Publications by authors named "Jessie Villanueva"

37 Publications

Targeting mTOR signaling overcomes acquired resistance to combined BRAF and MEK inhibition in BRAF-mutant melanoma.

Oncogene 2021 Sep 24;40(37):5590-5599. Epub 2021 Jul 24.

Department of Biology, School of Arts & Sciences, University of Pennsylvania, Philadelphia, PA, USA.

Targeting MAPK pathway using a combination of BRAF and MEK inhibitors is an efficient strategy to treat melanoma harboring BRAF-mutation. The development of acquired resistance is inevitable due to the signaling pathway rewiring. Combining western blotting, immunohistochemistry, and reverse phase protein array (RPPA), we aim to understanding the role of the mTORC1 signaling pathway, a center node of intracellular signaling network, in mediating drug resistance of BRAF-mutant melanoma to the combination of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) therapy. The mTORC1 signaling pathway is initially suppressed by BRAFi and MEKi combination in melanoma but rebounds overtime after tumors acquire resistance to the combination therapy (CR) as assayed in cultured cells and PDX models. In vitro experiments showed that a subset of CR melanoma cells was sensitive to mTORC1 inhibition. The mTOR inhibitors, rapamycin and NVP-BEZ235, induced cell cycle arrest and apoptosis in CR cell lines. As a proof-of-principle, we demonstrated that rapamycin and NVP-BEZ235 treatment reduced tumor growth in CR xenograft models. Mechanistically, AKT or ERK contributes to the activation of mTORC1 in CR cells, depending on PTEN status of these cells. Our study reveals that mTOR activation is essential for drug resistance of melanoma to MAPK inhibitors, and provides insight into the rewiring of the signaling networks in CR melanoma.
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http://dx.doi.org/10.1038/s41388-021-01911-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8445818PMC
September 2021

Melanoma models for the next generation of therapies.

Cancer Cell 2021 May 4;39(5):610-631. Epub 2021 Feb 4.

Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, and Department of Oncology, Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.

There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.
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http://dx.doi.org/10.1016/j.ccell.2021.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8378471PMC
May 2021

Targeting telomerase for cancer therapy.

Oncogene 2020 09 30;39(36):5811-5824. Epub 2020 Jul 30.

Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA.

Telomere maintenance via telomerase reactivation is a nearly universal hallmark of cancer cells which enables replicative immortality. In contrast, telomerase activity is silenced in most adult somatic cells. Thus, telomerase represents an attractive target for highly selective cancer therapeutics. However, development of telomerase inhibitors has been challenging and thus far there are no clinically approved strategies exploiting this cancer target. The discovery of prevalent mutations in the TERT promoter region in many cancers and recent advances in telomerase biology has led to a renewed interest in targeting this enzyme. Here we discuss recent efforts targeting telomerase, including immunotherapies and direct telomerase inhibitors, as well as emerging approaches such as targeting TERT gene expression driven by TERT promoter mutations. We also address some of the challenges to telomerase-directed therapies including potential therapeutic resistance and considerations for future therapeutic applications and translation into the clinical setting. Although much work remains to be done, effective strategies targeting telomerase will have a transformative impact for cancer therapy and the prospect of clinically effective drugs is boosted by recent advances in structural models of human telomerase.
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http://dx.doi.org/10.1038/s41388-020-01405-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7678952PMC
September 2020

PLX3397 inhibits the accumulation of intra-tumoral macrophages and improves bromodomain and extra-terminal inhibitor efficacy in melanoma.

Pigment Cell Melanoma Res 2020 03 11;33(2):372-377. Epub 2019 Dec 11.

Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA.

Bromodomain and extra-terminal inhibitors (BETi) delay tumor growth, in part, through tumor cell intrinsic alterations and initiation of anti-tumor CD8+ T-cell responses. By contrast, BETi effects on pro-tumoral immune responses remain unclear. Here, we show that the next-generation BETi, PLX51107, delayed tumor growth to differing degrees in Braf V600E melanoma syngeneic mouse models. These differential responses were associated with the influx of tumor-associated macrophages during BETi treatment. Tumors that were poorly responsive to PLX51107 showed increased influx of colony-stimulating factor-1 receptor (CSF-1R)-positive tumor-associated macrophages. We depleted CSF-1R+ tumor-associated macrophages with the CSF-1R inhibitor, PLX3397, in combination with PLX51107. Treatment with PLX3397 enhanced the efficacy of PLX51107 in poorly responsive Braf V600E syngeneic melanomas in vivo. These findings suggest that tumor-associated macrophage accumulation limits BETi efficacy and that co-treatment with PLX3397 can improve response to PLX51107, offering a potential novel combination therapy for metastatic melanoma patients.
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http://dx.doi.org/10.1111/pcmr.12845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028511PMC
March 2020

The next-generation BET inhibitor, PLX51107, delays melanoma growth in a CD8-mediated manner.

