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With the combination of KRASG12C and EGFR inhibitors, KRAS is becoming a druggable target in colorectal cancer. However, secondary resistance limits its efficacy. Using cell lines, patient-derived xenografts, and patient samples, we detected a heterogeneous pattern of putative resistance alterations expected primarily to prevent inhibition of ERK signaling by drugs at progression. Serial analysis of patient blood samples on treatment demonstrates that most of these alterations are detected at a low frequency except for KRASG12C amplification, a recurrent resistance mechanism that rises in step with clinical progression. Upon drug withdrawal, resistant cells with KRASG12C amplification undergo oncogene-induced senescence, and progressing patients experience a rapid fall in levels of this alteration in circulating DNA. In this new state, drug resumption is ineffective as mTOR signaling is elevated. However, our work exposes a potential therapeutic vulnerability, whereby therapies that target the senescence response may overcome acquired resistance. Significance: Clinical resistance to KRASG12C–EGFR inhibition primarily prevents suppression of ERK signaling. Most resistance mechanisms are subclonal, whereas KRASG12C amplification rises over time to drive a higher portion of resistance. This recurrent resistance mechanism leads to oncogene-induced senescence upon drug withdrawal and creates a potential vulnerability to senolytic approaches.

With the combination of KRASG12C and EGFR inhibitors, KRAS is becoming a druggable target in colorectal cancer. However, secondary resistance limits its efficacy. Using cell lines, patient-derived xenografts, and patient samples, we detected a heterogeneous pattern of putative resistance alterations expected primarily to prevent inhibition of ERK signaling by drugs at progression. Serial analysis of patient blood samples on treatment demonstrates that most of these alterations are detected at a low frequency except for KRASG12C amplification, a recurrent resistance mechanism that rises in step with clinical progression. Upon drug withdrawal, resistant cells with KRASG12C amplification undergo oncogene-induced senescence, and progressing patients experience a rapid fall in levels of this alteration in circulating DNA. In this new state, drug resumption is ineffective as mTOR signaling is elevated. However, our work exposes a potential therapeutic vulnerability, whereby therapies that target the senescence response may overcome acquired resistance.Significance:Clinical resistance to KRASG12C–EGFR inhibition primarily prevents suppression of ERK signaling. Most resistance mechanisms are subclonal, whereas KRASG12C amplification rises over time to drive a higher portion of resistance. This recurrent resistance mechanism leads to oncogene-induced senescence upon drug withdrawal and creates a potential vulnerability to senolytic approaches.This article is highlighted in the In This Issue feature, p. 1

Generation of acquired resistance PDX model

Characterization of acquired resistance in vitro models

Oncoprint of baseline alterations in CRC patients who developed resistance to KRAS G12C and EGFR inhibitors

C106 resistant cells drug withdrawal and Patient #12 MSK-Impact data

Patients Progression Cell Free DNA data

Patients Characteristics

C106 cell lines single cell sequencing analysis

Recently, screens for mediators of resistance to FLT3 and ABL kinase inhibitors in leukemia resulted in the discovery of LZTR1 as an adapter of a Cullin-3 RING E3 ubiquitin ligase complex responsible for the degradation of RAS GTPases. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations was identified in clonal hematopoietic conditions. Here we identify that loss of LZTR1, or leukemia-associated mutants in the LZTR1 substrate and RAS GTPase RIT1 that escape degradation, drives hematopoietic stem cell (HSC) expansion and leukemia in vivo. Although RIT1 stabilization was sufficient to drive hematopoietic transformation, transformation mediated by LZTR1 loss required MRAS. Proteolysis targeting chimeras (PROTAC) against RAS or reduction of GTP-loaded RAS overcomes LZTR1 loss-mediated resistance to FLT3 inhibitors. These data reveal proteolysis of noncanonical RAS proteins as novel regulators of HSC self-renewal, define the function of RIT1 and LZTR1 mutations in leukemia, and identify means to overcome drug resistance due to LZTR1 downregulation.Significance:Here we identify that impairing proteolysis of the noncanonical RAS GTPases RIT1 and MRAS via LZTR1 downregulation or leukemia-associated mutations stabilizing RIT1 enhances MAP kinase activation and drives leukemogenesis. Reducing the abundance of GTP-bound KRAS and NRAS overcomes the resistance to FLT3 kinase inhibitors associated with LZTR1 downregulation in leukemia.This article is highlighted in the In This Issue feature, p. 2221

