Combined BRAF, MEK, and heat-shock protein 90 inhibition in advanced BRAF V600-mutant melanoma.

BACKGROUND: Resistance to BRAF and MEK inhibitors in BRAF V600-mutant melanoma is common. Multiple resistance mechanisms involve heat-shock protein 90 (HSP90) clients, and a phase 1 study of vemurafenib with the HSP90 inhibitor XL888 in patients with advanced melanoma showed activity equivalent to that of BRAF and MEK inhibitors. METHODS: Vemurafenib (960 mg orally twice daily) and cobimetinib (60 mg orally once daily for 21 of 28 days) with escalating dose cohorts of XL888 (30, 45, 60, or 90 mg orally twice weekly) was investigated in a phase 1 trial of advanced melanoma, with a modified Ji dose-escalation design. RESULTS: Twenty-five patients were enrolled. After two dose-limiting toxicities (DLTs) (rash and acute kidney injury) in the first cohort, lower doses of vemurafenib (720 mg) and cobimetinib (40 mg) were investigated with the same XL888 doses. Three DLTs (rash) were observed in 12 patients in the XL888 60-mg cohort, and this was determined as the maximum tolerated dose. Objective responses were observed in 19 patients (76%), and the median progression-free survival was 7.6 months, with a 5-year progression-free survival rate of 20%. The median overall survival was 41.7 months, with a 5-year overall survival rate of 37%. Single-cell RNA sequencing was performed on baseline and on-treatment biopsies; treatment was associated with increased immune cell influx (CD4-positive and CD8-positive T cells) and decreased melanoma cells. CONCLUSIONS: Combined vemurafenib and cobimetinib plus XL888 had significant toxicity, requiring frequent dose reductions, which may have contributed to the relatively low progression-free survival despite a high tumor response rate. Given overlapping toxicities, caution must be used when combining HSP90 inhibitors with BRAF and MEK inhibitors.

GP100 expression is variable in intensity in melanoma.

Drugs or cellular products that bind to gp100 are being investigated for treatment of cutaneous melanoma. The relative specificity of gp100 expression in melanocytes makes it an attractive target to harness for therapeutic intent. For example, Tebentafusp, a bispecific gp100 peptide-HLA-directed CD3 T cell engager, has generated significant enthusiasm in recent years due to its success in improving outcomes for uveal melanoma and is being studied in cutaneous melanoma. However, the extent and intensity of gp100 expression in advanced cutaneous melanoma has not been well studied. Here, we interrogated a large cohort of primary and metastatic melanomas for gp100 expression by immunohistochemistry. Expression in metastatic samples was globally higher and almost uniformly positive, however the degree of intensity was variable. Using a quantitative immunofluorescence method, we confirmed the variability in expression. As gp100-binding drugs are assessed in clinical trials, the association between activity of the drugs and the level of gp100 expression should be studied in order to potentially improve patient selection.

Dose-escalation trial of combination dabrafenib, trametinib, and AT13387 in patients with BRAF-mutant solid tumors.

