Transcriptional Reprogramming of Super-Enhancer Associated Oncogenes Following Inhibition of Cyclin-Dependent Kinase-9 (CDK9) in Aggressive Non-Hodgkin Lymphoma (NHL)
Authors: Thieme Elana , Sun Duanchen , Sharma Geeta G , Bruss Nur , Liu Tingting , Coleman Daniel J , Xia Zheng , Danilov Alexey V. 
Published: November 2021

Abstract
Introduction. CDKs are Ser/Thr kinases that associate with specific cyclins during the cell cycle to regulate its progression. We and others have shown that selective targeting of CDK9, a component of the positive transcription elongation factor b (pTEFb) complex which facilitates RNA transcription, is a promising approach in NHL leading to downmodulation of MYC and MCL1 (Hashiguchi et al, 2019). However, cells also exhibited resistance to CDK9i in vitro and in vivo. Here we studied the transcriptional and epigenetic effects of CDK9 inhibition using the novel selective CDK9 inhibitor AZD4573 which is under evaluation in clinical trials.
Methods. A panel of NHL cell lines with variable sensitivity to CDK9i (OCI-LY3/19, SUDHL4/10/16, VAL, U2932) and primary NHL cells were employed. Response to CDK9i was characterized using RNA-Seq, ChIP-Seq and ATAC-Seq.
Results. Treatment with AZD4573 potently inhibited proliferation of NHL cell lines and primary cells (IC 50 range 5-30 nM). RNA-Seq of cells treated with CDK9i demonstrated that up to 6,000 genes were significantly downregulated in NHL cells treated with CDK9i for 3 h, leading to downregulation of MAPK, PI3K, NFκB and TNFR signaling. However, following the initial nadir at 3 h, a subset of genes recovered transcription to at least baseline level (e.g., PIM1, MYC, IRF8, BCL6, BCL2L1, CXCR4 etc.), confirmed by RT-PCR and immunoblotting. Recovery was more prominent in less sensitive cells (OCI-LY3; by 6-8 h) and occurred despite continued p-RNAPII Ser2 blockade. ChIP-Seq analysis confirmed increased global RNAPII pausing at promoters in cells treated with AZD4573, including at recovery genes.
Next, we conducted ChIP-Seq for H3K4me3 and H3K27ac in OCI-LY3 and VAL cells. We observed a global decrease in H3K4me3 signal across the promoters and gene bodies with AZD4573 treatment. Meanwhile, H3K27ac enhancer analysis showed a global decrease in VAL cells, but was stable/increased in OCI-LY3 cells, including at certain recovery genes (e.g., MYC and BCL2L1).
BRD4 was previously shown to compensate for loss of CDK9 function to recover transcription of the MYC oncogene in HeLa cells, thereby compensating for loss of pTEFb activity. We observed increased interaction between BRD4 and RNAPII in OCI-LY3 cells treated with AZD4573 (by IP) and increased global BRD4 binding at transcription start sites (ChIP-Seq). Combination of BET bromodomain inhibitor JQ1 and CDK9i reduced expression of recovery genes including MYC and synergistically restricted proliferation of NHL cells.
BRD4 inhibition leads to preferential loss of transcription at super enhancer (SE)-associated genes. Therefore we leveraged our H3K27ac ChIP-Seq data to elucidate the SE landscape in NHL cells. Enhancers were ranked by signal density and amplitude, followed by assignment of the closest gene. SE domains were identified near to genes known to contribute to lymphomagenesis. Notably the majority of the recovery genes had highly ranked SE association in the tested cell lines.
Next, ATAC-Seq analysis of NHL cells revealed a significant number of genes with differential accessibility following CDK9i. Upon an 8 h treatment with AZD4573, 11121 and 4367 hyper-accessible chromatin regions were identified in VAL and OCI-LY3 cells, respectively, including MYC and BCL2L1 loci. E-box motif was found accessible across many genes at this timepoint.
To investigate recovery genes as targets, we used PIM kinase inhibitors SGI1776 (PIM1) and AZD1208 (pan-PIM). Both drugs demonstrated synergy with AZD4573 in a panel of 4 cell lines and primary samples.
Finally, to further elucidate genes and pathways which mediate susceptibility to CDK9i, we conducted a genome-wide loss of function CRISPR-Cas9 library screen. Cas9-expressing OCI-LY3 cells were transduced with a lentivirus encoding a CRISPR library comprised of ~5 unique sgRNA per gene. Knockout of genes in the PI3K/mTOR and DNA repair pathways sensitized cells to AZD4573. Confirming this result, combination treatment with AZD8835 (PI3K inhibitor) synergistically suppressed proliferation of the NHL cell lines and primary cells treated with AZD4573.
Conclusions. CDK9i leads to transcriptional reprogramming including the upregulation of a subset of SE-associated oncogenes. Targeting recovery oncogenes may enhance sensitivity to CDK9i.

Disclosures
Danilov: Gilead Sciences: Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; SecuraBio: Research Funding; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; TG Therapeutics: Consultancy, Research Funding; Takeda Oncology: Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding; Rigel Pharm: Honoraria.