The loss of killer cell Ig-like receptor ligands (KIR-Ls) due to the copy number-neutral loss of heterozygosity of chromosome 6p (6pLOH) in leukocytes of patients with acquired aplastic anemia (AA) ...may alter the susceptibility of the affected leukocytes to NK cell killing in vivo. We studied 408 AA patients, including 261 who were heterozygous for KIR-Ls, namely C1/C2 or Bw6/Bw4, for the presence of KIR-L-missing KIR-L(-) leukocytes. KIR-L(-) leukocytes were found in 14 (5.4%, C1
= 4, C2
= 3, and Bw4
= 7) of the 261 patients, in whom corresponding KIR(+) licensed NK cells were detected. The incidence of 6pLOH in the 261 patients (18.0%) was comparable to that in 147 patients (13.6%) who were homozygous for KIR-L genes. The percentages of HLA-lacking granulocytes (0.8-50.3%, median 15.2%) in the total granulocytes of the patients with KIR-L(-) cells were significantly lower than those (1.2-99.4%, median 55.4%) in patients without KIR-L(-) cells.
and
were only possessed by three of the 14 patients, two of whom had C2/C2 leukocytes after losing C1 alleles. The expression of the KIR3DS1 ligand HLA-F was selectively lost on KIR-L(-) primitive hematopoietic stem cells derived from 6pLOH(+) induced pluripotent stem cells in one of the KIR3DS1(+) patients. These findings suggest that human NK cells are able to suppress the expansion of KIR-L(-) leukocytes but are unable to eliminate them partly due to the lack of activating KIRs on NK cells and the low HLA-F expression level on hematopoietic stem cells in AA patients.
Abstract 2903
Poster Board II-879
FMS-like tyrosine kinase 3 (FLT3), a class III receptor tyrosine kinase, is one of the most frequently mutated genes in hematological malignancies. The most common ...mutations of FLT3 are internal tandem duplications (ITDs) within the juxtamembrane domain: these mutations occur in 20% to 30% of patients with AML and are closely associated with a poor prognosis. In a small number of patients with myeloproliferative neoplasms (MPNs), FLT3 has been reported to fuse to ETV6 (TEL) and contribute to leukemogenesis, but the leukemogenic mechanism of ETV6/FLT3 remains unclear. We encountered a case of ETV6/FLT3 fusion in a patient with MPN complicated with T-cell lymphoblastic lymphoma. In this case, both myeloid and lymphoma cells shared the same chromosomal translocation, t(12;13)(p13;q12), and allogeneic hematopoietic stem cell transplantation led to complete remission for 3 years. Full-length ETV6/FLT3 fusion cDNA was cloned from the patient's bone marrow cells. Sequence analysis of the PCR product revealed that, in contrast to the finding of previously reported two cases of ETV6/FLT3-positive MPN, ETV6 exon 6 was fused to FLT3 exon 14 and that the fused portion of ETV6 contained 2 potential Grb2-binding sites (Vu et al., Leukemia 2006; Walz et al., Blood 2007a). The ETV6/FLT3 conferred IL-3-independent growth to Ba/F3 and 32Dcl3 cells. Using a dominant negative approach, we showed that both STAT5 and Ras played important roles in ETV6/FLT3-mediated transformation of the hematopoietic cell lines. To investigate the role of the ETV6/FLT3 fusion protein in vivo, we used a murine bone marrow transplant model. Retroviral transduction of the ETV6/FLT3 into primary murine bone marrow cells resulted in a CML-like myeloproliferative disease (MPD) with complete penetrance in the transplanted mice. The disease progressed to cause death at a median of 18 days after transplantation (n = 16). The transplanted mice developed severe leukocytosis (159 × 103 /μl to 417 × 103 /μl), splenomegaly, and extensive infiltration of myeloid cells in the bone marrow, spleen, liver, and peripheral blood. ETV6/FLT3-induced MPD was oligoclonal and only 2 of the 9 secondary transplant recipients developed similar MPD when 5 × 106 spleen cells from 3 independent diseased mice were used as donors. We assayed the mutant forms of the ETV6/FLT3 to test their ability to transform hematopoietic cells. Induction of MPD required the oligomerization domain of ETV6 and the tyrosine kinase activity of FLT3. Mice that received the double tyrosine-to-phenylalanine mutant of ETV6/FLT3 at