Journal articles on the topic 'Delayed OCRI'
To see the other types of publications on this topic, follow the link: Delayed OCRI.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 30 journal articles for your research on the topic 'Delayed OCRI.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Deguchi, T., DG Kim, and H. Kamioka. "CO2low-level laser therapy has an early but not delayed pain effect during experimental tooth movement." Orthodontics & Craniofacial Research 20 (June 2017): 172–76. http://dx.doi.org/10.1111/ocr.12158.
Full text
2
Hermann, Nuno V., Tron A. Darvann, and Sven Kreiborg. "Delayed maturation and reduced crown width of the permanent first mandibular molar in all subgroups of cleft lip and palate." Orthodontics & Craniofacial Research 23, no. 4 (May 19, 2020): 432–38. http://dx.doi.org/10.1111/ocr.12386.
Full text
3
Keller-Wood, M., E. Leeman, J. Shinsako, and M. F. Dallman. "Steroid inhibition of canine ACTH: in vivo evidence for feedback at the corticotrope." American Journal of Physiology-Endocrinology and Metabolism 255, no. 3 (September 1, 1988): E241—E246. http://dx.doi.org/10.1152/ajpendo.1988.255.3.e241.
Full textAbstract:
We infused submaximal feedback doses of either dexamethasone (DEX; 0.1 microgram.kg-1.min-1) or corticosterone and cortisol (B+F; 1.5 micrograms.kg-1.min-1) intravenously for 40 min into conscious dogs and measured the adrenocorticotropic hormone (ACTH) responses to hypoglycemia induced by insulin (0.1 U/kg) or to ovine corticotropin-releasing factor (oCRF; 1 microgram/kg); both agents were injected at 120 min. The dose of DEX was chosen to produce suppression of the ACTH response to oCRF equivalent to that produced by B+F. The purpose of the study was to determine 1) whether CRF- and hypoglycemia-induced ACTH secretion are equally inhibited by glucocorticoid treatment and 2) whether DEX and B+F have differential effects in the inhibition of stress-induced ACTH secretion. We found that peak ACTH responses to hypoglycemia and CRF were equally inhibited by DEX (36 +/- 6 and 52 +/- 9%, respectively). The peak ACTH responses to hypoglycemia and CRF were also equally inhibited after B+F infusion (45 +/- 13 and 65 +/- 5%, respectively). There was no significant interaction between the steroid administered and the stimulus given in controlling the ACTH response (by 2-way analysis of variance). The results suggest that pituitary feedback is of primary importance in suppression of canine ACTH secretion by delayed feedback and that the natural and synthetic steroids both act at this site.
4
Spagnuolo, Paul A., Jiayi Hu, Rose Hurren, Xiaoming Wang, Marcela Gronda, Mahadeo A. Sukhai, Ashley Di Meo, et al. "The antihelmintic flubendazole inhibits microtubule function through a mechanism distinct from Vinca alkaloids and displays preclinical activity in leukemia and myeloma." Blood 115, no. 23 (June 10, 2010): 4824–33. http://dx.doi.org/10.1182/blood-2009-09-243055.
Full textAbstract:
Abstract On-patent and off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication given their prior toxicity testing. To identify such compounds, we conducted chemical screens and identified the antihelmintic flubendazole. Flubendazole induced cell death in leukemia and myeloma cell lines and primary patient samples at nanomolar concentrations. Moreover, it delayed tumor growth in leukemia and myeloma xenografts without evidence of toxicity. Mechanistically, flubendazole inhibited tubulin polymerization by binding tubulin at a site distinct from vinblastine. In addition, cells resistant to vinblastine because of overexpression of P-glycoprotein remained fully sensitive to flubendazole, indicating that flubendazole can overcome some forms of vinblastine resistance. Given the different mechanisms of action, we evaluated the combination of flubendazole and vinblastine in vitro and in vivo. Flubendazole synergized with vinblastine to reduce the viability of OCI-AML2 cells. In addition, combinations of flubendazole with vinblastine or vincristine in a leukemia xenograft model delayed tumor growth more than either drug alone. Therefore, flubendazole is a novel microtubule inhibitor that displays preclinical activity in leukemia and myeloma.
5
Pichard, Alexandre, Sara Marcatili, Jihad Karam, Julie Constanzo, Riad Ladjohounlou, Alan Courteau, Marta Jarlier, et al. "The therapeutic effectiveness of 177Lu-lilotomab in B-cell non-Hodgkin lymphoma involves modulation of G2/M cell cycle arrest." Leukemia 34, no. 5 (December 13, 2019): 1315–28. http://dx.doi.org/10.1038/s41375-019-0677-4.
Full textAbstract:
AbstractSome patients with B-cell non-Hodkin lymphoma Lymphoma (NHL) become refractory to rituximab (anti-CD20 antibody) therapy associated with chemotherapy. Here, the effect of the anti-CD37 antibody-radionuclide conjugate lutetium-177 (177Lu)-lilotomab (Betalutin®) was investigated in preclinical models of NHL. In SCID mice bearing DOHH2 (transformed follicular lymphoma, FL) cell xenografts, 177Lu-lilotomab significantly delayed tumor growth, even at low activity (100 MBq/kg). In athymic mice bearing OCI-Ly8 (diffuse large B-cell lymphoma, DLBCL) or Ramos (Burkitt’s lymphoma) cell xenografts, 177Lu-lilotomab activity had to be increased to 500 MBq/kg to show a significant tumor growth delay. Clonogenic and proliferation assays showed that DOHH2 cells were highly sensitive to 177Lu-lilotomab, while Ramos cells were the least sensitive, and U2932 (DLBCL), OCI-Ly8, and Rec-1 (mantle cell lymphoma) cells displayed intermediate sensitivity. The strong 177Lu-lilotomab cytotoxicity observed in DOHH2 cells correlated with reduced G2/M cell cycle arrest, lower WEE-1- and MYT-1-mediated phosphorylation of cyclin-dependent kinase-1 (CDK1), and higher apoptosis. In agreement, 177Lu-lilotomab efficacy in vitro, in vivo, and in patient samples was increased when combined with G2/M cell cycle arrest inhibitors (MK-1775 and PD-166285). These results indicate that 177Lu-lilotomab is particularly efficient in treating tumors with reduced inhibitory CDK1 phosphorylation, such as transformed FL.
6
Choi, Sang-Jae, and Sung-Hun Lim. "Enhancement on the Fault Ride through Capability of Power Distribution Systems Linked by Distributed Generation due to the Impedance of Superconducting Fault Current Limiters." Energies 12, no. 24 (December 17, 2019): 4810. http://dx.doi.org/10.3390/en12244810.
Full textAbstract:
Recently, studies on connecting distributed generation (DG) to power distribution systems through DC links have been actively conducted. When a fault in feeder of this power distribution system occurs, a voltage dip can happen in the grid. In order to prevent voltage dips, there are several solutions such as the application of a superconducting fault current limiter (SFCL). If a SFCL with a larger impedance is applied, the voltage dip of the grid is effectively prevented. However, this action can bring about the malfunction or the delayed operation of the over-current relay (OCR) due to the decreased fault current, which causes another problem of protection coordination between the protective relays. On the other hand, if the impedance of the SFCL is too low, excessive reactive power is supplied by the fault ride-through (FRT) regulation and the active power is reduced. This causes an active power imbalance on the DC link and increases the DC link’s voltage. As previous solutions to prevent the rise of DC links’ voltage, the deloading method and the application of a chopper resistor have been suggested. In this paper, a technique called active power tracking control (APTC), was proposed to suppress the rise of DC links’ voltage. Case studies considering the impedance of SFCL in the constructed power distribution system were carried out, and the rise of DC links’ voltage could be effectively suppressed without any significant delay in the operation of the OCR. This study is expected to solve both the voltage dip of the grid and the rise of DC links’ voltage when distributed generation is connected to a grid.
7
Pinelis, Vsevolod, Irina Krasilnikova, Zanda Bakaeva, Alexander Surin, Dmitrii Boyarkin, Andrei Fisenko, Olga Krasilnikova, and Igor Pomytkin. "Insulin Diminishes Superoxide Increase in Cytosol and Mitochondria of Cultured Cortical Neurons Treated with Toxic Glutamate." International Journal of Molecular Sciences 23, no. 20 (October 20, 2022): 12593. http://dx.doi.org/10.3390/ijms232012593.
Full textAbstract:
Glutamate excitotoxicity is involved in the pathogenesis of many disorders, including stroke, traumatic brain injury, and Alzheimer’s disease, for which central insulin resistance is a comorbid condition. Neurotoxicity of glutamate (Glu) is primarily associated with hyperactivation of the ionotropic N-methyl-D-aspartate receptors (NMDARs), causing a sustained increase in intracellular free calcium concentration ([Ca2+]i) and synchronous mitochondrial depolarization and an increase in intracellular superoxide anion radical (O2–•) production. Recently, we found that insulin protects neurons against excitotoxicity by decreasing the delayed calcium deregulation (DCD). However, the role of insulin in O2–• production in excitotoxicity still needs to be clarified. The present study aims to investigate insulin’s effects on glutamate-evoked O2–• generation and DCD using the fluorescent indicators dihydroethidium, MitoSOX Red, and Fura-FF in cortical neurons. We found a linear correlation between [Ca2+]i and [O2–•] in primary cultures of the rat neuron exposed to Glu, with insulin significantly reducing the production of intracellular and mitochondrial O2–• in the primary cultures of the rat neuron. MK 801, an inhibitor of NMDAR-gated Ca2+ influx, completely abrogated the glutamate effects in both the presence and absence of insulin. In experiments in sister cultures, insulin diminished neuronal death and O2 consumption rate (OCR).
8
Ramachandiran, Sampath, Arsene Adone, Xiangxue Guo, Albert Liao, Uston Robert Sunay, Lee Ratner, Haian Fu, Izidore S. Lossos, Harold Saavedra, and Leon Bernal-Mizrachi. "Genomic Stability: A Novel Function of NF-Kb in Lymphomas." Blood 114, no. 22 (November 20, 2009): 3240. http://dx.doi.org/10.1182/blood.v114.22.3240.3240.
Full textAbstract:
Abstract Abstract 3240 Poster Board III-177 Lymphomas are heterogeneous diseases comprising multiple clinical and biological subgroups. Several studies have shown that genomic instability and constitutive activation of the NF-kB pathway are key features for lymphoma development (Shen M,Hematologica 2007). However, it remains unclear whether changes in activation of the canonical and non-canonical NF-kB pathways (rel-A/p50 and rel-B/p52, respectively) reflect a response to genomic instability and therefore promotes lymphomagenesis. To answer this question, we first demonstrated in two lymphoma cell lines (Daudi and OCI-Ly3) that DNA damage induced by Doxorubicin (2 mcg/mL) resulted in nuclear localization of rel-A and Rel-B. Then, to determine the role of each NF-kB pathway in DNA repair and centrosome duplication we compared the number of cells positive for phospo-H2aX (pH2aX) and centrosomes numbers (measured by gamma-tubulin) in p105-siRNA (canonical) and P100-siRNA (non-canonical) with luciferase-siRNA (control) expressing cells. Our results showed that the expression of p105 and p100 siRNAs increase the number of pH2aX (+) cells compared to control. Subsequently, a time course measuring pH2aX (+) cells was performed after treating p105, P100 and luciferase siRNA OCI-ly3 expressing cell lines with Doxorubicin (2 mcg/mL). In cells expressing luciferase siRNA, pH2aX (+) cells peak (60%) at 60 minutes (min) and return to normal at 120 min, in p105 siRNA –cells pH2aX peak at 90 min (90% + cells) and then decrease similarly to luciferase siRNA cells. P100 siRNA cells demonstrate a continuous increased in pH2aX (+) cells up to 80%. In addition, p100 siRNA expression was associated with centrosome amplification (>2 centrosomes in 20-30% of the cells vs. < 8% in p105 or Luciferase siRNA expressing cells). Also, the expression of NF-kB siRNAs delayed doxorubicin-induced phosphorylation of p53 (serine 15 – target of ATM) and CHK2. To evaluate whether the genomic instability caused by both NF-kB siRNAs affects tumor development, we performed xenograft experiments. Our results demonstrated that NF-kB siRNAs not only slow down tumor initiation but prevented tumor development (p105 siRNA= 8 days delayed and 22% were tumor free and p100 siRNA= 13.5 days delayed and 55% were tumor free compared to luciferase siRNA). To investigate these findings in primary tissues we measured the number of phospo-H2aX (+) cells and the levels of rel-A and rel-B nuclear localization in 40 primary lymphoma tumor samples. Our results demonstrated that phospo-H2aX levels inversely correlated with rel-B nuclear localization (r=-0.58, p<0.0001). To identify possible explanations for these results, gene expression analysis was performed in cells expressing NF-kB siRNAs. Our results demonstrated that p105 siRNA regulated genes involved in DNA repair (PPP2R5C, ING5, SYF2, SYF2, XRCC6, etc) and p100 siRNA regulated genes involved in both DNA repair and centrosome duplication (GADD45 alpha, cyclin G, REDD1, PCBP4, etc) consistent with our results above. Quantitative PCR for some these genes during a doxorubicin-time course confirmed GADD45 alpha, cyclin G, PCBP4 and SFRS6 to be induced. We explored further the role of GADD45 alpha in lymphomas and found that knock down of this protein increase doxorubicin sensitivity by 50-fold. Overall this study demonstrated that activation of each NF-kB pathway is essential for maintaining genomic stability and therefore promoting tumor resistance to chemotherapy in lymphomas. In addition, we identified that GADD45 alpha is important target of the non-canonical NF-kB pathway for mediating genomic stability. These findings provide the rationale for designing novel agents aiming at targeting key genes involved in genomic stability. Disclosures No relevant conflicts of interest to declare.
