Human Melanoma Tumor Research Articles

Inhibition of Pannexin 1 Reduces the Tumorigenic Properties of Human Melanoma Cells.

Pannexin 1 (PANX1) is a channel-forming glycoprotein expressed in many tissues including the skin. PANX1 channels allow the passage of ions and molecules up to 1 kDa, including ATP and other metabolites. In this study, we show that PANX1 is highly expressed in human melanoma tumors at all stages of disease progression, as well as in patient-derived cells and established melanoma cell lines. Reducing PANX1 protein levels using shRNA or inhibiting channel function with the channel blockers, carbenoxolone (CBX) and probenecid (PBN), significantly decreased cell growth and migration, and increased melanin production in A375-P and A375-MA2 cell lines. Further, treatment of A375-MA2 tumors in chicken embryo xenografts with CBX or PBN significantly reduced melanoma tumor weight and invasiveness. Blocking PANX1 channels with PBN reduced ATP release in A375-P cells, suggesting a potential role for PANX1 in purinergic signaling of melanoma cells. In addition, cell-surface biotinylation assays indicate that there is an intracellular pool of PANX1 in melanoma cells. PANX1 likely modulates signaling through the Wnt/β-catenin pathway, because β-catenin levels were significantly decreased upon PANX1 silencing. Collectively, our findings identify a role for PANX1 in controlling growth and tumorigenic properties of melanoma cells contributing to signaling pathways that modulate melanoma progression.

Harnessing autophagy to overcome mitogen-activated protein kinase kinase inhibitor-induced resistance in metastatic melanoma.

SummaryBackgroundPatients with malignant melanoma often relapse after treatment with BRAF and/or mitogen‐activated protein kinase kinase (MEK) inhibitors (MEKi) owing to development of drug resistance.ObjectivesTo establish the temporal pattern of CD271 regulation during development of resistance by melanoma to trametinib, and determine the association between development of resistance to trametinib and induction of prosurvival autophagy.MethodsImmunohistochemistry for CD271 and p62 was performed on human naevi and primary malignant melanoma tumours. Western blotting was used to analyse expression of CD271, p62 and LC3 in melanoma subpopulations. Flow cytometry and immunofluorescence microscopy was used to evaluate trametinib‐induced cell death and CD271 expression. MTS viability assays and zebrafish xenografts were used to evaluate the effect of CD271 and autophagy modulation on trametinib‐resistant melanoma cell survival and invasion, respectively.Results CD271 and autophagic signalling are increased in stage III primary melanomas vs. benign naevi. In vitro studies demonstrate MEKi of BRAF‐mutant melanoma induced cytotoxic autophagy, followed by the emergence of CD271‐expressing subpopulations. Trametinib‐induced CD271 reduced autophagic flux, leading to activation of prosurvival autophagy and development of MEKi resistance. Treatment of CD271‐expressing melanoma subpopulations with RNA interference and small‐molecule inhibitors to CD271 reduced the development of MEKi resistance, while clinically applicable autophagy modulatory agents – including Δ9‐tetrahydrocannabinol and Vps34 – reduced survival of MEKi‐resistant melanoma cells. Combined MEK/autophagy inhibition also reduced the invasive and metastatic potential of MEKi‐resistant cells in an in vivo zebrafish xenograft.ConclusionsThese results highlight a novel mechanism of MEKi‐induced drug resistance and suggest that targeting autophagy may be a translatable approach to resensitize drug‐resistant melanoma cells to the cytotoxic effects of MEKi.

Human breast tumor-infiltrating CD8+ T cells retain polyfunctionality despite PD-1 expression

Functional CD8+ T cells in human tumors play a clear role in clinical prognosis and response to immunotherapeutic interventions. PD-1 expression in T cells involved in chronic infections and tumors such as melanoma often correlates with a state of T-cell exhaustion. Here we interrogate CD8+ tumor-infiltrating lymphocytes (TILs) from human breast and melanoma tumors to explore their functional state. Despite expression of exhaustion hallmarks, such as PD-1 expression, human breast tumor CD8+ TILs retain robust capacity for production of effector cytokines and degranulation capacity. In contrast, melanoma CD8+ TILs display dramatic reduction of cytokine production and degranulation capacity. We show that CD8+ TILs from human breast tumors can potently kill cancer cells via bi-specific antibodies. Our data demonstrate that CD8+ TILs in human breast tumors retain polyfunctionality, despite PD-1 expression, and suggest that they may be harnessed for effective immunotherapies.

