bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3000: Defining the subclonal landscape of high-risk neuroblastoma
Authors: Harenza Jo Lynne , Diamond Maura A. , Oldridge Derek A. , Padovan-Merher Olivia , Raman Pichai , Mosse Yael P. , Maris John M.
Published: July 2017

We recently showed that relapsed neuroblastoma (NB) tumors harbor an increased somatic mutational burden, enriched for ALK or RAS-MAPK activating lesions, nominating targeted therapeutic strategies for a condition that is largely incurable. We also showed that these mutations can be present in subclonal tumor cell populations at diagnosis, suggesting that they may contribute to acquired chemoradiotherapy resistance. We hypothesize that subclonal mutations in NB oncogenes are common at diagnosis, are biomarkers for treatment failure, and can be targeted in conjunction with standard chemotherapy to improve patient survival. Here, we focus on the first aspect of this broad hypothesis by presenting the subclonal landscape of diagnostic, high-risk neuroblastomas. A meta-analysis of our previously published primary/relapse (N=23), whole exome (N=232) and whole genome (N=159) diagnostic data, and recent sequencing study of 78 diagnostic and 67 relapse cases were used to inform design of a custom amplicon panel for ultra-deep sequencing. The 28-gene panel covers 57 mutations in frequently mutated genes. Here, 250 high-risk primary tumors were sequenced to an average depth of 50,000X. More subclonal mutations were called (mean=36) compared to clonal alterations (mean=3), demonstrating the complex subclonal architecture in these tumors. We validated five clonal ALK mutations we originally discovered and identified 15 additional pathogenic ALK mutations with mutant allele frequencies (MAFs) ranging from 0.03-23%. Known ALK activating mutations were found at codons 1170, 1174, 1196, 1245, 1275 in 15 of 38 diagnostic primary tumors. This 39.5% frequency is much higher than the 14% frequency defined by us using Sanger or other sequencing methods. Improvement of subclonal mutation detection of actionable, relapse-specific driver mutations demonstrates the clinical utility of utilizing this sequencing methodology at diagnosis to enable timely improvement of outcomes for children with high-risk refractory NB.

Citation Format: Jo Lynne Harenza, Maura A. Diamond, Derek A. Oldridge, Olivia Padovan-Merher, Pichai Raman, Yael P. Mosse, John M. Maris. Defining the subclonal landscape of high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3000. doi:10.1158/1538-7445.AM2017-3000
Abstract 3015: Selective inhibition of mutant KRAS cell and tumor growth by PHT-7.3, an inhibitor of the KRas signaling nanocluster protein Cnk1
Authors: Delaney Roisin , Maruggi Marco , Indarte Martin , Lemos Robert , Grandjean Geoff , Kirkpatrick Lynn , Powis Garth
Published: July 2017

Activating mutations of KRas is the most common proto-oncogenic event in human cancer but there remains no effective therapy for patients harboring mutated KRas (mut-KRas). Despite intense efforts, tight nucleotide binding, few defined pockets, and redundant localization signals have impeded the development of compounds that bind or inhibit KRas. We have identified connector enhancer of kinase suppressor of Ras 1 (Cnk1) as a critical mediator for growth driven by mut-KRas in human cancer cells. Cnk1 co-localizes with mutant KRas at the membrane and deletion of Cnk1 abrogates KRas activation and the activation of the Ras effectors Ral and Rho. Cnk1 deletion caused cells with mutant KRas to accumulate at the G1 checkpoint similar to selective deletion of mutant KRas itself. Following a screen and initial structural optimization a small molecule probe compound PHT-7.3 was identified and shown to bind selectively to the pleckstrin homology PH domain of Cnk1 preventing Cnk1 and mut-KRas co-localization. PHT-7.3 inhibited mut-KRas but not wt-KRas non small cell lung cancer (nsclc) cell growth, and selectively blocks mut-KRas downstream signaling in cells. PHT-7.3 exhibited cytostatic antitumor activity in the mut-KRas(G12S) A549 and mut-KRas(G12V) H441 nsclc xenografts, but not in the wt-KRas H1975 nsclc xenograft. Mut-KRas downstream signaling was inhibited by PHT-7.3 in the xenografts with downregulation of activated Rho and Ral signaling. PHT-7.3 showed further increased antitumor activity in A549 xenografts in combination with erlotinib or trametinib. Thus, the work identifies the PH domain of Cnk1 as a druggable target whose inhibition selectively blocks mutant-KRas activation, and PHT-7.3 as a lead agent in the development of therapies for KRas tumors.

Citation Format: Roisin Delaney, Marco Maruggi, Martin Indarte, Robert Lemos, Geoff Grandjean, Lynn Kirkpatrick, Garth Powis. Selective inhibition of mutant KRAS cell and tumor growth by PHT-7.3, an inhibitor of the KRas signaling nanocluster protein Cnk1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3015. doi:10.1158/1538-7445.AM2017-3015
Abstract 3020: Novel therapeutic targets in head and neck cancer
Authors: Kondratyev Maria , Pesic Aleksandra , Ketela Troy , Sayad Azin , Marastoni Stephano , Virtanen Carl , Ailles Laurie , Samadian Soroush , Bashkurov Mikhail , Koritzinsky Marianne , Wouters Brad
Published: July 2017

