Chemoresistance In Melanoma Research Articles

Growth Hormone Upregulates Melanoma Drug Resistance and Migration via Melanoma-Derived Exosomes.

Drug resistance in melanoma is a major hindrance in cancer therapy. Growth hormone (GH) plays a pivotal role in contributing to the resistance to chemotherapy. Knocking down or blocking the GH receptor has been shown to sensitize the tumor cells to chemotherapy. Extensive studies have demonstrated that exosomes, a subset of extracellular vesicles, play an important role in drug resistance by transferring key factors to sensitize cancer cells to chemotherapy. In this study, we explore how GH modulates exosomal cargoes from melanoma cells and their role in drug resistance. We treated the melanoma cells with GH, doxorubicin, and the GHR antagonist, pegvisomant, and analyzed the exosomes released. Additionally, we administered these exosomes to the recipient cells. The GH-treated melanoma cells released exosomes with elevated levels of ABC transporters (ABCC1 and ABCB1), N-cadherin, and MMP2, enhancing drug resistance and migration in the recipient cells. GHR antagonism reduced these exosomal levels, restoring drug sensitivity and attenuating migration. Overall, our findings highlight a novel role of GH in modulating exosomal cargoes that drive chemoresistance and metastasis in melanoma. This understanding provides insights into the mechanisms of GH in melanoma chemoresistance and suggests GHR antagonism as a potential therapy to overcome chemoresistance in melanoma treatment.

Abstract PR009: Discovery of covalent NRAS inhibitors targeting the Palmitoylation site through imaging-based high-throughput screening

Abstract NRAS mutations play a crucial role as oncogenic drivers of tumor cell growth and chemo-resistance in melanoma and acute myeloid leukemia (AML). In melanoma, NRAS mutations occur in approximately 25% of cases, and are generally mutually exclusive with BRAF-mutations. NRAS mutant tumors often exhibit an aggressive phenotype, such as thicker lesions, increased mitotic activity, and higher rates of lymph node metastasis. Surgical resection of NRAS mutant melanoma has a poor prognosis with a lower median overall survival (OS) compared to non-NRAS-mutant melanoma (8.2 vs. 15.1 months for stage IV disease). Current treatment strategies for stage III/IV non-resectable metastatic NRAS mutant melanoma include immunotherapy and indirect targeting of NRAS associated signaling pathways, highlighting the urgent need to develop small molecule NRAS inhibitors. NRAS palmitoylation is essential for its oncogenic function by regulating its trafficking and anchoring to the plasma membrane. Unlike other RAS isoforms, NRAS relies on a single palmitoylation site (C181) for its plasma membrane localization and this unique dependency makes NRAS more sensitive to palmitoylation inhibitors. To identify selective NRAS inhibitors that covalently modify C181 and prevent its palmitoylation, we employed an imaging-based NRAS translocation high-throughput screen using a library of 23,000 covalent molecules. We identified a set of covalent inhibitors that selectively target NRAS translocation but do not affect HRAS localization. We further confirmed the direct binding of these compound hits to NRAS at its palmitoylation site (C181) using intact mass spectrometry and chemo-proteomic peptide mapping techniques. Moreover, we validated the functional impact of these compound hits by demonstrating their selective inhibition of cell growth in mutant NRAS-driven melanoma cell lines. In conclusion, our study presents the first direct inhibitor for NRAS, discovered through a high-throughput covalent screening approach. This finding holds promising implications for the development of novel targeted therapies for NRAS mutant tumors. Citation Format: Zhao Wang, Keith Moore, Simon Ng, Chao Wang, Ronald Tomlinson, Huay-Keng Loke, Jamie Rice, Ivan Cornella. Discovery of covalent NRAS inhibitors targeting the Palmitoylation site through imaging-based high-throughput screening [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR009.

OTUD4 regulates metastasis and chemoresistance in melanoma by stabilizing Snail1.

Melanoma is the most aggressive form of skin cancer with rapidly increased incidence worldwide especially in the Caucasian population. Surgical excision represents the curative treatment choice in patients with early-stage disease. However, the therapeutic outcomes in patients with metastatic melanoma remains unsatisfactory. Thus, understanding molecular mechanisms contributing to metastasis and chemoresistance is critical for new improved therapies of melanoma. Snail1, an important epithelial-mesenchymal transition transcription factors (EMT-TFs), is critical to induce the EMT process, thereby contributing to cancer metastasis. However, the involvement of Snail1 in melanoma metastasis remains elusive and the underlying mechanism to regulate Snail1 in melanoma needs to be further investigated. Here, we identified OTUD4 as a novel deubiquitinase of Snail1 in melanoma. Moreover, the depletion of OTUD4 in melanoma cells markedly inhibited Snail1 stability and Snail1-driven malignant phenotypes both in vitro and in vivo. Overall, our study establishes OTUD4 as a novel therapeutic target in metastasis and chemoresistance of melanoma by stabilizing Snail1 and provides a rationale for potential therapeutic strategies of melanoma.

