IGFBP7 is not required for B-RAF-induced melanocyte senescence.

Induction of senescence permanently restricts cellular proliferation after oncogenic stimulation thereby acting as a potent barrier to tumor development. The relevant effector proteins may therefore be fundamental to cancer development. A recent study identified IGFBP7 as a secreted factor mediating melanocyte senescence induced by oncogenic B-RAF, which is found commonly in cutaneous nevi. In contrast to the previous report, we demonstrate that B-RAF signaling does not induce IGFBP7 expression, nor the expression of the IGFBP7 targets, BNIP3L, SMARCB1, or PEA15, in human melanocytes or fibroblasts. We also found no correlation between B-RAF mutational status and IGFBP7 protein expression levels in 22 melanoma cell lines, 90 melanomas, and 46 benign nevi. Furthermore, using a lentiviral silencing strategy we show that B-RAF induces senescence in melanocytes and fibroblasts, irrespective of the presence of IGFBP7. Therefore, we conclude that the secreted protein IGFBP7 is dispensable for B-RAF(V600E)-induced senescence in human melanocytes.

Ex vivo tissue modelling informs drug selection for rare cancers.

The identification and therapeutic targeting of actionable gene mutations across many cancer types has resulted in improved response rates in a minority of patients. The identification of actionable mutations is usually not sufficient to ensure complete nor durable responses, and in rare cancers, where no therapeutic standard of care exists, precision medicine indications are often based on pan-cancer data. The inclusion of functional data, however, can provide evidence of oncogene dependence and guide treatment selection based on tumour genetic data. We applied an ex vivo cancer explant modelling approach, that can be embedded in routine clinical care and allows for pathological review within 10 days of tissue collection. We now report that ex vivo tissue modelling provided accurate longitudinal response data in a patient with BRAFV600E -mutant papillary thyroid tumour with squamous differentiation. The ex vivo model guided treatment selection for this patient and confirmed treatment resistance when the patient's disease progressed after 8 months of treatment.

Charge Conversional Biomimetic Nanocomplexes as a Multifunctional Platform for Boosting Orthotopic Glioblastoma RNAi Therapy.

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.

MHC Class I Deficiency in Solid Tumors and Therapeutic Strategies to Overcome It.

It is now well accepted that the immune system can control cancer growth. However, tumors escape immune-mediated control through multiple mechanisms and the downregulation or loss of major histocompatibility class (MHC)-I molecules is a common immune escape mechanism in many cancers. MHC-I molecules present antigenic peptides to cytotoxic T cells, and MHC-I loss can render tumor cells invisible to the immune system. In this review, we examine the dysregulation of MHC-I expression in cancer, explore the nature of MHC-I-bound antigenic peptides recognized by immune cells, and discuss therapeutic strategies that can be used to overcome MHC-I deficiency in solid tumors, with a focus on the role of natural killer (NK) cells and CD4 T cells.

Co-targeting bromodomain and extra-terminal proteins and MCL1 induces synergistic cell death in melanoma.

The treatment of melanoma has been markedly improved by the introduction of targeted therapies and checkpoint blockade immunotherapy. Unfortunately, resistance to these therapies remains a limitation. Novel anticancer therapeutics targeting the MCL1 anti-apoptotic protein have shown impressive responses in haematological cancers but are yet to be evaluated in melanoma. To assess the sensitivity of melanoma to new MCL1 inhibitors, we measured the response of 51 melanoma cell lines to the novel MCL1 inhibitor, S63845. Additionally, we assessed combination of this drug with inhibitors of the bromodomain and extra-terminal (BET) protein family of epigenetic readers, which we postulated would assist MCL1 inhibition by downregulating anti-apoptotic targets regulated by NF-kB such as BCLXL, BCL2A1 and XIAP, and by upregulating pro-apoptotic proteins including BIM and NOXA. Only 14% of melanoma cell lines showed sensitivity to S63845, however, combination of S63845 and I-BET151 induced highly synergistic apoptotic cell death in all melanoma lines tested and in an in vivo xenograft model. Cell death was dependent on caspases and BAX/BAK. Although the combination of drugs increased the BH3-only protein, BIM, and downregulated anti-apoptotic proteins such as BCL2A1, the importance of these proteins in inducing cell death varied between cell lines. ABT-199 or ABT-263 inhibitors against BCL2 or BCL2 and BCLXL, respectively, induced further cell death when combined with S63845 and I-BET151. The combination of MCL1 and BET inhibition appears to be a promising therapeutic approach for metastatic melanoma, and presents opportunities to add further BCL2 family inhibitors to overcome treatment resistance.

