Jamie L. Maciaszek

The genomic landscape of pediatric and young adult T-lineage acute lymphoblastic leukemia

Genetic alterations that activate NOTCH1 signaling and T cell transcription factors, coupled with inactivation of the INK4/ARF tumor suppressors, are hallmarks of T-lineage acute lymphoblastic leukemia (T-ALL), but detailed genome-wide sequencing of large T-ALL cohorts has not been carried out. Using integrated genomic analysis of 264 T-ALL cases, we identified 106 putative driver genes, half of which had not previously been described in childhood T-ALL (for example, CCND3, CTCF, MYB, SMARCA4, ZFP36L2 and MYCN). We describe new mechanisms of coding and noncoding alteration and identify ten recurrently altered pathways, with associations between mutated genes and pathways, and stage or subtype of T-ALL. For example, NRAS/FLT3 mutations were associated with immature T-ALL, JAK3/STAT5B mutations in HOXA1 deregulated ALL, PTPN2 mutations in TLX1 deregulated T-ALL, and PIK3R1/PTEN mutations in TAL1 deregulated ALL, which suggests that different signaling pathways have distinct roles according to maturational stage. This genomic landscape provides a logical framework for the development of faithful genetic models and new therapeutic approaches.

Sickle cell trait human erythrocytes are significantly stiffer than normal.

Atomic force microscopy (AFM) allows for high-resolution topography studies of biological cells and measurement of their mechanical properties in physiological conditions. In this work, AFM was employed to measure the stiffness of abnormal human red blood cells from human subjects with the genotype for sickle cell trait. The determined Youngs modulus was compared with that obtained from measurements of erythrocytes from healthy subjects. The results showed that Youngs modulus of pathological erythrocytes was approximately three times higher than in normal cells. Observed differences indicate the effect of the polymerization of sickle hemoglobin as well as possible changes in the organization of the cell cytoskeleton associated with the sickle cell trait.

Microelasticity of red blood cells in sickle cell disease

Translation of cellular mechanics findings is crucial in many diseases, including Alzheimer’s disease, Parkinson’s disease, type II diabetes, malaria, sickle cell disease, and cancer. Atomic force microscopy (AFM) is appropriate for measuring mechanical properties of living and fixed cells due to its high force sensitivity and its ability to measure local and overall properties of individual cells under physiological conditions. A systemic force–displacement curve analysis is reported on the quantification of material stiffness via AFM using two theoretical models derived from the Hertz model. This analysis was applied to red blood cells from patients with sickle cell disease to determine the Young’s modulus of these cells in the oxygenated and deoxygenated state. Sickle cell disease pathophysiology is a consequence of the polymerization of sickle hemoglobin in red blood cells upon partial deoxygenation and the impaired flow of these cells in the microcirculation. A model is presented for a four-sided pyramidal indenter that is subsequently shown to have a better fit to the obtained data than that using a model of a parabolic indenter. It is concluded that deoxygenation and therapeutic treatment have a significant impact on the stiffness. This analysis presents a new approach to addressing medical disorders.

Topography of Native SK Channels Revealed by Force Nanoscopy in Living Neurons

The spatial distribution of ion channels is an important determinant of neuronal excitability. However, there are currently no quantitative techniques to map endogenous ion channels with single-channel resolution in living cells. Here, we demonstrate that integration of pharmacology with single-molecule atomic force microscopy (AFM) allows for the high-resolution mapping of native potassium channels in living neurons. We focus on calcium-activated small conductance (SK) potassium channels, which play a critical role in brain physiology. By linking apamin, a toxin that specifically binds to SK channels, to the tip of an AFM cantilever, we are able to detect binding events between the apamin and SK channels. We find that native SK channels from rat hippocampal neurons reside primarily in dendrites as single entities and in pairs. We also show that SK channel dendritic distribution is dynamic and under the control of protein kinase A. Our study demonstrates that integration of toxin pharmacology with single-molecule AFM can be used to quantitatively map individual native ion channels in living cells, and thus provides a new tool for the study of ion channels in cellular physiology.

