Patient-derived Melanoma Cells Research Articles

Abstract IA03: The Hippo pathway in melanoma

Abstract Melanoma is usually driven by mutations in BRAF or NRAS that trigger hyperactivation of mitogen-activated protein kinase (MAPK) signaling. However, MAPK targeted therapies are not sustainably effective in most patients. Accordingly, characterizing mechanisms that cooperatively drive melanoma progression is key to improving patient outcomes. One possible mechanism is the Hippo signaling pathway, which regulates cancer progression via its central oncoproteins YAP and TAZ, although it is relatively rarely affected by direct mutation. As YAP hyperactivation occurs in uveal melanoma, we investigated this oncogene in cutaneous melanoma. YAP protein expression was elevated in most benign nevi and primary cutaneous melanomas but present at only very low levels in normal melanocytes. In patient-derived xenografts and melanoma cell lines, we observed variable reliance of cell viability on Hippo pathway signaling that was independent of classical melanoma driver mutations such as BRAF and NRAS. YAP activity was enriched in invasive melanoma cell lines and was necessary for their invasive properties in culture. In in vivo studies, we found that expression of hyperactive YAP induced spontaneous melanoma metastasis in mice. Finally, in genotyping studies of melanoma, we observed the first ever hyperactivating YAP mutations in a human cancer, manifest as seven distinct missense point mutations that caused serine to alanine transpositions. Strikingly, these mutate four of five known serine residues targeted by the Hippo pathway, and we show that they lead to hyperactivation of YAP function. Our studies highlight the YAP oncoprotein as a potential therapeutic target in select subgroups of melanoma patients, although successful treatment with anti-YAP therapies will depend on identification of biomarkers other than YAP protein expression. Citation Format: Xiaomeng Zhang, Lie Yang, Ismael Vergara, Pacman Szeto, Youfang Zhang, Anthony Papenfuss, Mark Shackleton, Kieran Harvey. The Hippo pathway in melanoma [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA03.

Insulin Reduces the Efficacy of Vemurafenib and Trametinib in Melanoma Cells.

BackgroundDespite the progress made in the clinical management of metastatic melanoma, a patient’s response to treatment cannot be fully predicted, and intrinsic or acquired resistance that is developed in most melanoma patients warrants further research efforts. In addition to genetic factors, microenvironmental input should be considered to explain the diversity of response to treatment among melanoma patients. In this study, we evaluated the impact of insulin on patient-derived BRAFV600E melanoma cells, either untreated or treated with vemurafenib or trametinib, inhibitors of BRAFV600 and MEK1/2, respectively.MethodsCells were cultured in serum-free conditions, either with or without insulin. The activity of the MAPK/ERK and PI3K/AKT pathways was assessed by Western blotting, cell viability, and percentages of Ki-67- and NGFR-positive cells by flow cytometry. Transcript levels were analyzed using qRT-PCR, and γ-H2AX levels by immunoblotting and confocal microscopy. A luminescence-based assay was used to measure glutathione content.ResultsWhile insulin did not influence the MAPK/ERK pathway activity, it had a strong influence on melanoma cells, in which this pathway was suppressed by either vemurafenib or trametinib. In the presence of insulin, both drugs were much less efficient in 1) inhibiting proliferation and reducing the percentage of Ki-67-positive cells, and 2) inducing apoptosis and phosphorylation of histone H2AX in melanoma cells. Changes induced by vemurafenib and trametinib in glutathione homeostasis and DNA repair gene expression were also attenuated by insulin. Moreover, insulin impaired the combined effects of targeted drugs and doxorubicin in melanoma cells. In addition to insulin-induced PI3K/AKT activity, which was either transient or sustainable depending on the cell line, an insulin-triggered increase in the percentage of cells expressing NGFR, a marker of neural crest stem-like cells, may contribute to the reduced drug efficacy.ConclusionOur results demonstrate the role of insulin in reducing the efficacy of vemurafenib and trametinib. This needs clinical assessment.

Targeting the innate immunoreceptor RIG-I overcomes melanoma-intrinsic resistance to T cell immunotherapy.

Understanding tumor resistance to T cell immunotherapies is critical to improve patient outcomes. Our study revealed a role for transcriptional suppression of the tumor-intrinsic HLA class I (HLA-I) antigen processing and presentation machinery (APM) in therapy resistance. Low HLA-I APM mRNA levels in melanoma metastases before immune checkpoint blockade (ICB) correlated with nonresponsiveness to therapy and poor clinical outcome. Patient-derived melanoma cells with silenced HLA-I APM escaped recognition by autologous CD8+ T cells. However, targeted activation of the innate immunoreceptor RIG-I initiated de novo HLA-I APM transcription, thereby overcoming T cell resistance. Antigen presentation was restored in interferon-sensitive (IFN-sensitive) but also immunoedited IFN-resistant melanoma models through RIG-I-dependent stimulation of an IFN-independent salvage pathway involving IRF1 and IRF3. Likewise, enhanced HLA-I APM expression was detected in RIG-Ihi (DDX58hi) melanoma biopsies, correlating with improved patient survival. Induction of HLA-I APM by RIG-I synergized with antibodies blocking PD-1 and TIGIT inhibitory checkpoints in boosting the antitumor T cell activity of ICB nonresponders. Overall, the herein-identified IFN-independent effect of RIG-I on tumor antigen presentation and T cell recognition proposes innate immunoreceptor targeting as a strategy to overcome intrinsic T cell resistance of IFN-sensitive and IFN-resistant melanomas and improve clinical outcomes in immunotherapy.

