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The protocol details an in vitro murine carcinoma model of non-genetic defective transcription elongation. Here, chronic inhibition of CDK9 is used to repress productive elongation of RNA Pol II along pro-inflammatory response genes to mimic and study the clinically observed TEdeff phenomenon, present in about 20% of all cancer types.
We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription elongation (TE)—we call such cancers as TEdeff. Notably, TEdeff cancers are characterized by spurious transcription and faulty mRNA processing in a large set of genes, such as interferon/JAK/STAT and TNF/NF-κB pathways, leading to their suppression. The TEdeff subtype of tumors in renal cell carcinoma and metastatic melanoma patients significantly correlate with poor response and outcome in immunotherapy. Given the importance of investigating TEdeff cancers—as it portends a significant roadblock against immunotherapy—the goal of this protocol is to establish an in vitro TEdeff mouse model to study these widespread, non-genetic transcriptional abnormalities in cancers and gain new insights, novel uses for existing drugs, or find new strategies against such cancers. We detail the use of chronic flavopiridol mediated CDK9 inhibition to abrogate phosphorylation of serine 2 residue on the C-terminal repeat domain (CTD) of RNA polymerase II (RNA Pol II), suppressing the release of RNA Pol II into productive transcription elongation. Given that TEdeff cancers are not classified under any specific somatic mutation, a pharmacological model is advantageous, and best mimics the widespread transcriptional and epigenetic defects observed in them. The use of an optimized sublethal dose of flavopiridol is the only efficacious strategy in creating a generalizable model of non-genetic widespread disruption in transcription elongation and mRNA processing defects, closely mimicking the clinically observed TEdeff characteristics. Therefore, this model of TEdeff can be leveraged to dissect, cell-autonomous factors enabling them in resisting immune-mediated cell attack.
A key rate-limiting step in the expression of nearly all active genes is the transition of RNA polymerase II (RNA Pol II) from promoter-proximal pausing to productive elongation1,2. Given that epigenetic dysregulation of transcriptional elongation assists in the progression of multiple human malignancies defined as TEdeff, leading to suboptimal signaling in the pro-inflammatory response pathways amounting to a poor response and outcome to immunotherapy3, the overarching goal of this protocol is to establish a useful in vitro model to study these widespread non-genetic transcr....
The Institutional Animal Care and Use Committee and Institutional Biosafety Committee of the Cincinnati Children’s Research Foundation approved all animal experimental procedures (IACUC protocol #2017-0061 and IBC protocol #IBC2016-0016), and these experiments were carried out in accordance with standards as described in the NIH Guide to the Care and Use of Laboratory Animals.
1. Chronic inhibition of RNA Pol II by flavopiridol treatment—basic strategy
Here, we provide a detailed scheme (Figure 1) to establish a TEdeff cell model obtained by chronic sub-lethal (Figure 2) treatment with flavopiridol at 25 nM. In Figure 3, on 3 days of treatment with flavopiridol, B16 OVA cells show partial characteristics of TEdeff but after one week of treatment, B16/F10 OVA cells show a profound loss of phosphorylation at serine 2 position on.......
RNA Pol II elongation control has emerged as a decisive lever for regulating stimulus-responsive gene expression to the benefit of malignant cells5,7,8. Overcoming promoter-proximal pausing to elongation and subsequent mRNA production requires the kinase activity of P-TEFb9,10,11. Our model utilizes flavopiridol (25 nM), an inhibitor of.......
This work was in part supported by NCI (CA193549) and CCHMC Research Innovation Pilot awards to Kakajan Komurov, and Department of Defense (BC150484) award to Navneet Singh. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the Department of Defense. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
....Name | Company | Catalog Number | Comments |
hhis6FasL | Cell Signaling | 5452 | |
10X TBS | Bio-Rad | 170-6435 | |
12 well plates | Falcon | 353043 | |
20% methanol | Fisher Chemical | A412-4 | |
24-well plates | Falcon | 351147 | |
4–18% SDS polyacrylamide gel | Bio-Rad | 4561086 | |
4% Paraformaldehyde | Thermo Fisher Scientific | AAJ19943K2 | |
5% dry milk | Bio-Rad | 170-6404 | |
7-Methylguanosine antibody | BioVision | 6655-30T | |
96-well plates | Cellstar | 655180 | |
AF647-conjugated mouse CD8 | Biolegend | 100727 | |
antibiotic and antimycotic | Gibco | 15240-062 | |
anti-His antibody | Cell Signaling | 2366 P | |
Anti-Rabit | Cell Signaling | 7074 | Dilution 1:5000 |
Anti-Rat | Cell Signaling | 7077S | Dilution 1:5000 |
Bradford assay Kit | Bio-Rad | 5000121 | |
BSA | ACROS Organics | 24040-0100 | |
BV421-conjugated mouse CD45 | Biolegend | 109831 | |
crystal violet | Sigma | C3886-100G | |
DMEM | Gibco | 11965-092 | |
Dynabeads Oligo (dT)25 | Ambion | 61002 | |
FBS | Gibco | 45015 | |
Fixable Live/Dead staining dye e780 | eBioscience | 65-0865-14 | |
Flavopiridol | Selleckchem | S1230 | |
H3k36me3 | Abcam | ab9050 | Dilution 1:2000 |
IFN-α | R&D systems | 12100-1 | |
IFN-γ | R&D systems | 485-MI-100 | |
IMDM | Gibco | 12440053 | |
Immobilon Western Chemiluminescent HRP Substrate | Millipore | WBKLS0500 | |
MojoSort Mouse CD8 T Cell Isolation Kit | Biolegend | 480007 | |
NF-κB | Cell Signaling | 8242s | Dilution 1:1000 |
PBS | Gibco | 14190-144 | |
p-NF-κB | Cell Signaling | 3033s | Dilution 1:1000 |
p-Ser2-RNAPII | Active Motif | 61083 | Dilution 1:500 |
p-Ser5-RNAPII | Active Motif | 61085 | Dilution 1:1000 |
p-STAT1 | Cell Signaling | 7649s | Dilution 1:1000 |
RiboMinu Eukaryote Kit | Ambion | A10837-08 | |
RIPA buffer | Santa Cruz Biotechnology | sc-24948 | |
RNAPII | Active Motif | 61667 | Dilution 1:1000 |
STAT1 | Cell Signaling | 9175s | Dilution 1:1000 |
TNF-α | R&D systems | 410-MT-010 | |
total H3 | Cell Signaling | 4499 | Dilution 1:2000 |
Tri reagent | Sigma | T9424 | |
Triton | Sigma | T8787-50ML | |
Tween 20 | AA Hoefer | 9005-64-5 | |
β-Actin | Cell Signaling | 12620S | Dilution 1:5000 |
β-ME | G Biosciences | BC98 |
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