A blood drop through the pore: nanopore sequencing in hematology.

The development of new sequencing platforms, technologies, and bioinformatics tools in the past decade fostered key discoveries in human genomics. Among the most recent sequencing technologies, nanopore sequencing (NS) has caught the interest of researchers for its intriguing potential and flexibility. This up-to-date review highlights the recent application of NS in the hematology field, focusing on progress and challenges of the technological approaches employed for the identification of pathologic alterations. The molecular and analytic pipelines developed for the analysis of the whole-genome, target regions, and transcriptomics provide a proof of evidence of the unparalleled amount of information that could be retrieved by an innovative approach based on long-read sequencing.

Clonal dynamics and copy number variants by single-cell analysis in leukemic evolution of myeloproliferative neoplasms.

Transformation from chronic (CP) to blast phase (BP) in myeloproliferative neoplasm (MPN) remains poorly characterized, and no specific mutation pattern has been highlighted. BP-MPN represents an unmet need, due to its refractoriness to treatment and dismal outcome. Taking advantage of the granularity provided by single-cell sequencing (SCS), we analyzed paired samples of CP and BP in 10 patients to map clonal trajectories and interrogate target copy number variants (CNVs). Already at diagnosis, MPN present as oligoclonal diseases with varying ratio of mutated and wild-type cells, including cases where normal hematopoiesis was entirely surmised by mutated clones. BP originated from increasing clonal complexity, either on top or independent of a driver mutation, through acquisition of novel mutations as well as accumulation of clones harboring multiple mutations, that were detected at CP by SCS but were missed by bulk sequencing. There were progressive copy-number imbalances from CP to BP, that configured distinct clonal profiles and identified recurrences in genes including NF1, TET2, and BCOR, suggesting an additional level of complexity and contribution to leukemic transformation. EZH2 emerged as the gene most frequently affected by single nucleotide and CNVs, that might result in EZH2/PRC2-mediated transcriptional deregulation, as supported by combined scATAC-seq and snRNA-seq analysis of the leukemic clone in a representative case. Overall, findings provided insights into the pathogenesis of MPN-BP, identified CNVs as a hitherto poorly characterized mechanism and point to EZH2 dysregulation as target. Serial assessment of clonal dynamics might potentially allow early detection of impending disease transformation, with therapeutic implications.

Nano-GLADIATOR: real-time detection of copy number alterations from nanopore sequencing data.

MOTIVATION: The past few years have seen the emergence of nanopore-based sequencing technologies which interrogate single molecule of DNA and generate reads sequentially. RESULTS: In this paper, we demonstrate that, thanks to the sequentiality of the nanopore process, the data generated in the first tens of minutes of a typical MinION/GridION run can be exploited to resolve the alterations of a human genome at a karyotype level with a resolution in the order of tens of Mb, while the data produced in the first 6-12 h allow to obtain a resolution comparable to currently available array-based technologies, and thanks to a novel probabilistic approach are capable to predict the allelic fraction of genomic alteration with high accuracy. To exploit the unique characteristics of nanopore sequencing data we developed a novel software tool, Nano-GLADIATOR, that is capable to perform copy number variants/alterations detection and allelic fraction prediction during the sequencing run ('On-line' mode) and after experiment completion ('Off-line' mode). We tested Nano-GLADIATOR on publicly available ('Off-line' mode) and on novel whole genome sequencing dataset generated with MinION device ('On-line' mode) showing that our tool is capable to perform real-time copy number alterations detection obtaining good results with respect to other state-of-the-art tools. AVAILABILITY AND IMPLEMENTATION: Nano-GLADIATOR is freely available at https://sourceforge.net/projects/nanogladiator/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Endothelial-to-Mesenchymal Transition in Bone Marrow and Spleen of Primary Myelofibrosis.

Primary myelofibrosis is characterized by the development of fibrosis in the bone marrow that contributes to ineffective hematopoiesis. Bone marrow fibrosis is the result of a complex and not yet fully understood interaction among megakaryocytes, myeloid cells, fibroblasts, and endothelial cells. Here, we report that >30% of the endothelial cells in the small vessels of the bone marrow and spleen of patients with primary myelofibrosis have a mesenchymal phenotype, which is suggestive of the process known as endothelial-to-mesenchymal transition (EndMT). EndMT can be reproduced in vitro by incubation of cultured endothelial progenitor cells or spleen-derived endothelial cells with inflammatory cytokines. Megakaryocytes appear to be implicated in this process, because EndMT mainly occurs in the microvessels close to these cells, and because megakaryocyte-derived supernatant fluid can reproduce the EndMT switch in vitro. Furthermore, EndMT is an early event in a JAK2-V617F knock-in mouse model of primary myelofibrosis. Overall, these data show for the first time that microvascular endothelial cells in the bone marrow and spleen of patients with primary myelofibrosis show functional and morphologic changes that are associated to the mesenchymal phenotype.

