Abstract 1535: Novel lncRNA ENSG00000254887 is a key factor for MYC-driven cell proliferation
Authors: García-Caballero Daniel , Hart Jonathan R. , Vogt Peter K. 
Published: June 2022

Abstract
Long non-coding RNAs (lncRNAs) are widely expressed, and many of them are involved in gene regulation. In a CRISPRi study, we looked for lncRNAs required for MYC-driven proliferation in human lymphoid cells and found a novel lncRNA, ENSG00000254887, among the top candidates. In the genome, ENSG00000254887 is head-to-head with a coding gene, POU2F2. To our knowledge, ENSG00000254887 has not been described in the literature yet. We aim to characterize the importance of ENSG00000254887 in cancer development and its mechanisms of action. We performed a ChIP analysis to confirm the direct interaction of MYC with ENSG00000254887 in Ramos cells, a Burkitt’s lymphoma cell line. To knock down the expression, we generated a library of single guide RNAs (sgRNAs) covering the whole length of ENSG00000254887. We transduced Cas9-expressing Ramos cells with this library and cultured them for 14 days. We then measured the differential depletion of guides using next generation sequencing. To validate these results, we performed CRISPR growth competition assays with individual GFP-expressing sgRNA constructs, and the cell growth was measured by flow cytometry. We corroborated the importance of ENSG00000254887-regulated expression by two additional approaches: downregulation by small-hairpin RNA (shRNA) and upregulation by expression of an ectopic copy of the gene. We have been able to retrieve the genomic sequence of ENSG00000254887 using an anti-MYC antibody, thus confirming the presence of binding sites for this transcription factor. Using CRISPR, we have mapped several sites along the ENSG00000254887 sequence where Cas9-introduced changes lead to loss of function and reduce the ability of the cells to proliferate. We selected three of the most efficient sgRNAs used for CRISPR for further analysis and observed that they induced a reduction in the expression of both ENSG00000254887 and the nearby coding gene, POU2F2. This significant result was validated using shRNA targeting the sequence of the lncRNA. On the contrary, the overexpression of ENSG00000254887 from an inducible ectopic copy has no effect on the expression of POU2F2. In conclusion, the expression of ENSG00000254887 is critical for MYC-driven cell proliferation and can be altered through Cas9-introduced changes. A downregulation of this lncRNA is always accompanied by a reduction in the expression of the nearby coding gene, POU2F2. The absence of any effect on POU2F2 when we overexpress ENSG00000254887 from an ectopic copy indicates that this lncRNA might be acting in cis. However, further research is needed to explain this fact and establish the molecular mechanisms. Acknowledgments: This work was supported by the National Cancer Institute of the National Institutes of Health under award R35 CA197582 (PKV).
Citation Format: Daniel García-Caballero, Jonathan R. Hart, Peter K. Vogt. Novel lncRNA ENSG00000254887 is a key factor for MYC-driven cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1535.