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Uveal melanoma is the most common primary intraocular malignancy in adults and has a high incidence of metastatic disease. Current treatments have shown limited clinical activity in patients with uveal melanoma with metastasis and there is an urgent need for new effective therapies. Recent findings have shown that women with uveal melanoma have better survival rates than men. The G protein–coupled estrogen receptor-1 (GPER) has distinct functions from those of the classic estrogen receptors ERα/β and its activation by specific agonists has tumor-suppressive roles in several cancers. However, the role of GPER had not previously been investigated in uveal melanoma. We demonstrated that downregulation of GPER in uveal melanoma cells decreased expression of p53 and stimulated cell growth. In contrast, the clinical GPER agonist, LNS8801, upregulated p53 and p21, induced melanocytic differentiation markers, inhibited cell proliferation and cell migration, and induced apoptosis. Furthermore, LNS8801 treatment arrested the cells in G2–M-phase of the cell cycle with concomitant activation of mitotic markers and disruption of the mitotic spindle apparatus. LNS8801 significantly inhibited tumor growth of uveal melanoma xenografts in vivo, suggesting that GPER agonists may be a novel treatment for uveal melanoma. Significance: Current treatments against metastatic uveal melanoma have shown limited clinical activity and there is an urgent need for effective therapies. Here, we demonstrate that the GPER agonist LNS8801 induced both GPER-dependent and GPER-independent effects and elicited potent anticancer activities in vitro and in vivo. Our results complement and support the ongoing clinical trial of LNS8801 in advanced uveal melanoma.

Uveal melanoma is the most common primary intraocular malignancy in adults and has a high incidence of metastatic disease. Current treatments have shown limited clinical activity in patients with uveal melanoma with metastasis and there is an urgent need for new effective therapies. Recent findings have shown that women with uveal melanoma have better survival rates than men. The G protein–coupled estrogen receptor-1 (GPER) has distinct functions from those of the classic estrogen receptors ERα/β and its activation by specific agonists has tumor-suppressive roles in several cancers. However, the role of GPER had not previously been investigated in uveal melanoma. We demonstrated that downregulation of GPER in uveal melanoma cells decreased expression of p53 and stimulated cell growth. In contrast, the clinical GPER agonist, LNS8801, upregulated p53 and p21, induced melanocytic differentiation markers, inhibited cell proliferation and cell migration, and induced apoptosis. Furthermore, LNS8801 treatment arrested the cells in G2–M-phase of the cell cycle with concomitant activation of mitotic markers and disruption of the mitotic spindle apparatus. LNS8801 significantly inhibited tumor growth of uveal melanoma xenografts in vivo, suggesting that GPER agonists may be a novel treatment for uveal melanoma.Significance:Current treatments against metastatic uveal melanoma have shown limited clinical activity and there is an urgent need for effective therapies. Here, we demonstrate that the GPER agonist LNS8801 induced both GPER-dependent and GPER-independent effects and elicited potent anticancer activities in vitro and in vivo. Our results complement and support the ongoing clinical trial of LNS8801 in advanced uveal melanoma.

The mitotic effect of LNS8801 is GPER-independent

Migration Assay after nintedanib treatment

PDF File - 542K, Effect of GNAQ knockdown on downstream signaling pathways. 92.1 cells were transfected with control (Ctr) and GNAQ siRNA (Gq3) as in Materials and Methods

Sarcomas are rare cancers that make up about 1% of all cancers in adults; however, they occur more commonly among children and young adolescents. Sarcomas are genetically complex and are often difficult to treat given the lack of clinical efficacy of any of the currently available therapies. Receptor tyrosine kinases (RTK) such as c-Kit, c-Met, PDGFR, IGF-1R, as well as FGFR have all been reported to be involved in driving tumor development and progression in adult and pediatric soft-tissue sarcoma. These driver kinases often act as critical determinants of tumor cell proliferation and targeting these signal transduction pathways remains an attractive therapeutic approach. Nintedanib, a potent triple angiokinase inhibitor, targets PDGFR, VEGFR, and FGFR pathways critical for tumor angiogenesis and vasculature. In this study, we evaluated the preclinical efficacy of nintedanib in soft-tissue sarcoma cell lines. Nintedanib treatment resulted in significant antiproliferative effect in vitro in cell lines with high expression of RTK drug targets. Furthermore, treatment with nintedanib showed significant downregulation of downstream phosphorylated AKT and ERK1/2. Finally, treatment with nintedanib resulted in significant tumor growth suppression in mouse xenograft model of synovial sarcoma. Notably, both the in vitro and in vivo efficacy of nintedanib was superior to that of imatinib, another multikinase inhibitor, previously tested with minimal success in clinical trials in sarcoma. Overall, the data from this study provide a strong rationale to warrant further clinical exploration of this drug in patients with synovial sarcoma. Mol Cancer Ther; 17(11); 2329–40. ©2018 AACR.

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PDF file - 1199KB, AEB071/ BYL719 combination promotes apoptosis in GNAQ mutant xenograft model. At the end of treatments, two animals from each cohort were sacrificed and the tumors were assessed for apoptosis by TUNEL staining. See Methods for the details of this analysis.

PDF file - 399KB, AEB071/BYL719 synergistically inhibits GNAQ/GNA11 mutant tumor cell viability after 5 days of drug exposure. Chou-Toulay plots are shown with X-axis representing Fa, or fractional activity, and Y-axis representing combination index (CI) with 1, and =1 indicating synergistic, antagonistic, and additive effects, respectively. Each point represents a different combination of drug concentrations used in the assay. Concentrations for AEB071 treatment include 0, 125, 250, 500, 1000 nM. Concentrations for BYL719 treatment include 0, 250, 500, 1000, 2000 nM. A table is also included showing Combination Indexes for each combination of AEB071 and BYL719 used in each cell line.

Western blot analysis for RTK and non-RTK targets

G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (sotrastaurin) and PI3K/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11-mutant cells with AEB071 versus no activity in wild-type cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of myristoylated alanine-rich C-kinase substrate, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal antiproliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11-mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ- and GNA11-mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ-mutant model. These findings suggest a new therapy treatment option for G-protein–mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy. Mol Cancer Ther; 13(5); 1044–53. ©2014 AACR.

