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The urbanization rate in China has been increasing in recent years, and along with the increasing intensity of human engineering activities, ecological and geological (eco-geological) degradation have become key factors impeding sustainable urban development. Taking the concentrated mineral exploitation area of Tonghua City as an example, the distribution of mines in the area is concentrated and the spatial heterogeneity is significant. This paper includes 14 evaluation indicators in three aspects: eco-geological environment background, anthropogenic and mining engineering activities, and environmental pollution. Then, based on game theory combined with ANP-CV (Analytic Network Process and Coefficient of Variation), two empowerment methods, GIS spatial calculation is used to evaluate the eco-geological environment quality (EEQ). The results showed that the EEQ was divided into grades I–V from high to low, with areas of 21.13%, 30.35%, 27.00%, 14.30%, and 7.22%, respectively; the EEQ of the Hun River basin has a high spatial autocorrelation and low EEQ, and the EEQ grade of mines was divided on this basis; the hot spot analysis is useful for determining the EEQ, as well as for allocating mine restoration resources in a sensible manner. Finally, we propose countermeasures to improve EEQ, and this study can provide a scientific basis for ecological construction and geological environmental protection in Tonghua City.

With an incidence of ~50%, the absence or reduced protein level of p53 is much more common than TP53 mutations in acute myeloid leukemia (AML). AML with FLT3-ITD (internal tandem duplication) mutations has an unfavorable prognosis and is highly associated with wt-p53 dysfunction. While TP53 mutation in the presence of FLT3-ITD does not induce AML in mice, it is not clear whether p53 haploinsufficiency or loss cooperates with FLT3-ITD in the induction of AML. Here, we generated FLT3-ITD knock-in; p53 knockout (heterozygous and homozygous) double-transgenic mice and found that both alterations strongly cooperated in the induction of cytogenetically normal AML without increasing the self-renewal potential. At the molecular level, we found the strong upregulation of Htra3 and the downregulation of Lin28a, leading to enhanced proliferation and the inhibition of apoptosis and differentiation. The co-occurrence of Htra3 overexpression and Lin28a knockdown, in the presence of FLT3-ITD, induced AML with similar morphology as leukemic cells from double-transgenic mice. These leukemic cells were highly sensitive to the proteasome inhibitor carfilzomib. Carfilzomib strongly enhanced the activity of targeting AXL (upstream of FLT3) against murine and human leukemic cells. Our results unravel a unique role of p53 haploinsufficiency or loss in the development of FLT3-ITD + AML.

With an incidence of ~50%, the absence or reduced protein level of p53 is much more common than TP53 mutations in acute myeloid leukemia (AML). AML with FLT3-ITD (internal tandem duplication) mutations has an unfavorable prognosis and is highly associated with wt-p53 dysfunction. While TP53 mutation in the presence of FLT3-ITD does not induce AML in mice, it is not clear whether p53 haploinsufficiency or loss cooperates with FLT3-ITD in the induction of AML. Here, we generated FLT3-ITD knock-in; p53 knockout (heterozygous and homozygous) double-transgenic mice and found that both alterations strongly cooperated in the induction of cytogenetically normal AML without increasing the self-renewal potential. At the molecular level, we found the strong upregulation of Htra3 and the downregulation of Lin28a, leading to enhanced proliferation and the inhibition of apoptosis and differentiation. The co-occurrence of Htra3 overexpression and Lin28a knockdown, in the presence of FLT3-ITD, induced AML with similar morphology as leukemic cells from double-transgenic mice. These leukemic cells were highly sensitive to the proteasome inhibitor carfilzomib. Carfilzomib strongly enhanced the activity of targeting AXL (upstream of FLT3) against murine and human leukemic cells. Our results unravel a unique role of p53 haploinsufficiency or loss in the development of FLT3-ITD + AML.

