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Supplementary Figure Legends

Figure S1. Generation of the LSL-N-RasQ61R allele. Figure S2. Conditional expression of N-Ras mutants does not alter melanocyte morphology. Figure S3. In both the heterozygous and homozygous state, NRasG12D fails to induce melanoma formation. Figure S4. Melanocyte-specific N-RASQ61R expression causes high penetrance nevus formation. Figure S5. Characteristics of Ras-mutant murine melanocytes and melanomas. Figure S6. Establishment and validation of TpLN61R/61R tumor cell lines. Figure S7. Binding of NRAS61R-mant-GMPPNP to PI3Kγ and RAF-RBD. Figure S8. NRASG12D and NRASQ61R similarly bind BRAF as measured by isothermal titration calorimetry. Figure S9. Proliferation of NRAS mutant human melanoma cell lines is not codon-specific. Figure S10. Activation of ERK and AKT in melanocytes is not codon-specific. Table S1. Frequency of canonical RAS mutations in human cancer. Table S2. Comparative stability of GMPPCP- and GDP- bound NRAS variants. Table S3. Summary of prior RAS-driven murine melanoma models.

Supplementary Figure S6. AIF knockdown did not affect the slow-cycling predominant MAPKi-addiction phenotype

Top 200 genes by KS.

Melanoma resistant to MAPK inhibitors (MAPKi) displays loss of fitness upon experimental MAPKi withdrawal and, clinically, may be resensitized to MAPKi therapy after a drug holiday. Here, we uncovered and therapeutically exploited the mechanisms of MAPKi addiction in MAPKi-resistant BRAFMUT or NRASMUT melanoma. MAPKi-addiction phenotypes evident upon drug withdrawal spanned transient cell-cycle slowdown to cell-death responses, the latter of which required a robust phosphorylated ERK (pERK) rebound. Generally, drug withdrawal–induced pERK rebound upregulated p38–FRA1–JUNB–CDKN1A and downregulated proliferation, but only a robust pERK rebound resulted in DNA damage and parthanatos-related cell death. Importantly, pharmacologically impairing DNA damage repair during MAPKi withdrawal augmented MAPKi addiction across the board by converting a cell-cycle deceleration to a caspase-dependent cell-death response or by furthering parthanatos-related cell death. Specifically in MEKi-resistant NRASMUT or atypical BRAFMUT melanoma, treatment with a type I RAF inhibitor intensified pERK rebound elicited by MEKi withdrawal, thereby promoting a cell death–predominant MAPKi-addiction phenotype. Thus, MAPKi discontinuation upon disease progression should be coupled with specific strategies that augment MAPKi addiction.Significance: Discontinuing targeted therapy may select against drug-resistant tumor clones, but drug-addiction mechanisms are ill-defined. Using melanoma resistant to but withdrawn from MAPKi, we defined a synthetic lethality between supraphysiologic levels of pERK and DNA damage. Actively promoting this synthetic lethality could rationalize sequential/rotational regimens that address evolving vulnerabilities. Cancer Discov; 8(1); 74–93. ©2017 AACR.See related commentary by Stern, p. 20.This article is highlighted in the In This Issue feature, p. 1

Supplementary Methods, Figure Legends

Supplementary Figure S1. Generation of GEMM-derived cell lines. Supplementary Figure S2. QPCR and Western blot analyses of DTYMK expression in 634 cells upon indicated shDtymks transduction. Supplementary Figure S3. Lkb1-wt 634 and Lkb1-null t4 cells transduced with shGFP, shDtymk-1 or shDtymk-3 and then cultured with or without additional 100 microM dTTP in medium for 4 days. Supplementary Figure S4. Lkb1-wt (634, 855, and 857) and Lkb1-null (t2, t4, and t5) cell lines (A) and human LKB1-wt (H358 and Calu-1) and LKB1-deficient H2122 and A549) NSCLC cell lines (B) were treated with CHEK1 inhibitors, AZD7762 and CHIR124, for the indicated dose and time, and then lysed for Western blot analysis of gammaH2AX. Supplementary Figure S5. Immunohistochemical analyses assessing gammaH2AZ. Supplementary Figure S6. QPCR and Western blot analyses of DTYMK expression in LKB1-wt Calu-1 NSCLC cells. Supplementary Figure S7. A schematic model is proposed based on our current data. Supplementary Figure S8. QPCR analyses of Dtymk (A) and Chek1 (B) transcript levels in Lkb1-wt (634, 855, and 857) and Lkb1-null (t2, t4, and t5) cell lines. Supplementary Figure S9. QPCR analysis of TK1 expression in 634 cells upon the indicated shTK1 transduction for 3 days. Supplementary Figure S10. Gene expression levels of DTYMK in normal lung vs. lung adenocarcinoma from the Oncomine Cancer Microarray database.