Aspirin Suppresses PGE2 and Activates AMP Kinase to Inhibit Melanoma Cell Motility, Pigmentation, and Selective Tumor Growth In Vivo.

There are conflicting epidemiologic data on whether chronic aspirin (ASA) use may reduce melanoma risk in humans. Potential anticancer effects of ASA may be mediated by its ability to suppress prostaglandin E2 (PGE2) production and activate 5'-adenosine monophosphate-activated protein kinase (AMPK). We investigated the inhibitory effects of ASA in a panel of melanoma and transformed melanocyte cell lines, and on tumor growth in a preclinical model. ASA and the COX-2 inhibitor celecoxib did not affect melanoma cell viability, but significantly reduced colony formation, cell motility, and pigmentation (melanin production) in vitro at concentrations of 1 mmol/L and 20 μmol/L, respectively. ASA-mediated inhibition of cell migration and pigmentation was rescued by exogenous PGE2 or Compound C, which inhibits AMPK activation. Levels of tyrosinase, MITF, and p-ERK were unaffected by ASA exposure. Following a single oral dose of 0.4 mg ASA to NOD/SCID mice, salicylate was detected in plasma and skin at 4 hours and PGE2 levels were reduced up to 24 hours. Some human melanoma tumors xenografted into NOD/SCID mice were sensitive to chronic daily ASA administration, exhibiting reduced growth and proliferation. ASA-treated mice bearing sensitive and resistant tumors exhibited both decreased PGE2 in plasma and tumors and increased phosphorylated AMPK in tumors. We conclude that ASA inhibits colony formation, cell motility, and pigmentation through suppression of PGE2 and activation of AMPK and reduces growth of some melanoma tumors in vivo This preclinical model could be used for further tumor and biomarker studies to support future melanoma chemoprevention trials in humans. Cancer Prev Res; 11(10); 629-42. ©2018 AACR.

Targeting the Warburg effect via LDHA inhibition engages ATF4 signaling for cancer cell survival.

Nutrient restriction reprograms cellular signaling and metabolic network to shape cancer phenotype. Lactate dehydrogenase A (LDHA) has a key role in aerobic glycolysis (the Warburg effect) through regeneration of the electron acceptor NAD+ and is widely regarded as a desirable target for cancer therapeutics. However, the mechanisms of cellular response and adaptation to LDHA inhibition remain largely unknown. Here, we show that LDHA activity supports serine and aspartate biosynthesis. Surprisingly, however, LDHA inhibition fails to impact human melanoma cell proliferation, survival, or tumor growth. Reduced intracellular serine and aspartate following LDHA inhibition engage GCN2-ATF4 signaling to initiate an expansive pro-survival response. This includes the upregulation of glutamine transporter SLC1A5 and glutamine uptake, with concomitant build-up of essential amino acids, and mTORC1 activation, to ameliorate the effects of LDHA inhibition. Tumors with low LDHA expression and melanoma patients acquiring resistance to MAPK signaling inhibitors, which target the Warburg effect, exhibit altered metabolic gene expression reminiscent of the ATF4-mediated survival signaling. ATF4-controlled survival mechanisms conferring synthetic vulnerability to the approaches targeting the Warburg effect offer efficacious therapeutic strategies.

Molecular properties of gp100-reactive T-cell receptors drive the cytokine profile and antitumor efficacy of transgenic host T cells.

To study the contribution of T-cell receptors (TCR) to resulting T-cell responses, we studied three different human αβ TCRs, reactive to the same gp100-derived peptide presented in the context of HLA-A*0201. When expressed in primary CD8 T cells, all receptors elicited classic antigen-induced IFN-γ responses, which correlated with TCR affinity for peptide-MHC in the order T4H2>R6C12>SILv44. However, SILv44 elicited superior IL-17A release. Importantly, in vivo, SILv44-transgenic T cells mediated superior antitumor responses to 888-A2+human melanoma tumor cells upon adoptive transfer into tumor-challenged mice while maintaining IL-17 expression. Modeling of the TCR ternary complexes suggested architectural differences between SILv44 and the other complexes, providing a potential structural basis for the observed differences. Overall, the data reveal a more prominent role for the T-cell receptor in defining host T-cell physiology than traditionally assumed, while parameters beyond IFN-γ secretion and TCR affinity ultimately determine the reactivity of tumor-reactive T cells.