sites 589 and 591 (Y589/591F) in the juxtamembrane domain of FLT3, which are critical for FLT3-ITD-induced MPD, also developed a similar MPD phenotype. Unlike FLT3-ITDs, Y589/591F mutation did not abrogate STAT5 activation in Ba/F3 and 32Dcl3 cells transformed by ETV6/FLT3. A recent study has shown that direct binding of Grb2 to tyrosine 768, 955, and 969 of FLT3 is important for FLT3-ITD-mediated proliferation and survival of hematopoietic cells. Tyrosine 314 in exon 5 of ETV6 has also been reported as the principal Grb2-binding site that contributes to leukemogenesis via oncogenic ETV6 fusion proteins such as ETV6/ABL. Thus, we next investigated the role of Grb2 binding in ETV6/FLT3-mediated leukemogenesis. Using coimmunoprecipitation assays, we demonstrated that Grb2 also binds to the tyrosine 314 and 354 of ETV6 of the ETV6/FLT3, in addition to the tyrosine 768, 955, and 969 of FLT3. Both ETV6/FLT3-Y314/354F and ETV6/FLT3-Y768/955/969F retained their interaction with Grb2 and induced rapidly fatal MPD when they were transduced into primary murine bone marrow cells. On the other hand, the ETV6/FLT3 mutant at all the binding sites of Grb2 (Y314/354/768/955/969F) significantly attenuated MPD development in mice. Simultaneous mutation of these 5 tyrosine residues completely abolished the binding of Grb2 and resulted in a marked decrease in the binding and phosphorylation of Gab2 and impaired activation of STAT5 and Akt in Ba/F3 cells. These results indicate that tyrosine 589 and 591 of FLT3 are dispensable for the ETV6/FLT3-induced MPD phenotype, and suggest that both ETV6 and FLT3 portions contribute to the ETV6/FLT3-mediated leukemogenesis by binding directly to Grb2. Our observations provide deep insights into the oncogenic signaling induced by active FLT3 mutants as well as provide a potential target for therapies.
No relevant conflicts of interest to declare.
BackgroundUsing pluripotent stem cell-derived cardiomyocytes (PSC-CMs) for clinical application of cardiac transplantation, large quantities of purified PSC-CMs are required. Moreover, genetic ...modifications and contamination of non-CMs should be avoided. Although cell sorting with antibodies against specific cell surface proteins is one of the promising strategies, it is time-consuming and harbors potential risk of immunogenic reaction and local inflammation. We have shown that synthetic mRNAs encoding a fluorescent protein tagged with complementary sequences against specifically expressed microRNAs (miRNA-switches) can be efficiently detected and used for purification. Combining miRNA-switch and magnetic-activated cell sorting (MACS) technologies, we evaluated the efficiency of large scale purification of human induced PSC-CMs (iPSC-CMs).MethodsWe used CD4 as a selection marker for MACS and miR-208a as a specific miRNA of CMs. We synthesized a CD4 mRNA and transfected it into differentiated cells from iPSCs to confirm CD4 expressing on the surface of the transfected cells. We also synthesized a miRNA switch encoding CD4 tagged with the complementary sequence against miR-208a (CD4-208a switch) and transfected it into iPSC-derived differentiated cells. This CD4-208a switch allowed the distinction of CMs from non-CMs by the expression of CD4. Non-CMs could be removed by MACS while a co-transfected puromycin resistance carrying mRNA allowed the removal of untransfected cells with puromycin. Purified cells were then transplanted into NOG mouse hearts with myocardial infarction by direct injections into the myocardium to show their successful engraftment.ResultsAfter transfection of the CD4 mRNA into differentiated cells from iPSCs, 78±5% expressed CD4 on the cell surface. We also confirmed that the CD4-208a switch separated CMs and non-CMs as expected. Combination of MACS and puromycin selection purified iPSC-CMs from 69±5% to 97±2% as assessed by troponin T staining. Purified cells were shown to be engrafted in mouse hearts.ConclusionsWe demonstrated that the synthetic CD4-208a switch efficiently purifies iPSC-CMs in large scale, suggesting that this technology can be clinically applied for stem cell-based cell therapy.
The Hippo pathway is now recognized as a major signaling network for development and organ size control in mammals as well as in Drosophila melanogaster. We previously identified human kpm/Lats2, the ...key serine/threonine kinase in the Hippo pathway, and reported that its overexpression induced cell cycle arrest and apoptosis. Down-regulation of kpm/Lats2 expression has been described in various malignancies. It is of particular importance in hematology that low expression of kpm/Lats2 has been reported to be associated with poor prognosis in acute lymphoblastic leukemia. In the present study, we first measured the expression level of kpm/Lats2 mRNA in various hematological malignancies and found that it was markedly decreased in adult T cell leukemia (ATL) and NK cell leukemia/lymphoma, both of which are known to be highly resistant to conventional chemotherapy. In order to investigate the relationship between down-regulation of kpm/Lats2 expression and chemo-resistance, we made kpm/Lats2-knockdown sublines from KG-1a, an AML-derived cell line, and ED-40515+, an ATL-derived cell line, by using shRNA-expression retrovirus vector targeting kpm/Lats2. Silencing of kpm/Lats2 expression in both leukemic cell lines did not change the rate of cell growth but rendered them resistant to DNA damage-inducing agents such as DOX and ETP. Expression of p21 and PUMA was strongly induced by these agents in control cells, despite defective p53, but was only slightly induced in kpm/Lats2-knockdown cells. DNA damage-induced nuclear accumulation of p73, a member of p53 family, was clearly observed in control cells but hardly detected in kpm/Lats2-knockdown cells. ChIP assay showed that p73 was recruited to the puma gene promoter in control cells but not in kpm/Lats2-knockdown cells after DNA damage stress. The analyses with transient co-expression of Kpm/Lats2, YAP2, and p73 showed that Kpm/Lats2 contributed to the stability of YAP2 and p73, which was dependent on the kinase function of Kpm/Lats2 and YAP2 phosphorylation at serine 127. These results strongly suggest that Kpm/Lats2 is involved in the fate of p73 and the induction of p73-target genes that underlie chemo-sensitivity of leukemic cells.
BackgroundRecently, mutations in CALM genes (CALM1-3) encoding calmodulin (CaM) are reported to be associated with severe early-onset arrhythmias known as the calmodulinopathy. Calmodulin modulates ...various proteins including several ion channels in cardiomyocytes. KCNQ1 channel is known to have the interaction with CaM, which regulates the channel gating, assembly and surface localization. However, the interaction between mutant CaM causing calmodulinopathy and KCNQ1 channel in cardiomyocytes remains unknown.PurposeThe present study aimed to evaluate the KCNQ1 channel function in human iPS cell-derived cardiomyocytes (hiPSC-CMs) generated from calmodulinopathy patient.MethodsThe hiPSC clones were generated from a 12-year-old boy with long-QT syndrome (LQTS) carrying a missense CALM2 mutation (c.293A>G, p.N98S), whose ECG showed marked QT prolongation in epinephrine stress test. After cardiac differentiation, the electrophysiological characteristics of N98S-hiPSC-CMs were analyzed and compared with those of control derived from healthy individual.ResultsIn KCNQ1 channel current (IKS) analysis, 500 nM isoproterenol (ISO) significantly increased peak current of control at 0, 20, 40 mV test potentials. In contrast, the response of peak current to ISO was impaired in N98S-hiPSC-CMs (Fig.1). The action potential durations at 90% repolarization (APD90) of N98S-hiPSC-CMs were significantly prolonged compared to those of control regardless of the presence or absence of ISO at 1.33 Hz pacing (Baseline and ISOControl, 249.4 ± 29.2 ms and 169.8 ± 11.7 ms vs N98S, 378.7 ± 19.8 ms and 321.2 ± 22.0 ms; Fig. 2). There was significant difference in the percentage of APD90 shortening between control and N98S.ConclusionThis study elucidated the KCNQ1 channel dysfunction using hiPSC model that may explain the clinical phenotype of the LQTS patient carrying the CALM2-N98S mutation.
BackgroundCalmodulin is a ubiquitous Ca-sensor molecule and an identical calmodulin protein is encoded by the 3 distinct genes, CALM1-3. Recently, mutations in CALM are reported to be associated with ...severe early-onset long-QT syndrome (LQT). We have established CALM2-related LQT patient-derived iPSC model which exhibited prolonged action potential duration (APD) due to impaired inactivation of L-type Ca currents (ICaL). It has been suggested that heterozygous CALM mutations could exert a dominant-negative effect, therefore, we aimed to assess the mutant allele-specific disruption by genome editing for the treatment of CALM-related LQT.MethodsWe generated hiPSC from a 12-year-old boy with LQT carrying a CALM2-N98S mutation. In order to disrupt mutated calmodulin, we designed guide RNA for targeting the mutant allele sequence. The Cas9/guide RNA expression vectors were transfected in LQT-hiPSCs and genome-edited clones were confirmed by sequencing. LQT- and LQT-KO-hiPSCs were differentiated into cardiomyocytes (CMs), and action potentials (APs) and ICaL were analyzed by patch-clamp technique.ResultsWe successfully obtained mutant allele-specific knockout clones using the CRISPR-Cas9 system. As a result of electrophysiological analyses, the decreased beating rate and prolonged APDs in CALM2-related hiPSC-CMs were rescued in LQT-KO-hiPSC-CMs (Fig. A). The parameters in LQT-KO clones were similar to those in control generated by a healthy volunteer. In addition, the impaired inactivation of L-type Ca channels in CALM2-related LQT was ameliorated in LQT-KO clones (Fig. B).ConclusionMutant allele-specific disruption using CRISPR-Cas9 system rescued the electrophysiological abnormalities in CALM2-related LQT iPSC model, which provides us new insights into a promising therapeutic approach using the latest genome-editing technology for inherited cardiac diseases especially caused by dominant-negative mechanism.
We report a 67-year-old man who developed pure red cell aplasia (PRCA) during therapy for epilepsy with sodium valproate since April 2004. He was admitted to our hospital because of severe anemia (Hb ...5.0g/dl, reticulocyte 0.1%) in August 2004. A bone marrow examination showed marked erythroid hypoplasia and a diagnosis of drug-induced PRCA was made. Because the discontinuation of valproate for one month failed to increase the number of reticulocytes and frequent blood transfusions were necessary, cyclosporine therapy was initiated. Within a week, substantial recovery of the numbers of reticulocytes was obtained, the cyclosporine had, however, to be changed to prednisolone due to the refusal of the patient to continue with it, resulting in the exacerbation of his anemia. After three weeks, cyclosporine therapy was resumed, which achieved rapid and remarkable recovery of red blood cells (Hb 8.9g/dl, reticulocyte 4.9%) within one month. Sixteen cases of valproate-induced PRCA have been reported in the literature and all cases except one recovered only by discontinuing or reducing the administration of valproate. However, our case required cyclosporine therapy in addition to the discontinuation of valproate. These results suggest that not only the direct toxic effect on erythropoiesis but also T lymphocyte-mediated immunological mechanism was involved in the pathogenesis of valproate-induced PRCA.
PDF
