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Despite the recent advances in melanoma therapy, the need for new targets and novel approaches to therapy is urgent. We previously reported melanoma actives that work via binding and downregulating spliceosomal proteins hnRNPH1 and H2. In a separate study, we reported that these compounds were non-toxic to Balb/C mice at 50 mg/kg suggesting their utility in in vivo studies. In the present study, we aimed to determine the therapeutic potential of these compounds by testing them in A375 cell-line derived xenograft in nu/nu mice. Animals were randomized into four groups (n=12/group) to receive subcutaneous administration of vehicle, 10 mg/kg vemurafenib, and 25 mg/kg 2155-14 and 2155-18 three times per week for 15 days along with a control group. The results revealed that both 2155-14 and 2155-18 significantly decreased the growth of A375 tumors, which was comparable to vemurafenib. These results were confirmed by tumor volume, weight, and histopathological examination. In conclusion, these results suggest a therapeutic potential of targeting spliceosomal proteins hnRNPH1 and H2.

BackgroundAlthough the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations are effective in advanced melanoma it remains unclear whether their mechanisms of action and resistance overlap.MethodsWe used single cell (sc) RNA-seq, flow cytometry and IHC analysis of responding SM1 and B16 melanoma flank tumors and SM1 brain metastases to explore the mechanism of action of the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combination. CD4+ and CD8+ T cell depletion and ELISPOT assays were used to demonstrate the unique role of CD4+ T cell help in the anti-tumor effects of the anti-PD-1+LAG-3 combination. Tetramer assays confirmed the loss of CD8+ tumor-reactive T cells in brain tumors resistant to the anti-PD-1+LAG-3 combination.ResultsThe anti-PD-1+CTLA-4 combination was associated with the infiltration of FOXP3+ regulatory CD4+ cells (Tregs), fewer activated CD4+ T cells and the accumulation of a subset of IFNγ secreting cytotoxic CD8+ T cells, whereas the anti-PD-1+LAG-3 combination led to the accumulation of CD4+ T helper cells that expressed CXCR4, TNFSF8, IL21R and a subset of CD8+ T cells with reduced expression of cytotoxic markers. T cell depletion studies showed a requirement for CD4+ T cells for the anti-PD-1+LAG-3 combination, but not the PD-1-CTLA-4 combination at both flank and brain tumor sites. In anti-PD-1+LAG-3 treated tumors, CD4+ T cell depletion was associated with fewer activated (CD69+) CD8+ T cells, impaired IFNγ release and increased numbers of myeloid-derived suppressor cells (MDSCs) but, conversely, increased numbers of activated CD8+ T cells and IFNγ release in anti-PD-1+CTLA-4 treated tumors. Analysis of relapsing melanoma brain metastases from anti-PD-1+LAG-3 treated mice showed an increased accumulation of MDSCs and a loss of gp100+ tumor reactive CD8+ T cells. An analysis of the inferred cell-cell interactions from the scRNA-seq data suggested the MDSCs interacted with multiple subsets of T cells in a bi-directional manner.ConclusionsTogether these studies suggest that these two clinically relevant ICI combinations have differential effects upon CD4+ T cell polarization, which in turn, impacted cytotoxic CD8+ T cell function. Further insights into the mechanisms of action/resistance of these clinically-relevant ICI combinations will allow therapy to be further personalized.

Immunotherapy (IT) and targeted therapy (TT) are both effective against melanoma, but their combination is frequently toxic. Here, we investigated whether the sequence of IT (anti–PD-1)→ TT (ceritinib–trametinib or dabrafenib–trametinib) was associated with improved antitumor responses in mouse models of BRAF- and NRAS-mutant melanoma. Mice with NRAS-mutant (SW1) or BRAF-mutant (SM1) mouse melanomas were treated with either IT, TT, or the sequence of IT→TT. Tumor volumes were measured, and samples from the NRAS-mutant melanomas were collected for immune-cell analysis, single-cell RNA sequencing (scRNA-seq), and reverse phase protein analysis (RPPA). scRNA-seq demonstrated that the IT→TT sequence modulated the immune environment, leading to increased infiltration of T cells, monocytes, dendritic cells and natural killer cells, and decreased numbers of tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells. Durable responses to the IT→TT sequence were dependent on T-cell activity, with depletion of CD8+, but not CD4+, T cells abrogating the therapeutic response. An analysis of transcriptional heterogeneity in the melanoma compartment showed the sequence of IT→TT enriched for a population of melanoma cells with increased expression of MHC class I and melanoma antigens. RPPA analysis demonstrated that the sustained immune response induced by IT→TT suppressed tumor-intrinsic signaling pathways required for therapeutic escape. These studies establish that upfront IT improves the responses to TT in BRAF- and NRAS-mutant melanoma models.

