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Patients with BRAF-mutant melanoma show significant responses to combined BRAF and MEK inhibition, but most relapse within 2 years. A major reservoir for drug resistance is minimal residual disease (MRD), comprised of drug-tolerant tumor cells laying in a dormant state. Towards exploiting potential therapeutic vulnerabilities of MRD, we established a genetically engineered mouse model of BrafV600E-driven melanoma MRD wherein genetic BrafV600Eextinction leads to strong but incomplete tumor regression. Transcriptional time-course analysis after BrafV600Eextinction revealed that after an initial surge of immune activation, tumors later became immunologically “cold” after MRD establishment. Computational analysis identified candidate T-cell recruiting chemokines that may be central players in the process, being strongly upregulated initially and steeply decreasing as the immune response faded. Therefore, we hypothesized that sustaining the chemokine signaling could impair MRD maintenance through increased recruitment of effector T-cells. We show that intratumoral administration of recombinant Cxcl9, either naked or loaded in microparticles, significantly impaired MRD relapse in BRAF-inhibited tumors, including several complete responses after microparticle-delivered rCxcl9 combined with BRAF and MEK-inhibition. Our experiments constitute a proof of concept that chemokine-based microparticle delivery systems are a potential strategy to forestall tumor relapse and thus improve the clinical success of frontline treatment methods.

KRAS and NRAS mutations occur in 45% of colorectal cancers, with combined MAPK pathway and CDK4/6 inhibition identified as a potential therapeutic strategy. In the current study, this combinatorial treatment approach was evaluated in a co-clinical trial in patient-derived xenografts (PDX), and safety was established in a clinical trial of binimetinib and palbociclib in patients with metastatic colorectal cancer with RAS mutations. Across 18 PDX models undergoing dual inhibition of MEK and CDK4/6, 60% of tumors regressed, meeting the co-clinical trial primary endpoint. Prolonged duration of response occurred predominantly in TP53 wild-type models. Clinical evaluation of binimetinib and palbociclib in a safety lead-in confirmed safety and provided preliminary evidence of activity. Prolonged treatment in PDX models resulted in feedback activation of receptor tyrosine kinases and acquired resistance, which was reversed with a SHP2 inhibitor. These results highlight the clinical potential of this combination in colorectal cancer, along with the utility of PDX-based co-clinical trial platforms for drug development. Significance: This co-clinical trial of combined MEK-CDK4/6 inhibition in RAS mutant colorectal cancer demonstrates therapeutic efficacy in patient-derived xenografts and safety in patients, identifies biomarkers of response, and uncovers targetable mechanisms of resistance.

Supplementary Tables S1-S3

Supplementary Figures S1-S5

Patients with BRAF-mutant melanoma show substantial responses to combined BRAF and MEK inhibition, but most relapse within 2 years. A major reservoir for drug resistance is minimal residual disease (MRD), comprised of drug-tolerant tumor cells laying in a dormant state. Towards exploiting potential therapeutic vulnerabilities of MRD, we established a genetically engineered mouse model of BrafV600E-driven melanoma MRD wherein genetic BrafV600E extinction leads to strong but incomplete tumor regression. Transcriptional time-course analysis after BrafV600E extinction revealed that after an initial surge of immune activation, tumors later became immunologically “cold” after MRD establishment. Computational analysis identified candidate T-cell recruiting chemokines as strongly upregulated initially and steeply decreasing as the immune response faded. Therefore, we hypothesized that sustaining chemokine signaling could impair MRD maintenance through increased recruitment of effector T cells. We found that intratumoral administration of recombinant Cxcl9 (rCxcl9), either naked or loaded in microparticles, significantly impaired MRD relapse in BRAF-inhibited tumors, including several complete pathologic responses after microparticle-delivered rCxcl9 combined with BRAF and MEK inhibition. Our experiments constitute proof of concept that chemokine-based microparticle delivery systems are a potential strategy to forestall tumor relapse and thus improve the clinical success of first-line treatment methods.

List of genes which are differentially expressed in melanomas with PTEN loss.

