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Acquired aplastic anemia and severe congenital neutropenia (SCN) are bone marrow (BM) failure syndromes of different origin, however, they share a common risk for secondary leukemic transformation. Here, we present a patient with severe aplastic anemia (SAA) evolving to secondary chronic neutrophilic leukemia (CNL; SAA-CNL). We show that SAA-CNL shares multiple somatic driver mutations in CSF3R, RUNX1, and EZH2/SUZ12 with cases of SCN that transformed to myelodysplastic syndrome or acute myeloid leukemia (AML). This molecular connection between SAA-CNL and SCN progressing to AML (SCN-AML) prompted us to perform a comparative transcriptome analysis on nonleukemic CD34high hematopoietic stem and progenitor cells, which showed transcriptional profiles that resemble indicative of interferon-driven proinflammatory responses. These findings provide further insights in the mechanisms underlying leukemic transformation in BM failure syndromes.

Zusammenfassung. Moderne Leukämie-Diagnostik ist eine integrative Leistung und beinhaltet die Kombination von Befunden aus Morphologie (zytologisch und histologisch) sowie der durchflusszytometrischen Immunphänotypisierung und der genetischen Analytik, welche mittels verschiedener Techniken von der grobmorphologischen Chromosomenanalyse bis zur Beurteilung von Punktmutationen reicht. Diese Untersuchungen sind in weiten Teilen quantitativ und erlauben nicht nur eine genaue Beurteilung und Prognosestellung zur vorliegenden Krankheit, sondern auch die Möglichkeit, im Verlauf Resterkrankung zu detektieren. Die verschiedenen Techniken werden hier vorgestellt.

Patients with acute myeloid leukaemia (AML) often achieve remission after therapy, but subsequently die of relapse1 that is driven by chemotherapy-resistant leukaemic stem cells (LSCs)2,3. LSCs are defined by their capacity to initiate leukaemia in immunocompromised mice4. However, this precludes analyses of their interaction with lymphocytes as components of anti-tumour immunity5, which LSCs must escape to induce cancer. Here we demonstrate that stemness and immune evasion are closely intertwined in AML. Using xenografts of human AML as well as syngeneic mouse models of leukaemia, we show that ligands of the danger detector NKG2D—a critical mediator of anti-tumour immunity by cytotoxic lymphocytes, such as NK cells6–9—are generally expressed on bulk AML cells but not on LSCs. AML cells with LSC properties can be isolated by their lack of expression of NKG2D ligands (NKG2DLs) in both CD34-expressing and non-CD34-expressing cases of AML. AML cells that express NKG2DLs are cleared by NK cells, whereas NKG2DL-negative leukaemic cells isolated from the same individual escape cell killing by NK cells. These NKG2DL-negative AML cells show an immature morphology, display molecular and functional stemness characteristics, and can initiate serially re-transplantable leukaemia and survive chemotherapy in patient-derived xenotransplant models. Mechanistically, poly-ADP-ribose polymerase 1 (PARP1) represses expression of NKG2DLs. Genetic or pharmacologic inhibition of PARP1 induces NKG2DLs on the LSC surface but not on healthy or pre-leukaemic cells. Treatment with PARP1 inhibitors, followed by transfer of polyclonal NK cells, suppresses leukaemogenesis in patient-derived xenotransplant models. In summary, our data link the LSC concept to immune escape and provide a strong rationale for targeting therapy-resistant LSCs by PARP1 inhibition, which renders them amenable to control by NK cells in vivo. Leukaemic stem cells in acute myeloid leukaemia are defined by a lack of expression of NKG2D ligands, which mediates their ability to evade surveillance by NK cells.

This translational study aimed at gaining insight into the effects of lenalidomide in acute myeloid leukemia (AML). Forty-one AML patients aged 66 or older of the Swiss cohort of the HOVON-103 AML/SAKK30/10 study were included. After randomization, they received standard induction chemotherapy with or without lenalidomide. Bone marrow biopsies at diagnosis and before the 2nd induction cycle were obtained to assess the therapeutic impact on leukemic blasts and microenvironment. Increased bone marrow angiogenesis, as assessed by microvessel density (MVD), was found at AML diagnosis and differed significantly between the WHO categories. Morphological analysis revealed a higher initial MVD in AML with myelodysplasia-related changes (AML-MRC) and a more substantial decrease of microvascularization after lenalidomide exposure. A slight increase of T-bet-positive TH1-equivalents was identifiable under lenalidomide. In the subgroup of patients with AML-MRC, the progression-free survival differed between the two treatment regimens, showing a potential but not significant benefit of lenalidomide. We found no correlation between the cereblon genotype (the target of lenalidomide) and treatment response or prognosis. In conclusion, addition of lenalidomide may be beneficial to elderly patients suffering from AML-MRC, where it leads to a reduction of microvascularization and, probably, to an intensified specific T cell-driven anti-leukemic response. Supplementary Information The online version contains supplementary material available at 10.1007/s00277-021-04467-2.

ISSN:0006-4971 ISSN:1528-0020

Background: RUNX1 is a crucial transcription factor for hematological stem cells and well-known for its association with acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML). Besides the translocation t(8; 21) that leads to the RUNX1-RUNX1T1 fusion, somatic mutations of RUNX1 have been discovered. Methods: Four bone marrow trephine biopsies of patients with CD79a-positive and/or PAX5-positive acute leukemias were investigated by immunohistochemistry (IHC), karyotyping, and next-generation sequencing-based genetic analysis. Data were then compared to a historical collective of AML (n = 42) and 42 cases of AML newly diagnosed at our institution between June 2017 and May 2018. Results: We report on 4 cases of acute leukemia with an equivocal immunophenotype showing expression of CD79a and/or PAX5, which led to a preliminary histopathologic classification as probable ALL/unclassifiable acute leukemia. All cases were positive for CD34 and TdT but negative for several myeloid markers on IHC. Mutational analysis revealed point mutations and indels of RUNX1 and further mutations typical for AML such as TET2, DNMT3A, and SRSF2, and 2 cases had tetrasomy 13 characteristic of RUNX1 mutant AML. Conclusion: Aberrant CD79a and/or PAX5 expression can be found in AML cases with RUNX1 mutations even without the translocation t(8; 21). Our series shows the expression of CD79a and PAX5 to be a potential pitfall in the classification of RUNX1 mutant acute leukemia.

