PF576 NONSENSE‐MEDIATED MRNA DECAY IS A THERAPEUTIC TARGET IN MULTIPLE MYELOMA

Abstract

Background:Nonsense‐mediated mRNA decay (NMD) is a translation‐coupled cellular quality control system that degrades mRNAs containing premature termination codons (PTCs) and regulates the expression of 5–10% of normal mRNAs. NMD prevents translation of misfolded proteins, thereby halting the potential activation of the unfolded protein response (UPR). Cancer cells display increased alternative splicing events and splicing factors are frequently mutated in cancer. This suggests that NMD, and its link with ER‐stress, might be therapeutically targetable in cancer. This notion was investigated, based on preliminary data showing that CC‐115, a known inhibitor of mTOR kinase (TORK) and DNA‐PK, also inhibits a key player in NMD activation.Aims:To explore the application of CC‐115 as a specific inhibitor of NMD in cancer.Methods:A comparative screen in 141 cancer cell lines was performed with CC‐115 versus specific inhibitors of TORK and DNA‐PK, using inhibition of proliferation (Cell‐Titer Glow) and cell death (apoptosis) as read‐out. Additional targets of CC‐115 were determined by ActivX KiNativ analysis, and confirmed by knockdown and additional kinase inhibitors. Dependence of CC‐115 sensitivity on specific genes was determined by CRISPR/Cas9 technology in various multiple myeloma (MM) cell lines. RNA sequencing was used for identification of potential targets in 3 sensitive and 3 resistant cell lines and confirmed by qPCR. Activity in vivo was tested in xenograft tumors.Results:ActivX KiNativ analysis revealed NMD inhibition as an additional target of CC‐115. NMD inhibition with CC‐115 resulted in a decreased phosphorylation of UPF1 and a dose dependent increase of NMD sensitive transcripts. CC‐115 activated the PERK branch of the UPR, as evidenced by increased mRNA expression of ATF4, ATF3 and CHOP, while HSPA5 and sXBP1 were not affected. Cell death analyses among various B cell lines showed that MM cell lines were in general highly sensitive to CC‐115. Activity of CC‐115 correlated strongly with cell death by the known ER‐stress inducer, thapsigargin. Cell death by CC‐115 occurred via the mitochondrial pathway of apoptosis, as it depended on caspase activity and the presence of Bax‐Bak. RNA sequencing indicated that CC‐115‐sensitive cell lines had a lower baseline expression of cytoskeleton and vesicle transport genes and a higher expression of genes involved in SMAD and BMP signaling. Gene set enrichment analysis revealed an overrepresentation of MYC downstream targets, oxidative phosphorylation and UPR genes in CC‐115 sensitive vs. resistant cell lines, and confirmed the inhibitory effects of CC‐115 on NMD. Lastly, antitumor effects of CC‐115 were superior to specific TORK inhibition in xenograft mice, without general toxicity.Summary/Conclusion:Hematologic tumors with high protein production are highly sensitive to CC‐115, a clinically exploitable inhibitor of NMD. This might be particularly interesting in hematologic malignancies such as multiple myeloma, that depend on NMD to avoid excessive protein stress.