bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3651: Natural killer cells eradicate galectin-1 deficient glioma in the absence of adaptive immunity
Authors: Baker Gregory J. , Yadav Viveka Nand , Chockley Peter , Doherty Robert , Ritt Michael , Sivaramakrishnan Sivaraj , Castro Maria G. , Lowenstein Pedro R.
Published: May 2015

Natural killer (NK) cells safeguard against early tumor formation by seeking out and destroying transformed target cells in a process referred to as NK immunosurveillance. While it is clear that malignant brain tumors such as glioblastoma (GBM) evade NK-mediated tumor suppression, the precise mechanisms by which this occurs remain unknown. We now show that shRNA-mediated knockdown of the β-galactoside-binding lectin, galectin-1 (gal-1), in malignant glioma cells leads to the failure to form lethal intracranial tumors in RAG1-/- mice, a mouse strain devoid of adaptive immunity. However, gal-1 deficient glioma growth is fully restored on implantation into the brain of severely immunocompromised NOD-scid IL2Rg null mice, which lack both adaptive and innate immune function, thus implicating the innate immune response in the early rejection of gal-1 deficient glioma. Immunodepletion of NK cells in RAG1-/- or C57BL/6J mice using anti-asialo GM1 or anti-NK1.1 antibodies permit the growth of large gal-1 deficient gliomas, while macrophage depletion with clodronate liposomes only permits limited tumor growth. This combined result suggests that NK cells and macrophages may work together to achieve gal-1 deficient glioma rejection. Antigen-specific IFN-γ ELISpot assays using splenocytes from immunocompetent C57BL/6J mice indicate that gal-1 deficient glioma is cleared prior to the onset of an adaptive anti-tumor immune response. Flow cytometric analysis of brain tumor-infiltrating immune cells reveal that gal-1 deficient gliomas contain significantly more macrophages and granzyme B+ NK cells compared to gal-1 expressing gliomas. In-vitro experiments further show that gal-1 deficient glioma cells are inherently over 3-times more sensitive to NK-mediated tumor lysis, fail to suppress pro-inflammatory (M1) microglial activation, and secrete pro-inflammatory cytokines IL-1β, IL-12p70, and CXCL2. We conclude that glioma-derived gal-1 is a powerful inhibitor of NK-mediated cytotoxicity in-vivo, and predict that its suppression will be of therapeutic value in the treatment of human malignant brain tumors by dramatically heightening anti-tumor NK immunosurveillance.

Citation Format: Gregory J. Baker, Viveka Nand Yadav, Peter Chockley, Robert Doherty, Michael Ritt, Sivaraj Sivaramakrishnan, Maria G. Castro, Pedro R. Lowenstein. Natural killer cells eradicate galectin-1 deficient glioma in the absence of adaptive immunity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3651. doi:10.1158/1538-7445.AM2014-3651
Abstract 3652: Surface expression and shedding of NKG2D ligands by (metastasizing) tumor cells is altered by platelets leading to impaired NK cell immunosurveillance
Authors: Raab Stefanie , Kropp Korbinian Nepomuk , Steinle Alexander , Klein Gerd , Kopp Hans-Georg , Salih Helmut R.
Published: May 2015

NK cells play an important role in anti-tumor immunity. The mechanisms leading to NK cell activation are described by the ‘missing-self’ and ‘induced-self' hypotheses, implying that cells with low or absent expression of MHC class I and stress-induced expression of ligands for activating receptors such as NKG2D (NKG2DL), are targeted by NK cells. Besides the direct interaction with their target cells, NK activity is also influenced by various other hematopoietic cells. In mouse models, thrombocytopenia impairs metastasis, and this is reversed by additional depletion of NK cells. Hence, the knowledge of the underlying molecular mechanisms by which platelets influence NK cells is still fragmentary. We already showed that release of TGF-β by platelets upon their interaction with (metastasizing) tumor cells downmodulates the activating receptor NKG2D on NK cells (Kopp et al, Cancer Res. 2009; Placke, J Innate Immun. 2011). Moreover, platelets transfer ´healthy´ MHC class I to the tumor cell surface. Thus, platelets may facilitate metastasis by interfering with both, ‘induced and missing self' NK cell recognition. Here we provide evidence for a yet unknown mechanism by which platelets further impair NKG2D-mediated immunosurveillance. Tumor cells were incubated with platelets from healthy donors resulting in coating of tumor cells and activation of the platelets, or treated with platelet-derived soluble factors (releasate) obtained either by tumor cell-induced platelet activation (TCIPA) or the platelet agonist thrombin. Presence of platelet derived factors derived either from coating of tumor cells or contained in platelet releasate substantially reduced surface expression of NKG2DL on tumor cells. This was paralleled by enhanced levels of soluble NKG2DL in culture supernatants, indicating that platelet-derived factors mediate NKG2DL shedding from the tumor cell surface. Diminished NKG2DL surface expression resulted in diminished NKG2D-dependent natural cytotoxicity of NK cells, as revealed by blocking experiments using NKG2D antibody and NKG2DL-specific F(ab)2 fragments targeting the specifically modulated NKG2DL. Our data thus identify induction of NKG2DL shedding as novel mechanism by which interaction of platelets with metastasizing tumor cells impairs NK cell immunosurveillance.