Diffuse large B-cell lymphoma is the most common malignant lymphoma in adults. By gene-expression profiling, this lymphoma is divided in three cell-of-origin subtypes with distinct molecular and clinical features. Most lymphomas arise sporadically, yet familial clustering is known, suggesting a genetic contribution to disease risk. Familial lymphoma cases are a valuable tool to investigate risk genes. We studied a Swiss/Japanese family with 2 sisters affected by a primary mediastinal B-cell lymphoma and a non-germinal center diffuse large B-cell lymphoma not otherwise specified, respectively. The somatic landscape of both lymphomas was marked by alterations affecting multiple components of the JAK-STAT pathway. Consequently, this pathway was constitutively activated as evidenced by high pJAK2 as well as increased nuclear pSTAT3 and pSTAT6 in malignant cells. Potential lymphoma risk variants were identified by whole exome sequencing of the germline DNA derived from siblings and unaffected family members. This analysis revealed a pathogenic variant in TIRAP, an upstream regulator of NF-κB, in both affected siblings and their mother. We observed increased B-cell proliferation in family members harboring the TIRAP p.R81C variant. B-cell proliferation correlated with TIRAP and NF-κB target gene expression, suggesting enhanced NF-κB pathway activity in TIRAP p.R81C individuals. TIRAP knockdown reduced B-cell survival and NF-κB target gene expression, particularly in individuals with TIRAP p.R81C. Functional studies revealed significantly increased NF-κB activity and resistance to stress-induced cell-death by TIRAP p.R81C. The identification of an inherited TIRAP variant provides evidence for a novel link between genetic alterations affecting the NF-κB pathway and lymphomagenesis.

Myeloproliferative neoplasms (MPN) are characterized by abnormal growth of functional and differentiated blood cells in the bone marrow. The generic term MPN includes, among others, polycythemia ve...

A novel platform of fluorescently labeled nanocarriers (NCs) is herein proposed based on amphiphilic linear-dendritic polymeric hybrids. These sophisticated polymers were synthesized with a high degree of structural control at a macro-molecular level, displayed hydrophobic cholesterol compartments as chain-terminus groups of the dendritic block and hydrophilic bifunctional linear poly(ethylene glycol) (PEG) block. Spherical supramolecular assemblies with therapeutically relevant properties were successfully achieved including (i) sizes in the region of 100 to 200 nm; (ii) narrow dispersity profile with values close to 0.12; and (iii) self-assembly down to nanomolar concentrations. The modular nature of the NCs permitted the encapsulation of single or dual anticancer drugs and in parallel provide intracellular fluorescent traceability. As polymer therapeutics, the NCs were proven to penetrate the cancerous cell membranes and deliver the cargo of drugs into the nuclei as well as the cytoplasm and mitochondr...

The β-3 sympathomimetic agonist BRL37344 restored nestin-positive cells within the stem cell niche, and thereby normalized blood counts and improved myelofibrosis in a mouse model of JAK2-V617F-positive myeloproliferative neoplasms. We therefore tested the effectiveness of mirabegron, a β-3 sympathomimetic agonist, in a phase II trial including 39 JAK2-V617F-positive patients with myeloproliferative neoplasms and a mutant allele burden more than 20%. Treatment consisted of mirabegron 50 mg daily for 24 weeks. The primary end point was reduction of JAK2-V617F allele burden of 50% or over, but this was not reached in any of the patients. One patient achieved a 25% reduction in JAK2-V617F allele burden by 24 weeks. A small subgroup of patients showed hematologic improvement. As a side study, bone marrow biopsies were evaluated in 20 patients. We found an increase in the nestin+ cells from a median of 1.09 (interquartile range 0.38-3.27)/mm2 to 3.95 (interquartile range 1.98-8.79)/mm2 (P

The β-3 sympathomimetic agonist BRL37344 restored nestin-positive cells within the stem cell niche, and thereby normalized blood counts and improved myelofibrosis in a mouse model of JAK2-V617F-positive myeloproliferative neoplasms. We therefore tested the effectiveness of mirabegron, a β-3 sympathomimetic agonist, in a phase II trial including 39 JAK2-V617F-positive patients with myeloproliferative neoplasms and a mutant allele burden more than 20%. Treatment consisted of mirabegron 50 mg daily for 24 weeks. The primary end point was reduction of JAK2-V617F allele burden of 50% or over, but this was not reached in any of the patients. One patient achieved a 25% reduction in JAK2-V617F allele burden by 24 weeks. A small subgroup of patients showed hematologic improvement. As a side study, bone marrow biopsies were evaluated in 20 patients. We found an increase in the nestin+ cells from a median of 1.09 (interquartile range 0.38-3.27)/mm2 to 3.95 (interquartile range 1.98-8.79)/mm2 (P

A new study reveals that leukemia stem cells (LSCs) in acute myeloid leukemia downregulate natural killer cell‐activating receptor ligands to evade immune surveillance via the transcriptional co‐factor PARP1. The inhibition of PARP1 sensitizes LSCs to immunotherapy, highlighting its potential as a therapeutic target.