Spatial and Temporal Changes in PD-L1 Expression in Cancer: The Role of Genetic Drivers, Tumor Microenvironment and Resistance to Therapy.

Immunotherapies blocking immune inhibitory receptors programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) on T-cells have dramatically improved patient outcomes in a range of advanced cancers. However, the lack of response, and the development of resistance remain major obstacles to long-term improvements in patient outcomes. There is significant interest in the clinical use of biomarkers to improve patient selection, and the expression of PD-1 ligand 1 (PD-L1) is often reported as a potential biomarker of response. However, accumulating evidence suggests that the predictive value of PD-L1 expression in tumor biopsies is relatively low due, in part, to its complex biology. In this review, we discuss the biological consequences of PD-L1 expression by various cell types within the tumor microenvironment, and the complex mechanisms that regulate PD-L1 expression at the genomic, transcriptomic and proteomic levels.

Neoadjuvant dabrafenib combined with trametinib for resectable, stage IIIB-C, BRAFV600 mutation-positive melanoma (NeoCombi): a single-arm, open-label, single-centre, phase 2 trial.

BACKGROUND: Adjuvant dabrafenib plus trametinib therapy improves relapse-free survival in patients with resected stage III melanoma. We aimed to ascertain the proportion of patients who would have a pathological response and a response according to Response Evaluation Criteria in Solid Tumors (RECIST) after neoadjuvant dabrafenib plus trametinib therapy for resectable clinical stage III melanoma. METHODS: NeoCombi was a single-arm, open-label, single-centre, phase 2 study done at Melanoma Institute Australia (Sydney, NSW, Australia). Eligible patients were adults (aged ≥18 years) with histologically confirmed, resectable, RECIST-measurable, clinical stage IIIB-C (American Joint Committee on Cancer [AJCC] 7th edition), BRAFV600-mutant melanoma, and had an Eastern Cooperative Oncology Group performance status of 1 or lower. Patients received 150 mg dabrafenib orally, twice daily, plus 2 mg trametinib orally, once daily, for 52 weeks (12 weeks of neoadjuvant therapy before complete resection of the pre-therapy tumour bed, and 40 weeks of adjuvant therapy thereafter). CT and PET scans were done at baseline and before resection. The primary outcomes were the proportion of patients achieving a complete pathological response and the proportion of patients achieving a response according to RECIST at week 12, analysed as per protocol. This trial is registered with ClinicalTrials.gov, NCT01972347, and follow-up of patients is ongoing. FINDINGS: Between Aug 20, 2014, and April 19, 2017, 40 patients were screened, of whom 35 eligible patients were enrolled, received neoadjuvant dabrafenib plus trametinib, and underwent resection. At the data cutoff (Sept 24, 2018), median follow-up was 27 months (IQR 21-36). At resection, 30 (86%) patients achieved a RECIST response; 16 (46%; 95% CI 29-63) had a complete response and 14 (40%; 24-58) had a partial response. Five patients (14%; 95% CI 5-30) had stable disease, and no patients progressed. After resection and pathological evaluation, all 35 patients achieved a pathological response, of whom 17 (49%; 95% CI 31-66) patients had a complete pathological response and 18 (51%; 95% CI 34-69) had a non-complete pathological response. Treatment-related serious adverse events occurred in six (17%) of 35 patients and grade 3-4 adverse events occurred in ten (29%) patients. No treatment-related deaths were reported. INTERPRETATION: Neoadjuvant dabrafenib plus trametinib therapy could be considered in the management of RECIST-measurable resectable stage III melanoma as it led to a high proportion of patients achieving a complete response according to RECIST and a high proportion of patients achieving a complete pathological response, with no progression during neoadjuvant therapy. FUNDING: GlaxoSmithKline; Novartis; National Health and Medical Research Council, Australia; and Melanoma Institute Australia.

