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The chemokine receptor, CXCR4, is involved in cancer growth, invasion, and metastasis. Several promising CXCR4 antagonists have been shown to halt tumor metastasis in preclinical studies, and clinical trials evaluating the effectiveness of these agents in patients with cancer are ongoing. However, the impact of targeting CXCR4 specifically on immune cells is not clear. Here, we demonstrate that genetic deletion of CXCR4 in myeloid cells (CXCR4) enhances the antitumor immune response, resulting in significantly reduced melanoma tumor growth. Moreover, CXCR4 mice exhibited slowed tumor progression compared with CXCR4 mice in an inducible melanocyte mouse model. The percentage of Fas ligand (FasL)-expressing myeloid cells was reduced in CXCR4 mice as compared with myeloid cells from CXCR4 mice. In contrast, there was an increased percentage of natural killer (NK) cells expressing FasL in tumors growing in CXCR4 mice. NK cells from CXCR4 mice also exhibited increased tumor cell killing capacity , based on clearance of NK-sensitive Yac-1 cells. NK cell-mediated killing of Yac-1 cells occurred in a FasL-dependent manner, which was partially dependent upon the presence of CXCR4 neutrophils. Furthermore, enhanced NK cell activity in CXCR4 mice was also associated with increased production of IL18 by specific leukocyte subpopulations. These data suggest that CXCR4-mediated signals from myeloid cells suppress NK cell-mediated tumor surveillance and thereby enhance tumor growth. Systemic delivery of a peptide antagonist of CXCR4 to tumor-bearing CXCR4 mice resulted in enhanced NK-cell activation and reduced tumor growth, supporting potential clinical implications for CXCR4 antagonism in some cancers. .
©2018 American Association for Cancer Research.
Little is known about the in vivo impacts of targeted therapy on melanoma cell abundance and protein expression. Here, 21 antibodies were added to an established melanoma mass cytometry panel to measure 32 cellular features, distinguish malignant cells, and characterize dabrafenib and trametinib responses in BRAF melanoma. Tumor cells were biopsied before neoadjuvant therapy and compared to cells surgically resected from the same site after 4 weeks of therapy. Approximately 50,000 cells per tumor were characterized by mass cytometry and computational tools t-SNE/viSNE, FlowSOM, and MEM. The resulting single-cell view of melanoma treatment response revealed initially heterogeneous melanoma tumors were consistently cleared of Nestin-expressing melanoma cells. Melanoma cell subsets that persisted to week 4 were heterogeneous but expressed SOX2 or SOX10 proteins and specifically lacked surface expression of MHC I proteins by MEM analysis. Traditional histology imaging of tissue microarrays from the same tumors confirmed mass cytometry results, including persistence of NES- SOX10+ S100β+ melanoma cells. This quantitative single-cell view of melanoma treatment response revealed protein features of malignant cells that are not eliminated by targeted therapy.
© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Targeted therapy is an effective standard of care in BRAF-mutated malignant melanoma. However, the duration of tumor remission varies unpredictably among patients, and relapse is almost inevitable. Here, we examine the responses of several BRAF-mutated melanoma cell lines (including isogenic subclones) to BRAF inhibitors. We observe complex response dynamics across cell lines, with short-term responses (<100 h) varying from cell line to cell line. In the long term, however, we observe equilibration of all drug-treated populations into a nonquiescent state characterized by a balanced rate of death and division, which we term the "idling" state, and to our knowledge, this state has not been previously reported. Using mathematical modeling, we propose that the observed population-level dynamics are the result of cells transitioning between basins of attraction within a drug-modified phenotypic landscape. Each basin is associated with a drug-induced proliferation rate, a recently introduced metric of an antiproliferative drug effect. The idling population state represents a new dynamic equilibrium in which cells are distributed across the landscape such that the population achieves zero net growth. By fitting our model to experimental drug-response data, we infer the phenotypic landscapes of all considered melanoma cell lines and provide a unifying view of how BRAF-mutated melanomas respond to BRAF inhibition. We hypothesize that the residual disease observed in patients after targeted therapy is composed of a significant number of idling cells. Thus, defining molecular determinants of the phenotypic landscape that idling populations occupy may lead to "targeted landscaping" therapies based on rational modification of the landscape to favor basins with greater drug susceptibility.
Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Antagonists of MDM2-p53 interaction are emerging anti-cancer drugs utilized in clinical trials for malignancies that rarely mutate p53, including melanoma. We discovered that MDM2-p53 antagonists protect DNA from drug-induced damage in melanoma cells and patient-derived xenografts. Among the tested DNA damaging drugs were various inhibitors of Aurora and Polo-like mitotic kinases, as well as traditional chemotherapy. Mitotic kinase inhibition causes mitotic slippage, DNA re-replication, and polyploidy. Here we show that re-replication of the polyploid genome generates replicative stress which leads to DNA damage. MDM2-p53 antagonists relieve replicative stress via the p53-dependent activation of p21 which inhibits DNA replication. Loss of p21 promoted drug-induced DNA damage in melanoma cells and enhanced anti-tumor activity of therapy combining MDM2 antagonist with mitotic kinase inhibitor in mice. In summary, MDM2 antagonists may reduce DNA damaging effects of anti-cancer drugs if they are administered together, while targeting p21 can improve the efficacy of such combinations.
Copyright © 2017. Published by Elsevier B.V.
Melanoma is the deadliest form of skin cancer and presents a significant health care burden in many countries. In addition to ultraviolet radiation in sunlight, the main causal factor for melanoma, genetic factors also play an important role in melanoma susceptibility. Although genome-wide association studies have identified many single nucleotide polymorphisms associated with melanoma, little is known about the proportion of disease risk attributable to these loci and their distribution throughout the genome. Here, we investigated the genetic architecture of melanoma in 1,888 cases and 990 controls of European non-Hispanic ancestry. We estimated the overall narrow-sense heritability of melanoma to be 0.18 (P < 0.03), indicating that genetics contributes significantly to the risk of sporadically-occurring melanoma. We then demonstrated that only a small proportion of this risk is attributable to known risk variants, suggesting that much remains unknown of the role of genetics in melanoma. To investigate further the genetic architecture of melanoma, we partitioned the heritability by chromosome, minor allele frequency, and functional annotations. We showed that common genetic variation contributes significantly to melanoma risk, with a risk model defined by a handful of genomic regions rather than many risk loci distributed throughout the genome. We also demonstrated that variants affecting gene expression in skin account for a significant proportion of the heritability, and are enriched among melanoma risk loci. Finally, by incorporating skin color into our analyses, we observed both a shift in significance for melanoma-associated loci and an enrichment of expression quantitative trait loci among melanoma susceptibility variants. These findings suggest that skin color may be an important modifier of melanoma risk. We speculate that incorporating skin color and other non-genetic factors into genetic studies may allow for an improved understanding of melanoma susceptibility and guide future investigations to identify melanoma risk genes.
The treatment and prognosis of metastatic melanoma has changed substantially since the advent of novel immune checkpoint inhibitors (ICI), agents that enhance the anti-tumor immune response. Despite the success of these agents, clinically actionable biomarkers to aid patient and regimen selection are lacking. Herein, we summarize and review the evidence for candidate biomarkers of response to ICIs in melanoma. Many of these candidates can be examined as parts of a known molecular pathway of immune response, while others are clinical in nature. Due to the ability of ICIs to illicit dramatic and durable responses, well-validated biomarkers that can be effectively implemented in the clinic will require strong negative predictive values that do not limit patients with who may benefit from ICI therapy.
Copyright © 2017 Elsevier Ltd. All rights reserved.
The class III PI3K Vacuolar protein sorting 34 (Vps34) plays a role in both canonical and noncanonical autophagy, key processes that control the presentation of antigens by dendritic cells (DCs) to naive T lymphocytes. We generated DC-specific -deficient mice to assess the contribution of Vps34 to DC functions. We found that DCs from these animals have a partially activated phenotype, spontaneously produce cytokines, and exhibit enhanced activity of the classic MHC class I and class II antigen-presentation pathways. Surprisingly, these animals displayed a defect in the homeostatic maintenance of splenic CD8α DCs and in the capacity of these cells to cross-present cell corpse-associated antigens to MHC class I-restricted T cells, a property that was associated with defective expression of the T-cell Ig mucin (TIM)-4 receptor. Importantly, mice deficient in the Vps34-associated protein Rubicon, which is critical for a noncanonical form of autophagy called "Light-chain 3 (LC3)-associated phagocytosis" (LAP), lacked such defects. Finally, consistent with their defect in the cross-presentation of apoptotic cells, DC-specific -deficient animals developed increased metastases in response to challenge with B16 melanoma cells. Collectively, our studies have revealed a critical role of Vps34 in the regulation of CD8α DC homeostasis and in the capacity of these cells to process and present antigens associated with apoptotic cells to MHC class I-restricted T cells. Our findings also have important implications for the development of small-molecule inhibitors of Vps34 for therapeutic purposes.
