CB-839 modulates cellular metabolism in AML and potently suppresses AML cell growth when combined with 5-azacitidine

Abstract Glutamine (Gln) is required for growth and proliferation of several tumor types including AML. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes conversion of Gln to glutamate (Glu), which provides carbons for the TCA cycle and regulates redox homeostasis through production of glutathione and NADH. CB-839 is a highly selective, reversible, allosteric inhibitor of GLS. In this study we studied metabolic and cellular consequences of GLS inhibition in AML cells cultured in normoxic or hypoxic conditions. First, we performed metabolic analysis of HL-60 cells co-cultured with bone marrow-derived mesenchymal stem cells (MSCs). Consistent with the known mechanism of GLS inhibition, CB-839 caused a rapid and extensive decrease in intracellular Glu in both HL60 and MSC and a corresponding increase in intracellular Gln in both cell types. Unexpectedly, CB-839-treated cells exhibited a rapid increase in intracellular and extracellular concentrations of multiple amino acids, possibly reflecting inhibition of global protein synthesis. CB-839 suppressed Cys consumption from the extracellular compartment and caused rapid increase in intracellular taurine in HL-60 cells, suggesting altered redox homeostasis. CB-839 inhibited cellular growth of HL-60 and MV4;11 AML cells cultured alone or co-cultured with MSC, demonstrating activity under conditions mimicking BM microenvironment. We have previously shown that the leukemic bone marrow microenvironment is highly hypoxic (Benito PLoS One 2011), and hypoxia has been reported to induce production of the L-enantiomer of 2-HG (L-2HG) (Intlekofer Cell Metabolism 2015). In AML cells, hypoxia selectively induced the production of L-2HG (measured by LC-MS/MS) in HL-60 (6.2+/- fold) and OCI-AML3 cells (2.9+/- fold) with wt-IDH. That increase in L-2HG was potently inhibited by CB-839. AML cells produced very little D-2HG, and neither hypoxia nor CB-839 significantly affected D-2HG levels. We recently reported that CB-839 increased hydroxymethylation (hmc) levels using a HELP-GT assay (Velez ASH 2015), and the implications of those observations are the subject of ongoing studies. Prompted by the observation of increased hmc in response to CB-839 treatment, we next examined the efficacy of CB-839 in combination with the DNMT3A inhibitor 5-azacitidine (5-AZA). Treatment with 1μM CB-839 and escalating doses of 5-AZA caused additive or synergistic inhibition of cellular growth after 5 days of culture, both under normoxia and hypoxia, in AML cell lines (OCI-AML3, HL-60, MV4;11) and primary AML cells (n = 6). In summary, GLS inhibition causes AML growth arrest by multiple mechanisms, including inhibition of protein synthesis and disruption of redox homeostasis. Gln contributes to hypoxia-induced production of L-2HG and possibly epigenome regulation in AML, and concomitant blockade of GLS by CB-839 and DNMT3A with 5-AZA potently suppresses AML cell growth. Citation Format: Tianyu Cai, Philip L. Lorenzi, Dinesh Rakheja, Michael Pontikos, Lina Han, Qi Zhang, Helen Ma, Thomas D. Horvath, Amit K. Verma, Marina Konopleva. GLS inhibitor CB-839 modulates cellular metabolism in AML and potently suppresses AML cell growth when combined with 5-azacitidine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1004.