Supplementary Figures S1-S13

This file contains supplementary methods, supplementary figure legends, supplementary table legends and supplementary references.

Targeting cyclin-dependent kinases 4/6 (CDK4/6) represents a therapeutic option in combination with BRAF inhibitor and/or MEK inhibitor (MEKi) in melanoma; however, continuous dosing elicits toxicities in patients. Using quantitative and temporal in vivo reporting, we show that continuous MEKi with intermittent CDK4/6 inhibitor (CDK4/6i) led to more complete tumor responses versus other combination schedules. Nevertheless, some tumors acquired resistance that was associated with enhanced phosphorylation of ribosomal S6 protein. These data were supported by phospho-S6 staining of melanoma biopsies from patients treated with CDK4/6i plus targeted inhibitors. Enhanced phospho-S6 in resistant tumors provided a therapeutic window for the mTORC1/2 inhibitor AZD2014. Mechanistically, upregulation or mutation of NRAS was associated with resistance in in vivo models and patient samples, respectively, and mutant NRAS was sufficient to enhance resistance. This study utilizes an in vivo reporter model to optimize schedules and supports targeting mTORC1/2 to overcome MEKi plus CDK4/6i resistance.Significance: Mutant BRAF and NRAS melanomas acquire resistance to combined MEK and CDK4/6 inhibition via upregulation of mTOR pathway signaling. This resistance mechanism provides the preclinical basis to utilize mTORC1/2 inhibitors to improve MEKi plus CDK4/6i drug regimens. Cancer Discov; 8(5); 568–81. ©2018 AACR.See related commentary by Sullivan, p. 532.See related article by Romano et al., p. 556.This article is highlighted in the In This Issue feature, p. 517

Supplementary Figure 1. Western blots. Supplementary Figure 2. Graphs showing tumor volume from mice following intradermal injection of (A) HMEL-BRAF^V600E cells, (B) WM115 cells, (C) 1205 Lu cells, and (D) pMEL-NRAS^G12D overexpressing GFP, RNF2 wild-type or catalytic mutant derivatives (R70C or I53S). Supplementary Figure 3. (A) RNF expression in the melanoma expression data for normal skin, nevi, and primary tumors. Supplementary Figure 4. (A) Heat map showing clustering of top 10 percent deregulated genes in duplicates of expression data from RNF2^WT overexpressing compared to GFP overexpressing HMEL-BRAF^V600E cells. Supplementary Figure 5. Chromatin state analysis on HMEL-BRAF^V600E cells.

Description of states in 45-state chromatin state model.This table describes the biological annotation of each chromatin state in 50-state chromatin model from HMEL-BRAFV600E cells from ChromHMM program.

Supplementary Figure 1. Immune profiling of pre-treatment, on-treatment and progression CTLA-4 blockade samples by immunohistochemistry. Supplementary Figure 2. Myeloid cell profiling of pre-treatment, on-treatment and progression CTLA-4 blockade samples by immunohistochemistry. Supplementary Figure 3. Increased contact between CD8 T cells and CD68 myeloid cells in non-responding patients to anti-CTLA-4 and anti-PD-1 therapy at pre-treatment CTLA-4 blockade, pre-treatment PD-1 blockade, and on-treatment PD-1 blockade time points. Supplementary Figure 4. Immune profiling of pre anti-PD-1, on-treatment anti-PD-1 and progression anti-PD-1 samples by immunohistochemistry. Supplementary Figure 5. Longitudinal increase in CD8, PD-1, and PD-L1 expression in responders to anti-PD-1 therapy. Supplementary Figure 6. Relative increase in CD8 T cell infiltrate at tumor center in responders to anti-PD-1 on treatment. Supplementary Figure 7. Significant increase in immune infiltrate between responders and non-responders to PD-1 blockade in absence of prior anti-CTLA-4 therapy. Supplementary Figure 8. Immune profiling of myeloid cells atpre-treatment and on-treatment PD-1 blockade time pointsby immunohistochemistry. Supplementary Figure 9. Heatmap of 54 NanoString samples. Supplementary Figure 10. Gene-specific NanoString concordance with immune profiling by IHC in pre-treatment, on-treatment and progression CTLA-4 blockade samples. Supplementary Figure 11. Gene-specific NanoString concordance with immune profiling by IHC in pre-treatment, on-treatment and progression PD-1 blockade samples. Supplementary Figure 12. Prior CTLA-4 blockade is not required for PD-1 early on-treatment profile. Supplementary Figure 13. Hierarchical clustering of gene expression across 54 samples confirms lack of batch effect.