Figure S1. Characterization of cells and CDK4/6 inhibitors; Figure S2. CDK6 phosphorylates serine residues of the regulatory domain of NFAT4 (NFATc3); Figure S3. Analysis of lung tumor immune infiltrates after CDK4/6 inhibition from KrasG12D (Kras), KrasG12DLkb1 (KL) or KrasG12DTrp53fl/fl (KP) mice; Figure S4. T cell proliferation and cytokine/chemokine profiling of KrasG12DTrp53fl/fl GEMM mice; Figure S5. Tumor antigen experienced T cells are more sensitive to CDK4/6 inhibition; Figure S6. Short-term CDK4/6 inhibition alters the cell cycle status of tumor infiltrating T cells; Figure S7. CDK4/6 inhibition induces changes in the expression of activation and suppression marker genes in tumor-infiltrating T cells; Figure S8. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody elicits anti-tumor immunity; Figure S9. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody on established tumor; Figure S10. Effect of TCR stimulation and CDK4/6 inhibition on phosphorylation of NFkB; Supplementary Table S1; Supplmentary Table S2; Supplementary Table S3: Selected genes reported to be regulated by NFAT

Supplementary Figures S1 - S10, Tables S1 - S2

NRAS mutation at codons 12, 13, or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an NrasQ61R knock-in allele to similarly designed KrasG12D and NrasG12D alleles. With concomitant p16INK4a inactivation, KrasG12D or NrasQ61R expression efficiently promoted melanoma in vivo, whereas NrasG12D did not. In addition, NrasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of NrasQ61R and NrasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, NrasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity, and increased stability when compared with NrasG12D. This work identifies a faithful model of human NRAS-mutant melanoma, and suggests that the increased melanomagenecity of NrasQ61R over NrasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways.Significance: This work explains the curious predominance in human melanoma of mutations of codon 61 of NRAS over other oncogenic NRAS mutations. Using conditional “knock-in” mouse models, we show that physiologic expression of NRASQ61R, but not NRASG12D, drives melanoma formation. Cancer Discov; 4(12); 1418–29. ©2014 AACR.This article is highlighted in the In This Issue feature, p. 1355

MEKi Experiment Overview

Purpose: To use genetically engineered mouse models (GEMM) and orthotopic syngeneic murine transplants (OST) to develop gene expression-based predictors of response to anticancer drugs in human tumors. These mouse models offer advantages including precise genetics and an intact microenvironment/immune system.Experimental Design: We examined the efficacy of 4 chemotherapeutic or targeted anticancer drugs, alone and in combination, using mouse models representing 3 distinct breast cancer subtypes: Basal-like (C3(1)-T-antigen GEMM), Luminal B (MMTV-Neu GEMM), and Claudin-low (T11/TP53−/− OST). We expression-profiled tumors to develop signatures that corresponded to treatment and response, and then tested their predictive potential using human patient data.Results: Although a single agent exhibited exceptional efficacy (i.e., lapatinib in the Neu-driven model), generally single-agent activity was modest, whereas some combination therapies were more active and life prolonging. Through analysis of RNA expression in this large set of chemotherapy-treated murine tumors, we identified a pair of gene expression signatures that predicted pathologic complete response to neoadjuvant anthracycline/taxane therapy in human patients with breast cancer.Conclusions: These results show that murine-derived gene signatures can predict response even after accounting for common clinical variables and other predictive genomic signatures, suggesting that mice can be used to identify new biomarkers for human patients with cancer. Clin Cancer Res; 19(17); 4889–99. ©2013 AACR.