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due to frequent recurrences the biology of which remains unclear. Using functional genomic technologies, we identified new therapeutic targets for metastatic disease in HNSCC. Whole genome shRNA screens were conducted in matched sets of cell lines derived from primary tumors and respective metastatic sites, identifying genes essential for cell survival only following metastasis. To test if knockdown of selected targets inhibits metastasis in a therapeutic setting, we established orthotropic model of HNSCC that metastasize to lymph nodes in the mouse. Components of Notch pathway were identified as essential for survival of cells derived from metastatic sites. Whole exome sequencing identified a novel mutation in one of the EGF domains of Notch3 that was acquired only in the metastatic line. Utilizing CRISPR methodology, we established that “fixing” the mutation results in reversal of metastatic phenotype of the cells, making them Notch independent similar to their primary tumor counterparts. Mutations in EGF domains have been reported to influence interaction with specific ligands, dictating which ligand can activate Notch signaling. Our data indicate that a distinct set of target genes is induced upon interaction between Notch3 and Jag2 ligand. Furthermore, our results indicate that suppression of Notch3 improves survival in mice bearing orthotropic tumors derived from the metastatic HNSCC lines. Overall, our data demonstrate that metastatic cells from head and neck tumors acquire dependency on Notch3 signaling. Novel treatments targeting components of this pathway may prove effective in targeting metastatic cells alone or in combination with conventional therapies.

Citation Format: Maria Kondratyev, Aleksandra Pesic, Troy Ketela, Azin Sayad, Stephano Marastoni, Carl Virtanen, Laurie Ailles, Soroush Samadian, Mikhail Bashkurov, Marianne Koritzinsky, Brad Wouters. Novel therapeutic targets in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3020. doi:10.1158/1538-7445.AM2017-3020
Abstract 3021: CCR5 inhibition: macrophage repolarization therapy for colorectal cancer
Authors: Halama Niels , Zoernig Inka , Berthel Anna , Kahlert Christoph , Klupp Fee , Suarez-Carmona Meggy , Brand Karsten , Krauss Juergen , Lasitschka Felix , Ulrich Alexis , Weitz Juergen , Schneider Martin , Buechler Markus , Zitvogel Laurence , Herrmann Thomas , Benner Axel , Kunz Christina , Luecke Stephan , Springfeld Christoph , Falk Christine S. , Jaeger Dirk
Published: July 2017

In patients with metastatic colorectal cancer (CRC), the local immune response influences the clinical course. An in-depth analysis of the invasive margin of human CRC liver metastases revealed a distinct immunological microenvironment. Within this microenvironment, two distinct subsets of myeloid cells induce an influx of T cells into the invasive margin via CXCL9/CXCL10. CCL5 is produced by these T cells and stimulates pro-tumoral effects via CCR5, creating an exploitive loop. CCR5 was found on macrophages, lymphocytes and on the vast majority of tumor cells. Inhibition of CCR5 in patient-derived functional in vitro organotypic culture models showed a promising macrophage repolarization with anti-tumoral effects. These effects are mediated by activation of an antiviral program in macrophages, leading to interferon and reactive oxygen species production and subsequent selective tumor cell death. These anti-tumoral effects were confirmed in a phase I trial with a CCR5 antagonist in 14 patients with liver metastases of advanced refractory CRC. Treatment with the oral CCR5 Inhibitor was very well tolerated and objective responses were seen, especially in combination with previously ineffective chemotherapy. Biopsies revealed mitigation of tumor-promoting inflammation within the tumor tissue, confirming the validity of the explant model and highlighting the feasbility of this approach. It furthermore shows the proof-of-concept for macrophage repolarization in cancer patients.

Citation Format: Niels Halama, Inka Zoernig, Anna Berthel, Christoph Kahlert, Fee Klupp, Meggy Suarez-Carmona, Karsten Brand, Juergen Krauss, Felix Lasitschka, Alexis Ulrich, Juergen Weitz, Martin Schneider, Markus Buechler, Laurence Zitvogel, Thomas Herrmann, Axel Benner, Christina Kunz, Stephan Luecke, Christoph Springfeld, Christine S. Falk, Dirk Jaeger. CCR5 inhibition: macrophage repolarization therapy for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3021. doi:10.1158/1538-7445.AM2017-3021
Abstract 3024: CD4+ outperform CD8+ central memory-derived CAR T cells, mediating persistent antitumor responses and long-term eradication of glioblastoma
Authors: Wang Dongrui , Starr Renate , Aguilar Brenda , Brito Alfonso , Chang Brenda , Sarkissian Aniee , Badie Behnam , Barish Michael E. , Forman Stephen J. , Brown Christine E.
Published: July 2017

Purpose: To compare antitumor effector function mediated by CD4+ and CD8+ chimeric antigen receptor (CAR)-T cells targeting IL13 receptor α2 (IL13Rα2) for treatment of glioblastoma (GBM).

Experimental Design: CD4+ and CD8+ IL13Rα2-specific CAR-T cells, derived from enriched central memory T cells (Tcm), were evaluated for their antitumor potential both in vitro by recursive challenge with GBM cells and in vivo in established orthotopic GBM xenograft models. Further, GBM-stimulated CD4+ and CD8+ CAR-T cells were examined for their exhaustion- and memory-associated phenotypes at transcriptional and protein levels, which would account for their differential antitumor activity.