Pigment Cell Melanoma Res 2019 09 20;32(5):687-696. Epub 2019 May 20.

Department of Cancer Biology, Thomas Jefferson University, Philadelphia, Pennsylvania.

Epigenetic agents such as bromodomain and extra-terminal region inhibitors (BETi) slow tumor growth via tumor intrinsic alterations; however, their effects on antitumor immunity remain unclear. A recent advance is the development of next-generation BETi that are potent and display a favorable half-life. Here, we tested the BETi, PLX51107, for immune-based effects on tumor growth in BRAF V600E melanoma syngeneic models. PLX51107 delayed melanoma tumor growth and increased activated, proliferating, and functional CD8+ T cells in tumors leading to CD8+ T-cell-mediated tumor growth delay. PLX51107 decreased Cox2 expression, increased dendritic cells, and lowered PD-L1, FasL, and IDO-1 expression in the tumor microenvironment. Importantly, PLX51107 delayed the growth of tumors that progressed on anti-PD-1 therapy; a response associated with decreased Cox2 levels, decreased PD-L1 expression on non-immune cells, and increased intratumoral CD8+ T cells. Thus, next-generation BETi represent a potential first-line and secondary treatment strategy for metastatic melanoma by eliciting effects, at least in part, on antitumor CD8+ T cells.
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http://dx.doi.org/10.1111/pcmr.12788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697571PMC
September 2019

Tailoring Chemotherapy for the African-Centric S47 Variant of TP53.

Cancer Res 2018 10 16;78(19):5694-5705. Epub 2018 Aug 16.

Program in Molecular and Cellular Oncogenesis, The Wistar Institute, Philadelphia, Pennsylvania.

The tumor suppressor is the most frequently mutated gene in human cancer and serves to restrict tumor initiation and progression. Single-nucleotide polymorphisms (SNP) in and pathway genes can have a marked impact on p53 tumor suppressor function, and some have been associated with increased cancer risk and impaired response to therapy. Approximately 6% of Africans and 1% of African Americans express a p53 allele with a serine instead of proline at position 47 (Pro47Ser). This SNP impairs p53-mediated apoptosis in response to radiation and genotoxic agents and is associated with increased cancer risk in humans and in a mouse model. In this study, we compared the ability of wild-type (WT) and S47 p53 to suppress tumor development and respond to therapy. Our goal was to find therapeutic compounds that are more, not less, efficacious in S47 tumors. We identified the superior efficacy of two agents, cisplatin and BET inhibitors, on S47 tumors compared with WT. Cisplatin caused dramatic decreases in the progression of S47 tumors by activating the p53/PIN1 axis to drive the mitochondrial cell death program. These findings serve as important proof of principle that chemotherapy can be tailored to p53 genotype. A rare African-derived radioresistant p53 SNP provides proof of principle that chemotherapy can be tailored to TP53 genotype. .
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http://dx.doi.org/10.1158/0008-5472.CAN-18-1327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168343PMC
October 2018

COMBATING NRAS MUTANT MELANOMA: FROM BENCH TO BEDSIDE.

Melanoma Manag 2017 Dec 21;4(4):183-186. Epub 2017 Nov 21.

Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA.

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http://dx.doi.org/10.2217/mmt-2017-0023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959291PMC
December 2017

Exploiting TERT dependency as a therapeutic strategy for NRAS-mutant melanoma.

Oncogene 2018 07 26;37(30):4058-4072. Epub 2018 Apr 26.

Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA.

