Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML.

Morito Kurata,G Willemijn Veldhuijzen,Justine M Jones,David A Largaespada,Natalie K Aumann,Susan K Rathe,Natashay J Bailey,Branden S Moriarity

doi:10.1038/srep36199

Abstract

Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML.

Highlights

Acute myeloid leukemias are myeloid proliferative disease associated with a very poor prognosis in general[1]
Acute myeloid leukemia (AML) is a heterogeneous disease with a diverse collection of mutations driving AML progression in each patient, making it problematic to establish a standard protocol for treating the disease effectively
We sought to clarify which drug resistant mechanism is more dominant in acquired resistance to cytosine arabinoside (Ara-C) resistant

Summary

Introduction

Acute myeloid leukemias are myeloid proliferative disease associated with a very poor prognosis in general[1]. We interrogated the response of 2 Dck-defective murine cells and their Ara-C sensitive parental lines to 446 FDA approved drugs. Glucocorticoid prednisolone can induce apoptosis in cells by binding to the glucocorticoid receptor (GR) and continues to play an important role in the treatment of acute lymphoblstic leukemia (ALL) and lymphoid malignancy but not AML5. Whole genome CRISPR libraries are powerful tools for genome-scale loss-of-function screening This system has been previously shown to be highly effective at identifying drug resistant genes in vitro. Shale et al showed the genes responsible for resistance to the BRAF protein kinase inhibitor vemurafenib (PLX)[7] We used these CRISPR/Cas[9] libraries for exploring Ara-C drug resistance in human AML cell lines to determine which genes are critically important for Ara-C resistance in leukemia relapse. We introduced selective pressure using Ara-C to create drug resistant cells capable of resistant to Ara-C but survival and self-renewal to reflect relapse processes

Methods

Results

Conclusion