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Uveal melanoma (UM) is a rare cancer arising from melanocytes in the uveal tract of the eye. Despite effective primary treatment, there is no approved therapy for metastatic UM and prognosis and survival remain poor. Over 90% of UM are driven by mutations affecting the Gα subunits encoded by the GNAQand GNA11genes. These mutations activate downstream and targetable signaling pathways, including the protein kinase C (PKC) cascade. PKC inhibitors have been used in clinical trials for metastatic UM but have shown limited efficacy. In this study, we examined the signaling and functional effects of two PKC inhibitors (AEB071 and IDE196) in a panel of UM cell models. In response to PKC inhibition, all UM cell lines showed potent suppression of PKC activity, but this was not sufficient to predict PKC inhibitor sensitivity and only two UM cell lines showed substantial PKC inhibitor-induced cell death. The differences in UM cell responses to PKC inhibition were not attributable to the degree or timing of PKC suppression or inhibition of the downstream mitogen-activated protein kinase (MAPK) or phosphatidylinositol-3-kinase (PI3K) pathways. Instead, UM cell show complex, PKC-independent signaling pathways that contribute to their survival and resistance to targeted therapies.

Blue nevi are benign, melanocytic neoplasms that show a range of clinical and morphologic patterns and include common/dendritic, cellular, and atypical cellular subtypes. Like other nevi, they most commonly occur in skin but can occasionally involve lymph nodes where they may be misinterpreted as representing metastatic melanoma. Moreover, whether benign blue nevi can metastasize to lymph nodes and their natural history and prognostic significance has been the subject of great controversy. To date, few cases of nodal blue nevi have been reported in the literature, and those reports have had limited clinical follow-up and supporting molecular data. This study sought to determine the clinical, pathologic, and molecular features of blue nevi involving lymph nodes, clarify their clinical significance, provide evidence for understanding their pathogenesis, and highlight potential pitfalls in the interpretation of lymph nodes with an ultimate aim of improving patient care. Thirteen cases of blue nevi involving lymph nodes were identified in the archives of Royal Prince Alfred Hospital, Sydney, Australia (1984-2018). A detailed assessment of the clinical and pathologic features of each case was performed, including an evaluation of all available immunohistochemical stains. Extended clinical follow-up was available for 9 patients. Droplet digital polymerase chain reaction for GNAQ Q209L, Q209P and GNA11 Q209L mutations was performed on 7 cases of blue nevi within lymph nodes together with matching cutaneous (presumed primary) blue nevi in 2 cases. All cases showed typical histologic features of blue nevi. BAP1 was retained in all cases (n=7). There were no recurrence or metastasis of blue nevus in any case on long-term clinical follow-up (n=9, median follow-up, 12 y). The majority of cases (n=5 of 7 evaluated) had GNAQ and GNA11 driver mutations. The 2 patients with a matched primary cutaneous blue nevus and regionally associated nodal blue nevus had the same GNAQ Q209L mutation in both sites in each patient. We conclude that blue nevi can involve lymph nodes and are associated with benign clinical behavior, and probably represent so-called “benign” metastasis. Awareness of these lesions is important when evaluating lymph nodes to avoid misdiagnosis as metastatic melanoma.

Nanotechnology-based RNA interference (RNAi) has shown great promise in overcoming the limitations of traditional clinical treatments for glioblastoma (GBM). However, because of the complexity of brain physiology, simple blood–brain barrier (BBB) penetration or tumor-targeting strategies cannot entirely meet the demanding requirements of different therapeutic delivery stages. Herein, we developed a charge conversional biomimetic nanoplatform with a three-layer core–shell structure to programmatically overcome persistent obstacles in siRNA delivery to GBM. The resulting nanocomplex presents good biocompatibility, prolonged blood circulation, high BBB transcytosis, effective tumor accumulation, and specific uptake by tumor cells in the brain. Moreover, red blood cell membrane (RBCm) disruption and effective siRNA release can be further triggered elegantly by charge conversion from negative to positive in the endo/lysosome (pH 5.0–6.5) of tumor cells, leading to highly potent target-gene silencing with a strong anti-GBM effect. Our study provides an intelligent biomimetic nanoplatform tailored for systemically siRNA delivery to GBM, leveraging Angiopep-2 peptide-modified, immune-free RBCm and charge conversional components. Improved therapeutic efficacy, higher survival rates, and minimized systemic side effects were achieved in orthotopic U87MG-luc human glioblastoma tumor-bearing nude mice.

