bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B12: Using Lrig1CreERT2/+ stem cell driver mice to model colorectal cancer
Authors: Poulin Emily
Published: May 2015

Genetically engineered mouse (GEM) models of human cancer provide important insights into mechanisms of disease development. Colorectal cancer is a common malignancy, whereas small intestinal cancer is much less frequent. We recently developed a mouse model of colonic neoplasia, using the highly efficient inducible stem cell driver, leucine-rich repeats and immunoglobulin-like domains protein 1 (Lrig1CreERT2), and a floxed allele of the tumor suppressor gene, adenomatous polyposis coli (Apcfl/+) (Cell 149: 146-158, 2012). Lrig1CreERT2/+;Apcfl/+ mice develop multiple, large highly dysplastic adenomas in the distal colon within 50 days of tamoxifen induction; the proximal colon is spared and smaller tumors are detected in the small intestine. We hypothesized an additional oncogenic event would advance tumorigenesis in this model. To investigate this hypothesis, using a Kras mutation (G12D) as the second event, we developed a new inducible mouse model: Lrig1CreERT2/+;Apcfl/+;KrasLSL-G12D/+. Due to broad Lrig1 expression, systemic Kras activation through intraperitoneal tamoxifen injection results in debilitating oral and facial tumors within three to five weeks, precluding longer-term intestinal studies. To overcome the rapid onset of this phenotype, we administered 4-hydroxytamoxifen (4-OHT) enemas to achieve local tamoxifen delivery. This method delayed onset of oral tumor development, even preventing it in some mice. However, we noted Lrig1CreERT2/+;Apcfl/+;KrasLSL-G12D/+ mice rarely developed colonic tumors, compared to Lrig1CreERT2/+;Apcfl/+ control mice. We hypothesized Kras activation before complete Apc loss of heterozygosity provides a barrier to tumor formation. Therefore, we administered 4-OHT enemas to Lrig1CreERT2/+;Apcfl/fl;KrasLSL-G12D/+ mice to determine whether Kras activation could advance tumorigenesis in the setting of loss of both Apc alleles. In a preliminary experiment, we observed that Kras activation caused a more severe phenotype in the colonic epithelium of Lrig1CreERT2/+;Apcfl/fl mice compared to Lrig1CreERT2/+;Apcfl/fl control mice. Both genotypes developed skin lesions that affected the health of animals, requiring sacrifice as early as 28 days after tamoxifen administration. Further characterization of this model will determine whether invasion in the colonic lesions can occur under these genetic conditions.

Citation Format: Emily Poulin. Using Lrig1CreERT2/+ stem cell driver mice to model colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B12. doi: 10.1158/1557-3125.RASONC14-B12
Abstract B14: Human Mut T homolog 1 (MTH1) as a novel facilitator of KRAS-driven lung cancer
Authors: Patel Asmita , Halvorsen Katherine , Balkan Wayne , Cohen Alexander , Reiner Teresita , Robbins David , Nguyen Dao , Rai Priyamvada
Published: May 2015

Non-small cell lung carcinoma (NSCLC), a chemoresistant form of lung cancer, is characterized by oncogenic KRAS mutations that confer multiple tumor-promoting traits. Several of these traits are mediated by oncogene-induced reactive oxygen species (ROS). However, oncogenic ROS also produce DNA damage, a major effector of tumor suppressor mechanisms. We previously reported overexpression of the mammalian 8-oxo-dGTP triphosphatase, human MutT Homolog 1 (MTH1), inhibits RAS oncogene-induced senescence (OIS) and any associated DNA damage without substantially affecting ROS levels. Here we report that MTH1 expression is elevated in NSCLC tumors relative to adjacent normal tissue, particularly in tumors with high KRAS expression. We show that MTH1 overexpression facilitates transformation of lung epithelial cells by oncogenic KRAS. We further report that MTH1 suppression in NSCLC cell lines reduces proliferation and tumorigenicity either via DNA strand break-induced elevation of the p53/p21 pathway or by inhibition of Akt activation in p53-nonfunctional tumor cells. Collectively our results indicate MTH1 as a novel potential chemotherapeutic target in NSCLC with activating KRAS mutations.

Citation Format: Asmita Patel, Katherine Halvorsen, Wayne Balkan, Alexander Cohen, Teresita Reiner, David Robbins, Dao Nguyen, Priyamvada Rai. Human Mut T homolog 1 (MTH1) as a novel facilitator of KRAS-driven lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B14. doi: 10.1158/1557-3125.RASONC14-B14
Abstract B40: Blocking Ras chaperons for cancer therapy
Authors: Kloog Yoel , Haklai Roni , Elad-Sfadia Galit
Published: May 2015

