Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Summary

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

Abstract

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

Introduction

Therapeutic options in acute myeloid leukemia (AML) have remained unchanged for nearly the past five decades, with cytarabine (AraC) and anthracyclines as the cornerstone for treating the disease. One of the challenges to the success of AML therapy is the resistance of leukemic stem cells to chemotherapy, leading to disease relapse^1,2. Epigenetic regulation plays a vital role in cancer pathogenesis and drug resistance, and several epigenetic factors have emerged as promising therapeutic targets^3,4,5. Epigenetic regula....

Protocol

Follow the Institutional Biosafety Committee (IBSC) guidelines and use the proper facility to handle lentivirus (BSL-2). Personnel should be appropriately trained in the handling and disposal of lentivirus. This protocol follows the biosafety guidelines of Christian Medical College, Vellore.

1. Selection of the most potent promoter to obtain persistent and prolonged expression of the shRNAs

NOTE: It is essential to perform a transduction experiment using lentiviral vectors with different promoters that express fluorescence proteins to identify the promoter that provides a stable and long-term expre....

Results

The overall screening workflow is depicted in Figure 1A. In vitro cytotoxicity of the MV4-11 AraC R (48 h) revealed the IC50 to cytarabine in the MV4-11 AraC R to be higher than the MV4-11 P (Figure 1B). This cell line was used in the study as the model for screening the epigenetic factors responsible for cytarabine resistance.

Figure 2A shows the linearized pZIP vector maps with.......

Discussion

RNA interference (RNAi) is extensively used for functional genomics studies, which include siRNA and shRNA screening. The benefit of shRNA is that they can be incorporated into plasmid vectors and integrated into genomic DNA, resulting in stable expression and, thus, more prolonged knockdown of the target mRNA. A pooled shRNA library screening is robust and cost-effective compared to the conventional arrayed screens (siRNA). Identifying the essentiality of a specific class of proteins in a genome-wide screen can be cumbe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
100 bp Ladder Hyper Ladder	BIOLINE	BIO-33025
1kb Ladder Hyper Ladder	BIOLINE	BIO-33056
Agarose	Lonza Seachekm	50004
Betaine (5mM)	Sigma	B03001VL
Boric Acid	Qualigens	12005
Cell culture plasticware	Corning	as appicable
Cytosine β-D-arabinofuranoside hydrochloride	Sigma	C1768-500MG
DMEM	MP BIO	91233354
DMSO	Wak Chemie GMBH	Cryosure DMSO 10ml
EDTA	Sigma	E5134
Ethidium Bromide	Sigma	E1510-10 mL
Fetal Bovine Serum	Thermo Fisher Scientific	16000044
Gel/PCR Purification Kit	MACHEREY-NAGEL	REF 740609.50
Gibco- RPMI 1640	Thermo Fisher Scientific	23400021
Glacial Acetic Acid	Thermo	Q11007
hCMV GFP Plasmid	Transomics	TransOmics Promoter selection KIT
hEF1a GFPlasmid	Transomics	TransOmics Promoter selection KIT
HEK 293T	ATCC	CRL-11268
HL60 cell line	ATCC	CCL-240
KOD Hot Start Polymerase	Merck	71086
Molm13 cell line	Ellen Weisberg Lab, Dana Farber Cancer Institute, Boston, MA, USA	Dana Farber Cancer Institute, Boston, MA, USA
MV4-11 cell line	ATCC	CRL-9591
Penicillin streptomycin	Thermo Fisher Scientific	15140122
psPAX2 and pMD2.G	Addgene	Addgene plasmid no.12260 & Addgene plasmid no. 12259
Qubit dsDNA HS Assay Kit	Invitrogen	REF Q32854
SFFV  GFP Plasmid	Transomics	TransOmics Promoter selection KIT
shERWOOD-UltrmiR shRNA Library from Transomics	Transomics	Cat No. TLH UD7409; Lot No: A116.V 132.14
Trans-IT-LTI Mirus	Mirus	Mirus Catalog Number: MIR2300
Tris	MP Biomedicals	0219485591
Trypan Blue	Sigma-Aldrich	T8154-100ML
Ultra centrifuge Tubes	Beckman Coulter	103319
Equipments
5% CO2 incubator	Thermo Fisher
BD Aria III cell sorter	Becton Dickinson
Beckman Coulter Optima L-100K- Ultracentrifugation	Beckman coulter
Centrifuge	Thermo Multiguge 3SR+
ChemiDoc Imaging system (Fluro Chem M system)	Fluro Chem
Leica AF600	Leica
Light Microscope	Zeiss Axiovert 40c
Nanodrop	Thermo Scientific
Qubit 3.0 Fluorometer	Invitrogen
Thermal Cycler	BioRad