Summary Familial erythrocytosis with elevated erythropoietin levels is frequently caused by mutations in genes that regulate oxygen-dependent transcription of the gene encoding erythropoietin (EPO). We identified a mutation in EPO that cosegregated with disease with a logarithm of the odds (LOD) score of 3.3 in a family with autosomal dominant erythrocytosis. This mutation, a single-nucleotide deletion (c.32delG), introduces a frameshift in exon 2 that interrupts translation of the main EPO messenger RNA (mRNA) transcript but initiates excess production of erythropoietin from what is normally a noncoding EPO mRNA transcribed from an alternative promoter located in intron 1. (Funded by the Gebert Ruf Foundation and others.)

Herein we develop a facile synthetic strategy for the functionalization of well-defined polyether copolymers with control over the number and location of catechol groups. Previously, the functionalization of polyethylene oxide (PEO)-based polymers with catechols has been limited to functionalization of the chain ends only, hampering the synthesis of adhesive and antifouling materials based on this platform. To address this challenge, we describe an efficient and high-yielding route to catechol-functionalized polyethers, which could allow the effects of polymer architecture, molecular weight, and catechol incorporation on the adhesive properties of surface-anchored PEO to be studied. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2015, 53, 2685–2692

We herein report the development of crosslinked polyether particles as a reactive platform for the preparation of functional microgels. Thiol-ene crosslinking of poly(allyl glycidyl ether) in miniemulsion droplets - stabilized by a surface active, bio-compatible polyethylene glycol block copolymer - resulted in colloidal gels with a PEG corona and an inner polymeric network containing reactive allyl units. The stability of the allyl groups allows the microgels to be purified and stored before a second, subsequent thiol-ene functionalization step allows a wide variety of pH- and chemically-responsive groups to be introduced into the nanoparticles. The facile nature of this synthetic platform enables the preparation of microgel libraries that are responsive to different triggers but are characterized by the same size distribution, surface functionality, and crosslinking density. In addition, the utilization of a crosslinker containing cleavable ester groups renders the resulting hydrogel particles degradable at elevated pH or in the presence of esterase under physiological conditions.

An amphiphilic block copolymer surfactant is used to impart peripheral surface functionality to polymer nanoparticles synthesized via emulsion polymerization. Particles ranged in size from ca. 55 nm by SEM (ca. 82 nm by DLS) to just over 200 nm. Particles displaying latent functionality were readily functionalized directly after polymerization using a fluorescent dye.

Histamine functionalized block copolymers based on poly(allyl glycidyl ether)-b-poly(ethylene oxide) (PAGE-b-PEO) were prepared with different ratios of histamine and octyl or benzyl groups using UV-initiated thiol-ene click chemistry. At neutral pH, the histamine units are uncharged and hydrophobic, while in acidic environments, such as in the endosome, lysosomes, or extracellular sites of tumours, the histamine groups are positively charged and hydrophilic. pH responsible polymer drug delivery systems is a promising route to site specific delivery of drugs and offers the potential to avoid side effects of systemic treatment. Our detailed in vitro experiments of the efficacy of drug delivery and the intracellular localization characteristics of this library of NPs in 2D and 3D cultures of breast cancer revealed that the 50% histamine-modified polymer loaded with DOX exhibited rapid accumulation in the nucleus of free DOX within 2 h. Confocal studies showed enhanced mitochondrial localization and lysosomal escape when compared to controls. From these combined studies, it was shown that by accurately tuning the structure of the initial block copolymers, the resulting self-assembled NPs can be designed to exploit histamine as an endosomal escape trigger and the octyl/benzyl units give rise to a hydrophobic core resulting in highly efficacious drug delivery systems (DDS) with control over intracellular localization. Optimization and rational control of the intracellular localization of both DDS and the parent drug can give nanomedicines a substantial increase in efficacy and should be explored in future studies.

Investigation of cancer cell lines is important for oncology research to characterize and understand mechanisms of cellular signaling and survival strategies in cancer. We analyzed the mutational profile of 11 diffuse large B-cell lymphoma (DLBCL) cell lines using a customized high throughput sequencing panel. We compared our data to previously published mutation data to better characterize these cell lines and establish consensus on the mutational status of some functionally relevant genes. With our targeted sequencing approach we detected 61 somatic mutations. The most frequently affected gene was TP53. MYD88 mutations were only seen in activated B-cell like cell lines. Overall comparison across different datasets revealed that just around 38% of mutations are reliable and can be validated by at least two independent observations whereas 24% of mutations could not be validated. Our analysis reveals considerable discrepancies regarding the mutational profile of some well-established cell lines.

Myeloproliferative neoplasms (MPNs) are diseases caused by mutations in the haematopoietic stem cell (HSC) compartment. Most MPN patients have a common acquired mutation of Janus kinase 2 (JAK2) gene in HSCs that renders this kinase constitutively active, leading to uncontrolled cell expansion. The bone marrow microenvironment might contribute to the clinical outcomes of this common event. We previously showed that bone marrow nestin(+) mesenchymal stem cells (MSCs) innervated by sympathetic nerve fibres regulate normal HSCs. Here we demonstrate that abrogation of this regulatory circuit is essential for MPN pathogenesis. Sympathetic nerve fibres, supporting Schwann cells and nestin(+) MSCs are consistently reduced in the bone marrow of MPN patients and mice expressing the human JAK2(V617F) mutation in HSCs. Unexpectedly, MSC reduction is not due to differentiation but is caused by bone marrow neural damage and Schwann cell death triggered by interleukin-1β produced by mutant HSCs. In turn, in vivo depletion of nestin(+) cells or their production of CXCL12 expanded mutant HSC number and accelerated MPN progression. In contrast, administration of neuroprotective or sympathomimetic drugs prevented mutant HSC expansion. Treatment with β3-adrenergic agonists that restored the sympathetic regulation of nestin(+) MSCs prevented the loss of these cells and blocked MPN progression by indirectly reducing the number of leukaemic stem cells. Our results demonstrate that mutant-HSC-driven niche damage critically contributes to disease manifestation in MPN and identify niche-forming MSCs and their neural regulation as promising therapeutic targets.

