TY - JOUR
T1 - Deconvoluting mechanisms of acquired resistance to RAF inhibitors in BRAFV600E-Mutant Human Glioma
AU - Schreck, Karisa C.
AU - Morin, Andrew
AU - Zhao, Guisheng
AU - Allen, Amy N.
AU - Flannery, Patrick
AU - Glantz, Michael
AU - Green, Adam L.
AU - Jones, Chris
AU - Jones, Kenneth L.
AU - Kilburn, Lindsay B.
AU - Nazemi, Kellie J.
AU - Samuel, David
AU - Sanford, Bridget
AU - Solomon, David A.
AU - Wang, Jiawan
AU - Pratilas, Christine A.
AU - Nicolaides, Theodore
AU - Levy, Jean M.Mulcahy
N1 - Funding Information:
This work is supported by Curing Kids Cancer Collaborative Research award, to K.C. Schreck, T. Nicolaides, C.A. Pratilas, and J.M. Mulcahy Levy; American Academy of Neurology Institute Clinical Research Training Scholarship, K.C. Schreck; Maryland Cigarette Restitution Fund, K.C. Schreck; Making Headway Foundation, T. Nicolaides and G. Zhao; Boettcher Foundation Scholar Award, J.M. Mulcahy Levy; Morgan Adams Foundation, J.M. Mulcahy Levy; NIH/National Institute of Neurological Disorders and Stroke (R01NS107313), J.M. Mulcahy Levy; Functional Genomics Facility (Denver, Colorado), which is supported by the Cancer Center Support Grant (P30CA046934), J.M. Mulcahy Levy; NIH Director’s Early Independence Award from the Office of the Director, NIH (DP5 OD021403), D.A. Solomon.
Funding Information:
K.C. Schreck reports personal fees from Springworks Therapeutics outside the submitted work. L.B. Kilburn reports other support from Novartis, Genentech, DayOne Biopharmaceuticals, Regeneron Pharmaceuticals, Bristol Myers Squibb, and Epizyme outside the submitted work; in addition, L.B. Kilburn reports stock ownership by spouse in Onconova Therapeutics. C.A. Pratilas reports grants from Kura Oncology and Novartis and personal fees from Genentech outside the submitted work. T. Nicolaides reports grants from Curing Kids Cancer Foundation during the conduct of the study. J.M. Mulcahy Levy reports grants from Curing Kids Cancer Collaborative Research Award, Boettcher Foundation Scholar Award, Morgan Adams Foundation, NIH-NINDS, and NIH Cancer Center Support Grant during the conduct of the study. No disclosures were reported by the other authors.
Funding Information:
Cells were maintained in a humidified incubator at 37°C and 5% CO2. The B76 cell line was generated from a cultured tumor sample obtained during surgery, and grown in OptiMEM Reduced Serum media sup- plemented with 15% FBS and 1% penicillin/streptomycin (21). The DBTRG-5MG cell line (RRID:CVCL_1169) was obtained from the ATCC and grown in RPMI1640 supplemented with 10% FBS and 1% penicillin/streptomycin. The AM38 cell line (RRID:CVCL_1070) was purchased from the Japan Health Sciences Foundation Health Science Research Resources Bank, and grown in MEM supplemented with 20% FBS and 1% penicillin/streptomycin. MAF-794 and MAF-905_3 cell lines were generated from tumor surgically removed from patients at Children’s Hospital Colorado (Aurora, CO), and grown in OptiMEM Reduced Serum media supplemented with 15% FBS and 1% penicillin/streptomycin (21). The BT40 cell line was obtained from the Brain Tumor Research Center (BTRC) Tissue Bank at the UCSF and grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Vemurafenib-resistant MAF-749 and AM38 cell lines were previously established and characterized (22). pLentiCRISPR-E was a gift from the laboratory of Phillip Abbosh (Addgene #78852, RRID:Addgene_78852). CRISPR constructs against three distinct regions of the NF1 gene were obtained from ABM (pLenti-U6-NF1sgRNA-SFFV-CAS9-2A-Pur). Lentivirus was produced in 293T cells and DBTRG-5MG cells were infected with the filtered supernatant. Cell lines were stably selected in puromycin prior to use. pBABE-PTEN-puro was a gift from the laboratory of William Sellers (Addgene plasmid #10785, RRID:Addgene_10785). TTIGFP-MLUEX plasmids encoding MEK1 and MEK1 p.E203K were a gift from the laboratory of Neal Rosen. Cells were first infected with pMSCF-rtTA3-PGK-Hygro and selected in hygromycin. Stable cultures were then infected with TTIGFP construct and selected in puromycin. GFP was induced by exposure to doxycycline for 24 hours and cells were GFP sorted to create a stable, doubly infected population, as described previously (23). Cultures were maintained in media containing TET-free FBS. Short hairpin (shRNA) targeting CRAF1 (TRCN0000001068, GAGACATGAAATC-CAACAATA) was purchased from the Functional Genomics Facility which is supported by the Cancer Center Support Grant (P30CA046934). For CBL knockdown, Silencer Select Cbl-siRNA (sense: CCAUCGU-GGUAGAUCCGUUtt, antisense: AACGGAUCUACCACGAUGGgt, catalog no. 4390824, assay ID s2478) and Silencer Select negative control No. 1 siRNA (catalog no. 4390843) purchased from Thermo Fisher Scientific were transfected in AM38 or BT40 cells using Lipofectamine RNAiMAX reagent (catalog no. 13778030) according to manufacturer’s protocol. Twenty-five pmol of siRNA were used to transfect cells in a 6-well plate.
