The Role And Mechanism Of CaMKⅡγ In Chronic Myeloid Leukemia Blast Crisis

BackgroundChronic myeloid leukemia (CML) is a hematopoietic stem cell disease caused by the BCR-ABL hybrid gene. All of CML has Ph chromosome, as well as t (9; 22) (q34; q11) karyotype. p210BCR-ABL protein, coded by BCR-ABL fusion gene, can strongly activate tyrosine kinase.It can phosphorylate a series of signal proteins continuously, affect cell proliferation, differentiation, apoptosis and adhesion,and the occurrence of CML. Therefore, BCR-ABL fusion gene is considered to be the molecular basis of CML,and the monitoring indicator of the diagnosis, curative observation and prognosis of CML.Chronic myeloid leukemia can be divided into three phases:chronic phase (CP), accelerated phase (AP) and blast crisis (BC), based on clinical characteristics and laboratory findings.CML-BC is the final phase in the evolution of CML, and behaves like an acute leukemia, with rapid progression and short survival.Although it is known that BCR-ABL initiates the disease, the secondary molecular and genetic abnormalities likely contribute to progression of the disease to BC.However, the mechanism(s) of BC phase progression are still lacking, thereby identifying the molecular switches that control the transition of CML from CP to BC is of particular importance.Previous studies have shown that CML progression from a CP to a deadly BC is supported by self-renewing leukemic stem cells (LSCs),but the critical drivers triggering BC of CML remain elusive. Our previous studies demonstrated that the calcium-calmodulin-dependent kinase Ⅱγ (CaMKIIy), a critical regulator of multiple cancer signaling pathways involved in proliferation of leukemia cells,is a critical target of berbamine that can eradicate imatinib-resistant CML BC cells as well as LSCs.These findings prompt us to investigate whether CaMKIIy is involved in the molecular basis of CML BC.Methods and results1. CaMK Ⅱγ is essential in the development of the blast crisis and leukemia stem cell self-renewal of CMLTo determine whether CaMK Ⅱγ is required for the development of CML blast crisis, we first evaluated the impact of CaMKⅡγ deficiency on the development of Bcr-Abl-induced CML in a mouse model. Bcr-Abl oncogene induced CaMK Ⅱγ-/- CML in mice, but CaMKⅡγ-/- leukemia cell burdens in peripheral blood and spleen were much lower than those of CaMKⅡγ+/+ CML. The survival time of mice bearing CaMKⅡγ-/- leukemia was markedly prolonged as compared with mice bearing CaMKⅡγ+/+ leukemia. Significant splenomegaly was observed in all CaMKⅡγ+/+ leukemia mice (n=7) as compared with CaMK Ⅱγ Ⅱγ-/- leukemia mice and normal mice (n=7). These data indicate that genetic deletion of CaMK Ⅱγ greatly inhibits disease progression and improves survival of CML mice. The critical role of leukemia stem cells in maintaining CML blast crisis prompted us to investigate the effects of CaMKⅡγ on the functional role of CML LSCs in vivo. We assayed CML LSC (GFP+ Lin-*c-kit+Sca-1+) levels and normal HSC (GFP- Lin-c-kit+ Sca-1+) levels in bone marrow and spleen samples of Bcr-Abl-induced CML mice at day 25 after bone marrow transplantation. Flow cytometry analysis data showed that CaMK Ⅱγ deficiency reduced LSC levels by 21.5-fold in bone marrows and 5.1-fold in spleen, respectively.Unlike WT CaMK Ⅱγ that impaired normal HSCs of leukemia mice, CaMK Ⅱγ deficiency had little effect on normal HSCs in bone marrow and spleen of the same animals. These results indicate that CaMK Ⅱγ is required for the self-renewal and expansion of LSCs, in particular for LT-LSCs, but not for normal HSCs.2. CAMKⅡγ is essential for the survival of human CML BC cell clones and CD34+cellsTo determine whether CaMK Ⅱγ is essential for the survival of human CML BC cell clones, we knock down CaMK Ⅱγ in human CML BC K562 cells.The results showed a marked growth inhibition of K562 cell colonies by CaMK Ⅱγ specific shRNA. To assess whether CaMK Ⅱγ promotes the growth of human CML BC cell clones, we next examined the effects of CaMK Ⅱγ expression on the colony-forming ability of tumor cells using colony-forming assay. In contrast to control, CaMK Ⅱγ overexpression led to a significant increase in the number of colonies. Of note, the colonies induced by CaMK Ⅱγ overexpression were larger in size than those of the controls. Consistently, overexpression of CaMK Ⅱγ also markedly augmented the number of mitotic cells in leukemia colony as compared to control. These results imply that CaMK Ⅱγ may enhance the colony-forming ability and proliferative capacity of leukemic cells through promoting self-renewal of leukemia stem cells. To test this hypothesis, we next examined the effects of CaMK Ⅱγ expression on CD34+cells of K562 cells and found that overexpression of CaMK Ⅱγ highly increased the number of CD34+cells whereas down-regulation of CaMK Ⅱγ decreased the number of CD34+ cells, suggesting that CaMK Ⅱγ has an important role in both colony-forming ability and expansion of CD34+leukemia cells.3. Aberrant activation of CaMK Ⅱγis associated with tumor growth, progression and overall survivalTo assess whether CaMK Ⅱγ promotes human CML progression in vivo, we established human CML blast crisis xenograft models with high (K562/ADR) or low expression (K562) levels of CaMK Ⅱγ, and then compared the tumor growth and survival rate of tumor-bearing mice. In agreement with in vitro observations, we observed that K562/ADR cells with high CaMK Ⅱγ expression exhibited a higher tumor growth rate and a faster decrease of the body weight of tumor-bearing