Results: While CD8+ CAR-T cells mediated robust short-term cytolytic effector function, they became rapidly exhausted and lost antitumor potential after repeated stimulation with tumor cells. CD4+ CAR-T cells, by contrast, persisted and retained effector potency even after repetitive tumor challenge. CD4-mediated cytotoxicity acted through the granzyme B/perforin pathway and was independent of the CD8+ cells. Activated CD4+ CAR-T cells augmented the proliferation of CD8+ cells, but failed to protect the CD8+ cells against stimulation-induced exhaustion. In NSG mice bearing GBM xenografts, CD4+ CAR-T cell treatment led to long-term tumor-free survival, while CD8+ CAR-T cell treated mice showed short-term tumor regression followed by antigen-positive relapse and associated T cell exhaustion. Upon GBM stimulation, CD4+ cells expressed less co-inhibitory receptors and more memory-associated markers than the CD8+ T cell subset. Further, a comprehensive transcriptional analysis revealed that when activated, CD4+ cells retained expression of genes involved in survival- and self-renewal-related pathways, indicating a less exhausted status and a better functional persistence in comparison to the CD8+ cells.

Conclusion: We observed a superior anti-GBM activity mediated by CD4+ CAR-T cells over the CD8+ subset, featured by their ability to maintain long-term immune response and reduce stimulation-induced exhaustion. It is thus inferred that the frequency of CD4+ cells could be an important criteria for evaluating the efficacy of CAR-T cells as immunotherapeutic products.

Citation Format: Dongrui Wang, Renate Starr, Brenda Aguilar, Alfonso Brito, Brenda Chang, Aniee Sarkissian, Behnam Badie, Michael E. Barish, Stephen J. Forman, Christine E. Brown. CD4+ outperform CD8+ central memory-derived CAR T cells, mediating persistent antitumor responses and long-term eradication of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3024. doi:10.1158/1538-7445.AM2017-3024
Abstract 3027: Dissecting mechanisms of anti-PD-1 therapy with massively parallel single-cell RNA-sequencing
Authors: Miller Brian C. , Wadsworth Marc H. , Bi Kevin , Hughes Travis K. , Manguso Robert , Sharpe Arlene H. , Shalek Alex K. , Haining Nicholas
Published: July 2017

Anti-PD-1 therapy is an important new treatment option for many different types of malignancies, but overall response rates are less than 40%. Limited understanding of how anti-PD-1 treatment changes the tumor immune microenvironment is a barrier to identifying rational combination therapies and understanding mechanisms of immunotherapy resistance. To overcome this barrier, we set out to understand the mechanisms by which anti-PD-1 therapy augments the anti-tumor immune response using single-cell genomics. We have developed a massively parallel single-cell RNA-sequencing platform (“Seq-Well”) that uses a fabricated chip with nearly 100,000 nanowells into which barcoded beads and individual cells are distributed prior to lysis and RNA capture. We used this platform to comprehensively define the global expression profile of all major immune lineages in the tumor microenvironment in a mouse tumor model of immunotherapy. Mice were implanted with the B16F10 melanoma transplantable model of cancer and treated with anti-PD-1 or control antibodies. Tumors were harvested and CD45+ tumor-infiltrating leukocytes isolated by FACS. In a single experiment we were able to sequence the transcriptomes of over 600 individual cells, allowing us to clearly distinguish different immune lineages within the tumor microenvironment. We detect two transcriptionally distinct populations of CD8+ T cells, one that is highly proliferative (as marked by Ki-67), and one that has higher expression of cytotoxic markers (i.e. perforin). The Ki-67hi population is enriched for a gene expression signature from terminally exhausted CD8+ T cells in chronic viral infection, suggesting that this is a more exhausted subset. Treatment with anti-PD-1 globally alters the tumor microenvironment, including enriching for CD8+ T cells in the Prfhi subpopulation compared with the Ki-67hi more terminally exhausted population. Studies to understand changes in the immune infiltrate of immunotherapy resistant tumors are currently ongoing. In conclusion, massively parallel single-cell RNA-sequencing allows us to dissect the mechanisms by which checkpoint blockade controls tumor growth, revealing shifts in the differentiation state of exhausted CD8+ T cells induced by checkpoint blockade.

Citation Format: Brian C. Miller, Marc H. Wadsworth, Kevin Bi, Travis K. Hughes, Robert Manguso, Arlene H. Sharpe, Alex K. Shalek, Nicholas Haining. Dissecting mechanisms of anti-PD-1 therapy with massively parallel single-cell RNA-sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3027. doi:10.1158/1538-7445.AM2017-3027
Abstract 3029: An in vivo role for Argonaute 2 in KRAS driven pancreatic cancer
Authors: Shankar Sunita , Tien Jean , Dommeti Vijaya L. , Zelenka-Wang Sylvia , Apel Ingrid A. , Shi Jiaqi , Kumar-Sinha Chandan , Chinnaiyan Arul M.
Published: July 2017

Oncogenic mutations in RAS provide a compelling yet intractable therapeutic target. Using co-immunoprecipitation mass spectrometry, we recently identified an interaction between RAS and Argonaute 2 (AGO2), the core component of the RNA silencing machinery exerting translational control of mRNA transcripts. RAS and AGO2 co-sediment and co-localize in intracellular endomembrane bound organelles. The AGO2 N-terminal domain directly binds the KRAS Switch II region, irrespective of GDP/GTP bound to RAS. Functionally, we observed that AGO2 was required for maximal oncogenic KRAS levels and AGO2 knock-down attenuates cell proliferation in mutant KRAS-dependent cells. Intriguingly, AGO2 mediated microRNA processing (RISC activity) was attenuated in cancer cells expressing mutant KRAS compared to those expressing wild type RAS. Yet, these initial investigations using cell line models do not reveal a clear role for AGO2 in KRAS driven oncogenesis. Therefore, we probed the role of AGO2 in the well-established pancreatic ductal adenocarcinoma (PDAC) mouse model driven by oncogenic KRAS.