RW7213 cell lines sequencing analysis

Supplementary Figure from Impaired Proteolysis of Noncanonical RAS Proteins Drives Clonal Hematopoietic Transformation

Patients Longitudinal Cell Free DNA data

Supplementary Figure S5. Response to W+T treatment in the L858R/T790M EGFR genetically engineered mouse model (LT GEMM). (A) Immunohistochemical analysis LT GEMMs treated with two doses of WZ4002, trametinib or a combination thereof assessing phosphorylation of EGFR, AKT and ERK1/2 at 100 nM. (B) TUNEL staining analysis of LT GEMMs treated with two doses of WZ4002, trametinib or a combination thereof at the 50 and 100 nM. (C) Tumor volume of individual LT GEMMs treated with 50 mg/kg WZ4002, 2.5 mg/kg trametinib and the combination up to 20 weeks (13). (D) Representative MRI images from each treatment.

Supplementary Fig 6

The incidence of esophagogastric cancer is rapidly rising, but only a minority of patients derive durable benefit from current therapies. Chemotherapy as well as anti-HER2 and PD-1 antibodies are standard treatments. To identify predictive biomarkers of drug sensitivity and mechanisms of resistance, we implemented prospective tumor sequencing of patients with metastatic esophagogastric cancer. There was no association between homologous recombination deficiency defects and response to platinum-based chemotherapy. Patients with microsatellite instability–high tumors were intrinsically resistant to chemotherapy but more likely to achieve durable responses to immunotherapy. The single Epstein–Barr virus–positive patient achieved a durable, complete response to immunotherapy. The level of ERBB2 amplification as determined by sequencing was predictive of trastuzumab benefit. Selection for a tumor subclone lacking ERBB2 amplification, deletion of ERBB2 exon 16, and comutations in the receptor tyrosine kinase, RAS, and PI3K pathways were associated with intrinsic and/or acquired trastuzumab resistance. Prospective genomic profiling can identify patients most likely to derive durable benefit to immunotherapy and trastuzumab and guide strategies to overcome drug resistance.Significance: Clinical application of multiplex sequencing can identify biomarkers of treatment response to contemporary systemic therapies in metastatic esophagogastric cancer. This large prospective analysis sheds light on the biological complexity and the dynamic nature of therapeutic resistance in metastatic esophagogastric cancers. Cancer Discov; 8(1); 49–58. ©2017 AACR.See related commentary by Sundar and Tan, p. 14.See related article by Pectasides et al., p. 37.This article is highlighted in the In This Issue feature, p. 1

Supplementary Figure S2 PDF file - 112K, PLX4032-treated BRAF mutant thyroid cancer cells show induction or high basal expression of several RTKs compared to melanoma cell lines. A, gene expression profiles (Illumina HT-12 v4) of thyroid (SW1736; red) vs melanoma (SK-MEL-28; green) cells at the indicated times after treatment with 2 �gM PLX4032. Graphs represent the median profiles of gene clusters that are significantly different between the lines. B, table shows KEGG terms significantly over-represented in the stated gene clusters vs the rest of the genome by Fisher's exact test. The p-values are adjusted by FDR (B & H). C, Technical validation of differentially expressed RTKs identified by expression profiling analysis of SW1736 vs SK-3 MEL-28 cells. Cells were treated with 2 �gM PLX4032 for 1, 6 or 48 h. Points represent RTK/GAPDH Q-RT-PCR values of triplicates assays (mean +/- SD)

Supplementary Figure S3. Biochemical analysis of WZ4002 + trametinib (W+T) resistant and sensitive cell lines. Each cell line was treated with 100 nM WZ4002 (W) or 30 nM trametinib (T) or a combination (C) thereof for 8 hours and total or phospho-EGFR, ERK1/2, AKT and S6 were assessed by western blot. Sensitivity to six week W+T treatment is indicated below the image. Hsp90 was used as a loading control.