MP78-17 PROGNOSTIC VALUE OF TISSUE BASED BIOMARKER SIGNATURE IN CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Epidemiology & Evaluation/Staging II1 Apr 2016MP78-17 PROGNOSTIC VALUE OF TISSUE BASED BIOMARKER SIGNATURE IN CLEAR CELL RENAL CELL CARCINOMA Ahmed Haddad, Jun-Hang Luo, Laura-Maria Krabbe, Oussama Darwish, Bishoy Gayed, Ramy Youssef, Payal Kapur, Dinesh Rakheja, Yair Lotan, Arthur Sagalowsky, and Vitaly Margulis Ahmed HaddadAhmed Haddad More articles by this author , Jun-Hang LuoJun-Hang Luo More articles by this author , Laura-Maria KrabbeLaura-Maria Krabbe More articles by this author , Oussama DarwishOussama Darwish More articles by this author , Bishoy GayedBishoy Gayed More articles by this author , Ramy YoussefRamy Youssef More articles by this author , Payal KapurPayal Kapur More articles by this author , Dinesh RakhejaDinesh Rakheja More articles by this author , Yair LotanYair Lotan More articles by this author , Arthur SagalowskyArthur Sagalowsky More articles by this author , and Vitaly MargulisVitaly Margulis More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1973AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Despite apparently curative surgery, a significant proportion of patients with clear cell renal cell carcinoma (ccRCC) will experience disease recurrence. We utilized multiple tissue based biomarkers to develop a prognostic tool to improve risk stratification in ccRCC patients. METHODS 367 patients with non-metastatic ccRCC were included. The cohort was divided into a training and validation set. Using tissue microarrays, immunostaining was performed for 24 biomarkers representative of key pathways in ccRCC. Using LASSO Cox regression, we identified several markers which were used to construct a risk classifier for risk of disease recurrence. RESULTS Median follow-up was 63.5 months (IQR 24.0-85.3 months). LASSO Cox regression selected 5 out of 24 markers for the risk classifier: N-cadherin, E-cadherin, Ki67, cyclin D1 and phosphorylated eukaryotic initiation factor 4E binding protein-1 (p-4EB1). Patients were classified as either low or high risk of disease recurrence according to the expression of these markers. In the training set, 5-year RFS was 90.4% for the low risk group and 74.3% for the high risk group (HR 2.70, 95%CI 1.15-6.36, p=0.023). In the validation set, 5-year RFS was 93.9% for the low risk group and 76.2% for the high risk group (HR 4.20, 95%CI 1.54-11.48, p=0.005). The risk classifier increased the accuracy of the clinicopathologic prognostic model. CONCLUSIONS We developed a 5-marker based prognostic tool that can effectively classify ccRCC patients into groups at low and high risk of disease recurrence. This tool, if prospectively validated, could provide individualized risk estimation for patients after surgical treatment for ccRCC. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1033 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Ahmed Haddad More articles by this author Jun-Hang Luo More articles by this author Laura-Maria Krabbe More articles by this author Oussama Darwish More articles by this author Bishoy Gayed More articles by this author Ramy Youssef More articles by this author Payal Kapur More articles by this author Dinesh Rakheja More articles by this author Yair Lotan More articles by this author Arthur Sagalowsky More articles by this author Vitaly Margulis More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract IA12: Mechanisms of tumorigenesis due to somatic mutations in DICER1 and DROSHA in childhood kidney cancers

Abstract Wilms tumor is the most common pediatric kidney cancer and the fourth most common childhood cancer overall. Wilms tumor is treated with a combination of surgery, chemotherapy, and radiation, and, while most children are cured, patients suffer adverse late effects of therapy and survival remains poor in those with advanced-stage disease. Known driver mutations (in WT1, WTX, and β-catenin) account for only one-third of Wilms tumor cases. To better define the genomic landscape of Wilms tumor, we performed whole-exome sequencing of 44 Wilms tumors to identify somatic mutations. Through this approach, we identified recurrent somatic mutations in MYCN, DROSHA, and DICER1, which were mutually exclusive with known Wilms tumor driver mutations in WT1 and β-catenin. DROSHA and DICER1 somatic hotspot mutations occur at or near critical metal-binding residues in RNase III domains and partially or completely disrupt enzyme activity. Examination of miRNA expression in tumors, in vitro processing assays, and genomic editing in human cells demonstrate that DICER1 and DROSHA mutations influence miRNA processing through distinct mechanisms. While DICER1 RNase IIIB mutations preferentially impair processing of miRNAs that derive from the 5′ arm of pre-miRNA hairpins, DROSHA RNase IIIB mutations globally inhibit miRNA biogenesis through a dominant-negative mechanism. Despite their distinct mechanisms of action, DROSHA and DICER1 mutations result in decreased expression of several common tumor-suppressing miRNAs, including multiple members of the let-7 and miR-15/16 families. We also identified a patient carrying a germline DICER1 loss-of-function allele who subsequently developed multiple malignancies with distinct DICER1 RNase IIIB somatic hotspot mutations, suggesting that DICER1 can act as both a tumor-suppressor and an oncogene depending on the nature of the mutation. These results provide new insights into the mechanisms through which mutations in miRNA biogenesis components reprogram miRNA expression in human cancer and suggest that mutations causing impairment of miRNA processing define a distinct subclass of Wilms tumors. Citation Format: Kenneth S. Chen, Dinesh Rakheja, Yangjian Liu, Abhay A. Shukla, Joshua T. Mendell, James F. Amatruda. Mechanisms of tumorigenesis due to somatic mutations in DICER1 and DROSHA in childhood kidney cancers. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr IA12.

