We conducted a comprehensive analysis of 28 recurrently mutated genes in acute myeloid leukemia (AML) in 271 patients with de novo AML. Co-mutations were frequently detected in the intermediate ...cytogenetic risk group, at an average of 2.76 co-mutations per patient. When assessing the prognostic impact of these co-mutations in the intermediate cytogenetic risk group, overall survival (OS) was found to be significantly shorter (P=0.0006) and cumulative incidence of relapse (CIR) significantly higher (P=0.0052) in patients with complex molecular genetic abnormalities (CMGAs) involving three or more mutations. This trend was marked even among patients aged ⩽65 years who were also FLT3-ITD (FMS-like tyrosine kinase 3 internal tandem duplications)-negative (OS: P=0.0010; CIR: P=0.1800). Moreover, the multivariate analysis revealed that CMGA positivity was an independent prognostic factor associated with OS (P=0.0007). In stratification based on FLT3-ITD and CEBPA status and 'simplified analysis of co-mutations' using seven genes that featured frequently in CMGAs, CMGA positivity retained its prognostic value in transplantation-aged patients of the intermediate cytogenetic risk group (OS: P=0.0002. CIR: P<0.0001). In conclusion, CMGAs in AML were found to be strong independent adverse prognostic factors and simplified co-mutation analysis to have clinical usefulness and applicability.
Gene mutations were found in acute myeloid leukemia (AML) and their importance has been noted. To clarify the importance and stability of mutations, we examined gene mutations in paired samples at ...diagnosis and relapse of 34 adult AML patients. Five acquired gene mutations were detected at relapse. Of the 45 gene mutations at diagnosis, 11 of them were lost at relapse. The acquired mutations at relapse were all class I mutations as Fms-like tyrosine kinase 3 (FLT3) and rat sarcoma viral oncogene homolog (RAS) mutations. The disappeared mutations at relapse were 3 of 11 internal tandem duplications of FLT3 (FLT3-ITD) (27.3%), 3 of 3 FLT3 tyrosine kinase domain (FLT3-TKD) (100%), 3 of 13 Nucleophosmin 1 (23.1%) and 2 of 5 CCAAT/enhancer-binding protein-α (40%) mutations. However, epigenetics-modifying gene (DNMT3a, TET2 and IDH1/2) mutations had no change between diagnosis and relapse samples, and may become minimal residual disease marker. The frequency of FLT3-ITD at relapse in patients with DNMT3a mutation at diagnosis is significantly higher than those in patients without them (P=0.001). Moreover, the high frequency of FLT3-ITD at relapse is also seen in AML cases that initially present with any epigenetics-modifying gene mutations (P<0.001). Our results indicate that epigenetics-modifying gene mutations may cause genetic instability and induce FLT3-ITD, leading to resistance to therapy and relapse.
Recently, c-kit mutations have been reported as a novel adverse prognostic factor of acute myeloid leukemia with t(8;21)(q22;q22) translocation (t(8;21) AML). However, much remains unclear about its ...clinical significance. In this study, we developed a highly sensitive mutation detection method known as mutation-biased PCR (MB-PCR) and investigated the relationship between c-kit mutations and prognosis. When c-kit mutations were analyzed for 26 cases of t(8;21) AML using the direct sequence (DS) and MB-PCR, the latter had a much higher detection rate of c-kit mutations at initial presentation (DS 5/26(19.2%) vs MB-PCR 12/26(46.2%)). Interestingly for the three cases, in which c-kit mutations were observed only at relapse with the DS, c-kit mutations were detected at initial presentation using the MB-PCR. This result suggests that a minor leukemia clone with c-kit mutations have resistance to treatment and are involved in relapse. In univariate analyses, the presence of a c-kit mutation using DS was not an adverse prognostic factor (P = 0.355), but was a factor when using MB-PCR (P = 0.014). The presence of c-kit mutations with MB-PCR was also an independent adverse prognostic factor by multivariate analyses (P = 0.006). We conclude that sensitivity of c-kit mutation detection method is important to predict prognosis for t(8;21) AML.
Mixed-lineage leukemia (MLL)/AF4-positive ALL is associated with a poor prognosis even after allogeneic hematopoietic SCT (allo-HSCT). We reported previously that MLL/AF4-positive ALL shows ...resistance to TNF-α, which is the main factor in the GVL effect, by upregulation of S100A6 expression followed by interference with the p53-caspase 8-caspase 3 pathway in vitro. We examined whether inhibition of S100A6 can induce an effective GVL effect on MLL/AF4-positive ALL in a mouse model. MLL/AF4-positive ALL cell lines (SEM) transduced with lentiviral vectors expressing both S100A6 siRNA and luciferase (SEM-Luc-S100A6 siRNA) were produced. SEM-Luc-S100A6 siRNA cells and SEM-Luc-control siRNA cells were injected into groups of five SCID mice (1 × 10(7)/body). After confirmation of engraftment of SEM cells by in vivo imaging, the mice in each group were injected with 4.8 × 10(7) human PBMCs. SEM-Luc-S100A6 siRNA-injected mice showed significantly longer survival periods than SEM-Luc-control siRNA-injected mice (P=0.002). SEM-Luc-S100A6 siRNA-injected mice showed significantly slower tumor growth than those injected with SEM-Luc-control siRNA (P<0.0001). These results suggested that inhibition of S100A6 may be a promising therapeutic target for MLL/AF4-positive ALL in combination with allo-HSCT.
