Karina J. Yoon

The BET bromodomain inhibitor JQ1 suppresses growth of pancreatic ductal adenocarcinoma in patient-derived xenograft models

The primary aim of this study was to evaluate the antitumor efficacy of the bromodomain inhibitor JQ1 in pancreatic ductal adenocarcinoma (PDAC) patient-derived xenograft (tumorgraft) models. A secondary aim of the study was to evaluate whether JQ1 decreases expression of the oncogene c-Myc in PDAC tumors, as has been reported for other tumor types. We used five PDAC tumorgraft models that retain specific characteristics of tumors of origin to evaluate the antitumor efficacy of JQ1. Tumor-bearing mice were treated with JQ1 (50 mg/kg daily for 21 or 28 days). Expression analyses were performed with tumors harvested from host mice after treatment with JQ1 or vehicle control. An nCounter PanCancer Pathways Panel (NanoString Technologies) of 230 cancer-related genes was used to identify gene products affected by JQ1. Quantitative RT–PCR, immunohistochemistry and immunoblots were carried out to confirm that changes in RNA expression reflected changes in protein expression. JQ1 inhibited the growth of all five tumorgraft models (P<0.05), each of which harbors a KRAS mutation; but induced no consistent change in expression of c-Myc protein. Expression profiling identified CDC25B, a regulator of cell cycle progression, as one of the three RNA species (TIMP3, LMO2 and CDC25B) downregulated by JQ1 (P<0.05). Inhibition of tumor progression was more closely related to decreased expression of nuclear CDC25B than to changes in c-Myc expression. JQ1 and other agents that inhibit the function of proteins with bromodomains merit further investigation for treating PDAC tumors. Work is ongoing in our laboratory to identify effective drug combinations that include JQ1.

Development and Histopathological Characterization of Tumorgraft Models of Pancreatic Ductal Adenocarcinoma

Pancreatic cancer is the one of the deadliest of all malignancies. The five year survival rate for patients with this disease is 3-5%. Thus, there is a compelling need for novel therapeutic strategies to improve the clinical outcome for patients with pancreatic cancer. Several groups have demonstrated for other types of solid tumors that early passage human tumor xenograft models can be used to define some genetic and molecular characteristics of specific human tumors. Published studies also suggest that murine tumorgraft models (early passage xenografts derived from direct implantation of primary tumor specimens) may be useful in identifying compounds with efficacy against specific tumor types. Because pancreatic cancer is a fatal disease and few well-characterized model systems are available for translational research, we developed and characterized a panel of pancreatic tumorgraft models for biological evaluation and therapeutic drug testing. Of the 41 primary tumor specimens implanted subcutaneously into mice, 35 produced viable tumorgraft models. We document the fidelity of histological and morphological characteristics and of KRAS mutation status among primary (F0), F1, and F2 tumors for the twenty models that have progressed to the F3 generation. Importantly, our procedures produced a take rate of 85%, higher than any reported in the literature. Primary tumor specimens that failed to produce tumorgrafts were those that either contained <10% tumor cells or that were obtained from significantly smaller primary tumors. In view of the fidelity of characteristics of primary tumor specimens through at least the F2 generation in mice, we propose that these tumorgraft models represent a useful tool for identifying critical characteristics of pancreatic tumors and for evaluating potential therapies.

N-glycosylation of ICAM-2 is required for ICAM-2-mediated complete suppression of metastatic potential of SK-N-AS neuroblastoma cells