Oncolytic Tanapoxvirus Expressing Interleukin-2 is Capable of Inducing the Regression of Human Melanoma Tumors in the Absence of T Cells.

Oncolytic viruses (OVs), which preferentially infect cancer cells and induce host anti-tumor immune responses, have emerged as an effective melanoma therapy. Tanapoxvirus (TANV), which possesses a large genome and causes mild self-limiting disease in humans, is potentially an ideal OV candidate. Interleukin-2 (IL-2), a T-cell growth factor, plays a critical role in activating T cells, natural killer (NK) cells and macrophages in both the innate and adaptive immune system. We aimed to develop a recombinant TANV expressing mouse IL-2 (TANVΔ66R/mIL- 2), replacing the viral thymidine kinase (TK) gene (66R) with the mouse (m) mIL-2 transgene resulting in TANVΔ66R/mIL-2. Human melanoma tumors were induced in female athymic nude mice by injecting SKMEL- 3 cells subcutaneously. Mice were treated with an intratumoral injection of viruses when the tumor volumes reached 45 ± 4.5 mm3. In cell culture, expression of IL-2 attenuated virus replication of not only TANVΔ66R/ mIL-2, but also TANVGFP. It was demonstrated that IL-2 inhibited virus replication through intracellular components and without activating the interferon-signaling pathway. Introduction of mIL-2 into TANV remarkably increased its anti-tumor activity, resulting in a more significant regression than with wild-type (wt) TANV and TANVΔ66R. Histopathological studies showed that extensive cell degeneration with a significantly increased peri-tumor accumulation of mononuclear cells in the tumors treated with TANVΔ66R/mIL-2, compared to wtTANV or TANVΔ66R. We conclude that TANVΔ66R/mIL-2 is potentially therapeutic for human melanomas in the absence of T cells, and IL-2 expression resulted in an overall increase of therapeutic efficacy.

FBXW7 regulates a mitochondrial transcription program by modulating MITF.

FBXW7 is well characterized as a tumor suppressor in many human cancers including melanoma; however, the mechanisms of tumor-suppressive function have not been fully elucidated. We leveraged two distinct RNA sequencing datasets: human melanoma cell lines (n=10) with control versus silenced FBXW7 and a cohort of human melanoma tumor samples (n=51) to define the transcriptomic fingerprint regulated by FBXW7. Here, we report that loss of FBXW7 enhances a mitochondrial gene transcriptional program that is dependent on MITF in human melanoma and confers poor patient outcomes. MITF is a lineage-specific master regulator of melanocytes and together with PGC-1alpha is a marker for melanoma subtypes with dependence for mitochondrial oxidative metabolism. We found that inactivation of FBXW7 elevates MITF protein levels in melanoma cells. In vitro studies examining loss of FBXW7 and MITF alone or in combination showed that FBXW7 is an upstream regulator for the MITF/PGC-1 signaling.

BRAF Inhibitors and Radiation Do Not Act Synergistically to Inhibit WT and V600E BRAF Human Melanoma.

Recent evidence suggests that melanoma patients treated with BRAF inhibitors experience radiosensitization with an increased frequency of side-effects. This could also imply increased effectiveness when treating melanoma. To test whether the BRAF inhibitors dabrafenib and vemurafenib together with ionizing radiation more effectively inhibit melanoma cells, primary human melanoma tumor cell lines expressing wild-type (WT) or mutant V600E BRAF were analyzed by cell survival, cell death, and cell-cycle testing. All melanoma cell lines examined were radioresistant in these assays. BRAF inhibitor treatment alone suppressed cell survival more effectively than radiation in all the mutant V600E BRAF cell lines, and vemurafenib, but not dabrafenib, also inhibited cell survival in the WT BRAF cell lines at clinically relevant concentrations. However, when cells were treated with BRAF inhibitor followed by radiation, there was no increased effect on the suppression of cell survival. Vemurafenib induced more necrosis than radiation in most melanoma cell lines, irrespective of BRAF status, but this effect was not additive with the combination treatment. BRAF inhibitors and radiation had variable, but independent effects on the induction of cell-cycle arrest. These results suggest that BRAF inhibitors and ionizing radiation do not act synergistically to inhibit the growth of primary human melanoma cells.

