FLT3-ITD–, but not BCR/ABL-transformed cells require concurrent Akt/mTor blockage to undergo apoptosis after histone deacetylase inhibitor treatment

Blood ◽  
2006 ◽  
Vol 107 (5) ◽  
pp. 2094-2097 ◽  
Author(s):  
Dali Cai ◽  
Ying Wang ◽  
Oliver G. Ottmann ◽  
Peter J. Barth ◽  
Andreas Neubauer ◽  
...  
Keyword(s):  
Philadelphia Chromosome ◽  
Mtor Inhibitors ◽  
Apoptosis Resistance ◽  
Akt Activation ◽  
Lymphatic Leukemia ◽  
All Trans Retinoic Acid ◽  
Deacetylase Inhibitor ◽  
Pathway Inhibition ◽  
Induced Apoptosis

Leukemias are differentially sensitive to histone deacytelase inhibitor (HDI)–induced apoptosis, but molecular reasons for this remain unclear. We here show that BCR/ABL-, but not FMS-like tyrosine kinase 3 (FLT3)–internal tandem duplication (ITD)–transformed 32D cells or primary acute myeloid leukemia (AML) blasts undergo apoptosis after treatment with the HDI valproic acid (VPA) plus all-trans retinoic acid (VPA/ATRA). A particular VPA/ATRA responsiveness of Philadelphia chromosome–positive (Ph+) acute lymphatic leukemia (ALL) was confirmed in a therapy-refractory patient in vivo. HDI-stimulated apoptosis in Ph+ cells was caspase dependent, but independent from Akt pathway inhibition. Conversely, separate blockage of the Akt/mTor-signaling pathway was a prerequisite for overcoming apoptosis resistance to VPA/ATRA in FLT3-ITD cells, and primary AML blasts (n = 9). In conclusion, constitutive Akt activation causes apoptosis resistance to VPA/ATRA in AML, but not in Ph+ leukemia. This warrants the application of HDI-based therapies in poor-risk Ph+ ALL, and the use of Akt/mTor inhibitors to overcome HDI resistance in AML.

MTOR Inhibitors Activate the AKT Kinase in Multiple Myeloma Cells by Upregulating the IGF-1/IRS-1/PI-3 Kinase Cascade.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3350-3350 ◽  
Author(s):  
Yijiang Shi ◽  
HuaJun Yan ◽  
Patrick Frost ◽  
Bao Hoang ◽  
Joseph Gera ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitors ◽  
Xenograft Model ◽  
Mtor Inhibitors ◽  
Pi3 Kinase ◽  
Serine Phosphorylation ◽  
G1 Arrest ◽  
Akt Activation ◽  
Induced Apoptosis

Abstract MTOR inhibitors, such as rapamycin and CCI-779, have shown pre-clinical potential as therapy for multiple myeloma (MM). By inhibiting expression of cell cycle proteins, these agents induce G1 arrest. However, by also inhibiting an mTOR-dependent phosphorylation of insulin receptor substrate-1 (IRS-1), they may alter its subcellular localization and/or prevent its degradation which could enhance IGF-1 signaling and downstream PI3-kinase/AKT activation. This may be a particular problem in MM where IGF-1-induced activation of AKT is an important anti-apoptotic cascade. We, thus, studied PI3-kinase/AKT activation in MM cells treated with mTOR inhibitors. Rapamycin enhanced basal AKT activity, AKT phosphorylation and PI3-kinase activity in MM cell lines. Both PTEN-null as well as PTEN-wild type myeloma lines were similarly affected. Rapamycin also significantly prolonged activation of AKT induced by exogenous IGF-1. CCI-779, used in a xenograft model, also resulted in MM cell AKT activation in vivo. Blockade of IGF-1 receptor function prevented rapamycin’s activation of AKT. Furthermore, rapamycin prevented serine phosphorylation of IRS-1 and IRS-1 degradation. Though similarly blocking IRS-1 degradation, proteasome inhibitors did not activate MM cell AKT. Although rapamycin sensitized MM cells for dexamethasone-induced apoptosis, it protected against PS-341-induced apoptosis. Thus, mTOR inhibitors activate PI3-K/AKT in MM cells and activation depends on basal IGF-1/IGF-R signaling. As activated AKT may protect against apoptosis, future use of mTOR inhibitors in myeloma patients will have to carefully consider the types of anti-myeloma agents used in combination.