9
Brookes, Paul, Andrew Tompkins, Kimberly Morse, Shannon Hilchey, Suhail Salim, Denise Ray, Richard Phipps, and Steven H. Bernstein. "The Triterpenoids 2-cyano-3,12-dioxooleana-1,9-dien-28-oic Acid (CDDO) and Their Imidazole (CDDO-Im) and Dinitrile Derivatives (DI-CDDO) Elicit Apoptosis through a Novel Mitochondrial Pathway." Blood 106, no. 11 (November 16, 2005): 2426. http://dx.doi.org/10.1182/blood.v106.11.2426.2426.
Full textAbstract:
Abstract We have recently shown that B-cell non Hodgkin’s lymphoma express the transcription factor PPARγ and undergo apoptosis upon exposure to PPARγ ligands. The synthetic triterpenoid CDDO is a specific ligand for PPARγ, and CDDO and its derivatives, CDDO-Im and DI-CDDO, induce diffuse large cell lymphoma (DLCL) death (OCI Ly10 and OCI Ly19 cells), with a potency of DI-CDDO>CDDO-Im>CDDO, suggesting that such agents have therapeutic potential in lymphoma. The natural PPARγ ligand, 15d-PGJ2 (which also elicits DLCL death), has previously been shown to inhibit mitochondrial complex I, enhance mitochondrial reactive oxygen species (ROS) generation, and react with protein thiols. Given that CDDO is structurally similar to 15d-PGJ2 we hypothesized that CDDO-induced cell death may similarly be mediated via complex I inhibition, ROS generation, thiol oxidation, and opening of a large membrane pore complex in the mitochondrial membrane, termed the “permeability transition” (PT) pore. Studies on isolated rat liver mitochondria however showed that none of the CDDO-derivatives inhibited complex I activity or affected mitochondrial protein thiols. However, all three compounds did induce PT pore opening and mitochondrial swelling, with a concurrent loss of mitochondrial membrane potential, in a Ca2+ dependent manner (potency DI-CDDO>CDDO-Im>CDDO). This is consistent with a previously shown role for Ca2+ in CDDO-induced cell death. Interestingly, this mitochondrial swelling was not inhibited by the classical PT pore inhibitor cyclosporin A (CsA). This is supported by our findings that the induction of OCI-Ly19 cell death by CDDO was also not inhibited by CsA, or by another classical PT pore inhibitor, nortriptyline. These phenomena may be partially explained by invoking the “unregulated PT pore”. In addition to the classical PT pore, a non-CsA sensitive “unregulated PT pore” also exists, which is generated by the aggregation of misfolded mitochondrial membrane proteins that are induced by oxidants and thiol reactive agents. That exposure of mitochondria to CDDO results in the formation of “unregulated PT pores” is supported by our findings that the proteosome inhibitor PS341, potentiates CDDO-induced cell death, suggesting the involvement of a protein folding response. The temporal role of ROS in CDDO-induced cell death was also investigated, and it was found that the antioxidant N-acetyl-cysteine did not inhibit PT pore opening, but did inhibit cell death. This is consistent with our observation that ROS generation in isolated mitochondria was not immediately triggered by CDDO, but rather increased at delayed time points, placing it downstream of PT pore opening. This proposes the following novel model of a direct mitochondrial effect of CDDO and its derivatives: \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[CDDO{\rightarrow}\ mitochondrial\ protein\ misfolding\ {\rightarrow}\ unregulated\ PT\ pore\ formation\ {\rightarrow}\ ROS\ {\rightarrow}\ cell\ death\] \end{document} In summary: CDDO and its derivatives have direct effects on mitochondria, and represent novel therapeutic approaches for the treatment of patients with DLCL; and combinations of CDDO and its derivatives with proteosome inhibitors represent a rational combination to test in the context of clinical trials.
10
Han, Lina, Antonio Cavazos, Natalia Baran, Qi Zhang, Vinitha Mary Kuruvilla, Jason P. Gay, Ningping Feng, et al. "Mitochondrial Oxphos As Survival Mechanism of Minimal Residual AML Cells after Induction Chemotherapy : Survival Benefit By Complex I Inhibition with Iacs-010759." Blood 134, Supplement_1 (November 13, 2019): 5161. http://dx.doi.org/10.1182/blood-2019-124475.
Full textAbstract:
Acute myeloid leukemia (AML) is initiated and maintained by a relatively rare leukemia stem cells (LSCs) capable of self-renewal and proliferation. Recent data showed that LSCs (Lagadinou et al. Cell Stem Cell 2013) and residual cytarabine (Ara-C)-resistant AML cells (representing minimal residual disease, MRD) (Farge et al. Cancer Discovery 2017) are highly dependent on mitochondrial function for survival. This unique metabolic biology makes chemoresistant LSCs and AML cells vulnerable to pharmacological blockade of the oxidative phosphorylation (OXPHOS). We have reported that a novel OXPHOS inhibitor IACS-010759 potently inhibits mitochondrial complex I, suppresses OXPHOS and selectively inhibits the growth of AML cells in vitro and in vivo (Molina et al. Nat Med 2018). In this study, we aimed to determine the effects of OXPHOS inhibition with IACS-010759 on residual AML cells surviving standard chemotherapy (Doxorubicin/Ara-C, DA) in cell line and patient-derived xenograft (PDX) AML models. Consistent with our hypothesis, OCI-AML3 cells treated with DA in vitro induced elevated levels of reactive oxygen species, higher mitochondrial mass and membrane potential (Fig. 1A), indicating reliance on the mitochondrial metabolism. Further, Ara-C treatment resulted in significantly increased basal and maximal oxygen consumption rates (OCR) (36%±8%, p=0.03; 36%±3%, p=0.003, respectively) compared to control. In turn, targeting OXPHOS with IACS-010759 at 30 nM fully inhibited basal and Ara-C-induced OCR. These findings indicate that chemotherapy fosters mitochondrial respiration in AML, which could be abrogated by OXPHOS inhibitor. To test the efficacy of combining IACS-010759 (5 mg/kg) and standard chemotherapy (Doxorubicin: 1.5 mg/kg; Ara-C: 50 mg/kg) in vivo, we injected NRG mice with genetically engineered OCI-AML3/Luc/GFP cells. Bioluminescent imaging demonstrated significantly reduced leukemia burden in DA/IACS-010759 combination group compared to vehicle on days 15 and 42 (p<0.01) (Fig. 1B). DA/IACS-010759 combination significantly extended survival, compared to the vehicle or single-agent treatment arms (Fig. 1C). Mouse body weight monitoring indicated that therapy was well tolerated We next examined the efficacy of IACS-010759 on leukemia cells surviving chemotherapy in a chemosensitive PDX AML model of minimal residual disease (Fig. 1D). Treatment of mice inoculated with a human AML PDX harboring FLT3-ITD mutation with DA reduced circulating leukemia burden (0.8 ± 0.6% vs 45.8 ± 8.2% blasts in vehicle-treated mice, p=0.001). The residual AML cells in DA-treated mice expanded and caused rapidly progressive leukemia (78.2 ± 6.2% vs 95.3 ± 1.0% in vehicle-treated mice, p=0.047) on week 6 post DA. Daily oral treatment of mice with IACS-010759 (7.5 mg/kg) as a single agent reduced leukemia burden, and delayed leukemia recurrence when administered post completion of DA (Fig. 1E). A SPADE tree was built based on 13 surface markers and colored by expression intensity of CD34 using CyTOF mass cytometry data (Fig. 1F). The data demonstrated reduced frequency of CD34+CD38lowCD123+ AML LSCs and increase in CD11c+ differentiated cells in both IACS and IACS/DA groups (Fig. 1G&H). In contrast, chemotherapy alone failed to significantly reduce fractions of LSCs or induce differentiation. Proliferation measured by Ki67 was greatly reduced by IACS/DA combination in all populations including LSCs (1.4 ± 0.3% vs 5.5 ± 0.4% in vehicle group, p<0.01). The expression of Hypoxia-Inducible Factor 1α (HIF-1α) was downregulated, consistent with the decreased oxygen consumption induced by IACS-010759 (not shown). In conclusion, minimal residual AML cells surviving chemotherapy depend on OXPHOS for survival. OXPHOS inhibition with complex I inhibitor IACS-010759 is effective in reducing LSCs and MRD, alone and in combination with chemotherapy in vivo. Our data advocate for combining IACS-010759 with chemotherapy for improved control of MRD upon identification of a recommended Phase II dose in a clinical trial of IACS-010759 in AML (NCT02882321). Disclosures Zhang: The University of Texas M.D.Anderson Cancer Center: Employment. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Kantarjian:Pfizer: Honoraria, Research Funding; Cyclacel: Research Funding; AbbVie: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Immunogen: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Ariad: Research Funding; Novartis: Research Funding; Agios: Honoraria, Research Funding; BMS: Research Funding; Astex: Research Funding; Amgen: Honoraria, Research Funding; Jazz Pharma: Research Funding. Daver:Jazz: Consultancy; Hanmi Pharm Co., Ltd.: Research Funding; Agios: Consultancy; Immunogen: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Celgene: Consultancy; Karyopharm: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Forty-Seven: Consultancy; Novartis: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Astellas: Consultancy; Servier: Research Funding; NOHLA: Research Funding; Glycomimetics: Research Funding; Otsuka: Consultancy. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy. Konopleva:Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding.
11
Barghout, Samir H., Parasvi Patel, Xiaoming Wang, G. Wei Xu, Simon Kavanagh, Marcela Gronda, Rose Hurren, et al. "TAK-243 Is a Selective UBA1 Inhibitor That Displays Preclinical Activity in Acute Myeloid Leukemia (AML)." Blood 130, Suppl_1 (December 7, 2017): 814. http://dx.doi.org/10.1182/blood.v130.suppl_1.814.814.