List of antibodies with catalog numbers

Figure legends for supplemental figures

PDF file - 405KB, AEB071/ BYL719 combination inhibits in vivo tumor growth in a GNA11 mutant xenograft model. A, Suppression of PKC/ ERK and PI3Kalpha/AKT pathways in Omm1 GNA11. Cells were treated with indicated AEB071/BYL719 concentrations for 2, 6 and 24 hrs and Western blots were then performed. alpha-Tubulin was used as a loading control. MARCKS and S6 inhibition was present but no AKT inhibition occurred with combination. ERK was inhibited by BYL719. B, AEB071/BYL719 inhibited tumor growth in a Omm1 xenograft model. AEB071/BYL719 inhibited tumor growth in a xenograft model with the GNA11 mutant Omm1 cell line. 6-8 week athymic female mice were subcutaneously injected with Omm1 cells. Drug treatments began after tumors reached 100 mm3. Mice with tumors were treated three times daily with AEB071 (80mg/kg/d) or once daily with BYL719 (50 mg/kg/d) or in combination for 5 days each week for a total of 3 weeks. Tumors were measured with calipers every 2 to 3 days. Tumor volume was compared between groups of mice at various points in time AEB071 or BYL719 alone. Toxicity was measured by weight loss.

Cell proliferation assay on Day 6

Bromodomain and extraterminal protein inhibitors (BETi) are epigenetic therapies aimed to target dysregulated gene expression in cancer cells. Despite early successes of BETi in a range of malignancies, the development of drug resistance may limit their clinical application. Here, we evaluated the mechanisms of BETi resistance in uveal melanoma, a disease with little treatment options, using two approaches: a high-throughput combinatorial drug screen with the clinical BET inhibitor PLX51107 and RNA sequencing of BETi-resistant cells. NF-κB inhibitors synergistically sensitized uveal melanoma cells to PLX51107 treatment. Furthermore, genes involved in NF-κB signaling were upregulated in BETi-resistant cells, and the transcription factor CEBPD contributed to the mechanism of resistance. These findings suggest that inhibitors of NF-κB signaling may improve the efficacy of BET inhibition in patients with advanced uveal melanoma.Significance:These findings provide evidence that inhibitors of NF-κB signaling synergize with BET inhibition in in vitro and in vivo models, suggesting a clinical utility of these targeted therapies in patients with uveal melanoma.

Synergistic effects of the NF-kB inhibitor QNZ in combination with PLX51107 in the BETi-resistant UM cells

PLX51107 inhibits the viability of UM cell lines in vitro and in vivo

The combination PLX51107+PTL induces apoptosis in the BETi-resistant cells. p-p65 expression does not change in liver biopsies of UM patients treated with PLX51107

UM cells resistant to PLX51107 show cross-resistance to other BET inhibitors, and a slower rate of growth

Purpose: Dysregulated cyclin-dependent kinases are important to the growth of some sarcomas. Flavopiridol is a pan-CDK inhibitor that has been shown to potentiate chemotherapy. As such, we explored the potentiation of doxorubicin by flavopiridol in sarcoma, in vitro and in vivo, and conducted a phase I trial of flavopiridol with doxorubicin in patients with advanced sarcomas.Experimental Design: Sarcoma cell lines and xenografts were treated with flavopiridol alone and in combination with doxorubicin. In the phase I study, doxorubicin and flavopiridol were administered on two flavopiridol schedules; a 1-hour bolus and split dosing as a 30-minute bolus followed by a 4-hour infusion.Results: Preclinically, flavopiridol potentiated doxorubicin. In vivo, doxorubicin administered 1 hour before flavopiridol was more active than doxorubicin alone. Clinically, 31 patients were enrolled on protocol and flavopiridol was escalated to target dose in two schedules (90 mg/m2 bolus; 50 mg/m2 bolus + 40 mg/m2 infusion) both in combination with doxorubicin (60 mg/m2). Dose-limiting toxicities were neutropenia, leukopenia, and febrile neutropenia but no maximum tolerated dose was defined. Flavopiridol pharmacokinetics showed increasing Cmax with increasing dose. Response Evaluation Criteria in Solid Tumors (RECIST) responses included two partial responses, however, stable disease was seen in 16 patients. Of 12 evaluable patients with progressive well- and dedifferentiated liposarcoma, eight had stable disease greater than 12 weeks.Conclusions: The sequential combination of doxorubicin followed by flavopiridol is well tolerated on both schedules. Disease control was observed in well- and dedifferentiated liposarcoma specifically, a disease in which CDK4 is known to be amplified. Clin Cancer Res; 18(9); 2638–47. ©2012 AACR.

PTL inhibits NF-kB activity and induces apoptosis in combination with PLX51107 in Omm1.3 cells

List of RNAs used in this study

PDF file - 66K, Cumulative grade 2 or greater hematologic toxicity throughout all cycles.

BETi-resistant cutaneous and uveal melanoma cells have different gene expression profiles

The NF-kB inhibitor PDTC has synergistic activity with the BET inhibitor PLX51107

XPO1 (Exportin-1) is the nuclear export protein responsible for the normal shuttling of several proteins and RNA species between the nucleocytoplasmic compartment of eukaryotic cells. XPO1 recognizes the nuclear export signal (NES) of its cargo proteins to facilitate its export. Alterations of nuclear export have been shown to play a role in oncogenesis in several types of solid tumor and hematologic cancers. Over more than a decade, there has been substantial progress in targeting nuclear export in cancer using selective XPO1 inhibitors. This has resulted in recent approval for the first-in-class drug selinexor for use in relapsed, refractory multiple myeloma and diffuse large B-cell lymphoma (DLBCL). Despite these successes not all patients respond effectively to XPO1 inhibition and there has been lack of biomarkers for response to XPO1 inhibitors in the clinic. Using hematologic malignancy cell lines and samples from patients with myelodysplastic neoplasms treated with selinexor, we have identified XPO1, NF-κB(p65), MCL-1 and p53 protein levels as protein markers of response to XPO1 inhibitor therapy. These markers could lead to the identification of response upon XPO1 inhibition for more accurate decision making in the personalized treatment of cancer patients undergoing treatment with selinexor.

