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IL15 is a pleiotropic cytokine with multiple roles that improve immune responses to tumor cells. Oncolytic viruses (OV) specifically lyse tumors and activate immune responses. Systemic administration of IL15 or its complex with the IL15Rα and chimeric antigen receptor (CAR) natural killer (NK) cells are currently being tested in the clinic. Here, we generated a herpes simplex 1–based OV-expressing human IL15/IL15Rα sushi domain fusion protein (named OV-IL15C), as well as off-the-shelf EGFR-CAR NK cells, and studied their monotherapy and combination efficacy in vitro and in multiple glioblastoma (GBM) mouse models. In vitro, soluble IL15/IL15Rα complex was secreted from OV-IL15C–infected GBM cells, which promoted GBM cytotoxicity and improved survival of NK and CD8+ T cells. Frozen, readily available off-the-shelf EGFR-CAR NK cells showed enhanced killing of tumor cells compared with empty vector–transduced NK cells. In vivo, OV-IL15C significantly inhibited tumor growth and prolonged survival of GBM-bearing mice in the presence of CD8+ T cells compared with parental OV. OV-IL15C plus EGFR-CAR NK cells synergistically suppressed tumor growth and significantly improved survival compared with either monotherapy, correlating with increased intracranial infiltration and activation of NK and CD8+ T cells and elevated persistence of CAR NK cells in an immunocompetent model. Collectively, OV-IL15C and off-the-shelf EGFR-CAR NK cells represent promising therapeutic strategies for GBM treatment to improve the clinical management of this devastating disease. Significance: The combination of an oncolytic virus expressing the IL15/IL15Rα complex and frozen, ready-to-use EGFR-CAR NK cells elicits strong antitumor responses in glioblastoma.

Non-small cell lung cancer (NSCLC) remains one of the major causes of cancer related mortality worldwide, yet clinical advances with checkpoint inhibitors suggest that an anti-tumor immune response can be harnessed to prolong survival in NSCLC patients. We recently described the identification of a tumor reactive natural killer (NK) cell population by its in vivo upregulation of PD-L1 in the presence of tumor, referred to here as TRACK-NK cells. PD-L1(+) NK cells are more potent than PD-L1(-) NK cells against tumor cell targets. Further, the administration of anti-PD-L1 antibody (atezolizumab) functions as an immune agonist on TRACK-NK cells resulting in significantly enhanced degranulation (P < 0.001) and secretion of IFN-gamma (P < 0.001) in the presence of tumor. Here we investigated the functional activity and product safety of allogeneic, off-the-shelf TRACK-NK cells retrovirally transduced to express soluble (s) IL-15 to treat NSCLC without or with the addition of atezolizumab. TRACK-NK cells derived from umbilical cord blood NK cells were expanded over 1,000-fold ex-vivo in 17 days with ~50% of the final product engineered to express sIL15 and cytokine-activated to express PD-L1. The phenotype of the final TRACK-NK cell product showed a significant increase in the expression of PD-L1, CD25 and CD69 when compared to non-transduced (P < 0.01) or NK cells transduced to express sIL15 without cytokine activation (s15 NK cells) (P < 0.01). Following cryopreservation, our thawed TRACK-NK cell product demonstrated high recovery (>85%) and viability (>80%). In a Real Time Cell Analysis (RTCA) in vitro cytotoxicity assay against a human NSCLC cell line (A549), TRACK-NK cells were 10 times more potent than non-transduced (NT) NK cells (P < 0.001) and 2 times more potent than s15 NK cells (P < 0.01). To assess the efficacy of our frozen, allogeneic off-the-shelf TRACK-NK cells in vivo, we utilized immunodeficient mice bearing human A549 metastatic NSCLC. Intravenous infusion of TRACK-NK cells resulted in significantly more suppression of luciferase-A549 NSCLC growth in mice compared to mice treated with NT NK cells (P < 0.05) or s15 NK cells (P < 0.01). In terms of dose response, 1 × 107 TRACK-NK cells per dose delivered every other day for a total of 4 doses showed significantly better tumor control compared to a dose of 5 × 106 TRACK-NK cells (P < 0.001) but similar control compared to 2 × 107 TRACK-NK cells. The combination of TRACK-NK cells plus atezolizumab showed better control of NSCLC growth in vivo compared to TRACK-NK cells alone (P < 0.01). Finally, we evaluated the safety of our TRACK-NK product in immunodeficient mice without tumor engraftment and did not observe any significant change in body weight, body temperature, liver enzymes, kidney function, or blood counts compared to mice treated with vehicle control. No cytokine release syndrome was observed. In summary, our studies using human allogeneic off-the-shelf TRACK-NK cells proved to be safe and non-toxic, demonstrated enhanced cytotoxicity against NSCLC in vitro and enhanced control of tumor growth in vivo, further improved with atezolizumab. As the intravenous infusion of activated NK cells naturally traffic to lung, our TRACK-NK platform will move forward to the clinic for the treatment of relapsed and refractory NSCLC patients. Citation Format: Ting Lu, Anthony G. Mansour, Rui Ma, Christian Bustillos, Zhiyao Li, Shoubao Ma, Zhenlong Li, Hanyu Chen, Kun-Yu Teng, Jianying Zhang, Michael A. Caligiuri, Jianhua Yu. Tumor-reactive and anti-PD-L1 co-stimulated killer cells (TRACK-NK) for immunotherapy of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB211.

