Abstract
Leukemia initiating cells (LIC) contribute to therapeutic resistance through mutations in cellular self-renewal and survival pathways. NOTCH1 mutations are common in T-cell acute ...lymphoblastic leukemia (T-ALL). However, the role of NOTCH1 activation in human LIC propagation has not been established. Pediatric T-ALL serially transplantable LIC were found to be enriched in the CD34+CD4− and CD34+CD7− fractions of newly diagnosed patient samples. More recently, a CD7+CD1a− glucocorticoid resistant LIC population, capable of engrafting leukemia in NOD/SCID IL2R gamma null (NSG) mice, was identified in primary adult T-ALL. To identify the molecularly characterized potential LIC populations in pediatric T-ALL without proceeding in vitro culture and examine the role of NOTCH1 activation in LIC propagation. 12 pediatric T-ALL samples were sequenced for NOTCH1 mutation examination. Humanized LIC mouse models were established and dosed with either NOTCH1 mAb or IgG1 mAb control at 10 mg/kg intraperitoneally every 4 days for 6 doses. Mice were sacrificed one day after the last dose, and hematopoietic organs were collected for FACS analysis. To further define the LIC populations in pediatric T-ALL, CD34+CD38+CD2+CD7+Lin− and CD34+CD38+CD2+CD7−Lin− cells were isolated from T-ALL primary patients’ blood by FACS sorting and transplanted into neonatal RAG2−/−γc−/− mice to determine their leukemic engraftment potential. Serial transplantations were done for testing the LIC self-renewal capacity. Mouse hematopoietic organs were collected for FACS analysis, mouse brains were sectioned for human cells examination by immunohistochemistry. NOTCH1 and its downstream gene expressions were examined by q-RT-PCR between the T-ALL CD34+ and CD34− populations. Six of 12 pediatric T-ALL patient samples were found NOTCH1 mutation. Mice transplanted with CD34+ and CD34+CD2+CD7+ or CD34+CD2+CD7− cells developed a T-ALL-like disease characterized by pale BM and enlarged spleen, thymus and liver. Human CD34+ enriched cells from NOTCH1 mutated T-ALL maintained leukemic engraftment while an equivalent number of CD34+ cells from NOTCH1 wild type T-ALL did not. T-ALL CD34+ progenitors from NOTCH1 mutated T-ALL have a significant higher engraftment in BM when compared with those from NOTCH1 wild type T-ALL. CD34+CD2+CD7+ and CD34+CD2+CD7− populations are more prominent in NOTCH1 mutated samples. Both the human CD34+ and CD34+CD2+CD7+ populations were significantly reduced in BM when treated with hN1 mAb in vivo. NOTCH1 and its downstream genes expression were significantly reduced in NOTCH1 mutated CD34+ cells when compared with CD34− cells. Human T-ALL LIC have enhanced NOTCH1 expression; CD34+CD2+CD7+ and CD34+CD2+CD7− subpopulations are enriched for LIC activity in pediatric T-ALL; A selective hN1 mAb inhibits human T-ALL LIC survival and self-renewal in vivo.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1011. doi:1538-7445.AM2012-1011
Abstract 1507
Leukemia initiating cells (LIC) contribute to therapeutic resistance as a result of their capacity to accumulate mutations in pathways, such as the NOTCH1 receptor signaling pathway, ...that promote self-renewal and survival within specific niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in driving therapeutic resistance. However, the role of NOTCH1 activation in human T-ALL LIC propagation and LIC sensitivity to selective NOTCH1 receptor inhibition has not been examined. The difficulties in maintaining primary cultures of leukemia cells have hampered investigations into the biology of T-ALL LIC and underscore the need for a direct transplantation model to characterize human LIC in vivo and as a paradigm for screening candidate drugs that inhibit self-renewal pathways active in T-ALL LIC. Pediatric T-ALL serially transplantable LIC were found to be enriched in the CD34+CD4− and CD34+CD7− fractions of newly diagnosed patient samples. More recently, a CD7+CD1a− glucocorticoid resistant LIC population, capable of engrafting leukemia in NOD/SCID IL2Rƒn gamma null (NSG) mice, was identified in primary adult T-ALL without an in vitro expansion. In this study, we identified and molecularly characterized potential LIC populations in pediatric T-ALL without preceding in vitro culture and examined the role of NOTCH1 activation