Long Noncoding RNA PVT1 and MYC Are Co-Regulated By Bromodomain Protein BRD4 in Multiple Myeloma and Associated with Disease Progression

Background: Special region called superenhancer where Bromodomain-containing protein 4 (BRD4) complex densely accumulates attracts attention. MYC is a transcription factor highly expressing in many types of cancer and plays an important role on carcinogenesis. MYC possesses superenhancer and its expression is inhibited by BRD4 inhibitor JQ1. Recent transcriptome-wide analyses have revealed an overwhelming amount of transcribed but not translated non-coding RNAs capable of influencing diverse cellular processes such as proliferation, apoptosis, and motility. Long non-coding RNA (lnc RNAs), which are commonly defined as transcripts >200 nt in length, have emerged as a class of key regulatory RNA. LncRNAs are deregulated in diverse human cancers and associated with disease progression; however little is available in multiple myeloma (MM). LncRNA PVT1 locates adjacent to MYC is also reported to be associated with carcinogenesis and abnormal PVT1 fusing NBEA or WWOX in MM with 8q24 abnormality has been reported, but its roles and regulation machinery remain uncertain.

Methods: 140 of MM patients, 58 of MGUS patients, 6 of control BM are subjected to the study after obtaining informed consent. The study was approved by IRB following Declaration of Helsinki. PVT1 and MYC expression were determined by RQ-PCR in RNA was extracted from purified CD138+ plasma cells from bone marrow (BM) mononuclear cells. Whole transcriptome analysis by next generation sequencer (NGS) using Illumina NextSeq 500 was performed in part of the samples. MM cell lines KMS11, KMS12PE, OPM2, RPMI8226 were treated with bromodomain BRD4 inhibitor JQ1, CPI203 or MYC inhibitor 10058-F4, then growth rate and MYC/PVT1 expression was analyzed by RQ-PCR and NGS. To analyze the function and role of PVT1, siRNA for PVT1 was transfected to the cell lines. MYC protein expression was determined by western blot.

Results: The expression level of PVT1 and MYC were significantly higher in MM (mean for PVT1 2.58, MYC 0.74) than MGUS (mean for PVT1 0.88, MYC 0.06) and control (mean for PVT1 0.06, MYC 0.07) (p<0.001, p<0.001). NGS confirmed that PVT1 was an gene expressed higher in MM than in MGUS. PVT1 and MYC expressions in MM did not differ in between different karyotypes (p=0.79 and 0.5) and stages according to ISS (p=0.21 and 0.95).No correlation between PVT1 expression level and poor cytogenetics. Interestingly the levels of PVT1 and MYC was positively correlated (r=0.423, p<0.0001 in control, MGUS and MM, r=0.484, p<0.001 in MM only). PVT1 expression was also significantly correlated with BMI1 and EZH2 (r=.0.63, p<0.001 and r=0.65, p<0.001). BRD4 inhibitor JQ1 and CPI203 inhibited cell proliferation of MM cell lines KMS11, KMS12PE, OPM2 and RPMI8226. JO1 and CPI203 almost completely shut down both PVT1 expression in KMS11, KMS12PE, OPM2 except in RPMI8226. RNA sequencing by NGS confirmed that PVT1 was one of the genes significantly downregulated by BRD4 blockade. MYC expression also diminished in the cell lines except RPMI8226 in both transcripts and protein level by JQ1. In contrast, both MYC siRNA and MYC inhibitor 10054-F4 which inhibits MYC transcriptional activity by disrupting MYC-MAX complex did not change PVT1 expression level at all. PVT1 knockdown by siRNA showed no appreciable phenotypic change so far. Interetingly overall survival of the MM patients with higher PVT1 expression tended to be shorter with median 2.7 years compared to 5.0 years (p=0.09).

Conclusions: Positive correlation between MYC and PVT1 in patients, synchronous downregulation of MYC and PVT1 by JQ1 and CPI203, no effect of MYC inhibitor for PVT1 expression suggest that these two genes expressions are co-regulated by BRD4 complex.Association between higher PVT1 expression and MM, poor prognosis suggests its pathogenetic role. PVT1 is known to stabilize MYC protein by inhibiting Th58 phosphorylation, and to enhance MYC transcription by binding to the promoter. Thus, PVT1 and MYC may cooperately contribute to MM pathogenesis and progression, and our discovery may support the rationale targeting BRD4.