Page 234 - Haematologica May 2020

P.J. Teoh et al.
With regards to ADAR1, interestingly, we observed that among the 1q21(amp) cells, P150 isoform expression was prominently higher in IL6-responsive and -dependent cells, while the expression of P110 isoform did not vary much (Figure 1B). Importantly, we also observed a consis- tent positive correlation between ADAR1 and IL6R in patients from several clinical datasets (Figure 1C) suggest- ing that the ADAR1-P150 isoform, just like IL6R, could be involved in IL6 signaling.
To test this hypothesis, we treated human MM cell lines with IL6 for different periods and investigated the expres- sion profile of various markers involved in IL6-signaling. We observed that IL6 caused an induction of STAT3 sig- naling in MM cells regardless of the cells’ 1q21 status, albeit at different levels. While there was a gradual and time-dependent accumulation of IL6R in the 1q21(WT) cells, the 1q21(amp) cells showed only a minimal increase, largely due to its already heightened endogenous level, even in the unstimulated state (Figure 1D). As expected, IL6 led to robust upregulation of phospho-STAT3 in both cell types; however, close scrutiny of the time kinetics revealed that 1q21(amp) cells had more rapid and sustain- able phospho-STAT3 induction than the 1q21(WT), con- comitantly with its downstream target MCL1. This indi- cated that the STAT3 signaling in 1q21(amp) cells was hypersensitized, potentially in relation to the constitutive- ly expressed IL6R.
With regards to ADAR1 expression, the P150 isoform was evidently upregulated upon IL6 stimulation, whereas, P110 seemed to show minimal changes, at both the pro- tein (Figure 1E) and mRNA levels (Online Supplementary Figure S1C), irrespective of 1q21 status. An antibody spe- cific for the P150 isoform was also used to confirm our observations. We further validated our observations by starving the IL6-dependent-cells lines XG6 and XG7 of IL6, which demonstrated depleted P150, and an attenua- tion of IL6R, concomitantly with MCL1; P110 was again mildly affected (Figure 1F and Online Supplementary Figure S1D). The specific correlation of IL6 induction with P150 was also recapitulated when the rescue of IL6 stimulation in a IL6-sensitive cell line, U266, returned P150 expression to its basal level (Figure 1G). In corroboration, in vitro IL6 starvation of CD138+ cells harvested from patients’ sam- ples (n=2) compromised the STAT3 pathway, concomi- tantly with P150 downregulation (Online Supplementary Figure S1E). Collectively, these data suggest that besides potently activating the STAT3 pathway, IL6 has a role in mediating ADAR1-P150 expression in MM cells.
STAT3 transcriptionally regulates ADAR1-P150 expression
Considering that IL6 robustly activated STAT3 signal- ing, we next proceeded to investigate the direct role of STAT3 transcription factor on ADAR1-P150. Utilizing mouse liver cells with STAT3-WT+/+ and null-/- genotypes, we observed that the absence of STAT3 (STAT3-/-) grossly compromised the upregulation of IL6-induced-P150 (Figure 2A). Pharmacological inhibition of STAT3 in H929 cells with two specific inhibitors (STA-21 and LLL12) caused a gradual reduction of P150 expression in a time- and dosage-dependent manner (Figure 2B), concomitantly with phospho-STAT3 and MCL1. At the mRNA level, knocking down STAT3 and overexpressing the constitu- tively-activated-STAT3 (CA-Y705), but not the dominant negative mutant derivative, caused a reduction and an
increase of P150 mRNA, respectively (Figure 2C, D). These data strongly suggest that STAT3 plays a role in reg- ulating the transcription and expression of P150.
Indeed, our hypothesis was validated when the ChIP- qPCR assay showed an enrichment of STAT3 protein on the promoter regions of P150 and its known target MCL1 (Figure 2E). There was no change in the binding enrich- ment of STAT3 on the IL6R promoter (Online Supplementary Figure S2B). A luciferase reporter assay pro- vided further evidence of STAT3’s transcriptional regula- tion of P150 where IL6 stimulation (Figure 2F) and the overexpression of STAT3-CA (but not the dominant neg- ative mutant) (Figure 2G) resulted in enhancement of the luciferase signal as compared to their respective controls.
P150 is important for the growth and proliferation of multiple myeloma cells
The above results demonstrated that P150 overexpres- sion in MM can arise from the combined effects of 1q21(amp) (Figure 1) and constitutive activation of STAT3 (Figure 2). This double-pronged regulation of P150 suggests that aberrant expression of the isoform may have important biological and functional effects. To elucidate the oncogenic role of IL6/STAT3-induced-P150 in MM, we cloned P150-specific sequences into a pCDNA-overexpressing plasmid and transfected them into OCIMY5 cells, after which we performed various functional assays. Effective overexpression of P150 but not P110 (Figure 3A) allowed us to study the P150-specif- ic oncogenic properties. P150 enrichment in the cells con- ferred a growth advantage (Figure 3B), increased colony- forming ability (Figure 3C) and a more proliferative cell cycle profile (cellular enrichment in S/G2 phase) (Figure 3D). Conversely, knocking down P150 in U266 cells (Figure 3A) was detrimental to the cells’ survival, with the P150-depleted cells demonstrating a slower growth rate, loss of colony-forming ability and a higher fraction of cells undergoing apoptosis (sub-G1 population enrich- ment in the cell cycle and increased annexin-V positivity) (Figure 3B-E). These phenotypes were replicated in P150- overexpressing KMS12BM cells (Online Supplementary Figure S3) and shP150-H929 cells (Online Supplementary Figure S4).
To further ensure that our observations thus far were P150-specific, we performed a rescue experiment by intro- ducing the P150 isoform back into the shP150 cells (Figure 3F). The re-introduction of P150 clearly led to a rescue of growth (Figure 3G) and colony-forming ability (Figure 3H), suggesting that the growth disadvantage observed in the shP150 cells in Figure 3B-E was, in part, mediated by the lack of P150.
ADAR1-P150 in turn mediates IL6-induced STAT3 signaling
Interestingly, we also observed evidence of STAT3 activ- ity being mediated by ADAR1. In the ADAR1 knockdown cells, IL6 delayed the activation of STAT3 signaling, as shown by slower induction of phospho-STAT3 and its direct target, MCL1. Up to 8 h were needed for the mech- anism to start in U266-shADAR1, whereas, U266-shCtr already demonstrated STAT3 activation at 4 h after IL6 stimulation. This was completely reversed in the OCIMY5 cells which overexpressed ADAR1 (Figure 4A). Importantly, this protein expression profile was commen- surate with the cellular phenotypes, in which the more
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