BACKGROUND: Combination BRAF and MEK inhibitor therapy is an active regimen in patients who have BRAF V600E-mutated tumors; however, the clinical efficacy of this therapy is limited by resistance. Preclinically, the addition of heat shock protein 90 (HSP90) inhibition improves the efficacy of BRAF inhibitor therapy in both BRAF inhibitor-sensitive and BRAF inhibitor-resistant mutant cell lines. METHODS: Cancer Therapy Evaluation Program study 9557 (ClinicalTrials.gov identifier NCT02097225) is a phase 1 study that was designed to assess the safety and efficacy of the small-molecule HSP90 inhibitor, AT13387, in combination with dabrafenib and trametinib in BRAF V600E/K-mutant solid tumors. Correlative analyses evaluated the expression of HSP90 client proteins and chaperones. RESULTS: Twenty-two patients with metastatic, BRAF V600E-mutant solid tumors were enrolled using a 3 + 3 design at four dose levels, and 21 patients were evaluable for efficacy assessment. The most common tumor type was colorectal cancer (N = 12). Dose-limiting toxicities occurred in one patient at dose level 3 and in one patient at dose level 4; specifically, myelosuppression and fatigue, respectively. The maximum tolerated dose was oral dabafenib 150 mg twice daily, oral trametinib 2 mg once daily, and intravenous AT13387 260 mg/m2 on days 1, 8, and 15. The best response was a partial response in two patients and stable disease in eight patients, with an overall response rate of 9.5% (90% exact confidence interval [CI], 2%-27%), a disease control rate of 47.6% (90% CI, 29%-67%), and a median overall survival of 5.1 months (90% CI, 3.4-7.6 months). There were no consistent proteomic changes associated with response or resistance, although responders did have reductions in BRAF expression, and epidermal growth factor receptor downregulation using HSP90 inhibition was observed in one patient who had colorectal cancer. CONCLUSIONS: HSP90 inhibition combined with BRAF/MEK inhibition was safe and produced evidence of modest disease control in a heavily pretreated population. Additional translational work may identify tumor types and resistance mechanisms that are most sensitive to this approach.

Melanoma brain metastases: Biological basis and novel therapeutic strategies.

The development of brain metastases is the deadliest complication of advanced melanoma and has long been associated with a dismal prognosis. The recent years have seen incredible progress in the development of therapies for melanoma brain metastases (MBM), with both targeted therapies (the BRAF-MEK inhibitor combination) and immune checkpoint inhibitors (the anti-CTLA-4, anti-PD-1 combination) showing impressive levels of activity. Despite this, durations of response for these therapies remain lower at intracranial sites of metastasis compared to extracranial metastases and it has been suggested that there are unique features of the brain microenvironment that contribute to therapeutic escape. In this review, we outline the latest research into the biology and pathophysiology of melanoma brain metastasis development and progression. We then discuss the current status of clinical trial that are open to patients with MBM and end by describing the ongoing challenges for the field.

ITCH as a potential therapeutic target in human cancers.

The ITCH/AIP4 ubiquitin E3 ligase was discovered independently by two groups searching for atrophin-1 interacting proteins and studying the genetics of mouse coat color alteration, respectively. ITCH is classified as a NEDD4 family E3 ligase featured with the C-terminal HECT domain for E3 ligase function and WW domains for substrate recruiting. ITCH deficiency in the mouse causes severe multi-organ autoimmune disease. Its roles in maintaining a balanced immune response have been extensively characterized over the past two and a half decades. A wealth of reports demonstrate a multifaceted role of ITCH in human cancers. Given the versatility of ITCH in catalyzing both proteolytic and non-proteolytic ubiquitination of its over fifty substrates, ITCH's role in malignancies is believed to be context-dependent. In this review, we summarize the downstream substrates of ITCH, the functions of ITCH in both tumor cells and the immune system, as well as the implications of such functions in human cancers. Moreover, we describe the upstream regulatory mechanisms of ITCH and the efforts have been made to target ITCH using small molecule inhibitors.

Translational pathology, genomics and the development of systemic therapies for acral melanoma.

Acral melanomas arise on the non-hair bearing skin of the palms, soles and in the nail beds. These rare tumors comprise 2-3 % of all melanomas, are not linked to UV-exposure, and represent the most frequent subtype of melanomas in patients of Asian, African and Hispanic origin. Although recent work has revealed candidate molecular events that underlie acral melanoma development, this knowledge is not yet been translated into efficacious local, regional, or systemic therapies. In the current review, we describe the clinical characteristics of acral melanoma and outline the genetic basis of acral melanoma development. Further discussion is given to the current status of systemic therapy for acral melanoma with a focus on ongoing developments in both immunotherapy and targeted therapy for the treatment of advanced disease.