17-Aminogeldanamycin Inhibits Constitutive Nuclear Factor-Kappa B (NF-κB) Activity in Patient-Derived Melanoma Cell Lines.

Melanoma remains incurable skin cancer, and targeting heat shock protein 90 (HSP90) is a promising therapeutic approach. In this study, we investigate the effect of 17-aminogeldanamycin, a potent HSP90 inhibitor, on nuclear factor-kappa B (NF-κB) activity in BRAFV600E and NRASQ61R patient-derived melanoma cell lines. We performed time-lapse microscopy and flow cytometry to monitor changes in cell confluence and viability. The NF-κB activity was determined by immunodetection of phospho-p65 and assessment of expression of NF-κB-dependent genes by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Constitutive activity of p65/NF-κB was evident in all melanoma cell lines. Differences in its level might be associated with genetic alterations in CHUK, IL1B, MAP3K14, NFKBIE, RIPK1, and TLR4, while differences in transcript levels of NF-κB-inducible genes revealed by PCR array might result from the contribution of other regulatory mechanisms. 17-Aminogeldanamycin markedly diminished the level of phospho-p65, but the total p65 protein level was unaltered, indicating that 17-aminogeldanamycin inhibited activation of p65/NF-κB. This conclusion was supported by significantly reduced expression of selected NF-κB-dependent genes: cyclin D1 (CCND1), C-X-C motif chemokine ligand 8 (CXCL8), and vascular endothelial growth factor (VEGF), as shown at transcript and protein levels, as well as secretion of IL-8 and VEGF. Our study indicates that 17-aminogeldanamycin can be used for efficient inhibition of NF-κB activity and the simultaneous diminution of IL-8 and VEGF levels in the extracellular milieu of melanoma.

Preclinical development of a novel, highly selective PI3Kδ inhibitor, IOA-244, for the treatment of solid malignancies

Abstract Background Inhibiting PI3Kδ preferentially targets regulatory T cells and myeloid derived suppressor cells, breaking tumour-induced immune tolerance and restoring anti-tumour immunity. Bioinformatics and protein expression studies have shown that PIK3CD / PI3Kδ is highly expressed in certain solid malignancies, and therefore inhibition of PI3Kδ may modulate tumour growth and development via intrinsic as well as immune driven effects. Methods In vitro proliferation of primary human T cells demonstrated the selective effects of PI3Kδ inhibition on Treg cells. In vivo immune mediated effects of IOA-244 were tested using the CT26 mouse model. High PI3Kδ expressing MDA-MB-231 breast cancer cells, as well as a patient derived melanoma model with high PIK3CD profile were tested in T cell deficient hosts. Results IOA-244 is a novel, highly selective PI3Kδ inhibitor, with the unique property of being ATP non-competitive. IOA-244 showed a selective and concentration-dependent suppression of human Treg cell proliferation whilst leaving CD8+ T cell proliferation intact. In mouse models with a full immune system, IOA-244 inhibited tumour growth when combined with either anti-PD-1 or anti-PD-L1. Furthermore, the composition of the tumour infiltrating lymphocytes showed a marked decrease in the suppressor cell populations, i.e. Tregs and MDSCs. In nude mice we demonstrated that IOA-244 can effectively inhibit the growth of PI3Kδ-expressing breast cancer cells as a monotherapy. To support the clinical development of IOA-244 we demonstrate that patient derived melanoma cells with a high expression of PIK3CD are susceptible to treatment with IOA-244 in a mouse patient-derived xenograft model. Conclusions Solid tumours with a high Treg:CD8 ratio and / or a high protein expression of PI3Kδ may be amenable to treatment with IOA-244. Based on these and other supporting data, IOA-244 has received clinical trial approval in two European countries and phase I clinical testing is expected to start by the end of 2020. Legal entity responsible for the study iOnctura SA. Funding iOnctura SA. Disclosure Z. Johnson: Leadership role: iOnctura SA. L. Van der Veen: Leadership role: iOnctura SA. M. Lahn: Leadership role: iOnctura SA. All other authors have declared no conflicts of interest.

