Publications by authors named "Meenhard Herlyn"

347 Publications

ICAM-1-mediated adhesion is a prerequisite for exosome-induced T cell suppression.

Dev Cell 2022 Jan 25. Epub 2022 Jan 25.

Department of Biology, School of Arts & Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Tumor-derived extracellular vesicles (TEVs) suppress the proliferation and cytotoxicity of CD8 T cells, thereby contributing to tumor immune evasion. Here, we report that the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) co-localizes with programmed death ligand 1 (PD-L1) on the exosomes; both ICAM-1 and PD-L1 are upregulated by interferon-γ. Exosomal ICAM-1 interacts with LFA-1, which is upregulated in activated T cells. Blocking ICAM-1 on TEVs reduces the interaction of TEVs with CD8 T cells and attenuates PD-L1-mediated suppressive effects of TEVs. During this study, we have established an extracellular vesicle-target cell interaction detection through SorTagging (ETIDS) system to assess the interaction between a TEV ligand and its target cell receptor. Using this system, we demonstrate that the interaction of TEV PD-L1 with programmed cell death 1 (PD-1) on T cells is significantly reduced in the absence of ICAM-1. Our study demonstrates that ICAM-1-LFA-1-mediated adhesion between TEVs and T cells is a prerequisite for exosomal PD-L1-mediated immune suppression.
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http://dx.doi.org/10.1016/j.devcel.2022.01.002DOI Listing
January 2022

Costimulation of γδTCR and TLR7/8 promotes Vδ2 T-cell antitumor activity by modulating mTOR pathway and APC function.

J Immunother Cancer 2021 12;9(12)

Department of Pathology and Laboratory Medicine, The University of Pennsylvania, Philadelphia, Pennsylvania, USA

Background: Gamma delta (γδ) T cells are attractive effector cells for cancer immunotherapy. Vδ2 T cells expanded by zoledronic acid (ZOL) are the most commonly used γδ T cells for adoptive cell therapy. However, adoptive transfer of the expanded Vδ2 T cells has limited clinical efficacy.

Methods: We developed a costimulation method for expansion of Vδ2 T cells in PBMCs by activating γδ T-cell receptor (γδTCR) and Toll-like receptor (TLR) 7/8 using isopentenyl pyrophosphate (IPP) and resiquimod, respectively, and tested the functional markers and antitumoral effects in vitro two-dimensional two-dimensional and three-dimensional spheroid models and in vivo models. Single-cell sequencing dataset analysis and reverse-phase protein array were employed for mechanistic studies.

Results: We find that Vδ2 T cells expanded by IPP plus resiquimod showed significantly increased cytotoxicity to tumor cells with lower programmed cell death protein 1 (PD-1) expression than Vδ2 T cells expanded by IPP or ZOL. Mechanistically, the costimulation enhanced the activation of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt)-the mammalian target of rapamycin (mTOR) pathway and the TLR7/8-MyD88 pathway. Resiquimod stimulated Vδ2 T-cell expansion in both antigen presenting cell dependent and independent manners. In addition, resiquimod decreased the number of adherent inhibitory antigen-presenting cells (APCs) and suppressed the inhibitory function of APCs by decreasing PD-L1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression in these cells during in vitro Vδ2 T-cell expansion. Finally, we showed that human Vδ2 T cells can be expanded from PBMCs and spleen of humanized NSG mice using IPP plus resiquimod or ZOL, demonstrating that humanized mice are a promising preclinical model for studying human γδ T-cell development and function.

Conclusions: Vδ2 T cells expanded by IPP and resiquimod demonstrate improved anti-tumor function and have the potential to increase the efficacy of γδ T cell-based therapies.
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http://dx.doi.org/10.1136/jitc-2021-003339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8705233PMC
December 2021

NUMB as a Therapeutic Target for Melanoma.

J Invest Dermatol 2021 Dec 7. Epub 2021 Dec 7.

The Wistar Institute, Philadelphia, Pennsylvania, USA.

The upregulation of the adaptor protein NUMB triggers melanocytic differentiation from multipotent skin stem cells, which share many properties with aggressive melanoma cells. Although NUMB acts as a tumor suppressor in various human cancer types, little is known about its role in melanoma. In this study, we investigated the role of NUMB in melanoma progression and its regulatory mechanism. Analysis of The Cancer Genome Atlas melanoma datasets revealed that high NUMB expression in melanoma tissues correlates with improved patient survival. Moreover, NUMB expression is downregulated in metastatic melanoma cells. NUMB knockdown significantly increased the invasion potential of melanoma cells in a three-dimensional collagen matrix in vitro and in the lungs of a mouse model in vivo; it also significantly upregulated the expression of the NOTCH target gene CCNE. Previous studies suggested that Wnt signaling increases NUMB expression. By mimicking Wnt stimulation through glycogen synthase kinase-3 inhibition, we increased NUMB expression in melanoma cells. Furthermore, a glycogen synthase kinase-3 inhibitor reduced the invasion of melanoma cells in a NUMB-dependent manner. Together, our results suggest that NUMB suppresses invasion and metastasis in melanoma, potentially through its regulation of the NOTCH‒CCNE axis and that the inhibitors that upregulate NUMB can exert therapeutic effects in melanoma.
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http://dx.doi.org/10.1016/j.jid.2021.11.027DOI Listing
December 2021

Pathway signatures derived from on-treatment tumor specimens predict response to anti-PD1 blockade in metastatic melanoma.