Neoadjuvant systemic therapy in melanoma: recommendations of the International Neoadjuvant Melanoma Consortium.

Advances in the treatment of metastatic melanoma have improved responses and survival. However, many patients continue to experience resistance or toxicity to treatment, highlighting a crucial need to identify biomarkers and understand mechanisms of response and toxicity. Neoadjuvant therapy for regional metastases might improve operability and clinical outcomes over upfront surgery and adjuvant therapy, and has become an established role for drug development and biomarker discovery in other cancers (including locally advanced breast cancer, head and neck squamous cell carcinomas, gastroesophageal cancer, and anal cancer). Patients with clinically detectable stage III melanoma are ideal candidates for neoadjuvant therapy, because they represent a high-risk patient population with poor outcomes when treated with upfront surgery alone. Neoadjuvant therapy is now an active area of research for melanoma with numerous completed and ongoing trials (since 2014) with disparate designs, endpoints, and analyses under investigation. We have, therefore, established the International Neoadjuvant Melanoma Consortium with experts in medical oncology, surgical oncology, pathology, radiation oncology, radiology, and translational research to develop recommendations for investigating neoadjuvant therapy in melanoma to align future trial designs and correlative analyses. Alignment and consistency of neoadjuvant trials will facilitate optimal data organisation for future regulatory review and strengthen translational research across the melanoma disease continuum.

Label-Free Fluorescent Poly(amidoamine) Dendrimer for Traceable and Controlled Drug Delivery.

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.

Liquid biomarkers in melanoma: detection and discovery.

A vast array of tumor-derived genetic, proteomic and cellular components are constantly released into the circulation of cancer patients. These molecules including circulating tumor DNA and RNA, proteins, tumor and immune cells are emerging as convenient and accurate liquid biomarkers of cancer. Circulating cancer biomarkers provide invaluable information on cancer detection and diagnosis, prognosticate patient outcomes, and predict treatment response. In this era of effective molecular targeted treatments and immunotherapies, there is now an urgent need to implement use of these circulating biomarkers in the clinic to facilitate personalized therapy. In this review, we present recent findings in circulating melanoma biomarkers, examine the challenges and promise of evolving technologies used for liquid biomarker discovery, and discuss future directions and perspectives in melanoma biomarker research.

HDAC inhibitors restore BRAF-inhibitor sensitivity by altering PI3K and survival signalling in a subset of melanoma.

Mutations in BRAF activate oncogenic MAPK signalling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non-genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signalling. Here, we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient- and in vivo-derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat in 2D and 3D culture systems synergistically induced caspase-dependent apoptotic cell death. Key changes induced by HDAC inhibition included decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases, and cell line dependent upregulation of pro-apoptotic BIM or NOXA together with reduced expression of anti-apoptotic proteins. Independent of these changes, panobinostat reduced c-Myc and pre-treatment of cells with siRNA against c-Myc reduced BRAFi/HDACi drug-induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance mechanisms are due to activation of MAPK-independent pathways.

Immune cell profiling in the age of immune checkpoint inhibitors: implications for biomarker discovery and understanding of resistance mechanisms.

Immunotherapy has changed the landscape of cancer treatment. The introduction of immune checkpoint inhibitors has seen tremendous success in improving overall survival of patients with advanced metastatic cancers and has now become the standard of care for multiple tumor types. However, efficacy of immune checkpoint blockade appears to be limited to immunogenic cancers, and even amongst immune-reactive cancers, response rates are low and variable between patients. Recent data have also demonstrated the rapid emergence of resistance to immune checkpoint inhibitors, with some patients progressing on treatment within one year. Significant research efforts are now directed at identifying predictive biomarkers and mechanisms of resistance to immune checkpoint blockade. These studies are underpinned by comprehensive and detailed profiling of the immune milieu. In this review, we discuss the utility and efficacy of immune cell profiling to uncover biomarkers of response and mechanisms of resistance to immune checkpoint inhibitors.