Expanded detail on MIB MS experiment

Structural reorganization and amelanotic intratumoral nests are evident in persistent melanoma on MEKi.

PDF file, 53K, MP1U Dosing Regimens.

Kinome and transcriptome responses to combined BRAF and MEK inhibition in murine tumors, patient samples, and human cell lines

XLSX file - 16K, List of mouse C3(1)-T-antigen gene expression microarrays used to derive the murine gene lists.

PDF file - 42K, Summary of drugs used in this study.

PDF file - 68K

PDF file - 806K, Supplemental Figure 1. MRI scans at the indicated time points showing tumor burden in EML4-ALK lung cancer mice treated by crizotinib. Multiple scans from a total of 4 mice are shown. Supplemental Figure 2. Representative MRI images showing that EML4-ALK F1174L mutant lung cancers respond to 17-DMAG and TAE684. Note the development of acquired resistance to 17-DMAG and TAE684 after prolonged treatment. Supplemental Figure 3. Acquired resistance to 17-DMAG and TAE684 in mice bearing tumors driven by the EML4-ALK F1174L mutant can be overcome by the combination of the two drugs.

Supplementary Table 1. Clinical and histologic characteristics of cutaneous melanocytic nevi, primary melanomas, and melanoma metastases and their relationship to ITK protein levels

PDF file - 1539K, Kaplan-Meier analyses for the prediction of Distant Relapse Free Survival.

Graphical representation and model of results

Legends for Supplementary Figures 1 and 2

mRNAseq pathway signature analysis reveals gene expression reprogramming on MEKi.

BRAFV600E and PTEN-loss are required for tumor growth, which is temporally controlled and reproducible at the cell level.

Purpose: To extend the results of a phase III trial in patients with non–small cell lung cancer with adenocarcinomas harboring EML4-ALK fusion.Experimental Design: We conducted a co-clinical trial in a mouse model comparing the ALK inhibitor crizotinib to the standard-of-care cytotoxic agents docetaxel or pemetrexed.Results: Concordant with the clinical outcome in humans, crizotinib produced a substantially higher response rate compared with chemotherapy, associated with significantly longer progression-free survival. Overall survival was also prolonged in crizotinib- compared with chemotherapy-treated mice. Pemetrexed produced superior overall survival compared with docetaxel, suggesting that this agent may be the preferred chemotherapy in the ALK population. In addition, in the EML4-ALK–driven mouse lung adenocarcinoma model, HSP90 inhibition can overcome both primary and acquired crizotinib resistance. Furthermore, HSP90 inhibition, as well as the second-generation ALK inhibitor TAE684, demonstrated activity in newly developed lung adenocarcinoma models driven by crizotinib-insensitive EML4-ALK L1196M or F1174L.Conclusions: Our findings suggest that crizotinib is superior to standard chemotherapy in ALK inhibitor–naïve disease and support further clinical investigation of HSP90 inhibitors and second-generation ALK inhibitors in tumors with primary or acquired crizotinib resistance. Clin Cancer Res; 20(5); 1204–11. ©2013 AACR.

XLSX file - 60K, Gene lists obtained from a study of carboplatin/paclitaxel treated tumors.

PDF file - 46K, Univariate and Multivariate Analysis for pCR.

