Stacey J. Adam

MYC oncogene overexpression drives renal cell carcinoma in a mouse model through glutamine metabolism

Significance The absence of appropriate transgenic animal models of renal cell carcinomas (RCCs) has made it difficult to identify and test new therapies for this disease. We developed a new transgenic mouse model of a highly aggressive form of RCC in which tumor growth and regression is conditionally regulated by the MYC oncogene. Using desorption electrospray ionization–mass-spectrometric imaging, we found that certain glycerophosphoglycerols and metabolites of the glutaminolytic pathway were higher in abundance in RCC than in normal kidney tissue. Up-regulation of glutaminolytic genes and proteins was identified by genetic analysis and immunohistochemistry, therefore suggesting that RCC tumors are glutamine addicted. Pharmacological inhibition of glutaminase slowed tumor progression in vivo, which may represent a novel therapeutic route for RCC. The MYC oncogene is frequently mutated and overexpressed in human renal cell carcinoma (RCC). However, there have been no studies on the causative role of MYC or any other oncogene in the initiation or maintenance of kidney tumorigenesis. Here, we show through a conditional transgenic mouse model that the MYC oncogene, but not the RAS oncogene, initiates and maintains RCC. Desorption electrospray ionization–mass-spectrometric imaging was used to obtain chemical maps of metabolites and lipids in the mouse RCC samples. Gene expression analysis revealed that the mouse tumors mimicked human RCC. The data suggested that MYC-induced RCC up-regulated the glutaminolytic pathway instead of the glycolytic pathway. The pharmacologic inhibition of glutamine metabolism with bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide impeded MYC-mediated RCC tumor progression. Our studies demonstrate that MYC overexpression causes RCC and points to the inhibition of glutamine metabolism as a potential therapeutic approach for the treatment of this disease.

Twist1 Suppresses Senescence Programs and Thereby Accelerates and Maintains Mutant Kras-Induced Lung Tumorigenesis

KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer mouse model and in human lung tumors that the inhibition of Twist1 restores a senescence program inducing the loss of a neoplastic phenotype. The Twist1 gene encodes for a transcription factor that is essential during embryogenesis. Twist1 has been suggested to play an important role during tumor progression. However, there is no in vivo evidence that Twist1 plays a role in autochthonous tumorigenesis. Through two novel transgenic mouse models, we show that Twist1 cooperates with KrasG12D to markedly accelerate lung tumorigenesis by abrogating cellular senescence programs and promoting the progression from benign adenomas to adenocarcinomas. Moreover, the suppression of Twist1 to physiological levels is sufficient to cause Kras mutant lung tumors to undergo senescence and lose their neoplastic features. Finally, we analyzed more than 500 human tumors to demonstrate that TWIST1 is frequently overexpressed in primary human lung tumors. The suppression of TWIST1 in human lung cancer cells also induced cellular senescence. Hence, TWIST1 is a critical regulator of cellular senescence programs, and the suppression of TWIST1 in human tumors may be an effective example of pro-senescence therapy.

STK38 is a Critical Upstream Regulator of MYC’s Oncogenic Activity in Human B-cell lymphoma

The MYC protooncogene is associated with the pathogenesis of most human neoplasia. Conversely, its experimental inactivation elicits oncogene addiction. Besides constituting a formidable therapeutic target, MYC also has an essential function in normal physiology, thus creating the need for context-specific targeting strategies. The analysis of post-translational MYC activity modulation yields novel targets for MYC inactivation. Specifically, following regulatory network analysis in human B-cells, we identify a novel role of the STK38 kinase as a regulator of MYC activity and a candidate target for abrogating tumorigenesis in MYC-addicted lymphoma. We found that STK38 regulates MYC protein stability and turnover in a kinase activity-dependent manner. STK38 kinase inactivation abrogates apoptosis following B-cell receptor activation, whereas its silencing significantly decreases MYC levels and increases apoptosis. Moreover, STK38 knockdown suppresses growth of MYC-addicted tumors in vivo, thus providing a novel viable target for treating these malignancies.

Lymphomas that recur after MYC suppression continue to exhibit oncogene addiction

The suppression of oncogenic levels of MYC is sufficient to induce sustained tumor regression associated with proliferative arrest, differentiation, cellular senescence, and/or apoptosis, a phenomenon known as oncogene addiction. However, after prolonged inactivation of MYC in a conditional transgenic mouse model of Eμ-tTA/tetO-MYC T-cell acute lymphoblastic leukemia, some of the tumors recur, recapitulating what is frequently observed in human tumors in response to targeted therapies. Here we report that these recurring lymphomas express either transgenic or endogenous Myc, albeit in many cases at levels below those in the original tumor, suggesting that tumors continue to be addicted to MYC. Many of the recurring lymphomas (76%) harbored mutations in the tetracycline transactivator, resulting in expression of the MYC transgene even in the presence of doxycycline. Some of the remaining recurring tumors expressed high levels of endogenous Myc, which was associated with a genomic rearrangement of the endogenous Myc locus or activation of Notch1. By gene expression profiling, we confirmed that the primary and recurring tumors have highly similar transcriptomes. Importantly, shRNA-mediated suppression of the high levels of MYC in recurring tumors elicited both suppression of proliferation and increased apoptosis, confirming that these tumors remain oncogene addicted. These results suggest that tumors induced by MYC remain addicted to overexpression of this oncogene.

