Targeted genomic CRISPR-Cas9 screen identifies MAP4K4 as essential for glioblastoma invasion

Abstract

Among high-grade brain tumors, glioblastoma is particularly difficult to treat, in part due to its highly infiltrative nature which contributes to the malignant phenotype and high mortality in patients. In order to better understand the signaling pathways underlying glioblastoma invasion, we performed the first large-scale CRISPR-Cas9 loss of function screen specifically designed to identify genes that facilitate cell invasion. We tested 4,574 genes predicted to be involved in trafficking and motility. Using a transwell invasion assay, we discovered 33 genes essential for invasion. Of the 11 genes we selected for secondary testing using a wound healing assay, 6 demonstrated a significant decrease in migration. The strongest regulator of invasion was mitogen-activated protein kinase 4 (MAP4K4). Targeting of MAP4K4 with single guide RNAs or a MAP4K4 inhibitor reduced migration and invasion in vitro. This effect was consistent across three additional patient derived glioblastoma cell lines. Analysis of epithelial-mesenchymal transition markers in U138 cells with lack or inhibition of MAP4K4 demonstrated protein expression consistent with a non-invasive state. Importantly, MAP4K4 inhibition limited migration in a subset of human glioma organotypic slice cultures. Our results identify MAP4K4 as a novel potential therapeutic target to limit glioblastoma invasion.

P-selectin axis plays a key role in microglia immunophenotype and glioblastoma progression

“…6B, C). Within these clusters and following differential expression analysis we found genes related to cancer invasion such as Col3a1, Map4k4, Chd7 and Tubb2b [27][28][29] , genes associated with proliferation and tumor progression such as Pdgfa and the oncogenes Fos, Jun and Myc [30][31][32][33] , and angiogenesis genes as VEGFa, Tcf4, Timp1 and Timp3 [34][35][36][37] (Fig. 6D).…”

An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

“…Follow up experiments utilizing CRISPR/Cas9 methods with less side effects to confirm results obtained from stable Cas9 cell lines may be warranted. Despite the disadvantages, stable Cas9 cell lines have been effectively used to identify genes responsible for specific phenotypes in other vertebrate cell lines 54 , 56 and are a valuable model of choice for initial high-throughput screens to identify the most suitable targets. Using this approach, key information such as cellular phenotype and gRNA efficiency can be obtained in a high-throughput manner to inform subsequent experiments that validate specific targets are associated with phenotypes of interest.…”

Selective protection of human cardiomyocytes from anthracycline cardiotoxicity by small molecule inhibitors of MAP4K4

“…These findings are consistent with the proven benefits of blocking MAP4K4 to promote cell survival not only in hPSC-CMs subjected to alternative death signals (H 2 O 2 , menadione, hypoxia-reoxygenation) 11 , but also in adult mouse myocardium after experimental myocardial infarction 11 , human islet cells in palmitate-induced apoptosis 52 , and hPSC-derived motor neurons in amyotrophic lateral sclerosis 53 . Whereas this highly generalizable benefit speaks to the likely utility of MAP4K4 inhibitors beyond merely DOX-induced cardiotoxicity, such results also point to a reciprocal concern, that MAP4K4 inhibitors, if capable of acting as a broad survival signal, might interfere with tumour cell killing, notwithstanding current interest in the cancer field regarding the participation of MAP4K4 in tumor angiogenesis, tumor cell motility, and metastasis 54 – 59 .…”