Tumor cells treated by immunogenic cell death (ICD) inducers emit danger associated molecular patterns (DAMP), including but not limited to calreticulin (CALR), which translocates from the ER lumen to the surface of the cellular membrane where it serves as de novo uptake signal for antigen presenting cells of the immune system. CALR is exposed at an early stage of ICD and dictates tumor antigen transfer and therefore the immunogenicity of cancer cell death. Here, we provide a bi-color flow cytometry protocol for the quantification of ICD-associated CALR cell surface exposure in fixed samples. As compared to the detection of surface exposed CALR by confocal microscopy, the present flow cytometry-based analysis is cost-efficient and does not require sophisticated equipment. Moreover, the staining panel can be extended to a multicolor analysis for the parallel assessment of additional parameters.

Parkinson's disease (PD) is associated with olfactory defects in addition to dopaminergic degeneration. Dopaminergic signalling is necessary for subventricular zone (SVZ) proliferation and olfactory bulb (OB) neurogenesis. Alpha-synuclein (α-syn or Snca) modulates dopaminergic neurotransmission, and SNCA mutations cause familial PD, but how α-syn and its mutations affect adult neurogenesis is unclear. To address this, we studied a bacterial artificial chromosome transgenic mouse expressing the A30P SNCA familial PD point mutation on an Snca-/- background. We confirmed that the SNCA-A30P transgene recapitulates endogenous α-syn expression patterns and levels by immunohistochemical detection of endogenous α-syn in a wild-type mouse and transgenic SNCA-A30P α-syn protein in the forebrain. The number of SVZ stem cells (BrdU+GFAP+) was decreased in SNCA-A30P mice, whereas proliferating (phospho-histone 3+) cells were decreased in Snca-/- and even more so in SNCA-A30P mice. Similarly, SNCA-A30P mice had fewer Mash1+ transit-amplifying SVZ progenitor cells but Snca-/- mice did not. These data suggest the A30P mutation aggravates the effect of Snca loss in the SVZ. Interestingly, calbindin+ and calretinin (CalR)+ periglomerular neurons were decreased in both Snca-/-, and SNCA-A30P mice but tyrosine hydroxylase+ periglomerular OB neurons were only decreased in Snca-/- mice. Cell death decreased in the OB granule layer of Snca-/- and SNCA-A30P mice. In the same region, CalR+ numbers increased in Snca-/- and SNCA-A30P mice. Thus, α-syn loss and human A30P SNCA decrease SVZ proliferation, cell death in the OB and differentially alter interneuron numbers. Similar disruptions in human neurogenesis may contribute to the olfactory deficits, which are observed in PD.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by an excessive production of pro-inflammatory cytokines resulting in chronic inflammation and genomic instability. Besides the driver mutations in JAK2, MPL, and CALR genes, the deregulation of miRNA expression may also contribute to the pathogenesis of PMF. To this end, we recently reported the upregulation of miR-382-5p in PMF CD34+ cells. In order to unveil the mechanistic details of the role of miR-382-5p in pathogenesis of PMF, we performed gene expression profiling of CD34+ cells overexpressing miR-382-5p. Among the downregulated genes, we identified superoxide dismutase 2 (SOD2), which is a predicted target of miR-382-5p. Subsequently, we confirmed miR-382-5p/SOD2 interaction by luciferase assay and we showed that miR-382-5p overexpression in CD34+ cells causes the decrease in SOD2 activity leading to reactive oxygen species (ROS) accumulation and oxidative DNA damage. In addition, our data indicate that inhibition of miR-382-5p in PMF CD34+ cells restores SOD2 function, induces ROS disposal, and reduces DNA oxidation. Since the pro-inflammatory cytokine transforming growth factor-β1 (TGF-β1) is a key player in PMF pathogenesis, we further investigated the effect of TGF-β1 on ROS and miR-382-5p levels. Our data showed that TGF-β1 treatment enhances miR-382-5p expression and reduces SOD2 activity leading to ROS accumulation. Finally, inhibition of TGF-β1 signaling in PMF CD34+ cells by galunisertib significantly reduced miR-382-5p expression and ROS accumulation and restored SOD2 activity. As a whole, this study reports that TGF-β1/miR-382-5p/SOD2 axis deregulation in PMF cells is linked to ROS overproduction that may contribute to enhanced oxidative stress and inflammation. Our results suggest that galunisertib may represent an effective drug reducing abnormal oxidative stress induced by TGF-β1 in PMF patients. DATABASE LINKING: GEO: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103464.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) in which dysregulation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways is the major pathogenic mechanism. Most patients with PMF carry a driver mutation in the JAK2, MPL (myeloproliferative leukemia), or CALR (calreticulin) genes. Mutations in epigenetic regulators and RNA splicing genes may also occur, and play critical roles in PMF disease progression. Based on revised World Health Organization diagnostic criteria for MPNs, both screening for driver mutations and bone marrow biopsy are required for a specific diagnosis. Clinical trials of JAK2 inhibitors for PMF have revealed significant efficacy for improving splenomegaly and constitutional symptoms. However, the currently available drug therapies for PMF do not improve survival. Although allogeneic stem cell transplantation is potentially curative, it is associated with substantial treatment-related morbidity and mortality. PMF is a heterogeneous disorder and decisions regarding treatments are often complicated, necessitating the use of prognostic models to determine the management of treatments for individual patients. This review focuses on the clinical aspects and outcomes of a cohort of Japanese patients with PMF, including discussion of recent advances in the management of PMF.