The Plasticity of Myeloma Plasma Cells as Expressed by Dedifferentiation into an Immature, Resilient, Apoptosis-Resistant Phenotype.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 633-633 ◽  
Author(s):  
Shmuel Yaccoby ◽  
Kenichiro Yata ◽  
Yun Ge ◽  
Bart Barlogie ◽  
Guido Tricot ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Rabbit Model ◽  
Common Mechanism ◽  
Proliferative Potential ◽  
Apoptotic Cells ◽  
Apoptosis Resistance ◽  
Hematopoietic Stem ◽  
Induced Apoptosis

Abstract Progression of myeloma (MM) is considered to be a multistage and dynamic process of cell differentiation, survival, proliferation and dissemination. We have previously demonstrated the proliferative potential of purified CD45lowCD38high mature MM cells in SCID-hu mice (Yaccoby & Epstein, Blood, 1999), the ability of CD138-selected MM cells to produce myeloma in our novel SCID-rabbit model (Yata et al., ASH, 2003), and the interdependence of MM bone disease and tumor growth whereby MM cells induce osteoclast activity and are dependent on osteoclasts in vivo (Yaccoby et al., BJH, 2002) and ex vivo (Yaccoby et al., Cancer Res., 2004). The aim of this study was to determine the osteoclast-induced phenotypic changes associated with survival of MM cells in long term co-culture. CD138-selected (>95% purity) MM cells from 16 patients were co-cultured with human osteoclasts for up to 20 weeks. The pre-cultured baseline cells were typically CD45low/inermediateCD38high, CD19−CD34−. At the end of long term co-culture (>6 weeks) MM cells had BrdU labeling index (LI) of 2.5±2.0 and their viability was 97%±1%. The phenotype of co-cultured MM cells consistently shifted to a less mature phenotype, with CD45 expression increasing from CD45low to CD45intermmediata/high and reduced expression of CD38 from CD38high to subpopulations with CD38intermediate levels, as determined by flow cytometry and confirmed by qRT-PCR. Further flow analysis revealed that co cultured MM cells also expressed low levels of CD19 and CD34, and identified a small subpopulation of CD138lowCD45high MM cells. Morphologically, the co-cultured MM cells uniformly gained plasmablastic characteristics when compared to pre-cultured cells. Previous reports suggested that IL-6 was important for maintaining subpopulation of CD45-expressing MM cells. However, blocking IL-6 activity in co-cultures with anti-IL6 and anti-IL6R neutralizing antibodies (5 μg/ml, each) did not affect the immature phenotype of MM cells. Intriguingly, long term co-culture of normal CD34+ hematopoietic stem cells (HSCs) with osteoclasts results in loss of CD34 expression, suggesting a common mechanism for osteoclast-induced MM PC and HSC plasticity. To investigate if the observed phonotypic changes are associated with apoptosis resistance, we determined the effects of 3 days exposure to the pro-apoptotic agent dexamethasone (DEX, 10−7 M) on MM cells cultured alone or in co-cultures (n=5), at initiation (baseline) and after 6 weeks of co-cultures. The percent apoptotic cells was determined by trypan blue exclusion and annexin V flow cytometry. When baseline MM cells were cultured alone, DEX significantly increased the percent of apoptotic cells over that spontaneous rate (p<0.01). In contrast, when MM cells recovered from co-cultures after 6 weeks they survived and were resistant to DEX-induced apoptosis (16%±11% and 24%±21% apoptotic cells in the absence and presence of DEX, respectively). As reported, osteoclasts supported survival of MM cells at baseline and after 6 weeks of co-culture (p<0.01), and protected MM PCs from DEX-induced apoptosis. Our data demonstrate the phenotypic plasticity of primary myeloma cells, whereby mature MM cells are reprogrammed and acquire autonomous survival properties after co-culture with osteoclasts. We hypothesize that in vivo these cells are dormant, resistant to spontaneous and drug-induced apoptosis, and could be responsible for relapse.