Citation Format: Stefanie Raab, Korbinian Nepomuk Kropp, Alexander Steinle, Gerd Klein, Hans-Georg Kopp, Helmut R. Salih. Surface expression and shedding of NKG2D ligands by (metastasizing) tumor cells is altered by platelets leading to impaired NK cell immunosurveillance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3652. doi:10.1158/1538-7445.AM2014-3652
Abstract 3669: IDH mutant glial cell resistance to natural killer cell cytotoxicity
Authors: Zhang Xiaoran , Chang Yigang , Li Aofei , Glorioso Joseph C. , Grandi Paola , Lotze Mike , Amankulor Nduka
Published: May 2015

Mutations in IDH 1 and 2 are common in gliomas. Methylation-related epigenetic repression of gene expression is a feature of IDH mutant (IDHmut) tumors. Epigenetic repression of NK cell ligands is a common occurrence in cancer, but it is unknown if this occurs in gliomas. Here, we demonstrate transcriptional repression of NKG2D activating ligands (NKG2DLs) in IDHmut tumors, and correlate this with decreased susceptibility to natural killer (NK) cell-mediated cytolysis in vitro We compared IDHmut and IDHwt gene expression values for annotated immune response genes and examined NKG2DL HM450 mean methylation values in glioma patients using TCGA data. cDNA was synthesized from cultured IDHwt or IDHmut astrocytes. NKG2DL expression was assayed using primers for NKG2D ligands ULBP1, ULBP2, ULBP3 and RAET1G. IDHwt astrocytes, U87 cells and IDHmut astrocytes were cultured with activated NK cells. Cytotoxicity was measured by flow cytometry. mRNA expression values for NKG2DLs ULBP1 and ULBP3 were significantly reduced in IDHmut gliomas. Conversely, methylation values were significantly increased. NKG2DL hierarchical clustering of gene expression of shows genotype-based expression clusters. IDHmt astrocytes expressed a two to eight-fold reduction in the relative gene expression of NKG2DLs compared with IDHwt astrocyte. Cytotoxicity is significantly lower in IDHmut astrocytes/NK cell co-cultures compared with IDHmut/NK or U87/NK co-cultures. Our data demonstrates evidence for the transcriptional repression of NKG2DLs in IDHmut gliomas in silico and in vitro. Reduced NKG2D levels in IDHmut cells correlates with impaired NK-mediated cytotoxicity and suggests a role for NK cell resistance in IDH mutant tumors. Further studies are in progress to utilize HSV vectors that express ULBP3 to determine whether restoration of ULBP3 expression in cancer cells will enhance susceptibility to NK activation and tumor cell killing. Results of these experiments will be reported.

Citation Format: Xiaoran Zhang, Yigang Chang, Aofei Li, Joseph C. Glorioso, Paola Grandi, Mike Lotze, Nduka Amankulor. IDH mutant glial cell resistance to natural killer cell cytotoxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3669. doi:10.1158/1538-7445.AM2014-3669
Abstract 3671: Myeloid-derived suppressive cells require education from tumor-evoked Bregs to mediate metastasis
Authors: Bodogai Monica , Chang Catalina Lee , Biragyn Arya
Published: May 2015

Myeloid-derived suppressive cells (MDSC) are key metastasis-promoting regulatory cells. They are drastically expanded in WT BALB/c mice with highly aggressive 4T1 cancer and thought to promote metastasis by inactivating antitumor effector immune cells. Surprisingly, despite comparable expansion of MDSC, in B-cell deficient congenic mice 4T1 cancer fails to metastasize to lungs, questioning the importance of MDSC. We found that this is due to the lack of a new subset of tumor-evoked regulatory B cells (tBregs) 1,2 that induces metastasis-promoting regulatory T cells (Tregs) 3. The mechanism of this process is that cancer initiates a chain of metastasis-promoting events, such as converts normal B cells into tBregs utilizing metabolites of 5-lipooxygenase, which in turn use TGFβ to generate FoxP3+ Tregs from non-regulatory CD4+ T cells. The Tregs then inactivate effector NK and CD8+ T cells that kill the metastasizing cancer cells in the lungs. To reconcile these results with the reported importance of MDSC, we speculated that metastasis-promoting activity of MDSC requires tBregs. Here we report that tBregs educate a small subset of MDSC rendering them regulatory. As a result, the ability of 4T1 cancer to metastasize in B-cell KO mice can be restored by the adoptive transfer of the regulatory subset of MDSC from tumor-bearing WT mice. In tumor-bearing B-cell KO mice, however, tBregs are not generated and thus MDSC from these mice cannot support lung metastasis. Overall, these results further underscore the importance of tBregs by uncovering a previously unknown regulatory step that MDSC can only promote metastasis if educated by cancer-induced tBregs.

This work was supported by the National Institute on Aging, the Intramural Research Program

1 Olkhanud, P. B. et al. Tumor-evoked regulatory B cells promote breast cancer metastasis by converting resting CD4 T cells to T-regulatory cells. Cancer research 71, 3505-3515, doi:10.1158/0008-5472.CAN-10-4316 (2011).

2 Bodogai, M. et al. Anti-CD20 antibody promotes cancer escape via enrichment of tumor-evoked regulatory B cells expressing low levels of CD20 and CD137L. Cancer research, doi:10.1158/0008-5472.CAN-12-4184 (2013).

3 Olkhanud, P. B. et al. Breast cancer lung metastasis requires expression of chemokine receptor CCR4 and regulatory T cells. Cancer Res. 69, 5996-6004 (2009).