Full textAbstract:
Abstract Introduction: Ubiquitin-like Modifier Activating Enzyme 1 (UBA1; UAE) is the initiating enzyme in the ubiquitylation cascade in which proteins are tagged with ubiquitin moieties to regulate their degradation or function. Compared to normal hematopoietic cells, AML cell lines and primary AML cells have equal levels of UBA1 protein, but increased requirement for this enzyme. TAK-243 is a potent and selective inhibitor of UBA1 and we determined the preclinical activity, biological effects and mechanisms of resistance to the drug in AML. Results: TAK-243 reduced growth and viability of human AML cell lines (OCI-AML2, TEX, U937 and NB4) in a concentration- and time-dependent manner with IC50's ranging from 15-40 nM after treatment for 48 hours. In primary AML samples, most (n=18/21) were sensitive to TAK-243 with an IC50<75 nM at 48 hours of incubation. These samples included patients with high-risk cytogenetics, FLT3 mutations, and patients refractory to induction chemotherapy. We also compared the effects of TAK-243 on primary AML cells (n=6) and normal hematopoietic cells (n=6) and demonstrated that TAK-243 preferentially inhibited the clonogenic growth of AML cells over normal (19-fold reduction CFU-leukemia vs CFU-GM (normal), p ≤ 0.01). Binding of TAK-243 to UBA1 and related E1 enzymes was measured in intact AML cells using the cellular thermal shift assay (CETSA). In AML cell lines and primary AML samples, TAK-243 bound UBA1 at concentrations associated with cell death, but bound other E1 enzymes UBA3 and UBA6 only at much higher concentrations. Next, we evaluated the biological effects of UBA1 inhibition by TAK-243. At concentrations associated with cell death, TAK-243 decreased the abundance of poly- and mono-ubiquitylated proteins in OCI-AML2 cells and primary AML samples. In addition, TAK-243 treatment increased PERK phosphorylation, CHOP, XBP1s and ATF4 which are markers of proteotoxic stress and unfolded protein response. TAK-243 inhibited DNA double strand break (DSB) repair as evidenced by reduced recruitment of 53BP1 to DSBs and sustained γH2AX foci after 3 Gy of irradiation. We assessed the preclinical efficacy and toxicity of TAK-243 in mouse models of AML. OCI-AML2 cells were injected subcutaneously (sc) into SCID mice, and when tumors were palpable, mice were treated with TAK-243 (20 mg/kg sc twice weekly). TAK-243 significantly delayed tumor growth in mice (T/C=0.02) with no toxicity as evidenced by no changes in mouse body weight, serum chemistry, or organ histology. In tumors and organs isolated from the above treated mice, TAK-243 preferentially reduced levels of mono- and poly-ubiquitylated proteins in tumors over normal tissues. As an additional model, primary AML cells from 2 patients were injected into the femurs of NOD-SCID mice. Two weeks after injection, mice were treated with TAK-243 (20 mg/kg sc twice weekly). After 3 weeks of treatment, mice were sacrificed, and AML engraftment in the non-injected femur was measured by flow cytometry. TAK-243 reduced primary AML tumor burden in both tested samples without toxicity. Using secondary transplantations, we demonstrated that TAK-243 had targeted the leukemic stem cells. To understand mechanisms of resistance to TAK-243, we selected a population of TAK-243-resistant OCI-AML2 by culturing cells with increasing concentrations of the drug. Persisting cells were 33-fold more resistant to TAK-243 compared to wild-type cells (IC50 757 vs 23 nM), but had a normal rate of proliferation and remained equally sensitive to bortezomib, daunorubicin, mitoxantrone and the NEDD8-activating enzyme inhibitor pevonedistat. Using CETSA, we showed reduced binding of TAK-243 to UBA1 in the resistant cells. We sequenced UBA1 exons 12-16 and 23-24 that span the adenylation domain. Resistant cells had a missense mutation in exon 16 resulting in substitution of tyrosine with cysteine at codon 583 (Y583C). Y583 in human UBA1 corresponds to Y551 in yeast Uba1, which makes a favorable interaction with TAK-243 in its Uba1 binding site. Therefore, Y583C substitution is predicted to interfere with TAK-243 binding to UBA1. Conclusions: TAK-243 is a potent and selective UBA1 inhibitor that displays preferential activity towards AML cells over normal hematopoietic cells. Acquired mutations affect drug binding and may be a clinically relevant mechanism of resistance. These data support conducting a clinical trial of TAK-243 in patients with AML. Disclosures Hyer: Takeda Pharmaceuticals International Co.: Employment. Berger: Takeda Pharmaceuticals International Co.: Employment. Traore: Takeda Pharmaceuticals International Co.: Employment. Sintchak: Takeda Pharmaceuticals International Co.: Employment. Milhollen: Takeda Pharmaceuticals International Co.: Employment. Schimmer: Takeda Pharmaceuticals: Research Funding; Medivir: Research Funding; Novartis Pharmaceuticals: Honoraria.
12
Mohamed Khalil, Sohaila, Swagata Goswami, Xiaokui Mo, and Natarajan Muthusamy. "Protein Phosphatase 2A Activation Dependent Down Regulation of PGC1-Alpha and FOXO1 Phosphorylation with OSU-2S in Human Lymphoma Cells." Blood 134, Supplement_1 (November 13, 2019): 3977. http://dx.doi.org/10.1182/blood-2019-129224.
Full textAbstract:
Metabolic reprogramming has been recognized to provide survival advantage in cancer cells. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that is involved in regulation of multiple cellular pathways including metabolic events. OSU-2S, is a novel PP2A activator that exhibited potent anti-cancerous properties against human and canine lymphoma cell lines and primary human and canine lymphoma patient samples. It has been shown to activate PP2A in Ramos human lymphoma cell line leading to cytotoxicity that is prevented by inhibition of PP2A with okadaic acid (OA). Peroxisome proliferative activated receptor-gamma co-activator 1 (PPARGC1, also known as PGC1a), is a transcriptional co-activator that serves as a positive regulator of mitochondrial biogenesis and respiration, gluconeogenesis as well as many other metabolic processes such as lipid and energy metabolism. FOXO1 is a transcription factor that directly binds to the promoters of PGC1a and gluconeogenic genes involved in activation of gluconeogenesis. Activated PP2A has been shown to directly interact with FOXO1 and dephosphorylate it, leading to its delayed nuclear translocation. Given the role of PP2A in dephosphorylation of pFOX01, a regulator of PGC1a gene transcription, we hypothesized that PP2A activator OSU-2S, will down regulate PGC-1a expression through PP2A dependent FOXO1 regulation. Consistent with this hypothesis OSU-2S treatment inhibited PGC1a mRNA and protein expression in Jeko, OCI-ly18 and OCI-ly19 and raji lymphoma cell lines 24 hours post treatment. OSU-2S mediated downregulation of PGC1a and mitochondria biogenesis genes (NRF1, ERR alpha and TFAM) are dependent on PP2A activation as concentrations of OA that inhibited PP2A activation abrogated OSU-2S which induced up regulation of PGC1a and mitochondria biogenesis genes . To determine if the OSU-2S mediated inhibition of PGC1a expression is associated with its PP2A dependent modulation of phosphoFOXO1(pFOXO1), we tested the effect of OSU-2S on pFOXO1. Treatment of lymphoma cells with OSU-2S induced 60-70% decrease in pFOXO1 compared to vehicle control P =0.0001)], that is correlated with the decrease in PGC1a protein expression. Importantly OA mediated inhibition of PP2A, prevented OSU-2S-induced FOXO1 dephosphorylation. These studies suggest a role of OSU-2S induced modulation of metabolic regulator PGC1a via PP2A dependent dephosphorylation of FOXO1. Importantly, OSU-2S-induced PGC1a reduction resulted in decreased mitochondrial biogenesis as evidenced by ~43 % decrease in mitochondrial mass and ATP generation that led to reduced energy production as determined by Nonyl Acridine Orange dye staining followed by flow cytometry analysis. Interestingly, OSU-2S decreased expression of genes involved in mitochondrial biogenesis including NRF1a, ERR1a and TFAM by 75, 65 and 60% respectively P<0.0001. Ongoing mechanistic studies are aimed to define the molecular basis of OSU-2S induced transcriptional regulation of PGC1a and other genes involved in mitochondrial biogenesis in human lymphoma cell lines and primary cells. (This work was supported by NIH-R01 CA197844-02. SMK is a recipient of Egyptian Cultural and Educational Bureau (ECEB) Award). SG is a recipient of Pelotonia Graduate Fellowship) Keywords: PGC1- alpha, OSU-2S, PP2A, FOXO1, metabolism, lymphoma Disclosures Muthusamy: Ohio State University: Patents & Royalties: OSU-2S.
13
Liu, Tingting, Vi Lam, Duanchen Sun, Elana Thieme, Tamilla Nechiporuk, Daniel Bottomly, Olga V. Danilova, et al. "Abstract A02: Probing the roles of SUMOylation in B-cell lymphoma cells by using a selective SUMO inhibitor subasumstat (TAK-981)." Blood Cancer Discovery 3, no. 5_Supplement (September 6, 2022): A02. http://dx.doi.org/10.1158/2643-3249.lymphoma22-a02.
Full textAbstract:
Abstract SUMOylation is a post-translational modification that leads to covalent attachment of small ubiquitin-like modifiers (SUMO) proteins to a target protein. SUMOylation is essential for the regulation of genomic integrity, gene expression and intracellular signaling. Many tumors exhibit deregulation of SUMOylation pathway. TAK-981, a novel selective inhibitor of SUMO-activating enzyme (SAEi) has entered early phase II clinical trials. Here we studied SAEi in pre-clinical models of non-Hodgkin lymphoma (NHL). TAK-981 was provided by Takeda (Lexington, MA). Diffuse large B-cell lymphoma (DLBCL) cell lines and primary mantle cell lymphoma (MCL) cells were assayed for survival, mitochondrial function and metabolic phenotype. In vivo experiments were performed using DLBCL cell line xenografts and MCL PDX models. Treatment with TAK-981 restricted growth of DLBCL lines and led to rapid protein de-SUMOylation in a dose-dependent manner. TAK-981 induced pronounced DNA break in DLBCL cells (comet assay) and cell cycle arrest (propidium iodide). RNA-Seq and ATAC-Seq analysis of TAK-981-treated OCI-LY3 and U-2932 cells revealed upregulation of the NFκB, and OXPHOS signaling pathways. We noted that mitochondrial proteins were heavily de-SUMOylated upon treatment of NHL cells with TAK-981. In vitro exposure to TAK-981 resulted in mitochondrial membrane depolarization, loss of mitochondrial integrity (EM) and a change of mitochondrial dynamics (ICC for p-Drp1), accompanied by rapid accumulation of reactive oxygen species. Seahorse respirometry revealed dramatic downmodulation of OXPHOS. Metabolomic profiling of DLBCL cells treated with TAK981 exhibited downmodulation of TCA substrates. Exposure of primary MCL cells to TAK-981 in stromal conditions (CD40L- or BAFF expressing stroma) induced apoptosis and reduced OXPHOS. Next, we conducted genome-wide CRISPR-Cas9 loss-of-function library screens and identified that loss of genes in the NFκB, TP53 and DNA damage pathways contributed to resistance to SAEi in DLBCL cells. By means of genetic knockout we demonstrated that TP53 and its transcriptional target BAX contributed to TAK-981-induced G2 arrest and apoptosis, further implicating mitochondrial dysfunction in sensitivity to SAEi. For in vivo experiments, OCI-LY3 and U-2932 cells were inoculated subcutaneously in NSG mice. Once tumors reached 100 mm3, mice received twice-weekly IV doses of 7.5 mg/kg TAK-981 (over 5 weeks), leading to resolution of flank tumors and improved survival compared with control. Finally, we used an MCL PDX model where MCL cells were inoculated intravenously. Once cells became detectable in the blood, mice were treated as above. Treatment with TAK-981 resulted in delayed tumor expansion in blood and spleen and extended animal survival. TAK-981 exposed splenocytes exhibited a reduction of maximal respiration by Seahorse. In summary, pharmacologic SAEi with TAK-981 demonstrated pre-clinical activity in NHL models in vitro and in vivo, accompanied by DNA damage, mitochondrial dysfunction and metabolic reprogramming. Citation Format: Tingting Liu, Vi Lam, Duanchen Sun, Elana Thieme, Tamilla Nechiporuk, Daniel Bottomly, Olga V Danilova, Nur Bruss, Martina Cusan, Lili Wang, Shannon K McWeeney, Jeffrey W Tyner, Steve Kurtz, Zheng Xia, Alexey Danilov. Probing the roles of SUMOylation in B-cell lymphoma cells by using a selective SUMO inhibitor subasumstat (TAK-981) [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A02.
14
Benito, Juliana, Yuexi Shi, Barbara Szymanska, Hernan Carol, Ingrid Bohem, Hongbo Lu, Sergej Konoplev, et al. "Targeting the Leukemia-Associated Hypoxic Microenvironment with Hypoxia-Activated Prodrug PR-104." Blood 116, no. 21 (November 19, 2010): 868. http://dx.doi.org/10.1182/blood.v116.21.868.868.
