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Dennie T Frederick - One of the best experts on this subject based on the ideXlab platform.

  • abstract pr04 a melanoma transcriptional state distinction influences sensitivity to mapk pathway inhibitors
    Clinical Cancer Research, 2015
    Co-Authors: Cory M Johannessen, Ravid Straussman, Dennie T Frederick, Michal Barzilyrokni, David J Konieczkowski, Omar O Abudayyeh, Zachary A Cooper, Adriano Piris, David E Fisher, Jill P Mesirov
    Abstract:

    The deployment of cancer therapeutics that exploit oncogenic dependencies has yielded remarkable advances in patient treatment. However, the therapeutic benefit of these approaches is transient and the majority of patients develop resistance within several months. BRAF V600E -mutant malignant melanoma provides an illustrative example of this phenomenon: treatment with RAF and MEK inhibitors yields clinical responses in 50-80% of patients. However, 10-20% fail to respond to treatment (intrinsic resistance) and patients that do respond become drug resistant within ∼9 months (acquired resistance), presenting a formidable and unsolved clinical challenge. It remains incompletely understood why a subset of BRAF V600 -mutant melanoma patients (10-20%) fail to respond to MAPK-pathway inhibition. Here, we show that RAF inhibitor sensitive and resistant BRAF V600 -mutant melanomas display distinct transcriptional profiles. RAF-inhibitor sensitive cell lines are distinguishable by expression and activity of the melanocytic lineage transcription factor MITF, whereas intrinsically drug-resistant cell lines are defined by expression of the receptor tyrosine kinase AXL and elevated levels of NF-κB signaling. In vitro, these signatures were sufficient to predict MAPK-pathway inhibitor responsiveness in independent panels of melanoma cell lines. MITF-low, AXL/NF-κB high melanomas were resistant to single-agent RAF, MEK and ERK and combined RAF/MEK inhibition. In treatment-naive patient biopsies, markers of the drug sensitive transcriptional states were associated with improved therapeutic responses to combined RAF/MEK inhibitors in BRAF V600 -mutant melanoma. Moreover, in cell lines, NF-κB activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF and AXL/NF-κB activity can influence intrinsic resistance to MAPK pathway inhibitors in BRAF V600 -mutant melanoma. More broadly, these data suggest that the transcriptional context in which an oncogenic event arises can have a profound impact on the establishment of oncogene-dependencies and associated drug susceptibilities. Citation Format: Cory Johannessen, David Konieczkowski, Omar Abudayyeh, Jong Wook Kim, Zachary Cooper, Adriano Piris, Dennie Frederick, Michal Barzily-Rokni, Ravid Straussman, Rizwan Haq, David Fisher, Jill Mesirov, William Hahn, Keith Flaherty, Jennifer Wargo, Pablo Tamayo, Levi Garraway. A melanoma transcriptional state distinction influences sensitivity to MAPK pathway inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR04.

  • a melanocyte lineage program confers resistance to map kinase pathway inhibition
    Nature, 2013
    Co-Authors: Dennie T Frederick, Cory M Johannessen, Laura A Johnson, Federica Piccioni, Aisha Townes, Melanie Donahue, Rajiv Narayan
    Abstract:

    Expression of more than 15,500 genes individually in a melanoma cell line treated with RAF, MEK, ERK or combined RAF–MEK inhibitors reveals a cyclic-AMP-dependent melanocytic signalling network associated with drug resistance; this may represent a novel therapeutic target for melanoma treatment. Mutations that cause constitutive activation of BRAF/MEK signalling can promote the development of melanoma. Although patients can benefit from therapies that target this pathway, they usually develop resistance. A number of resistance mechanisms have been uncovered in recent years. In an unbiased screen for genes whose expression promotes resistance to BRAF and MEK inhibitors in melanoma cells carrying BRAF mutations, Levi A. Garraway and colleagues find that cyclic AMP signalling, a pathway important for the development and survival of the melanocyte lineage, can confer resistance. Although further work will be needed to show if and how cAMP is activated in melanoma patients who develop resistance, it may represent a novel therapeutic target for melanoma treatment. Malignant melanomas harbouring point mutations (Val600Glu) in the serine/threonine-protein kinase BRAF (BRAF(V600E)) depend on RAF–MEK–ERK signalling for tumour cell growth1. RAF and MEK inhibitors show remarkable clinical efficacy in BRAF(V600E) melanoma2,3; however, resistance to these agents remains a formidable challenge2,4. Global characterization of resistance mechanisms may inform the development of more effective therapeutic combinations. Here we carried out systematic gain-of-function resistance studies by expressing more than 15,500 genes individually in a BRAF(V600E) melanoma cell line treated with RAF, MEK, ERK or combined RAF–MEK inhibitors. These studies revealed a cyclic-AMP-dependent melanocytic signalling network not previously associated with drug resistance, including G-protein-coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB). Preliminary analysis of biopsies from BRAF(V600E) melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF–MEK inhibition but restored in relapsing tumours. Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF. Combined treatment with MAPK-pathway and histone-deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance. Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF–MEK–ERK inhibition, which may be overcome by combining signalling- and chromatin-directed therapeutics.