S1: IT monotherapy in SW1 model S2: IT->dabrafenib-trametinib therapy in BRAF mutant melanoma. S3: IT->TT sequence in NRAS#5 model S4: Expanded heatmap of all immune cells S5: IT->TT increases accumulation of activated CD8+ T cells S6: The TT->IT sequence on T cells, Tregs and MDSCS S7: Expanded heatmap of T and NK cells S8: IT->TT increases accumulation of activated CD8+ T cells, pathway analysis. S9: Expanded heatmap of myeloid cell data S10: IT->TT sequence increases expression of MHC I mRNAs S11: TT affects melanoma antigens S12: IC50 curves showing resistance to TT S13: Model showing mechanism of IT->TT sequence ST1: tumor weights at the end of the experiment

File containing 12 Supplemental Figures. Figure 1 shows a kinome array on WM209 cells, Figure 2 shows detail from the kinome array. Figure 3 shows the effects of the drugs upon p-c-JUN in the BRAF/NRAS WT cell lines. Figure 4 is a time course of pS6 and pERK inhibition following drug treatment. Figure 5 is the effects of drug upon SPRY-2. Figure 6 shows the structure of c-ceritinib. Figure 7 is the IGF1R expression in the cell line panel. Figure 8 is an RTK array following ceritinib treatment. Figure 9 is the effects of IGF1R siRNA upon cell growth. Figure 10 shows the effects of ceritinib and trametinib upon FAK expression. Figure 11 shows the effects of FER silencing upon the growth of the melanoma cells. Figure 12 is the effects of an ACK1 inhibitor and a MEK inhibitor on cell growth.

Supplemental Figure 1. Characterization of the resistant cell lines. Supplemental Figure 2. Quantification of the cell invasion from figure 1D. Supplemental Figure 3. Model showing ligand dependent and independent ephrin signaling. Supplemental Figure 4. Acute drug treatment does not induce ligand independent ephrin signaling. Supplemental Figure 5. Quantification of matrigel invasion data from Figure 2E. Supplemental Figure 6. knockdown of EphA2 does not resensitize to BRAF inhibition. Supplemental Figure 7. PI3K and AKT inhibitors block AKT signaling. Supplemental Figure 8. Removal of drug increases cell doubling time. Supplemental Figure 9. Removal of drug reverses the resistance phenotype. Supplemental Figure 10. Removal of drug reverses EphA2 signaling and melanoma invasion. Supplemental Figure 11. Time course of EphA2 induction following chronic BRAF inhibition Supplemental Table 1. Levels of EphA2 staining in primary and metastatic melanoma lesions Supplemental Table 2. Increased EphA2 staining observed in melanoma patient specimens on BRAF inhibitor trial Supplemental Table 3. Incidence of new metastases in patients treated with either vemurafenib or dacarbazine.

The HSP90 inhibitor XL888 is effective at reversing BRAF inhibitor resistance in melanoma, including that mediated through acquired NRAS mutations. The present study has investigated the mechanism of action of XL888 in NRAS-mutant melanoma. Treatment of NRAS-mutant melanoma cell lines with XL888 led to an inhibition of growth, G2–M phase cell-cycle arrest, and the inhibition of cell survival in three-dimensional spheroid and colony formation assays. In vitro, HSP90 inhibition led to the degradation of ARAF, CRAF, Wee1, Chk1, and cdc2 and was associated with decreased mitogen-activated protein kinase (MAPK), AKT, mTOR, and c-jun NH2 kinase (JNK) signaling. Apoptosis induction was associated with increased BIM expression and a decrease in the expression of the prosurvival protein Mcl-1. The critical role of increased BIM and decreased Mcl-1 expression in the survival of NRAS-mutant melanoma cell lines was shown through siRNA knockdown and overexpression studies. In an animal xenograft model of NRAS-mutant melanoma, XL888 treatment led to reduced tumor growth and apoptosis induction. Important differences in the pattern of client degradation were noted between the in vivo and in vitro studies. In vivo, XL888 treatment led to degradation of CDK4 and Wee1 and the inhibition of AKT/S6 signaling with little or no effect observed upon ARAF, CRAF, or MAPK. Blockade of Wee1, using either siRNA knockdown or the inhibitor MK1775, was associated with significant levels of growth inhibition and apoptosis induction. Together, these studies have identified Wee1 as a key target of XL888, suggesting novel therapeutic strategies for NRAS-mutant melanoma. Mol Cancer Ther; 12(6); 901–12. ©2013 AACR.

Supplemental Tables 1 and 2 and Figures 1-3. Supplemental Table 1: Cell line resistance mechanisms Supplemental Table 2: Ic50 values for BI-847325 on the cell line panel Supplemental Figure 1: BI-847325 blocks the proliferation and induces apoptosis in BRAF-mutant melanoma Supplemental Figure 2: MEK and Aurora kinase inhibitors do not decrease expression of Mcl-1 and MEK. Supplemental Figure 3: Inhibition of the proteasome does not reverse the effects of BI-847325 upon MEK expression.