Dosimetry Prediction for Clinical Translation of 64Cu-Pembrolizumab ImmunoPET Targeting Human PD-1 Expression

The immune checkpoint programmed death 1 receptor (PD-1) expressed on some tumor-infiltrating lymphocytes, and its ligand (PD-L1) expressed on tumor cells, enable cancers to evade the immune system. Blocking PD-1 with the monoclonal antibody pembrolizumab is a promising immunotherapy strategy. Thus, noninvasively quantifying the presence of PD-1 expression in the tumor microenvironment prior to initiation of immune checkpoint blockade may identify the patients likely to respond to therapy. We have developed a 64Cu-pembrolizumab radiotracer and evaluated human dosimetry. The tracer was utilized to image hPD-1 levels in two subcutaneous mouse models: (a) 293 T/hPD-1 cells xenografted into NOD-scid IL-2Rγnull mice (NSG/293 T/hPD-1) and (b) human peripheral blood mononuclear cells engrafted into NSG bearing A375 human melanoma tumors (hNSG/A375). In each mouse model two cohorts were evaluated (hPD-1 blockade with pembrolizumab [blk] and non-blocked [nblk]), for a total of four groups (n = 3–5/group). The xenograft-to-muscle ratio in the NSG/293 T/hPD-1 model at 24 h was significantly increased in the nblk group (7.0 ± 0.5) compared to the blk group (3.4 ± 0.9), p = 0.01. The radiotracer dosimetry evaluation (PET/CT ROI-based and ex vivo) in the hNSG/A375 model revealed the highest radiation burden to the liver. In summary, we validated the 64Cu-pembrolizumab tracer’s specific hPD-1 receptor targeting and predicted human dosimetry.

Abstract PL02-02: Immunopeptidomics: Accelerating the development of personalized cancer immunotherapy

Abstract Cancer immunotherapy amplifies the inherent capacity of cytotoxic T cells to eliminate tumor cells by recognizing human leukocyte antigen-binding peptides (HLAp) presented specifically on tumors but not on normal cells. Recent data show that recognition of mutated neoantigens plays a key role. Currently, mass spectrometry (MS) is the only unbiased methodology to comprehensively uncover the repertoire of HLAp presented in vivo. Recently, we have developed an in-depth MS-based immunopeptidomics approach combined with exome sequencing analysis to directly identify neoantigens from human melanoma tumors (Bassani-Sternberg et al., Nat Commun 2016). This approach is not feasible for substantial number of patients for whom not enough tissue material is available. So far, the discovery of neoantigens relies mainly on prediction-based interrogation of the "mutanome." In a proof-of-concept study we showed that incorporation of deconvoluted HLAp data in ligand prediction algorithms can improve their accuracy (Bassani-Sternberg and Gfeller, J Immunol 2016). In addition, we assembled a comprehensive HLAp database in term of number of peptides and diversity of HLA-I molecules. We showed that by taking advantage of co-occurring HLA-I alleles, we can rapidly and accurately identify HLA-I binding motifs and map them to their corresponding alleles without any a priori knowledge of HLA-I binding specificity. Our novel and scalable approach uncovers new motifs for several alleles that up to now had no known ligands. HLA-ligand predictors trained on such data substantially improve neoantigen predictions in four melanoma and two lung cancer patients, indicating that unbiased HLAp data are ideal for in silico identification of neoantigens (Bassani-Sternberg et al., PLOS Comp Biol 2017). Furthermore, our immunopeptidomics database revealed hotspots that are sub-sequences of proteins frequently presented. We introduce two immunopeptidomics MS-based features to guide prioritization of neoantigens: the number of peptides matching a protein in our database and the overlap of the predicted wild-type peptide with other peptides in our database. We show as a proof of concept that our immunopeptidomics MS-based features improved neoantigen prioritization by up to 50% (Mueller et al., submitted). Overall, our work shows that, in addition to providing huge training data to improve the HLA binding prediction, immunopeptidomics also captures other aspects of the natural in vivo presentation that significantly improve prediction of clinically relevant neoantigens. Citation Format: Michal Bassani-Sternberg, Chloe Chong, Fabio Marino, HuiSong Pak, David Gfeller, Markus Müller, George Coukos. Immunopeptidomics: Accelerating the development of personalized cancer immunotherapy [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 PL02-02.