Targeting RAS is one of the greatest challenges in cancer therapy. Oncogenic mutations in NRAS are present in over 25% of melanomas and patients whose tumors harbor NRAS mutations have limited therapeutic options and poor prognosis. Thus far, there are no clinical agents available to effectively target NRAS or any other RAS oncogene. An alternative approach is to identify and target critical tumor vulnerabilities or non-oncogene addictions that are essential for tumor survival. We investigated the consequences of NRAS blockade in NRAS-mutant melanoma and show that decreased expression of the telomerase catalytic subunit, TERT, is a major consequence. TERT silencing or treatment of NRAS-mutant melanoma with the telomerase-dependent telomere uncapping agent, 6-thio-2'-deoxyguanosine (6-thio-dG), led to rapid cell death, along with evidence of both telomeric and non-telomeric DNA damage, increased ROS levels, and upregulation of a mitochondrial antioxidant adaptive response. Combining 6-thio-dG with the mitochondrial inhibitor Gamitrinib attenuated this adaptive response and more effectively suppressed NRAS-mutant melanoma. Our study uncovers a robust dependency of NRAS-mutant melanoma on TERT, and provides proof-of-principle for a new combination strategy to combat this class of tumors, which could be expanded to other tumor types.
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http://dx.doi.org/10.1038/s41388-018-0247-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6062502PMC
July 2018

Co-targeting BET and MEK as salvage therapy for MAPK and checkpoint inhibitor-resistant melanoma.

EMBO Mol Med 2018 05;10(5)

Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA

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 -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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http://dx.doi.org/10.15252/emmm.201708446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5938620PMC
May 2018

The landscape of BRAF transcript and protein variants in human cancer.

Mol Cancer 2017 04 28;16(1):85. Epub 2017 Apr 28.

Oncogenomics Unit, Core Research Laboratory, Istituto Toscano Tumori (ITT), AOUP, CNR-IFC, Via Moruzzi 1, 56124, Pisa, Italy.

Background: The BRAF protein kinase is widely studied as a cancer driver and therapeutic target. However, the regulation of its expression is not completely understood.

Results: Taking advantage of the RNA-seq data of more than 4800 patients belonging to 9 different cancer types, we show that BRAF mRNA exists as a pool of 3 isoforms (reference BRAF, BRAF-X1, and BRAF-X2) that differ in the last part of their coding sequences, as well as in the length (BRAF-ref: 76 nt; BRAF-X1 and BRAF-X2: up to 7 kb) and in the sequence of their 3'UTRs. The expression levels of BRAF-ref and BRAF-X1/X2 are inversely correlated, while the most prevalent among the three isoforms varies from cancer type to cancer type. In melanoma cells, the X1 isoform is expressed at the highest level in both therapy-naïve cells and cells with acquired resistance to vemurafenib driven by BRAF gene amplification or expression of the Δ[3-10] splicing variant. In addition to the BRAF-ref protein, the BRAF-X1 protein (the full length as well as the Δ[3-10] variant) is also translated. The expression levels of the BRAF-ref and BRAF-X1 proteins are similar, and together they account for BRAF functional activities. In contrast, the endogenous BRAF-X2 protein is hard to detect because the C-terminal domain is selectively recognized by the ubiquitin-proteasome pathway and targeted for degradation.

Conclusions: By shedding light on the repertoire of BRAF mRNA and protein variants, and on the complex regulation of their expression, our work paves the way to a deeper understanding of a crucially important player in human cancer and to a more informed development of new therapeutic strategies.
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http://dx.doi.org/10.1186/s12943-017-0645-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5410044PMC
April 2017

Chemically Linked Vemurafenib Inhibitors Promote an Inactive BRAF Conformation.

ACS Chem Biol 2016 10 6;11(10):2876-2888. Epub 2016 Sep 6.

Department of Biochemistry and Biophysics and the Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania , 421 Curie Blvd., Philadelphia, Pennsylvania 19104, United States.

The BRAF kinase, within the mitogen activated protein kinase (MAPK) signaling pathway, harbors activating mutations in about half of melanomas and to a significant extent in many other cancers. A single valine to glutamic acid substitution at residue 600 (BRAF) accounts for about 90% of these activating mutations. While BRAF-selective small molecule inhibitors, such as debrafenib and vemurafenib, have shown therapeutic benefit, almost all patients develop resistance. Resistance often arises through reactivation of the MAPK pathway, typically through mutation of upstream RAS, downstream MEK, or splicing variants. RAF kinases signal as homo- and heterodimers, and another complication associated with small molecule BRAF inhibition is drug-induced allosteric activation of a wild-type RAF subunit (BRAF or CRAF) of the kinase dimer, a process called "transactivation" or "paradoxical activation." Here, we used BRAF and vemurafenib as a model system to develop chemically linked kinase inhibitors to lock RAF dimers in an inactive conformation that cannot undergo transactivation. This structure-based design effort resulted in the development of Vem-BisAmide-2, a compound containing two vemurafenib molecules connected by a bis amide linker. We show that Vem-BisAmide-2 has comparable inhibitory potency as vemurafenib to BRAF both in vitro and in cells but promotes an inactive dimeric BRAF conformation unable to undergo transactivation. The crystal structure of a BRAF/Vem-BisAmide-2 complex and associated biochemical studies reveal the molecular basis for how Vem-BisAmide-2 mediates selectivity for an inactive over an active dimeric BRAF conformation. These studies have implications for targeting BRAF/RAF heterodimers and other kinase dimers for therapy.
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http://dx.doi.org/10.1021/acschembio.6b00529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5108658PMC
October 2016

