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Supplementary Figure 3. Kaplan-Meier plots for progression-free survival (PFS) and overall survival (OS).

Supplementary data on genomics

Supplementary table with clinical data

Purpose: Metastatic melanoma is largely unresponsive to DNA-damaging chemotherapy agents, although WTp53 is frequently detected. Several isoforms of p53 have been discovered, some of which inhibit p53 function. We therefore examined whether p53 isoforms were present in melanoma and whether they may contribute to aberrant p53 function in melanoma.Experimental Design: We studied the expression and subcellular localization of p53 and its isoforms in a panel of human melanoma cell lines using Western blot, two-dimensional electrophoresis, and reverse transcription-PCR. We also characterized the relationship between the expression of p53, p53 isoforms, and p53 target genes following treatment with the DNA-damaging agent cisplatin.Results: We report that p53β and Δ40p53 were expressed in the majority of melanoma cell lines at the mRNA level, but were absent or expressed at low levels in fibroblasts and melanocytes, suggesting that their expression may play a role in melanoma development. Analysis by two-dimensional gel electrophoresis revealed that p53β was expressed at the protein level in melanoma cells. Both p53 and the small molecular weight forms of p53 were aberrantly expressed between the nuclear and cytosolic fractions of melanoma cell lines, compared with normal fibroblasts. Treatment with cisplatin had differential effects on WTp53 and the small molecular weight form of p53 that were cell line dependent. Δ40p53 was shown to inhibit, whereas p53β was shown to enhance, p53-dependent transcription of p21 and PUMA.Conclusions: p53β and Δ40p53 are expressed in melanoma and this may have important implications for understanding resistance of melanoma to DNA-damaging chemotherapy.

Supplemental Table

PDF file - 85K, Maintenance of NOXA levels in ABT-737 treated xenografts.

Supplemental Figure

PDF file - 7.8MB, Effects of ABT-737 on growth of 3D melanoma spheroids derived from C8161/NOXA clone C6 (A) ABT-737 was applied at the concentrations shown for 3 days. Note the dose-dependent inhibition of proliferation and invasion as well as decrease of viable cells (calcein-AM) and increase of dead cells (ethidium bromide). Bar: 500 �m. (B) Untreated spheroids; (C) treated with ABT-737 from day 0 at the concentrations shown with replenishment every 3 days for 10 days or (D) treated likewise from day 7. Bars: 500 �m.

Supplementary material

Purpose: Therapeutic blockade of immune checkpoints has revolutionized cancer treatment. Durable responses, however, occur in less than half of those treated, and efforts to improve treatment efficacy are confounded by a lack of understanding of the characteristics of the cells that initiate antitumor immune response.Patients and Methods: We performed multiparameter flow cytometry and quantitative multiplex immunofluorescence staining on tumor specimens from immunotherapy-naïve melanoma patients and longitudinal biopsy specimen obtained from patients undergoing anti–PD-1 therapy.Results: Increased numbers of CD69+CD103+ tumor-resident CD8+ T cells were associated with improved melanoma-specific survival in immunotherapy-naïve melanoma patients. Local IL15 expression levels strongly correlated with these tumor-resident T-cell numbers. The expression of several immune checkpoints including PD-1 and LAG3 was highly enriched in this subset, and these cells significantly expanded early during anti–PD-1 immunotherapy.Conclusions: Tumor-resident CD8+ T-cell numbers are more prognostic than total CD8+ T cells in metastatic melanoma. In addition, they are likely to initiate response to anti–PD-1 and anti–LAG-3 treatments. We propose that the immune profile of these cells prior to treatment could inform strategies for immune checkpoint blockade. Clin Cancer Res; 24(13); 3036–45. ©2018 AACR.

PDF file - 75K, ffects of NOXA knockdown on sensitization to ABT-737 by flavopiridol.

Supplementary Table S1 from Small Molecular Weight Variants of p53 Are Expressed in Human Melanoma Cells and Are Induced by the DNA-Damaging Agent Cisplatin

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PDF file - 75K, Expanded immunoblot analysis.