Immunophenotypic analysis using multiparameter flow cytometry is an indispensable tool for diagnosis and management of acute leukemia. Mouse models have been widely used for medical research for more than 100 years and are indispensable for leukemia research. However, immunophenotypic analysis of murine leukemia was not always performed in published studies, and blast gating for isolation of blasts was shown only in very few studies. No systemic characterization of all types of murine acute leukemia in large cohorts by flow cytometry has been reported. In this study, we used flow cytometry to comprehensively characterize murine acute leukemia in a large cohort of mice. We found that murine T-lymphoblastic leukemia/lymphoma (T-ALL) exhibits a distinctive “blast gate” (CD45bright) with CD45/side scatter gating that differs from the “blast gate” (CD45dim) of human T-ALL. By contrast, murine B-lymphoblastic leukemia and acute myeloid leukemia show the same blast region (CD45dim) as human leukemia. Using blast cell gating, we for first time detected T-ALL development in FLT3-ITD knock-in mice (incidence: 23%). These leukemic cells were selectively killed by the FLT3 inhibitors crenolanib and midostaurin in vitro. These data suggest that FLT3-ITD plays a potential role in the pathogenesis of T-ALL and that FLT3-ITD inhibition is a therapeutic option in the management of patients with T-ALL. Our gating strategy for immunophenotypic analysis can be used for leukemogenesis and preclinical gene therapy studies in mice and may improve the quality of such analyses.

Although the patients with t(8;21) acute myeloid leukemia (AML) have a favorable prognosis compared with other non-acute promyelocytic leukemia AML patients, only ~50% patients with this relatively favorable subtype can survive for 5 years and refractory/relapse is common in clinical practice. So it is necessary to find novel agents to treat this type of AML. In this study, the effects and the mechanisms of plumbagin and recombinant soluble tumor necrosis factor‑α-related apoptosis-inducing ligand (rsTRAIL) on leukemic Kasumi‑1 cells were primarily investigated. Plumbagin and/or rsTRAIL could significantly inhibit the growth of Kasumi‑1 cells and induce apoptosis in vitro and in vivo. Plumbagin enhanced TRAIL-induced apoptosis of Kasumi‑1 cells in association with mitochondria damage, caspase activation, upregulation of death receptors (DRs) and decreased cFLIP expression. The effects of plumbagin on the expression of DR5, Bax and cFLIP could be partially abolished by the reactive oxygen species (ROS) scavenger NAC. Glutathione (GSH) depletion by plumbagin increased the production of ROS. In vivo, there was no obvious toxic pathologic change in the heart, liver and kidney tissues in any of the groups. Comparing with the control mice, a significantly increased number of apoptotic cells were observed in the combined treated mice by flow cytometry. Plumbagin also increased the expression of DR4 and DR5 in cells of xenograft tumors. Collectively, our results suggest that both plumbagin and rsTRAIL could be used as a single agent or synergistical agents to induce apoptosis of leukemic Kasumi‑1 cells in vitro and in vivo.

FLT3-ITD (internal tandem duplication) is a late event in the pathogenesis of acute myeloid leukemia (AML). Identification of early cooperating events for FLT3 mutations may improve our understanding of the pathogenesis of AML and lead to a more efficient treatment and improved outcome for AML patients. p53 alteration can be found in up to 70% of AML patients with complex karyotypes, while studies from our group and others show a relatively low incidence of p53 mutation (generally around 10%) in other AML patients. Dysfunction of p53 pathway resulting from overexpressed MDM2/MDM4 is more often found than p53 mutations in patients with de novo AML. An early mouse study identified the FLT3 gene to be preferentially mutated by insertional mutagenesis in p53 knock-out but not in p53 wild-type tumors. Importantly, p53 mutation appears to be an early event in the pathogenesis of AML. In this study, we investigated whether p53 haploinsufficiency or loss cooperates with FLT3-ITD in the induction of AML. To this end, we crossed FLT3-ITD knock-in mice with p53 knockout mice to generate mice harboring both ITD/ITD and p53 knockout mutations. ITD/ITD; p53+/- (FLT3-ITD homozygous and p53 heterozygous) mice became moribund much faster than mice with ITD/ITD alone or p53+/- alone. The median survival of ITD/ITD;p53+/- mice (n=69) was 313 days, which was significantly shorter than that of ITD/ITD mice (littermates of ITD/ITD; p53+/- mice, 583 days, n=16), p53+/- mice (521 days, n=30), and WT mice (862 days, n=10) (p50% ITD/ITD; p53-/- mice and ITD/ITD; p53+/- mice with acute leukemia demonstrated a bi-clone disease, with co-existence of AML and ALL. Unexpectedly, all analyzed murine AMLs with complete or heterozygous loss of p53 (n=9) showed normal karyotypes. p53 haploinsufficiency or loss did not increase self-renewal of hematopoietic stem/progenitor cells as defined by serial replanting assays in vitro and limiting dilution transplantation of leukemic cells in vivo. However, animals with only AML in the ITD/ITD; p53+/- group showed a significant increase of CMPs in comparison with ITD/ITD mice with CMML, P53+/- and WT mice, e.g. a 66-fold increase of CMPs in #1376 mouse compared with WT mice. This suggests that the block of differentiation from CMPs to GMPs may have contributed to the development of AML. P53 haploinsufficiency or loss reduced the sensitivity of murine FLT3-ITD leukemia to crenolanib in vitro (IC50: 313.3nM, 461.4nM, and 185.8nM for ITD/ITD;p53+/-, ITD/ITD;p53-/-, and ITD/ITD leukemia, respectively), but not their sensitivity to midostaurin (IC50: 117.3nM, 110.2nM, and 201.3nM). To develop an efficient therapy for p53-mutated AML, we firstly tried to combine FLT3 inhibitors and MDM2 antagonist idasanutlin, FLT3 inhibitor and Bcl2 inhibitor venetoclax. Unfortunately, these did not enhance induction of apoptosis in leukemic cells from ITD/ITD;p53+/- mice. However, exposure to the proteasome inhibitor carfilzomib had a strong cytotoxic effect against ITD/ITD; p53+/- and ITD/ITD;p53-/-, leukemic cells (IC50: 5.2nM and 17.1nM vs. 6.8nM for ITD/ITD leukemia). In addition, the combination of carfilzomib with midostaurin showed an additive cytotoxicity in murine ITD/ITD; p53+/- leukemia as well as in primary leukemic cells from most patients with AML (n=10). Taken together, our data indicate a strong cooperating effect of FLT3-ITD and p53 haploinsufficiency or loss in the induction of AML and ALL. Our data emphasize more careful analysis of p53 deregulation in AML. Targeting FLT3 in combination with drugs working independently of p53 status, such as proteasome inhibitor carfilzomib, might improve outcomes of AML patients. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees.