mice. Moreover, tumor-bearing mice with high CaMK Ⅱγ expression displayed a significantly shorter survival time. Altogether, these results strongly suggest that CaMK Ⅱγ may promote the progression of CML blast crisis. Our findings indicate that CaMK Ⅱγ is critical in regulating transition of CML from chronic phase to accelerated phase and blast crisis. To further validate these data in primary human CML, we next examined whether CaMK Ⅱγ is aberrantly activated during human CML progression. Phosphorylated CaMK Ⅱγ (p-CaMKⅡγ), the activated form of CaMK Ⅱγ, was examined in 15 chronic phase (CP),12 accelerated phase (AP) and 19 blast crisis CML (BC) samples using western blot, and found to be expressed at significantly higher levels in blast crisis CML and accelerated phase CML samples, but weaekly or undetectable in chronic phase CML. To determine whether this aberrant activation of CaMK Ⅱγ in tumor cells of CML at blast crisis is associated with CML LSCs, we also examined expression levels of β-catenin, a well-known central regulator of CML LSC self-renewal. Consistent with these data, Western blot analysis showed that expression levels of CaMK Ⅱγ was correlated with P-catenin levels in CML patient samples, suggesting that CaMK Ⅱγ might play an important role in β-catenin-driven LSC expansion. The highly activation of CaMK Ⅱγ in CML at blast crisis but not in chronic phase, together with the fact that CaMK Ⅱγ is essential for the self-renewal of LSC and P-catenin activation, indicates that this kinase is indeed involved in progression of CML from chronic phase to an aggressive accelerated and blast crisis phases.4. CaMKⅡγ-driven blast crisis is caused by lowering the expression of nuclear p27To elucidate underlying mechanisms responsible for CaMKⅡγ-driven blast crisis, we next examined whether CaMKⅡγ expression has an effect on the cyclin-dependent kinase (CDK) inhibitor p27Kipl (p27), a critical brake that maintains stem cell quiescence. The results showed that overexpression of CaMKⅡγ dramatically augmented the phosphorylated p27T187 protein levelsand reduced nuclear p27 accumulation. Similar results were observed in 293-T cells expressing EGFP-CaMKⅡγ. These data suggest that CaMKⅡγ supports the expansion of LSCs by virtue of its ability to phosphorylate p27 at T187, which in turns results in decrease of nuclear p27 through proteasome-dependent degradation. To obtain biochemical evidence, we performed co-immunoprecipitation assays. Total cellular proteins, which were isolated from K562 cells expressing flag-CaMKⅡγ, were incubated with flag-antibody(a-flag) or phospho-p27 antibody (α-p-p27), and the immune complexes were then purified, separated by SDS-PAGE, and analyzed with Western blotting with p-p27 antibody or flag antibody or CaMKⅡγ antibody. We found that p-p27 protein was present in a protein complex immunoprecipitated by the flag antibody. As expected, CaMKⅡγ protein was also present in the complex in a reciprocal immunoprecipitate using p-p27 antibody. These findings, together with previous studies that phosphorylation of p27 at T187 promotes the decrease of nuclear p27, which in turns leads to abrogate the quiescence of p27 for CML LSC. To validate these observations, we evaluated whether CaMKⅡγ expression has an impact on quiescent cells using FCM. Consistent with above results, CaMKⅡγ expression increased mitotic cells, whereas quiescent cells (GO/Gl cells) were reduced from 43.9%(control) to 36.1%(CaMK Ⅱγ overexpression), whereas proliferating cells (S+G2/M) increased from 52.7% to 62.2%(CaMK Ⅱγ overexpression), suggesting that CaMKⅡγ can wake up dormant leukemia cells via abrogating p27-mediated cell quiescence.To further confirm the role of CaMKⅡγ in leukemia cells, we analyzed subcellular localization of this kinase, and found that CaMK Ⅱγ protein level greatly differed in the cells at different phases of the cell cycle. We observed that CaMK Ⅱγ was present in dormant cells (Go phase) although its level was low. Interestingly, once cells enter proliferating status, CaMKⅡγ protein expression level dramatically increased with cell-cycle progression, peaked in early S/G2 phase cells, and then decreased. These results suggest that CaMKⅡγ might play important roles in promoting the transition of both G0-G1 and S-G2/M in cell cycles, which is responsible for the growth promotion of CML cells by CaMK Ⅱγ.In summary, we have identified an important role of CaMKⅡγ in CML blast crisis. On the basis of our findings, we propose that CML is triggered by Bcr-Abl expression in an HSC clone but that progression to blast crisis might be driven by aberrant activation of CaMK Ⅱγ via promoting the self-renewal and expansion of leukemia stem cells. Our data also indicate that CaMK Ⅱγ phosphorylates p27 at T187, which decreases the nuclear p27 levels and reactivates dormant leukemia stem cells. Overall, these findings support a novel concept that aberrant activation of CaMK Ⅱγ is required for the emergence of CML blast crisis. Thus, targeting the CaMK II y pathway might represent a novel diagnostic and therapeutic approach.Conclusions1. Aberrant activation of CaMKⅡγ promotes self-renewal and replication of leukemia stem cells,plays an important role in CML chronic phase to blastic phase.2. CaMKⅡγ reactivates dormant leukemia stem cells by phosphorylation of p27, and reduces the level of nuclear p27.3. CaMKⅡγ promotes the growth of CML cells by accelerating transition of both Go-G1and S-G2/M in cell cycles.3. CaMK Ⅱγ may become a new target for a new diagnosis and treatment of CML.