Purpose: The purpose of this study was to assess the association of baseline tumor size (BTS) with other baseline clinical factors and outcomes in pembrolizumab-treated patients with advanced melanoma in KEYNOTE-001 (NCT01295827).Experimental Design: BTS was quantified by adding the sum of the longest dimensions of all measurable baseline target lesions. BTS as a dichotomous and continuous variable was evaluated with other baseline factors using logistic regression for objective response rate (ORR) and Cox regression for overall survival (OS). Nominal P values with no multiplicity adjustment describe the strength of observed associations.Results: Per central review by RECIST v1.1, 583 of 655 patients had baseline measurable disease and were included in this post hoc analysis. Median BTS was 10.2 cm (range, 1–89.5). Larger median BTS was associated with Eastern Cooperative Oncology Group performance status 1, elevated lactate dehydrogenase (LDH), stage M1c disease, and liver metastases (with or without any other sites; all P ≤ 0.001). In univariate analyses, BTS below the median was associated with higher ORR (44% vs. 23%; P < 0.001) and improved OS (HR, 0.38; P < 0.001). In multivariate analyses, BTS below the median remained an independent prognostic marker of OS (P < 0.001) but not ORR. In 459 patients with available tumor programmed death ligand 1 (PD-L1) expression, BTS below the median and PD-L1–positive tumors were independently associated with higher ORR and longer OS.Conclusions: BTS is associated with many other baseline clinical factors but is also independently prognostic of survival in pembrolizumab-treated patients with advanced melanoma. Clin Cancer Res; 24(20); 4960–7. ©2018 AACR.See related commentary by Warner and Postow, p. 4915

PDF file - 46K, Sensitivity of NOXA-transduced melanoma cells to DNA-damaging agents.

Figure legends for supplemental figures

PDF file - 3.7MB, Spheroids derived from untransfected or NOXA-overexpressing MM200 or C8161 melanoma cells.

Purpose: To compare the efficacy and tolerability of the mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) 1/2 inhibitor selumetinib versus temozolomide in chemotherapy-naive patients with unresectable stage III/IV melanoma.Experimental Design: This phase II, open-label, multicenter, randomized, parallel-group study examined the effect of 100 mg oral selumetinib twice daily in 28-day cycles versus oral temozolomide (200 mg/m2/d for 5 days, then 23 days off-treatment). The primary endpoint was progression-free survival.Results: Two hundred patients were randomized. Progression-free survival did not differ significantly between selumetinib and temozolomide (median time to event 78 and 80 days, respectively; hazard ratio, 1.07; 80% confidence interval, 0.86–1.32). Objective response was observed in six (5.8%) patients receiving selumetinib and nine (9.4%) patients in the temozolomide group. Among patients with BRAF mutations, objective responses were similar between selumetinib and temozolomide groups (11.1% and 10.7%, respectively). However, five of the six selumetinib partial responders were BRAF mutated. Frequently reported adverse events with selumetinib were dermatitis acneiform (papular pustular rash; 59.6%), diarrhea (56.6%), nausea (50.5%), and peripheral edema (40.4%), whereas nausea (64.2%), constipation (47.4%), and vomiting (44.2%) were reported with temozolomide.Conclusions: No significant difference in progression-free survival was observed between patients with unresectable stage III/IV melanoma unselected for BRAF/NRAS mutations, who received therapy with selumetinib or temozolomide. Five of six patients with partial response to selumetinib had BRAF mutant tumors. Clin Cancer Res; 18(2); 555–67. ©2011 AACR.

Supplementary Figure 4. Decision tree used to categorize the pattern of PD-L1 positive staining based on the immune cell infiltrate, percentage of tumor cells expressing PD-L1 and the location of staining within the samples.

