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 regulatory mechanisms affect proliferation and survival under continuous exposure to chemotherapeutic drugs. Studies in non-hematological malignancies have reported that a small fraction of cells that overcome the drug effect undergo various epigenetics modifications, resulting in those cells' survival^6,7. However, the role of epigenetic factors in mediating acquired resistance to cytarabine in AML has not been explored.

High-throughput screening is an approach to drug discovery that has gained global importance over time and has become a standard method in different aspects to identify potential targets in cellular mechanisms, for pathway profiling, and at the molecular level^8,9. The synthetic lethality concept involves the interaction between two genes where the perturbation of either gene alone is viable but of both genes simultaneously results in the loss of viability¹⁰. Exploiting synthetic lethality in cancer treatment could help identify and mechanistically characterize robust synthetic lethal genetic interactions¹¹. We have adopted a combinatorial approach of high-throughput shRNA screening with synthetic lethality to identify the epigenetic factors responsible for acquired cytarabine resistance in AML.

Acute leukemias driven by chromosomal translocation of the mixed-lineage leukemia gene (MLL or KMT2A) are known to be associated with poor survival in patients. The resulting chimeric products of MLL gene rearrangements, i.e., MLL fusion proteins (MLL-FPs), can transform hematopoietic stem/progenitor cells (HSPCs) into leukemic blasts with the involvement of multiple epigenetic factors. These epigenetic regulators constitute a complicated network that dictates the maintenance of the leukemia program and, therefore, could form potential therapeutic targets. In this context, we used the MV4-11 cell line (harboring the MLL fusion gene MLL-AF4 with the FLT3-ITD mutation; termed as MV4-11 P) to develop the acquired cytarabine resistant cell line, termed as MV4-11 AraC R. The cell line was exposed to increasing doses of cytarabine with intermittent recovery from the drug treatment, known as a drug holiday. The half-maximal inhibitory concentration (IC50) was assessed by in vitro cytotoxicity assay.

We used the pooled epigenetic shRNA Library (see Table of Materials) driven by the hEF1a promoter with a pZIP lentiviral backbone. This library comprises shRNAs targeting 407 epigenetic factors. Each factor has 5-24 shRNAs, with a total of 5,485 shRNAs, including five non-targeting control shRNAs. The modified "UltrmiR" miR-30 scaffold has been optimized for efficient primary shRNA biogenesis and expression^12,13.

The outline of this experiment is illustrated in Figure 1A. The current protocol focuses on RNAi screening using the epigenetic factor shRNA library in the MV4-11 AraC R cell line (Figure 1B), a suspension cell line. This protocol can be used to screen any targeted library in any drug-resistant cell line of one's choice. It should be noted that the transduction protocol will be different for adherent cells.

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 expression of shRNAs in the cell line selected for the experiment. The most commonly used promoters for this purpose are hEF1α (human elongation factor 1α), hCMV (human cytomegalovirus), and SSFV (spleen focus-forming virus) promoters that express green fluorescence protein (GFP) (Figure 2A).

1. Perform the cell count using the trypan blue exclusion method. Suspend the MV4-11 AraC R cells in 10% RPMI medium (RPMI with 10% FBS supplemented with 100 U/mL penicillin and 100 U/mL streptomycin) containing 8 µg/mL polybrene. Seed 1 x 10⁶ cells in 1.5 mL of medium/well of a six-well plate in triplicates.
2. Add different volumes of concentrated lentiviruses (for example, 10 µL, 20 µL, and 40 µL depending on the titer of the lentiviruses) to each well. Gently swirl the plate to mix the contents.
3. Perform spinfection by centrifugation at 920 x g at 37 °C for 90 min.
4. Immediately incubate the plates in a CO₂ incubator (5% CO₂ at 37 °C).
5. Observe the GFP expression after 48 h under a fluorescence microscope to ensure successful transduction.
6. Quantify the GFP expression by flow cytometry after 72 h to evaluate the transduction efficiency.
7. Culture the cells for a total of 2 weeks and monitor the GFP expression periodically by flow cytometry. The reduction in GFP expression measured by the mean fluorescence intensity and the percentage of GFP+ cells indicate promoter silencing.
8. Sort the GFP+ cells on the 10th day and culture the cells for 1 week after sorting. Check the GFP expression periodically during the culture.
9. Use the untransduced cells to set the gate. A population beyond the 1 x 10¹ scale toward the right on the x-axis is considered a positive population for GFP.
10. Choose the promoter that shows a persistent expression of GFP in the prolonged culture of the cells.