Mechanisms and strategies to overcome resistance to molecularly targeted therapy for melanoma.

The identification of driver mutations in melanoma has changed the field of cancer treatment. BRAF and NRAS mutations are predominant in melanoma and lead to overactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways. Selective inhibitors targeting key effectors of the MAPK pathway have revolutionized the treatment of patients with advanced metastatic BRAF-mutant melanoma. However, resistance to therapy is almost universal and remains a major challenge in clinical care, with the majority of patients progressing within 1 year. Dissecting the mechanisms of resistance to targeted therapies may offer new insights into strategies for overcoming resistance. This review describes the efficacy of therapies targeting the MAPK and PI3K/AKT signaling pathways in melanoma, details the mechanisms contributing to drug resistance, and discusses current approaches to improving outcomes further. Cancer 2017;123:2118-29. © 2017 American Cancer Society.

The Role of Sumoylation in Senescence.

Cellular senescence is a program initiated by many stress signals including aberrant activation of oncogenes, DNA damage, oxidative lesions and telomere attrition. Once engaged senescence irreversibly limits cellular proliferation and potently prevents tumor formation in vivo. The precise mechanisms driving the onset of senescence are still not completely defined, although the pRb and p53 tumor suppressor pathways converge with the SUMO cascade to regulate cellular senescence. Sumoylation translocates p53 to PML nuclear bodies where it can co-operate with many sumoylated co-factors in a program that activates pRb and favors senescence. Once activated pRb integrates various proteins, many of them sumoylated, into a repressor complex that inhibits the transcription of proliferation-promoting genes and initiates chromatin condensation. Sumoylation is required for heterochromatin formation during senescence and may act as a scaffold to stabilize the pRb repressor complex. Thus, SUMO is a critical component of a tumor-suppressor network that limits aberrant cell proliferation and tumorigenesis.

Single-cell RNA sequencing in melanoma: what have we learned so far?

Over the past decade, there have been remarkable improvements in the treatment and survival rates of melanoma patients. Treatment resistance remains a persistent challenge, however, and is partly attributable to intratumoural heterogeneity. Melanoma cells can transition through a series of phenotypic and transcriptional cell states that vary in invasiveness and treatment responsiveness. The diverse stromal and immune contexture of the tumour microenvironment also contributes to intratumoural heterogeneity and disparities in treatment response in melanoma patients. Recent advances in single-cell sequencing technologies have enabled a more detailed understanding of melanoma heterogeneity and the underlying transcriptional programs that regulate melanoma cell diversity and behaviour. In this review, we examine the concept of intratumoural heterogeneity and the challenges it poses to achieving long-lasting treatment responses. We focus on the significance of next generation single-cell sequencing in advancing our understanding of melanoma diversity and the unique insights gained from single-cell studies.

PKC-independent PI3K signalling diminishes PKC inhibitor sensitivity in uveal melanoma.

Protein kinase C (PKC) is activated downstream of gain-of-function GNAQ or GNA11 (GNAQ/GNA11) mutations in over 90% of uveal melanoma (UM). Phase I clinical trials of PKC inhibitors have shown modest response rates with no survival benefit in metastatic UM. Although PKC inhibitors actively suppress mitogen-activated protein kinase (MAPK) signalling in UM, the effect on other UM signalling cascades is not well understood. We examined the transcriptome of UM biopsies collected pre- and post-PKC inhibitor therapy and confirmed that MAPK, but not PI3K/AKT signalling, was inhibited early during treatment with the second-generation PKC inhibitor IDE196. Similarly, in GNAQ/GNA11-mutant UM cell models, PKC inhibitor monotherapy effectively suppressed MAPK activity, but PI3K/AKT signalling remained active, and thus, concurrent inhibition of PKC and PI3K/AKT signalling was required to synergistically induce cell death in a panel of GNAQ/GNA11-mutant UM cell lines. We also show that re-activation of MAPK signalling has a dominant role in regulating PKC inhibitor responses in UM and that PI3K/AKT signalling diminishes UM cell sensitivity to PKC inhibitor monotherapy. Thus, combination therapies targeting PKC and PKC-independent signalling nodes, including PI3K/AKT activity, are required to improve responses in patients with metastatic UM.