Abstract 3038: IDH1 facilitates melanoma cell survival under metabolic stress

Abstract Introduction: Novel immunotherapies and targeted therapies have generated remarkable responses for patients with melanoma relative to historic treatments, but these responses are only seen in about 50% of patients. This suggests that treatment resistance is an ongoing challenge for a significant number of patients with melanoma, leaving an urgent need for improved therapeutic strategies. In our previous studies, we demonstrated that oxidative stress has an important role in drug resistance, and that wild type isocitrate dehydrogenase 1 (IDH1) is a major source of cytosolic NADPH that maintains redox homeostasis of cells under hypoxic and metabolic stress. This critical enzyme has not been thoroughly evaluated in melanoma. Herein, we explored the expression and function of IDH1 in human melanoma and the role of IDH1 in regulating melanoma metabolism. Methods: We evaluated IDH1 expression in primary and metastatic melanoma using The Cancer Genome Atlas (TCGA). We performed IDH1 knockdown by siRNA oligos, and evaluated cell viability by Trypan blue and PicoGreen assays under normal and nutrient-deprived conditions. Cellular reactive oxygen species (ROS) levels were determined by the DCFDA method. Metastatic activity of these cells was measured by transwell migration assays. In order to determine the impact of IDH1 in cellular metabolism, metabolomics profiling was performed by using LC-MS. These experiments were performed in A375 and SK-Mel 28 cell lines. Results: Analysis of TCGA data from melanoma samples showed IDH1 is highly overexpressed in primary and metastatic melanoma, and higher levels are associated with decreased progression-free survival in patients. Further, we validated that IDH1 is overexpressed in tumors by comparing normal skin tissue versus tumor samples by IHC and protein expression arrays. Silencing IDH1 impaired cell proliferation and migration (vs. control) in a nutrient-deprived microenvironment, but this effect was not seen in nutrient abundance. Metabolomics revealed that inhibiting IDH1 significantly decreased NADPH, α-ketoglutarate (αKG), and GSH levels with a corresponding increase in ROS levels and an impairment of mitochondrial function. In addition, silencing IDH1 sensitized melanoma cells to temozolomide (TMZ), a DNA-alkylating agent, as indicated by a decrease in relative cell survival compared with controls. Conclusion: IDH1 plays a critical role in tumorigenesis and chemoresistance in melanoma. Our data show that IDH1 inhibition sensitizes melanoma cells to TMZ therapy. Our study suggests that IDH1 is a potential target in melanoma as a monotherapy or in combination with existing chemotherapies. Citation Format: Mehrdad Zarei, Ali Vaziri-Gohar, Jonathan Hue, Omid Hajihassani, Erryk Katayama, Hallie Graor, Jordan Winter, Luke Rothermel. IDH1 facilitates melanoma cell survival under metabolic stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3038.

Intermittent treatment of BRAFV600E melanoma cells delays resistance by adaptive resensitization to drug rechallenge

Patients with melanoma receiving drugs targeting BRAFV600E and mitogen-activated protein (MAP) kinase kinases 1 and 2 (MEK1/2) invariably develop resistance and face continued progression. Based on preclinical studies, intermittent treatment involving alternating periods of drug withdrawal and rechallenge has been proposed as a method to delay the onset of resistance. The beneficial effect of intermittent treatment has been attributed to drug addiction, where drug withdrawal reduces the viability of resistant cells due to MAP kinase pathway hyperactivation. However, the mechanistic basis of the intermittent effect is incompletely understood. We show that intermittent treatment with the BRAFV600E inhibitor, LGX818/encorafenib, suppresses growth compared with continuous treatment in human melanoma cells engineered to express BRAFV600E, p61-BRAFV600E, or MEK2C125 oncogenes. Analysis of the BRAFV600E-overexpressing cells shows that, while drug addiction clearly occurs, it fails to account for the advantageous effect of intermittent treatment. Instead, growth suppression is best explained by resensitization during periods of drug removal, followed by cell death after drug readdition. Continuous treatment leads to transcriptional responses prominently associated with chemoresistance in melanoma. By contrast, cells treated intermittently reveal a subset of transcripts that reverse expression between successive cycles of drug removal and rechallenge and include mediators of cell invasiveness and the epithelial-to-mesenchymal transition. These transcripts change during periods of drug removal by adaptive switching, rather than selection pressure. Resensitization occurs against a background of sustained expression of melanoma resistance genes, producing a transcriptome distinct from that of the initial drug-naive cell state. We conclude that phenotypic plasticity leading to drug resensitization can underlie the beneficial effect of intermittent treatment.