Citation Format: Monica Bodogai, Catalina Lee Chang, Arya Biragyn. Myeloid-derived suppressive cells require education from tumor-evoked Bregs to mediate metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3671. doi:10.1158/1538-7445.AM2014-3671
Abstract 3689: Minocycline regulates hypoxia-inducible factor-1α expression through the induction of its degradation in ovarian cancer: in vitro and in vivo studies
Authors: Ataie-Kachoie Parvin , Morris David L. , Pourgholami Mohammad H.
Published: May 2015

Experimental data suggest therapeutic advantage from selective disruption of the hypoxia response in cancer. We recently found that minocycline decreases tumor VEGF expression in ovarian tumor xenografts in mice and herein report a companion laboratory study to test if this effect was the result of hypoxia-inducible factor (HIF) inhibition. Human ovarian carcinoma cell lines (OVCAR-3, SKOV-3 and A2780) were treated with minocycline in normoxic and hypoxic conditions and also in the presence of hypoxia-mimetic agent deferoxamine in vitro. Two different in vivo experiments investigated miocycline effects after single dose (30 mg/kg, 4 or 24 h) or longer exposure in nude mice bearing OVCAR-3 xenografts. Treatment with minocycline inhibited the accumulation of HIF-1alpha in a concentration-dependent fashion in vitro. A similar effect of minocycline was seen in vivo. Furthermore, the inhibtory effect of minocycline on HIF-1alpha was found to be associated with a significant decrease in HIF-1alpha half-life showing that minocycline enhances degradtion of HIF-1alpha. These data warrents further evaluation of minocycline in the treatment of ovarian cancer.

Citation Format: Parvin Ataie-Kachoie, David L. Morris, Mohammad H. Pourgholami. Minocycline regulates hypoxia-inducible factor-1α expression through the induction of its degradation in ovarian cancer: in vitro and in vivo studies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3689. doi:10.1158/1538-7445.AM2014-3689
Abstract 369: Network-based stratification of thyroid cancer
Authors: Hofree Matan , Shen John Paul Y. , Ideker Trey
Published: May 2015

Many forms of cancer consist of multiple subtypes with different molecular causes and clinical outcomes. Somatic tumor genomes provide a rich new source of data for uncovering these subtypes, but have proven difficult to compare as two tumors share little in their genetic landscapes. Recently, we have introduced a method called ‘network-based stratification’ (NBS) which integrates somatic tumor genomes with gene networks. This approach allows for stratification of cancer into informative subtypes by clustering together patients who have mutations within similar network regions. Here we apply NBS to a combination of whole-exome somatic mutation data, copy number aberrations and gene fusion events in a cohort of thyroid cancer (TC) patients, an as yet unpublished part of The Cancer Genome Atlas (TCGA). Using NBS combined with the HumanNet gene interaction network we were able to discover a robust cluster assignment of thyroid patients into three subtypes. These subtypes are found to significantly associate with histological type (X2 P = 5.42x10-22) and are also found to be predictive multiple clinical variables such as predicted risk of recurrence (X2 P = 9.95x10-6, estimated based on standard (TNM) staging by AJCC Cancer Staging Manual. 7th ed.), extra-thyroidal expansion(X2 P = 4.68x10-7), lymph node status (pathological N stage, X2 P = 9.25x10-4), and pathological T stage (X2 P = 4.46x10-3). In each subtype we highlight the characteristically perturbed genes and subnetworks (Table 1) and further explore the association of mRNA and protein expression to these subtypes. The study demonstrates the utility of combining genomic profiles with interaction networks, enabling the discovery of clinically meaningful mutation based subtypes and provides genetic based subtypes with potential clinical implications.

Characteristically perturbed genes and subnetworksTypeCharacteristic histologyFrequent mutationSelected enriched terms in frequently perturbed subnetworks1FollicularCHEK2, CTNND2, DICER1, HRAS, KRAS, MUC5B, MYH13, NRAS, NTRK1, PAX8, PPARG, PTEN, SLC12A4, SLITRK3, TG, ZBTB22Ras protein signal transduction, Activation of MAPK activity2ClassicCCDC6, ETV6, MALAT1, NCOA4, NTRK3, RETProtein tyrosine kinase activity, Transmembrane receptor protein kinase activity, Regulation of blood vessel size3Classic and Tall cellBRAFStress activated MAPK cascade, Immune response-regulating signaling pathway, FGR signaling pathway, JACK-STAT cascade, activation of pro-apoptotic gene products

Citation Format: Matan Hofree, John Paul Y. Shen, Trey Ideker. Network-based stratification of thyroid cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 369. doi:10.1158/1538-7445.AM2014-369
Abstract 3690: MMP-1 and pro-MMP-10 as potential urinary pharmacodynamic biomarkers of FGFR3-targeted therapy in patients with bladder cancer
Authors: Lin Benjamin C. , Du Xiangnan , Wang Qian-Rena , Li Hao , Ingalla Ellen , Tien Janet , Rooney Isabelle , Ashkenazi Avi , Penuel Elicia , Qing Jing
Published: May 2015

Aberrant activation of fibroblast growth factor receptor 3 (FGFR3) via overexpression or mutation occurs frequently in bladder cancer, and experimental therapeutic agents have entered into clinical trials to evaluate the potential activity of FGFR3-targeted therapy in this disease. However, a convenient accessible biomarker indicative of targeted pathway modulation in vivo drug activity is urgently needed. Through transcriptional profiling of bladder carcinoma cells expressing inducible FGFR3 short hairpin RNAs, we identified two matrix metalloproteinases (MMPs), MMP-1 and MMP-10, as potential downstream targets of FGFR3. We found that FGFR3 signaling promotes expression and secretion of MMP-1 and pro-MMP-10 in a MEK-dependent fashion. FGFR3 inhibition by an investigational anti-FGFR3 human monoclonal antibody, R3Mab, in bladder cancer xenograft models substantially blocked tumor progression and reduced the protein levels of human MMP-1 and pro-MMP-10 in tumor tissues as well as in mouse serum. Furthermore, we detected elevated levels of both MMP-1 and pro-MMP-10 in the urine of patients with advanced bladder cancer. In a phase 1 dose-escalation trial, R3Mab administration resulted in an acute reduction of urinary MMP-1 and pro-MMP-10 levels in bladder cancer patients. Together, these findings reveal a critical role of FGFR3 in regulating MMP-1 and pro-MMP-10 expression and secretion, and identify urinary MMP-1 and pro-MMP-10 as potential pharmacodynamic biomarkers for FGFR3-targeted therapy in bladder cancer.