Nat Commun 2021 10 15;12(1):6023. Epub 2021 Oct 15.

Department of Neurosurgery, Duke University School of Medicine, Durham, NC, 27710, USA.

Both genomic and transcriptomic signatures have been developed to predict responses of metastatic melanoma to immune checkpoint blockade (ICB) therapies; however, most of these signatures are derived from pre-treatment biopsy samples. Here, we build pathway-based super signatures in pre-treatment (PASS-PRE) and on-treatment (PASS-ON) tumor specimens based on transcriptomic data and clinical information from a large dataset of metastatic melanoma treated with anti-PD1-based therapies as the training set. Both PASS-PRE and PASS-ON signatures are validated in three independent datasets of metastatic melanoma as the validation set, achieving area under the curve (AUC) values of 0.45-0.69 and 0.85-0.89, respectively. We also combine all test samples and obtain AUCs of 0.65 and 0.88 for PASS-PRE and PASS-ON signatures, respectively. When compared with existing signatures, the PASS-ON signature demonstrates more robust and superior predictive performance across all four datasets. Overall, we provide a framework for building pathway-based signatures that is highly and accurately predictive of response to anti-PD1 therapies based on on-treatment tumor specimens. This work would provide a rationale for applying pathway-based signatures derived from on-treatment tumor samples to predict patients' therapeutic response to ICB therapies.
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http://dx.doi.org/10.1038/s41467-021-26299-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8519947PMC
October 2021

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery.

J Vis Exp 2021 09 27(175). Epub 2021 Sep 27.

The Wistar Institute;

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2Rγnull (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34 cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34 injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.
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http://dx.doi.org/10.3791/62906DOI Listing
September 2021

Epigenetic Silencing of BMP6 by the SIN3A-HDAC1/2 Repressor Complex Drives Melanoma Metastasis via FAM83G/PAWS1.

Mol Cancer Res 2021 Oct 5. Epub 2021 Oct 5.

Center for Discovery and Innovation, Hackensack University Medical Center, Nutley, New Jersey.

Aberrant epigenetic transcriptional regulation is linked to metastasis, a primary cause of cancer-related death. Dissecting the epigenetic mechanisms controlling metastatic progression may uncover important insights to tumor biology and potential therapeutic targets. Here, we investigated the role of the SIN3A histone deacetylase 1 and 2 (SIN3A-HDAC1/2) complex in cancer metastasis. Using a mouse model of melanoma metastasis, we found that the SIN3A-HDAC1/2 transcription repressor complex silences BMP6 expression, causing increased metastatic dissemination and tumor growth via suppression of BMP6-activated SMAD5 signaling. We further discovered that FAM83G/PAWS1, a downstream effector of BMP6-SMAD5 signaling, contributes critically to metastatic progression by promoting actin-dependent cytoskeletal dynamics and cell migration. Pharmacologic inhibition of the SIN3A-HDAC1/2 complex reduced the numbers of melanoma cells in the circulation and inhibited metastatic tumor growth by inducing disseminated cell dormancy, highlighting the SIN3A-HDAC1/2 repressor complex as a potential therapeutic target for blocking cancer metastasis. IMPLICATIONS: This study identifies the novel molecular links in the metastatic progression to target cytoskeletal dynamics in melanoma and identifies the SIN3A-HDAC1/2 complex and FAM83G/PAWS1 as potential targets for melanoma adjuvant therapy.
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http://dx.doi.org/10.1158/1541-7786.MCR-21-0289DOI Listing
October 2021

A Modified Nucleoside 6-Thio-2'-Deoxyguanosine Exhibits Antitumor Activity in Gliomas.

Clin Cancer Res 2021 12 30;27(24):6800-6814. Epub 2021 Sep 30.

Department of Computer Science, Ying Wu College of Computing, New Jersey Institute of Technology, Newark, New Jersey.

Purpose: To investigate the therapeutic role of a novel telomere-directed inhibitor, 6-thio-2'-deoxyguanosine (THIO) in gliomas both and .