Targeted therapy options are currently lacking for the heterogeneous population of patients whose melanomas lack BRAF or NRAS mutations (∼35% of cases). We undertook a chemical biology screen to identify potential novel drug targets for this understudied group of tumors. Screening a panel of 8 BRAF/NRAS-WT melanoma cell lines against 240 targeted drugs identified ceritinib and trametinib as potential hits with single-agent activity. Ceritinib enhanced the efficacy of trametinib across the majority of the BRAF/NRAS-WT cell lines, and the combination showed increased cytotoxicity in both three-dimensional spheroid culture and long-term colony formation experiments. Coadministration of ceritinib and trametinib led to robust inhibition of tumor growth in an in vivo xenograft BRAF/NRAS-WT melanoma model; this was not due to ALK inhibition by ceritinib. Mechanistic studies showed the ceritinib–trametinib combination to increase suppression of MAPK and TORC1 signaling. Similar results were seen when BRAF/NRAS-WT melanoma cells were treated with a combination of trametinib and the TORC1/2 inhibitor INK128. We next used mass spectrometry–based chemical proteomics and identified known and new ceritinib targets, such as IGF1R and ACK1, respectively. Validation studies suggested that ceritinib could suppress mTORC1 signaling in the presence of trametinib through inhibition of IGF1R and/or ACK1 in a cell line–dependent manner. Together, our studies demonstrated that combining a specific inhibitor (trametinib) with a more broadly targeted agent (ceritinib) has efficacy against tumors with heterogeneous mutational profiles. Mol Cancer Ther; 17(1); 73–83. ©2017 AACR.

Resistance to BRAF inhibitors is a major clinical problem. Here, we evaluate BI-847325, an ATP-competitive inhibitor of MEK and Aurora kinases, in treatment-naïve and drug-resistant BRAF-mutant melanoma models. BI-847325 potently inhibited growth and survival of melanoma cell lines that were both BRAF inhibitor naïve and resistant in 2D culture, 3D cell culture conditions, and in colony formation assays. Western blot studies showed BI-847325 to reduce expression of phospho-ERK and phospho-histone 3 in multiple models of vemurafenib resistance. Mechanistically, BI-847325 decreased the expression of MEK and Mcl-1 while increasing the expression of the proapoptotic protein BIM. Strong suppression of MEK expression was observed after 48 hours of treatment, with no recovery following >72 hours of washout. siRNA-mediated knockdown of Mcl-1 enhanced the effects of BI-847325, whereas Mcl-1 overexpression reversed this in both 2D cell culture and 3D spheroid melanoma models. In vivo, once weekly BI-847325 (70 mg/kg) led to durable regression of BRAF-inhibitor naïve xenografts with no regrowth seen (>65 days of treatment). In contrast, treatment with the vemurafenib analog PLX4720 was associated with tumor relapse at >30 days. BI-847325 also suppressed the long-term growth of xenografts with acquired PLX4720 resistance. Analysis of tumor samples revealed BI-847325 to induce apoptosis associated with suppression of phospho-ERK, total MEK, phospho-Histone3, and Mcl-1 expression. Our studies indicate that BI-847325 is effective in overcoming BRAF inhibitor resistance and has long-term inhibitory effects upon BRAF-mutant melanoma in vivo, through a mechanism associated with the decreased expression of both MEK and Mcl-1. Mol Cancer Ther; 14(6); 1354–64. ©2015 AACR.

This file contains the raw data from the drug screen

PDF file - 295K, Supplemental Figure 1: XL888 (0.3 μM) treatment induces the nuclear accumulation of FOXO3a. Supplemental Figure 2: XL888 decreases Mcl-1 mRNA expression in NRAS mutant melanoma. Supplemental Figure 3: BAK is not required for XL888-mediated apoptosis. Supplemental Figure 4: The BH3 family protein inhibitor ABT-737 enhances the pro-apoptotic effects of XL888 in the WM1366 and WM1361A cell lines. Supplemental Figure 5: The effects of XL888 (300nM) and ABT-737 (1 μM) on the viability of NRAS mutant melanoma spheroids. Supplemental Table 1: List of heat shock proteins used on the LC-MRM by UniPROT ID and gene name.

