Contributors: You Na Ha

Date: 2019-12-13

... Treatment of acute myeloid leukemia (AML) remains inefficient due to drug resistance and relapse, particularly in patients with FMS-like tyrosine kinase (FLT)3-internal tandem duplication (ITD). Marine-derived natural products have recently been used for drug development against AML. We show here that petromurin C, isolated from the culture extract of the marine-derived fungus Aspergillus candidus KUFA0062, which was isolated from the marine sponge Epipolasis sp., induces early autophagy followed by apoptotic cell death via activation of the intrinsic cell death pathway concomitant with mitochondrial stress and downregulation of Mcl-1 in FLT3-ITD mutated MV4-11 cells. Moreover, petromurin C synergized with the clinically-used FLT3 inhibitor gilteritinib at sub-toxic concentrations. Altogether our results suggest that petromurin C could act as an anti-leukemic agent alone or in combination with gilteritinib.

Contributors: Sungmi Song

Date: 2019-12-10

... Abstract Chronic myeloid leukemia (CML) results from a t(9;22) (q34; q11) translocation, also called Philadelphia chromosome (Ph). This reciprocal translocation causes a constitutively-activated tyrosine kinase BCR-ABL fusion gene. By comparing imatinib-sensitive and -resistant CML cell models, we investigated the molecular mechanisms by which tetrahydrobenzimidazole derivative TMQ0153 triggered caspase-dependent apoptosis at low concentrations accompanied by loss of mitochondrial membrane potential (MMP) and increase of cytosolic free Ca2+ levels. Interestingly, at higher concentrations, TMQ0153 induced necroptotic cell death with accumulation of ROS, both preventable by N-acetyl-L-cysteine (NAC) pretreatment. At necroptosis-inducing concentrations, we observed increased ROS and decreased ATP and GSH levels, concomitant with protective autophagy induction. Inhibitors such as bafilomycin A1 (baf-A1) and siRNA against beclin 1 abrogated autophagy, sensitized CML cells against TMQ0153 and enhanced necroptotic cell death. Importantly, TMQ153-induced necrosis led to cell surface exposure of calreticulin (CRT) and ERp57 as well as the release of extracellular ATP and high mobility group box (HMGB1) demonstrating the immunological cell death markers by TMQ0153. We validated the anti-cancer potential of TMQ0153 by in vivo inhibition of K562 microtumor formation in zebrafish. Taken together, our findings provide evidence that cellular stress and redox modulation by TMQ0153 concentration-dependently leads to different cell death modalities including controlled necrosis in CML cell models.

Contributors: Sungmi Song

Date: 2019-12-10

... Abstract Chronic myeloid leukemia (CML) results from a t(9;22) (q34; q11) translocation, also called Philadelphia chromosome (Ph). This reciprocal translocation causes a constitutively-activated tyrosine kinase BCR-ABL fusion gene. By comparing imatinib-sensitive and -resistant CML cell models, we investigated the molecular mechanisms by which tetrahydrobenzimidazole derivative TMQ0153 triggered caspase-dependent apoptosis at low concentrations accompanied by loss of mitochondrial membrane potential (MMP) and increase of cytosolic free Ca2+ levels. Interestingly, at higher concentrations, TMQ0153 induced necroptotic cell death with accumulation of ROS, both preventable by N-acetyl-L-cysteine (NAC) pretreatment. At necroptosis-inducing concentrations, we observed increased ROS and decreased ATP and GSH levels, concomitant with protective autophagy induction. Inhibitors such as bafilomycin A1 (baf-A1) and siRNA against beclin 1 abrogated autophagy, sensitized CML cells against TMQ0153 and enhanced necroptotic cell death. Importantly, TMQ153-induced necrosis led to cell surface exposure of calreticulin (CRT) and ERp57 as well as the release of extracellular ATP and high mobility group box (HMGB1) demonstrating the immunological cell death markers by TMQ0153. We validated the anti-cancer potential of TMQ0153 by in vivo inhibition of K562 microtumor formation in zebrafish. Taken together, our findings provide evidence that cellular stress and redox modulation by TMQ0153 concentration-dependently leads to different cell death modalities including controlled necrosis in CML cell models.

Contributors: Sungmi Song

Date: 2019-12-10

... Abstract Chronic myeloid leukemia (CML) results from a t(9;22) (q34; q11) translocation, also called Philadelphia chromosome (Ph). This reciprocal translocation causes a constitutively-activated tyrosine kinase BCR-ABL fusion gene. By comparing imatinib-sensitive and -resistant CML cell models, we investigated the molecular mechanisms by which tetrahydrobenzimidazole derivative TMQ0153 triggered caspase-dependent apoptosis at low concentrations accompanied by loss of mitochondrial membrane potential (MMP) and increase of cytosolic free Ca2+ levels. Interestingly, at higher concentrations, TMQ0153 induced necroptotic cell death with accumulation of ROS, both preventable by N-acetyl-L-cysteine (NAC) pretreatment. At necroptosis-inducing concentrations, we observed increased ROS and decreased ATP and GSH levels, concomitant with protective autophagy induction. Inhibitors such as bafilomycin A1 (baf-A1) and siRNA against beclin 1 abrogated autophagy, sensitized CML cells against TMQ0153 and enhanced necroptotic cell death. Importantly, TMQ153-induced necrosis led to cell surface exposure of calreticulin (CRT) and ERp57 as well as the release of extracellular ATP and high mobility group box (HMGB1) demonstrating the immunological cell death markers by TMQ0153. We validated the anti-cancer potential of TMQ0153 by in vivo inhibition of K562 microtumor formation in zebrafish. Taken together, our findings provide evidence that cellular stress and redox modulation by TMQ0153 concentration-dependently leads to different cell death modalities including controlled necrosis in CML cell models. Conclusion: Our findings indicate that TMQ0153-induced ROS act as a rheostat determining the onset of apoptotic- or autophagy-related controlled necrotic cell death in CML.

