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Objective: To engineer a BCMA mutant resistant to γ-secretase cleavage in order to stabilize wild-type BCMA expression after γ-secretase cleavage and to generate target cells for measuring BCMA CAR-T cell cytotoxicity. Methods: Our study aimed to engineer a mutant BCMA protein (BCMA-CD8α TM) that could resist γ-secretase cleavage by replacing the transmembrane domain of the wild-type BCMA protein with the human CD8α sequence. Four different types of cells in which this mutant gene was expressed excessively were engineered, including U266 (U266BCMA Mut), K562 (K562BCMA Mut), SKOV3 (SKOV3BCMA Mut), and CHO (CHOBCMA Mut) cells. BCMA CAR Jurkat cells, loaded with the NFAT-EGFP reporter gene (BCMA-CAR-Jurkat-Reporter), were engineered and cocultured with U266BCMA Mut cells. The expression level of EGFP was detected by FCM in order to indicate the activation level of NFAT. The cytotoxicity of BCMA CAR-T cells against Luciferase-labeled K562BCMA Mut cells was detected by the luciferase assay. Additionally, real-time cell analysis (RTCA) technique was employed to detect the cytotoxicity of BCMA CAR-T cells against SKOV3BCMA Mut and CHOBCMA Mut cells. Results: Application of γ-secretase inhibitor LY411575 to inhibit γ-secretase activity significantly enhanced the expression level of BCMA on the surface of wild-type U266 cells, and the average fluorescence intensity was increased by more than 10 times. However, the expression level of BCMA gradually decreased after removal of inhibitors (P<0.01). BCMA-CD8α TM mutant could resist the cleavage of γ-secretase and expressed stably on the surface of U266 cells (P>0.05). U266 cells and U266 cells overexpressing BCMA-CD8α TM were co-incubated with BCMA-CAR-Jurkat-Reporter cells, both of which could activate the Reporter system and enhance the expression of EGFP, but the effect was more significant in U266 cells overexpressing BCMA-CD8α TM (P<0.01). BCMA-CD8α TM mutants were successfully overexpressed in 3 BCMA-negative target cells, namely K562, SKOV3 and CHO cells, and the expression level of the mutant was only slightly increased under LY411575 treatment. Luciferase assay results showed that under different target-effect ratios BCMA CAR-T cells could all be specific and efficient in killing K562 cells overexpressing BCMA-CD8α TM. RTCA results showed that under different target ratios BCMA CAR-T cells could all effectively recognize and kill SKOV3 and CHO cells overexpressing BCMA-CD8α TM, but Mock-T cells with the same target ratio had no such effect. Conclusion: The BCMA-CD8α TM mutant engineered in this study can resist γ-secretase cleavage and exhibits stable surface expression on various target cells, thus offering various methods for evaluating the efficacy and specificity of BCMA CAR-T cell cytotoxicity in vitro. [ABSTRACT FROM AUTHOR]

The article discusses findings of a study on effects of realgar (As4S4) on degradation of promyelocytic leukemia gene (PML)-RARA fusion protein. It states role of PML-RARA as a therapy target of acute promyelocytic leukemia (APL), and mentions that arsenic trioxide cures APL by binding to the PML moiety and triggering the degradation of PML-RARA oncoprotein. It notes that most of the identified arsenic-resistance mutations were found to be located in the PML B2 domain of PML-RARA.

Background: The genome-wide single-nucleotide polymorphisms (SNPs) profiles can be used as diagnostic markers for human cancers. The associations between mitogen-activated protein kinase kinase kinase 1 (MAP3K1) SNPs rs889312 A>C, rs16886165 T>G and breast cancer risk have been widely evaluated, but the results were inconsistent. To derive a conclusive assessment of the associations, we performed a meta-analysis by combining data from all eligible case-control studies up to date. Methods: By searching PubMed, ISI web of knowledge, Embase and Cochrane databases, we identified all eligible studies published before September 2013. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of associations in fixed-effect or random-effect model. False-positive report probability (FPRP) was calculated to confirm the significance of the results. Results: A total of 59670 cases in 20 case-control studies were included in this meta-analysis. Significant associations with breast cancer risk were observed for SNPs rs889312 and rs16886165 polymorphisms with a per-allele OR of 1.11 (95% CI: 1.09–1.13) and 1.14 (95% CI: 1.09–1.20) respectively. For rs889312, in subgroup analysis by ethnicity, significant associations were identified in Europeans and Asians, but not in Africans. When stratified by estrogen receptor (ER) expression status, rs889312 was associated with both ER-positive and ER-negative breast cancers. Results from the FPRP analyses were consistent with and supportive to the above results. Conclusions: The present meta-analysis suggests that rs889312-C allele and rs16886165-G allele might be risk factors for breast cancer, especially in Europeans and Asians. [ABSTRACT FROM AUTHOR]

