A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TE^deff) in Cancers

Given that TEdeff cancers are a significant roadblock against immunotherapy, our pharmacological model is an advantageous tool because it suitably mimics the widespread transcriptional and epigenetic defects observed in such cancers, and it allows one to study these non-genetic abnormalities to gain new insights, find novel uses for existing drugs, or find new strategies against such cancers. This is an easy-to-establish and a generalizable model to study transcriptional elongation defects in cancers, enabling one to study tumor-immune interactions both in vitro and in vivo. This method can also be applied to human carcinoma lines.

We have tested this on T47D and CAL51 for a short term, giving rise to similar TEdeff-like characteristics. The protocol we describe here gives a basic framework to minimize the known variables critical for the generation of TEdeff-like features by chronic CDK9 inhibition. However, care must be taken to optimize the exact sublethal dose of flavopiridol for other murine lines.

The impact of variation in cell plating density, culture conditions, cytokine stimulation conditions may vary for different cell murine lines. Begin by extracting polyA-positive RNA from half of the previously ribosomal RNA-depleted samples using oligo dT magnetic beads. Resuspend the beads in the vial by vortexing for 30 seconds, and transfer 200 microliters of the beads to a tube.

Add an equal volume of binding buffer, and mix the contents. Place the tube in a magnet for one minute, and discard the supernatant. Then, remove the tube from the magnet, and resuspend the washed beads in 100 microliters of binding buffer.

Adjust the volume of the ribosomal RNA-depleted sample to 100 microliters with 10-millimolar Tris-HCl at pH 7.5. Add 100 microliters of binding buffer to the sample. Heat the mixture to 65 degrees Celsius for two minutes to disrupt secondary RNA structures, and then immediately place it on ice.

Add the 200 microliters of total RNA to the 100 microliters of washed beads, and mix thoroughly on a rotor for five minutes. Place the tube on the magnet for one to two minutes, carefully remove all supernatant, then remove the tube from the magnet, and add 200 microliters of washing buffer. Carefully mix the sample by pipetting up and down a few times, return the tube to the magnet for one minute, and remove the supernatant.

Then, use a spectrophotometer to measure the purity and concentration of the isolated bead-bound mRNA. Use the remaining half of the ribosomal RNA-depleted sample as input to protein A columns to immunoprecipitate five-prime capped RNAs with monoclonal 7-methylguanosine antibody. Wash the protein A magnetic beads from the RIP kit according to the manufacturer's protocol to pre-bind the antibody to the beads, of the 7-methylguanosine antibody to the bead suspended in 100 microliters of wash buffer from the kit.

Incubate the mixture at room temperature for 30 minutes while rotating at low speed. Then centrifuge the tubes briefly, and put them on the magnetic separator. Remove and discard the supernatant, then take the tubes off the magnetic separator, and resuspend the beads with 500 microliters of wash buffer.

Vortex the tubes, centrifuge them briefly, and place them back on the magnetic separator, and again discard the supernatant. Then, add 120 nanograms of ribosomal RNA-depleted RNA to the antibody-bound beads along with one microliter of RNase inhibitor, and incubate the mixture at room temperature for 60 to 90 minutes with mild agitation. After the incubation, spin down the sample at 300 times g for 10 seconds, and transfer the supernatant with the uncapped mRNA to a new microcentrifuge tube.

Repeat the wash two more times with 100 microliters of wash buffer, and pool the collected supernatant. Elute the capped mRNA from the beads by adding 300 microliters of urea lysis buffer prepared according to the manuscript directions and heating the mixture to 65 degrees Celsius for two to three minutes. Combine 300 microliters of the eluted sample with 300 microliters of phenol:chloroform:isoamyl alcohol, invert to mix, and leave for 10 minutes.

Gently mix the sample again, and centrifuge for two minutes. Carefully pipette the top layer to a fresh tube, and discard the bottom layer. Add 300 microliters of 2-propanol and 30 microliters of three-molar sodium acetate to the sample, invert it a few times, and put it in minus 20 degrees Celsius for 20 hours.

