Search

Aneuploidy and resulting gene copy number alterations (CNAs) are important hallmarks of human cancers. Since CNAs are not associated with dosage compensation in mRNA expression, cancer cells with a high CNA burden must harbor mechanisms to mitigate proteotoxic stress resulting from stoichiometric imbalance and accumulation of unfolded proteins (1). Here, we show that aneuploid human cancer cells exhibit discordance between CNAs and protein levels due to compensation at the proteome level, mainly concerning multi-protein complexes. Moreover, we identify the N-recognin ubiquitin ligase UBR4 as a critical mediator of protein interactome homeostasis that is essential for viability, specifically in highly aneuploid cancersin vitroandin vivo. UBR4 prunes the proteome to ensure the balanced expression of protein complex members. Inactivation of UBR4 in highly aneuploid cancer cells causes a convergence of copy number and protein levels and induces proteotoxic stress pathways. UBR4 inhibition may present a broadly applicable therapeutic strategy for cancer and other diseases driven by aneuploidy.One-Sentence SummaryThe N-recognin ubiquitin ligase UBR4 as a critical mediator of protein interactome homeostasis that is essential for viability in aneuploid cancers.

FGFR inhibitors are approved for the treatment of advanced cholangiocarcinoma harboring FGFR2 fusions. However, the response rate is moderate, and resistance emerges rapidly due to acquired secondary FGFR2 mutations or due to other less-defined mechanisms. Here, we conducted high-throughput combination drug screens, biochemical analysis, and therapeutic studies using patient-derived models of FGFR2 fusion–positive cholangiocarcinoma to gain insight into these clinical profiles and uncover improved treatment strategies. We found that feedback activation of EGFR signaling limits FGFR inhibitor efficacy, restricting cell death induction in sensitive models and causing resistance in insensitive models lacking secondary FGFR2 mutations. Inhibition of wild-type EGFR potentiated responses to FGFR inhibitors in both contexts, durably suppressing MEK/ERK and mTOR signaling, increasing apoptosis, and causing marked tumor regressions in vivo. Our findings reveal EGFR-dependent adaptive signaling as an important mechanism limiting FGFR inhibitor efficacy and driving resistance and support clinical testing of FGFR/EGFR inhibitor therapy for FGFR2 fusion–positive cholangiocarcinoma. Significance: We demonstrate that feedback activation of EGFR signaling limits the effectiveness of FGFR inhibitor therapy and drives adaptive resistance in patient-derived models of FGFR2 fusion–positive cholangiocarcinoma. These studies support the potential of combination treatment with FGFR and EGFR inhibitors as an improved treatment for patients with FGFR2-driven cholangiocarcinoma. This article is highlighted in the In This Issue feature, p. 1171

Biological importance of Gab1 in HNSCC cells

Supplementary Table from EGFR Inhibition Potentiates FGFR Inhibitor Therapy and Overcomes Resistance in FGFR2 Fusion–Positive Cholangiocarcinoma

Preclinical and clinical studies have evidenced that effective targeted therapy treatment designed against receptor tyrosine kinases (RTKs) in different solid tumor paradigms is predicated on simultaneous inhibition of both the PI3K and MEK intracellular signaling pathways. Indeed, reactivation of either pathway results in resistance to these therapies. Recently, oncogenic phosphatase SHP2 inhibitors have been developed with some now reaching clinical trials. To expand on possible indications for SHP099, we screened over 800 cancer cell lines covering over 25 subsets of cancer. We found head and neck squamous cell carcinoma (HNSCC) was the most sensitive adult subtype of cancer to SHP099. We found that, in addition to the MEK pathway, SHP2 inhibition blocks the PI3K pathway in sensitive HNSCCs, resulting in downregulation of mTORC signaling and antitumor effects across several HNSCC mouse models, including an human papillomavirus (HPV+) patient-derived xenograft. Importantly, we found low levels of the RTK ligand epiregulin identified HNSCCs that were sensitive to SHP2 inhibitor, and, adding exogenous epiregulin mitigated SHP099 efficacy. Mechanistically, epiregulin maintained SHP2–GAB1 complexes in the presence of SHP2 inhibition, preventing downregulation of the MEK and PI3K pathways. In the presence of SHP2 inhibitor, HNSCCs are highly dependent on GAB1 for their survival and knockdown of GAB1 is sufficient to block the ability of epiregulin to rescue MEK and PI3K signaling. These data connect the sensitivity of HNSCC to SHP2 inhibitors and to a broad reliance on GAB1-SHP2, revealing an important and druggable signaling axis. Overall, SHP2 inhibitors are being heavily developed and may have activity in HNSCCs, and in particular those with low levels of epiregulin.Significance:This work identifies a novel role of SHP2 inhibitor by dual downregulation of PI3K and MEK pathways, through loss of GAB1 activation and disruption of GAB1 complexes in low-epiregulin HNSCC.

