miR-196b target screen reveals mechanisms maintaining leukemia stemness with therapeutic potential.

We have shown that antagomiR inhibition of miRNA miR-21 and miR-196b activity is sufficient to ablate MLL-AF9 leukemia stem cells (LSC) in vivo. Here, we used an shRNA screening approach to mimic miRNA activity on experimentally verified miR-196b targets to identify functionally important and therapeutically relevant pathways downstream of oncogenic miRNA in MLL-r AML. We found Cdkn1b (p27Kip1) is a direct miR-196b target whose repression enhanced an embryonic stem cell–like signature associated with decreased leukemia latency and increased numbers of leukemia stem cells in vivo. Conversely, elevation of p27Kip1 significantly reduced MLL-r leukemia self-renewal, promoted monocytic differentiation of leukemic blasts, and induced cell death. Antagonism of miR-196b activity or pharmacologic inhibition of the Cks1-Skp2–containing SCF E3-ubiquitin ligase complex increased p27Kip1 and inhibited human AML growth. This work illustrates that understanding oncogenic miRNA target pathways can identify actionable targets in leukemia

Comprehensive characterization of human bone marrow microenvironment shows age-related changes

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Setd2 regulates quiescence and differentiation of adult hematopoietic stem cells by restricting RNA polymerase II elongation

SET domain containing 2 (Setd2), encoding a histone methyltransferase, is associated with many hematopoietic diseases when mutated. By generating a novel exon 6 conditional knockout mouse model, we describe an essential role of Setd2 in maintaining the adult hematopoietic stem cells. Loss of Setd2 results in leukopenia, anemia, and increased platelets accompanied by hypocellularity, erythroid dysplasia, and mild fibrosis in bone marrow. Setd2 knockout mice show significantly decreased hematopoietic stem and progenitor cells except for erythroid progenitors. Setd2 knockout hematopoietic stem cells fail to establish long-term bone marrow reconstitution after transplantation because of the loss of quiescence, increased apoptosis, and reduced multiple-lineage terminal differentiation potential. Bioinformatic analysis revealed that the hematopoietic stem cells exit from quiescence and commit to differentiation, which lead to hematopoietic stem cell exhaustion. Mechanistically, we attribute an important Setd2 function in murine adult hematopoietic stem cells to the inhibition of the Nsd1/2/3 transcriptional complex, which recruits super elongation complex and controls RNA polymerase II elongation on a subset of target genes, including Myc. Our results reveal a critical role of Setd2 in regulating quiescence and differentiation of hematopoietic stem cells through restricting the NSDs/SEC mediated RNA polymerase II elongation

Twist1 Suppresses Senescence Programs and Thereby Accelerates and Maintains Mutant Kras-Induced Lung Tumorigenesis

KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer mouse model and in human lung tumors that the inhibition of Twist1 restores a senescence program inducing the loss of a neoplastic phenotype. The Twist1 gene encodes for a transcription factor that is essential during embryogenesis. Twist1 has been suggested to play an important role during tumor progression. However, there is no in vivo evidence that Twist1 plays a role in autochthonous tumorigenesis. Through two novel transgenic mouse models, we show that Twist1 cooperates with KrasG12D to markedly accelerate lung tumorigenesis by abrogating cellular senescence programs and promoting the progression from benign adenomas to adenocarcinomas. Moreover, the suppression of Twist1 to physiological levels is sufficient to cause Kras mutant lung tumors to undergo senescence and lose their neoplastic features. Finally, we analyzed more than 500 human tumors to demonstrate that TWIST1 is frequently overexpressed in primary human lung tumors. The suppression of TWIST1 in human lung cancer cells also induced cellular senescence. Hence, TWIST1 is a critical regulator of cellular senescence programs, and the suppression of TWIST1 in human tumors may be an effective example of pro-senescence therapy

Multiomic profiling identifies predictors of survival in African American patients with acute myeloid leukemia