Citation Format: Benjamin C. Lin, Xiangnan Du, Qian-Rena Wang, Hao Li, Ellen Ingalla, Janet Tien, Isabelle Rooney, Avi Ashkenazi, Elicia Penuel, Jing Qing. MMP-1 and pro-MMP-10 as potential urinary pharmacodynamic biomarkers of FGFR3-targeted therapy in patients with bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3690. doi:10.1158/1538-7445.AM2014-3690
Abstract 3692: Preclinical studies of a mutant p53 reactivating drug in pancreatic cancer
Authors: Yu Xin , Li Zhe , Eng Oliver S. , Tsang Ashley T. , Lin Hongxia , Gounder Murugesan , Carpizo Darren R.
Published: May 2015

Pancreatic cancer therapy suffers from a lack of effective chemotherapy. TP53 is second only to Kras as the most commonly mutated gene in pancreatic cancer with point mutations occurring in 75% of patients. Recently, our laboratory identified NSC319726 (726) as an allele specific p53 mutant reactivating compound (Yu, et al. 2012, Cancer Cell). This compound selectively kills cancer cells that express the p53-R175H mutation by restoring wildtype structure and function to this mutant. It does not reactivate the DNA contact mutant, p53-R273H or p53-R248H. This established 726 as a lead compound for mutant p53 targeted drug development. We are currently investigating the potential of 726 as novel anti-cancer drug through pre-clinical studies in pancreatic cancer. We have investigated the pharmacokinetics (PK) and pharmacodynamics (PD) of 726 in murine pancreatic cancer cell lines derived from the KPC mouse model expressing mutant KrasG12D and different alleles of TP53 (WT, null, p53R172H, p53R270H). Using LC/MS/MS we developed a sensitive assay for detection of 726 in mouse plasma. PK studies indicate a half-life of approximately 4 hours with an AUC of 199.78 ng/ml/h for a single 1mg/kg dose given Intravenously. Treatment of KPC cell lines with 726 indicates that the murine p53R172H protein is reactivated as indicated by loss of mutant conformation and induction of p53 target gene transcription. Cell growth inhibition assays reveal that the cell line KPCp53-R172H is sensitive to 726 while the KPCp53-R270H is not. We have developed a PD assay for 726 consisting of cleaved caspase-3 immunostaining and measurement of transcriptional levels of p53 targets PUMA, Noxa and p21. Using these assays we have tested several doses of 726 and found that we detect activity of 726 in KPCp53-R172H xenograft tumors while at the same time relatively no activity in KPCp53-R270H tumors. These same assays were also performed on KPC autochthanous tumors and again reveal activity of 726 in the KPCp53-R172H mice. Efficacy studies in the KPCp53-R172H xenografts reveal potent tumor growth inhibition at both IV (1mg/kg) and IP (5mg/kg) dosing while no evidence of growth inhibition in KPCp53-R270H. These studies confirm that 726 is an allele-specific p53 mutant reactivating compound and holds promise for future applications in pancreatic cancer.

Citation Format: Xin Yu, Zhe Li, Oliver S. Eng, Ashley T. Tsang, Hongxia Lin, Murugesan Gounder, Darren R. Carpizo. Preclinical studies of a mutant p53 reactivating drug in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3692. doi:10.1158/1538-7445.AM2014-3692
Abstract 3703: PDGFRα up-regulation mediated by Sonic Hedgehog Pathway activation leads to BRAF inhibitor resistance in melanoma cells with BRAF mutation
Authors: Sabbatino Francesco , Wang Yangyang , Wang Xinhui , Flaherty Keith T. , Pepin David , Scognamiglio Giosuè , Yu Ling , Cooper Zachary A. , Pepe Stefano , Kirkwood John M. , Frederick Dennie T. , Wargo Jennifer A. , Ferrone Soldano , Ferrone Cristina R.
Published: May 2015