Cell line information and SHP099 screen data

Supplementary Figure from EGFR Inhibition Potentiates FGFR Inhibitor Therapy and Overcomes Resistance in FGFR2 Fusion–Positive Cholangiocarcinoma

FGFR inhibitors are approved for the treatment of advanced cholangiocarcinoma harboring FGFR2 fusions. However, the response rate is moderate, and resistance emerges rapidly due to acquired secondary FGFR2 mutations or due to other less-defined mechanisms. Here, we conducted high-throughput combination drug screens, biochemical analysis, and therapeutic studies using patient-derived models of FGFR2 fusion–positive cholangiocarcinoma to gain insight into these clinical profiles and uncover improved treatment strategies. We found that feedback activation of EGFR signaling limits FGFR inhibitor efficacy, restricting cell death induction in sensitive models and causing resistance in insensitive models lacking secondary FGFR2 mutations. Inhibition of wild-type EGFR potentiated responses to FGFR inhibitors in both contexts, durably suppressing MEK/ERK and mTOR signaling, increasing apoptosis, and causing marked tumor regressions in vivo. Our findings reveal EGFR-dependent adaptive signaling as an important mechanism limiting FGFR inhibitor efficacy and driving resistance and support clinical testing of FGFR/EGFR inhibitor therapy for FGFR2 fusion–positive cholangiocarcinoma.Significance:We demonstrate that feedback activation of EGFR signaling limits the effectiveness of FGFR inhibitor therapy and drives adaptive resistance in patient-derived models of FGFR2 fusion–positive cholangiocarcinoma. These studies support the potential of combination treatment with FGFR and EGFR inhibitors as an improved treatment for patients with FGFR2-driven cholangiocarcinoma.This article is highlighted in the In This Issue feature, p. 1171

ATP-competitive fibroblast growth factor receptor (FGFR) kinase inhibitors, including BGJ398 and Debio 1347, show antitumor activity in patients with intrahepatic cholangiocarcinoma (ICC) harboring activating FGFR2 gene fusions. Unfortunately, acquired resistance develops and is often associated with the emergence of secondary FGFR2 kinase domain mutations. Here, we report that the irreversible pan-FGFR inhibitor TAS-120 demonstrated efficacy in 4 patients with FGFR2 fusion–positive ICC who developed resistance to BGJ398 or Debio 1347. Examination of serial biopsies, circulating tumor DNA (ctDNA), and patient-derived ICC cells revealed that TAS-120 was active against multiple FGFR2 mutations conferring resistance to BGJ398 or Debio 1347. Functional assessment and modeling the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profiles observed clinically for each inhibitor. Our findings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and ctDNA analysis, may prolong the duration of benefit from FGFR inhibition in patients with FGFR2 fusion–positive ICC.Significance:ATP-competitive FGFR inhibitors (BGJ398, Debio 1347) show efficacy in FGFR2-altered ICC; however, acquired FGFR2 kinase domain mutations cause drug resistance and tumor progression. We demonstrate that the irreversible FGFR inhibitor TAS-120 provides clinical benefit in patients with resistance to BGJ398 or Debio 1347 and overcomes several FGFR2 mutations in ICC models.This article is highlighted in the In This Issue feature, p. 983

Peptide quantification from MIB column isolation of the active kinome in DMSO- versus dasatinib-treated HuCCT1, RBE, and SNU-1079 cells.