Towards this end, we generated LSL-KrasG12D; Pft1a Cre; AGO2flox/flox (KCA) mice. Preliminary analysis (8-10 weeks of age) suggests that compared to LSL-KRASG12D (KC) mice, homozygous loss of AGO2 results in increased KRAS driven Acinar to Ductal Metaplasia (ADM) phenotype considered as precursors of PanIN (pancreatic intraepithelial neoplasia) and PDAC. This mirrors the phenotype of loss of Dicer in the same model further reinforcing a role for microRNAs in restraining KRAS oncogenic programs. Yet unlike Dicer, loss of AGO2 alone did not cause any gross or histological changes in the development of the mouse pancreata. In an effort to understand the underlying mechanisms that are regulated by AGO2, we also studied the activated levels of a variety of signaling molecules in pancreata obtained from different genotypes. Primary analysis indicates that wild type RAS levels are elevated during AGO2 loss and has a bearing on PDAC initiation. Using both Western blot and Immunohistochemical analyses, we demonstrate a previously unknown and critical role for AGO2 in KRAS-MAPK signaling pathway.

Citation Format: Sunita Shankar, Jean Tien, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Ingrid A. Apel, Jiaqi Shi, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An in vivo role for Argonaute 2 in KRAS driven pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3029. doi:10.1158/1538-7445.AM2017-3029
Abstract 3032: Direct regulation of alternative splicing by Smad3 through PCBP1 is essential to the tumor-promoting role of transforming growth factor beta1
Authors: Tripathi Veenu , Sixt Katherine M. , Xu Xuan , Zhang Ying E.
Published: July 2017

In advanced stages of cancers, TGF- β promotes tumor progression in conjunction with inputs from receptor tyrosine kinase pathways. However, the mechanisms that underpin the signaling cooperation and convert TGF- β from a potent growth inhibitor to a tumor promoter are not fully understood. In this work we report that TGF- β directly regulates alternative splicing of cancer stem cell marker CD44 through a phosphorylated T179 of SMAD3-mediated interaction with RNA-binding protein PCBP1. We show that TGF- β and EGF respectively induce SMAD3 and PCBP1 to colocalize in SC35 positive nuclear speckles, and the two proteins interact in the variable exon region of CD44 pre-mRNA to inhibit spliceosome assembly in favor of expressing the mesenchymal isoform CD44s over the epithelial isoform CD44E. We further show that the SMAD3-mediated alternative splicing is essential to the tumor-promoting role of TGF- β and has a global influence on protein products of genes instrumental to epithelial to mesenchymal transition and metastasis.

Citation Format: Veenu Tripathi, Katherine M. Sixt, Xuan Xu, Ying E. Zhang. Direct regulation of alternative splicing by Smad3 through PCBP1 is essential to the tumor-promoting role of transforming growth factor beta1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3032. doi:10.1158/1538-7445.AM2017-3032
Abstract 3034: A programmed ribosomal frameshifting defect potentiates the transforming activity of the JAK2-V617F mutation
Authors: Khan Yousuf A. , Sulima Sergey O. , Kendra Joe , Advani Vivek M. , Jones John E. , Briggs Joseph , Keersmaecker Kim de , Dinman Jonathan D.
Published: July 2017

The JAK-STAT pathway is one of the major mechanisms through which cellular proliferation, differentiation, migration, survival and apoptosis are regulated in response to external stimuli. Promiscuous activation of this pathway is a major driver in the pathogenesis of myeloproliferative neoplasms, often characterized by clonal expansion of mature myeloid cells. The JAK2-V617F allele is the most common and well characterized mutation linked to this class of leukemias. A change of G to T at nucleotide 1849 of the coding sequence (CDS) results in substitution of valine to phenylalanine at codon 617. JAK2-V617F mutant cells have strongly increased activation of JAK-STAT signaling, which can partially be explained by its ability abolish inhibition of kinase activity and increase the Km of the enzyme. It is however unclear if these effects are sufficient to explain the strong activation of the JAK-STAT pathway induced by the V617F mutation. Here, we demonstrate that this mutation destabilizes a programmed -1 ribosomal frameshift (-1 PRF) signal in the JAK2 mRNA, leading to decreased rates of -1 PRF promoted by this element. There is an inverse relationship between -1 PRF and mRNA stability. Thus, decreased -1 PRF results in increased abundance of the JAK2 mRNA and increased JAK2 protein expression. Prior studies established that elevated expression levels of JAK proteins are sufficient to transform cells in vitro, suggesting that -1 PRF may normally help to limit JAK2 expression. Silent protein coding changes in the -1 PRF signal slippery sequence ablating JAK2 frameshifting also increased JAK2 expression and phenocopied the transforming activity of JAK2-V617F in cell culture. Further, the combination of the V617F and slippery site mutations conferred an additive effect on cellular transformation. The presence at least three additional -1 PRF signals in the JAK2 mRNA suggests that -1 PRF plays a significant role in controlling JAK2 expression.

