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P.J. Teoh et al.
   Cytogenetic abnormalities remain one of the main hall- marks of MM. 1q21 amplification [1q21(amp)], the most commonly identified chromosomal aberration, present in 36-48% of newly diagnosed cases of MM, is an important prognostic marker.15-18 Previous studies revealed that CKS1B and PMSD4 are genes that putatively drive disease aggressiveness in 1q21(amp) cases;18-20 nevertheless, bio- logical and functional reports on these genes conferring oncogenic phenotypes are lacking. In reality, the critical genes within the minimally amplified region have yet to be fully characterized.
IL6R, the gene encoding the transmembrane receptor protein for IL6 is located on chromosome 1q21. Although it has been shown that IL6R has a role in predicting patients’ outcome,5,21,22 it remains unknown whether IL6R expression is associated with 1q21(amp) or could cause the hyperactivation of STAT3 signaling that could poten- tially contribute to adverse disease manifestations.
Importantly, our group and Lazzari et al. have recently reported that overexpression of ADAR1, an RNA editing enzyme, residing in the 1q21 region near the aforemen- tioned IL6R, also has significant prognostic value in MM.23,24 The protein exists in two distinct isoforms, name- ly the short P110 and the long P150 isoforms. While P110 is constitutively expressed in the nucleus, P150 expression is interferon-inducible and can shuttle between the nucle- us and cytoplasm due to the presence of a nuclear export signal on its extended amino terminal domain.25-27 Although interferon and IL6 are both members of the cytokine family and can be upregulated upon infection and inflammation, a possible cross-talk between IL6/STAT3 signaling and ADAR1 expression has not been thoroughly investigated. Aberrant adenosine-to-inosine editing events arising from deregulated ADAR1 been widely reported in cancers,27-31 however, the general knowledge on its upstream regulators and the mecha- nisms mediating its overexpression have remained elu- sive, not to mention in MM.
In view of both IL6R and ADAR1 being located in close proximity on 1q21 and having been independently report- ed to be prognostically important for MM, we sought to delineate their potential collaboration in the pathogenesis of MM and to determine how they are associated with STAT3 signaling. Here, we report that 1q21(amp) leads to elevated expression of IL6R and ADAR1. ADAR1-P150 and STAT3 form a regulatory feedback loop mediating the growth and proliferation of MM cells; the convergence of regulatory signals from both IL6R and ADAR1-P150 con- fers hyperactivation of STAT3 signaling, potentially driv- ing the malicious evolution of MM. Critically, MM patients with concurrent overexpression of both proteins had a poorer prognosis than those who had no abnormal- ity or a single one.
Methods
Patients’ samples and human multiple myeloma cell lines
Primary samples from the healthy volunteers and MM patients were collected after obtaining informed consent, according to con- ditions stated by the Institutional Review Board, National University Hospital. All human MM cell lines used have been pre- viously characterized.32 Isolation of patients’ samples and culture conditions for them and human MM cell lines are described in the Online Supplementary Information. The purity of CD138+ cells of the
patients’ samples was checked with anti-κ and anti-λ immunoflu- orescence staining (Online Supplementary Figure S5).
Gene expression profiling, genomic hybridization and biostatistics
Details of the multiple gene expression profiling and array com- parative genomic hybridization datasets analyzed are provided in Online Supplementary Table S1. Analyses of IL6R/ADAR1 copy number and expression and STAT3 signature/index are described in the Online Supplementary Information. Statistical analyses between two groups in the patients’ datasets were performed with the Wilcoxon test. P-values between two groups from in vitro studies were computed with an independent t-test, assuming a normal distribution of the means. All experiments were repeated at least twice (biological replicates) and the error bars in the graphs represent the means ± standard deviations.
ADAR1-P150, IL6R and STAT3 knockdown and overexpression
We knocked down ADAR1-P150 and IL6R in human MM cell lines with specific shRNA through lentivirus infection. shRNA sequences are available in the Online Supplementary Information (Online Supplementary Table S3). pCDNA-Flag-tagged vector encoding for P150 protein was transfected into MM cells using an electroporation method (Neon, Invitrogen). siRNA against STAT3 and pIRES-STAT3 plasmids were also introduced into MM cells via Neon transfection. Based on the basal level of the proteins (Figure 1B), we picked H929 and U266 cell lines for P150 and IL6R knockdown and the OCIMY5 and KMS12BM lines for P150 over- expression.
Assays
Details of the chromatin immunoprecipitation (ChIP), luciferase, co-immunoprecipitation, immunofixation and func- tional assays (cell growth, colony formation and cell cycle) are available in the Online Supplementary Information. Blots and images presented are representative of multiple repeats (at least two) of the experiments.
RNA-sequencing and analysis of global RNA editing events
Total RNA from IL6-treated MM cells was isolated using the RNeasy kit (Qiagen) according to the manufacturer’s protocol. Whole transcriptome sequencing was done on an Illumina Hi-Seq- 4000 platform (Supplier). Detailed information about variant call- ing and RNA editing analysis are provided in the Online Supplementary Information.
Results
ADAR1-P150 and IL6R are associated with 1q21 copy number and IL6 stimulation
Since IL6R and ADAR1 genes are located in close prox- imity (Online Supplementary Figure S1A), we postulated that 1q21(amp) could lead to concomitant increases in the expression of both. In silico analyses revealed that 1q21 status was indeed closely associated with IL6R and ADAR1 levels in diverse patients’ datasets and human MM cell lines (Figure 1A and Online Supplementary Figure S1B). Quantitative polymerase chain reaction (qPCR) and west- ern blotting confirmed the upregulation of ADAR1 and IL6R at the mRNA and protein levels, respectively, in cell lines with 1q21(amp) compared to the levels in the wild- type (WT) line (Figure 1B).
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