The interferon-γ (IFNγ)/signal transducer and activator of transcription 1 (Stat1) pathway shows higher activity in patients with essential thrombocythemia (ET) than in polycythemia vera (PV) and was proposed to be promoting the ET phenotype. We explored the phenotypic consequences of Stat1 deficiency on the effects of Janus kinase 2 (JAK2)-V617F in vivo by crossing mice expressing JAK2-V617F with Stat1 knockout mice. JAK2-V617F;Stat1(-/-) double transgenic mice showed higher red cell parameters and lower platelet counts compared with JAK2-V617F;Stat1(+/+) mice. Bone marrow transplantation reproduced these phenotypic changes in wild-type recipients, demonstrating that the effect of Stat1 is cell-intrinsic and does not require a Stat1-deficient microenvironment. Deletion of Stat1 increased burst-forming unit-erythroid and reduced colony-forming unit-megakaryocyte colony formation driven by JAK2-V617F, but was not sufficient to completely normalize the platelet count. Gata1, a key regulator of megakaryopoiesis and erythropoiesis, was decreased in Stat1-deficient platelets. V617F transgenic mice with thrombocytosis had higher serum levels of IFNγ than normal controls and patients with ET showed higher IFNγ serum levels than patients with PV. Together, these results support the concept that activating Stat1 in the presence of JAK2-V617F, for example, through IFNγ, constrains erythroid differentiation and promotes megakaryocytic development, resulting in ET phenotype.

Myeloproliferative neoplasms (MPNs) are a group of clonal disorders characterized by aberrant hematopoietic proliferation and an increased tendency toward leukemic transformation. We used targeted next-generation sequencing (NGS) of 104 genes to detect somatic mutations in a cohort of 197 MPN patients and followed clonal evolution and the impact on clinical outcome. Mutations in calreticulin (CALR) were detected using a sensitive allele-specific polymerase chain reaction. We observed somatic mutations in 90% of patients, and 37% carried somatic mutations other than JAK2 V617F and CALR. The presence of 2 or more somatic mutations significantly reduced overall survival and increased the risk of transformation into acute myeloid leukemia. In particular, somatic mutations with loss of heterozygosity in TP53 were strongly associated with leukemic transformation. We used NGS to follow and quantitate somatic mutations in serial samples from MPN patients. Surprisingly, the number of mutations between early and late patient samples did not significantly change, and during a total follow-up of 133 patient years, only 2 new mutations appeared, suggesting that the mutation rate in MPN is rather low. Our data show that comprehensive mutational screening at diagnosis and during follow-up has considerable potential to identify patients at high risk of disease progression.

Xenografts in immune suppressed mice are routinely used for in vivostudies on human acute myeloid leukemia (AML). However, a significant proportion of primary AML samples (»50-60% of cases) were engrafting in mice with particularly latency. Here, we report that changing the transplantation time-point from day (5:00 pm) to night (4:00 am) accelerates the engraftment of human AML cells in NOD/SCID/IL2Rgnull(NSG) mice. Equal numbers of freshly thawed AML cells from the same donor (n=5 patients) were transplanted into sublethally irradiated gender-matched 6 to 8 weeks old mice at day or at night (n=3-5 mice per condition and patient). Transplanted mice were monitored for leukemic engraftment in peripheral blood (PB), bone marrow (BM) and other hematopoietic organs using flow cytometry and immunohistochemistry. Enhanced leukemic engraftment was observed in night vs. day transplanted mice for each AML case (Fig. A). Interestingly, in 2/5 samples, engraftment was exclusively detected with the night transplant condition. Limiting dilutions showed that night transplantation allows leukemia (Fig. B) induction from lower cell numbers. Results were confirmed in a syngeneic MLL-PTD/FLT3-ITD mouse leukemia model (Fig. C): mice analyzed at the same time-point after night versus day transplantation showed enhanced splenomegaly (401.2 mg vs. 234.5 mg, p Intriguingly, mice transplanted at night showed stress-related behavioral abnormalities. We thus hypothesized that night transplantation induced catecholamine release may affect homing and leukemogenesis. ELISAs indeed revealed enhanced epinephrine levels in PB (318.00±12.96 vs. 122.20±47.11 ng/mL, p Surprisingly, even though all our AML samples express the homing molecule CXCR4, blocking CXCR4 (anti-CXCR4 antibody, 50mM) prior to transplantation could not counteract epinephrine-driven homing (0.032±0.017 vs. 0.038±0.025% leukemic cells in BM, p=0.53), indicating that the stimulating effect of adrenergic signaling on homing was independent of the SDF1-CXCR4 axis. Notably, transplantations of healthy human cord-blood CD34+ HSPCs or of mouse BM Lin- cells revealed no difference in the hematopoietic reconstitution in night vs. day transplantations (Fig. G). This indicates that the observed mechanisms are leukemia specific. To explore further potential mechanisms, RNAseq was performed on MS-5 cells, a mouse stromal cell line, treated with either epinephrine or vehicle control. Preliminary analyses indicate upregulated vitamin D receptor (VDR) expression (log2FC=1.18, p=1.32x1012) as potentially involved in catecholamine-induced homing of leukemic cells. We conclude that enhanced catecholamine levels (e.g. induced by night transplantation) accelerate leukemia development from AML cells, by enhancing the homing of leukemic cells to BM niches via SDF1-CXCRR4 independent, leukemic specific mechanisms. Figure Disclosures No relevant conflicts of interest to declare.

