The CDK7 Inhibitor THZ1 Alters RNA Polymerase Dynamics at the 5' and 3' Ends of Genes

The t(8;21) is one of the most frequent chromosomal translocations in acute myeloid leukemia (AML). This translocation encodes a fusion protein containing the DNA binding domain of RUNX1, a master regulator of hematopoiesis, linked to the majority of the myeloid translocation gene on chromosome 8 (MTG8, also known as eight-twenty-one or ETO; gene name RUNX1T1). This chimeric fusion protein binds to the RUNX1 DNA elements to control gene expression by recruiting transcriptional cofactors via the MTG8/ETO moiety. In addition, MTG/ETO family proteins co-purified in a complex that also contained "E proteins" and the cyclin dependent kinase CDK9, a component of the positive transcription elongation factor b (P-TEFb).

P-TEFb contains Cyclin T1 and CDK9 and several potential CDK9 inhibitors have shown promise in the clinics for t(8;21) AML, such as flavopiridol (alvocidib). Transcription initiation, promoter-proximal pausing of RNA polymerase II (RNAPII), and transcription elongation are events that are highly regulated for genes involved in proliferation and differentiation such as MYC and BCL2 . CDK7 phosphorylates Ser5 of the C-terminal domain of RNAPII to trigger promoter-proximal pausing of RNA polymerase II, whereas CDK9 phosphorylates Ser2 of RNAPII and the negative elongation factors DSIF and NELF to release the polymerase into the body of the gene.

In this study, we used inhibitors of CDK7 and CDK9 to probe for transcriptional vulnerabilities in t(8;21) AML. We used both broad spectrum (flavopiridol, dinacicilib) and a specific CDK9 inhibitor PHA767491 along with THZ1 (CDK7 inhibitor). Since CDK7 is also a CAK (CDK activating kinase) that activates cell-cycle CDKs and mutations in Cyclin D2 are common in t(8;21) AML, we also tested the highly selective CDK4/6 inhibitor palbociclib (PD-0332991). We observed that the t(8;21)-containing AML cell lines SKNO-1 and Kasumi-1, as well as primary AML patient cells, were extremely sensitive to all the CDK7/9 inhibitors, but not palbociclib. We used precision global run-on transcription sequencing (PROseq) to define the mechanism of action of these compounds. This technique creates high resolution genomic maps of the active RNA polymerases to define the effects on RNA polymerase dynamics. CDK9 inhibitors caused wide spread RNA Polymerase II promoter proximal pausing at nearly 80% of the expressed genes, as well as at enhancers where there was a general impairment of production of eRNAs and lncRNAs. Conversely, inhibition of CDK7, a critical component of TFIIH caused a loss of paused RNA polymerase II at promoters, which was associated with an increase of polymerases in the body of genes. While RUNX1 was reported to be regulated by CDK7 in T-ALL, there was no impact on the expression of RUNX1 or the fusion protein in t(8;21)-containing cells. Although enhancers were not affected, super enhancers were modestly affected by THZ1. Interestingly, within a subset of genes that showed increased RNA polymerase accumulation within the gene body, CDK7 inhibition altered 3' end transcription termination dynamics within 1 hr and this effect remained even at 2 hr. A time course of THZ1 treatment at 0, 15', 30', and 60' suggested that CDK7 inhibition altered RNA polymerase dynamics at 5'ends of the genes, and it's these effects that eventually affected transcription termination at the 3' ends of the genes.