Creation of Philadelphia chromosome by CRISPR/Cas9-mediated double cleavages on BCR and ABL1 genes as a model for initial event in leukemogenesis

The Philadelphia (Ph) chromosome was the first translocation identified in leukemia. It is supposed to be generated by aberrant ligation between two DNA double-strand breaks (DSBs) at the BCR gene located on chromosome 9q34 and the ABL1 gene located on chromosome 22q11. Thus, mimicking the initiation process of translocation, we induced CRISPR/Cas9-mediated DSBs simultaneously at the breakpoints of the BCR and ABL1 genes in a granulocyte-macrophage colony-stimulating factor (GM-CSF) dependent human leukemia cell line. After transfection of two single guide RNAs (sgRNAs) targeting intron 13 of the BCR gene and intron 1 of the ABL1 gene, a factor-independent subline was obtained. In the subline, p210 BCR::ABL1 and its reciprocal ABL1::BCR fusions were generated as a result of balanced translocation corresponding to the Ph chromosome. Another set of sgRNAs targeting intron 1 of the BCR gene and intron 1 of the ABL1 gene induced a factor-independent subline expressing p190 BCR::ABL1. Both p210 and p190 BCR::ABL1 induced factor-independent growth by constitutively activating intracellular signaling pathways for transcriptional regulation of cell cycle progression and cell survival that are usually regulated by GM-CSF. These observations suggested that simultaneous DSBs at the BCR and ABL1 gene breakpoints are initiation events for oncogenesis in Ph+ leukemia. (200/200 words)

Inhibition of the Hepatic Uptake of 99mTc-Tetrofosmin Using an Organic Cation Transporter Blocker

The accumulation of high levels of 99mTc-tetrofosmin (99mTc-TF) in the hepatobiliary system can lead to imaging artifacts and interference with diagnosis. The present study investigated the transport mechanisms of 99mTc-TF and attempted to apply competitive inhibition using a specific inhibitor to reduce 99mTc-TF hepatic accumulation. In this in vitro study, 99mTc-TF was incubated in HEK293 cells expressing human organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, OATP2B1, organic anion transporter 2 (OAT2), organic cation transporter 1 (OCT1), OCT2, and Na+-taurocholate cotransporting polypeptide with or without each specific inhibitor to evaluate the contribution of each transporter to 99mTc-TF transportation. In vivo studies, dynamic planar imaging, and single photon emission computed tomography (SPECT) experiments with rats were performed to observe alterations to 99mTc-TF pharmacokinetics using cimetidine (CMT) as an OCT1 inhibitor. Time–activity curves in the liver and heart were acquired from dynamic data, and the 99mTc-TF uptake ratio was calculated from SPECT. From the in vitro study, 99mTc-TF was found to be transported by OCT1 and OCT2. When CMT-preloaded rats and control rats were compared, the hepatic accumulation of the 99mTc-TF was reduced, and the time to peak heart count shifted to an earlier stage. The hepatic accumulation of 99mTc-TF was markedly suppressed, and the heart-to-liver ratio increased 1.6-fold. The pharmacokinetics of 99mTc-TF were greatly changed by OCT1 inhibitor. Even in humans, the administration of OCT1 inhibitor before cardiac SPECT examination may reduce 99mTc-TF hepatic accumulation and contribute to the suppression of artifacts and the improvement of SPECT image quality

KIR3DL1 Allotype-Dependent Modulation of NK Cell Immunity against Chronic Myeloid Leukemia

Tyrosine kinase inhibitor (TKI)–treated chronic myeloid leukemia (CML) patients with increased NK cell number have a better prognosis, and thus, NK cells may suppress CML. However, the efficacy of TKIs varies for reasons yet to be fully elucidated. As NK cell activity is modulated by interactions between their killer cell Ig-like receptors (KIRs) and HLAs of target cells, the combination of their polymorphisms may have functional significance. We previously showed that allelic polymorphisms of KIR3DL1 and HLAs were associated with the prognosis of TKI-treated CML patients. In this study, we focus on differential NK cell activity modulation through KIR3DL1 allotypes. KIR3DL1 expression levels varied according to their alleles. The combination of KIR3DL1 expression level and HLA-Bw4 motifs defined NK cell activity in response to the CML-derived K562 cell line, and Ab-mediated KIR3DL1 blocking reversed this activity. The TKI dasatinib enhanced NK cell activation and cytotoxicity in a KIR3DL1 allotype-dependent manner but did not significantly decrease effector regulatory T cells, suggesting that it directly activated NK cells. Dasatinib also enhanced NK cell cytotoxicity against K562 bearing the BCR-ABL1 T315I TKI resistance–conferring mutation, depending on KIR3DL1/HLA-Bw4 allotypes. Transduction of KIR3DL1*01502 into the NK cell line NK-92 resulted in KIR3DL1 expression and suppression of NK-92 activity by HLA-B ligation, which was reversed by anti-KIR3DL1 Ab. Finally, KIR3DL1 expression levels also defined activation patterns in CML patient–derived NK cells. Our findings raise the possibility of a novel strategy to enhance antitumor NK cell immunity against CML in a KIR3DL1 allotype-dependent manner