2. Preparation of pooled lentiviral human epigenetic factor shRNA library

1. Culture 293T cells (at a low passage number with a good proliferation rate) in three 10 cm cell culture dishes with 8 mL of 10% DMEM medium (DMEM with 10% FBS containing 100 U/ml penicillin and 100 U/ml streptomycin) in each plate.
2. Once the cells attain 60% confluency, replace the medium with a complete medium.
3. Prepare the transfection mix (as outlined in Table 1) that contains serum-free medium, lentiviral packaging (psPAX2) and envelope plasmid (pMD2.G), pooled library plasmids, and finally, the transfection reagent.
4. Tap the mixture to mix it well and incubate at room temperature for 15 min.
5. Add the transfection mixture to the 293T cells and mix gently by swirling the plates. Incubate the plates in a CO₂ incubator (5% CO₂ at 37 °C). Change the medium after 24 h.
6. After 48 h, check for GFP expression under a fluorescence microscope to ensure high transfection efficiency in the 293T cells.
7. Collect the virus supernatants after 48 h, 60 h, and 72 h and store them at 4 °C. Add fresh medium (8 mL) at each time point after collecting the virus.
8. Pool the virus supernatants. The total volume of the pooled viruses will be: ~8 mL/plate x 3 collections= ~24 mL/plate; ~24 mL/plate x 3 plates = ~72 mL from 3 plates.
9. Filter the pooled viruses with the 0.4 µm filter.
10. For obtaining a high titer of viruses, concentrate the pooled viruses by ultracentrifugation. Transfer the filtered virus supernatant to two 70 Ti tubes (32 mL/tube) and centrifuge at 18,000x g for 2 h with slow acceleration and deceleration. Use a rotor and centrifuge precooled to 4 °C.
11. Take out the tubes from the centrifuge gently and remove the supernatant completely.
12. Using a micropipette, gently resuspend the pellet in 400 µL of DMEM (without FBS and antibiotics). Incubate on the ice for 1 h and mix the pellets from both tubes.
13. Aliquot the viruses and freeze at −80 °C.
    NOTE: The tubes and viruses must be kept on ice during steps 2.10.-2.13. Avoid frothing while resuspending the virus with the plain medium. The medium should be maintained at 4 °C.
14. Calculate the concentration (x) of virus as below:
    Total volume before concentration = 72 mL (72,000 µL)
    Volume after concentrating = 400 µL
    The concentration = 72,000/400= 180x

3. Estimation of the transduction efficiency of lentiviruses

1. Transduce 293T cells with the concentrated virus in different volumes (2 µL, 4 µL, 8 µL) to confirm the successful preparation of lentiviruses.
   NOTE: If concentrated viruses show high transduction efficiencies (>50%) in small volumes (1-2 µL), it is advisable to dilute the concentrated virus in the desired amounts (100x to 75x).
2. Dilute the concentrated virus to 100x with DMEM (without FBS and antibiotics) to perform titration experiments in the MV4-11 AraC R cell line.
3. Seed 1 x 10⁶ cells (MV4-11 AraC R cell line in 1.5 mL of 10% RPMI medium containing 8 µg/mL polybrene) in one well of a six-well plate. Prepare four wells for transducing with different volumes of viruses.
4. Add four different volumes (for example, 1 µL, 1.5 µL, 2 µL, and 2.5 µL) of 100x viruses.
5. Perform spinfection by centrifugation of the plate at 920 x g for 90 min at 37 °C.
6. After 72 h, measure the percentage of GFP+ cells by flow cytometry.
7. Determine the volume of the viruses to obtain 30% transduction efficiency. This low transduction efficiency is to ensure single shRNA integration per cell.