Targeting Notch enhances the efficacy of ERK inhibitors in BRAF-V600E melanoma.

Oncotarget 2016 11;7(44):71211-71222

The Wistar Institute, Melanoma Research Center, Philadelphia, PA, USA.

The discovery of activating BRAF mutations in approximately 50% of melanomas has led to the development of MAPK pathway inhibitors, which have transformed melanoma therapy. However, not all BRAF-V600E melanomas respond to MAPK inhibition. Therefore, it is important to understand why tumors with the same oncogenic driver have variable responses to MAPK inhibitors. Here, we show that concurrent loss of PTEN and activation of the Notch pathway is associated with poor response to the ERK inhibitor SCH772984, and that co-inhibition of Notch and ERK decreased viability in BRAF-V600E melanomas. Additionally, patients with low PTEN and Notch activation had significantly shorter progression free survival when treated with BRAF inhibitors. Our studies provide a rationale to further develop combination strategies with Notch antagonists to maximize the efficacy of MAPK inhibition in melanoma. Our findings should prompt the evaluation of combinations co-targeting MAPK/ERK and Notch as a strategy to improve current therapies and warrant further evaluation of co-occurrence of aberrant PTEN and Notch activation as predictive markers of response to therapy.
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http://dx.doi.org/10.18632/oncotarget.12078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342073PMC
November 2016

Playing Polo-Like Kinase in NRAS-Mutant Melanoma.

J Invest Dermatol 2015 Oct;135(10):2352-2355

The Wistar Institute, Molecular and Cellular Oncogenesis Program & Melanoma Research Center, Philadelphia, Pennsylvania, USA. Electronic address:

NRAS-mutant melanomas are extremely aggressive and highly resistant to currently available therapeutic modalities. Hence, new targets and therapeutic strategies for NRAS-driven melanomas are needed. As blocking NRAS directly has not been possible thus far, targeting downstream NRAS effectors, such as MAPK/ERK kinase (MEK), is being evaluated as an alternative therapeutic approach. However, blocking this pathway alone has limited efficacy. In this issue, Posch et al. report on a combination approach co-targeting polo-like kinase 1 and MEK in NRAS-mutant melanomas. This combination triggers a dual blockade of the cell cycle machinery, leading to apoptosis, and providing a new strategy to treat NRAS-mutant melanoma.
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http://dx.doi.org/10.1038/jid.2015.253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568563PMC
October 2015

Development of organometallic S6K1 inhibitors.

J Med Chem 2015 Jan 13;58(1):305-14. Epub 2014 Nov 13.

The Wistar Institute , 3601 Spruce Street, Philadelphia, Pennsylvania 19104, United States.

Aberrant activation of S6 kinase 1 (S6K1) is found in many diseases, including diabetes, aging, and cancer. We developed ATP competitive organometallic kinase inhibitors, EM5 and FL772, which are inspired by the structure of the pan-kinase inhibitor staurosporine, to specifically inhibit S6K1 using a strategy previously used to target other kinases. Biochemical data demonstrate that EM5 and FL772 inhibit the kinase with IC50 value in the low nanomolar range at 100 μM ATP and that the more potent FL772 compound has a greater than 100-fold specificity over S6K2. The crystal structures of S6K1 bound to staurosporine, EM5, and FL772 reveal that the EM5 and FL772 inhibitors bind in the ATP binding pocket and make S6K1-specific contacts, resulting in changes to the p-loop, αC helix, and αD helix when compared to the staurosporine-bound structure. Cellular data reveal that FL772 is able to inhibit S6K phosphorylation in yeast cells. Together, these studies demonstrate that potent, selective, and cell permeable S6K1 inhibitors can be prepared and provide a scaffold for future development of S6K inhibitors with possible therapeutic applications.
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http://dx.doi.org/10.1021/jm5011868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4289024PMC
January 2015

Targeting ER stress-induced autophagy overcomes BRAF inhibitor resistance in melanoma.

