ALBERT

Description: The focusing inversion of gravity and magnetic potential-field data using the randomized singular value decomposition (RSVD) method is considered. This approach facilitates tackling the computational challenge that arises in the solution of the inversion problem that uses the standard and accurate approximation of the integral equation kernel. We have developed a comprehensive comparison of the developed methodology for the inversion of magnetic and gravity data. The results verify that there is an important difference between the application of the methodology for gravity and magnetic inversion problems. Specifically, RSVD is dependent on the generation of a rank [Formula: see text] approximation to the underlying model matrix, and the results demonstrate that [Formula: see text] needs to be larger, for equivalent problem sizes, for the magnetic problem compared to the gravity problem. Without a relatively large [Formula: see text], the dominant singular values of the magnetic model matrix are not well approximated. We determine that this is due to the spectral properties of the matrix. The comparison also shows us how the use of the power iteration embedded within the randomized algorithm improves the quality of the resulting dominant subspace approximation, especially in magnetic inversion, yielding acceptable approximations for smaller choices of [Formula: see text]. Further, we evaluate how the differences in spectral properties of the magnetic and gravity input matrices also affect the values that are automatically estimated for the regularization parameter. The algorithm is applied and verified for the inversion of magnetic data obtained over a portion of the Wuskwatim Lake region in Manitoba, Canada.

Description: Natural remanent magnetization acts as a record of the previous orientations of the earth’s magnetic field, and it is an important feature when studying geologic phenomena. The so-called IDQ curve is used to describe the relationship between the inclination ( I) and declination ( D) of remanent magnetization and the Köenigsberger ratio ( Q). Here, we construct the IDQ curve using data on ground and airborne magnetic anomalies. The curve is devised using modified approaches for estimating the total magnetization direction, e.g., identifying the maximal position of minimal reduced-to-the-pole fields or identifying correlations between total and vertical reduced-to-the-pole field gradients. The method is tested using synthetic data, and the results indicate that the IDQ curve can provide valuable information on the remanent magnetization direction based on available data on the Köenigsberger ratio. Then, the method is used to interpret field data from the Yeshan region in eastern China, where ground anomalies have been produced by igneous rocks, including diorite and basalt, which occur along with magnetite and hematite ore bodies. The IDQ curves for 24 subanomalies are constructed, and these curves indicate two main distribution clusters of remanent magnetization directions corresponding to different structural units of magma intrusion and help identify the lithologies of the magnetic sources in areas covered by Quaternary sediments. The estimated remanent magnetization directions for Cenozoic basalt are consistent with measurements made in paleomagnetism studies. The synthetic and field data indicate that the IDQ curve can be used to efficiently estimate the remanent magnetization direction from a magnetic anomaly, which could help with our understanding of geologic processes in an area.