Glycosaminoglycans (GAGs), such as heparan sulfate and chondroitin sulfate, are an important component of the tumor microenvironment (TME). These carbohydrates are attached to proteoglycans at the surface of tumor cells and play a vital role in malignant transformation. Heparan sulfate (HS) glycosaminoglycans have been demonstrated to play a pleiotropic role in regulating the activity of important cellular receptors and downstream signaling pathways, and are implicated in cancer cell proliferation, angiogenesis, invasion, and metastasis. Heparinase, an endoglycosidase that specifically cleaves heparan sulfate side chains of heparan sulfate proteoglycans is expressed in several sarcoma subtypes. We measured heparinase activity in several cell lines and found that A673, CHP100, CHLA9 and RD-ES Ewing’s sarcoma cell lines have significantly higher levels of heparinase activity compared to the heparinase-deficient CHO-K1 psgD-677 cell line. Interference with heparinase or heparin sulfate function(s) may represent a therapeutic approach in sarcoma. To this end, we tested these cell lines by measuring cell growth using a cell viability assay, in the presence or absence of GTC3300P, a small molecule inhibitor of protein interaction with glycosaminoglycan. We demonstrate that the Ewing’s sarcoma cell lines exhibit potent and significant inhibition in cell growth in vitro, in the presence of drug. Western blot analysis shows induction of cleaved Poly-ADP Ribose Polymerase (PARP), yH2AX, as well as cleaved caspase-3, known markers of apoptosis and down regulation of mTORC1 targets such as phospho-S6 (p-S6), in Ewing’s sarcoma. Apoptosis was confirmed by the induction of sub-G1 population in Ewing’s sarcoma by cell cycle analysis. Previous reports have indicated that heparinase activity is strongly implicated in cell migration and invasion and is associated with tumor metastasis, angiogenesis and inflammation. This was confirmed by LC-MS/MS proteomics and bioinformatics (gene ontology and gene set enrichment) analysis of these multiple cell lines, which identified alterations in numerous biomarkers in these pathways, including multiple ribosomal protein subunits, spliceosomes components, and complement coagulation cascade. We further conducted a cell migration assay and demonstrate that GTC3300 significantly inhibits cell migration in Ewing’s sarcoma cell lines, but not in heparinase-deficient psg677 cells. Taken together our data strongly suggests that targeting protein interaction with glycosaminoglycans including heparan sulfate in Ewing’s sarcoma represents a novel approach and merits further investigation Citation Format: Tahir N. Sheikh, Elgilda Musi, Alex J. Rai, Gary K. Schwartz. Development of small molecule inhibitors of protein interaction with glycosaminoglycans. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4977.

Soft tissue sarcomas are heterogeneous mesenchymal neoplasms and account for 1% of all cancers in adults. Over 50 sarcomas subtypes have been classified and many have very limited treatment options which include surgery, chemotherapy and radiation. Two of the most common sarcomas are liposarcoma and leiomyosarcoma which are often treated with chemotherapies such as doxorubicin, dacarbazine and gemcitabine with low response rates. There is now more than ever an urgent need for new therapies for sarcoma. PCNA (Proliferating Cell Nuclear Antigen) is considered to be a key regulator of DNA and cell cycle control. In addition, it has been implicated to have roles in metabolism, cellular signaling and some immunological functions. PCNA forms complexes with proteins bearing a novel PCNA-interacting Peptide Motif called APIM (AlkB homolog 2 PCNA Interacting Motif). This binding usually occurs during stress responses such as those achieved during cancer therapy or cancer development. By preventing stress proteins from repairing or defending themselves, cancer therapies can be made more effective. We examined the in vitro effects of ATX-101, a PCNA-APIM protein interaction blocking peptide, on liposarcoma cell lines LS141, DDLS, SW872, 93T449 and 94T778 and leiomyosarcoma cell lines SK-LMS, SK-UT1 and SK-UT-1B. Cell survival, as measured by cell proliferation assays, indicated IC50s of 7.5-15µM with ATX-101. Enhanced combination effects with ATX-101 and chemotherapies such as doxorubicin, irinotecan and gemcitabine were also observed by proliferation assay. Synergism, with ATX-101 and gemcitabine, was indicated with SynergyFinder analysis. Cell cycle changes exhibited increases of S and G2 phases when treated with ATX-101 and gemcitabine. This correlated with increase of cell cycle proteins, cyclin B1 and cyclin A2. Western blot observations included increase of apoptotic marker, cleaved caspase 3, and DNA damage marker, pH2A.X. In addition, immunofluorescence assay showed increasing pH2A.X with ATX-101 and gemcitabine. In vivo studies revealed ATX-101 enhanced gemcitabine decrease of tumor volume in leiomyosarcoma cell line xenograft, SK-UT1. Tumor target inhibition by western blot showed increased pH2A.X and cleaved PARP along with decreased RAD51 API protein with combination treatment. Taken these observations and results, there is strong evidence of combining gemcitabine with ATX-101 in liposarcoma and leiomyosarcoma. Citation Format: Elgilda Musi, Tahir Sheikh, Matthew Ingham, Sminu Bose, Gary K. Schwartz. ATX-101, a peptide drug targeting PCNA, enhances the effect of gemcitabine in liposarcoma and leiomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6174.