ER stress promotes antitumor effects in BRAFi/MEKi resistant human melanoma induced by natural compound 4-nerolidylcathecol (4-NC).

Melanoma accounts for only 4% of malignant neoplasms of the skin, but is considered the most serious because it is highly deadly. Mutations in the MAPK (Ras-Raf-MEK-ERK) pathway is closely linked to the lack of control of cell proliferation. Especially in melanoma, this pathway has become a target for the development of oncogene-targeted therapies, such as the potent inhibitors of v-Raf murine sarcoma viral oncogene homolog B (BRAFi) and mitogen-activated protein kinase kinase (MEKi). Very high rates of response have been achieved, but most patients are relapsed due to the development of resistance, justifying the constant search for new therapeutic compounds. Early results from our group indicated that 4-nerolidylcatechol (4-NC), a catechol compound extracted from Pothomorphe umbellata, induces DNA damage, ROS production, increased p53 expression culminating in apoptosis in melanoma but with no data regarding the 4-NC effects in cells resistant to BRAFi or MEKi. Therefore, here we evaluated the role of 4-NC alone or in combination with BRAFi/MEKi in resistant melanoma cells. Double-resistant cells were generated and characterized by MAPK pathway reactivation. 4-NC alone or in combination (30 µM) with MAPK inhibitors was cytotoxic, inhibited colony formation and decreased invasiveness in two and three-dimensional cell culture models of treatment-naïve, BRAFi-resistant and BRAF/MEKi double-resistant melanoma cells. Apoptosis induction was demonstrated in resistant and double-resistant melanoma cell lines after 4-NC treatments. 4-NC showed important ability to induce apoptosis via Endoplasmatic Reticulum (ER) stress and specifically BiP and CHOP that had increased protein expression in all melanoma cell lines proving to be part of the ER stress pathway activation. CHOP knockdown slightly but enough increases cellular viability following 4-NC treatment indicating that apoptosis observed is partially dependent on CHOP. In summary, we show that 4-NC is a compound with activity against cutaneous melanoma, including resistant cells to clinically approved therapies.

SinCHet: a MATLAB toolbox for single cell heterogeneity analysis in cancer.

SUMMARY: Single-cell technologies allow characterization of transcriptomes and epigenomes for individual cells under different conditions and provide unprecedented resolution for researchers to investigate cellular heterogeneity in cancer. The SinCHet ( gle ell erogeneity) toolbox is developed in MATLAB and has a graphical user interface (GUI) for visualization and user interaction. It analyzes both continuous (e.g. mRNA expression) and binary omics data (e.g. discretized methylation data). The toolbox does not only quantify cellular heterogeneity using S hannon P rofile (SP) at different clonal resolutions but also detects heterogeneity differences using a D statistic between two populations. It is defined as the area under the P rofile of S hannon D ifference (PSD). This flexible tool provides a default clonal resolution using the change point of PSD detected by multivariate adaptive regression splines model; it also allows user-defined clonal resolutions for further investigation. This tool provides insights into emerging or disappearing clones between conditions, and enables the prioritization of biomarkers for follow-up experiments based on heterogeneity or marker differences between and/or within cell populations. AVAILABILITY AND IMPLEMENTATION: The SinCHet software is freely available for non-profit academic use. The source code, example datasets, and the compiled package are available at http://labpages2.moffitt.org/chen/software/ . CONTACT: ann.chen@moffitt.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Activity-Based Protein Profiling Shows Heterogeneous Signaling Adaptations to BRAF Inhibition.

