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Flaherty, Keith

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Flaherty

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Keith

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Flaherty, Keith
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    Evolution of Delayed Resistance to Immunotherapy in a Melanoma Responder
    (Springer Nature, 2021-05-03) Liu, David; Lin, Jia-Ren; Robitschek, Emily; Kasumova, Gyulnara; Heyde, Alexander; Shi, Alvin; Kraya, Adam; Zhang, Gao; Moll, Tabea; Frederick, Dennie; Chen, Yu-An; Schapiro, Denis; Ho, Li-Lun; Bi, Kevin; Sahu, Avinash; Mei, Shaolin; Miao, Benchun; Sharova, Tatyana; Alvarez-Breckenridge, Christopher; Stocking, Jackson; Kim, Tommy; Fadden, Riley; Lawrence, Donald; Hoang, Mai; Cahill, Daniel; Maleh Mir, Mohsen; Nowak, Martin; Brastianos, Priscilla; Lian, Christine; Ruppin, Eytan; Izar, Benjamin; Herlyn, Meenhard; Van Allen, Eliezer; Nathanson, Katherine; Flaherty, Keith; Sullivan, Ryan; Kellis, Manolis; Sorger, Peter; Boland, Genevieve
    Despite initial responses, most melanoma patients develop resistance to immune checkpoint blockade (ICB). To understand the evolution of resistance, we studied 37 tumor samples over 9 years from a metastatic melanoma patient with exceptional response followed by delayed recurrence and death. Phylogenetic analysis revealed co-evolution of 7 lineages with multiple convergent, but independent resistance-associated alterations (RAAs). All recurrent tumors emerged from a lineage characterized by loss of chromosome 15q, with post-treatment clones acquiring additional genomic driver events. Deconvolution of bulk RNAseq and highly-multiplexed immunofluorescence (t-CyCIF) revealed differences in immune composition amongst different lineages. Imaging revealed a vasculogenic mimicry phenotype in NGFR-High tumor cells with high PD-L1 expression in close proximity to immune cells. Rapid autopsy demonstrated 2 distinct NGFR spatial patterns with high polarity and proximity to immune cells in subcutaneous tumors versus a diffuse spatial pattern in lung tumors, suggesting different roles of this neural crest-like program in different tumor microenvironments. Broadly, this study establishes a high-resolution map of the evolutionary dynamics of resistance to ICB, characterizes a de-differentiated, neural crest tumor population in melanoma immunotherapy resistance, and describes site specific differences in tumor-immune interactions via longitudinal analysis of a melanoma patient with an unusual clinical course.
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    Landscape of Targeted Anti-Cancer Drug Synergies in Melanoma Identifies a Novel BRAF-VEGFR/PDGFR Combination Treatment
    (Public Library of Science, 2015) Friedman, Adam A.; Amzallag, Arnaud; Pruteanu-Malinici, Iulian; Baniya, Subash; Cooper, Zachary A.; Piris, Adriano; Hargreaves, Leeza; Igras, Vivien; Frederick, Dennie T.; Lawrence, Donald; Haber, Daniel; Flaherty, Keith; Wargo, Jennifer A.; Ramaswamy, Sridhar; Benes, Cyril; Fisher, David
    A newer generation of anti-cancer drugs targeting underlying somatic genetic driver events have resulted in high single-agent or single-pathway response rates in selected patients, but few patients achieve complete responses and a sizeable fraction of patients relapse within a year. Thus, there is a pressing need for identification of combinations of targeted agents which induce more complete responses and prevent disease progression. We describe the results of a combination screen of an unprecedented scale in mammalian cells performed using a collection of targeted, clinically tractable agents across a large panel of melanoma cell lines. We find that even the most synergistic drug pairs are effective only in a discrete number of cell lines, underlying a strong context dependency for synergy, with strong, widespread synergies often corresponding to non-specific or off-target drug effects such as multidrug resistance protein 1 (MDR1) transporter inhibition. We identified drugs sensitizing cell lines that are BRAFV600E mutant but intrinsically resistant to BRAF inhibitor PLX4720, including the vascular endothelial growth factor receptor/kinase insert domain receptor (VEGFR/KDR) and platelet derived growth factor receptor (PDGFR) family inhibitor cediranib. The combination of cediranib and PLX4720 induced apoptosis in vitro and tumor regression in animal models. This synergistic interaction is likely due to engagement of multiple receptor tyrosine kinases (RTKs), demonstrating the potential of drug- rather than gene-specific combination discovery approaches. Patients with elevated biopsy KDR expression showed decreased progression free survival in trials of mitogen-activated protein kinase (MAPK) kinase pathway inhibitors. Thus, high-throughput unbiased screening of targeted drug combinations, with appropriate library selection and mechanistic follow-up, can yield clinically-actionable drug combinations.