PDF file - 528K, Supplementary Figure 1. BMS908662 enhances human T cell activation in vitro in a concentration-dependent manner. Supplementary Figure 2. BMS908662 inhibits the growth of BRAF mutant tumor cells in vitro. Supplementary Figure 3. BMS908662 enhances murine T cell activation in vitro in a concentration-dependent manner. Supplementary Figure 4. Detection of ERK phosphorylation in activated T cells. Supplementary Figure 5. Gating strategies for OT-1 and ex vivo phosphoflow. Supplementary Figure 6. PLX4720 enhances human T cell activation in vitro in a concentration-dependent manner.

PDF file - 480K

PDF file 338K, Phospho-flow data

Supplementary Figures 1-7

Supplemental Figure 1: BRAFV600E mutant allele burden in cell-free DNA (cfDNA) of urine and plasma from treatment naïve patients based on BRAFV600E tissue genotype result.

Concordance between copy-number inferred from next generation sequencing and protein overexpression by IHC or gene amplification by FISH.

PDF file - 356K

PDF file - 105K, Tables describing the somatic SNPs and indels identified by WGS, SV validation results, PCR primers used for validation, and tumor measurements from the patient treated with TAK-733

Supplementary Figure 1. Somatic mutations detected in histiocytic neoplasms. Supplementary Figure 2. Frequencies of known activating kinase mutations in histiocytic neoplasms. Supplementary Figure 3. ARAF mutations and variants of unknown significance detected in BRAFV600E-wildtype, non-Langerhans Cell Histiocytic neoplasms. Supplementary Figure 4. Clinical and histological images of the non-Langerhans cell histiocytosis (non-LCH) lesions from index patients with kinase fusions identified by RNA-seq. Supplementary Figure 5. Gene expression analysis of histiocytic neoplasms by RNA-seq. Supplementary Table 1. Characteristics of patient samples with histiocytic neoplasms used in this study and genomic analysis utilized for each sample. Supplementary Table 2. Whole exome sequencing metrics. Supplementary Table 3. Somatic variants identified by whole exome and whole transcriptome sequencing and validated by droplet-digital PCR and/or custom-capture targeted next-generation sequencing with variant allele frequencies (VAFs). Supplementary Table 4. List of the top 1% of differentially expressed genes across histiocytic samples from mRNA sequencing. Supplementary Table 5. PCR primers with M13F2 and M13R2 tails (blue) for Sanger sequencing used in the targeted sequencing recurrence testing for MAP2K1 exons 2 and 3 and all coding regions of ARAF.

Supplemental Figure 2: Radiographic, histologic, and molecular evaluation of KRASG12S mutant patient with Erdheim-Chester Disease (ECD).

