Treatment outcome and germline predictive factors of ropeginterferon alpha-2b in myeloproliferative neoplasm patients.

BACKGROUND: Studies have shown that some single nucleotide polymorphisms (SNPs) could serve as excellent markers in foretelling the treatment outcome of interferon (IFN) in myeloproliferative neoplasms (MPN). However, most work originated from western countries, and data from different ethnic populations have been lacking. METHODS: To gain insights, targeted sequencing was performed to detect myeloid-associated mutations and SNPs in eight loci across three genes (IFNL4, IFN-γ, and inosine triphosphate pyrophosphatase [ITPA]) to explore their predictive roles in our cohort of 21 ropeginterferon alpha-2b (ROPEG)-treated MPN patients, among whom real-time quantitative PCR was also performed periodically to monitor the JAK2V617F allele burden in 19 JAK2V617F-mutated cases. RESULTS: ELN response criteria were adopted to designate patients as good responders if they achieved complete hematological responses (CHR) within 1 year (CHR1) or attained major molecular responses (MMR), which occurred in 70% and 45% of the patients, respectively. IFNL4 and IFN-γ gene SNPs were infrequent in our population and were thus excluded from further analysis. Two ITPA SNPs rs6051702 A>C and rs1127354 C>A were associated with an inferior CHR1 rate and MMR rate, respectively. The former seemed to be linked to grade 2 or worse hepatotoxicity as well, although the comparison was of borderline significance only (50%, vs. 6.7% in those with common haplotype, p = 0.053). Twelve patients harbored 19 additional somatic mutations in 12 genes, but the trajectory of these mutations varied considerably and was not predictive of any response. CONCLUSIONS: Overall, this study provided valuable information on the ethnics- and genetics-based algorithm in the treatment of MPN.

The predictive role of platelet count for bleeding in patients with hepatitis B virus and hepatitis C virus infection.

The impact of platelet count on bleeding in hepatitis B virus (HBV) and hepatitis C virus (HCV)-infected patients is unclear. We aimed to evaluate the relationship between platelet count and bleeding in patients with viral hepatitis. We selected patients with HBV and HCV infection. All esophagogastroduodenoscopy, colonoscopy, and brain imaging reports were reviewed to document upper gastrointestinal bleeding (UGIB), lower gastrointestinal bleeding (LGIB), and central nervous system bleeding (CNSB), respectively. We analyzed risk factors for first bleeding events by using Cox proportional hazards models. Incidence rate ratios (IRRs) were used to compare bleeding incidences between viral types and platelet levels. A total of 2522 HCV and 2405 HBV patients were enrolled. The HCV-to-HBV IRRs of UGIB, LGIB, and CNSB were significant at 1.797, 2.255, and 2.071, respectively. The common risk factors in both groups were thrombocytopenia, hypoalbuminemia, high alkaline phosphatase level, and cirrhosis for UGIB, whereas thrombocytopenia and hypoalbuminemia for LGIB. Hypoalbuminemia was the only risk for CNSB. After adjusting platelet count, the higher bleeding rates in the HCV patients diminished. Using a reference platelet count less than 100 x 10 9 /l, bleeding risk elevated at platelet count less than 70 x 10 9 /l and less than 40 x 10 9 /l for UGIB and LGIB in the HCV patients, respectively, compared with less than 60 x 10 9 /l for UGIB in the HBV patients. The incidence of CNSB was not related to platelet levels. HCV patients had a higher risk for major bleeding. Thrombocytopenia was a significant predictor. Monitoring and management of thrombocytopenia in addition to cirrhotic status was important in these patients.

The Impact of Human Platelet Antigen Allele on Antiplatelet Antibodies and Cryoglobulins in Patients with Primary Immune Thrombocytopenia and Hepatitis C Virus-Associated Immune Thrombocytopenia.

Background And Objectives: Human platelet antigens (HPAs) are alloantigens associated with antiplatelet alloantibodies and the risk of immune thrombocytopenia (ITP). However, few studies have investigated associations among HPAs, antiplatelet autoantibodies, and cryoglobulins. Methods: We enrolled 43 patients with primary ITP, 47 with hepatitis C virus-associated ITP (HCV-ITP), 21 with hepatitis B virus-associated ITP (HBV-ITP), 25 controls with HCV, and 1013 normal controls. We analyzed HPA allele frequencies, including HPA1-6 and 15, antiplatelet antibodies binding to platelet glycoprotein (GP) IIb/IIIa, Ia/IIa, Ib/IX, IV, human leukocyte antigen class I, cryoglobulin IgG/A/M, and their associations with thrombocytopenia. Results: In the ITP cohort, HPA2ab, rather than HPA2aa, predicted a low platelet count. HPA2b was associated with the risk of developing ITP. HPA15b was correlated with multiple antiplatelet antibodies. In HCV-ITP patients, HPA3b was correlated with anti-GPIIb/IIIa antibodies. HCV-ITP patients with anti-GPIIb/IIIa antibodies had a higher positive rate of cryoglobulin IgG and IgA compared with those without anti-GPIIb/IIIa antibodies. Overlapping detection was also found among other antiplatelet antibodies and cryoglobulins. Like the antiplatelet antibodies, cryoglobulins were associated with clinical thrombocytopenia, implying their close relationship. Finally, we extracted cryoglobulins to confirm the exhibition of cryoglobulin-like antiplatelet antibodies. In contrast, in primary ITP patients, HPA3b was correlated with cryoglobulin IgG/A/M rather than anti-GPIIb/IIIa antibodies. Conclusion: HPA alleles were associated with antiplatelet autoantibodies and had different impacts in primary ITP and HCV-ITP patients. HCV-ITP was considered to be a symptom of mixed cryoglobulinemia in HCV patients. The pathophysiology may differ between these two groups.