Full textAbstract:
Abstract Abstract 868 Interactions between leukemia cells and the bone marrow (BM) microenvironment are known to promote leukemia cell survival and confer resistance to drugs commonly used in the management of this disease. We investigated the protective role of hypoxia in the BM microenvironment. We observed a marked expansion of hypoxic niches in the bone marrow of immunodeficient mice engrafted with the acute lymphoblastic leukemia (ALL) cell line Nalm6 and with primary ALL cells, as detected by the reductive 2-nitroimidazole compound pimonidazole (PIM), which forms stable adducts in hypoxic regions. We further demonstrated induction of the chemokine receptor CXCR4, and of the enzyme carbonic anhydrase 9 (CAIX), both targets of Hypoxia-Inducible Factor 1α (HIF-1a), in hypoxic areas of BM from mice harboring the Nalm6 xenografts. Furthermore, we used a CML blast crisis model to evaluate the time course of hypoxia expansion in the BM. In C57Bl6/J mice engrafted with murine HSCs co-expressing BCR/ABL and Nup98 we observed a time-dependent increase in PIM positive areas which coincided with the presence of GFP positive cells. In line with these findings, HIF-1α was highly expressed in BM biopsies from newly diagnosed ALL patients (n=15) but was significantly reduced when the patients achieved complete remission (CR). Culture under hypoxic conditions (1% O2) conferred resistance of pre-B ALL cells REH, Nalm-6, and of AML OCI-AML3 leukemic cells against several chemotherapeutic agents including vincristine, methotrexate and idarubicin (% of Annexin V(+) cells at 21% vs 1%O2; REH plus 1ng/ml vincristine:88.5+/−2.3 vs 18.1+/−10.3; REH plus 0.25uM etoposide: 86.4 +/−7 vs 14.4+/−8.6; Nalm6 plus 10ng/ml methotrexate: 33.4+/−2.7 vs 4.6+/−1; OCI-AML3 plus 50ng/ml idarubicin: 28.45+/−5 vs 12.76+/−1) . Taken together, these results provide rationale for examining the potential of hypoxia-activated pro-drugs to eliminate leukemia progenitor cells within hypoxic niches. To this end, we tested the hypoxia-activated prodrug PR104, a dinitrobenzamide nitrogen mustard that is reduced to its active metabolites under hypoxic conditions (Patterson et al., Clin Can Res 2007). In vitro, PR-104 induced cell death in three different leukemia cell lines (Nalm6, REH and the AML line Molm13) selectively under hypoxic (pO2 1%) conditions. The anti-leukemic efficacy of PR-104 as a single agent was next examined in several in vivo leukemia models. Administration of PR-104 prolonged survival and decreased leukemia burden of 1) NOD/Scid/IL2Rg-KO (NOG) mice injected with cells from primary refractory FLT3-mutated AML; 2) NOG mice injected with leukemic cells from an infant with MLL-rearranged B-lineage ALL; and 3) NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice injected with Nalm6-luciferase ALL cells. Next, we evaluated anti-tumor effects of PR-104 at clinically relevant doses (200, 100 and 50 mg/kg) and at maximal tolerated dose (550 mg/kg) in two ALL xenograft models (a T-lineage ALL and a B-cell precursor ALL) (Figure 1, studies supported by NCI NO1CM42216 and by PPTP contract NO1-CM91001-03). Compared to vehicle control, PR-104 significantly delayed progression of the T-ALL xenografts at all doses tested and at three of the four doses (550, 200 and 100 mg/kg) of B-cell pre-ALL, which resulted in significantly increased event-free survival (EFS) of mice in the treatment groups. Altogether, these findings strongly suggest that targeting hypoxia is feasible. If successful, this approach may significantly impact leukemia therapy and ultimately improve patient survival. This concept is currently being tested in an ongoing Phase I clinical trial of PR-104 in relapsed/refractory AML patients. Disclosures: Wilson: PROACTA: Equity Ownership. Konopleva:PROACTA: PI on clin trial funded by Proacta.
15
Yang, Ye, Zhimin Gu, Hongwei Xu, Junwei Huang, Jie He, Yi Tao, Guido J. Tricot, and Fenghuang Zhan. "Targeting BTK As a Treatment For Multiple Myeloma Stem Cells." Blood 122, no. 21 (November 15, 2013): 271. http://dx.doi.org/10.1182/blood.v122.21.271.271.
Full textAbstract:
Abstract Background Multiple myeloma stem cells (MMSCs), characterized by drug-resistance and self-renewal, are very likely responsible for relapse. Though the phenotypic features of MMSCs are still controversial, it is believed that MMSCs are enriched in plasma cell progenitors. Bruton’s tyrosine kinase (BTK) plays a central role in B-cell development and plasma cell differentiation. BLAB/c. CBA/N mice carrying the defective BTK gene are resistant to pristine-induced plasmacytomagenesis suggesting activation of BTK is essential for plasma tumor initiation. In this study, we explore the role of BTK in maintaining MMSCs. Materials and methods Real-time-PCR was used to detect the BTK expression in MMSCs and compared to the bulk cells. BTK-related self-renewal and drug resistance were evaluated by clonogenic formation assays, using 10,000 MM cells from the BTK over-expressing ARP1 and OPM2 MM cell lines, treated with bortezomib (1nM, 10nM), as well as doxorubicin (50nM, 100nM), etoposide (50nM, 100nM), and verapamil (50nM). Flow cytometry was performed to detect the side population (SP) fraction and apoptosis in MM cells. Co-immunoprecipitation assay and western blot were utilized for the mechanistic BTK studies. Effect of BTK on MM progression in vivo was assessed in the 5TGM1 myeloma mice and NOD/SCID mice models. Results Our results revealed that BTK, iPS genes, Wnt and Hedgehog signaling pathways were significantly increased in MMSCs (CD138- and SP fraction) compared to bulk myeloma cells in both MM cell lines and primary MM cells. We also demonstrated that over-expression of BTK in the low-expressing MM cell lines, ARP1 and OPM2 increased the SP fraction, clonogenic potential and drug-resistance, while knockdown of BTK in high-expressing MM cell lines, OCI-MY5 and H929 abrogated these characteristics. Our in vivo study indicated that over-expression of BTK in ARP1 cells accelerates tumor formation in NOD/SCID mice compared to the EV control cells. Targeting BTK by shRNA or BTK inhibitor, CGI-1747 delayed MM tumor formation and MM progression in NOD/SCID mice and 5TGM1 model, respectively. Mechanistic studies identified that BTK activates the AKT pathway, inducing the drug-resistant genes, p-Bcl-2 and ABCB1, and inhibiting GSK3β leading to activation of the Wnt/ β-Catenin pathway. Inhibition of AKT by a specific inhibitor decreased Wnt and Hh proteins by western blot. Conclusion BTK plays an important role on maintaining MM stemness and drug-resistance through activating AKT, Wnt and Hh signaling pathways. Targeting BTK in MM may be a promising strategy to eliminate MMSCs. Disclosures: No relevant conflicts of interest to declare.
16
Sukhai, Mahadeo A., Xiaoming Li, Rose Hurren, Xiaoming Wang, Skrtic Marko, Hong Sun, Marcela Gronda, et al. "The Anti-Malarial Mefloquine Demonstrates Preclinical Activity In Leukemia and Myeloma, and Is Dependent Upon Toll-Like Receptor Signaling for Its Cytotoxicity." Blood 116, no. 21 (November 19, 2010): 290. http://dx.doi.org/10.1182/blood.v116.21.290.290.
Full textAbstract:
Abstract Abstract 290 Known drugs with previously unrecognized anti-cancer activity can be rapidly repurposed for this new indication, given their prior safety and toxicity testing. To identify such compounds, we compiled and screened an in-house library of on-patent and off-patent drugs and screened them to identify agents cytotoxic to hematologic malignancies. From this screen, we identified mefloquine, a quinoline licensed for malaria treatment and prophylaxis. In secondary assays, leukemia and myeloma cell lines were treated with mefloquine for 72 hours and cell viability measured by MTS. Mefloquine decreased the viability of 10/10 human and murine leukemia (LD50 <8.0 μM) and 9/9 human myeloma (LD50 <5.0 μM) cell lines; cell death was confirmed by Annexin V staining. Mefloquine also reduced the viability of 6/6 primary AML samples with LD50 < 5 μ M. These concentrations of mefloquine appear pharmacologically achievable based on prior studies conducted in the context of malaria treatment. In contrast to the effects on malignant cells, mefloquine was significantly less cytotoxic to normal hematopoietic cells (LD50 31.83 ± 5.38 μM) and murine monocyte-derived dendritic cells (LD50 17.56 ± 2.69 μM), Given its in vitro activity, we evaluated the effects of oral mefloquine in mouse xenograft models of leukemia and myeloma. Sublethally irradiated SCID mice were injected subcutaneously with OCI-AML2 or K562 human leukemia cells, MDAY-D2 murine leukemia cells, or LP1 human myeloma cells, and treated with 50 mg/kg mefloquine, or vehicle alone, by gavage. Oral mefloquine delayed tumor growth by up to 60% in all 4 mouse models without toxicity at doses that appear pharmacologically relevant to humans based on scaling for body surface area. Mefloquine's mechanism of action as an anti-malarial agent is unknown. Therefore, to determine the mechanism by which mefloquine induced cell death in malignant cells, we performed gene expression oligonucleotide array analysis of mefloquine-treated OCI-AML2 cells. At times preceding cell death, mefloquine altered the expression of genes associated with Toll-like receptor (TLR) signaling. For example, we detected 4.5-fold up-regulation of STAT1 and >10-fold up-regulation of its downstream targets, including OAS1, IFIT3 and TRIM22, by 24 hr after treatment. Upregulation of additional TLR targets IRF1, IRF7 and IL-8 was also noted by 8 hours after treatment. Mefloquine also induced early activation of NF-κB with a 2.5± 0.2-fold increase noted after 1 hr, using an ELISA-based DNA binding assay. In contrast to TLR activation in malignant cells, changes in TLR targets were not detected in mefloquine-resistant normal dendritic cells, suggesting that mefloquine's effects on TLR signaling were specific to malignant cells. We next investigated whether TLR activation was functionally important for mefloquine's cytotoxicity in malignant cells. STAT1 activity was required for mefloquine-mediated cell death, as U4A bladder sarcoma cells lacking JAK1 were resistant to mefloquine (LD50 14.6± 4.9 μM), compared to the mefloquine sensitive parental line (LD50 2.3± 0.4 μM). TLR signaling requires the immediate downstream adapter proteins MyD88 and TRIF1. To assess the functional importance of TLR activation for mefloquine induced cell death, we knocked down MyD88 and TRIF1 with siRNA. Double knockdown of MyD88 and TRIF1 completely abrogated mefloquine-induced cell death in K562 leukemia cells at concentrations where control cells exhibited up to 80% loss of viability. TLR signaling and up-regulation of STAT1 can increase reactive oxygen species (ROS) generation. Therefore, we measured ROS generation in leukemia cells after mefloquine treatment. Mefloquine increased ROS production in leukemia cells in a dose-dependent manner within 24 hr. Co-treatment with the ROS scavenger N-Acetyl-L-Cysteine abrogated mefloquine-induced ROS production and cell death. Mefloquine-induced ROS production was also abrogated in MyD88 and TRIF1 double knockdown cells. Our data suggest that the known anti-malarial mefloquine displays preclinical activity in leukemia and myeloma through a mechanism related to TLR activation. Thus, these results highlight TLR activation as a novel therapeutic strategy for the treatment of leukemia and myeloma. Moreover, given its prior toxicology and pharmacology testing, mefloquine could be rapidly advanced into clinical trial for patients with leukemia and myeloma. Disclosures: No relevant conflicts of interest to declare.
17
Tiedemann, Rodger E., Yuan Xiao Zhu, Jessica Schmidt, Hongwei Yin, Quick Que, David Azorsa, Louise M. Perkins, et al. "Kinome-Wide RNAi Studies in Human Multiple Myeloma Identify a Lymphoid Restricted Kinase GRK6 as a Selectively Vulnerable Target That Regulates STAT3/MCL1." Blood 114, no. 22 (November 20, 2009): 601. http://dx.doi.org/10.1182/blood.v114.22.601.601.