Despite the unprecedented clinical success of autologous chimeric antigen receptor (CAR) T cell therapy in B cell leukemia and lymphoma patients, CAR T cell therapy for acute myeloid leukemia (AML) patients has lagged, in part due to the time required for autologous CAR T cell preparation in the face of the rapid relapse of AML following remission, and the possibility of on-target off-tumor hematopoietic toxicity. In this study we investigated the functional activity of an allogeneic, off-the-shelf FLT3 CAR natural killer (NK) cells expressing soluble (s) IL-15 (FLT3 CAR_s15 NK) to treat FLT3+ AML. Cord blood NK cells were first transduced with FLT3 CAR intracellular signaling constructs containing either CD28/CD3ζ or 2B4/CD3ζ. Each displayed similar cytotoxicity against FLT3+ AML in vitro (p < 0.05) and in vivo (p < 0.01) when compared to mock transduced NK cells. Cord blood NK cells transduced with either soluble (s) IL-15 (s15 NK cells) or membrane (m) bound IL-15 showed comparable extended survival in vivo and superior to NK cells transduced with sIL-15/IL-15Rα (p < 0.01) or mIL-15/IL-15Rα (p < 0.01). However, only those NK cells transduced with soluble IL-15 were able to activate neighboring non-transduced (NT) NK cells and T cells in a paracrine fashion. The FLT3 CAR_s15 NK cells were expanded ex-vivo to over 1500-fold in 16 days with ~50% of the NK cells expressing both the FLT3 CAR and sIL-15. These FLT3 CAR_s15 NK cells were then subjected to a cytotoxicity assay against the FLT3+ AML cell line (MOLM-13) and were 10 times more potent than NT NK cells (p < 0.01) and 1.7 times more potent than s15 NK cells (p < 0.05). Likewise, when co-cultured with the FLT3+ AML cell line, FLT3 CAR_s15 NK cells produced a greater amount of IFN-γ compared to NT NK cells (p < 0.01) or compared to s15 NK cells (p < 0.05). Furthermore, following cryopreservation, our thawed FLT3 CAR_s15 NK cells demonstrated high recovery (~90%) and viability (> 85%), maintaining their FLT3 CAR expression at ~50% with a phenotype and cytotoxicity that was nearly equivalent to fresh FLT3 CAR_s15 NK cells. Armed with these data we assessed the efficacy of our allogeneic, off-the-shelf FLT3 CAR_s15 NK cells in vivo using immunodeficient mice engrafted with the MOLM-13 FLT3+ cell line. Repeated infusions of FLT3 CAR_s15 NK cells provided an improved median survival compared to placebo group (36 vs 24 days; p < 0.001) or compared to identical infusions of s15 NK cells (36 vs 31 days; p < 0.05) with similar results obtained in a patient-derived xenograft model. Finally, to assess toxicity against normal peripheral blood mononuclear cells (PBMC), including FLT3+ dendritic cells, and against hematopoietic stem cells (HSCs) we performed in vitro and in vivo studies, respectively, using our FLT3 CAR_s15 NK cells. We did not observe an untoward cytotoxicity of PBMC in vitro when compared to NT or s15 NK cells and no disruption of HSC differentiation in vivo when compared to placebo group. In summary, our in vitro and in vivo studies with allogeneic off-the-shelf cord blood derived FLT3 CAR_s15 NK cells demonstrated enhanced cytotoxicity and IFN-γ secretion against FLT3+ AML in vitro and enhanced the survival of mice engrafted with FLT3+ AML without any evidence of PBMC or HSC toxicity. We believe these data collectively support a rationale for investigating this novel form of allogeneic, off-the-shelf FLT3 CAR_s15 NK cell therapy in patients stricken with FLT3+ AML. Citation Format: Anthony G. Mansour, Kun-Yu Teng, Zhiyao Li, Zheng Zu, Hanyu Chen, Aliya Ali, Jianying Zhang, Ting Lu, Shoubao Ma, Michael A. Caligiuri, Jianhua Yu. Off-the-shelf cord blood FLT3 CAR-NK cells for immunotherapy of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB102.

Background Real-world data (RWD) has been increasingly used in drug development. A reliable evaluation of efficacy for current treatments is challenging with very limited publication data of clinical trials available. The RWD provides a unique value and an alternative way to achieve such goal. Use data from metastatic Breast Cancer (mBC) patients in the real world clinical practice, we examine the treatment patterns and durations of different endocrine monotherapies in various clinical settings. Methods We obtained Flatiron Health subject-level mBC data set which consists a group of 20645 mBC patients. The analysis set consisted of subjects who were ER+/Her2-/(PgR+ or PgR-), received at least one prior line of CDK4/6 inhibitor (mono or combo) and one prior line of endocrine therapy (mono or combo) in the mBC settings, and had an ECOG performance score Result Out of all subjects in the mBC RWD database, 263 subjects met the inclusion criteria and were included in the analysis set. Among all 263 ER+/Her2- subjects, the median TTD of endocrine monotherapies was 3.0 months (95% CI: 2.8-3.7 months, N=263) in all subjects, 3.7 months (95% CI: 3.0-4.3 months, N=186) in PgR+ group and 2.1 months (95% CI: 1.9-2.9 months, N=77) in PgR- group. Among 186 ER+/PgR+/Her2- subjects, 1) the median TTD of endocrine monotherapies was 3.3 months (95% CI: 2.8-4.6 months, N=153) in ECOG=3 prior treatment lines group. Conclusion By investigating the RWD, we observed that the PgR status is prognostic in post CDK setting: endocrine monotherapies in PgR+ group has longer TTD compared to PgR- group in ER+/Her2- mBC subjects. By further looking into ER+/PgR+/Her2- subjects, we observed different treatment durations of endocrine therapies in various clinical scenarios, which may provide a helpful understanding of the mBC monotherapy treatments. Citation Format: Tenghui Chen, Zhaojie Zhang, Lei Gao, Catherine Scholz, Antonio Gualberto, Lihua Yu, Kun Yu. Using real-world data to evaluate the performance of endocrine therapies in ER+/Her2- metastatic breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5247.