The role of mutations in <i>NF1</i> gene in sporadic carcinogenesis

The review article presents data on somatic inactivation of NF1 gene as a cause of sporadic malignant neoplasms. The re- lationship between the features of specific tumors in neurofibromatosis type 1 and specific types of sporadic neoplasms, in which mutations in NF1 gene are found, are presented. Evidence for the role of somatic mutations in NF1 gene in the development of chemoresistance in melanoma, neuroblastoma, ovarian and breast cancer, and lung cancer is described (only if there are no mutations of known protooncogenes). To overcome the resistance of these neoplasms, inhibitors of mitogen-activated protein kinase have been proposed, the effectiveness of which has been proven in the treatment of plexiform neurofibromas. The review presents evidence of the relationship between NF1 and microRNA, which can be used for targeted therapy of both neurofibromatosis type 1 and sporadic neoplasms with mutations of this gene. Prospects for gene therapy of these diseases are considered.

Effects of Growth Hormone in Human Melanoma Drug Resistance

Melanoma, an extremely drug resistant form of cancer, displays elevated levels of growth hormone (GH) and growth hormone receptor (GHR) expression. We have described a GH dependent mechanism of chemoresistance in melanoma through a series of earlier studies. In GHR overexpressing human melanoma, GH action drives active drug efflux and phenotype switching by upregulating ATP-binding cassette transporters (ABC-transporters) and epithelial-to-mesenchymal transition (EMT) markers. Inhibition of GHR activation reverses these effects, increases drug retention, and sensitizes the melanoma cells to chemotherapy. GH action in melanoma also appears to drive drug sequestration in melanosomes as we present here. GH action was found to modulate the levels of melanocyte-inducing transcription factor (MITF), the master regulator of melanosome synthesis and melanoma. Additionally, MITF target gene levels and drug sequestering ABC transporter expression were also elevated by GH treatment in vitro and in vivo in mouse tumor xenografts. This was corroborated by in silico analyses of tumor databases. Furthermore, we observed a dose dependent increase in tyrosinase activity and melanogenesis in human melanoma cells with GH, in the presence of chemotherapy, which was suppressed by siRNA mediated GHR inhibition. Thus, we describe an array of molecular mechanisms by which GH drives chemoresistance in melanoma. Therefore, GHR inhibition combined with chemotherapy should lead to better tumor clearance and improve overall survival.

Growth Hormone Receptor Inhibition Sensitizes Human Pancreatic Cancer to Chemotherapy Treatments

Human growth hormone (GH) and its cognate growth hormone receptor (GHR) have been established to have a distinct role in promoting the progression of several types of human cancers. We had earlier described a newfound role of the GH-GHR axis in driving chemoresistance in melanoma by upregulating drug efflux by ABC multidrug transporter expression and a phenotype switch by induction of epithelial-to-mesenchymal transition (EMT). Here we present an in-depth analysis of this role of GH-GHR in the highly therapy resistant human pancreatic cancer which has a 5-year survival rate of only 10% in 2020. Using human and mouse pancreatic cancer cells and RNA and protein expression analyses by RT-qPCR, ELISA, and western-blot, we identified that (i) GH upregulates specific ABC-transporter expressions in a drug-context specific manner, (ii) GH upregulates EMT transcription factors, (iii) GH activates specific oncogenic signaling pathways, and (iii) GH action increases cytochrome P450 members involved in hepatic drug metabolism. The GH antagonist, Pegvisomant, significantly inhibited these effects. Additionally, we confirmed the effects of these molecular changes by specific assays. For example, GH increases basement membrane invasion, viability of circulating tumor cells, and drug efflux; while inhibition of GHR by pegvisomant in pancreatic cancer cells reversed this aggressive tumor phenotype and sensitized the tumor cells to chemotherapy. Cell viability assays confirmed a decreased IC50 of gemcitabine, doxorubicin, and erlotinib in pancreatic cancer cells treated with pegvisomant and an increase in IC50 cells treated with GH. We further verified our results using in silico analyses of TCGA datasets for pancreatic cancer - which provided robust confirmation of our experimental findings. Presently we are validating our observation in nude mice with human pancreatic cancer cell xenografts. In conclusion, our in vitro results confirm that GHR antagonism can drastically sensitize human pancreatic cancer cells by blocking mechanisms of drug resistance, thus providing a valuable window for improved efficacy of available chemo- and targeted therapy.

Ocoxin Increases the Antitumor Effect of BRAF Inhibition and Reduces Cancer Associated Fibroblast-Mediated Chemoresistance and Protumoral Activity in Metastatic Melanoma.