Experimental Design: A panel of human and mouse glioma cell lines was used to test therapeutic efficacy of THIO using cell viability assays, flow cytometric analyses, and immunofluorescence. Integrated analyses of RNA sequencing and reverse-phase protein array data revealed the potential antitumor mechanisms of THIO. Four patient-derived xenografts (PDX), two patient-derived organoids (PDO), and two xenografts of human glioma cell lines were used to further investigate the therapeutic efficacy of THIO.

Results: THIO was effective in the majority of human and mouse glioma cell lines with no obvious toxicity against normal astrocytes. THIO as a monotherapy demonstrated efficacy in three glioma cell lines that had acquired resistance to temozolomide. In addition, THIO showed efficacy in four human glioma cell lines grown as neurospheres by inducing apoptotic cell death. Mechanistically, THIO induced telomeric DNA damage not only in glioma cell lines but also in PDX tumor specimens. Integrated computational analyses of transcriptomic and proteomic data indicated that THIO significantly inhibited cell invasion, stem cell, and proliferation pathways while triggering DNA damage and apoptosis. Importantly, THIO significantly decreased tumor proliferation in two PDO models and reduced the tumor size of a glioblastoma xenograft and a PDX model.

Conclusions: The current study established the therapeutic role of THIO in primary and recurrent gliomas and revealed the acute induction of telomeric DNA damage as a primary antitumor mechanism of THIO in gliomas.
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http://dx.doi.org/10.1158/1078-0432.CCR-21-0374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8678347PMC
December 2021

TOP1 modulation during melanoma progression and in adaptative resistance to BRAF and MEK inhibitors.

Pharmacol Res 2021 11 22;173:105911. Epub 2021 Sep 22.

Skin Biology Group, Clinical Chemistry and Toxicology Department, School of Pharmaceutical Sciences, University of Sao Paulo, FCF/USP, Brazil. Electronic address:

In melanomas, therapy resistance can arise due to a combination of genetic, epigenetic and phenotypic mechanisms. Due to its crucial role in DNA supercoil relaxation, TOP1 is often considered an essential chemotherapeutic target in cancer. However, how TOP1 expression and activity might differ in therapy sensitive versus resistant cell types is unknown. Here we show that TOP1 expression is increased in metastatic melanoma and correlates with an invasive gene expression signature. More specifically, TOP1 expression is highest in cells with the lowest expression of MITF, a key regulator of melanoma biology. Notably, TOP1 and DNA Single-Strand Break Repair genes are downregulated in BRAFi- and BRAFi/MEKi-resistant cells and TOP1 inhibition decreases invasion markers only in BRAFi/MEKi-resistant cells. Thus, we show three different phenotypes related to TOP1 levels: i) non-malignant cells with low TOP1 levels; ii) metastatic cells with high TOP1 levels and high invasiveness; and iii) BRAFi- and BRAFi/MEKi-resistant cells with low TOP1 levels and high invasiveness. Together, these results highlight the potential role of TOP1 in melanoma progression and resistance.
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http://dx.doi.org/10.1016/j.phrs.2021.105911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8729257PMC
November 2021

Protein Signatures of NK Cell-Mediated Melanoma Killing Predict Response to Immunotherapies.

Cancer Res 2021 11 13;81(21):5540-5554. Epub 2021 Sep 13.

Molecular Physiology, Institute of Cardiovascular Physiology, University Medical Center, Georg August University, Göttingen, Germany.

Despite impressive advances in melanoma-directed immunotherapies, resistance is common and many patients still succumb to metastatic disease. In this context, harnessing natural killer (NK) cells, which have thus far been sidelined in the development of melanoma immunotherapy, could provide therapeutic benefits for cancer treatment. To identify molecular determinants of NK cell-mediated melanoma killing (), we quantified NK-cell cytotoxicity against a panel of genetically diverse melanoma cell lines and observed highly heterogeneous susceptibility. Melanoma protein microarrays revealed a correlation between and the abundance and activity of a subset of proteins, including several metabolic factors. Oxidative phoshorylation, measured by oxygen consumption rate, negatively correlated with melanoma cell sensitivity toward , and proteins involved in mitochondrial metabolism and epithelial-mesenchymal transition were confirmed to regulate . Two- and three-dimensional killing assays and melanoma xenografts established that the PIK/AKT/mTOR signaling axis controls via regulation of NK cell-relevant surface proteins. A "protein-killing-signature" based on the protein analysis predicted of additional melanoma cell lines and the response of patients with melanoma to anti-PD-1 checkpoint therapy. Collectively, these findings identify novel NK cell-related prognostic biomarkers and may contribute to improved and personalized melanoma-directed immunotherapies. SIGNIFICANCE: NK-cell cytotoxicity assays and protein microarrays reveal novel biomarkers of NK cell-mediated melanoma killing and enable development of signatures to predict melanoma patient responsiveness to immunotherapies.
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http://dx.doi.org/10.1158/0008-5472.CAN-21-0164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8727679PMC
November 2021

TRIM15 and CYLD regulate ERK activation via lysine-63-linked polyubiquitination.