Full textAbstract:
Abstract Abstract 601 A paucity of validated kinase targets in human multiple myeloma (MM) has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in MM tumor lines bearing common t(4;14), t(14;16) and t(11;14) translocations to identify critically vulnerable kinases in MM tumor cells without regard to preconceived mechanistic notions. Primary screening was performed in duplicate using an 1800-oligo siRNA library in a single-siRNA-per-well format. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. After 96 hours, viability was measured by ATP-dependent luminescence. Fifteen kinases were consistently vulnerable in MM cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. While several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly the G-protein coupled receptor kinase, GRK6, appeared selectively vulnerable in MM. GRK6 inhibition is selectively lethal to KMS11, OPM1, H929, KMS18 and OCI-MY5 myeloma cells and has minimal effect on 293, MCF7, SF767, A375 or A549 epithelial cells. Persistent expression of FLAG-GRK6 via cDNA rescued KMS11 cells from the lethal effect of a 3'UTR-targeted GRK6 siRNA, but not from control siRNA, validating identification of GRK6 as an essential myeloma survival kinase. Furthermore, concordant results were obtained using four different exon-based GRK6 siRNA, all of which induced GRK6 silencing and similar inhibition of KMS11 proliferation and viability. Significantly, GRK6 is ubiquitously expressed in lymphoid tissues and myeloma, but by comparison appears absent or only weakly expressed in most primary human somatic tissues. From co-immunoprecipitation experiments we demonstrate that GRK6 is highly expressed in myeloma cells via direct association with the HSP90 chaperone. Inhibition of HSP90 with geldanamycin blocks GRK6 protein expression. Importantly, direct GRK6 silencing causes rapid and selective suppression of STAT3 phosphorylation that is associated with sustained reductions in total MCL1 protein levels and MCL1 phosphorylation (within 24 hours), providing a potent mechanism for the cytotoxicity of GRK6 inhibition in MM tumor cells. GF109203X is an inhibitor of both protein kinase C and of GRK6 that causes near total inhibition of these kinases in vitro at distinct concentrations of 0.1μM and 1-10μM respectively. Notably, GF109203X was substantially cytotoxic to 10/14 myeloma tumor lines at concentrations most consistent with GRK6 inhibition (5-20μM), and was selectively more cytotoxic to myeloma tumor cells than to non-myeloma cell lines (P=0.01), highlighting the potential of GRK6 as a pharmaceutical target for selective therapeutic intervention in myeloma. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma. Disclosures: Perkins: MMRC: Employment. Reeder:Celgene: Research Funding; Millennium: Research Funding. Fonseca:Otsuka: Consultancy; BMS: Consultancy; Amgen: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy.
18
Andersen, Courtney L., Azadeh Cheraghchi-Bashi, Patricia Jaaks, Elizabeth A. Coker, Kathleen Burke, Justin Cidado, Paul Smith, et al. "Abstract 4024: Combining selumetinib with BH3 mimetics enhances activity in MAPK-activated acute myeloid leukemia." Cancer Research 82, no. 12_Supplement (June 15, 2022): 4024. http://dx.doi.org/10.1158/1538-7445.am2022-4024.
Full textAbstract:
Abstract Mitogen-activated protein kinase (MAPK) pathway alterations comprise some of the most frequent mutations in newly diagnosed acute myeloid leukemia (AML). Moreover, MAPK pathway alterations are also emerging as potential mechanisms of resistance to targeted therapy in AML including FLT3 inhibitors and venetoclax. In an ex vivo pharmacologic analysis of primary AML samples, sensitivity to the MEK inhibitor selumetinib (ARRY-142886) was enriched in patient samples resistant to venetoclax. Clinical activity of MAPK pathway inhibitors such as selumetinib has been explored in AML but monotherapy responses were modest. Another MEK inhibitor, cobimetinib, is currently being tested in combination with venetoclax in AML. We sought to understand whether combining selumetinib with BH3 mimetics such as venetoclax would improve efficacy in AML models. We evaluated combination activity of selumetinib plus venetoclax or the MCL1 inhibitor AZD5991 in a panel of AML cell lines. Cells were exposed to a 6x6 matrix of both agents for 72hrs and then viability assessed using CellTiter-Glo. Combination benefit was assessed using highest single agent (HSA) analysis. Selumetinib+AZD5991 demonstrated strong combination benefit (HSA score >0.1, Emax >0.5) in 4/19 AML cell lines. Selumetinib+venetoclax showed strong combination activity in 6/19 lines. Three lines showed benefit with both combinations. Many of these cell lines harbor MAPK pathway mutations including OCI-AML5 (SOS1N233Y, NF1K1385R), ML-2 (KRASA146T), HL-60 (NRASQ61L), and Nomo-1 (KRASG13D). Selumetinib treatment also led to robust BIM induction in vitro. Nomo-1 xenografts were evaluated for in vivo sensitivity to venetoclax (100 mg/kg qd PO), 5-azacytidine (1mg/kg BID q8h 3days on/4days off IP), AZD5991 (30mpk bid q2h qw IV), selumetinib (10 mg/kg bid q8h PO), as well as combinations of venetoclax+5-aza, AZD5991+selumetinib, and venetoclax+selumetinib. Venetoclax and venetoclax+5-aza treatment were ineffective. Selumetinib monotherapy led to 63% tumor growth inhibition (TGI) but tumors eventually grew out through treatment. The combination of selumetinib+venetoclax slightly improved efficacy (81% TGI) but markedly delayed tumor outgrowth (selumetinib monotherapy arm reached mean tumor volume >1000 mm3 on day 17, selumetinib+venetoclax reached average of ~966 mm3 on day 28). Reducing venetoclax dose (30mg/kg qd) or frequency (100mg/kg 3days on/4days off) maintained most of the combination efficacy. Selumetinb+AZD5991 also strongly improved efficacy compared to either monotherapy (88% TGI, mean tumor volume did not exceed ~400mm3 by day 28). Together these data suggest potential for combining MEK inhibitors with BH3 mimetics in AML. Work is ongoing to further understand how scheduling impacts combination efficacy, evaluate the triple combination of selumetinib+BH3 mimetic+azacytidine, and assess efficacy in disseminated models. Citation Format: Courtney L. Andersen, Azadeh Cheraghchi-Bashi, Patricia Jaaks, Elizabeth A. Coker, Kathleen Burke, Justin Cidado, Paul Smith, Corinne Reimer, Raoul Tibes, Mathew Garnett, Jerome Mettetal. Combining selumetinib with BH3 mimetics enhances activity in MAPK-activated acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4024.
19
Tibes, Raoul, Francis Giles, Teresa McQueen, Donald A. Bergstrom, Steven J. Freedman, and Michael Andreeff. "Translational In Vivo and In Vitro Studies in Patients (pts) with Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML) and Myeloproliferative Disease (MPD) Treated with MK-0457 (MK), a Novel Aurora Kinase, Flt3, JAK2, and Bcr-Abl Inhibitor." Blood 108, no. 11 (November 16, 2006): 1362. http://dx.doi.org/10.1182/blood.v108.11.1362.1362.
Full textAbstract:
Abstract Introduction: MK-0457 (VX-680) is a small molecule kinase inhibitor of the Aurora kinases A/B, wild type and mutant (T315I) Bcr-Abl, Flt3, and Jak2 (V617F) with preclinical activity in solid and hematological tumor models. We report correlative laboratory studies from pts with AML, CML and MPD enrolled in a Phase I/II study with MK-0457, on a 5-day continuous intravenous infusion (CIV), dose escalation schedule given at 2 to 3 week intervals. Methods: Specimens from 6 MPD (all Jak2 (V617F) positive), 6 CML pts in accelerated/blastic phase (3/6 positive for T315I) and 3 AML pts with at least two study time points were assessed for apoptosis (AnnexinV (AV)/PI by FACS) and cell cycle (PI) changes in vivo. A CD34+ progenitor sub-population was studied. PBMC isolated prior to therapy were cultured in vitro with MK-0457, either alone or in co-culture with human, BM-derived mesenchymal stem cells (MSC). Concentration and time dependent apoptosis and cell cycle changes were determined. Results: At day 5, there was no significant induction of apoptosis in 5/6 MPD pts compared to baseline (BL) (mean for total cell population 37 vs 27%; gated myeloid subpopulation 37 vs 38%). All pts had a hematological response and, except one, a slight decrease in Jak2 (V617F) transcript levels. In vivo, CML pts showed a heterogeneous response. 3/5 pts had an increase (7, 98, 103 %) in AV/PI positive cells and 2/5 pts a decrease (−28, −74%) at day 5, one of which in lymphoid BC, exhibited delayed massive apoptosis at day 11 (470% increase from BL). The other pt without evidence of apoptosis induction achieved a hematological response with disappearance of the T315I clone, relapsing with high Bcr-Abl transcript levels thereafter. A pt with partial cytogenetic and molecular response had high BL apoptosis (50%) that remained high (58%) at 90 days when taken off study due to side effects (muscle cramps/pain). In vitro, a dose dependent rise in specific apoptosis was seen in MPD cells between 48–144 hrs, reaching statistical significance at 50–400 nM MK (p=0.017−0.008, two tailed t-test, unequal variance) and in CML cells at 24, 48, 72 hrs (mean increase at 400nM MK of 17, 14, 28%, respectively in CML, p≤0.05 for mean of all time points). In AML, 2/3 pts had increased numbers of AV/PI positive cells on day 5, declining to BL levels on day 11 in one pt. In vitro, AML specimens (from non-study pts) showed less pronounced apoptosis than MPD and CML cells, with similar effects in CD34 cells. Co-incubation with MSC protected cells of AML and MPD pts at all drug concentrations, reaching significance at ≥100nM in MPD pts (p=0.03). In cell line experiments, HL-60 and OCI-AML cells showed striking G2/M block, polyploidization and apoptosis. Conclusion: In preliminary experiments, MK-0457 induces apoptosis in AML and CML pts in vivo, shows a time and dose dependent increase of apoptosis in vitro in MPD, CML and AML pts cells and leads to accumulation in G2/M phase. Co-culture with human MSC protect cells from MPD and AML pts in vitro. The clinical trial is ongoing and molecular profiling studies are underway.
20
Teh, Tse-Chieh, Nhu-Y. Nguyen, Stefan Glaser, Donia Moujalled, Giovanna Pomilio, David CS Huang, Mark Guthridge, and Andrew H. Wei. "Eradication of Acute Myeloid Leukemia Is Enhanced By Combined Bcl-2 and Mcl-1 Targeting." Blood 124, no. 21 (December 6, 2014): 988. http://dx.doi.org/10.1182/blood.v124.21.988.988.