Simple Summary Uveal melanoma is a rare subset of melanoma characterized by the presence of early initiating GNAQ/11 mutations, with downstream activation of several pathways which are thought to contribute to cell growth. Based on clinical and preclinical data supporting targeting of protein kinase C (PKC) and the phosphatidylinositol 3-kinase (PI3K) pathway, we conducted a phase Ib study to assess the safety of combined sotrastaurin, a PKC inhibitor, and alpelisib, a PI3K inhibitor. We found that sotrastaurin and alpelisib can be safely administered, however there was no evidence of clinical efficacy. Abstract Uveal melanoma (UM) is a rare subset of melanoma characterized by the presence of early initiating GNAQ/11 mutations, with downstream activation of the PKC, MAPK, and PI3Kα pathways. Activity has been observed with the PKC inhibitors sotrastaurin (AEB071) and darovasertib (IDE196) in patients with UM. Inhibition of the PI3K pathway enhances PKC inhibition in in vivo models. We therefore conducted a phase Ib study of sotrastaurin in combination with the PI3Kα inhibitor alpelisib to identify a tolerable regimen that may enhance the activity of PKC inhibition alone. Patients with metastatic uveal melanoma (n = 24) or GNAQ/11 mutant cutaneous melanoma (n = 1) were enrolled on escalating dose levels of sotrastaurin (100–400 mg BID) and alpelisib (200–350 mg QD). The primary objective was to identify the maximum tolerated dose (MTD) of these agents when administered in combination. Treatment-related adverse events (AE) occurred in 86% (any grade) and 29% (Grade 3). No Grade 4–5-related AEs occurred. Dose Level 4 (sotrastaurin 200 mg BID and alpelisib 350 mg QD) was identified as the maximum tolerated dose. Pharmacokinetic analysis demonstrated increasing concentration levels with increasing doses of sotrastaurin and alpelisib, without evidence of interaction between agents. Pharmacodynamic assessment of pMARCKS and pAKT protein expression with drug exposure suggested modest target inhibition that did not correlate with clinical response. No objective responses were observed, and median progression-free survival was 8 weeks (range, 3–51 weeks). Although a tolerable dose of sotrastaurin and alpelisib was identified with pharmacodynamic evidence of target inhibition and without evidence of a corresponding immunosuppressive effect, limited clinical activity was observed.

// Elgilda Musi 1 , Gary K. Schwartz 1 , 2 , Jae Hyuk Yoo 3 , Shannon J. Odelberg 3 , 4 , 6 , Dean Y. Li 3 , 5 , 6 , 7 , Michael Y. Bonner 8 , Ponniah Selvakumar 9 , Shikha Rao 8 , Linda C. Gilbert 8 , 10 , Justin Elsey 8 and Jack L. Arbiser 8 , 10 1 Department of Medicine, Columbia University Medical Center, New York, New York, USA 2 Herbert Irving Comprehensive Cancer Center, Columbia University College of Medicine, New York, New York, USA 3 Department of Medicine, Program in Molecular Medicine, University of Utah, Salt Lake City, Utah, USA 4 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, Utah, USA 5 Department of Human Genetics, University of Utah, Salt Lake City, Utah, USA 6 Department of Internal Medicine, Division of Cardiovascular Medicine, University of Utah, Salt Lake City, Utah, USA 7 Department of Oncological Sciences, University of Utah, Salt Lake City, Utah, USA 8 Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia, USA 9 Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada 10 Veterans Affairs Medical Center, Decatur, Georgia, USA Correspondence to: Jack L. Arbiser, email: jarbise@emory.edu Keywords: melanoma; chemotherapy Received: April 09, 2019 Accepted: June 05, 2019 Published: July 09, 2019 ABSTRACT Uveal melanoma is a rare but often lethal malignancy and is the leading cause of death due to an ophthalmic condition. Uveal melanoma is often diagnosed at a late stage and has a strong propensity to hepatic metastasis. Recently, the most common driver mutations in uveal melanoma have been identified, predominantly in the G-proteins GNAQ. This pattern differs from that of cutaneous melanoma in which Braf and Nras predominate. There are no current clinically used agents that target GNAQ mutations, unlike the use of Braf inhibitors in cutaneous melanoma. We tested the novel agent Tris DBA palladium and found that it was markedly more effective against GNAQ mutant melanomas than wild type uveal melanomas. Given that ARF6 has recently been discovered as a node in GNAQ mutations, we evaluated the efficacy of Tris DBA palladium on ARF6 signaling and found that it was effective in inhibiting ARF6 activation. Finally, Tris DBA palladium was orally effective against GNAQ mutant melanoma in vivo . Tris DBA Palladium deserves further evaluation as a systemic agent for uveal melanoma.

Bromodomain and extraterminal protein inhibitors (BETi) are epigenetic therapies aimed to target dysregulated gene expression in cancer cells. Despite early successes of BETi in a range of malignancies, the development of drug resistance may limit their clinical application. Here, we evaluated the mechanisms of BETi resistance in uveal melanoma, a disease with little treatment options, using two approaches: a high-throughput combinatorial drug screen with the clinical BET inhibitor PLX51107 and RNA sequencing of BETi-resistant cells. NF-κB inhibitors synergistically sensitized uveal melanoma cells to PLX51107 treatment. Furthermore, genes involved in NF-κB signaling were upregulated in BETi-resistant cells, and the transcription factor CEBPD contributed to the mechanism of resistance. These findings suggest that inhibitors of NF-κB signaling may improve the efficacy of BET inhibition in patients with advanced uveal melanoma. Significance: These findings provide evidence that inhibitors of NF-κB signaling synergize with BET inhibition in in vitro and in vivo models, suggesting a clinical utility of these targeted therapies in patients with uveal melanoma.

Supplemental Digital Content is available in the text., Uveal melanoma is a rare form of melanoma with particularly poor outcomes in the metastatic setting. In contrast with cutaneous melanoma, uveal melanoma lacks BRAF mutations and demonstrates very low response rates to immune-checkpoint blockade. Our objectives were to study the transcriptomics of metastatic uveal melanoma with the intent of assessing gene pathways and potential molecular characteristics that might be nominated for further exploration as therapeutic targets. We initially analyzed transcriptional data from The Cancer Genome Atlas suggesting PI3K/mTOR and glycolysis as well as IL6 associating with poor survival. From tumor samples collected in a prospective phase II trial (A091201), we performed a transcriptional analysis of human metastatic uveal melanoma observing a novel role for epithelial-mesenchymal transition associating with survival. Specifically, we nominate and describe initial functional validation of neuropillin-1 from uveal melanoma cells as associated with poor survival and as a mediator of proliferation and migration for uveal melanoma in vitro. These results immediately nominate potential next steps in clinical research for patients with metastatic uveal melanoma.