Citation Format: Yousuf A. Khan, Sergey O. Sulima, Joe Kendra, Vivek M. Advani, John E. Jones, Joseph Briggs, Kim de Keersmaecker, Jonathan D. Dinman. A programmed ribosomal frameshifting defect potentiates the transforming activity of the JAK2-V617F mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3034. doi:10.1158/1538-7445.AM2017-3034
Abstract 3050: Polymorphisms in the arntl2 promoter affect breast cancer metastasis susceptibility
Authors: Ha Ngoc-Han , Hunter Kent
Published: July 2017

Breast cancer mortality is primarily due to metastatic lesions rather than primary tumors, yet relatively little is known regarding the mechanisms of metastatic breast cancer, making it difficult to identify patients who are at risk for metastatic disease. Our hypothesis suggests that inherited germline mutations contribute to metastatic disease and that these single nucleotide polymorphisms (SNPs) could be used to predict outcome in breast cancer patients. To investigate the effect of inherited SNPs on metastasis, we used a mouse genetics approach comparing strains with high (FVB) and low (MOLF) metastatic phenotypes and identified Arntl2, a circadian rhythm transcription factor, as a gene whose differential expression predicted outcome in breast cancer patients. To identify SNP differences in Arntl2 between MOLF and FVB, we performed whole genome sequencing of MOLF and compared it to the FVB genome. Overlapping the data with DNase hypersensitivity sites revealed 10 SNPs in the predicted promoter of Arntl2. To test the causative role of the SNPs on Arntl2 expression in vivo, metastatic cell lines were engineered using the CRISPR-Cas9 approach to specifically replace the FVB Arntl2 promoter with that of MOLF. In agreement with our hypothesis, substitution of the MOLF promoter reduced Arntl2 transcript levels and subsequently decreased lung metastases in orthotopic implantation assays. In vitro pulldown experiments with strain-specific promoter probes revealed potential differential binding of chromatin modifier proteins, demonstrating the significance of the SNPs in regulating Arntl2 transcription. Finally, analysis of SNPs associated with Arntl2 expression in a cohort of Chinese breast cancer patients revealed significant correlation of Arntl2 expression with overall survival, validating this gene as a marker in humans. Since Arntl2 is a transcription factor, current studies are focused on identifying Arntl2-regulated genes to investigate downstream pathways involved in metastasis. This study has important implications regarding the role of circadian rhythm in cancer progression and provides a potential mechanism to explain the increased risk of breast cancers in nightshift workers. Furthermore, this provides the first evidence that transcriptional control elements can be engineered using CRISPR-Cas9 to establish the causative role of SNPs in inherited susceptibility to cancer metastasis.

Citation Format: Ngoc-Han Ha, Kent Hunter. Polymorphisms in the arntl2 promoter affect breast cancer metastasis susceptibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3050. doi:10.1158/1538-7445.AM2017-3050
Abstract 3051: Mechanism of early dissemination and metastasis in Her2+ mammary cancer
Authors: Harper Kathryn , Sosa Maria Soledad , Entenberg David , Hosseini Hedayatollah , Cheung Julie , Nobre Rita , Avivar-Valderas Alvaro , Nagi Chandandaneep , Girnius Nomeda , Davis Roger , Farias Eduardo , Condeelis John , Klein Christoph A. , Aguirre-Ghiso Julio A.
Published: July 2017

Metastasis is the leading cause of cancer related deaths and these lesions develop from disseminated cancer cells (DCC) that can remain dormant. Metastasis initiating cells are thought to originate from a subpopulation present in progressed invasive tumors. However, DCCs detected in patients before the manifestation of breast cancer metastasis contain fewer genetic abnormalities than primary tumors or than DCCs from patients with metastases. These findings and those in pancreatic cancer and melanoma models argued that dissemination might occur during early stages of tumor evolution. Yet, the mechanisms that might allow early-disseminated cancer cells (eDCC) to complete all steps of metastasis were unknown. Here we show that in early lesions (EL), before any overt primary tumor (PT) masses are detected, there is a sub-population of Her2+/P-p38lo/P-ATF2lo/TWISThi/E-cadherinlo early cancer cells that are invasive and disseminate to target organs. Intra-vital imaging and organoid studies of early lesions revealed that Her2+ eDCC precursors locally invaded, intravasated and lodged in target organs. Her2+ eDCCs activated a Wnt-dependent EMT-like dissemination program but without complete loss of epithelial phenotype that was reversed by Her2 or Wnt inhibition. Surprisingly, while the majority of eDCCs are TWISThi/E-cadherinlo and dormant, they eventually initiate metastasis. Our work identifies a mechanism for early dissemination whereby Her2 aberrantly activates a program similar to mammary ductal branching that spawns eDCCs capable of forming metastasis after a dormancy phase.