The IL-17/IL-17R axis has controversial roles in cancer, which may be explained by tumor-specific results. Here, we describe a novel molecular mechanism underlying IL-17RC-controlled tumor-specific proliferation. Triggered by IL-17RC knockdown (KD), B16 melanoma and 4T1 carcinoma cells inversely altered homeostatic tumor proliferation and tumor growth in vitro and in vivo. In contrast to the existing dogma that IL-17RC-dependent signaling activates the JNK pathway, IL-17RC KD in both tumor cell lines caused aberrant expression and activation of different JNK isoforms along with markedly diminished levels of the ubiquitin-editing enzyme A20. We demonstrated that differential up-regulation of JNK1 and JNK2 in the two tumor cell lines was responsible for the reciprocal regulation of c-Jun activity and tumor-specific proliferation. Furthermore, we showed that A20 reconstitution of IL-17RCKD clones with expression of full-length A20, but not a truncation-mutant, reversed aberrant JNK1/JNK2 activities and tumor-specific proliferation. Collectively, our study reveals a critical role of IL-17RC in maintaining baseline A20 production and a novel role of the IL-17RC-A20 axis in controlling JNK isoform-dependent tumor-specific homeostatic proliferation.
Quantitative assessment of key proteins that control the tumor-immune interface is one of the most formidable analytical challenges in immunotherapeutics. We developed a targeted MS platform to quantify programmed cell death-1 (PD-1), programmed cell death 1 ligand 1 (PD-L1), and programmed cell death 1 ligand 2 (PD-L2) at fmol/microgram protein levels in formalin fixed, paraffin-embedded sections from 22 human melanomas. PD-L1 abundance ranged 50-fold, from ∼0.03 to 1.5 fmol/microgram protein and the parallel reaction monitoring (PRM) data were largely concordant with total PD-L1-positive cell content, as analyzed by immunohistochemistry (IHC) with the E1L3N antibody. PD-1 was measured at levels up to 20-fold lower than PD-L1, but the abundances were not significantly correlated (r = 0.062, = 0.264). PD-1 abundance was weakly correlated (r = 0.3057, = 0.009) with the fraction of lymphocytes and histiocytes in sections. PD-L2 was measured from 0.03 to 1.90 fmol/microgram protein and the ratio of PD-L2 to PD-L1 abundance ranged from 0.03 to 2.58. In 10 samples, PD-L2 was present at more than half the level of PD-L1, which suggests that PD-L2, a higher affinity PD-1 ligand, is sufficiently abundant to contribute to T-cell downregulation. We also identified five branched mannose and N-acetylglucosamine glycans at PD-L1 position N192 in all 22 samples. Extent of PD-L1 glycan modification varied by ∼10-fold and the melanoma with the highest PD-L1 protein abundance and most abundant glycan modification yielded a very low PD-L1 IHC estimate, thus suggesting that N-glycosylation may affect IHC measurement and PD-L1 function. Additional PRM analyses quantified immune checkpoint/co-regulator proteins LAG3, IDO1, TIM-3, VISTA, and CD40, which all displayed distinct expression independent of PD-1, PD-L1, and PD-L2. Targeted MS can provide a next-generation analysis platform to advance cancer immuno-therapeutic research and diagnostics.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Histopathological interpretation of proliferative nodules occurring in association with congenital melanocytic nevi can be very challenging due to their similarities with congenital malignant melanoma and malignant melanoma arising in association with congenital nevi. We hereby report a diagnostically challenging case of congenital melanocytic nevus with proliferative nodules and ulcerations, which was originally misdiagnosed as congenital malignant melanoma. Subsequent histopathological examination in consultation by one of the authors (R.L.) and mass spectrometry imaging analysis rendered a diagnosis of congenital melanocytic nevus with proliferative nodules. In this case, mass spectrometry imaging, a novel method capable of distinguishing benign from malignant melanocytic lesions on a proteomic level, was instrumental in making the diagnosis of a benign nevus. We emphasize the importance of this method as an ancillary tool in the diagnosis of difficult melanocytic lesions.