Background Real-world data (RWD) has been increasingly used for drug development. RWD coupled with genomic data provides an opportunity to identify the association between certain genomic alterations and response to current standard of care (SOC). Such analyses may help identify predictive biomarkers to select patients who will benefit most from SOC, and/or potential therapeutic nodes that can be pharmacologically targeted to overcome resistance to SOC. Methods We obtained Flatiron CGDB Breast Cancer (as of Q3 2020 release) data set. This data set includes clinical and genomic data from 6,918 breast cancer patients. Time to treatment discontinuation (TTD) was used as real world outcome. We performed two types of genomic-outcome association analysis: 1) Use Cox proportional hazards regression model to identify which baseline mutation (mutation occurred prior to the treatment) could predict the outcome; and 2) use Fisher exact test to identify which mutations are enriched in post-treatment tumors samples, compared to pre-treatment tumor samples. Result We found TP53 mutation is associated with shorter TTD for Palbociclib + Aromatase inhibitor (AI); Palbociclib + Fulvestrant; and endocrine monotherapy. It is consistent with the reports that TP53 mutation is poor prognostic factor (ref). Interestingly, ESR1 mutation is associated with shorter TTD for Palbociclib + AI and AI monotherapy; but not associated with TTD for Palbociclib + Fulvestrant or Fulvestrant monotherapy. On the other hand, MYC amplification is associated shorter TTD for Palbociclib + Fulvestrant or Fulvestrant monotherapy. We found that genes associated with Palbociclib + Endocrine are by and large same as those associated with endocrine monotherapy; suggesting the association is largely driven by endocrine treatment. Comparison of post-treatment vs. pre-treatment analysis found that ESR1 mutation is enriched in post treatment of AI; but not Fulvestrant. Conclusion We demonstrated that real-world clinicogenomic data could identify the biologically meaningful genomic biomarkers that are associated with real world response. This study warrants the further investigation of novel genomic biomarkers identified from the analysis. Citation Format: Kun Yu, Tenghui Chen, Manav Korpal, Ping Zhu, Zhaojie Zhang, Lihua Yu, Yonghong Xiao. Real-world data to identify the genomic association with response to CDK4/6i + Endocrine therapy in ER+ breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4123.

Activation of the fibroblast growth factor receptor FGFR4 by FGF19 drives hepatocellular carcinoma (HCC), a disease with few, if any, effective treatment options. While a number of pan-FGFR inhibitors are being clinically evaluated, their application to FGF19-driven HCC may be limited by dose-limiting toxicities mediated by FGFR1–3 receptors. To evade the potential limitations of pan-FGFR inhibitors, we generated H3B-6527, a highly selective covalent FGFR4 inhibitor, through structure-guided drug design. Studies in a panel of 40 HCC cell lines and 30 HCC PDX models showed that FGF19 expression is a predictive biomarker for H3B-6527 response. Moreover, coadministration of the CDK4/6 inhibitor palbociclib in combination with H3B-6527 could effectively trigger tumor regression in a xenograft model of HCC. Overall, our results offer preclinical proof of concept for H3B-6527 as a candidate therapeutic agent for HCC cases that exhibit increased expression of FGF19. Cancer Res; 77(24); 6999–7013. ©2017 AACR.

Pancreatic cancer (PC) is one of the most lethal forms of cancer with a 5-year overall survival rate of 9%, routinely presenting as a late-stage incurable cancer that responds only modestly to standard chemotherapy. It is the third leading cause of cancer-related deaths. Approximately 60-80% of PC express prostate stem cell antigen (PSCA), as do gastric, bladder, prostate and some lung cancers. We therefore developed a chimeric antigen receptor (CAR) directed against PSCA for transduction into human natural killer (NK) cells. NK cells spontaneously kill both liquid and solid tumors without regards to expression of MHC self-antigens. In vivo, human NK cells utilize endogenous IL-15 to develop, survive, expand and activate against tumor cells. We therefore incorporated a soluble (s), secretable form of IL-15 into the PSCA CAR construct itself, followed by transduction into human NK cells obtained from umbilical cord blood with ~50% transduction efficiency. Transduced NK cells expressed the CAR directed against PSCA and secreted measurable amounts of human IL-15 protein in vitro. The PSCA CAR NK cells could be expanded ex vivo over 1,000-fold in approximately 16 days and retain their expression and their capacity to specificity kill PSCA(+) tumor cell targets in vitro. Indeed, when directed against the PSCA(+) human pancreatic tumor cell line (Capan-1), PSCA CAR NK cells produced significantly greater TNFα (P < 0.0001), IFNγ (P < 0.0001), and CD107a (P < 0.05), when compared to PSCA CAR NK cells against the PSCA(-) human pancreatic tumor cell line PANC-1. Moreover, we showed that the co-expression of sIL-15 with PSCA CAR NK cells significantly enhances their cytotoxic function against pancreatic tumor cells compared to PSCA CAR NK cells without expression of sIL-15 (P < 0.01) determined by a real-time cytolysis assay (RTCA) over 3.5 days. Encouraged by these in vitro data, we developed a model of metastatic human pancreatic cancer in immunodeficient mice using the PSCA(+) Capan-1 cell line. Compared to NK cells only expressing sIL-15, repeated infusions of human PSCA CAR NK cells from a viably frozen source resulted in a significantly prolonged survival (P < 0.001), including clearance of metastatic disease. In summary, our in vitro and in vivo studies utilizing viably frozen human PSCA CAR NK cells co-expressing sIL-15 demonstrate significant efficacy in prolonging survival against a human pancreatic tumor cell line without evidence of systemic toxicity, providing a rationale to move this novel form of cell therapy into the clinic for PSCA(+) solid tumors. Citation Format: Kun-Yu Teng, Anthony Mansour, Zhu Zheng, Lei Tien, Yi Zheng, Zhiyao Li, Jianying Zhang, Saul J. Priceman, Michael A. Caligiuri, Jianhua Yu. A potent human CAR NK cell therapy directed against pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB154.

