Summary In acute myeloid leukemia (AML), the cell of origin, nature and biological consequences of initiating lesions and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukemic phase. Here, highly purified hematopoietic stem cells (HSC), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3a mutations (DNMT3amut) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3amut-bearing HSC exhibited multilineage repopulation advantage over non-mutated HSC in xenografts, establishing their identity as pre-leukemic-HSC (preL-HSC). preL-HSC were found in remission samples indicating that they survive chemotherapy. Thus DNMT3amut arises early in AML evolution, likely in HSC, leading to a clonally expanded pool of preL-HSC from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance.

In acute myeloid leukaemia, long-term survival is poor as most patients relapse despite achieving remission. Historically, the failure of therapy has been thought to be due to mutations that produce drug resistance, possibly arising as a consequence of the mutagenic properties of chemotherapy drugs. However, other lines of evidence have pointed to the pre-existence of drug-resistant cells. For example, deep sequencing of paired diagnosis and relapse acute myeloid leukaemia samples has provided direct evidence that relapse in some cases is generated from minor genetic subclones present at diagnosis that survive chemotherapy, suggesting that resistant cells are generated by evolutionary processes before treatment and are selected by therapy. Nevertheless, the mechanisms of therapy failure and capacity for leukaemic regeneration remain obscure, as sequence analysis alone does not provide insight into the cell types that are fated to drive relapse. Although leukaemia stem cells have been linked to relapse owing to their dormancy and self-renewal properties, and leukaemia stem cell gene expression signatures are highly predictive of therapy failure, experimental studies have been primarily correlative and a role for leukaemia stem cells in acute myeloid leukaemia relapse has not been directly proved. Here, through combined genetic and functional analysis of purified subpopulations and xenografts from paired diagnosis/relapse samples, we identify therapy-resistant cells already present at diagnosis and two major patterns of relapse. In some cases, relapse originated from rare leukaemia stem cells with a haematopoietic stem/progenitor cell phenotype, while in other instances relapse developed from larger subclones of immunophenotypically committed leukaemia cells that retained strong stemness transcriptional signatures. The identification of distinct patterns of relapse should lead to improved methods for disease management and monitoring in acute myeloid leukaemia. Moreover, the shared functional and transcriptional stemness properties that underlie both cellular origins of relapse emphasize the importance of developing new therapeutic approaches that target stemness to prevent relapse.

SummaryTo investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance.

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Transcription factor IKZF1 (IKAROS) acts as a critical regulator of lymphoid differentiation and is frequently deleted or mutated in B-cell precursor acute lymphoblastic leukemia. IKZF1 gene defects are associated with inferior treatment outcome in both childhood and adult B-cell precursor acute lymphoblastic leukemia and occur in more than 70% of BCR-ABL1-positive and BCR-ABL1-like cases of acute lymphoblastic leukemia. Over the past few years, much has been learned about the tumor suppressive function of IKZF1 during leukemia development and the molecular pathways that relate to its impact on treatment outcome. In this review, we provide a concise overview on the role of IKZF1 during normal lymphopoiesis and the pathways that contribute to leukemia pathogenesis as a consequence of altered IKZF1 function. Furthermore, we discuss different mechanisms by which IKZF1 alterations impose therapy resistance on leukemic cells, including enhanced cell adhesion and modulation of glucocorticoid response.

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Deletions and mutations affecting lymphoid transcription factor IKZF1 (IKAROS) are associated with an increased relapse risk and poor outcome in B-cell precursor acute lymphoblastic leukemia. However, additional genetic events may either enhance or negate the effects of IKZF1 deletions on prognosis. In a large discovery cohort of 533 childhood B-cell precursor acute lymphoblastic leukemia patients, we observed that single-copy losses of BTG1 were significantly enriched in IKZF1-deleted B-cell precursor acute lymphoblastic leukemia (P=0.007). While BTG1 deletions alone had no impact on prognosis, the combined presence of BTG1 and IKZF1 deletions was associated with a significantly lower 5-year event-free survival (P=0.0003) and a higher 5-year cumulative incidence of relapse (P=0.005), when compared with IKZF1-deleted cases without BTG1 aberrations. In contrast, other copy number losses commonly observed in B-cell precursor acute lymphoblastic leukemia, such as CDKN2A/B, PAX5, EBF1 or RB1, did not affect the outcome of IKZF1-deleted acute lymphoblastic leukemia patients. To establish whether the combined loss of IKZF1 and BTG1 function cooperate in leukemogenesis, Btg1-deficient mice were crossed onto an Ikzf1 heterozygous background. We observed that loss of Btg1 increased the tumor incidence of Ikzf1+/− mice in a dose-dependent manner. Moreover, murine B cells deficient for Btg1 and Ikzf1+/− displayed increased resistance to glucocorticoids, but not to other chemotherapeutic drugs. Together, our results identify BTG1 as a tumor suppressor in leukemia that, when deleted, strongly enhances the risk of relapse in IKZF1-deleted B-cell precursor acute lymphoblastic leukemia, and augments the glucocorticoid resistance phenotype mediated by the loss of IKZF1 function.

2Abbreviations: 2D, two-dimensional; 3D, three-dimensional; AFS, atomic force spectroscopy; ECM, extracellular matrix; H2B, histone-2B; MMP matrix metalloproteinase; NII, nuclear irregularity index; NT, non-targeting control; PDMS, polydimethylsiloxane SummaryNuclear deformability during cancer cell invasion and metastasis is critically regulated by lamin A. Here, researchers showed that lamin B2 also contributes to nuclear mechanics, implicating cooperating lamin networks regulating nuclear integrity, migration efficacy, and metastatic tumor progression. AbstractInterstitial tumor cell invasion depends upon complex mechanochemical adaptation of both cell body and the rigid nucleus in response to extracellular tissue topologies. Nuclear mechanics during cell migration through confined environments is controlled by A-type lamins, however, the contribution of B-type lamins to the deformability of the nucleus remains unclear. Using systematic expression regulation of different lamin isoforms, we applied multi-parameter wet-lab and in silico analysis to test their impact on nuclear mechanics, shape regulation, and cancer cell migration. Modulation of lamin A/C and B2 but not B1 isoforms controlled nuclear deformation and viscoelasticity, whereby lamin B2 generally followed lamin A/C-mediated effects. Cell migration rates were altered by 5 to 9fold in dense collagen environments and synthetic devices, with accelerated rates after lamin downregulation and reverse effects after lamin upregulation, with migration rates strongly depending on nuclear shape change. These findings implicate cooperation of lamin B2 with lamin A/C in regulating nuclear mechanics for shape adaptation and migration efficacy.