To address the clinical relevance of small DNA variants in chronic myeloid neoplasms (CMNs), an Association for Molecular Pathology Working Group comprehensively reviewed published literature, summarized key findings that support clinical utility, and defined critical gene inclusions for high-throughput sequencing testing panels. This review highlights the biological complexity of CMNs [including myelodysplastic syndromes, myeloproliferative neoplasms, entities with overlapping features (myelodysplastic syndromes/myeloproliferative neoplasms), and systemic mastocytosis], the genetic heterogeneity within diagnostic categories, and similarities between apparently disparate diagnostic entities. The founding variant's hematopoietic differentiation compartment, specific genes and variants present, order of variant appearance, individual subclone dynamics, and therapeutic intervention all contribute to the clinicopathologic features of CMNs. Selection and efficacy of targeted therapies are increasingly based on DNA variant profiles present at various time points; therefore, high-throughput sequencing remains critical for patient management. The following genes are a minimum recommended list to provide relevant clinical information for the management of most CMNs: ASXL1, BCOR, BCORL1, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NF1, NPM1, NRAS, PHF6, PPM1D, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1, and ZRSR2. This list is not comprehensive for all myeloid neoplasms and will evolve as insights into effects of combinations of relevant biomarkers on specific clinicopathologic characteristics of CMNs accumulate.

In the 2016 revised classification of myeloproliferative neoplasms pre-fibrotic primary myelofibrosis (pre-PMF) was recognized as a separate entity, distinct from essential thrombocythemia (ET). Owing that the majority of cases falling in the pre-PMF category were previously diagnosed as ET, one may question about the need to re-evaluate the results of epidemiologic, clinical, and molecular studies, and the results of clinical trials in the two entities. Based on a critical review of recently published studies, pre-PMF usually presents with a distinct clinical and hematological presentation and higher frequency of constitutional symptoms. JAK2V617F and CALR mutations in pre-PMF patients are superimposable to ET, whereas non-driver high-risk mutations are enriched in pre-PMF compared with ET. Thrombosis is not significantly different, whereas bleeding is more frequent in pre-PMF. Median survival is significantly shorter in pre-PMF and 10-year cumulative rates progression to overt myelofibrosis is 0-1% vs. 10-12%, and leukemic transformation is 1-2% vs. 2-6%, in ET and pre-fibrotic-PMF, respectively. Most patients fall in the lower prognostic IPSS group in which observation alone can be recommended. Patients at intermediate risk may require a symptom-driven treatment for anemia, splenomegaly or constitutional symptoms while cytoreductive drugs are indicated in the high-risk category.