ARC Is Regulated by MAPK and PI3K and Confers Drug Resistance and Survival Advantage to AML in Vitro and in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 893-893
Author(s):  
Po Yee Mak ◽  
Duncan H Mak ◽  
Yuexi Shi ◽  
Vivian Ruvolo ◽  
Rodrigo Jacamo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Apoptosis Resistance ◽  
Control Mscs ◽  
Extrinsic Apoptosis ◽  
Induced Apoptosis ◽  
And Control

Abstract Abstract 893 ARC (Apoptosis repressor with caspase recruitment domain) is a unique antiapoptotic protein that has been shown to suppress the activation of both intrinsic and extrinsic apoptosis. We previously reported that ARC is one of the most potent adverse prognostic factors in AML and that high ARC protein expression predicted shorter survival and poor clinical outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report how ARC is regulated and its role in inhibition of AML apoptosis and in cell survival. We provide evidence that ARC expression is regulated by MAPK and PI3K signaling. Inhibition of MAPK and PI3K pathways decreased ARC mRNA and protein levels in AML cells. ARC expression in AML cells is upregulated in co-cultures with bone marrow-derived mesenchymal stromal cells (MSCs) and the upregulation is suppressed in the presence of MAPK or PI3K inhibitors. To investigate the role of ARC in apoptosis resistance in AML, we generated stable ARC overexpressing (O/E) KG-1 and stable ARC knock down (K/D) OCI-AML3 and Molm13 cells and treated them with Ara-C and agents selectively inducing intrinsic (ABT-737) or extrinsic (TRAIL) apoptosis. We found that ARC O/E cells are more resistant and ARC K/D cells more sensitive to Ara-C, ABT-737, and TRAIL-induced apoptosis: EC50s of Ara-C, ABT-737, or TRAIL treatment at 48 hours for ARC O/E KG-1 and control cells were 1.5 ± 0.1 μM vs. 83.5 ± 4.6 nM, 2.2 ± 0.2 μM vs. 60.2 ± 3.1 nM, or 0.97 ± 0.03 μg/mL vs. 0.17 ± 0.08 μg/mL, respectively and for ARC K/D OCI-AML3 and control cells were 0.33 ± 0.02 μM vs. 3.4 ± 0.2 μM, 0.24 ± 0.01 μM vs. 1.3 ± 0.1 μM, or 0.13 ± 0.09 μg/mL vs. 0.36 ± 0.03 μg/mL, respectively. Bone marrow microenvironment is known to play critical roles in AML disease progression and in protecting leukemia cells from various therapeutic agent-induced apoptosis. Leukemia cells were co-cultured with MSCs in vitro study to mimic the in vivo condition. ARC was found to be highly expressed in MSCs and stable ARC K/D MSCs were generated. AML cell lines and primary patient samples were co-cultured with ARC K/D or control MSCs and treated with Ara-C, ABT-737, or TRAIL. Interestingly, ARC K/D MSCs lost their protective activity for leukemia cells treated with these agents. EC50s for OCI-AML3 cells co-cultured with ARC K/D or control MSCs for 48 hours treated with Ara-C, ABT-737, or TRAIL were 1.0 ± 0.04 μM vs. 4.5 ± 0.2 μM, 0.15 ± 0.06 μM vs. 0.53 ± 0.02 μM, or 1.4 ± 0.8 μg/mL vs. 8.1 ± 0.3 μg/mL, respectively. In addition, ARC O/E KG-1 cells grew faster and ARC K/D OCI-AML3 and Molm13 cells and ARC K/D MSCs grew slower than their respective controls. We then injected KG-1 cells into mice and found that NOD-SCID mice harboring ARC O/E KG-1 had significantly shorter survival than mice injected with the vector control KG-1 (median 84 vs. 111 days) as shown in the figure. Collectively, results demonstrate that ARC plays critical roles in AML. ARC is regulated by MSCs through various signaling pathways in AML cells, protects leukemia cells from apoptosis induced by chemotherapy and by agents selectively inducing intrinsic and extrinsic apoptosis. ARC regulates leukemia cell growth in vitro and in vivo. The results suggest that ARC is a potential target for AML therapy. In addition, targeting ARC in MSCs suppresses microenvironmental protection of AML cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals SMARCA4/2 loss inhibits chemotherapy-induced apoptosis by restricting IP3R3-mediated Ca2+ flux to mitochondria