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    Loss of cohesin complex components STAG2 or STAG3 confers resistance to BRAF inhibition in melanoma
    (2016) Shen, Che-Hung; Kim, Sun Hye; Trousil, Sebastian; Frederick, Dennie T.; Piris, Adriano; Yuan, Ping; Cai, Li; Gu, Lei; Li, Man; Lee, Jung Hyun; Mitra, Devarati; Fisher, David; Sullivan, Ryan; Flaherty, Keith; Zheng, Bin
    The protein kinase V-Raf murine sarcoma viral oncogene homolog B (BRAF) is an oncogenic driver and therapeutic target in melanoma. Inhibitors of BRAF (BRAFi) have shown high response rates and extended survival in melanoma patients bearing tumors that express BRAF Val600 mutations, but a vast majority of these patients develop drug resistance. Here we show that loss of Stromal antigen 2 or 3 (STAG2 or STAG3), which encode subunits of the cohesin complex, in melanoma cells results in resistance to BRAFi. We identified loss-of-function mutations in STAG2 as well as decreased expression of STAG2 or STAG3 proteins in several tumor samples from patients with acquired resistance to BRAFi and in BRAFi-resistant melanoma cell lines. Knockdown of STAG2 or STAG3 decreased sensitivity of Val600Glu BRAF-mutant melanoma cells and xenograft tumors to BRAFi. Loss of STAG2 inhibited CCCTC-binding factor (CTCF)-mediated expression of dual specificity phosphatase 6 (DUSP6), leading to reactivation of ERK signaling. Our studies unveil a previously unknown genetic mechanism of BRAFi resistance and provide new insights into the tumor suppressor function of STAG2 and STAG3.
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    Overcoming sorafenib evasion in hepatocellular carcinoma using CXCR4-targeted nanoparticles to co-deliver MEK-inhibitors
    (Nature Publishing Group, 2017) Chen, Yunching; Liu, Ya-Chi; Sung, Yun-Chieh; Ramjiawan, Rakesh R.; Lin, Ts-Ting; Chang, Chih-Chun; Jeng, Kuo-Shyang; Chang, Chiung-Fang; Liu, Chun-Hung; Gao, Dong-Yu; Hsu, Fu-Fei; Duyverman, Annique M.; Kitahara, Shuji; Huang, Peigen; Dima, Simona; Popescu, Irinel; Flaherty, Keith; Zhu, Andrew; Bardeesy, Nabeel; Jain, Rakesh; Benes, Cyril; Duda, Dan
    Sorafenib is a RAF inhibitor approved for several cancers, including hepatocellular carcinoma (HCC). Inhibition of RAF kinases can induce a dose-dependent “paradoxical” upregulation of the downstream mitogen-activated protein kinase (MAPK) pathway in cancer cells. It is unknown whether “paradoxical” ERK activation occurs after sorafenib therapy in HCC, and if so, if it impacts the therapeutic efficacy. Here, we demonstrate that RAF inhibition by sorafenib rapidly leads to RAF dimerization and ERK activation in HCCs, which contributes to treatment evasion. The transactivation of RAF dimers and ERK signaling promotes HCC cell survival, prevents apoptosis via downregulation of BIM and achieves immunosuppression by MAPK/NF-kB-dependent activation of PD-L1 gene expression. To overcome treatment evasion and reduce systemic effects, we developed CXCR4-targeted nanoparticles to co-deliver sorafenib with the MEK inhibitor AZD6244 in HCC. Using this approach, we preferentially and efficiently inactivated RAF/ERK, upregulated BIM and down-regulated PD-L1 expression in HCC, and facilitated intra-tumoral infiltration of cytotoxic CD8+ T cells. These effects resulted in a profound delay in tumor growth. Thus, this nano-delivery strategy to selectively target tumors and prevent the paradoxical ERK activation could increase the feasibility of dual RAF/MEK inhibition to overcome sorafenib treatment escape in HCC.