Purpose: Anticancer drug development is inefficient, but genetically engineered murine models (GEMM) and orthotopic, syngeneic transplants (OST) of cancer may offer advantages to in vitro and xenograft systems.Experimental Design: We assessed the activity of 16 treatment regimens in a RAS-driven, Ink4a/Arf-deficient melanoma GEMM. In addition, we tested a subset of treatment regimens in three breast cancer models representing distinct breast cancer subtypes: claudin-low (T11 OST), basal-like (C3-TAg GEMM), and luminal B (MMTV-Neu GEMM).Results: Like human RAS-mutant melanoma, the melanoma GEMM was refractory to chemotherapy and single-agent small molecule therapies. Combined treatment with AZD6244 [mitogen-activated protein–extracellular signal-regulated kinase kinase (MEK) inhibitor] and BEZ235 [dual phosphoinositide-3 kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor] was the only treatment regimen to exhibit significant antitumor activity, showed by marked tumor regression and improved survival. Given the surprising activity of the “AZD/BEZ” combination in the melanoma GEMM, we next tested this regimen in the “claudin-low” breast cancer model that shares gene expression features with melanoma. The AZD/BEZ regimen also exhibited significant activity in this model, leading us to testing in even more diverse GEMMs of basal-like and luminal breast cancer. The AZD/BEZ combination was highly active in these distinct breast cancer models, showing equal or greater efficacy compared with any other regimen tested in studies of over 700 tumor-bearing mice. This regimen even exhibited activity in lapatinib-resistant HER2+ tumors.Conclusion: These results show the use of credentialed murine models for large-scale efficacy testing of diverse anticancer regimens and predict that combinations of PI3K/mTOR and MEK inhibitors will show antitumor activity in a wide range of human malignancies. Clin Cancer Res; 18(19); 5290–303. ©2012 AACR.

Supplementary Figure 2. Ingenuity Pathway Analysis Results from RPPA assay and effects of ITK activity on specific cellular proteins by westerns and RPPA results.

Purpose: IL2 inducible T-cell kinase (ITK) promoter CpG sites are hypomethylated in melanomas compared with nevi. The expression of ITK in melanomas, however, has not been established and requires elucidation.Experimental Design: An ITK-specific monoclonal antibody was used to probe sections from deidentified, formalin-fixed paraffin-embedded tumor blocks or cell line arrays and ITK was visualized by IHC. Levels of ITK protein differed among melanoma cell lines and representative lines were transduced with four different lentiviral constructs that each contained an shRNA designed to knockdown ITK mRNA levels. The effects of the selective ITK inhibitor BI 10N on cell lines and mouse models were also determined.Results: ITK protein expression increased with nevus to metastatic melanoma progression. In melanoma cell lines, genetic or pharmacologic inhibition of ITK decreased proliferation and migration and increased the percentage of cells in the G0–G1 phase. Treatment of melanoma-bearing mice with BI 10N reduced growth of ITK-expressing xenografts or established autochthonous (Tyr-Cre/Ptennull/BrafV600E) melanomas.Conclusions: We conclude that ITK, formerly considered an immune cell–specific protein, is aberrantly expressed in melanoma and promotes tumor development and progression. Our finding that ITK is aberrantly expressed in most metastatic melanomas suggests that inhibitors of ITK may be efficacious for melanoma treatment. The efficacy of a small-molecule ITK inhibitor in the Tyr-Cre/Ptennull/BrafV600E mouse melanoma model supports this possibility. Clin Cancer Res; 21(9); 2167–76. ©2015 AACR.

Supplementary Table 6. Significant pathways identified by Ingenuity Pathway Analysis for Reverse Phase Protein Array (RPPA) results of RPMI 8322

XLSX file - 32K, Modules/expression signatures analysis of carboplatin/paclitaxel treated C3(1)-T-antigen mouse tumors.

Supplementary Tables 3 and 4. Supplementary Table 3. ITK protein expression levels in cell lines by single fluorescent immunocytochemistry Supplementary Table 4. Statistical analysis of shRNA and BI 10N effects on the proliferation and motility rates of melanoma cell lines

Sup Fig Legends

Achievement of reproducible spatial and temporal control of melanoma growth with

PDF file - 3255K, Hierarchical clustering analysis of the untreated and responding murine chemotherapy signature using 337 human breast tumors.

Supplementary Figure 1. IHC staining for co-localization of immune markers with cells expressing ITK and determining the presence of ITK in immune cell subtypes infiltrating the tumor and the effect of ITK ablation on the cell cycle of the melanoma cells

Supplementary Figures 1-2, Tables 1 and 3-7 from Lack of Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Signaling Shows a New Type of Melanoma

Supplementary Table 2 from Lack of Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Signaling Shows a New Type of Melanoma