LAUSR

Background: N6-methyladenosine (m6A) RNA methylation is a dynamic reversible epitranscriptomic modification that includes methyl transferases (writes m6A), demethylases (erases m6A) and reader proteins, which proofreads m6A marks of a specific site of transcripts. Recent studies have suggested that RNA methylation affects several fundamental cellular and molecular functions including mRNA splicing, stability, export, stem cell fate, circadian rhythms, DNA repair and cell survival. The objective of this study is to establish the mechanisms by which RNA demethylase ALKBH5 (AlkB homolog 5) facilitates tumor growth and progression. Methods: To establish the significance of RNA methylation in children’s cancers, we performed siRNA screen targeting m6A writers, erasers, and readers in osteosarcoma (OS). We used several OS cell lines including 143B, MG63, SaOS2, U2OS and multiple patient derived OS cell lines. Mechanistic studies were conducted using ALKBH5 KO and knockdown cells and by measuring the status of Autophagy and UPR associated proteins using Western blot analysis, confocal and electron microscopy. Results: Our results revealed that depletion of ALKBH5, a demethylase that erases the m6A mark from the target gene, altered the autophagy in OS cells. Interestingly, we found that level of LC3, which is the universal marker for autophagy, was significantly increased in OS cells. RNA seq analysis showed that depletion of ALKBH5 significantly altered several autophagy related genes in the OS cells. To better understand the molecular mechanism by which ALKBH5 regulates autophagy, we investigated the Endoplasmic Reticulum (ER) stress-induced Unfolded Protein Response (UPR) pathway, which is a known activator of autophagy. We discovered that ER stress induced UPR signaling pathway is highly activated in ALKBH5 depleted cancer cells. Further, mechanistic studies suggested that ALKBH5 promoted ER homeostasis by controlling the expression of ER lipid raft associated 1 (ERLIN1), which binds to the activated inositol 1, 4, 5,-triphosphate receptor and facilitates its degradation via ERAD to maintain calcium flux between ER and mitochondria. Using functional studies and electron microscopy, we show that ALKBH5-ERLIN1-IP3R-dependent calcium signaling modulates the activity of AMP kinase, and consequently mitochondrial biogenesis. These findings thus reveal that ALKBH5 serves an important role in maintaining ER homeostasis and cellular fitness. Conclusion: These findings provide novel insight into how m6A may promote cancer cell growth by regulating the crosstalk among ER signaling, UPR, autophagy, and mitochondrial function. Our study is the first to show that RNA methylation plays an important role in osteosarcoma by regulating autophagy via UPR. Citation Format: Panneerdoss Subbarayalu, Daisy Medina, Pooja Yadav, Santosh Timilsina, Kunal Baxi, Ratna Vadlamudi, Yidong Chen, Manjeet Rao. ALKBH5 promotes cancer growth by regulating ER homeostasis via UPR, autophagy, and mitochondrial function. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3528.