RAF inhibitors selectively block extracellular signal–regulated kinase (ERK) signaling in BRAF-mutant melanomas and have defined a genotype-guided approach to care for this disease. RAF inhibitors have the opposite effect in BRAF wild-type tumor cells, where they cause hyperactivation of ERK signaling. Here, we predict that RAF inhibitors can enhance T-cell activation, based on the observation that these agents paradoxically activate ERK signaling in BRAF wild-type cells. To test this hypothesis, we have evaluated the effects of the RAF inhibitor BMS908662 on T-cell activation and signaling in vitro and in vivo. We observe that T-cell activation is enhanced in a concentration-dependent manner and that this effect corresponds with increased ERK signaling, consistent with paradoxical activation of the pathway. Furthermore, we find that the combination of BMS908662 with cytotoxic T-lymphocyte antigen 4 (CTLA-4) blockade in vivo potentiates T-cell expansion, corresponding with hyperactivation of ERK signaling in T cells detectable ex vivo. Finally, this combination demonstrates superior antitumor activity, compared with either agent alone, in two transplantable tumor models. This study provides clear evidence that RAF inhibitors can modulate T-cell function by potentiating T-cell activation in vitro and in vivo. Paradoxical activation of ERK signaling in T cells offers one mechanism to explain the enhanced antitumor activity seen when RAF inhibitors are combined with CTLA-4 blockade in preclinical models. Cancer Immunol Res; 2(1); 70–79. ©2013 AACR.

Supplementary Figure S4. PC9 GR4 xenograft response to W+T treatment. Mice were treated with two doses (A) or four doses (B) of WZ4002, trametinib or a combination thereof. Tumors were lysed, subjected to immunoblot and phosphorylation of EGFR, AKT, ERK1/2 and S6 were assessed. Hsp90 was used as a loading control. (C) Mice were photographed 31 days following cessation of drug treatment.

PDF file 103K, Supplementary tables and supplementary figure legends

The major histocompatibility complex I (MHC-1) presents antigenic peptides to tumor-specific CD8+ T cells. The regulation of MHC-I by kinases is largely unstudied, even though many patients with cancer are receiving therapeutic kinase inhibitors. Regulators of cell-surface HLA amounts were discovered using a pooled human kinome shRNA interference–based approach. Hits scoring highly were subsequently validated by additional RNAi and pharmacologic inhibitors. MAP2K1 (MEK), EGFR, and RET were validated as negative regulators of MHC-I expression and antigen presentation machinery in multiple cancer types, acting through an ERK output–dependent mechanism; the pathways responsible for increased MHC-I upon kinase inhibition were mapped. Activated MAPK signaling in mouse tumors in vivo suppressed components of MHC-I and the antigen presentation machinery. Pharmacologic inhibition of MAPK signaling also led to improved peptide/MHC target recognition and killing by T cells and TCR-mimic antibodies. Druggable kinases may thus serve as immediately applicable targets for modulating immunotherapy for many diseases. Cancer Immunol Res; 4(11); 936–47. ©2016 AACR.

PDF file - 1.3MB, Supplementary data on MAPK1 copy number and expression, combination treatment with WZ4002 and PI3K inhibitors, impact of MEK1 K57N on WZ4002 sensitivity, Mapk1 copy number analyses and NF1 and dusp expression in drug sensitive and resistant mouse tumors

Supplementary Figure S2. Comparison of WZ4002 + crizotinib and WZ4002 + trametinib combinations in TKI resistant cell lines. HCC827 GR6 and HCC827 + HGF were treated with DMSO control (Media), 100 nM WZ4002, 30 nM trametinib, 100 nM crizotinib or WZ4002 combinations weekly. Positive wells were tallied weekly (mean +/- SD, n = 3 biological replicates of 60 wells each). Arrow indicates that confluence was not reached at 9 weeks.

Supplementary Table 1. List of cell lines used in this study.

PDF file - 76K, Includes a description of the patient sample used for WGS in addition to the methods for somatic SNP, indel, SV, and CNV determination by WGS