We have previously developed a new concept and a new class of Ras inhibitors that affect specifically the active forms of Ras. This concept is based on the knowledge that the farnesyl moiety common to all Ras proteins might not only serve as a lipophilic lipid anchor but also confer functional specificity on Ras. This raised the possibility that the farnesyl group may act as part of a recognition unit for specific anchorage lipids or protein(s) that interact with Ras in the cell membrane. Such binding partners of Ras have been identified where it was shown that galectin-1 and galectin-3 discovered first as lectin binding proteins, act respectively as specific binding partners of farnesylated active H-Ras and K-Ras. Recently Nucleoline was identified as a specific chaperon of active N-Ras. The galectins possess a putative hydrophobic binding pocket-the putative farnesyl binding site. Galectin-1 was shown to drive H-Ras.GTP nanocluster formation and to be an integral component of the H-Ras-GTP nanocluster, a site at which Raf is activated. Similarly, galectin-3 drives K-Ras.GTP nanoclustring and robust K-Ras signaling. Ras signaling to ERK is strongly diminished in Galectin -3 knockout MEFS and in thyroid cancer cells that lack Galectin - 3. Together these findings mark the farnesyl-binding pockets as outstanding targets for Ras directed therapy, assuming that compounds that would block such sites will act as Ras inhibitors. The Ras inhibitor, S-trans-trans farnesylthiosalicylic acid (Salirasib, FTS) was indeed designed to mimic the farnesyl moiety in the carboxy terminal of Ras and was found to induce accelerated dislodgement of Ras from the cell membrane and subsequently degradation of the protein. FTS was also found to be highly efficient in inhibiting growth of many types of cancer cell lines that exhibit high Ras.GTP levels both, in vitro and in vivo. More recent phase 2 clinical trials showed that oral Salirasib treatment of pancreatic and of non-small-lung cancer patients increased significantly survival of the patients with no toxic effects. We propose to use Salirasib for the treatment of pancreatic and of non-small-lung carcinoma.

Note: This abstract was not presented at the conference.

Citation Format: Yoel Kloog, Roni Haklai, Galit Elad-Sfadia. Blocking Ras chaperons for cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B40. doi: 10.1158/1557-3125.RASONC14-B40
Abstract B53: Utilizing bimolecular fluorescent complementation to identify inhibitors of RAL and KRAS
Authors: Ifediba Marytheresa , Burnett David , Hovda Nicholas , Burnett William , VanBrocklin Matthew
Published: May 2015

We have identified RAL activity to be vital for NSCLC survival independent of KRAS or EGFR mutation status in a large panel of adenocarcinomas and have developed a novel high throughput screening assay based on Bimolecular Fluorescent Complementation (BiFC) in order to identify small molecules that impair RAL function. By coupling RALA and RALB to the C-terminus of the Venus protein (VC) and coexpressing either with direct binding effector RALBP1 fused to the N-terminus of the Venus protein (VN) results in a fluorescent signal when RAL-RALBP1 binding occurs in cells. This allows for the interrogation of small molecules to disrupt RAL-RALBP1 binding interactions (resulting in loss of fluorescence) in a real time, high throughput manner. Our preliminary screen with a natural compound library identified 14 small molecules that demonstrated high activity in disrupting both RALA- and RALB-RALBP1 binding and fluorescence. We are currently investigating these compounds' mode of action and have recently expanded our assay to screen for molecules that impair mutant KRAS activity (VC-KRAS / RAF1-VN). BiFC provides a rapid, more physiologic method to identify molecules that can disrupt protein-protein interactions of targets considered ‘undruggable’ including membrane bound small GTPases such as RAL and KRAS.

Citation Format: Marytheresa Ifediba, David Burnett, Nicholas Hovda, William Burnett, Matthew VanBrocklin. Utilizing bimolecular fluorescent complementation to identify inhibitors of RAL and KRAS. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B53. doi: 10.1158/1557-3125.RASONC14-B53
Abstract IA06: Discovery of a first-in-class ERK 1,2 inhibitor
Authors: Samatar Ahmed A.
Published: May 2015

The RAS /RAF /MEK / ERK signal transduction pathway is frequently activated in several human cancers, due to a gain of function mutations in KRAS, NRAS, or BRAF. Because of the high frequency of activating mutations, this pathway has become a focus for targeted therapy. A wide spectrum of MEK and RAF inhibitors have been developed to inhibit components of the pathway. Vemurafenib and dabrafenib, selective BRAF inhibitors, and trametinib, an allosteric MEK inhibitor, have shown clinical efficacy in melanoma patients. However, the majority of responses to RAF inhibitors are transient and acquired resistance is often associated with pathway reactivation involving the downstream extracellular-signal-regulated kinases 1 and 2 (ERK1/2). Pathway blockade at ERK, the last signaling node prior to MAPK transcriptional programming, may not only be efficacious in MAPK-activated tumors but might also have utility in RAF or MEK inhibitor resistant settings. This abstract describes the identification and characterization of SCH772984, a potent and selective ATP competitive inhibitor of ERK1/2 which displays behaviors of both type I and type II kinase inhibitors. SCH772984 has nanomolar cellular potency on tumor cells with mutations in RAF, NRAS, or KRAS and induces tumor regressions in xenograft models at tolerated doses. SCH772984 effectively inhibited MAPK signaling and cell proliferation in RAF or MEK inhibitor resistant models as well as in the context of RAF/MEK combination resistance. Together these data support the clinical development of ERK inhibitors, not only in patients with MAPK activated tumors, but also in patients who have developed acquired resistance to RAF or MEK inhibitors or resistance to the recently described combination of these agents. Strategies to overcome acquired resistance to pathway inhibitors will be also described.