Genomic profiles and prognostic biomarkers in patients with acute myeloid leukemia (AML) from ancestry-diverse populations are underexplored. We analyzed the exomes and transcriptomes of 100 patients with AML with genomically confirmed African ancestry (Black; Alliance) and compared their somatic mutation frequencies with those of 323 self-reported white patients with AML, 55% of whom had genomically confirmed European ancestry (white; BeatAML). Here we find that 73% of 162 gene mutations recurrent in Black patients, including a hitherto unreported PHIP alteration detected in 7% of patients, were found in one white patient or not detected. Black patients with myelodysplasia-related AML were younger than white patients suggesting intrinsic and/or extrinsic dysplasia-causing stressors. On multivariable analyses of Black patients, NPM1 and NRAS mutations were associated with inferior disease-free and IDH1 and IDH2 mutations with reduced overall survival. Inflammatory profiles, cell type distributions and transcriptional profiles differed between Black and white patients with NPM1 mutations. Incorporation of ancestry-specific risk markers into the 2022 European LeukemiaNet genetic risk stratification changed risk group assignment for one-third of Black patients and improved their outcome prediction.</p

Why Single-Cell Sequencing Has Promise in MDS

Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases characterized by ineffective hematopoiesis. The risk of MDS is associated with aging and the accumulation of somatic mutations in hematopoietic stem cells and progenitors (HSPC). While advances in DNA sequencing in the past decade unveiled clonal selection driven by mutations in MDS, it is unclear at which stage the HSPCs are trapped or what prevents mature cells output. Single-cell-sequencing techniques in recent years have revolutionized our understanding of normal hematopoiesis by identifying the transitional cell states between classical hematopoietic hierarchy stages, and most importantly the biological activities behind cell differentiation and lineage commitment. Emerging studies have adapted these powerful tools to investigate normal hematopoiesis as well as the clonal heterogeneity in myeloid malignancies and provide a progressive description of disease pathogenesis. This review summarizes the potential of growing single-cell-sequencing techniques, the evolving efforts to elucidate hematopoiesis in physiological conditions and MDS at single-cell resolution, and discuss how they may fill the gaps in our current understanding of MDS biology.</jats:p

A Gene Dosage Requirement for Transcription Factor Gfi1 in the Regulation of Myelopoiesis and Myeloproliferative Disorders.

Abstract The generation of mature myeloid lineage cells from hematopoietic stem cells (HSCs) requires a precise synergy between cytokine signaling and lineage-specific transcription factors. Gfi1 (Growth Factor Independence 1) is a zinc finger transcription factor that is necessary for normal myelopoiesis. Gfi1 knockout mice display an abnormal ratio of phenotypic common myeloid progenitors (CMP) and granulocyte/monocyte progenitors (GMP), and such mice completely lack mature neutrophils. In contrast, the Gfi1 heterozygote mouse presents no obvious phenotype. Here we show a gene dosage requirement for Gfi1 in the differentiation of mature myeloid cells. While bone marrow from wild type littermates generates granulocytic, monocytic and mixed methylcellulose colonies, Gfi1 knockout bone marrow cells yield mainly monocytic colonies. In comparison to wild type littermates, Gfi1+/− bone marrow cells generate lower numbers of granulocytic colonies and higher numbers of monocytic colonies. Interestingly, methylcellulose colonies from both Gfi1 knockout mice and heterozygotes display increased serial replating capacity in comparison to wild type littermates. These data suggest that Gfi1 gene dosage may control self renewal and may relate to oncogenic transformation. Activating mutations in K-Ras are common genetic abnormalities in human acute myeloid leukemia and myeloproliferative disease (MPD). However, expression of activated Ras from endogenous regulatory sequences (knock-in) results in an MPD of varying lethality. The severity of the K-Ras-induced MPD depends on unknown genetic modifiers, as lethality is increased in a Balb/c genetic background. Given the effect of Gfi1 gene dosage on serial replating, we have analyzed the effect of Gfi1 gene dosage on the activated Ras knock-in mouse model of MPD. Preliminary data indicate that lowering Gfi1 gene dosage modifies the phenotype of activated Ras MPD by dramatically increasing the number of immature circulating myeloid progenitors. These results reveal that Gfi1 is a modifier of Ras induced disease pathogenesis.</jats:p