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yibo Xue ◽  
Jordan L. Morris ◽  
Kangning Yang ◽  
Zheng Fu ◽  
Xianbing Zhu ◽  
...  
Keyword(s):  
Chemotherapy Resistance ◽  
Chromatin Accessibility ◽  
Chemotherapy Response ◽  
Apoptosis Induction ◽  
Lung Cancers ◽  
Deacetylase Inhibitor ◽  
Chromatin Remodeling Complexes ◽  
Induced Apoptosis

AbstractInactivating mutations in SMARCA4 and concurrent epigenetic silencing of SMARCA2 characterize subsets of ovarian and lung cancers. Concomitant loss of these key subunits of SWI/SNF chromatin remodeling complexes in both cancers is associated with chemotherapy resistance and poor prognosis. Here, we discover that SMARCA4/2 loss inhibits chemotherapy-induced apoptosis through disrupting intracellular organelle calcium ion (Ca2+) release in these cancers. By restricting chromatin accessibility to ITPR3, encoding Ca2+ channel IP3R3, SMARCA4/2 deficiency causes reduced IP3R3 expression leading to impaired Ca2+ transfer from the endoplasmic reticulum to mitochondria required for apoptosis induction. Reactivation of SMARCA2 by a histone deacetylase inhibitor rescues IP3R3 expression and enhances cisplatin response in SMARCA4/2-deficient cancer cells both in vitro and in vivo. Our findings elucidate the contribution of SMARCA4/2 to Ca2+-dependent apoptosis induction, which may be exploited to enhance chemotherapy response in SMARCA4/2-deficient cancers.

Pre-Clinical Efficacy of Combined Therapy with Novel β-Catenin Antagonist BC2059 and Histone Deacetylase Inhibitor Against Cultured and Primary AML Blast Progenitor Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 287-287
Author(s):  
Kapil Bhalla ◽  
Warren Fiskus ◽  
Stephen K. Horrigan ◽  
Sunil Abhyankar ◽  
Joseph McGuirk ◽  
...  
Keyword(s):  
Cyclin D1 ◽  
Progenitor Cells ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitor ◽  
Nuclear Accumulation ◽  
Hematopoietic Stem ◽  
Tail Vein ◽  
Deacetylase Inhibitor ◽  
Induced Apoptosis