Supplementary Figure Legends PDF file 97K, Complete legends for all 5 Supplementary Figures

Supplementary Table S1. ALK inhibitors in clinical development. Supplementary Table S2. Summary of clinical characteristics of ALK-positive patients undergoing biopsies at the time of ALK inhibitor resistance. Supplementary Table S3. Paired, post-crizotinib and post second-generation ALK inhibitor biopsies. Supplementary Table S4. Treatment course of ALK-positive patients undergoing post-ceritinib biopsies. Supplementary Table S5. Treatment course of ALK-positive patients undergoing post-alectinib biopsies. Supplementary Table S6. Treatment course of ALK-positive patients undergoing post-brigatinib biopsies. Supplementary Table S7. Resistant biopsies with {greater than or equal to}2 resistance mutations. Supplementary Table S8. Mutations in ceritinib-resistant biopsies. Supplementary Table S9. Mutations in alectinib-resistant biopsies. Supplementary Table S10. Mutations in brigatinib-resistant biopsies. Supplementary Table S11. Single-nucleotide variants identified in patient-derived cell lines. Supplementary Table S12. EMT in ceritinib-resistant biopsies. Supplementary Table S13. Cells seeded for survival assays. Supplementary Table S14. Cells seeded for growth assays.

GSK-J4 inhibits histone demethylation of EWS-FLI1 target genes

suppl tables

This file contains legends and information about supplemental material.

BH3 mimetic drugs, which inhibit prosurvival BCL2 family proteins, have limited single-agent activity in solid tumor models. The potential of BH3 mimetics for these cancers may depend on their ability to potentiate the apoptotic response to chemotherapy and targeted therapies. Using a novel class of potent and selective MCL1 inhibitors, we demonstrate that concurrent MEK + MCL1 inhibition induces apoptosis and tumor regression in KRAS-mutant non–small cell lung cancer (NSCLC) models, which respond poorly to MEK inhibition alone. Susceptibility to BH3 mimetics that target either MCL1 or BCL-xL was determined by the differential binding of proapoptotic BCL2 proteins to MCL1 or BCL-xL, respectively. The efficacy of dual MEK + MCL1 blockade was augmented by prior transient exposure to BCL-xL inhibitors, which promotes the binding of proapoptotic BCL2 proteins to MCL1. This suggests a novel strategy for integrating BH3 mimetics that target different BCL2 family proteins for KRAS-mutant NSCLC.Significance:Defining the molecular basis for MCL1 versus BCL-xL dependency will be essential for effective prioritization of BH3 mimetic combination therapies in the clinic. We discover a novel strategy for integrating BCL-xL and MCL1 inhibitors to drive and subsequently exploit apoptotic dependencies of KRAS-mutant NSCLCs treated with MEK inhibitors.See related commentary by Leber et al., p. 1511.This article is highlighted in the In This Issue feature, p. 1494

Supplementary Figure S1. IDHm ICC are insensitive to IDH inhibition. Supplementary Figure S2. Similarity matrix for drug response of BTC cell lines. Supplementary Figure S3. Hypersensitivity of ICC cells harboring endogenous IDH mutations to dasatinib. Supplementary Figure S4. Torin 1 inhibits growth of ICC cell lines. Supplementary Figure S5. Critical role for SRC in IDHm ICC cells. Supplementary Figure S6. Ectopic expression of mutant IDH has only a modest effect on dasatinib sensitivity.

Supplemental Figure 3. Chemotherapy and venetoclax combination cell viability assays. Cell viability assays were performed for 72h treatment of cyclophosphamide and topotecan, venetoclax, or combination at indicated concentrations, with bliss synergy scores in (A) MYCN-amplified, and (B) MYCN-wild-type cell lines. (C) Plot of Bliss sum of cell lines in A-B, calculated by the summation of the bliss synergy scores across the doses presented.