Poly(amidoamine) dendrimer (PAMAM) is well-known for its high efficiency as a drug delivery vehicle. However, the intrinsic cytotoxicity and lack of a detectable signal to facilitate tracking have impeded its practical applications. Herein, we have developed a novel label-free fluorescent and biocompatible PAMAM derivative by simple surface modification of PAMAM using acetaldehyde. The modified PAMAM possessed a strong green fluorescence, which was generated by the C=N bonds of the resulting Schiff Bases via n−π* transition, while the intrinsic cytotoxicity of PAMAM was simultaneously ameliorated. Through further PEGylation, the fluorescent PAMAM demonstrated excellent intracellular tracking in human melanoma SKMEL28 cells. In addition, our PEGylated fluorescent PAMAM derivative achieved enhanced loading and delivery efficiency of the anticancer drug doxorubicin (DOX) compared to the original PAMAM. Importantly, the accelerated kinetics of DOX-encapsulated fluorescent PAMAM nanocomposites in an acidic environment facilitated intracellular drug release, which demonstrated comparable cytotoxicity to that of the free-form doxorubicin hydrochloride (DOX·HCl) against melanoma cells. Overall, our label free fluorescent PAMAM derivative offers a new opportunity of traceable and controlled delivery for DOX and other drugs of potential clinical importance.

IMPORTANCE: Longitudinal circulating tumor DNA (ctDNA) has been shown to predict response and survival in patients with metastatic melanoma treated with anti–programmed cell death 1 (PD-1) antibodies. Pseudoprogression, defined as radiologic finding of disease progression prior to response, has been a challenge to clinicians. OBJECTIVE: To establish whether ctDNA at baseline and up to week 12 of treatment can differentiate between the radiologic findings of pseudoprogression and true progression in patients with metastatic melanoma. DESIGN, SETTING, AND PARTICIPANTS: This explorative biomarker study examined circulating BRAF and NRAS mutations in a cohort of 125 patients with melanoma receiving PD-1 antibodies alone or in combination with ipilimumab between July 3, 2014, and May 24, 2016. Pseudoprogression was defined retrospectively as radiologic progression not confirmed as progressive disease at the next radiologic assessment. Plasma samples of ctDNA at baseline and while receiving treatment were taken for analysis prospectively over the first 12 weeks of treatment. Favorable ctDNA profile (undetectable ctDNA at baseline or detectable ctDNA at baseline followed by >10-fold decrease) and unfavorable ctDNA profile (detectable ctDNA at baseline that remained stable or increased) were correlated with response and prognosis. MAIN OUTCOMES AND MEASURES: Early differentiation of pseudoprogression from true progression using longitudinal ctDNA profile. RESULTS: According to guidelines by Response Evaluation Criteria in Solid Tumors (RECIST), progressive disease occurred in 29 of the 125 patients (23.2%). Of the 29 patients, 17 (59%) were 65 years or younger, 18 (62%) were men, 9 (31%) had pseudoprogression, and 20 (69%) had true progression. Of the 9 patients (7%) with confirmed pseudoprogression, all patients had a favorable ctDNA profile. At a median follow-up of 110 weeks, 7 of 9 patients (78%) were alive. All but 2 patients with true progression had an unfavorable ctDNA profile. Sensitivity of ctDNA for predicting pseudoprogression was 90% (95% CI, 68%-99%) and specificity was 100% (95% CI, 60%-100%). The 1-year survival for patients with RECIST-defined progressive disease and favorable ctDNA was 82% vs 39% for unfavorable ctDNA (hazard ratio [HR], 4.8; 95% CI, 1.6-14.3; P = .02). Overall survival was longer in patients with a partial response (54 of 125 patients [43%]) compared with patients with progressive disease and a favorable ctDNA profile (11 of 125 patients [9%]; HR, 0.09; 95% CI, 0.01-0.80; P < .01). CONCLUSIONS AND RELEVANCE: The results demonstrate that ctDNA profiles can accurately differentiate pseudoprogression from true progression of disease in patients with melanoma treated with PD-1 antibodies. Results of this blood test performed at regular intervals during systemic treatment reflect tumor biology and have potential as a powerful biomarker to predict long-term response and survival.