BackgroundCell adhesion molecules (CAMs) are expressed ubiquitously. Each of the four families of CAMs is comprised of glycosylated, membrane-bound proteins that participate in multiple cellular processes including cell-cell communication, cell motility, inside-out and outside-in signaling, tumorigenesis, angiogenesis and metastasis. Intercellular adhesion molecule-2 (ICAM-2), a member of the immunoglobulin superfamily of CAMs, has six N-linked glycosylation sites at amino acids (asparagines) 47, 82, 105, 153, 178 and 187. Recently, we demonstrated a previously unknown function for ICAM-2 in tumor cells. We showed that ICAM-2 suppressed neuroblastoma cell motility and growth in soft agar, and induced a juxtamembrane distribution of F-actin in vitro. We also showed that ICAM-2 completely suppressed development of disseminated tumors in vivo in a murine model of metastatic NB. These effects of ICAM-2 on NB cell phenotype in vitro and in vivo depended on the interaction of ICAM-2 with the cytoskeletal linker protein α-actinin. Interestingly, ICAM-2 did not suppress subcutaneous growth of tumors in mice, suggesting that ICAM-2 affects the metastatic but not the tumorigenic potential of NB cells. The goal of the study presented here was to determine if the glycosylation status of ICAM-2 influenced its function in neuroblastoma cells.MethodsBecause it is well documented that glycosylation facilitates essential steps in tumor progression and metastasis, we investigated whether the glycosylation status of ICAM-2 affected the phenotype of NB cells. We used site-directed mutagenesis to express hypo- or non-glycosylated variants of ICAM-2, by substituting alanine for asparagine at glycosylation sites, and compared the impact of each variant on NB cell motility, anchorage-independent growth, interaction with intracellular proteins, effect on F-actin distribution and metastatic potential in vivo.ResultsThe in vitro and in vivo phenotypes of cells expressing glycosylation site variants differed from cells expressing fully-glycosylated ICAM-2 or no ICAM-2. Most striking was the finding that mice injected intravenously with NB cells expressing glycosylation site variants survived longer (P ≤ 0.002) than mice receiving SK-N-AS cells with undetectable ICAM-2. However, unlike fully-glycosylated ICAM-2, glycosylation site variants did not completely suppress disseminated tumor development.ConclusionsReduced glycosylation of ICAM-2 significantly attenuated, but did not abolish, its ability to suppress metastatic properties of NB cells.

JQ1 Induces DNA Damage and Apoptosis, and Inhibits Tumor Growth in a Patient-Derived Xenograft Model of Cholangiocarcinoma

Cholangiocarcinoma (CCA) is a fatal disease with a 5-year survival of <30%. For a majority of patients, chemotherapy is the only therapeutic option, and virtually all patients relapse. Gemcitabine is the first-line agent for treatment of CCA. Patients treated with gemcitabine monotherapy survive ∼8 months. Combining this agent with cisplatin increases survival by ∼3 months, but neither regimen produces durable remissions. The molecular etiology of this disease is poorly understood. To facilitate molecular characterization and development of effective therapies for CCA, we established a panel of patient-derived xenograft (PDX) models of CCA. We used two of these models to investigate the antitumor efficacy and mechanism of action of the bromodomain inhibitor JQ1, an agent that has not been evaluated for the treatment of CCA. The data show that JQ1 suppressed the growth of the CCA PDX model CCA2 and demonstrate that growth suppression was concomitant with inhibition of c-Myc protein expression. A second model (CCA1) was JQ1-insensitive, with tumor progression and c-Myc expression unaffected by exposure to this agent. Also selective to CCA2 tumors, JQ1 induced DNA damage and apoptosis and downregulated multiple c-Myc transcriptional targets that regulate cell-cycle progression and DNA repair. These findings suggest that c-Myc inhibition and several of its transcriptional targets may contribute to the mechanism of action of JQ1 in this tumor type. We conclude that BET inhibitors such as JQ1 warrant further investigation for the treatment of CCA. Mol Cancer Ther; 17(1); 107–18. ©2017 AACR.

Dysregulated human Tyrosyl-DNA phosphodiesterase I acts as cellular toxin

Tyrosyl-DNA phosphodiesterase I (TDP1) hydrolyzes the drug-stabilized 3’phospho-tyrosyl bond formed between DNA topoisomerase I (TOPO1) and DNA. TDP1-mediated hydrolysis uses a nucleophilic histidine (Hisnuc) and a general acid/base histidine (Hisgab). A Tdp1Hisgab to Arg mutant identified in patients with the autosomal recessive neurodegenerative disease SCAN1 causes stabilization of the TDP1-DNA intermediate. Based on our previously reported Hisgab-substitutions inducing yeast toxicity (Gajewski et al. J. Mol. Biol. 415, 741-758, 2012), we propose that converting TDP1 into a cellular poison by stabilizing the covalent enzyme-DNA intermediate is a novel therapeutic strategy for cancer treatment. Here, we analyzed the toxic effects of two TDP1 catalytic mutants in HEK293 cells. Expression of human Tdp1HisnucAla and Tdp1HisgabAsn mutants results in stabilization of the covalent TDP1-DNA intermediate and induces cytotoxicity. Moreover, these mutants display reduced in vitro catalytic activity compared to wild type. Co-treatment of Tdp1mutant with topotecan shows more than additive cytotoxicity. Overall, these results support the hypothesis that stabilization of the TDP1-DNA covalent intermediate is a potential anti-cancer therapeutic strategy.