Control of mutant BRAF(V600E) metastatic melanoma by BRAF inhibitor (BRAF-I) is limited by intrinsic and acquired resistance. Up-regulation of growth factor receptor such as platelet-derived growth factor receptor (PDGFR)β or insulin-like growth factor receptor (IGFR) is among the mechanisms underlying BRAF-I resistance of melanoma cells. Here we demonstrate for the first time that up-regulation of PDGFRα, a growth factor receptor which markedly differs in its functional properties from its family member PDGFRβ, causes BRAF-I resistance in vitro and in vivo. PDGFRα inhibition by PDGFRα-specific short hairpin RNA (shRNAs) and by clinically available PDGFRα inhibitors (sunitinib, imatinib and crenolanib) restores and increases melanoma cells' sensitivity to BRAF-I in vitro and in vivo. This effect reflects the inhibition of ERK and AKT activation which is associated with BRAF-I resistance of melanoma cells. PDGFRα up-regulation is mediated by Sonic Hedgehog Homolog (Shh) pathway activation which is induced by BRAF-I treatment. Similarly to PDGFRα inhibition, Shh inhibition by the novel small molecule LDE225 restores and increases melanoma cells' sensitivity to BRAF-I in vitro and in vivo. These effects are mediated by PDGFRα down-regulation and by inhibition of ERK and AKT activation. The clinical relevance of these data is indicated by the association of PDGFRα up-regulation in melanoma matched biopsies of BRAF-I +/- MEK inhibitor treated patients with shorter time to disease progression and less tumor regression. These findings suggest that monitoring patients for early PDGFRα up-regulation will facilitate the identification of those who may benefit from the treatment with BRAF-I in combination with clinically approved PDGFRα or Shh inhibitors

Citation Format: Francesco Sabbatino, Yangyang Wang, Xinhui Wang, Keith T. Flaherty, David Pepin, Giosuè Scognamiglio, Ling Yu, Zachary A. Cooper, Stefano Pepe, John M. Kirkwood, Dennie T. Frederick, Jennifer A. Wargo, Soldano Ferrone, Cristina R. Ferrone. PDGFRα up-regulation mediated by Sonic Hedgehog Pathway activation leads to BRAF inhibitor resistance in melanoma cells with BRAF mutation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3703. doi:10.1158/1538-7445.AM2014-3703
Abstract 3704: Novel panRAF inhibitors active in melanomas that are resistant to BRAF-selective, or BRAF-selective/MEK inhibitor combinations
Authors: Girotti Maria R. , Lopes Filipa , Preece Natasha , Niculescu-Duvaz Dan , Zambon Alfonso , Davies Lawrence , Whittaker Steven , Saturno Grazia , Viros Amaya , Pedersen Malin , Suijkerbuijk Bart MJM , Menard Delphine , Mcleary Robert , Johnson Louise , Fish Laura , Ejiama Sarah , Sanchez-Laorden Berta , Carragher Neil , Macleod Kenneth , Ashton Garry , Marusiak Anna , Fusi Alberto , Brognard John , Frame Margaret , Lorigan Paul , Springer Caroline J. , Marais Richard
Published: May 2015

The protein kinase BRAF is mutated ∼40% of human melanomas. BRAF is a component of the RAS/RAF/MEK/ERK pathway and BRAF or MEK inhibitors increase progression-free and overall survival in melanoma patients with BRAF mutations. However, most patients relapse with acquired resistance and ∼20% of patients present intrinsic resistance and do not respond to these drugs. We describe here two novel compounds that target mutant BRAF and wild-type CRAF. Our compounds inhibited the growth of melanoma cells that were resistant to BRAF-selective inhibitors. ERK pathway reactivation is responsible for resistance to BRAF targeted therapies in ∼60% of the patients and in ∼25% of patients resistance is driven by acquisition of mutations in NRAS. We show that our compounds inhibited the growth of melanoma cells that were resistant to BRAF-selective inhibitors due to pathway reactivation mediated by different mechanisms. We show that the drugs were active against patient derived xenografts (PDXs) from patients with acquired or intrinsic resistance to BRAF-selective inhibitors and in whose tumors resistance was associated with ERK pathway reactivation. Further, our compounds are active in a PDX from a patient whose tumor developed acquired resistance to a combination of a BRAF-selective plus a MEK inhibitor and associated with acquisition of an NRAS mutation. Thus, our panRAF inhibitors can inhibit melanomas with different mechanisms of acquired or intrinsic resistance to BRAF-selective and BRAF-selective/MEK inhibitor combinations, potentially providing first-line treatment for naïve patients and second-line treatments for a range of relapsed patients.

Citation Format: Maria R. Girotti, Filipa Lopes, Natasha Preece, Dan Niculescu-Duvaz, Alfonso Zambon, Lawrence Davies, Steven Whittaker, Grazia Saturno, Amaya Viros, Malin Pedersen, Bart MJM Suijkerbuijk, Delphine Menard, Robert Mcleary, Louise Johnson, Laura Fish, Sarah Ejiama, Berta Sanchez-Laorden, Neil Carragher, Kenneth Macleod, Garry Ashton, Anna Marusiak, Alberto Fusi, John Brognard, Margaret Frame, Paul Lorigan, Caroline J. Springer, Richard Marais. Novel panRAF inhibitors active in melanomas that are resistant to BRAF-selective, or BRAF-selective/MEK inhibitor combinations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3704. doi:10.1158/1538-7445.AM2014-3704
Abstract 3707: The multitargeted kinase inhibitor dasatinib induces DNA damage, Hippo pathway engagement and senescence in lung cancer cell lines that possess kinase-inactivating BRAF mutations
Authors: Sen Banibrata , Peng Shaohua , Mazumdar Tuhina , Byers Lauren A. , Kadara Humam , Johnson Faye M.
Published: May 2015