Whereas the prevalence of several cancer types is decreasing, skin malignancies are growing more common every year. Malignant melanoma is the most aggressive form of skin cancer with high metastatic capacity. In most cases, malignant melanoma shows acquired therapy resistance. We evaluated the ability of Ocoxin, a natural compound-based antioxidant and anti-inflammatory nutritional complement, to exert an antitumor effect in melanoma. To do so, the cytotoxicity of Ocoxin in a panel of BRAF-mutated murine and human melanoma cell lines was tested alone and in combination with BRAF inhibitor Vemurafenib. Our results revealed a potent cytotoxic effect of Ocoxin against melanoma cells and a synergic effect when combined with Vemurafenib, reducing viability and increasing apoptosis. Besides, Ocoxin interferes with the cell cycle, impairs the inherent and fibroblast-mediated melanoma cell migration, and reduces resistance to BRAF inhibition. Proteomic analysis revealed reduced tumor secretion of inflammatory factors Galectin-1, Osteopontin, CCL5, and CCL9 upon treatment with Ocoxin. Moreover, RNASeq showed that Ocoxin downregulated the cell cycle and proliferation-related genes. In vivo, Ocoxin reduced the number of lung metastasis of YUMM-1.7 melanoma cells. Therefore, Ocoxin arises as a good candidate for clinical trials analyzing the beneficial effects in patients suffering from this cutaneous malignancy.

Growth Hormone Upregulates Melanocyte-Inducing Transcription Factor Expression and Activity via JAK2-STAT5 and SRC Signaling in GH Receptor-Positive Human Melanoma.

Growth hormone (GH) facilitates therapy resistance in the cancers of breast, colon, endometrium, and melanoma. The GH-stimulated pathways responsible for this resistance were identified as suppression of apoptosis, induction of epithelial-to-mesenchymal transition (EMT), and upregulated drug efflux by increased expression of ATP-binding cassette containing multidrug efflux pumps (ABC-transporters). In extremely drug-resistant melanoma, ABC-transporters have also been reported to mediate drug sequestration in intracellular melanosomes, thereby reducing drug efficacy. Melanocyte-inducing transcription factor (MITF) is the master regulator of melanocyte and melanoma cell fate as well as the melanosomal machinery. MITF targets such as the oncogene MET, as well as MITF-mediated processes such as resistance to radiation therapy, are both known to be upregulated by GH. Therefore, we chose to query the direct effects of GH on MITF expression and activity towards conferring chemoresistance in melanoma. Here, we demonstrate that GH significantly upregulates MITF as well as the MITF target genes following treatment with multiple anticancer drug treatments such as chemotherapy, BRAF-inhibitors, as well as tyrosine-kinase inhibitors. GH action also upregulated MITF-regulated processes such as melanogenesis and tyrosinase activity. Significant elevation in MITF and MITF target gene expression was also observed in mouse B16F10 melanoma cells and xenografts in bovine GH transgenic (bGH) mice compared to wild-type littermates. Through pathway inhibitor analysis we identified that both the JAK2-STAT5 and SRC activities were critical for the observed effects. Additionally, a retrospective analysis of gene expression data from GTEx, NCI60, CCLE, and TCGA databases corroborated our observed correlation of MITF function and GH action. Therefore, we present in vitro, in vivo, and in silico evidence which strongly implicates the GH–GHR axis in inducing chemoresistance in human melanoma by driving MITF-regulated and ABC-transporter-mediated drug clearance pathways.

Abstract 3732: Targeted photodynamic therapy enhances the therapeutic efficacy of combination therapy (PDT and chemotherapy) on chemoresistant melanoma cells