Checkpoint kinase 1 inhibitor + low-dose hydroxyurea efficiently kills BRAF inhibitor- and immune checkpoint inhibitor-resistant melanomas.

Treatment of melanomas with targeted and immunotherapies has proven effective, but resistance to both treatments is a common outcome leaving a high proportion of patients without effective alternative treatment options. Replication stress is a common feature of melanomas, and this is effectively targeted using a combination of checkpoint kinase 1 (CHK1) inhibitor and low-dose hydroxyurea (LDHU). This combination also promotes inflammatory and anti-tumour immune responses in vivo. Melanoma cell lines resistant to BRAF inhibitor (BRAFi) or immune checkpoint inhibitors (ICI) retain their sensitivity to CHK1i + LDHU, with sensitivity similar to that of parental tumours. In vivo, BRAFi-resistant and BRAFi-sensitive parental tumours produce an identical immune response with treatment.

Membrane and cytoplasmic marker exchange between malignant neoplastic cells and fibroblasts via intermittent contact: increased tumour cell diversity independent of genetic change.

We previously demonstrated that human osteosarcoma cells (SAOS-2) induce contact-dependent apoptosis in endothelium, and expected similar apoptosis in human gingival fibroblasts (h-GF) using SAOS-2 alkaline phosphatase (AP) to identify cells. However, h-GF apoptosis did not occur, despite reduction in AP-negative h-GF number (p < 0.01) and enhancement of this by h-GF TNFα pretreatment (p < 0.01). We suggest that TNFα-enhanced transfer of membrane AP from SAOS-2 to h-GF would explain these data. This idea was investigated using fluorescence prelabelled cells and confocal laser scanning microscopy. Co-cultures of membrane-labelled h-GF (marker-DiO) and SAOS-2 (marker-DiD) generated dual-labelled cells, primarily at the expense of single labelled h-GF (p < 0.001), suggesting predominant membrane transfer from SAOS-2 to h-GF. However, opposite directional transfer predominated when membrane labels were reversed; SAOS-2 further expressed green fluorescent protein (GFP) in cytoplasm and nuclei, and h-GF additionally bore nuclear label (Syto59) (p < 0.001). Cytoplasmic exchange was investigated using h-GF prelabelled with cytoplasmic DDAO-SE and nuclear Syto59, co-cultured with SAOS-2 expressing GFP in cytoplasm and nuclei, and predominant cytoplasmic marker transferred from h-GF to SAOS-2 (p < 0.05). Pretreating h-GF with TNFα increased exchange of membrane markers (p < 0.04) but did not affect either cell surface area profile or circularity. Dual-labelled cells had a morphological phenotype differing from SAOS-2 and h-GF (p < 0.001). Time-lapse microscopy revealed extensive migration of SAOS-2 and cell process contact with h-GF, with the appearance of SAOS-2 indulging in 'cellular sipping' from h-GF. Similar exchange of membrane was seen between h-GF and with other cell lines (melanoma MeIRMu, NM39, WMM175, MM200-B12; osteosarcoma U20S; ovarian carcinoma cells PE01, PE04 and COLO316), while cytoplasmic sharing was also seen in all cell lines other than U20S. We suggest that in some neoplasms, cellular sipping may contribute to phenotypic change and the generation of diverse tumour cell populations independent of genetic change, raising the possibility of a role in tumour progression.

IFN-γ Signaling Sensitizes Melanoma Cells to BH3 Mimetics.

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.