Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive activation of JAK/STAT signaling through mutations in JAK2, CALR, or MPL is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis.

A short hairpin RNA library screening was performed on JAK2^V617F-mutant HEL cells. Nuclear-cytoplasmic transport (NCT) genes including RAN and RANBP2 were among top candidates. JAK2^V617F-mutant cell lines, human primary myelofibrosis CD34⁺ cells, and a retroviral JAK2^V617F-driven myeloproliferative neoplasms mouse model were used to determine the effects of inhibiting NCT with selective inhibitors of nuclear export compounds KPT-330 (selinexor) or KPT-8602 (eltanexor).

JAK2^V617F-mutant HEL, SET-2, and HEL cells resistant to JAK inhibition are exquisitely sensitive to RAN knockdown or pharmacologic inhibition by KPT-330 or KPT-8602. Inhibition of NCT selectively decreased viable cells and colony formation by myelofibrosis compared with cord blood CD34⁺ cells and enhanced ruxolitinib-mediated growth inhibition and apoptosis, both in newly diagnosed and ruxolitinib-exposed myelofibrosis cells. Inhibition of NCT in myelofibrosis CD34⁺ cells led to nuclear accumulation of p53. KPT-330 in combination with ruxolitinib-normalized white blood cells, hematocrit, spleen size, and architecture, and selectively reduced JAK2^V617F-mutant cells in vivo.

Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.

Impaired sonic hedgehog (Shh) signaling is involved in the pathology of cortical formation found in neuropsychiatric disorders. However, its role in the specification of human cortical progenitors is not known. Here, we report that Shh is expressed in the human developing cortex at mid-gestation by radial glia cells (RGCs) and cortical neurons. We used RGC cultures, established from the dorsal (cortical) telencephalon of human brain at mid-gestation to study the effect of Shh signaling. Cortical RGCs in vitro maintained their regional characteristics, expressed components of Shh signaling, and differentiated into Nkx2.1, Lhx6, and calretinin-positive (CalR(+)) cells, potential cortical interneuron progenitors. Treatment with exogenous Shh increased the pool of Nkx2.1(+) progenitors, decreased Lhx6 expression, and suppressed the generation of CalR(+) cells. The blockade of endogenous Shh signaling increased the number of CalR(+) cells, but did not affect Nkx2.1 expression, implying the existence of parallel Shh-independent pathways for cortical Nkx2.1 regulation. These results support the idea that, during human brain development, Shh plays an important role in the specification of cortical progenitors. Since direct functional studies in humans are limited, the in vitro system that we established here could be of great interest for modeling the development of human cortical progenitors.

Background: Bladder cancer (BCa) has the highest incidence of aggressive malignant tumors in the urogenital system and is the ninth most common cancer worldwide. Immune function-related genes (IFRGs), which are plentiful in immune cells and the immune microenvironment (IME), have the potential to assess prognosis and predict the efficacy of immunotherapy. A complete and significant immunogenomic analysis based on abundant BCa genetic samples from The Cancer Genome Atlas (TCGA) will provide insight into the field.

Results: A total of 57 differentially expressed IFRGs were significantly associated with the clinical outcomes of patients with BCa. Functional enrichment analysis showed that these genes actively participated in the KEGG pathway of human cytomegalovirus infection. Based on the IFRGs (CALR, MMP9, PAEP, RBP7, STAT1, CACYBP, ANHAK, RAC3, SLIT2, EDNRA, IGF1, NAMPT, NTF3, PPY, ADRB2 and SH3BP2), the risk scores were calculated to predict survival and reveal the relationships with age, sex, grade, staging, T-stage, N-stage, and M-stage. Interestingly, IFRG-based risk scores (IRRSs) reflected the infiltration of several types of immune cells. The expression of CACYBP was more significant in grade 3, T3 and T4 stages than in earlier grades and T-stages.

Conclusion: Our results highlighted some sIFRGs with remarkable clinical relevance, showed the driving factors of the immune repertoire, and illustrated the significance of IFRG-based individual immune features in the identification, monitoring, and prognosis of patients with BCa.