Damages to the enteric nervous system caused by diabetes mellitus (DM) are frequently attributed to oxidative and nitrosative stress. We aimed to investigate the effect of Resveratrol (RSV) (10 mg/kg) on oxidative and nitrosative stress in the intestinal wall and morphoquantitative aspects of the myenteric plexus of the duodenum, jejunum and ileum in diabetic rats. Twenty-four rats were distributed into four groups (n = 6/group): control (C group), control treated with RSV (CR group), diabetic (D group), and diabetic treated with RSV (DR group) for 120 days. Immunohistochemical staining techniques for the general neuronal population, nitrergic and calretinin neuronal subpopulations, enteric glial cells and glial fibrillary acid protein were performed in the myenteric plexus. Furthermore, parameters of oxidative and nitrosative stress were analyzed in the intestinal wall. RSV attenuated oxidative and nitrosative stress and prevented neuronal loss and hypertrophy of the HuC/D-IR, nNOS-IR and CALR-IR neuronal subpopulations in the DR group compared with the D group (P < 0.05). In addition, RSV prevented the increase in glial fibrillary acid protein fluorescence in the DR group compared with the D (P < 0.05). These results suggest that RSV has antioxidant and neuroprotective effects in myenteric plexus in rats with experimental DM.

Myelofibrosis is one of the BCR-ABL-negative clonal disorders that collectively are known as myeloproliferative neoplasms (MPNs). It is caused by the proliferation of clonal hematopoietic stem cells, which over time leads to characteristic clinical features. The disease presentation is heterogeneous, however, with 30% of patients initially asymptomatic. This variation in clinical phenotype warrants careful risk stratification to guide appropriate management, and prognostic risk scores are continually being refined. Considerable advancements have been made in the understanding of MPN pathogenesis, in particular recognition of the driver mutations JAK2 V617F, CALR, and MPL, which has led to the development of ruxolitinib, an inhibitor of Janus kinase 1 (JAK1) and JAK2 that has transformed therapy for myelofibrosis. Although ruxolitinib decreases symptoms and is associated with a survival advantage, it has no clear disease-altering activity, and allogeneic hematopoietic stem cell transplant remains the sole curative option for myelofibrosis. Ongoing studies are evaluating newer JAK inhibitors, combinations of ruxolitinib with other targeted drugs, and targeted therapies that do not inhibit JAK. This review provides further detail regarding the clinical features, pathogenesis, risk stratification, and current management of myelofibrosis, including older and newer targeted treatments.

Despite advances in the various treatment options for esophageal squamous cell carcinoma (ESCC), its prognosis is still very poor with a 5-year survival rate of only 14-22%. Recently, among the various therapeutic approaches, the focus has shifted to immunotherapy, specifically immunotherapy involving dendritic cells (DCs), which depends on their maturation and antigen presentation to effector immune cells. Recent studies have suggested that melanoma-associated antigen 3 (MAGE-A3) is a potential immunotherapeutic target and also a candidate for the development of an anti-tumor vaccine. Calreticulin (CALR) has been shown to support induction of DC maturation. Therefore, in this study, we overexpressed MAGE-A3 and CALR on DCs and studied their potential to generate anti-tumor immune responses. We observed that adenovirus (Ad)-infected DCs overexpressing CALR and MAGE-A3 showed enhanced expression of CD80, CD83, CD86, and HLA-DR markers. Also, these DCs secreted higher levels of interleukin (IL)-12, which induces the T helper type 1 cell (Th1) response, and a lower level of IL-10, a negative regulator of the Th1 response. Furthermore, CALR/MAGE-A3-infected DCs stimulated CD8(+) cytotoxic T lymphocytes, which in turn secreted higher levels of interferon-γ, which induced cytotoxic effects on ESCC cells expressing MAGE-A3. In conclusion, our results revealed the potential of CALR/MAGE-A3-infected DCs to elicit a MAGE-A3-specific anti-tumor immunogenic response in ESCC. This proof-of-principle study may promote the future design and development of DC-based effective immunotherapy against ESCC.

Background: Gliomas are the most common primary malignant brain tumors and have a poor prognosis. Early detection of gliomas is crucial to improve patient outcomes. Urine accumulates systematic body changes and thus serves as an excellent early biomarker source.

Methods: At the biomarker discovery phase, we performed a self-controlled proteomics analysis by comparing urine samples collected from five glioma patients at the time of tumor diagnosis and after surgical removal of the tumor. At the biomarker validation phase, we further validated some promising proteins using parallel reaction monitoring (PRM)-based targeted proteomics in another cohort, including glioma, meningioma, and moyamoya disease patients as well as healthy controls.

Results: Using label-free proteome quantitation (LFQ), we identified twenty-seven urinary proteins that were significantly changed after tumor resection, many of which have been previously associated with gliomas. The functions of these proteins were significantly enriched in the autophagy and angiogenesis, which are associated with glioma development. After targeted proteomics validation, we identified a biomarker panel (AACT, TSP4, MDHM, CALR, LEG1, and AHSG) with an area under the curve (AUC) value of 0.958 for the detection of gliomas. Interestingly, AACT, LEG1, and AHSG are also potential cerebrospinal fluid or blood biomarkers of gliomas.

Conclusions: Using LFQ and PRM proteome quantification, we identified candidate urinary protein biomarkers with the potential to detect gliomas. This study will also provide clues for future biomarker studies involving brain diseases.

Keywords: Biomarkers; Glioma; Proteomics; Urine.

BACKGROUND

Genetic factors play a role in the etiology of BCR-ABL-negative myeloproliferative neoplasms (MPNs). This study explored the relationship between mutations in the Janus kinase 2 gene ( JAK2), MPL, and the calreticulin gene ( CALR) in Uygur and Han Chinese patients with BCR-ABL fusion gene-negative MPN and corresponding clinical features.

METHODS

A total of 492 BCR-ABL-negative MPN patients treated in our hospital from May 2013 to August 2016 were enrolled. Genomic DNA was extracted from peripheral blood and used for PCR amplification and DNA sequencing. Mutations including JAK2 V617F, MPL W515L/K, and those in JAK2 exon 12 and CALR were analyzed and compared with patient clinical characteristics.

RESULTS

Of the 492 MPN patients, 169 were Uygur and 323 were Han. In these two patient groups, JAK2 mutations were detected in 39.64% and 52.63%, respectively, CALR mutations were detected in 10.06% and 20.43%, respectively, and MPL mutations were detected in 0.93% of Han patients. The age, white blood cell count, platelet levels, and hemoglobin levels in JAK2 in Han patients were higher than those in Uygur patients.

CONCLUSIONS

Han MPN patients harboring JAK2 mutations had higher level of age, WBC, PLT, and Hb than Uyghur patients with the same mutations.

Background: The discovery of somatic acquired mutations of JAK2 (V617F) in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) has not only improved rational disease classification and prognostication but also brings new understanding insight into the pathogenesis of diseases. Dosage effects of the JAK2 (V617F) allelic burden in Ph-negative MPNs may partially influence clinical presentation, disease progression, and treatment outcome. Material and Methods: Pyrosequencing was performed to detect JAK2 (V617F) and MPL (W515K/L) and capillary electrophoresis to identify CALR exon 9.0 mutations in 100.0 samples of Ph-negative MPNs (38.0 PV, 55 ET, 4 PMF, and 3 MPN-U). Results: The results showed somatic mutations of JAK2 (V617F) in 94.7% of PV, 74.5% of ET, 25.0% of PMF, and all MPN-U. A high proportion of JAK2 (V617F) mutant allele burden (mutational load>> 50.0%) was predominantly observed in PV when compared with ET. Although a high level of JAK2 (V617F) allele burden was strongly associated with high WBC counts in both PV and ET, several hematological parameters (hemoglobin, hematocrit, and platelet count) were independent of JAK2 (V617F) mutational load. MPL (W515K/L) mutations could not be detected whereas CALR exon 9.0 mutations were identified in 35.7% of patients with JAK2 negative ET and 33.3% with JAK2 negative PMF. Conclusions: The JAK2 (V617F) allele burden may be involved in progression of MPNs. Furthermore, a high level of JAK2 (V617F) mutant allele appears strongly associated with leukocytosis in both PV and ET.

UNASSIGNED

Familial partial lipodystrophy of the Dunnigan type is one of the most common inherited lipodystrophies variables. These individuals have important metabolic disorders that cause predisposition to various diseases. In this study we aimed to demonstrate the relation between the metabolic abnormalities, inflammatory profile and the expression of genes involved in the activation of the endoplasmic reticulum stress (ERS) in subjects with FPLD.

UNASSIGNED

We evaluated 14 female FPLD patients and compared with 13 female healthy individuals. The subjects were paired with their respective BMI and age and categorized into two groups: Familial partial lipodystrophy of the Dunnigan type (FPLD) and control. Patients were fasted for 12 h before blood collection for measurement of HbA1c, glucose, insulin, lipids and inflammatory markers. Subcutâneous adipose tissue was collected by puncture aspiration of submental region during ambulatorial surgical aesthetic procedure.

UNASSIGNED

We demonstrate that patients with FPLD show increased HbA1c (p < 0.01), fasting glucose (p < 0.002) and triglycerides (p < 0.005) while HDL/cholesterol (p < 0.001) was lower when compared to healthy individuals. We found that 64.2% FPLD patients had metabolic syndrome according to International Diabetes Federation definition. We also observe increased AUC of glucose (p < 0.001) and insulin during oGTT, featuring a frame of hyperglycemia and hyperinsulinemia, suggesting insulin resistance. Also we found hyperactivation of several genes responsible for ERS such as ATF-4 (p < 0.01), ATF-6 (p < 0.01), EIF2α3K (p < 0.005), CCT4 (p < 0.001), CHOP (p < 0.01), CALR (p < 0.001) and CANX (p < 0.005), that corroborate the idea that diabetes mellitus and metabolic syndrome are associated with direct damage to the endoplasmic reticulum homeostasis. Ultimately, we note that individuals with lipodystrophy have an increase in serum interleukins, keys of the inflammatory process, as IL-1β, TNF-α and IL-6 (p < 0.05 all), compared with healthy individuals, which can be the trigger to insulin resistance in this population.

UNASSIGNED

Individuals with FPLD besides having typical dysfunctions of metabolic syndrome, show a hyperactivation of ERS associated with increased systemic inflammatory profile, which together may explain the complex clinical aspect of this diseases.Trial registration HCRP no 6711/2012.

Recurrent mutations in calreticulin (CALR) are present in approximately 20% of patients with myeloproliferative neoplasms (MPN). Since its discovery in 2013, we now have a more precise understanding of how mutant CALR, an endoplasmic reticulum chaperone protein, activates the JAK/STAT signaling pathway via a pathogenic binding interaction with the thrombopoietin receptor, MPL, to induce MPN. In this Spotlight, we review the current understanding of the biology underpinning mutant CALR driven MPN, discuss clinical implications, and highlight future therapeutic approaches.

A high number of circulating CD34+ cells has been advocated to distinguish primary myelofibrosis from other Philadelphia-negative myeloproliferative neoplasms. We re-evaluated the diagnostic interest of measuring circulating CD34+ cells in 26 healthy volunteers and 256 consecutive patients at diagnosis for whom a myeloproliferative neoplasm was suspected. The ROC curve analysis showed that a number of CD34+ <10/μl excludes the diagnosis of primary myelofibrosis with a sensitivity of 97 % and a specificity of 90 % (area under the curve: 0.93 [0.89-0.98]; p < 0.001). Patients with PMF harboring a CALR mutation had more circulating CD34+ cells than patients with either a JAK 2 or MPL mutation (p = 0.02 and p < 0.01, respectively). These results suggest that this fast, simple, non-invasive, and standardized test is of particular interest to exclude the diagnosis of primary myelofibrosis.

Vibrio parahaemolyticus is the leading cause of seafood-associated gastroenteritis. Type III secretion system 1 (T3SS1) is one of the virulence determinants of this bacteria. T3SS1 expression is regulated by ToxR and CalR. ToxR represses the transcription of T3SS1 genes via activation of CalR, which acts as a transcriptional repressor of T3SS1 genes. However, the transcriptional regulation mechanisms have not been elucidated. As showing in the present work, ToxR binds to the promoter DNA region of calR to activate its transcription. CalR occupies the promoter-proximal regions of each detected target operons in T3SS1 loci to repress their transcription, and thereby inhibiting T3SS1-dependent cytotoxicity. Moreover, a feedback CalR inhibits toxR and its own gene in a direct manner. Collectively, this work reported an interesting gene regulatory network involving the reciprocal regulation of ToxR and CalR, and their regulation on T3SS1 genes transcription in V. parahaemolyticus.

Background: Mitoxantrone (MTX) is a synthetic compound used as a second line chemotherapeutic drug for prostate cancer. It has been reported to trigger immunogenic cell death (ICD) in animal model studies, but the underlying mechanism is not fully understood yet, especially not in prostate cancer cells.

Methods: ICD was determined by assessing the release of damage-associated molecular patterns (DAMPs) in the prostate cancer-derived cell lines LNCaP, 22RV1 and PC-3. Short hairpin RNAs (shRNAs) were used to knock down target gene expression. Phagocytosis was assessed using a dual labeling technology in dendric cells co-cultured with cancer cells. The PERK gene promoter was cloned for dual luciferase assays. Chromatin immunoprecipitation (ChIP) was used to determine p53 protein-DNA binding activity. Immunocompetent mice and murine RM-1 prostate cancer cells were used for vaccination experiments.

Results: MTX treatment induced typical characteristics of DAMP release, including increased cell surface exposure of calreticulin (CALR), and extracellular release of ATP and high mobility group box-1 (HMGB1) protein. MTX also enhanced phagocytosis by dendritic cells. Moreover, MTX treatment increased eukaryotic initiation factor 2α (eIF2α) S51 phosphorylation, which was reduced when PERK and GCN2 were silenced using shRNAs. In addition, PERK or GCN2 silencing significantly reduced MTX-induced release of DAMPs in vitro and anti-tumor immunity in vivo. MTX treatment also resulted in dendritic cell activation in mice, which was attenuated when PERK or GCN2 were silenced in cancer cells used for vaccination. Further analysis revealed that PERK and GCN2 expression was enhanced by MTX treatment, of which PERK, but not GCN2, was enhanced via a p53-dependent mechanism.

Conclusion: MTX triggers ICD by activating eIF2α via PERK/GCN2 upregulation in prostate cancer cells. MTX-induced PERK expression upregulation depends on the p53 pathway, while that of GCN2 requires further investigation.

Keywords: GCN2; Immunogenic cell death; Mitoxantrone; PERK; Prostate cancer; eIF2α; p53.

Myeloproliferative neoplasms (MPNs) are generally acquired as a result of a somatic stem cell mutation leading to clonal expansion of myeloid precursors. In addition to sporadic cases, familial MPN occurs when one or several MPN affect different relatives of the same family. MPN driver mutations (JAK2, CALR, MPL) are somatically acquired also in familial cases, so a genetic predisposition to acquire one of the MPN driver mutations would be inherited, even though the causative germline mutations underlying familial MPN remain largely unknown. Recently some germline variants [ATG2B and GSKIP duplication, RBBP6 mutations, SH2B3 (LNK) mutations], which can cause familial MPN, have been reported but these mutations are rare and do not explain most familial cases. Patients with familial MPN show the same clinical features and suffer the same complications as those with sporadic disease. This review aims to offer up-to-date information regarding the genetics of familial MPN.

Patients with high-risk myeloproliferative neoplasms (MPNs), and in particular myelofibrosis (MF), can be cured only with allogeneic hematopoietic stem cell transplantation (HSCT). Because MPNs and JAK2^V617F-mutated cells show genomic instability, stalled replication forks, and baseline DNA double-strand breaks, DNA repair inhibition with poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors represents a potential novel therapy. Because the alkylating agent busulfan is integral in conditioning regimens for HSCT and leads to stalled replication forks through DNA strand cross-linking, we hypothesized that PARP inhibition with veliparib in combination with busulfan may lead to synergistic cytotoxicity in MPN cells. We first treated 2 MPN cell lines harboring the JAK2^V617F mutation (SET2 and HEL) with veliparib at increasing concentrations and measured cell proliferation. SET2 and HEL cells were relatively sensitive to veliparib (IC₅₀ of 11.3 μM and 74.2 μM, respectively). We next treated cells with increasing doses of busulfan in combination with 4 μM veliparib and found that the busulfan IC₅₀ decreased from 27 μM to 4 μM in SET2 cells and from 45.1 μM to 28.1 μM in HEL cells. The mean combination index was .55 for SET2 cells and .40 for HEL cells. Combination treatment of SET2 cells caused G2M arrest in 53% of cells, compared with 30% with veliparib alone and 35% with busulfan alone. G2M arrest was associated with activation of the ATR-Chk1 pathway, as shown by an immunofluorescence assay for phosphorylated Chk1 (p-Chk1). We then tested in vivo the effect of combined low doses of busulfan and veliparib in a JAK2^V617F MPN-AML xenotransplant model. Vehicle- and veliparib-treated mice had similar median survival of 39 and 40 days, respectively. Combination treatment increased median survival from 47 days (busulfan alone) to 50 days (P = .02). Finally, we tested the combined effect of busulfan and veliparib on CD34⁺ cells obtained from the bone marrow or peripheral blood of 5 patients with JAK2^V617F-mutated and 2 patients with CALR-mutated MF. MF cells treated with the combination of veliparib and busulfan showed reduced colony formation compared with busulfan alone (87% versus 68%; P = .001). In contrast, treatment of normal CD34⁺ cells with veliparib did not affect colony growth. Here we show that in vivo confirmation that treatment with the PARP-1 inhibitor veliparib and busulfan results in synergistic cytotoxicity in MPN cells. Our data provide the rationale for testing novel pretransplantation conditioning regimens with combinations of PARP-1 inhibition and reduced doses of alkylators, such as busulfan and melphalan, for high-risk MPNs or MPN-derived acute myelogenous leukemia.

Myeloproliferative neoplasms (MPNs) are hematopoietic stem cell disorders with dysregulated myeloid blood cell production and propensity for transformation to acute myeloid leukemia, thrombosis, and bleeding. Acquired mutations in JAK2, MPL, and CALR converge on hyperactivation of Janus kinase 2 (JAK2) signaling as a central feature of MPN. Accordingly, JAK2 inhibitors have held promise for therapeutic targeting. After the JAK1/2 inhibitor ruxolitinib, similar JAK2 inhibitors as fedratinib are entering clinical use. While patients benefit with reduced splenomegaly and symptoms, disease-modifying effects on MPN clone size and clonal evolution are modest. Importantly, response to ruxolitinib may be lost upon treatment suggesting the MPN clone acquires resistance. Resistance mutations, as seen with other tyrosine kinase inhibitors, have not been described in MPN patients suggesting that functional processes reactivate JAK2 signaling. Compensatory signaling, which bypasses JAK2 inhibition, and other processes contribute to intrinsic resistance of MPN cells restricting efficacy of JAK2 inhibition overall. Combinations of JAK2 inhibition with pegylated interferon-α, a well-established therapy of MPN, B-cell lymphoma 2 inhibition, and others are in clinical development with the potential to enhance therapeutic efficacy. Novel single-agent approaches targeting other molecules than JAK2 are being investigated clinically. Special focus should be placed on myelofibrosis patients with anemia and thrombocytopenia, a delicate patient population at high need for options. The extending range of new treatment approaches will increase the therapeutic options for MPN patients. This calls for concomitant improvement of our insight into MPN biology to inform tailored therapeutic strategies for individual MPN patients.

Megakaryocytes give rise to platelets, which have a wide variety of functions in coagulation, immune response, inflammation, and tissue repair. Dysregulation of megakaryocytes is a key feature of in the myeloproliferative neoplasms, especially myelofibrosis. Megakaryocytes are among the main drivers of myelofibrosis by promoting myeloproliferation and bone marrow fibrosis. In vivo targeting of megakaryocytes by genetic and pharmacologic approaches ameliorates the disease, underscoring the important role of megakaryocytes in myeloproliferative neoplasms. Here we review the current knowledge of the function of megakaryocytes in the JAK2, CALR, and MPL-mutant myeloproliferative neoplasms.