Dear Editor, A 25-year-old otherwise healthy patient was referred by his family physician for investigation of polyglobulia. His hemoglobin was 210 g/L (reference value 140– 180 g/L) with a hematocrit of 60 % (reference 38– 52 %). The family history was remarkable, as four relatives, including the patient’s mother, were affected by polyglobulia (Fig. 1). Clinical investigation was inconspicuous; in particular, no splenomegaly was noticed. After a total of six phlebotomies performed by his family physician within 8 months, the patient’s hemoglobin and hematocrit were 192 g/L and 56 %, respectively. All other blood values and the peripheral blood smear were otherwise unremarkable. A relative polyglobulia could be excluded by history and clinical examination. As the patient’s family history was indicative, we supposed a hereditary hemoglobinopathy with increased oxygen affinity. However, a high-performance liquid chromatography was unremarkable and the partial pressure of oxygen at which the hemoglobin is 50 % saturated (P50) was normal. Other obvious secondary causes of polyglobulia were also excluded. Erythropoietin (EPO) level was suppressed (

Myeloproliferative neoplasms (MPN) are a group of stem cell disorders predominantly occurring in elderly,1 whereas children are affected at much lower frequencies.2 Therefore, less is known about the mutational spectrum and the biology of childhood MPN.3, 4 Lower incidence of JAK2-V617F has been reported in childhood essential thrombocythemia (ET) and polycythemia vera (PV),5, 6 and in recent studies fewer CALR mutations were found in children with ET.7, 8, 9, 10

Histamine functionalized poly(allyl glycidyl ether)-b-poly(ethylene glycol)-b-poly(allyl glycidyl ether) (PAGE-PEO-PAGE) triblock copolymers represent a new class of physically cross-linked, pH-responsive hydrogels with significant potential for biomedical applications. These telechelic triblock copolymers exhibited abrupt and reversible hydrogelation above pH 7.0 due to a hudrophilic/hydrophobic transition of the histamine units to form a network of hydrophobic domains bridged by a hydrophilic PEO matrix. These hydrophobic domains displayed improved ordering upon increasing pH and self-assembled into a body centered cubic lattice at pH 8.0, while at lower concentrations formed well-defined micelles. Significantly, all materials were found to be non-toxic when evaluated on three different cell lines and suggests a range of medical and biomedical applications.

One approach to investigate gene function, by silencing the activity of certain proteins, is the usage of double stranded decoy oligodeoxynucleotides (ds decoy ODNs). Decoy, in this sense, is ds ODNs bearing the consensus binding sequence for a DNA-binding protein. This can be used in clinical settings to attenuate the effect of overexpressed transcription factors in tumor cells. We here choose to target the oncogenic protein Myc. Since oligonucleotides are poorly internalized to cells, a cell-penetrating peptide, TP10, was coupled to the Myc decoy, using two different strategies. Either TP10 was simply mixed with ds decoy ODNs forming complexes through non-covalent electrostatic interactions, or by having a nona-nucleotide overhang in one of the decoy strands, and adding a complementary PNA sequence coupled to an NLS sequence and TP10, which could hybridize to the Myc decoy. By using these strategies, uptake was significantly enhanced, especially with the co-incubation approach. Interestingly, various endocytosis inhibitors had no effect on the uptake pattern, suggesting that uptake of these complexes is not mediated via endocytosis. Finally, a decreased proliferative capacity was observed when treating the neuroblastoma cell line N2a with TP10-PNA conjugate hybridized to Myc decoy compared to naked Myc decoy and untreated cells. A dose-dependent decrease in proliferation was also observed in MCF-7 cells, when using both strategies. These results suggest an alternative way to efficiently deliver ds ODNs into cells using the cell-penetrating peptide TP10 and prevent tumor growth by targeting the oncogenic protein Myc.

© 2015 American Chemical Society. We describe robustly anchored triblock copolymers that adopt loop conformations on surfaces and endow them with unprecedented lubricating and antifouling properties. The triblocks have two end blocks with catecholanchoring groups and a looping poly(ethylene oxide) (PEO) midblock. The loops mediate strong steric repulsion between two mica surfaces. When sheared at constant speeds of ∼2.5 μm/s, the surfaces exhibit an extremely low friction coefficient of ∼0.002-0.004 without any signs of damage up to pressures of ∼2-3 MPa that are close to most biological bearing systems. Moreover, the polymer loops enhance inhibition of cell adhesion and proliferation compared to polymers in the random coil or brush conformations. These results demonstrate that strongly anchored polymer loops are effective for high lubrication and low cell adhesion and represent a promising candidate for the development of specialized highperformance biomedical coatings.

The pathogenesis of acquired myeloperoxidase (MPO) deficiency, a rare phenomenon observed in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), is unknown. MPO is a glycoprotein (GP) chaperoned by calreticulin (CALR) in the endoplasmic reticulum. Mutations in CALR are frequently found in patients with myelofibrosis (MF) and essential thrombocythemia (ET) with nonmutated Janus kinase 2 (JAK2). We hypothesized that acquired MPO deficiency in MPN could be associated with the presence of CALR mutations. A cohort of 317 patients with MPN (142 polycythemia vera [PV], 94 ET, and 81 MF) was screened for MPO deficiency. MPO deficiency was observed in 6/81 MF patients (7.4%), but not in PV or ET patients. Susceptibility to infections had been documented in 2/6 (33%) MPO-deficient patients. Five out of 6 patients with MPO deficiency carried a homozygous CALR mutation and were also deficient in eosinophilic peroxidase (EPX). In contrast, 1 patient with MF, a JAK2-V617F mutation, and MPO deficiency, carried 2 previously reported MPO mutations and showed normal EPX activity. Patients with homozygous CALR mutations had reduced MPO protein, but normal MPO messenger RNA (mRNA) levels supporting a posttranscriptional defect in MPO production. Finally, we demonstrate in vitro that in the absence of CALR, immature MPO protein precursors are degraded in the proteasome. Therefore, 4 decades after the first description of acquired MPO deficiency in MPN, we provide the molecular correlate associated with this phenomenon and evidence that CALR mutations can affect the biosynthesis of GPs.

