scholarly journals Genomic Instability and Cyto-Genotoxic Damage in Animal Species

2021 ◽  
Author(s):  
María Evarista Arellano-García ◽  
Olivia Torres-Bugarín ◽  
Maritza Roxana García-García ◽  
Daniel García-Flores ◽  
Yanis Toledano-Magaña ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Death ◽  
Genomic Instability ◽  
Farm Animals ◽  
Degenerative Diseases ◽  
Micronuclei Test ◽  
Blood Erythrocyte ◽  
Health Disorders ◽  
Micronuclei Assay

Genomic instability is a condition that may be associated with carcinogenesis and/or physiological disorders when genetic lesions are not repaired. Besides, wild, captive, and domesticated vertebrates are exposed to xenobiotics, leading to health disorders due to cytogenotoxicity. This chapter provides an overview of tests to assess cytogenotoxicity based on micronuclei (MNi) formation. Bone marrow micronuclei test (BmMNt), peripheral blood erythrocyte micronuclei test (PBMNt), and lymphocyte cytokinesis blocking micronuclei assay (CBMN) are discussed. The most illustrative studies of these techniques applied in different vertebrates of veterinary interest are described. The values of spontaneous basal micronuclei in captive, experimental, and farm animals (rodents, hamsters, pigs, goats, cattle, horses, fish) are summarized. In addition, a flow cytometry technique is presented to reduce the time taken to record MNi and other cellular abnormalities. Flow cytometry is helpful to analyze some indicators of genomic instability, such as cell death processes and stages (necrosis, apoptosis) and to efficiently evaluate some biomarkers of genotoxicity like MNi in BmMNt, PBMNt, and CBMN. The intention is to provide veterinary professionals with techniques to assess and interpret cytogenotoxicity biomarkers to anticipate therapeutic management in animals at risk of carcinogenesis or other degenerative diseases.

scholarly journals Commonly Used Chemotherapy Drugs Differentially Determine Microenvironment-Mediated Protection, Via Mitochondrial Transfer, to B-Precursor Acute Lymphoblastic Leukaemia Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2690-2690
Author(s):  
Richard J Burt ◽  
Aditi Dey ◽  
Kenton Cheuk Yan Ng ◽  
Erik Sahai ◽  
Adele K Fielding
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Death ◽  
Acute Lymphoblastic Leukaemia ◽  
Lymphoblastic Leukaemia ◽  
Healthy Donor ◽  
Low Dose ◽  
Chemotherapy Drugs ◽  
Mitochondrial Transfer