Citation Format: Kathryn Harper, Maria Soledad Sosa, David Entenberg, Hedayatollah Hosseini, Julie Cheung, Rita Nobre, Alvaro Avivar-Valderas, Chandandaneep Nagi, Nomeda Girnius, Roger Davis, Eduardo Farias, John Condeelis, Christoph A. Klein, Julio A. Aguirre-Ghiso. Mechanism of early dissemination and metastasis in Her2+ mammary cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3051. doi:10.1158/1538-7445.AM2017-3051
Abstract 3052: Defining the role of EMT in breast cancer dormancy and metastasis in vivo
Authors: Jung Hae-Yun , Yeung Kay T. , Lee Tiffany , Tsai Jeff H. , Yang Jing
Published: July 2017

Breast cancer patients can develop metastases from dormant tumor cells years after primary tumor resection and treatment. Despite the clinical significance of metastatic latency, the exact cellular and molecular events regulating tumor dormancy remains largely unknown due to challenges in detecting single dormant tumor cells in vivo and characterizing the dormant tumor microenvironment. A developmental program termed Epithelial Mesenchymal Transition (EMT) has been shown to play an important role in tumor cell dissemination, tumor dormancy, and chemoresistance. During EMT, stationary epithelial cells lose their epithelial characteristics, including adherent junctions and apical-basal polarity, and acquire mesenchymal shape and properties. These cells gain the ability to migrate into the circulation, lodge into a distant organ, and form dormant tumor niches. We have previously shown that reversal of EMT is a key driving force in dormant tumor cell proliferation and macrometastasis formation in a skin carcinoma mouse model. Here, we present a novel metastatic breast cancer mouse model in which HER2-driven primary breast tumor cells are labelled with red fluorescent protein with inducible EMT transcription factor TWIST1. We established this mouse model by crossing the TetON-TWIST1 mice with the MMTV-rtTA mice to generate the MMTV-rtTA/TetON-TWIST1 double transgenic mice. To generate HER2-driven breast tumors in these mice, we administered concentrated lentivirus that expresses both the HER2 gene and the tdTomato fluorescent marker under one promoter via intraductal injection. This allows for rapid and synchronized development of primary tumors which recapitulate the formation of sporadic human breast cancer which arises in single cells in otherwise normally developed breast tissue. TWIST1 expression in the luminal layer of the mammary gland was then induced via oral doxycycline. Upon expression of TWIST1, Her2-driven breast tumor cells were found in circulation and in the lungs as micrometastases. We determined that disseminated breast tumor cells mostly remained in dormancy until TWIST expression was switched off by doxycycline withdrawal. We showed that tumor dormancy is partly due to the inability to reverse EMT and regain proliferative capacity. Lastly, using our mouse model to track disseminated breast cancer cells, we will characterize the associated dormant residing niche to further our understanding of tumor dormancy in connection with EMT and immune escape.

Citation Format: Hae-Yun Jung, Kay T. Yeung, Tiffany Lee, Jeff H. Tsai, Jing Yang. Defining the role of EMT in breast cancer dormancy and metastasis in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3052. doi:10.1158/1538-7445.AM2017-3052
Abstract 3053: Stability and stemness of the hybrid epithelial-mesenchymal phenotype
Authors: Jolly Mohit Kumar , Jia Dongya , Tripathi Satyendra C. , Mooney Steve , Celiktas Muge , Hanash Samir M. , Mani Sendurai A. , Pienta Kenneth J. , Ben-Jacob Eshel , Levine Herbert
Published: July 2017

Transitions between epithelial and mesenchymal phenotypes – EMT and MET – are hallmarks of cellular plasticity during embryonic development and cancer metastasis. During these transitions, cells can also adopt a hybrid epithelial/mesenchymal (hybrid E/M) phenotype enabling them to migrate collectively as observed during gastrulation, wound healing, and clusters of Circulating Tumor Cells (CTCs). The hybrid E/M phenotype has largely been tacitly assumed to be 'metastable', i.e. transient state. Here, we integrate mathematical modeling with in vitro experiments to identify certain 'phenotypic stability factors' (PSFs) - GRHL2, OVOL2 and ΔNP63α that can stabilize a hybrid E/M phenotype. We show that H1975 (NSCLC cell line) cells can display a hybrid E/M phenotype stably and migrate collectively, a behavior that is impaired by knockdown of GRHL2 or OVOL2. Further, our computational model predicts that these PSFs can also associate hybrid E/M phenotype with high tumor-initiating potential, a prediction strengthened by the observation that the higher levels of one or more of these PSFs may predict poor patient outcome. Overall, our results suggest that a hybrid E/M phenotype need not be 'metastable', and bolster the notion that a hybrid E/M phenotype, but not necessarily full EMT, associates with aggressive tumor progression.

Citation Format: Mohit Kumar Jolly, Dongya Jia, Satyendra C. Tripathi, Steve Mooney, Muge Celiktas, Samir M. Hanash, Sendurai A. Mani, Kenneth J. Pienta, Eshel Ben-Jacob, Herbert Levine. Stability and stemness of the hybrid epithelial-mesenchymal phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3053. doi:10.1158/1538-7445.AM2017-3053
Abstract 3055: A permanent lung imaging window reveals, for the first time, the steps of extravasation, seeding and growth of early metastatic dissemination
Authors: Voiculescu Sonia E. , Wang Yarong , Oktay Maja , Condeelis John , Entenberg David
Published: July 2017

Metastatic disease is the major cause of cancer mortality and is responsible for over 1/2 million deaths each year in the U.S. alone. Of the three most common sites of metastasis observed clinically (bone, lung and liver), the most difficult to study using intravital microscopy is the lung; a delicate organ in perpetual motion. We have recently developed and validated an implantable, permanent, lung imaging window which allows high-resolution multiphoton imaging of the intact, breathing (without ventilation), murine lung over days to weeks of repeated imaging. This window does not use vacuum to immobilize the lung tissue thereby avoiding well know artifacts associated with vacuum lung windows. Using our new window, we have been able to visualize, with single cell resolution, the steps of metastasis in a clinically relevant, spontaneously-metastasizing murine breast cancer model. We present the first direct visualization of tumor cell arrival, extravasation, and progression to micro-metastases. Further, we have observed, for the first time, the activity of the tripartite structure called Tumor MicroEnvironment of Metastasis (TMEM) in metastatic lung lesions directly demonstrating the mechanism of dissemination from metastatic lung tumors. Using this approach to imaging live lung tissue serially, within a single animal, we are investigating the mechanisms underlying the fate of tumor cells arriving in the lung with the ultimate goal of directly identifying signals behind seeding and survival of metastatic tumor cells and their responses to interventions in real time.

