CRISPR/Cas9 screens Reveal Dasatinib Targets of Inhibiting T cell Activation and Proliferation

Abstract

< 250 words) 19Immune response by T cells is essential for a healthy body against cancer, infection, and pathophysiological alteration. The 20 activation and expansion of T cells can be inhibited by dasatinib, a tyrosine inhibitor, thus improving the outcome of diseases, 21 such as autoimmune disease, graft-versus-host disease, and transplant rejection. The underlying mechanism of inhibition by 22 dasatinib is elusive. Here, we designed and synthesized a CRISPR/Cas9 screening library that includes 6,149 genes. Using 23 the library, we performed dasatinib CRISPR/cas9 screening in Jurkat cell, a T lymphocyte cell. We firstly identified survival 24 essential genes for Jurkat cells. Comparing with other CRISPR/Cas9 screenings, we obtained Jurkat cell specific essential 25 genes. By comparing dasatinib treatment to control, we identified a set of dasatinib targets, which includes known targets: 26 CSK, LCK, ZAP70, and previously unknown targets: ZFP36L2, LRPPRC, CFLAR, PD-1, CD45 et al. Visualizing these 27 target genes on T cell receptor signaling pathway, we found several genes could be inhibited by dasatinib. Furthermore, we 28 introduced a framework, 9-square, to classify genes and found a group of genes that are associated with dasatinib resistance, 29 possibly linking the side effects of dasatinib. These data reveal a set of dasatinib targets and demonstrate the molecular 30 potential functions of dasatinib. Identification of dasatinib targets will broaden our understanding to its molecular mechanism, 31 and thus benefits to clinical outcome. 32 33 34 53 as Src family. Dasatinib as salvage therapy for sclerotic chronic graft-vs-host disease was reported. Dasatinib is a first-line 54 therapy for chronic myelogenous leukemia (CML) patients [3]. CML is a hematopoietic stem cell disorder frequently caused 55 by chromosomal abnormality, called Philadelphia (Ph) chromosome, a translocation between the abelson (ABL) tyrosine 56 kinase gene at chromosome 9 and the breakpoint cluster region (BCR) at chromosome 22, which forms a fusion oncogene 57 BCR-ABL. Dasatinib is a potent multi-kinase inhibitor that can target and inhibit the BCR-ABL, SRC family (SRC, LCK, 58 HCK, YES, FYN, FGR, BLK, LYN, FRK), receptor tyrosine kinases (c-KIT, PDGFR, DDR1/2, c-FMS, ephrin receptors), 59 and TEC family kinases (TEC and BTK). The Src family kinases are involved in T cell activation, signal transductions, 60 blocking cell duplications, adhesion et al [6]. Dasatinib potently inhibits Src family kinases, diminishing metastatic spread 61 3 of tumor cells, trigging apoptosis of tumor cells, and inhibiting T cell over-activation [7]. These discoveries suggest that 62 dasatinib can inhibit multiple targets and consequently determine cancer cell fate. 63 64 The CRISPR/Cas9 is a gene-editing technology that can cause double-strand breaks (DSBs) by single-guide RNAs (sgRNAs) 65 binding. A sgRNA usually contains 18-20 nucleotides complementary to its target and guides the Cas9 enzyme to a specific 66 DNA location where Cas9 induces a DSB. Repai...

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