Simultaneous enumeration of cancer and immune cell types from bulk tumor gene expression data.

Immune cells infiltrating tumors can have important impact on tumor progression and response to therapy. We present an efficient algorithm to simultaneously estimate the fraction of cancer and immune cell types from bulk tumor gene expression data. Our method integrates novel gene expression profiles from each major non-malignant cell type found in tumors, renormalization based on cell-type-specific mRNA content, and the ability to consider uncharacterized and possibly highly variable cell types. Feasibility is demonstrated by validation with flow cytometry, immunohistochemistry and single-cell RNA-Seq analyses of human melanoma and colorectal tumor specimens. Altogether, our work not only improves accuracy but also broadens the scope of absolute cell fraction predictions from tumor gene expression data, and provides a unique novel experimental benchmark for immunogenomics analyses in cancer research (http://epic.gfellerlab.org).

1662P - Synergistic antitumor effects of OT-101 (trabedersen), a transforming growth factor-beta 2 (TGF-β2) antisense oligonucleotide (ASO) and chemotherapy in preclinical tumor models

Background: Overexpression of TGF-β2 has been implicated in the malignant progression of tumors by inducing immunosuppression, proliferation, angiogenesis and metastasis. OT-101 (Trabedersen) is a phosphorothioate ASO designed to specifically target human TGF-β2 mRNA. Herein, we report the synergizing effect of OT-101 with chemotherapy in multiple human tumor xenograft models for further exploration of clinical combination strategies. Methods: OT-101 was administered as single agent (1-64 mg/kg, qdx3/wk or qdx21) and in combination with Gemcitabine (GEM, 15 mg/kg, qdx2/wk), Dacarbazine (DTIC, 1-10 mg/kg, qdx4/wk) or Paclitaxel (PTX, 10 mg/kg, qdx5) to nude mice (10/subgroup) bearing either (i) orthotopic human L3.6pl pancreatic cancer (PAC), (ii) human metastatic C8161 melanoma, (iii) SC glioblastoma (U87) or (iv) SC ovarian (SKOV-3) tumors. Mice were monitored for adverse effects, body weight loss, tumor size and survival outcome. Lymph node and liver surface and micro-metastases as well as size and weight of the pancreatic tumors were determined. Tumor sections were stained with anti-BrdUrd and CD31 antibodies to determine tumor cell proliferation and vascularization, respectively. Results: OT-101 significantly reduced tumor growth (p = 0.0084), lymph node metastasis (p = 0.023), and tumor angiogenesis (p < 0.0001) versus untreated control in the PAC model. OT-101 demonstrated synergy in tumor growth inhibition and increased survival in human malignant melanoma (C8161, p = 0.038, vs. DTIC alone), glioblastoma (U87, p = 0.001 vs. PTX) and ovarian (SKOV-3, p < 0.05 vs. PTX) cancer models when combined with either DTIC (C8161) or PTX (U87 and SKOV-3). No synergy was observed with GEM (PAC). The combination regimen tested was effective and tolerable. Significant antitumor activity was achieved at HED of 80 mg/m2/day which is well below the optimized clinical dose used for IV infusion of patients at 140 mg/m2/day. Conclusions: The preclinical data laid the groundwork for establishing combination therapies in the clinic. Of interest is the preferential synergy between OT-101 and PTX or DTIC, but not with GEM. Legal entity responsible for the study: Autotelic Inc Funding: None Disclosure: All authors have declared no conflicts of interest.

Paracrine Activin-A Signaling Promotes Melanoma Growth and Metastasis through Immune Evasion

The secreted growth factor Activin-A of the transforming growth factor β family and its receptors can promote or inhibit several cancer hallmarks including tumor cell proliferation and differentiation, vascularization, lymphangiogenesis and inflammation. However, a role in immune evasion and its relationship with tumor-induced muscle wasting and tumor vascularization, and the relative contributions of autocrine versus paracrine Activin signaling remain to be evaluated. To address this, we compared the effects of truncated soluble Activin receptor IIB as a ligand trap, or constitutively active mutant type IB receptor versus secreted Activin-A or the related ligand Nodal in mouse and human melanoma cell lines and tumor grafts. We found that although cell-autonomous receptor activation arrested tumor cell proliferation, Activin-A secretion stimulated melanoma cell dedifferentiation and tumor vascularization by functional blood vessels, and it increased primary and metastatic tumor burden and muscle wasting. Importantly, in mice with impaired adaptive immunity, the tumor-promoting effect of Activin-A was lost despite sustained vascularization and cachexia, suggesting that Activin-A promotes melanoma progression by inhibiting antitumor immunity. Paracrine Activin-A signaling emerges as a potential target for personalized therapies, both to reduce cachexia and to enhance the efficacy of immunotherapies.