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    A first‐in‐human phase I, multicenter, open‐label, dose‐escalation study of the oral RAF/VEGFR‐2 inhibitor (RAF265) in locally advanced or metastatic melanoma independent from BRAF mutation status
    (John Wiley and Sons Inc., 2017) Izar, Benjamin; Sharfman, William; Hodi, F. Stephen; Lawrence, Donald; Flaherty, Keith; Amaravadi, Ravi; Kim, Kevin B.; Puzanov, Igor; Sosman, Jeffrey; Dummer, Reinhard; Goldinger, Simone M.; Lam, Lyhping; Kakar, Shefali; Tang, Zhongwen; Krieter, Oliver; McDermott, David; Atkins, Michael B.
    Abstract To establish the maximum tolerated dose (MTD), dose‐limiting toxicities (DLT), safety profile, and anti‐tumor efficacy of RAF265. We conducted a multicenter, open‐label, phase‐I, dose‐escalation trial of RAF265, an orally available RAF kinase/VEGFR‐2 inhibitor, in patients with advanced or metastatic melanoma. Pharmacokinetic (PK) analysis, pharmacodynamics (PD) and tumor response assessment were conducted. We evaluated metabolic tumor response by 18[F]‐fluorodeoxyglucose‐positron‐emission tomography (FDG‐PET), tissue biomarkers using immunohistochemistry (IHC), and modulators of angiogenesis. RAF265 has a serum half‐life of approximately 200 h. The MTD was 48 mg once daily given continuously. Among 77 patients, most common treatment‐related adverse effects were fatigue (52%), diarrhea (34%), weight loss (31%) and vitreous floaters (27%). Eight of 66 evaluable patients (12.1%) had an objective response, including seven partial and one complete response. Responses occurred in BRAF‐mutant and BRAF wild‐type (WT) patients. Twelve of 58 (20.7%) evaluable patients had a partial metabolic response. On‐treatment versus pretreatment IHC staining in 23 patients showed dose‐dependent p‐ERK inhibition. We observed a significant temporal increase in placental growth factor levels and decrease in soluble vascular endothelial growth factor receptor 2 (sVEGFR‐2) levels in all dose levels. RAF265 is an oral RAF/VEGFR‐2 inhibitor that produced antitumor responses, metabolic responses, and modulated angiogenic growth factor levels. Antitumor activity occurred in patients with BRAF‐mutant and BRAF‐WT disease. Despite low activity at tolerable doses, this study provides a framework for the development of pan‐RAF inhibitors and modulators of angiogenesis for the treatment of melanoma.
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    Mechanisms of resistance to immune checkpoint inhibitors
    (Nature Publishing Group, 2018) Jenkins, Russell; Barbie, David A; Flaherty, Keith
    Immune checkpoint inhibitors (ICI) targeting CTLA-4 and the PD-1/PD-L1 axis have shown unprecedented clinical activity in several types of cancer and are rapidly transforming the practice of medical oncology. Whereas cytotoxic chemotherapy and small molecule inhibitors (‘targeted therapies’) largely act on cancer cells directly, immune checkpoint inhibitors reinvigorate anti-tumour immune responses by disrupting co-inhibitory T-cell signalling. While resistance routinely develops in patients treated with conventional cancer therapies and targeted therapies, durable responses suggestive of long-lasting immunologic memory are commonly seen in large subsets of patients treated with ICI. However, initial response appears to be a binary event, with most non-responders to single-agent ICI therapy progressing at a rate consistent with the natural history of disease. In addition, late relapses are now emerging with longer follow-up of clinical trial populations, suggesting the emergence of acquired resistance. As robust biomarkers to predict clinical response and/or resistance remain elusive, the mechanisms underlying innate (primary) and acquired (secondary) resistance are largely inferred from pre-clinical studies and correlative clinical data. Improved understanding of molecular and immunologic mechanisms of ICI response (and resistance) may not only identify novel predictive and/or prognostic biomarkers, but also ultimately guide optimal combination/sequencing of ICI therapy in the clinic. Here we review the emerging clinical and pre-clinical data identifying novel mechanisms of innate and acquired resistance to immune checkpoint inhibition.