Inhibition of p38/MK2 Signaling Prevents Vascular Invasion of Melanoma

Melanoma cells can switch between distinct gene expression profiles, resulting in proliferative or invasive phenotypes. Signaling pathways involved in this switch were analyzed by gene expression profiling of a cohort of 22 patient-derived melanoma cell lines. CDH1 negativity was identified as a surrogate marker for the invasive phenotype. CDH1 expression could be turned on and off by modulating activity of p38 or its downstream target MK2, suggesting that this pathway controls melanoma progression. Mechanistically, MK2 inhibition prevented melanoma-induced vascular barrier disruption, reduced the expression of PODXL and DEL-1, and prevented vascular dissemination invivo. PODXL and DEL-1 expression in patients with melanoma were associated with poor survival and thus can be used as prognostic markers. Downstream targets of MK2 may thus serve as candidate therapeutics.

Physiologically Relevant Oxygen Concentration (6% O2) as an Important Component of the Microenvironment Impacting Melanoma Phenotype and Melanoma Response to Targeted Therapeutics In Vitro.

Cancer cell phenotype largely depends on oxygen availability. The atmospheric oxygen concentration (21%) used in in vitro studies is much higher than in any human tissue. Using well-characterized patient-derived melanoma cell lines, we compared: (i) activities of several signaling pathways, and (ii) the effects of vemurafenib and trametinib in hyperoxia (21% O2), normoxia (6% O2) and hypoxia (1% O2). A high plasticity of melanoma cells in response to changes in oxygen supplementation and drug treatment was observed, and the transcriptional reprograming and phenotypic changes varied between cell lines. Normoxia enhanced the expression of vascular endothelial growth factor (VEGF), glucose metabolism/transport-related genes, and changed percentages of NGFR- and MITF-positive cells in cell line-dependent manner. Increased protein stability might be responsible for high PGC1α level in MITFlow melanoma cells. Vemurafenib and trametinib while targeting the activity of MAPK/ERK pathway irrespective of oxygen concentration, were less effective in normoxia than hyperoxia in reducing levels of VEGF, PGC1α, SLC7A11 and Ki-67-positive cells in cell line-dependent manner. In conclusion, in vitro studies performed in atmospheric oxygen concentration provide different information on melanoma cell phenotype and response to drugs than performed in normoxia, which might partially explain the discrepancies between results obtained in vitro and in clinical settings.

Plasticity of Drug-Naïve and Vemurafenib- or Trametinib-Resistant Melanoma Cells in Execution of Differentiation/Pigmentation Program.

Melanoma plasticity creates a plethora of opportunities for cancer cells to escape treatment. Thus, therapies must target all cancer cell subpopulations bearing the potential to contribute to disease. The role of the differentiation/pigmentation program in intrinsic and acquired drug resistance is largely uncharacterized. MITF level and expression of MITF-dependent pigmentation-related genes, MLANA, PMEL, TYR, and DCT, in drug-naïve and vemurafenib- or trametinib-treated patient-derived melanoma cell lines and their drug-resistant counterparts were analysed and referred to genomic alterations. Variability in execution of pigmentation/differentiation program was detected in patient-derived melanoma cell lines. Acute treatment with vemurafenib or trametinib enhanced expression of pigmentation-related genes in MITF-Mhigh melanoma cells, partially as the consequence of transcriptional reprograming. During development of resistance, changes in pigmentation program were not unidirectional, but also not universal as expression of different pigmentation-related genes was diversely affected. In selected resistant cell lines, differentiation/pigmentation was promoted and might be considered as one of drug-tolerant phenotypes. In other resistant lines, dedifferentiation was induced. Upon drug withdrawal (“drug holiday”), the dedifferentiation process in resistant cells either was enhanced but reversed by drug reexposure suggesting involvement of epigenetic mechanisms or was irreversible. The irreversible dedifferentiation might be connected with homozygous loss-of-function mutation in MC1R, as MC1RR151C +/+ variant was found exclusively in drug-naïve MITF-Mlow dedifferentiated cells and drug-resistant cells derived from MITFhigh/MC1RWT cells undergoing irreversible dedifferentiation. MC1RR151C +/+ variant might be further investigated as a parameter potentially impacting melanoma patient stratification and aiding in treatment decision.

Melanoma-Derived Extracellular Vesicles Bear the Potential for the Induction of Antigen-Specific Tolerance.