Citation Format: Sonia E. Voiculescu, Yarong Wang, Maja Oktay, John Condeelis, David Entenberg. A permanent lung imaging window reveals, for the first time, the steps of extravasation, seeding and growth of early metastatic dissemination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3055. doi:10.1158/1538-7445.AM2017-3055
Abstract 3056: Genetic deletion of insulin-like growth factor binding protein-7 (IGFBP7) promotes hepatocellular carcinoma (HCC): a novel role of IGFBP7 in regulating antitumor immune surveillance
Authors: Akiel Maaged A. , Guo Chunqing , Li Xia , Rajasekaran Devaraja , Mendoza Rachel G. , Jariwala Nidhi H. , Robertson Chadia L. , Subler Mark A. , Windle Jolene J. , Fisher Paul B. , Sarkar Devanand
Published: July 2017

In the US, the incidence and mortality rates of hepatocellular carcinoma (HCC) are alarmingly increasing since no effective therapy is available for the advanced disease. Activation of IGF signaling is a major oncogenic event in diverse cancers, including HCC. Insulin-like growth factor binding protein-7 (IGFBP7) inhibits IGF signaling by binding to IGF-1 receptor (IGF-1R) and functions as a potential tumor suppressor for hepatocellular carcinoma (HCC). IGFBP7 abrogates tumors by inducing cancer-specific senescence and apoptosis and inhibiting angiogenesis. We now document that Igfbp7 knockout (Igfbp7-/-) mouse shows constitutive activation of IGF signaling, presents with pro-inflammatory and immunosuppressive microenvironment, and develops spontaneous tumors in lungs and liver and markedly accelerated carcinogen-induced HCC. Loss of Igfbp7 resulted in increased proliferation and decreased senescence in hepatocytes and mouse embryonic fibroblasts that could be blocked by an IGF-1 receptor inhibitor. A significant inhibition of genes regulating immune surveillance was observed in Igfbp7-/- livers which was associated with marked inhibition in antigen cross-presentation by Igfbp7-/- dendritic cells. IGFBP7 overexpression inhibited growth of HCC cells in syngeneic immunocompetent mice which could be abolished by depletion of CD4+ or CD8+ T lymphocytes. Our studies unravel modulation of immune response as a novel component of pleiotropic mechanisms by which IGFBP7 suppresses HCC. Even though HCC has an immunosuppressive milieu, immune targeted therapies are beginning to demonstrate significant objective responses in clinical trials. IGFBP7 might be an effective anti-HCC therapeutic by directly inhibiting cancer cells and stimulating an anti-tumor immune response.

Citation Format: Maaged A. Akiel, Chunqing Guo, Xia Li, Devaraja Rajasekaran, Rachel G. Mendoza, Nidhi H. Jariwala, Chadia L. Robertson, Mark A. Subler, Jolene J. Windle, Paul B. Fisher, Devanand Sarkar. Genetic deletion of insulin-like growth factor binding protein-7 (IGFBP7) promotes hepatocellular carcinoma (HCC): a novel role of IGFBP7 in regulating antitumor immune surveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3056. doi:10.1158/1538-7445.AM2017-3056
Abstract 3062: Metformin inhibits TGFβ-induced stromal ECM remodeling to impede invasion in ovarian cancer
Authors: Hart Peter C. , Sheikh Shermeen , Lengyel Ernst , Romero Iris
Published: July 2017

Recently, our group has elucidated that metformin, the commonly used medication for type 2 diabetes, has anti-ovarian cancer (OvCa) effects in preclinical models and use of the drug is associated with improved OvCa survival in retrospective epidemiological studies. While the cytotoxic effects of metformin in ovarian cancer cell lines is well demonstrated, the impact of metformin on tumor-stromal interactions in the tumor microenvironment is not well defined. Currently, our work has focused on the impact of metformin on the tumor microenvironment and cancer cell behavior in physiologic models of tumor metastasis. Using a 3D organotypic model, we demonstrate that OvCa cell invasion is inhibited by metformin. In evaluating the molecular mechanism mediating this effect we show that OvCa cells co-cultured with stromal cells or stimulated with TGFβ1 upregulate the production of the extracellular matrix (ECM) proteins fibronectin and collagen 1 and that this effect is attenuated by metformin. Interestingly, metformin’s inhibition of ECM production was not dependent on AMP-activated kinase (AMPK), a common metformin target. To expand on this finding we performed label-free shotgun proteomics and identified collagens, fibronectin and TGFBR2 to be among the proteins most highly repressed in response to metformin exposure in OvCa cells. Finally, we identified that this repression of ECM remodeling by metformin was associated with an overall reduction in TGFBR2 expression and consequent SMAD/STAT3 activity. Taken together, our data indicate that metformin may have novel targets in the tumor microenvironment where it targets pro-tumorigenic effects of the stromal compartment by repressing TGFβ-dependent stromal cell ECM secretion, thereby inhibiting ovarian cancer invasion and metastasis.