Abstract Cutaneous melanoma remains refractory to current therapies such as chemotherapy. Failure of treatment has been attributed to enhanced self-renewal capacity and the over-expression of ABC transporters by therapy resistant populations. A need therefore exists to identify and evaluate new approaches. Alternative combinatorial approaches comprising chemotherapeutics, photodynamic therapy (PDT) and targeted-PDT have been proposed to overcome resistance. Objective This study set to investigate the efficacy of novel (targeted-PDT-IR700) and standard combinatorial effect of PDT and chemotherapy (Hypericin-PDT and DTIC) as an adjunctive combination therapy treatment to decrease cellular chemoresistance in melanoma. This was assessed in an in vitro model, to show differences in killing of melanoma cells. Methodology Single chain variable fragments (scFv) of panitumumab were cloned into a transient eukaryotic SNAP expression vector co-expressing cytosolic proteins and transfected into HEK293T cells. Purified recombinant protein was confirmed by SDS-PAGE and Western blot analyses. Benzylguanine (BG) modified IR700 was irreversibly conjugated in a 1:1 stochiometric ratio to the 1711(scFv)-SNAP using the self-labelling property of the suicide enzyme SNAP-tag. Binding activity and cytotoxicity was documented on EGFR expressing melanoma cells using flow cytometry, confocal microscopy and XTT assays. Cytotoxic activity, self-renewal capacity and ABC transporters expression (ABCB5 and ABCG2) after the combination therapy (HYP-PDT and DTIC) was determined using XTT, clonogenic assays and flow cytometry. Results Binding and internalization of purified 1711(scFv)-SNAP conjugated to BG-modified Alexa 488 or IR700 was successfully confirmed by confocal microscopy and flow cytometry. Cell viability showed cell specific cytotoxicity of the photosensitizer labeled fusion proteins upon light induction, at nanomolar concentrations which were lower than those used for the non-targeted combinatorial approach (HYP-PDT and DTIC). Moreover, DTIC resistant melanoma cells were less sensitive to therapeutic treatment than their non-resistant counterpart. This correlated with an increased self-renewal capacity and ABC transporter expression in DTIC resistant melanoma in comparison to their non-resistant counterpart. This study describes a proof of concept of selective targeting and elimination of EGFR expressing melanoma cells by targeted photoimmunotherapy using a panitumumab 1711-scFv fused to SNAP tag and conjugated to near infrared photosensitizer IR700 which improve therapeutic efficacy with lower concentration and reduced sides effect. This work highlights the efficacy of targeted PDT and suggest that it is highly applicable to improve the current clinical arsenal of therapies. Note: This abstract was not presented at the meeting. Citation Format: Lester M. Davids, Fleury Nsole Biteghe, Eden Padayachee, Stefan Barth. Targeted photodynamic therapy enhances the therapeutic efficacy of combination therapy (PDT and chemotherapy) on chemoresistant melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3732.

Nodal induced by hypoxia exposure contributes to dacarbazine resistance and the maintenance of stemness in melanoma cancer stem‑like cells

Nodal signaling has a critical role in the processes of embryogenesis and is necessary for maintaining cell stemness. However, its upregulation in melanoma is positively correlated with malignant potential, including cancer progression, metastasis and recurrence, and a recent report has revealed its role in promoting self‑renewal capacity in melanoma. Our study aimed to explore the effects of hypoxia exposure, which is one of the main causes of chemoresistance in melanoma, on the physiological processes of melanoma cancer stem‑like cells (CSCs) via regulating Nodal. A375 CSCs were enriched by culturing in serum‑free medium (SFM) and were analyzed for the expression levels of Nodal and its correlated proteins by semi‑quantitative western blotting. Lentiviral‑packaged Nodal coding sequence or short‑hairpin RNA (shRNA) was employed. After hypoxia exposure, the effects on glucose uptake, ATP production and O2 consumption were detected, and whether Nodal contributed to the proliferation, invasion, colony formation, self‑renewal capacity and chemoresistance was evaluated. We demonstrated that hypoxia exposure induced Nodal expression and activated the Smad2/3 pathway in A375 CSCs. Hypoxic‑induced Nodal partially promoted dacarbazine resistance, promoted invasion and self‑renewal capacity, but not proliferation which was further confirmed using Nodal knockdown. Blockage of Nodal signaling activity with the small‑molecule inhibitor SB431542 partially reversed Nodal‑induced chemoresistance. Nodal knockdown further sensitized A375 CSCs to dacarbazine after SB431542 pretreatment, indicating the involvement of proNodal in dacarbazine resistance. The introduction of mut‑proNodal induced chemoresistance further confirming the role of proNodal in this process. Taken together, our results demonstrated that Nodal induced by hypoxia exposure induced a malignant phenotype and chemoresistance in A375 CSCs, and proNodal also contributed to these processes, indicating that Nodal may be a potential therapeutic target for melanoma.

Abstract A134: Eeyarestatin I sensitizes melanoma cells to cisplatin-induced cell death