Introduction: Calmette-Guerin (BCG)-unresponsive non-muscle invasive bladder cancer (NMIBC) is a significant unmet medical need. We report pharmacological, pharmacodynamic and pharmacogenomic characterization of E7766, a novel STING (stimulator of interferon genes) agonist, as a potent immunotherapy in BCG-insensitive orthotopic murine NMIBC models via intravesical administration. To our knowledge, this is the first demonstration of activity of a STING agonist in NMIBC tumor model. Methods: E7766 was examined for antitumor activity in two orthotopic murine NMIBC tumor models via intravesical administration and comparatively characterized in STING activation activity in primary human PBMC cells with defined STING genotypes. Local and systemic pharmacodynamic biomarker changes in tumor-bearing model were identified. Results: E7766, from a structurally novel class of macrocyclic STING agonists, showed specific and potent agonist activity in both human and mouse STING. In human PBMCs, E7766 demonstrated potent and consistent activity across seven tested human STING genotypes. By contrast, a reference cyclic dinucleotide STING agonist showed substantial variability, and was inactive in the REF-REF genotype. Intravesical administration of E7766 to two orthotopic murine models for BCG-insensitive NMIBC showed a dose-dependent and curative activity without serious adverse effects. In contrast, anti-PD1 treatment was ineffective in both models. Tumor-free animal derived from E7766 treatment completely rejected rechallenge of the same tumor cells, demonstrating establishment of effective tumor-specific memory immune response in those compound-treated animals. The anti-tumoral activity was accompanied with activation of IFN pathway, T cell infiltration, NK activity and antigen presentation in bladder wall, and robust induction of IFNβ, CXCL10 and other downstream effectors of STING activation inside the bladder cavity and in urine. Notably, the IFNβ gene induction was found in bladder, but not in blood from treated animals indicative of a primarily local STING activation by intravesical E7766, which was consistent with low systemic bioavailability of the compound via the administration route. Conclusions: These preclinical studies demonstrated a potent anti-tumor activity and induction of tumor-specific memory response by intravesically administered STING agonist E7766 in orthotopic murine models for BCG- and anti-PD1-insensitive NMIBC. A clinical study for intravesical E7766 in NMIBC patients has been initiated (NCT04109092) in North America. Citation Format: Kuan-Chun Huang, Chi Zhang, Kun Yu, Dae-Shik Kim, Vaishali Dixit, Renee Hukkanen, Hyeong-Wook Choi, Janna Hutz, Frank Fang, Xingfeng Bao. Demonstration of E7766, a novel STING agonist, as a potent immunotherapy in BCG-insensitive non-muscle invasive bladder cancer models via intravesical administration [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 592.

Background: Natural Killer (NK) cells are a type of immune cells that belong to the innate immune system and executes a rapid immune reaction against cancer cells and viruses without prior activation. The huge success of chimeric antigen receptor (CAR)-T cells has motivated scientists to genetically modify human NK cells with CARs to further improve their tumor killing capacity. To engineer NK cells with a CAR, NK cells can be isolated from peripheral blood and umbilical cord blood. NK cells can also be derived from induced pluripotent stem cells (iPSCs) to produce a homogenous and standardized group of NK cells for the treatment of cancer. Here, we test the feasibility to engineer our EGFR-CAR construct into iPSC-derived hematopoietic stem cells (iPSC-HSC) to generate EGFR-CAR NK cells that will eradicate glioblastoma (GBM). Material and Methods: iPSCs-derived HSCs were differentiated into NK cells using co-culture with a feeder stroma of OP9-DL1 cells in the presence of human cytokines and NK cells were assessed by flow cytometry. Expanded NK cells (with K562 feeder cells expressing mb-IL-21 and 4-1BB) and HSCs from peripheral blood were efficiently transduced with a retroviral vector, carrying a CAR targeting EGFR. CAR expression was assessed by flow cytometry. After transduction, the CAR-NK cells were collected for cytotoxicity assay to test their efficacy. CAR-HSCs were differentiated into CAR-NK cells utilizing OP9-DL1 stroma cells. Results: Our results demonstrated that iPSCs can be fully differentiated into NK cells expressing typical NK cell surface markers including CD56, NKp46, CD16 and NKG2A, etc. In addition, HSCs can be engineered with EGFR-CAR without affecting their differentiation into CAR-NK cells. Our engineered EGFR-CAR NK cells showed an enhanced antitumor activity against GBM compared to NK cells without the EGFR-CAR. Conclusion: Collectively, our study suggests the feasibility of engineering CAR-EGFR NK cells from iPSCs for effective “off the shelf” immunotherapy. Our approach can be utilized to generate an unlimited number of CAR-NK cells to treat patients with cancer such as GBM in the clinical setting. Citation Format: Melissa Yu, Anthony G. Mansour, Kun-Yu Teng, Guoqiang Sun, Yanhong Shi, Michael A. Caligiuri. iPSC-derived natural killer cells expressing EGFR-CAR against glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3313.

Asian nonsmoking populations have a higher incidence of lung cancer compared with their European counterparts. There is a long-standing hypothesis that the increase of lung cancer in Asian never-smokers is due to environmental factors such as second-hand smoke. We analyzed whole-genome sequencing of 30 Asian lung cancers. Unsupervised clustering of mutational signatures separated the patients into two categories of either all the never-smokers or all the smokers or ex-smokers. In addition, nearly one third of the ex-smokers and smokers classified with the never-smoker–like cluster. The somatic variant profiles of Asian lung cancers were similar to that of European origin with G.C>T.A being predominant in smokers. We found EGFR and TP53 to be the most frequently mutated genes with mutations in 50% and 27% of individuals, respectively. Among the 16 never-smokers, 69% had an EGFR mutation compared with 29% of 14 smokers/ex-smokers. Asian never-smokers had lung cancer signatures distinct from the smoker signature and their mutation profiles were similar to European never-smokers. The profiles of Asian and European smokers are also similar. Taken together, these results suggested that the same mutational mechanisms underlie the etiology for both ethnic groups. Thus, the high incidence of lung cancer in Asian never-smokers seems unlikely to be due to second-hand smoke or other carcinogens that cause oxidative DNA damage, implying that routine EGFR testing is warranted in the Asian population regardless of smoking status. Cancer Res; 74(21); 6071–81. ©2014 AACR.