Abstract B39: NQO1 as a therapeutic target for atypical teratoid/ rhabdoid tumor

Abstract Atypical Teratoid/ Rhabdoid Tumor (ATRT) is an aggressive cancer that accounts for up to 20% of infant brain tumors. Even with intense chemotherapy and radiation, approximately 75% of children die from their disease at a median of 10 months after diagnosis. We found that the pathognomonic genetic lesion of ATRTs and related malignant rhabdoid tumors (MRTs), loss of the SWI/SNF chromatin remodeling complex member INI1/hSNF5/SMARCB1, is associated with high expression of NAD(P)H:quinone oxidoreductase 1 (NQO1), a two-electron oxidoreductase. Thus, we hypothesized that ATRTs will be vulnerable to drugs such as ß-lapachone (ß-lap, ARQ761 in clinical form) that selectively kill NQO1-expressing cells via mu-calpain mediated cell necrosis. A panel of ATRT cell lines exhibited overall high NQO1 expression and sensitivity to ß-lap (IC50 1.6-4 uM). A two-hour pulse with ß-lap caused a burst of ROS-mediated DNA damage and PARP1 hyperactivation, resulting in NAD+ and ATP depletion. Lack of NQO1 expression or inhibition of NQO1 with dicoumarol abrogated sensitivity to ß-lap. Low expression of NQO1 in normal brain and other pediatric tissues suggests a wide therapeutic window for ß-lap, and a survival advantage was observed after treatment in a heterotopic mouse xenograft model with several mice achieving apparent cures. Furthermore, the mechanism of ß-lap results in synergy with standard of care agents such as ionizing radiation, increasing its potential for clinical applications. Citation Format: Julia C. Meade, Zachary R. Moore, Sarai H. Stuart, Agnieszka Cholka, Richard L. Boriack, Jingying Xu, Anat Erdreich-Epstein, Dinesh Rakheja, David A. Boothman, James F. Amatruda, Theodore W. Laetsch. NQO1 as a therapeutic target for atypical teratoid/ rhabdoid tumor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B39.

Abstract B03: EGFR and mTORC1 are novel therapeutic targets in nonseminomatous germ cell tumors

Abstract This study aims to identify novel therapeutic targets for nonseminomatous pediatric germ cell tumors (GCTs). GCTs are the most common cancer in young men, and affect both children and adolescents. They are histologically classified into two types: seminomatous GCTs (SGCTs), which are undifferentiated, and non-seminomatous GCTs (NSGCTs), which exhibit differentiation. Although cisplatin treatment is effective for many types of GCTs, cisplatin resistance, which is especially common in NSGCTs, confers poor prognosis for affected patients. However, because the signaling pathways and genes responsible for the development of different types of GCTs are not well-understood, few targeted therapies exist for GCTs, and no specific therapies exist for NSGCTs. Therefore, novel therapies to specifically target NSGCTs are needed. To determine a targetable pathway that is activated in GCTs, we used quantitative RT-PCR to measure the expression of growth factor receptors in pediatric GCTs, immunohistochemistry (IHC) on a panel of clinically annotated germ cell tumors, and Western blot as well as cell viability assays on NSGCT cell lines (NCCIT and NTERA-2) to determine the effect of inhibition of two signaling pathways on cell viability. Our RT-PCR results showed that multiple members of the EGF and FGF receptor families are expressed at higher levels in NSGCTs than SGCTs. In addition, we found that EGF and FGF2 stimulate Ras-MAPK as well as PI3K/mTOR signaling in NSGCT cell lines. Based on IHC staining of phosphorylated ERK1/2, mTOR, and S6 ribosomal protein, we showed that both the Ras-MAPK and PI3K-mTOR pathways are activated at higher levels in NSGCTs than SGCTs. The results suggested that inhibiting EGFR as well as mTORC1 may be effective in impairing the growth and survival of NSGCT cell lines. To test this hypothesis, we examined the effects of two small molecule inhibitors of EGFR and mTOR signaling, erlotinib and rapamycin, respectively, on the survival of NCCIT and NTERA-2 cell lines. We verified by Western blot that erlotinib inhibits components of the Ras-MAPK pathway in NSGCT cell lines, while rapamycin inhibits components of the PI3K-mTOR pathway. Cell survival experiments showed that while treatment with rapamycin or erlotinib alone decreased cell viability, sufficient reduction of survival could only be achieved with high concentrations that are not clinically feasible. However, a combination treatment of erlotinib and rapamycin synergistically inhibited the growth of the two NSGCT cell lines at clinically achievable concentrations. Our findings showed that NSGCTs are dependent on EGFR and mTOR signaling in vitro, and suggest that targeting these signaling pathways may be a promising therapy to specifically target chemoresistant NSGCTs. Citation Format: Albert Budhipramono, Dinesh Rakheja, Kenneth S. Chen, Nicholas Fustino, Abhay Shukla, Jonathan Wickiser, Theodore Laetsch, James F. Amatruda. EGFR and mTORC1 are novel therapeutic targets in nonseminomatous germ cell tumors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B03.