Mutations in JAK2 exon 12 are frequently found in patients with polycythemia vera (PV) that do not carry a JAK2-V617F mutation. The majority of these patients display isolated erythrocytosis. We generated a mouse model that expresses JAK2-N542-E543del, the most frequent JAK2 exon 12 mutation found in PV patients. Mice expressing the human JAK2-N542-E543del (Ex12) showed a strong increase in red blood cell parameters but normal neutrophil and platelet counts, and reduced overall survival. Erythropoiesis was increased in the bone marrow and spleen, with normal megakaryopoiesis and absence of myelofibrosis in histopathology. Erythroid progenitors and precursors were increased in hematopoietic tissues, but the numbers of megakaryocytic precursors were unchanged. Phosphorylation Stat3 and Erk1/2 proteins were increased, and a trend toward increased phospho-Stat5 and phospho-Stat1 was noted. However, Stat1 knock out in Ex12 mice induced no changes in platelet or red cell parameters, indicating that Stat1 does not play a central role in mediating the effects of Ex12 signaling on megakaryopoiesis or erythropoiesis. Ex12 mice showed decreased expression of hepcidin and increased expression of transferrin receptor-1 and erythroferrone, suggesting that the strong erythroid phenotype in Ex12 mutant mice is favored by changes in iron metabolism that optimize iron availability to allow maximal production of red cells.

Four types of drug nanoparticles (NPs) based on amphiphilic hyperbranched block copolymers were developed for the delivery of the chemotherapeutic doxorubicin (DOX) to breast cancer cells. These carriers have their hydrophobic interior layer composed of the hyperbranched aliphatic polyester, Boltorn (R) H30 or Boltorn (R) H40, that are polymers of poly 2,2-bis (methylol) propionic acid (bis-MPA), while the outer hydrophilic shell was composed of about 5 poly(ethylene glycol) (PEG) segments of 5 or 10 kDa molecular weight. A chemotherapeutic drug DOX, was further encapsulated in the interior of these polymer micelles and was shown to exhibit a controlled release profile. Dynamic light scattering and transmission electron microscopy analysis confirmed that the NPs were uniformly sized with a mean hydrodynamic diameter around 110 nm. DOX-loaded H30-PEG10k NPs exhibited controlled release over longer periods of time and greater cytotoxicity compared with the other materials developed against our tested breast cancer cell lines. Additionally, flow cytometry and confocal scanning laser microscopy studies indicated that the cancer cells could internalize the DOX-loaded H30-PEG10k NPs, which contributed to the sustained drug release, and induced more apoptosis than free DOX did. These findings indicate that the H30-PEG10k NPs may offer a very promising approach for delivering drugs to cancer cells. (C) 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 50: 280-288, 2012

The mouse is an increasingly important system for the study of both normal and aberrant hematopoiesis. As a model organism, the mouse recapitulates much of human hematopoiesis; however, there are some important differences. Here, the basic approaches for analyzing hematopoiesis in mice are described. In particular, methods are provided for the collection and analysis of peripheral blood, flow cytometry analysis of peripheral blood, bone marrow, and spleen cells, and isolation and transplantation of bone marrow stem cells. Curr. Protoc. Mouse Biol. 1:323-346 © 2011 by John Wiley & Sons, Inc.

In the last decades, the fabrication of ordered nano- and microporous structures has attracted increasing interest due to their specific properties and multiple possible applications in electronics, as templates or in the biological field. The development of such materials has been favored by the introduction of the simple breath-figure templating method in the 1990’s. In order to fully exploit the potential of these porous materials, the use of advanced functional molecules as precursors is essential. One suitable class of molecules is the well-defined linear-dendritic hybrids (LD hybrids) family. The structural variations, multiple end-groups and possible amphiphilicity of these molecules are significant advantages that could lead to highly sophisticated functional materials with potential usage in biology. Therefore, this project was directed towards the synthesis of advanced LD hybrids and the evaluation of their ability to form ordered functional porous films.A degradation and toxicity study was initially conducted on polyester-based 2,2-bis(methylol)propionic acid (bis-MPA) dendrimers under physiological conditions to support the potential usage of these molecules for biological purposes. The materials were found to undergo a relatively fast depolymerization process at pH 7.5. Moreover, the initial dendrimer and its decomposition products were proven to be non-toxic for immune competent cells, allowing for the utilization of these molecules for biological applications.A linear-dendritic-linear hybrid library was successfully synthesized from biocompatible poly(ethylene glycol) (PEG), poly(e-caprolactone) (PCL) and bis-MPA building blocks using a combination of ring-opening polymerization (ROP)and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The materials, consisting of one long PEG block connected to the focal point of the dendron and several PCL arms attached at its periphery, were used to construct ordered porous films using the breath figure method. The polymeric architecture strongly affected the ordering of the films with a more regular morphology obtained from a more flexible polymer. Changing the semi-crystalline PCL to amorphous polylactide (PLA) also permitted the formation of porous arrays. Interestingly, films obtained from inverted structures possessing one long PCL block and several short PEG chains, also presented a regular morphology. Moreover they could be activated to exhibit multiple surface hydroxyl groups.To increase the number of orthogonal synthetic methodologies available for the preparation of advanced macromolecules, high molecular weight dendritic macrothiols were synthesized. These molecules were efficiently coupled to a number of core molecules via thiol-ene coupling, generating a comprehensive library of dendritic materials. This approach represents an attractive alternative to the commonly used, but potentially toxic, CuAAC.Exploiting the obtained results, a final LD hybrid was synthesized from atom transfer radical polymerization (ATRP) of 2-hydroxyethyl methacrylate (HEMA) derivatives and thiol-ene coupling (TEC) with macrothiols. This macromolecule was successfully utilized to form functional ordered porous arrays and the availability of peripheral alkyne functional groups was demonstrated by efficient coupling with fluorescent Rhodamine-B. The HEMA-backbone allowed for the introduction of cross-linkable azide groups that were used to significantly improve the thermal stability of the films from 50 °C to 200 °C. These materials have the potential to be used in applications such as catalysis, in medicine and as sensors.