Immunotherapy targeting PD-1 and/or CTLA4 leads to durable responses in a proportion of patients with melanoma. However, many patients will not respond to these immune checkpoint inhibitors, and up to 60% of responding patients will develop treatment resistance. We describe a vulnerability in melanoma driven by immune cell activity that provides a pathway towards additional treatment options. This study evaluated short-term melanoma cell lines (referred to as PD1 PROG cells) derived from melanoma metastases that progressed on PD-1 inhibitor–based therapy. We show that the cytokine IFN-γ primes melanoma cells for apoptosis by promoting changes in the accumulation and interactions of apoptotic regulators MCL-1, NOXA, and BAK. The addition of pro-apoptotic BH3 mimetic drugs sensitized PD1 PROG melanoma cells to apoptosis in response to IFN-γ or autologous immune cell activation. These findings provide translatable strategies for combination therapies in melanoma.

Colorectal cancer (CRC) is the third most diagnosed and the second lethal cancer worldwide. Approximately 30–50% of CRC are driven by mutations in the KRASoncogene, which is a strong negative predictor for response to anti‐epidermal growth factor receptor (anti‐EGFR) therapy. Examining the phenotype of KRASmutant and wild‐type (WT) CRC cells in response to anti‐EGFR treatment may provide significant insights into drug response and resistance. Herein, surface‐enhanced Raman spectroscopy (SERS) assay was applied to phenotype four cell surface proteins (EpCAM, EGFR, HER2, HER3) in KRASmutant (SW480) and WT (SW48) cells over a 24‐day time course of anti‐EGFR treatment with cetuximab. Cell phenotypes were obtained using Raman reporter‐coated and antibody‐conjugated gold nanoparticles (SERS nanotags), where a characteristic Raman spectrum was generated upon single laser excitation, reflecting the presence of the targeted surface marker proteins. Compared to the KRASmutant cells, KRASWT cells were more sensitive to anti‐EGFR treatment and displayed a significant decrease in HER2 and HER3 expression. The SERS results were validated with flow cytometry, confirming the SERS assay is promising as an alternative method for multiplexed characterization of cell surface biomarkers using a single laser excitation system. Examining the phenotypes of KRAS mutant and wild‐type colorectal cancer cells during anti‐EGFR treatment may provide significant insights into drug resistance. Herein, we applied surface‐enhanced Raman spectroscopy (SERS) assay to detect four cell surface proteins on KRAS mutant and wild‐type cells over a 24‐day treatment, where SERS assay achieved multiplexed characterization of cell surface markers using a single laser excitation.

The p14ARF tumour suppressor is frequently targeted for inactivation in many human cancers and in individuals predisposed to cutaneous melanoma. The functions of p14ARF are closely linked with its subcellular distribution. Nucleolar p14ARF dampens ribosome biosynthesis and nucleoplasmic forms of p14ARF activate the p53 pathway and induce cell cycle arrest. p14ARF can also be recruited to mitochondria where it interacts with many mitochondrial proteins, including Bcl-xL and p32 to induce cell death. It has been suggested that the movement of p14ARF to mitochondria requires its interaction with p32, but we now show that the ARF-p32 interaction is not necessary for the accumulation of p14ARF in mitochondria. Instead, highly hydrophobic domains within the amino-terminal half of p14ARF act as mitochondrial import sequences. We suggest that once this hydrophobic pocket is exposed, possibly in a stimulus-dependent manner, it accelerates the mitochondrial import of p14ARF. This allows the interaction of p14ARF with mitochondrial proteins, including p32 and enables p53-independent cell death.