Abstract Abstract 287 The canonical WNT-β-catenin pathway is essential for self-renewal, growth and survival of AML stem and early blast progenitor cells (BPCs). Deregulated WNT signaling in AML inhibits polyubiquitylation and proteasomal degradation of β-catenin by the multi-protein complex. The inactivation of the degradation complex for β-catenin results in the preservation, nuclear translocation and activity of β-catenin in AML BPCs. This is especially notable in AML BPCs expressing FLT3-ITD, where increased activity of PI3K/AKT phosphorylates and inactivates GSK3β, thereby inhibiting phospho-degradation, increasing stability and nuclear accumulation of β-catenin. Activated FLT3 kinase has also been shown to directly phosphorylate β-catenin and promote its stabilization and nuclear localization. As a co-activator, in the nucleus β-catenin interacts with the bipartite T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factor, which increases the expression of pro-growth and pro-survival genes, including cyclin D1, Myc and survivin. Aberrant expression of LEF1 in hematopoietic stem cells has been shown to induce AML. β-catenin is also required for the HOXA9 and MEIS1 or MLL-AF9-mediated transformation of hematopoietic stem/progenitor cells. Thus, reactivation of β-catenin is required for maintenance of leukemic transformation making it an attractive drug target in AML. Here, we determined the in vitro and in vivo anti-AML activity of BC2059 (β-Cat Pharmaceuticals), a potent, small molecule, anthraquinone oxime-analog, against cultured (HL-60, OCI-AML3 and MV4-11 cells) and primary AML BPCs with or without the expression of FT3-ITD. First, as compared to the normal human CD34+ cells, FLT3-ITD expressing AML BPCs showed increased levels of β-catenin, mostly in the nucleus, as determined by confocal immunofluorescence microscopy. Exposure to 100 nM of BC2059 (BC) induced β-catenin degradation through the proteasome, as well as attenuated the nuclear and cytoplasmic levels of β-catenin in the cultured and primary AML BPC. Chromatin immunoprecipitation with anti-β-catenin antibody demonstrated that treatment with BC2059 (BC) reduced the binding of β-catenin to the WNT response elements (WRE) in the promoter DNA of its target genes, including Myc, cyclin D1 and survivin. Estimation of the intracellular luciferase levels in AML cells transfected with the TOP/FLASH versus FOP/FLASH construct showed that treatment with BC2059 significantly reduced only the TOP-FLASH luciferase activity, indicating that BC inhibits the expression of genes with promoters containing WRE elements. This was associated with reduced mRNA and protein levels of cyclin D1, MYC and survivin. Treatment with BC dose-dependently induced apoptosis of cultured and primary AML BPCs (up to 70%), including apoptosis of the CD34+CD38-Lin- AML BPCs. In contrast, BC induced apoptosis in < 10% of normal CD34+ progenitor cells. We have previously reported that treatment with the histone deacetylase inhibitor panobinostat (PS) attenuates p-FLT3, p-AKT and p-GSK3β levels in AML BPCs expressing FLT3-ITD. Consistent with this, here, we determined that co-treatment with BC and PS (10 to 20 nM) synergistically induced apoptosis of cultured and primary AML BPCs. Against primary AML BPCs expressing FLT3-ITD, co-treatment with the FLT3 kinase inhibitor AC220 (100 nM) further augmented BC mediated depletion of the cytoplasmic and nuclear levels of β-catenin and significantly enhanced BC-induced apoptosis (p < 0.01). This was associated with induction of BIM and p27 with depletion of MCL-1 levels. Following tail vein infusion and establishment of AML by OCI-AML3 cells in NOD-SCID mice, treatment with BC (5 or 10 mg/kg b.i.w, IV) for three weeks demonstrated improved survival of the mice compared to the vehicle control treated mice (p <0. 001). Survival was further improved upon co-treatment with BC and PS (5 mg/kg IP, MWF). BC treatment (5 or 10 mg/kg IV) also dramatically improved survival of the NOD/SCID/IL2Rγ-depleted mice with established human AML following tail-vein injection of primary AML BPCs expressing FLT3 ITD. Mice did not experience any toxicity or weight loss. These findings support a compelling rationale for further development and in vivo testing of the BC-based combination with PS and FLT3 antagonist against human AML BPCs. Disclosures: Horrigan: BetaCat Pharmaceuticals: Employment. Sharma:BetaCat Pharmaceuticals: Equity Ownership.

Combined effect of all-trans retinoic acid and arsenic trioxide in acute promyelocytic leukemia cells in vitro and in vivo

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 264-269 ◽  
Author(s):  
Yongkui Jing ◽  
Long Wang ◽  
Lijuan Xia ◽  
Guo-qiang Chen ◽  
Zhu Chen ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Induced Apoptosis

