Pharmacological Downregulation of De Novo Active and Essential lncRNA SMILO via Inhibition of FLI1 Transcription Factor in Multiple Myeloma. [ChIP-Seq]
Background: Despite the advances in therapeutic strategies, Multiple Myeloma (MM) remains incurable. Our recent research has detected 1556 de novo active chromatin regions in MM in comparison to healthy B cells. These regions were associated to 1059 coding genes and 89 lncRNAs, which play important roles in MM pathobiology (Ordoñez. Genome Research 2020; Carrasco-León. Leukemia 2021). The modulation of these genes could be essential for the development of novel therapeutic strategies to improve the response of MM patients.  Aims: We aimed to pharmacologically regulate these de novo active genes in an attempt to modulate the abnormal transcriptome of MM. Methods: Given that de novo chromatin activation marks were mainly located at acetylated promoter and enhancer regions, JQ1 Bromodomain and Extraterminal Domain Inhibitor (BETi) was used. Three MM cell lines (KMS-11, RPMI8226 and KMS-12) were treated at 1µM during 72h and then, RNA-seq, ChIP-seq of H3K27ac and ATAC-seq were performed. For the identification of the regulatory proteins of de novo active genes Reverse-ChIP was carried out. Protein inhibitions were conducted using CRISPR-Cas9, and protein and mRNA levels were analyzed by Western Blot and RT-qPCR, respectively. Results: After JQ1 treatment, the RNA-seq revealed a significant downregulation of more than ten percent of de novo active genes, indicating that JQ1 can negatively modify the expression of de novo active genes in MM. Interestingly, SMILO, a de novo active and essential lncRNA in MM, presented one of the greatest inhibitions, surpassing even described BETi targets (Fig. 1A). Considering that pharmacological regulation of the lncRNAs has been widely proposed as a therapeutic approach, we decided to use JQ1 to further explore the mechanism of regulation of this lncRNA. Through ChIP-seq and ATAC-seq analysis, we verified that this decrease was not due to changes in chromatin activation and accessibility produced by JQ1 in MM cells, suggesting that JQ1 may displace the transcriptional machinery, such as Transcription Factors (TFs), from de novo active regions and specifically from those related to SMILO. Interestingly, the Reverse-ChIP study revealed the binding of 16 proteins in the SMILO region. Among these, we selected as our candidate the TF FLI1, because FLI1: 1) was overexpressed in MM in comparison to healthy B cells; 2) presented a positive correlation of expression with SMILO; 3) MM patients with higher expression of FLI1 presented worse Progression Free Survival (PFS) and Overall Survival (OS) both in univariate and multivariate analyses considering classical genetic alterations of MM (Fig. 1B) and 4) FLI1 showed a significant decrease in H3K27ac at the promoter region after JQ1 treatment (Fig. 1C), which correlated with a great decrease in FLI1 protein levels. To ascertain that FLI1 is causally involved in the expression of SMILO, FLI1 was silenced using CRISPR-Cas9 technology, which substantially depleted FLI1 protein levels giving rise to a significant reduction of the expression of SMILO (Fig. 1D). All these results explain the mechanism by which JQ1 downregulates SMILO via the downregulation of the TF FLI1 (Fig. 1E).  Summary/Conclusion: Our study highlights the innovative ability of BETi molecules to modulate the expression of lncRNAs. We underscore the involvement of FLI1 in the regulation of SMILO, a de novo active and essential lncRNA in MM, opening the door to the development of novel therapeutic strategies based on RNA directed therapies. Overall design: To investigate the pharmacological regulation of de novo active genes in Multiple Myeloma, we treated three human MM cell lines with the JQ1 Bromodomain and Extraterminal Inhibitor. KMS-111, KMS-12 and RPMI8226 cell lines were treated at 1µM and 72h. We performed the experiment in duplicate and then, the  changes in the transcriptome were analysed by RNA-seq.