Abstract Murine models of Acute Lymphoblastic Leukaemia (ALL) suggest relapse arises not from intrinsic chemoresistance by genetically distinct cells, but from a subset of cells protected within a specific niche. To confirm the existence of such a niche in patients with ALL and elucidate the mechanism by which stromal cells (MSC) protect ALL cells we isolated MSC from bone marrow of 70 B-ALL patients enrolled on the UKALL14 trial. Immunostaining for f-actin, gene expression profiling and cytokine/chemokine quantification showed that a significant proportion of bone marrow specimens, especially after treatment with a cytarabine (AraC)-containing block, contained MSC with an activated phenotype, analagous to cancer associated fibroblasts (ALL-CAF). We demonstrated that primary ALL cells, ALL cell lines, AraC and daunorubicin (but not vincristine (VCR) or dexamethasone (dex)) generated ALL-CAF de novo from both healthy donor MSC and the MSC cell line HS27a. Notably, the chemotherapy drugs induced distinct morphological changes and differential alpha-smooth muscle expression (figure 1a). Control of oxidative stress via modification of reactive oxygen species (ROS) presented a potential unifying explanation for ALL-CAF generation by both ALL cells and chemotherapy. Using flow cytometry we demonstrated that AraC significantly increased ROS in the B-ALL cell line, SEM, in monoculture but in co-culture with HS27a, ROS was significantly lowered and was not impacted by AraC. The MSC co-culture-mediated reduction in ROS in co-culture corresponded to a significant reduction in cell death (10.5% vs 36%, p = 0.0001). By contrast, VCR did not impact ROS significantly and Dex reduced it. Both were significantly more effective than AraC at inducing SEM cell death in co-culture (VCR 20.2% vs AraC 10.5%, p = 0.0003; Dex 39.1% vs AraC 10.5%, p = 0.0007), despite inducing the same degree of cell death in monoculture. We hypothesised that mitochondrial transfer between ALL-CAF and B-ALL cells could provide a generalised mechanism to overcome the deleterious impact of cell-intrinsic and chemotherapy-driven ROS in B-ALL cells. A 'mitotracker' flow cytometry assay showed differential mitochondrial transfer from HS27a to B-ALL cells, in proportion to the baseline ROS levels. We confirmed that mitochondria could also be transferred from healthy donor MSC co-cultured with primary patient ALL cells. Furthermore, AraC, but not VCR or Dex, significantly enhanced mitochondrial transfer, and did so in a dose-dependent manner. To rule out passive transfer of dye, we used the murine stromal line MS5 as an alternative mitochondria donor to SEM cells. Murine mitochondrial, but not nuclear, DNA was clearly seen in flow-sorted SEM cells after co-culture with MS5, at baseline and at higher levels after AraC therapy. We also directly visualised the transfer of mitochondria along tunnelling nanotubes (TNT) by time-lapse confocal imaging (figure 1b). To confirm that mitochondrial transfer was essential in MSC 'rescue' of ALL cells, we generated HS27a cells deficient in mitochondria following prolonged culture with low dose ethidium bromide. The mitochondrially-deficient cells retained viability as well as the ability to become ALL-CAF but were clearly defective in their ability to rescue SEM ALL cells from AraC induced cell death. To confirm the functional impact of mitochondrial transfer via TNT, we used actin inhibitor latrunculin B (LatB) and the microtubule damaging agent nocodazole which both significantly blocked the phenomenon. Both LatB and nocodazole significantly restored AraC-related cytotoxicity. Colchicine, another microtubule damaging agent had a similar impact to nocodazole. VCR completely overcame the protective impact of HS27a on AraC cytotoxicity and was additive with AraC in the co-culture system. We have shown that CAF-like MSC provide support to ALL cells under oxidative stress by mitochondrial transfer via TNT. This is disrupted by microtubule damaging agents and conditions provoking their formation are mitigated by Dex, both mainstays of ALL therapy. Our data may explain the ineffectiveness of ROS-inducing chemotherapy at eradicating disease at the niche and provides an explanation of why low dose, microtubule damaging agents such as VCR used in maintenance therapy are effective in ALL. Our findings have immediate implications for the design and scheduling of current combination chemotherapies for ALL. Disclosures No relevant conflicts of interest to declare.

Targeting CD99 in T-Cell Neoplasms with Monoclonal Antibodies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2749-2749
Author(s):  
Montreh Tavakkoli ◽  
Dong H. Lee ◽  
Benjamin Durham ◽  
Stephen S. Chung ◽  
Christopher Y. Park
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Death ◽  
T Cell ◽  
Cell Lines ◽  
Functional Role ◽  
Normal Peripheral Blood ◽  
T Cell Lymphomas ◽  
Cd34 Cells

