Transcription factor 3 is dysregulated in megakaryocytes in myelofibrosis.

Transcription factor 3 (TCF3) is a DNA transcription factor that modulates megakaryocyte development. Although abnormal TCF3 expression has been identified in a range of hematological malignancies, to date, it has not been investigated in myelofibrosis (MF). MF is a Philadelphia-negative myeloproliferative neoplasm (MPN) that can arise de novo or progress from essential thrombocythemia [ET] and polycythemia vera [PV] and where dysfunctional megakaryocytes have a role in driving the fibrotic progression. We aimed to examine whether TCF3 is dysregulated in megakaryocytes in MPN, and specifically in MF. We first assessed TCF3 protein expression in megakaryocytes using an immunohistochemical approach analyses and showed that TCF3 was reduced in MF compared with ET and PV. Further, the TCF3-negative megakaryocytes were primarily located near trabecular bone and had the typical "MF-like" morphology as described by the WHO. Genomic analysis of isolated megakaryocytes showed three mutations, all predicted to result in a loss of function, in patients with MF; none were seen in megakaryocytes isolated from ET or PV marrow samples. We then progressed to transcriptomic sequencing of platelets which showed loss of TCF3 in MF. These proteomic, genomic and transcriptomic analyses appear to indicate that TCF3 is downregulated in megakaryocytes in MF. This infers aberrations in megakaryopoiesis occur in this progressive phase of MPN. Further exploration of this pathway could provide insights into TCF3 and the evolution of fibrosis and potentially lead to new preventative therapeutic targets.

What is the context? We investigated TCF3 (transcription factor 3), a gene that regulates megakaryocyte development, for genomic and proteomic changes in myelofibrosis.Myelofibrosis is the aggressive phase of a group of blood cancers called myeloproliferative neoplasms, and abnormalities in development and maturation of megakaryocytes is thought to drive the development of myelofibrosis.What is new? We report detection of three novel TCF3 mutations in megakaryocytes and decreases in TCF3 protein and gene expression in primary megakaryocytes and platelets from patients with myelofibrosis.This is the first association between loss of TCF3 in megakaryocytes from patients and myelofibrosis.What is the impact? TCF3 dysregulation may be a novel mechanism that is responsible for the development of myelofibrosis and better understanding of this pathway could identify new drug targets.

Novel modes of MPL activation in triple-negative myeloproliferative neoplasms.

The identification of a somatic mutation associated with myeloid malignancy is of diagnostic importance in myeloproliferative neoplasms (MPNs). Individuals with no mutation detected in common screening tests for variants in JAK2, CALR, and MPL are described as 'triple-negative' and pose a diagnostic challenge if there is no other evidence of a clonal disorder. To identify potential drivers that might explain the clinical phenotype, we used an extended sequencing panel to characterise a cohort of 44 previously diagnosed triple-negative MPN patients for canonical mutations in JAK2, MPL and CALR at low variant allele frequency (found in 4/44 patients), less common variants in the JAK-STAT signalling pathway (12 patients), or other variants in recurrently mutated genes from myeloid malignancies (18 patients), including hotspot variants of potential clinical relevance in eight patients. In one patient with thrombocytosis we identified biallelic germline MPL variants. Neither MPL variant was activating in cell proliferation assays, and one of the variants was not expressed on the cell surface, yet co-expression of both variants led to thrombopoietin hypersensitivity. Our results highlight the clinical value of extended sequencing including germline variant analysis and illustrate the need for detailed functional assays to determine whether rare variants in JAK2 or MPL are pathogenic.

Clinical impact of NPM1-mutant molecular persistence after chemotherapy for acute myeloid leukemia.

Monitoring of NPM1 mutant (NPM1mut) measurable residual disease (MRD) in acute myeloid leukemia (AML) has an established role in patients who are treated with intensive chemotherapy. The European LeukemiaNet has defined molecular persistence at low copy number (MP-LCN) as an MRD transcript level <1% to 2% with a <1-log change between any 2 positive samples collected after the end of treatment (EOT). Because the clinical impact of MP-LCN is unknown, we sought to characterize outcomes in patients with persistent NPM1mut MRD after EOT and identify factors associated with disease progression. Consecutive patients with newly diagnosed NPM1mut AML who received ≥2 cycles of intensive chemotherapy were included if bone marrow was NPM1mut MRD positive at the EOT, and they were not transplanted in first complete remission. One hundred patients were followed for a median of 23.5 months; 42% remained free of progression at 1 year, either spontaneously achieving complete molecular remission (CRMRD-; 30%) or retaining a low-level NPM1mut transcript (12% for ≥12 months and 9% at last follow-up). Forty percent met the criteria for MP-LCN. Preemptive salvage therapy significantly prolonged relapse-free survival. Risk factors associated with disease progression were concurrent FLT3-internal tandem duplication at diagnosis and suboptimal MRD response (NPM1mut reduction <4.4-log) at EOT.