4. Transduction of pooled epigenetic shRNA library in the drug-resistant cell line

1. Maintain the target cell line MV4-11 AraC R at a 0.5 x 10⁶ cells/mL density. Ensure the cells are at the exponential growth phase in culture.
2. Calculate the number of cells to be taken for the experiment as below based on the number of viral integrants.
   Number of cells required for transduction = 5,485 (number of shRNAs) × 500 (fold shRNA representation) / 0.25 (MOI) = 10,982,000 ~11 x 10⁶ cells
3. Resuspend 1.1 x 10⁷ cells in 16 mL of 10% RPMI medium.
4. Add polybrene (8 µg/mL) and mix well. Then, add the required volume of viruses to obtain 30% transduction efficiency as calculated in the previous section.
5. Seed all the cells on a six-well plate at a density of 1 x 10⁶ cells/1.5 mL of 10% RPMI medium per well.
6. Centrifuge the plate for 90 min at 920 x g at 37 °C.
7. Incubate the plate overnight in a CO₂ incubator (5% CO₂ at 37 °C).
8. After 24 h, change the medium and transfer the transduced cells to a T-75 flask.
9. After 48 h, check the GFP under a fluorescence microscope to ensure successful transduction.
10. After 72 h, quantitate the percentage of GFP+ cells by flow cytometry.

5. Enrichment of GFP positive cells

NOTE: Expand the transduced cells by culturing them at a density of 0.5 x 10⁶ cells/mL for 5-7 days. These cells are a mixed population of transduced and untransduced, selected based on GFP by sorting, as mentioned in the next step.

1. Perform flow-sorting of GFP+ transduced cells with the flow-sorting settings set as high purity and low yield. Use the untransduced cells to set the gate. A population beyond 1 x 10² toward the right is considered a positive population.
2. Collect the sorted cells in 5% RPMI (RPMI with 5% FBS supplemented with 100 U/mL penicillin and 100 U/mL streptomycin).
3. Culture the sorted cells in a 10% RPMI medium.
4. After 72 h, perform the post-sorting estimation of the percentage of GFP+ cells to ensure >95% sorting efficiency.
   NOTE: Make sure to obtain ~3-5 x 10⁶ GFP positive cells after sorting to obtain 450x to ~500x representation of the shRNA library.

6. Dropout screening to identify epigenetic factors mediating drug resistance

1. Culture the shRNA library transduced cells in 10% RPMI for up to 5 days. Cryopreserve the transduced cells for future experiments, if required, before the drug treatment.
2. Centrifuge 1 x 10⁷ cells in duplicates at 280 x g for 5 min at 25 °C. Discard the supernatant, and store the pellets at −80 °C. These samples will serve as the baseline reference for the epigenetic shRNA library.
3. Culture the remaining transduced cells as duplicates (R1 and R2), each maintained at a cell count that provides a 500x representation of the library.
4. Treat one of the duplicates with the drug (10 µM cytarabine) (R2) and the other without the drug treatment (R1).
5. Change the medium for the flasks with and without the drug every 72 h. Repeat the medium change 3x for a cumulative drug exposure of 9 days.
6. After 9 days of drug treatment, check the viability of the cells by the trypan blue exclusion method.
7. Centrifuge the remaining cells at 280 x g for 5 min at room temperature. Resuspend the cells in sterile PBS and wash the cells. Centrifuge at 280 x g for 5 min at room temperature.
8. Discard the supernatant and store the pellets at −80 °C for DNA extraction.