J Clin Invest 2014 Mar 24;124(3):1406-17. Epub 2014 Feb 24.

Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAF(V600E) melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAF(V600E) melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.
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http://dx.doi.org/10.1172/JCI70454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3934165PMC
March 2014

Hypoxia induces phenotypic plasticity and therapy resistance in melanoma via the tyrosine kinase receptors ROR1 and ROR2.

Cancer Discov 2013 Dec 8;3(12):1378-93. Epub 2013 Oct 8.

1Tumor Metastasis and Microenvironment Program and 2Molecular and Cellular Oncogenesis Program, The Wistar Institute; 3Abramson Cancer Center, University of Pennsylvania, Philadelphia; 4Lehigh Valley Health Network, Allentown, Pennsylvania; 5The National Institute on Aging, NIH, Baltimore, Maryland; and 6Dana-Farber/Harvard Cancer Center, Boston, Massachusetts.

Unlabelled: An emerging concept in melanoma biology is that of dynamic, adaptive phenotype switching, where cells switch from a highly proliferative, poorly invasive phenotype to a highly invasive, less proliferative one. This switch may hold significant implications not just for metastasis, but also for therapy resistance. We demonstrate that phenotype switching and subsequent resistance can be guided by changes in expression of receptors involved in the noncanonical Wnt5A signaling pathway, ROR1 and ROR2. ROR1 and ROR2 are inversely expressed in melanomas and negatively regulate each other. Furthermore, hypoxia initiates a shift of ROR1-positive melanomas to a more invasive, ROR2-positive phenotype. Notably, this receptor switch induces a 10-fold decrease in sensitivity to BRAF inhibitors. In patients with melanoma treated with the BRAF inhibitor vemurafenib, Wnt5A expression correlates with clinical response and therapy resistance. These data highlight the fact that mechanisms that guide metastatic progression may be linked to those that mediate therapy resistance.

Significance: These data show for the fi rst time that a single signaling pathway, the Wnt signaling pathway, can effectively guide the phenotypic plasticity of tumor cells, when primed to do so by a hypoxic microenvironment. Importantly, this increased Wnt5A signaling can give rise to a subpopulation of highly invasive cells that are intrinsically less sensitive to novel therapies for melanoma, and targeting the Wnt5A/ROR2 axis could improve the efficacy and duration of response for patients with melanoma on vemurafenib.
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http://dx.doi.org/10.1158/2159-8290.CD-13-0005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918498PMC
December 2013

Concurrent MEK2 mutation and BRAF amplification confer resistance to BRAF and MEK inhibitors in melanoma.

Cell Rep 2013 Sep 19;4(6):1090-9. Epub 2013 Sep 19.

Molecular and Cellular Oncogenesis Program, Melanoma Research Center, The Wistar Institute, Philadelphia, PA 19104, USA. Electronic address:

Although BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF-V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it.
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http://dx.doi.org/10.1016/j.celrep.2013.08.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3956616PMC
September 2013

Overcoming intrinsic multidrug resistance in melanoma by blocking the mitochondrial respiratory chain of slow-cycling JARID1B(high) cells.

Cancer Cell 2013 Jun;23(6):811-25

Department of Dermatology, The Saarland University Hospital, D-66421 Homburg/Saar, Germany.

Despite success with BRAFV600E inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multidrug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (oxidative phosphorylation) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1B(high) subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anticancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation.
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http://dx.doi.org/10.1016/j.ccr.2013.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3810180PMC
June 2013

Highlights of the 2012 Congress of the Society for Melanoma Research, 8-11 November 2012, Hollywood, CA.

Melanoma Res 2013 Jun;23(3):237-40

Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

The 2012 Congress of the Society for Melanoma Research was attended by researchers with widespread expertise in basic, translational, and clinical research. Exciting research has led to the discovery of therapies to target mutations found in melanoma; however, it is clear that much still needs to be learned about how to use these therapies and the role of the microenvironment in therapy resistance and melanoma progression. This summary highlights recent discoveries in genetics and epigenetics, biology, immunotherapy, and targeted therapies for melanoma discussed at this year's meeting.
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http://dx.doi.org/10.1097/CMR.0b013e3283610528DOI Listing
June 2013

The anti-melanoma activity of dinaciclib, a cyclin-dependent kinase inhibitor, is dependent on p53 signaling.