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Purpose: Drug resistance in melanoma is commonly attributed to ineffective apoptotic pathways. Inhibiting antiapoptotic BCL-2 and its relatives is an attractive strategy for sensitizing lymphoid malignancies to drugs but it has been largely unsuccessful for melanoma and other solid tumors. ABT-737, a small-molecule BH3-mimetic, selectively inhibits BCL-2, BCL-XL, and BCL-w and shows promise for treating leukemia, lymphoma, and small-cell lung cancer. Melanoma cells are insensitive to ABT-737, but MCL-1 inhibition reportedly increases the sensitivity of other tumors to the compound.Experimental Design: The efficacy of MCL-1 and BFL-1 inhibition for sensitizing melanoma cells to ABT-737 was investigated by short hairpin RNA–mediated knockdown or overexpression of their antagonist NOXA in two-dimensional cell culture, a three-dimensional organotypic spheroid model, and an in vivo model.Results:MCL-1 downregulation or NOXA overexpression strongly sensitized melanoma cells to ABT-737 in vitro. NOXA-inducing cytotoxic drugs also strongly sensitized melanomas to ABT-737 but, surprisingly, not vice versa. The drugs most suitable are not necessarily those normally used to treat melanoma. Resistance to ABT-737 occurred quickly in three-dimensional melanoma spheroids through reduced NOXA expression, although experiments with both xenografts and three-dimensional spheroids suggest that penetration of ABT-737 into tumor masses may be the principal limitation, which may be obviated through use of more diffusible BH3-mimetics.Conclusion: Sensitization of tumors to BH3-mimetics by cytotoxic drugs that induce NOXA is a therapeutic strategy worth exploring for the treatment of melanoma and other solid cancers. Clin Cancer Res; 18(3); 783–95. ©2011 AACR.

Supplementary Figure 1. Heat map of patients grouped based on baseline (PRE) PD-L1, change in (Δ) PD-L1 expression, Δ tumor infiltrating lymphocytes (TILs) and Δ PD-1+ lymphocytes from PRE to early during treatment (EDT).

Supplementary Table 2. The percentage of PD-L1 expressing tumor/immune cells and the intensity of staining in these samples used to derive the immuno-reactive score (IRS) for the 40 patients. *Immune cells= lymphocytes and macrophages.

PDF file - 1.3MB, Growth of xenografts, where the hosts were treated i.p. with 75 mg/kg ABT-737 or vehicle daily from 7 days post-engraftment for 21 days, 10 mice/group.

PDF file - 1.6MB, Sensitization of MM200 and Me4405 cells in 2D culture to ABT-737 by flavopiridol.

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Purpose:Immune components of the tumor microenvironment (TME) have been associated with disease outcome. We prospectively evaluated the association of an immune-related gene signature (GS) with clinical outcome in melanoma and non–small cell lung cancer (NSCLC) tumor samples from two phase III studies.Experimental Design:The GS was prospectively validated using an adaptive signature design to optimize it for the sample type and technology used in phase III studies. One-third of the samples were used as “training set”; the remaining two thirds, constituting the “test set,” were used for the prospective validation of the GS.Results:In the melanoma training set, the expression level of eight Th1/IFNγ-related genes in tumor-positive lymph node tissue predicted the duration of disease-free survival (DFS) and overall survival (OS) in the placebo arm. This GS was prospectively and independently validated as prognostic in the test set. Building a multivariate Cox model in the test set placebo patients from clinical covariates and the GS score, an increased number of melanoma-involved lymph nodes and the GS were associated with DFS and OS. This GS was not associated with DFS in NSCLC, although expression of the Th1/IFNγ-related genes was associated with the presence of lymphocytes in tumor samples in both indications.Conclusions:These findings provide evidence that expression of Th1/IFNγ genes in the TME, as measured by this GS, is associated with clinical outcome in melanoma. This suggests that, using this GS, patients with stage IIIB/C melanoma can be classified into different risk groups.