The novel FMS-like tyrosine kinase 3 (FLT3)-N676K point mutation within the FLT3 kinase domain-1 was recently identified in 6 % of de novo acute myeloid leukemia (AML) patients with inv(16). Because FLT3-N676K was encountered almost exclusively in inv(16) AML, we investigated the transforming potential of FLT3-N676K, the cooperation between FLT3-N676K and core binding factor ß-smooth muscle myosin heavy chain (CBFß-SMMHC) (encoded by the inv(16) chimeric gene CBFB-MYH11) in inducing acute leukemia, and tested the sensitivity of FLT3-N676K-positive leukemic cells to FLT3 inhibitors. Retroviral expression of FLT3-N676K in myeloid 32D cells induced AML in syngeneic C3H/HeJ mice (n = 11/13, median latency 58 days), with a transforming activity similar to FLT3-internal tandem duplication (ITD) (n = 8/8), FLT3-TKD D835Y (n = 8/9), and FLT3-ITD-N676K (n = 9/9) mutations. Three out of 14 (21.4 %) C57BL/6J mice transplanted with FLT3-N676K-transduced primary hematopoietic progenitor cells developed acute leukemia (latency of 68, 77, and 273 days), while no hematological malignancy was observed in the control groups including FLT3-ITD. Moreover, co-expression of FLT3-N676K/CBFß-SMMHC did not promote acute leukemia in three independent experiments (n = 16). In comparison with FLT3-ITD, FLT3-N676K induced much higher activation of FLT3 and tended to trigger stronger phosphorylation of MAPK and AKT. Importantly, leukemic cells carrying the FLT3-N676K mutant in the absence of an ITD mutation were highly sensitive to FLT3 inhibitors AC220 and crenolanib, and crenolanib even retained activity against the AC220-resistant FLT3-ITD-N676K mutant. Taken together, the FLT3-N676K mutant is potent to transform murine hematopoietic stem/progenitor cells in vivo. This is the first report of acute leukemia induced by an activating FLT3 mutation in C57BL/6J mice. Moreover, further experiments investigating molecular mechanisms for leukemogenesis induced by FLT3-N676K mutation and clinical evaluation of FLT3 inhibitors in FLT3-N676K-positive AML seem warranted.

