Inhibition of the phosphatidylinositol-3-kinase pathway is selectively cytotoxic to primitive and mature acute myeloid leukemia progenitor cells while sparing their normal counterparts

Acute myeloid leukemia (AML) is a disorder of the hematopoietic system in which there is an overproduction of immature myeloblast (blast) cells in the bone marrow and suppression of normal hematopoiesis. Like normal hematopoiesis, AML is structured hierarchically and is maintained by rare progenitor cells capable of self-renewal and production of large numbers of leukemic blasts. These rare cells, leukemia-initiating cells (L-IC), are capable of engraftment and proliferation in immunocompromised mice. Dysregulation of signal transduction pathways are frequent occurrences in AML, leading to enhanced cell proliferation and resistance to apoptosis. Pathways which are frequently constitutively active in AML blasts include the phosphatidylinosityl-3-kinase (PI3K) and FMS-like tyrosine kinase 3 (FLT3) receptor signalling pathways. Constitutive PI3K activation, as measured by Akt phosphorylation, was detected in the majority of the 36 AML patient samples tested. The PI3K signalling intermediate integrin linked kinase (ILK) was ubiquitously expressed, and FLT3 abundantly expressed although at variable levels. Furthermore, in the five samples tested, ILK and FLT3, as well as PI3K pathway activation, were also detected in CD34+CD38- sorted and quiescent AML cells, populations enriched for L-IC. siRNA inhibition of ILK caused a decrease in AML colony forming cell (CFC) production. To further test the effects of ILK inhibition on AML, the small molecule inhibitor QLT0267 was used. In vitro kinase assays showed that QLT0267 inhibits both ILK and FLT3 kinases to a similar degree. Treatment of AML blasts with QLT0267 caused dose-dependent killing of AML-CFC, with AML samples containing FLT3 mutations being more sensitive to the inhibitor, although some samples with wildtype FLT3 were also effectively killed. Treatment of AML cells with QLT0267 also decreased survival of both primitive leukemic progenitor cells and L-IC in a dose-dependent fashion. In contrast, QLT0267 showed little toxicity toward normal bone marrow progenitors detected in the same assays. When combined with cytarabine or daunorubicin, QLT0267 worked synergistically to kill AML-CFC from a proportion of AML samples tested. Thus, FLT3, ILK, and constitutive PI3K activation are present in L-ICs, and these candidate leukemic stem cells can be eliminated by inhibition of these pathways, while their normal hematopoietic counterparts are spared.Medicine, Faculty ofMedical Genetics, Department ofGraduat

Structural and functional properties of the human Notch-1 ligand binding region

We present NMR structural and dynamics analysis of the putative ligand binding region of human Notch-1, comprising EGF-like domains 11–13. Functional integrity of an unglycosylated, recombinant fragment was confirmed by calcium-dependent binding of tetrameric complexes to ligand-expressing cells. EGF modules 11 and 12 adopt a well-defined, rod-like orientation rigidified by calcium. The interdomain tilt is similar to that found in previously studied calcium binding EGF pairs, but the angle of twist is significantly different. This leads to an extended double-stranded ? sheet structure, spanning the two EGF modules. Based on the conservation of residues involved in interdomain hydrophobic packing, we propose this arrangement to be prototypical of a distinct class of EGF linkages. On this premise, we have constructed a model of the 36 EGF modules of the Notch extracellular domain that enables predictions to be made about the general role of calcium binding to this region

Alberta Spinal Muscular Atrophy Newborn Screening—Results from Year 1 Pilot Project

Spinal muscular atrophy (SMA) is a progressive neuromuscular disease caused by biallelic pathogenic/likely pathogenic variants of the survival motor neuron 1 (SMN1) gene. Early diagnosis via newborn screening (NBS) and pre-symptomatic treatment are essential to optimize health outcomes for affected individuals. We developed a multiplex quantitative polymerase chain reaction (qPCR) assay using dried blood spot (DBS) samples for the detection of homozygous absence of exon 7 of the SMN1 gene. Newborns who screened positive were seen urgently for clinical evaluation. Confirmatory testing by multiplex ligation-dependent probe amplification (MLPA) revealed SMN1 and SMN2 gene copy numbers. Six newborns had abnormal screen results among 47,005 newborns screened during the first year and five were subsequently confirmed to have SMA. Four of the infants received SMN1 gene replacement therapy under 30 days of age. One infant received an SMN2 splicing modulator due to high maternally transferred AAV9 neutralizing antibodies (NAb), followed by gene therapy at 3 months of age when the NAb returned negative in the infant. Early data show that all five infants made excellent developmental progress. Based on one year of data, the incidence of SMA in Alberta was estimated to be 1 per 9401 live births

MicroRNA-223 dose levels fine tune proliferation and differentiation in human cord blood progenitors and acute myeloid leukemia

A precise understanding of the role of miR-223 in human hematopoiesis and in the pathogenesis of acute myeloid leukemia (AML) is still lacking. By measuring miR-223 expression in blasts from 115 AML patients, we found significantly higher miR-223 levels in patients with favorable prognosis, whereas patients with low miR-223 expression levels were associated with worse outcome. Furthermore, miR-223 was hierarchically expressed in AML subpopulations, with lower expression in leukemic stem cell-containing fractions. Genetic depletion of miR-223 decreased the leukemia initiating cell (LIC) frequency in a myelomonocytic AML mouse model, but it was not mandatory for rapid-onset AML. To relate these observations to physiologic myeloid differentiation, we knocked down or ectopically expressed miR-223 in cord-blood CD34+ cells using lentiviral vectors. Although miR-223 knockdown delayed myeloerythroid precursor differentiation in vitro, it increased myeloid progenitors in vivo following serial xenotransplantation. Ectopic miR-223 expression increased erythropoiesis, T lymphopoiesis, and early B lymphopoiesis in vivo. These findings broaden the role of miR-223 as a regulator of the expansion/differentiation equilibrium in hematopoietic stem and progenitor cells where its impact is dose- and differentiation-stage-dependent. This also explains the complex yet minor role of miR-223 in AML, a heterogeneous disease with variable degree of myeloid differentiation

MicroRNA-223 dose levels fine tune proliferation and differentiation in human cord blood progenitors and acute myeloid leukemia

A precise understanding of the role of miR-223 in human hematopoiesis and in the pathogenesis of acute myeloid leukemia (AML) is still lacking. By measuring miR-223 expression in blasts from 115 AML patients, we found significantly higher miR-223 levels in patients with favorable prognosis, whereas patients with low miR-223 expression levels were associated with worse outcome. Furthermore, miR-223 was hierarchically expressed in AML subpopulations, with lower expression in leukemic stem cell–containing fractions. Genetic depletion of miR-223 decreased the leukemia initiating cell (LIC) frequency in a myelomonocytic AML mouse model, but it was not mandatory for rapid-onset AML. To relate these observations to physiologic myeloid differentiation, we knocked down or ectopically expressed miR-223 in cord-blood CD34(+) cells using lentiviral vectors. Although miR-223 knockdown delayed myeloerythroid precursor differentiation in vitro, it increased myeloid progenitors in vivo following serial xenotransplantation. Ectopic miR-223 expression increased erythropoiesis, T lymphopoiesis, and early B lymphopoiesis in vivo. These findings broaden the role of miR-223 as a regulator of the expansion/differentiation equilibrium in hematopoietic stem and progenitor cells where its impact is dose- and differentiation-stage-dependent. This also explains the complex yet minor role of miR-223 in AML, a heterogeneous disease with variable degree of myeloid differentiation