Epinephrine Modulates BCAM/Lu and ICAM-4 Expression on the Sickle Cell Trait Red Blood Cell Membrane

Collapse and sudden death in physical training are the most serious complications of sickle cell trait (SCT). There is evidence that erythrocytes in SCT patients aggregate during strenuous exercise, likely because of adhesive interactions with the extracellular matrix (ECM) and endothelial cells, and because of their irregular viscoelastic properties. This results in inflammation, blood flow impairment, and vaso-occlusive events. However, the exact role of stress conditions and how they lead to these complications is virtually unknown. Using single-molecule atomic force microscopy experiments, we found that epinephrine, a hormone that is secreted under stressful conditions, increases both the frequency and strength of adhesion events between basal cell adhesion molecule (BCAM/Lu) and ECM laminin, and between intercellular adhesion molecule-4 (ICAM-4) and endothelial α(v)β(3), compared with nonstimulated SCT erythrocytes. Increases in adhesion frequency provide significant evidence of the role of epinephrine in BCAM/Lu-laminin and ICAM-4-α(v)β(3) bonding, and suggest mechanisms of vaso-occlusion during physical exertion in SCT.

Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin.

Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding.

Dietary supplementation with docosahexanoic acid (DHA) increases red blood cell membrane flexibility in mice with sickle cell disease.

Humans and mice with sickle cell disease (SCD) have rigid red blood cells (RBCs). Omega-3 fatty acids, such as docosahexanoic acid (DHA), may influence RBC deformability via incorporation into the RBC membrane. In this study, sickle cell (SS) mice were fed natural ingredient rodent diets supplemented with 3% DHA (DHA diet) or a control diet matched in total fat (CTRL diet). After 8weeks of feeding, we examined the RBCs for: 1) stiffness, as measured by atomic force microscopy; 2) deformability, as measured by ektacytometry; and 3) percent irreversibly sickled RBCs on peripheral blood smears. Using atomic force microscopy, it is found that stiffness is increased and deformability decreased in RBCs from SS mice fed CTRL diet compared to wild-type mice. In contrast, RBCs from SS mice fed DHA diet had markedly decreased stiffness and increased deformability compared to RBCs from SS mice fed CTRL diet. Furthermore, examination of peripheral blood smears revealed less irreversibly sickled RBCs in SS mice fed DHA diet as compared to CTRL diet. In summary, our findings indicate that DHA supplementation improves RBC flexibility and reduces irreversibly sickled cells by 40% in SS mice. These results point to potential therapeutic benefits of dietary omega-3 fatty acids in SCD.

AKAP-Dependent Modulation of BCAM/Lu Adhesion on Normal and Sickle Cell Disease RBCs Revealed by Force Nanoscopy

Human normal and sickle red blood cells (RBCs) adhere with high affinity to the alpha5 chain of laminin (LAMA5) via the basal cell adhesion molecule/Lutheran (BCAM/Lu) receptor, which is implicated in vasoocclusive episodes in sickle cell disease and activated through the cyclic adenosine monophosphate (cAMP) signaling pathway. However, the effect of the cAMP pathway on the expression of active BCAM/Lu receptors at the single-molecule level is unknown. We established an in vitro technique, based on atomic force microscopy, which enables detection of single BCAM/Lu proteins on the RBC surface and measures the unbinding force between BCAM/Lu and LAMA5. We showed that the expression of active BCAM/Lu receptors is higher in homozygous sickle RBCs (SS-RBCs) than normal RBCs and that it is critically dependent on the cAMP signaling pathway on both normal and SS-RBCs. Of importance, we illustrated that A-kinase anchoring proteins are crucial for BCAM/Lu receptor activation. Furthermore, we found that SS-RBCs from hydroxyurea-treated patients show a lower expression of active BCAM/Lu receptors, a lower unbinding force to LAMA5, and insignificant stimulation by epinephrine as compared to SS-RBCs from untreated patients. To our knowledge, these findings may lead to novel antiadhesive targets for vasoocclusive episodes in sickle cell disease.