Contributors: Wheaton Schroeder, Rajib Saha

Date: 2019-12-02

... Dataset related to the paper of the same title submitted for publication in the journal iScience. The abstract is as follows.

Contributors: Tetsuji Okada

Date: 2019-11-23

... DSA files of human (N to Z, by gene name) : UniProt ID is used for a protein to which no gene name is assigned.

Contributors: Tetsuji Okada

Date: 2019-11-22

... DSA files of human (A to M, by gene name) : UniProt ID is used for a protein to which no gene name is assigned.

Contributors: Saroj Chakraborty

Date: 2019-11-12

... Pediatric hypertension is recognized as an emerging global health concern. While new guidelines are developed for facilitating clinical management, the reasons for the prevalence of hypertension in children remain unknown. Genetics and environmental factors do not fully account for the growing incidence of pediatric hypertension. Because stable bacterial flora in early life are linked with health outcomes later in life, we hypothesized that reshaping of gut microbiota in early developmental stages of life affects blood pressure (BP) of pediatric subjects. To test this hypothesis, we administered amoxicillin, the most commonly prescribed pediatric antibiotic, to alter gut microbiota of young, genetically hypertensive rats (study 1) and dams during gestation and lactation to reshape microbiota of offspring (study 2). Reshaping of microbiota, with reductions in Firmicutes/Bacteriodetes ratio observed in Amoxicillin treated young rats and in dams. Amoxicillin treated rats also had lower blood pressure compared to the untreated rats. In the young rats treated with amoxicillin, the lowering effect on blood pressure persisted even after the antibiotics were discontinued. Similarly, the offspring from the dams treated with amoxicillin also showed lower systolic blood pressure compared to the control rats. Remarkably, in all cases, a decrease in BP was associated with lowering of Veillonellaceae, which are succinate-producing bacteria. Elevated plasma succinate is reported in hypertension. Accordingly, serum succinate was measured and found lower in animals treated with amoxicillin. Our results demonstrate a direct correlation between succinate-producing gut microbiota and early development of hypertension, and indicate that reshaping gut microbiota, especially by depleting succinate-producing microbiota early in life may have long-term benefits for hypertension-prone individuals.

Contributors: Yasuhiro Funahashi

Date: 2019-10-25

... To identify the phosphorylation sites of Npas4, GST-Npas4-390-489 aa, GST-Npas4 490-597 aa or GST-Npas4 598-701 aa was expressed in COS7 cells with or without the coexpression of MAP2K1-CA. Cells were lysed in lysis buffer [20 mM Tris/HCl, 1 mM EDTA, 150 mM NaCl, 1% NP-40, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (PhosStop, Roche), pH 7.5], and sonicated 3 times for 5 sec. After centrifugation at 16,000×g at 4°C for 10 min, the soluble supernatant was incubated in 30 µl of glutathione-Sepharose 4B beads (GE Healthcare) for 1 hr at 4°C with rotation. The beads were then washed three times with lysis buffer and an additional three times with wash buffer (20 mM Tris/HCl, 1 mM EDTA, and 150 mM NaCl, pH 7.5). The bound proteins were extracted from the beads using urea solution, reduced via incubation in 5 mM dithiothreitol for 30 min, and alkylated using 10 mM iodoacetamide for 1 h in the dark. The proteins were digested with Trypsin/Lys-C (Promega) or Glu-C (Promega)/Asp-N (FUJIFILM Wako). Demineralization was performed using SPE c-tips according to the manufacturer’s instructions. The peptides were analyzed by LC−MS using an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific Inc). Dual-phosphorylated peptide DLVCTPPYTPHQPGGCAFLFSLHEPFQTHLPPPSSSLQE, containing T423 and T427 phosphorylation sites, was identified from digested fragments of GST-Npas4-390-489 aa; LPPSPSSPGNGDCTLLALAQLR, containing S577 and S580 phosphorylation sites, was identified from digested fragments of GST-Npas4 490-597 aa; and GLLTPEASPVKQSFFHYTEKE, containing T611 and S615 phosphorylation sites, was identified from digested fragments of GST-Npas4 598-701 aa. Selected ion monitoring (SIM) analysis revealed that the amount of these dual-phosphorylation was significantly increased by coexpression with MAP2K1-CA. These results suggest that Npas4 is phosphorylated by MAPK at T423, T427, S577, S580, T611 and S615 sites.

Contributors: Laisuo Su

Date: 2019-10-05

... The data here includes necessary information for the MWR-assisted surface engineering project we worked on.