Loss of KMT2C reprograms the epigenomic landscape in hPSCs resulting in NODAL overexpression and a failure of hemogenic endothelium specification

Germline or somatic variation in the family of KMT2 lysine methyltransferases have been associated with a variety of congenital disorders and cancers. Notably

Change in Physical and Mental Quality-of-Life between the Short- and Mid-Term Periods after Cervical Laminoplasty for Cervical Spondylotic Myelopathy: A Retrospective Cohort Study with Minimum 5 Years Follow-up

The mid-term surgical outcomes of cervical spondylotic myelopathy (CSM), evaluated using the cervical Japanese Orthopedic Association (cJOA) score, are reported to be satisfactory. However, there remains room for improvement in quality-of-life (QOL), especially after short-term follow-up. We aimed to demonstrate changes in mental and physical QOL between short- and mid-term follow-ups and determine the predictive factors for deterioration of QOL. In this retrospective cohort study, 80 consecutive patients underwent laminoplasty for CSM. The outcome measures were Short Form-36 Physical Component Summary (PCS), Mental Component Summary (MCS), and cJOA scores. PCS and MCS scores were compared at the 2- and 5-year postoperative time points. Additionally, a multivariate logistic regression model was used to identify the predictive factors for deterioration. Significant factors in the logistic regression analysis were analyzed using receiver-operating characteristic curves. The results showed that MCS scores did not deteriorate after 2 years postoperatively (p = 0.912). Meanwhile, PCS significantly declined between 2 and 5 years postoperatively (p = 0.008). cJOA scores at 2 years postoperatively were significantly associated with PCS deterioration at 2-year follow-up. In conclusion, only physical QOL might show deterioration after short-term follow-up. Such deterioration is likely in patients with a cJOA score <13.0 at 2 years postoperatively

Short-Term Risk Factors for Distal Junctional Kyphosis after Spinal Reconstruction Surgery in Patients with Osteoporotic Vertebrae

Study Design Level 3 retrospective cohort case-control study. Purpose This study aimed to investigate the risk factors for distal junctional kyphosis (DJK) caused by osteoporotic vertebral fractures following spinal reconstruction surgery, with a focus on the sagittal stable vertebra. Overview of Literature Despite the rarity of reports on DJK in this setting, DJK was reported to reduce when the lower instrumented vertebra (LIV) was extended to the sagittal stable vertebra in the posterior corrective fixation for Scheuermann’s disease. Methods This study included 46 patients who underwent spinal reconstruction surgery for thoracolumbar osteoporotic vertebral fractures and kyphosis and were followed up for 1 year postoperatively. DJK was defined as an advanced kyphosis angle >10° between the LIV and one lower vertebra. The patients were divided into groups with and without DJK. The risk factors of the two groups, such as patient background, surgery-related factors, radiographic parameters, and clinical outcomes, were analyzed. Results The DJK and non-DJK groups included 14 and 32 patients, respectively, without significant differences in patient background. Those with instability in the distal adjacent LIV disc had a significantly higher risk of DJK occurrence (28.6% vs. 3.2%, p=0.027). DJK occurrence significantly increased in those with the sagittal stable vertebra not included in the fixation range (57.1% vs. 18.8%, p=0.020). Other preoperative radiographic parameters were not significantly different. Instability in the distal adjacent LIV disc (adjusted odds ratio, 14.50; p=0.029) and the exclusion of the sagittal stable vertebra from the fixation range (adjusted odds ratio, 5.29; p=0.020) were significant risk factors for DJK occurrence. Conclusions Regarding spinal reconstruction surgery in patients with osteoporotic vertebral fractures, instability in the distal adjacent LIV disc and the exclusion of the sagittal stable vertebra from the fixation range were risk factors for DJK occurrence in the short term