Abstract CD99 is a 32-kDa glycoprotein involved in leukocyte migration and homotypic cell aggregation. Since its initial discovery as a marker on acute lymphoblastic leukemia (ALL), few studies have investigated its potential targeting and biological role in this disease. We have shown that CD99 is up-regulated in malignant stem cells in acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS), and that monoclonal antibodies (mAbs) targeting CD99 induce cell death. Given that targeting CD99 holds promise in AML/MDS, we sought to determine whether it is an effective target in other hematologic malignancies. We began by screening 15 T-, B-, and plasma cell lines as well as normal peripheral blood and umbilical cord CD34+ cells for CD99 expression by flow cytometry. CD99 expression was 7- and 10-fold higher on 1/1 T-ALL and 1/2 anaplastic large cell lymphoma (ALCL) cell lines compared with CD34+ cells, and 2- and 3-fold higher relative to normal peripheral blood T cells, respectively. However, it was minimally expressed in 11/12 B cell lymphomas, plasma cell dyscrasias, and peripheral T cell neoplasms. CD99 expression (degree, localization) was also assessed on 264 lymphoma patient samples by immunohistochemistry (IHC) using the CD99 mAb, 12E7. We found that 11/20 (55%) T-lymphoblastic lymphomas, 7/16 (44%) angioimmunoblastic T-cell lymphomas, 4/13 (31%) ALCLs, 10/63 (16%) peripheral T-cell lymphomas, and 0/3 (0%) of NK/T cell lymphomas express CD99 by IHC, while only 1/70 (1.4%) diffuse large B cell lymphomas, 2/24 (8%) mantle cell lymphomas, 2/17 (12%) follicular lymphomas, 4/22 (18%) chronic lymphocytic leukemias, and 3/16 (19%) marginal zone lymphomas express CD99. Staining was predominately moderate and cytoplasmic. Using a BioGPS dataset from T-ALL patient bone marrow samples, CD99 transcript was found to be up-regulated in T-ALL bone marrow (n=117) relative to normal bone marrow (n=7) (p<0.0001), and was expressed at similar levels at diagnosis (n=14) and relapse (n=14), suggesting it is stably expressed and may be a candidate therapeutic target. To test whether CD99 mAbs are cytotoxic to T-ALL and ALCL cell lines, cells were incubated with 5µg/ml CD99 mAb in the presence of 7µg/ml anti-IgG antibody, and cell survival was assessed by flow cytometry following 72-hours relative to IgG isotype control. 4/5 T-ALL cell lines (KOPTK1, Loucy, CCRF HSB-2, PF283) were sensitive to the cytotoxicity of CD99 mAb, mediating 30-96% cell death (p≤0.003), with 2/4 cell lines displaying 90-96% cytotoxicity. Remarkably, incubating CD99 mAb with a primary T-ALL patient sample induced 100% cell death within 48 hours of treatment (p<0.0001). 1/2 ALCL cell lines (Karpas-299) were sensitive to cytotoxic CD99 mAb (46% cell death, p=0.02). Furthermore, CD99 mAb treatment induced Annexin V positivity, and cell death occurred independent of complement and within 3 hours of treatment. To determine whether CD99 mAb cytotoxicity depends on the level of CD99 expression, we stably transduced KOPTK1 cells with an optimized CD99 shRNA (199-fold reduction in CD99 mean fluorescence intensity [MFI]), stably transduced CD99-low Mac2A (ALCL) cells with TetOn CD99 (17-fold increase in CD99 MFI), and analyzed the cells for cytotoxicity following 24-hour incubations with CD99 mAbs. CD99 mAb-induced cell death increased from 4.4% to 88% upon overexpressing CD99, and decreased from 89% to 20% upon knocking down CD99, suggesting that cell death is dependent on the level of CD99 expression. To elucidate the functional role of CD99 in T-cell neoplasms, we xenografted KOPTK1 cells expressing CD99 shRNA into sublethally irradiated NOD/SCID/IL-2Rgc-null (NSG) mice. Animals transplanted with CD99 knockdown showed no improved survival compared to controls (n=4 and 5 in each group, respectively). We further evaluated the potential oncogenic role of CD99 in vitro, and observed no effect of CD99 knock down in KOPTK1 or overexpression in Mac2A on cell cycle status or proliferation by PI staining and cell counting. Our data indicate that CD99 is expressed in a subset of T-lineage neoplasms. While there is no evidence for a functional role of CD99 in the growth or survival of T-ALL and ALCL, CD99 can be targeted by CD99 mAbs to induce apoptosis with rapid kinetics and in a manner that is dependent on levels of CD99 expression and independent of complement. Thus, CD99 is a promising target in the treatment of a subset of T-cell neoplasms. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Unfolded Protein Response Mediates Resistance in AML and Its Therapeutic Targeting Enhances TKI Induced Cell Death in FLT3-ITD + AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2246-2246
Author(s):  
Timo Jaquet ◽  
Christian Preisinger ◽  
Marlena Bütow ◽  
Stefan Tillmann ◽  
Nicolas Chatain ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Death ◽  
Board Of Directors ◽  
Research Funding ◽  
Bone Marrow Niche ◽  
Advisory Committees ◽  
Clonogenic Potential ◽  
The Unfolded Protein Response ◽  
Support Research