Familial dominant epilepsy and mild pachygyria associated with a constitutional LIS1 mutation.

We describe a mother and son with focal epilepsy, mild cognitive impairment, and pachygyria, which was parieto-occipital in the mother and with remarkable posterior greater than anterior severity in the son. Overall clinical manifestations, although overlapping in type, were likewise slightly more severe in the son. Using targeted resequencing through a gene panel for malformations of cortical development, we identified the c.655 T > A [p.(Trp219Arg)] novel missense variant in the LIS1 gene, segregating in the proband and in his mother. Western Blot analysis, qPCR gene expression and RT-PCR disclosed no significant differences between proband, his parents, and controls. Epilepsy and mild cognitive impairment can be the only clinical presentation of constitutional LIS1 mutations, which can therefore be inherited if the associated phenotype implies limited or no reproductive disadvantage. Parents of patients harboring LIS1 mutations should be assessed for their mutation carrier status.

Ubiquity of cis-Halide → Isocyanide Direct Interligand Interaction in Organometallic Complexes.

We recently reported a density functional theory (DFT) analysis of the Nb(V)-C bond in various NbCl5(L) complexes, discovering that the carbon ligand L receives electronic density from the metal (classical back-donation) and from the chlorides in the cis position (direct interligand interaction). Here we report the synthesis and the structural characterization of two new coordination compounds of niobium pentahalides, i.e., NbX5(CNXyl) (X = Cl, Br; Xyl = 2,6-C6H3Me2), and the corresponding DFT analyses of the Nb(V)-C bond using the Natural Orbitals for Chemical Valence-Charge Displacement (NOCV-CD) approach, confirming the presence of a cis-halide → isocyanide direct interligand interaction. To verify whether the latter is limited to Nb complexes or not, we performed a NOCV-CD analysis on a series of several organometallic complexes based on Ti(IV), Nb(V), Ta(V), Rh(III), Pd(II), and Au(III), all of which bear one halide ligand and m-xylyl-isocyanide in a mutual cis position, revealing that the cis-halide → isocyanide interaction is always present.

Synthesis and Antiproliferative Activity of New Ruthenium Complexes with Ethacrynic-Acid-Modified Pyridine and Triphenylphosphine Ligands.

Pyridine- and phosphine-based ligands modified with ethacrynic acid (a broad acting glutathione transferase inhibitor) were prepared and coordinated to ruthenium(II)-arene complexes and to a ruthenium(III) NAMI-A type complex. All the compounds (ligands and complexes) were fully characterized by analytical and spectroscopic methods and, in one case, by single-crystal X-ray diffraction. The in vitro anticancer activity of the compounds was studied, with the compounds displaying moderate cytotoxicity toward the human ovarian cancer cell lines. All the complexes led to similar levels of residual GST activity in the different cell lines, irrespective of the stability of the Ru-ligand bond.

Co-targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms.

Aberrant JAK2 signalling plays a central role in myeloproliferative neoplasms (MPN). JAK2 inhibitors have proven to be clinically efficacious, however, they are not mutation-specific and competent enough to suppress neoplastic clonal haematopoiesis. We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated. To this end we investigated the efficacy of BEZ235, a dual PI3K/mTOR inhibitor, alone and in combination with the JAK1/JAK2 inhibitor ruxolitinib, in different preclinical models of MPN. Single-agent BEZ235 inhibited the proliferation and induced cell cycle arrest and apoptosis of mouse and human JAK2V617F mutated cell lines at concentrations significantly lower than those required to inhibit the wild-type counterpart, and preferentially prevented colony formation from JAK2V617F knock-in mice and patients' progenitor cells compared with normal ones. Co-treatment of BEZ235 and ruxolitinib produced significant synergism in all these in-vitro models. Co-treatment was also more effective than single drugs in reducing the extent of disease and prolonging survival of immunodeficient mice injected with JAK2V617F-mutated Ba/F3-EPOR cells and in reducing spleen size, decreasing reticulocyte count and improving spleen histopathology in conditional JAK2V617F knock-in mice. In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN.