Citation Format: Ahmed A. Samatar. Discovery of a first-in-class ERK 1,2 inhibitor. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA06. doi: 10.1158/1557-3125.RASONC14-IA06
Abstract PR04: Cooperation between Ras network mutations in cancer
Authors: Stites Edward C. , Trampont Paul C. , Haney Lisa B. , Walk Scott F. , Ravichandran Kodi S.
Published: May 2015

Cancer genome sequencing often identifies mutations within the Ras signaling network other than the well-established KRAS, NRAS, and BRAF oncogene mutations and NF1 tumor suppressor gene mutations. The extent to which a novel mutation in a commonly mutated pathway can be assumed to have cancer promoting potential, and whether or not certain contextual conditions are required for the mutation to have cancer promoting effects, is not well understood. We investigated this problem with a computational model of the Ras signaling network, with focused cell-based experiments, and with statistical analysis of large cancer genomic data sets. We first applied our previously developed computational model of the Ras signaling network to identify potential synergistic combinations within the Ras network. For example, our modeling suggested that mutations to the tumor suppressor gene NF1 can amplify the effects of other Ras pathway mutations, including the effects of weakly-activating, noncanonical, Ras mutants. Experiments comparing the strength of signal produced by noncanonical Ras mutants expressed in neurofibromin deficient and wild-type cells confirmed this prediction. If such synergistic effects contribute to the development of cancer, we reasoned that there should be an increased rate of co-occurrence between the mutations that the model predicted to have cooperative potential. Analysis of large cancer genomic data sets from the Cancer Cell Line Encyclopedia and The Cancer Genome Atlas identified increased rates of co-occurrence between mutations predicted to display synergy. Overall, our study suggests that there may be combinations of Ras pathway mutations that serve the role of cancer “driver”.

This abstract is also presented as Poster B33.

Citation Format: Edward C. Stites, Paul C. Trampont, Lisa B. Haney, Scott F. Walk, Kodi S. Ravichandran. Cooperation between Ras network mutations in cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr PR04. doi: 10.1158/1557-3125.RASONC14-PR04
Abstract A24: Network-based inference of resistance mechanisms to estrogen inhibitors in ER+ breast cancer
Authors: Alvarez Mariano Javier , Shen Yao , Bisikirska Brygida , Realubit Ronald , Pampou Sergey , Karan Charles , Califano Andrea
Published: May 2015

Anti-estrogen therapy is the standard of adjuvant care for luminal breast cancer. However, more than 30% of the cases either do not respond to the therapy or develop resistance and progress to metastatic disease. In this work we use a network-based approach to identify the regulatory motifs driving resistance to estrogen inhibitors and to predict small molecule compounds that can abrogate the activity of such regulatory modules, thus reverting the resistance phenotype. This is similar to what we have accomplished in glucocorticoid resistance in T-ALL (Piovan et.al (2013) Cancer Cell 24(6):766). To accomplish this goal, we established genome-wide expression signatures characteristic of the resistant phenotype (ER-GES) both from patients' data and cell lines. Then, we used the VIPER algorithm to interrogate a breast carcinoma-specific transcriptional network (BRCAnet) assembled from 844 TCGA tumor samples with these signatures, to infer master regulators of resistance. Finally we leveraged the connectivity map (CMAP) dataset, containing expression profiles from MCF7 cells exposed to 1,300 small molecule compounds, to identify the effectors of each profiled compounds. By comparing drug effectors with master regulators of ER-inhibitors chemoresistance, we identified small molecule compounds predicted to revert resistant cells to a sensitive state.

We reasoned that a compound able to reverse such resistance should induce synergistic activity when combined with anti-estrogen agents, like tamoxifen. To validate our prediction in an unbiased way, we assessed the synergistic nature of the interaction between tamoxifen and 100 compounds (sensitizer set) selected to have the most orthogonal mechanism of action, using 4 x 4 dilutions assays, with cell viability as readout. Synergistic interactors were identified by combination index (CI < 0.9) and Excess over BLISS (EOB > 10%). Interestingly, compounds predicted to rescue sensitivity to ER inhibitors in cell lines were highly enriched in those that were experimentally validated to be synergistic with tamoxifen. Taken together, these results suggest that network-based analysis is effective in capturing drivers of chemoresistance to estrogen deprivation, as well as the specific compounds that can rescue sensitivity by inhibiting them.