Abstract Introduction: The unfolded protein response (UPR) is a stress sensing signaling network that is activated upon endoplasmic reticulum (ER) stress, a condition characterized by an accumulation of mis- and unfolded proteins in the ER. To retain a functional cell metabolism, UPR activation increases protein folding and degradation. Acute myeloid leukemia (AML) stem cells are prone to develop ER stress, due to their oncogene-driven metabolism and the bone marrow niche, where they face stressors like hypoxia or nutrient fluctuations. Our preliminary work showed enhanced UPR gene expression levels, especially of IRE1α and XBP1, in different AML subtypes. Patients with high XBP1 mRNA expression had an inferior overall survival rate compared to patients with low XBP1 mRNA expression. Aims: We studied the role of elevated UPR signaling in AML therapy resistance and assessed the therapeutic potential of IRE1α-XBP1 inhibitor STF-083010 (STF) as a new strategy in different AML subtypes, including FLT3-ITD + AML. Methods: Human MV4-11 (FLT3-ITD), RS4-11 (FLT3 wildtype; WT), NB-4 (PML-RARα), THP-1 (MLLr) cells, and murine 32D cells transduced with FLT3-ITD or FLT3 WT were analyzed via western blot and RT-PCR. Metabolic activity was assessed by MTT assay, cell death and apoptosis were measured with propidium iodide (PI) or Annexin V staining using flow cytometry. FLT3 cell surface expression was measured via flow cytometry. The clonogenic potential was determined in CFU assays, using patient-derived mononuclear and CD34 + cells. For hypoxic experiments, MV4-11 cells were cultivated under hypoxia (3 % O 2) and cells were subjected to phosphoproteomic analysis, which was performed by mass spectrometry. Conditional Mx1-Cre/XBP1 fl/fl knockout mice were generated and deletion of XBP1 was induced by IP injection of Polyinosinic-polycytidylic acid (Poly(I:C)). Bone marrow and spleen cells were analyzed via flow cytometry and RT-PCR. Results: Treatment with FLT3 TKI AC220 specifically enhanced IRE1α mRNA (9.3-fold, p&lt;0.05) and increased IRE1α protein in 32D FLT3-ITD cells. Likewise, the percentage of dead cells was significantly elevated in 32D FLT3-ITD upon IRE1α inhibition by STF compared to 32D FLT3 WT cells. Treatment with STF prevented XBP1 splicing and reduced the metabolic activity of human AML cell lines in a dose-dependent manner. Furthermore, IRE1α inhibition significantly induced apoptosis in human MV4-11 (6-fold, p&lt;0.05), NB-4 (8-fold, p&lt;0.01) and THP-1 (7-fold, p&lt;0.01) cells and reduced their clonogenic potential. The combination of STF and AC220 strongly enhanced the percentage of apoptotic cells in MV4-11 cells compared to single treatments (by 3-fold, p&lt;0.001). This strong induction of cell death was specific for FLT3-ITD + MV4-11 cells and not observed in FLT3 WT + RS4-11 cells. Similarly, the clonogenic potential of MV4-11 cells and FLT3-ITD + AML mononuclear patient cells was significantly decreased by the combinatorial treatment, while healthy donor cells were not affected. Likewise, conditional XBP1 knockout did not significantly alter normal hematopoiesis in mice. Hypoxia further enhanced IRE1α signaling in MV4-11 cells and strongly reduced the efficacy of AC220 (normoxia: 58.4-fold induction of dead cells, p&lt;0.01; hypoxia: 2.2-fold induction, p&gt;0.05). Analysis of phosphoproteomics revealed a less active FLT3 signaling under hypoxia. Intriguingly, the combination of IRE1α and FLT3 inhibition overcame the resistance towards AC220 under hypoxia and significantly induced cell death. Conclusion: IRE1α-XBP1 signaling is activated in different AML subtypes including FLT3-ITD + and is further enhanced by hypoxia present in the bone marrow niche. Targeting IRE1α in FLT3-ITD + cells effectively decreases clonogenic growth and induces apoptosis. Our data demonstrate that hypoxia-mediated resistance against AC220 can be overcome by simultaneous IRE1α inhibition. Genetic deletion of XBP1 does not harm steady-state murine hematopoiesis, rendering XBP1 an excellent therapeutic target. Disclosures Koschmieder: CTI: Membership on an entity's Board of Directors or advisory committees, Other; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Baxalta: Membership on an entity's Board of Directors or advisory committees, Other; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); AOP Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Shire: Honoraria, Other; Image Biosciences: Other: Travel support; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Geron: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Karthos: Other: Travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Alexion: Other: Travel support; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Abbvie: Other: Travel support; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Brümmendorf: Bristol Myers: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Repeat Diagnostics: Research Funding; Takepart Media: Honoraria.