Abstract 1613: β-lapachone: a novel targeted therapy for atypical teratoid rhabdoid tumors (ATRTs)

Abstract Atypical teratoid rhabdoid tumors (ATRTs) are aggressive malignant neoplasms that occur in the CNS, most commonly in children <3 years of age. Despite intensive chemo-radiotherapy and surgery, the outcomes for patients with this disease remain dismal, with ∼75% of patients dying from their disease at a median of 10 months after diagnosis. Thus, it is critical to identify and validate novel ATRT targets for new therapies. We found that the pathognomonic genetic lesion of ATRTs, loss of the SWI/SNF chromatin remodeling complex member INI1/hSNF5/SMARCB1, leads to high expression of NAD(P)H:quinone oxidoreductase 1 (NQO1), a two-electron oxidoreductase. Thus, we hypothesized that ATRTs will be vulnerable to drugs, such as ß-lapachone (ß-lap, ARQ761 in clinical form), that selectively kill NQO1-expressing cells. A panel of ATRT cell lines exhibited overall high NQO1 expression and low micromolar sensitivity to ß-lap (IC50 1.6-4 uM), with the exception of cell lines that were homozygous for the C609T NQO1 (*2) polymorphism, which exhibited no NQO1 activity and were resistant to ß-lap treatment (IC50 >15 uM). A two-hour pulse with ß-lap caused a burst of ROS-mediated DNA damage and PARP1 hyperactivation, resulting in NAD+ and ATP depletion and caspase-independent, mu-calpain-mediated apoptosis. Co-treatment with dicoumarol, a specific inhibitor of NQO1, prevented these effects by blocking the NQO1-dependent bioactivation of ß-lap. Low expression of NQO1 in normal brain and other pediatric tissues suggests a wide therapeutic window for ß-lap. These findings encourage the continued study of ß-lap and other NQO1 bioactivated therapeutics for the treatment of this recalcitrant disease. This work was supported by NIH/NCI R01 CA102972 to DAB and a REACH award from the Alex's Lemonade Stand Foundation to JFA and TWL. Citation Format: Zachary R. Moore, Sarai Stuart, Agnieszka Cholka, Ashwin Venkataraman, Jingying Xu, Anat Erdreich-Epstein, Dinesh Rakheja, David A. Boothman, James F. Amatruda, Theodore W. Laetsch. β-lapachone: a novel targeted therapy for atypical teratoid rhabdoid tumors (ATRTs). [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 1613. doi:10.1158/1538-7445.AM2015-1613

Abstract A22: Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis in Wilms tumors

Abstract Wilms tumor is the most common pediatric kidney cancer and the fourth most common childhood cancer overall. It is treated with a combination of surgery, chemotherapy, and radiation, and while most children are cured, survival remains poor in those with advanced-stage disease. Known driver mutations (in WT1, WTX, and CTNNB1) account for only one-third of Wilms tumor cases. To better define the genomic landscape of Wilms tumor, we performed whole-exome sequencing of 44 Wilms tumors. We also sequenced matched germline DNA by whole-exome sequencing (15 cases) or targeted Sanger sequencing (29 cases) to identify somatic mutations. Through this approach, we identified recurrent somatic mutations in MYCN, DROSHA, and DICER1, which were mutually exclusive with known Wilms tumor mutations in WT1 and CTNNB1. The ribonucleases DROSHA and DICER1 perform two key steps in the biogenesis of microRNAs (miRNAs), which are ∼22-nt non-coding RNAs that regulate the stability and translation of target mRNAs. DROSHA and DICER1 mutations occurred at or near conserved metal-binding residues in their ribonuclease (RNase) IIIB domains. In vitro processing assays modeling these mutations showed that they partially or completely disrupted the ribonuclease activity of DROSHA and DICER1. In addition, next-generation sequencing of small RNAs in these tumors revealed that the DICER1 mutations preferentially impaired processing of miRNAs derived from the 5′ arm of pre-miRNA hairpins, including the let-7 tumor suppressor miRNA family. Processing of 5′-derived miRNAs is specifically dependent on DICER1 RNase IIIB activity. Although DROSHA-mutant tumors did not exhibit skewed expression of 5′ vs. 3′-derived miRNAs, expression of let-7 and other putative tumor suppressor miRNAs was reduced in these tumors as well. While germline and somatic mutations in DICER1 have been reported in some cancers, this is the first report of recurrent DROSHA mutations in any human tumor type. Moreover, these results suggest that impairment of miRNA processing by somatic mutation of DROSHA and DICER1 defines a novel subclass of Wilms tumor. Citation Format: Dinesh Rakheja, Kenneth S. Chen, Yangjian Liu, Abhay A. Shukla, Sara Hildebrand, Vanessa Schmid, Xiaoyong Sun, Xin Feng, Tsung-Cheng Chang, Shama Khokhar, Nitin J. Karandikar, James S. Malter, Joshua T. Mendell, James F. Amatruda. Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis in Wilms tumors. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A22.