Using high-throughput sequencing, an increased number of gene mutations has been identified in cancer. Among the up to hundreds of acquired mutations in cancer clones, only a few cooperating mutations are believed to be needed for initiation of the malignant disease. Recently, we reported a single amino acid substitution at position 676 (N676K) within the FLT3 kinase domain as the sole cause of resistance to PKC412 in one patient with FLT3-ITD associated acute myeloid leukemia (AML). The FLT3-N676K mutation was more recently identified independently in up to 6% of de novo AML patients with inv(16) by other groups. As FLT3-TKD mutations are strongly associated with inv(16) in AML and particularly FLT3-N676K was found almost exclusively in AML patients with inv(16), this prompted us to investigate the transforming activity of FLT3-N676K and to test whether FLT3-N676K would cooperate with inv(16) to promote AML. First, we analyzed in vivo leukemogenesis mediated by FLT3-N676K. Retroviral expression of FLT3-N676K in myeloid 32D cells induced AML in syngeneic C3H/HeJ mice (n=11/13, latency ~8 weeks), with a transforming activity similar to FLT3-ITD (n=8/8), FLT3-TKD D835Y (n=8/9), and FLT3-ITD-N676K (n=9/9) mutations. Three out of 14 C57BL/6J mice transplanted with FLT3-N676K-transduced primary lineage negative (Lin-) bone marrow cells died of acute leukemia (latency of 68, 77, and 273 days), while none of 16 animals in the control groups including FLT3-ITD and CBFß-SMMHC developed any hematological malignancy. Secondly, co-expression of FLT3-N676K and CBFß-SMMHC did not promote acute leukemia in 3 independent experiments using C3H/HeJ and C57BL/6J mice (n=16). So far only 1 out of 11 C57BL/6J mice co-expressing FLT3-N676K and CBFß-SMMHC developed acute leukemia (AML with latency of 166 days). In comparison with FLT3-ITD, FLT3-N676K tended to result in stronger phosphorylation of FLT3, MAPK and AKT, and diseased animals carrying FLT3-N676K demonstrated much lower frequency of leukemic stem cells in the majority of analyzed cases. Importantly, leukemic cells co-expressing FLT3-N676K and CBFß-SMMHC were still highly sensitive to the FLT3 inhibitor AC220. Taken together, we show that FLT3-N676K mutant is potent to transform murine hematopoietic stem/progenitor cells in vivo independently of the inv(16) chimeric gene CBFB-MYH11. This is the first report of acute leukemia induced by an activating FLT3 mutation in C57BL/6J mice. Moreover, our data suggest that targeting FLT3-N676K mutation may be an attractive treatment option for FLT3-N676K-positive patients without concurrent ITD. Our data emphasize more careful analysis of the cooperating network of mutations identified in AML by high-throughput sequencing. This work was supported by DJCLS (grant: 13/22) and the Deutsche Forschungsgemeinschaft (grant: Li 1608/2-1). KH and ZP were supported by the China Scholarship Council (2011638024 and 201406100008). Disclosures Heidel: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Commercially available solid phase extraction (SPE) cartridges were combined with GC–MS for fast cleanup and fractionation of biodiesel/diesel samples in oil fingerprinting analysis. Several commercially available SPE cartridges (e.g., silica gel/cyanopropyl (SiO2/C3–CN) and extractable petroleum hydrocarbons (EPH) fractionation cartridges), were employed for the fractionation of biodiesel/diesel blends into aliphatic, aromatic hydrocarbons, and fatty acid methyl esters (FAMEs). The SiO2/C3–CN (1 g/0.5 g) SPE cartridge could not separate biodiesel from petroleum hydrocarbons effectively, however, the 5 g EPH SPE cartridge successfully separated blended samples into aliphatic, aromatic and FAME fractions by eluting with 15 mL of hexane, 15 mL of dichloromethylene (DCM)/hexane (1:1, v/v), and 18 mL of DCM, respectively. No cross-elution was observed among aliphatic, aromatic and FAME fractions when the loading mass of blends was less than 8 mg. The relative standard deviations (RSD) for five replicate SPE–GC–MS analyses of 5 mg of diesel blended with 20% biodiesel (volume/volume) and soybean oil ranged from 3.4% to 33.2%, 4.3% to 18.7% and 5.4% to 29.2% for selected alkane, polycyclic aromatic hydrocarbons (PAHs) and FAMEs, respectively. The final recovery rates of surrogates for aliphatic, aromatic and FAME fractions ranged from 82.5 to 91.1%. This developed method was successfully used for the fingerprinting analysis of three simulated blend samples. The concentration profiles of target compounds and the blended level are both comparable to those obtained by the conventional silica gel column–GC–MS method.