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Acute myeloid leukemia may be characterized by a fraction of leukemia stem cells (LSCs) that sustain disease propagation eventually leading to relapse. Yet, the contribution of LSCs to early therapy resistance and AML regeneration remains controversial. We prospectively identify LSCs in AML patients and xenografts by single-cell RNA sequencing coupled with functional validation by a microRNA-126 reporter enriching for LSCs. Through nucleophosmin 1 ( NPM1 ) mutation calling or chromosomal monosomy detection in single-cell transcriptomes, we discriminate LSCs from regenerating hematopoiesis, and assess their longitudinal response to chemotherapy. Chemotherapy induced a generalized inflammatory and senescence-associated response. Moreover, we observe heterogeneity within progenitor AML cells, some of which proliferate and differentiate with expression of oxidative-phosphorylation (OxPhos) signatures, while others are OxPhos (low) miR-126 (high) and display enforced stemness and quiescence features. miR-126 (high) LSCs are enriched at diagnosis in chemotherapy-refractory AML and at relapse, and their transcriptional signature robustly stratifies patients for survival in large AML cohorts. Relapse within acute myeloid leukaemia may be driven by the presence of leukaemia stem cells. Here, the authors use single cell RNA-seq seq to characterise leukemia stem cells, and show miR-126 as a potential marker of resistance.

In vivo gene therapy to the liver using lentiviral vectors (LV) may represent a one-and-done therapeutic approach for monogenic diseases. Increasing LV gene therapy potency is crucial for reducing the effective doses, thus alleviating dose-dependent toxicities and facilitating manufacturing. LV-mediated liver transduction may be enhanced by positively selecting LV-transduced hepatocytes after treatment ( a posteriori ) or by augmenting the initial fraction of LV-targeted hepatocytes (a priori). We show here that the a posteriori enhancement increased transgene output without expansion of hepatocytes bearing LV genomic integrations near cancer genes, in mouse models of hemophilia, an inherited coagulation disorder. Furthermore, we enhanced hepatocyte transduction a priori in mice by transiently inhibiting antiviral pathways and/or through a fasting regimen. The most promising transduction-enhancer combination synergized with phagocytosis-shielded LV, resulting in a remarkable 40-fold increase in transgene output. Overall, our work highlights the potential of minimally invasive, cost-effective treatments capable of improving the potency of in vivo LV gene therapy to hepatocytes, in order to expand its applicability and ease clinical translation. Lentiviral vectors are promising gene delivery vehicles to target hepatocytes in vivo, but restriction factors limit their efficiency. Here, the authors counteract many of these restrictions, amplifying lentiviral gene transfer into hepatocytes, strengthening its translational potential.

Self-inactivating (SIN) lentiviral vectors (LV) have an excellent therapeutic potential as demonstrated in preclinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could still pose a significant risk in clinical applications. Taking advantage of novel in vivo genotoxicity assays, we tested a battery of LV constructs, including some with clinically relevant designs, and found that oncogene activation by promoter insertion is the most powerful mechanism of early vector-induced oncogenesis. SIN LVs disabled in their capacity to activate oncogenes by promoter insertion were less genotoxic and induced tumors by enhancer-mediated activation of oncogenes with efficiency that was proportional to the strength of the promoter used. On the other hand, when enhancer activity was reduced by using moderate promoters, oncogenesis by inactivation of tumor suppressor gene was revealed. This mechanism becomes predominant when the enhancer activity of the internal promoter is shielded by the presence of a synthetic chromatin insulator cassette. Our data provide both mechanistic insights and quantitative readouts of vector-mediated genotoxicity, allowing a relative ranking of different vectors according to these features, and inform current and future choices of vector design with increasing biosafety.

Hematopoietic stem cell gene therapy (GT) using a γ-retroviral vector (γ-RV) is an effective treatment for Severe Combined Immunodeficiency due to Adenosine Deaminase deficiency. Here, we describe a case of GT-related T-cell acute lymphoblastic leukemia (T-ALL) that developed 4.7 years after treatment. The patient underwent chemotherapy and haploidentical transplantation and is currently in remission. Blast cells contain a single vector insertion activating the LIM-only protein 2 ( LMO2) proto-oncogene, confirmed by physical interaction, and low Adenosine Deaminase (ADA) activity resulting from methylation of viral promoter. The insertion is detected years before T-ALL in multiple lineages, suggesting that further hits occurred in a thymic progenitor. Blast cells contain known and novel somatic mutations as well as germline mutations which may have contributed to transformation. Before T-ALL onset, the insertion profile is similar to those of other ADA-deficient patients. The limited incidence of vector-related adverse events in ADA-deficiency compared to other γ-RV GT trials could be explained by differences in transgenes, background disease and patient’s specific factors. Leukaemia development has been reported as an associated risk of haematopoietic stem cell gene therapy (HSPC-GT) using retroviral vectors in different diseases. Here, the authors show a case of T-cell acute lymphoid leukaemia in a patient with Adenosine Deaminase-deficient Severe Combined Immunodeficiency (ADA-SCID) treated with retroviral gene therapy.

Within the chromatin, distal elements interact with promoters to regulate specific transcriptional programs. Histone acetylation, interfering with the net charges of the nucleosomes, is a key player in this regulation. Here, we report that the oncoprotein SET is a critical determinant for the levels of histone acetylation within enhancers. We disclose that a condition in which SET is accumulated, the severe Schinzel-Giedion Syndrome (SGS), is characterized by a failure in the usage of the distal regulatory regions typically employed during fate commitment. This is accompanied by the usage of alternative enhancers leading to a massive rewiring of the distal control of the gene transcription. This represents a (mal)adaptive mechanism that, on one side, allows to achieve a certain degree of differentiation, while on the other affects the fine and corrected maturation of the cells. Thus, we propose the differential in cis -regulation as a contributing factor to the pathological basis of SGS and possibly other the SET-related disorders in humans. The usage of specific distal regulatory regions within the genome is critical for fate specification and cell maturation. Here, the authors show that the accumulation of the oncoprotein SET, as occurring in the rare Schinzel-Giedion syndrome, and associated histone hypo-acetylation interfere with normal enhancer repertoire employed during brain development.

Within the chromatin, distal elements interact with promoters to regulate specific transcriptional programs. Histone acetylation, interfering with the net charges of the nucleosomes, is a key player in this regulation. Here, we report that the onco-protein SET is a critical determinant for the levels of histone acetylation within enhancers. We disclose that conditions in which SET is accumulated, including the severe Schinzel-Giedion Syndrome (SGS), are characterized by a failure in the usage of the distal regulatory regions typically employed during fate commitment. This is accompanied by the usage of alternative enhancers leading to a massive rewiring of the distal control of the gene transcription. This represents a (mal)adaptive mechanism that, on one side, allows to achieve a certain degree of differentiation, while on the other affects the fine and corrected maturation of the cells. Thus, we propose the differential in cis-regulation as a contributing factor to the pathological basis of the SET-related disorders in humans, including SGS, neurodevelopmental disorders, myeloproliferative diseases, and cancer.