Patients with BRAF V600E mutant melanoma are typically treated with targeted BRAF kinase inhibitors, such as vemurafenib and dabrafenib. Although these drugs are initially effective, they are not curative. Most of the focus to date has been upon genetic mechanisms of acquired resistance; therefore, we must better understand the global signaling adaptations that mediate escape from BRAF inhibition. In the current study, we have used activity-based protein profiling (ABPP) with ATP-analogue probes to enrich kinases and other enzyme classes that contribute to BRAF inhibitor (BRAFi) resistance in four paired isogenic BRAFi-naïve/resistant cell line models. Our analysis showed these cell line models, which also differ in their PTEN status, have considerable heterogeneity in their kinase ATP probe uptake in comparing both naïve cells and adaptations to chronic drug exposure. A number of kinases including FAK1, SLK, and TAOK2 had increased ATP probe uptake in BRAFi resistant cells, while KHS1 (M4K5) and BRAF had decreased ATP probe uptake in the BRAFi-resistant cells. Gene ontology (GO) enrichment analysis revealed BRAFi resistance is associated with a significant enhancement in ATP probe uptake in proteins implicated in cytoskeletal organization and adhesion, and decreases in ATP probe uptake in proteins associated with cell metabolic processes. The ABPP approach was able to identify key phenotypic mediators critical for each BRAFi resistant cell line. Together, these data show that common phenotypic adaptations to BRAF inhibition can be mediated through very different signaling networks, suggesting considerable redundancy within the signaling of BRAF mutant melanoma cells.

Managing leptomeningeal melanoma metastases in the era of immune and targeted therapy.

Melanoma frequently metastasizes to the brain, with CNS involvement being clinically evident in ∼30% of patients (as high as 75% at autopsy). In ∼5% cases melanoma cells also metastasize to the leptomeninges, the sub-arachnoid space and cerebrospinal fluid (CSF). Patients with leptomeningeal melanoma metastases (LMM) have the worst prognosis and are characterized by rapid disease progression (mean survival 8-10 weeks) and a death from neurological causes. The recent years have seen tremendous progress in the development of targeted and immune therapies for melanoma that has translated into an increased survival benefit. Despite these gains, the majority of patients fail therapy and there is a suspicion that the brain and the leptomeninges are a "sanctuary" sites for melanoma cells that escape both targeted therapy and immunologic therapies. Emerging evidence suggests that (1) Cancer cells migrating to the CNS may have unique molecular properties and (2) the CNS/leptomeningeal microenvironment represents a pro-survival niche that influences therapeutic response. In this Mini-Review, we will outline the clinical course of LMM development and will describe how the intracranial immune and cellular microenvironments offer both opportunities and challenges for the successful management of this disease. We will further discuss the latest data demonstrating the potential use of BRAF inhibitors and immune therapy in the management of LMM, and will review future potential therapeutic strategies for the management of this most devastating complication of advanced melanoma.

Evaluating kinase ATP uptake and tyrosine phosphorylation using multiplexed quantification of chemically labeled and post-translationally modified peptides.

Cancer biologists and other healthcare researchers face an increasing challenge in addressing the molecular complexity of disease. Biomarker measurement tools and techniques now contribute to both basic science and translational research. In particular, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for multiplexed measurements of protein biomarkers has emerged as a versatile tool for systems biology. Assays can be developed for specific peptides that report on protein expression, mutation, or post-translational modification; discovery proteomics data rapidly translated into multiplexed quantitative approaches. Complementary advances in affinity purification enrich classes of enzymes or peptides representing post-translationally modified or chemically labeled substrates. Here, we illustrate the process for the relative quantification of hundreds of peptides in a single LC-MRM experiment. Desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are used as examples. These targeted quantification panels can be applied to further understand the biology of human disease.

Evaluating melanoma drug response and therapeutic escape with quantitative proteomics.

The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 µg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor ß signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic.

Phosphoproteomic analysis of basal and therapy-induced adaptive signaling networks in BRAF and NRAS mutant melanoma.