Background: Cancer-induced immunosuppression is antigen-specific rather than systemic and the mechanisms for the antigen specificity are incompletely understood. Here we explore the option that tumor-associated antigens (TAAs) may be transferred to antigen-presenting cells (APCs), together with immunosuppressive molecules, through cancer-derived small extracellular vesicles (sEVs), such as exosomes. Stimulation of a suppressive phenotype in the very same APCs that take up TAAs may yield antigen-specific tolerance. Methods: sEVs isolated from patient-derived or well-established melanoma cell lines were used to demonstrate the transfer of major histocompatibility complex (MHC) molecules to the surface of APCs. The immunosuppressive influence of sEVs was assessed by flow cytometry analysis of activation markers, cytokine expression, and mixed lymphocyte reactions. Results: MHC class I molecules were transferred from melanoma cells to the cell surface of APCs by sEVs. Concomitantly, CD86 and CD40 co-stimulatory molecules were down-regulated and IL-6 production was strongly induced. TGF-β transported by sEVs contributed to the promotion of a suppressive phenotype of APCs. Conclusion: The presented results indicate the existence of a hitherto undescribed mechanism that offers an explanation for antigen-specific tolerance induction mediated by cancer-derived sEVs.

Abstract 3946: Tumor checkpoint inhibitor profiling for an optimal clinical response

Abstract The last three decades have seen a steady rise in melanoma, the most aggressive form of skin cancer. The discovery of checkpoint inhibitors has revolutionized the treatment landscape for melanoma; anti-CTLA4 and anti-PD1 showing great successes in clinic. While checkpoint molecules and co-stimulatory molecules are traditionally present on cells involved in immune activation, mounting evidence suggests that tumor cells also express these molecules. Our laboratory has characterized five patient-derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM, and GLM-2 and screened them for the expression of a comprehensive list of 29 co-stimulatory and co-inhibitory molecules compared to normal adult melanocytes. We see a differential mRNA expression of many of these immune-regulatory molecules, including BTLA, HVEM, CD160, CD226 and TIM1. Western blots and immunofluorescence confirmed the presence of these molecules at the protein level. A flow cytometry analysis demonstrated that BTLA, HVEM, CD160, TIM1 and CD226 are present on the membrane of these patient derived melanoma cells; implying that they are capable of engaging their respective ligands and exerting a functional role in immunomodulation. These findings were additionally validated in melanoma tissues, obtained from patients, by immunohistochemistry. Interestingly, treatment of MEL-2, MEL-V, KFM and GLM-2, our BRAFV600E containing patient derived cells, with BRAFV600E inhibitor PLX4032 led to the upregulation of these molecules. This upregulation was coupled with an increase in transcription factor MITF, a binding site for which is present in the promoter region of all the upregulated molecules. Co-culture experiments with immune cells, demonstrate a modulatory role played by the HVEM/BTLA/CD160 axis molecules that are present on tumor cells. The presence of these additional immune-regulatory molecules signify the existence of a robust tolerance mechanism induced by these checkpoint inhibitors. Our studies underscore the importance of characterizing tumor profiles to enable the selection of an optimal treatment regimen combining targeted small molecule inhibitors, and checkpoint inhibitors to maximize T cell killing, and tumor clearance. Citation Format: Rachana R. Maniyar, Sanjukta Chakraborty, Ghada Ben Rahoma, John J. Degliuomini, Marc Wallack, Jan Geliebter, Raj K. Tiwari. Tumor checkpoint inhibitor profiling for an optimal clinical response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3946.

Somatic Hypermutation of the YAP Oncogene in a Human Cutaneous Melanoma.

Melanoma is usually driven by mutations in BRAF or NRAS, which trigger hyperactivation of MAPK signaling. However, MAPK-targeted therapies are not sustainably effective in most patients. Accordingly, characterizing mechanisms that co-operatively drive melanoma progression is key to improving patient outcomes. One possible mechanism is the Hippo signaling pathway, which regulates cancer progression via its central oncoproteins YAP and TAZ, although is thought to be only rarely affected by direct mutation. As YAP hyperactivation occurs in uveal melanoma, we investigated this oncogene in cutaneous melanoma. YAP protein expression was elevated in most benign nevi and primary cutaneous melanomas but present at only very low levels in normal melanocytes. In patient-derived xenografts and melanoma cell lines, we observed variable reliance of cell viability on Hippo pathway signaling that was independent of TAZ activity and also of classical melanoma driver mutations such as BRAF and NRAS. Finally, in genotyping studies of melanoma, we observed the first ever hyperactivating YAP mutations in a human cancer, manifest as seven distinct missense point mutations that caused serine to alanine transpositions. Strikingly, these mutate four serine residues known to be targeted by the Hippo pathway and we show that they lead to hyperactivation of YAP. IMPLICATIONS: Our studies highlight the YAP oncoprotein as a potential therapeutic target in select subgroups of melanoma patients, although successful treatment with anti-YAP therapies will depend on identification of biomarkers additional to YAP protein expression.