Abstract LB-313: Development of a novel anti-MYC therapeutic with efficacy against liver and kidney cancers.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnOne of the most attractive, yet elusive, targets for cancer therapy is the MYC oncogene. MYC is overexpressed in the majority of human cancers, and we and others have shown that suppression of this oncogene is sufficient to induce sustained tumor regression, a phenomenon known as oncogene addiction. However, no viable therapy has been developed as yet that targets this crucial regulator of the tumor phenotype. Here, we report the development of a novel antisense therapeutic that targets the MYC oncogene. This inhibitor can potently suppress MYC expression in primary tumors in vivo, as demonstrated by immunohistochemistry and real-time PCR. Importantly, we show that treatment with the anti-MYC drug significant reduces tumor onset and progression in transgenic mouse models of primary hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC) cancer. We imaged tumor growth via MRI and utilized quantitative image analysis to demonstrate the inhibition of HCC and RCC progression, confirming our results on total tumor burden histologically after four weeks of drug treatment. Additionally, this antisense therapy against MYC can also attenuate the growth of human HCC cells in vivo, irrespective of the inducing oncogene. In contrast with other reported MYC inhibitors, our approach results in no discernible toxicity due to MYC suppression on any organs examined in the treated animals. Thus, we have established a novel therapy that directly inhibits the MYC oncogene, representing a potentially exciting advance in the field of targeted cancer therapeutics.nnCitation Format: David I. Bellovin, Aleksey Yevtodiyenko, Stacey J. Adam, Hanan Fernando, Julia Arzeno, Meital Gabay, Dean W. Felsher. Development of a novel anti-MYC therapeutic with efficacy against liver and kidney cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-313. doi:10.1158/1538-7445.AM2013-LB-313nnNote: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2975: Transgenic mouse model of Twist1-induced metastasis reveals genes highly prognostic for human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a top global cancer health concern due to its high rate of mortality and increasing incidence. HCC lethality is largely attributable to its poor response to existing therapy and frequent diagnosis at late stage of disease, consequently resulting in HCC that progresses to local invasion and distant metastasis. Expression of Twist1 has been correlated with cancer invasion and metastasis, but a causal role has yet to be established for autochthonous tumors. Here, we report a new conditional transgenic mouse model of Twist1- and MYC-induced primary hepatocellular carcinoma (HCC). Expression of MYC alone produced primary tumors, but failed to induce metastasis, while Twist1 co-expression with MYC caused extra-hepatic metastases to the lymph nodes, spleen, peritoneum, and lungs. Moreover, Twist1 expression caused an increase in circulating tumor cells (CTCs). Combined Twist1 and MYC inactivation resulted in sustained regression of both primary and metastatic HCC as shown by X-ray computed tomography (micro-CT) and bioluminescence imaging (BLI) and the suppression of metastasis as measured by a loss of CTCs. Importantly, gene expression profiling illustrated that our mouse model of Twist1/MYC-induced liver cancer was representative of human HCC. Moreover, we exploited the Twist1-induced progression to identify a gene signature in Twist1/MYC tumors that is highly predictive of metastasis in human HCC patients, as well as prognostic for overall survival. Hence, we show that Twist1 expression can causally induce stepwise disease progression to metastasis in primary HCC, allowing for the identification of a gene signature that is highly predictive of clinical outcome in human liver cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2975. doi:1538-7445.AM2012-2975

Abstract 4879: Distinct roles of p53 and p19ARF in MYC-dependent tumor oncogene addiction

Acute T-cell lymphoblastic leukemia (T-ALL) is a pediatric malignancy in which the MYC oncogene is frequently overexpressed. Although, T-ALL responds to standard therapies, in 20% of cases, treatment fails to induce sustained remission, resulting in reduced patient survival. Inhibition or restoration of the mutant gene products that drive tumorigenesis can reverse the aberrant cell growth and result in elimination of tumor cells through the phenomenon of oncogene addiction. In order to recapitulate tumor regression and recurrence, we have developed conditional mouse models of MYC-dependent T-ALL, thereby allowing for the investigation of therapeutic resistance. Recently, we have shown oncogene addiction in our MYC-driven murine model of T-ALL occurs through both tumor cell-intrinsic mechanisms, such as cellular senescence and apoptosis, and tumor cell-extrinsic, host-dependent mechanisms, including inhibition of angiogenesis, immune system-dependent tumor clearance, and autocrine/paracrine survival signaling. We now present evidence that loss of either p53 or p19ARF facilitates tumor recurrence through distinct mechanisms. p53 utilizes host-dependent mechanisms by allowing tumors to maintain angiogenesis, while p19ARF employs tumor-cell intrinsic mechanisms to circumvent senescence upon oncogene inactivation in vivo. To further interrogate the mechanisms that underlie p53- and p19ARF-dependent tumor recurrence, we performed gene expression analysis on MYC wild-type, MYC/p53-/-, and MYC/p19-/- tumors before and after MYC inactivation. Utilizing novel computational techniques to compare gene expression from our T-ALL cells to gene expression of normal hematopoietic differentiation from the Immunologic Genome Project, we have delineated the regulatory pathways underlying the effects of tumor suppressor loss in human MYC-driven lymphoma cells in the presence of targeted MYC-inactivation. We have also utilized expression analysis to establish gene signatures from unique to our MYC/p53-/- and MYC/p19-/- murine T-ALL models and shown them to be prognostic for specific human T-ALL subtypes, as well as patient relapse-free survival. Overall, we have utilized whole transcriptomic analysis to further define the underlying cell signaling by which loss of p53 and p19 facilitate tumor recurrence and demonstrated that loss of these tumor suppressors can predict outcome in human patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4879. doi:1538-7445.AM2012-4879