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    Tumor-associated B-cells induce tumor heterogeneity and therapy resistance
    (Nature Publishing Group UK, 2017) Somasundaram, Rajasekharan; Zhang, Gao; Fukunaga-Kalabis, Mizuho; Perego, Michela; Krepler, Clemens; Xu, Xiaowei; Wagner, Christine; Hristova, Denitsa; Zhang, Jie; Tian, Tian; Wei, Zhi; Liu, Qin; Garg, Kanika; Griss, Johannes; Hards, Rufus; Maurer, Margarita; Hafner, Christine; Mayerhöfer, Marius; Karanikas, Georgios; Jalili, Ahmad; Bauer-Pohl, Verena; Weihsengruber, Felix; Rappersberger, Klemens; Koller, Josef; Lang, Roland; Hudgens, Courtney; Chen, Guo; Tetzlaff, Michael; Wu, Lawrence; Frederick, Dennie Tompers; Scolyer, Richard A.; Long, Georgina V.; Damle, Manashree; Ellingsworth, Courtney; Grinman, Leon; Choi, Harry; Gavin, Brian J.; Dunagin, Margaret; Raj, Arjun; Scholler, Nathalie; Gross, Laura; Beqiri, Marilda; Bennett, Keiryn; Watson, Ian; Schaider, Helmut; Davies, Michael A.; Wargo, Jennifer; Czerniecki, Brian J.; Schuchter, Lynn; Herlyn, Dorothee; Flaherty, Keith; Herlyn, Meenhard; Wagner, Stephan N.
    In melanoma, therapies with inhibitors to oncogenic BRAFV600E are highly effective but responses are often short-lived due to the emergence of drug-resistant tumor subpopulations. We describe here a mechanism of acquired drug resistance through the tumor microenvironment, which is mediated by human tumor-associated B cells. Human melanoma cells constitutively produce the growth factor FGF-2, which activates tumor-infiltrating B cells to produce the growth factor IGF-1. B-cell-derived IGF-1 is critical for resistance of melanomas to BRAF and MEK inhibitors due to emergence of heterogeneous subpopulations and activation of FGFR-3. Consistently, resistance of melanomas to BRAF and/or MEK inhibitors is associated with increased CD20 and IGF-1 transcript levels in tumors and IGF-1 expression in tumor-associated B cells. Furthermore, first clinical data from a pilot trial in therapy-resistant metastatic melanoma patients show anti-tumor activity through B-cell depletion by anti-CD20 antibody. Our findings establish a mechanism of acquired therapy resistance through tumor-associated B cells with important clinical implications.
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    Academic Cancer Center Phase I Program Development
    (AlphaMed Press, 2017) Frankel, Arthur E.; Flaherty, Keith; Weiner, George J.; Chen, Robert; Azad, Nilofer S.; Pishvaian, Michael J.; Thompson, John A.; Taylor, Matthew H.; Mahadevan, Daruka; Lockhart, A. Craig; Vaishampayan, Ulka N.; Berlin, Jordan D.; Smith, David C.; Sarantopoulos, John; Riese, Matthew; Saleh, Mansoor N.; Ahn, Chul; Frenkel, Eugene P.
    Abstract Multiple factors critical to the effectiveness of academic phase I cancer programs were assessed among 16 academic centers in the U.S. Successful cancer centers were defined as having broad phase I and I/II clinical trial portfolios, multiple investigator‐initiated studies, and correlative science. The most significant elements were institutional philanthropic support, experienced clinical research managers, robust institutional basic research, institutional administrative efforts to reduce bureaucratic regulatory delays, phase I navigators to inform patients and physicians of new studies, and a large cancer center patient base. New programs may benefit from a separate stand‐alone operation, but mature phase I programs work well when many of the activities are transferred to disease‐oriented teams. The metrics may be useful as a rubric for new and established academic phase I programs.