Distinct Cellular Mechanisms Underlie Anti-CTLA-4 and Anti-PD-1 Checkpoint Blockade

Immune-checkpoint blockade is able to achieve durable responses in a subset of patients; however, we lack a satisfying comprehension of the underlying mechanisms of anti-CTLA-4- and anti-PD-1-induced tumor rejection. To address these issues, we utilized mass cytometry to comprehensively profile the effects of checkpoint blockade on tumor immune infiltrates in human melanoma and murine tumor models. These analyses reveal a spectrum of tumor-infiltrating Tcell populations that are highly similar between tumor models and indicate that checkpoint blockade targets only specific subsets of tumor-infiltrating Tcell populations. Anti-PD-1 predominantly induces the expansion of specific tumor-infiltrating exhausted-like CD8 Tcell subsets. In contrast, anti-CTLA-4 induces the expansion of an ICOS+ Th1-like CD4 effector population in addition to engaging specific subsets of exhausted-likeCD8 Tcells. Thus, our findings indicate that anti-CTLA-4 and anti-PD-1 checkpoint-blockade-induced immune responses are driven by distinct cellular mechanisms.

CD3xPDL1 bi-specific T cell engager (BiTE) simultaneously activates T cells and NKT cells, kills PDL1+ tumor cells, and extends the survival of tumor-bearing humanized mice.

Bi-specific T cell engagers (BiTEs) activate T cells through CD3 and target activated T cells to tumor-expressed antigens. BiTEs have shown therapeutic efficacy in patients with liquid tumors; however, they do not benefit all patients. Anti-tumor immunity is limited by Programmed Death 1 (PD1) pathway-mediated immune suppression, and patients who do not benefit from existing BiTES may be non-responders because their T cells are anergized via the PD1 pathway. We have designed a BiTE that activates and targets both T cells and NKT cells to PDL1+ cells. In vitro studies demonstrate that the CD3xPDL1 BiTE simultaneously binds to both CD3 and PDL1, and activates healthy donor CD4+ and CD8+ T cells and NKT cells that are specifically cytotoxic for PDL1+ tumor cells. Cancer patients’ PBMC are also activated and cytotoxic, despite the presence of myeloid-derived suppressor cells. The CD3xPDL1 BiTE significantly extends the survival time and maintains activated immune cell levels in humanized NSG mice reconstituted with human PBMC and carrying established human melanoma tumors. These studies suggest that the CD3xPDL1 BiTE may be efficacious for patients with PDL1+ solid tumors, in combination with other immunotherapies that do not specifically neutralize PD1 pathway-mediated immune suppression.

Abstract 5895: Targeting RhoA-regulated gene transcription in drug-resistant melanoma