scholarly journals Inhibition of GSK-3 Signalling Enhances Sensitivity of Non-Promyelocitic Acute Myeloid Leukemia (AML) Cell to Chemotherapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1582-1582 ◽  
Author(s):  
Paul Takam Kamga ◽  
Giada Dal Collo ◽  
Adriana Cassaro ◽  
Annalisa Adamo ◽  
Alessandro Gatti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Death ◽  
Western Blot ◽  
Metabolic Regulation ◽  
Mononuclear Cells ◽  
Chemotherapeutic Agents ◽  
Response To Treatment ◽  
Dependent Manner ◽  
Gsk 3Β

Abstract Background: GSK-3 is a serine-threonine kinase involved in metabolic regulation as well as in the control of many pathways associated to cancer development, including Notch Wnt/β-catenin, Hedgehog, and AKT. It has been demonstrated that association of GSK-3 inhibitors with All-trans-retinoic acid (ATRA) significantly improves ATRA-mediated differentiation and cell death of acute promyelocytic (APL) leukaemia cells. However, little is currently known about the contribution of GSK-3 role to non-promyelocytic AML cell response to treatment with chemotherapeutic agents. Aims: In this study, we aim to validate GSK-3 signalling as potent successful therapeutic target in non-promyelocytic AML. For this purpose we tested different GSK-3 for their ability to influence AML cells proliferation and response to Cytarabine (Ara-C) or Idarubicin treatments. Methods: GSK-3 expression was analyzed by Western blot or flow cytometry inAML cell lines (HL-60, THP1, U937) or primary non-promyelocyticAML blast cells (30 samples). AML cellscultured alone or in presence ofhuman bone marrow mesenchymal stromal cells (hBM-MSCs) were treated with GSK-3 inhibitors, including LiCL, AR-A014418, SB 216763, in association or not with Cytarabine (Ara-C) or Idarubicin. Cell proliferation and cell death were measured by CFSE dilution and TOPRO-3/Annexin-V staining, respectively. Results: Flow cytometry and Western blot analysis in AML samples revealed high expression levels of all GSK-3forms, including total GSK-3α, (Ser21) GSK-3α, total GSK-3β, and (Ser 21) GSK-3β; theseforms were all down-modulated when AML cells were cultured in presence of hBM-MSCs, thus suggesting that GSK-3 plays an important role in transducting micro-environmental signals in AML cells interacting with bone marrow stroma. The treatment of AML cells with increasing concentrations of each GSK-3 inhibitors decreased AML cell viability in a dose-dependent manner; interestingly, hBM-MSCs or peripheral blood mononuclear cells were less sensitive to GSK-3inhibitors. The addition of each inhibitor increased dramatically the AML cell apoptotic rate induced by the addition of Ara-C or Idarubicin in vitro. Notably, LiCl and AR-A014418 were capable of abrogating hBM-MSC-mediated AML cell resistance to apoptosis induced by Ara-C or Idarubicin. Conclusion: Overall our data clearly demonstrated that inhibition of GSK-3 reduced proliferation and chemoresistance of non promyelocytic AML cells. Thus GSK-3 inhibition represents a therapeutic strategy not only for APL but also for other AML subtypes. Disclosures Bonifacio: Ariad Pharmaceuticals: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Amgen: Consultancy.

scholarly journals RAC1 Inhibitor EHT1864 and Venetoclax Overcome Midostaurin Resistance in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1277-1277
Author(s):  
Andoni Garitano-Trojaola ◽  
Ana Sancho ◽  
Ralph Goetz ◽  
Susanne Walz ◽  
Hardikkumar Jetani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Death ◽  
Actin Filaments ◽  
Myeloid Leukemia ◽  
Actin Polymerization ◽  
Cell Stiffness ◽  
Adhesion Forces ◽  
Intracellular Expression ◽  
Acute Myeloid