PLoS One 2013 18;8(3):e59588. Epub 2013 Mar 18.

The Wistar Institute, Philadelphia, Pennsylvania, United States of America.

Although cyclin dependent kinase (CDK)-2 is known to be dispensable for the growth of most tumors, it is thought to be important for the proliferation of melanoma cells, where its expression is controlled by the melanocyte-lineage specific transcription factor MITF. Treatment of a panel of melanoma cells with the CDK inhibitor dinaciclib led to a concentration-dependent inhibition of growth under both 2D adherent and 3D organotypic cell culture conditions. Dinaciclib targeted melanoma cell lines regardless of cdk2 or MITF levels. Inhibition of growth was associated with a rapid induction of G2/M cell arrest and apoptosis. Treatment of human melanoma mouse xenografts with dinaciclib led to tumor regression associated with reduced retinoblastoma protein phosphorylation and Bcl-2 expression. Further mechanistic studies revealed that dinaciclib induces p53 expression whilst simultaneously downregulating the expression of the anti-apoptotic factors Mcl-1 and XIAP. To clarify the role of p53 activation in the dinaciclib-induced cell death, we generated melanoma cell lines in which p53 expression was knocked down using a shRNA lentiviral vector. Knockdown of p53 completely abolished the induction of apoptosis seen following dinaciclib treatment as shown by a lack of annexin-V staining and caspase-3 cleavage. Altogether, these data show that dinaciclib induces apoptosis in a large panel of melanoma cell lines through a mechanism requiring p53 expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059588PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3601112PMC
September 2013

The novel SMAC mimetic birinapant exhibits potent activity against human melanoma cells.

Clin Cancer Res 2013 Apr 12;19(7):1784-94. Epub 2013 Feb 12.

Tumor Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA 19104, USA.

Purpose: Inhibitor of apoptosis proteins (IAP) promote cancer cell survival and confer resistance to therapy. We report on the ability of second mitochondria-derived activator of caspases mimetic, birinapant, which acts as antagonist to cIAP1 and cIAP2, to restore the sensitivity to apoptotic stimuli such as TNF-α in melanomas.

Experimental Design: Seventeen melanoma cell lines, representing five major genetic subgroups of cutaneous melanoma, were treated with birinapant as a single agent or in combination with TNF-α. Effects on cell viability, target inhibition, and initiation of apoptosis were assessed and findings were validated in 2-dimensional (2D), 3D spheroid, and in vivo xenograft models.

Results: When birinapant was combined with TNF-α, strong combination activity, that is, neither compound was effective individually but the combination was highly effective, was observed in 12 of 18 cell lines. This response was conserved in spheroid models, whereas in vivo birinapant inhibited tumor growth without adding TNF-α in in vitro resistant cell lines. Birinapant combined with TNF-α inhibited the growth of a melanoma cell line with acquired resistance to BRAF inhibition to the same extent as in the parental cell line.

Conclusions: Birinapant in combination with TNF-α exhibits a strong antimelanoma effect in vitro. Birinapant as a single agent shows in vivo antitumor activity, even if cells are resistant to single agent therapy in vitro. Birinapant in combination with TNF-α is effective in a melanoma cell line with acquired resistance to BRAF inhibitors.
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http://dx.doi.org/10.1158/1078-0432.CCR-12-2518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3618495PMC
April 2013

A modified HSP70 inhibitor shows broad activity as an anticancer agent.

Mol Cancer Res 2013 Mar 9;11(3):219-29. Epub 2013 Jan 9.

Program in Molecular and Cellular Oncogenesis, The Wistar Institute, Philadelphia, PA 19104, USA.