445. A Delectable Lentiviral Vector System for Testing Oncogene Addiction in Hematological Malignancies

Mouse models of hematological malignancies can be established by overexpressing oncogenes in hematopoietic cells followed by transplantation. It is often unclear whether the oncogene is only necessary for initiating the malignancies or if it is also required for maintenance, known as oncogene addiction. An addicted oncogene could then serve as target for therapy. We have developed a dual lentiviral vector system to distinguish these possibilities. We chose the oncogene LMO2 as our model because it is involved in a number of leukemias caused by gamma-retroviral vector insertion in gene therapy of SCID-X1 and Wiskott-Aldrich syndrome. Our previous studies showed that overexpression of LMO2 using a gamma-retroviral vector in early thymic progenitors (ETP) caused a differentiation block at the DN2 stage of development and subsequent transplant of these cells gave rise to leukemia with 100% penetrance in mice. We inserted the LoxP sequence into the 3’ U3 region of a self-inactivating CL20 lentiviral backbone and subcloned a MSCV-LMO2-i-GFP expression cassette into the vector (Fig 1A) to make a CL20-tLMO2-i-GFP vector. To deliver Cre recombinase in an inducible fashion, we constructed a second lentiviral vector in which a MSCV-CreERT2-i-mCherry expression cassette is inserted. Vectors were made by transiently transfecting 293T cells and the unconcentrated titer of these vectors were 2E6 and 3E6 TU/ml, respectively. We first tested these vectors in NIH3T3 cells. NIH3T3 cells transduced with these two vectors were subsequently sorted for GFP+mCherry+ double positive cells. We then treated the cells with various concentrations of 4-hydroxytamoxifen (4-OHT) to induce nuclear entry of Cre recombinase in order to delete the MSCV-LMO2-i-GFP expression cassette. Seven days later, 79.3% of the cells lost GFP expression in the group that was treated with 100nM 4-OHT (Fig 1B), indicating efficient cassette deletion. The loss of LMO2 protein is also confirmed by Western blot (Fig 1C). We then transduced ETP cells with the CL20-tLMO2-i-GFP vector and cultured the cells on an OP9-DL1 stroma expressing the Delta-like ligand DL1 to induce differentiation. At day 14, 90.5% of the transduced cells were blocked at the DN2 stage, demonstrating that this deletable lentiviral vector was able to express sufficient levels of LMO2 to induce the DN2 block (Fig 1D). These cells have been transplanted into sublethally irradiated Rag2-/-IL2rg-/- mice to establish the leukemia. One leukemia has been established with a GFP+ tumor, enlarged spleen of 0.534g and an erythroid phenotype as analyzed via flow cytometry. Tumor cells have been transduced with CreERT2 vector for secondary transplant and treatment with tamoxifen to establish whether LMO2 is required for tumor maintainence. In summary, we have established a dual lentiviral vector system that can be used conveniently to study oncogene addiction in hematological malignancies. View Large Image | Download PowerPoint Slide

Red Blood Cell Surface Receptor Expression of BCAM/Lu is Regulated by Protein Kinase A Activity

Irregular sickle red blood cells (RBCs) can contribute to the pathogenesis of vasoocclusion and other complications of sickle cell disease (SCD) via abnormal adherence to the vascular endothelium. It has previously been demonstrated that epinephrine enhances SCD RBC adhesion by activating the BCAM/Lu and ICAM-4 surface receptors [1–2]. Epinephrine acts on the RBC β2-adrenergic receptor, thereby activating Gas proteins that stimulate adenylyl cyclase (AC). This enzyme catalyzes the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP), leading to protein kinase A (PKA) activation, an intermediate step in the upregulation of BCAM/Lu and ICAM-4 mediated adhesion. The interaction of BCAM/Lu with the α5 chain of laminin may contribute to vaso-occlusive events in SCD due to overexpression of BCAM on SCD RBCs.© 2013 ASME