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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 relapse1,2. Epigenetic regulation plays a vital role in cancer pathogenesis and drug resistance, and several epigenetic factors have emerged as promising therapeutic targets3,4,5. 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 us.......

Representative 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.......

Acknowledgements

This study is funded in part by a Department of Biotechnology grant BT/PR8742/AGR/36/773/2013 to SRV; and Department of Biotechnology India BT/COE/34/SP13432/2015 and Department of Science and Technology, India: EMR/2017/003880 to P.B. RVS and P.B. are supported by Wellcome DBT India Alliance IA/S/17/1/503118 and IA/S/15/1/501842, respectively. S.D. is supported by the CSIR-UGC fellowship, and S.I. is supported by an ICMR senior research fellowship. We thank Abhirup Bagchi, Sandya Rani, and the CSCR Flow Cytometry Core Facility staff for their help. We also thank MedGenome Inc. for helping with the high-throughput sequencing and data analysis.

....

Materials

NameCompanyCatalog NumberComments
Reagents
100 bp Ladder Hyper LadderBIOLINEBIO-33025
1kb Ladder Hyper LadderBIOLINEBIO-33056
AgaroseLonza Seachekm50004
Betaine (5mM)SigmaB03001VL
Boric AcidQualigens12005
Cell culture plasticwareCorningas appicable
Cytosine β-D-arabinofuranoside hydrochlorideSigmaC1768-500MG
DMEMMP BIO91233354
DMSOWak Chemie GMBHCryosure DMSO 10ml
EDTASigmaE5134
Ethidium BromideSigmaE1510-10 mL
Fetal Bovine SerumThermo Fisher Scientific16000044
Gel/PCR Purification KitMACHEREY-NAGELREF 740609.50
Gibco- RPMI 1640Thermo Fisher Scientific23400021
Glacial Acetic AcidThermoQ11007
hCMV GFP PlasmidTransomicsTransOmics Promoter selection KIT
hEF1a GFPlasmidTransomicsTransOmics Promoter selection KIT
HEK 293TATCCCRL-11268
HL60 cell lineATCCCCL-240
KOD Hot Start PolymeraseMerck71086
Molm13 cell lineEllen Weisberg Lab, Dana Farber Cancer Institute, Boston, MA, USADana Farber Cancer Institute, Boston, MA, USA
MV4-11 cell lineATCCCRL-9591
Penicillin streptomycinThermo Fisher Scientific15140122
psPAX2 and pMD2.GAddgeneAddgene plasmid no.12260 & Addgene plasmid no. 12259
Qubit dsDNA HS Assay KitInvitrogenREF Q32854
SFFV  GFP PlasmidTransomicsTransOmics Promoter selection KIT
shERWOOD-UltrmiR shRNA Library from TransomicsTransomicsCat No. TLH UD7409; Lot No: A116.V 132.14
Trans-IT-LTI MirusMirusMirus Catalog Number: MIR2300
TrisMP Biomedicals0219485591
Trypan BlueSigma-AldrichT8154-100ML
Ultra centrifuge TubesBeckman Coulter 103319
Equipments
5% CO2 incubatorThermo Fisher
BD Aria III cell sorterBecton Dickinson
Beckman Coulter Optima L-100K- UltracentrifugationBeckman coulter
CentrifugeThermo Multiguge 3SR+
ChemiDoc Imaging system (Fluro Chem M system)Fluro Chem
Leica AF600Leica
Light MicroscopeZeiss Axiovert 40c
NanodropThermo Scientific
Qubit 3.0 FluorometerInvitrogen
Thermal CyclerBioRad

References

  1. Döhner, H., Weisdorf, D. J., Bloomfield, C. D. Acute myeloid leukemia. The New England Journal of Medicine. 373 (12), 1136-1152 (2015).
  2. De Kouchkovsky, I., Abdul-Hay, M. Acute myeloid leukemia: A comprehensive review and 2016 update.

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