After the incubation, centrifuge the sample for 10 minutes at four degrees Celsius. Carefully discard the supernatant, and dry the pellet at room temperature for five minutes. Resuspend the pellet in nuclease-free water, and measure the purity and concentration of the RNA with a spectrophotometer.

Isolate CD8-positive cells according to the manuscript directions, then resuspend the cells in commercially available magnetic separation system buffer. Add 100 microliters of antibody cocktail for every one milliliter of cells, and incubate the cells on ice for 15 minutes. Then, add 100 microliters of magnetic beads per every 100 microliters of antibody cocktail, and leave the cells on ice for another 15 minutes.

After the incubation, add seven milliliters of magnetic separation buffer to the cells, aliquot three to four milliliters to a fresh tube, and place the tube on a magnetic for five minutes. Decant the liquid with the CD8-positive cells to a fresh tube on ice. Then, add the remaining three to four milliliters of cells to the magnetic beads, and place the tube on the magnet for five minutes.

Decant the second batch of CD8-positive cells to the tube with the first batch. Seed engineered adherent fibroblast SAMBOK cells along with the co-stimulatory molecule at 75, 000 cells per well in 24-well plates, and culture them in a 37-degree Celsius, 5%carbon dioxide humidified incubator. After 24 hours, wash the APC monolayer once with Iscove's modified Dulbecco's medium, and add 0.5 times 10 to the six naive cells in two milliliters of medium supplemented according to the manuscript directions.

Culture the cells for 20 hours, then gently harvest the nonadherent OT-I cells by collecting the media and pelleting the cells at 191 times g for two minutes. Count the cells, and seed them at a one-to-one ratio in a co-culture with B16/F10, untreated B16/F10-OVA, and B16/F10-OVA cells pretreated with flavopiridol. Culture the cells for 20 hours in a 37-degree Celsius, 5%carbon dioxide humidified incubator, then remove the OT-I CD8-positive cells, and wash the adherent B16/F10-OVA cells in PBS.

Trypsinize the three groups of attached cells in 05%EDTA with trypsin for five minutes, and then pellet them at 191 times g for five minutes. Incubate the harvested B16/F10-OVA cells in cold PBS with viability dye and relevant labeled antibodies, then analyze the viability with flow cytometry. This protocol has been used to establish a transcription elongation defective cell model that shows a profound loss of phosphorylation at the serine 2 position on the C-terminal repeat domain of RNA polymerase II and a significant decrease in H3K36me3, which is implicated in defining exon boundaries and inhibiting runaway cryptic transcriptions.

The cells show critical mRNA processing defects with increasing ratios of improperly capped and non-polyadenylated mRNAs. Furthermore, specific repression of key inflammatory response pathway genes and FasL-mediated cell death are observed in this cell model. An exploratory assay to test if the cell model confers resistance to cytotoxic T-cell attack shows that chronic flavopiridol-induced transcription elongation defects can bestow a means of escape from an anti-tumor immune attack.

Flavopiridol-treated B16/F10 cells stably overexpressing the OVA gene were not susceptible to CD8-positive cytotoxic T cells, which have a selective toxicity towards OVA-expressing cells. Cells not pretreated with flavopiridol underwent major cell death, while parental B16/F10 that do not express the OVA antigen survived. It is important to remember that reduction in phosphoserine 2 and H3K36 trimethylation level on 25-nanomolar flavopiridol treatment does not guarantee a reduction in both phopsho-STAT1 and phospho-NF-kappaB level.

Each mouse carcinoma line is unique, and JAK1 and CCNT1 may rescue the effect of flavopiridol. Additionally, this model can be used in vivo to monitor the resistance offered by TEdeff cancers against innate and adaptive anti-tumor immune responses. For example, anti-asialo treatments could be used to regulate the activity of NK cells, and immune checkpoint therapy could be administered to TEdeff tumor-bearing mice.

The tumor-infiltrating lymphocyte, or TIL, load is an indicator of success in immunotherapy. Our model has paved the way for us to explore the extent of activation and exhaustion of TILs in the TEdeff cancer microenvironment both before and after immunotherapy.