Citation Format: Mariano Javier Alvarez, Yao Shen, Brygida Bisikirska, Ronald Realubit, Sergey Pampou, Charles Karan, Andrea Califano. Network-based inference of resistance mechanisms to estrogen inhibitors in ER+ breast cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A24.
Abstract A31: Revisited use of tamoxifen for improving melanoma therapy
Authors: Morad Samy A.F , Bridges Lance C. , Cabot Myles C.
Published: May 2015

The rapid increase in the incidence of malignant melanoma, a highly aggressive, drug-resistant cancer, has not been accompanied by significant therapeutic breakthroughs for decades, despite the introduction of biochemotherapies. The Dartmouth regimen, of which tamoxifen is a standard component, has also had little impact on outcome. Therefore, examination of alternative melanoma vulnerabilities is essential. Sphingolipid (SL) metabolism is an area of cancer science that has recently risen to prominence. This is because ceramide, the aliphatic backbone of SL's, can act as a powerful tumor censor. However, cancer cells rapidly inactivate ceramide via two major metabolic routes: glycosylation to form glucosylceramide and hydrolysis, which generates sphingosine 1-phosphate, a mitogenic SL. These actions severely blunt ceramide anticancer properties, thwarting potential utility in treatment. Our previous work has shown that tamoxifen is a potent inhibitor of ceramide metabolism at glycosylation and hydrolysis. Herein, we exploit these unique actions and pair tamoxifen with a cell-permeable ceramide, C6-ceramide, and demonstrate that this novel drug duo, unlike the single agents, was highly effective in inhibiting melanoma cell (A375 and cell lines established from patients) proliferation, inducing caspase-dependent apoptosis, promoting cell cycle arrest, diminishing cellular adhesion and migration, reducing cell surface expression of integrins, and downregulating expression of the main source of drug resistance in melanoma, survivin, all essential elements of a drug regimen useful for limiting melanoma growth. Because integrin expression often correlates with conversion of melanoma growth from radial to vertical, we propose that tamoxifen can serve as an effective adjuvant to C6-ceramide for countering metastatic potential. Whereas the role of tamoxifen in the Dartmouth regimen has always remained elusive, its role as a ceramide sensitizer is clear and presents novel inroads for improving melanoma therapy.

Citation Format: Samy A.F Morad, Lance C. Bridges, Myles C. Cabot. Revisited use of tamoxifen for improving melanoma therapy. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A31.
Abstract A39: A platform to assess multiple therapy options simultaneously in a patient's own tumor
Authors: Klinghoffer Richard , Moreno-Gonzalez Alicia , Carleton Michael , Grenley Marc , Hatton Beryl , Frazier Jason , Kerwin William , Tretyak Ilona , Hedin Nathan , Dey Joyoti , Casalini Joseph , Ditzler Sally , Olson James , Caffo Nathan
Published: May 2015

Assessment of anti-cancer drug efficacy is an imprecise and challenging undertaking. Early candidate selection is typically based on results from systemically treated animal models and later by performance in human trials where patients are exposed to often toxic levels of drug, prior to obtaining readouts of tumor response. In both of these testing models, only one drug can be tested at a time. Using these methods, over 90% of candidate new oncology drugs fail to provide benefit for patients in human clinical trials.

To improve the predictive value of preclinical candidate selection in animal models and enable a new type of pre-Phase 1 toxicity-sparing comparative drug efficacy study in humans, amenable for use in the solid tumor clinic, we have developed a technology platform called CIVO™. This platform allows for simultaneous assessment of multiple drugs or drug combinations directly in a single solid tumor to assess efficacy, resistance and drug synergies.

In this study, precise, controlled delivery of classic chemotherapy drugs vincristine and doxorubicin induced spatially defined (ranging 0.3 – 2.0 mm in diameter), readily detectable, and mechanism-specific cellular changes around sites of tumor microinjection across three xenograft models of lymphoma. The extent of apoptosis induced via CIVO™ microdosing of each drug (<1/100th the effective dose used to treat human patients) correlated with drug effect on tumor growth mediated by conventional systemic drug dosing. Consistent with utility for detecting pre-existing tumor resistance to certain drugs, CIVO™ microdosing predicted diminished responses to both vincristine and doxorubicin in tumors derived from cells that had previously acquired resistance to doxorubicin. This lack of efficacy was confirmed by systemic treatment of the resistant tumors. The CIVO™ platform is concurrently being evaluated for correlation to systemic treatment in immune-intact canine patients with autochthonous tumors.

The data presented here generated in drug-responsive and non-responsive solid tumors in the preclinical setting sets the stage for future application of this technology to demonstrate tumor responsiveness to novel drug candidates in the context of human patients.

Citation Format: Richard Klinghoffer, Alicia Moreno-Gonzalez, Michael Carleton, Marc Grenley, Beryl Hatton, Jason Frazier, William Kerwin, Ilona Tretyak, Nathan Hedin, Joyoti Dey, Joseph Casalini, Sally Ditzler, James Olson, Nathan Caffo. A platform to assess multiple therapy options simultaneously in a patient's own tumor. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A39.
Abstract A57: Effects of pre-exposure to siRNA on silencing response: Do cells become resistant to siRNA silencing?
Authors: Aliabadi Hamidreza Montazeri , Mahdipoor Parvin , Kucharsky Cezary , Chan Nicole , Uludag Hasan
Published: May 2015

Development of resistance against cytotoxic effect of different generations of anticancer drugs is considered inevitable and remains a major concern for cancer therapy. An alternative therapeutic approach based on protein silencing via RNA interference (RNAi) have shown considerable promise; however, our information about the factors affecting siRNA efficiency and the limitations to their efficacy are limited. This study focused on determining the possibility of development of resistance against siRNA treatment as a result of repeated exposure to siRNA that could affect cellular internalization of the particles, silencing efficiency at mRNA level, and ultimately, the observed cellular response. Two approaches to siRNA exposure were undertaken: one involving a single high concentration exposure to eradicate siRNA-responsive cells, and one involving multiple exposures with a gradually increasing siRNA concentration to study the potential intracellular adaptations. Our results demonstrated a temporary decrease in silencing efficiency at mRNA level without significant alteration in siRNA cellular uptake. We conclude that cells would respond to repeated siRNA exposure in a similar fashion after a temporary initial alteration; however, we could not rule out the possibility of a change in phenotypical response (e.g., number of viable cells) as a result of adjustments in (or selection of cells reliant on) alternative pathways.

Citation Format: Hamidreza Montazeri Aliabadi, Parvin Mahdipoor, Cezary Kucharsky, Nicole Chan, Hasan Uludag. Effects of pre-exposure to siRNA on silencing response: Do cells become resistant to siRNA silencing? [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A57.
Abstract B05: Recurrence and hormone therapy resistance of cervical cancer in a transgenic mouse model
Authors: Mehta Fabiola , Chung Sang-Hyuk
Published: May 2015

Persistent HPV infection and other cofactors are required for the progression of cervical cancer. Oral contraceptive use or multiple parities increases risk for cervical cancer in HPV-positive women. These risk factors have hormonal balance skewed toward estrogen (i.e., an increase in estrogen levels is much greater than an increase in progesterone levels). It is consistent that transgenic mice expressing HPV16 E6 and E7 (K14E6/K14E7) develop cervical cancer only when treated with physiological levels of estrogen. Using this validated mouse model, we demonstrated that estrogen receptor alpha (ERα) antagonists (e.g., raloxifene) or a progesterone receptor (PR) agonist [e.g. medroxyprogesterone acetate (MPA)] is efficient in treating cervical cancer. Cervical cancer recurs after raloxifene therapy, and recurring disease remains sensitive to raloxifene. The present study investigates whether cervical cancer recurs after stopping MPA treatment. Consistent with our prior results, K14E6/K14E7 mice (7 of 7) developed cervical cancer after 6-month estrogen treatment (initial cancer), which regressed in 14 of 14 mice upon 2-month MPA treatment (i.e., complete response). When the mice were re-treated for 2 months with estrogen after MPA therapy, cervical cancer was observed in 6 of 6 mice (100%). As a control, a group of mice were left untreated for first 6 months, and then treated with MPA for 2 months followed by 2-month estrogen treatment. We defined cervical disease in this group as newly arising disease. Cancer incidence in this group (25%; 4 of 16 mice) was significantly lower. These results indicate that cervical cancer recurs if MPA treatment is discontinued. Interestingly, recurrence was also observed in 8 of 8 mice without readministration of estrogen, indicating that recurring cancers are independent of exogenous estrogen (we do not know whether they depend on endogenous estrogen). Strikingly, when mice bearing recurring cancers were re-treated with MPA, cancer incidence (100%, 5 of 5 mice) and cancer multiplicity remained similar to a control group, and moderate reduction in cancer size was observed. These results indicate that recurring cervical cancers acquired resistance to PR agonist therapy. This resistance was not due to loss of PR because PR status in recurring cancers was similar to that in initial cancers. Further study is warranted to determine a mechanism of therapy resistance in this mouse model, which is anticipated to facilitate development of a more efficient therapy for the disease.

Citation Format: Fabiola Mehta, Sang-Hyuk Chung. Recurrence and hormone therapy resistance of cervical cancer in a transgenic mouse model. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B05.
Abstract B14: Cisplatin induced activation of the epidermal growth factor receptor as a mechanism for epigenetic silencing in ovarian cancer cell platinum resistance
Authors: Granados Michaela Lee , Hudson Laurie G. , Samudio-Ruiz Sabrina Leigh
Published: May 2015

The 5-year survival rate for women diagnosed with ovarian cancer remains dismally unchanged at 44%. Resistance to platinum compounds in ovarian cancer imparts a major obstacle to increasing patient survival. The epidermal growth factor receptor (EGFR) plays a role in the regulation of growth and differentiation in normal cells, and proliferation in malignant cells. It is known that disregulation of the EGFR pathway, including prolonged receptor activation, is associated with platinum resistance in cellular models and our lab revealed a novel mechanism by which constitutive EGFR activation regulates DNA methyltransferase (DNMT) activity and DNA methylation. Platinum resistance is associated with epigenetic downregulation (via DNA methylation) of genes involved in platinum drug response, particularly genes important for drug uptake. Given the established link between platinum resistance and alterations to DNA methylation, our studies investigated the effects of acute cisplatin treatment on EGFR activation and DNMT activity. We hypothesized that acquired chemoresistance resulting from cisplatin treatment is initially triggered by cisplatin-induced EGFR activation and subsequent alterations to DNMT activity. We show activation of EGFR upon treatment of OVCA433 cells with cisplatin. Functional EGFR activation following cisplatin treatment was also observed by evaluating activation of downstream targets, Jak2 and Akt. Hence, cisplatin facilitates activation of EGFR and its downstream signaling pathways. Moreover, there was also a significant increase in DNMT activity in cisplatin treated cells when compared to untreated passage controls. These observations unmask a potential mechanism by which initial exposure to cisplatin can activate EGFR signaling and cause epigenetic alterations that may contribute to the development of platinum resistance. By understanding the underlying mechanism for development of platinum resistance and discovering targets of methylation, we may be able to enhance sensitivity to platinum based chemotherapeutics.

Citation Format: Michaela Lee Granados, Laurie G. Hudson, Sabrina Leigh Samudio-Ruiz. Cisplatin induced activation of the epidermal growth factor receptor as a mechanism for epigenetic silencing in ovarian cancer cell platinum resistance. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B14.
Abstract B25: A comparison of resistance mechanisms to 1st, 2nd, and 3rd generation EGFR TKIs in NSCLC
Authors: Paul Sarah M. , Ye Xioafen , Modrusan Zora , Seshagiri Somasekar , Yauch Robert , Shames David S.
Published: May 2015

Reversible EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib and gefitinib offer significant clinical benefit to patients with EGFR mutation positive NSCLC compared to chemotherapy alone. In nearly all cases resistance emerges after approximately a year on therapy, often due to the EGFR T790M (50-60% of cases). Irreversible pan-HER TKIs, including dacomitnib and afatinib were developed in part to address the emergence of T790M mutations. Both molecules showed promise, but the T790M mutation is still a dominant resistance mechanism and the number and severity of adverse events in the clinic underscore the unmet need for better targeted treatments for NSCLC patients. Third generation TKIs—AZ9291 and CO-1686—were designed against activating EGFR mutations in order to spare the wild type pathway and developed for efficacy in the T790M setting. However, resistance to the new class of EGFR TKIs is inevitable, prompting us to investigate the impact of erlotinib resistance mechanisms on AZ9291 and ask what new mechanisms are involved. NSCLC cell lines harboring activating EGFR mutations with (H1975) or without T790M (HCC4006 and HCC827) were exposed to erlotinib, dacomitnib, or AZ9291 at IC 75 or greater for three months to generate resistance. The PTEN null line H1650 was found to be resistant to all three TKIs without long-term drug exposure, implicating constitutive PIK3 pathway signaling as one mechanism of resistance. In addition, AZ9291 resistance in H1975 resulted in an epithelial to mesenchymal transition (EMT) at the phenotypic level and a corresponding increase in vimentin staining, with at least a 1000-fold increase in IC 50. Preliminary results indicate both acquired and de novo mechanisms account for AZ9291 resistance in H1975. AZ9291 resistance in HCC827 resulted in resistance to erlotinib and dacomitnib, implicating either additional mutations in EGFR or in downstream genes. Expression of E-cadherin was markedly increased in the AZ9291 resistant HCC827 line. An analysis of EGFR mutation status, PTEN status, Met expression, and EMT gene expression and methylation signatures will be presented, and the possible implications for patients will be discussed.

Citation Format: Sarah M. Paul, Xioafen Ye, Zora Modrusan, Somasekar Seshagiri, Robert Yauch, David S. Shames. A comparison of resistance mechanisms to 1st, 2nd, and 3rd generation EGFR TKIs in NSCLC. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B25.
Abstract B42: Adaptive drug resistance is associated with phenotypic reprogramming and hypersensitivity to HDAC inhibitors
Authors: Samulitis Betty k. , Pond Erika , Pond Kelvin , Cress Anne E. , Dorr Robert T. , Landowski Terry H.
Published: May 2015

Pancreatic cancer is the fourth leading cause of cancer death in the United States. One of the major reasons for the poor prognosis of pancreatic cancer is its high resistance to gemcitabine and other currently available chemotherapeutic agents. In the present study, we have developed gemcitabine-resistant cell lines to investigate the mechanisms by which adaptive resistance occurs during pancreatic tumor progression. Gemcitabine-resistant (GR) cell lines were established from the MiaPaCa2 cell line. Increasing drug selection pressure resulted in the establishment of populations that proliferate in the continuous presence of 300 nm (GR300), 800 nM (GR800), or 2µM (GR2000) gemcitabine. Using a SCID mouse xenograft mouse model, we demonstrate that the gemcitabine resistant phenotype is maintained in vivo. Growth inhibition profiles demonstrate cross resistance to Ara-c (cytarabine), and 2-chlorodeoxyadenosine (2CDA/cladribine), but no resistance to DNA damaging agents or microtubule inhibitors. Most strikingly, the gemcitabine resistant subclones demonstrated a dose dependent hypersensitivity to the Class I histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) (IC50: MP 1.6uM; GR300 1.0uM; GR800 0.2uM; GR2000 0.08uM). This collateral hypersensitivity extended to additional Class I HDAC inhibitors including MS-275 (Entinostat) and Trichostatin A, but not to the Class IIa selective agent MC1568. There was no significant difference in basal levels of histone acetylation, but the gemcitabine resistant clones demonstrated a greater increase in histone acetylation in response to HDAC treatment. Cell morphology of the gemcitabine resistant cells demonstrate an EMT-like phenotype, however evaluation of classic EMT markersidentified no significant changes in expression compared to the parental cell line. Invasive potential was measured using matrigel invasion assays, and found to be increased in a dose dependent manner. These data demonstrate that selection for drug resistance co-selects for an invasive phenotype with EMT-like characteristics. In addition, the resistance phenotype that emerges under drug pressure is associated with a phenotypic reprogramming that can be exploited to devise new therapeutic options for patients with advanced pancreatic adenocarcinoma.

Citation Format: Betty k. Samulitis, Erika Pond, Kelvin Pond, Anne E. Cress, Robert T. Dorr, Terry H. Landowski. Adaptive drug resistance is associated with phenotypic reprogramming and hypersensitivity to HDAC inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B42.
Abstract B54: Characterizing pathway vulnerabilities and tumor heterogeneity through signaling-associated complexes
Authors: Smith Matthew A. , Clark Leah , Hall Richard , Fisher Kate , Yoshida Takeshi , Vuaroqueaux Vincent , Fiebig Heinz-Herbert , Chen Ann , Haura Eric B.
Published: May 2015

Signaling pathways controlling cellular proliferation are major targets of cancer therapeutics, however, accurate assessment of activation status of targetable signaling cascades remains a challenge. We have developed proximity ligation assay (PLA)-based methodologies to detect signaling-associated complexes for a variety of receptor tyrosine kinases, intracellular adaptors and phosphorylation events in formalin-fixed, paraffin-embedded tissues. By assessing the degree of functional association of oncogenic kinases (such as EGFR and ALK) with intracellular adaptor molecules, it is possible to characterize the activation status of targetable kinase activity and monitor variations across tissue types, within tissues and in response to perturbations. Here, we present data for a panel of PLAs using cell lines, patient specimens and patient-derived xenografts models. We show that EGFR-GRB2 PLA reflects erlotinib pharmacodynamics and has predictive capacity for response to both erlotinib and cetuximab. We demonstrate that PLA can identify alterations in multiple bypass signaling cascades associated with therapeutic escape using both in vitro models of acquired resistance and patient specimens. We observe striking intratumoral variation in abundance of signaling-associated complexes, likely reflecting influences from the tumor microenvironment and phenotypic heterogeneity. We are currently expanding our PLA panel to include emerging therapeutic targets (MET and FGFR1) and evaluating performance of these assays to characterize signaling complexes in circulating tumor cells obtained from advanced stage non-small cell lung cancer patients. Collectively, our data suggests that PLA-based methodologies can be harnessed to explore the cancer interactome and can be important tools to enable precision medicine.

Citation Format: Matthew A. Smith, Leah Clark, Richard Hall, Kate Fisher, Takeshi Yoshida, Vincent Vuaroqueaux, Heinz-Herbert Fiebig, Ann Chen, Eric B. Haura. Characterizing pathway vulnerabilities and tumor heterogeneity through signaling-associated complexes. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B54.
Abstract IA05: Circulating tumor cells in lung cancer: Biomarkers, biology, and mouse models to study drug resistance
Authors: Dive Caroline , Morrow Chris , Brady Ged , Blackhall Fiona
Published: May 2015

SCLC patients (∼75%) respond well initially to standard chemotherapy (platinum/etoposide) but drug resistant disease relapse ensues rapidly. Most Small Cell Lung Cancer (SCLC) patients present with metastases, consequently tumor resection is rare and tumor biopsy is rarely obtained at disease relapse to explore drug resistance mechanisms. Targeted therapies have yet to impact SCLC patient outcomes, new drug targets and biomarkers measured in minimally invasive samples are required. Using EpCam dependent CellSearch technology, we showed that CTCs are prevalent in SCLC and CTC number is an independent prognosticator. SCLC CTC number is pharamcodynamic and CTCs are amenable to biomarker assay development (protein expression, omic profiling, FISH etc.) to be applied in clinical trials. We also developed unique patient CTC derived mouse models (CDX) to study SCLC biology, search for new drug targets, develop biomarkers and explore drug resistance mechanisms. Successful CDX engraftment has been observed so far in ∼45% cases. The molecular landscape of CDX can be compared to that of single, isolated CTCs from the same patient. CDX are readily passaged and their response to cisplatin/etoposide closely mimics the donor patient. CDX offer unparalleled opportunity to interrogate drug resistance mechanisms as paired models can be generated at presentation (chemosensitive) and again at relapse (drug resistance). Our data demonstrate that serial blood sampling with CDX and CTC molecular analysis could facilitate delivery of precision medicine to improve outcomes in SCLC. A program of evaluation of novel therapeutics in CDX models, the development of orthotopic CDX SCLC and NSCLC CDX are underway.

Citation Format: Caroline Dive, Chris Morrow, Ged Brady, Fiona Blackhall. Circulating tumor cells in lung cancer: Biomarkers, biology, and mouse models to study drug resistance. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr IA05.
Abstract IA15: Strategies to overcome acquired resistance to kinase inhibitors
Authors: Bollag Gideon
Published: May 2015

Current therapies for oncogenic kinase-driven cancers often involve kinase inhibitors. While the efficacy of these therapies is often striking, eventual relapse due to acquired resistance is common.

For example, first-generation BRAF inhibitors are making a major impact on the disease-course in BRAF-mutant cancers and metastatic melanoma in particular. Nonetheless, many patients relapse and relapsed tumors generally display BRAF pathway re-activation. In order to improve the durability and tolerability of therapy, Plexxikon has been pursuing multiple strategies. Since the well-documented RAF inhibitor paradox is implicated as a contributor to drug resistance as well as unwanted side effects, we have designed a series of compounds that exhibit potent anti-BRAF efficacy without causing paradoxical pathway activation in RAS mutant cells. These compounds, dubbed Paradox Breakers, are also active against certain BRAF isoforms that are resistant to inhibition by first-generation compounds. Furthermore, we believe that macrophages in the tumor microenvironment are likely to enable resistance to BRAF inhibition. Therefore, we are pursuing studies to co-target BRAF in tumors and macrophages in the tumor microenvironment.

Furthermore, inhibiting oncogenic KIT mutations is a validated therapeutic approach to treat gastrointestinal stromal tumors (GIST). The use of clinically approved kinase inhibitors such as imatinib frequently reveals secondary or acquired mutations that render these first-generation KIT inhibitors ineffective. Some of the acquired mutations reside in exon 17, coding for amino acids in the activation loop of the folded protein. This set of mutations has been shown to constitutively activate the receptor. Using a scaffold-based drug discovery approach, we have successfully identified a novel KIT exon 17 mutant-targeted inhibitor that shows minimal cross-reactivity in a kinome wide screen. This inhibitor shows robust efficacy in a patient-derived, imatinib-resistant GIST xenograft (Molecular Response).

Plans to develop these approaches into clinical projects will be discussed.

Citation Format: Gideon Bollag (on behalf of the Plexxikon team). Strategies to overcome acquired resistance to kinase inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr IA15.
RAI2: Linking Retinoic Acid Signaling with Metastasis Suppression
Authors: Esposito Mark , Kang Yibin
Published: May 2015

Summary: Considerable evidence points to the importance of disseminated tumor cells, which are commonly detected in the bone marrow and display features of cellular plasticity, in predicting the clinical outcome of breast cancer. In this issue of Cancer Discovery, Werner and colleagues report on the discovery of retinoic acid–induced 2 (RAI2) as a differentiation factor that suppresses early metastatic spread of estrogen receptor–positive breast cancer. Cancer Discov; 5(5); 466–8. ©2015 AACR.

See related article by Werner et al., p. 506
CHD5 downregulation associated with poor prognosis in epithelial ovarian cancer in Hong Kong Chinese women
Authors: Wong Raymond R. , Chan Loucia K.Y. , Tsang Teresa B.T. , Lee Coral W.S. , Cheung T.H. , Yim S.F. , Siu Nelson S.S. , Lee Sophia N.C. , Chim Stephen S.C. , Wong Y.F. , Chung Tony K.H.
Published: May 2015

Background: CHD5 gene located on 1p36, encodes a non-histone chromosomal protein, chromodomain helicase DNA binding protein 5. Recently, CHD5 has been shown to be a tumor suppressor gene candidate. This study was to investigate the involvement of CHD5 in epithelial ovarian cancer and its clinico-pathological significance.

Methods: CHD5 expression in ovarian cancer and its normal counterpart was determined by quantitative RT-PCR. The methylation of CHD5 promoter was evaluated by methylation specific PCR. The correlation of CHD5 expression to patient age, tumor histological type and grade, clinical stage and status, recurrence and patient survival time was analyzed statistically.

Results: CHD5 expression was down at least two-fold in 32 of 72 (41%) invasive epithelial ovarian carcinomas when compared to 12 normal controls in Hong Kong Chinese women. Hypermethylation in two promoter regions of CHD5 was not detected in any of the 20 tumor samples available to be analyzed. CHD5 down-regulation was correlated to clinical status (P<0.05), but not to patient age, tumor type and grade, recurrence and clinical stage (P>0.05). Kaplan-Meier survival analysis showed that patients with CHD5 down-regulation in their tumors were associated with shorter disease-free and total survival times compared to those without CHD5 down-regulation (P<0.05). Cox proportional hazard regression analysis indicated that down-regulation of CHD5 is an independent adverse prognostic factor in ovarian cancer.

Conclusion: This preliminary study shows that CHD5 s down-regulated in a certain part of ovarian cancers and appears to be an adverse predictor candidate of ovarian cancer disease-free and total survival in Hong Kong Chinese women. If this role of CHD5 dysregulation can be confirmed in more prospective studies with a large number of patients, it is also worthwhile to further evaluate if CHD5 can be as an individualized molecular therapeutic target for epithelial ovarian cancer.