Whole exome sequencing identified sixty-five coding mutations in four neuroblastoma tumors

Neuroblastoma is a pediatric tumor characterized by histologic heterogeneity, and accounts for ~15% of childhood deaths from cancer. The five-year survival for patients with high-risk stage 4 disease has not improved in two decades. We used whole exome sequencing (WES) to identify mutations present in three independent high-risk stage 4 neuroblastoma tumors (COA/UAB-3, COA/UAB -6 and COA/UAB -8) and a stage 3 tumor (COA/UAB-14). Among the four tumors WES analysis identified forty-three mutations that had not been reported previously, one of which was present in two of the four tumors. WES analysis also corroborated twenty-two mutations that were reported previously. No single mutation occurred in all four tumors or in all stage 4 tumors. Three of the four tumors harbored genes with CADD scores ≥20, indicative of mutations associated with human pathologies. The average depth of coverage ranged from 39.68 to 90.27, with >99% sequences mapping to the genome. In summary, WES identified sixty-five coding mutations including forty-three mutations not reported previously in primary neuroblastoma tumors. The three stage 4 tumors contained mutations in genes encoding protein products that regulate immune function or cell adhesion and tumor cell metastasis.

Targeting PIM kinase as a therapeutic strategy in human hepatoblastoma

Increasing incidence coupled with poor prognosis and treatments that are virtually unchanged over the past 20 years have made the need for the development of novel therapeutics for hepatoblastoma imperative. PIM kinases have been implicated as drivers of tumorigenesis in multiple cancers, including hepatocellular carcinoma. We hypothesized that PIM kinases, specifically PIM3, would play a role in hepatoblastoma tumorigenesis and that PIM kinase inhibition would affect hepatoblastoma in vitro and in vivo. Parameters including cell survival, proliferation, motility, and apoptosis were assessed in human hepatoblastoma cells following PIM3 knockdown with siRNA or treatment with the PIM inhibitor AZD1208. An in vivo model of human hepatoblastoma was utilized to study the effects of PIM inhibition alone and in combination with cisplatin. PIM kinases were found to be present in the human hepatoblastoma cell line, HuH6, and in a human hepatoblastoma patient-derived xenograft, COA67. PIM3 knockdown or inhibition with AZD1208 decreased cell survival, attachment independent growth, and motility. Additionally, inhibition of tumor growth was observed in a hepatoblastoma xenograft model in mice treated with AZD1208. Combination therapy with AZD1208 and cisplatin resulted in a significant increase in animal survival when compared to either treatment alone. The current studies showed that PIM kinase inhibition decreased human hepatoblastoma tumorigenicity both in vitro and in vivo, implying that PIM inhibitors may be useful as a novel therapeutic for children with hepatoblastoma.

Sphingolipid metabolism and drug resistance in ovarian cancer

Despite progress in understanding molecular aberrations that contribute to the development and progression of ovarian cancer, virtually all patients succumb to drug resistant disease at relapse. Emerging data implicate bioactive sphingolipids and regulation of sphingolipid metabolism as components of response to chemotherapy or development of resistance. Increases in cytosolic ceramide induce apoptosis in response to therapy with multiple classes of chemotherapeutic agents. Aberrations in sphingolipid metabolism that accelerate the catabolism of ceramide or that prevent the production and accumulation of ceramide contribute to resistance to standard of care platinum- and taxane-based agents. The aim of this review is to highlight current literature and research investigating the influence of the sphingolipids and enzymes that comprise the sphingosine-1-phosphate pathway on the progression of ovarian cancer. The focus of the review is on the utility of sphingolipid-centric therapeutics as a mechanism to circumvent drug resistance in this tumor type.

