No abstract

Leukemia-initiating cells (LICs

Acute myeloid leukemia (AML) induces bone marrow (BM) failure in patients, predisposing them to life-threatening infections and bleeding. The mechanism by which AML mediates this complication is unknown but one widely accepted explanation is that AML depletes the BM of hematopoietic stem cells (HSCs) through displacement. We sought to investigate how AML affects hematopoiesis by quantifying residual normal hematopoietic subpopulations in the BM of immunodeficient mice transplanted with human AML cells with a range of genetic lesions. The numbers of normal mouse HSCs were preserved whereas normal progenitors and other downstream hematopoietic cells were reduced following transplantation of primary AMLs, findings consistent with a differentiation block at the HSC-progenitor transition, rather than displacement. Once removed from the leukemic environment, residual normal hematopoietic cells differentiated normally and outcompeted steady-state hematopoietic cells, indicating that this effect is reversible. We confirmed the clinical significance of this by ex vivo analysis of normal hematopoietic subpopulations from BM of 16 patients with AML. This analysis demonstrated that the numbers of normal CD34 + CD38− stem-progenitor cells were similar in the BM of AML patients and controls, whereas normal CD34 + CD38+ progenitors were reduced. Residual normal CD34+ cells from patients with AML were enriched in long-term culture, initiating cells and repopulating cells compared with controls. In conclusion the data do not support the idea that BM failure in AML is due to HSC depletion. Rather, AML inhibits production of downstream hematopoietic cells by impeding differentiation at the HSC-progenitor transition.ematopoiesis is tightly regulated under normal circumstances to ensure adequate production of mature blood cells. At steady state hematopoietic stem cells (HSCs) are relatively quiescent and the majority of proliferation occurs downstream of HSCs. Hematopoietic stresses such as bleeding or bone marrow (BM) damage from chemotherapy induce HSCs to enter the cell cycle to replenish mature blood cells (1-3).BM failure (reduced production of neutrophils, red cells, and platelets) is almost universal at diagnosis of acute myeloid leukemia (AML) and contributes significantly to morbidity and mortality by inducing severe infections and bleeding. These complications often compromise the delivery of intensive chemotherapy and lead to a high frequency of induction death (4).A common assumption is that marrow failure occurs due to displacement of normal hematopoietic cells from the marrow by AML cells, resulting in depletion of normal hematopoietic cells. However, AML has a more profound impact on BM function than many other types of hematologic malignancy (e.g., chronic lymphocytic leukemia, follicular lymphoma) even where there is a similar degree of diffuse infiltration of BM by leukemia/ lymphoma cells.To clarify how AML suppresses normal hematopoiesis, we investigated the impact of AML on residual normal hematopoietic subpopul...

Key Points• Most AMLs lack ASS1, which allows synthesis of arginine, and so depend on exogenous sources.• Depletion of arginine via ADI-PEG 20 reduces the burden of primary AML in vivo and in vitro.The strategy of enzymatic degradation of amino acids to deprive malignant cells of important nutrients is an established component of induction therapy of acute lymphoblastic leukemia.Here we show that acute myeloid leukemia (AML) cells from most patients with AML are deficient in a critical enzyme required for arginine synthesis, argininosuccinate synthetase-1 (ASS1). Thus, these ASS1-deficient AML cells are dependent on importing extracellular arginine. We therefore investigated the effect of plasma arginine deprivation using pegylated arginine deiminase (ADI-PEG 20) against primary AMLs in a xenograft model and in vitro. ADI-PEG 20 alone induced responses in 19 of 38 AMLs in vitro and 3 of 6 AMLs in vivo, leading to caspase activation in sensitive AMLs. ADI-PEG 20-resistant AMLs showed higher relative expression of ASS1 than sensitive AMLs. This suggests that the resistant AMLs survive by producing arginine through this metabolic pathway and ASS1 expression could be used as a biomarker for response. Sensitive AMLs showed more avid uptake of arginine from the extracellular environment consistent with their auxotrophy for arginine. The combination of ADI-PEG 20 and cytarabine chemotherapy was more effective than either treatment alone resulting in responses in 6 of 6 AMLs tested in vivo. Our data show that arginine deprivation is a reasonable strategy in AML that paves the way for clinical trials. (Blood. 2015;125(26): 4060-4068)