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death. Therapies that target specific genetic aberrations are very effective in a subset on NSCLC. An effective approach for discovering novel therapeutic targets is to characterize responsive tumors. In this regard, we discovered a novel, kinase-inactivating BRAF mutation in the tumor of a patient with a marked and durable response to the multi-targeted kinase inhibitor dasatinib. Likewise, dasatinib induces senescence in NSCLC cells with endogenous kinase-inactivating BRAF mutations (KIBRAF). To identify the mechanism underlying this effect, we used reverse phase protein array, Western blotting and gene expression analysis on NSCLC cell lines with KIBRAF or WTBRAF incubated with dasatinib. We found that ATM, ATR, Rad 51, pH2AX, CDC6, CHEK1 (Chk1), pLATS and TAZ, were differentially regulated in mutant vs. WT cell lines. Quantitative real-time PCR analysis revealed that CHEK1, TAZ and TAZ's downstream targets were decreased following incubation with dasatinib in KIBRAF cells. Overexpression of CHEK1 or TAZ in KIBRAF NSCLC cells resulted in dasatinib resistance and decreased dasatinib-induced senescence. Given the differential effect on CHEK1 and pH2AX, we studied the effect of dasatinib on DNA damage using the COMET assay and found that dasatinib induces a significant increase in COMET-positive KIBRAF NSCLC cells. These data suggest that the inhibition of Chk1 along with inhibition of Hippo pathway results in the dasatinib sensitivity in NSCLC with KIBRAF.

Citation Format: Banibrata Sen, Shaohua Peng, Tuhina Mazumdar, Lauren A. Byers, Humam Kadara, Faye M. Johnson. The multitargeted kinase inhibitor dasatinib induces DNA damage, Hippo pathway engagement and senescence in lung cancer cell lines that possess kinase-inactivating BRAF mutations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3707. doi:10.1158/1538-7445.AM2014-3707
Abstract 3708: microRNAs involved in BRAF inhibitor resistance
Authors: Koetz Lisa , Sokolova Elena , Brown Brian D. , Hernando Eva
Published: May 2015

BRAF inhibitors (BRAFi) have shown remarkable clinical activity in advanced metastatic melanomas with mutant BRAF(V600E). However, there is considerable variability in both the extent and the duration of patient responses, and acquired resistance invariably emerges. To date, a number of mechanisms underlying acquired resistance to BRAF inhibitors have been identified, most of which reactivate ERK signaling while simultaneously engaging parallel survival pathways. However, these mechanisms account for only a fraction of progressed melanoma and do not explain the large variability of initial response rates and time to progression of melanoma patients subjected to these therapies. We hypothesize that in addition to genetic events, non-genetic alterations such as microRNA changes are critical in mediating time to progression in melanoma tumors treated with BRAF inhibitors. In order to identify microRNAs involved in BRAF inhibitor resistance we performed a functional screen approach utilizing a novel lentiviral-based library of microRNA ‘decoys’. These constructs are better able to stably suppress endogenous microRNA activity than the hairpin RNAs traditionally used. This pooled parallel loss-of-microRNA function analysis in BRAF(V600E) mutant melanoma cells showed enrichment and loss of specific decoys after drug treatment, corresponding to microRNAs that may enhance or compromise the response, respectively. MicroRNA decoys that are positively or negatively selected during drug treatment are being validated using single decoy constructs for their ability to modulate BRAFi response. MicroRNA target genes will be determined by a combination of microarray analysis, in silico predictive algorithms, and 3′UTR-luciferase reporter assay. MicroRNAs and target genes are being validated on human samples from treated patients. Those potentially involved in processes/pathways known to influence drug resistance (i.e. survival genes) will be examined for their ability to mimic or counteract microRNA effects in BRAFi resistance when silenced or overexpressed, respectively. Identification of novel modulators of drug resistance provides the opportunity to design rational combinatorial approaches that will enhance and prolong the effect of these therapies, with the ultimate goal of achieving cures.

Citation Format: Lisa Koetz, Elena Sokolova, Brian D. Brown, Eva Hernando. microRNAs involved in BRAF inhibitor resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3708. doi:10.1158/1538-7445.AM2014-3708
Abstract 3710: The role of eIF4E in promoting melanoma cell proliferation and maintaining acquired resistance to Vemurafenib in melanoma
Authors: Zhan Yao , Dahabieh Michael S. , Pettersson Filippa , Dobocan Monica C. , Boutchou Marie Noel M. , Kempen Leon Van , Rincon Sonia V. del , Miller Wilson H.
Published: May 2015

In eukaryotic cells, mRNA translation is restricted by the translation initiation step, in which eIF4E has been regarded as a master regulator. Overexpression of eIF4E has been found in different cancer types; however, whether eIF4E is involved in melanoma tumorigenesis and resistance to standard of care in melanoma remains unclear. We hypothesize that eIF4E promotes melanoma cell proliferation and facilitates the development of acquired resistance to the B-raf inhibitor, Vemurafenib. From our preliminary data, we show that eIF4E is overexpressed in a panel of malignant melanoma cell lines compared to immortalized melanocytes Mel ST. By genetically knocking down eIF4E via siRNA, we can significantly repress the proliferation of a subset of melanoma cell lines and decrease the level of the eIF4E downstream target Cyclin D1. In addition, the B-raf inhibitor Vemurafenib can drastically inhibit 4EBP-1 phosphorylation in BRAFV600E melanoma cell lines, and further cap binding analysis confirmed that Vemurafenib stabilizes the eIF4E - 4EBP-1 association in these cell lines.. In contrast, 4EBP-1 is highly phosphorylated in all Vemurafenib resistant cell lines, compared to the parental A375, even in the presence of Vemurafenib. Furthermore, cap binding assay show that, unlike their parental counterpart, four out of six resistant lines exhibit weak eIF4E - 4EBP-1 association even in the presence of Vemurafenib. This may imply that more free eIF4E is available for cap-dependent translation initiation in Vemu resistant lines compared to the parental A375. Besides, eIF4E activity contributes to the proliferation of some melanoma cell lines with acquired resistance to Vemurafenib, since siRNA-silencing eIF4E decreases their proliferation. In conclusion, our data show that eIF4E promotes proliferation of some melanoma cell lines and may be involved in maintaining the survival of some melanoma cell lines with acquired vemurafenib resistance. These data suggest that therapeutically targeting eIF4E may be a viable means of inhibiting melanoma cell proliferation and overcoming Vemurafenib resistance.

Citation Format: Yao Zhan, Michael S. Dahabieh, Filippa Pettersson, Monica C. Dobocan, Marie Noel M. Boutchou, Leon Van Kempen, Sonia V. del Rincon, Wilson H. Miller, Jr.. The role of eIF4E in promoting melanoma cell proliferation and maintaining acquired resistance to Vemurafenib in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3710. doi:10.1158/1538-7445.AM2014-3710
Abstract 3718: Adaptive drug escape of EML4-ALK lung cancer against targeted ALK inhibitor involving TGFβ2-mitochondrial priming
Authors: Yin Lihong , Zhang Wei , Shi Ivy , Feng Yan , Bagai Rakesh , Lindner Daniel , Ma Patrick C.
Published: May 2015

Lung cancer is the second most common malignancy with the highest cancer-mortality rate. EML4-ALK chromosomal translocation is now known to represent a highly actionable unique molecular target in approximately 5% of non-small cell lung cancer (NSCLC) patients. The ALK tyrosine kinase inhibitor (TKI) crizotinib induces remarkably high response rates in molecularly-selected NSCLC patients harboring EML4-ALK driven tumors, and is being used in first-line therapy. Nonetheless, responsive tumors ultimately develop acquired drug resistance. We have recently investigated non-mutational mechanisms of tumor resistant escape emerging under early TKI treatment time-window. Our recent studies in EGFR-mutant NSCLC in early adaptive drug escape against EGFR TKI identified an autocrine upregulation of TGFβ2 within these survivor cells. Here, we found that TGFβ2 was elevated significantly in the EML4-ALK-driven H3122 cells undergoing early adaptive drug escape against ALK TKI (TAE684) at treatment day 14. Interestingly, we found that the augmented TGFβ signaling was specific to TGFβ2, but not TGFβ1 or TGFβ3. We also found correlative enhanced mitochondrial BCL-2/BCL-xL prosurvival signaling in the day 14 TKI surviving H3122 cells. The upregulated TGFβ2-BCL-2/BCL-xL prosurvival mitochondrial priming was further validated in H3122 xenografts by IHC. Exogenous TGFβ2 stimulation in vitro for 7 days induced a dose-dependent upregulated BCL-2 and BCL-xL expression. Inhibiting mitochondrial BCL-2/BCL-xL priming using dual BCL-2/BCL-xL inhibitor ABT-263 as BH3-mimetic led to significant reduction in early adaptive drug escape induced by ALK TKIs. Our study showed that EML4-ALK NSCLC could escape ALK TKI early in treatment through cell plasticity that involved TGFβ2-mitochondrial priming in an upregulated prosurvival state. We also found that the early adaptive drug-escaping cancer cells possessed inherently increased EMT-ness (upregulated vimentin, AXL, GAS6) and cancer stemness (upregulated OCT4, NANOG). These findings provide deeper insights into the process of drug resistance emergence and highlighted potential window-of-opportunities to therapeutically target early adaptive escape to eradicate targeted drug resistance and impact long term survival.

Citation Format: Lihong Yin, Wei Zhang, Ivy Shi, Yan Feng, Rakesh Bagai, Daniel Lindner, Patrick C. Ma. Adaptive drug escape of EML4-ALK lung cancer against targeted ALK inhibitor involving TGFβ2-mitochondrial priming. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3718. doi:10.1158/1538-7445.AM2014-3718
Abstract 3721: ALK inactivation induced acquired resistance to alectinib in lung cancer harboring EML4-ALK fusion gene
Authors: Isozaki Hideko , Ichihara Eiki , Yasugi Masayuki , Nobuaki Ochi , Hotta Katsuyuki , Takigawa Nagio , Sendo Toshiaki , Tanimoto Mitsune , Kiura Katsuyuki
Published: May 2015

Dramatic clinical responses to a first generation anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor (TKI) crizotinib have been observed in patients with advanced non-small cell lung cancer (NSCLC) haboring ALK fusion gene. Alectinib (CH5424802) (Chugai Pharmaceutical Co., Ltd.) is a second generation ALK-TKI could partially overcome acquired resistance to crizotinib in vitro (Cancer Cell 19, 2011: 679-90), and showed promising result in phase I/II clinical trials with high response rate (93.5 %) and less toxicity than crizotinib. However, even this promising ALK-TKI, the problem of acquired resistance is inevitable.

To elucidate resistant mechanisms, we established an alectinib-resistant cell line H2228/CHR from H2228 that have echinoderm microtubule associated protein like 4 (EML4)-ALK fusion gene, by continuous exposure to this agent. ALK fusion gene disappeared in H2228/CHR, which was confirmed Western blotting, IHC, qRT-PCR and FISH. H2228/CHR cells presented higher phosphorylated levels of epidermal growth factor receptor (EGFR) than the parental H2228 cells and became more sensitive to EGFR-TKI erlotinib.

Next, we established alectinib-resistant ABC-11/CHR cells from ABC-11, a cell line established from pleural effusion of EML4-ALK positive ALK-TKI naïve patient. In ABC-11/CHR cells, phosphorylated levels of ALK was markedly decreased and phosphorylated levels of MET was increased.In addition, autocrine of HGF was increased in ABC-11/CHR cells. ABC-11/CHR showed sensitivity to ALK/MET tyrosine kinase inhibitor, crizotinib in vitro and in vivo model.

In conclusion, inactivation of ALK signaling induced acquired resistance to alectinib in NSCLC harboring ALK fusion gene. Signaling switch from ALK to EGFR or MET was observed in the alectinib-resistant cells. We revealed to overcome such acquired resistance using erlotinib or crizotinib.

Citation Format: Hideko Isozaki, Eiki Ichihara, Masayuki Yasugi, Ochi Nobuaki, Katsuyuki Hotta, Nagio Takigawa, Toshiaki Sendo, Mitsune Tanimoto, Katsuyuki Kiura. ALK inactivation induced acquired resistance to alectinib in lung cancer harboring EML4-ALK fusion gene. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3721. doi:10.1158/1538-7445.AM2014-3721
Abstract 3729: An individualized approach to bladder cancer treatment using patient-derived cell lines to predict response to chemotherapeutic agents
Authors: Barlow LaMont , Chua Chee Wai , Lei Ming , DeCastro G. Joel , Badani Ketan , Benson Mitchell , McKiernan James , Shen Michael
Published: May 2015

Introduction: Chemotherapy (both intravesical and systemic) can reduce the risk of recurrence and progression in various stages of bladder cancer. However, recurrence after treatment failure is associated with an increased risk of progression. There are currently no established methods for predicting patient-specific responses to treatment prior to drug selection. In our studies, we have developed a novel protocol for efficient establishment of cell lines from primary human bladder tumors, which enables in vitro drug sensitivity assays using chemotherapeutic agents.

Methods: Using an Institutional Review Board-approved tissue acquisition protocol, informed consent was obtained prior to specimen acquisition for all samples. Specimens were obtained during standard transurethral resection of papillary bladder tumors. Following generation of a single-cell suspension, epithelial cells were isolated using immunomagnetic cell separation and used for establishment of adherent cell cultures using a novel protocol. We performed immunohistochemistry on parental tissue as well as cultured cells to confirm that the urothelial cancer phenotype was maintained during serial passaging. For sensitivity assays, cultured cells were passaged and treated with chemotherapeutic agents, followed by assessment of cell viability using MTT assays.

Results: To date, seven specimens from patients with papillary urothelial carcinoma have been obtained, resulting in the establishment of six independent adherent cell lines. All established lines have been serially passaged (as high as P10) without significant decline in growth rate, and maintained expression of CK7, uroplakin III, p53, and Ki67 in patterns similar to parental tissue. Cells from line #7 were treated with mitomycin C, docetaxel, gemcitabine, and rapamycin at three different equivalent concentrations, resulting in a unique sensitivity profile that was reproduced in a replicate experiment performed at a subsequent passage.

Conclusions: We have established a novel protocol for culture and rapid expansion of primary cells from human bladder tumors for assays of drug response. Ultimately, we envision that this approach will provide a basis for the design of patient-specific therapeutic regimens for bladder cancer.

Citation Format: LaMont Barlow, Chee Wai Chua, Ming Lei, G. Joel DeCastro, Ketan Badani, Mitchell Benson, James McKiernan, Michael Shen. An individualized approach to bladder cancer treatment using patient-derived cell lines to predict response to chemotherapeutic agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3729. doi:10.1158/1538-7445.AM2014-3729
Abstract 3733: SilenciX®, novel stable knock-down cellular models to screen new molecular targets through the synthetic lethality approach
Authors: Mennesson Eric , Renault Anne-Marie , Fixe Isabelle , GRILLON Catherine , Kiéda Claudine , Normand Nadia
Published: May 2015

PARP inhibitors are promising therapeutic drugs for cancers harboring BRCA1 and BRCA2 mutations. PARP is involved in the Base Excision Repair (BER) DNA repair pathway. Tumors with BRCA mutations are deficient in Homologous Recombination (HR), another DNA repair pathway. To maintain DNA integrity and prevent cell death, at least one one of the BER or HE pathways must be functional, while the other may be inhibited or silenced (eg. mutation, drug).

In drug discovery, Synthethic Lethality (SL) used a cancer mutation and a drug in combination to cause the tumor cell's death. SL is thus a promising approach to find new druggable targets by controlling the HR and BER DNA repair pathways in order to selectively kill mutated cancer cells. However, the main technical hurdle in SL approaches is to easily identify cancer-specific molecular interaction in various cancer types, while using robust and validated experimental cellular models.

In this presentation we demonstrate how the SilenciX® technology meets SL experimental requirements. BRCA1 and BRCA2 Silencix® cells were treated with PARP1 inhibitors (Olaparib, Veliparib, Rucaparib) and with Gemcitabine.

All inhibitors except Gemcitabine were synthetic lethal in BRCA1 SilenciX® cell lines.

To mimic the cancer micro-environement, the model was tested under hypoxic conditions.

We conclude that SilenciX® cell lines are ingenious gene-specific Knock Down engineered cell lines using a new pEBV derived plasmid, avoiding integration and off-target effects, suitable for a broad range of synthetic lethality studies.

Citation Format: Eric Mennesson, Anne-Marie Renault, Isabelle Fixe, Catherine GRILLON, Claudine Kiéda, Nadia Normand. SilenciX®, novel stable knock-down cellular models to screen new molecular targets through the synthetic lethality approach. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3733. doi:10.1158/1538-7445.AM2014-3733