Alterations in the ARF tumour suppressor protein (also known as p14ARF in humans and p19ARF in the mouse) occur frequently in cancer and are associated with susceptibility to melanoma, pancreatic cancer and nervous system tumours. ARF proteins interact with the E2F-1, -2 and -3 transcription activators to inhibit their transcriptional activity and induce their degradation via the 26S proteasome pathway. The impact of ARF on the E2F proteins may provide a mechanism for p53-independent ARF activity on cell cycle progression and tumour susceptibility. In this report we explored the effects of ARF on E2F ubiquitination and degradation in relationship to cell cycle effects and p53 status. We now show that ARF induced the rapid ubiquitination and degradation of E2F-1 only in the presence of functional p53. E2F-1 continued to be ubiquitinated following ARF induction in cycling p53-wild-type, p21-null cells, showing that effects of ARF were not simply a result of p14ARF induced cell-cycle arrest. Importantly, these data establish that the ARF-E2F-1 pathway is an extension of the p53-mdm2-ARF tumour suppressor network and is unlikely to constitute a p53-independent pathway for ARF function.

The p14ARF tumour suppressor regulates a series of cell cycle regulatory proteins to promote cellcycle arrest in response to abnormal hyperproliferative growth stimuli. p14ARF alterations arecommon in human cancers and, when inherited, confer susceptibility to cutaneous melanoma. Wenow propose that the mechanism of p14ARF action may involve the covalent modification of itsbinding partners with the small ubiquitin-related protein SUMO-1. In particular, we demonstratethat p14ARF interacts with the SUMO E2 conjugating enzyme, Ubc9 and enhances the sumoylationof its binding partners, hdm2, E2F-1, HIF-1?, TBP-1 and p120E4F. Furthermore, p14ARF-inducedsumoylation is abrogated by a subset of melanoma-associated p14ARF mutations. These resultsprovide a mechanism for p14ARF action through a common modification of diverse bindingpartners.

Expression of the p14ARF tumour suppressor is induced by hyperproliferative signals produced by RAS, MYC and other oncogenes. p14ARF quenches inappropriate mitogenic signaling by activating the p53 pathway, and the frequent loss of p14ARF in human cancer diminishes the duration and level of the p53 response. Consistent with this role, p14ARF accumulation can induce potent cell cycle arrest, but its role in promoting apoptosis has not been well established. Therefore we investigated the effects of p14ARF on the survival and growth of several human cell types. To avoid the toxicity associated with adenoviral-based vectors, we established inducible expression of p14ARF in p53-intact and p53-deficient human cell lines. As expected, transient and inducible expression of p14ARF induced rapid cell cycle arrest only in tumour cells expressing intact p53. Further, p14ARF expression did not promote apoptosis in primary human fibroblasts, or in any human tumour cell line tested, irrespective of p53 status. Instead, p14ARF expression sensitized cells to apoptosis in the presence of inhibitors of topoisomerase II (adriamycin) and transcription (DRB). Thus, loss of p14ARF would be an important step in the selection of apoptotic resistant tumour cells.

The INK4a/ARFlocus encodes two cell cycle regulatory proteins, the cyclin-dependent kinase inhibitor, p16INK4a, and the p53 activator, p14ARF. Germline mutations in this locus are associated with melanoma susceptibility in 20–40% of multiple case melanoma families. Many of these mutations specifically impair p16INK4a, whereas mutations uniquely targeting p14ARFare rare. Nevertheless, the importance of p14ARFhas not been excluded because more than 40% ofINK4a/ARFalterations affect p16INK4aand p14ARF. We now report that p14ARFis functionally impaired in melanoma kindreds carryingINK4a/ARFmutations. Of the seven INK4a/ARFmutations tested, three altered the subcellular distribution of p14ARFand diminished the ability of p14ARFto activate the p53 pathway. This work establishes the importance of p14ARFin melanoma predisposition.