Full textAbstract:
Abstract Toxicity and drug resistance has impeded clinical improvements in acute myeloid leukaemia (AML). We hypothesized that directly targeting pro-survival proteins with BH3-mimetics may have therapeutic rationale in AML and potentially sensitize chemoresistant cases to chemotherapy. This work will therefore assess which Bcl-2 family members mediate pro-survival activity in AML and the potential benefit of combining BH3-mimetics with standard AML drugs. Immunoblotting of fresh primary AML samples revealed strong Bcl-2 expression in 12/13 (92%) cases, Bcl-xL expression in 3/13 (23%) cases and Mcl-1 expression in 8/13 cases (62%). Sensitivity to ABT-199 (targets Bcl-2) or ABT-737 (targets Bcl-2, -xL, -w) was assessed by incubation of freshly harvested AML samples in RPMI + 20% FBS for 48 hrs and viable cells enumerated by exclusion of Sytox Blue using flow cytometry. There were 2 discrete populations identified; one that was sensitive to ABT-199 with an LC50 (concentration of drug to kill 50% of AML blasts) < 10nM (5/13 cases; 39%) and a more resistant population (LC50 > 10μM in 6/13 cases, 46%). A strong correlation in the sensitivity of AML cells to ABT-199 and ABT-737 was observed (r=0.9628, p < 0.0001), suggesting Bcl-xL was not a dominant survival factor in AML. To identify relevant Bcl-2 pro-survival targets for elimination of AML in vivo, MV4;11 cells were engineered to express under doxycycline control 1) BimSwt targeting all pro-survival proteins, 2) BimS2A targeting Mcl-1 only, 3) BimSBad targeting Bcl-2, -x and –w; or 4) an inert BimS4E, which served as a negative control. 1x 105 MV4;11 cells per mouse were xenografted into cohorts of 6 NSG mice and doxycycline-rich water introduced on day 5 after transplantation. Mice transplanted with MV4;11 cells expressing the inert BimS4E succumbed to leukemia by ~ day 40. Expression of BimSBad or BimS2A delayed, but did not prevent leukemia-related death, whereas mice engrafted with MV4;11 cells enforced to express BimSwt were alive and leukemia-free after 100 days (Figure 1A). Similar outcomes were demonstrated using an OCI-AML xenograft model (not shown). As on-target toxicity to platelets is an undesirable consequence of targeting Bcl-xL (Mason, Cell 2006), the effectiveness of targeting only Bcl-2 and Mcl-1 in vivo was next investigated. Mice engrafted with MV4;11 cells expressing BimS2A (to target Mcl-1) were fed doxycycline water from day 5 onwards. In addition, the selective Bcl-2 inhibitor ABT-199 75mg/kg was administered daily by oral gavage between days 5-12 post engraftment. Strikingly, this combination strategy led to eradication of AML by day 100 in all mice, in contrast to delayed, but inevitable leukemic death in most mice receiving treatment directed at either Bcl-2 (ABT-199) or Mcl-1 (Bims2A) alone (Figure 1B). These results suggest that AML eradication in vivo is best achieved through combined targeting of Bcl-2 and Mcl-1. In the absence of direct inhibitors of Mcl-1, standard AML drugs were examined for their capacity to suppress Mcl-1. In the MV4;11 cell line, anthracyclines such as idarubicin rapidly (< 3 hrs) suppressed Mcl-1 at concentrations between 300nM and 1μM. In contrast, 100μM cytarabine did not suppress Mcl-1 after exposure for 6 hrs. Further studies in primary AML cells demonstrated synergy between ABT-199 and idarubicin in ABT-199 resistant AML cells. We conclude that 1) a subset of primary AML cells is highly sensitive to Bcl-2 inhibition, 2) that eradication of AML in vivo is best through combined Bcl-2/Mcl-1 targeting and 3) that anthracyclines are effective at suppressing Mcl-1 and could be combined with ABT-199 to treat patients with AML resistant to Bcl-2 targeted therapy. A Figure 1 A) Cohorts of 6 NSG mice were xenografted with MV4;11 cells expressing BimS ligand variants under doxycycline control. Doxycycline water was fed to mice from day 5+ and survival assessed. B. Cohorts of 6 NSG mice were xenografted with MV4;11 cells expressing BimS2A under doxycycline control. Mice were allocated to treatment with either vehicle or ABT-199 on days 5-12 +/- doxycycline water from day 5+ and survival assessed. All mice in the arm combining Doxycycline (to suppress Mcl-1) and ABT-199 (to target Bcl-2) were alive and leukemia-free on day 100. Figure 1. A) Cohorts of 6 NSG mice were xenografted with MV4;11 cells expressing BimS ligand variants under doxycycline control. Doxycycline water was fed to mice from day 5+ and survival assessed. B. Cohorts of 6 NSG mice were xenografted with MV4;11 cells expressing BimS2A under doxycycline control. Mice were allocated to treatment with either vehicle or ABT-199 on days 5-12 +/- doxycycline water from day 5+ and survival assessed. All mice in the arm combining Doxycycline (to suppress Mcl-1) and ABT-199 (to target Bcl-2) were alive and leukemia-free on day 100. B Figure 2 Figure 2. Disclosures Huang: AbbVie: Consultancy, Honoraria. Wei:Abbvie: Consultancy, Honoraria.
21
Skrtic, Marko, Shrivani Sriskanthadevan, Bozhena Livak, Marinella Gebbia, Xiaoming Wang, Zezhou Wang, Rose Hurren, et al. "Inhibition of Mitochondrial Translation As a Therapeutic Strategy for Acute Myeloid Leukemia (AML)." Blood 118, no. 21 (November 18, 2011): 233. http://dx.doi.org/10.1182/blood.v118.21.233.233.
Full textAbstract:
Abstract Abstract 233 To identify novel therapeutic strategies that can eliminate AML and AML stem cells, we screened a library of on and off-patent drugs for candidates that could reduce the viability of engineered human AML cell lines that display the stem cell properties of differentiation, self-renewal, and marrow repopulation. This screen identified the anti-microbial agent tigecycline (TIG) as a top candidate with an LD50 of 3 to 8 uM, on 5 human AML cell lines. A lethal action was also demonstrated on 13 of 20 1°AML samples with similar potency (LD50 <5 uM). In contrast, normal hematopoietic cells, including the CD34+ subset, were more resistant (LD50 >10 uM). We also found that 5 mM TIG reduced the clonogenic growth of 1°AML samples by 93±4% and was effective in reducing the ability of AML cells to regenerate disease in transplanted immunodeficient mice. In contrast, 5 uM TIG had no effect on the clonogenic growth or repopulating potential of normal human hematopoietic cells. To determine the mechanism of action of TIG, we used Haplo-Insufficiency Profiling, a functional chemical genomic screen, in S. cerevisiae. The Gene Ontology component that was the most enriched for TIG was the mitochondrial ribosome. We subsequently demonstrated that TIG inhibited mitochondrial but not cytoplasmic translation in AML cell lines and in 1°AML samples. Consistent with the inhibition of mitochondrial translation, TIG decreased the enzyme activity of Complex I and IV, which contain mitochondrially-translated subunits, but not complex II (nuclear-encoded subunits only). TIG also decreased oxygen consumption and decreased mitochondrial-membrane potential in AML cell lines and 1°AML samples, but not normal hematopoietic cells. Interestingly, unlike many mitochondrial inhibitors, TIG did not increase ROS production in AML cells. Additional experiments demonstrated that inhibition of mitochondrial translation was functionally important for the anti-leukemia activity of TIG. Next, we asked whether genetic strategies in leukemia cells would produce similar anti-leukemic effects as obtained with TIG. Knockdown of the mitochondrial-elongation factor EF-Tu mimicked the ability of TIG to inhibit mitochondrial translation, decrease mitochondrial membrane potential, decrease complex I and IV activity and induce cell death in AML cells. Also, EF-Tu knockdown did not increase ROS production. To investigate the basis of the hypersensitivity of AML cells to mitochondrial translation inhibition, we assessed baseline mitochondrial characteristics of 1°AML cells and their normal counterparts. 1°AML cells (including CD34+CD38- AML cells) had higher intrinsic mitochondrial-biogenesis (mtDNA copy number, mitochondrial mass) than normal CD34+ hematopoietic cells. Furthermore, rates of oxygen consumption were higher in 1°AML cells as compared to normal hematopoietic cells. Baseline mitochondrial-mass in AML cells also predicted in vitro toxicity to TIG, as 1° AML cells with higher mitochondrial mass were more sensitive to TIG (r = −0.71, p <0.05). To assess the anti-leukemia efficacy of mitochondrial translation inhibition in vivo, we investigated human AML cells in mouse xenograft models. TIG significantly delayed tumor growth of OCI-AML2 xenografts in SCID compared to untreated control mice. We then assessed the effect of TIG on AML stem cells defined by their ability to sustain leukemic cell growth in vivo. NOD/SCID mice engrafted with human AML cells and then treated with TIG showed a decrease in human AML cells by up to 77% without toxicity including alterations in liver and muscle enzymes. In contrast, NOD/SCID mice engrafted with normal cord blood did not show reduced engraftment after TIG treatment. Importantly, the human AML cells harvested from the bone marrow of the TIG-treated 1° mice generated fewer leukemic cells in secondary mice, compared to the AML cells harvested from control (untreated) primary mice, thus demonstrating an in vivo effect on the AML stem cells. In conclusion, mitochondrial translation inhibition selectively kills AML vs. normal cells, including those defined functionally as AML progenitors and stem cells. This selectivity appears attributable to the higher rate of mitochondrial biogenesis found in AML cells. Given these results and the known pharmacology and toxicology of TIG in humans, targeting mitochondrial translation inhibition as a therapeutic strategy in AML is attractive. Disclosures: Off Label Use: Tigecycline is currently used as an a broad spectrum antibiotic, and is here discussed as an AML agent.
22
Burda, Pavel, Nikola Curik, Nina Dusilkova, Giorgio L. Papadopoulos, John Strouboulis, Anna T. Jonasova, and Tomas Stopka. "Erythroid Transcription Factor GATA-1 Binds and Represses PU.1 Gene – Candidate Mechanism Of Epigenetic Repression Of PU.1 and Inefficient Erythropoiesis In MDS." Blood 122, no. 21 (November 15, 2013): 1558. http://dx.doi.org/10.1182/blood.v122.21.1558.1558.
Full textAbstract:
Abstract Introduction Myelodysplastic syndrome (MDS) is often manifested by anemia due to ineffective erythropoiesis. Upon transformation to MDS/AML the uniform population of leukemic blasts overgrow dysplastic bone marrow. Hematopoiesis is regulated by transcription factors GATA-1 and PU.1 that interact and mutually inhibit each other in progenitor cells to guide multilineage commitment and subsequent lineage differentiation. Expression of PU.1 is controlled by several transcription factors including PU.1 itself at distal URE enhancer. It has been well established that underexpression of PU.1 in progenitor cells leads to AML (Rosenbauer F et al. 2004). In addition, co-expression of PU.1 and GATA-1 in AML-erythroleukemia (EL) blasts prevents induction of differentiation programs regulated by these transcription factors. In our laboratory, we recently observed that MDS/AML erythroblasts display repressive histone modifications and methylation status of PU.1 gene that respond to 5-azacitidine leading to inhibited blast cell proliferation and stimulated myeloid differentiation (Curik N et al. 2012). Inhibition of transcriptional activity of PU.1 protein by GATA-1 has been reported (Nerlov C et al. 2000) however it is not known whether GATA-1 can inhibit PU.1 gene in human early erythroblasts directly. Hypothesis GATA-1 inhibits PU.1 levels directly and modulates its transcriptional outcome in early erythroblasts. We also hypothesize that GATA-1-mediated repression of PU.1 transcription is delayed and this may play a role in ineffective erythropoiesis. Material and Methods Cell lines: MDS-derived OCI-M2 EL and other two human ELs (HEL, K562) and one murine EL (MEL); all co-expressing GATA-1 and PU.1. Patients: MDS patients (N=5) with rather advanced disease; MDS/AML (4) and RAEBI (1). Four received AZA; response: PR (2), SD (2) with HI. Median OS>24 Mo. For chromatin immunoprecipitation (ChIP) analysis either cell lines or CD19/CD3-depleted bone marrow cells were used. Results Direct association of GATA-1 with PU.1 gene was demonstrated in all three human ELs using ChIP. Occupancy of GATA-1 was detected upstream the PU.1 promoter and distally at GATA-1 binding sites or at PU.1 binding sites together with PU.1. Comparable data documenting occupancy of GATA-1 at PU.1 gene were observed also in MEL cells and in normal murine fetal erythroblasts using ChIP-sequencing. To test how GATA-1 regulates PU.1 expression we overexpressed GATA-1 in erythroblasts and tested expression of PU.1, histone H3 modification (near GATA-1 occupancy) and cell growth. We found that GATA-1 inhibited PU.1 expression, facilitated enrichment of repressive modifications at PU.1 gene (H3K9Me, H3K27Me) while depleted activation modifications (H3K9Ac, H3K4Me), and also inhibited cell growth. Next, we tested effects of GATA-1 knockdown using siRNA. Indeed, inhibition of GATA-1 expression in erythroblasts leads to increase in PU.1 level as well as of its targets (CEBPA, MAC1). Using Luciferase assay we confirmed that both endogenously produced PU.1 and GATA-1 are capable to stimulate exogenously inserted reporters. Next, we compared chromatin structure of PU.1 gene between data from ELs, normal controls and high risk MDS. Our data revealed that PU.1 gene in MDS is enriched with repressive modifications (H3K9Me, H3K27Me) while depleted with activation modifications (H3K9Ac, H3K4Me) suggesting defects in dynamic regulation of PU.1 expression in MDS. Conclusion Our data from ELs provide a) evidence of GATA-1-mediated repression of PU.1 gene in erythroblasts and that b) manipulation of GATA-1 affected PU.1 level in opposite direction. In high risk MDS, the chromatin structure of PU.1 gene displays accumulation of repressive epigenetic marks that are responsive to AZA. We think that during early erythroid differentiation GATA-1 binds and represses PU.1 gene, however this is not fully completed in MDS and therefore erythroid differentiation is not efficient. Grants: P301/12/P380, P305/12/1033, NT14174-3/2013, UNCE204021, FR-TI2/509, SVV-2013-266509, PRVOUK-P24/LF1/3 Disclosures: No relevant conflicts of interest to declare.
23
Gouni, Sushanth, Paolo Strati, Jason Westin, Loretta J. Nastoupil, Raphael E. Steiner, Ranjit Nair, Luis Fayad, et al. "Use of Medium Potency Statins Is Associated with Improved Outcomes after Frontline RCHOP in Patients with Diffuse Large B-Cell Lymphoma (DLBCL)." Blood 136, Supplement 1 (November 5, 2020): 45. http://dx.doi.org/10.1182/blood-2020-136010.