Introduction Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease characterized by clonal expansion of immature myeloid progenitor cells in the bone marrow (BM). Mutations of the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 30% of AML cases, with Internal Tandem Duplications (ITD) being the most common type of mutation. Several FLT3 specific inhibitors (TKI) have been developed such as quizartinib, crenolanib and midostaurin (recently approved for clinical use). Nevertheless FLT3-ITD is associated with unfavorable prognosis and patients develop drug resistance with the underlying mechanisms remaining largely unexplained. Recently, changes within the actin cytoskeleton were associated with drug resistance development in various cancers. FLT3-ITD mutations are associated with RAC1 activation. RAC1 belongs to the family of RHO GTPases and enhances the actin polymerization by inducing the expression of N-WASP or WAVE2 and ARP2/3 complex. Therefore, we investigated actin cytoskeleton rearrangements through RAC1 activation as a potential mechanism contributing to Midostaurin resistance in AML. Material and methods First, we developed two Midostaurin resistant AML cell lines (MID-RES, MV4-11 and MOLM-13). Single cell measurements of Cell Stiffnes, cell adhesion forces between tumor and HS5 stroma cells and Actin filaments were performed by Atomic Force Microscopy (FluidFM®) and SIM microscopy, respectively. RAC1 activation was measured by RAC1 activation kit provided by Cytoskeleton. FLT3 surface and intracellular expression was measured by Flow cytometry and western blot, respectively. Cell death was analyzed by Annexin/PI staining in flow cytometry. Results The MID-RES cell lines MV4-11/MOLM-13 showed higher FLT3 surface and intracellular expression compared to their MID sensitive parental cells. In line with our expectations, we observed RAC1 activation, as well as an up-regulation of actin polymerization positive regulators such as N-WASP, WAVE2, PFN1 and ARP2/3 complex and the inhibition of actin polymerization negative regulator P-ser3 CFL1 in MID-RES cells. FLT3 receptor knock down by siRNAs reversed the MID resistance and reduced RAC1 activation and actin polymerization inducers expression. Likewise, bioinformatic analysis from publicly available microarray expression data (E-MTAB-3444), confirmed positive correlation between actin polymerization inducers and FLT3 signaling expression in 178 FLT3-ITD (r=0,67) and 461 FLT3 WT(r= 0,57) de novoAML patients. RAC1 induced Actin polymerization positively correlates with actin filaments growth and cell stiffness, which was observed in our MID-RES cells, higher load of actin filaments and increased cell stiffness. The combination between RAC1 specific inhibitor, EHT1864 and Midostaurin synergistically induces cell death in MID-RES cells by arresting cell cycle in G0/G1 phase and activating apoptosis. Beside, this combination reduced the adhesion forces to stroma cells, decreased the expression of PFN1/N-WASP/ARP2 and consequently reduced drastically the number of actin filaments and cell stiffness in MID-RES cells. EHT1864 and Midostaurin (alone and in combination) were not toxic in PBMCs obtained from healthy donors. Interestingly, this combination increase >45 % cell death in cells obtained from refractory FLT3-mutated AML patient (this patient was relapsed (≥ 50% residual blasts in the bone marrow)under Chemotherapy+Midostaurin combination).The specific knock down of PFN1/N-WASP/ARP2 with siRNAs equally reversed the resistance to Midostaurin. Of note, RAC1 regulates the anti-apoptotic BCL2. Indeed, EHT1864 in combination with Midostaurin reduced anti-apoptotic family BCL2/MCL1 expression and increases the pro-apoptotic proteins BAX/PUMA. As expected, our MID-RES cells showed higher sensitivity to BCL2 inhibitor Venetoclax, than their parental cells. The combinations EHT1864+venetoclax, venetoclax+midostaurin and venetoclax+Midostaurin+EHT1864 synergistically induced cell death in MID-RES cells. Conclusion Actin polymerization inducers N-WASP, ARP2/3 complex and PFN1 may provide a valuable approach to overcome Midostaurin resistance in AML. Our data further suggest that the addition of BCL2 inhibition through EHT1864 and venetoclax could represent an interesting strategy to potentiate the activity of Midostaurin in FLT3 mutated AML. Disclosures Duell: Regeneron Pharmaceuticals, Inc.: Research Funding. Rosenwald:MorphoSys: Consultancy.

