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.

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.

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.

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.

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.

RAS/CBL mutations predict resistance to JAK inhibitors in myelofibrosis and are associated with poor prognostic features.

The dysregulation of the JAK/STAT pathway drives the pathogenesis of myelofibrosis (MF). Recently, several JAK inhibitors (JAKis) have been developed for treating MF. Select mutations (MTs) have been associated with impaired outcomes and are currently incorporated in molecularly annotated prognostic models. Mutations of RAS/MAPK pathway genes are frequently reported in cancer and at low frequencies in MF. In this study, we investigated the phenotypic, prognostic, and therapeutic implications of NRASMTs, KRASMTs, and CBLMTs (RAS/CBLMTs) in 464 consecutive MF patients. A total of 59 (12.7%) patients had RAS/CBLMTs: NRASMTs, n = 25 (5.4%); KRASMTs, n = 13 (2.8%); and CBLMTs, n = 26 (5.6%). Patients with RAS/CBLMTs were more likely to present with high-risk clinical and molecular features. RAS/CBLMTs were associated with inferior overall survival compared with patients without MTs and retained significance in a multivariate model, including the Mutation-Enhanced International Prognostic Score System (MIPSS70) risk factors and cytogenetics; however, inclusion of RAS/CBLMTs in molecularly annotated prognostic models did not improve the predictive power of the latter. The 5-year cumulative incidence of leukemic transformation was notably higher in the RAS/CBLMT cohort. Among 61 patients treated with JAKis and observed for a median time of 30 months, the rate of symptoms and spleen response at 6 months was significantly lower in the RAS/CBLMT cohort. Logistic regression analysis disclosed a significant inverse correlation between RAS/CBLMTs and the probability of achieving a symptom or spleen response that was retained in multivariate analysis. In summary, our study showed that RAS/CBLMTs are associated with adverse phenotypic features and survival outcomes and, more important, may predict reduced response to JAKis.

Reprogramming of Amino Acid Transporters to Support Aspartate and Glutamate Dependency Sustains Endocrine Resistance in Breast Cancer.

Endocrine therapy (ET) is the standard of care for estrogen receptor-positive (ER+) breast cancers. Despite its efficacy, ∼40% of women relapse with ET-resistant (ETR) disease. A global transcription analysis in ETR cells reveals a downregulation of the neutral and basic amino acid transporter SLC6A14 governed by enhanced miR-23b-3p expression, resulting in impaired amino acid metabolism. This altered amino acid metabolism in ETR cells is supported by the activation of autophagy and the enhanced import of acidic amino acids (aspartate and glutamate) mediated by the SLC1A2 transporter. The clinical significance of these findings is validated by multiple orthogonal approaches in a large cohort of ET-treated patients, in patient-derived xenografts, and in in vivo experiments. Targeting these amino acid metabolic dependencies resensitizes ETR cells to therapy and impairs the aggressive features of ETR cells, offering predictive biomarkers and potential targetable pathways to be exploited to combat or delay ETR in ER+ breast cancers.