Isolation of biologically active small peptide from KSHV LANA protein sequence, which induces CHD4 degradation to promote cell differentiation and inhibits leukemic cell growth (SLAM-seq)

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a latent infection, in which most viral genes are silenced except a few latent proteins such as latency-associated nuclear antigen (LANA). In latent viral chromatin, viral genes are poised to be transcribed, and KSHV LANA plays a major role in maintaining such transcription status. In the poised chromatins, LANA recruits cellular CHD4 (Chromodomain Helicase DNA binding protein 4) and suppresses inducible viral gene promoters. The CHD4 is known to regulate cell differentiation by restricting enhancer-promoter interactions and its mutation or overexpression deregulates the host cell transcription program and therefore associates with tumorigenesis. Here, we identified a putative CHD4 inhibitor from the LANA amino acid sequence from the LANA-CHD4 interaction surface. The small peptide interacts with CHD4 at its PHD domain with 14 nM KD (dissociation constant). The introduction of the peptide into the primary effusion lymphomas induces caspase-mediated CHD4 cleavage and subsequently triggered cell apoptosis and autophagy. A series of MTT assays demonstrated that the peptide preferentially killed lymphoma and leukemia cell lines at low micromolar concentrations, while peripheral mononuclear cells or adhesion cell lines were found to be more resistant to the peptide treatment. A monocyte cell differentiation model demonstrated that pre-treatment with the peptide at a low dose substantially enhanced transitioning into M2 macrophage (by reducing CHD4 occupancies from gene promoters), and globally altered the repertories of phorbol myristate acetate target genes in U937 cells. Finally, the PEL xenograft mouse model inhibited tumor growth without measurable side effects, and PEL cells isolated from xenograft tumors showed reduced CHD4 and LANA expression with terminally differentiated phenotype. We propose that the peptide isolated from the KSHV LANA sequence is biologically active and may function as a CHD4 inhibitor. Overall design: The goal of these studies was to utilize thiol(SH )-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) in order to define the direct transcriptional effects of treatment with the VGN73 peptide. This was performed in the context of the BC3, BCBL-1, and Raji cell line models. Replicate cultures from each cell line were treated with either vehicle control, wild-type VGN73 peptide (at the IC50 for each cell line), or mutant VGN73 peptide (at the equivalent concentrations to the IC50 of wild type peptide for each cell line) for 30 minutes, and then incubated for an additional 1.5 hours in the presence of 4-Thiouridine (s4U; 300 uM) in order to label nascent transcripts. Following treatment, the cells were harvested for isolation of total RNA (containing s4Uracil-labeled transcripts) and followed by library preparation and next-generation sequencing (NGS).