Focused CRISPR-Cas9 genetic screening reveals USO1 as a vulnerability in B-cell acute lymphoblastic leukemia

Amit Kumar Jaiswal,David Casero,Tasha L Lin,Dinesh S Rao,Tiffany M Tran,Michael O Alberti,Hellen Truong

doi:10.1038/s41598-021-92448-w

Abstract

Post-transcriptional gene regulation, including that by RNA binding proteins (RBPs), has recently been described as an important mechanism in cancer. We had previously identified a set of RBPs that were highly dysregulated in B-cell acute lymphoblastic leukemia (B-ALL) with MLL translocations, which carry a poor prognosis. Here, we sought to functionally characterize these dysregulated RBP genes by performing a focused CRISPR dropout screen in B-ALL cell lines, finding dependencies on several genes including EIF3E, EPRS and USO1. Validating our findings, CRISPR/Cas9-mediated disruption of USO1 in MLL-translocated B-ALL cells reduced cell growth, promoted cell death, and altered the cell cycle. Transcriptomic analysis of USO1-deficient cells revealed alterations in pathways related to mTOR signaling, RNA metabolism, and targets of MYC. In addition, USO1-regulated genes from these experimental samples were significantly and concordantly correlated with USO1 expression in primary samples collected from B-ALL patients. Lastly, we found that loss of Uso1 inhibited colony formation of MLL-transformed in primary bone marrow cells from Cas9-EGFP mice. Together, our findings demonstrate an approach to performing focused sub-genomic CRISPR screens and highlight a putative RBP vulnerability in MLL-translocated B-ALL, thus identifying potential therapeutic targets in this disease.

Highlights

Post-transcriptional gene regulation, including that by RNA binding proteins (RBPs), has recently been described as an important mechanism in cancer
Comparing the results across cell lines, we found that single guide RNAs (sgRNAs) targeting three genes, USO1, EIF3E and EPRS were significantly depleted in SEM cells (p < 0.001), when compared to NALM6 cells (Fig. 1E)
We sought to understand whether overexpression of putative RBPs, which we identified previously, contributes to the pathogenesis of MLL-AF4+ B-ALL15

Summary

Introduction

Post-transcriptional gene regulation, including that by RNA binding proteins (RBPs), has recently been described as an important mechanism in cancer. Patients with MLL-rearranged B-ALL have a dismal prognosis, with 5-year event-free survival rates hovering at 33.6% for infants[7] and 50% for older children and a dults[8] Most of these patients are resistant to conventional treatment with chemotherapy and steroids[9], with bone marrow transplantation being the only curative therapeutic a lternative[10]. We performed a sub-genomic CRISPR/Cas[9] dropout screen using 36 highly upregulated RBPs in primary human B-ALL and identified several novel vulnerabilities that included three putative RBPs. Of these, USO1, a putative RBP and a known regulator of vesicular transport, was identified as a MLL-AF4 target gene. CRISPR/Cas9-mediated disruption of USO1 significantly altered cell growth and the cell cycle in B-ALL cell lines; as well as inhibited the colony forming potential of MLL-transformed primary murine bone marrow cells. Our studies provide a comprehensive rubric to functionally evaluate putative targets identified from expression profiling, and the identification of a novel potential target in MLL-rearranged leukemia

Methods

Results

Conclusion