in LIC propagation. To further define the T-ALL LIC, CD34+CD2+CD7+ or CD34+CD2+CD7− cells were isolated from T-ALL primary patients’ blood by FACS sorting and transplanted into neonatal RAG2−/− gamma chain−/− mice to determine their leukemic engraftment potential. Limiting dilution experiments were performed with cells from six T-ALL patient samples. Mice transplanted with CD34+CD2+CD7+ or CD34+CD2+CD7− cells developed a T-ALL-like disease characterized by pale bone marrow and enlarged spleen, thymus and liver. Hematopoietic organs were analyzed by flow cytometry and showed engraftment of bone marrow, spleen, thymus and liver. Furthermore, the disease could be serially transplanted. LIC were uniquely susceptible to targeted inhibition in vivo with a therapeutic human NOTCH1 negative regulatory region selective monoclonal antibody (mAb) while normal human hematopoietic progenitors were spared thereby highlighting the cell type and context specific effects of NOTCH signaling. Both the NOTCH1 mAb treatment and lentiviral shRNA knockdown of NOTCH1 reduced NOTCH1, HES1 and c-MYC transcript levels, underscoring the selectivity of NOTCH1 mAb inhibition of NOTCH signaling. These results demonstrate that CD34+CD2+CD7+ and CD34+CD2+CD7− subpopulations are enriched for LIC activity in pediatric T-ALL. Moreover, inhibition of NOTCH signaling by either mAb or shRNA-mediated Notch1 knockdown might be another strategy to target the LIC in T-ALL.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract 3739
Leukemia stem cells (LSC) represent a frequently dormant self-renewing population integral to the initiation, maintenance, and progression of human chromic myeloid leukemia (CML). The ...current standard of care dasatinib, a BCR-ABL targeted tyrosine kinase inhibitor (TKI), effectively eradicates the bulk of CML cells but frequently fails to affect the LSC population that is thought to drive CML relapse. Members in the BCL2 family are proteins that regulate apoptosis, 6 of which regulate cell survival. Each of these 6 members has a long and short isoform with opposing functions; generally, long isoforms promote cell survival while the short isoforms promote apoptosis. Previously, we demonstrated that upregulation of pro-survival BCL2 proteins in CML LSC contributes to chemotherapy resistance and LSC quiescence in protective hematopoietic niches. LSC found in different hematopoietic niches differ in their response to TKI treatment. Niche affects LSC cell cycle, either by maintaining quiescence or by promoting rapid cell cycling. Quiescent cells are a hurdle for traditional chemotherapy, which usually targets rapidly cycling cells, leaving the quiescent LSC untouched. We hypothesize that the inhibition of pro-survival BCL2 protein family members will sensitize LSC to dasatinib therapy and therefore prevent CML relapse.
We tested a novel pan pro-survival BCL2 family protein inhibitor, sabutoclax, delivered by intravenous injection either alone or in combination with oral dasatinib in immunodeficient RAG2−/-gc−/- mice engrafted with BC CML patient samples. After treatment, LSC burden, self-renewal, and cell cycle status were quantified using FACS. Our results showed a reduction in the LSC burden in combination treated mice when compared to mice that received either drug alone. Mice treated with the combination regimen were found to have fewer quiescent human leukemic cells than their counterparts that received single agent treatments. Immunofluorescence staining confirmed the reduction of quiescent cells in the bone marrow after combination treatment when compared to single agent or vehicle treatments. We validated the molecular targets by using human specific splice isoform primers to perform RT-qPCR on FACS sorted LSC and showed a reduction in the BCL2 long to short isoform ratio in sabutoclax versus vehicle treated animals, indicating a skewing towards the pro-apoptotic splice variant. Together, these results indicate that the combination strategy with a pan pro-survival BCL2 family inhibitor and a tyrosine kinase inhibitor may be the foundation for a promising clinical strategy to effectively eliminate LSC and prevent cancer progression and relapse.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract 2772
While advanced malignancies in Chronic Myeoloid Leukemia (CML) are diverse in phenotype, they often exhibit stem cell properties including enhanced survival, quiescence and self-renewal ...potential. The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. While DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity may also be generated by aberrant RNA editing mediated by adenosine deaminase acting on dsRNA (ADAR) family, which have been shown to promote an embryonic transcriptional program and regulate fetal and adult hematopoietic stem cell (HSC) self-renewal as well as stem cell responses to inflammation. In this study, whole transcriptome sequencing of normal, chronic phase (CP) and functionally validated blast crisis (BC) chronic myeloid leukemia (CML) progenitors revealed increased inflammatory pathway gene expression in concert with BCR-ABL amplification, enhanced expression of interferon-responsive ADAR1 and a propensity for increased A-to-I RNA editing during CML progression. Mechanistic studies demonstrated that lentivirally enforced ADAR1 p150 expression induced expression of the myeloid-skewing transcription factor PU.1 and skewed cell fate towards granulocyte-macrophage progenitors - the initiating LSC population in BC CML. Moreover, lentiviral ADAR1 knockdown reduced BC LSC self-renewal capacity in RAG2−/−gc−/− mice. These data shed new light on the role of ADAR1-directed RNA editing in myeloid progenitor reprogramming and self-renewal potential of malignant progenitors that drive disease progression and therapeutic resistance in CML, and provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We have used flux-transmission correlations in Lyα forests to measure the imprint of baryon acoustic oscillations (BAO). The study uses spectra of 157 783 quasars in the redshift range 2.1 ≤ z ≤ 3.5 ...from the Sloan Digital Sky Survey (SDSS) data release 12 (DR12). Besides the statistical improvements on our previous studies using SDSS DR9 and DR11, we have implemented numerous improvements in the analysis procedure, allowing us to construct a physical model of the correlation function and to investigate potential systematic errors in the determination of the BAO peak position. The Hubble distance, DH = c/H(z), relative to the sound horizon is DH(z = 2.33) /rd = 9.07 ± 0.31. The best-determined combination of comoving angular-diameter distance, DM, and the Hubble distance is found to be DH0.7DM0.3 /rd = 13.94 ± 0.35. This value is 1.028 ± 0.026 times the prediction of the flat-ΛCDM model consistent with the cosmic microwave background (CMB) anisotropy spectrum. The errors include marginalization over the effects of unidentified high-density absorption systems and fluctuations in ultraviolet ionizing radiation. Independently of the CMB measurements, the combination of our results and other BAO observations determine the open-ΛCDM density parameters to be ΩM = 0.296 ± 0.029, ΩΛ = 0.699 ± 0.100 and Ωk = −0.002 ± 0.119.
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FMFMET, NUK, UL, UM, UPUK
We present a measurement of baryon acoustic oscillations (BAO) in the cross-correlation of quasars with the Lyα-forest flux transmission at a mean redshift of z = 2.40. The measurement uses the ...complete Sloan Digital Sky Survey (SDSS-III) data sample: 168 889 forests and 234 367 quasars from the SDSS data release DR12. In addition to the statistical improvement on our previous study using DR11, we have implemented numerous improvements at the analysis level enabling a more accurate measurement of this cross-correlation. We have also developed the first simulations of the cross-correlation that allow us to test different aspects of our data analysis and to search for potential systematic errors in the determination of the BAO peak position. We measure the two ratios DH(z = 2.40) /rd = 9.01 ± 0.36 and DM(z = 2.40) /rd = 35.7 ± 1.7, where the errors include marginalization over the non-linear velocity of quasars and the cross-correlation of metals and quasars, among other effects. These results are within 1.8σ of the prediction of the flat-ΛCDM model describing the observed cosmic microwave background anisotropies. We combine this study with the Lyα-forest auto-correlation function, yielding DH(z = 2.40) /rd = 8.94 ± 0.22 and DM(z = 2.40) /rd = 36.6 ± 1.2, within 2.3σ of the same flat-ΛCDM model.