Full textAbstract:
Background: Pre-clinical studies show that statins may improve the efficacy of chemoimmunotherapy in patients with DLBCL, through interference with cell membrane-initiated signaling pathways. Clinical retrospective studies, however, yield conflicting data, due to heterogeneous properties of statins, including potency and hydrophilicity. Methods: This is a retrospective analysis of patients with previously untreated, advanced stage DLBCL, non-double hit, treated with frontline R-CHOP between 01/01/2000 and 09/01/2019 (data cut-off 04/15/2020) at MD Anderson Cancer Center, and for whom data regarding statin use at time of initiation of treatment were available. Lugano 2014 response criteria were applied retrospectively for response assessment. Cellular cholesterol levels were analyzed in 6 DLBCL cell lines using an Amplex red fluorometric assay. A doxorubicin (DXR)-resistant cell line was generated exposing SUDHL4 cells to escalating doses of DXR; a DXR-resistant DLBCL patient-derived xenograft (PDX) model was established through serial transplantation and exposure to DXR. Results: 271 patients were included in the analysis, 182 (67%) were older than 60 years, 134 (49%) were male, 212 (72%) had stage IV disease, and 217 (80%) had an IPI score > 3; upon pathological review, 38 (36%) cases were non-GCB type, and 18 (28%) were double-expressors; 214 (79%) were able to complete all planned 6 cycles of RCHOP. Seventy-nine (29%) patients received statins at time of initiation of chemoimmunotherapy: 15 patients received low potency statin, 51 medium and 13 high; 18 patients received hydrophilic statins and 61 lipophilic. Patients receiving statins were significantly older as compared to patients who did not (p<0.001); no other significant difference in baseline characteristics was observed when comparing the 2 groups. Overall, 265 out of 271 patients were evaluable for response, as 6 stopped treatment because of toxicity before first response assessment. Among these, ORR was 95% (252/265) and CR rate was 62% (165/265). ORR rate was identical in patients who were treated with statin and those who did not (95% both, p=1). After a median follow-up of 77 months (95% CI, 70-84 months), 119 patients progressed/died, median PFS was not reached and 6-year PFS was 57%. 6-year PFS rate according to statin intensity was: 48% (low), 72% (medium), 57% (high). PFS. 6-year PFS rate was 64% for hydrophilic and 72% for lipophilic statins. Patients treated with statins had a trend for longer PFS (p=0.06), significantly longer for patients receiving medium potency statins (p=0.04). No significant difference in PFS was observed when comparing patients treated with lipophilic statins to all others (not reached vs 84 months, p=0.22). To confirm these clinical data, in-vitro and in-vivo studies were performed. Six cell lines were tested: 4 with high cholesterol content (SUDHL4, HBL1, HT, and U2932; 5.0-8.0 µg/mg protein), and 2 with low cholesterol content (DOHH2 and OCI-LY19; 1.5-2.0 µg/mg protein); the latter showed the highest sensitivity to DXR-mediated killing. The combination of lovastatin and DXR (10nM) was tested in all 4 cell lines with high cholesterol content, resulting in more cell death than either treatment alone. Lovastatin (at the nanomolar range) resensitized DXR-resistant SUDHL4 cells to DXR. Finally, in a DXR-resistant PDX model, the combination of lovastatin and DXR resulted in delayed tumor growth as compared to chemotherapy alone. Conclusions: Use of medium potency statins is associated with improved outcomes after frontline RCHOP in patients with DLBCL. This was further confirmed in functional in-vitro and in-vivo studies. Future interventional studies, aimed at improving outcomes in these patients using this novel combination, are warranted. Disclosures Westin: Amgen: Consultancy; 47: Research Funding; Kite: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Morphosys: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Curis: Consultancy, Research Funding; Astra Zeneca: Consultancy, Research Funding. Nastoupil:Gamida Cell: Honoraria; Merck: Research Funding; TG Therapeutics: Honoraria, Research Funding; Karus Therapeutics: Research Funding; Janssen: Honoraria, Research Funding; LAM Therapeutics: Research Funding; Novartis: Honoraria, Research Funding; Bayer: Honoraria; Celgene: Honoraria, Research Funding; Genentech, Inc.: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Gilead/KITE: Honoraria. Neelapu:Bristol-Myers Squibb: Other: personal fees, Research Funding; Merck: Other: personal fees, Research Funding; Kite, a Gilead Company: Other: personal fees, Research Funding; Pfizer: Other: personal fees; Celgene: Other: personal fees, Research Funding; Novartis: Other: personal fees; Karus Therapeutics: Research Funding; N/A: Other; Takeda Pharmaceuticals: Patents & Royalties; Acerta: Research Funding; Cellectis: Research Funding; Poseida: Research Funding; Precision Biosciences: Other: personal fees, Research Funding; Legend Biotech: Other; Adicet Bio: Other; Allogene Therapeutics: Other: personal fees, Research Funding; Cell Medica/Kuur: Other: personal fees; Calibr: Other; Incyte: Other: personal fees; Unum Therapeutics: Other, Research Funding. Landgraf:NCI/NIH: Research Funding. Vega:NCI: Research Funding.
24
Lam, Vi, Xiaoguang Wang, Scott R. Best, Nur Bruss, Tingting Liu, Olga Danilova, Tanya Siddiqi, Nathan Pennock, and Alexey V. Danilov. "Pharmacologic Inhibition of SUMO-Activating Enzyme Potentiates Interferon Response and T Cell-Mediated Anti-Tumor Immunity in Chronic Lymphocytic Leukemia (CLL) and Lymphoma Models." Blood 138, Supplement 1 (November 5, 2021): 3719. http://dx.doi.org/10.1182/blood-2021-146347.
Full textAbstract:
Abstract Introduction: CLL is characterized by deficient immunity which clinically manifests as increased predisposition towards malignancies and infectious complications. T-cells from patients with CLL exhibit a skewed repertoire with predominance of Tregs as well as impaired immune synapse formation and cytotoxic function. Small ubiquitin-like modifier (SUMO) family proteins regulate a variety of cellular processes, including nuclear trafficking, gene transcription and cell cycle progression, via post-translational modification of target proteins. Sumoylation regulates NFκB signaling, IFN response and NFAT activation, processes indispensable in immune cell activation. Despite this, the role of sumoylation in T cell biology in context of cancer is not known. TAK-981 is a small molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein, thereby preventing its transfer to the SUMO-conjugating enzyme (Ubc9). Here, we investigated the immunomodulatory effects of TAK-981 in CLL. Methods: T cells from patients with CLL were purified using Dynabeads. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for 7 days in control or differentiation media. For gene expression profiling (GEP; Clariom S), RNA was harvested after 3 and 24 hours of TCR engagement from FACS-sorted naïve CD4+ T cells. For in vivo immunization experiments, CD4+KJ1-26+ cells were inoculated IV into BALB/cJ mice. Mice received 100 µg IV ovalbumin ± R848 followed by TAK-981 7.5 mg/kg or vehicle control IV twice weekly for 10 days prior to spleen collection. Both recipient and transplanted splenocytes were analyzed. For analysis of tumor-infiltrating lymphocytes (TILs), BALB/c mice were injected with 1x10 6 A20 lymphoma cells and treated as above. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA). Results: T cells from patients with CLL demonstrated high baseline protein sumoylation that slightly increased following TCR engagement (αCD3/CD28). Treatment with TAK-981 significantly downregulated SUMO1 and SUMO2/3-modified protein levels yet did not disrupt early TCR signaling as evidenced by sustained ZAP70, p65/NFκB and NFAT activation detected by immunoblotting, immunocytochemistry and GEP. Treatment with TAK-981 resulted in dose-dependent upregulation of the early activation marker CD69 in CD4 + T cells following 72 and 96 hours of TCR stimulation vs. control. Meanwhile, expression of CD25, HLA-DR and CD40L was delayed in the presence of TAK-981. Interestingly, CD38, an IFN response target, was induced two-fold in TAK-981-treated cells after 24 hours and persisted at high levels at subsequent timepoints. T cell proliferation was reduced in the presence of high (1 μM) but not low/intermediate concentrations of TAK-981, accompanied by reduced S phase entry and decreased synthesis of IL-2. However, T cells did not undergo apoptosis under those conditions. Targeting SAE in either control or Th1/Treg polarizing conditions facilitated an increase in IFNγ and loss of FoxP3 expression (accompanied by decreased IL-2/STAT5), suggesting a shift towards Th1 and away from Treg phenotype, respectively. GEP (Reactome, GSEA) confirmed a dramatically upregulated IFN response in TAK-981-treated CD4 + naïve T cells. Furthermore, targeting SAE enhanced degranulation (CD107a), IFNγ and perforin secretion in cytotoxic CD8+ T cells and potentiated T cell cytotoxicity in allogeneic assays with lymphoma cells (OCI-LY3, U2932) as targets. Consistent with our in vitro data, OVA-stimulated transplanted transgenic KJ1-26+ splenocytes, as well as total CD4+ T cells from recipient mice treated with TAK-981 in vivo exhibited a significant reduction in expression of FoxP3 and an increased production of IFNγ (Figure 1). In the A20 syngeneic model, treatment with TAK-981 similarly downregulated FoxP3 expression in CD4+ TILs and induced IFNγ secretion in CD8+ TILs. Conclusion. Using a combination of in vitro and in vivo experiments, we demonstrate that pharmacologic targeting of sumoylation with TAK-981 does not impair proximal TCR signaling in T cells obtained from patients with CLL, but leads to rebalancing toward healthy immune T cell subsets via induction of IFN response and downmodulation of Tregs. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies. Figure 1 Figure 1. Disclosures Siddiqi: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; TG Therapeutics: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncternal: Research Funding; Janssen: Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Danilov: SecuraBio: Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy, Research Funding; Rigel Pharm: Honoraria; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding.
25
Bhattacharya, Seemana, Sujan Piya, Qi Zhang, Natalia Baran, Teresa McQueen, R. Eric Davis, Nicholas Cosford, Marina Y. Konopleva, Michael Andreeff, and Gautam Borthakur. "Targeting Autophagy Kinase ULK1 Can Reverse Bcl2 Inhibitor (ABT-199) Induced Autophagy to Overcome Acquired Resistance in Acute Myeloid Leukemia." Blood 132, Supplement 1 (November 29, 2018): 3934. http://dx.doi.org/10.1182/blood-2018-99-114817.