Nat Cell Biol 2021 09 8;23(9):978-991. Epub 2021 Sep 8.

Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

The extracellular-signal-regulated kinases ERK1 and ERK2 (hereafter ERK1/2) represent the foremost mitogenic pathway in mammalian cells, and their dysregulation drives tumorigenesis and confers therapeutic resistance. ERK1/2 are known to be activated by MAPK/ERK kinase (MEK)-mediated phosphorylation. Here, we show that ERK1/2 are also modified by lysine-63 (K63)-linked polyubiquitin chains. We identify the tripartite motif-containing protein TRIM15 as a ubiquitin ligase and the tumour suppressor CYLD as a deubiquitinase of ERK1/2. TRIM15 and CYLD regulate ERK ubiquitination at defined lysine residues through mutually exclusive interactions as well as opposing activities. K63-linked polyubiquitination enhances ERK interaction with and activation by MEK. Downregulation of TRIM15 inhibits the growth of both drug-responsive and drug-resistant melanomas. Moreover, high TRIM15 expression and low CYLD expression are associated with poor prognosis of patients with melanoma. These findings define a role of K63-linked polyubiquitination in the ERK signalling pathway and suggest a potential target for cancer therapy.
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http://dx.doi.org/10.1038/s41556-021-00732-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8440396PMC
September 2021

Neural Crest-Like Stem Cell Transcriptome Analysis Identifies LPAR1 in Melanoma Progression and Therapy Resistance.

Cancer Res 2021 10 30;81(20):5230-5241. Epub 2021 Aug 30.

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

Metastatic melanoma is challenging to clinically address. Although standard-of-care targeted therapy has high response rates in patients with BRAF-mutant melanoma, therapy relapse occurs in most cases. Intrinsically resistant melanoma cells drive therapy resistance and display molecular and biologic properties akin to neural crest-like stem cells (NCLSC) including high invasiveness, plasticity, and self-renewal capacity. The shared transcriptional programs and vulnerabilities between NCLSCs and cancer cells remains poorly understood. Here, we identify a developmental LPAR1-axis critical for NCLSC viability and melanoma cell survival. LPAR1 activity increased during progression and following acquisition of therapeutic resistance. Notably, genetic inhibition of LPAR1 potentiated BRAFi ± MEKi efficacy and ablated melanoma migration and invasion. Our data define LPAR1 as a new therapeutic target in melanoma and highlights the promise of dissecting stem cell-like pathways hijacked by tumor cells. SIGNIFICANCE: This study identifies an LPAR1-axis critical for melanoma invasion and intrinsic/acquired therapy resistance.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8530965PMC
October 2021

Comprehensive characterization of 536 patient-derived xenograft models prioritizes candidatesfor targeted treatment.

Nat Commun 2021 08 24;12(1):5086. Epub 2021 Aug 24.

Department of Medicine, Washington University in St. Louis, St. Louis, MO, USA.

Development of candidate cancer treatments is a resource-intensive process, with the research community continuing to investigate options beyond static genomic characterization. Toward this goal, we have established the genomic landscapes of 536 patient-derived xenograft (PDX) models across 25 cancer types, together with mutation, copy number, fusion, transcriptomic profiles, and NCI-MATCH arms. Compared with human tumors, PDXs typically have higher purity and fit to investigate dynamic driver events and molecular properties via multiple time points from same case PDXs. Here, we report on dynamic genomic landscapes and pharmacogenomic associations, including associations between activating oncogenic events and drugs, correlations between whole-genome duplications and subclone events, and the potential PDX models for NCI-MATCH trials. Lastly, we provide a web portal having comprehensive pan-cancer PDX genomic profiles and source code to facilitate identification of more druggable events and further insights into PDXs' recapitulation of human tumors.
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http://dx.doi.org/10.1038/s41467-021-25177-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8384880PMC
August 2021

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

Oncogene 2021 09 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

Evolution of delayed resistance to immunotherapy in a melanoma responder.

Nat Med 2021 06 3;27(6):985-992. Epub 2021 May 3.

Department of Pathology, Harvard Medical School, Brigham and Woman's Hospital, Boston, MA, USA.

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 patient with metastatic melanoma with complete clinical response to ICB followed by delayed recurrence and death. Phylogenetic analysis revealed co-evolution of seven lineages with multiple convergent, but independent resistance-associated alterations. 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 RNA sequencing and highly multiplexed immunofluorescence (t-CyCIF) revealed differences in immune composition among different lineages. Imaging revealed a vasculogenic mimicry phenotype in NGFR tumor cells with high PD-L1 expression in close proximity to immune cells. Rapid autopsy demonstrated two 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 patient with melanoma with an unusual clinical course.
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http://dx.doi.org/10.1038/s41591-021-01331-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8474080PMC
June 2021

Exploiting Allosteric Properties of RAF and MEK Inhibitors to Target Therapy-Resistant Tumors Driven by Oncogenic BRAF Signaling.