Uveal melanoma is one of the most common intraocular malignancies of the adult eye. GNAQ/GNA11 genetic alterations are present in 85% of cases resulting in activation of the MAPK pathway. Single agent approaches targeting the MAPK pathway and histone deacetylases (HDACs) have shown very modest activity in uveal melanoma. We hypothesize that a combination strategy may be an effective, novel therapeutic approach in uveal melanoma. We examined the activity of ASTX029, a novel dual-mechanism ERK inhibitor, which inhibits both the catalytic activity of ERK and its phosphorylation by MEK, and belinostat, a pan-HDAC inhibitor, in one GNAQQ209L- (92.1) and GNAQQ209P- (OMM1.3) and two GNA11Q209L-mutant (MP41, OMM1) uveal melanoma cell lines. The uveal melanoma cell lines were sensitive to ASTX029 with IC50 values between 50 to 100nM and to belinostat with IC50 values between 100 to 250nM. Combination index analysis of ASTX029 and belinostat by the Chou-Talalay method indicated synergism, with CI values ranging from 0.34 to 0.97. We explored other drug candidates for combinatorial approaches including the BCL-2 inhibitor navitoclax, but no combination effect was observed. After 48h of combination treatment with ASTX029 and belinostat, we examined the biochemical effects by western blot which demonstrated a sustained decrease in levels of p-ERK with the combination treatment. Western blot analysis confirmed reduction of downstream markers of MAPK pathway activity including cell cycle markers p-RB (780), cyclin D1 and c-myc with combination treatment. Apoptosis was evident from an increase in the subG1 population by flow cytometry analysis, which correlated with BIM induction and PARP cleavage on western blot. We also observed increased surface expression of immunological markers HLA-A, B, C (MHC Class 1), PD-L1, gp100 and MART-1. These results support that a combination strategy targeting ERK and HDAC in uveal melanoma should be investigated further. Citation Format: Elgilda Musi, Kim-Hien Dao, Chris Hindley, Gary K. Schwartz. Targeting of ERK and HDAC in the treatment of uveal melanoma [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 2668.

Sarcomas are rare cancers that make up about 1% of all cancers in adults; however, they occur more commonly among children and young adolescents. Sarcomas are genetically complex and are often difficult to treat given the lack of clinical efficacy of any of the currently available therapies. Receptor tyrosine kinases (RTK) such as c-Kit, c-Met, PDGFR, IGF-1R, as well as FGFR have all been reported to be involved in driving tumor development and progression in adult and pediatric soft-tissue sarcoma. These driver kinases often act as critical determinants of tumor cell proliferation and targeting these signal transduction pathways remains an attractive therapeutic approach. Nintedanib, a potent triple angiokinase inhibitor, targets PDGFR, VEGFR, and FGFR pathways critical for tumor angiogenesis and vasculature. In this study, we evaluated the preclinical efficacy of nintedanib in soft-tissue sarcoma cell lines. Nintedanib treatment resulted in significant antiproliferative effect in vitro in cell lines with high expression of RTK drug targets. Furthermore, treatment with nintedanib showed significant downregulation of downstream phosphorylated AKT and ERK1/2. Finally, treatment with nintedanib resulted in significant tumor growth suppression in mouse xenograft model of synovial sarcoma. Notably, both the in vitro and in vivo efficacy of nintedanib was superior to that of imatinib, another multikinase inhibitor, previously tested with minimal success in clinical trials in sarcoma. Overall, the data from this study provide a strong rationale to warrant further clinical exploration of this drug in patients with synovial sarcoma. Mol Cancer Ther; 17(11); 2329–40. ©2018 AACR.

Soft tissue sarcomas are heterogeneous mesenchymal neoplasms and account for 1% of all cancers in adults. Over 50 sarcomas subtypes have been classified and many have limited treatment options some of which include surgery, chemotherapy and radiation. 3-phosphoinositide dependent protein kinase 1 (PDK1) is overexpressed in many cancers, suggesting a role for PDK1 in cancer progression. In addition, PDK1 has been identified as a key modifier of acquired resistance to CDK4/6 inhibitors. In order to interrogate this in sarcoma, we elected to test SNS-510 a potent, orally bioavailable PDK1 inhibitor against a broad range of sarcoma cell lines (leiomyosarcoma, dedifferentiated liposarcoma, Ewing's sarcoma, osteosarcoma, chondrosarcoma and synovial sarcoma) as well as dedifferentiated liposarcoma cell lines with acquired resistance to ribociclib, which are also cross resistant to palbociclib. All the cell lines expressed PDK1 and, as measured by WST-8 cell proliferation assay, nearly all the cell lines were sensitive to the drug, with IC50s generally between 250 to 500nM. Leiomyosarcoma cell lines (SK-LMS, SK-UT1, SK-UT1b) were seen to be most sensitive to SNS-510 with IC50s determined to be between 50-100nM. In contrast, synovial sarcoma cell lines (SYO-1 and HSSY-II) exhibited relative resistance to SNS-510 with IC50s determined to be > 500nM. As assessed by western blot, SNS-510 exhibited a dose dependent inhibition of the PDK-1/RSK2/AKT pathway with inhibition of p-RSK2(244), p-AKT(308), and pS6(235/236). SNS-510 induced a dose-dependent G1 cell cycle arrest. This was associated with inhibition of p-RB(780) and suppression of cyclin D1 and cyclin E1. The exception to this was the synovial cell lines for which there was no inhibition of p-RB despite inhibition of p-RSK, p-AKT and p-S6. When combined with palbociclib in both the CDK4/6 sensitive (LS141 and DDLS) and CDK4/6 resistant liposarcoma cells (RES.LS141 and RES.DDLS), we observed enhanced inhibition of proliferation with a synergistic effect, as defined by the Chou-Talalay method. These results would suggest a role for PDK-1 targeting with SNS-510 in sarcoma and consideration of combination strategies, especially with CDK4/6 inhibitors in both CDK4/6 sensitive and resistant dedifferentiated liposarcoma. Citation Format: Elgilda Musi, Pietro Taverna, Gary K. Schwartz. PDK-1 targeting with SNS-510 in sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1067.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Approximately 50% of the patients develop hepatic metastasis and the median survival rate is 12 months. Currently available drugs have shown limited clinical activity in patients with UM and there is an urgent need for new effective therapies. UM is characterized by activating mutations of G protein q/11, mutations in BAP1, and amplification of the oncogene MYC. Epigenetic dysregulation plays a critical role in UM pathogenesis, and we have demonstrated that Gnaq/11 mutant UM cells are addicted to BRD4 activity. BRD4 is a key regulator of transcriptional elongation by recruiting positive transcriptional elongation factor complex (PTEFb) to chromatin and activating RNA polymerase II-dependent transcription. While BET inhibitors have shown potent activity in pre-clinical studies, single-agent BET inhibitors exhibited limited efficacy in advanced UM clinical trials. MAPK signaling in constitutively active in Gnaq/11 mutant UM cells and previous studies have shown that the MAPK pathway mediates resistance to BET inhibition. Therefore, we sought to combine MEK and BET inhibitors in a panel of UM cells. We found synergistic activities with a variety of structurally distinct MEK inhibitors including trametinib and binimetinib in combination with BET inhibitors, such as I-BET and PLX2853. These combinations induced a greater reduction in cell viability and significant apoptosis compared to either monotherapy, in both parental and BET inhibitor-resistant cells. Western blot studies were performed to evaluate the mechanism of the combination treatment with BET and MEK inhibition and demonstrated a marked downregulation of p-ERK, MYC, Bcl-xL and upregulation of BIM with the combination therapy. Similarly, the combination treatment resulted in greater inhibition of tumor growth compared to either BET or MEK inhibition alone in a Gnaq mutant xenograft model. In conclusion, our studies suggest that co-targeting of MEK and BET proteins may be required to maximize the responses of UM cells to BET inhibitors. Clinical trials with the combination of BET and MEK inhibitors are warranted. Citation Format: Grazia Ambrosini, Elgilda Musi, Gary K. Schwartz. BET bromodomain inhibition synergizes with MEK inhibitors in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1115.

