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

Summary

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 TE^deff phenomenon, present in about 20% of all cancer types.

Abstract

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 TE^deff. Notably, TE^deff 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 TE^deff 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 TE^deff cancers—as it portends a significant roadblock against immunotherapy—the goal of this protocol is to establish an in vitro TE^deff 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 TE^deff 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 TE^deff characteristics. Therefore, this model of TE^deff can be leveraged to dissect, cell-autonomous factors enabling them in resisting immune-mediated cell attack.

Introduction

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 elongation^1,2. Given that epigenetic dysregulation of transcriptional elongation assists in the progression of multiple human malignancies defined as TE^deff, 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....

Protocol

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

1. Seed B16/F10 mous.......

Discussion

RNA Pol II elongation control has emerged as a decisive lever for regulating stimulus-responsive gene expression to the benefit of malignant cells^5,7,8. Overcoming promoter-proximal pausing to elongation and subsequent mRNA production requires the kinase activity of P-TEFb^9,10,11. Our model utilizes flavopiridol (25 nM), an inhibitor of.......

Materials

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