The stress-induced HSP70 is an ATP-dependent molecular chaperone that plays a key role in refolding misfolded proteins and promoting cell survival following stress. HSP70 is marginally expressed in nontransformed cells, but is greatly overexpressed in tumor cells. Silencing HSP70 is uniformly cytotoxic to tumor but not normal cells; therefore, there has been great interest in the development of HSP70 inhibitors for cancer therapy. Here, we report that the HSP70 inhibitor 2-phenylethynesulfonamide (PES) binds to the substrate-binding domain of HSP70 and requires the C-terminal helical "lid" of this protein (amino acids 573-616) to bind. Using molecular modeling and in silico docking, we have identified a candidate binding site for PES in this region of HSP70, and we identify point mutants that fail to interact with PES. A preliminary structure-activity relationship analysis has revealed a derivative of PES, 2-(3-chlorophenyl) ethynesulfonamide (PES-Cl), which shows increased cytotoxicity and ability to inhibit autophagy, along with significantly improved ability to extend the life of mice with pre-B-cell lymphoma, compared with the parent compound (P = 0.015). Interestingly, we also show that these HSP70 inhibitors impair the activity of the anaphase promoting complex/cyclosome (APC/C) in cell-free extracts, and induce G2-M arrest and genomic instability in cancer cells. PES-Cl is thus a promising new anticancer compound with several notable mechanisms of action.
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http://dx.doi.org/10.1158/1541-7786.MCR-12-0547-TDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3606282PMC
March 2013

Functional profiling of live melanoma samples using a novel automated platform.

PLoS One 2012 28;7(12):e52760. Epub 2012 Dec 28.

BioMarker Strategies, 855 N. Wolfe St., Baltimore, MD 21205, USA.

Aims: This proof-of-concept study was designed to determine if functional, pharmacodynamic profiles relevant to targeted therapy could be derived from live human melanoma samples using a novel automated platform.

Methods: A series of 13 melanoma cell lines was briefly exposed to a BRAF inhibitor (PLX-4720) on a platform employing automated fluidics for sample processing. Levels of the phosphoprotein p-ERK in the mitogen-activated protein kinase (MAPK) pathway from treated and untreated sample aliquots were determined using a bead-based immunoassay. Comparison of these levels provided a determination of the pharmacodynamic effect of the drug on the MAPK pathway. A similar ex vivo analysis was performed on fine needle aspiration (FNA) biopsy samples from four murine xenograft models of metastatic melanoma, as well as 12 FNA samples from patients with metastatic melanoma.

Results: Melanoma cell lines with known sensitivity to BRAF inhibitors displayed marked suppression of the MAPK pathway in this system, while most BRAF inhibitor-resistant cell lines showed intact MAPK pathway activity despite exposure to a BRAF inhibitor (PLX-4720). FNA samples from melanoma xenografts showed comparable ex vivo MAPK activity as their respective cell lines in this system. FNA samples from patients with metastatic melanoma successfully yielded three categories of functional profiles including: MAPK pathway suppression; MAPK pathway reactivation; MAPK pathway stimulation. These profiles correlated with the anticipated MAPK activity, based on the known BRAF mutation status, as well as observed clinical responses to BRAF inhibitor therapy.

Conclusion: Pharmacodynamic information regarding the ex vivo effect of BRAF inhibitors on the MAPK pathway in live human melanoma samples can be reproducibly determined using a novel automated platform. Such information may be useful in preclinical and clinical drug development, as well as predicting response to targeted therapy in individual patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052760PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3532357PMC
June 2013

Control of tumor bioenergetics and survival stress signaling by mitochondrial HSP90s.

Cancer Cell 2012 Sep;22(3):331-44

Prostate Cancer Discovery and Development Program, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA.

Tumors successfully adapt to constantly changing intra- and extracellular environments, but the wirings of this process are still largely elusive. Here, we show that heat-shock-protein-90-directed protein folding in mitochondria, but not cytosol, maintains energy production in tumor cells. Interference with this process activates a signaling network that involves phosphorylation of nutrient-sensing AMP-activated kinase, inhibition of rapamycin-sensitive mTOR complex 1, induction of autophagy, and expression of an endoplasmic reticulum unfolded protein response. This signaling network confers a survival and proliferative advantage to genetically disparate tumors, and correlates with worse outcome in lung cancer patients. Therefore, mitochondrial heat shock protein 90s are adaptive regulators of tumor bioenergetics and tractable targets for cancer therapy.
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http://dx.doi.org/10.1016/j.ccr.2012.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3615709PMC
September 2012

Intratumoral heterogeneity as a therapy resistance mechanism: role of melanoma subpopulations.

Adv Pharmacol 2012 ;65:335-59

Molecular and Cellular Oncogenesis Program, Melanoma Research Center, The Wistar Institute, Philadelphia, USA.