Summary: Papillary thyroid carcinoma usually presents as a multifocal disease; and tumors often recur in the contralateral thyroid lobe, raising questions concerning their clonal origins. The clonality of tumors appearing simultaneously in both lobes or recurring in the contralateral lobe remains unknown. Accordingly, we examined 25 pairs of bilateral papillary thyroid carcinomas (synchronous or metachronous) and 15 matched metastatic lymph nodes. BRAF gene mutation analysis combined with X-chromosome inactivation was used to evaluate these tumors'' clonal origins. Genomic DNA was prepared from paraffin-embedded tissues after microdissection. In total, 62 tumors yielded DNA of adequate quality. Eighteen (18/21, 85.7%) of 21 informative cases showed concordant BRAF status in tumors from both thyroid lobes, being either BRAF mutation positive (12 patients) or BRAF mutation negative (6 patients). Metastatic lymph nodes in 12 patients (12/15, 80%) had a complete concordance of BRAF state with their primaries. Of the 18 studied female patients, 11 were suitable for X-chromosome inactivation assay. Nine cases (9/11, 81.1%) showed the same pattern of inactivation in bilateral tumors. A close correlation was found between BRAF mutation and X-chromosome inactivation analysis. In conclusion, our data provide evidence that bilateral, recurrent, and metastatic papillary thyroid carcinomas often arise from a single clone and that intrathyroidal metastasis may play an important role in the development of bilateral tumors, as well as in the recurrence of this malignancy. [Copyright &y& Elsevier]

Risk-stratification of acute myeloid leukemia (AML) based on (cyto)genetic aberrations, including hotspot mutations, deletions and point mutations have evolved substantially in recent years. With the development of next-generation sequence technology, more and more novel mutations in the AML were identified. Thus, to unravel roles and mechanism of novel mutations would improve prognostic and predictive abilities. In this study, two novel germline JAK2 His608Tyr (H608Y) and His608Asn (H608N) mutations were identified and the molecular basis of these mutations in the leukemiagenesis of AML was elucidated. Our results indicated that JAK2 H608Y and H608N mutations disrupted the hydrogen bond between Q656 and H608 which reduced the JH2 domain's activity and abolished interactions between JH1 and JH2 domains, forced JAK2 into the active conformation, facilitated the entrance of substrates and thus caused JAK2 hyperactivation. Further studies suggested that JAK2 H608Y and H608N mutations enhanced the cell proliferation and inhibited the differentiation of Ba/F3 and MV4-11 cells via activating the JAK2-STAT5 signaling pathway. Moreover, rescue experiments demonstrated that mutations repaired the hydrogen bond between Q656 and H608 displayed opposite results. Thus, this study revealed the molecular basis of JAK2 H608Y and H608N mutations in the pathology of AML. • Two novel JAK2 H608Y and H608N germline mutations were identified in 6.5 % of AML by NGS. • JAK2 H608Y and H608N mutations abolished the interaction between Q656 and H608 led to JAK2 hyperactivation. • H608Y and H608N mutations reduced JH2 domain's activity and forced JAK2 into the open, active state. • JAK2 H608Y and H608N mutations maintained cell proliferation of Ba/F3 cells without IL-3. • JAK2 H608Y and H608N mutations promoted the pathology of AML via activating JAK2-STAT5 signaling pathway. [ABSTRACT FROM AUTHOR]

Although roles of somatic JAK2 mutations in clonally myeloproliferative neoplasms (MPNs) are well established, roles of germline JAK2 mutations in the pathogenesis of MPNs remain unclear. Recently, a novel activating, germline JAK2 F556V mutation was identified and involved in the pathogenesis of MPNs, but, its pathogenesis mechanism was still unknown. In this study, homology models of JAK2 demonstrated that F556 located between two threonine residues which interacted with ATP phosphate groups by hydrogen bonds, Thr555 with the γ-phosphate and Thr557 with the β-phosphate in the active site of JAK2's JH2 domain. Moreover, the hydrogen bond between Thr557 and Arg715 played vital roles in sustaining the structural conformation of JH2's active site and JH1-JH2 domains' interactions. When F556 was replaced by other amino acids except Trp, the hydrogen bond, JH2 domain's structural conformation and JH1-JH2 domains' interactions disrupted for changing the helix between β2 and β3 strands which finally caused JAK2 activation. Mechanistic and functional studies showed that JAK2 F556V mutation disrupted JAK2 JH2 domain's activity, caused JAK2-STAT5 pathway activation and promoted the proliferation of BaF3 cells. Thus, our results herein may provide clues to understand the pathogenesis mechanism of JAK2 F556V mutation in the MPNs. [ABSTRACT FROM AUTHOR]