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    Co‐targeting BET and MEK as salvage therapy for MAPK and checkpoint inhibitor‐resistant melanoma
    (John Wiley and Sons Inc., 2018) Echevarría‐Vargas, Ileabett M; Reyes‐Uribe, Patricia I; Guterres, Adam N; Yin, Xiangfan; Kossenkov, Andrew V; Liu, Qin; Zhang, Gao; Krepler, Clemens; Cheng, Chaoran; Wei, Zhi; Somasundaram, Rajasekharan; Karakousis, Giorgos; Xu, Wei; Morrissette, Jennifer JD; Lu, Yiling; Mills, Gordon B; Sullivan, Ryan; Benchun, Miao; Frederick, Dennie T; Boland, Genevieve; Flaherty, Keith; Weeraratna, Ashani T; Herlyn, Meenhard; Amaravadi, Ravi; Schuchter, Lynn M; Burd, Christin E; Aplin, Andrew E; Xu, Xiaowei; Villanueva, Jessie
    Abstract Despite novel therapies for melanoma, drug resistance remains a significant hurdle to achieving optimal responses. NRAS‐mutant melanoma is an archetype of therapeutic challenges in the field, which we used to test drug combinations to avert drug resistance. We show that BET proteins are overexpressed in NRAS‐mutant melanoma and that high levels of the BET family member BRD4 are associated with poor patient survival. Combining BET and MEK inhibitors synergistically curbed the growth of NRAS‐mutant melanoma and prolonged the survival of mice bearing tumors refractory to MAPK inhibitors and immunotherapy. Transcriptomic and proteomic analysis revealed that combining BET and MEK inhibitors mitigates a MAPK and checkpoint inhibitor resistance transcriptional signature, downregulates the transcription factor TCF19, and induces apoptosis. Our studies demonstrate that co‐targeting MEK and BET can offset therapy resistance, offering a salvage strategy for melanomas with no other therapeutic options, and possibly other treatment‐resistant tumor types.
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    Distinct histone modifications denote early stress-induced drug tolerance in cancer
    (Impact Journals LLC, 2018) Al Emran, Abdullah; Marzese, Diego M.; Menon, Dinoop Ravindran; Stark, Mitchell S.; Torrano, Joachim; Hammerlindl, Heinz; Zhang, Gao; Brafford, Patricia; Salomon, Matthew P.; Nelson, Nellie; Hammerlindl, Sabrina; Gupta, Deepesh; Mills, Gordon B.; Lu, Yiling; Sturm, Richard A.; Flaherty, Keith; Hoon, Dave S. B.; Gabrielli, Brian; Herlyn, Meenhard; Schaider, Helmut
    Besides somatic mutations or drug efflux, epigenetic reprogramming can lead to acquired drug resistance. We recently have identified early stress-induced multi-drug tolerant cancer cells termed induced drug-tolerant cells (IDTCs). Here, IDTCs were generated using different types of cancer cell lines; melanoma, lung, breast and colon cancer. A common loss of the H3K4me3 and H3K27me3 and gain of H3K9me3 mark was observed as a significant response to drug exposure or nutrient starvation in IDTCs. These epigenetic changes were reversible upon drug holidays. Microarray, qRT-PCR and protein expression data confirmed the up-regulation of histone methyltransferases (SETDB1 and SETDB2) which contribute to the accumulation of H3K9me3 concomitantly in the different cancer types. Genome-wide studies suggest that transcriptional repression of genes is due to concordant loss of H3K4me3 and regional increment of H3K9me3. Conversely, genome-wide CpG site-specific DNA methylation showed no common changes at the IDTC state. This suggests that distinct histone methylation patterns rather than DNA methylation are driving the transition from parental to IDTCs. In addition, silencing of SETDB1/2 reversed multi drug tolerance. Alterations of histone marks in early multi-drug tolerance with an increment in H3K9me3 and loss of H3K4me3/H3K27me3 is neither exclusive for any particular stress response nor cancer type specific but rather a generic response.