Abstract Melanoma is the most deadly form of skin cancer with a high metastatic potential. Initial stages of the disease can be surgically treated, but the treatment options for advanced stages are very limited. Although progress has been made with the development of some target- and immune-therapy, improving melanoma patients' outcomes, the cost of these therapies is reaching prohibiting values. Not all melanoma patients, mainly those treated in public health systems, have access to these new therapies. Indeed, they are treated with conventional chemotherapy, such as dacarbazine or cisplatin, which promotes very low therapeutic responses. In light of this, we sought to investigate in the present work an alternative to improve the response of conventional chemotherapy to treat melanomas. A previous proteomic study from our group revealed that human melanoma cells treated with cisplatin accumulated the endoplasmic reticulum (ER) chaperone GRP78, an important sensor of ER stress and regulator of unfolded protein response (UPR). UPR signaling is activated in response to ER stress, inducing survival pathways to restore ER homeostasis or activating cell death pathways under severe or persistent ER stress conditions. Adaptation and selection of melanoma cells to ER stress can be one of the drivers of chemoresistance in melanoma. To investigate ER stress adaptation, we assessed in the current work the transcription factor CHOP, which mediates UPR-induced apoptosis. Using a TMA (tissue microarray) platform, we observed the nuclear exclusion of CHOP in primary and metastatic human melanomas when compared to nevus tissues. In the face of these results, we hypothesized that by inducing more ER stress we could surpass the adaptive levels of ER stress, favoring nuclear CHOP accumulation, culminating in the sensitization of melanoma cells to cisplatin. To induce ER stress, we tested an ERAD (ER-associated degradation) inhibitor that blocks the exit of unfolded proteins from ER to degradation in the cytosol, eeyarestatin I (EerI). We observed that treatment of the human melanoma cells CHL-01 with EerI favored the accumulation of GRP78 in the cytosol and CHOP in the nucleus. Notably, the combination of EerI and cisplatin resulted in a significant increase in cell death as compared to single treatments. Our results suggest that approaches to surpass adaptive levels to ER stress, such as EerI treatment, are a promising strategy to chemosensitize melanoma cells. Supported by FAPESP (1998/14247-6); (Redoxoma, CNPq 573530/2008-4) and FAPESP (2010/08284-0). Citation Format: Renata de Freitas Saito, Andréia Hanada Otake, Margarita M. Cortez, Robbert J. Gillies, Roger Chammas. Eeyarestatin I sensitizes melanoma cells to cisplatin-induced cell death [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A134.

Can Integrin Agonists Have Cards to Play against Cancer? A Literature Survey of Small Molecules Integrin Activators.

The ability of integrins to activate and integrate intracellular communication illustrates the potential of these receptors to serve as functional distribution hubs in a bi-directional signal transfer outside-in and inside-out of the cells. Tight regulation of the integrin signaling is paramount for normal physiological functions such as migration, proliferation, and differentiation, and misregulated integrin activity could be associated with several pathological conditions. Because of the important roles of integrins and their ligands in biological development, immune responses, leukocyte traffic, haemostasis, and cancer, their potential as therapeutic tools is now widely recognized. Nowadays extensive efforts have been made to discover and develop small molecule ligands as integrin antagonists, whereas less attention has been payed to agonists. In recent years, it has been recognized that integrin agonists could open up novel opportunities for therapeutics, which gain benefits to increase rather than decrease integrin-dependent adhesion and transductional events. For instance, a significant factor in chemo-resistance in melanoma is a loss of integrin-mediated adhesion; in this case, stimulation of integrin signaling by agonists significantly improved the response to chemotherapy. In this review, we overview results about small molecules which revealed an activating action on some integrins, especially those involved in cancer, and examine from a medicinal chemistry point of view, their structure and behavior.

Abstract B07: Differential roles for AKT isoforms in BRAF mutant melanoma

Abstract Despite recent advances, melanoma remains the deadliest form of skin cancer, and new therapeutic strategies are urgently needed. The MAP kinase (MAPK) and PI3K/AKT pathways are central to both disease progression and chemoresistance in melanoma, with dual targeting approaches yielding promising results that would benefit from increased target specificity and selectivity. The AKT family of serine/threonine kinases comprises three highly homologous and functionally distinct isoforms (AKT1, AKT2, and AKT3) that play unique roles in melanoma but have not yet been leveraged for isoform-specific targeting. Our lab has engineered a unique mouse model of melanoma, in which mutant BRAFV600E is constitutively targeted to mouse melanocytes, resulting in spontaneous malignant disease upon additional loss of tumor suppressor genes, and faithfully recapitulates the human disease. We have identified differential and dynamic activation of AKT isoforms in our mouse melanoma model, which supports the hypothesis that AKT isoforms may play both non-redundant and compensatory roles in melanoma progression. To systematically address this question, we have genetically ablated each AKT isoform individually in melanoma-prone mice in order to elucidate the individual and unique contribution of each isoform to melanoma progression. In parallel, we performed in vitro studies using shRNA mediated conditional knockdown of AKTs in a wide variety of human melanoma cell lines. Our results support an isoform-specific role for AKT1 in promoting melanoma cell proliferation, while AKT2 stimulates cell migration. Further studies utilizing the precision genome editing technology CRISPR/Cas9 allows us to interrogate the effects of AKT isoform specific knockout (KO) on in vivo tumor formation. This work moves toward establishing the contribution of AKT isoforms to tumor progression in melanoma, using both human melanoma cell lines and mouse models, to advance our understanding of the role of AKT in melanomagenesis. With AKT inhibitors in clinical trials for melanoma, our findings could yield novel therapeutic strategies and increase the efficiency of existing therapies to improve treatment options and outcome for this devastating disease. Citation Format: Siobhan K. McRee, Jodie R. Pietruska, Elisabeth Dignan, Philip W. Hinds. Differential roles for AKT isoforms in BRAF mutant melanoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B07.