Supplementary Data from Single-cell Characterization of the Cellular Landscape of Acral Melanoma Identifies Novel Targets for Immunotherapy

Purpose:The clinical use of MEK inhibitors in uveal melanoma is limited by the rapid acquisition of resistance. This study has used multiomics approaches and drug screens to identify the pan-HDAC inhibitor panobinostat as an effective strategy to limit MEK inhibitor resistance.Experimental Design: Mass spectrometry–based proteomics and RNA-Seq were used to identify the signaling pathways involved in the escape of uveal melanoma cells from MEK inhibitor therapy. Mechanistic studies were performed to evaluate the escape pathways identified, and the efficacy of the MEK-HDAC inhibitor combination was demonstrated in multiple in vivo models of uveal melanoma.Results:We identified a number of putative escape pathways that were upregulated following MEK inhibition, including the PI3K/AKT pathway, ROR1/2, and IGF-1R signaling. MEK inhibition was also associated with increased GPCR expression, particularly the endothelin B receptor, and this contributed to therapeutic escape through ET-3–mediated YAP signaling. A screen of 289 clinical grade compounds identified HDAC inhibitors as potential candidates that suppressed the adaptive YAP and AKT signaling that followed MEK inhibition. In vivo, the MEK-HDAC inhibitor combination outperformed either agent alone, leading to a long-term decrease of tumor growth in both subcutaneous and liver metastasis models and the suppression of adaptive PI3K/AKT and YAP signaling.Conclusions:Together, our studies have identified GPCR-mediated YAP activation and RTK-driven AKT signaling as key pathways involved in the escape of uveal melanoma cells from MEK inhibition. We further demonstrate that HDAC inhibition is a promising combination partner for MEK inhibitors in advanced uveal melanoma.

Supplemental Table 5: Cox proportional hazard model analysis identifies immune cell subpopulations associated with better survival outcomes

Supplementary Figures 1-11. 1. CSF cytology of leptomeningeal patient cohort. 2. Additional clinical timelines of leptomeningeal patient cohort 3. Patient MRIs 4. RNAseq Workflow 5. Differential responses to BRAFi treatment in CSF 6. RNAseq shows CSF from Patient 4 to elicit transcriptional modulation of melanoma cell response to BRAF inhibition 7. CSF from LMM patients elicits transcriptional changes in melanoma cells 8. CSF protects melanoma cells from BRAF inhibitor therapy 9. CSF activates AKT in WM164 10. No-LMM CSF does not induce expression of TGFbeta or activation of AKT 11. ELISA assay showing the levels of TGFβ1 in CSF specimens from extrordi- nary responder (Patient 1) and non-responders.

Activating mutations in BRAF, a key mediator of RAS signaling, are present in approximately 50% of melanoma patients. Pharmacologic inhibition of BRAF or the downstream MAP kinase MEK is highly effective in treating BRAF-mutant melanoma. In contrast, RAS pathway inhibitors have been less effective in treating epithelial malignancies, such as lung cancer. Here, we show that treatment of melanoma patients with BRAF and MEK inhibitors (MEKi) activated tumor NF-κB activity. MEKi potentiated the response to TNFα, a potent activator of NF-κB. In both melanoma and lung cancer cells, MEKi increased cell-surface expression of TNFα receptor 1 (TNFR1), which enhanced NF-κB activation and augmented expression of genes regulated by TNFα and IFNγ. Screening of 289 targeted agents for the ability to increase TNFα and IFNγ target gene expression demonstrated that this was a general activity of inhibitors of MEK and ERK kinases. Treatment with MEKi led to acquisition of a novel vulnerability to TNFα and IFNγ-induced apoptosis in lung cancer cells that were refractory to MEKi killing and augmented cell-cycle arrest. Abolishing the expression of TNFR1 on lung cancer cells impaired the antitumor efficacy of MEKi, whereas the administration of TNFα and IFNγ in MEKi-treated mice enhanced the antitumor response. Furthermore, immunotherapeutics known to induce expression of these cytokines synergized with MEKi in eradicating tumors. These findings define a novel cytokine response modulatory function of MEKi that can be therapeutically exploited.Significance:Lung cancer cells are rendered sensitive to MEK inhibitors by TNFα and IFNγ, providing a strong mechanistic rationale for combining immunotherapeutics, such as checkpoint blockers, with MEK inhibitor therapy for lung cancer.See related commentary by Havel, p. 5699

Table showing frequency distribution of cell cycle phases (G1, S, G2) from 3 biological replicates in indicated cell lines (A549, LKR, PC-9, HCC44, H23).

Esophageal squamous cell carcinoma (ESCC) is an exceptionally drug-resistant tumor with a 5-year survival rate 2-M-phase cell cycle arrest and p53-independent apoptosis associated with caspase cleavage and Noxa induction. Bortezomib also showed excellent activity in organotypic culture and in vivo models of ESCC. Biochemically, bortezomib treatment activated the p38 and c-Jun NH2-termnial kinase stress-activated mitogen-activated protein kinase (MAPK) pathways and induced phospho-H2AX activity. Although H2AX is known to cooperate with c-Jun NH2-termnial kinase to induce apoptosis following UV irradiation, knockdown of H2AX did not abrogate bortezomib-induced apoptosis. Instead, blockade of p38 MAPK signaling, using either small interfering RNA or a pharmacologic inhibitor, reversed bortezomib-induced apoptosis and the up-regulation of Noxa. Radiation therapy is known to activate the p38 MAPK pathway and is a mainstay of ESCC treatment strategies. In a final series of studies, we showed that the coadministration of bortezomib with irradiation led to enhanced p38 MAPK activity and a significant reduction in colony formation. We therefore suggest that p38 MAPK pathway activation is an excellent potential therapeutic strategy in ESCC. It is further suggested that bortezomib could be added to existing ESCC therapeutic regimens. [Mol Cancer Ther 2008;7(9):2866–75]