Supplementary Figure Legends

Supplementary Figures

Supplementary Figure Legends

Supplementary Tables S1-S5

Supplementary Table S1a. Patient Cohort Clinical Summary. Supplementary Table S1b. Patient clinical characteristics. Supplementary Table S1c. Immune profiling by IHC sample log. Supplementary Table S1d. Nanostring 54 sample log. Supplementary Table S2. Summary of immune profiling by immunohistochemistry. Supplementary Table S3. Summary of immune profiling by 4 additional myeloid markers. Supplementary Table S4. Summary of immune profiling by IHC of additional CTLA-4 blockade-naïve samples. Supplementary Table S5. Nanostring Gene List. Supplementary Table S6a. Nanostring summary 54 samples. Supplementary Table S6b. Differentially upregulated and downregulated genes in pre-treatment anti-CTLA-4 samples. Supplementary Table S6c. Differentially upregulated and downregulated genes in on-treatment anti-CTLA-4 samples. Supplementary Table S6d. Differentially upregulated and downregulated genes in pre-treatment anti-PD-1 samples. Supplementary Table S6e. Differentially upregulated and downregulated genes in on-treatment anti-PD-1 samples. Supplementary Table S7. Differentially upregulated and downregulated genes from pre- to on-treatment anti-CTLA-4. Supplementary Table S8. Differentially upregulated and downregulated genes from pre- to on-treatment anti-PD-1. Supplementary Table S9a. Nanostring gene list - chip used for comparison of CTLA-4 blockade-experienced vs -naive anti-PD1 samples (28 samples). Supplementary Table S9b. NanoString additional 28 samples to compare CTLA-4 blockade-experienced vs -naive anti-PD1 samples. Supplementary Table S9c. NanoString normalized data of additional 28 samples to compare CTLA-4 blockade-experienced vs -naive anti-PD1 samples. Supplementary Table S10. Commonly differentially regulated genes between pre- to on-treatment CTLA-4 blockade and PD-1 blockade. Supplementary Table S11a. Fold changes of significant change by anti-PD-1 therapy for paired samples. Supplementary Table S11b. Frequency of significant change by anti-PD1 therapy for paired samples.

Materials and Methods

Binding sites for RNF2 in HMEL-RNF2WT cells. This file contains list of all chromosomal locations that show enrichment by MACS for RNF2 binding by V5 ChIP-Seq in HMEL-RNF2 cells.

Supplementary Table S1. Patient characteristics of the single-agent anti-PD-1 cohort. Supplementary Table S2. List of primers used for gene expression analysis by real-time PCR. Supplementary Table S3. List of autophagy-related genes for overexpressing experiment.

Supplementary Figures

Supplementary Table

Metastatic melanoma is challenging to clinically address. Although standard-of-care targeted therapy has high response rates in patients with BRAF-mutant melanoma, therapy relapse occurs in most cases. Intrinsically resistant melanoma cells drive therapy resistance and display molecular and biologic properties akin to neural crest-like stem cells (NCLSC) including high invasiveness, plasticity, and self-renewal capacity. The shared transcriptional programs and vulnerabilities between NCLSCs and cancer cells remains poorly understood. Here, we identify a developmental LPAR1-axis critical for NCLSC viability and melanoma cell survival. LPAR1 activity increased during progression and following acquisition of therapeutic resistance. Notably, genetic inhibition of LPAR1 potentiated BRAFi ± MEKi efficacy and ablated melanoma migration and invasion. Our data define LPAR1 as a new therapeutic target in melanoma and highlights the promise of dissecting stem cell–like pathways hijacked by tumor cells.Significance:This study identifies an LPAR1-axis critical for melanoma invasion and intrinsic/acquired therapy resistance.