Safety and efficacy of everolimus, a mTOR inhibitor, as single agent in a phase 1/2 study in patients with myelofibrosis.

In addition to dysregulated JAK/STAT signaling, activation of the AKT/mTOR pathway occurs in myelofibrosis, a myeloproliferative neoplasm with no approved therapies. We conducted a phase 1/2 study with everolimus, an mTOR inhibitor, in 39 high- or intermediate-risk primary or postpolycythemia vera/postessential thrombocythemia myelofibrosis subjects. Responses were evaluated in 30 patients of phase 2. No dose-limiting toxicity was observed in phase 1 up to 10 mg/d. When this dose was used in phase 2, grade ≥ 3 toxicities were infrequent; the commonest toxicity was grade 1-2 stomatitis. Rapid and sustained splenomegaly reduction of > 50% and > 30% occurred in 20% and 44% of subjects, respectively. A total of 69% and 80% experienced complete resolution of systemic symptoms and pruritus. Response in leukocytosis, anemia, and thrombocytosis occurred in 15%-25%. Clinical responses were not associated with reduced JAK2V617F burden, circulating CD34(+) cells, or cytokine levels, whereas CCDN1 mRNA and phospho-p70S6K level, known targets of mTOR, and WT1 mRNA were identified as possible biomarkers associated with response. Response rate was 60% when European Network for Myelofibrosis criteria were used (8 major, 7 moderate, 3 minor responses) or 23% when IWG-MRT criteria (1 partial response, 6 clinical improvements) were used. These results provide proof-of-concept that targeting mTOR pathway in myelofibrosis may be clinically relevant.

Resolving complex structural variants via nanopore sequencing.

The recent development of high-throughput sequencing platforms provided impressive insights into the field of human genetics and contributed to considering structural variants (SVs) as the hallmark of genome instability, leading to the establishment of several pathologic conditions, including neoplasia and neurodegenerative and cognitive disorders. While SV detection is addressed by next-generation sequencing (NGS) technologies, the introduction of more recent long-read sequencing technologies have already been proven to be invaluable in overcoming the inaccuracy and limitations of NGS technologies when applied to resolve wide and structurally complex SVs due to the short length (100-500 bp) of the sequencing read utilized. Among the long-read sequencing technologies, Oxford Nanopore Technologies developed a sequencing platform based on a protein nanopore that allows the sequencing of "native" long DNA molecules of virtually unlimited length (typical range 1-100 Kb). In this review, we focus on the bioinformatics methods that improve the identification and genotyping of known and novel SVs to investigate human pathological conditions, discussing the possibility of introducing nanopore sequencing technology into routine diagnostics.

Inhibition of ERK1/2 signaling prevents bone marrow fibrosis by reducing osteopontin plasma levels in a myelofibrosis mouse model.

Clonal myeloproliferation and development of bone marrow (BM) fibrosis are the major pathogenetic events in myelofibrosis (MF). The identification of novel antifibrotic strategies is of utmost importance since the effectiveness of current therapies in reverting BM fibrosis is debated. We previously demonstrated that osteopontin (OPN) has a profibrotic role in MF by promoting mesenchymal stromal cells proliferation and collagen production. Moreover, increased plasma OPN correlated with higher BM fibrosis grade and inferior overall survival in MF patients. To understand whether OPN is a druggable target in MF, we assessed putative inhibitors of OPN expression in vitro and identified ERK1/2 as a major regulator of OPN production. Increased OPN plasma levels were associated with BM fibrosis development in the Romiplostim-induced MF mouse model. Moreover, ERK1/2 inhibition led to a remarkable reduction of OPN production and BM fibrosis in Romiplostim-treated mice. Strikingly, the antifibrotic effect of ERK1/2 inhibition can be mainly ascribed to the reduced OPN production since it could be recapitulated through the administration of anti-OPN neutralizing antibody. Our results demonstrate that OPN is a novel druggable target in MF and pave the way to antifibrotic therapies based on the inhibition of ERK1/2-driven OPN production or the neutralization of OPN activity.

Targeting the PI3K pathway in myeloproliferative neoplasms.

