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C. Hoareau-Aveilla et al.
often a disseminated disease and pediatric patients are prone to chemoresistance or to relapse after stopping the treatment. All relapses occur in patients with advanced disease and there is still no gold standard for treating relapses.
In children and adolescents, more than 90% of sys- temic ALCL cases harbor a chromosomal translocation t(2;5)(p23;q35) that leads to the expression of the NPM- ALK chimeric tyrosine kinase. This kinase consists of an N-terminal nucleophosmin (NPM) domain fused to the ALK cytoplasmic domain.3,4 ALK is a tyrosine kinase receptor whose expression is normally restricted to neu- ral progenitor cells during development.5 At diagnosis, ALCL patients are grouped into risk groups based on the presence of NPM-ALK transcripts in peripheral blood and/or bone marrow6 and the quantification of anti-ALK antibody in peripheral blood,7 and chemotherapy treat- ment is adapted accordingly.
In ALCL, oncogenic NPM-ALK signaling is mediated by different pathways, such as RAS/ERK and JNK/STAT pathways, which play major roles in lymphomagenesis by controlling key cellular processes such as cell cycle progression.8,9 Studies showed increased proliferation of Rat1a fibroblasts upon ectopic expression of NPM-ALK as a consequence of accelerated entry into the S phase of the cell cycle. Furthermore, this was associated with a significant upregulation of cyclin A and cyclin D1 and increased levels of the mitogenic proto-oncogenes Jun, Fos, and Myc.10 Moreover, in 293T and Jurkat cells, it has been shown that forced expression of NPM-ALK induces JNK (JUN N-terminal kinase) and Jun phospho- rylation, thereby up-regulating the activity of AP1 tran- scription factors. This results in uncontrolled cell cycle progression and cell growth due to the downregulation of p21 and the concomitant upregulation of cyclin A and cyclin D3.11 The ectopic expression of constitutively active NPM-ALK in Ba/F3 mouse cells leads to FOXO3A phosphorylation by Akt, upregulation of cyclin D2 expression and downregulation of p27 and Bim-1 expression to promote cell survival and cell cycle pro- gression.12 In Ba/F3 mouse cells, NPM-ALK, via the PI3K/Akt pathway, also controls cell division cycle 25 A (Cdc25A), a key regulator of the G1 phase and the G1/S transition.13
Many microRNAs (miRNAs) modulate several major proliferation pathways by controlling critical regulators such as Cyclin-CDK complexes.14 miRNAs are single- stranded small non-coding RNAs that are pivotal in physiological and pathological processes such as devel- opment, cell proliferation and apoptosis. In general, by binding to specific targets with distinct degrees of com- plementarity, miRNAs exhibit a negative regulatory role at the post-transcriptional level through the inhibition of translation and/or degradation of their messenger RNA targets. There is growing evidence to show that differ- entially expressed miRNAs are associated with tumor types and cancer development.15 Indeed, several miRNAs display defective expression patterns in tumors, consequently altering oncogenic or tumor sup- pressive targets. miRNAs such as miR-16, miR-17-92, miR-21, miR-26a, miR-29a, miR-96, miR-101, miR- 135b, miR-146a, miR-150, miR-155 and miR-219 are dysregulated and serve as oncogenes or tumor suppres- sors in NPM-ALK+ ALCL.16-20 Most of these miRNAs have been found to be down-regulated (miR-16, miR-21,
miR-26a, miR-29a, miR-96, miR-101, miR-146a, miR- 150, miR-155 et miR-219) in NPM-ALK+ ALCL. Our lab- oratory showed, for the first time, that NPM-ALK+ ALCL cell lines and primary tissues express low levels of several miRNAs mediated by the hypermethylation of their gene promoter.17,21 Both NPM-ALK and STAT3 activities contributed to epigenetic silencing in NPM- ALK+ ALCL cell lines and biopsy specimens by up-regu- lating and recruiting DNMT1 to the promoter of miR- 29a, miR-125b and miR-150.17,19,21 The repressive methy- lation catalyzed by DNMT1 can be partially reversed by treatment with 5-aza-2’-deoxycytidine (5-aza-dC, decitabine, Dacogen,® SuperGen Inc., Dublin, CA, USA), a DNMT inhibitor. This DNA-demethylating agent has been shown to restore miR-497 expression, which is suppressed in HT29 colorectal cancer cells.22 In addition, miR-497 downregulation has been consistent- ly demonstrated in a variety of solid tumor types such as hepatocellular carcinoma, ovarian cancer, colorectal ade- nomas, and in multiple myeloma cells.22,23 MiR-497, a highly conserved miRNA encoded by the first intron of the MIR497HG gene on human chromosome 17p13.110 belongs to the miR-15/16 family (miR-15a, miR-15b, miR-16-1/2, miR-195, miR-424 and miR-497) sharing the same seed sequence AGCAGCA.24 Downregulation of miR-497 controls cell cycle progression by regulating cell cycle regulators such as Cyclin A2, Cyclin D1, Cyclin D2, Cyclin D3 and Cdc25a. In a previous study, using microarray miRNA-expression profiling, we showed that miR-195 and miR-497 was differentially expressed in NPM-ALK+ ALCL lymph node primary tis- sues compared to reactive lymph nodes of healthy donors.21 As miR-195 and miR-497 are encoded as a cluster within the same host gene, MIR497HG (a highly conserved miRNA cluster),25 we sought to simultane- ously study the roles of miR-195 and miR-497 in NPM- ALK+ ALCL tumorigenesis. Accordingly, we measured miR-195 and miR-497 expression in human NPM-ALK+ ALCL primary biopsies and cell lines. First, we studied the biological functions of these miRNAs in human NPM-ALK+ ALCL cells. We showed that overexpression of miR-497 inhibits cellular growth and causes cell cycle arrest. We identified cyclin E1, E2F3 and CDK6 as the main miR-497-targets responsible for the observed phe- notype. Several CDK4/6 inhibitors have been developed [PD-0332991/palbociclib (Pfizer), LEE011/ribociclib (Novartis), and LY2835219/abemaciclib (Lilly)] and are currently being tested in clinical trials for patients with solid tumors and B lymphomas.26 Previous studies have demonstrated that palbociclib caused cycle arrest and apoptosis in T-cell leukemia in vitro and delayed disease progression in mouse models of T-cell acute lym- phoblastic leukemia.27 However, this class of drugs has not been tested in T-cell lymphomas. Thus, we demon- strate, for the first time, the sensitivity of NPM-ALK+ cell lines to CDK4/6 inhibition using palbociclib. Furthermore, we show that a scoring system based on CDK6, E2F3 and CCNE1 expression could help identify relapsing patients with a very short survival. Altogether, our results indicate that miR-497 functions as a tumor suppressor and that some of the miR-497 functional downstream targets could be used as predictors of clini- cal outcomes. Amongst them, CDK6 could be a thera- peutic target for the development of future treatments for NPM-ALK+ ALCL.
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haematologica | 2019; 104(2)