Pharmacogenetic 3C interaction associated with the PDGFRA gene as a chromatin conformation marker for treatment with tyrosine kinase inhibitors.

Abstract

e23054 Background: IDH1 mutations detected in glioma cells impair the insulator function between FIPL1L1 and PDGFRA at 4q12 ( Flavahan et al. 2016). We have used a high-resolution chromosome-conformation capture 3C analysis platform, EpiSwitch, and quantitative PCR, to map, evaluate, and quantify the TKI-sensitive conformational juxtaposition between FIP1L1 and PDGFRA. Loss of the insulator function in glioma prompted us to investigate the same interaction in the context of insulator loss with interstitial deletions at 4q12 in eosinophilic leukemias and AML. Methods: We tested a total of 72 primers in temperature gradient PCRs, with concentration matched negative controls, using the AML cell lines EOL-1 and HL-60. Products were sequenced in forward and reverse order. Dual label 5’FAM-BHQ1-3’hydrolysis probe assays, entirely specific for the PCR products, targeted the junction region of the 3C fragments. A reference 3C interaction was used as an internal copy number control for 3C library production. Results: EpiSwitch predicted and identified six 3C FIP1L1-PDGFRA interactions in different sequence orientations, within the 3D organization of the PDGFRA locus. The interaction D7 identified by the EpiSwitch qPCR assay was detected reproducibly in EOL-1 cells and glioblastoma tissue using both single step PCR and qPCR. An imatinib-sensitive AML cell line EOL-1 was used as a positive control for qPCR assays. Both AML and glioma cell lines tested positive using the assay as did glioma patient biopsies. The glioblastoma cell line DBTRG-05MG also tested positive for the D7 interaction at a maximum of 8.92 copies per 20 ng of the template. Conclusions: We confirmed and characterized, at high resolution, the conformational deregulation of FIP1L1 and PDGFRA in glioma. Additionally, our group detected the interaction in TKI-sensitive leukemia cell lines. The analysis of 3C microstructural alterations is consistent with latest insights into epigenetic regulation of PDGFRA. It provides a promising approach to the stratification of patients for tyrosine kinase inhibitor treatment, which could not be provided diagnostically with conventional sequencing approaches.