Sphingosine Kinase Inhibition Has Pre-Clinical Activity in Acute Lymphoblastic Leukemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3573-3573
Author(s):  
Craig T. Wallington-Beddoe ◽  
David Ho ◽  
Kenneth Francis Bradstock ◽  
Linda J. Bendall
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Term Survival ◽  
Intracellular Location ◽  
Ic50 Values

Abstract Abstract 3573 Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer, which usually responds to chemotherapy. Long-term survival in adults is poor with most developing disease relapse, whilst Ph+ ALL has a particularly poor prognosis. Sphingosine 1-phosphate (S1P) is a lipid mediator of diverse cellular functions, most notably of lymphocyte trafficking, angiogenesis, cell proliferation and survival. S1P is produced intracellularly by the sphingosine kinases (SK) of which there are two isoforms, SK1 and SK2. SK1 is over expressed in a number of malignancies and evidence points overwhelmingly to a pro-survival role. The role of SK2 is much less well defined and appears to be dependent on its intracellular location with some reports of opposite effects to those of SK1. Inhibition of either SK1 or SK2 is currently under investigation as a novel anti-cancer strategy and potent anti-leukemic effects are likely. Application of the combined SK1/SK2 inhibitor SKI II and the selective SK2 inhibitor ABC294640 to ALL cells produced a reduction in cellular proliferation as measured by 3H-thymidine incorporation in all cell lines (REH, NALM6, LK63, ALL1, 2070 and TOM1) tested with IC50 values of 1μM – 7μM for SKI II and <40μM for ABC294640. Viability, measured by flow cytometry using annexin V and propidium iodide (PI) staining, was also reduced in all cell lines except the Ph+ ALL1 and 2070 cells treated with SKI II with IC50 values ranging from 2μM to >10μM for SKI II and 50–60μM for ABC294640. SKI II resulted in caspase-dependent cell death, as determined by flow cytometric assessment of intracellular caspase-3 cleavage and apoptotic morphology on light microscopy, with cell death prevented by pre-incubation with 100μM of the pan-caspase inhibitor Z-VAD-FMK. However, ABC294640 induced caspase-3 cleavage at lower than expected levels and cell death was not prevented by Z-VAD-FMK. Both agents significantly reduced intracellular S1P concentrations by 24 hours as determined by ELISA, thereby confirming the ability of these compounds to inhibit SK1 or SK2 activity. A search for agents that synergized with the SK inhibitors revealed that when Ph+ ALL cells were treated with the combination of imatinib and either ABC294640 or SKI II, a further reduction in cell death occurred than with either agent alone, thereby enhancing the therapeutic effect of ABC294640 and overcoming resistance seen with SKI II alone. Furthermore, the combination of mildly cytotoxic concentrations of ABC294640 and the novel pan histone deacetylase inhibitor AR-42 were found to significantly increase leukemic cell death at 24 and 48 hours in Ph+ and negative ALL cells. In vivo assessment of the SK inhibitors was determined by injecting NOD/SCID IL2gc−/− mice with 2–5 million human B-ALL cells and treating with 100 mg/kg/day ABC294640 or vehicle by intraperitoneal injection for 21 days after which all animals were sacrificed. Assessment of leukemia in blood, bone marrow and spleen was determined by flow cytometry using antibodies to human CD19 and murine CD45. Significant reductions in the levels of leukemia in all examined tissues were found in ABC294640-treated animals using three different human ALL xenografts, including the Ph+ positive xenograft 2070. Average absolute levels of leukemia in the bone marrow of ABC294640-treated mice for xenografts 2070, 1345 and 0398 were reduced by 40% (p = 0.00007), 55% (p = 0.004) and 72% (p = 0.000001) respectively. No overt toxicity was noted. SK inhibition, resulting in reduced intracellular S1P, is an exciting novel anti-leukemic strategy potentially adding to the repertoire of non-chemotherapeutic agents for the treatment of ALL. Combinations of SK inhibitors with newer targeted agents show promise of greater leukemia reduction. Disclosures: No relevant conflicts of interest to declare.