Abstract A17: Silencing of Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) impairs growth and proliferation of hepatoblastoma cells

Abstract Background: Hepatoblastoma (HB) is the most common primary, malignant liver tumor in children, occurring primarily in children under the age of 4 years. Although chemotherapy in conjunction with surgical resection has improved the prognosis for patients with HB, disease-free survival rates remain less than 50% for patients with advanced, metastatic or relapsed disease, highlighting the need for novel treatments. Additional subsets of tumors with a worse prognosis include those with histologic features of hepatocellular carcinoma (HCC) and tumors which develop in children older than 4 years. Nuclear factor erythroid-2–related factor 2 (NFE2L2, also known as Nrf2), a key transcriptional regulator for the cellular response to oxidative stress in normal cells, has been shown to contribute to malignant phenotypes of cancers. Recently, activating mutations in NFE2L2 have been reported in HCC and also in HB by our research team (Trevino et al1). These activating mutations of NFE2L2 and/or high expression of NFE2L2 have been observed notably in several pediatric liver tumors with high-risk features associated with a poor outcome. We hypothesize that NFE2L2 activation/overexpression may contribute to an aggressive tumor phenotype, and investigated its role in HB cell proliferation. Methods: Hep293TT cells, previously established using primary tumor tissues from a highly aggressive hepatoblastoma from a 5-year-old Caucasian female, were transfected with NFE2L2 small hairpin RNAs (shRNAs) to determine the effect of NFE2L2 suppression on cell proliferation and to identify potential NFE2L2-regulated targets. Analyses of Hep293TT cell proliferation and cell cycle kinetics were conducted using cell viability assays (CellTiter-Glo Luminescence), propidium iodide staining and FACS analysis. A comprehensive transcriptome analyses of Hep293TT cells was carried out by performing RNA sequencing (RNA-seq) using an Illumina HiSeq 2000. Results: RNA-seq analysis was performed on a set of pediatric cancer cell lines including Hep293TT, as well as reference liver cell lines HepG2 and Hep3B. After normalization, the analysis revealed high expression of NFE2L2 in Hep293TT cells with fold change of 2.01 relative to the mean expression level from the pediatric cancer cell line panel (14 cell lines). Compared to other liver cancer cell lines, fold change of 1.28 and 1.5 were measured from Hep3B and HepG2, respectively. Differential expression between NFE2L2 knockdown and parental cell will further demonstrate the molecular and functional influence of NFE2L2. Furthermore, knockdown of NFE2L2 inhibited cell proliferation in Hep293TT cells by inducing cycle arrest. Conclusions: Our results suggest that NFE2L2 may contribute to cell growth and proliferation in hepatoblastoma. Further studies are warranted to determine if NFE2L2 status can be used to stratify highly aggressive pediatric liver tumors and establish NFE2L2 as a potentially novel target for the treatment of HBs. 1Trevino et al, AACR Annual Meeting, 2012, Abstract #4592 This work was supported by grants from the Cancer Prevention and Research Institute of Texas RP101195 and NIH R21 CA122565. Citation Format: Hima Bansal, Tina Chen, Zhao Lai, Donald Parsons, Lisa Trevino, Dolores Lopez-Terrada, Milton Finegold, Dinesh Rakheja, Sarah Comerford, Robert Hammer, Bradley Pollock, Jaclyn Hung, Sanjay Bansal, Yidong Chen, Gail Tomlinson. Silencing of Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) impairs growth and proliferation of hepatoblastoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A17.