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations play key roles in the pathology of myeloproliferative neoplasms (MPNs). Recently, germline JAK2 mutations are also associated with triple-negative MPNs. A novel germline mutation JAK2 V625F is reported to be involved in a subset of MPNs patients. However, the pathogenesis of this mutation caused MPN is still unclear. In this study, the homology models of JAK2 V625F showed that the newly formed interaction between F625 and Y613 disrupted the JAK2 JH1-JH2 domain interactions was responsible for its activation, when F625 and Y613 interaction was disrupted, its activity significantly decreased. While, when this interaction was repaired whether by forming hydrogen bond or salt bond, it would cause JAK2 activation. Biochemical studies also demonstrated that JAK2 V625F mutation led to JAK2-STAT5 pathway activation and promoted the proliferation of BaF3 cells. Thus, our results herein provide clues to understand the mechanism JAK2 V625F mutation caused MPNs and give information for the development of JAK2 mutation specific inhibitors. [ABSTRACT FROM AUTHOR]

Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by ATP, yielding the phosphoarginine. Amino acid residues in the guanidine specificity (GS) region play important roles in the guanidine-recognition. However, little is known about roles of amino acid residue G66 in the GS region in proteins folding, activity and structural stability. In this study, a series of G66 mutations were constructed to investigate its roles in AK's structural stability and activity. Our studies revealed that mutations in this conserved site could cause pronounced loss of activity, conformational changes and structural stability. Spectroscopic experiments indicate that G66 mutations influences AK transition from the molten globule intermediate to the native state in folding process. These results provided herein may suggest that amino acid residue G66 may play a relatively important role in AK's activity and structural stability. [ABSTRACT FROM AUTHOR]

Constitutive NF-κB activation is required for survival of activated B cell-like subtype of diffuse large B cell lymphoma (ABC-DLBCL). However, current NF-κB targeting strategies lack cancer cell specificity. Here, we identified a novel inhibitor, piperlongumine, features direct binding to NF-κB p65 subunit and suppression of p65 nuclear import. This was accompanied by NF-κB reporter activity suppression and NF-κB target gene downregulation. Moreover, mutation of Cys 38 to Ser in p65 abolished this effect of piperlongumine on inhibition of p65 nuclear import. Furthermore, we show that piperlongumine selectively inhibited proliferation and induced apoptosis of ABC-DLBCL cells. Most notably, it has been reported that piperlongumine did not affect normal cells even at high doses and was nontoxic to animals. Hence, our current study provides new insight into piperlongumine's mechanism of action and novel approach to ABC-DLBCL target therapy. [ABSTRACT FROM AUTHOR]

Highlights: [•] JAK2 R683S (G) mutations are presumed to be a biomarker for MPDs. [•] R683 located in the linker between the N and C lobes of JH2 domain which was important for JAK2 auto-inhibition. [•] Mutations R683G, R683S and R683E increased JAK2 activity and led them into partially unfolded state. [•] This study provides clues in understanding the mechanism of JAK2 R683S (G) mutations caused B-ALL. [Copyright &y& Elsevier]

Abstract: Creatine kinase (CK) is a key enzyme for cellular energy metabolism, catalyzing the reversible phosphoryl transfer from phosphocreatine to ADP in vertebrates. Due to its important physiological functions, the acquired CK somatic mutations are closely correlated to diseases. In this study, the E79G point mutation was identified in two acute myocardial infarction patients with muscle CK activity deficiency. The crystal structure of CK indicates that the E79 and K138 interaction plays key roles in sustaining the recognition between N-terminal and C-terminal domains. Mutations of these residues caused pronounced loss of activity, conformational changes and distinct substrate synergism alteration. Moreover, spectroscopic spectra experiments suggested that mutations disrupting this hydrogen bond impaired the secondary and tertiary structure of CK. Meanwhile, protein folding experiments implied that mutations lead them to the partially unfolded state which made them easier to be inactivated and unfolded under environmental stresses. Furthermore, this partially unfolded state upon environmental stresses might gradually decrease the CK level in the patients. Thus, these results might provide clues in the mechanism of CK deficiency diseases. [Copyright &y& Elsevier]