Publisher Copyright:
© 2021 The Authors.
PY - 2021/11/15
Y1 - 2021/11/15
N2 - Purpose: Selective RAF-targeted therapy is effective in some patients with BRAFV600E-mutated glioma, though emergent and adaptive resistance occurs through ill-defined mechanisms. Experimental Design: Paired pre-/post- RAF inhibitor (RAFi)- treated glioma samples (N = 15) were obtained and queried for treatment-emergent genomic alterations using DNA and RNA sequencing (RNA-seq). Functional validation of putative resistance mechanisms was performed using established and patient-derived BRAFV600E-mutant glioma cell lines. Results: Analysis of 15 tissue sample pairs identified 13 alterations conferring putative resistance were identified among nine paired samples (including mutations involving ERRFI1, BAP1, ANKHD1, and MAP2K1). We performed functional validation of mechanisms of resistance, including loss of NF1, PTEN, or CBL, in BRAFV600E-mutant glioma lines, and demonstrate they are capable of conferring resistance in vitro. Knockdown of CBL resulted in increased EGFR expression and phosphorylation, a possible mechanism for maintaining ERK signaling within the cell. Combination therapy with a MEKi or EGFR inhibitor was able to overcome resistance to BRAFi, in NF1 knockdown and CBL knockdown, respectively. Restoration of wild-type PTEN in B76 cells (PTEN-/-) restored sensitivity to BRAFi. We identified and validated CRAF upregulation as a mechanism of resistance in one resistant sample. RNA-seq analysis identified two emergent expression patterns in resistant samples, consistent with expression patterns of known glioma subtypes. Conclusions: Resistance mechanisms to BRAFi in glioma are varied and may predict effective precision combinations of targeted therapy, highlighting the importance of a personalized approach.
AB - Purpose: Selective RAF-targeted therapy is effective in some patients with BRAFV600E-mutated glioma, though emergent and adaptive resistance occurs through ill-defined mechanisms. Experimental Design: Paired pre-/post- RAF inhibitor (RAFi)- treated glioma samples (N = 15) were obtained and queried for treatment-emergent genomic alterations using DNA and RNA sequencing (RNA-seq). Functional validation of putative resistance mechanisms was performed using established and patient-derived BRAFV600E-mutant glioma cell lines. Results: Analysis of 15 tissue sample pairs identified 13 alterations conferring putative resistance were identified among nine paired samples (including mutations involving ERRFI1, BAP1, ANKHD1, and MAP2K1). We performed functional validation of mechanisms of resistance, including loss of NF1, PTEN, or CBL, in BRAFV600E-mutant glioma lines, and demonstrate they are capable of conferring resistance in vitro. Knockdown of CBL resulted in increased EGFR expression and phosphorylation, a possible mechanism for maintaining ERK signaling within the cell. Combination therapy with a MEKi or EGFR inhibitor was able to overcome resistance to BRAFi, in NF1 knockdown and CBL knockdown, respectively. Restoration of wild-type PTEN in B76 cells (PTEN-/-) restored sensitivity to BRAFi. We identified and validated CRAF upregulation as a mechanism of resistance in one resistant sample. RNA-seq analysis identified two emergent expression patterns in resistant samples, consistent with expression patterns of known glioma subtypes. Conclusions: Resistance mechanisms to BRAFi in glioma are varied and may predict effective precision combinations of targeted therapy, highlighting the importance of a personalized approach.
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U2 - 10.1158/1078-0432.CCR-21-2660
DO - 10.1158/1078-0432.CCR-21-2660
M3 - Article
C2 - 34433654
AN - SCOPUS:85119911641
SN - 1078-0432
VL - 27
SP - 6197
EP - 6208
JO - Clinical Cancer Research
JF - Clinical Cancer Research
IS - 22
ER -