Platelets in myeloproliferative neoplasms have a distinct transcript signature in the presence of marrow fibrosis.

Marrow fibrosis is a significant complication of myeloproliferative neoplasms (MPN) that affects up to 20% of patients and is associated with a poor prognosis. The pathological processes that lead to fibrotic progression are not well understood, but megakaryocytes have been implicated in the process. The aim of this study was to determine whether platelets, derived from megakaryocytes, have transcriptomic alterations associated with fibrosis. Platelets from MPN patients with and without fibrosis and non-malignant control individuals were assessed using next generation sequencing. Results from the initial training cohort showed discrete changes in platelet transcripts in the presence of marrow fibrosis. We identified more than 1000 differentially expressed transcripts from which a putative 3-gene fibrotic platelet signature (CCND1, H2AX [previously termed H2AFX] and CEP55) could be identified. This fibrosis-associated signature was assessed blinded on platelets from an independent test MPN patient cohort. The 3-gene signature was able to discriminate between patients with and without marrow fibrosis with a positive predictive value of 71% (93% specificity, 71% sensitivity). This demonstrates that assessment of dysregulated transcripts in platelets may be a useful monitoring tool in MPN to identify progression to marrow fibrosis. Further, sequential monitoring could have clinical applications for early prediction of progression to fibrosis.

PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice.

B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.

The Diagnostic, Prognostic, and Therapeutic Utility of Molecular Testing in a Patient with Waldenstrom's Macroglobulinemia.

The application of molecular genomics and our understanding of its clinical implications in the diagnosis, prognostication and treatment of lymphoproliferative disorders has rapidly evolved over the past few years. Of particular importance are indolent B-cell malignancies where tumour cell survival and proliferation are commonly driven by mutations involving the B-cell receptor and downstream signalling pathways. In addition, the increasing number of novel therapies and targeted agents have provided clinicians with new therapeutic options with the aim of exploiting such mutations. In this case report, we highlight one such success story involving the diagnostic impact of the MYD88L265P mutation in Waldenstrom's macroglobulinemia (WM), its prognostic implications and effect on choice of therapy in the era of novel therapies.

Molecular synergy underlies the co-occurrence patterns and phenotype of NPM1-mutant acute myeloid leukemia.

NPM1 mutations define the commonest subgroup of acute myeloid leukemia (AML) and frequently co-occur with FLT3 internal tandem duplications (ITD) or, less commonly, NRAS or KRAS mutations. Co-occurrence of mutant NPM1 with FLT3-ITD carries a significantly worse prognosis than NPM1-RAS combinations. To understand the molecular basis of these observations, we compare the effects of the 2 combinations on hematopoiesis and leukemogenesis in knock-in mice. Early effects of these mutations on hematopoiesis show that compound Npm1cA/+;NrasG12D/+ or Npm1cA;Flt3ITD share a number of features: Hox gene overexpression, enhanced self-renewal, expansion of hematopoietic progenitors, and myeloid differentiation bias. However, Npm1cA;Flt3ITD mutants displayed significantly higher peripheral leukocyte counts, early depletion of common lymphoid progenitors, and a monocytic bias in comparison with the granulocytic bias in Npm1cA/+;NrasG12D/+ mutants. Underlying this was a striking molecular synergy manifested as a dramatically altered gene expression profile in Npm1cA;Flt3ITD , but not Npm1cA/+;NrasG12D/+ , progenitors compared with wild-type. Both double-mutant models developed high-penetrance AML, although latency was significantly longer with Npm1cA/+;NrasG12D/+ During AML evolution, both models acquired additional copies of the mutant Flt3 or Nras alleles, but only Npm1cA/+;NrasG12D/+ mice showed acquisition of other human AML mutations, including IDH1 R132Q. We also find, using primary Cas9-expressing AMLs, that Hoxa genes and selected interactors or downstream targets are required for survival of both types of double-mutant AML. Our results show that molecular complementarity underlies the higher frequency and significantly worse prognosis associated with NPM1c/FLT3-ITD vs NPM1/NRAS-G12D-mutant AML and functionally confirm the role of HOXA genes in NPM1c-driven AML.

Preventing chemotherapy-induced myelosuppression by repurposing the FLT3 inhibitor quizartinib.

We describe an approach to inhibit chemotherapy-induced myelosuppression. We found that short-term exposure of mice to the FLT3 inhibitor quizartinib induced the transient quiescence of multipotent progenitors (MPPs). This property of quizartinib conferred marked protection to MPPs in mice receiving fluorouracil or gemcitabine. The protection resulted in the rapid recovery of bone marrow and blood cellularity, thus preventing otherwise lethal myelosuppression. A treatment strategy involving quizartinib priming that protected wild-type bone marrow progenitors, but not leukemic cells, from fluorouracil provided a more effective treatment than conventional induction therapy in mouse models of acute myeloid leukemia. This strategy has the potential to be extended for use in other cancers where FLT3 inhibition does not adversely affect the effectiveness of chemotherapy. Thus, the addition of quizartinib to cancer treatment regimens could markedly improve cancer patient survival and quality of life.