Cancer Discov 2021 07 10;11(7):1716-1735. Epub 2021 Feb 10.

Department of Oncological Sciences, The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York.

Current clinical RAF inhibitors (RAFi) inhibit monomeric BRAF (mBRAF) but are less potent against dimeric BRAF (dBRAF). RAFi equipotent for mBRAF and dBRAF have been developed but are predicted to have lower therapeutic index. Here we identify a third class of RAFi that selectively inhibits dBRAF over mBRAF. Molecular dynamic simulations reveal restriction of the movement of the BRAF αC-helix as the basis of inhibitor selectivity. Combination of inhibitors based on their conformation selectivity (mBRAF- plus dBRAF-selective plus the most potent BRAF-MEK disruptor MEK inhibitor) promoted suppression of tumor growth in BRAF therapy-resistant models. Strikingly, the triple combination showed no toxicities, whereas dBRAF-selective plus MEK inhibitor treatment caused weight loss in mice. Finally, the triple combination achieved durable response and improved clinical well-being in a patient with stage IV colorectal cancer. Thus, exploiting allosteric properties of RAF and MEK inhibitors enables the design of effective and well-tolerated therapies for BRAF tumors. SIGNIFICANCE: This work identifies a new class of RAFi that are selective for dBRAF over mBRAF and determines the basis of their selectivity. A rationally designed combination of RAF and MEK inhibitors based on their conformation selectivity achieved increased efficacy and a high therapeutic index when used to target BRAF tumors...
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http://dx.doi.org/10.1158/2159-8290.CD-20-1351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8295204PMC
July 2021

Melanoma models for the next generation of therapies.

Cancer Cell 2021 05 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

Inhibition of endothelin-B receptor signaling synergizes with MAPK pathway inhibitors in BRAF mutated melanoma.

Oncogene 2021 03 26;40(9):1659-1673. Epub 2021 Jan 26.

Drug Discovery Biology, Idorsia Pharmaceuticals Ltd., Hegenheimermattweg 91, 4123, Allschwil, Switzerland.

The clinical benefit of MAPK pathway inhibition in melanoma patients carrying BRAF mutations is temporal. After the initial response to treatment, the majority of tumors will develop resistance and patients will relapse. Here we demonstrate that the endothelin-endothelin receptor B (ETBR) signaling pathway confers resistance to MAPK pathway inhibitors in BRAF mutated melanoma. MAPK blockade, in addition to being anti-proliferative, induces a phenotypic change which is characterized by increased expression of melanocyte-specific genes including ETBR. In the presence of MAPK inhibitors, activation of ETBR by endothelin enables the sustained proliferation of melanoma cells. In mouse models of melanoma, including patient-derived xenograft models, concurrent inhibition of the MAPK pathway and ETBR signaling resulted in a more effective anti-tumor response compared to MAPK pathway inhibition alone. The combination treatment significantly reduced tumor growth and prolonged survival compared to therapies with MAPK pathway inhibitors alone. The phosphoproteomic analysis revealed that ETBR signaling did not induce resistance towards MAPK pathway inhibitors by restoring MAPK activity, but instead via multiple alternative signaling pathways downstream of the small G proteins GNAq/11. Together these data indicate that a combination of MAPK pathway inhibitors with ETBR antagonists could have a synergistically beneficial effect in melanoma patients with hyperactivated MAPK signaling pathways.
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http://dx.doi.org/10.1038/s41388-020-01628-xDOI Listing
March 2021

Tumor-infiltrating mast cells are associated with resistance to anti-PD-1 therapy.

Nat Commun 2021 01 12;12(1):346. Epub 2021 Jan 12.

The Wistar Institute, Philadelphia, PA, USA.

Anti-PD-1 therapy is used as a front-line treatment for many cancers, but mechanistic insight into this therapy resistance is still lacking. Here we generate a humanized (Hu)-mouse melanoma model by injecting fetal liver-derived CD34 cells and implanting autologous thymus in immune-deficient NOD-scid IL2Rγ (NSG) mice. Reconstituted Hu-mice are challenged with HLA-matched melanomas and treated with anti-PD-1, which results in restricted tumor growth but not complete regression. Tumor RNA-seq, multiplexed imaging and immunohistology staining show high expression of chemokines, as well as recruitment of FOXP3 Treg and mast cells, in selective tumor regions. Reduced HLA-class I expression and CD8/Granz B T cells homeostasis are observed in tumor regions where FOXP3 Treg and mast cells co-localize, with such features associated with resistance to anti-PD-1 treatment. Combining anti-PD-1 with sunitinib or imatinib results in the depletion of mast cells and complete regression of tumors. Our results thus implicate mast cell depletion for improving the efficacy of anti-PD-1 therapy.
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http://dx.doi.org/10.1038/s41467-020-20600-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804257PMC
January 2021

Conservation of copy number profiles during engraftment and passaging of patient-derived cancer xenografts.