Abstract Much of the recent focus of melanoma targeted therapy has been on the ERK pathway, which is aberrantly activated in approximately 90% of melanoma tumors. Over half of these express BRAFV600E. Current targeted therapies such as Vemurafenib (BRAFV600E inhibitor), or a combination therapy of BRAF + MEK inhibitors show profound initial effects in a majority of BRAFV600E expressing tumors. However, these responses are often short-lived and resistance typically develops within months. The goal of this work is to identify pharmacologically targetable resistance mechanisms so that effective combination therapies can be developed. Preliminary bioinformatics analysis suggests that the RhoA subfamily of Rho GTPases is activated in BRAFi-resistant cancer cell lines and BRAFi/MEKi-resistant human melanoma tumors. Thus, we hypothesize that RhoA promotes BRAFi resistance, so simultaneously targeting RhoA and BRAF may reverse drug resistance. Using cell line models of acquired BRAFi-resistance we demonstrated that the RhoA pathway is activated in a subset of resistant cell lines. The cell lines with increased RhoA activation have increased sensitivity to multiple ROCK inhibitors. In addition to regulating the cytoskeleton, RhoA can also regulate gene transcription through activation of multiple transcriptional co-activators, including MRTF and YAP. MRTF and YAP regulated gene transcription through their role as transcriptional co-activators and their interaction with chromatin remodeling complexes. MRTF and YAP are both activated in BRAFi-resistant cell lines. Indirectly targeting MRTF with CCG-222740 or YAP with the YES1 inhibitor Dasatinib effectively kills BRAFi-resistant melanoma cells. Sox10 was previously identified as a gene which promotes BRAFi resistance. Expression of Sox10 is downregulated, and Sox9 is upregulated, 100-1000-fold exclusively in the resistant cell lines which have RhoA activation. Further, a Sox10 gene signature is inversely correlated with a RhoA/C signature in the TCGA dataset and melanoma scRNA-seq data. Sox10 loss is a major drug resistance melanoma in melanoma. These data suggest that targeting RhoA-regulated gene transcription may be an effective mechanism to target Sox9High/Sox10Low cells, and ultimately prevent or reverse drug resistance. Taken together these data suggest that MRTF and YAP promote Vemurafenib resistance in Sox9High/Sox10Low melanoma cells. Citation Format: Sean A. Misek, Kathleen A. Gallo, Richard R. Neubig. Targeting RhoA-regulated gene transcription in drug-resistant 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 5895.

Abstract 2800: Transforming growth factor-beta 2 (TGF-β2) antisense oligonucleotide (ASO) OT-101 synergizes with chemotherapy in preclinical tumor models

Abstract Background: Overexpression of TGF-β2 has been implicated in the malignant progression of tumors by inducing immunosuppression, proliferation, angiogenesis and metastasis. Clinical failures of TGF-β inhibitors in targeting the tumor promoting arm of TGF-β signaling is attributed to multiple isoforms and receptor functions. OT-101 (Trabedersen) is a phosphorothioate ASO designed to specifically target human TGF-β2 mRNA with superior clinical activity in patients with TGF-β2-overexpressing tumors. Herein, we report the synergizing effect of OT-101 with chemotherapy in multiple human tumor xenograft models for further exploration of clinical combinations. Methods: The in vivo efficacy studies of intraperitoneal (IP, 16 or 50 mg/kg) or subcutanous (SC, 1-64 mg/kg) repeated administration (qdx3/wk or qdx21) of OT-101 as single agent and in combination with Gemcitabine (15 mg/kg, qdx2/wk, IP), Dacarbazine (1-10 mg/kg, qdx4/wk, IP) or Paclitaxel (10 mg/kg, qdx5, IV) were evaluated in nude mice (10/subgroup) bearing either (i) orthotopic human L3.6pl pancreatic cancer (PAC), (ii) human metastatic C8161 melanoma, (iii) subcutaneous glioblastoma (U87-MG) or (iv) subcutaneous ovarian (SK-OV-3) tumors. Mice were monitored thrice a week for adverse effects, body weight loss, tumor size and survival outcome. The incidence of lymph node and liver surface and micro-metastases as well as size and weight of the pancreatic tumors were determined. Tumor sections were stained with anti-5-bromo-2'-deoxy-uridine antibody to determine tumor cell proliferation and with anti-CD31/PECAM-1 antibody to determine vascularization. Results: OT-101 significantly reduced tumor growth (p = 0.0084), lymph node metastasis (p = 0.023), and tumor angiogenesis (p &amp;lt;0.0001) in the orthotopic PAC model. Mean tumor vessel density was significantly reduced (p &amp;lt;0.0001) in all groups in comparison to untreated control. OT-101 demonstrated synergy in tumor growth inhibition and increased survival in human malignant melanoma (C8161, p = 0.038, vs. Dacarbazine alone), glioblastoma (U87-MG, p = 0.001 vs. Paclitaxel) and ovarian (SKOV-3, p &amp;lt;0.05 vs. Paclitaxel) cancer models when combined with either Dacarbazine (C8161) or Paclitaxel (U87-MG and SK-OV-3). No syngergy was observed with Gemcitabine (PAC). The combination regimen tested was effective and tolerable. Significant antitumor activity was achieved at human dose equivalent of 80 mg/m2/day which is well below the optimized clinical dose used for IV infusion of patients at 140 mg/m2/day. Conclusions: The preclinical data lay the groundwork for further exploration of combination therapy in the clinic as well as demonstrating that TGF-β2 is a druggable target. Of interest is the preferential synergy between OT-101 and Paclitaxel or Dacarbazine, but not with Gemcitabine. Citation Format: Osmond D'Cruz, Cynthia Lee, Vuong Trieu, Larn Hwang. Transforming growth factor-beta 2 (TGF-β2) antisense oligonucleotide (ASO) OT-101 synergizes with chemotherapy in preclinical tumor models [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 2800. doi:10.1158/1538-7445.AM2017-2800