Abstract 5070: Identification of DKK2 as a candidate gene that facilitates tumor reoccurrence upon MYC inactivation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnThe MYC oncogene is prevalently activated in human hematopoietic cancers, especially T-cell acute lymphoblastic leukemia (T-ALL) and Burkitts lymphoma. Utilizing the Tet-system to generate a conditional model of MYC-induced T-ALL, our laboratory has demonstrated that MYC inactivation induces sustained tumor regression. However, after prolonged MYC inactivation some tumors eventually escape MYC dependence and reoccur. Our results support the hypothesis that tumors escape complete dependence on MYC by acquiring additional genetic or epigenetic changes, leading to altered gene function that may substitute totally or partially for MYC. Specifically, using Spectral Karyotypic Analysis (SKY) and array Comparative Genomic Hybridization (aCGH) we have obtained evidence that the Wnt/ β-catenin related gene, DKK2, may play a role in the mechanism by which tumors escape dependence upon MYC. We demonstrate DKK2 facilitates the ability of MYC-driven lymphomas to reoccur even in the presence of sustained oncogene inactivation. We have evidence that cellular context may play a pivotal role in DKK2s ability to aid tumor reoccurrence. Our data also suggest a mechanism by which DKK2 functions, alone or in conjunction with other Wnt/β-catenin pathway members, to maintain tumorigenesis or facilitate tumor reoccurrence in murine and human MYC-driven tumors in the presence of sustained MYC inactivation. Discovering that DKK2 overexpression may aid in lymphoma reoccurrence is a highly novel finding as DKK2 has never been implicated as an oncogene in lymphoma. Results presented here provide critical new insights into the mechanism by which MYC-driven cancers may obtain chemotherapeutic resistance and reoccur even in the presence of sustained drug therapy.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5070.

Abstract 5036: NF-κB pathway inhibition delays the onset of MYC-induced liver cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnLiver cancer is one of the most lethal forms of cancer worldwide. Hence, it is critical to identify pathways that may be essential for the genesis of liver cancer and can thus be considered as targets for therapeutic intervention. Previously, our laboratory developed a conditional transgenic murine model of MYC-induced liver cancer that is dependent upon the expression of this oncogene and regresses following its inactivation. Here, we present evidence of an important role for the nuclear factor-kappa B (NF-κB) pathway in MYC-induced hepatocarcinogenesis. In order to examine whether NF-κB activation is necessary for MYC-induced liver cancer, we established a transgenic system in which MYC can be activated in hepatocytes concomitantly with a mutant form of IκB, thereby inhibiting the NF-κB pathway. Our approach has revealed that suppression of NF-κB activation can significantly inhibit MYC-induced hepatocellular carcinoma (HCC) in adult hosts. Indeed, animals with hepatocyte-inhibited NF-κB remain tumor free up to 8 months after oncogene activation compared to mortality in 100% within 6 months in animals expressing MYC alone. Moreover, inhibition of NF-κB reduces disease penetrance from 100% to 75%. In hosts that do develop liver tumors, there is evidence for activation of the NF-κB pathway, suggesting a necessity for NF-κB activation in MYC-induced HCC. Importantly, suppression of NF-κB activation in neonatal hosts is not only able to similarly suppress MYC-induced tumor formation, but animals that develop liver cancer in this context demonstrate a strikingly altered tumor phenotype. In addition, MYC inactivation results in a concomitant decrease in expression of NF-κB target genes, suggesting a direct role of MYC in the activation of NF-κB in hepatocytes. The inhibition of NF-κB did not result in increased apoptosis upon MYC activation. Rather, NF-κB activation may be important for MYC to evade oncogene-induced senescence, thereby facilitating tumorigenesis. Our data suggests that the inhibition of the NF-κB pathway may be sufficient to abrogate MYCs ability to induce tumorigenesis in the liver and thereby establish NF-κB activation as an attractive therapeutic target for liver cancer.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5036.