Leukemia-associated somatic mutations drive distinct patterns of age-related clonal hemopoiesis.

Clonal hemopoiesis driven by leukemia-associated gene mutations can occur without evidence of a blood disorder. To investigate this phenomenon, we interrogated 15 mutation hot spots in blood DNA from 4,219 individuals using ultra-deep sequencing. Using only the hot spots studied, we identified clonal hemopoiesis in 0.8% of individuals under 60, rising to 19.5% of those ≥90 years, thus predicting that clonal hemopoiesis is much more prevalent than previously realized. DNMT3A-R882 mutations were most common and, although their prevalence increased with age, were found in individuals as young as 25 years. By contrast, mutations affecting spliceosome genes SF3B1 and SRSF2, closely associated with the myelodysplastic syndromes, were identified only in those aged >70 years, with several individuals harboring more than one such mutation. This indicates that spliceosome gene mutations drive clonal expansion under selection pressures particular to the aging hemopoietic system and explains the high incidence of clonal disorders associated with these mutations in advanced old age.

Loss of c-Cbl E3 ubiquitin ligase activity enhances the development of myeloid leukemia in FLT3-ITD mutant mice.

Mutations in the Fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase (RTK) occur frequently in acute myeloid leukemia (AML), with the most common involving internal tandem duplication (ITD) within the juxtamembrane domain. Fms-like tyrosine kinase 3-ITD mutations result in a mislocalized and constitutively activated receptor, which aberrantly phosphorylates signal transducer and activator of transcription 5 (STAT5) and upregulates the expression of its target genes. c-Cbl is an E3 ubiquitin ligase that negatively regulates RTKs, including FLT3, but whether it can downregulate mislocalized FLT3-ITD remains to be resolved. To help clarify this, we combined a FLT3-ITD mutation with a loss-of-function mutation in the RING finger domain of c-Cbl that abolishes its E3 ligase activity. Mice transplanted with hematopoietic stem cells expressing both mutations rapidly develop myeloid leukemia, indicating strong cooperation between the two. Although the c-Cbl mutation was shown to cause hyperactivation of another RTK, c-Kit, it had no effect on enhancing FLT3-ITD protein levels or STAT5 activation. This indicates that c-Cbl does not downregulate FLT3-ITD and that the leukemia is driven by independent pathways involving FLT3-ITD's activation of STAT5 and mutant c-Cbl's activation of other RTKs, such as c-Kit. This study highlights the importance of c-Cbl's negative regulation of wild-type RTKs in suppressing FLT3-ITD-driven myeloid leukemia.

Acute myeloid leukaemia: a paradigm for the clonal evolution of cancer?

Acute myeloid leukaemia (AML) is an uncontrolled clonal proliferation of abnormal myeloid progenitor cells in the bone marrow and blood. Advances in cancer genomics have revealed the spectrum of somatic mutations that give rise to human AML and drawn our attention to its molecular evolution and clonal architecture. It is now evident that most AML genomes harbour small numbers of mutations, which are acquired in a stepwise manner. This characteristic, combined with our ability to identify mutations in individual leukaemic cells and our detailed understanding of normal human and murine haematopoiesis, makes AML an excellent model for understanding the principles of cancer evolution. Furthermore, a better understanding of how AML evolves can help us devise strategies to improve the therapy and prognosis of AML patients. Here, we draw from recent advances in genomics, clinical studies and experimental models to describe the current knowledge of the clonal evolution of AML and its implications for the biology and treatment of leukaemias and other cancers.

Vascular endothelial growth factor: the key mediator in pleural effusion formation.

Pleural effusion is common in clinical practice. Increased vascular permeability and leakage play a principal role in the development of exudative pleural effusions. In vitro and in vivo evidence have solidly established vascular endothelial growth factor (VEGF), a potent inducer of vascular permeability, as a crucial mediator in pleural fluid formation. VEGF is present in high quantities in human effusions. In the pleural space, mesothelial cells, infiltrating inflammatory cells, and (in malignant pleuritis) cancer cells contribute to the VEGF accumulation in the pleural fluids. Pleural fluid VEGF is biologically active and may promote tumor growth and chemotaxis. Strategies to antagonize the VEGF activity at various target points of its signaling pathway have shown success in vitro and in animal models of malignant pleural or peritoneal effusions. Novel agents targeting VEGF activities are undergoing clinical trials. Regulation of VEGF activity and vascular permeability represent a rapidly expanding field of research, which is likely to provide further insight in the pathophysiology of pleural fluid formation.