Purpose: NRAS mutations in malignant melanoma are associated with aggressive disease requiring rapid antitumor intervention, but there is no approved targeted therapy for this subset of patients. In clinical trials, the MEK inhibitor (MEKi) binimetinib displayed modest antitumor activity, making combinations a requisite. In a previous study, the BRAF inhibitor (BRAFi) vemurafenib was shown to induce endoplasmic reticulum (ER) stress that together with inhibition of the RAF–MEK–ERK (MAPK) pathway amplified its proapoptotic activity in BRAF-mutant melanoma. The present study investigated whether this effect might extent to NRAS-mutant melanoma, in which MAPK activation would be expected.Experimental Design and Results: BRAFi increased pERK, but also significantly increased growth inhibition and apoptosis induced by the MEKi in monolayer, spheroids, organotypic, and patient-derived tissue slice cultures of NRAS-mutant melanoma. BRAFi such as encorafenib induced an ER stress response via the PERK pathway, as detected by phosphorylation of eIF2α and upregulation of the ER stress–related factors ATF4, CHOP, and NUPR1 and the proapoptotic protein PUMA. MEKi such as binimetinib induced the expression of the proapoptotic protein BIM and activation of the mitochondrial pathway of apoptosis, the latter of which was enhanced by combination with encorafenib. The increased apoptotic rates caused by the combination treatment were significantly reduced through siRNA knockdown of ATF4 and BIM, confirming its critical roles in this process.Conclusions: The data presented herein encourage further advanced in vivo and clinical studies to evaluate MEKi in combination with ER stress inducing BRAFi as a strategy to treat rapidly progressing NRAS-mutant melanoma. Clin Cancer Res; 23(20); 6203–14. ©2017 AACR.

Supplementary Fig. S4 from Multiple signaling pathways must be targeted to overcome drug resistance in cell lines derived from melanoma metastases

The fold changes in gene expression between the vemurafenib treated and vehicle treated cells for each cell culture condition

Supplemental Figure 1. Expression of T, NK, B and Plasma cell markers by metastatic site. Supplemental Figure 2. Expression of myeloid cell markers by metastatic site and gene expression associated with classical and alternative macrophage activation. Supplemental Figure 3. Baseline and therapy induced changes in the LMM immune landscape. Supplemental Figure 4. Baseline and therapy-mediated changes to the MBM immune landscape. Supplemental Figure 5. Expression of MHC class I and II in melanoma cells from samples with and without DC3s. Supplemental Figure 6. Cell-cell interactions between DC3s and T cells defined by SingleCellR. Supplemental Figure 7. Detection of DC3 cells in clinical samples using multiplexed IHC. Supplemental Figure 8. Validation analysis of samples from melanoma metastases in a publicly available validation dataset. Supplemental Figure 9. Expression of key markers that distinguish the major subsets of myeloid cells. Supplemental Figure 10. Correlation of DC3 gene signature and PC loading for DC3 gene signature in TCGA. Supplemental Figure 11. Overall survival for melanoma patients with LMM metastases whose CSF samples contained high DC3 cells vs. low DC3 cells. Supplemental Figure 12. Correlation of individual genes from the DC3 gene signature to overall survival and their univariate cox proportional-hazards models in TCGA dataset.

Although >66% of melanomas harbor activating mutations in BRAF and exhibit constitutive activity in the mitogen-activated protein kinase/extracellular signal–regulated kinase kinase (MEK)/extracellular signal–regulated kinase signaling pathway, it is unclear how effective MEK inhibition will be as a sole therapeutic strategy for melanoma. We investigated the anticancer activity of MEK inhibition in a panel of cell lines derived from radial growth phase (WM35) and vertical growth phase (WM793) of primary melanomas and metastatic melanomas (1205Lu, 451Lu, WM164, and C8161) in a three-dimensional spheroid model and found that the metastatic lines were completely resistant to MEK inhibition (U0126 and PD98059) but the earlier stage cell lines were not. Similarly, these same metastatic melanoma lines were also resistant to inhibitors of the phosphatidylinositol 3-kinase/Akt pathway (LY294002 and wortmannin). Under adherent culture conditions, the MEK inhibitors blocked growth through the induction of cell cycle arrest and up-regulation of p27, but this was readily reversible following inhibitor washout. However, when the phosphatidylinositol 3-kinase and MEK inhibitors were combined, the growth and invasion of the metastatic melanoma three-dimensional spheroids were blocked. Taken together, these results suggest that the most aggressive melanomas are resistant to strategies targeting one signaling pathway and that multiple signaling pathways may need to be targeted for maximal therapeutic efficacy. It is further suggested that BRAF mutational status is not predictive of response to MEK inhibition under three-dimensional culture conditions. [Mol Cancer Ther 2006;5(5):1136–44]