Linear dendritic hybrid materials enable a range of architectural variations which offers novel possibilities in the tailoring of polymeric materials. In this study dendrons based on the 2,2-bis(methylol)propionic acid (bis-MPA) building block, bearing click chemistry moieties in the core and peripheral hydroxyl functionalities, have been used as macroinitiators for ring opening polymerization of e-caprolactone. A library of star branched polymers with poly(e-caprolactone) chains was initially constructed using dendrons up to 4th generation. In a second step, the popular CuAAC or thiol–ene click reaction was efficiently used to attach poly(ethylene glycol) chains of different lengths to the core. Potential applications of the resulted amphiphilic linear dendritic hybrids were investigated. Both self-assembled micelles loaded with doxorubicin anticancer drug and ordered honeycomb membranes with enhanced surface area were successfully fabricated and characterized.

Patients with acute myeloid leukaemia (AML) often achieve remission but subsequently die of relapse driven by chemotherapy resistant leukemic stem cells (LSCs). To initiate and maintain cancer, LSCs must also escape immunosurveillance. However, in vivo studies on human LSCs largely disregard lymphocyte mediated anti-tumor immunity due to the use of immunocompromised mice. Here we investigate the immunosurveillance mediated by NKG2D, a danger detector expressed by cytotoxic lymphocytes such as natural killer (NK) cells that recognizes stress-induced ligands (NKG2DL) of the MIC and ULBP protein families on AML cells. Staining of n=175 de novo AML with antibodies against MICA, MICB and ULB2/5/6 or an NKG2D-Fc chimeric protein recognizing pan-NKG2DL expression revealed NKG2DL to heterogeneously express among leukemic cells of the same patient (Fig. 1a). As expected, NKG2DLpos AML cells were efficiently cleared by natural killer (NK) cells, while NKG2DLneg leukemic cells escaped NK cell lysis. Interestingly, these NKG2DLneg AML cells also showed immature morphology, enhanced in vitro clonogenicity (39±47 colonies vs. 1±4, p Mechanistically, expression of poly-ADP-ribose polymerase 1 (PARP1) was identified as enriched in NKG2DLneg compared to NKG2DLpos leukemic subpopulations, and PARP1 inhibition (PARPi) using either siRNAs or pharmacological inhibitors such as AG-14361, veliparib, talazoparib or olaparib, increased NKG2DL mRNA transcripts between 6 and >50 fold. PARP1 binding sites were identified by in silico analysis in NKG2DL promoters and binding was confirmed by chromatin immunoprecipitation in the promoters of MICA and MICB. Importantly, treatment with PARPi induced NKG2DL surface expression on LSCs in vitro and in vivo and co-treatment with PARPi and NK cells efficiently suppressed leukemogenesis in patient derived xenograft (PDX) models (Fig. 1c). These data suggest that PARP1 inhibition sensitizes LSCs to NK cell mediated elimination. Finally, NKG2DL surface expression was found to inversely correlate with favorable molecular AML characteristics (favorable ELN risk group vs. other: p=0.034; inv(16) versus other: p=0.023), complete remission rates after induction chemotherapy (all patients: p=0.002, patients In summary, our data link the concept of LSCs to immune escape in human AML and propose the absence of immunostimulatory NKG2DL as a novel method to identify LSCs across genetic AML subtypes (including CD34 negative AMLs). This LSC specific mechanism of immune evasion could be overcome by treatment with PARP1 inhibitors, which in conjunction with functional NK cells holds promise to eradicate LSCs and promote immune-mediated cure of AML. Fig. 1: Human AML contain NKG2DLpos as well as NKG2DLneg subpopulations but only latter display leukemia initiation capacity (a: left, analysis of n=175 AML cases using NKG2D-Fc staining, right: exemplary flow cytometry plots; b: leukemic infiltration and survival in mice transplanted with NKG2DLneg or NKG2DLpos AML cells sorted from the same AML cases). PARP1 inhibition with AG-14361 up-regulates NKG2DL on CD34+ LSCs, and in vivo co-treatment with AG-14361 and polyclonal allogeneic NK cells suppresses leukemogenesis in PDX models (c). Figure. Figure. Disclosures Salih: Several patent applications: Patents & Royalties: e.g. EP3064507A1.