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FMFMET, NUK, UL, UM, UPUK
Objective Hypoxic-ischemic white mater brain injury commonly occurs in neonates with hypoplastic left heart syndrome (HLHS). Approximately one half of HLHS survivors will exhibit neurobehavioral ...symptoms believed to be associated with this injury, although the exact timing of the injury is unknown. Methods Neonates with HLHS were recruited for pre- and postoperative monitoring of cerebral oxygen saturation, cerebral oxygen extraction fraction, and cerebral blood flow using 2 noninvasive optical-based techniques: diffuse optical spectroscopy and diffuse correlation spectroscopy. Anatomic magnetic resonance imaging was performed before and approximately 1 week after surgery to quantify the extent and timing of the acquired white matter injury. The risk factors for developing new or worsened white matter injury were assessed using uni- and multivariate logistic regression. Results A total of 37 neonates with HLHS were studied. On univariate analysis, neonates who developed a large volume of new, or worsened, postoperative white matter injury had a significantly longer time to surgery ( P = .0003). In a multivariate model, a longer time between birth and surgery, delayed sternal closure, and greater preoperative cerebral blood flow were predictors of postoperative white matter injury. Additionally, a longer time to surgery and greater preoperative cerebral blood flow on the morning of surgery correlated with lower cerebral oxygen saturation ( P = .03 and P = .05, respectively) and greater oxygen extraction fraction ( P = .05 for both). Conclusions A longer time to surgery was associated with new postoperative white matter injury in otherwise healthy neonates with HLHS. The results suggest that earlier Norwood palliation might decrease the likelihood of acquiring postoperative white matter injury.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The placenta is a complex organ that influences prenatal growth and development, and through fetal programming impacts postnatal health and well-being lifelong. Little information exists on placental ...pathology in the presence of congenital heart disease (CHD). Our objective is to characterize the placenta in CHD and investigate for distinctions based on type of malformation present. Placental pathology from singleton neonates prenatally diagnosed and delivered at > 37 weeks gestation was analyzed. Placental findings of absolute weight, placental weight-to-newborn birth weight ratio, chorangiosis, villus maturity, thrombosis, and infarction were recorded and analyzed based on four physiological categories of CHD: (1) single ventricle-aortic obstruction, (2) single ventricle-pulmonic obstruction, (3) two-ventricle anomalies, and (4) transposition of the great arteries (TGA). Associations between fetal Doppler assessments of middle cerebral/umbilical arterial flow and placental findings were investigated. A total of 120 cases of complex CHD were analyzed. Overall placental-to-birth weight ratios were < 10th percentile for 77% and < 3rd percentile for 49% with abnormalities of chorangiosis (18%), hypomature villi (15%), thrombosis (41%), and infarction (17%) common. There was no association between fetal Doppler flow measures and placental abnormalities. Newborns with TGA had the greatest degree of placental abnormality. Placentas of newborns with CHD are smaller than expected and manifest a number of vascular abnormalities, with TGA most prominent. Fetal Doppler does not correlate with these abnormalities. Studies investigating the relationship between placental abnormalities and postnatal outcomes may offer insight into the fetal origins of outcome variability in CHD.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Previous research has noted the transformation of the American parties since the 1970s, as exhibited in their increased ideological polarization and transformation on social issues like civil rights, ...abortion, and the environment. We contribute to the literature on party change by theoretically stressing the decentralized and individualistic nature of American parties, while using a measure of party change that is based on legislative behavior beyond roll call voting. Our paper uses social network analysis to analyze the parties from the 93rd to 110th Congresses, utilizing bill cosponsorship to define connections between members. Our analysis illustrates how the core of the party, that is, who are most central in the cosponsorship network, has changed over time. We find evidence that party centrality influenced retirement decisions, thereby reinforcing and contributing to party change.
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NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Background Preoperative brain injury is common in neonates with complex congenital heart disease. Increasing evidence suggests a complex interaction of prenatal and postnatal risk factors for ...development of brain white matter injury, called periventricular leukomalacia (PVL), in neonates with complex congenital heart disease. To date, there remains a limited understanding of the risk factors contributing to preoperative PVL in hypoplastic left heart syndrome (HLHS). Methods Neonates with HLHS or HLHS variants from 3 prospective magnetic resonance imaging studies (2003-2010) were selected for this cohort. Preoperative brain magnetic resonance imaging was performed the morning of the surgery. Stepwise multilogistic regression of patient characteristics, mode of delivery (cesarean section vs vaginal), time of diagnosis (prenatal vs postnatal), HLHS subtypes, brain total maturation score, time to surgery, individual averaged daily preoperative blood gases, and complete blood cell count values was used to determine significant associations. Results A total of 57 neonates with HLHS were born at 38.7 ± 2.3 weeks; 86% (49/57) had a prenatal diagnosis, with 31% (18/57) delivered by cesarean section. HLHS with aortic atresia (AA) was common in this cohort, 71% (41/57). Preoperative PVL was identified in 19% (11/57). Male patients with AA ( P = .004) were at higher risk for PVL. Lower total brain maturation score was also identified as a strong predictor for preoperative PVL ( P = .005). Conclusions In neonates with HLHS, nonmodifiable patient-related factors, including male sex with AA (lack of antegrade blood flow) and lower total brain maturation score, placed neonates at the greatest risk for preoperative white matter injury.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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