Endogenous Retrovirus Derepression Drives Ectopic UGT2B17 Overexpression in Multiple Myeloma Cells: Molecular Sequelae and Pathophysiological Implications

Introduction: UGT2B17 is a phase II detoxification enzyme which catalyzes the glucuronidation of C19 steroid hormones and lipid-soluble drugs. UGT2B17 gene is ubiquitously expressed in hepatic tissues as well as in steroid target tissues including breast, uterus, and prostate. UGT2B17 mRNA levels directly correlate with functional glucuronidation activity toward vorinostat (Gruber et al., Blood 2013), which is used against a variety of cancers including multiple myeloma (MM).

Progression from monoclonal gammopathy of undetermined significance to MM is associated with increased genomic hypomethylation. Importantly it is transposable elements (TEs), remaining stochastically methylated in normal cells, that lend themselves predominantly susceptible to hypomethylation during myelomatogenesis. Hypomethylated TEs could subsequently acquire the potential to exert a significant impact on host gene expression. Herein we present evidence that an aberrantly activated long terminal repeat (LTR) of the ERV1 family of endogenous retroviruses (ERVs) drives the lineage-inordinate expression of UGT2B17 in MM cells.

Methods: UGT2B17 high-throughput sequencing of polyadenylated RNA (RNA-Seq) data across 27 normal tissues from 95 human individuals, generated from the PRJEB4337 BioProject, were analyzed via the National Center for Biotechnology Information (NCBI) portal. Publicly available UGT2B17 RNA-Seq data coupled with ribosome profiling (Ribo-Seq) data across 17 cell types were downloaded from GWIPS-viz ribo-seq genome browser. UGT2B17 Transcription factor (TF) chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-seq) data as well as ChIP-Seq data on histone H3 protein subunit modified at lysine position 4 with trimethylation (H3K4me3) were downloaded from the Encyclopedia of DNA Elements (ENCODE) Regulation supertrack, publicly available in UCSC Genome Browser Database. RepeatMasker software was employed to reveal the presence of integrated TEs in the genomic segment comprising UGT2B17 on chromosome 4.

Results: Data analysis from the PRJEB4337 BioProject reveals low-level ectopic UGT2B17 transcription initiation in 5 of 27 normal human tissues (stomach, colon, appendix, spleen, and white blood cells) from a novel exon (E0) which is located ~7500 nucleotides (nts) upstream of the canonical UGT2B17 first exon (E1). Notably, H3K4me3 ChIP-Seq data indicate the presence of a highly potent promoter sequence immediately upstream of E0. RepeatMasker scan reveals that the genomic segment that comprises UGT2B17 E0 was evolutionary derived from a human-specific Harlequin-int LTR of the ERV1 family.

Data from the GWIPS-viz genome browser show that the LTR-driven UGT2B17 transcription is minimal in 15 cell types, including normal somatic cells, cancer cells, and embryonic stem cells. In stark contrast, specifically in EBV-immortalized lymphocytes and the MM.1 myeloma cell line, UGT2B17 E0 presents higher transcription rate and also higher ribosome coverage compared to the UGT2B17 E1.

TF ChIP-seq data from ENCODE reveal that UGT2B17 E0 comprises a binding site for RNA polymerase II subunit A (POLR2A) which, amongst several cell types examined, is functional specifically in B-lineage cells. Furthermore the intragenic segment that is located between E0 and E1 comprises a high-affinity binding site for RELA proto-oncogene. Of note, adjacent to the POLR2A and RELA binding sites, located are two binding sites for the CCCTC-binding factor (CTCF), which is known to act as a transcriptional insulator restricting activation of ectopic promoters.

Conclusion: A retroviral-derived promoter that exhibits only basal activity in most normal or malignant somatic cells has the potential to initiate transcription of the adjacent UGT2B17 locus specifically in EBV-immortalized lymphocytes and MM cells. It is highly likely that ectopic UGT2B17 expression, restricted in B-lineage transformed and cancer cells, is induced by a lineage-specific aberration of CTCF function. Furthermore, the RELA binding site may serve as a potent enhancer of UGT2B17 inordinate transcription.

The UGT2B17 LTR-driven misexpression in B-lineage transformed and cancer cell types may impact the effectiveness of vorinostat-based interventions in MM patients, a tantalizing scenario warranting further interrogation in primary MM samples.