Mouse xenografts are routinely used for in vivo studies on human acute myeloid leukemia (AML) cells. However, a significant proportion of primary AML samples (~50-60% of cases) are not able to repopulate mice, or respectively require a particularly long time (up to 9 months) to show detectable engraftment in such models. Here we report that changing the transplantation time-point from day (5:00 pm) to night (4:00 am) strongly improved the engraftment of human AML cells in NOD/SCID/IL2Rgnull (NSG) mice. Sublethally irradiated gender-matched NSG mice of 6-8 weeks of age (n=40) were transplanted with primary AML cells (n=5 patients). For each sample, equal numbers of freshly thawed AML cells were injected via the tail vein at both time-points; transplanted mice were afterwards monitored for human leukemic engraftment in peripheral blood (PB) and bone marrow (BM) using multicolor flow cytometry and immunohistochemistry. In all n=5 analyzed AML patient samples, transplantation at night strongly promoted leukemogenesis, with 2/5 samples showing engraftment only in the night transplant condition and 3/5 samples showing engraftment in both but significantly higher leukemic burden in night vs. day transplanted mice (Fig. 1a), although similar leukemia-specific mutations were retrieved in mice of the two conditions by next generation sequencing analysis (Fig. 1b). Limiting dilution experiments (n=2 AML samples) further confirmed these findings and showed that in fact lower numbers of AML cells were required to initiate leukemia when cells were transplanted at night versus day times. Given that the immediate process that happens after intravenous injection of AML cells into mice involves homing to BM niches, we hypothesized that the time-point of transplantation influences engraftment by modulating homing. Therefore, we performed homing assays as described for healthy hematopoietic stem/progenitor cells (HSPCs); we injected equally treated human CFSE labeled AML cells via tail vein injection at day versus night time points and analyzed murine BMs 8 hours later for the presence of human leukemic cells. Indeed, significantly higher BM colonization by leukemic cells was observed after night vs. day time injections (0.31±0.09 vs. 0.13±0.18, p Since mice transplanted at night showed stress-related behavioral abnormalities, we hypothesized that this transplantation method might induce catecholamine release. ELISAs indeed revealed enhanced epinephrine levels in PB (318.00±12.96 vs. 122.20±47.11 ng/mL, p We conclude that enhanced catecholamine levels can accelerate leukemia development from AML cells transplanted in xenograft models by facilitating BM niche colonization by AML cells. Catecholamines might in general promote leukemia progression by favoring the retention of leukemic versus healthy HSCs in BM niches. Disclosures No relevant conflicts of interest to declare.

Presently, the majority of reports deal with combining chemical reactions, in a stepwise fashion, to obtain well-defined polymers. In the future, chemists need to address new challenges such as increase in the range of available efficient reactions, developing libraries of compatible one-pot reactions, and the application of obtained materials in key industries. Indeed, the rising importance of the click concept has now devised robust synthetic approaches in various fields of research. The unique selectivity of the click reaction is today a new found toolbox for scientists to investigate one-pot multi-step systems. Several accelerated protocols have elegantly been reported to obtain a library of advanced polymers.

In prion diseases, the cellular prion protein (PrP(C)) becomes misfolded into the pathogenic scrapie isoform (PrP(Sc)) responsible for prion infectivity. We show here that peptides derived from the prion protein N terminus have potent antiprion effects. These peptides are composed of a hydrophobic sequence followed by a basic segment. They are known to have cell-penetrating ability like regular cell-penetrating peptides (CPPs), short peptides that can penetrate cellular membranes. Healthy (GT1-1) and scrapie-infected (ScGT1-1) mouse neuronal hypothalamic cells were treated with various CPPs, including the prion protein-derived CPPs. Lysates were analyzed for altered protein levels of PrP(C) or PrP(Sc). Treatment with the prion protein-derived CPPs mouse mPrP(1-28) or bovine bPrP(1-30) significantly reduced PrP(Sc) levels in prion-infected cells but had no effect on PrP(C) levels in noninfected cells. Further, presence of prion protein-derived CPPs significantly prolonged the time before infection was manifested when infecting GT1-1 cells with scrapie. Treatment with other CPPs (penetratin, transportan-10, or poly-L-arginine) or prion protein-derived peptides lacking CPP function (mPrP(23-28,) mPrP(19-30,) or mPrP(23-50)) had no effect on PrP(Sc) levels. The results suggest a mechanism by which the signal sequence guides the prion protein-derived CPP into a cellular compartment, where the basic segment binds specifically to PrP(Sc) and disables formation of prions.

The membrane leakage caused by the cell penetrating peptide Tp10, a variant of transportan, was studied in large unilamellar vesicles with the entrapped fluorophore calcein. The vesicles were composed of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. A significant decrease in membrane leakage was found when the 55kDa streptavidin protein was attached to Tp10. When a 5.4kDa peptide nucleic acid molecule was attached, the membrane leakage was comparable to that caused by Tp10 alone. The results suggest that direct membrane effects may cause membrane translocation of Tp10 alone and of smaller complexes, whereas these effects do not contribute for larger cargoes.

Cell-penetrating peptides (CPPs) are peptides able to promote uptake of various cargos, including proteins and plasmids. Advances in recent years imply the uptake to be endocytic, where the current hurdle for efficient intracellular delivery is material being retained in the endosomes. In this study we wanted to compare the ability of various established CPPs to deliver siRNA and induce gene silencing of luciferase, with a novel designed penetratin analog having endosomolytic properties, using a noncovalent strategy. In principal, the penetratin analog EB1 will, upon protonation in the early-late endosomes, be able to form an amphipathic alpha helix resulting in permeabilization of the endosomal membrane. We demonstrate that even though all CPPs evaluated in this study can form complexes with siRNA, there is not a direct relationship between the complex formation ability and delivery efficacy. More important, although all CPPs significantly promote siRNA uptake, in some cases no gene silencing effect can be observed unless endosomal escape is induced. We find the designed endosomolytic peptide EB1 to be far more effective both in forming complexes and transporting biologically active siRNA than its parent peptide penetratin. We believe that developing CPPs with increased endosomolytical properties is a necessary step toward achieving biological effects at low concentrations for future in vivo applications.

The structural flexibility and efficacy of thiourea−amine catalysts for the supramolecular activation and ring-opening polymerization (ROP) of lactide are described. The nature of the hydrogen bonding group and its strength as well as the steric congestion have been altered, leading to shorter polymerization times, better control, and pathways to influence the stereochemistry of the resulting polymer. The tolerance to functionality and the mild conditions of the ROP mechanism allow for block copolymer synthesis by combination of nitroxide-mediated polymerization as well as reversible addition fragmentation and chain transfer polymerization using dual-headed initiators. Tandem hydrogen bond activation to organocatalyze ROP of lactide is an effective, versatile means to generate polymers with predictable molecular weights, narrow polydispersities, control of microstructure and a variety of complex architectures and block copolymers.

A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1-24) and a basic domain (KKRPKP, residues 25-30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp-DNA-gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide's ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases.