Lenalidomide Effectively Induces Philadelphia-Chromosome Positive ALL Cells into Apoptosis in Synergy with Tyrosine Kinase Inhibitor

[Background] Lenalidomide (LMD) has been effectively used for the treatment of hematologic malignancies such as multiple myeloma and 5q-MDS. Recently, LMD was shown to bind the celebron (CRBN) E3 ubiquitin ligase complex and degrade Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3) via ubiquitin-proteasome system in myeloma cells, which leads to decrease in expression of downstream targets such as IRF4 and c-Myc. The IKZF1 is an essential transcription factor for the development of all lymphoid lineages particularly in the development of B- cell progenitors (BCP), and its gene consists of 8 exons including exons 4-6 and exon 8 encoding DNA-binding and dimerization domains, respectively. Genomic heterozygous deletions in IKZF1 are found in ~15% childhood BCP-ALL and the most frequent deletions affect the whole gene or exons 4-7 resulting in haploinsufficiency or expression of a dominant negative isoform. The IKZF1 deletions are now a hallmark for refractory nature of BCP-ALL linking to an unfavorable clinical outcome, and the frequency of IKZF1deletion is remarkably high (~70%) in Philadelphia-chromosome positive (Ph+) ALL cases. It is thus of great interest to examine LMD's effects on Ph+ALL cells.

[Materials mad Methods] A total of 7 Ph+ALL cell lines with IKZF1 deletions (4 in exons 4-7, 2 in exons 2-7, and 1 in exons 1-8) were used to examine LMD's effects. The synergic effects of LMD with tyrosine kinase inhibitor (TKI; imatinib, dasatinib) were also examined. The ex vivo studies using NOD/Shi-scid, IL-2Rg^null (NOG) mice model were performed.

[Results] 1. We first performed [³H]-thymidine uptake assays in the presence or absence of 20µM LMD for 3-6 days, and found that thymidine uptakes were variably inhibited (8~65%) after LMD treatment and two of sensitive cell lines showed a marked inhibition in a dose and a time dependent manners of LMD (~75%). Of importance, their thymidine uputakes were further inhibited (~95%) by LMD in the presence of the imatinib in a dose dependent manner (0.05-0.5nM). 2. To access the inhibition of thymidine uptake, we performed alamarBlue cytotoxic assays using the sensiitive cell line KOPN57bi, and found that the cell numbers modestly decreased by LMD (20 µM) or imatinib (0.5nM) , and markedly decreased (up to 35% at day 4) by LMD+imatinib. In flow cytometric analysis, KOPN57bi treated with LMD or imatinib showed cell cycle arrest alone, while the cell line treated with LMD+imatinib showed a marked increase in the subdiploid (42.3%), Annexin V-positive (72.0%), and active caspase 3-positive (31.8%) populations at day 4 after trreatment. The pancaspase inhibitor Z-VAD-FMK completely inhibited induction of cell death (apoptosis) by LMD+imatinib. These results were also obtained by use of another TKI dasatinib instead of imatinib. 3. We examined changes in expression of IKZF1 on Western blot using anti-C-terminal IKZF1 antibody in KOPN57bi (deleted in exons 4-7) 24h after LMD and/or TKI treatment, and found that several alternative splicing forms translated from intact IKZF1 disappear by LMD, but expression of a dominant negative Ik6 isoform was not affected by LMD presumably due to a lack of the CRBN binding site (residue Gln 146:exon5) in Ik6 isoform. IRF4 was also down-regulated by LMD and further in the presence of TKI. 4. We performed ex vivo studies using NOG mice. After intravenous injection of KOPN57bi (2x10⁶cells), mice were orally administrated with saline (control, n=4), LMD (30mg/kg/d, n=5), imatinib (150mg/kg/d, n=5) , or LMD+imatinib (n=4) for 10 days (day 0,1,2,3,4, 7,8, 9,10,and 11) and percentages of human CD45 positive population in bone marrow mononuclear cells were examined at day 14. Human CD45+ cells showed 29.7±4.3% (mean±SEM) and 30.3±1.5% in control and imatinib-treated mice, respectively, whereas they were significantly (p=0.0002) decreased to 2.1±0.4% in LMD-treated mice. Of importance, they were further decreased to 0.3±0.4% in LMD+imatinib treated mice (p=0.0051), suggesting that combined treatment of LMD and imatinib should be very effective in xenograft mice models.

[Conclusion] LMD renderd Ph+ALL cells a marked cell cycle arrest by completely abrogating the remaining IKZF1 function, and effectively induced them into apoptosis in synergy with TKI. Orally administration of LMD and TKI might become a high QOL treatment option for the treatment of Ph+ALL patients particularly for those with a higher age and organ dysfunctions.