Purpose: To evaluate the expression of tumor PD-L1 and changes in tumor-infiltrating lymphocyte (TIL) populations in patients with metastatic melanoma treated with targeted MAPK inhibitors.Experimental Design: Ninety-three tumors were analyzed from 40 patients treated with a BRAF inhibitor alone (BRAFi; n = 28) or combination of BRAF and MEK inhibitors (Combi; n = 12). Tumors were excised before treatment (PRE), early during treatment (EDT), and at progression (PROG). Immunohistochemical staining was performed for CD4, CD8, CD68, FOXP3, LAG3, PD-1, and PD-L1 and correlated with clinical outcome.Results: Patients' tumors that were PD-L1 positive at baseline showed a significant decrease in PD-L1 expression at PROG (P = 0.028), whereas patients' tumors that were PD-L1 negative at baseline showed a significant increase in PD-L1 expression at PROG (P = 0.008) irrespective of treatment with BRAFi or Combi. Overall PD-L1 expression highly correlated with TIL immune markers. BRAFi-treated patients showed significant increases in CD4+, CD8+, and PD-1+ lymphocytes from PRE to EDT (P = 0.001, P = 0.001, P = 0.017, respectively), and Combi-treated patients showed similar increases in CD4+ and CD8+ lymphocytes from PRE to EDT (P = 0.017, P = 0.021).Conclusions: The addition of MEKi to BRAFi did not result in significant reduction in immune infiltration in EDT biopsies. This provides support for conducting trials that combine MAPKi with immune checkpoint inhibitors in the hope of improving complete and durable response rates. PD-L1 expression at PROG on MAPK inhibitors varied according to baseline expression suggesting that combining MAPKi with immunotherapies concurrently may be more effective in patients with PD-L1 expression and TILs in baseline melanoma samples. Clin Cancer Res; 21(14); 3140–8. ©2015 AACR.See related commentary by Cooper et al., p. 3102

Supplementary Figure 2. LAG3 and FoxP3 expressing lymphocytes at PRE, EDT and PROG. Patients treated with either BRAFi (A-B) or Combi (C-D) showed no significant changes in LAG3+ and FoxP3+ lymphocytes at any time point.

Supplementary Table 1. Patterns of PD-L1 staining at various time points of MAPK inhibitor treatment (n=55 PD-L1 positive patients).

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Supplementary Figure 1 from Nucleotide Excision Repair Gene Expression after Cisplatin Treatment in Melanoma

Supplementary Figure 3 from Inhibition of MEK Sensitizes Human Melanoma Cells to Endoplasmic Reticulum Stress-Induced Apoptosis

Past studies have shown that activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK is a common cause for resistance of melanoma cells to death receptor–mediated or mitochondria-mediated apoptosis. We report in this study that inhibition of the MEK/ERK pathway also sensitizes melanoma cells to endoplasmic reticulum (ER) stress-induced apoptosis, and this is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperon glucose-regulated protein 78 (GRP78) expression. Treatment with the ER stress inducer tunicamycin or thapsigargin did not induce significant apoptosis in the majority of melanoma cell lines, but resistance to these agents was reversed by the MEK inhibitor U0126 or MEK1 small interfering RNA (siRNA). Induction of apoptosis by ER stress when MEK was inhibited was caspase dependent with caspase-4, caspase-9, and caspase-3 being involved. Caspase-4 seemed to be the apical caspase in that caspase-4 activation occurred before activation of caspase-9 and caspase-3 and that inhibition of caspase-4 by a specific inhibitor or siRNA blocked activation of caspase-9 and caspase-3, whereas inhibition of caspase-9 or caspase-3 did not inhibit caspase-4 activation. Moreover, overexpression of Bcl-2 inhibited activation of caspase-9 and caspase-3 but had minimal effect on caspase-4 activation. Inhibition of MEK/ERK also resulted in down-regulation of GRP78, which was physically associated with caspase-4, before and after treatment with tunicamycin or thapsigargin. In addition, siRNA knockdown of GRP78 increased ER stress-induced caspase-4 activation and apoptosis. Taken together, these results seem to have important implications for new treatment strategies in melanoma by combinations of agents that induce ER stress and inhibitors of the MEK/ERK pathway. [Cancer Res 2007;67(20):9750–61]