Abstract All-trans retinoic acid (tRA) and arsenic trioxide (As2O3) induce non–cross-resistant complete clinical remission in patients with acute promyelocytic leukemia with t(15;17) translocation and target PML-RARα, the leukemogenic protein, by different pathways suggesting a possible therapeutic synergism. To evaluate this possibility, this study examined the effect of As2O3 on tRA-induced differentiation and, conversely, the effect of tRA on As2O3-induced apoptosis. As2O3 at subapoptotic concentrations (0.5 μM) decreased tRA-induced differentiation in NB4 cells but synergized with atRA to induce differentiation in tRA-resistant NB4 subclones MR-2 and R4 cells as measured by nitroblue tetrazolium reduction and tRA-inducible genes (TTGII, RARβ, RIG-E). tRA cleaved PML-RARα into distinct fragments in NB4 but not in tRA-resistant MR-2 or R4 cells, whereas As2O3 completely degraded PML-RARα in all 3 cell lines. As2O3-induced apoptosis was decreased by tRA pretreatment of NB4 cells but not of R4 cells and was associated with a strong induction of Bfl-1/A1 expression, a Bcl-2 protein family member. Severe combined immunodeficient mice bearing NB4 cells showed an additive survival effect after sequential treatment, but a toxic effect was observed after simultaneous treatment with tRA and As2O3. These data suggest that combined As2O3 and tRA treatment may be more effective than single agents in tRA-resistant patients. Although in vitro data do not always translate to in vivo response, toxicity and potential drug antagonism may be diminished by decreasing the concentration of As2O3 when given at the same time with therapeutic levels of tRA.

Complete remission through blast cell differentiation inPLZF/RARα-positive acute promyelocytic leukemia: in vitro and in vivo studies

Blood ◽  
2002 ◽  
Vol 100 (3) ◽  
pp. 1065-1067 ◽  
Author(s):  
Maria C. Petti ◽  
Francesco Fazi ◽  
Massimo Gentile ◽  
Daniela Diverio ◽  
Paolo De Fabritiis ◽  
...  
Keyword(s):  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
In Vivo Studies ◽  
Atra Treatment ◽  
Genetic Studies ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Deacetylase Inhibitor

Abstract Acute leukemia with the t(11;17) expressing the PLZF-RARαgene fusion is a rare variant of acute promyelocytic leukemia (APL) that has been associated with poor clinical response to all-trans retinoic acid (ATRA) treatment. However, some recent reports have put into question the absolute refractoriness of this leukemia to ATRA. We describe here a patient withPLZF/RARα APL who was treated at relapse with ATRA and low-dose hydroxyurea. Complete hematologic remission was obtained through differentiation of leukemic blasts, as proven by morphologic, immunophenophenotypic, and genetic studies carried out in sequential bone marrow samples. Moreover, in vitro studies indicated that blast differentiation was potentiated by the addition of the histone deacetylase inhibitor tricostatin A, but not of hydroxyurea, to ATRA. Our findings indicate that the maturation block may be overcome and terminal differentiation obtained in this leukemia subset and support the view that sensitivity/refractoriness of this form to ATRA should be revisited.

Combination of the mTOR inhibitor rapamycin and CC-5013 has synergistic activity in multiple myeloma

Blood ◽  
2004 ◽  
Vol 104 (13) ◽  
pp. 4188-4193 ◽  
Author(s):  
Noopur Raje ◽  
Shaji Kumar ◽  
Teru Hideshima ◽  
Kenji Ishitsuka ◽  
Dharminder Chauhan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mtor Inhibitor ◽  
Mtor Inhibitors ◽  
Mitogen Activated Protein Kinase ◽  
Synergistic Activity ◽  
Growth And Survival ◽  
Mitogen Activated Protein ◽  
Induced Apoptosis

Abstract Previous studies have demonstrated the in vitro and in vivo activity of CC-5013 (Revlimid), an immunomodulatory analog (IMiD) of thalidomide, in multiple myeloma (MM). In the present study, we have examined the anti-MM activity of rapamycin (Rapamune), a specific mTOR inhibitor, combined with CC-5013. Based on the Chou-Talalay method, combination indices of less than 1 were obtained for all dose ranges of CC-5013 when combined with rapamycin, suggesting strong synergism. Importantly, this combination was able to overcome drug resistance when tested against MM cell lines resistant to conventional chemotherapy. Moreover, the combination, but not rapamycin alone, was able to overcome the growth advantage conferred on MM cells by interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1), or adherence to bone marrow stromal cells (BMSCs). Combining rapamycin and CC-5013 induced apoptosis of MM cells. Differential signaling cascades, including the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3′-kinase/Akt kinase (PI3K/Akt) pathways, were targeted by these drugs individually and in combination, suggesting the molecular mechanism by which they interfere with MM growth and survival. These studies, therefore, provide the framework for clinical evaluation of mTOR inhibitors combined with IMiDs to improve patient outcome in MM.

