Poster abstracts
Poster number 77 submitted by Jeongjin Kim
Discovery and characterization of a novel telomerase alternative splicing isoform that protects lung cancer cells from chemotherapy induced cell death
Jeongjin J. Kim (School of Kinesiology, University of Michigan, Ann Arbor, Michigan), Alexander Ahn (School of Kinesiology, University of Michigan, Ann Arbor, Michigan), Jeffrey Y. Ying (School of Kinesiology, University of Michigan, Ann Arbor, Michigan), Andrew T. Ludlow (School of Kinesiology, University of Michigan, Ann Arbor, Michigan)
Abstract:
Telomerase reverse transcriptase (TERT) is a core protein component of the telomerase holoenzyme complex and forms various isoforms by alternative splicing (AS). While some isoform functions are known beyond telomere synthesis, a full catalog of TERT isoforms and their functions remains elusive. Thus, we aimed to discover TERT isoforms and identify their functions. By identifying TERT AS isoforms and determining their functions we may be able to find a novel way to target TERT in cancers without impacting TERT positive stem cells. To achieve our goal, we performed single molecule long-read RNA sequencing with a TERT specific cDNA library. Novel TERT isoforms were discovered, one of which we call TERT Delta 2-4, which lacks exons 2-4, was selected for further studies. Stable cell lines expressing TERT Delta 2-4 were established in Calu-6 cells (telomerase expressing cells) and U2OS (telomerase deficient cells). siRNAs targeting TERT exon1/5 junction were used to knockdown TERT Delta 2-4. Telomerase activity, cell growth, and telomere length changes were measured following overexpression or knockdown of TERT Delta 2-4. Clonogenic assays were used to determine the effect of TERT Delta 2-4 on cells’ resistance to apoptosis. 45 TERT isoforms with 13 known and 32 novel were observed from four cell lines (iPSC, Calu-6, A549, and H1299). Among the novel TERT isoforms, TERT Delta 2-4 was relatively highly abundant and predicted to be translated into protein. Neither overexpression nor knockdown of TERT Delta 2-4 affected telomerase activity, cell growth rate, or telomere length overtime. However, clonogenicity and resistance to cisplatin were enhanced by TERT Delta 2-4 overexpression and reduced by knockdown, supporting the role of TERT Delta 2-4 in enhancing resistance to apoptosis. The novel TERT isoform Delta 2-4 enhances resistance to apoptosis and knockdown of the isoform made lung cancer cells more sensitive to cisplatin treatment. TERT Delta 2-4 could be a potential therapeutic target to enhance the efficacy of lung cancer drugs without the side-effects in stem cells of direct telomerase inhibitors.
References:
1. Slusher, Aaron L., Jeongjin JJ Kim, and Andrew T. Ludlow. "The role of alternative rna splicing in the regulation of htert, telomerase, and telomeres: Implications for cancer therapeutics." Cancers 12.6 (2020): 1514.
2. Hrdličková, Radmila, Jiří Nehyba, and Henry R. BoseJr. "Alternatively spliced telomerase reverse transcriptase variants lacking telomerase activity stimulate cell proliferation." Molecular and cellular biology (2012).
3. Fleisig, H. B., et al. "Telomerase reverse transcriptase expression protects transformed human cells against DNA-damaging agents, and increases tolerance to chromosomal instability." Oncogene 35.2 (2016): 218-227.
4. Penev, Alex, et al. "Alternative splicing is a developmental switch for hTERT expression." Molecular cell 81.11 (2021): 2349-2360.
Keywords: Alternative RNA splicing, TERT, Telomerase