Abstract LB-131: Elevated (L) -2-hydroxyglutarate promotes loss of 5-hydroxymethylcytosine in clear cell renal cancer

Abstract We undertook an unbiased metabolomics profile of clear cell renal cell carcinoma (ccRCC), the most common histological subtype, to gain deeper insight into the metabolic basis of this disease. Here, we demonstrate elevations of the metabolite 2-hydroxyglutarate (2HG) in kidney cancer. Elevation of 2HG, specifically the D- enantiomer, has been linked to transformation and identified in the context of gain of function mutations of isocitrate dehydrogenase (IDH1 and IDH2). Notably, enantiomeric resolution demonstrates that, in contrast to IDH1/IDH2 mutant tumors, kidney tumors demonstrate elevations of the L enantiomer of 2HG (L-2HG). Moreover, high 2HG tumors demonstrate reduced DNA levels of 5-hydroxymethylcytosine (5hmC) consistent with the ability of L-2HG to inhibit TET (Ten Eleven Translocation) enzymes which convert 5-methylcystoine to 5-hmC. Finally, we demonstrate that these changes are mediated by the reduced expression of L-2HG dehydrogenase (L2HGDH) in ccRCC. Here, we report elevations of the putative oncometabolite L-2HG in the most common subtype of kidney cancer and describe a novel mechanism for the regulation of DNA 5hmC levels. Our findings provide new insight into the metabolic basis for the epigenetic landscape of renal cancer. Citation Format: Eun-Hee Shim, Carolina B. Livi, John Knight, Ross P. Holmes, Dinesh Rakheja, Sadanan Velu, Eun-Young Kho, Balachandra Chenna, Shane L. Rea, Daniel Benson, Richard Kirkman, Arindam Ghosh, Qiuhua Li, Sejong Bae, Shi Wei, Karen L. Block, Sunil Sudarshan. Elevated (L) -2-hydroxyglutarate promotes loss of 5-hydroxymethylcytosine in clear cell renal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-131. doi:10.1158/1538-7445.AM2014-LB-131

Abstract LB-204: Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis in Wilms tumors

Abstract Wilms tumor is the most common pediatric kidney cancer and the fourth most common childhood cancer overall. It is treated with a combination of surgery, chemotherapy, and radiation, and, while most children are cured, survival remains poor in those with advanced-stage disease. Known driver mutations (in WT1, WTX, and CTNNB1) account for only one-third of Wilms tumor cases. To better define the genomic landscape of Wilms tumor, we performed whole-exome sequencing of 44 Wilms tumors. We also sequenced matched germline DNA by whole-exome sequencing (15 cases) or targeted Sanger sequencing of candidate mutations (29 cases) to identify somatic mutations. Through this approach, we identified recurrent somatic mutations in MYCN, DROSHA, and DICER1 which were mutually exclusive with known Wilms tumor mutations in WT1 and CTNNB1. The ribonucleases DROSHA and DICER1 perform two key steps in the biogenesis of microRNAs (miRNAs), which are ∼22-nt non-coding RNAs that regulate the stability and translation of target mRNAs. DROSHA and DICER1 mutations occurred at or near conserved metal-binding residues in their ribonuclease (RNase) IIIB domains. In vitro processing assays modeling these mutations showed that they partially or completely disrupt the ribonuclease activity of DROSHA and DICER1. In addition, next-generation sequencing of small RNAs in these tumors revealed that the DICER1 mutations preferentially impaired processing of miRNAs derived from the 5′ arm of pre-miRNA hairpins, including the let-7 tumor suppressor miRNA family. Although DROSHA-mutant tumors did not exhibit skewed expression of 5′ vs. 3′-derived miRNAs, expression of let-7 and other putative tumor suppressor miRNAs was reduced in these tumors as well. While germline and somatic mutations in DICER1 have been reported in some cancers, this is the first report of recurrent DROSHA mutations in any human tumor type. Moreover, these results suggest that impairment of miRNA processing by somatic mutation of DROSHA and DICER1 defines a novel subclass of Wilms tumor. Citation Format: Dinesh Rakheja, Kenneth S. Chen, Yangjian Liu, Tsung-Cheng Chang, Abhay A. Shukla, James Malter, Joshua T. Mendell, James F. Amatruda. Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis in Wilms tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-204. doi:10.1158/1538-7445.AM2014-LB-204