Cell-free DNA (cfDNA) in the circulating blood plasma of patients with cancer contains tumour-derived DNA sequences that can serve as biomarkers for guiding therapy, for the monitoring of drug resistance, and for the early detection of cancers. However, the analysis of cfDNA for clinical diagnostic applications remains challenging because of the low concentrations of cfDNA, and because cfDNA is fragmented into short lengths and is susceptible to chemical damage. Barcodes of unique molecular identifiers have been implemented to overcome the intrinsic errors of next-generation sequencing, which is the prevailing method for highly multiplexed cfDNA analysis. However, a number of methodological and pre-analytical factors limit the clinical sensitivity of the cfDNA-based detection of cancers from liquid biopsies. In this Review, we describe the state-of-the-art technologies for cfDNA analysis, with emphasis on multiplexing strategies, and discuss outstanding biological and technical challenges that, if addressed, would substantially improve cancer diagnostics and patient care.

Supplementary materials and methods

Table S5, related to Figure S5C

Time-lapse imaging of A375(Fucci2)-BR cells treated with the control.

Supplementary Figure from Targeting RAS Mutant Colorectal Cancer with Dual Inhibition of MEK and CDK4/6

Contains Supplementary materials and methods, Supplementary Figures S1-S9, Table S2, Table S4, Supplemental References Figure S1, related to Figure 1. Cell surface marker screening of A375P cells treated with vehicle (DMSO) or PLX. Figure S2, related to Figure 1. CD36 is a biomarker of the early stage of MAPK inhibition in melanoma. Figure S3, related to Figure 1. MAPK inhibition induces and maintains the CD36+ population in melanoma cells Figure S4, related to Figure 2. MAPKi induce the expression of genes involved in lipid catabolism in BRAFV600E melanomas. Figure S5, related to Figure 3. MAPK inhibition decreased the glycolytic flux in BRAF-mutated melanoma cells. Figure S6, related to Figure 4. Phenotypic and metabolic characterization of CD36 WT, CD36 KO, CD36+ and CD36- A375P cells. Figure S7, related to Figure 5. PPARα does not regulate CD36 expression. Figure S8, related to Figure 6. Role of glycolysis and FAO inhibitors in proliferation and resistance to MAPKi Figure S9, related to Figure 6. Effects of MAPK, glycolysis and FAO inhibition on mouse and tumor weight. Table S2, related to Figure 1, Figure 3, Figure 6, Figure S3 and Figure S6. Primer list. Table S4, related to Figure 1 and Figure S2. Cell lines, their mutational status and their responsiveness to MAPK inhibitors.

Table S1 Mean density values of crystal violet staining of 16 melanoma cell lines treated with 6-thio-dG and PLX4720. Table S2 RNA-seq data related to A375 cells treated with 6-thio-dG. Table S3 RNA-seq data related to A375 cells treated with BIBR 1532. Table S4 Apoptotic index and ssGSEA scores of 11 gene sets in 18 CCLE melanoma cell lines. Table S5 RPPA data related to A375 cells treated with 6-thio-dG. Table S6 Mean density values of crystal violet staining of 6 therapy-resistant melanoma cell lines treated with 6-thio-dG. Table S7 RPPA data related to the comparison between LOX-IMVI parental and BR cells. Table S8 RPPA data related to LOX-IMVI parental cells treated with 6-thio-dG. Table S9 RPPA data related to LOX-IMVI BR cells treated with 6-thio-dG. Table S10 RPPA data related to LOX-IMVI BR xenografts treated with 6-thio-dG. Table S11 The clinical information from patients with metastatic melanoma treated with immunotherapies.

