Lymphomagenic Roles of MYC, E2F and Mir-150-5p in Plasmablastic Lymphoma: Therapeutic and Prognostic Implications

Introduction: Plasmablastic lymphoma (PBL) is an uncommon non-Hodgkin lymphoma which is characterized by an aggressive clinical behavior, very poor prognosis and which mostly affects immunosuppressed individuals. Although different studies have described genetic alterations in PBL, its transcriptome and epigenome have been scarcely studied. Our goal was to describe the gene and miRNA expression profiles and to identify new therapeutic targets and new prognostic factors in PBL.

Methods: Formalin fixed paraffin embedded (FFPE) samples from 66 patients with PBL and samples from 14 reactive adenopathies, as a control group, were included for miRNA and gene expression studies (microarrays and RNA-seq, respectively). Enrichment analyses of miRNAs and genes differentially expressed, using hallmark data set, were carried out. The role of miR-150-5p in PBL lymphomagenesis was studied in vitro upon introduction of specific miRNA mimic in the PBL-1 cell line (Mine S, Sci Rep. 2017). Expression of the miRNA and its target genes was analyzed by RT-qPCR and Western Blot. Its role in cell proliferation, cell cycle progression and apoptosis induction, was assessed respectively by MTT assay and flow cytometry.

Results: Patients with PBL showed 36 differentially expressed miRNAs compared to controls. Two expression profiles of these miRNAs were identified in PBL, related with the presence of MYC-translocation. The functional enrichment analysis of genes targeted by deregulated miRNAs revealed an enrichment of MYC and E2F targets, genes involved in G2M checkpoint and mTORC1 signaling pathway. This latter observation was confirmed by gene set enrichment analysis (GSEA) of genes differentially expressed in PBL (Fig.1). Different genes are shared among the top 3 enriched hallmark gene sets (Fig.2), this data suggests that both MYC and E2F are involved in the deregulation of cell cycle in PBL. Additionally, the upregulation of E2F1, E2F3 and E2F8 correlates significantly with an increased expression of MKI67 (Ki67), indicating that E2F exerts a crucial role in the abnormal proliferation in PBL.

To identify deregulated miRNA-target interactions related with the top 3 enriched hallmark gene sets, we studied the correlation between the most differentially expressed miRNAs (miR-148a-3p, -4417, -150-5p, -342-5p, -342-3p, -3609) and their differentially expressed target genes. Only miR-150-5p showed a significant negative correlation with the anti-apoptotic gene BIRC5, coding for survivin, in PBL patients (p. 0.0063) (Fig.2). Of interest, high expression of BIRC5 and low expression of miR-150-5p correlated with high expression of MKI67 (p. 0.0006 and p. 0.04, respectively) indicating that the deregulation of miR-150-5p and BIRC5 may favor the proliferation of PBL cells. Low expression of miR-150-5p (lower than median= 6.56) was associated with shorter overall survival (p <0.0001), suggesting that this miRNA may represent a potential prognostic factor in PBL. We further studied the role of miR-150-5p in PBL lymphomagenesis by mimicking this miRNA in PBL-1 cell line. Upregulation of miR-150-5p decreased the expression of survivin at both mRNA and protein level, promoted apoptosis and reduced viability and proliferation, pointing out a pro-apoptotic role of miR-150-5p via survivin inhibition. Consistent with the known role of survivin in the control of mitosis, the upregulation of miR-150-5p and the consequent reduction in survivin levels produced a slight arrest of cells in G1 cell cycle phase and lower numbers of cells in G2-M phase. Finally, a positive tendency between BIRC5 and E2F3 expression was observed (p. 0.06), being this latter gene negatively correlated with miR-150-5p (p. 0.017). Indeed, a greater expression of E2F3 was encountered in those patients with low expression of miR-150-5p. Thus, these results suggest that E2F3 could repress miR-150-5p and mediate apoptosis via BIRC5 induction.

Conclusions: We identified miR-150-5p as a new promising prognostic factor, and MYC and E2F as important regulators of global gene expression in PBL. Among these genes, E2F3 could represent a lymphomagenic-driver in PBL by repressing miR-150-5p and inducing BIRC5 expression.

Baptista:Swedish Orphan Biovitrum: Current Employment. Roué:TG Therapetics: Research Funding; Kancera: Research Funding. Abrisqueta:BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sandoz: Honoraria; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. González Barca:Janssen: Honoraria, Other: Travel, Accommodations, Expenses; AbbVie: Honoraria, Other: Travel, Accommodations, Expenses; Takeda: Honoraria; EUSA Pharma: Honoraria; AstraZeneca: Honoraria; Roche: Other: Travel, Accommodations, Expenses. Climent:EUSA-Pharma: Speakers Bureau. Salar:Gilead: Research Funding; Incyte: Speakers Bureau; Abbvie: Research Funding; Roche: Research Funding, Speakers Bureau; Beigene: Honoraria; BMS/Celgene: Honoraria; Janssen: Honoraria, Speakers Bureau. Sancho:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Bristol Myers Squibb: Honoraria; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Kern Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees; BeiGene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Eli Lilly & Company: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi Biomedicine: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lenz:AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Constellation: Consultancy, Honoraria; ADC Therapeutics: Consultancy, Honoraria; Miltenyi: Consultancy, Honoraria; Takeda: Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria, Research Funding, Speakers Bureau; Genmab: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Bayer: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; University Hospital Münster: Current Employment; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau. Navarro:Gilead: Research Funding; EUSA Pharma: Honoraria; AstraZeneca: Honoraria; AstraZeneca: Consultancy; Novartis: Consultancy; Novartis: Honoraria; EUSA Pharma: Research Funding; BluePrint Medicines: Consultancy.