Malignant melanoma is an aggressive form of skin cancer whose incidence continues to increase worldwide. Increased exposure to sun, ultraviolet radiation, and the use of tanning beds can increase the risk of melanoma. Early detection of melanomas is the key to successful treatment mainly through surgical excision of the primary tumor lesion. But in advanced stage melanomas, once the disease has spread beyond the primary site to distant organs, the tumors are difficult to treat and quickly develop resistance to most available forms of therapy. The advent of molecular and cellular techniques has led to a better characterization of tumor cells revealing the presence of heterogeneous melanoma subpopulations. The discovery of gene mutations and alterations of cell-signaling pathways in melanomas has led to the development of new targeted drugs that show dramatic response rates in patients. Single-agent therapies generally target one subpopulation of tumor cells while leaving others unharmed. The surviving subpopulations will have the ability to repopulate the original tumors that can continue to progress. Thus, a rational approach to target multiple subpopulations of tumor cells with a combination of drugs instead of single-agent therapy will be necessary for long-lasting inhibition of melanoma lesions. In this context, the recent development of immune checkpoint reagents provides an additional armor that can be used in combination with targeted drugs to expand the presence of melanoma reactive T cells in circulation to prevent tumor recurrence.
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http://dx.doi.org/10.1016/B978-0-12-397927-8.00011-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3677516PMC
February 2013

Cytotoxicity of new duplex drugs linking 3'-C-ethynylcytidine and 5-fluor-2'-deoxyuridine against human melanoma cells.

Int J Cancer 2012 Nov 15;131(9):2165-74. Epub 2012 Mar 15.

Department of Gynecology and Obstetrics, the National Centre of Tumor Disease, University of Heidelberg, Heidelberg, Germany.

Melanoma is an increasingly common and potentially fatal malignancy of the skin and some mucous membranes. As no cure exists for metastatic disease, there is an urgent need for novel drugs. 2'-Deoxy-5-fluorouridylyl-(3'-5')-3'-C-ethynylcytidine [5-FdU(3'-5')ECyd] and 3'-C-ethynylcytidinylyl-(5' → 1-O)-2-O-octadecyl-sn-glycerylyl-(3-O → 5')-2'-deoxy-5-fluorouridine [ECyd-lipid-5-FdU] represent cytostatic active duplex drugs, which can be metabolized into various active antimetabolites. We evaluated the cytotoxicity of these heterodinucleoside phosphate analogs, their corresponding monomers ECyd and 5-FdU and combinations thereof on six metastatic melanoma cell lines and six ex vivo patient-derived melanoma cells in comparison to current standard cytostatic agents and the BRAF V600E inhibitor Vemurafenib. In vitro (real-time)-proliferation assays demonstrated that 5-FdU(3'-5')ECyd and ECyd-lipid-5-FdU had a high cytotoxic efficacy causing 75% melanoma cell death at concentrations in the nanomolar and micromolar range. Cytotoxicity was conducted by induction of DNA cleavage indicating apoptotic cells. Chicken embryotoxicity demonstrated that the duplex drugs were less toxic than 5-FdU at 0.01 μM. In vivo the duplex drug 5-FdU(3'-5')ECyd was efficacious in the murine LOX IMVI melanoma xenograph model on administration of 11.2 mg/kg/injection every fourth day. Both duplex drugs are promising novel cytostatic agents for the treatment of malignant melanoma meriting clinical evaluation.
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http://dx.doi.org/10.1002/ijc.27476DOI Listing
November 2012

Resistance to BRAF inhibitors: unraveling mechanisms and future treatment options.

Cancer Res 2011 Dec;71(23):7137-40

Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.

The mitogen-activated protein kinase (MAPK) pathway has emerged as a central target for melanoma therapy due to its persistent activation in the majority of tumors. Several BRAF inhibitors aimed at curbing MAPK pathway activity are currently in advanced stages of clinical investigation. However, their therapeutic success is limited by the emergence of drug resistance, as responses are transient and tumors eventually recur. To develop effective and long-lasting therapies for melanoma patients, it is essential to understand the mechanisms underlying resistance to BRAF inhibitors. Here, we briefly review recent preclinical studies that have provided insight into the molecular mechanisms of resistance to BRAF inhibitors and discuss potential strategies to treat drug-resistant melanomas.
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http://dx.doi.org/10.1158/0008-5472.CAN-11-1243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3588168PMC
December 2011
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