Abstract 826: Global DNA methylation levels regulate PD-L1 expression in melanoma

Abstract The programmed death-ligand 1 (PD-L1) receptor is an important immune checkpoint and is often upregulated in cancer cells to allow immune evasion. In melanoma, the patients with PD-L1 expression and absense of tumour infiltrating lymphocytes (TILs) (i.e. “constitutive PD-L1 or PD-L1CON”) show worse response rates and prognosis than patients with PD-L1 expression and the presence of TILs (i.e. “inducible PD-L1 or PD-L1IND”). However, how PD-L1 expression is regulated in melanoma cells remains elusive. Understanding the mechanisms of how PD-L1 is regulated is important for predicting responses for anti-PD-L1 treatment and for developing new combinatorial therapies. We hypothesised that epigenetic state regulates constitutive and inducible PD-L1 expression in melanoma. To address the hypothesis, we have generated whole-genome scale DNA methylomes (using reduced representation bisulfite sequencing) and transcriptomes (RNA-Seq) for patient derived melanoma cell lines (in PD-L1IND and PD-L1CON groups). We discovered extensive global hypomethylation in the constitutive lines, particularly pronounced in intergenic repeat regions and gene bodies. A high proportion of hypomethylated regions exhibited dichotomous methylation patterns indicating a common regulatory mechanism between the PD-L1IND and PD-L1CON lines. RNA-Sequening data indicated that the hypomethylated state of the PD-L1CON cells was correlated with higher upregulation of the differentially expressed genes at a global-scale and the upregulated genes were associated with cancer hallmark properties. The upregulated genes exhibited signatures of viral mimicry and cytosolic sensing of dsRNA similar to what has been observed after DNA methyltransferase inhibitor (DNMTi) treatment in cancers. DNMTi treatment increased PD-L1 transcription in the PD-L1IND cell lines. PD-L1CON samples showed greater resistance to DNMTi compared to PD-L1 negative samples. This was observed at protein-coding genes, transposable elements and long non-coding RNAs. Moreover, genes involved in the innate immune pathway was strongly increased upon DNMTi mediated hypomethylation in the PD-L1IND cell lines (FDR-adjusted P-value = 0.02), however there was no expression change in the PD-L1 positive cells (Adjusted P-value = 1.0). Furthermore, we found that the IRF1 transcription factor, which plays a critical role in the innate immune pathway and also binds to the PD-L1 promoter, is significantly upregulated upon DNMTi in the PD-L1 negative cell lines (Adjusted P-value = 0.01) but not in the PD-L1 positive cells (Adjusted P-value = 0.9). These results show that global hypomethylation levels regulate PD-L1 expression in melanoma. We believe these results are the first to show that DNA methylation levels play a role in regulating PD-L1 on melanoma and suggest they may have important implications for combined treatments targeting methylation (DNMTi) and PD1/PD-L1 (anti-PD1 antibodies). Citation Format: Aniruddha Chatterjee, Antonio Ahn, Euan J. Rodger, Peter A. Stockwell, Matthew Parry, Jyoti Motwani, Stuart J. Gallagher, Elena Shklovskaya, Jessamy Tiffen, Peter Hersey, Michael R. Eccles. Global DNA methylation levels regulate PD-L1 expression in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 826.

Abstract SY45-04: Development of therapeutic strategies by resolving the tumor ecosystem

