CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro.

A GGGGCC24+ hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), fatal neurodegenerative diseases with no cure or approved treatments that substantially slow disease progression or extend survival. Mechanistic underpinnings of neuronal death include C9ORF72 haploinsufficiency, sequestration of RNA-binding proteins in the nucleus, and production of dipeptide repeat proteins. Here, we used an adeno-associated viral vector system to deliver CRISPR/Cas9 gene-editing machineries to effectuate the removal of the HRE from the C9ORF72 genomic locus. We demonstrate successful excision of the HRE in primary cortical neurons and brains of three mouse models containing the expansion (500-600 repeats) as well as in patient-derived iPSC motor neurons and brain organoids (450 repeats). This resulted in a reduction of RNA foci, poly-dipeptides and haploinsufficiency, major hallmarks of C9-ALS/FTD, making this a promising therapeutic approach to these diseases.

A human fetal liver-derived infant MLL-AF4 acute lymphoblastic leukemia model reveals a distinct fetal gene expression program.

Although 90% of children with acute lymphoblastic leukemia (ALL) are now cured, the prognosis for infant-ALL remains dismal. Infant-ALL is usually caused by a single genetic hit that arises in utero: an MLL/KMT2A gene rearrangement (MLL-r). This is sufficient to induce a uniquely aggressive and treatment-refractory leukemia compared to older children. The reasons for disparate outcomes in patients of different ages with identical driver mutations are unknown. Using the most common MLL-r in infant-ALL, MLL-AF4, as a disease model, we show that fetal-specific gene expression programs are maintained in MLL-AF4 infant-ALL but not in MLL-AF4 childhood-ALL. We use CRISPR-Cas9 gene editing of primary human fetal liver hematopoietic cells to produce a t(4;11)/MLL-AF4 translocation, which replicates the clinical features of infant-ALL and drives infant-ALL-specific and fetal-specific gene expression programs. These data support the hypothesis that fetal-specific gene expression programs cooperate with MLL-AF4 to initiate and maintain the distinct biology of infant-ALL.

Heterogeneous disease-propagating stem cells in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a poor-prognosis childhood leukemia usually caused by RAS-pathway mutations. The cellular hierarchy in JMML is poorly characterized, including the identity of leukemia stem cells (LSCs). FACS and single-cell RNA sequencing reveal marked heterogeneity of JMML hematopoietic stem/progenitor cells (HSPCs), including an aberrant Lin-CD34+CD38-CD90+CD45RA+ population. Single-cell HSPC index-sorting and clonogenic assays show that (1) all somatic mutations can be backtracked to the phenotypic HSC compartment, with RAS-pathway mutations as a "first hit," (2) mutations are acquired with both linear and branching patterns of clonal evolution, and (3) mutant HSPCs are present after allogeneic HSC transplant before molecular/clinical evidence of relapse. Stem cell assays reveal interpatient heterogeneity of JMML LSCs, which are present in, but not confined to, the phenotypic HSC compartment. RNA sequencing of JMML LSC reveals up-regulation of stem cell and fetal genes (HLF, MEIS1, CNN3, VNN2, and HMGA2) and candidate therapeutic targets/biomarkers (MTOR, SLC2A1, and CD96), paving the way for LSC-directed disease monitoring and therapy in this disease.

Discovery of a CD10-negative B-progenitor in human fetal life identifies unique ontogeny-related developmental programs.

Human lymphopoiesis is a dynamic lifelong process that starts in utero 6 weeks postconception. Although fetal B-lymphopoiesis remains poorly defined, it is key to understanding leukemia initiation in early life. Here, we provide a comprehensive analysis of the human fetal B-cell developmental hierarchy. We report the presence in fetal tissues of 2 distinct CD19+ B-progenitors, an adult-type CD10+ve ProB-progenitor and a new CD10-ve PreProB-progenitor, and describe their molecular and functional characteristics. PreProB-progenitors and ProB-progenitors appear early in the first trimester in embryonic liver, followed by a sustained second wave of B-progenitor development in fetal bone marrow (BM), where together they form >40% of the total hematopoietic stem cell/progenitor pool. Almost one-third of fetal B-progenitors are CD10-ve PreProB-progenitors, whereas, by contrast, PreProB-progenitors are almost undetectable (0.53% ± 0.24%) in adult BM. Single-cell transcriptomics and functional assays place fetal PreProB-progenitors upstream of ProB-progenitors, identifying them as the first B-lymphoid-restricted progenitor in human fetal life. Although fetal BM PreProB-progenitors and ProB-progenitors both give rise solely to B-lineage cells, they are transcriptionally distinct. As with their fetal counterparts, adult BM PreProB-progenitors give rise only to B-lineage cells in vitro and express the expected B-lineage gene expression program. However, fetal PreProB-progenitors display a distinct, ontogeny-related gene expression pattern that is not seen in adult PreProB-progenitors, and they share transcriptomic signatures with CD10-ve B-progenitor infant acute lymphoblastic leukemia blast cells. These data identify PreProB-progenitors as the earliest B-lymphoid-restricted progenitor in human fetal life and suggest that this fetal-restricted committed B-progenitor might provide a permissive cellular context for prenatal B-progenitor leukemia initiation.