Description: Introduction: TBLR1-RARα is the tenth fusion gene of acute promyelocytic leukemia (APL) first identified in a rare case of APL with t(3;17)(q26;q21) chromosomal translocation in our previous study. The characteristics of its basic structure and functions had been clarified in our previous study. In this study, we successfully established a novel TBLR1-RARα leukemia mouse model (TR mouse) which fully recapitulated the most relevant features of human APLs. The therapeutic effects of retinoic acid (ATRA), arsenic trioxide (As2O3), cytarabine (Ara-C) and histone deacetylase inhibitors (HDACi) on TR mice were examined. The differentially expressed genes (DEGs) between TR mice and normal mice were compared to explore the possible mechanisms and better therapeutic targets for this kind of APL. Methods: pMSCV-TBLR1-RARα-Flag-IRES-GFP (MSCV-TR) and pMSCV-IRES-GFP (vehicle) retroviral plasmids were constructed and transfected 293T packaging cells to produce retroviruses. Lin- cells from C57BL/6 mice bone marrow were purified and infected with MSCV-TR and vehicle retroviral supernatant. For in vitro assay, the GFP+ lin- cells sorted and incubated with or without different concentrations of ATRA were analyzed for the differentiation and proliferation capacity by cell morphology, myeloid markers (CD11b and GR-1) and colony formation assay. For the in vivo experiment, GFP+ lin- cells transfected with indicated retroviral vectors were injected intravenously to lethally irradiated C57BL/6 mice to establish an APL mouse model. Cell surface markers were analyzed by flow cytometry. In treatment assays, GFP+ spleen cells from TR leukemia mice were injected intravenously into recipient mice. The mice were randomly separated into groups and received different treatment with ATRA, As2O3, As2O3 in combination with ATRA, Ara-C, Ara-C in combination with ATRA, chidamide and NL101, respectively. The percentage of GFP+ cells in peripheral blood and body weight were measured dynamically. The survival time of every group was recorded and compared. RNA-seq assay was used to identify DEGs between TR mice and normal mice. Results: In vitro assays indicated that TBLR1-RARα could either block the differentiation of HSCs at a relatively early stage or enhanced the clonogenic potential of cells. The TBLR1-RARα leukemia mouse model was successfully established. During the ten-month observational period, 3 out of 15 mice transplanted with TBLR1-RARα expressing cells developed an APL-like disease. Development of leukemia was not observed in any of the mice in control group. All the leukemia mice had a body weight loss as well as splenomegaly and hepatomegaly. The phenotype analysis revealed that the progenitor markers Sca-1, CD34 and C-kit were positive, the myeloid lineage markers Gr-1 and CD11b were also positive, erythroid lineage marker Ter119 was weekly positive, but the lymphatic lineage marker B220, CD3,CD4 and CD8 were all negative. TR mice treated with 1.5-2.5 mg/kg ATRA alone or together with 2.0 mg/kg As2O3 didn't survive longer than that of control group, although in vitro differentiation experiment showed that the leukemia cells were sensitive to ATRA. Leukemic mice receiving Ara-C treatment had a much longer survive time. Surprisingly, HDAC inhibitors (12.5 and 25 mg/kg chidamide and 30 mg/kg NL-101) could significantly prolong the survival time of TR mice. Thousands of DEGs had been identified between TR mice and wild type mice, which were widely involved in multiple pathways and participated in various biological functions. Conclusion: The TBLR1-RARα leukemia mouse model was successfully established for the first time, and its main characteristics were clarified. Although the leukemia cells were sensitive to ATRA in vitro, TR mice didn't benefit from ATRA or As2O3 treatment in vivo. Besides Ara-C, HDAC inhibitors, such as chidamide and NL-101 exhibited potency therapeutic values for TR mice, which provided a new strategy for this kind of refractory APL. What' more, lots of genes that might be related with the process of leukemogenesis and new therapeutic targets for TR leukemia were identified. This model would serve as a versatile tool to study the mechanisms of leukemogenesis and help to design better strategies for APLs in further studies. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Hybrid anticancer drugs are of great therapeutic interests as they can potentially overcome the flaws of conventional chemotherapy drugs and improve their efficacy. Histone deacetylase inhibitors (HDACi) and DNA damaging agents have showed synergistic effects in recent studies. In this study, we reported a novel hybrid NL-101 that combines chemo-active groups from suberoylanilide hydroxamic acid (SAHA) and bendamustine, the typical HDACi and alkylating agent respectively.The anticancer effect of NL-101 and its possible mechanisms were investigated in human leukemia cell lines and primary leukemia cells. Methods MTT assay was performed to determine the proliferation of Kasumi-1 and NB4 cells treated with NL-101. Cell cycle distribution and apoptosis rate were detected by flow cytometry. Western-blot analysis was used to analyze the level of acetylated H3 as well as apoptotic-related proteins including γ-H2AX, PARP, caspase-3, Bax, Bcl-2 and Bcl-xL. Bone marrow mononuclear cells of AML patients were isolated by density gradient centrifugation. Wright staining and Western blot were performed to determine the inducing apoptosis effect. Results NL-101 inhibited the proliferation of leukemia cell lines Kasumi-1 and NB4 cells with similar IC50 to that of SAHA. Cell cycle analysis indicated that NL-101 induced S phase arrest. As expected, apoptotic cell death was observed in response to NL-101 treatment. After treatment with 2 µmol/L NL-101 for 48 hours, the apoptosis rate of Kasumi-1 and NB4 cells were (60.19±12.01)% and (49.43±11.61)%, respectively. Western blot analysis showed that NL-101 exposure could induce the accumulation of acetylated Histone H3 and γ-H2AX as the biomarker of DNA double-strand breaks. Anti-apoptotic protein Bcl-xL involved in mitochondrial death pathway was also decreased. Moreover, NL-101 induced apoptosis with a low micromolar IC50 in various leukemia cell lines but not in nonmalignant cell line HEK293. The efficacy of NL-101 was also tested in human primary leukemia cells and all the treated samples exhibited apoptosis confirmed by the morphological examination and expression of apoptotic markers. Conclusions The novel SAHA-bendamustine hybrid NL-101 inhibited the proliferation and induced apoptotic cell death of leukemia cell lines and primary leukemia cells. It presented the properties of both HDAC inhibition and DNA damaging. Down-regulation of Bcl-xL was also involved in the apoptosis induction. These results indicated that NL-101 might be a potential compound for the treatment of leukemia. Disclosures Wang: Bristol Myers Squibb: Consultancy; Novartis: Consultancy.

Description: Introduction iASPP played an important role in leukemogenesis in our previous study. In order to clarify its mechanism, a yeast two-hybrid screen was performed to find the binding partner of iASPP. In this study, we reported FHL2 as a novel binding partner of iASPP. The biological functions of the communication between FHL2 and iASPP were detected, and its possible mechanisms were investigated in human leukemia cell lines. Methods A yeast two-hybrid screen was performed to identify FHL2 as a novel binding partner of iASPP. Immunofluorescence, Co-IP and Western blot analysis were used to confirm the communication between FHL2 and iASPP. After FHL2 or iASPP was knocked down in K562 and Kasumi-1 cells by lentiviral, MTT assay and flow cytometry were performed to detect the proliferation, cell cycle distribution and apoptosis rate of leukemic cells, meanwhile Western blot analysis was used to analyze the level of cell cycle- and apoptotic-related proteins. Dual luciferase assay was conducted to investigate the transcriptional activity of p53 on Bax when iASPP and FHL2 were overexpressed or FHL2 was knocked down. Results FHL2 was highly expressed in K562 and Kasumi-1 cells. FHL2 and iASPP interacted with each other and co-localized in both nucleus and cytoplasm. Either FHL2 or iASPP silenced could reduce cell proliferation, induce cell cycle arrest at G0/G1 phases, and increase cell apoptosis. Western blot analysis showed that the level of p21 increased and anti-apoptotic protein Bcl-2 was reduced. Interestingly, when FHL2 was knocked down, the protein expression level of iASPP also decreased. Similarly, the expression of FHL2 would reduce when iASPP was silenced. Dual luciferase assay suggested that iASPP could reduce the transcriptional activity of p53 on Bax, furthermore, when FHL2 was knocked down at the same time, the transcriptional activity of p53 was rescued. Conclusions The interaction between FHL2 and iASPP in AML was observed for the first time. Cell proliferation reducing, cell cycle arresting at G0/G1 phases, and cell apoptosis increasing occurred in either FHL2 knockdown or iASPP knockdown. Moreover, iASPP and FHL2 participated in the regulation of the transcriptional activation function of p53. These results indicated that FHL2 might be a novel potential target for AML treatment. Disclosures No relevant conflicts of interest to declare.

Description: Liver injury associated with veno-occlusive disease and graft-versus-host disease (GVHD) is a frequent and severe complication of hematopoietic stem cell transplantation, and remains an important cause of transplant-related mortality. Bone marrow derived mesenchymal stem cells (MSCs) have been evaluated for the prevention and treatment of refractory GVHD. However, poor cell viability has limited the therapeutic capacity of mesenchymal stromal cell therapy in vivo. In this study, we genetically engineered C57BL/6 mouse bone marrow MSCs using ex vivo retroviral transduction to overexpress Akt1, a serine threonine kinase and pro-survival signal protein, and tested the hypothesis that Akt1-expressing MSCs (Akt1-MSCs) are more resistant to apoptosis and can ameliorate acute liver injury induced by concanavalin A (ConA) in BALB/c mice. Cell proliferation and apoptosis analyses showed that, under both regular culture and high concentration IFN-γ (100 ng/mL) stimulation conditions, Akt1-GFP-MSCs had proliferation and survival (anti-apoptotic) advantages with down-regulated apoptosis pathways, compared to control GFP-MSCs. Twenty-four hours after receiving lethal dose of ConA (40 mg/kg, intravenous) (N=10 each group), no mouse survived, with or without 1x106 Akt1-MSCs or GFP-MSCs administration (intravenous); however, 3 and 1 survived in the 5×106 Akt1-MSCs group and 5×106 GFP-MSCs groups, respectively. In subsequent sub-lethal dose ConA (20 mg/kg) experiments, compared to GFP-MSCs, mice received Akt1-MSCs administration had significantly lower serum AST, ALT, TNF-α and IFN-γ levels and less histopathological abnormalities. In addition, Akt1-MSCs treated mice had significantly higher serum concentrations of IL-10, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). In vivo imaging showed that, hepatic fluorescence signal in sub-lethal ConA+Akt1-MSCs group was significantly stronger than ConA+GFP-MSCs group on day 0, and persisted up to 14 days, whereas the signal in ConA+GFP-MSCs, Akt1-MSCs and GFP-MSCs groups was negligible on both day 7 and day 14. Thus, bone marrow derived MSCs genetically enhanced with Akt1 had survival advantage in vitro and in vivo, and have the potential to be a potent therapy for prevention and amelioration of GVHD-associated liver impairment. Further translational pre-clinical studies are ongoing to further determine the efficacy, dosage and timing of administration of Akt1-MSCs in animal models. Disclosures No relevant conflicts of interest to declare.

Description: E-proteins are critical transcription factors in B-cell lymphopoiesis. E2A, 1 of 3 E-protein–encoding genes, is implicated in the induction of acute lymphoblastic leukemia through its involvement in the chromosomal translocation 1;19 and consequent expression of the E2A-PBX1 oncoprotein. An interaction involving a region within the N-terminal transcriptional activation domain of E2A-PBX1, termed the PCET motif, which has previously been implicated in E-protein silencing, and the KIX domain of the transcriptional coactivator CBP/p300, critical for leukemogenesis. However, the structural details of this interaction remain unknown. Here we report the structure of a 1:1 complex between PCET motif peptide and the KIX domain. Residues throughout the helical PCET motif that contact the KIX domain are important for both binding KIX and bone marrow immortalization by E2A-PBX1. These results provide molecular insights into E-protein–driven differentiation of B-cells and the mechanism of E-protein silencing, and reveal the PCET/KIX interaction as a therapeutic target for E2A-PBX1–induced leukemia.

Description: Introduction: As the important suprressor of P53, iASPP was found to be overexpressed in leukemia, and functioned as oncogene that inhibited apoptosis of leukemia cells. Sertad1 is identified as one of the proteins that can bind with iASPP in our previous study by two-hybrid screen. Sertad1 is highly expressed in carcinomas from pancreatic, lung and ovarian tissues, which considered Sertad1 as an oncoprotein. In this study, our findings revealed that Sertad1 could interact with iASPP in the cytoplasm near nuclear membrane, which could block iASPP to enter into nucleus to interact with P53, and inhibited the function of iASPP eventually. Methods: Co-immunoprecipitation and fluorescence confocal microscopic imaging were used to confirm the interaction between iASPP and Sertad1, the exact binding domains and the subcellular colocalization.The plasmids of iASPP and Sertad1 were transfected alone or co-transfected into K562 cells, the stable subclones that highly expressed iASPP, Sertad1 or both of them were then established by limiting dilution and named as K562-iASPPhi, K562-Sertad1hi, and K562-Douhi, respectively. The cell proliferation, cell cycle and apoptosis of above subclones were investigated by flow cytometry. Further, silence of the above two proteins was performed to confirm their functions. Immunoblotting analysis and immunofluorescence were performed to explore the possible mechanisms of difference between the biological functions of the above subclones. Results: Sertad1 expression level varied in leukemic cell lines and AML patients irrespectively of iASPP and P53. Interaction between iASPP and Sertad1 did exist in 293 cell and leukemic cells, both iASPP and Sertad1 scattered in the cytoplasm and nucleus, and their colocalizations were mainly in the cytoplasm, which encircled the nucleus. iASPP binds directly to Sertad1 through its PHD-bromo domain, C-terminal domain and Cyclin-A domain in a reduced order, and Serta domain failed to bind to iASPP. Overexpression of iASPP in K562 cells (iASPPhi) could result in the increased cell proliferation, cell cycle arrest in G2/M phase and resistance to apoptosis induced by chemotherapy drugs. While overexpression of iASPP and Sertad1 at the same time (Douhi) could slow down the cell proliferation, lead the cells more vulnerable to the chemotherapy drugs. As figure showed, in K562-Douhi cells, both iASPP and Sertad1 were obviously located in the cytoplasm, which encircled the nuclei, the subcellular colocalization was nearly outside the nuclei. The immunoblotting analysis further supported the conclusions. The resistance of iASPP to chemotherapeutic drug was accompanied by Puma protein expression in a p53-independent manner. By knocking down the expersssion of iASPP and Sertad separately, we found that iASPP is dispensable for maintenance of anti-apoptotic function and Sertad1 is indispensable for cell cycle in leukemic cells. Conclusions: In normal situation, the protein iASPP and Sertad1 scatter in the nucleus and cytoplasm, mainly in the cytoplasm. As convinced by our study, iASPP was overexpressed in the leukemia cell lines and primary AML patients, it could function as oncogene through its binding with P53 protein in the nucleus, inhibit the function of P53. When iASPPhi cells were exposed to apoptosis stimuli, Puma protein could play an important role in this process, irrespective of the expression level of P53. But when iASPP and Sertad1 were both overexpressed in the leukemic cells, Sertad1 could tether iASPP outside the nucleus mainly through its PHD-bromo domain, prevent it from inhibiting P53 function, suppress the leukemic cell growth and stimulate cell apoptosis by rescuing the P53 eventually. Our data provided a new insight to overcome iASPP protein, namely through its binding partners, when the similar proteins or drugs that can tether iASPP outside the nucleus such as Sertad1 are transfected into the leukemic cells, it may restore p53 function to eliminate the leukemic cells. Figure 1 Figure 1. Disclosures Wang: Novartis: Consultancy; Bristol Myers Squibb: Consultancy.