bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B095: CARM1 methylates BAF155 and perturbs chromatin remodeling machinery to enhance tumor progression
Authors: Wang Lu
Published: May 2015

CARM1 belongs to the type I protein arginine (R) methyltransferase (PRMT) family and asymmetrically di-methylates proteins on arginines. Because CARM1 is highly expressed in high grade breast tumors and was shown to transactivate cancer-relevant transcription factors including ERa, NF-kb, p53 and E2F1, it has been strongly implicated in tumorigenesis. To fully characterize functions of CARM1 in breast cancer, we knocked-out CARM1 from several breast cancer cell lines using Zinc-Finger Nuclease technology. Western blot using an asymmetric di-methylated R antibody revealed striking differences between CARM1 KO and parental MCF7 cell lysates, indicating that CARM1 methylates a significant number of substrates in breast cancer cells. BAF155, a core subunit of SWI/SNF chromatin remodeling complex, was identified as a CARM1 substrate by mass spectrometry. Further biochemical analyses validated that BAF155 was methylated by CARM1 in vivo and in vitro and methylation was mapped to a single site, R1064. We next generated BAF155R1064K knock-in MCF7 and MDA-mb-231 cells and showed that methylation of BAF155 is essential for breast cancer cell growth, colony formation and migration. Further, we developed an antibody that selectively recognized methylated form of BAF155 (me-BAF155) and observed strong nuclear me-BAF155 staining in invasive ductal carcinoma but not in the normal breast tissue. Importantly, me-BAF155 immunostaining positively correlates with tumor stage and metastasis. To understand the molecular mechanism by which BAF155 methylation affects tumorigenesis, we performed ChIP-seq using ChIP-validated BAF155 antibody in the BAF155WT or BAF155R1064K knock-in MCF7 cells. Interestingly, C-Myc pathway genes were only identified in WT but not mutant BAF155 regulated categories. Individual ChIP assays validated differential binding of BAF155WT or BAF155R1064K to specific target genes and importantly, the mRNA levels of these genes were affected correspondingly. This result suggests that BAF155 methylation affects gene expression by altering genomic association of BAF155, and perhaps the SWI/SNF complex, to chromatin regions. Collectively, our study revealed novel mechanisms by which CARM1 perturbs the chromatin remodeling machinery to enhance tumor progression.

Citation Format: Lu Wang. CARM1 methylates BAF155 and perturbs chromatin remodeling machinery to enhance tumor progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B095.
Abstract B098: Tumor cell-derived IL-4 suppresses tumor growth and metastasis
Authors: Zhang Connie Shengnan , Greer Peter A.
Published: May 2015

Interleukin 4 (IL-4) is a cytokine that, among other actions, can induce macrophages to undergo alternative activation and polarize to M2 macrophages which have been reported to promote tumorigenesis and metastasis. IL-4 also promotes the formation of multinucleated giant cells from macrophages in vitro and participates in the development of the foreign body reaction in vivo.

AC2M2 cells, a highly metastatic mouse carcinoma cell line, were transduced with retroviruses expressing IL-4 (IL-4-AC2M2) or empty vector control (EV-AC2M2). Co-culture experiments showed that AC2M2-derived IL-4 polarized macrophages to express the M2 marker, arginase 1. EV- and IL-4-AC2M2 cells grew at the same rate in vitro. However, mice injected with IL-4-AC2M2 cells grew tumors at a significantly reduced rate as compared to control mice injected with EV-AC2M2 cells in an orthotopic mouse mammary tumor engraftment model. IL-4 expression also correlated with elimination of lung metastasis. Reduced primary tumor growth and complete abolishment of lung metastasis in the IL-4 group correlated with a 30-fold increase of tumor associated macrophage populations and macrophage phagocytosis of tumor cells.

Thus, tumor-derived IL-4 suppressed tumorigenesis and lung metastasis by activating macrophage phagocytosis in the tumor microenvironment; suggesting that IL-4 could be a good candidate for immunotherapy.

Note:This abstract was not presented at the conference.

Citation Format: Connie Shengnan Zhang, Peter A. Greer. Tumor cell-derived IL-4 suppresses tumor growth and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B098.
Abstract IA02: Tumor plasticity and reactive stroma in breast cancer metastasis
Authors: Kang Yibin
Published: May 2015

Metastasis represents the most devastating stage of cancer progression. The multi-step cascade of cancer metastasis is driven by both intrinsic changes of cancer cell properties, as well as the stromal reaction to tumor cells. Epithelial-mesenchymal transition (EMT) is a complex process that is associated with dramatic changes in cell adhesion, polarity, and migratory properties, and is intimately associated both with cell fate transitions during embryonic development and with the acquisition of invasive properties during early steps of cancer metastasis. We found that the transcription factor Elf5, a key regulator of alveologenesis in the mammary gland, regulates EMT in both mammary gland development and metastasis. This specific role for Elf5 in suppressing EMT and metastasis was uncovered via analyses of Elf5 conditional knockout animals, various in vitro and in vivo models of EMT and metastasis, an MMTV-neu transgenic model of mammary gland tumor progression, and clinical breast cancer samples. Furthermore, we demonstrate that this role of Elf5 is mediated by the direct transcriptional repression of Snail2/Slug, a master regulator of mammary stem cells and a known inducer of EMT. These findings establish a broad molecular function of Elf5 that extends from being a key cell lineage regulator during normal mammary gland development to serving as a suppressor of EMT and metastasis in breast cancer.

