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C. Hoareau-Aveilla et al.
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Figure 8. Anaplastic lymphoma kinase (ALK)-positive(+) lymphoma cells are sensitive to CDK6 inhibitor. NPM-ALK+ ALCL SU-DHL1 (A-D), COST (E-H) and KARPAS- 299 (KARPAS) (I-L) cells were treated or not (H2O) with palbociclib, an CDK4/6 inhibitor. Cell count at three different time points (0, 1, 2 days) (A, E and I), cell cycle distribution (B, F and J), caspase 3/7 activity (C, G and K) and cell proliferation rate (D, H and L) were measured. Data represent means±SEM (bars) from 3 inde- pendent experiments. *P<0.05; **P<0.001; ***P<0.0001; ns: not significant; unpaired two-tailed Student’s t-test with Welch’s correction. Data represent means±Standard Error of Mean (bars) from 3 independent experiments, *P<0.05; **P<0.001; unpaired two-tailed Student’s t-test with Welch’s correction.
but, as expected, not in KARPAS-299 cells (Online Supplementary Figure S7). In addition, the silencing of E2F3 mRNA did not affect caspase activity in both NPM-ALK+ COST and KARPAS-299 cells (Online Supplementary Figure S7). Moreover, regardless of the siRNA transfected, cell viability following RNAi delivery was never significantly modified in NPM-ALK+ cells (data not shown). Thus, indi- vidual silencing of three miR-497-targets, CDK6, CCNE1 and E2F3, can reproduce (only to a lesser extent) the miR- 497-induced phenotype (Figure 4A and B). Consequently, in order to improve the effects observed in COST and KARPAS-299 cells, we decided to pool siRNAs targeting CCNE1, CDK6 and E2F3. We transfected KARPAS-299 or COST cells with siRNAs (pools of three siRNAs/targets) targeting each of the miR-497-down-regulated transcripts. In order to check whether the knockdown of gene expres- sion had been efficiently achieved, we performed qPCR to detect the mRNA (Online Supplementary Figure S8). We then analyzed transfected cells for their cell cycle distribu- tions. Similar to transfection of miR-497 mimics, siRNA pools induced significant accumulation of KARPAS-299 and COST cells in G0/G1 fraction (Figure 7D and G). Of note, the same result was observed in COST and SU- DHL1 cells, 24 h post transfection with the miR-497 mimic (Online Supplementary Figure S9A and B). The reduc- tion in cell proliferation in the knockdown NPM-ALK+ cells was observed by cell counting (Figure 7E and H). In addition, compared to the control condition (si-CTL), CCNE1, CDK6, E2F3 silencing induced apoptosis in COST cells thereby inhibiting cell growth (Figure 7G and
K) but, as expected, not in KARPAS-299 cells (Figure 7K and H). Taken together, these findings demonstrate that miR-497 controls multiple targets that collaborate to regu- late cell cycle progression and cell growth.
ALK-positive cells are responsive to CDK4/6 inhibitors
The new generation of selective CDK4/6 inhibitors, with anti-proliferative activity in Rb-proficient cells, target tumor types in which CDK4/6 has a pivotal role in the G1- to-S-phase cell cycle transition with improved effective- ness and fewer adverse effects. Palbociclib is an orally bioavailable CDK4/6 inhibitor recently approved by the US Food and Drug Administration for the treatment of metastatic breast cancer.32 As all three NPM-ALK+ cell lines, SU-DHL1, COST and KARPAS-299 expressed CDK4, CDK6 and Rb (Online Supplementary Figure S4), we evaluated the effect of palbociclib treatment. We observed that all NPM-ALK+ cell lines are sensitive to this molecule (Figure 8A, E and I). In addition, we showed that palboci- clib markedly inhibited the proliferation of NPM-ALK+ cells through the induction of G0/G1 cell-cycle arrest (Figure 8B, F and J), increased apoptosis (Figure 8C, G and K) and reduced cell growth (Figure 8D, H and L). As a con- trol of palbociclib efficacy, we observed a decrease in Cdk4/6-specific phosphorylation of the Rb protein as shown by Western blotting (Online Supplementary Figure S10A). Densitometric analysis of the mean relative inten- sity for pRB and RB corroborate Western blotting results (Online Supplementary Figure S10B). As palbociclib is a selective cyclin D kinase 4/6 inhibitor, NPM-ALK+ ALCL
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haematologica | 2019; 104(2)