It is well established that the liver specific miR-122, a bona fide tumor suppressor, plays critical role in lipid homeostasis [1]. However, its role, if any, in amino acid metabolism has not been explored. Since glutamine is an important energy source, we monitored glutamine metabolism in the wild type (WT) and miR-122 knockout (KO) mice by SIRM (Stable Isotope Resolved Metabolomics) studies. To this end, 6-8 weeks old mice were injected with [U-13C,15N]-L-glutamine (Gln) (7 mg/mouse, 2x at 15 min interval) and sacrificed 15 min after the last injection. Analysis of polar metabolites from snap-frozen liver tissues by NMR and IC-MS showed elevated 13C-glutamine, -glutamate, -α-ketoglutarate, -isocitrate and -citrate levels without significant changes in succinate, malate or fumarate levels in KO livers suggest enhanced glutaminolysis and a break in the latter half of the Krebs cycle in miR-122 depleted livers. Reduced conversion of [13C,15N-glutamine] to 13C3- and or/ 13C6-glucose-6-phosphate and -fructose-6-phosphate implicates reduced gluconeogenesis from glutamine in KO livers while that to 13C-glutathiones suggests decreased synthesis and/or increased utilization. To elucidate the underlying mechanism, we focused on HITS-CLIP and RNA-seq data of the WT and KO mice, which identified functional miR-122 targets in the liver transcriptome [2]. The results showed that Gls is a direct miR-122 target, which was validated by luciferase reporter assay. Importantly, Gls expression was suppressed in glutamine dependent hepatoma cells by ectopic expression of miR-122 mimic. These results explain that the increased conversion of [U-13C,15N]-L-glutamine to glutamate and other Krebs cycle metabolites is likely due to the upregulation of Gls in miR-122 depleted livers. TCGA-LIHC database analysis showed that GLS expression in tumors inversely correlated with miR-122 expression. Upregulation of GLS protein level in primary HCCs relative to the matched benign liver tissues suggests enhanced use of glutamine as energy source by the tumor cells. Collectively, these results show that miR-122 modulates glutamine metabolism both in vitro and in vivo. These results implicate therapeutic potential of glutaminase inhibitors in patients bearing HCCs that bear low levels of miR-122. (supported in part, by R01CA193244 grant from NIH and Pelotonia Idea grant) 1. Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver. J Clin Invest. 2012 Aug;122(8):2871-83. 2. Argonaute CLIP Defines a Deregulated miR-122-Bound Transcriptome that Correlates with Patient Survival in Human Liver Cancer. Mol Cell. 2017 Aug 3;67(3):400-410.e7. Citation Format: Dipanwita Sengupta, Teresa Cassel, Kun-yu Teng, Peng Hu, Teresa Fan, Kalpana Ghoshal. miR-122 regulates hepatic glutamine metabolism by directly targeting mitochondrial glutaminase [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 4505.

Endocrine resistance is a major challenge in the management of estrogen receptor (ER)-positive breast cancers. Although multiple mechanisms leading to endocrine resistance have been proposed, the poor outcome of patients developing resistance to endocrine therapy warrants additional studies. Here we show that noncanonical Hedgehog (Hh) signaling is an alternative growth promoting mechanism that is activated in tamoxifen-resistant tumors. Importantly, phosphoinositide 3-kinase inhibitor/protein kinase B (PI3K/AKT) pathway plays a key role in regulating Hh signaling by protecting key components of this pathway from proteasomal degradation. The levels of Hh-signaling molecules SMO and GLI1 and the targets were significantly elevated in tamoxifen-resistant MCF-7 cells and T47D cells. Serial passage of the resistant cells in mice resulted in aggressive tumors that metastasized to distant organs with concurrent increases in Hh marker expression and epithelial mesenchymal transition. RNAi-mediated depletion of SMO or GLI1 in the resistant cells resulted in reduced proliferation, clonogenic survival and delayed G1–S transition. Notably, treatment of resistant cells with PI3K inhibitors decreased SMO and GLI1 protein levels and activity that was rescued upon blocking GSK3β and proteasomal degradation. Furthermore, treatment of tamoxifen-resistant xenografts with anti-Hh compound GDC-0449 blocked tumor growth in mice. Importantly, high GLI1 expression correlated inversely with disease-free and overall survival in a cohort of 315 patients with breast cancer. In summary, our results describe a signaling event linking PI3K/AKT pathway with Hh signaling that promotes tamoxifen resistance. Targeting Hh pathway alone or in combination with PI3K/AKT pathway could therefore be a novel therapeutic option in treating endocrine-resistant breast cancer. Cancer Res; 72(19); 5048–59. ©2012 AACR.

Background Although successful discovery and applications of genome editing system, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/associated nuclease 9), has been reported in basic science research, it has been unclear what happen to patients when moving CRISPR/Cas9 technology to bedside. We performed the first-in-man clinical trial (NCT02793856), phase I, to assess the safety of CRISPR/Cas9-mediated knockout of the programmed cell death 1 (PD-1) gene in autologous T lymphocytes in patients with metastatic non-small cell lung cancer (NSCLC). Methods Using a 3 + 3 + 3 dose-escalation study, we assigned patients with advanced NSCLC with PD-L1 expression positive on tumor cells that had progressed after the 3rd line standard therapeutic regimens. Pre-A cohort was used to enroll 2 patients who received PD-1 deficient gene-edited T cells (PD-1- T) with 2x107 cells/kg in one cycle via CRISPR/Cas9 for safety concern. Then 3 cohorts (A, B, C) enrolled 3 patients receiving total dose of PD-1- T cells with 1 ×107/kg, 2 x 107/kg, 4×107/kg each cycle every 4 weeks, respectively. The primary outcome was safety. Secondary end points were objective response rate, 8 weeks progression-free survival. In exploratory analyses, next-generation sequencing (NGS) was performed on PD-1 editing region of T cell and the CDR3 region of the T-cell receptor beta chain. Results 8 patients received totally 16 cycles of PD-1- T cell infusion in cohort Pre-A, A and B. 13 adverse events (AEs) related to the PD-1- T cell infusion occurred (84.6% of grade 1 and 15.4% of grade 2). Most common AEs were acute fever and hepatic dysfunction (both 15.4%). No DLT and 3-5 AEs were found. 92.3% (12/13) AEs presented within 1st cycle. 7 patients were response evaluable. 2/4 of patients receiving 2 x 107/kg PD-1- T cell experienced SD while other 3/3 patients with lower dose had disease progression. 8-week PFS were 28.6%. Of exploratory data, numerous novel T-cell clonotypes were detected in PD-1- T cell products but not in peripheral bloods before cell infusion. Intriguingly, the persisting novel T cell clones were found in patient peripheral bloods with the frequency of 0.005%-3.05% during the cell therapy, indicating the existence of potential responsive T cell clones. Conclusion The patients in this trial seemed safety when conducing the PD-1 deficient engineered T cells with CRISPR/Cas9 system. Further study should be performed to explore the effective dose and the related immune response. Citation Format: You Lu, Meijuan Huang, Tao Deng, Xiaojuan Zhou, Kun Yu, Maozhi Liang, Lei Deng, Jianxin Xue, Xin Yi, Zhenyu Ding, Youling Gong, Jiang Zhu, Yongsheng Wang, Yuqi Wang, Jin Song, Ruizhan Tong, Li Li, Jingwen Huang, Feifei Na, Min Zhao, Chong Chen, Yuquan Wei, Weimin Li. A phase I trial of PD-1 deficient engineered T cells with CRISPR/Cas9 in patients with advanced non-small cell lung cancer with PD-L1 expression [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 CT133.