Nat Genet 2021 01 7;53(1):86-99. Epub 2021 Jan 7.

Center for Cancer Biology, VIB, Leuven, Belgium.

Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, affecting the accuracy of PDX modeling of human cancer. Here, we exhaustively analyze copy number alterations (CNAs) in 1,451 PDX and matched patient tumor (PT) samples from 509 PDX models. CNA inferences based on DNA sequencing and microarray data displayed substantially higher resolution and dynamic range than gene expression-based inferences, and they also showed strong CNA conservation from PTs through late-passage PDXs. CNA recurrence analysis of 130 colorectal and breast PT/PDX-early/PDX-late trios confirmed high-resolution CNA retention. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multiregion samples within patients. Our study demonstrates the lack of systematic copy number evolution driven by the PDX mouse host.
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http://dx.doi.org/10.1038/s41588-020-00750-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7808565PMC
January 2021

The State of Melanoma: Emergent Challenges and Opportunities.

Clin Cancer Res 2021 05 7;27(10):2678-2697. Epub 2021 Jan 7.

University of Pittsburgh, Pittsburgh, Pennsylvania.

Five years ago, the Melanoma Research Foundation (MRF) conducted an assessment of the challenges and opportunities facing the melanoma research community and patients with melanoma. Since then, remarkable progress has been made on both the basic and clinical research fronts. However, the incidence, recurrence, and death rates for melanoma remain unacceptably high and significant challenges remain. Hence, the MRF Scientific Advisory Council and Breakthrough Consortium, a group that includes clinicians and scientists, reconvened to facilitate intensive discussions on thematic areas essential to melanoma researchers and patients alike, prevention, detection, diagnosis, metastatic dormancy and progression, response and resistance to targeted and immune-based therapy, and the clinical consequences of COVID-19 for patients with melanoma and providers. These extensive discussions helped to crystalize our understanding of the challenges and opportunities facing the broader melanoma community today. In this report, we discuss the progress made since the last MRF assessment, comment on what remains to be overcome, and offer recommendations for the best path forward.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-4092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8127340PMC
May 2021

IspH inhibitors kill Gram-negative bacteria and mobilize immune clearance.

Nature 2021 01 23;589(7843):597-602. Epub 2020 Dec 23.

Vaccine and Immunotherapy Center, The Wistar Institute, Philadelphia, PA, USA.

Isoprenoids are vital for all organisms, in which they maintain membrane stability and support core functions such as respiration. IspH, an enzyme in the methyl erythritol phosphate pathway of isoprenoid synthesis, is essential for Gram-negative bacteria, mycobacteria and apicomplexans. Its substrate, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), is not produced in metazoans, and in humans and other primates it activates cytotoxic Vγ9Vδ2 T cells at extremely low concentrations. Here we describe a class of IspH inhibitors and refine their potency to nanomolar levels through structure-guided analogue design. After modification of these compounds into prodrugs for delivery into bacteria, we show that they kill clinical isolates of several multidrug-resistant bacteria-including those from the genera Acinetobacter, Pseudomonas, Klebsiella, Enterobacter, Vibrio, Shigella, Salmonella, Yersinia, Mycobacterium and Bacillus-yet are relatively non-toxic to mammalian cells. Proteomic analysis reveals that bacteria treated with these prodrugs resemble those after conditional IspH knockdown. Notably, these prodrugs also induce the expansion and activation of human Vγ9Vδ2 T cells in a humanized mouse model of bacterial infection. The prodrugs we describe here synergize the direct killing of bacteria with a simultaneous rapid immune response by cytotoxic γδ T cells, which may limit the increase of antibiotic-resistant bacterial populations.
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http://dx.doi.org/10.1038/s41586-020-03074-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8776033PMC
January 2021

ARID2 Deficiency Correlates with the Response to Immune Checkpoint Blockade in Melanoma.

J Invest Dermatol 2021 06 15;141(6):1564-1572.e4. Epub 2020 Dec 15.

Division of Dermatology, Department of Internal Related, Graduate School of Medicine, Kobe University, Kobe, Japan.

