Fig. 4: CCND3 protects B-ALL from apoptosis in a kinase-independent manner and confers resistance to palbociclib.

A B-ALL cell lines were treated with 1 µM palbociclib for 3 days or transduced with either a scrambled (control) or a CCND3-shRNA containing vector, harboring a fluorescent marker (RFP). The RFP⁺ cells were sorted by FACS 4 days post transduction and cultured for 3 more days. Apoptosis was analyzed by PI/annexin V staining, with help of flow cytometry. Data shown as mean ± SD, n = 3. Statistical analysis was performed by student’s t-test. *=p < 0.05 B CCND3 protein expression level was analyzed by immunoblot 3 days after treatment with 1 µM palbociclib and 4 days after transduction with either scrambled control or CCND3 targeting shRNA. Image is representative of n = 3. C NALM-6 cells were transduced with SFFV-CCND3 vector expressing wtCCND3 and a fluorescent marker (EGFP) or by control vector (EV). CCND3 overexpression was controlled by immunoblot. D Cell growth of CCND3 overexpressing NALM-6 cells and EV control expressing NALM-6 cells was determined by calculating GFP⁺ cells relative to the absolute cell number of living cells. Data shown as mean ± SD, n = 3. Statistical analysis was performed by Student’s t-test. *=p < 0.05, ***=p < 0.005. E, F Cells were cultured in the presence of increasing (from 0.01 to 1.7 µM) concentrations of palbociclib for 3 months. After reaching 1.7 µM concentration with cells growing at a speed comparable to the vehicle-treated control cells, their sensitivity to palbociclib was measured using MTT Assay (E) and CCND3 and CCND2 expression at the same time point was measured by immunoblot (F). Image is representative for n = 3. Statistical analysis was performed by student’s t-test. **=p < 0.01, ****=p < 0.001. G Control (Ctrl) and palbociclib-resistant (PR) NALM-6 and RS4;11 cells were transduced with either a scrambled shRNA (scr) or shRNA against CCND3 (shC3), co-expressing RFP. The proportion of RFP⁺ cells was monitored throughout 9 days, starting 4 days post transduction (Day 0). Data shown as mean ± SD, n = 3.