Abstract Solid tumors are complex ecosystem with inherent genetic and phenotypic variability, and interactions among cancer cells with the tumor microenvironment that shape the development, progression and the response or resistance to targeted- and immunotherapies. This heterogeneity is poorly captured with profiling of bulk tumor tissues that provides an average of these signals without recapitulating cellular interactions and phenotypes. Single-cell genomics technologies, such as single-cell RNA-sequencing (scRNA-seq), represent powerful tools to resolve tumor composition and dissect interactions within the ecosystem that may determine drug resistance and reveal vulnerabilities that can be therapeutically targeted. Using scRNA-seq we profiled thousands of malignant and non-malignant cells from patients with metastatic melanoma. We found that all tumors, irrespective of prior drug exposures, contained cells expressing a cell state characterized by high expression of AXL and low expression of MITF, the master regulator of the melanocytic lineage. The AXL-high/MITF-low cell state conferred intrinsic resistance to RAF/MEK inhibitor therapy in BRAF-mutated melanoma models, and led to the emergence of a small number of pre-existing AXL-high/MITF-low cells with treatment. Interrogation of tumor-infiltrating lymphocytes (TILs) provided insights into their diversity and expression of various immune checkpoints, such as PD-1, CTLA-4, LAG3, TIGIT and others, indicating their co-expression on CD8 and CD4 cells. While these markers indicated dysfunction of the T cells, we found concurrent expression of activating markers, including key cytokines such as interferon gamma. We dissected the relationship between activation, dysfunction, and clonality in these cells, and identified markers that saliently characterized the dysfunction state across different patients. Several complement factors, including C3, were predicted to modulate the infiltration and exclusion of T cells from the TME, and we validated the relationship of these in independent melanoma patient tissue sections. We performed scRNA-seq on additional melanoma patients to assemble a total of 31 samples, of which 15 were isolated from patients with resistance to immune checkpoint inhibitors (ICI) (anti-PD-1 or anti-CTLA4 therapy or a combination of both). We performed a genome-scale inference of cancer cell-mediated drivers of T cell exclusion, a major mechanisms of ICI resistance. Next, we directly measured cell-intrinsic expression of pathways associated with immune evasion. Strikingly, the same cell state mediated both, T cell exclusion and immune evasion, indicating that one coherently regulated cancer cell program could be responsible for ICI resistance in patients. We validated the spatial impact of this resistance program by multiplexed immunofluorescence of immune infiltrates for predicted drivers of T cell exclusion in matching formalin-fixed, paraffin-embedded (FFPE) patient specimens. The signature was prognostic for survival in the melanoma TCGA cohort, indicating that the program can be intrinsically expressed. To test the predictive value of this signature, we compiled two validation cohorts, including one with 112 melanoma patients undergoing pre-treatment biopsies followed by anti-PD-1 therapy, and 26 additional patients of which some had sequential biopsies. RNA-sequencing of these cohorts revealed that the resistance program was predictive of progression-free survival, discriminated objective responders (OR), including complete response vs. partial response from patients progressive disease, and the duration of OR, supporting the possibility that the program captures both intrinsic and acquired resistance to ICI. Using an in silico prediction across >600 cell lines screened against 131 drugs, we found that cells with high expression of the resistance program were selectively susceptible to inhibition by CDK4/6 inhibitors, such as palbociclib or abemaciclib. We experimentally validated that abemaciclib reversed the resistance program in vitro, enhanced T cell responses in an ex vivo model of patient-derived melanoma cells and autologous T cells, and when combined with ICI, induced a high rate of complete responses in an otherwise ICI resistant syngeneic in vivo model of melanoma. Along with other studies in the field, our study suggest a potentially clinically active synergistic effect of combining CDK4/6 inhibitors with ICI. Together, these studies highlight the possibility of developing rationale therapeutic strategies to overcome drug resistance by systematic assessment of patient tumor ecosystems. Citation Format: Benjamin Izar, Livnat Jerby-Arnon, Itay Tirosh, Parin Shah, Michael C. Cuoco, Christopher Rodman, Mei-Ju Su, Johannes C. Melms, Rachel Leeson, Abhay Kanodia, Jia-Ren Lin, Genevieve M. Boland, Gao Zhang, F. Stephen Hodi, Peter K. Sorger, Kai W. Wucherpfennig, Eli M. Van Allen, Dirk Schadendorf, Orit Rozenblatt-Rosen, Levi A. Garraway, Asaf Rotem, Charles H. Yoon, Aviv Regev. Development of therapeutic strategies by resolving the tumor ecosystem [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY45-04.

Bispecific Antibody Approach for Improved Melanoma-Selective PD-L1 Immune Checkpoint Blockade

Reactivation of functionally-impaired anticancer T cells by programmed cell death protein 1 (PD-1) and programmed cell death receptor ligand-1 (PD-L1)-blocking antibodies shows prominent therapeutic benefit in advanced melanoma and patients with non-small cell lung cancer. However, current PD-L1-blocking antibodies lack intrinsic tumor selectivity. Therefore, efficacy may be reduced resulting from on-target and off-tumor binding to PD-L1-expressing normal cells. This may lead to indiscriminate activation of antigen-experienced T cells, including those implicated in autoimmune-related adverse events. To direct PD-L1 blockade to chondroitin sulfate proteoglycan 4 (CSPG4)-expressing cancers and to reactivate anticancer T cells more selectively, we constructed bispecific antibody PD-L1xCSPG4. CSPG4 is an established target antigen that is selectively overexpressed on malignant melanoma and various other difficult-to-treat cancers. PD-L1xCSPG4 showed enhanced capacity for CSPG4-directed blockade of PD-L1 on cancer cells. Importantly, treatment of mixed cultures containing primary patient-derived CSPG4-expressing melanoma cells and autologous tumor-infiltrating lymphocytes with PD-L1xCSPG4 significantly enhanced activation status, IFN-γ production, and cytolytic activity of anticancer T cells. In conclusion, tumor-directed blockade of PD-L1 by PD-L1xCSPG4 may improve efficacy and safety of PD-1/PD-L1 checkpoint blockade for treatment of melanoma and other CSPG4-overexpressing malignancies.

A novel anti-melanoma SRC-family kinase inhibitor.

