Page 297 - 2021_02-Haematologica-web

Letters to the Editor
ALK copies/10,000 ABL1 in ALK-high patients. Interestingly, most of the patients who relapsed belonged to the ALK-high subgroup. The median time of relapse of these ALK-high patients was 5 months, in striking con- trast to the time of relapse of ALK-low cases (30 months). By means of a gene set enrichment analysis and in vitro functional studies, we showed a differential gene expres- sion signature in ALK-high versus -low patients. Moreover, we identified signaling pathways preferential- ly associated to the different phenotype and clinical course of ALK-high and ALK-low cases. In particular, the less aggressive phenotype observed in the ALK-low group was characterized by IL-2 signaling retention, while higher ALK expression levels were shown to induce cyclins and Aurora Kinases overexpression in the ALK-high subgroup. These data also suggest that cells expressing high ALK levels could acquire a selective growth advantage as compared to ALK-low cells.6
To further elucidate the molecular mechanisms respon- sible for the heterogeneous aggressiveness of ALK+ ALCL, and to better characterize these two previously uncov- ered disease entities, we performed array-based miRNA profiling in 16 pediatric cases previously classified according to their ALK endogenous expression (n=11 ALK-high, n=5 ALK-low) (Online Supplementary Table S1). By supervised analysis, we identified 19 microRNAs sig- nificantly upregulated in the ALK-low subgroup (False Discovery Rate <0.05) (Online Supplementary Table S2).
Some of these, such as miR-155, miR-146a and miR-497, had previously been described as differentially expressed in ALK+ compared to ALK-ALCL.7
Since high ALK expression was previously associated with a more aggressive ALCL phenotype,6 miRNAs upregulated in the ALK-low cases could probably act as tumor suppressors. In line with this concept, Hoareau- Aveilla et al. found that the overexpression of miR-497 in ALK+ cell lines (corresponding to ALK-high patients) inhibited ALCL cell growth and induced cell cycle arrest.8
In this study, we focused our attention on miR-939, that was the most differentially expressed miRNA between ALK-high and ALK-low tumor cells (Figure 1A). We first evaluated miR-939 expression by quantitative reverse transcription PCR (qRT-PCR) in an extended panel of 49 ALCL primary tumor samples collected in the last 15 years in our lab from the Italian Association of Pediatric Hematology and Oncology (Associazione Italiana di Ematologia e Oncologia Pediatrica, AIEOP) centers. Unfortunately, despite a long period of recruit- ment, this larger cohort included only 20% of ALK-low cases, and miR-939 expression difference, observed with the array-based miRNA profiling, was less appreciable. Of note, three ALK-low cases had very high levels of miR-939 (Figure 1B). Although whether the expression of miR-939 is actually higher in ALK-low profile patients could only be confirmed by an analysis of a very large cohort of ALCL pediatric cases, it does seem evident that
ABC
D
F
E
Figure 2. miR-939 impairs tumor cell migration and invasion. (A) miR-939 basal expression levels in anaplastic large cell lymphoma (ALCL) cell lines. Data have been calculated according to the comparative δCt method and compared to reactive lymph nodes tissue (LN, n=11). miR-16 was used as endogenous control. Karpas299 and SUP-M2 cells were transiently transfected with miR-939 mimic, or negative control (NC) or with the inhibitor and used to analyze migration (B) and clonogenicity (C). The results are shown as fold change of negative control values. Two or three independent experiments were performed in triplicate and mean results are shown. *P<0.05; **P<0.001. (D) Western blotting analysis of JUNB and JUN proteins in Karpas299 and SUP-M2 cell lines transfected with miR-939 mimic or negative control at indicated post-transfection time points. γ-tubulin was used as loading control. Vertical lines have been inserted to indicate repositioned gel lanes. (E) The JUNB 3’UTR sequence containing the miR-939 putative binding site has been subcloned into pmirGLO reporter plasmid. Karpas299 and SUP-M2 cells transfected with miR-939 mimic and the reporter plasmid containing wild-type or mutated miR-939 binding site were tested by luciferase assay 24 hours post transfection. **P<0.001; ****P<0.0001; ns: non-significant. (F) miR-939 binding site on 3’-UTR of JUNB. The putative binding sequences have been predicted using the on-line tool available at http://www.microrna.org.
haematologica | 2021; 106(2)
611