Targeting mitosis-regulating genes in cisplatin-sensitive and -resistant melanoma cells: A live-cell RNAi screen displays differential nucleus-derived phenotypes.

Chemoresistance in malignant melanoma remains an unresolved clinical issue. In the search for novel molecular targets, a live-cell high-content RNAi screen based on gene expression data was performed in cisplatin-sensitive and cisplatin-resistant MeWo melanoma cells, Mel-28 cells and a melanocyte cell line. Cells were exposed to 91 siRNAs and distinct nucleus-derived phenotypes such as cell division, cell death and migration phenotypes were detected by time-lapse microscopy over 60 h. Using this approach, cisplatin-sensitive and cisplatin-resistant melanoma cells were compared by automated image analysis and visual inspection. In cisplatin-sensitive MeWo melanoma cells, 14 genes were identified that showed distinct phenotype abnormalities after exposure to gene-specific siRNAs. In cisplatin-resistant MeWo cells, five genes were detected. Nine genes were detected whose knock-down led to differential nuclear phenotypes in cisplatin-sensitive and -resistant cells. In Mel-28 cells, nine genes were identified which induced nuclear phenotypes including all eight genes which were identified in cisplatin-resistant MeWo cells. An analogous RNAi screen on melanocytes revealed no detectable phenotype abnormalities after RNAi. Pathway analysis showed in cisplatin-sensitive MeWo cells and Mel-28 cells an enrichment of at least three genes in major mitotic pathways. We hereby show that siRNA screening may help to identify tumor-specific genes leading to phenotype abnormalities. These genes may serve as potential therapeutic targets in the treatment of melanoma.

Abstract 2414: A phase I/II clinical trial to evaluate the efficacy of combined epigenetic therapy for improving temozolomide chemosensitivity in metastatic melanoma.

Abstract Background: Cancer epigenomes are characterized by global changes in DNA methylation and covalent histone modification patterns. The disruption of epigenetic mechanisms is a hallmark of cancer development and has strong implications in cancer treatment. Emerging evidence indicates that epigenetics may also play a key role in the development of chemoresistance in melanoma. Keeping the toxicity to a minimum may allow for repeated dosing when combine epigenetic and cytotoxic therapy in the treatment of solid tumors. We proposed combining the histone deacetylase inhibitor panobinostat and the demethylator decitabine to epigenetically regulate apoptotic process and overcome temozolomide resistance in metastatic melanoma Methods: Eligible patients must be ≥18, stage IV melanoma, and naïve or previously treated, with good performance status (ECOG Performance Status of ≤ 2) and normal organ functions. The study includes the dose escalation of Decitabine and Panobinostat followed by expansion cohorts. Decitabine (0.1mg/kg SQ, 0.2mg/kg SQ) on days 1,3,5,8,10,12, Panobinostat (10mg PO, 20mg PO, 30mg PO) Q96h starting day 8, and Temozolomide 150mg/m2 PO daily on days 9-13 of each 42 day cycle. Temozolomide was increased to 200mg/m2 in the absence of grade 2 thrombocytopenia. Primary endpoints of phase I study are to determine the toxicity, safety and maximum tolerated dose (MTD) of the Decitabine, Panobinostat and Temozolomide combination. Results: To date, the phase I study of this trial is completed. 17 patients were enrolled (1st cohort: 5; 2nd cohort: 4; 3rd cohort: 4; 4th cohort: 4). M:F 11:6. Median age: 56 (32-77); ECOG PS: 1; 82% of the patients received at least one cycle (n=14). Median number of cycles given: 2 (0-6). To date, no DLTs have occurred. The MTD was not reached. The only G4 adverse event (AE) is neutropenia on a patient in cohort 3 and it resolved within 3 days. G3 AEs included lymphopenia (n=4, 23%), anemia (n=2, 12%), leukopenia (n=2, 12%) and fatigue (n=2, 12%). Common G2 toxicities were leukocyte (n=5, 30%), neutropenia (n=4, 23%), nausea (n=4, 23%) and lymphopenia (n=3, 18%). Conclusions: The combination of decitabine, panobinostat, and temozolomide appears to be safe and well-tolerated. The recommended dosing regimen for phase II studies is Decitabine 0.2 mg/kg SQ 3x weekly, on days 1,3,5,8,10,12, Panobinostat 30mg PO Q96h starting day 8, and Temozolomide 200mg/m2 PO daily on days 9-13 of each 42 day cycle. Citation Format: Chang Xia, Jeremy M. Deutsch, Mohammed M. Milhem, Brian J. Smith, Douglus Laux, Raymond Hohl. A phase I/II clinical trial to evaluate the efficacy of combined epigenetic therapy for improving temozolomide chemosensitivity in metastatic melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2414. doi:10.1158/1538-7445.AM2013-2414 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Sensitization to the mitochondrial pathway of apoptosis augments melanoma tumor cell responses to conventional chemotherapeutic regimens