Incidence of hepatocellular carcinoma (HCC) that represents ~90% of all cases of primary liver cancer is on the rise worldwide. Sorafenib, the only approved targeted therapy, prolongs median survival only by ∼3 months. Thus, new therapeutic strategies are urgently needed. In this study, we investigated therapeutic potential of zerumbone, a sesquiterpene from an edible ginger, against HCC. Zerumbone inhibited proliferation and clonogenic survival of HCC cells by blocking glycolysis and pentose phosphate pathway, arresting cells at G2/M phase of cell cycle and inducing apoptosis. To uncover the underlying molecular mechanisms of its action on HCC cells, we employed several unbiased approaches. First, phosphokinase array showed significant inhibition PI3K/AKT/mTOR and STAT3 signaling in zerumbone-treated HCC cells. Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) of the microarray data revealed that zerumbone-deregulated genes showed positive correlation with PI3/AKT/mTOR and STAT3 pathways and those involved in metabolism, apoptosis and cell cycle. Notably, the alteration in expression of genes regulated by PI3/AKT/mTOR or STAT3 pathways and downregulated by zerumbone in HCCs is associated with poor patient survival. These results implicate that treatment with this dietary sesquiterpene might lead to a better outcome in HCC patients. Indeed, zerumbone treatment significantly inhibited growth and lung metastasis of orthotopic HCC xenografts in NSG mice. These findings reveal novel therapeutic strategies targeting cancer metabolism. Collectively, these observations underscore the therapeutic potential of zerumbone in HCC. Citation Format: Nissar A. Wani, Bo Zhang, Kun-yu Teng, Juan Barajas, Kalpana Ghoshal, Rafael Brüschweiler, Samson T. Jacob. Inhibition of metabolic reprogramming by zerumbone alters the tumorigenic potential of hepatocellular 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 2521. doi:10.1158/1538-7445.AM2017-2521

Hepatocellular carcinoma (HCC), the second cancer-related deadly disease, commonly arises in the setting of chronic liver inflammation. CC chemokine ligand 2 (CCL2), a chemokine that recruits CCR2-positive immune cells to promote inflammation, is highly upregulated in HCC patients. Here, we examined the therapeutic efficacy of inhibitors of the Ccl2-Ccr2 axis against hepatitis and HCC in the miR-122 knockout (aka KO) mouse model, in which upregulation of Ccl2 promotes liver inflammation that progresses to HCC with age. To determine whether blocking the Ccl2-Ccr2 axis has any therapeutic potential, we treated KO mice with Ccl2 neutralizing antibody (nab) or a small molecule inhibitor of Ccr2 and monitored liver pathology. Both agents were able to reduce the bridging inflammation in these mice by reducing the population of CD11highGr1+ cells, and expression of IL-6 and TNF-alpha in KO livers. Furthermore, treatment of tumor-bearing KO mice with Ccl2 nab for 8 weeks significantly reduced the recruitment of CD11bhighGr1+ monocytes, IL-6 and TNF-alpha production, and HCC incidence as well as tumor burden. The down-regulation of pStat3 and c-Myc (IL-6 downstream) as well as NF-kappaB (TNF-alpha downstream) upon targeting Ccl2 positively correlated with tumor growth suppression. In addition, inhibition of tumorigenesis by Ccl2 nab also correlated with enhanced cytotoxicity and IFN-gamma expression of NK cells isolated from the tumor microenvironment. Collectively, our results demonstrate that blocking the Ccl2-Ccr2 axis can be an effective therapeutic approach against both hepatitis and HCC. Citation Format: Kun-Yu Teng. Inhibition of liver inflammation and tumorigenesis by blocking Ccl2-Ccr2 axis in a mouse model. [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 588.

Rationale: Endocrine therapy is the cornerstone in the management of patients with estrogen receptor positive (ER+) breast cancer (BC). While endocrine therapy is very effective, 30-40% of patients will develop resistance. Our lab has previously shown that the PI3K/AKT pathway activates the Sonic Hedgehog pathway, stabilizing the activated transcription factor, GLI-1, and contributing to the development of endocrine resistance (Ramaswamy B et al: Can Res:August 2012). The purpose of our current study is to elucidate how GLI-1 modulates its target genes that contribute to endocrine-resistance. Methodology: Ectopic expression of GLI-1 in MCF7 cells was followed by microarray gene expression analysis. The GLI-1 signature characterized by differential expression of several BC associated genes was next validated in vitro using real-time PCR. We next utilized the Hatzis data set to determine whether these signature genes correlated with GLI-1 expression in BC patients and whether expression of these genes could be predictive of metastatic recurrence. Results: Several known GLI-1 targets and novel genes were found to be differentially expressed in GLI-1 overexpressing MCF-7 cells. The expression profile of the majority of these genes followed a similar pattern in the endocrine resistant cell lines, as determined by real-time PCR. Our analysis with the Hatzis data set revealed that several genes were positively correlated with GLI-1 expression (ATM, BRCAI, EGFR, ESR2, SERPINE1, SNAI1, IL6) and several genes were negatively correlated with GLI-1 expression (IGF1R, MUC1, PGR, SLC-39A6, THBS1, XBP1). Of these signature genes, co-expression of GLI-1 & EGFR and GLI-1 & SNAI1 were predictive of metastatic recurrence in the luminal A subtype only. Western blot analysis showed an increase in total EGFR and EGFR phosphorylation upon GLI-1 overexpression in MCF7 cells suggesting cross-talk between Hedgehog pathway and EGFR signaling. Conclusions: The Luminal A breast cancer subtype is the most common molecular subtype of breast cancer. Although associated with good prognosis, many patients with this subtype ultimately have a recurrence. Interestingly we observed that combined expression of EGFR and GLI-1 was predictive of metastatic recurrence in patients with the luminal A subtype. Prior trials of targeting EGFR with endocrine therapy have not been very successful. We propose that dual targeting of EGFR and hedgehog pathways could improve clinical outcomes of ER+ BC and warrants further investigation. Citation Format: Mark A. Rudolph, Stephen Sizemore, Yuanzhi Lu, Kun-yu Teng, Alec Gonos, Bhuvaneswari Ramaswamy, Sarmila Majumder. Targeting Hedgehog pathway and EGFR signaling to overcome endocrine resistance. [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 3463. doi:10.1158/1538-7445.AM2015-3463