Soft tissue sarcomas are heterogeneous mesenchymal neoplasms and account for 1% of all cancers in adults. One of the most common diagnosed soft tissue sarcomas is well-differentiated (WD)/dedifferentiated (DD) liposarcoma which is characterized by amplification of CDK4. We have previously reported that the CDK4 inhibitor palbociclib induces cell cycle arrest in the CDK4 amplified, LS141 and DDLS liposarcoma cell lines in vitro (Barretina J. et al Nature Genet. 2010) and in clinical trial results palbociclib results in prolonged progression free survival in patients with WD/DD-liposarcoma (Dickson M Et al. J Clin Oncol. 2013). We have now studied the mechanisms of CDK4/6 resistance in liposarcoma by exposing LS141 and DDLS cell lines to increasing concentrations of the CDK4/6 inhibitor, ribociclib, for an extended time period of 1-2 months. Cell survival, as measured by cell proliferation assays, indicated IC50s of 10µM for CDK4/6 Resistant LS141 and 15 µM for CDK4/6 Resistant DDLS, which is more than ten times higher than the sensitive parental cells. We also compared the established CDK4/6 resistant cell lines to palbociclib and found that resistance was similar to the ribociclib effect. Cell cycle changes to CDK4/6 resistant cell lines exhibited no effect of G1 arrest when exposed to higher concentrations of the drug. We also examined the effect of ribociclib and palbociclib on p-RB after treatment in sensitive parental and CDK4/6 resistant liposarcoma cell lines and showed that inhibition of p-Rb occurred at lower doses of 250-500 nM in the sensitive cell lines compared to higher doses of 2-8 µM in the resistant cell lines. Overexpression of cyclin E1 and p-CDK2 were prominent in the resistant cell lines when examined by western blot. siRNA suppression of cyclin E showed a reversal of resistance as determined by proliferation assays. We also observed induction of immunological markers including PD-L1, HLA-A2 and B2M (MHC-1 class) with CDK4/6 inhibitor treatment in the sensitive cell lines. Flow cytometry showed HLA-A2 and B2M increased in the resistant LS141 while B2M only increased in the resistant DDLS compared to their parental cell lines. We also observed PD-L1 increased in the resistant LS141 while it decreased in the resistant DDLS when exposed to the drug over long periods of time. Cytokine production in the CDK4/6 resistant liposarcoma was also seen with IL-8 increasing in resistant LS141 and IL-6 increasing in resistant DDLS cell line. Taken together these results indicate that cyclin E1/CDK2 activation is a mechanism of resistance to CDK4/6 inhibitors in WD/DD-liposarcoma and that CDK4/6 inhibition induces immunologic effects including induction of PD-L1 that are modified over time in CDK4/6 resistant liposarcoma cells. These results would support CDK2/4/6 targeting in this disease and the development of combination trials with PD-L1 inhibitors. Citation Format: Elgilda Musi, Parag P. Patwardhan, Matthew Ingham, Gary K. Schwartz. Pre-clinical evaluation of CDK4/6 inhibitor resistance and immunologic modification in liposarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5289.

Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10–18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma.

// Grazia Ambrosini 1 , Ashley D. Sawle 1 , Elgilda Musi 1 , Gary K. Schwartz 1 1 The Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY, USA Correspondence to: Grazia Ambrosini, e-mail: ga2391@columbia.edu Keywords: BRD4, JQ1, Gnaq/11, Bcl-xL, Rad51 Received: June 18, 2015 Accepted: August 24, 2015 Published: September 05, 2015 ABSTRACT Uveal melanoma (UM) is an aggressive intraocular malignancy with limited therapeutic options. Both primary and metastatic UM are characterized by oncogenic mutations in the G-protein alpha subunit q and 11. Furthermore, nearly 40% of UM has amplification of the chromosomal arm 8q and monosomy of chromosome 3, with consequent anomalies of MYC copy number. Chromatin regulators have become attractive targets for cancer therapy. In particular, the bromodomain and extra-terminal (BET) inhibitor JQ1 has shown selective inhibition of c-Myc expression with antiproliferative activity in hematopoietic and solid tumors. Here we provide evidence that JQ1 had cytotoxic activity in UM cell lines carrying Gnaq/11 mutations, while in cells without the mutations had little effects. Using microarray analysis, we identified a large subset of genes modulated by JQ1 involved in the regulation of cell cycle, apoptosis and DNA repair. Further analysis of selected genes determined that the concomitant silencing of Bcl-xL and Rad51 represented the minimal requirement to mimic the apoptotic effects of JQ1 in the mutant cells, independently of c-Myc. In addition, administration of JQ1 to mouse xenograft models of Gnaq-mutant UM resulted in significant inhibition of tumor growth. Collectively, our results define BRD4 targeting as a novel therapeutic intervention against UM with Gnaq/Gna11 mutations.