Supplementary Figure 2

The effects of selective phosphoinositide 3-kinase (PI3K) and AKT inhibitors were compared in human tumor cell lines in which the pathway is dysregulated. Both caused inhibition of AKT, relief of feedback inhibition of receptor tyrosine kinases, and growth arrest. However, only the PI3K inhibitors caused rapid induction of cell death. In seeking a mechanism for this phenomenon, we found that PI3K inhibition, but not AKT inhibition, causes rapid inhibition of wild-type RAS and of RAF–MEK–ERK signaling. Inhibition of RAS–ERK signaling is transient, rebounding a few hours after drug addition, and is required for rapid induction of apoptosis. Combined MEK and AKT inhibition also promotes cell death, and in murine models of HER2+ cancer, either pulsatile PI3K inhibition or combined MEK and AKT inhibition causes tumor regression. We conclude that PI3K is upstream of RAS and AKT and that pulsatile inhibition of both pathways is sufficient for effective antitumor activity.Significance: We show that the RAS–ERK pathway is a key downstream effector pathway of oncogenic PI3K. Coordinate downregulation of AKT and ERK is necessary for induction of apoptosis and antitumor activity and can be accomplished with pulsatile dosing, which will likely decrease toxicity and allow administration of therapeutic doses. Cancer Discov; 4(3); 334–47. ©2014 AACR.This article is highlighted in the In This Issue feature, p. 259

Supplementary Fig 3

PDF file 88K, Spleen length size

Supplementary Figure S1: Representative H&E, phospho-ERK and FISH images of pre-dabrafenib (3) and post-dabrafenib (4) tumors. Supplementary Figure S2: WES analysis of copy number variation in pre- and postdabrafenib tumors. Supplementary Figure S3: Whole copy number profiles from WES of pre- and posttreatment tumors. Supplementary Figure S4: Homology alignment of BRAF p. L514 with residues in other tyrosine kinases. Supplementary Figure S5: Relative frequency of BRAF variant alleles in SK-BT-DR cells determined by individual clone sequencing. Supplementary Figure S6: BRAF V600E/L514V reduces dabrafenib sensitivity in NIH- 3T3 cells, related to Figure 2. Supplementary Figure S7: BRAF V600E/L514V confers biochemical resistance to dabrafenib over a time course, related to Figure 2G. Supplementary Figure S8: Comparison of IC50, IC75 and IC90 of dabrafenib against A375 cells expressing BRAF V600E and BRAF V600E/L514V, related to Figure 2H. Supplementary Figure S9: The BRAF V600E/L514V double mutant promotes homodimerization, related to Figure 3A and B. Supplementary Figure S10: BRAF L514V alone is hypoactive and associated with decreased ERK signaling that is not sensitive to dabrafenib. Supplementary Figure S11: BRAF V600E/L514V is inhibited by dabrafenib in a purified kinase assay, indicating that it is not a gatekeeper mutation. Supplementary Figure S12: Quantitation of p-MEK and p-ERK immunoblots, related to Figure 4B. Supplementary Figure S13: Comparison of IC50, IC75 and IC90 of trametinib against A375 expressing BRAF V600E and BRAF V600E/L514V, related to Figure 4C. Supplementary Figure S14: The BRAF V600E/L514V mutant mediates resistance to dabrafenib that cannot be completely overcome by trametinib or dabrafenib plus trametinib, related to Figure 4D. Supplementary Figure S15: V5, p-MEK, total MEK immunoblots, and quantitation of p-ERK immunoblots, related to Figure 5A. Supplementary Figure S16: Novel RAF dimer inhibitors, MEK inhibitor and ERK inhibitor equipotently inhibit cell growth in BRAF V600E and V600E/L514V expressing cells. Supplementary Figure S17: Novel RAF dimer inhibitor, MEK inhibitor and ERK inhibitor equipotently inhibit ERK signaling in BRAF V600E and V600E/L514V expressing cells. Supplementary Figure S18: BGB3245 binds mutant BRAF V600E monomer and second site of V600E/L514V dimer with similar affinity. Supplementary Table S1: Mutations identified by WES of pre-dabrafenib and postdabrafenib tumors. Supplementary Table S2: Secondary mutations associated with acquired resistance and occurring in residues homologous with L514 in BRAF. Supplementary Table S3: BRAF L514V allele frequency determined by ddPCR.

Supplementary Fig 2