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide with increasing incidence and mortality. Risk factors for this deadly disease include acquisition of the Hepatitis B or C virus, alcohol use, and non-alcoholic steatohepatitis (NASH). Due to their ability to regulate multiple genes and their therapeutic potential, miRNAs (miRs) have received considerable attention for their role as tumor suppressors or oncogenes. In our previous study to identify miRs involved in the pathogenesis of HCC, we have demonstrated consistent upregulation of miR-155, a pro-inflammatory microRNA, in a CDAA diet-induced mouse model of HCC that mimics the progression of steatohepatitis to HCC in humans (B.Wang et.al, Hepatology. 2009). Importantly, increased miR-155 level in humans is an independent predictor of poor prognosis and survival and also correlates with invasion in HCC patients. Upregulation of miR-155 at very early stages of HCC suggested that it plays a causal role in NASH and HCC and that its absence will result in decreased inflammation, fibrosis, and eventually, development of HCC. The overall goal of this study is to establish the role of miR-155 in the pathogenesis of HCC for future development of anti-miR therapy. For this purpose, male C57BL6 mice and miR-155KO mice were fed the chow diet (control) or the CDAA diet for 4 or 8 weeks. Blood and liver were then harvested for serologic, pathologic, and biochemical studies. Additionally, body and liver weight were also measured at the time of sacrifice. Analysis of the ratio of liver weight to body weight demonstrated a small but significant increase of this ratio (in the absence of any increase in body weight) in the miR-155KO mice compared to the control mice. As expected, reduced inflammation was seen in the miR-155KO mice that correlated with decreased NF-κB activity. Histopathological analysis also revealed increased steatosis in the KO mice suggesting that miR-155 plays a role in hepatic lipid metabolism. Global gene expression analysis performed on RNA isolated from livers was consistent with reduced inflammation and enhanced steatosis in the miR-155KO mice. Importantly, changes in expression of genes associated with hepatic necrosis, fibrosis and cancer indicated reduced liver damage in miR-155KO mice suggesting that these mice will be less prone to tumorigenesis. Reduced expression of the p65 subunit of NF-κB in the miR-155KO mice was identified as one of the mechanisms of decreased NF-κB activity. Currently studies are underway to explore further the mechanism of regulation of p65 expression/NF-κB activity and lipid metabolism in HCC by miR-155. Citation Format: Tasneem Motiwala, Mufaddal Mustafa, Huban Kutay, Rachael C. Sullivan, Kun-Yu Teng, Vivek Chowdhary, Lianbo Yu, Kalpana Ghoshal, Samson T. Jacob. Elucidation of the role of microRNA-155 in a murine model of non-alcoholic steatohepatitis. [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 4359. doi:10.1158/1538-7445.AM2014-4359

The Nottingham prognostic index (NPI) is a widely used clinicopathological staging system for breast cancer prognostication. Using a step-wise classification approach where breast tumor expression profiles were first divided into general “molecular subtypes” [estrogen receptor (ER)+, ER−, ERBB2+], followed by an independent analysis of each subtype, we identified a 62-gene expression signature (NPI-ES) highly correlated to the NPI in ER+ tumors. The NPI-ES classified the majority of ER+ tumors into two distinct groups with high confidence and was significantly correlated to NPI status in two independent sets of ER+ tumors derived from different centers. The NPI-ES is comparable to the classical NPI in identifying patients likely to exhibit a poor clinical prognosis, as well as to a recently described “prognosis signature” for breast cancer. Our findings demonstrate how expression profiling can complement classical staging systems employing histopathological parameters scored over a continuous range of values.

Comparative genomic hybridization (CGH), microsatellite instability (MSI) assays, and expression microarrays were used to molecularly subclassify a common set of gastric tumor samples. We identified a number of novel genomic aberrations associated with gastric cancer and discovered that gastric tumors could be grouped by their expression profiles into three broad classes: "tumorigenic," "reactive," and "gastric-like." Patients with gastric-like tumors exhibited a significantly better overall survival than patients belonging to the other two classes (P0.05). A novel supervised learning methodology for multiclass prediction was used to identify optimal predictor gene sets that accurately predicted the class of an unknown tumor sample. These predictor sets may prove useful in the development of new diagnostic applications for gastric cancer staging and prognostication.