Mutation-Driven S100A8 Overexpression Confers Aberrant Phenotypes in Type 1 CALR-Mutated MPN.

Numerous pathogenic CALR exon 9 mutations have been identified in myeloproliferative neoplasms (MPN), with type 1 (52bp deletion; CALRDEL) and type 2 (5bp insertion; CALRINS) being the most prevalent. Despite the universal pathobiology of MPN driven by various CALR mutants, it is unclear why different CALR mutations result in diverse clinical phenotypes. Through RNA sequencing followed by validation at the protein and mRNA levels, we found that S100A8 was specifically enriched in CALRDEL but not in CALRINS MPN-model cells. The expression of S100a8 could be regulated by STAT3 based on luciferase reporter assay complemented with inhibitor treatment. Pyrosequencing demonstrated relative hypomethylation in two CpG sites within the potential pSTAT3-targeting S100a8 promoter region in CALRDEL cells as compared to CALRINS cells, suggesting that distinct epigenetic alteration could factor into the divergent S100A8 levels in these cells. The functional analysis confirmed that S100A8 non-redundantly contributed to accelerated cellular proliferation and reduced apoptosis in CALRDEL cells. Clinical validation showed significantly enhanced S100A8 expression in CALRDEL-mutated MPN patients compared to CALRINS-mutated cases, and thrombocytosis was less prominent in those with S100A8 upregulation. This study provides indispensable insights into how different CALR mutations discrepantly drive the expression of specific genes that contributes to unique phenotypes in MPN.

The Genomic Landscape in Philadelphia-Negative Myeloproliferative Neoplasm Patients with Second Cancers.

Patients with myeloproliferative neoplasms (MPNs) are characterized by systemic inflammation. With the indolent nature of the diseases, second cancers (SCs) have emerged as a challenging issue in afflicted patients. Epidemiological studies have confirmed the excessive risk of SCs in MPNs, but little is known about their molecular basis. To explore further, we used whole exome sequencing to explore the genetic changes in the granulocytes of 26 paired MPN patients with or without SC. We noticed that MPN−SC patients harbor genomic variants of distinct genes, among which a unique pattern of co-occurrence or mutual exclusiveness could be identified. We also found that mutated genes in MPN−SC samples were enriched in immune-related pathways and inflammatory networks, an observation further supported by their increased plasma levels of TGF-ß and IL-23. Noteworthily, variants of KRT6A, a gene capable of mediating tumor-associate macrophage activity, were more commonly detected in MPN−SC patients. Analysis through OncodriveCLUST disclosed that KRT6A replaces JAK2V617F as the more prominent disease driver in MPN−SC, whereas a major mutation in this gene (KRT6A c.745T>C) in our patients is linked to human carcinoma and predicted to be pathogenic in COSMIC database. Overall, we demonstrate that inflammation could be indispensable in MPN−SC pathogenesis.

An Efficient Screen for Cell-Intrinsic Factors Identifies the Chaperonin CCT and Multiple Conserved Mechanisms as Mediating Dendrite Morphogenesis.

Dendritic morphology is inextricably linked to neuronal function. Systematic large-scale screens combined with genetic mapping have uncovered several mechanisms underlying dendrite morphogenesis. However, a comprehensive overview of participating molecular mechanisms is still lacking. Here, we conducted an efficient clonal screen using a collection of mapped P-element insertions that were previously shown to cause lethality and eye defects in Drosophila melanogaster. Of 280 mutants, 52 exhibited dendritic defects. Further database analyses, complementation tests, and RNA interference validations verified 40 P-element insertion genes as being responsible for the dendritic defects. Twenty-eight mutants presented severe arbor reduction, and the remainder displayed other abnormalities. The intrinsic regulators encoded by the identified genes participate in multiple conserved mechanisms and pathways, including the protein folding machinery and the chaperonin-containing TCP-1 (CCT) complex that facilitates tubulin folding. Mutant neurons in which expression of CCT4 or CCT5 was depleted exhibited severely retarded dendrite growth. We show that CCT localizes in dendrites and is required for dendritic microtubule organization and tubulin stability, suggesting that CCT-mediated tubulin folding occurs locally within dendrites. Our study also reveals novel mechanisms underlying dendrite morphogenesis. For example, we show that Drosophila Nogo signaling is required for dendrite development and that Mummy and Wech also regulate dendrite morphogenesis, potentially via Dpp- and integrin-independent pathways. Our methodology represents an efficient strategy for identifying intrinsic dendrite regulators, and provides insights into the plethora of molecular mechanisms underlying dendrite morphogenesis.

