Transcriptome Analysis at Locus-Specific Resolution Identified Alteration in Human Endogenous Retroviruses, a Class of Transposable Elements in Mantle Cell Lymphoma

Mantle Cell lymphoma (MCL) is an incurable non-Hodgkin lymphoma (NHL) and has the poorest overall prognoses. Despite intensive therapeutic approaches, the median progression-free survival is 3-5 years. Genetic alterations have been identified in MCL including chromosomal alterations and mutations. However, these changes are not solely responsible for MCL development. Recent studies highlight the biological relevance of human endogenous retroviruses (HERVs), a class of transposable elements (TE), in cancer biogenesis and their potential as novel diagnostic, prognostic, and treatment response biomarkers. However, studies on TE/HERV in lymphoid malignancies are scant and have not been examined in MCL. TEs account for 45% of the human genome, which is broken down into long-interspersed nuclear elements (LINEs), short-interspersed nuclear elements (SINEs), and long-terminal repeats (LTRs). HERV fall into the LTR family and are ancestral remnants of germline retroviral infections, accounting for 8-10% of the human genome.

To investigate HERV expression in MCL, we performed RNA sequencing (RNA-Seq) in CD19+ B-cells taken from patients with MCL (n=18), and healthy donors (n=10). HERV characterization was conducted by developing an in-house workflow that relied on the telescope, a statistical model to quantify HERVs with a locus-specific resolution. Among the 14,968 HERV genomic loci evaluated in this study, 3345 (22%) were expressed (mean Transcript Per Million (TPM) > 10) across all our MCL samples and, therefore, considered for the differential expression analysis. Such analysis was performed by comparing the MCL samples against the healthy donors. Specifically, a total of 406 and 683 HERVs {(Log2FC) > 0.58 and FDR < 0.05} were found up- and down-regulated in MCL, respectively. From a genomic standpoint, the differentially expressed HERVs were dispersed throughout the genome. However, some chromosomes (chr) such as chr16, chr17, chr19, and chr22, had higher percentages of differentially expressed HERVs calculated by the total number of HERV loci per chromosome. In addition, we identified an increased enrichment of HERV families including HERVL, HERV3, and HERVK in MCL patients vs healthy donors. Among, all the known HERV families, the HERVK family is the most recently integrated into the human genome and the most biologically active in generating retrovirus-like particles. We validated the HERVK RNA-Seq data, by quantitative RT-PCR and confirmed the downregulation of HERVK transcription in 4 MCL cell lines compared with peripheral blood cells from healthy donors. Using RNA-Seq data, we further identified significant downregulation of HERVK subfamilies such as HML2, HML3, and HML-6 in MCL compared to normal control. Mechanistically, HERV transcription is controlled by epigenetic mechanisms including DNA and histone modifications including methylation. We thus interrogated whether DNA methyltransferase inhibitor azacytidine (Aza) or G9a methyltransferase inhibitor (G9ai) controls the HERVK transcription in the MCL cells. We observed that low doses of Aza or G9ai, exhibit variable responses in modulating the transcription of HERVK among the MCL lines. To understand the variability of HERVK upregulation among MCL cells, we examined whether TP53 status affected HERVK expression after in-vitro epigenetic drug treatment. The mutant TP53 MCL cells exhibited significantly less upregulation of HERVK with both epigenetic drugs than the wild-type (wt) TP53 MCL cells. Interestingly, nutlin-3a (MDM2 inhibitor; inhibits MDM2-p53 interaction) treatment significantly increased the HERVK expression in the TP53 wt MCL cells, however, no effect on HERVK expression was seen in TP53 mutant MCL cells. Therapeutically, nutlin-3a was highly sensitive to TP53 wt MCL cells compared to TP53 mutant cells. Combining nutlin-3a with epigenetic drugs Aza or G9ai significantly decreased the proliferation and survival of wt TP53 cells compared to single agents. This is the first study to characterize the expression of HERVs at genomic locus-specific resolution in MCL patients. Our study provides a comprehensive, genome-wide picture of HERV families in MCL along with changes in HERVK after epigenetic treatment and implicates p53 in HERV-K transcriptional regulation. Overall, we provide a novel insight into the biology of HERVs in MCL pathogenesis and epigenetic therapies.

Chiappinelli:ROME Therapeutics, Daiichi Sankyo: Consultancy. Baiocchi:Codiak Biosciences: Research Funding; Agenus: Other: Involved in supply of drug (vaccine) and product development; Prelude Therapeutics: Other: Advisory Board, Research Funding; Viracta Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Other: Advisory Board; ATARABio: Consultancy, Other: Advisory Board.