Patient-derived xenograft (PDX) models have emerged as key model systems to understand the efficacy of anti-cancer agents. PDX systems have demonstrated superiority over cell lines with regards to higher histological resemblance to primary tumor, presence of stroma and also in mimicking response to therapeutic agents. However, the genomic evolution landscape of model establishment is not a well studied topic. In order to do that, we systematically collected materials from the first three passages of xenografts derived from eight gastric cancer patients from Seoul National University, Korea from 2008-2011. We subsequently generated comprehensive genomic profiles of the first three passages, parent primary tumor, and matched normal tissues for the eight patients. The genomic profiling data included whole-exome sequencing data (Agilent exome capture, paired-end sequencing on Illumina HiSeq 2000), mRNA expression (Affymetrix U133 Plus2.0), copy number data (Affymetrix SNP6), miRNA expression (Agilent miRNA array v16.0) and DNA methylation data (Illumina HumanMethylation27). The analysis is focused on two types of events: a) Cancer-related genomic changes derived from normal vs. tumor comparison, and their subsequent assessment in early passages; b) Passage-specific genomic changes derived from tumor vs. passages comparison. A further refinement of passage-specific genomic changes is done to differentiate ‘true’ passage-specific events from the ones that appear due to the difference in tumor purity that is lower in primary tumor due to the normal contamination. Overall, we describe the genomic landscape of evolution along xenograft establishment in gastric cancer and provide a comprehensive picture of genetic and genomic similarities (and differences) of xenografts to the primary tumor. This analysis will help us to better interpret the in vivo results emanating from experiments using xenografts and to translate the findings appropriately to the clinic. Citation Format: Kun Yu, Swee Seong Wong, Jason C. Ting, Thompson N. Doman, Yong Yue, Amit aggarwal, Gregory P. Donoho, Rebekka Krumbach, Heiner H. Feibig, Seong-Ho kong, Woo-ho Kim, Han-kwang Yang, Christoph Reinhard. Genomic evolution landscape of patient tumor derived xenograft of gastric cancer. [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 817. doi:10.1158/1538-7445.AM2013-817 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Aims: Approximately 30-40% of patients with estrogen receptor positive (ER+) invasive breast cancer relapse despite targeted endocrine therapy with tamoxifen (TAM). Alternative cell survival mechanisms aberrantly activated over time supports tumor growth, upon blockade of estrogen dependent pathways by TAM. We have earlier identified Hedgehog (Hhg) signaling pathway as an alternative survival pathway activated in TAM resistant ER+ tumors (SABC Abst# 851859, 2011; S-9, 2010). We sought to determine if activation of Hhg pathway is critical for survival of the resistant cells, the mechanism underlying activation of this pathway, and determine if targeting such tumors with Hhg inhibitor would improve outcomes. Methods: SMO and GLI1 were depleted from TAM-resistant MCF7 (OHTR) and T47D cells using siRNA. These cells were tested in presence and absence of TAM for i) viability by MTT assay, ii) clonogenic survival, iii) apoptosis and autophagy by Western Blotting of respective markers. Pathway specific inhibitors were used to determine cross talk between Hhg pathway and different signaling pathways. Effect of GDC-0449, a small molecule inhibitor of SMO was tested on OHTR cell induced xenografts in athymic nude mice by monitoring tumor growth. Correlation of GLI1 expression in primary breast tumor tissues (assessed by IHC) and disease-free survival (DFS) was studied in women with ER+ breast cancer (N=230). Results: A. Depletion of SMO or GLI1 resulted in reduced cell proliferation, clonogenic survival and increased autophagy in TAM-resistant cells. We observed loss of p53 in SMO and GLI1 depleted cells as well as in Myc depleted cells. Expression of Myc, a Hhg target is reduced in SMO/GLI1 depleted cells, resulting in loss of p53. B. PI3K inhibitor Ly294002, inhibited GLI1 driven promoter activity, with simultaneous loss of SMO and GLI1 protein. Treatment with LiCl (GSK3B inhibitor) and/or MG132 (proteasome inhibitor) rescued these proteins from degradation. C. Proliferation of OHTR cells was significantly inhibited with GDC-0449 alone or TAM both in vitro and in vivo. D. Further, primary human ER+ breast tumors with high expression of GLI1 were associated with poor DFS in node-positive patients (p 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 5606. doi:1538-7445.AM2012-5606

Background: The critical promoter element responsible for epigenetic silencing of retinoic acid receptor responder 1 (RARRES1) by methylation is unclear. Likewise, how aberrant methylation interplays effectors and thus affects breast neoplastic features remains largely unknown. Methodology/Principal Findings: We first compared methylation occurring at the sequences flanking the RARRES1 promoter in primary breast carcinomas to that in adjacent benign tissues. Surprisingly, tumor cores displayed significantly elevated methylation occurring solely at the upstream region, while the downstream element proximal to the transcription start site (+1) remained largely unchanged. Yet, hypermethylation at the former did not result in appreciable silencing effect. In contrast, the proximal sequence displayed full promoter activity and methylation of which remarkably silenced RARRES1 transcription. This phenomenon was recapitulated in breast cancer cell lines, in which methylation at the proximal region strikingly coincided with downregulation. We also discovered that CTCF occupancy was enriched at the unmethylayed promoter enriched with transcription-active histone markings. Furthermore, knocking-down CTCF expression hampered RARRES1 expression, suggesting CTCF positively regulated RARRES1 transcription presumably by binding to unmethylated promoter poised at transcription-ready state. Moreover, RARRES1 restoration not only impeded cell invasion but also promoted death induced by chemotherapeutic agents, denoting its tumor suppressive effects. Its role of attenuating invasion agreed with data generated from clinical specimens revealing that RARRES1 was generally downregulated in metastatic lymph nodes compared to the tumor cores. Conclusion/Significance: For the first time, this report delineated that silencing of RARRES1 by hypermethylation is occurring at a crucial proximal promoter element and is associated with a loss of binding to CTCF, an activator for RARRES1 expression. We also revealed the tumor suppressive roles exerted by RARRES1 in part by promoting breast epithelial cell death and by impeding cell invasion that is an important property for metastatic spread. 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 4044. doi:1538-7445.AM2012-4044

Gastric cancer is common in Asia, and advanced disease is typically incurable. Development of effective treatments for gastric cancer is thus a high priority to address this highly unmet clinical need. c-Met gene amplification resulting in c-Met overexpression and pathway activation has been highly linked to tumor progression and metastasis. It has been reported that approximately 10-20% of gastric tumors have increased copy numbers of the MET gene. c-Met- amplified gastric cancer cell lines that are dependent on an activated c-Met pathway for cell growth have been reported to be highly sensitive to growth inhibition by small molecule inhibitors of c-Met. We report that LA480, a humanized monoclonal antibody specific for c-Met, can directly inhibit HGF-independent in vitro cell proliferation of three c-Met- amplified Asian gastric cell lines (MKN45, SNU-5, and NUGC-4), in a dose-dependent manner. After 48 hours of treatment with LA480, 60-70% percent inhibition of 3H-thymidine incorporation was observed. For one of these gastric lines (NUGC-4), sensitivity to LA480 increases with continuous passage of the culture. This increased sensitivity to LA480 over time appears to be associated with increased expression of cell surface c-Met and increased constitutive phospho-Met expression. In addition to inhibiting proliferation of c-Met- amplified Asian gastric lines, LA480 also directly inhibited proliferation of a c-Met- amplified gastric cell line (Hs 746T) from a Caucasian patient. These data suggest that targeting c-Met amplification with LA480 may be a promising treatment for gastric cancer. Citation 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 2430.