Outer nuclear membrane protein Kuduk modulates the LINC complex and nuclear envelope architecture.

Linker of nucleoskeleton and cytoskeleton (LINC) complexes spanning the nuclear envelope (NE) contribute to nucleocytoskeletal force transduction. A few NE proteins have been found to regulate the LINC complex. In this study, we identify one, Kuduk (Kud), which can reside at the outer nuclear membrane and is required for the development of Drosophila melanogaster ovarian follicles and NE morphology of myonuclei. Kud associates with LINC complex components in an evolutionarily conserved manner. Loss of Kud increases the level but impairs functioning of the LINC complex. Overexpression of Kud suppresses NE targeting of cytoskeleton-free LINC complexes. Thus, Kud acts as a quality control mechanism for LINC-mediated nucleocytoskeletal connections. Genetic data indicate that Kud also functions independently of the LINC complex. Overexpression of the human orthologue TMEM258 in Drosophila proved functional conservation. These findings expand our understanding of the regulation of LINC complexes and NE architecture.

Glial cell adhesive molecule unzipped mediates axon guidance in Drosophila.

Axon guidance needs help from the glial cell system during embryogenesis. In the Drosophila embryonic central nervous system (CNS), longitudinal glia (LG) have been implicated in axon guidance but the mechanism remains unclear. We identified the protein encoded by the Drosophila gene unzipped (uzip) as a novel cell adhesion molecule (CAM). Uzip expressed in Drosophila S2 cells triggered cell aggregation through homophilic binding. In the embryonic CNS, Uzip was mainly produced by the LG but was also located at axons, which is consistent with the secretion of Uzip expressed in cultured cells. Although uzip mutants displayed no axonal defect, loss of uzip enhanced the axonal defects in the mutant of N-cadherin (CadN) and the Wnt gene family member wnt5. Overexpression of uzip could rescue the phenotype in the CadNuzip(D43) mutant. Thus, Uzip is a novel CAM from the LG regulating axon guidance.

Organogenesis and tumorigenesis: insight from the JAK/STAT pathway in the Drosophila eye.

The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway is one of the main signaling pathways in eukaryotic cells. This pathway is used during diverse growth and developmental processes in multiple tissues to control cell proliferation, differentiation, survival, and apoptosis. In addition to its role during development, the JAK/STAT pathway has also been implicated in tumorigenesis. Drosophila melanogaster is a powerful genetic tool, and its eyes have been used extensively as a platform to study signaling pathways. Many reports have demonstrated that the JAK/STAT pathway plays pleiotropic roles in Drosophila eye development. Its functions and activation are decided by its interplay with other signal pathways and the epigenetic status. In this review, we focus on the functions and regulation of the JAK/STAT pathway during eye development and provide some insights into the study of this pathway in tumorigenesis.

Reduction of Lobe leads to TORC1 hypoactivation that induces ectopic Jak/STAT signaling to impair Drosophila eye development.

The TOR and Jak/STAT signal pathways are highly conserved from Drosophila to mammals, but it is unclear whether they interact during development. The proline-rich Akt substrate of 40 kDa (PRAS40) mediates the TOR signal pathway through regulation of TORC1 activity, but its functions in TORC1 proved in cultured cells are controversial. The Drosophila gene Lobe (L) encodes the PRAS40 ortholog required for eye cell survival. L mutants exhibit apoptosis and eye-reduction phenotypes. It is unknown whether L regulates eye development via regulation of TORC1 activity. We found that reducing the L level, by hypomorphic L mutation or heterozygosity of the null L mutation, resulted in ectopic expression of unpaired (upd), which is known to act through the Jak/STAT signal pathway to promote proliferation during eye development. Unexpectedly, when L was reduced, decreasing Jak/STAT restored the eye size, whereas increasing Jak/STAT prevented eye formation. We found that ectopic Jak/STAT signaling and apoptosis are mutually dependent in L mutants, indicating that L reduction makes Jak/STAT signaling harmful to eye development. In addition, our genetic data suggest that TORC1 signaling is downregulated upon L reduction, supporting the idea that L regulates eye development through regulation of TORC1 activity. Similar to L reduction, decreasing TORC1 signaling by dTOR overexpression results in ectopic upd expression and apoptosis. A novel finding from our data is that dysregulated TORC1 signaling regulates the expression of upd and the function of the Jak/STAT signal pathway in Drosophila eye development.