Keywords: JAK/STAT; Megakaryocyte; Myeloproliferative neoplasms.

Systemic anticancer immunity can be reinstated via the induction of immunogenic cell death (ICD) in malignant cells. Thus, certain classes of cytotoxic compounds, for example, anthracyclines, oxaliplatin and taxanes are endowed with the capacity to act on cancer cells to ignite premortem stress pathways that lead to the surface exposure of calreticulin (CALR) and the cellular release of adenosine triphosphate, annexin A1, high mobility group B1 and type-1 interferons. Altogether, these alterations constitute the hallmarks of ICD. Here we report the design of a discovery pipeline for the identification of novel ICD inducers by means of a phenotypic screening platform. The use of fluorescent biosensors as proxies for the manifestation of ICD hallmarks has enabled the exploration of large collections of chemical compounds by automatized screening routines. Imaging-based assessment and phenotypic selection led to the identification of potential ICD inducers that could be validated further invitro and invivo, confirming that bona fide ICD inducers possess the capacity to induce immunological long-term memory and to confer resistance against rechallenge with syngeneic tumors. Machine learning algorithms analyzing the physicochemical properties of ICD inducers can assist in the preselection of compounds with potential ICD-stimulatory properties, further accelerating the screening efforts designed to develop new immunotherapeutic agents.

Development of cortical interneurons continues until the end of human pregnancy. Premature birth deprives the newborns from the supply of maternal estrogen and a secure intrauterine environment. Indeed, preterm infants suffer from neurobehavioral disorders. This can result from both preterm birth and associated postnatal complications, which might disrupt recruitment and maturation of cortical interneurons. We hypothesized that interneuron subtypes, including parvalbumin-positive (PV+), somatostatin-positive (SST+), calretinin-positive (CalR+), and neuropeptide Y-positive (NPY+) interneurons, were recruited in the upper and lower cortical layers in a distinct manner with advancing gestational age. In addition, preterm birth would disrupt the heterogeneity of cortical interneurons, which might be reversed by estrogen treatment. These hypotheses were tested by analyzing autopsy samples from premature infants and evaluating the effect of estrogen supplementation in prematurely delivered rabbits. The PV+ and CalR+ neurons were abundant, whereas SST+ and NPY+ neurons were few in cortical layers of preterm human infants. Premature birth of infants reduced the density of PV+ or GAD67+ neurons and increased SST+ interneurons in the upper cortical layers. Importantly, 17 β-estradiol treatment in preterm rabbits increased the number of PV+ neurons in the upper cortical layers relative to controls at postnatal day 14 (P14) and P21 and transiently reduced SST population at P14. Moreover, protein and mRNA levels of Arx, a key regulator of cortical interneuron maturation and migration, were higher in estrogen-treated rabbits relative to controls. Therefore, deficits in PV+ and excess of SST+ neurons in premature newborns are ameliorated by estrogen replacement, which can be attributed to elevated Arx levels. Estrogen replacement might enhance neurodevelopmental outcomes in extremely preterm infants.SIGNIFICANCE STATEMENT Premature birth often leads to neurodevelopmental delays and behavioral disorders, which may be ascribed to disturbances in the development and maturation of cortical interneurons. Here, we show that preterm birth in humans is associated with reduced population of parvalbumin-positive (PV+) neurons and an excess of somatostatin-expressing interneurons in the cerebral cortex. More importantly, 17 β-estradiol treatment increased the number of PV+ neurons in preterm-born rabbits, which appears to be mediated by an elevation in the expression of Arx transcription factor. Hence the present study highlights prematurity-induced reduction in PV+ neurons in human infants and reversal in their population by estrogen replacement in preterm rabbits. Because preterm birth drops plasma estrogen level 100-fold, estrogen replacement in extremely preterm infants might improve their developmental outcome and minimize neurobehavioral disorders.