Abstract Activity-dependent re-organizations of central synapses are thought to play important roles in learning and memory. Although the precise mechanisms of how neuronal activities modify synaptic ...connections remain to be elucidated, the activity-induced neuronal proteins such as Homer1a may contribute to the onset of synaptic remodeling. To further understand the physiological roles of Homer1a, we first examined prolonged effects of neuronal stimulation capable of inducing Homer1a on the distribution of a postsynaptic protein Homer1c by live imaging and immunostaining. We found that glutamate stimulation induced a biphasic change in the distribution of Homer1c, in which the postsynaptic clusters of Homer1c defused initially after 30 min to 1 h, and then reassembled more than the original level after 4–8 h. When other synaptic proteins (postsynaptic density-95 (PSD95), Filamentous actin (F-actin), glutamate receptors, synaptotagmin, synaptophysin and synapsin) were analyzed by immunocytochemical methods, the distribution of these proteins also showed a similar biphasic pattern, suggesting that glutamate stimulation induces a global alteration in synaptic structures. To further dissect the functions of Homer1a in the activity-induced synaptic remodeling, the short hairpin RNA (shRNA) vectors that specifically block the expression of endogenous Homer1a were constructed. When the shRNA of Homer1a was introduced to the cells, the activity-induced changes were almost completely suppressed. The expression of surface glutamate receptor 2 was also inhibited, suggesting that Homer1a may modulate the efficacy of synaptic transmission. Furthermore, we found that Homer1a contributes to the presynaptic remodeling in a retrograde manner. Our data indicate that Homer1a regulates the activity-induced biphasic changes of post- and pre-synaptic sites.
In the previous study, we demonstrated that the antisense oligodeoxynucleotides against calcineurin Aα and Aβ, catalytic subunits of Ca
2+/calmodulin-dependent protein phosphatase, produce a ...facilitatory effect on long-term potentiation induction in the hippocampal CA1 region in rats anesthetized with urethane. Here, we have studied how animals, in which the hippocampal long-term potentiation induction is enhanced by antisense oligodeoxynucleotides against calcineurin, perform in learning tasks that depend on hippocampal function. The rats received antisense oligodeoxynucleotides by bilateral ventricular administration via miniosmotic pumps. We tested four groups of rats, three infused with either antisense oligodeoxynucleotides, scramble oligodeoxynucleotides, or saline, and untreated rats, for two types of hippocampus-dependent learning, water maze and contextual fear conditioning. After the behavioral tests, we conducted a long-term potentiation induction test to determine whether long-term potentiation induction was enhanced. In contextual fear conditioning, rats in which long-term potentiation induction was enhanced by antisense oligodeoxynucleotides displayed significantly more conditioned freezing response than control rats. Rats with enhanced long-term potentiation induction showed no differences in shock sensitivity, general activity, or light–dark choice from control rats. In contrast with contextual fear conditioning, rats with enhanced long-term potentiation induction showed no difference in spatial learning performance on the water maze compared with control rats.
These results demonstrate that an enhancement in long-term potentiation induction produced by the inhibition of calcineurin leads to an increase in memory strength in specific forms of hippocampus-dependent learning.
Many studies have reported the roles played by regulated proteolysis in synaptic plasticity and memory, but the role of autophagy in neurons remains unclear. In mammalian cells, autophagy functions ...in the clearance of long-lived proteins and organelles and in adaptation to starvation. In neurons, although autophagy-related proteins (ATGs) are highly expressed, autophagic activity markers, autophagosome (AP) number, and light chain protein 3-II (LC3-II) are low compared with other cell types. In contrast, conditional knock-out of ATG5 or ATG7 in mouse brain causes neurodegeneration and behavioral deficits. Therefore, this study aimed to test whether autophagy is especially regulated in neurons to adapt to brain functions. In cultured rat hippocampal neurons, we found that KCl depolarization transiently increased LC3-II and AP number, which was partially inhibited with APV, an NMDA receptor (NMDAR) inhibitor. Brief low-dose NMDA, a model of chemical long-term depression (chem-LTD), increased LC3-II with a time course coincident with Akt and mammalian target of rapamycin (mTOR) dephosphorylation and degradation of GluR1, an AMPA receptor (AMPAR) subunit. Downstream of NMDAR, the protein phosphatase 1 inhibitor okadaic acid, PTEN inhibitor bpV(HOpic), autophagy inhibitor wortmannin, and short hairpin RNA-mediated knockdown of ATG7 blocked chem-LTD-induced autophagy and partially recovered GluR1 levels. After chem-LTD, GFP-LC3 puncta increased in spines and in dendrites when AP-lysosome fusion was blocked. These results indicate that neuronal stimulation induces NMDAR-dependent autophagy through PI3K-Akt-mTOR pathway inhibition, which may function in AMPAR degradation, thus suggesting autophagy as a contributor to NMDAR-dependent synaptic plasticity and brain functions.
Acquired memory initially depends on the hippocampus (HPC) for the process of cortical permanent memory formation. The mechanisms through which memory becomes progressively independent from the HPC ...remain unknown. In the HPC, adult neurogenesis has been described in many mammalian species, even at old ages. Using two mouse models in which hippocampal neurogenesis is physically or genetically suppressed, we show that decreased neurogenesis is accompanied by a prolonged HPC-dependent period of associative fear memory. Inversely, enhanced neurogenesis by voluntary exercise sped up the decay rate of HPC dependency of memory, without loss of memory. Consistently, decreased neurogenesis facilitated the long-lasting maintenance of rat hippocampal long-term potentiation in vivo. These independent lines of evidence strongly suggest that the level of hippocampal neurogenesis play a role in determination of the HPC-dependent period of memory in adult rodents. These observations provide a framework for understanding the mechanisms of the hippocampal-cortical complementary learning systems.
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