Full textAbstract:
Abstract Introduction Anti-apoptotic Bcl2 family members mediate resistance to therapies in acute myeloid leukemia (AML)1. The small molecule Bcl2 inhibitor ABT-199 (venetoclax) promotes mitochondria driven intrinsic apoptosis, and in combination with hypomethylating agents or chemotherapy, has been highly promising in the clinic as treatment of AML2-4. The response rate to ABT-199 is very impressive, but acquired resistance is a major problem. Compensatory upregulation of Mcl1 is an important mechanism of such acquired resistance to mitochondrial apoptosis5. Autophagy is vital for mitochondrial health, mediates resistance to apoptosis and is induced by Bcl2 inhibition6. We performed mechanistic studies to address our hypothesis that disabling autophagy by targeting the apical autophagy kinase ULK1 can reverse resistance to ABT-199. Methods ULK1 was genetically modified in OCIAML3 (human AML cell line), by shRNA knockdown (KD) or CRISPR-Cas9 knockout (KO). In addition, AML cell lines (including ABT-199 resistant) and patient samples were treated with ABT-199 and ULK1 inhibitor SBI-02069657. Combination index (CI) for drug synergy was calculated based on Chou-Talalay method8. Drug-treated or genetically manipulated cells were profiled by reverse phase protein array (RPPA), mass cytometry (CyTOF) and gene expression profiling (GEP). Autophagy was detected by LC3 quantification by western blot (WB) and flow cytometry, and monodansylcadaverine assay. Mitochondrial functions were analyzed by Seahorse Cell Mito Stress test, and MTG, TMRE and ROS assays (flow cytometry). For in vivo studies ULK1 KO and corresponding control cells were injected in NSG mice and monitored by bioluminescent imaging (BLI) and quantification of human CD45 cells. Results ABT-199 induced autophagy in OCIAML3 (increase by 175±27%, p=0.01 - LC3 flow; 4X increase in LC3 II/I ratio - WB). Apoptosis induction by ABT-199 was enhanced by ULK1 KD (36±1.9% over control, p<0.01) or KO (77±1.3%, p<0.01). ULK1 inhibitor SBI-0206965 demonstrated high synergy with ABT-199 at inducing apoptosis in OCIAML3 (CI = 0.51, p<0.001) and MOLM13 (CI = 0.24, p<0.001). The combination was also effective in eliminating bulk and CD34+ stem/progenitor cells in primary AML samples (CI - Bulk: 0.69; CD34: 0.74; p<0.05) (Fig 1). Mcl1 was significantly downregulated by ULK1 inhibitor alone and in combination with ABT-199. ULK1 inhibition lowered Mcl1 transcription, as measured by qRT-PCR: 43±0.03% with SBI-0206965 and 63±0.3% in KO cells (both p<0.01). SBI-0206965 inhibited Mcl1 transcription regulators Stat3 and Erk1/2 and enhanced DNA damage in combination with ABT-199 (WB) (Fig 2). Since ABT-199 modulates mitochondrial function, we examined the effect of inhibiting ULK1 in this context. By Seahorse assay, the combination decreased basal OCR and ATP production by 62 and 58% respectively, p<0.01. This was accompanied by an increase in membrane depolarization (TMRE change - OCIAML3: 62%, MOLM13: 82%; p<0.01) and mitochondrial ROS generation (62% increase, p=0.01) compared to control cells (Fig 3). Interestingly, ULK1 inhibition increased mitochondrial mass (30%, p=0.012) by MTG assay, which may be due to impaired mitophagy. Thus, the combination impairs mitochondrial metabolism and function, which results in the observed increase in ROS that may account for the observed DNA damage and apoptosis. CD44/44v is critical for mitigating ROS through reduced glutathione (GSH)9, and ULK1 inhibition lowered CD44/44v transcription (qRT-PCR) and intracellular GSH in AML cells. Corroborating our earlier data, the ABT-199 resistant cells (OCIAML2R & MOLM13R) show enhanced autophagy as compared to parental cells (OCIAML2: 83%, MOLM13: 35% increase; p=0.001 & 0.009). SBI-0206965 reversed ABT-199 induced autophagy and restored ABT-199 sensitivity in these cells (Fig 4). In a pilot in vivo experiment control and ULK1 KO cells were injected in NSG mice and leukemia engraftment was markedly delayed in the ULK1 KO group (Fig 5). The therapeutic combination study is ongoing. Conclusion Results indicate concomitant targeting of autophagy by ULK1 inhibition and Bcl2 inhibition by ABT-199 can overcome acquired resistance to ABT-199. Hence, with the emergence of Bcl2 inhibitors in frontline therapy for AML and efforts at developing ULK1 inhibitors, this study informs the development of novel apoptosis/autophagy targeting approaches to improve AML therapy. Disclosures Konopleva: Stemline Therapeutics: Research Funding; abbvie: Research Funding; Immunogen: Research Funding; cellectis: Research Funding. Andreeff:Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Jazz Pharma: Consultancy; SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Celgene: Consultancy; Reata: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding.
26
Tian, Miao, Qing Li, Yang Liu, Peng Zheng, Danyu Li, Yanpeng Zhao, Bing Wang, et al. "Relationship between delayed luminescence emission and mitochondrial status in Saccharomyces cerevisiae." Scientific Reports 12, no. 1 (January 10, 2022). http://dx.doi.org/10.1038/s41598-021-04290-9.
Full textAbstract:
AbstractDelayed luminescence (DL) is gradually used in various detection of biological systems as a rapid detection technique, however, its biological mechanism was still not clear. In this study, a new model of DL detection system for liquid biological samples is established to investigate the DL emission of Saccharomyces cerevisiae cells cultured in different glucose concentrations. We analyzed the relationship between the DL emission and cell growth, cell vitality, mitochondrial morphology, mitochondrial DNA (mtDNA) copy number, adenosine triphosphate (ATP), oxygen consumption rate (OCR), as well as mitochondria membrane potential (MMP) in S. cerevisiae cells cultured with 0.01, 0.05, 0.15, 3, 10 and 20 g/L glucose respectively. It was found that the DL emission had strong correlation with mitochondrial morphology, OCR, and MMP. The results suggested that DL is an indicator of mitochondria status under different glucose supply conditions, and may be an effective method to detect mitochondrial metabolism related disorders.
27
Toli, Jessica M., Minzhen He, Carolyn Suzuki, and Maha Abdellatif. "Abstract 331: Recovery of Mitochondrial Bioenergetics after Hypoxia via Regulating Pyruvate Dehydrogenase Kinase and Adenosine Monophosphate-activated Kinase." Circulation Research 115, suppl_1 (July 18, 2014). http://dx.doi.org/10.1161/res.115.suppl_1.331.
Full textAbstract:
Mitochondrial quality control is critical for the survival of cardiac myocytes during stress. The purpose of this study was to examine the effect of metabolic substrates and regulators of metabolism on mitochondrial bioenergetics, as an indicator of mitochondrial quality, and how these factors might influence the recovery of the cell’s bioenergetics after hypoxia/ischemia. By monitoring oxygen consumption rates (OCR), in real-time, in live neonatal rat myocytes and human cardiac myocyte-differentiated induced pluripotent stem cells, we found that both cell types can maintain basal OCR efficiently with any metabolic substrate; however, the neonatal cells require both glucose and fatty acid, while the human adult cells require fatty acid only, for mounting maximum reserve respiratory capacity (RRC). Our data also show that subjecting cardiac myocytes to hypoxia results in a reduction of the cells’ basal OCR and oxidative phosphorylation, and exhausts the RRC, which is accompanied by an increase in pyruvate dehydrogenase kinase (Pdk) 1 and 4. Except for normalization of Pdk1 levels, there was little or no recovery of these parameters after reoxygenation. We, thus, hypothesized, that inhibition of Pdks may help recovery of the cell’s bioenergetics. Indeed, our results show that by inhibiting Pdks with dichloroacetate (DCA) before or after hypoxia, the cells’ bioenergetics, including OCR, oxidative phosphorylation, and RRC in neonatal myocytes, and RRC in the human myocytes fully recover within 24 h. On the other hand, activating AMP-activated kinase (AMPK) resulted in delayed (96 h) improvement of the cells’ RRC that was accompanied by an increase in peroxisome proliferator-activated receptor gamma, coactivator 1α (3.5x), peroxisome proliferator-activated receptor-α (2x), and mitochondrial number (2x). These results led us to conclude that compromised mitochondrial quality can be rescued through mechanisms that regulate glucose or fatty acid oxidation by either inhibiting Pdks or activating AMPK, respectively, in rodent and human myocytes.
28
Burkert, Karsten, Hadiseh Taheri, Sarkawt Hamad, Matteo Oliverio, Gabriel Peinkofer, Jan-Wilhelm Kornfeld, Wacharee Harnying, et al. "Salicylic diamines selectively eliminate residual undifferentiated cells from pluripotent stem cell-derived cardiomyocyte preparations." Scientific Reports 11, no. 1 (January 27, 2021). http://dx.doi.org/10.1038/s41598-021-81351-z.
Full textAbstract:
AbstractClinical translation of pluripotent stem cell (PSC) derivatives is hindered by the tumorigenic risk from residual undifferentiated cells. Here, we identified salicylic diamines as potent agents exhibiting toxicity to murine and human PSCs but not to cardiomyocytes (CMs) derived from them. Half maximal inhibitory concentrations (IC50) of small molecules SM2 and SM6 were, respectively, 9- and 18-fold higher for human than murine PSCs, while the IC50 of SM8 was comparable for both PSC groups. Treatment of murine embryoid bodies in suspension differentiation cultures with the most effective small molecule SM6 significantly reduced PSC and non-PSC contamination and enriched CM populations that would otherwise be eliminated in genetic selection approaches. All tested salicylic diamines exerted their toxicity by inhibiting the oxygen consumption rate (OCR) in PSCs. No or only minimal and reversible effects on OCR, sarcomeric integrity, DNA stability, apoptosis rate, ROS levels or beating frequency were observed in PSC-CMs, although effects on human PSC-CMs seemed to be more deleterious at higher SM-concentrations. Teratoma formation from SM6-treated murine PSC-CMs was abolished or delayed compared to untreated cells. We conclude that salicylic diamines represent promising compounds for PSC removal and enrichment of CMs without the need for other selection strategies.
29
"Characterization of odours emitted by liquid waste treatment plants (LWTPs)." Issue 4 18, no. 4 (October 11, 2016): 721–27. http://dx.doi.org/10.30955/gnj.002077.
Full textAbstract:
<p>Odour emissions from liquid waste treatment plants (LWTPs) generally cause significant effects on the environment in terms of nuisance to exposed population. The particular and complex nature of the mixture of the volatile substances, its variability in time and the strong influence of the atmospheric conditions, are the elements that delayed their regulation and relative management.</p>
<p>Limited data are available in the technical and scientific literature, regarding the odour emissions characterization from liquid waste treatment plants. Moreover there isn’t a common strategy from the different European Countries in the regulation of their emissions.</p>
<p>Different methods can be used to measure odour emissions from environmental engineering plants, and currently, in Europe, the most used techniques for odour emissions characterization and quantification is the dynamic olfactometry, according to EN 13725:2003.</p>
<p>The aim of this study is the characterization of the odour emissions from different liquid waste treatment plants (LWTPs), trough a case study of two large real LWTPs, in order to identify the principal odour sources and to define their related odour emissions.</p>
<p>Odour Concentration Index (OCI) is proposed as a useful and simply odour management tool for the identification of the priority actions necessary to identify and control the main odorous sources. Relationship between the measured odour emissions and the types of treated liquid waste (identified in terms of EWC code, COD and NH4<sup>+</sup>) is also discussed.</p>
<p>Results show that the influent collection tank is the source with the highest detected odour emissions. OCI results are useful for the definition of a clear priority action for odour control, similar for both investigated plants. Between the characterized types of liquid waste treated by LWTPs the leachate (EWC 190703) show the maximum odour emissions.</p>
30
Zhang, Huiliang, Sara Bisetto, Shey-Shing Sheu, and Wang Wang. "Abstract 391: Endogenous Drp1 Modulates Cardiac Respiration Through mPTP and Independent of Fission." Circulation Research 117, suppl_1 (July 17, 2015). http://dx.doi.org/10.1161/res.117.suppl_1.391.
Full textAbstract:
Background: The cardiac mitochondria exhibit a stable morphology with a rather low level of dynamic changes. However, fission and fusion proteins, such as dynamin-related protein 1 (DRP1) are abundant in the heart. Whether these proteins bear other functions in the heart than mitochondrial dynamics regulation are largely unknown. We hypothesize that endogenous DRP1 in the heart regulates mitochondrial respiration independent of fission. Methods: Mitochondrial respiration was determined by measuring the OCR with Seahorse assay or Clark type electrode in adult rat cardiomyocytes or mitochondria isolated from adult mouse heart. Confocal imaging was used to quantify mitochondrial morphology in adult cardiomyocytes and H9C2 myoblasts. To evaluate the role of mitochondrial permeability transition pore (mPTP), we monitored superoxide flashes (SOF) and laser-induced mPTP openings, and used cyclophilin D knockout mice (CypD KO). Mitochondrial ROS and Ca2+ were also monitored. Results: Inhibiting the DRP1 GTPase activity by Mdivi-1 or overexpression of the dominant-negative mutant (DRP1-K38A) induced mild mitochondrial morphological changes in adult cardiomyocytes, and inhibited mitochondrial respiration. Modulation of fission/fusion by overexpressing DRP1 or treating cells with S3, a compound facilitates fusion, exhibited significant morphological changes, but failed to influence respiration. Therefore, endogenous DRP1 activity may regulate respiration in the heart and this effect is dissociated with morphological changes. Further, inhibiting DRP1 activity attenuated the frequency of SOF, indicating decreased transient mPTP openings, delayed laser-induced permanent mPTP opening, and increased mitochondrial Ca2+. Inhibiting DRP1 activity decreased mitochondrial ROS levels. The role of DRP1 inhibition on respiration absents in CypD KO myocytes, suggesting the involvement of mPTP in the modulation of respiration by endogenous DRP1. Conclusion: These results suggest that endogenous DRP1 positively regulates respiration in the heart. This effect is likely independent of its role in mitochondrial fission. DRP1 regulation of respiration may involve transient opening of mPTP and contribute to mitochondrial Ca2+ and ROS signaling.