We have previously shown that most melanoma cell lines are insensitive to endoplasmic reticulum (ER) stress–induced apoptosis, and this involves activation of the mitogen-activated protein/extracellular signal-regulated kinase (MEK)/ERK signaling pathway and expression of the apoptosis repressor with caspase recruitment domain (ARC) protein in the cells. In the present study, we show that up-regulation of the antiapoptotic Bcl-2 family member Mcl-1 is another mechanism critical for protection of melanoma cells against ER stress–induced apoptosis. Inhibition of Mcl-1 by small interference RNA (siRNA) rendered melanoma cells sensitive to apoptosis induced by the ER stress inducers thapsigargin and tunicamycin, but this sensitization was partially reversed by siRNA knockdown of PUMA or Noxa, as shown in Mcl-1–deficient melanoma cells. Both PUMA and Noxa were increased by ER stress through transcriptional up-regulation, but only up-regulation of Noxa was dependent on p53, whereas up-regulation of PUMA seemed to be mediated by a p53-independent mechanism(s). Up-regulation of Mcl-1 was also due to increased transcription that involved the IRE1α and activating transcription factor 6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway seemed to be necessary for optimal up-regulation of Mcl-1. Taken together, these results reveal the mechanisms of resistance of melanoma cells to apoptosis induction mediated by BH3-only proteins upon ER stress, and identify Mcl-1 as a target for the treatment of melanoma in combination with therapeutics that induce ER stress. [Cancer Res 2008;68(16):6708–17]

Supplementary Figure 2 from Inhibition of Endoplasmic Reticulum Stress–Induced Apoptosis of Melanoma Cells by the ARC Protein

Supplementary Figure 4 from Inhibition of MEK Sensitizes Human Melanoma Cells to Endoplasmic Reticulum Stress-Induced Apoptosis

Supplementary Figure 4 from Inhibition of Endoplasmic Reticulum Stress–Induced Apoptosis of Melanoma Cells by the ARC Protein

Supplementary Figure 2 from Inhibition of MEK Sensitizes Human Melanoma Cells to Endoplasmic Reticulum Stress-Induced Apoptosis

Supplementary Figure 1 from Inhibition of Endoplasmic Reticulum Stress–Induced Apoptosis of Melanoma Cells by the ARC Protein

Supplementary Figure 1 from Inhibition of MEK Sensitizes Human Melanoma Cells to Endoplasmic Reticulum Stress-Induced Apoptosis

Supplementary Figures 1-9 from Up-regulation of Mcl-1 Is Critical for Survival of Human Melanoma Cells upon Endoplasmic Reticulum Stress

Supplementary Figure 3 from Inhibition of Endoplasmic Reticulum Stress–Induced Apoptosis of Melanoma Cells by the ARC Protein

We have shown previously that most melanoma cell lines are insensitive to endoplasmic reticulum (ER) stress–induced apoptosis, but resistance can be reversed through activation of caspase-4 by inhibition of the MEK/ERK pathway. We report in this study that apoptosis was induced by the ER stress inducer thapsigargin or tunicamycin via a caspase-8–mediated pathway in the melanoma cell line Me1007, although the MEK/ERK pathway was activated in this cell line. The high sensitivity of Me1007 to ER stress–induced apoptosis was associated with low expression levels of the apoptosis repressor with caspase recruitment domain (ARC) protein that was expressed at relatively high levels in the resistant melanoma cell lines. Transfection of cDNA encoding ARC into Me1007 cells inhibited both caspase-8 activation and apoptosis induced by thapsigargin or tunicamycin. In contrast, inhibition of ARC by small interfering RNA knockdown sensitized the resistant melanoma cell lines to ER stress–induced apoptosis, which was inhibitable by blockage of caspase-8 activation. Both exogenous and endogenous ARC seemed to predominantly locate to the cytoplasm and mitochondria and could be coimmunoprecipitated with caspase-8. Taken together, ER stress can potentially activate multiple apoptosis signaling pathways in melanoma cells in a context-dependent manner. Whereas the MEK/ERK signaling pathway plays an important role in inhibiting ER stress–induced caspase-4 activation, ARC seems to be critical in blocking activation of casapse-8 in melanoma cells subjected to ER stress. [Cancer Res 2008;68(3):834–42]