KRAS and NRAS mutations occur in 45% of colorectal cancers, with combined MAPK pathway and CDK4/6 inhibition identified as a potential therapeutic strategy. In the current study, this combinatorial treatment approach was evaluated in a co-clinical trial in patient-derived xenografts (PDX), and safety was established in a clinical trial of binimetinib and palbociclib in patients with metastatic colorectal cancer with RAS mutations. Across 18 PDX models undergoing dual inhibition of MEK and CDK4/6, 60% of tumors regressed, meeting the co-clinical trial primary endpoint. Prolonged duration of response occurred predominantly in TP53 wild-type models. Clinical evaluation of binimetinib and palbociclib in a safety lead-in confirmed safety and provided preliminary evidence of activity. Prolonged treatment in PDX models resulted in feedback activation of receptor tyrosine kinases and acquired resistance, which was reversed with a SHP2 inhibitor. These results highlight the clinical potential of this combination in colorectal cancer, along with the utility of PDX-based co-clinical trial platforms for drug development.Significance:This co-clinical trial of combined MEK-CDK4/6 inhibition in RAS mutant colorectal cancer demonstrates therapeutic efficacy in patient-derived xenografts and safety in patients, identifies biomarkers of response, and uncovers targetable mechanisms of resistance.

Supplementary Table from Targeting RAS Mutant Colorectal Cancer with Dual Inhibition of MEK and CDK4/6

Purpose: Telomerase promoter mutations are highly prevalent in human tumors including melanoma. A subset of patients with metastatic melanoma often fail multiple therapies, and there is an unmet and urgent need to prolong disease control for those patients.Experimental Design: Numerous preclinical therapy-resistant models of human and mouse melanoma were used to test the efficacy of a telomerase-directed nucleoside, 6-thio-2′-deoxyguanosine (6-thio-dG). Integrated transcriptomics and proteomics approaches were used to identify genes and proteins that were significantly downregulated by 6-thio-dG.Results: We demonstrated the superior efficacy of 6-thio-dG both in vitro and in vivo that results in telomere dysfunction, leading to apoptosis and cell death in various preclinical models of therapy-resistant melanoma cells. 6-thio-dG concomitantly induces telomere dysfunction and inhibits the expression level of AXL.Conclusions: In summary, this study shows that indirectly targeting aberrant telomerase in melanoma cells with 6-thio-dG is a viable therapeutic approach in prolonging disease control and overcoming therapy resistance. Clin Cancer Res; 24(19); 4771–84. ©2018 AACR.See related commentary by Teh and Aplin, p. 4629

Purpose:Treatment of BRAFV600E-mutant melanomas with MAPK inhibitors (MAPKi) results in significant tumor regression, but acquired resistance is pervasive. To understand nonmutational mechanisms underlying the adaptation to MAPKi and to identify novel vulnerabilities of melanomas treated with MAPKi, we focused on the initial response phase during treatment with MAPKi.Experimental Design:By screening proteins expressed on the cell surface of melanoma cells, we identified the fatty acid transporter CD36 as the most consistently upregulated protein upon short-term treatment with MAPKi. We further investigated the effects of MAPKi on fatty acid metabolism using in vitro and in vivo models and analyzing patients' pre- and on-treatment tumor specimens.Results:Melanoma cells treated with MAPKi displayed increased levels of CD36 and of PPARα-mediated and carnitine palmitoyltransferase 1A (CPT1A)-dependent fatty acid oxidation (FAO). While CD36 is a useful marker of melanoma cells during adaptation and drug-tolerant phases, the upregulation of CD36 is not functionally involved in FAO changes that characterize MAPKi-treated cells. Increased FAO is required for BRAFV600E-mutant melanoma cells to survive under the MAPKi-induced metabolic stress prior to acquiring drug resistance. The upfront and concomitant inhibition of FAO, glycolysis, and MAPK synergistically inhibits tumor cell growth in vitro and in vivo.Conclusions:Thus, we identified a clinically relevant therapeutic approach that has the potential to improve initial responses and to delay acquired drug resistance of BRAFV600E-mutant melanoma.

