Abstract 4338: Introduction of CALR mutations to a human erythroleukemia cell line, HEL, by CRISPR/Cas9 systems

[Introduction]Mutations of the calreticulin gene (CALR) are frequently found in patients with myeloproliferative neoplasms (MPN). Five-nucleotide insertion (Ins5) and 52-nucleotide deletion (Del52) in the last exon of the CALR gene are common mutations, generating an altered CALR protein with a mutated peptide sequence at the carboxy end due to the frame-shifts. Cell-lines with such CALR mutations have not been reported yet. CRISPR/Cas9 system is the novel technique commonly used in genetic engineering. CRISPR/Cas9 technology allows us to “edit” genome in mammalian cells. We introduced 46-nucleotide deletion (46Del) in the last exon of the CALR gene in a human erythroleukemia cell line, HEL. This 46Del abnormality lead to generation of an altered CALR protein with the abnormal peptide sequence at the carboxy end as seen in MPN cells with 52Del or Ins5. We compared the CALR-engineered HEL cells with the parental cells in the presence/absence of a Jak2 kinase inhibitor, ruxolitinib (RUX). [Methods and Results]To introduce 46Del abnormality, we have designed two guide RNAs. We co-transfected vectors encoding the Cas9 with a fluorescence protein as a marker as well as the two guide RNAs into HEL cells using nucleoporation. Fluorescence protein positive cells were isolated and the single cells were cultured in 96-well plates for 3 weeks. Genomic DNA of each the clone was extracted, and genomic status of CALR was examined by PCR/sequencing. We have isolated one clone with 46Del (HEL-46DEL), of which expression of abnormal transcripts was confirmed by RT-PCR/sequencing. We also confirmed expressions of abnormal CALR protein by western blot analysis. Since the JAK2-STAT5 pathway is known to be activated in MPN cells, phosphorylation status of STAT5 in HEL and HEL-DEL46 was examined, showing comparable levels in each the line. Proliferation of HEL and HEL-DEL46 was examined with XTT assay, showing that the two lines proliferated similarly. RUX inhibited proliferation of HEL cells as well as HEL-DEL46. HEL cells have a JAK2 mutation which causes constitutive activation of the JAK2-STAT5 pathway. Introduction of the Del46 mutation into HEL cells might not enhance activation of the pathway because the JAK2 mutation already activates the pathway at the maximum level. Citation Format: Shunichiro Yasuda, Satoru Aoyama, Daisuke Watanabe, Norihiko Kawamata. Introduction of CALR mutations to a human erythroleukemia cell line, HEL, by CRISPR/Cas9 systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4338.