The growth of disseminated tumor cells at distant organs represents a critical final stage of metastasis development. Bone metastasis is a frequent occurrence in breast cancer, affecting more than 70% of late stage cancer patients. Increasing evidence suggest that the development of bone metastasis relies on the positive feedbacks from stromal components of the bone microenvironment. Using in vivo imaging technology, we showed that TGFβ is released from bone matrix upon bone destruction, and signals to breast cancer to further enhance their malignancy in developing bone metastasis. We furthered identified Jagged1 as a TGFβ target genes in tumor cells that engaged bone stromal cells through the activation of Notch signaling to provide a positive feedback to promote tumor growth and to activate osteoclast differentiation. More recently, we identified osteoclast-derived miRNAs as biomarkers and therapeutic targets of osteolytic bone metastasis. Importantly, pharmaceutical inhibitors against TGFβ and Notch signaling pathways and miRNAs blocking osteoclast differentiation significantly reduce the development of bone metastasis, suggesting novel avenues for clinical management of skeletal complications of breast cancer.

Citation Format: Yibin Kang. Tumor plasticity and reactive stroma in breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA02.
Abstract IA11: Dissecting cancer through the lens of development
Authors: Wahl Geoffrey Myles , Dravis Christopher , Johns Claire , Rodewald Rose , Spike Benjamin T. , Trejo Christy
Published: May 2015

Mammary stem cells (MaSCs) self-renew and generate the different epithelial cell types of the breast. Impressively, a single MaSC can regenerate an entire functional mouse mammary gland when transplanted into a cleared fat pad. Intriguingly, stem-like cells have been identified in some types of breast cancer using bioinformatic strategies. These observations raise a number of important questions: Do some breast cancers arise by mutations occurring in mammary stem cells, as such cells have a high capacity for self-renewal, proliferation and differentiation? Or, do breast cancers arise by de-differentiation of cells to a stem-like state, as such cells may be particularly able to generate the heterogeneity that fuels tumor progression? If stem cells contribute to cancer, what is the nature of such cells, and can we use this information to devise better treatment strategies or prognostic molecular signatures? Finally, cancer cells that seem particularly competent at generating tumors upon xenografting into nude mice have also been suggested to exhibit stem cell characteristics such as multi-lineage differentiation and self-renewal. Are these “cancer stem cells” mutant stem cells, or a different type of cell that is particularly tumorigenic in the xenograft assay? Here, we integrate approaches involving developmental, stem cell, and cancer biology to gain insight into these challenging questions.

We took a new direction to isolate and characterize MaSCs during fetal mammogenesis when undifferentiated cells are expected to be enriched. We microdissected and dissociated fetal mammary cells across different stages of development and conducted limiting dilution, orthotopic mammary transplantation analyses to determine fetal MaSC (fMaSC) frequency. We found that the fMaSCs are not measurable until mid-gestation, when an elongating duct that invades through the adjacent mesenchyme is formed. We used flow sorting to significantly enrich fMaSCs, and then used microarray and single cell RNA-seq to begin to interrogate the molecular pathways that drive their proliferation. We find that the pathways they express, and that are expressed by the fetal stroma that surround them, are enriched in several very aggressive types of human breast cancer, as well as in several genetically engineered mouse models. These data and powerful experimental strategies are opening new ways of understanding cancer from a developmental perspective, and for using this knowledge to develop new diagnostic and treatment strategies.

Citation Format: Geoffrey Myles Wahl, Christopher Dravis, Claire Johns, Rose Rodewald, Benjamin T. Spike, Christy Trejo. Dissecting cancer through the lens of development. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA11.
Abstract IA23: Molecular and clinical impact of APOBEC3B mutagenesis in breast cancer
Authors: Harris Reuben S.
Published: May 2015

Recent studies have revealed a role for the DNA cytosine deaminase APOBEC3B in breast cancer mutagenesis. First, APOBEC3B mRNA is up-regulated in the majority of primary breast tumors and breast cancer cell lines. Second, endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell line extracts. Third, APOBEC3B knockdown causes a corresponding decrease in genomic uracil concentrations, mutation frequencies, and C-to-T mutation levels in breast cancer cell lines. Fourth, the biochemical signature of APOBEC3B is evident in breast cancer mutation spectra. Fifth, breast carcinomas that express high levels of APOBEC3B have twice as many mutations as those expressing low levels. Finally, high levels of APOBEC3B associate with poorer prognostic outcomes including shorter survival durations. All of these data are consistent with a model in which APOBEC3B-catalyzed DNA cytosine deamination provides a chronic source of DNA damage and mutagenesis in breast carcinomas that helps to explain how some tumors evolve rapidly and manifest gross heterogeneity.