Metastatic malignant melanoma is highly resistant to chemotherapy, and the average survival rate is under 1 year. The only FDA-approved conventional chemotherapy (i.e., dacarbazine) targets melanoma tumor cells by inducing a form of cell death referred to as apoptosis. However, dacarbazine exhibits a response rate of ∼5%, and combination chemotherapies consisting of cisplatin, vinblastine, and dacarbazine often offer little clinical advantage over dacarbazine alone. Apoptosis is governed by the BCL-2 family of proteins, which is comprised of anti-apoptotic and pro-apoptotic members. To determine if the anti-apoptotic BCL-2 repertoire established the cell death threshold and chemoresistance in melanoma, a novel treatment strategy was designed to inhibit the anti-apoptotic BCL-2 members with ABT-737. Using various melanoma model systems, we determined the affects of ABT-737 on sensitivity to dacarbazine-based regimens. Strikingly, ABT-737 re-sensitized melanoma cell lines to common chemotherapeutics leading to marked BIM-mediated apoptosis. Cellular features of the ABT-737 combination treatments were loss of proliferation, mitochondrial fragmentation, nuclear condensation, phosphatidylserine exposure, and decreased clonogenic survival. We also observed significant anti-tumor activity in an in vivo melanoma model system. Our data indicate that ABT-737 may be a beneficial adjuvant therapy to improve melanoma response rates when conventional chemotherapy is the only option.

A phase I/II study to evaluate the ability of decitabine and panobinostat to improve temozolomide chemosensitivity in metastatic melanoma.

3056 Background: Epigenetic gene regulation is likely a contributing mechanism of cancer initiation and progression. Emerging evidence indicates that epigenetics may also play a key role in the development of chemoresistance in melanoma. We proposed combining the histone deacetylase inhibitor panobinostat (PT) and the demethylator decitabine (D) to overcome the development of epigenetic mediated temozolomide (TMZ) resistance in metastatic melanoma. Methods: Eligible patients must be ≥18, stage IV melanoma, and naïve or previously treated, with good performance status (ECOG ≤ 2) and normal organ functions. Patients with previous exposure to TMZ were allowed on study. The study includes the dose escalation of D and PT followed by expansion cohorts. D (0.1mg/kg SQ, 0.2mg/kg SQ) on days 1, 3, 5, 8, 10,12, PT (10mg PO, 20mg PO, 30mg PO) Q96h starting day 8, and TMZ 150mg/m2 PO daily on days 9-13 of each 42 day cycle. TMZ was increased to 200mg/m2 in the absence of grade 2 thrombocytopenia. Primary endpoints of phase I study are to determine the toxicity, safety and maximum tolerated dose (MTD) of the D, PT and TMZ combination. Results: To date, the phase I portion of this trial is completed. We report on the safety data for this combination. 17 patients received treatment (1st cohort: 5; 2nd cohort: 4; 3rd cohort: 4; 4th cohort: 4 ). M:F 11:6. Median age: 56 (32-77); Median ECOG PS: 1; 82% of the patients received at least one cycle (n=14). Median number of cycles given: 2 (0-6). To date, no DLTs have occurred. The MTD was not reached. The only grade (G) 4 adverse event (AE) is neutropenia on a patient in cohort 3 and it resolved within 3 days. G3 AEs included lymphopenia (n=4, 23%), anemia (n=2, 12%), leukopenia (n=2, 12%) and fatigue (n=2, 12%). Common G2 toxicities were leukopenia (n=5, 30%), neutropenia (n=4, 23%), nausea (n=4, 23%) and lymphopenia (n=3, 18%). Conclusions: The combination of D, PT, and TMZ appears to be safe and well-tolerated. The recommended dose for the phase II study remains to be determined.

Frequent somatic mutations in MAP3K5 and MAP3K9 in metastatic melanoma identified by exome sequencing

We sequenced 8 melanoma exomes to identify novel somatic mutations in metastatic melanoma. Focusing on the MAP3K family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modelling predicts that mutations in the kinase domain may affect the activity and regulation of MAP3K5/9 protein kinases. The position of the mutations and loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely inactivating. In vitro kinase assay shows reduction in kinase activity in MAP3K5 I780F and MAP3K9 W333X mutants. Overexpression of MAP3K5 or MAP3K9 mutant in HEK293T cells reduces phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA leads to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.