Open AccessPF214 CRISPR/CAS9 IN VIVO SCREEN REVEALS A CRUCIAL ROLE FOR TMEM63A IN ENGRAFTMENT OF PRIMARY HUMAN ACUTE MYELOID LEUKEMIA CELLS
Author(s) -
He L.,
Garg S.,
Xia J.,
Jagdhane P.,
MüllerTidow C.,
Pabst C.
Publication year - 2019
Publication title -
hemasphere
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.677
H-Index - 11
ISSN - 2572-9241
DOI - 10.1097/01.hs9.0000559072.23049.a5
Subject(s) - crispr , gene knockdown , biology , myeloid leukemia , in vivo , leukemia , small hairpin rna , guide rna , ex vivo , rna , cancer research , myeloid , microbiology and biotechnology , gene , computational biology , cas9 , immunology , genetics
Background: Acute myeloid leukemia (AML) is a hematologic malignancy, which is sustained by a small fraction of leukemia stem cells (LSCs). In previous work, we determined LSC frequencies of primary AML specimens by xenograft assays and correlated engraftment capacity with gene expression determined by RNA‐Sequencing (RNA‐Seq). Here, we performed a clustered regularly interspaced short palindromic repeats (CRISPR)/ C RISPR‐ a ssociated s ystem (Cas) in vivo screen targeting 189 selected genes to identify novel regulators of LSC activity. Aims: In this study, we aimed to identify crucial leukemia drivers and suppressors via single‐guide (sgRNA) library in vivo screening in a humanized NRGS mouse xenograft model. Methods: We performed a pooled, unbiased two‐step lentiviral single‐guide (sgRNA) library screen in vitro and simultaneously in mice. Furthermore, we performed knockdown (KD) experiments using small hairpin (sh) RNAs and single‐guide (sg) RNAs to confirm screen results in single assays. Lentivirally transduced AML cells were analyzed in vivo for engraftment capacity and simultaneously in vitro for proliferative output, differentiation, viability and cell cycle. RNA sequencing (RNA‐Seq) was used to determine how KD of the top candidate gene affected gene expression. Moreover, we tracked protein localization by immunofluorescence (IF) imaging and performed global proteomics experiments to gain insight into the mechanism of action. Results: We found that knockdown of TMEM63A inhibited cell proliferation, enhanced differentiation ex vivo and impaired engraftment capacity of AML cells in vivo . Gene Ontology (GO) term enrichment analysis on RNA‐Seq data showed enrichment for pathways associated with immune response. Summary/Conclusion: We found that knockdown of TMEM63A inhibited cell proliferation, enhanced differentiation ex vivo and impaired engraftment capacity of AML cells in vivo . Gene Ontology (GO) term enrichment analysis on RNA‐Seq data showed enrichment for pathways associated with immune response.