The SWI/SNF chromatin remodeler family includes the BAF and PBAF complexes. ARID2, encoding a PBAF complex subunit, is frequently mutated in melanoma independently of BRAF/RAS mutations. Emerging evidence shows that SWI/SNF complexes regulate tumor immunity; for instance, the loss of PBRM1, another PBAF complex subunit, enhances susceptibility to immune checkpoint inhibitors in melanoma. Notably, ARID2 mutations are more frequent in melanoma than PBRM1 mutations. However, the role of ARID2 as a modulator of tumor immunity remains unclear. In this study, we show that ARID2 knockout sensitizes melanoma to immune checkpoint inhibitors. Anti‒PD-L1 treatment restricts tumor growth in mice bearing ARID2-knockout melanoma cells, correlating with an increase in the infiltration of cytotoxic CD8 T cells. Furthermore, ARID2 deficiency leads to signal transducer and activator of transcription 1 upregulation, which subsequently causes increased expression of T-cell‒attracting chemokines such as CXCL9, CXCL10, and CCL5. These results demonstrate that ARID2 is an immunomodulator and a potential biomarker that indicates immune checkpoint inhibitor effectiveness in patients with melanoma.
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http://dx.doi.org/10.1016/j.jid.2020.11.026DOI Listing
June 2021

Large-Scale Characterization of Drug Responses of Clinically Relevant Proteins in Cancer Cell Lines.

Cancer Cell 2020 12 5;38(6):829-843.e4. Epub 2020 Nov 5.

Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Graduate Program in Quantitative and Computational Biosciences, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address:

Perturbation biology is a powerful approach to modeling quantitative cellular behaviors and understanding detailed disease mechanisms. However, large-scale protein response resources of cancer cell lines to perturbations are not available, resulting in a critical knowledge gap. Here we generated and compiled perturbed expression profiles of ∼210 clinically relevant proteins in >12,000 cancer cell line samples in response to ∼170 drug compounds using reverse-phase protein arrays. We show that integrating perturbed protein response signals provides mechanistic insights into drug resistance, increases the predictive power for drug sensitivity, and helps identify effective drug combinations. We build a systematic map of "protein-drug" connectivity and develop a user-friendly data portal for community use. Our study provides a rich resource to investigate the behaviors of cancer cells and the dependencies of treatment responses, thereby enabling a broad range of biomedical applications.
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http://dx.doi.org/10.1016/j.ccell.2020.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738392PMC
December 2020

Inhibiting insulin and mTOR signaling by afatinib and crizotinib combination fosters broad cytotoxic effects in cutaneous malignant melanoma.

Cell Death Dis 2020 10 20;11(10):882. Epub 2020 Oct 20.

Department of Oncology-Pathology, Karolinska Institutet, 171 64, Stockholm, Sweden.

Current treatment modalities for disseminated cutaneous malignant melanoma (CMM) improve survival, however disease progression commonly ensues. In a previous study we identified afatinib and crizotinib in combination as a novel potential therapy for CMM independent of BRAF/NRAS mutation status. Herein, we elucidate the underlying mechanisms of the combination treatment effect to find biomarkers and novel targets for development of therapy that may provide clinical benefit by proteomic analysis of CMM cell lines and xenografts using mass spectrometry based analysis and reverse phase protein array. Identified candidates were validated using immunoblotting or immunofluorescence. Our analysis revealed that mTOR/Insulin signaling pathways were significantly decreased by the afatinib and crizotinib combination treatment. Both in vitro and in vivo analyses showed that the combination treatment downregulated pRPS6KB1 and pRPS6, downstream of mTOR signaling, and IRS-1 in the insulin signaling pathway, specifically ablating IRS-1 nuclear signal. Silencing of RPS6 and IRS-1 alone had a similar effect on cell death, which was further induced when IRS-1 and RPS6 were concomitantly silenced in the CMM cell lines. Silencing of IRS-1 and RPS6 resulted in reduced sensitivity towards combination treatment. Additionally, we found that IRS-1 and RPS6KB1 expression levels were increased in advanced stages of CMM clinical samples. We could demonstrate that induced resistance towards combination treatment was reversible by a drug holiday. CD171/L1CAM, mTOR and PI3K-p85 were induced in the combination resistant cells whereas AXL and EPHA2, previously identified mediators of resistance to MAPK inhibitor therapy in CMM were downregulated. We also found that CD171/L1CAM and mTOR were increased at progression in tumor biopsies from two matched cases of patients receiving targeted therapy with BRAFi. Overall, these findings provide insights into the molecular mechanisms behind the afatinib and crizotinib combination treatment effect and leverages a platform for discovering novel biomarkers and therapy regimes for CMM treatment.
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http://dx.doi.org/10.1038/s41419-020-03097-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576205PMC
October 2020

Targeting Extracellular Matrix Remodeling Restores BRAF Inhibitor Sensitivity in BRAFi-resistant Melanoma.

Clin Cancer Res 2020 11 20;26(22):6039-6050. Epub 2020 Aug 20.

Department of Dermatology, University of Miami Miller School of Medicine, Miami, Florida.

Purpose: The extracellular matrix (ECM) is an intriguing, yet understudied component of therapy resistance. Here, we investigated the role of ECM remodeling by the collagenase, MT1-MMP, in conferring resistance of v-Raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutant melanoma to BRAF inhibitor (BRAFi) therapy.

