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CDK4/CDK6 inhibitors in ALK-positive lymphomas
SU-DHL1, COST and KARPAS-299 cells were transfected with either an irrelevant siRNA as the negative control (si- CTL) or a siRNA targeting CDK4 mRNA. In order to check whether the knockdown of gene expression had been efficiently achieved, we performed qPCR to detect the CDK4 mRNA (Online Supplementary Figure S11C, F and I). As silencing of CDK4 does not reduce cell proliferation in ALK+ lymphoma cells (Online Supplementary Figure S11A, D and G), nor induce apoptosis (Online Supplementary Figure S11B, E and H), we suggest that the action of palbiciclib is only CDK6-dependent.
Based on our findings that CDK6, CCNE1 and E2F3 are important for efficient proliferation of NPM-ALK+ ALCL cells, we hypothesized that the highest level of expression of these factors could be the most favorable for tumor development and possibly affect chemotherapy effective- ness. To test this hypothesis, we used qPCR to measure the expression of CDK6, CCNE1 and E2F3 in a cohort of NPM-ALK+ primary ALCL samples (n=55) obtained from chemotherapy-naive patients at diagnosis. Compared to normal reactive lymph nodes (RLNs) (n=19) and despite the presence of a mixed population of neoplastic and nor- mal cells, we observed a significant CDK6, CCNE1 and E2F3 gene overexpression in NPM-ALK+ samples (Online Supplementary Figure S12A). In order to evaluate the clinical significance of CCNE1, CDK6 and E2F3 overexpression, first using linear regression, we derived a model based on microarray data on expression levels of CCNE1, CDK6, E2F3 genes in NPM-ALK+ samples (n=55). The samples included NPM-ALK+ primary biopsies of patients who had experienced early relapse and those without relapse after three years of minimal follow up after chemotherapy cure. By applying the formulae obtained from linear regression, we derived a ‘score’ for each patient: score = (-0.08378 x CDK6) + (-0.41269 x CCNE1) + (0.37893 x E2F3). Secondly, a ROC curve was generated from CCNE1, CDK6, E2F3 genes-based score from 44 NPM-ALK+ ALCL samples, including 8 adults and 36 children, for whom event-free survival is known (Online Supplementary Figure S12B). The cut-off value and Air Under Curve (AUC) val- ues were determined as -0.038 and 0.65, respectively. Based on the optimal cut-off points from the ROC curve, patients were significantly categorized into two groups, high (n=25) and low (n=14) CCNE1, CDK6, E2F3 expres- sion (Online Supplementary Figure S12C and D). NPM-ALK+ ALCL patients with a low expression of CCNE1, CDK6, E2F3 had a poor prognosis (P<0.046) (Online Supplementary Figure S12C). Moreover, this trend can be observed for high (n=14) and low (n=22) expression in NPM-ALK+ pedi- atric ALCL (Online Supplementary Figure S12D). In addi- tion, for the same pediatric cases, we noted that CCNE1, CDK6, E2F3 gene-based score was increased in relapsing patients (n=23 vs. without relapse n=13) (Online Supplementary Figure S12E). As all patients were treated with chemotherapy following diagnosis, our data suggest that CDK4/6 inhibitors could benefit ALK+ patients har- boring resistant tumors.
Discussion
MiR-195 and miR-497 are important members of the miR-15/16 family and are aberrantly expressed in multiple diseases, such as solid cancers.23,33 Based on the expression of miRNAs, Steinhilber et al. have identified a gene signa-
ture including reduced expression of some miR-15/16 family members, such as miR-16, miR-195 and miR-497 that distinguish ALK+ from ALK– ALCL.34 Except miR-16,19 the functional characterization of miR-195 and miR-497, which are clustered on chromosome 17p13.1, has never been undertaken in ALK+ lymphoma cells.
MiR-195 and miR-497 downregulation has been consis- tently demonstrated in a variety of solid tumor types. miRNA-silencing in cancer is frequently related to abnor- mal promoter methylation.35 Accordingly, the downregu- lation of miR-195 and miR-497 has been reported to be linked to promoter-associated hypermethylation in solid cancers; demethylation following 5-aza-CdR treatment resulted in the re-expression of these two miRNAs.36,37 In the present work, we demonstrate for the first time that miR-195 and miR-497 are down-regulated in human NPM-ALK+ ALCL tissues and cell lines through a mecha- nism involving NPM-ALK and DNA hypermethylation. These results are highly reminiscent of what we previous- ly observed for three other miRNA genes, MIR29A, MIR125B and MIR150, the levels of which were also reduced in NPM-ALK+ ALCL as a consequence of the pro- moter hypermethylation.17,20,21 In addition, we observed that only ectopic expression of miR-497 markedly attenu- ates in vitro cell proliferation of NPM-ALK+ cells.
It is estimated that more than half of human genes are targeted by at least one miRNA. One miRNA may have multiple different mRNA targets and at the same time, one mRNA might be targeted by multiple miRNAs. miR-497 regulates the expression of a plethora of targets, which are involved in cell cycle, apoptosis, proliferation, etc.23 Recent studies have revealed that miR-497 can directly reduce the CCNE1 protein level to suppress tumor growth by induc- ing G1 arrest in breast cancer38 and hepatocellular carcino- ma.39 Indeed, CCNE1 protein binds to and activates cyclin- dependent kinase 2 (CDK2) to promote a cascade of events required for cell-cycle progression from G1 to S phase. Several other genes encoding cell-cycle regulators, including Cyclin D1/CCND1 and Cyclin D3/CCND3, Cyclin-dependent kinase 4 (CDK4) and CDK6, cell-cycle division factors CDC25A and/or E2F3 transcription factor, are also known targets of miR-497.23,40 The role of miR-497 in human cancer is not yet clear. Several studies have reported that increased miR-497 expression via miR-497 mimic transfection suppressed proliferation and increased apoptosis in various solid cancers, such as adrenocortical carcinoma41 and pancreatic cancer.42 Accordingly, miR-497 overexpression was found to block G0/G1 phase transi- tion in breast cancer cells43 and induce G1/S arrest in gas- tric cancer cells.37 In our study, we revealed for the first time that enhanced miR-497 expression in human NPM- ALK+ lymphoma cells could suppress cell proliferation, by inducing cell cycle arrest, and inhibit tumor growth in vivo. These biological effects are the consequence of simultane- ous downregulation of three mir-497 mRNA targets: CCNE1, E2F3 and CDK6, and their subsequent decreased protein level. Contribution of CCNE1 and E2F3 in uncon- trolled cell-cycle progression and oncogenesis have never been studied in human NPM-ALK+ ALCL models. Of note, only two publications report an abnormal CDK6 expres- sion in human NPM-ALK+ lymphoma cells. The amplifica- tion of the CDK6 locus in NPM-ALK+ SU-DHL1 cells and co-expression of high levels of CDK6 and CD31 (angio- genic marker) in NPM-ALK+ ALCL samples have been reported.31,44
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