INTRODUCTION: Decreasing efficacy over time and initial suboptimal response to Janus kinase (JAK) inhibitors such as ruxolitinib in a subset of patients are critical clinical challenges associated with myeloproliferative neoplasms (MPNs), primarily myelofibrosis. AREAS COVERED: The role of phosphatidylinositol-3 kinase (PI3K) in MPN disease progression and treatment resistance and as a potential therapeutic target in patients who experience loss of response to JAK inhibition is discussed. Understanding the complex signaling networks involved in the pathogenesis of MPNs has identified potentially novel therapeutic targets and treatment strategies, such as inhibiting other signaling pathways in addition to the JAK/signal transducer and activator of transcription (STAT) pathway. PI3K plays a crucial role downstream of JAK signaling in rescuing tumor cell proliferation, with PI3Kδ being particularly important in hematologic malignancies. Concurrent targeting of both PI3K and JAK/STAT pathways may offer an innovative therapeutic strategy to maximize efficacy. EXPERT OPINION: Based on our understanding of the underlying mechanisms and the role of PI3K pathway signaling in the loss of response or resistance to JAK inhibitor treatment and initial results from clinical studies, the combination of parsaclisib (PI3Kδ inhibitor) and ruxolitinib holds great clinical potential. If confirmed in larger clinical trials, parsaclisib may provide more treatment options and improve clinical outcomes for patients with MPNs.

Immunoblotting-assisted assessment of JAK/STAT and PI3K/Akt/mTOR signaling in myeloproliferative neoplasms CD34+ stem cells.

Philadelphia-negative myeloproliferative neoplasms (pH-MPNs) origin from the clonal expansion of hematopoietic stem cells with acquired mutations leading to uncontrolled proliferation of differentiated myeloid cells. The main entities of Ph-MPNs are represented by Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF) that are characterized by microvascular disorders, thrombosis and bleeding, splenomegaly secondary to extramedullary hematopoiesis, various degree of bone marrow fibrosis and a progressive risk of leukemic transformation. Somatic mutations in myeloid genes including JAK2, CALR, and MPL cause the constitutive activation of the Janus Kinase 2 (JAK)/signal transducer and activator of transcription (STAT) pathway that confers proliferative and differentiative advantage to mutated hematopoietic progenitors and ultimately drives the development of a Ph-MPNs phenotype. Beyond the JAK/STAT axis, a wide number of intracellular signaling pathways were found deregulated in Ph-MPNs including the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) constitutive activation. In this chapter, we provide a detailed protocol for the immunoblotting assisted assessment of Ph-MPNs pathways activation. This protocol can be easily adapted to study protein expression and phosphorylation of hematopoietic stem progenitors and differentiated cell lineages.

The Response to Oxidative Damage Correlates with Driver Mutations and Clinical Outcome in Patients with Myelofibrosis.

Myelofibrosis (MF) is the Philadelphia-negative myeloproliferative neoplasm characterized by the worst prognosis and no response to conventional therapy. Driver mutations in JAK2 and CALR impact on JAK-STAT pathway activation but also on the production of reactive oxygen species (ROS). ROS play a pivotal role in inflammation-induced oxidative damage to cellular components including DNA, therefore leading to greater genomic instability and promoting cell transformation. In order to unveil the role of driver mutations in oxidative stress, we assessed ROS levels in CD34+ hematopoietic stem/progenitor cells of MF patients. Our results demonstrated that ROS production in CD34+ cells from CALR-mutated MF patients is far greater compared with patients harboring JAK2 mutation, and this leads to increased oxidative DNA damage. Moreover, CALR-mutant cells show less superoxide dismutase (SOD) antioxidant activity than JAK2-mutated ones. Here, we show that high plasma levels of total antioxidant capacity (TAC) correlate with detrimental clinical features, such as high levels of lactate dehydrogenase (LDH) and circulating CD34+ cells. Moreover, in JAK2-mutated patients, high plasma level of TAC is also associated with a poor overall survival (OS), and multivariate analysis demonstrated that high TAC classification is an independent prognostic factor allowing the identification of patients with inferior OS in both DIPSS lowest and highest categories. Altogether, our data suggest that a different capability to respond to oxidative stress can be one of the mechanisms underlying disease progression of myelofibrosis.

Differentiation of crescent-forming kidney progenitor cells into podocytes attenuates severe glomerulonephritis in mice.