Purpose: Selinexor, a small molecule that inhibits nuclear export protein XPO1, has demonstrated efficacy in solid tumors and hematologic malignancies with the evidence of clinical activity in sarcoma as a single agent. Treatment options available are very few, and hence the need to identify novel targets and strategic therapies is of utmost importance. Experimental Design: The mechanistic effects of selinexor in sarcomas as a monotherapy and in combination with proteasome inhibitor, carfilzomib, across a panel of cell lines in vitro and few in xenograft mouse models were investigated. Results: Selinexor induced IκB nuclear localization as a single agent, and the effect was enhanced by stabilization of IκB when pretreated with the proteasome inhibitor carfilzomib. This stabilization and retention of IκB in the nucleus resulted in inhibition of NFκB and transcriptional suppression of the critical antiapoptotic protein, survivin. Treatment of carfilzomib followed by selinexor caused selinexor-sensitive and selinexor-resistant cell lines to be more sensitive to selinexor as determined by an increase in apoptosis. This was successfully demonstrated in the MPNST xenograft model with enhanced tumor suppression. Conclusions: The subcellular distributions of IκB and NFκB are indicative of carcinogenesis. Inhibition of XPO1 results in intranuclear retention of IκB, which inhibits NFκB and thereby provides a novel mechanism for drug therapy in sarcoma. This effect can be further enhanced in relatively selinexor-resistant sarcoma cell lines by pretreatment with the proteasome inhibitor carfilzomib. Because of these results, a human clinical trial with selinexor in combination with a proteasome inhibitor is planned for the treatment of sarcoma. Clin Cancer Res; 23(15); 4301–11. ©2017 AACR.

Uveal melanoma is one of most common intraocular malignancies of the adult eye. GNAQ/ GNA11 genetic alterations are present in 85% of diagnosed cases resulting in activation of MAPK pathway. Recently, MEK inhibitors, such as selumetinib have been used to treat GNAQ/GNA11-mutant uveal melanoma patients. However, selumetinib has shown modest effect compared to chemotherapy. As ERK is the primary downstream effector of the MAPK pathway, it is hypothesized that ERK inhibitors have the potential to improve clinical activity in these patient populations. We examined the effects of a selective and potent ERK inhibitor (Compound A), on several uveal melanoma cell lines including 4 GNAQ cell lines (92.1, Mel202, Omm1.3, MP46), 2 GNA11 cell lines (MP41, Omm1), 2 BRAF cell lines (OCM1A, OCM1) and a Wild Type (Mel290) cell line. The uveal melanoma cell lines were sensitive to ERK inhibition by Compound A, with IC50s concentrations determined to be between 50 to 250 nM. Compound A was more effective at inhibiting growth than the MEK inhibitor selumetinib at nanomolar concentrations. Compound A at 250 nM inhibited pERK1/2 within at least 2 hours of drug exposure. By 24 hours inhibition of pRB and suppression of cyclin D1 was detected in all the cell lines tested except the Wild type cell line Mel290. Cell cycle analysis of Compound A dosed at 250 nM for 24 hours showed significant G1 arrest (85-90% G1). These in vitro results support further evaluation of ERK inhibition in the treatment of uveal melanoma. Citation Format: Elgilda Musi, Grazia Ambrosini, Joanne Munck, Gary K. Schwartz. A novel ERK inhibitor in the treatment of uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5784.

SummaryBlood platelets provide the initial response to vascular endothelial injury, becoming activated as they adhere to the injured site. Activated platelets recruit leukocytes, and initiate proliferation and migration of vascular smooth muscle cells (SMC) within the injured vessel wall, leading to development of neointimal hyperplasia. Endothelial CD39/NTPDase1 and recombinant solCD39 rapidly metabolise nucleotides, including stimulatory ADP released from activated platelets, thereby suppressing additional platelet reactivity. Using a murine model of vascular endothelial injury, we investigated whether circulating human solCD39 could reduce platelet activation and accumulation, thus abating leukocyte infiltration and neointimal formation following vascular damage. Intraperitoneally-administered solCD39 ADP -ase activity in plasma peaked 1 hour (h) post-injection, with an elimination half-life of 43 h. Accordingly, mice were administered solCD39 or saline 1 h prior to vessel injury, then either sacrificed 24 h post-injury or treated with solCD39 or saline (three times weekly) for an additional 18 days. Twenty-four hours post-injury, solCD39-treated mice displayed a reduction in platelet activation and recruitment, P-selectin expression, and leukocyte accumulation in the arterial lumen. Furthermore, repeated administration of solCD39 modulated the late stage of vascular injury by suppressing leukocyte deposition, macrophage infiltration and smooth muscle cell (SMC) proliferation/migration, resulting in abrogation of neointimal thickening. In contrast, injured femoral arteries of saline-injected mice exhibited massive platelet thrombus formation, marked P-selectin expression, and leukocyte infiltration. Pronounced neointimal growth with macrophage and SMC accretion was also observed (intimal-to-medial area ratio 1.56 ± 0.34 at 19 days). Thus, systemic administration of solCD39 profoundly affects injury-induced cellular responses, minimising platelet deposition and leukocyte recruitment, and suppressing neointimal hyperplasia.

The new RuII chloroquine complexes [Ru(η6-arene)(CQ)Cl2] (CQ = chloroquine; arene = p-cymene 1, benzene 2), [Ru(η6-p-cymene)(CQ)(H2O)2][BF4]2 (3), [Ru(η6-p-cymene)(CQ)(en)][PF6]2 (en = ethylenediamine) (4), and [Ru(η6-p-cymene)(η6-CQDP)][BF4]2 (5, CQDP = chloroquine diphosphate) have been synthesized and characterized by use of a combination of NMR and FTIR spectroscopy with DFT calculations. Each complex is formed as a single coordination isomer: in compounds 1–4 chloroquine binds to ruthenium in the η1-N mode through the quinoline nitrogen atom whereas in complex 5 an unprecedented η6 bonding through the carbocyclic ring is observed. Compounds 1, 2, 3, and 5 are active against CQ-resistant (Dd2, K1 and W2) and CQ-sensitive (FcB1, PFB, F32 and 3D7) malaria parasites (Plasmodium falciparum); importantly, the potency of these complexes against resistant parasites is consistently higher than that of the standard drug chloroquine diphosphate. Complexes 1 and 5 also inhibit the growth of colon cancer cells, independently of the p53 status and of liposarcoma tumor cell lines with the latter showing increased sensitivity, especially to complex 1 (IC50 8 µM); this is significant because this type of tumor does not respond to currently employed chemotherapies.