We used single cell Q-PCR on a micro-fluidic platform (Fluidigm) to analyse clonal, genetic architecture and phylogeny in acute myeloid leukaemia (AML) using selected mutations. Ten cases of NPM1 c mutant AML were screened for 111 mutations that are recurrent in AML and cancer. Clonal architectures were relatively simple with one to six sub-clones and were branching in some, but not all, patients. NPM1 mutations were secondary or sub-clonal to other driver mutations ( DNM3TA, TET2, WT1 and IDH2 ) in all cases. In three of the ten cases, single cell analysis of enriched CD34 + /CD33 − cells revealed a putative pre-leukaemic sub-clone, undetectable in the bulk CD33 + population that had one or more driver mutations but lacked NPM1 c. Cells from all cases were transplanted into NSG mice and in most (8/10), more than one sub-clone (#2-5 sub-clones) transplanted. However, the dominant regenerating sub-clone in 9/10 cases was NPM1 + and this sub-clone was either dominant or minor in the diagnostic sample from which it was derived. This study provides further evidence, at the single cell level, for genetic variegation in sub-clones and stem cells in acute leukaemia and demonstrates both a preferential order of mutation accrual and parallel evolution of sub-clones.

Activation of the pseudoexon in the GHR gene can lead to a variety of GHI phenotypes. Therefore, screening for the presence of this mutation should be performed in all GHI patients without mutations in the coding exons.

No abstract

Key Points• Loss of imprinting occurs at the 14q32 domain in APL.• DNA methylation at the CTCF binding sites correlates with the overexpression of 14q32 miRNAs.Distinct patterns of DNA methylation characterize the epigenetic landscape of promyelocytic leukemia/retinoic acid receptor-a (PML-RARa)-associated acute promyelocytic leukemia (APL). We previously reported that the microRNAs (miRNAs) clustered on chromosome 14q32 are overexpressed only in APL. Here, using high-throughput bisulfite sequencing, we identified an APL-associated hypermethylation at the upstream differentially methylated region (DMR), which also included the site motifs for the enhancer blocking protein CCCTC-binding factor (CTCF). Comparing the profiles of diagnostic/ remission paired patient samples, we show that hypermethylation was acquired in APL in a monoallelic manner. The cytosine guanine dinucleotide status of the DMR correlated with expression of the miRNAs following a characteristic position-dependent pattern. Moreover, a signature of hypermethylation was also detected in leukemic cells from an established transgenic PML-RARA APL mouse model at the orthologous region on chromosome 12, including the CTCF binding site located upstream from the mouse miRNA cluster. These results, together with the demonstration that the region does not show DNA methylation changes during myeloid differentiation, provide evidence that 14q32 hypermethylation is implicated in the pathogenesis of APL. We propose a model in which loss of imprinting at the 14q32 domain leads to overexpression of the miRNAs in APL. (Blood. 2014;123(13):2066-2074 IntroductionAcute promyelocytic leukemia (APL) is a subclass of acute myeloid leukemia (AML) characterized by the balanced reciprocal translocation t(15;17)(q22;q11-12) resulting in the fusion between the promyelocytic leukemia gene (PML) and the retinoic acid receptor-a (RARA) gene. 1 The chimeric protein PML-RARa leads to a block of myeloid cell differentiation through constitutive repression of retinoic acid responsive genes.2 This is consistent with the typical accumulation of abnormal hematopoietic progenitor cells blocked at the promyelocyte stage. Accordingly, PML-RARa has been shown to induce APL in transgenic mice. 3 However, deregulation of the retinoic acid pathway is insufficient to initiate APL, 4 and several studies have shown additional genetic and epigenetic processes that accompany the expression of the PML-RARa protein, 5,6 also involving master transcription regulators 7 and modulators of chromatin structure. 8,9 MicroRNAs (miRNAs) are single-stranded small noncoding RNAs (sncRNAs) that negatively regulate the expression of target genes.10 They have been extensively associated with cancer as regulators of cell proliferation, differentiation, and apoptosis. 11 We have previously reported a signature of overexpressed miRNAs in APL.12 These miRNAs are clustered in the DLK1-DIO3 imprinted domain on chromosome 14q32, 13,14 and their specific upregulation in primary APL cells was also confirmed by other ...