Supplementary Table S1- Antibodies used for protein expression in western blotting experiments Supplementary Table S2 - Assays used for gene expression in RT-qPCR experiments Supplementary Table S3 - Antibodies used for protein expression in immunofluorescence experiments Supplementary Table S4 - Inhibitory concentration 50% (IC50) of MEKi after 72h in uveal melanoma cell lines (nM) Supplementary Table S5 - Members of the GSEA GO_REGULATION_OF_PHOSPHATIDYLINOSITOL_3_KINASE_SIGNALING family Supplementary Table S6 - Members of the GSEA GO_TRANSMEMBRANE_RECEPTOR_PROTEIN_KINASE_ACTIVITY family Supplementary Table S7 - Members of the GSEA GO_TRANSMEMBRANE_RECEPTOR_PROTEIN_TYROSINE_KINASE_ACTIVITY family Supplementary Table S8 - Members of the GSEA HIPPO_TARGETS family Supplementary Table S9 - Members of the GSEA REACTOME_GPCR_LIGAND_BINDING Family Supplementary Table S10 - Compounds used in the drug screening Supplementary Table S11 - Members of the GSEA HALLMARK_PI3K_AKT_MTOR_SIGNALING

Purpose:Melanoma brain metastases (MBM) and leptomeningeal melanoma metastases (LMM) are two different manifestations of melanoma CNS metastasis. Here, we used single-cell RNA sequencing (scRNA-seq) to define the immune landscape of MBM, LMM, and melanoma skin metastases.Experimental Design:scRNA-seq was undertaken on 43 patient specimens, including 8 skin metastases, 14 MBM, and 19 serial LMM specimens. Detailed cell type curation was performed, the immune landscapes were mapped, and key results were validated by IHC and flow cytometry. Association analyses were undertaken to identify immune cell subsets correlated with overall survival.Results:The LMM microenvironment was characterized by an immune-suppressed T-cell landscape distinct from that of brain and skin metastases. An LMM patient with long-term survival demonstrated an immune repertoire distinct from that of poor survivors and more similar to normal cerebrospinal fluid (CSF). Upon response to PD-1 therapy, this extreme responder showed increased levels of T cells and dendritic cells in their CSF, whereas poor survivors showed little improvement in their T-cell responses. In MBM patients, therapy led to increased immune infiltrate, with similar T-cell transcriptional diversity noted between skin metastases and MBM. A correlation analysis across the entire immune landscape identified the presence of a rare population of dendritic cells (DC3) that was associated with increased overall survival and positively regulated the immune environment through modulation of activated T cells and MHC expression.Conclusions:Our study provides the first atlas of two distinct sites of melanoma CNS metastases and defines the immune cell landscape that underlies the biology of this devastating disease.

Supplementary Methods for Xie et al, MEK inhibition modulates cytokine response to mediate therapeutic efficacy in lung cancer

Supplemental Figure 1: Gene ontology network analysis of uveal melanoma cells following MEKi treatment (24h) and ABPP. Supplemental Figure 2: ET-3 increases nuclear accumulation of YAP in 92.1, Mel270, MP41 and OMM1 cells. Supplemental Figure 3: ET-1 does not induce YAP reporter activity in 92.1 or Mel270 cells. Supplemental Figure 4: HDAC inhibitors increase the cytotoxic effects of MEK inhibition. Supplemental Figure 5: MEK inhibition leads to an increase in global protein acetylation in uveal melanoma cells. Supplemental Figure 6: MEKi, HDACi and the MEKi-HDACi combination do not affect the survival of primary uveal melanocytes. Supplemental Figure 7: HDACi suppresses MEKi-induced release of ET-3 from uveal melanoma cells. Supplemental Figure 8: Negative modulation of PI3K-AKT pathway by combinatory treatment with MEKi+HDACi. Supplemental Figure 9: The MEK-HDACi combination delivers durable responses in the 92.1 uveal melanoma xenograft model. Supplemental Figure 10: Macroscopic findings of liver tumor formation after inoculation of MP41 by tail vein injection. Supplemental Figure 11: Uncropped blots Supplemental Figure 12: Flow cytometry plots

Purpose: The clinical use of BRAF inhibitors is being hampered by the acquisition of drug resistance. This study shows the potential therapeutic use of the HSP90 inhibitor (XL888) in six different models of vemurafenib resistance.Experimental Design: The ability of XL888 to inhibit growth and to induce apoptosis and tumor regression of vemurafenib-resistant melanoma cell lines was shown in vitro and in vivo. A novel mass spectrometry–based pharmacodynamic assay was developed to measure intratumoral HSP70 levels following HSP90 inhibition in melanoma cell lines, xenografts, and melanoma biopsies. Mechanistic studies were carried out to determine the mechanism of XL888-induced apoptosis.Results: XL888 potently inhibited cell growth, induced apoptosis, and prevented the growth of vemurafenib-resistant melanoma cell lines in 3-dimensional cell culture, long-term colony formation assays, and human melanoma mouse xenografts. The reversal of the resistance phenotype was associated with the degradation of PDGFRβ, COT, IGFR1, CRAF, ARAF, S6, cyclin D1, and AKT, which in turn led to the nuclear accumulation of FOXO3a, an increase in BIM (Bcl-2 interacting mediator of cell death) expression, and the downregulation of Mcl-1. In most resistance models, XL888 treatment increased BIM expression, decreased Mcl-1 expression, and induced apoptosis more effectively than dual mitogen-activated protein–extracellular signal–regulated kinase/phosphoinositide 3-kinase (MEK/PI3K) inhibition.Conclusions: HSP90 inhibition may be a highly effective strategy at managing the diverse array of resistance mechanisms being reported to BRAF inhibitors and appears to be more effective at restoring BIM expression and downregulating Mcl-1 expression than combined MEK/PI3K inhibitor therapy. Clin Cancer Res; 18(9); 2502–14. ©2012 AACR.

Drug screening data used in Figure 2 are shown, including drug names, cell viability and expression levels of chemokines.

Supplementary Figure from Single-cell Characterization of the Cellular Landscape of Acral Melanoma Identifies Novel Targets for Immunotherapy

Supplemental Table 2: Sample and Patient Clinical Correlative Information

Melanoma cells have the ability to switch to a dedifferentiated, invasive phenotype in response to multiple stimuli. Here, we show that exposure of melanomas to multiple stresses including BRAF–MEK inhibitor therapy, hypoxia, and UV irradiation leads to an increase in histone deacetylase 8 (HDAC8) activity and the adoption of a drug-resistant phenotype. Mass spectrometry–based phosphoproteomics implicated HDAC8 in the regulation of MAPK and AP-1 signaling. Introduction of HDAC8 into drug-naïve melanoma cells conveyed resistance both in vitro and in vivo. HDAC8-mediated BRAF inhibitor resistance was mediated via receptor tyrosine kinase activation, leading to MAPK signaling. Although HDACs function at the histone level, they also regulate nonhistone substrates, and introduction of HDAC8 decreased the acetylation of c-Jun, increasing its transcriptional activity and enriching for an AP-1 gene signature. Mutation of the putative c-Jun acetylation site at lysine 273 increased transcriptional activation of c-Jun in melanoma cells and conveyed resistance to BRAF inhibition. In vivo xenograft studies confirmed the key role of HDAC8 in therapeutic adaptation, with both nonselective and HDAC8-specific inhibitors enhancing the durability of BRAF inhibitor therapy. Our studies demonstrate that HDAC8-specific inhibitors limit the adaptation of melanoma cells to multiple stresses including BRAF–MEK inhibition.Significance:This study provides evidence that HDAC8 drives transcriptional plasticity in melanoma cells in response to a range of stresses through direct deacetylation of c-Jun.

Supplemental Table 4: top genes that differentiate each cluster of myeloid cells

Purpose:The development of leptomeningeal melanoma metastases (LMM) is a rare and devastating complication of the late-stage disease, for which no effective treatments exist. Here, we performed a multi-omics analysis of the cerebrospinal fluid (CSF) from patients with LMM to determine how the leptomeningeal microenvironment shapes the biology and therapeutic responses of melanoma cells.Experimental Design:A total of 45 serial CSF samples were collected from 16 patients, 8 of these with confirmed LMM. Of those with LMM, 7 had poor survival (35 months). CSF samples were analyzed by mass spectrometry and incubated with melanoma cells that were subjected to RNA sequencing (RNA-seq) analysis. Functional assays were performed to validate the pathways identified.Results:Mass spectrometry analyses showed the CSF of most patients with LMM to be enriched for pathways involved in innate immunity, protease-mediated damage, and IGF-related signaling. All of these were anticorrelated in the extraordinary responder. RNA-seq analysis showed CSF to induce PI3K/AKT, integrin, B-cell activation, S-phase entry, TNFR2, TGFβ, and oxidative stress responses in the melanoma cells. ELISA assays confirmed that TGFβ expression increased in the CSF of patients progressing with LMM. CSF from poorly responding patients conferred tolerance to BRAF inhibitor therapy in apoptosis assays.Conclusions:These analyses identified proteomic/transcriptional signatures in the CSF of patients who succumbed to LMM. We further showed that the CSF from patients with LMM has the potential to modulate BRAF inhibitor responses and may contribute to drug resistance.See related commentary by Glitza Oliva and Tawbi, p. 2083

Supplementary Fig. S1 from Increased cyclin D1 expression can mediate BRAF inhibitor resistance in BRAF V600E–mutated melanomas

CCR Translation for This Article from The HSP90 Inhibitor XL888 Overcomes BRAF Inhibitor Resistance Mediated through Diverse Mechanisms

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Supplementary Table 1: Clinical data on samples stained for HDAC8 expression. S1) HDAC expression levels in melanoma cells S2) Cell lines that are resistant to BRAF inhibitors have upregulated c-Jun protein expression S3) Inhibiting HDAC6 does not resensitize BRAF/MEK resistant cells to BRAF inhibitors S4) BRAF/MEK inhibitor withdrawal decreases HDAC8 expression over time S5) Knocking down or elevating HDAC8 levels does not change cell proliferation S6) Expression of HDAC8 increased ERK activity and resistance upon treatment with BRAF-MEK inhibitors S7) Knocking down HDAC8 in BRAFi naïve cells increased apoptosis and BIM expression while decreasing ERK activity after BRAFi activity. S8) HDAC8 expression increases baseline RTK signaling S9) Inhibiting MET or FGFR3 partially resensitizes HDAC8 expressing cells to BRAF inhibitors S10) HDAC8 expression increases baseline c-Jun, p53, AKT and HSP60 signaling S11) Inhibiting c-Jun resensitizes HDAC8 expressing cells to a BRAF inhibitor S12) Protein sequences of c-Jun acetyl mutants S13) Acetylation-deficient c-JUN at residue 273 increases melanoma cell survival following BRAF inhibition S14) Acetylation-deficient c-Jun at residue 273 increases p53, Akt 1/2/3, STAT3, WNK1 and HSP60 S15) Acetylation-deficient c-JUN at residue 273 increases baseline EGFR signaling

Purpose:Acral melanoma is a rare subtype of melanoma that arises on the non–hair-bearing skin of the palms, soles, and nail beds. In this study, we used single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape of acral melanoma and identify novel immunotherapeutic targets.Experimental Design:We performed scRNA-seq on nine clinical specimens (five primary, four metastases) of acral melanoma. Detailed cell type curation was performed, the immune landscapes were mapped, and key results were validated by analysis of The Cancer Genome Atlas (TCGA) and single-cell datasets. Cell–cell interactions were inferred and compared with those in nonacral cutaneous melanoma.Results:Multiple phenotypic subsets of T cells, natural killer (NK) cells, B cells, macrophages, and dendritic cells with varying levels of activation/exhaustion were identified. A comparison between primary and metastatic acral melanoma identified gene signatures associated with changes in immune responses and metabolism. Acral melanoma was characterized by a lower overall immune infiltrate, fewer effector CD8 T cells and NK cells, and a near-complete absence of γδ T cells compared with nonacral cutaneous melanomas. Immune cells associated with acral melanoma exhibited expression of multiple checkpoints including PD-1, LAG-3, CTLA-4, V-domain immunoglobin suppressor of T cell activation (VISTA), TIGIT, and the Adenosine A2A receptor (ADORA2). VISTA was expressed in 58.3% of myeloid cells and TIGIT was expressed in 22.3% of T/NK cells.Conclusions:Acral melanoma has a suppressed immune environment compared with that of cutaneous melanoma from nonacral skin. Expression of multiple, therapeutically tractable immune checkpoints were observed, offering new options for clinical translation.

Supplemental Table 1: Flow Cytometry Antibodies

Supplementary Table Legend for Xie et al, MEK inhibition modulates cytokine response to mediate therapeutic efficacy in lung cancer

Suppl. Figure 1 Encorafenib combined with binimetinib leads to increased growth inhibition and increased induction of apoptosis in NRAS-mutant melanoma cells Suppl. Figure 2 The panRAF inhibitor Raf265 combined with binimetinib leads to growth inhibition, induction of apoptosis and ER stress genes in NRAS-mutant melanoma cells Suppl. Figure 3 Dabrafenib combined with trametinib leads to strong growth inhibition, induction of apoptosis and ER stress genes in NRAS-mutant melanoma cells Suppl. Figure 4 In normal skin cells encorafenib and binimetinib induce only weak growth inhibition and no significant levels of apoptosis Suppl. Figure 5 BRAFi combined with MEKi leads to an ER stress response involving the PERK pathway? Suppl. Figure 6 BRAFi combined with MEKi leads to activation of PUMA in patient-derived tumor slice cultures. Suppl. Figure 7 Binimetinib and encorafenib induced apoptosis is partly mediated by the anti-apoptotic BCL-2 family members and by the pro-apoptotic BH3-only protein NOXA . Suppl. Figure 8 NRAS mutated cells resistant to encorafenib, binimetinib or the combination of encorafenib and binimetinib.

Supplemental Table 3: top genes that differentiate each cluster of T/NK cells

Supplementary Figures for Xie et al, MEK inhibition modulates cytokine response to mediate therapeutic efficacy in lung cancer