Methods: Based on the TCGA dataset, we integrated the expression profiles of IFRGs and overall survival (OS) in 430 patients with BCa. Differentially expressed IFRGs and survival-related IFRGs (sIFRGs) were highlighted by calculating the difference algorithm and COX regression analysis in patients with BCa. Based on computational biology, the potential molecular mechanisms and characteristics of these IFRGs were also explored. Using multivariate Cox analysis, new risk scores based on immune-related genes were developed. The expression of CACYBP was verified by qPCR, western blot and immunohistochemistry. The relations between CACYBP and clinical features were proven by immunohistochemistry.

Keywords: CACYBP; bladder cancer; immunogen; prognosis; risk score.

OBJECTIVE

Whole genome interrogation by array-based comparative genomic hybridization has led to a rapidly increasing number of discoveries of novel microdeletion and/or microduplication syndromes. We here describe the clinical and cytogenomic correlates of a novel microdeletion/microduplication of 19p13.13.

METHODS

Among patients referred to the Cytogenetics laboratory for array-based comparative genomic hybridization analysis, we identified four with a deletion and one with a duplication within 19p13.13. Confirmatory fluorescence in situ hybridization and parental studies were performed. Detailed clinical findings and array profiles were reviewed and compared.

RESULTS

Patients with deletions of 19p13.13 share a unique constellation of phenotypic abnormalities. In addition to developmental disabilities, the microdeletion manifested in overgrowth, macrocephaly, and ophthalmologic and gastrointestinal findings; in contrast, the single microduplication manifested in growth delay and microcephaly.

CONCLUSIONS

The consistent constellation of clinical findings associated with copy number variation of this region warrants the designation of microdeletion/microduplication syndrome of 19p13.13. An approximately 311-340 Kb smallest region of overlap encompassing 16 genes was identified. Candidate genes include MAST1, NFIX, and CALR. Identification of this syndrome has led to recommendations for diagnostic work-up and follow-up of patients with this copy number variant. Integration of detailed clinical and array data is critical for advancing both patient care and human genomic research.

Hyperactive Janus associated kinase-signal transducers and activators of transcription (JAK-STAT) signaling has been shown to be integral to the pathogenesis of myelofibrosis (MF) regardless of the driver mutational status (JAK2V617F, JAK2 exon 12, MPL515L/K, CALR). Targeting of the JAK-STAT pathway has been the intense focus of therapeutic development and led to the approval of the JAK1/2 inhibitor, ruxolitinib. Despite the clear clinical success of ruxolitinib, dose limiting thrombocytopenia, treatment associated anemia and failure to effectively achieve bone marrow pathologic, cytogenetic and molecular remission remain shortcomings. JAK1 inhibition leads to depression in inflammatory cytokine expression associated with MF-related constitutional symptoms. The selective targeting of JAK1 may provide an opportunity to alleviate MF-related symptoms without anti-JAK2 therapy-related myelosuppression. Additionally, a JAK1 inhibitor may serve as an ideal candidate partner for combination therapeutic approaches in the treatment of MF. Current evaluation of selective JAK1 inhibition in MF will further clarify the relative contribution of aberrant JAK1 signaling to the pathogenesis of MF.

Myeloproliferative neoplasms (MPNs) include essential thrombocythemia, polycythemia vera (PV), and primary myelofibrosis (MF). Phenotype-driver mutations of JAK2, CALR, and MPL genes are present in MPNs and can be variably combined with additional mutations. Driver mutations entail a constitutive activation of the JAK2/STAT pathway, the key signaling cascade in MPNs. Among JAK2 inhibitors (JAKis), ruxolitinib (RUX) has been approved for the treatment of intermediate and high-risk MF and for PV inadequately controlled by or intolerant of hydroxyurea. Other JAKis, such as fedratinib and pacritinib, proved to be useful in MF. The primary end points in MF trials were spleen volume response (SVR) and symptom response, whereas in PV trials they were hematocrit control with or without spleen response. In advanced MF, RUX achieved a long lasting SVR of >35% in ∼60% of patients, establishing a new benchmark for MF treatment. RUX efficacy in early MF is also remarkable and toxicity is mild. In PV, RUX achieved hematocrit control in ∼60% of cases and SVR in 40%. Symptom relief was evident in both conditions. In the long-term, however, many MF patients lose their SVR. Indeed, the definition of RUX failure and the design of new trials in this setting are unmet needs. Decrease of hemoglobin/platelet levels and increased infection rates are the most common side effects of RUX, and nonmelanoma skin tumors need to be monitored while on treatment. In conclusion, the introduction of JAKis raises the bar of treatment goals in MF and PV.

With the discovery of the JAK2V617F mutation in patients with Philadelphia chromosome-negative (Ph(-)) myeloproliferative neoplasms (MPNs) in 2005, major advances have been made in the diagnosis of MPNs, in understanding of their pathogenesis involving the JAK/STAT pathway, and finally in the development of novel therapies targeting this pathway. Nevertheless, it remains unknown which mutations exist in approximately one-third of patients with non-mutated JAK2 or MPL essential thrombocythemia (ET) and primary myelofibrosis (PMF). At the end of 2013, two studies identified recurrent mutations in the gene encoding calreticulin (CALR) using whole-exome sequencing. These mutations were revealed in the majority of ET and PMF patients with non-mutated JAK2 or MPL but not in polycythemia vera patients. Somatic 52-bp deletions (type 1 mutations) and recurrent 5-bp insertions (type 2 mutations) in exon 9 of the CALR gene (the last exon encoding the C-terminal amino acids of the protein calreticulin) were detected and found always to generate frameshift mutations. All detected mutant calreticulin proteins shared a novel amino acid sequence at the C-terminal. Mutations in CALR are acquired early in the clonal history of the disease, and they cause activation of JAK/STAT signaling. The CALR mutations are the second most frequent mutations in Ph(-) MPN patients after the JAK2V617F mutation, and their detection has significantly improved the diagnostic approach for ET and PMF. The characteristics of the CALR mutations as well as their diagnostic, clinical, and pathogenesis implications are discussed in this review.

The 2005 discovery of the JAK2 mutation redefined the diagnosis and natural history of myeloproliferative neoplasms (MPNs). Most importantly, this improvement in the pathobiologic conceptualization has focused our evolution of this field from being defined as what it is not (e.g., Philadelphia [Ph]-negative) to what it is (e.g., JAK2-positive, CALR-positive) in the majority of MPN cases. In the ensuing 10 years, the field has experienced a paradigm shift in terms of understanding of the biologic basis of the development of MPNs, an explosion of knowledge of the genetics of MPNs, and has translated disease knowledge into effective targeted therapies. With greater uniformity and agreement on the diagnosis and differences among the individual MPNs, augmented by improved cytogenetic and molecular classification, attention has turned now to addressing the need for uniformity in risk stratification of patients in the clinic for both disease complications and disease transformation. This article will highlight the developments in the field with regard to risk stratification and prognostication in MPNs with focus on the clinical aspects of the patient who presents with either essential thrombocytosis (ET), polycythemia vera (PV), or myelofibrosis (MF).

This study analyzed the expression of differentiation markers (Calbindin D28K: CaBP; parvalbumin: PARV; calretinin: CalR), gamma-aminobutyric acid (GABA) markers (GABA, glutamic acid decarboxylases: GAD65, GAD67; and GABA transporters: GAT1, GAT3), and other markers (neurotensin: NT, and neurofilament-specific protein: SMI32) in the human thalamus at 8-23 gestation weeks (g.w.), focusing on the motor-related nuclei. From 8-13 g.w. mainly CaBP was expressed in the cells while fiber bundles traversing the thalamus in addition to CaBP expressed all GABA markers except GAD67. CaBP and PARV expression patterns in different nuclei changed over the time course studied, whereas NT was expressed consistently along the anterior-lateral curvature of the thalamus. CalR and SMI were detectable at 23 g.w. in the ventral parts of the dorsal thalamus. Most remarkably, punctate GAD65 immunoreactivity in the neuropil was confined to the nigro- and pallidothalamic afferent receiving nuclei from 16 to about 21 g.w., overlapping with that of CaBP in some of these nuclei (subdivisions of the ventral anterior and mediodorsal nuclei) and with PARV in others (centromedian nucleus). During this period, GAD65 immunoreactivity can be considered a marker of the basal ganglia afferent receiving territory in the motor thalamus. GAD67-positive local circuit neurons were first detected at 12-13 g.w. in the thalamic nuclei outside the basal ganglia afferent receiving territory. In the ventral anterior and centromedian nuclei, GAD-containing local circuit neurons were not conspicuous even at 22-23 g.w. The cells of the reticular nucleus expressed GAD67 and PARV from 12 g.w. on starting in the lateral-posterior regions. By 23 g.w., both markers were expressed in about two-thirds of the nucleus except for its most medial-anterior part. The results imply spatially and temporally differential expression of GABA and differentiation markers in the developing human thalamus.

Maternal deprivation (MD) is a well-established protocol used to investigate neurobiological changes that are associated with the etiology of and vulnerability to stress-related diseases in animal models. The resulting psychophysiological effects, the timing and duration of these adverse stimuli, and the method by which they exert their effects on the animals remain unclear. This study characterized differences in the hippocampal expression of glucocorticoid receptors (GRs) and the calcium-binding proteins calretinin (CALR) and calbindin-D28k (CALB) in male and female rats that underwent different MD paradigms during the stress hyporesponsive period (SHRP). Both GRs and the two calcium-binding proteins were much more abundant in females than in males. MD paradigms had a significant effect on CALR and CALB expression in both males and females but affected GR levels only in males. Additionally, expression of the two calcium-binding proteins in the hippocampus responded differently to MD-induced stress, especially in females. Taken together, these results indicate that females are able to modulate their response to stress better than males.

BACKGROUND

The management of patients with glioblastoma multiforme is difficult. Poor results have led to a search for novel therapeutic approaches. Gene therapy that could be both anti-invasive and antiangiogenic would be ideal. In this study, we constructed the recombinant adenoviral vector Ad-CALR/MAGE-A3 and evaluated its antitumor effects on glioblastoma in vitro and in vivo.

METHODS

In this study, CALR and MAGE-A3 genes were delivered to the glioblastoma cell line U87, using adenovirus (Ad-CALR/MAGE-A3). U87 glioblastoma cells were transfected with Ad-green fluorescent protein to identify the multiplicity of infection. The expressions of CALR and MAGE-A3 were detected by PCR and Western blot. Cell proliferation was measured by MTT assay. Cell apoptosis was assessed by Annexin-V FITC/PI double staining flow cytometry. The invasive potential of U87 cells was determined by Matrigel invasion assay. Tube formation assay was used to detect the effects on angiogenesis of human umbilical vein endothelial cells. Protein expressions of PI3K/AKT, Erk1/2 and MMP-2/-9 in transfected cells were detected by Western blot. In vivo, the effects of Ad-CALR/MAGE-A3 on tumor growth and angiogenesis of U87 glioblastoma xenografts in nude mice were investigated.

RESULTS

The expressions of CALR and MAGE-A3 in U87 cells resulted in the suppression of cell proliferation and invasion properties, and induced cell apoptosis. The Erk MAPK, PI3K/AKT pathways and expressions of MMP-2/-9 were inhibited in Ad-CALR/MAGE-A3-transfected cells. Outcomes of the tube formation assay confirmed the antiangiogenic effect of CALR. Moreover, in the in vivo model of glioblastoma, intratumoral injection of Ad-CALR/MAGE-A3 suppressed tumor growth and angiogenesis.

CONCLUSIONS

Although Ad-CALR/MAGE-A3 and Ad-CALR demonstrated antiangiogenic effects on U87 cells, the repression of invasion was significant only in Ad-CALR/MAGE-A3-treated cells. To our knowledge, this is the first description of a role for combined CALR and MAGE-A3 in the anti-invasion and antiangiogenesis of U87.

Paroxysmal nocturnal hemoglobinuria (PNH), a disease characterized by intravascular hemolysis, thrombosis, and bone marrow failure, is associated with mutations in the PIG-A gene, resulting in a deficiency of glycosylphosphatidylinositol-anchored proteins. Many hypotheses have been posed as to whether PNH and PIG-A mutations result in an intrinsic survival benefit of CD55(-)/CD59(-) cells or an extrinsic permissive environment that allows for their clonal expansion within the bone marrow compartment. Recent data have identified the concurrence of PIG-A mutations with additional genetic mutations associated with myeloproliferative disorders, suggesting that some presentations of PNH are the result of a stepwise progression of genetic mutations similar to other myelodysplastic or myeloproliferative syndromes. We report for the first time in the literature the development of clinically significant PNH in a patient with JAK2V617F-negative, CALR-positive essential thrombocythemia, providing further support to the hypothesis that the development of PNH is associated with the accumulation of multiple genetic mutations that create an intrinsic survival benefit for clonal expansion. This case study additionally highlights the utility of genomic testing in diagnosis and the understanding of disease progression in the clinical setting.

The consequence of presence versus absence of mucosal neurons is not consistently assessed. Here, we addressed two questions. First, based on resected gut specimens of 65 patients/body donors suffering from different diseases, counts of mucosal neurons per mm(2) were analysed with respect to age, gender and region. Second, we evaluated resected megacolonic specimens of four patients suffering from chronic Chagas' disease. Mucosal wholemounts were triple-stained for calretinin (CALR), peripherin (PER) and human neuronal protein Hu C/D (HU). Counts revealed no clear correlation between the presence of mucosal neurons and age, gender or region. Mucosal neurons were present in 30 of 36 specimens derived from males (83%) and in 20 of 29 from females (69%). The numbers per mm(2) increased from duodenum to ileum (1.7-10.8) and from ascending to sigmoid colon (3.2-9.9). Out of 149 small intestinal mucosal neurons, 47% were co-reactive for CALR, PER and HU (large intestine: 76% of 300 neurons) and 48% for PER and HU only (large intestine: 23%). In 12 megacolonic specimens (each 3 from 4 patients), all 23 mucosal neurons found (1.9 per mm(2)) displayed co-reactivity for CALR, PER and HU. We suggest that the presence or the absence of mucosal neurons is variable, ongoing studies will address our assumption that they correspond in their morphochemical characteristics to submucosal neurons. Furthermore, both the architecture and neuron number of the megacolonic mucosal plexus displayed no dramatic changes indicating that mucosal nerves might be less involved in chagasic/megacolonic neurodegeneration as known from the myenteric plexus.

Background: Myelofibrosis (MF) is a clonal disorder of hemopoietic stem/progenitor cells (HSPCs) with high prevalence in elderly patients and mutations in three driver genes (JAK2, MPL, or CALR). Around 10-15% of patients are triple-negative (TN) for the three driver mutations and display significantly worse survival. Circulating extracellular vesicles (EVs) play a role in intercellular signaling and are increased in inflammation and cancer. To identify a biomolecular signature of TN patients, we comparatively evaluated the circulating HSPCs and their functional interplay with the microenvironment focusing on EV analysis.

Methods: Peripheral blood was collected from MF patients (n = 29; JAK2^V617F mutation, n = 23; TN, n = 6) and healthy donors (HD, n = 10). Immunomagnetically isolated CD34⁺ cells were characterized by gene expression profiling analysis (GEP), survival, migration, and clonogenic ability. EVs were purified from platelet-poor plasma by ultracentrifugation, quantified using the Nanosight technology and phenotypically characterized by flow cytometry together with microRNA expression. Migration and survival of CD34⁺ cells from patients were also analyzed after in vitro treatments with selected inflammatory factors, i.e. (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, IL6) or after co-culture with EVs from MF patients/HD.

Results: The absolute numbers of circulating CD34⁺ cells were massively increased in TN patients. We found that TN CD34⁺ cells show in vitro defective functions and are unresponsive to the inflammatory microenvironment. Of note, the plasma levels of crucial inflammatory cytokines are mostly within the normal range in TN patients. Compared to JAK2^V617F-mutated patients, the GEP of TN CD34⁺ cells revealed distinct signatures in key pathways such as survival, cell adhesion, and inflammation. Importantly, we observed the presence of mitochondrial components within plasma EVs and a distinct phenotype in TN-derived EVs compared to the JAK2^V617F-mutated MF patients and HD counterparts. Notably, TN EVs promoted the survival of TN CD34⁺ cells. Along with a specific microRNA signature, the circulating EVs from TN patients are enriched with miR-361-5p.

Conclusions: Distinct EV-driven signals from the microenvironment are capable to promote the TN malignant hemopoiesis and their further investigation paves the way toward novel therapeutic approaches for rare MF.

Keywords: Extracellular vesicles; Inflammation, microRNAs; Myelofibrosis.

Mutations in CALR have recently been detected in JAK2-negative myeloproliferative neoplasms (MPNs) and are key pathological drivers in these diseases. CALR-mutated MPNs are shown to have numerous clinicopathological differences to JAK2-mutated MPNs. The basis of these differences is poorly understood. It is unknown whether these differences result directly from any differences in intracellular signalling abnormalities induced by JAK2/CALR mutations or whether they relate to other phenomena such as a differing spectrum of genetic lesions between the two groups. We aimed to review the clinicopathological and molecular features of CALR- and JAK2-mutated MPNs from samples referred for diagnostic testing using a custom-designed targeted next-generation sequencing (NGS) panel. Eighty-nine CALR-mutated cases were compared with 70 JAK2-mutated cases. CALR-mutated MPNs showed higher platelet counts and a female predominance as compared to JAK2-mutated MPNs in our cohort. We have also observed differences between CALR mutation subtypes in terms of disease phenotype, mutational frequency and allelic burden. Type 1 CALR mutations were found to be more common in myelofibrosis, associated with a higher frequency and number of additional mutations and a higher mutant allelic burden as compared to type 2 CALR mutations. Despite these biological differences, our molecular characterisation suggests that CALR- and JAK2-mutated MPNs are broadly similar in terms of the quantity, frequency and spectrum of co-occurring mutations and therefore observed biological differences are likely to not be heavily influenced by the nature and quantity of co-mutated genes.

Tunicamycin (TM) is an inducer of endoplasmic reticulum (ER) stress. However, which genes related to ER stress was induced in cardiomyocytes on a genome-wide scale remains poorly understood. Salubrinal and its derivatives are ER stress inhibitors. However, the cellular protection mechanisms remain unresolved. Neonatal rat cardiomyocytes were cultured from ventricles of one-day-old Wistar rats. Cells were exposed to salubrinal, its derivatives (PP1-12, PP1-24) or vehicle followed by TM treatment at different times. Total RNA was isolated from cells for RNA-sequencing analysis. The expressions of 189, 182, 556, 860, and 1314 genes were changed in cells exposed to TM for 1, 3, 6, 12, and 24 h. Five well-known UPR genes (Hspa5, Hsp90b1, Calr, Ddit3, and Atf4) were significantly increased in a time-dependent manner. Six not well-known genes (Hyou1, Herpud1, Manf, Creld2, Sdf2l1, and Slc3a2) were highlighted to be involved in ER stress. Compared with TM-only treated cells, the expressions of 36 genes upregulated by TM and 74 genes downregulated by TM were reversed by salubrinal. In comparison, 121 genes upregulated by TM and 92 genes downregulated by TM were reversed by PP1-12. Most genes altered by salubrinal are in the category of transcription (1 h) and cell cycle (24 h). Most genes altered by PP1-12 are in the category of response to ER stress (3 h) and cell cycle (24 h). Our findings help elucidate the mechanism for TM treatment and may be useful for future drug screens involved in ER stress.