7. Amplification of the integrated shRNAs by PCR

1. Extract DNA from the transduced baseline cells (T) (step 6.2.) and the cells treated with the drug (R2) and untreated with the drug (R1).
2. Check the concentration of the sample using a fluorometer.
3. Calculate the amount of DNA required for PCR as below:
   5,485 (no. of shRNAs) × 500 (shRNA representation) × 1 x 6.6⁻¹² (g/diploid genome) = 15 µg of DNA
4. Subject the samples to the first round of PCR.
   NOTE: The PCR reaction mixture is depicted in Table 2 with the thermal cycler conditions. Details of the primers are provided in Supplementary Table 1.
5. Set up multiple reactions containing 850 ng of DNA per tube (for a total of 43 reactions).
   NOTE: The first round of PCR amplifies the flanking regions of the shRNA resulting in an amplicon size of 397 bp.
6. Pool the PCR products and purify the PCR products using 3-4 columns of a standard gel/PCR purification kit.
   NOTE: Details are provided in Table 3.
7. Elute the product in 50 µL of buffer from each column and pool the elutes.
8. Quantify the DNA and store it at −20 °C.
   NOTE: The reagents are tabulated in Table 4 with the thermal cycler conditions. The second-round PCR utilizes the primers with the INDEX sequence required for multiplexing in NGS in a singleflow cell. So, each sample should be tagged with a different index. Primer sequences are provided in Supplementary Table 1.
9. Set up four reactions with 500 ng of the primary PCR product in a total reaction volume of 50 µL for each sample and the negative control.
10. Load the entire product for electrophoresis using 2% TBE agarose gel and confirm the band size using a 1 kb molecular ladder. The size of the amplicon is 399 bp.
11. Visualize the PCR products on the gel documentation system.
12. Excise the specific band and purify using a gel purification kit.
    NOTE: Calculations for purification with the kit are provided in Table 3.
13. Elute in a final volume of 30 µL of elution buffer. The approximate concentration of each eluent will be around 80-90 ng/µL with a total volume of 30 µL.

8. Next-generation sequencing and data analysis

1. Sequence the gel-purified product from step 7.13. on a next-generation sequencer (e.g., Illumina) to obtain the read count for the depleted shRNAs.
2. Trim the adaptor sequences of the generated FastQ reads using the Fastx-tool kit (version 0.7).
3. Align the filtered reads to reference sequences using a Bowtie aligner with the parameter of allowing two mismatches. Ensure the read length after adapter trimming is around 21 bp.
4. Load the files using the Samtools (version 1.9) program. Use the Flagstat option and calculate the alignment summary.
5. Load the trimmed fastQ files in CRISPRCloud2 (CC2) software (https://crispr.nrihub.org/) along with the reference shRNA sequences in the form of FASTA files and perform the analysis as per the instructions.
   1. Click on the Start Analysis link in the CC2.
   2. Select the screen type as Survival and Dropout screens. Set the number of groups and type the name for each group.
   3. Upload the reference library in FASTA file format. The data uploaded is processed. Click on the output URL to obtain the results.
      NOTE: This software uses a beta-binomial model with a modified Student's t-test to measure differences in sgRNA/shRNA levels, followed by Fisher's combined probability test to estimate the gene-level significance. It calculates estimated overall log₂-fold changes (Log2Fc) of shRNAs for the epigenetic factors between the treated (enriched/depleted) and the untreated samples (baseline) with "p-values" and "FDR values."
6. Ensure the FDR value is "Negative" for a Dropout screen and "Positive" for a Survival screen.
   NOTE: CC2 is an analysis platform for counting the reads and calculating the fold representation (enrichment or depletion) of shRNAs between samples.
7. Identify the significantly enriched and depleted shRNAs (FDR <0.05) from the CC2 results.
   NOTE: There are 5-24 shRNAs against each factor. Check the FDR values for each of the shRNAs; consider the FDR, which is <0.01, and take an average for the selected shRNAs. Consider the top 40 hits. Plot the graph for the selected factors based on Log2Fc or FDR negative values. Make sure the non-targeting shRNAs also have significant FDR values to ensure the authenticity of the data as they serve as control shRNAs.

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

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