The major drawback of melanoma therapy with BRAF and MAPK inhibitors is the innate and acquired drug resistance. We therefore explored alternative targets and developed a new compound, SAB298, that is a SRC-family kinase (SFK) inhibitor. The drug is cytotoxic to patient-derived melanoma cells regardless of oncogene expression and inhibits tumor growth in vivo. As expected, it inhibited SRC and PI3K activity, and had the additional property of ERBB2 inhibition, that lead to inactivation of the two ERK phosphatases PP2A and SHP2. In 57% of the melanoma cell lines tested, the consequent increase in ERK activity lead to proteolytic degradation of its substrate, the lineage specific transcription factor MITF, likely contributing to growth arrest. Treatment with a combination of SAB298 and AZD6244 (selumetinib), induced a synergistic growth inhibition, suggesting that the new compound could be used in the clinic as a substitute for, or in combination with MAPK inhibitors.

Down-regulation of FZD3 receptor suppresses growth and metastasis of human melanoma independently of canonical WNT signaling

Frizzled 3 receptor (FZD3) plays an important role in the homeostasis of the neural crest and its derivatives, which give rise to pigment-synthesizing cells, melanocytes. While the role for FZD3 in specification of the melanocytic lineage from neural crest is well established, its significance in the formation of melanoma, its associated malignancy, is less understood. In this study we identified FZD3 as a critical regulator of human melanoma tumorigenesis. Down-regulation of FZD3 abrogated growth, colony-forming potential, and invasive capacity of patient-derived melanoma cells. Xenotransplantation of tumor cells with down-regulated FZD3 levels originating from melanomas carrying the BRAF(V600) mutation uniformly suppressed their capacity for tumor and metastasis formation. FZD3 knockdown leads to the down-regulation of the core cell cycle protein components (cyclins D1, E2, B1, and CDKs 1, 2, and 4) in melanomas with a hyperactive BRAF oncogene, indicating a dominant role of this receptor during melanoma pathogenesis. Enriched pathway analysis revealed that FZD3 inhibits transcriptional networks controlled by CREB5, FOXD1, and ATF3, which suppress the activity of MAPK-mediated signaling. Thus, FZD3 establishes a positive-feedback mechanism that activates MAPK signal transduction network, critical to melanoma carcinogenesis. Importantly, high levels of FZD3 mRNA were found to be correlated with melanoma advancement to metastatic stages and limited patient survival. Changes in gene-expression patterns mediated by FZD3 activity occur in the absence of nuclear β-catenin function, thus representing an important therapeutic target for the melanoma patients whose disease progresses independent of canonical WNT signaling.

Synthetic lethality of a cell-penetrating anti-RAD51 antibody in PTEN-deficient melanoma and glioma cells.

PTEN is a tumor suppressor that is highly mutated in a variety of human cancers. Recent studies have suggested a link between PTEN loss and deficiency in the non-homologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair. As a means to achieve synthetic lethality in this context, we tested the effect of 3E10, a cell-penetrating autoantibody that inhibits RAD51, a key factor in the alternative pathway of DSB repair, homology dependent repair (HDR). We report here that treatment of PTEN-deficient glioma cells with 3E10 leads to an accumulation of DNA damage causing decreased proliferation and increased cell death compared to isogenic PTEN proficient controls. Similarly, 3E10 was synthetically lethal to a series of PTEN-deficient, patient-derived primary melanoma cell populations. Further, 3E10 was found to synergize with a small molecule inhibitor of the ataxia telangiectasia and Rad3-related (ATR) protein, a DNA damage checkpoint kinase, in both PTEN-deficient glioma cells and primary melanoma cells. These results point to a targeted synthetic lethal strategy to treat PTEN-deficient cancers through a combination designed to disrupt both DNA repair and DNA damage checkpoint signaling.

Abstract B174: Co-expression of stimulators and inhibitors of T-cell activation in melanoma

Abstract Melanoma, one of the most aggressive skin cancers, has steadily been on the rise over the last three decades. With limited treatment options, the recent impetus in immuno-oncology drugs, especially checkpoint inhibitors, has changed the treatment landscape. While anti-CTLA4 and anti-PD1 demonstrated success in clinic, the search for additional targets is needed to enable potent comprehensive curative combination therapies. Our laboratory has characterized and screened five primary patient-derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM, and GLM-2. While immunomodulatory molecules are canonically present on antigen presenting cells and T-cells, increasing evidence suggests that they are not expressed in isolation. In an effort to identify immunomodulatory molecules expressed on our primary melanoma cells, a comprehensive RT-PCR screen of 29 co-inhibitory and co-stimulatory molecules was carried out. Several molecules including CD160, CD226, TIM1, HVEM, and BTLA were seen to be differentially expressed in melanoma cells compared to normal melanocytes at the mRNA level. Western blots and immunocytochemistry validated the differential expression of these molecules at the protein level. 50-80% of melanoma cases are positive for the BRAFV600E mutation, and are treated with a small molecule inhibitor of the mutated BRAF, vemurafenib (PLX4032). Treatment of these cells with PLX4032 led to an upregulation of transcription factors MITF and AP-1, as well as immunomodulatory molecules, CD160, CD226, TIM1, HVEM, and BTLA, a phenomenon seen only in cells positive for the BRAFV600E lesion. MITF and AP-1, owing to the binding sites present in the promoter regions of these molecules, can drive their expression upon treatment with PLX4032. These additional immune-regulatory molecules of T-cell activation and/or immune tolerance mechanisms are potential targets for a combination therapy with PLX4032 in melanoma patients positive for the BRAFV600E genetic lesion. Our future directions aim to elucidate the role of these molecules on the tumor cells and devise an effective combination with small-molecule inhibitors and immunotherapies. Citation Format: Rachana Maniyar, Robert Freund, Aryan Malhotra, Sanjukta Chakraborty, Jan Geliebter, Marc Wallack, Raj K. Tiwari. Co-expression of stimulators and inhibitors of T-cell activation in melanoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B174.