Experimental Design: Publicly available RNA-sequencing data and reverse phase protein array were used to determine the relevance of MT1-MMP upregulation in BRAFi-resistant melanoma in patients, patient-derived xenografts, and cell line-derived tumors. Short hairpin RNA (shRNA)-mediated knockdown of MT1-MMP, inhibition via the selective MT1-MMP/MMP2 inhibitor, ND322, or overexpression of MT1-MMP was used to assess the role of MT1-MMP in mediating resistance to BRAFi.

Results: MT1-MMP was consistently upregulated in posttreatment tumor samples derived from patients upon disease progression and in melanoma xenografts and cell lines that acquired resistance to BRAFi. shRNA- or ND322-mediated inhibition of MT1-MMP synergized with BRAFi leading to resensitization of resistant cells and tumors to BRAFi. The resistant phenotype depends on the ability of cells to cleave the ECM. Resistant cells seeded in MT1-MMP uncleavable matrixes were resensitized to BRAFi similarly to MT1-MMP inhibition. This is due to the inability of cells to activate integrinβ1 (ITGB1)/FAK signaling, as restoration of ITGB1 activity is sufficient to maintain resistance to BRAFi in the context of MT1-MMP inhibition. Finally, the increase in MT1-MMP in BRAFi-resistant cells is TGFβ dependent, as inhibition of TGFβ receptors I/II dampens MT1-MMP overexpression and restores sensitivity to BRAF inhibition.

Conclusions: BRAF inhibition results in a selective pressure toward higher expression of MT1-MMP. MT1-MMP is pivotal to an ECM-based signaling pathway that confers resistance to BRAFi therapy.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-2773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7669662PMC
November 2020

Brain Metastasis Cell Lines Panel: A Public Resource of Organotropic Cell Lines.

Cancer Res 2020 10 8;80(20):4314-4323. Epub 2020 Jul 8.

Neurology Clinic and National Center for Tumor Diseases, University Hospital Heidelberg, and Clinical Cooperation Unit Neurooncology, German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.

Spread of cancer to the brain remains an unmet clinical need in spite of the increasing number of cases among patients with lung, breast cancer, and melanoma most notably. Although research on brain metastasis was considered a minor aspect in the past due to its untreatable nature and invariable lethality, nowadays, limited but encouraging examples have questioned this statement, making it more attractive for basic and clinical researchers. Evidences of its own biological identity (i.e., specific microenvironment) and particular therapeutic requirements (i.e., presence of blood-brain barrier, blood-tumor barrier, molecular differences with the primary tumor) are thought to be critical aspects that must be functionally exploited using preclinical models. We present the coordinated effort of 19 laboratories to compile comprehensive information related to brain metastasis experimental models. Each laboratory has provided details on the cancer cell lines they have generated or characterized as being capable of forming metastatic colonies in the brain, as well as principle methodologies of brain metastasis research. The Brain Metastasis Cell Lines Panel (BrMPanel) represents the first of its class and includes information about the cell line, how tropism to the brain was established, and the behavior of each model . These and other aspects described are intended to assist investigators in choosing the most suitable cell line for research on brain metastasis. The main goal of this effort is to facilitate research on this unmet clinical need, to improve models through a collaborative environment, and to promote the exchange of information on these valuable resources.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572582PMC
October 2020

SPANX Control of Lamin A/C Modulates Nuclear Architecture and Promotes Melanoma Growth.

Mol Cancer Res 2020 10 22;18(10):1560-1573. Epub 2020 Jun 22.

Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California.

Mechanisms regulating nuclear organization control fundamental cellular processes, including the cell and chromatin organization. Their disorganization, including aberrant nuclear architecture, has been often implicated in cellular transformation. Here, we identify Lamin A, among proteins essential for nuclear architecture, as SPANX (sperm protein associated with the nucleus on the X chromosome), a cancer testis antigen previously linked to invasive tumor phenotypes, interacting protein in melanoma. SPANX interaction with Lamin A was mapped to the immunoglobulin fold-like domain, a region critical for Lamin A function, which is often mutated in laminopathies. SPANX downregulation in melanoma cell lines perturbed nuclear organization, decreased cell viability, and promoted senescence-associated phenotypes. Moreover, SPANX knockdown (KD) in melanoma cells promoted proliferation arrest, a phenotype mediated in part by IRF3/IL1A signaling. SPANX KD in melanoma cells also prompted the secretion of IL1A, which attenuated the proliferation of naïve melanoma cells. Identification of SPANX as a nuclear architecture complex component provides an unexpected insight into the regulation of Lamin A and its importance in melanoma. IMPLICATIONS: SPANX, a testis protein, interacts with LMNA and controls nuclear architecture and melanoma growth.
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http://dx.doi.org/10.1158/1541-7786.MCR-20-0291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541784PMC
October 2020
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