NKG2D Ligand-Targeted Bispecific T-Cell Engagers Lead to Robust Antitumor Activity against Diverse Human Tumors.

Two new bispecific T-cell engaging (BiTE) molecules with specificity for NKG2D ligands were developed and functionally characterized. One, huNKG2D-OKT3, was derived from the extracellular portion of the human NKG2D receptor fused to a CD3ε binding single-chain variable fragment (scFv), known as OKT3. NKG2D has multiple ligands, including MICA, which are expressed by a variety of malignant cells. A second molecule, B2-OKT3, was created in the tandem scFv BiTE format that targets MICA on tumor cells and CD3ε on human T cells. Both BiTEs specifically activated T cells to kill human tumor cell lines. Cytotoxicity by B2-OKT3, but not huNKG2D-OKT3, is blocked by soluble rMICA. The huNKG2D-OKT3 induced greater T-cell cytokine production in comparison with B2-OKT3. No T-cell pretreatment was required for IFNγ production upon coculture of B2-OKT3 or huNKG2D-OKT3 with T cells and target cells. The effector memory T-cell compartment was the primary source of IFNγ, and culture of T cells and these BiTEs with plate-bound rMICA showed ligand density-dependent production of IFNγ from both CD4+ and CD8+ T cells. There was 2-fold more IFNγ produced per CD8+ T cell and 5-fold greater percentage of CD8+ T cells producing IFNγ compared with CD4+ T cells. In addition, both BiTEs elicited significant antitumor responses against human metastatic melanoma tumor samples using autologous or healthy donor T cells. These data demonstrate the robust antitumor activity of these NKG2D ligand-binding bispecific proteins and support their further development for clinical use. Mol Cancer Ther; 16(7); 1335-46. ©2017 AACR.

Shifting the Balance of Activating and Inhibitory Natural Killer Receptor Ligands on BRAFV600E Melanoma Lines with Vemurafenib.

Over 60% of human melanoma tumors bear a mutation in the BRAF gene. The most frequent mutation is a substitution at codon 600 (V600E), leading to a constitutively active BRAF and overactivation of the MAPK pathway. Patients harboring mutated BRAF respond to kinase inhibitors such as vemurafenib. However, these responses are transient, and relapses are frequent. Melanoma cells are efficiently lysed by activated natural killer (NK) cells. Melanoma cells express several stress-induced ligands that are recognized by activating NK-cell receptors. We have investigated the effect of vemurafenib on the immunogenicity of seven BRAF-mutated melanoma cells to NK cells and on their growth and sensitivity to NK-cell-mediated lysis. We showed that vemurafenib treatment modulated expression of ligands for two activating NK receptors, increasing expression of B7-H6, a ligand for NKp30, and decreasing expression of MICA and ULBP2, ligands for NKG2D. Vemurafenib also increased expression of HLA class I and HLA-E molecules, likely leading to higher engagement of inhibitory receptors (KIRs and NKG2A, respectively), and decreased lysis of vemurafenib-treated melanoma cell lines by cytokine-activated NK cells. Finally, we showed that whereas batimastat (a broad-spectrum matrix metalloprotease inhibitor) increased cell surface ULBP2 by reducing its shedding, vemurafenib lowered soluble ULBP2, indicating that BRAF signal inhibition diminished expression of both cell-surface and soluble forms of NKG2D ligands. Vemurafenib, inhibiting BRAF signaling, shifted the balance of activatory and inhibitory NK ligands on melanoma cells and displayed immunoregulatory effects on NK-cell functional activities. Cancer Immunol Res; 5(7); 582-93. ©2017 AACR.