Population Pharmacological Profiles Of AML Treatments In Patient Samples By Automated Flow Cytometry; A Bridge To Individualized Medicine

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1347-1347
Author(s):  
Joan Ballesteros ◽  
Pau Montesinos ◽  
Federico Moscardo ◽  
David Martinez ◽  
Miguel Angel Sanz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Death ◽  
Ex Vivo ◽  
Drug Combinations ◽  
In Vitro Test ◽  
Combination Treatments ◽  
Survival Index ◽  
The Individual ◽  
Pharmacological Profiles

Abstract Background To aid in the identification of effective treatments for individual patients, ex vivo assays for detecting cell death inducible by drugs for hematological malignancies have been in development for over 20 years. We have developed a novel approach incorporating 4 key innovations; incubating drugs in whole bone marrow sample without isolating leukocytes, using flow cytometry enables identification of the malignant cells selectively, an automated flow cytometry-based platform (ExviTech) decreases errors and enables full pharmacological characterization, and analyzing the data using pharmacodynamic population models. Aim The purpose of this study is to derive the ex vivo pharmacological profiles across the AML patient population of single drugs and combination treatments as a tool for individualized treatment selection. Patients and Methods Bone-marrow samples from 160 patients diagnosed with AML were sent to Vivia from 24 hospitals across Spain within 24 hrs. The plates were incubated for 48-hours prior to analysis with ExviTech, The percentage of leukemic cell death was determined via labeling with monoclonal antibodies and AnnexinV-FITC. A survival index is computed for each drug, the lower the survival index, the more effective the drug. Dose-response curves of cytarabine, idarubicin, daunorubicine, etoposide, mitoxantrone, fludarabine, clofarabine, and 6-thioguanine were measured in 160 patient samples. The added benefit of combining these drugs into 12 combination treatments was assessed by measuring their synergy in each individual patient. In 39 patients treated with CYT IDA we had clinical data of response, and then we performed a blinded interpretation of this in vitro test by an expert hematologist, to predict the clinical response based in this test result. Results There was a large range of interpatient variability in the response to a single drug and even larger in the synergism between drugs. The Population Pharmacological Profiles for an individual patient is shown on the figure below. The relative drug potency in terms of their percentile ranking within the population is shown in the left panel from 0 (weakest) to 100 (most potent). Green lines represent the individual patient potency relative to the population ranking, with confidence intervals. Third column lists when a drug leaves a significant % of leukemic cells alive, potential resistant clones. The panel on the right side shows the synergism of the drug combinations treatments shown as box-plots at 10-25-75-90% to highlight their distribution. The synergism value for an individual patient in each combination is shown in green, with confidence interval as parallel dotted green lines. This representation of the Pharmacological Profile of an individual patient sample quickly identifies extreme values, when a drug or combination is very sensitive (rightward shift green lines, green boxes) or very resistant (leftward shift green lines, red boxes). This patient showed average sensitivities for most drugs though highly resistant to Clofarabine (red box) that leaves 45% alive. However this patient showed lack of synergism in multiple treatments (right, red boxes). CYT and IDA show average potencies but lack of synergism, suggesting CYT-DAU might be a more efficient treatment. These representations lead to clear guidelines in >90% samples, and based on hematologist's interpretation of these guidelines show a clinical correlation with clinical responses to CYT-IDA of 84%. Conclusion We have developed an improved a methodology to measure the pharmacological activity of drugs and drug combinations in AML patient samples as well as modeling their pharmacological behavior. This information may be useful in selecting the optimal treatment for the individual patient, especially relapse/refractory patients in need of therapeutic alternatives. By testing the drugs used in the treatment protocols for AML directly on patient samples, a pharmacological based model has been developed to infer drug resistance or sensitivity, patient by patient. Disclosures: Ballesteros: Vivia Biotech: Equity Ownership. Primo:Vivia Biotech: Employment. Hernandez-Campo:Vivia Biotech: Employment. Rojas:Vivia Biotech: Employment. Liebana:Vivia Biotech: Employment. Lopez:Vivia Biotech: Employment. Iñaki:Vivia Biotech: Consultancy. Bennett:Vivia Biotech: Employment.

Development and Challenges of Nanotherapeutic Formulations for Targeting Mitochondrial Cell Death Pathways in Lung and Brain Degenerative Diseases

2020 ◽  
Vol 48 (3) ◽  
pp. 137-152
Author(s):  
Marko Manevski ◽  
Dinesh Devadoss ◽  
Ruben Castro ◽  
Lauren Delatorre ◽  
Adriana Yndart ◽  
...  
Keyword(s):  
Cell Death ◽  
Degenerative Diseases ◽  
Cell Death Pathways
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