Basal and kinase inhibitor driven adaptive signaling has been examined in a panel of melanoma cell lines using phosphoproteomics in conjunction with pathway analysis. A considerable divergence in the spectrum of tyrosine-phosphorylated peptides was noted at the cell line level. The unification of genotype-specific cell line data revealed the enrichment for the tyrosine-phosphorylated cytoskeletal proteins to be associated with the presence of a BRAF mutation and oncogenic NRAS to be associated with increased receptor tyrosine kinase phosphorylation. A number of proteins including cell cycle regulators (cyclin dependent kinase 1, cyclin dependent kinase 2, and cyclin dependent kinase 3), MAPK pathway components (Extracellular signal regulated kinase 1 and Extracellular signal regulated kinase 2), interferon regulators (tyrosine kinase-2), GTPase regulators (Ras-Rasb interactor 1), and controllers of protein tyrosine phosphorylation (dual specificity tyrosine (Y) phosphorylation regulated kinase 1A and protein tyrosine phosphatase receptor type A) were common to all genotypes. Treatment of a BRAF-mutant/phosphatase and tensin homologue (PTEN) null melanoma cell line with vemurafenib led to decreased phosphorylation of ERK, phospholipase C1, and ß-catenin with increases in receptor tyrosine kinase phosphorylation, signal transduction and activator of signaling 3, and glycogen synthase kinase 3α noted. In NRAS-mutant melanoma, MEK inhibition led to increased phosphorylation of epidermal growth factor receptor signaling pathway components, Src family kinases, and protein kinase Cδ with decreased phosphorylation seen in STAT3 and ERK1/2. Together these data present the first systems level view of adaptive and basal phosphotyrosine signaling in BRAF- and NRAS-mutant melanoma.

Rewired ERK-JNK signaling pathways in melanoma.

Constitutive activation of MEK-ERK signaling is often found in melanomas. Here, we identify a mechanism that links ERK with JNK signaling in human melanoma. Constitutively active ERK increases c-Jun transcription and stability, which are mediated by CREB and GSK3, respectively. Subsequently, c-Jun increases transcription of target genes, including RACK1, an adaptor protein that enables PKC to phosphorylate and enhance JNK activity, enforcing a feed-forward mechanism of the JNK-Jun pathway. Activated c-Jun is also responsible for elevated cyclin D1 expression, which is frequently overexpressed in human melanoma. Our data reveal that, in human melanoma, the rewired ERK signaling pathway upregulates JNK and activates the c-Jun oncogene and its downstream targets, including RACK1 and cyclin D1.

Resistance to Raf inhibition in cancer.

The use of small molecule BRAF inhibitors has revolutionized the treatment of advanced melanoma. Despite this, resistance is commonplace and associated with a median progression-free survival of >5 months. Major resistance mechanisms include reactivation of the MAPK pathway and increased PI3K/AKT signaling. Here we review some of the combination therapeutic strategies currently undergoing evaluation for the management of acquired drug resistance in melanoma.

Ex Vivo Culture of Circulating Tumor Cells in the Cerebral Spinal Fluid from Melanoma Patients to Study Melanoma-Associated Leptomeningeal Disease.

Melanoma-associated leptomeningeal disease (M-LMD) occurs when circulating tumor cells (CTCs) enter into the cerebral spinal fluid (CSF) and colonize the meninges, the membrane layers that protect the brain and the spinal cord. Once established, the prognosis for M-LMD patients is dismal, with overall survival ranging from weeks to months. This is primarily due to a paucity in our understanding of the disease and, as a consequence, the availability of effective treatment options. Defining the underlying biology of M-LMD will significantly improve the ability to adapt available therapies for M-LMD treatment or design novel inhibitors for this universally fatal disease. A major barrier, however, lies in obtaining sufficient quantities of CTCs from the patient-derived CSF (CSF-CTCs) to conduct preclinical experiments, such as molecular characterization, functional analysis, and in vivo efficacy studies. Culturing CSF-CTCs ex vivo has also proven to be challenging. To address this, a novel protocol for the culture of patient-derived M-LMD CSF-CTCs ex vivo and in vivo is developed. The incorporation of conditioned media produced by human meningeal cells (HMCs) is found to be critical to the procedure. Cytokine array analysis reveals that factors produced by HMCs, such as insulin-like growth factor-binding proteins (IGFBPs) and vascular endothelial growth factor-A (VEGF-A), are important in supporting CSF-CTC survival ex vivo. Here, the usefulness of the isolated patient-derived CSF-CTC lines is demonstrated in determining the efficacy of inhibitors that target the insulin-like growth factor (IGF) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, the ability to intrathecally inoculate these cells in vivo to establish murine models of M-LMD that can be employed for preclinical testing of approved or novel therapies is shown. These tools can help unravel the underlying biology driving CSF-CTC establishment in the meninges and identify novel therapies to reduce the morbidity and mortality associated with M-LMD.

