Abstract
Chronic lymphocytic leukemia (CLL) follows a variable clinical course mostly dependent upon genomic factors, with a subset of patients having low risk disease and others displaying rapid progression associated with clonal evolution. Epigenetic mechanisms such as DNA promoter hypermethylation were shown to have a role in CLL evolution where the acquisition of increasingly heterogeneous DNA methylation patters occurred in conjunction with clonal evolution of genetic aberrations and was associated with disease progression. However the role of epigenetic mechanisms regulated by the histone deacetylase group of transcriptional repressors in the progression of CLL has not been well characterized.
The histone deacetylases (HDACs) 1 and 2 are recruited onto gene promoters and form a complex with the histone demethylase KDM1. Once recruited, the complex mediate the removal of acetyl groups from specific lysines on histones (H3K9 and H3K14) thus triggering the demethylation of lysine 4 (H3K4me3) and the silencing of gene expression.
CLL is characterized by the dysregulation of numerous coding and non coding genes, many of which have key roles in regulating the survival or progression of CLL. For instance, our group showed that the levels of HDAC1 were elevated in high risk as compared to low risk CLL or normal lymphocytes and this over-expression was responsible for the silencing of miR-106b, mR-15, miR-16, and miR-29b which affected CLL survival by modulating the expression of key anti-apoptotic proteins Bcl-2 and Mcl-1.
To characterize the HDAC-repressed gene signature in high risk CLL, we conducted chromatin immunoprecipitation (ChIP) of the nuclear lysates from 3 high risk and 3 low risk CLL patients using antibodies against HDAC1, HDAC2 and KDM1 or non-specific IgG, sequenced and aligned the eluted DNA to a reference genome and determined the binding of HDAC1, HDAC2 and KDM1 at the promoters for all protein coding and microRNA genes. Preliminary results from this ChIP-seq showed a strong recruitment of HDAC1, HDAC2 and KDM1 to the promoters of several microRNA as well as protein coding genes in high risk CLL.
To further corroborate these data we performed ChIP-Seq in the same 6 CLL samples to analyze the levels of H3K4me2 and H3K4me3 around gene promoters before and after 6h exposure to the HDACi panobinostat. Our goal was to demonstrate that HDAC inhibition elicited an increase in the levels of acetylation on histones and triggered the accrual of H3K4me2 at the repressed promoter, events likely to facilitate the recruitment of RNA polymerase II to this promoter. Initial analysis confirmed a robust accumulation of H3K4me2 and H3K4me3 marks at the gene promoters of representative genes that recruited HDAC1 and its co-repressors in the previous ChIP-Seq analysis in high risk CLL patients.
Finally, 5 aggressive CLL samples were treated with the HDACi abexinostat for 48h and RNA before and after treatment was subjected to RNA-seq for small and large RNA to confirm that the regions of chromatin uncoiled by HDACi treatment were actively transcribed.
HDAC inhibition induced the expression of a large number of miRNA genes as well as key protein coding genes, such as miR-29b, miR-210, miR-182, miR-183, miR-95, miR-940, FOXO3, EBF1 and BCL2L11. Of note, some of the predicted or validated targets of the induced miRNAs were key facilitators in the progression of CLL, such as BTK, SYK, MCL-1, BCL-2, TCL1, and ROR1. Moreover, RNA-seq showed that the expression of these protein coding genes was reduced by 2-33 folds upon HDAC inhibition.
We plan to extend the RNA-seq to 5 CLL samples with indolent disease and combine all the data to identify a common signature of protein coding and miRNA genes that recruited the HDAC1 complex, accumulated activating histone modifications upon treatment with HDACi and altered gene and miRNA expression after HDAC inhibition in high risk CLL versus low risk CLL. The signature will be than validated on a large cohort of indolent and aggressive CLL patients.
Our final goal is to define a signature of coding and non coding genes silenced by HDACs in high risk CLL and its role in facilitating disease progression.
Disclosures
Woyach: Acerta: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding.
The Integrator complex cleaves nascent mRNAs to attenuate transcription