Supplementary Data from Targeting RAS Mutant Colorectal Cancer with Dual Inhibition of MEK and CDK4/6

Supplementary Fig. 1: Related to Fig. 2 (A) The percentage of apoptotic and dead cells indicated as PSVue 643+ cells in each of 12 BRAF-mutant melanoma cell lines treated with PLX4720 or 6-thio-dG at indicated doses for 120 hours. Cells were then harvested and co-stained with PSVue643 and Propidium iodide (PI). The average of 2 biological replicates was plotted. (B) SA-β-gal staining of 4 BRAF-mutant melanoma cell lines treated with 6-thio-dG at 5μM for 9 days. A representative image of 3 biological replicates was shown for each experimental sample. (C and D) Mouse weights of 1205Lu (C) and A375 (D) xenografts in each treatment group were shown. upplementary Fig. 2: Related to Fig. 4 (A) Long-term cell growth assay of 4 melanoma cell lines that acquired resistance to MAPKi treated with 6-thio-dG at indicated doses for 12 days. Cells were then fixed and stained with 3 crystal violet. A representative image of 2 biological replicates was shown for each experimental condition. (B-D) Tumor volumes of WM9 BR (B), UACC-903 BR (C) and A2058 CR (D) xenografts that were treated with the vehicle control and 6-thio-dG at indicated doses. (E) The ssGSEA plot of 11 MSigDB gene sets related to telomere and telomerase that were significantly altered in LOX-IMVI BR cells treated with BIBR 1532 (BIBR) or 6-thio-dG (6dG). (F-I) The heatmaps of RPPA data depicting 30 proteins that were most significantly down-regulated in four representative BR cell lines treated with 6-thio-dG, including A375 BR (F), WM3936-1 (G), 1205Lu BR (H) and WM1552C BR (I). (J) Western blotting of proteins that were downregulated in LOX-IMVI BR cells treated with 6-thio-dG (6dG). (K) The immnohistochemical analysis of AXL was performed in A375 tumors that were treated with the control, 6-thio-dG and Dabrafenib. Supplementary Fig. S3: Related to Fig. 5 (A-C) The heatmaps of two telomere transcriptional gene signatures, two melanoma-specific gene sets and two MAPK pathway-related gene sets in three datasets in which transcriptomes of paired pre- and post-treatment tumor biopsies derived from patients who progressed on MAPKi were profiled. 5 Supplementary Fig. S4: Related to Fig. 5 6 The heatmaps of two telomere transcriptional gene signatures, two melanoma-specific gene sets and two MAPK pathway-related gene sets in paired pre-, on- and post-treatment tumor biopsies derived from 16 patients who were treated with immunotherapies. Supplementary Fig. S5: Related to Fig. 6 (A-E) The heatmaps of RPPA data depicting 30 proteins that were most significantly downregulated in 5 short-term cultures or cell lines derived from immunotherapy-resistant tumors that were treated with 6-thio-dG, including 13-456-3-3 (A), 15-1761-1-2 (B), WM4265-1 (C), WM4265-2 (D) and G43 (E).

PDF file - 59KB, RAS/RAF mutations correlate with chr 10 status.

PDF file - 328KB, Correlation of DNA copy number aberrations to survival statistics.

XLSX file - 131KB, Genotypes and characteristics of human melanoma cell lines used in this study.

XLSX file - 68KB, The shRNA pools used in the iNRAS-463 primary in vivo screen.

PDF file - 57KB

Although many DNA aberrations in melanoma have been well characterized, including focal amplification and deletions of oncogenes and tumor suppressors, broad regions of chromosomal gain and loss are less well understood. One possibility is that these broad events are a consequence of collateral damage from targeting single loci. Another possibility is that the loss of large regions permits the simultaneous repression of multiple tumor suppressors by broadly decreasing the resident gene dosage and expression. Here, we test this hypothesis in a targeted fashion using RNA interference to suppress multiple candidate residents in broad regions of loss. We find that loss of chromosome regions 6q, 10, and 11q21-ter is correlated with broadly decreased expression of most resident genes and that multiple resident genes impacted by broad regional loss of chromosome 10 are tumor suppressors capable of affecting tumor growth and/or invasion. We also provide additional functional support for Ablim1 as a novel tumor suppressor. Our results support the hypothesis that multiple cancer genes are targeted by regional chromosome copy number aberrations. Cancer Res; 74(6); 1814–21. ©2014 AACR.