Abstract 316: FTY720 enhances the efficacy of paclitaxel and of carboplatin in drug-resistant ovarian cancer cells

Objectives: Our long-term goal is to develop effective therapies for ovarian cancer by targeting molecules that contribute to drug resistance in this tumor type. Preliminary RNA-seq data were generated using a patient-derived xenograft model. These data identified the sphingosine-1-phosphate (S1P) signaling pathway as one of the pathways most affected by carboplatin and paclitaxel, the current standards of care for ovarian cancer. We then examined whether a synthetic analog of sphingosine, FTY720, enhanced the cytotoxicity of platinum and taxane resistant ovarian cancer cell lines. Methods: To assess the efficacy of FTY720 + carboplatin and of FTY720 + paclitaxel, we exposed three pairs of parental and drug resistant human ovarian cancer cell lines to each combination. The cell lines used were: SKOV3-ip1 and SKOV3-TR (taxane resistant), A2780 and A2780-CP20 (platinum resistant), and HeyA8 and HeyA8-MDR (taxane-platinum resistant). We used alamarBlue cell proliferation assays to determine the efficacy of FTY720 as a single agent or in combination with paclitaxel or carboplatin, and immunoblots to assess the expression of proteins involved in the S1P pathway. Results: FTY720 as a single agent decreased cell viability in a dose-dependent manner in all three pairs of cell lines, with IC50 values ranging from 5 to 8 μM. When cells were exposed to the IC50 of FTY720 + a range of concentrations of carboplatin or paclitaxel, FTY720 had little effect in any of the three parental cell lines and in SKOV3-TR cells. Interestingly, however, FTY720 decreased the IC50 of carboplatin 15- to 16-fold and the IC50 of paclitaxel 20- to 90- fold in A2780-CP20 and HeyA8-MDR cells, respectively. We also observed that FTY720 altered the expression of sphingosine kinase2, S1P lyase, and acid ceramidase, each of which contributes to the ceramide-sphingosine-S1P rheostat that tightly regulates expression of S1P pathway proteins. Conclusions: Our data show that FTY720 sensitized drug resistant ovarian cancer cell lines A2780-CP20 and HeyA8-MDR to carboplatin and to paclitaxel 15- to 90-fold. The data suggest that the S1P pathway may contribute to carboplatin and paclitaxel resistance in this cell type. We propose that proteins of the S1P pathway merit further study as effective molecular targets in drug resistant ovarian cancer cells. Citation Format: Kelly M. Kreitzburg, Ronald D. Alvarez, Charles N. Landen, Karina J. Yoon. FTY720 enhances the efficacy of paclitaxel and of carboplatin in drug-resistant ovarian cancer cells. [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 316.

Abstract 1293: JQ1 sensitivity of patient-derived xenograft models of cholangiocarcinoma

Cholangiocarcinoma (CCA) is a lethal malignancy arising from cholangiocytes in any part of the biliary tree. The incidence of CCA has been on the rise worldwide, and the prognosis and clinical outcome have remained essentially unchanged for 30 years. The majority of patients are diagnosed at late stage, and surgery continues to be the only cure. Patients receive systemic chemotherapy with the first-line combination therapy comprising gemcitabine and cisplatin. Median survival for these patients is To determine gene products whose upregulation or downregulation is responsible for the differences in sensitivity to JQ1 among our CCA models, we generated expression profiles of tumors from vehicle control and JQ1 treated mice using NanoString technology (nCounter PanCancer Pathways panel). Our data demonstrate that JQ1 inhibited the expression of c-Myc to a greater extent in the sensitive models than in the insensitive model. Expression array data showed further that gene products involved in cell cycle and DNA repair pathways were also decreased by JQ1. Of particular interest were two transcriptional targets of c-Myc, Chk1 and BRCA2, each of which is involved in DNA damage response. Immunohistochemistry staining confirmed expression profile analyses. We conclude that the inhibition of cell cycle and DNA repair genes may contribute to the mechanism of action of JQ1 in CCA tumors. Citation Format: Aubrey L. Miller, Patrick L. Garcia, Tracy L. Gamblin, Leona N. Council, Xiangqin Cui, James E. Bradner, Eddy S. Yang, Karina J. Yoon. JQ1 sensitivity of patient-derived xenograft models of cholangiocarcinoma. [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 1293.