The development of resistance to treatments of melanoma is commonly associated with an upregulation of the MAPK pathway and the development of an undifferentiated state. Previous studies have suggested that melanoma with these resistance characteristics may be susceptible to innate death mechanisms such as pyroptosis triggered by the activation of inflammasomes. In this study, we have taken cell lines from patients before and after the development of resistance to BRAF V600 inhibitors and exposed the resistant melanoma to temozolomide (a commonly used chemotherapy) with and without chloroquine to inhibit autophagy. It was found that melanoma with an inflammatory undifferentiated state appeared susceptible to this combination when tested in vitro and in vivo against xenografts in nonobese diabetic scid gamma mice. Translation of the latter results into patients would promise durable responses in patients treated by the combination. The inflammasome and death mechanism involved appeared to vary between melanoma and involved either AIM2 or NLRP3 inflammasomes and gasdermin D or E. These preliminary studies have raised questions as to the selectivity for different inflammasomes in different melanoma and their selective targeting by chemotherapy. They also question whether the inflammatory state of melanoma may be used as biomarkers to select patients for inflammasome-targeted therapy.

ABSTRACTPurpose: Anti-PD-1 therapy has revolutionized the treatment and improved the survival of stage IV melanoma patients. However, almost half of the patients fail to respond due to immune evasive mechanism. A known mechanism is the downregulation of major histocompatibility complex (MHC) class I expression, which prevents T cell recognition of the tumor. This study determined the relationship between natural killer (NK) cell numbers and clinical response to anti-PD-1 therapy in metastatic melanoma.Experimental Design: Twenty-five anti-PD-1 treated metastatic melanoma patients were categorized into responders (complete response (CR)/partial response (PR)/stable disease (SD) ≥ 6 mo, n = 13) and non-responders (SD < 6 days/progressive disease (PD), n = 12) based on RECIST response. Whole transcriptome sequencing and multiplex immunofluorescent staining were performed on pre-treatment and on a subset of early during treatment tumor samples. Spatial distribution analysis was performed on multiplex immunofluorescent images to determine the proximity of NK cells to tumor cells. Flow cytometry was used to confirm NK phenotypes in lymph node metastases of treatment naïve melanoma patients (n = 5). Cytotoxic assay was performed using NK cells treated with anti-PD-1 or with isotype control and co-cultured with 3 different melanoma cell lines and with K562 cells (leukemia cell line).Results: Differential expression analysis identified nine upregulated NK cell specific genes (adjusted p < 0.05) in responding (n = 11) versus non-responding patients (n = 10). Immunofluorescent staining of biopsies confirmed a significantly higher density of intra- and peri-tumoral CD16+ and granzyme B + NK cells in responding patients (p < 0.05). Interestingly, NK cells were in closer proximity to tumor cells in responding PD-1 treated patients compared to non-responding patients. Patients who responded to anti-PD-1 therapy, despite MHC class I loss had higher NK cell densities than patients with low MHC class I expression. Lastly, functional assays demonstrated PD-1 blockade induces an increase in NK cells’ cytotoxicity.Conclusions: A higher density of tumoral NK cells is associated with response to anti-PD-1 therapy. NK cells may play an important role in mediating response to anti-PD-1 therapy, including in a subset of tumors downregulating MHC class I expression.

Mutations of the serine/threonine-protein kinase (BRAF) gene occur in approximately half of all patients with met-astatic melanoma. The BRAF inhibitors have proven highly active in treating BRAF mutant metastatic melanoma patients. Eventually, almost all patients develop resistance to BRAF inhibition and relapse.

Comment on: Haferkamp S, et al. Aging 2009; 1:542-56.

The transcriptional regulator c-JUN is a key mediator of the metastatic potential and drug resistance in melanoma.