Neonatal leukaemia.

Neonatal leukaemia is defined as occurring within the first 28 days of life and most, if not all, cases are congenital. With the exception of Down syndrome-associated transient abnormal myelopoiesis, which is not considered here, neonatal leukaemias are rare. In two-thirds of patients the disease manifests as an acute myeloid leukaemia, frequently with monocytic/monoblastic characteristics. Most other cases are acute lymphoblastic leukaemia, particularly B lineage, but some are mixed phenotype or blastic plasmacytoid dendritic cell neoplasms. The most frequently observed cytogenetic/molecular abnormality is t(4;11)(q21.3;q23.3)/KMT2A-AFF1 followed by t(1;22)(p13.3;q13.1)/RBM15-MKL1 and t(8;16)(p11.2;p13.3)/KAT6A-CREBBP. Common clinical features include prominent hepatosplenomegaly and a high incidence of skin involvement, sometimes in the absence of bone marrow disease. A distinctive feature is the occurrence of spontaneous remission in some cases, particularly in association with t(8;16). In this review, we summarise current knowledge of the clinical, cytogenetic and molecular features of neonatal leukaemia and discuss clinical management of these cases.

Niche-mediated depletion of the normal hematopoietic stem cell reservoir by Flt3-ITD-induced myeloproliferation.

Although previous studies suggested that the expression of FMS-like tyrosine kinase 3 (Flt3) initiates downstream of mouse hematopoietic stem cells (HSCs), FLT3 internal tandem duplications (FLT3 ITDs) have recently been suggested to intrinsically suppress HSCs. Herein, single-cell interrogation found Flt3 mRNA expression to be absent in the large majority of phenotypic HSCs, with a strong negative correlation between Flt3 and HSC-associated gene expression. Flt3-ITD knock-in mice showed reduced numbers of phenotypic HSCs, with an even more severe loss of long-term repopulating HSCs, likely reflecting the presence of non-HSCs within the phenotypic HSC compartment. Competitive transplantation experiments established that Flt3-ITD compromises HSCs through an extrinsically mediated mechanism of disrupting HSC-supporting bone marrow stromal cells, with reduced numbers of endothelial and mesenchymal stromal cells showing increased inflammation-associated gene expression. Tumor necrosis factor (TNF), a cell-extrinsic potent negative regulator of HSCs, was overexpressed in bone marrow niche cells from FLT3-ITD mice, and anti-TNF treatment partially rescued the HSC phenotype. These findings, which establish that Flt3-ITD-driven myeloproliferation results in cell-extrinsic suppression of the normal HSC reservoir, are of relevance for several aspects of acute myeloid leukemia biology.

Hemophagocytic lymphohistiocytosis responding to withdrawal of gluten: a case report.

BACKGROUND: This is the first documented case of a patient with hemophagocytic lymphohistiocytosis in association with coeliac disease. There was complete clinical and biochemical remission of hemophagocytic lymphohistiocytosis following the introduction of a gluten-free diet. CASE PRESENTATION: A 7-year-old white girl presented with fevers and maculopapular rash with a recent history of tonsillitis. Blood tests revealed thrombocytopenia (64×109/L), anemia (80 g/L), hypofibrinogenemia (1 g/L), and hyperferritinemia (71,378 µg/L). A bone marrow revealed evidence of hemophagocytosis, but the results of tests for the genetic or familial-associated hemophagocytic lymphohistiocytosis syndromes were negative. The results of screening tests for known secondary causes were negative. She was diagnosed as having hemophagocytic lymphohistiocytosis and following treatment with the hemophagocytic lymphohistiocytosis-2004 protocol these symptoms, in addition to the biochemical and hematological markers, completely resolved. She presented again 10 months later with fever, rash, and biochemical abnormalities suggestive of hemophagocytic lymphohistiocytosis. Her tissue transglutaminase was markedly raised and the results of blood tests revealed a genetic susceptibly to coeliac disease in the form of HLA-DQ2 positivity. She commenced a gluten-free diet and there was complete symptomatic and biochemical response without any further chemotherapy. She had further episodic rashes, each associated with the accidental intake of gluten. CONCLUSIONS: This is to the best of our knowledge the first documented case of hemophagocytic lymphohistiocytosis in association with coeliac disease. No other secondary cause found; she initially responded to chemoimmunotherapy specific for hemophagocytic lymphohistiocytosis but relapsed within a few months of cessation of treatment and then achieved complete remission on gluten withdrawal alone.