Citation Format: Reuben S. Harris. Molecular and clinical impact of APOBEC3B mutagenesis in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA23.
Abstract A34: Mammary-specific, doxycycline-regulated transposon mobilization for identifying driver genes in mouse models of breast cancer
Authors: Gestl Shelley A. , Gunther Edward J.
Published: May 2015

Transposon-mediated mutagenesis offers a powerful tool for discovering genetic changes that contribute to cancer pathogenesis. Tissue-specific transposon mobilization has been exploited widely to identify candidate cancer genes in a variety of tissues, including intestine, pancreas, and liver. These models typically use tissue-specific expression of the Cre recombinase to activate a latent transposase allele, which in turn mobilizes a mutagenic transposon. Notably, transposase expression cannot be halted once activated in these models, potentially leading to unwanted transposition events that either delete driver insertions or create additional passenger insertions. As an alternative strategy, we generated transgenic mice harboring the SB11 transposase under the control of tet operator sequences. Tet-O-SB11 founder lines were characterized that permit a range of transgene expression levels when crossed to mice carrying a mammary-specific transactivator, MMTV-rtTA. We show that efficient mobilization of the T2/Onc transposon in mammary tissue requires high-level SB11 expression, which strictly depends on doxycycline administration in our models. Furthermore, efficient T2/Onc mobilization enabled by high-level SB11 expression strongly cooperated with Wnt1 transgene expression to drive mammary tumorigenesis in vivo. Preliminary analysis of transposon integration sites in these mammary tumors confirms that transposon-mediated mutations serve as pathogenic driver events. Temporal regulation of transposase-mediated mutagenesis may offer a flexible tool for discovering driver events operative at various stages of tumor progression.

Citation Format: Shelley A. Gestl, Edward J. Gunther. Mammary-specific, doxycycline-regulated transposon mobilization for identifying driver genes in mouse models of breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A34.
Abstract B24: ATDC (Trim29) drives invasive bladder cancer formation
Authors: Palmbos Phillip , Wang Lidong , Yang Huibin , LeFlein Jake , Wilkinson John E. , Kumar-Sinha Chandan , Kunju L. Priya , Daignault Stephanie , Wu Xue-Ru , Lotan Yair , Liebert Monica , Ljungman Mats , Simeone Diane
Published: May 2015

Bladder cancer is a common and deadly malignancy, but incomplete understanding of the molecular events driving tumor development and metastasis has limited development of effective treatments. Ataxia-Telangiectasia Group D Complementing (ATDC; also named TRIM29) is highly expressed in many cancer types, including bladder and pancreas. To determine the role of ATDC in tumorigenesis, we developed a novel transgenic mouse model in which ATDC was overexpressed in multiple tissue types. The dominant phenotype in these mice was the development of both papillary and invasive bladder carcinoma beginning at approximately 8 months of age. In this model, invasive bladder tumors were histologically indistinguishable and shared a similar gene expression signature with human invasive bladder cancers. We also found that ATDC was highly expressed in ~70% human bladder cancers where it marked invasive tumors and correlated with worse survival after chemotherapy (p = 0.002). Furthermore, ATDC knockdown decreased proliferation, invasion and tumor growth in multiple human bladder cancer cell lines and in an in vivo orthotopic bladder tumor model. ATDC-induced bladder tumorigenesis was mediated by upregulation of DNA methyltransferase 3A (DNMT3A), which drove PTEN promoter methylation and silencing, events which promoted invasion and proliferation in both transgenic mice and human bladder cancers. These findings establish ATDC as a novel driver and therapeutic target in bladder cancer. In addition, the ATDC transgenic mouse model should serve as an important and unique new model to study bladder cancer development, progression and therapeutic responses.

Citation Format: Phillip Palmbos, Lidong Wang, Huibin Yang, Jake LeFlein, John E. Wilkinson, Chandan Kumar-Sinha, L. Priya Kunju, Stephanie Daignault, Xue-Ru Wu, Yair Lotan, Monica Liebert, Mats Ljungman, Diane Simeone. ATDC (Trim29) drives invasive bladder cancer formation. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B24.
Abstract IA10: Improving existing cancer therapy by targeting the tumor microenvironment
Authors: Pallasch Christian , Gilbert Luke , Hemann Michael
Published: May 2015

Many human cancers fail to respond to chemotherapy, and cancers that initially respond frequently acquire drug resistance and relapse. This process of tumor relapse is particularly confounding, as patients can be in remission for years following treatment prior to the reemergence of a cancer. Additionally, tumors that relapse have generally acquired resistance to the initial treatment. While conventional anti-cancer therapies have been in clinical use for decades, little is known about the mechanisms by which a tumor cell can survive treatment and persist in a patient for extended period of time. In fact, a major cause of cancer deaths is the inability to eradicate small sets of surviving tumor cells, termed “minimal residual disease” or “MRD”. Work from our laboratory has found that chemotherapy paradoxically elicits a pro-survival response in certain anatomical cites. Specifically, normal cells proximal to tumor cells secrete factors that counter the effects of the chemotherapy. This survival response likely serves to protect normal progenitor and stem cells to allow for tissue regeneration following damage, but it is coopted by tumor cells that find themselves in these specialized sites. Thus, effective cancer therapy may not only involve targeting cancer cells but also may require inhibiting survival signals emanating from surrounding cells. We have developed multiple models of MRD that have not only identified mechanisms of tumor cell persistence, but also strategies to sensitize chemorefractory tumors to front-line chemotherapy.

Citation Format: Christian Pallasch, Luke Gilbert, Michael Hemann. Improving existing cancer therapy by targeting the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr IA10.
Abstract IA11: Understanding drug responses and resistance mechanisms using imaging in live mice
Authors: Egeblad Mikala , Nakasone Elizabeth , Askautrud Hanne , Wysocki Robert , Fein Miriam , Kees Tim , Park Juwon , Park Jae-Hyun
Published: May 2015

It is well appreciated that factors intrinsic to the cancer cells, such as specific mutations, regulate response to chemotherapy. However, there is limited knowledge on the dynamics of cancer cell death in response to therapy in the naturally developing tumor microenvironment. Using intravital microscopy (microscopy in live mice) of tumors, we show that factors within the microenvironment, extrinsic to the cancer cells, support the development of chemoresistance by regulating drug distribution and the inflammatory response. Specifically, intravital microscopy of chemotherapy-treated mouse mammary carcinomas allowed us to follow drug distribution, cell death, and tumor-stroma interactions. We observed associations between vascular leakage and response to the chemotherapeutic drug doxorubicin, including improved response in matrix metalloproteinase-9 null mice that had increased vascular leakage. Furthermore, we observed CCR2-dependent infiltration of myeloid cells after treatment and that Ccr2 null host mice responded better to treatment with doxorubicin or cisplatin. These data show that the microenvironment contributes critically to drug response via regulation of vascular permeability and innate immune cell infiltration. These results have clinical implications, as myeloid cell infiltration is increased in human breast tumors after chemotherapy and the cellular composition of the immune infiltrate is a strong predictor of overall survival. Our data further suggest that the response to classical chemotherapeutic drugs can be improved by changing the tumor microenvironment with agents that modify matrix metalloproteinase activity and chemokine signaling. Thus, intravital imaging can be used to gain insights into drug responses in situ.

Citation Format: Mikala Egeblad, Elizabeth Nakasone, Hanne Askautrud, Robert Wysocki, Miriam Fein, Tim Kees, Juwon Park, Jae-Hyun Park. Understanding drug responses and resistance mechanisms using imaging in live mice. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr IA11.
Abstract IA12: The use of forward genetics for drug target discovery and the identification of mediators of therapy resistance
Authors: Adams David J.
Published: May 2015

In this talk I will outline how we've been using the extensive collection of cancer genomes generated by the TCGA and ICGC together with forward genetic screens in the mouse to discover new cancer genes. I will also discuss how further insights into the function of these genes, and importantly candidate therapies and combination treatments, have been derived using mouse model systems. Specifically, I will describe the identification of CUX1 as an important new driver of tumorigenesis, and a new mechanism of resistance to Braf-inhibitor treatment. Both of these stories illustrate the power of model systems to discover new cancer genes that were not immediately obvious from the human cancer genome alone, and how the mouse may be deployed to gain important insights into the function of these new cancer genes.

Citation Format: David J. Adams. The use of forward genetics for drug target discovery and the identification of mediators of therapy resistance. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr IA12.
Abstract IA5: Using cross-species analysis of genome-wide regulatory networks to identify drivers of cancer malignancy
Authors: Abate-Shen Cory
Published: May 2015

We have been interested in using systems biology approaches for cross-species interrogations of cancer phenotypes. To identify regulatory drivers of prostate cancer malignancy, we have performed cross-species analysis of complementary human and mouse gene regulatory networks (interactomes) assembled from molecular profiles of human tumors and genetically engineered prostate cancer mouse models, respectively. Genome-wide analysis of these interactomes reveals extensive conservation of their transcriptional programs, particularly those known to be essential for prostate tumorigenesis. Cross-species interactome interrogation and subsequent experimental validation have elucidated a synergistic interaction of FOXM1 and CENPF that drives prostate cancer malignancy. Silencing of both FOXM1 and CENPF, but not either individually, abrogates prostate tumor growth in vivo via cooperative regulation of key signaling pathways in prostate cancer progression. Furthermore, co-expression of FOXM1 and CENPF is a highly robust prognostic indicator of disease outcome. Taken together, these analyses suggest that genome-wide cross-species interrogation of regulatory networks represents a valuable new strategy to identify causal mechanisms of human cancer.

Citation Format: Cory Abate-Shen. Using cross-species analysis of genome-wide regulatory networks to identify drivers of cancer malignancy. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr IA5.
Abstract IA8: A new class of drugs active in T-ALL is revealed in a zebrafish screen
Authors: Gutierrez Alejandro , Pan Li , Groen Richard , Baleydier Frederic , Kentsis Alex , Marineau Jason , Grebliunaite Ruta , Kozakewich Elena , Reed Casie , Pflumio Francoise , Poglio Sandrine , Uzan Benjamin , Clemons Paul , Verplank Lynn , An Frank , Burbank Jason , Norton Stephanie , Tolliday Nicola , Steen Hanno , Weng Andrew P. , Yuan Huipin , Bradner James E. , Mitsiades Constantine , Look A. Thomas , Aster Jon C.
Published: May 2015

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer frequently associated with activating NOTCH1 mutations and dysregulation of MYC. We performed two complementary screens to identify novel agents with activity against T-ALL: i) a zebrafish screen for small molecules toxic to MYC-overexpressing thymocytes, and ii) a human T-ALL cell line screen for small molecules that synergize with Notch inhibitors. “Hits” common to both screens included perphenazine, a phenothiazine antipsychotic that induced apoptosis of fish, mouse, and human T-ALL cells. Using ligand-affinity chromatography coupled to mass spectrometry, we identified protein phosphatase 2A (PP2A) as the critical perphenazine target. In line with this finding, T-ALL cell lines treated with perphenazine underwent apoptosis associated with rapid dephosphorylation of multiple PP2A substrates, indicating that perphenazine binds and activates the PP2A tumor supressor. Moreover, shRNA knockdown of the scaffolding or catalytic subunits of PP2A attenuated the activity of perphenazine, indicating that PP2A is required for its antileukemic activity. Finally, treatment of primary human T-ALLs with pherphenazine suppressed cell growth and caused dephosphorylated of PP2A targets in vitro and in vivo. Our findings provide a mechanistic explanation for the recurrent “rediscovery” of phenothiazines as a class of drugs with anti-cancer effects and highlight the therapeutic potential of pharmacologic PP2A activation in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates.

Citation Format: Alejandro Gutierrez, Li Pan, Richard Groen, Frederic Baleydier, Alex Kentsis, Jason Marineau, Ruta Grebliunaite, Elena Kozakewich, Casie Reed, Francoise Pflumio, Sandrine Poglio, Benjamin Uzan, Paul Clemons, Lynn Verplank, Frank An, Jason Burbank, Stephanie Norton, Nicola Tolliday, Hanno Steen, Andrew P. Weng, Huipin Yuan, James E. Bradner, Constantine Mitsiades, A. Thomas Look, Jon C. Aster. A new class of drugs active in T-ALL is revealed in a zebrafish screen. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr IA8.
Abstract PR02: Targeting NOTCH1 and C-MYC in humanized models of relapsed and induction failure pediatric T-ALL
Authors: Roderick Justine E. , Tesell Jessica , Gutierrez Alejandro , Look Thomas , Qi Jun , Bradner James E. , Kelliher Michelle A.
Published: May 2015

Although prognosis has improved for children with T cell acute lymphoblastic leukemia (T-ALL), 20-30% of patients undergo induction failure (IF) or relapse. Leukemia-initiating cells (L-ICs) are hypothesized to be resistant to chemotherapy and to mediate relapse. We and others have shown that Notch1 directly regulates c-Myc and c-Myc is a known regulator of quiescence in stem and progenitor populations, leading us to examine whether c-Myc inhibition results in efficient targeting of T-ALL-initiating cells. We demonstrate that c-Myc suppression by shRNA or pharmacological approaches prevents leukemia initiation in mice by reducing the L-IC population. Consistent with its anti-L-IC activity in mice, treatment with the BET bromodomain BRD4 inhibitor JQ1 reduces C-MYC expression and inhibits the growth of relapsed and induction failure (IF) pediatric T-ALL samples in vitro. Although treatment of relapsed T-ALL cells with gamma secretase inhibitors (GSI) or JQ1 reduces, it does not abrogate C-MYC mRNA levels, indicating a role for dual NOTCH and MYC inhibition in relapsed disease. To test this possibility, we engrafted immunodeficient NSG mice with relapsed pediatric T-ALL cells and treated them with vehicle, GSI, JQ1 or with GSI and JQ1 and monitored survival. Although JQ1 or GSI treatment alone significantly prolonged survival, the combination therapy was more effective at extending survival. These findings reveal a requirement for MYC in mouse and human L-IC maintenance and provide evidence that NOTCH1 and MYC inhibition may be an effective therapy for relapsed/IF T-ALL patients.

This abstract is also presented as Poster A48.

Citation Format: Justine E. Roderick, Jessica Tesell, Alejandro Gutierrez, Thomas Look, Jun Qi, James E. Bradner, Michelle A. Kelliher. Targeting NOTCH1 and C-MYC in humanized models of relapsed and induction failure pediatric T-ALL. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR02.
Abstract PR06: Clonal evolution enhances leukemia propagating cell activity in T-cell acutelymphoblastic leukemia through AKT/mTORC1 pathway activation
Authors: Blackburn Jessica , Liu Sali , Dobrinski Kimberly , Martinez Sarah , Moore Finola , Lobbardi Riadh , Langenau David
Published: May 2015

Leukemia progression and relapse are driven by molecularly distinct and often-rare cancer cells called leukemia-propagating cells (LPCs). If LPCs are retained following treatment, they will ultimately initiate relapse disease. Despite the substantial number of genetic lesions that have been identified in relapse samples and the contention that these mutations likely modulate response to therapy, acquired mutations that increase the overall frequency of tumor propagating cells following continued clonal evolution have not been reported in any cancer to date. Here, we have developed a transgenic zebrafish model where single fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) cells are transplanted into genetically identical recipient fish and functionally assessed for differences in leukemia propagating cell frequency, growth, and dexamethasone resistance. While subclonal variation was observed within single cells from the same primary leukemia, a subset of clones continued to evolve genetic lesions and epigenetic modifications to enhance growth and overall LPC frequency. A majority of evolved clones acquired activated AKT signaling, which simultaneously increased the number of leukemia propagating cells through activating the mTORC1 pathway, enhanced growth by stabilizing Myc protein levels, and rendered T-ALL cells resistant to dexamethasone, which was reversed by combined treatment with an AKT inhibitor. These results were confirmed using large-scale transgenic epistasis experiments and limiting-dilution cell transplantation studies. In total, our data suggest that diagnosis clones can stochastically acquire mutations necessary to survive treatment and drive relapse even before a patient begins treatment, with acquired mutations being independently selected based on important cancer phenotypes including elevated growth rate and leukemia propagating potential. Moreover, our work also identifies combination therapies that utilize dexamethasone and AKT inhibitor can kill LPCs in a subset of refractory T-ALL. Finally, these are the first studies performed in any model to follow single cell evolution as it relates to relapse, utilizing in excess of 6,000 transplant recipient animals and opening new and exciting avenues of study to uncover genetic pathways that drive cancer malignancy.

This abstract is also presented as Poster A08.

Citation Format: Jessica Blackburn, Sali Liu, Kimberly Dobrinski, Sarah Martinez, Finola Moore, Riadh Lobbardi, David Langenau. Clonal evolution enhances leukemia propagating cell activity in T-cell acutelymphoblastic leukemia through AKT/mTORC1 pathway activation. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR06.
Abstract PR10: From melanocyte to melanoma: Identifying key molecular events at the onset of cancer
Authors: Kaufman Charles K. , Mosimann Christian , White Richard , Matos Dominic , Zon Leonard I.
Published: May 2015

A fundamental challenge in cancer biology is understanding the earliest events that switch a premalignant or premetastatic cell into a fully competent cancer cell. Driven by this question, we focused on the zebrafish gene crestin that marks the embryonic neural crest from the onset of somitogenesis through 72 hpf when it normally turns off; surprisingly, our lab previously discovered that crestin mRNA is reexpressed in melanoma tumors in adult zebrafish. We have developed a fluorescent reporter gene using the crestin promoter that recapitulates the embryonic expression pattern of crestin, and when bred into our BRAFV600E/p53 mutant melanoma zebrafish model, marks grossly visible melanoma tumors. Remarkably, we found that crestin:EGFP becomes active when lesions are only a few cells in number, potentially in the first cell of the melanoma. We are able to isolate crestin (+) patches at the few to single cell level. Using a crestin:CreERt2 inducible recombinase together with a lox-dependent fluorescent lineage tracer line ubi:switch, multiple neural crest descendant lineages including melanocytes can be traced. Further by expressing the melanocyte lineage master transcription factor mitf under the control of the crestin promoter, we can rescue melanocyte formation in mitf null embryos, and in the Na/BRAFV600E/p53 melanoma-prone mutant background, some adult fish develop darkly pigmented patches and melanoma tumors. These data support a model in which BRAFV600E/p53 mutated melanocytes stochastically reactivate an early melanocyte or neural crest progenitor program as read out by crestin reactivation at the onset of melanoma formation. Using our novel reporter, we are able to analyze in vivo the earliest steps of cancer formation as well as the neural crest lineage program.

This abstract is also presented as Poster A47.

Citation Format: Charles K. Kaufman, Christian Mosimann, Richard White, Dominic Matos, Leonard I. Zon. From melanocyte to melanoma: Identifying key molecular events at the onset of cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR10.
Abstract A05: The control of nuclear ERK activity by the DUSP phosphatases in colorectal cancer
Authors: Cagnol Sebastien , Rivard Nathalie
Published: May 2015

The RAS/RAF/MEK/ERK pathway is suspected to be strongly implicated in malignant transformation of colorectal epithelial cells since KRAS and BRAF genes are mutated in up to 60% of human colorectal cancers (CRCs). Despite intensive efforts made in the understanding of KRAS and BRAF functions in colorectal carcinogenesis, little is known about the oncogenic regulation of ERK activity in CRC cells, especially by the ERK-specific nuclear DUSP phosphatases. We have recently demonstrated in CRC cells that ERK1/2 activity is confined to the cytoplasm because of a pervanadate-dependent phosphatase activity. Accordingly, DUSP4 mRNA was found to be highly expressed, in a MEK-dependent manner, in CRC cells. Thus, DUSP4 and other nuclear DUSPs may function as part of a negative feedback mechanism in the control of the duration and magnitude of nuclear ERK activation during colorectal carcinogenesis. We thus speculate that the modification of nuclear ERK activity might strongly impact on the oncogenic properties of CRC.

Methods: Expression of DUSPs was analyzed by PCR in CRC cells and qPCR in human colorectal tumors from different stages. To elucidate the role of nuclear DUSPs, RNA interference was used to specifically silence their expression in human HT29 CRC cell line, a cell line exhibiting BRafV600E mutation. Inducible expression of a DUSP4 inactivated mutant (DUSP4C284S) was done in a model of IEC-6 cells that express an inducible form of BRaf oncogene (BRafV600E:ER).

Results: 1- DUSP2, DUSP4 and DUSP5 mRNAs were found to be highly expressed, in a MEK-dependent manner, in CRC cells. 2- DUSP4 and DUSP5 mRNA levels were markedly increased in colorectal tumors in comparison to healthy matched adjacent tissues. 3- Silencing of DUSP 2, 4 or 5 in HT29 cells increased ERK phosphorylation levels and markedly reduced colony formation in soft agar. 4- Furthermore, expression of DUSP4C284S in BRafV600E-expressing IEC-6 cells increased ERK phosphorylation into the nucleus and markedly reduced colony formation in soft agar.

Conclusion: We are currently searching by which mechanisms sustained nuclear ERK activity reduces colorectal tumor cell growth. Therefore, targeting nuclear DUSP activity could become a new strategy to treat CRC.

Citation Format: Sebastien Cagnol, Nathalie Rivard. The control of nuclear ERK activity by the DUSP phosphatases in 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 A05. doi: 10.1158/1557-3125.RASONC14-A05
Abstract A15: Megakaryocyte differentiation is regulated by p21-activated kinase 2
Authors: Kosoff Rachelle E. , Chernoff Jonathan
Published: May 2015

Megakaryocytes (MKs) mature and differentiate into multi-nucleated cells in the bone marrow, and migrate from the osteoblastic niche to the vascular niche by means of an SDF-1a gradient, culminating in the release of platelets into circulation. Failure to do so results in myeloproliferative disorders, which can manifest with extremely reduced platelet counts. Regulation of this process is controlled by MAPK signaling, cytoskeletal and microtubule-associated proteins and GTPases. p21-activated kinase 2 (Pak2) regulates the cytoskeleton through the MAPK and RhoA signaling pathways, both required for MK differentiation and cytokinesis. In our study, we demonstrate that Pak2 is essential for proper MK differentiation and polyploidization, in vitro and in vivo. Pak2 deletion, upon cre induction specifically in hematopoietic tissues (Mx1-cre), reduces peripheral blood platelet counts (thrombocytopenia) by 50%. Bone marrow histology demonstrates hyperplastic MKs coinciding with reduced platelet counts. Analysis of bone marrow by flow cytometry demonstrates altered MK differentiation, observed by elevated MK stem cell precursors in the bone marrow (CD150+CD41+). Increased stem cell potential of MKs deficient for Pak2 results in increased colony forming units (CFU-Mks). Additionally, Pak2-null MKs have increased levels of polyploidization. Signaling to MLC2 and GEF-H1 are altered in Pak2-deficient MKs, both previously shown to regulate MK differentiation and polyploidization, respectively. In summary, Pak2 is a negative regulator of MK stem cell differentiation as well as polyploidization. Since Pak2 negatively regulates polyploidization, this leads to therapeutic uses for Pak family inhibitors in the treatment for acute megakaryoblastic leukemia, a fatal disorder which presents with an accumulation of immature MKs, due to a failure to differentiate into mature MKs.

Citation Format: Rachelle E. Kosoff, Jonathan Chernoff. Megakaryocyte differentiation is regulated by p21-activated kinase 2. [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 A15. doi: 10.1158/1557-3125.RASONC14-A15
Abstract A23: Oncogenic Ras induces inflammatory cytokine production by upregulating squamous cell carcinoma antigen (SerpinB3)
Authors: Catanzaro Joseph , Sheshadri Namratha , Pan Ji-An , Sun Yu , Shi Chanjuan , Li Jinyu , Powers Scott , Crawford Howard , Zong Wei-Xing
Published: May 2015

Mounting evidence indicates that oncogenic Ras can modulate cell autonomous inflammatory cytokine production, although the underlying mechanism remains unclear. Here we show that squamous cell carcinoma antigen (SCCA), a member of the Serpin family of serine/cysteine protease inhibitors, is transcriptionally up-regulated by oncogenic Ras via MAPK and the ETS family transcription factor PEA3. Increased SCCA expression leads to inhibition of protein turnover, unfolded protein response (UPR), activation of NF-κB, and is essential for Ras-mediated cytokine production and tumor growth. Analysis of human colorectal and pancreatic tumor samples reveals a positive correlation between Ras mutation, enhanced SCCA expression, and IL-6 expression. These results indicate that SCCA is a Ras-responsive factor that plays an essential role in Ras-associated cytokine production and tumorigenesis.

Citation Format: Joseph Catanzaro, Namratha Sheshadri, Ji-An Pan, Yu Sun, Chanjuan Shi, Jinyu Li, Scott Powers, Howard Crawford, Wei-Xing Zong. Oncogenic Ras induces inflammatory cytokine production by upregulating squamous cell carcinoma antigen (SerpinB3). [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 A23. doi: 10.1158/1557-3125.RASONC14-A23
Abstract A39: Repurposing of AP-102 and AP-103 in the treatment of breast cancer via inhibiting the RAF/ERK pathway
Authors: Tsai Pei-Yi , Ding Yu-Sian , Hung Le-Mei , Ho Yueh-Feng , Peng Ling-Chiu , Liou Chiung-Wen , Chang Jia-Ming
Published: May 2015

Breast cancer is a serious health care issue with millions of women being diagnosed worldwide. Although RAS is mutated in only a minor fraction of breast cancers, the Ras signaling pathway is hyperactivated in half of these tumors. Tamoxifen is most commonly used as hormone therapy for breast cancer, which usually accompanied not serious but bothersome side effects. AP-102 and AP-103 are approved drugs in treating diabetes and antipyretic and analgesic, respectively. Repurposing approved drugs for cancer represents an opportunity to improve advance patients therapies based on the new findings and clinical treatment experience via new proposed signaling pathway. In this study, the antitumor efficacy of two drugs was evaluated on the estrogen-dependent ZR-75-1 human breast cancer xenograft model. The tumor-bearing mice were randomly divided into 5 groups and administrated with vehicle, AP-102, AP-103, AP-102 plus AP-103, and paclitaxel, respectively. Results showed that the tumor inhibition rate of AP-102, AP-103, AP-102/AP-103, and paclitaxel were 27.25%, 17.3%, 43.42%, and 14.25%, respectively. Mechanistic analyses revealed that combination of AP-102 and AP-103 is deduced by inhibiting RAF/ERK activation. Moreover, the mice treated with AP-102/AP-103 showed an increase of 36.5% in white blood cell count compared with the mice treated with vehicle. These data posed an apparent anticancer effect in combination of AP-102 and AP-103 with significant difference, which might be associated with innate immunity evidenced by increased WBC number. For in vitro cytotoxicity, ZR-75-1 cells showed more resistant to paclitaxel than MDA-MB-231 cells. This study provides the important information that combination of two drugs, AP-102 and AP-103, might have potential therapeutic use for tumor cells with highly expressed estrogen receptors or breast tumor resistance to chemo drugs.

Citation Format: Pei-Yi Tsai, Yu-Sian Ding, Le-Mei Hung, Yueh-Feng Ho, Ling-Chiu Peng, Chiung-Wen Liou, Jia-Ming Chang. Repurposing of AP-102 and AP-103 in the treatment of breast cancer via inhibiting the RAF/ERK pathway. [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 A39. doi: 10.1158/1557-3125.RASONC14-A39
Abstract A47: Targeting Ras - is there Ras-specific palmitoylation in human cells that can be targeted for cancer treatment?
Authors: Young Evelin C. , Zheng Ze-Yi , Wilkins Angela D. , Jeong Hee-Tae , Li Min , Olivier Lichtarge , Chang Eric C.
Published: May 2015

Ras GTPases act on the plasma membrane (PM) to mediate extracellular signaling and tumorigenesis. PM association for many Ras proteins depends on palmitoylation, which in budding yeast is catalyzed by the Erf2-Erf4 palmitoyl transferase. Despite this, there was no evidence that its human ortholog DHHC9 can similarly control Ras in vivo, and some studies even proclaimed that there was no Ras-specific palmitoylation. We performed an unbiased screen to seek fission yeast (Schizosaccharomyces pombe, sp) mutants with reduced PM Ras. Mutations affecting sp-erf2 to varying degrees were isolated 5 times; in contrast, deleting several other S. pombe palmitoyl transferases did not affect Ras. Sp-Erf2 localizes to the trans-Golgi, mediated by its third transmembrane domain and the Erf4 cofactor. In fission yeast, the human ortholog zDHHC9 fully rescues the phenotypes of sp-erf2 null cells, while expressing zDHHC14, another sp-Erf2-like human protein, did not rescue Ras1 mislocalization in these cells. Importantly, ZDHHC9 is widely overexpressed in human cancers, including several types that do not contain frequent oncogenic RAS mutations. Overexpressing ZDHHC9 in mammalian cells promotes, while repressing it diminishes, Ras PM localization, as well as transformation. These data strongly demonstrate that sp-Erf2/zDHHC9 palmitoylates Ras proteins in a highly selective manner in the trans-Golgi to facilitate PM targeting via the trans Golgi network. zDHHC9 overexpression can apparently increase Ras signaling outputs at the PM, such that tumor formation can occur in many tissues with no oncogenic mutations in RAS genes themselves. zDHHC9 may thus be a good drug target for the treatment of a wide range of cancers.

Citation Format: Evelin C. Young, Ze-Yi Zheng, Angela D. Wilkins, Hee-Tae Jeong, Min Li, Lichtarge Olivier, Eric C. Chang. Targeting Ras - is there Ras-specific palmitoylation in human cells that can be targeted for cancer treatment? [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 A47. doi: 10.1158/1557-3125.RASONC14-A47