Table S1: Crystallographic data collection and refinement statistics Table S2: Inhibitory activity against other BET bromodomains Table S3: Bromodomain profiling data Fig. S1: Detailed binding interactions of compounds 1 - 5 in BRD4-1 Fig. S2: Comparison of compound 2 and JQ1 in Brd4-1 Fig. S3: Chemical structures of other compounds used in this work. Fig. S4: Statistical evaluation of kinome profiling data using the Gini coefficient Fig. S5: Original flow cytometry data of MM1.S cells Fig. S6: MM1.S cells require stimulation with IL-6 to detect phospho-STAT3 levels Fig. S7: Effect of dual BRD4-kinase inhibitors on MV-4-11 cells. Fig. S8: Time dependent alteration of c-Myc and p21Cip1 levels in MM1.S cells. Fig. S9: Effect of dual BET-kinase inhibitors on ruxolitinib sensitive MPN cell lines Fig. S10: Dose-effect analysis of UKE-1 cells treated with drug combinations Fig. S11: Cell line sensitivity by tissue type

Supplementary Figure S3. JIB-04 inhibits translation initiation.

Supplemental Figure 2. PDX ex vivo model demonstrated cleavage of PARP with corresponding NOXA upregulation (A) Western blot of COG-N-561 ex vivo cell line treated overnight with antibodies as indicated.

Supplementary Methods include Enzyme Kinetic Assays and Computational Methods.

Supplementary Table 2. Lung cell lines homozygotes for KDM4A SNP-A482 are not enriched for mutations that could be linked to sensitivity to PI3K/mTOR inhibitors.

Gene symbols and names

PDF file140K, Supplementary figure legends

The sequences of the primers used for ChIP

Supplementary Figure S1. ALK G1202R versus ALK G1202del. Supplementary Figure S2. ALK G1202del in ALK-rearranged lung cancer. Supplementary Figure S3. Swimmer's plot - alectinib. Supplementary Figure S4. Evaluation of MGH075-2E in drug screen. Supplementary Figure S5. MGH067-1 harbors an ALK rearrangement. Supplementary Figure S6. MGH021-5A. Supplementary Figure S7. MGH051-2C and MGH084-1D. Supplementary Figure S8. MGH049-1A and MGH075-2E. Supplementary Figure S9. MGH034-2A.

Synergistic action of kinase and BET bromodomain inhibitors in cell killing has been reported for a variety of cancers. Using the chemical scaffold of the JAK2 inhibitor TG101348, we developed and characterized single agents which potently and simultaneously inhibit BRD4 and a specific set of oncogenic tyrosine kinases including JAK2, FLT3, RET, and ROS1. Lead compounds showed on-target inhibition in several blood cancer cell lines and were highly efficacious at inhibiting the growth of hematopoietic progenitor cells from patients with myeloproliferative neoplasm. Screening across 931 cancer cell lines revealed differential growth inhibitory potential with highest activity against bone and blood cancers and greatly enhanced activity over the single BET inhibitor JQ1. Gene drug sensitivity analyses and drug combination studies indicate synergism of BRD4 and kinase inhibition as a plausible reason for the superior potency in cell killing. Combined, our findings indicate promising potential of these agents as novel chemical probes and cancer therapeutics. Mol Cancer Ther; 16(6); 1054–67. ©2017 AACR.

PDF file - 93K, This file contains 9 supplementary tables including the full primary screening data and secondary testing of BETi in neuroblastoma, supporting tables for the expression profiling analyses, and the list of Affymetrix probes (MYCN) and shRNA sequences for BRD4 and MYCN.

Primer sequences

Supplementary Figure Legends

PDF file - 21944K

Supplementary Figure 3. Rapamycin treatment reduces KDM4A protein levels.

Legends for Suppl. Fig. 1-6

PDF file 764K, Supp. Figures 1-10 include data describing in vitro and in vivo experiments in support of Faber at al

Supplementary Figure 2 PDF file 74K, Mcl-1 is essential for cell survival in SCC cells and vorinostat induces redistribution of Bim from Mcl-1 to Bcl-2/Bcl-xl

Supplementary Table 3. List of drugs and primary targets used for the study.

Supplementary Figures