Citation Format: Peter C. Hart, Shermeen Sheikh, Ernst Lengyel, Iris Romero. Metformin inhibits TGFβ-induced stromal ECM remodeling to impede invasion in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3062. doi:10.1158/1538-7445.AM2017-3062
Abstract 3063: Predicting efficacy of c-Met targeting therapy in autocrine and paracrine tumor cell lines
Authors: Hughes Veronica S. , Siemann Dietmar W.
Published: July 2017

Tumor cells frequently harbor abnormalities in signaling pathways, leading to increased migration, invasion, survival, angiogenesis, and proliferation. C-Met is one such commonly aberrant pathway. Because of its effects on tumor cells, c-Met has been a target of interest for anti-metastatic therapies. Clinical trials of c-Met inhibition select patients based on total Met expression, or amplification of the Met gene. However, c-Met inhibitors target the activated pathway. We therefore predict that phospho-Met will be a better marker for patient sensitivity to, and hence selection for, c-Met inhibitor trials. C-Met is activated by the endogenous ligand Hepatocyte Growth Factor (HGF). In order to mimic the paracrine signaling during in vitro experiments, many studies add exogenous HGF at concentrations of 25-50 ng/mL. However, these concentrations may not be representative of in vivo conditions. HGF serum levels of healthy humans is 0.4 ng/mL. While HGF levels do rise in cancer patients, it is unusual to see serum levels higher than 1 ng/mL. Conditioned media from several cell lines (MDA-MB-231, PC-3, DU145, LNCaP, OS156, OS521, U87, U118, RKO, KHT, SCC7, and RIF) was collected to analyze for secretion of HGF in conditioned media. These cells were then examined for basal phosphorylation of Met. The cell lines that secreted HGF also had higher levels of phospho-Met in comparison to non-HGF-secreting cell lines. Importantly, levels of total Met did not predict for levels of phospho-Met. In migration and invasion assays, the HGF-secreting cell line KHT was found to be sensitive to the c-Met inhibitor BMS-777607.The non-HGF-secreting cell line DU145 only became sensitive to the inhibitor when high, non-physiologic exogenous levels of HGF (25-50 ng/mL) were added.These results suggest that autocrine activation of the c-Met pathway may be a factor in predicting sensitivity to c-Met inhibition. This may indicate the existence of a subset of patients most likely respond to c-Met inhibition therapy. Furthermore, consideration of exogenous HGF concentration should be considered when conducting preclinical trials of c-Met inhibitors, as addition of exogenous HGF may change the apparent efficacy of the inhibitor.

Citation Format: Veronica S. Hughes, Dietmar W. Siemann. Predicting efficacy of c-Met targeting therapy in autocrine and paracrine tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3063. doi:10.1158/1538-7445.AM2017-3063
Abstract 3066: Inhibition of hsp90 by auy922 preferentially kills mutant KRAS colon cancer cells by activating Bim through ER stress
Authors: Wang Chun Yan , Guo Su Tang , Wang Jia Yu , Yan Xu Guang , Farrelly Margaret , Zhang Yuan Yuan , Liu Fen , Yari Hamed , La Ting , Lei Fu Xi , Jin Lei , Jiang Chen Chen , Zhang Xu Dong
Published: July 2017

Colon cancer is one of the most common and deadly malignancies (1). Despite recent advances in early diagnosis and the development of molecularly targeted therapy, the overall survival of patients with metastatic colon cancers remains disappointing (1). This is often associated with resistance of colon cancer cells to systemic therapies resulting from oncogenic mutations of KRAS that drive activation of multiple downstream signalling pathways important for cell survival and proliferation. Here we report that mutant KRAS colon cancer cells are nevertheless more susceptible to apoptosis induced by the heat shock protein 90 (HSP90) inhibitor AUY922 than those carrying wild-type KRAS. Although AUY922 inhibited HSP90 activity with the comparable potency in colon cancer cells irrespective of their KRAS mutational statuses, those with activating mutations of KRAS were markedly more sensitive to AUY922-induced apoptosis. This was associated with upregulation of the BH3 only proteins Bim, Bik, and PUMA. However, only Bim appeared essential, in that knockdown of Bim abolished, whereas knockdown of Bik or PUMA only moderately attenuated apoptosis induced by AUY922. Mechanistic investigations revealed that endoplasmic reticulum (ER) stress was responsible for AUY922-induced upregulation of Bim, which was inhibited by a chemical chaperone or overexpression of GRP78. Conversely, siRNA knockdown of GRP78 or XBP-1 enhanced AUY922-induced apoptosis. In addition, AUY922 inhibited the growth of mutant KRAS colon cancer xenografts through activation of Bim that was similarly associated with ER stress. Taken together, these results suggest that AUY922 is a promising drug in the treatment of mutant KRAS colon cancers, and that agents that enhance the apoptosis-inducing potential of Bim may be useful to improve the therapeutic efficacy.

Citation Format: Chun Yan Wang, Su Tang Guo, Jia Yu Wang, Xu Guang Yan, Margaret Farrelly, Yuan Yuan Zhang, Fen Liu, Hamed Yari, Ting La, Fu Xi Lei, Lei Jin, Chen Chen Jiang, Xu Dong Zhang. Inhibition of hsp90 by auy922 preferentially kills mutant KRAS colon cancer cells by activating Bim through ER stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3066. doi:10.1158/1538-7445.AM2017-3066