Soft tissue sarcomas are heterogeneous mesenchymal neoplasms and account for 1% of all cancers in adults. Over 50 sarcomas subtypes have been classified and many have very limited treatment options. Chemotherapies such as doxorubicin, dacarbazine and gemcitabine have had very low response rates. Two of the most common subtypes of sarcomas are liposarcoma and leiomyosarcoma, also referred to as the L-sarcomas. Trabectedin, a marine derived compound from the sea sponge, Ecteinascidia turbinate, has recently been approved for the treatment of L-sarcomas which has shown a modest effect over common therapies. As a DNA damaging agent, we hypothesized that trabectedin would induce a G2 cell cycle arrest and subsequent inhibition of Wee1 would abrogate this cell cycle checkpoint and induce apoptosis. We therefore examined the effects of trabectedin followed by AZD1775, a Wee1 inhibitor, in a panel of 6 sarcoma cell lines including liposarcoma (LS141, DDLS) and leiomyosarcoma (SK-LMS, SK-UT1, SK-UT1b). IC50s concentrations of trabectedin were determined to be between 0.5 to 2nM, while IC50s for AZD1775 were in the range of 100-200 nM. In vitro results have shown an enhanced inhibition of cell viability with combination treatment in all cell lines at concentrations of 1nM trabectedin for 24hrs followed by 100nM AZD1775 for 48hrs. We also examined biochemical effects with sequential drug treatment (24hrs trabectedin followed by 24hrs AZD1775) which presented an increase of cyclin A1 and cyclin B1 indicative of G2 cell cycle arrest along with increase of phospho-H2AX, indicative of DNA damage by trabectedin, while subsequent treatment with the Wee1 inhibitor showed decrease of phosphorylation of CDC2 (Tyr15). Flow cytometric analysis indicated G2/M cell cycle arrest with trabectedin and abrogation of G2/M with AZD1775. These observations correlated with enhanced apoptosis as measured by biochemical PARP cleavage and DNA content (sub-G1 population). Taken together these results provide a foundation for the development of the sequential treatment of trabectedin followed by the Wee1 inhibitor, AZD775, in the L-sarcomas. Citation Format: Elgilda Musi, Grazia Ambrosini, Gary K. Schwartz. Sequential treatment with the Wee1 inhibitor, AZD1775, enhances the effect of trabectedin in the L-sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 291. doi:10.1158/1538-7445.AM2017-291

G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (sotrastaurin) and PI3K/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11-mutant cells with AEB071 versus no activity in wild-type cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of myristoylated alanine-rich C-kinase substrate, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal antiproliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11-mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ- and GNA11-mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ-mutant model. These findings suggest a new therapy treatment option for G-protein–mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy. Mol Cancer Ther; 13(5); 1044–53. ©2014 AACR.

Product R (Reticulose) is a peptide-nucleic acid immunomodulator recently shown to enhance the expression of mRNAs encoding pro-inflammatory cytokines. Interleukin 8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) are pro-inflammatory chemokines involved in immune cell mobilization and stimulation. To determine whether Product R acts by upregulating these chemokines, we assayed its effects on the expression of IL-8 and MCP-1 mRNAs and proteins by human monocytic U937 cells and by adherent peripheral blood mononuclear cells (PBMCs). U937 cells were cultured for 0-21 days in media containing 0-20% Product R or phosphate-buffered saline (PBS). Compared to control cultures, cells cultured in Product R expressed increased amounts of IL-8 and MCP-1 mRNAs, as measured by reverse transcriptase-polymerase chain reaction (RT-PCR). Product R also increased secretion of IL-8 and MCP-1, as measured by enzyme-linked immunosorbent assay (ELISA), and boosted secretion induced by bacterial lipopolysaccharide (LPS), in a time- and dose-dependent manner. In adherent PBMCs, Product R increased IL-8 and MCP-1 secretion, but reduced LPS-induced MCP-1 secretion. While mRNAs encoding the IL-8 receptor, CXCR2, and the MCP-1 receptor, CCR2, were increased in U937 cells cultured in 5-10% Product R, we observed no change in binding of receptor-specific antibodies. These findings suggest that Product R upregulates the expression of IL-8 and MCP-1, which may boost immune system activity in virally-infected patients.

Oncogenic mutations in GNAQ and GNA11 genes are found in 80% of uveal melanoma. These mutations result in the activation of the RAF/MEK signaling pathway culminating in the stimulation of ERK1/2 mitogen-activated protein kinases. In this study, using a siRNA strategy, we show that mutant GNAQ signals to both MEK and AKT, and that combined inhibition of these pathways with the MEK inhibitor selumetinib (AZD6244) and the AKT inhibitor MK2206 induced a synergistic decrease in cell viability. This effect was genotype dependent as autophagic markers like beclin1 and LC3 were induced in GNAQ-mutant cells, whereas apoptosis was the mechanism of cell death of BRAF-mutant cells, and cells without either mutation underwent cell-cycle arrest. The inhibition of MEK/ATK pathways induced activation of AMP-activated protein kinase (AMPK) in the GNAQ-mutant cells. The downregulation of AMPK by siRNA or its inhibition with compound C did not rescue the cells from autophagy, rather they died by apoptosis, defining AMPK as a key regulator of mutant GNAQ signaling and a switch between autophagy and apoptosis. Furthermore, this combination treatment was effective in inhibiting tumor growth in xenograft mouse models. These findings suggest that inhibition of MEK and AKT may represent a promising approach for targeted therapy of patients with uveal melanoma. Mol Cancer Ther; 12(5); 768–76. ©2013 AACR.