Crescentic glomerulonephritis is characterized by vascular necrosis and parietal epithelial cell hyperplasia in the space surrounding the glomerulus, resulting in the formation of crescents. Little is known about the molecular mechanisms driving this process. Inducing crescentic glomerulonephritis in two Pax2Cre reporter mouse models revealed that crescents derive from clonal expansion of single immature parietal epithelial cells. Preemptive and delayed histone deacetylase inhibition with panobinostat, a drug used to treat hematopoietic stem cell disorders, attenuated crescentic glomerulonephritis with recovery of kidney function in the two mouse models. Three-dimensional confocal microscopy and stimulated emission depletion superresolution imaging of mouse glomeruli showed that, in addition to exerting an anti-inflammatory and immunosuppressive effect, panobinostat induced differentiation of an immature hyperplastic parietal epithelial cell subset into podocytes, thereby restoring the glomerular filtration barrier. Single-cell RNA sequencing of human renal progenitor cells in vitro identified an immature stratifin-positive cell subset and revealed that expansion of this stratifin-expressing progenitor cell subset was associated with a poor outcome in human crescentic glomerulonephritis. Treatment of human parietal epithelial cells in vitro with panobinostat attenuated stratifin expression in renal progenitor cells, reduced their proliferation, and promoted their differentiation into podocytes. These results offer mechanistic insights into the formation of glomerular crescents and demonstrate that selective targeting of renal progenitor cells can attenuate crescent formation and the deterioration of kidney function in crescentic glomerulonephritis in mice.

Activated IL-6 signaling contributes to the pathogenesis of, and is a novel therapeutic target for, CALR-mutated MPNs.

Calreticulin (CALR), an endoplasmic reticulum-associated chaperone, is frequently mutated in myeloproliferative neoplasms (MPNs). Mutated CALR promotes downstream JAK2/STAT5 signaling through interaction with, and activation of, the thrombopoietin receptor (MPL). Here, we provide evidence of a novel mechanism contributing to CALR-mutated MPNs, represented by abnormal activation of the interleukin 6 (IL-6)-signaling pathway. We found that UT7 and UT7/mpl cells, engineered by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to express the CALR type 1-like (DEL) mutation, acquired cytokine independence and were primed to the megakaryocyte (Mk) lineage. Levels of IL-6 messenger RNA (mRNA), extracellular-released IL-6, membrane-associated glycoprotein 130 (gp130), and IL-6 receptor (IL-6R), phosphorylated JAK1 and STAT3 (p-JAK1 and p-STAT3), and IL-6 promoter region occupancy by STAT3 all resulted in increased CALR DEL cells in the absence of MPL stimulation. Wild-type, but not mutated, CALR physically interacted with gp130 and IL-6R, downregulating their expression on the cell membrane. Agents targeting gp130 (SC-144), IL-6R (tocilizumab [TCZ]), and cell-released IL-6 reduced proliferation of CALR DEL as well as CALR knockout cells, supporting a mutated CALR loss-of-function model. CD34+ cells from CALR-mutated patients showed increased levels of IL-6 mRNA and p-STAT3, and colony-forming unit-Mk growth was inhibited by either SC144 or TCZ, as well as an IL-6 antibody, supporting cell-autonomous activation of the IL-6 pathway. Targeting IL-6 signaling also reduced colony formation by CD34+ cells of JAK2V617F-mutated patients. The combination of TCZ and ruxolitinib was synergistic at very low nanomolar concentrations. Overall, our results suggest that target inhibition of IL-6 signaling may have therapeutic potential in CALR, and possibly JAK2V617F, mutated MPNs.

Nanopore sequencing for the screening of myeloid and lymphoid neoplasms with eosinophilia and rearrangement of PDGFRα, PDGFRß, FGFR1 or PCM1-JAK2.

Eosinophilia represents a group of diseases with heterogeneous pathobiology and clinical phenotypes. Among the alterations found in primary Eosinophilia, gene fusions involving PDGFRα, PDGFRß, FGFR1 or JAK2 represent the biomarkers of WHO-defined "myeloid and lymphoid neoplasms with eosinophilia". The heterogeneous nature of genomic aberrations and the promiscuity of fusion partners, may limit the diagnostic accuracy of current cytogenetics approaches. To address such technical challenges, we exploited a nanopore-based sequencing assay to screen patients with primary Eosinophilia. The comprehensive sequencing approach described here enables the identification of genomic fusion in 60 h, starting from DNA purified from whole blood.