  • tumour micro environment elicits innate resistance to raf inhibitors through hgf secretion
    Nature, 2012
    Co-Authors: Ravid Straussman, Ashli Davis, Margaret M. Mongare, Joshua Gould, Jinyan Du, Kevin Shee, Teppei Morikawa, Zhi Rong Qian, Michal Barzilyrokni, Dennie T Frederick
    Abstract:

    The secretion of hepatocyte growth factor by stromal cells in the tumour micro-environment can make melanoma resistant to RAF inhibitors, through the activation of the MET signalling pathway, but a combination of RAF and MET inhibitors can overcome this resistance. Targeted anticancer therapies are gaining ground in clinical applications as researchers begin to understand the genetic changes underlying tumorigenesis and the factors that determine an individual patient's response to a drug. However, resistance is a challenging problem in most clinical trials of targeted therapies. Two complementary papers show that the secretion of growth factors from the tumour microenvironment can cause resistance to a range of anticancer drugs, through their ability to drive tumour growth by activating redundant signalling pathways. Both papers provide evidence that stromal production of hepatocyte growth factor can confer resistance to a class of drugs called BRAF inhibitors, such as vemurafenib, in patients with melanoma, through activation of the MET signalling pathway. They show that a combination of BRAF and MET inhibitors can overcome resistance, suggesting that this combination should be tested in patients with melanoma. Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance1,2,3,4. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)–AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.

  • Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion
    Nature, 2012
    Co-Authors: Ravid Straussman, Michal Barzily-rokni, Ashli Davis, Margaret M. Mongare, Joshua Gould, Jinyan Du, Kevin Shee, Teppei Morikawa, Zhi Rong Qian, Dennie T Frederick
    Abstract:

    Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)-AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.

Ravid Straussman - One of the best experts on this subject based on the ideXlab platform.

  • abstract pr04 a melanoma transcriptional state distinction influences sensitivity to mapk pathway inhibitors
    Clinical Cancer Research, 2015
    Co-Authors: Cory M Johannessen, Ravid Straussman, Dennie T Frederick, Michal Barzilyrokni, David J Konieczkowski, Omar O Abudayyeh, Zachary A Cooper, Adriano Piris, David E Fisher, Jill P Mesirov
    Abstract:

    The deployment of cancer therapeutics that exploit oncogenic dependencies has yielded remarkable advances in patient treatment. However, the therapeutic benefit of these approaches is transient and the majority of patients develop resistance within several months. BRAF V600E -mutant malignant melanoma provides an illustrative example of this phenomenon: treatment with RAF and MEK inhibitors yields clinical responses in 50-80% of patients. However, 10-20% fail to respond to treatment (intrinsic resistance) and patients that do respond become drug resistant within ∼9 months (acquired resistance), presenting a formidable and unsolved clinical challenge. It remains incompletely understood why a subset of BRAF V600 -mutant melanoma patients (10-20%) fail to respond to MAPK-pathway inhibition. Here, we show that RAF inhibitor sensitive and resistant BRAF V600 -mutant melanomas display distinct transcriptional profiles. RAF-inhibitor sensitive cell lines are distinguishable by expression and activity of the melanocytic lineage transcription factor MITF, whereas intrinsically drug-resistant cell lines are defined by expression of the receptor tyrosine kinase AXL and elevated levels of NF-κB signaling. In vitro, these signatures were sufficient to predict MAPK-pathway inhibitor responsiveness in independent panels of melanoma cell lines. MITF-low, AXL/NF-κB high melanomas were resistant to single-agent RAF, MEK and ERK and combined RAF/MEK inhibition. In treatment-naive patient biopsies, markers of the drug sensitive transcriptional states were associated with improved therapeutic responses to combined RAF/MEK inhibitors in BRAF V600 -mutant melanoma. Moreover, in cell lines, NF-κB activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF and AXL/NF-κB activity can influence intrinsic resistance to MAPK pathway inhibitors in BRAF V600 -mutant melanoma. More broadly, these data suggest that the transcriptional context in which an oncogenic event arises can have a profound impact on the establishment of oncogene-dependencies and associated drug susceptibilities. Citation Format: Cory Johannessen, David Konieczkowski, Omar Abudayyeh, Jong Wook Kim, Zachary Cooper, Adriano Piris, Dennie Frederick, Michal Barzily-Rokni, Ravid Straussman, Rizwan Haq, David Fisher, Jill Mesirov, William Hahn, Keith Flaherty, Jennifer Wargo, Pablo Tamayo, Levi Garraway. A melanoma transcriptional state distinction influences sensitivity to MAPK pathway inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR04.

  • tumour micro environment elicits innate resistance to raf inhibitors through hgf secretion
    Nature, 2012
    Co-Authors: Ravid Straussman, Ashli Davis, Margaret M. Mongare, Joshua Gould, Jinyan Du, Kevin Shee, Teppei Morikawa, Zhi Rong Qian, Michal Barzilyrokni, Dennie T Frederick
    Abstract:

    The secretion of hepatocyte growth factor by stromal cells in the tumour micro-environment can make melanoma resistant to RAF inhibitors, through the activation of the MET signalling pathway, but a combination of RAF and MET inhibitors can overcome this resistance. Targeted anticancer therapies are gaining ground in clinical applications as researchers begin to understand the genetic changes underlying tumorigenesis and the factors that determine an individual patient's response to a drug. However, resistance is a challenging problem in most clinical trials of targeted therapies. Two complementary papers show that the secretion of growth factors from the tumour microenvironment can cause resistance to a range of anticancer drugs, through their ability to drive tumour growth by activating redundant signalling pathways. Both papers provide evidence that stromal production of hepatocyte growth factor can confer resistance to a class of drugs called BRAF inhibitors, such as vemurafenib, in patients with melanoma, through activation of the MET signalling pathway. They show that a combination of BRAF and MET inhibitors can overcome resistance, suggesting that this combination should be tested in patients with melanoma. Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance1,2,3,4. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)–AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.

  • abstract 4837 tumor microenvironment contributes to raf inhibitor resistance through hgf secretion
    Cancer Research, 2012
    Co-Authors: Ravid Straussman, Ashli Davis, Joshua Gould, Jinyan Du, Kevin Shee, Teppei Morikawa, Keith T Flaherty, Michal Barzilyronki, Shuji Ogino, Jennifer A Wargo
    Abstract:

    Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Drug resistance remains a vexing problem in the treatment of cancer patients. While many studies have focused on cell autonomous mechanisms of drug resistance, we hypothesized that the tumor microenvironment may confer innate resistance to therapy. We employed a novel co-culture assay to systematically assay the ability of 23 stromal cell types to influence the sensitivity of 45 cancer cell lines to 35 anti-cancer drugs testing more than 15,000 cancer-stroma-drug combinations. We found that stroma-mediated resistance is surprisingly common - particularly to targeted chemotherapies. We decided to further characterize the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibition because most patients exhibit some degree of innate resistance. Proteomic analysis showed that stromal secretion of the growth factor hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the MAPK and PI3K/AKT pathways, and immediate resistance to RAF inhibition. Immunohistochemistry confirmed tumor stromal HGF expression in 11 out of 15 patients with BRAF-mutant melanoma. Among these patients, only one experienced a complete response, and that patient was negative for stromal HGF, consistent with our prediction of HGF-mediated RAF-inhibitor resistance. In contrast, of 6 evaluable patients with stable disease 4 had particularly high levels of stromal HGF expression. Dual inhibition of RAF and MET in BRAF-mutant melanoma cell lines resulted in reversal of drug resistance, suggesting RAF/MET combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. Interestingly, the activation of MET in these cell lines was cell autonomous, caused, for example, by autocrine secretion of HGF and resulting in complete resistance to RAF inhibitors even in the absence of stromal cells. Treatment with a MET inhibitor re-sensitized these cell lines to RAF inhibitors suggesting the same RAF/MET combination therapy for non-melanoma BRAF mutant cancers as well. More generally, these studies indicate that the systematic dissection of tumor-microenvironment interactions may reveal important mechanisms underlying drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4837. doi:1538-7445.AM2012-4837

  • Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion
    Nature, 2012
    Co-Authors: Ravid Straussman, Michal Barzily-rokni, Ashli Davis, Margaret M. Mongare, Joshua Gould, Jinyan Du, Kevin Shee, Teppei Morikawa, Zhi Rong Qian, Dennie T Frederick
    Abstract:

    Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)-AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.

Levi A Garraway - One of the best experts on this subject based on the ideXlab platform.

  • abstract ng04 clinical acquired resistance to raf inhibitor combinations in braf mutant colorectal cancer through mapk pathway alterations
    Cancer Research, 2015
    Co-Authors: Ryan B Corcoran, Nikhil Wagle, Levi A Garraway, Leanne G Ahronian, Eliezer M Van Allen, Erin M Coffee, Eunice L Kwak, Jason E Faris, John A Iafrate, Jeffrey A Engelman
    Abstract:

    BRAFmutations occur in ∼10% of colorectal cancers (CRCs) and confer poor prognosis. While RAF inhibitor monotherapy leads to response rates of ∼60% in BRAF mutant melanoma, response rates in BRAF mutant CRC are disappointingly low (∼5%), suggesting a fundamental difference between these tumor types, despite the mutual presence of a BRAF V600 mutation. Previously, our group and others found that while RAF inhibitors lead to profound and sustained suppression of MAPK signaling in BRAF mutant melanoma cells, suppression of MAPK signaling by RAF inhibitors in BRAF mutant CRC cells is transient, and the MAPK pathway is rapidly reactivated despite the continued presence of drug. These data suggest that MAPK pathway suppression by RAF inhibitors alone may be inadequate in BRAF mutant CRC and that combinations of RAF inhibitors with other targeted agents might be required to achieve robust MAPK suppression and clinical responses. More recently, our group and others found that in many BRAF mutant CRCs, MAPK pathway reactivation is driven by feedback signaling through EGFR via RAS and CRAF. Importantly, we found that the combination of a RAF inhibitor and an EGFR inhibitor can lead to sustained MAPK suppression and improved efficacy, leading to marked tumor regressions in preclinical BRAF mutant CRC xenograft models. These data have led to clinical trials in BRAF mutant CRC patients evaluating RAF inhibitor combinations, including combinations of RAF+EGFR, RAF+MEK, and RAF+MEK+EGFR inhibitors. Initial data from these trials, reported at ASCO 2014, have shown encouraging efficacy, with some trials showing initial response rates of as much as 40%. However, as with all targeted therapies, clinical benefit is invariably limited by the emergence of acquired resistance to these therapies. In this study, we sought to identify clinically relevant mechanisms of acquired resistance to RAF inhibitor combinations in BRAF mutant CRC, in order to understand the signaling changes leading to resistance and to devise therapeutic strategies to overcome or prevent resistance. To do so, we obtained tumor biopsies from BRAF mutant CRC patients upon disease progression, after initial response or prolonged stable disease on RAF+EGFR, RAF+MEK, or RAF+MEK+EGFR inhibitor combinations. Matched pre-treatment, post-progression, and normal DNA were analyzed by whole exome sequencing (WES) and RNA sequencing. To supplement this approach, in vitro modeling of acquired resistance was performed in BRAF mutant CRC cell lines. In one BRAF mutant CRC patient with prolonged stable disease on a RAF+EGFR inhibitor combination, WES identified KRAS amplification in a progressing lesion. RNA sequencing confirmed KRAS transcript overexpression, and KRAS amplification (∼25-fold) was confirmed by FISH in the post-progression biopsy, but was absent in a pre-treatment biopsy. Interestingly, in resistant clones generated from BRAF mutant CRC cell lines selected with either RAF+EGFR or RAF+MEK inhibitor combinations, KRAS exon 2 mutations were identified. Either KRAS amplification or KRAS mutation led to sustained MAPK pathway activity and cross-resistance to either RAF+EGFR or RAF+MEK inhibitor combinations. However, an ERK inhibitor, either alone or in combination with a RAF inhibitor retained the ability to suppress the MAPK pathway and could overcome resistance. In a second patient with a minor response to a RAF+EGFR inhibitor combination, BRAF amplification was identified in a progressing lesion, which was confirmed by FISH and was not present in a pre-treatment biopsy of the same lesion. Previously, our laboratory found that amplification of mutant BRAF could cause resistance to RAF or MEK inhibitors in BRAF mutant CRC cells by abrogating the ability of these inhibitors to suppress MAPK signaling. Importantly, an ERK inhibitor, either alone or in combination with a RAF inhibitor could suppress MAPK signaling and overcome resistance in this setting. In one patient with a minor response to a RAF+MEK inhibitor combination, WES identified the presence of an ARAF Q489L mutation and a MEK1 F53L mutation in a single progressing lesion, suggesting possible intra-lesional heterogeneity of acquired resistance mechanisms. However, utilizing a cell line derived from the patient9s post-progression biopsy, we found that 30 out of 30 single cell clones harbored both the ARAF and MEK1 mutations, and that the MEK1 F53L seemed to function as the primary driver of acquired resistance in these resistant tumor cells. MEK1 F53L expression markedly abrogated the ability of RAF+MEK and RAF+EGFR inhibitor combinations to suppress MAPK signaling. Interestingly, an ERK inhibitor was able to effectively suppress MAPK signaling and overcome resistance. In a second patient with a minor response to RAF+MEK, WES identified a focal amplicon on chromosome 2 in a progressing lesion, encompassing the c-mer oncogene (MERTK) receptor tyrosine kinase and the MAPK-regulated checkpoint kinase BUB1. Post-progression biopsies from two patients who progressed after prolonged stable disease to the triple combination of a RAF+MEK+EGFR inhibitor combination are currently being analyzed by WES, and updated results will be presented. In summary, RAF inhibitor combinations are leading to promising initial response rates in recent clinical trials in BRAF mutant CRC patients. The frequent identification of alterations leading to reactivation of MAPK pathway signaling upon clinical acquired resistance to RAF+EGFR or RAF+MEK combinations underscores the MAPK pathway as a valid and critical target in BRAF mutant CRC. Importantly, we found that many of these resistance mechanisms could be overcome by an ERK inhibitor or ERK inhibitor-based combinations, suggesting that ERK inhibitors may be key components of therapeutic combinations to be explored in future clinical trials for BRAF mutant CRC. Further efforts to understand acquired resistance mechanisms will be vital to developing novel therapeutic strategies to overcome resistance and extend clinical benefit in this lethal CRC subtype. Citation Format: Ryan B. Corcoran, Leanne G. Ahronian, Eliezer Van Allen, Erin M. Coffee, Nikhil Wagle, Eunice L. Kwak, Jason E. Faris, A. John Iafrate, Levi A. Garraway, Jeffrey A. Engelman. Clinical acquired resistance to RAF inhibitor combinations in BRAF mutant colorectal cancer through MAPK pathway alterations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr NG04. doi:10.1158/1538-7445.AM2015-NG04

  • abstract c221 a genome wide rna interference screen identifies synthetic lethal interactions with the braf oncogene
    Molecular Cancer Therapeutics, 2013
    Co-Authors: Chengyin Min, Eva M Goetz, David J Konieczkowski, Christine Kwon, Marika Linja, Krishna Vasudevan, Barbara A Weir, Levi A Garraway
    Abstract:

    Oncogenic mutations in BRAF are found in 50-70% of malignant melanomas and in ∼8% of all cancers. Activating BRAF mutations confer exquisite dependency on RAF/MEK/ERK signaling and RAF and MEK inhibitors achieved striking clinical response in the majority of patients with BRAFV600E-mutant melanoma. However, nearly all patients progress to therapeutic resistance and there are limited treatment options for patients with relapsed tumors. Identification of genes that are essential only in the presence of BRAFV600E mutation may inform alternative as well as combinatorial therapeutic approaches. Here, we used systematic RNA interference to discover synthetic lethal interactions in melanoma cells carrying BRAFV600E mutation. In addition, Project Achilles at the Broad Institute reported systematic loss-of-function studies in more than 100 human cancer cell lines. We intersected the list of genes that are synthetic lethal with BRAFV600E mutation in our melanoma study and that of Project Achilles. As a result, we have identified novel “druggable” targets for cells driven by BRAFV600E mutation. Our extensive in vitro and in vivo validation has confirmed and established key synthetic lethal interactions with BRAFV600E mutation. One major mechanism of acquired resistance to RAF and MEK inhibition is reactivation of MEK/ERK pathway. Importantly, we demonstrated that these synthetic lethal genes are also essential in the context of acquired resistance to RAF and MEK inhibition. Together, our study has identified novel targets for the treatment of oncogenic BRAF-driven melanoma and suggests targeting these genes as novel therapeutic strategies to overcome acquired resistance to RAF and MEK inhibition. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C221. Citation Format: Chengyin Min, David J. Konieczkowski, Christine Kwon, Marika Linja, Krishna Vasudevan, Barbara Weir, Eva Goetz, Levi Garraway. A genome-wide RNA interference screen identifies synthetic lethal interactions with the BRAF oncogene. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C221.

  • abstract b27 probing synthetic lethal interactions with raf inhibition in braf mutant colorectal cancer
    Molecular Cancer Therapeutics, 2013
    Co-Authors: Steven Whittaker, Jessica Hsiao, Glenn S Cowley, David E Root, Levi A Garraway
    Abstract:

    Large-scale RNA interference screens have indicated that BRAF-mutant colorectal cancer (CRC) cell lines are dependent upon BRAF expression for proliferation. However, small molecule RAF inhibitors, such as vemurafenib, have so far failed to exhibit robust activity in clinical trials of patients with CRC, with a response rate of only 5%. Commensurate with this, CRC cell lines demonstrate intrinsic resistance to RAF inhibitors. Therefore, we designed an RNA interference screen to identify loss of function events that could synergize with pharmacologic RAF inhibition. We transduced a pooled lentiviral library encoding 90,000 shRNAs targeting over 16,500 genes into RAF-inhibitor-resistant RKO CRC cells. The shRNA-infected population was divided into two experimental arms, one treated with DMSO, the other treated with PLX4720. Following 16 population doublings, the abundance of each hairpin was assessed by PCR amplification of barcoded hairpin DNA, followed by massively parallel paired-end sequencing. The log-fold change in shRNA abundance between the PLX4720- and DMSO-treated controls was determined and RNAi gene enrichment (RIGER) used to rank individual shRNAs and nominate candidate genes that were required for survival of RKO cells exposed to PLX4720. We validated the 40 top-ranking genes that displayed synthetic lethality with RAF inhibition in an arrayed secondary screen. shRNAs targeting MET scored highly, consistent with recent reports implicating HGF-MET signaling in resistance to BRAF inhibition, validating our approach. Furthermore, we describe the characterization of additional genes that sensitize cells to inhibition of BRAF, suggesting novel therapeutic opportunities. By integrating these findings with other genomic and functional studies we aim to identify clinically actionable events that cause RAF-inhibitor resistance in colorectal cancers. Citation Format: Steven R. Whittaker, Flora Luo, Jessica Hsiao, Glenn S. Cowley, David E. Root, Levi A. Garraway. Probing synthetic lethal interactions with RAF inhibition in BRAF-mutant colorectal cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B27.

  • abstract 2953 anticipating new biochemical mechanisms of resistance to raf inhibition in melanoma
    Cancer Research, 2012
    Co-Authors: Rajee Antony, Caroline Emery, Levi A Garraway
    Abstract:

    Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL More than 50% of melanoma's harbor the V600E activating mutation in BRAF, which constitutively activates the Mek/Erk signaling cascade leading to tumor cell proliferation. The BRAF(V600E) mutation predicts responsiveness to RAF inhibitors, which led to FDA approval of the RAF inhibitor vemurafenib (PLX4032) in 2011. Although several mechanisms of resistance to RAF inhibition have been described, no resistance-associated point mutations involving the RAF genes themselves have been detected in the clinical setting thus far. This is in contrast to well described resistance mechanisms to receptor tyrosine kinase inhibitors, which frequently involve secondary mutations within the target kinase. To gain additional insights in this regard, we created randomly mutagenized cDNA library of BRAF (V600E) and it's homolog C-Raf and expressed individually in A375(V600E B-RAF) cells in the presence of PLX4720, an analogue of PLX4032. The resistance clones emerged from the screen were characterized by massive parallel sequencing and the mutant alleles were further recreated via site directed mutageneis in the respective cDNA's. However, no candidate mutations occuring in BRAF (V600E) could be validated as bona fide resistance alleles. In contrast, we identified 11 alleles that engendered sustained Mek phosphorylation in the setting of PLX4720 exposure. Upon ectopic expression, three of these alleles conferred profound pharmacologic resistance to PLX4720. Two of these resistance alleles clustered within the 14-3-3 consensus binding site of C-Raf, and the third occurred in the kinase domain (ATP binding domain, activation segment). 14-3-3 proteins are known to stabilize and activate C-Raf through phosphorylation of key serine and tyrosine residues. Toward this end, some C-Raf resistance alleles promoted increased BRAF heterodimerization and increased kinase activity. Each of these alleles remained sensitive to the Mek inhibitor AZD6244. On the basis of these findings, we speculate that mutations in C-Raf that alter 14-3-3 binding, promoted dimerization, and/or enhance intrinsic kinase activity may conceivably mediate resistance to RAF inhibition - particularly as more potent inhibitors become utilized. Ongoing work involves investigating the clinical relevance of this phenomenon. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2953. doi:1538-7445.AM2012-2953

  • braf mutation predicts sensitivity to mek inhibition
    Nature, 2006
    Co-Authors: David B Solit, Levi A Garraway, Christine A Pratilas, Ayana Sawai, Gad Getz, Andrea D Basso, Jose Lobo, Yuhong She
    Abstract:

    The kinase pathway comprising RAS, RAF, mitogen-activated protein kinase kinase (MEK) and extracellular signal regulated kinase (ERK) is activated in most human tumours, often through gain-of-function mutations of RAS and RAF family members. Using small-molecule inhibitors of MEK and an integrated genetic and pharmacologic analysis, we find that mutation of BRAF is associated with enhanced and selective sensitivity to MEK inhibition when compared to either 'wild-type' cells or cells harbouring a RAS mutation. This MEK dependency was observed in BRAF mutant cells regardless of tissue lineage, and correlated with both downregulation of cyclin D1 protein expression and the induction of G1 arrest. Pharmacological MEK inhibition completely abrogated tumour growth in BRAF mutant xenografts, whereas RAS mutant tumours were only partially inhibited. These data suggest an exquisite dependency on MEK activity in BRAF mutant tumours, and offer a rational therapeutic strategy for this genetically defined tumour subtype.

James A Fagin - One of the best experts on this subject based on the ideXlab platform.

  • relief of feedback inhibition of her3 transcription by raf and mek inhibitors attenuates their antitumor effects in braf mutant thyroid carcinomas
    Cancer Discovery, 2013
    Co-Authors: Cristina Monteroconde, Neal Rosen, Jeffrey A Knauf, Sergio Ruizllorente, Jose M Dominguez, Agnes Viale, Eric J Sherman, Mabel Ryder, Ronald Ghossein, James A Fagin
    Abstract:

    The RAF inhibitor vemurafenib (PLX4032) increases survival in patients with BRAF-mutant metastatic melanoma, but has limited efficacy in patients with colorectal cancers. Thyroid cancer cells are also comparatively refractory to RAF inhibitors. By contrast to melanomas, inhibition of MAPK signaling by vemurafenib is transient in thyroid and colorectal cancer cells. The rebound in ERK in thyroid cells is accompanied by increased HER3 signaling caused by induction of HER3 transcription through decreased promoter occupancy by the transcriptional repressors CtBP1 and 2, and by autocrine secretion of neuregulin-1. The HER kinase inhibitor lapatinib prevents MAPK rebound and sensitizes BRAF-mutant thyroid cancer cells to RAF or MEK inhibitors. This provides a rationale for combining ERK pathway antagonists with inhibitors of feedback-reactivated HER signaling in this disease. The determinants of primary resistance to MAPK inhibitors vary between cancer types, due to preferential upregulation of specific RTKs, and the abundance of their respective ligands.

  • braf mediates ret ptc induced mitogen activated protein kinase activation in thyroid cells functional support for requirement of the ret ptc ras braf pathway in papillary thyroid carcinogenesis
    Endocrinology, 2006
    Co-Authors: Norisato Mitsutake, Makoto Miyagishi, Shin Mitsutake, Nagako Akeno, Cleo Mesa, Jeffrey A Knauf, Lei Zhang, Kazunari Taira, James A Fagin
    Abstract:

    In human papillary thyroid cancers (PTCs), mutations of RET/PTC, NTRK, RAS, or BRAF are found in about two thirds of cases with practically no overlap, providing genetic evidence that constitutive signaling along RET-RAS-BRAF-MAPK is key to their development. The requirement for BRAF in RET/PTC-mediated MAPK activation and gene expression has not been tested functionally. There are three RAF isoforms: ARAF, BRAF, and CRAF. Compared with the others, ARAF is a much weaker stimulator of MAPK. To determine the key RAF isoform mediating RET/PTC-induced ERK phosphorylation, we stably transfected doxycycline-inducible RET/PTC3-expressing thyroid PCCL3 cells with small interfering RNA vectors to induce selective knockdown of BRAF or CRAF. Conditional RET/PTC3 expression induced comparable ERK phosphorylation in CRAF knockdown and control cells but negligible ERK phosphorylation in BRAF knockdown cells. Selective knockdown of BRAF prevented RET/PTC-dependent down-regulation of the sodium iodide symporter, a gene t...

  • braf mediates ret ptc induced mitogen activated protein kinase activation in thyroid cells functional support for requirement of the ret ptc ras braf pathway in papillary thyroid carcinogenesis
    Endocrinology, 2006
    Co-Authors: Norisato Mitsutake, Makoto Miyagishi, Shin Mitsutake, Nagako Akeno, Cleo Mesa, Jeffrey A Knauf, Lei Zhang, Kazunari Taira, James A Fagin
    Abstract:

    In human papillary thyroid cancers (PTCs), mutations of RET/PTC, NTRK, RAS, or BRAF are found in about two thirds of cases with practically no overlap, providing genetic evidence that constitutive signaling along RET-RAS-BRAF-MAPK is key to their development. The requirement for BRAF in RET/PTC-mediated MAPK activation and gene expression has not been tested functionally. There are three RAF isoforms: ARAF, BRAF, and CRAF. Compared with the others, ARAF is a much weaker stimulator of MAPK. To determine the key RAF isoform mediating RET/PTC-induced ERK phosphorylation, we stably transfected doxycycline-inducible RET/PTC3-expressing thyroid PCCL3 cells with small interfering RNA vectors to induce selective knockdown of BRAF or CRAF. Conditional RET/PTC3 expression induced comparable ERK phosphorylation in CRAF knockdown and control cells but negligible ERK phosphorylation in BRAF knockdown cells. Selective knockdown of BRAF prevented RET/PTC-dependent down-regulation of the sodium iodide symporter, a gene that confers key biological effects of RET/PTC in PTCs. Moreover, microarray analysis revealed numerous RET/PTC-regulated genes showing requirement of BRAF for appropriate expression. These data indicate that BRAF is required for RET/PTC-induced MAPK activation in thyroid cells and support the notion that BRAF inactivation may be an attractive target for PTCs.

Ashli Davis - One of the best experts on this subject based on the ideXlab platform.

  • tumour micro environment elicits innate resistance to raf inhibitors through hgf secretion
    Nature, 2012
    Co-Authors: Ravid Straussman, Ashli Davis, Margaret M. Mongare, Joshua Gould, Jinyan Du, Kevin Shee, Teppei Morikawa, Zhi Rong Qian, Michal Barzilyrokni, Dennie T Frederick
    Abstract:

    The secretion of hepatocyte growth factor by stromal cells in the tumour micro-environment can make melanoma resistant to RAF inhibitors, through the activation of the MET signalling pathway, but a combination of RAF and MET inhibitors can overcome this resistance. Targeted anticancer therapies are gaining ground in clinical applications as researchers begin to understand the genetic changes underlying tumorigenesis and the factors that determine an individual patient's response to a drug. However, resistance is a challenging problem in most clinical trials of targeted therapies. Two complementary papers show that the secretion of growth factors from the tumour microenvironment can cause resistance to a range of anticancer drugs, through their ability to drive tumour growth by activating redundant signalling pathways. Both papers provide evidence that stromal production of hepatocyte growth factor can confer resistance to a class of drugs called BRAF inhibitors, such as vemurafenib, in patients with melanoma, through activation of the MET signalling pathway. They show that a combination of BRAF and MET inhibitors can overcome resistance, suggesting that this combination should be tested in patients with melanoma. Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance1,2,3,4. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)–AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.

  • abstract 4837 tumor microenvironment contributes to raf inhibitor resistance through hgf secretion
    Cancer Research, 2012
    Co-Authors: Ravid Straussman, Ashli Davis, Joshua Gould, Jinyan Du, Kevin Shee, Teppei Morikawa, Keith T Flaherty, Michal Barzilyronki, Shuji Ogino, Jennifer A Wargo
    Abstract:

    Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Drug resistance remains a vexing problem in the treatment of cancer patients. While many studies have focused on cell autonomous mechanisms of drug resistance, we hypothesized that the tumor microenvironment may confer innate resistance to therapy. We employed a novel co-culture assay to systematically assay the ability of 23 stromal cell types to influence the sensitivity of 45 cancer cell lines to 35 anti-cancer drugs testing more than 15,000 cancer-stroma-drug combinations. We found that stroma-mediated resistance is surprisingly common - particularly to targeted chemotherapies. We decided to further characterize the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibition because most patients exhibit some degree of innate resistance. Proteomic analysis showed that stromal secretion of the growth factor hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the MAPK and PI3K/AKT pathways, and immediate resistance to RAF inhibition. Immunohistochemistry confirmed tumor stromal HGF expression in 11 out of 15 patients with BRAF-mutant melanoma. Among these patients, only one experienced a complete response, and that patient was negative for stromal HGF, consistent with our prediction of HGF-mediated RAF-inhibitor resistance. In contrast, of 6 evaluable patients with stable disease 4 had particularly high levels of stromal HGF expression. Dual inhibition of RAF and MET in BRAF-mutant melanoma cell lines resulted in reversal of drug resistance, suggesting RAF/MET combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. Interestingly, the activation of MET in these cell lines was cell autonomous, caused, for example, by autocrine secretion of HGF and resulting in complete resistance to RAF inhibitors even in the absence of stromal cells. Treatment with a MET inhibitor re-sensitized these cell lines to RAF inhibitors suggesting the same RAF/MET combination therapy for non-melanoma BRAF mutant cancers as well. More generally, these studies indicate that the systematic dissection of tumor-microenvironment interactions may reveal important mechanisms underlying drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4837. doi:1538-7445.AM2012-4837

  • Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion
    Nature, 2012
    Co-Authors: Ravid Straussman, Michal Barzily-rokni, Ashli Davis, Margaret M. Mongare, Joshua Gould, Jinyan Du, Kevin Shee, Teppei Morikawa, Zhi Rong Qian, Dennie T Frederick
    Abstract:

    Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)-AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.