Page 181 - Haematologica - Vol. 105 n. 6 - June 2020

DARS-AS1 promotes myeloma malignancy in hypoxia
vivo. We propose that the HIF-1α–DARS-AS1–RBM39 axis is a potential target for MM treatment.
DARS-AS1 is close to DARS in the genome (Online Supplementary Figure S4A). Numerous antisense lncRNA participate in physiological or pathological cellular processes through the regulation of the expression of its adjacent gene.21,22 By contrast, we found that the downreg- ulation of DARS-AS1 was not associated with the expres- sion of DARS at either transcriptional or protein levels (Online Supplementary Figure S4B-E). The DARS-AS1 gene has two annotated transcripts, NR_110199.1 and NR_110200.1, in the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/), with limited protein-coding potential (http://cpc.cbi.pku.edu.cn/) (Online Supplementary Figure S1E, F). The transcript of DARS-AS1 #2 (NR_110200.1) is not detectable in the myeloma cells. We, therefore, focused on DARS-AS1 #1 isoform in our gain- and loss-of-function studies.
Nutritional deficiency is common in the microenviron- ment of tumors. HIF-1, the protein that responds to low O2 concentrations, apparently leads to increased glycoly- sis, angiogenesis and drug resistance.1 In culture with low levels of glucose, the overexpression of DARS-AS1 sup- pressed myeloma cell apoptosis (Online Supplementary Figure S2D, E). More importantly, DARS-AS1 can promote the expression of HIF-1α. Thus, DARS-AS1 and HIF-1α may create a positive feedback, increasing the ability of myeloma cells to survive, although the underlying mecha- nism is not fully known. In addition, it would be interest- ing to examine the impact of DARS-AS1 on the prognosis of myeloma patients through the analysis of data from large numbers of samples.
The mass spectrometry and RNA pullown data revealed that DARS-AS1 interacts directly with RBM39, suppress- ing the ubiquitination and subsequent degradation of RBM39 protein, an RNA-binding protein. Dysregulation of these types of proteins, which can lead to aberrant expression of cancer-related genes, has been widely observed in cancer cells.23,24 Previous studies suggested that RBM39 is a proto-oncogene in multiple cancer types. Our results extend this notion, indicating that RBM39 is a novel oncogene in myeloma. First, a significantly higher mortality risk was observed in patients with high expres- sion of RBM39 than in those with lower expression. Second, activation of the mTOR signaling pathway reportedly plays a critical role in the pathogenesis of myeloma. Our results showed that the knockdown of RBM39 inhibited mTOR signaling, thus suggesting that RBM39 is critical for the proliferation and tumorigenesis of MM cells and may serve as a prognostic predictor for patients with MM. However, further investigations will be necessary to reveal the mechanisms of the regulation of the mTOR pathway by RBM39. Furthermore, similar results were observed in DARS-AS1 knockdown cells. The overexpression of RBM39 reversed the inhibition of mTOR signaling induced by the knockdown of DARS- AS1. Based on these data, we propose that the aberrant
expression of DARS-AS1 in cells may increase the levels of RBM39, thereby triggering continuous activation of mTOR signaling.
Previous studies showed that sulfonamides have anti- cancer effects by promoting an interaction between RBM39 and the E3 ubiquitin ligase DCAF15, leading to the degradation of RBM39.12 However, RBM39 is not the endogenous substrate of DCAF15. To the best of our knowledge, in the present study we, for the first time, dis- covered that RBM39 is a substrate of the E3 ubiquitin lig- ase RNF147. RNF147 is a member of the tripartite motif protein (TRIM) family and contains an N-terminal RING- domain, one or two B-boxes, and a coiled-coil region.25 We found that the RRM1 domain of RBM39, which contains the ubiquitin binding site domain (http://ubibrowser.ncpsb.org/, http://cplm.biocuckoo.org/), was responsible for the interaction with both DARS-AS1 and RNF147. Our results raise the possibility that DARS-AS1 may inhibit ubiquitination of RBM39 by competing with E3 ubiquitin ligase RNF147 for binding to RBM39. In fact, accumulating evidence shows that lncRNA can protect proteins from proteasome-mediated degradation. For instance, NKILA is a lncRNA that directly masks the phos- phorylation motifs of IκB, thereby inhibiting the degrada- tion of IκB and subsequently activating the NF-κB path- way.26 UPAT was found to inhibit the ubiquitination of epigenetic factor UHRF1 and play a critical role in the sur- vival and tumorigenicity of tumor cells.27 In addition, LINC00673, as a cancer suppressor, was able to promote PTPN11 degradation, which weakened SRC–ERK signal- ing and increased STAT1-dependent antitumor effects.28 Our findings, together with the aforementioned earlier results, indicate that there is a class of lncRNA that regu- late protein ubiquitination and degradation.
In conclusion, we demonstrate that DARS-AS1 has important functions in the hypoxic microenvironment of myeloma and may serve as a prognostic predictor for patients with MM. Our results indicate that the HIF- 1/DARS-AS1/RBM39 pathway might provide a positive feedback loop that augments the HIF-1 response in myelo- ma, and that targeting this pathway may be pivotal in the prevention or treatment of myeloma. Our findings shed new light on the orchestrated interactions between HIF-1 and lncRNA in maintaining myeloma cell survival under hypoxia.
Acknowledgments
We thank Guoqiang Chen at Shanghai Jiao Tong University School of Medicine for providing plasmids.
Funding
This work was supported in part by grants from the National Key Research and Development Program of China (n. 2017YFA0505200), National Natural Science Foundation of China (8167010684, 81570118), Shanghai Commission of Science and Technology (16ZR1421400), and the Science and Technology Committee of Shanghai (15401901800).
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