Leptomeningeal disease in melanoma: An update on the developments in pathophysiology and clinical care.

Leptomeningeal disease (LMD) remains a major challenge in the clinical management of metastatic melanoma patients. Outcomes for patient remain poor, and patients with LMD continue to be excluded from almost all clinical trials. However, recent trials have demonstrated the feasibility of conducting prospective clinical trials in these patients. Further, new insights into the pathophysiology of LMD are identifying rational new therapeutic strategies. Here we present recent advances in the understanding of, and treatment options for, LMD from metastatic melanoma. We also annotate key areas of future focus to accelerate progress for this challenging but emerging field.

Structural basis for mouse LAG3 interactions with the MHC class II molecule I-Ab.

The immune checkpoint protein, Lymphocyte activation gene-3 (LAG3), binds Major Histocompatibility Complex Class II (MHC-II) and suppresses T cell activation. Despite the recent FDA approval of a LAG3 inhibitor for the treatment of melanoma, how LAG3 engages MHC-II on the cell surface remains poorly understood. Here, we determine the 3.84 Å-resolution structure of mouse LAG3 bound to the MHC-II molecule I-Ab, revealing that domain 1 (D1) of LAG3 binds a conserved, membrane-proximal region of MHC-II spanning both the α2 and ß2 subdomains. LAG3 dimerization restricts the intermolecular spacing of MHC-II molecules, which may attenuate T cell activation by enforcing suboptimal signaling geometry. The LAG3-MHC-II interface overlaps with the MHC-II-binding site of the T cell coreceptor CD4, implicating disruption of CD4-MHC-II interactions as a mechanism for LAG3 immunosuppressive function. Lastly, antibody epitope analysis indicates that multiple LAG3 inhibitors do not recognize the MHC-II-binding interface of LAG3, suggesting a role for functionally distinct mechanisms of LAG3 antagonism in therapeutic development.

Modeling Melanoma Heterogeneity In Vitro: Redox, Resistance and Pigmentation Profiles.

Microenvironment and transcriptional plasticity generate subpopulations within the tumor, and the use of BRAF inhibitors (BRAFis) contributes to the rise and selection of resistant clones. We stochastically isolated subpopulations (C1, C2, and C3) from naïve melanoma and found that the clones demonstrated distinct morphology, phenotypic, and functional profiles: C1 was less proliferative, more migratory and invasive, less sensitive to BRAFis, less dependent on OXPHOS, more sensitive to oxidative stress, and less pigmented; C2 was more proliferative, less migratory and invasive, more sensitive to BRAFis, less sensitive to oxidative stress, and more pigmented; and C3 was less proliferative, more migratory and invasive, less sensitive to BRAFis, more dependent on OXPHOS, more sensitive to oxidative stress, and more pigmented. Hydrogen peroxide plays a central role in oxidative stress and cell signaling, and PRDXs are one of its main consumers. The intrinsically resistant C1 and C3 clones had lower MITF, PGC-1α, and PRDX1 expression, while C1 had higher AXL and decreased pigmentation markers, linking PRDX1 to clonal heterogeneity and resistance. PRDX2 is depleted in acquired BRAFi-resistant cells and acts as a redox sensor. Our results illustrate that decreased pigmentation markers are related to therapy resistance and decreased antioxidant defense.