Brain Distribution and Active Efflux of Three panRAF Inhibitors: Considerations in the Treatment of Melanoma Brain Metastases.

Targeted inhibition of RAF and MEK by molecularly targeted agents has been employed as a strategy to block aberrant mitogen-activated protein kinase (MAPK) signaling in melanoma. While the use of BRAF and MEK inhibitors, either as a single agent or in combination, improved efficacy in BRAF-mutant melanoma, initial responses are often followed by relapse due to acquired resistance. Moreover, some BRAF inhibitors are associated with paradoxical activation of the MAPK pathway, causing the development of secondary malignancies. The use of panRAF inhibitors, i.e., those that target all isoforms of RAF, may overcome paradoxical activation and resistance. The purpose of this study was to perform a quantitative assessment and evaluation of the influence of efflux mechanisms at the blood-brain barrier (BBB), in particular, Abcb1/P-glycoprotein (P-gp) and Abcg2/breast cancer resistance protein (Bcrp), on the brain distribution of three panRAF inhibitors: CCT196969 [1-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)phenyl)urea], LY3009120 1-(3,3-Dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl-2-(methylamino)pyrido(2,3-d)pyrimidin-6-yl)phenyl)urea, and MLN2480 [4-pyrimidinecarboxamide, 6-amino-5-chloro-N-[(1R)-1-[5-[[[5-chloro-4-(trifluoromethyl)-2-pyridinyl]amino]carbonyl]-2-thiazolyl]ethyl]-]. In vitro studies using transfected Madin-Darby canine kidney II cells indicate that only LY3009120 and MLN2480 are substrates of Bcrp, and none of the three inhibitors are substrates of P-gp. The three panRAF inhibitors show high nonspecific binding in brain and plasma. In vivo studies in mice show that the brain distribution of CCT196969, LY3009120, and MLN2480 is limited, and is enhanced in transgenic mice lacking P-gp and Bcrp. While MLN2480 has a higher brain distribution, LY3009120 exhibits superior in vitro efficacy in patient-derived melanoma cell lines. The delivery of a drug to the site of action residing behind a functionally intact BBB, along with drug potency against the target, collectively play a critical role in determining in vivo efficacy outcomes.

Whole-exome sequencing reveals novel genetic variants associated with diverse phenotypes of melanoma cells.

We have extensively studied the phenotypic heterogeneity of patient-derived melanoma cells. Here, whole-exome sequencing revealed novel variants of genes associated with the MAPK, NOTCH, Hippo, cell-cycle, senescence, and ubiquitin-dependent pathways, which could contribute to the observed phenotypic diversity between cell lines. Focusing on mutations in the MAPK pathway-associated genes, we found BRAF (BRAFV600E ) and RAS subtypes, including NRASQ61R and the rare HRASQ61R variant, and additional alterations potentially leading to different ERK1/2 activity. Both RASQ61R cell lines harbored a MEK1P124S variant and exerted a low level of phospho-MEK1/2. Activity of the MAPK pathway was further attenuated in NRASQ61R /MEKP124S cells by trametinib, and this effect was also shown in HRASQ61R /MEKP124S melanoma cells. The observed variability in doubling time might be a consequence of diverse MAPK and PI3K/AKT pathway activities, but not exclusively, as a senescence program was also executed to different extent in distinct melanoma cell lines. Low percentages of senescent cells might result from mutations in CDKN2A, E2F3, and EZH2, and a high c-MYC expression. Vemurafenib and trametinib induced senescence concomitantly with c-MYC downregulation and irrespectively of CDKN2A mutation, but the EZH2S412C variant might limit senescence induction. Damaging alterations in Hippo pathway-associated genes were accompanied with variability in the phosphorylation level of YAP1/TAZ and CTGF expression. Our study also suggests opposite activity of NOTCH2F1209V and NOTCH2N2002S variants. Additionally, we found a novel FBXW7V418M variant that retained its function in melanoma cells. The obtained molecular data might be further exploited in genotype-phenotype relationship studies and in identifying novel biomarkers and therapies for melanomas.