Intra-Pathway Inhibition of Upstream (PI3K) and Downstream (mTOR) Kinases Synergistically Induces Apoptosis in AML.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2477-2477
Author(s):  
Zhihong Zeng ◽  
Zeev Estrov ◽  
David Harris ◽  
Frank Giles ◽  
Michael Andreeff ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Mammalian Target Of Rapamycin ◽  
Leukemia Cell ◽  
Mtor Inhibitors ◽  
Phosphatidylinositol 3 Kinase ◽  
Growth And Survival ◽  
Downstream Target ◽  
Pathway Inhibition ◽  
Induced Apoptosis

Abstract Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway regulates the growth and survival of acute myeloid leukemia (AML). We hypothesized that targeting this pathway with both PI3K and mTOR inhibitors may greatly enhance the effectiveness of these two inhibitors in the treatment of AML. PI3KI1 is a novel PI3K inhibitor that induced apoptosis in AML cell lines and primary AML cells at an IC50 of 5μM. It directly inhibited AKT at Ser473, however had limited effects on pGSK3b and on the mTOR downstream target p70S6K at Thr389. Colony-forming assays demonstrated that PI3KI1 decreased the viability of primary AML samples but spared normal bone marrow progenitor cells. mTOR inhibitor CCI779 inhibited phosphorylation of downstream mTOR targets p70S6K and 4EBP, however showed only minor cytotoxicity to AML cell lines and primary samples, suggesting that inhibition of mTOR signaling is not sufficient to cause growth inhibition in the majority of AML. Combined use of PI3KI1 and CCI779 synergistically induced apoptosis in U937 cells, with a combination index of 0.061±0.02. Western blot analysis demonstrated enhanced suppression of pP70S6K, pAKT and p4EBP1(Thr70) when PI3KI1 and CCI779 were used in combination. In primary AML samples, combined inhibition of PI3K and mTOR pathways enhanced apoptosis induction in 8/12 samples, with true synergistic responses in 3 samples. Importantly, the combination, but not PI3KI1 or CCI779 alone, was able to overcome the growth advantage conferred to AML cell lines or primary AML samples by adherence to bone marrow stromal cells. Taken together, our results indicate that PI3K and mTOR are relevant molecular targets in AML and that intra-pathway inhibition of both, upstream and downstream proteins may be required for maximal inhibition of leukemia cell growth.

scholarly journals Regulatory T cells are less sensitive to glucocorticoid hormone induced apoptosis than CD4+ T cells

APOPTOSIS ◽  
2020 ◽  
Vol 25 (9-10) ◽  
pp. 715-729 ◽  
Author(s):  
Lilla Prenek ◽  
Tímea Litvai ◽  
Noémi Balázs ◽  
Réka Kugyelka ◽  
Ferenc Boldizsár ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Treg Cells ◽  
High Dose ◽  
Apoptosis Resistance ◽  
Apoptotic Pathway ◽  
Glucocorticoid Hormone ◽  
Induced Apoptosis

Abstract Earlier we have reported that thymic regulatory T cells (Treg) are resistant to in vivo glucocorticoid hormone (GC)-induced apoptosis, while the most GC-sensitive DP thymocytes died through the activation of mitochondrial apoptotic pathway. Here we analyzed the apoptosis-inducing effect of high dose (10–6 M) in vitro dexamethasone (DX) treatment in mouse thymic- and splenic Tregs and CD4+ T cells. Activation of both extrinsic and intrinsic apoptotic pathways started after 2 h of DX treatment in CD4 SP thymocytes and was 3 × higher than in CD4+ splenocytes, while in Treg cells, weak activation of the extrinsic apoptotic pathway started only after 3 h. We also investigated the expression of 21 apoptosis-related molecules using a protein array and found higher level of both pro-and anti-apoptotic molecules in Tregs compared to CD4+ T cells. 4 h in vitro DX treatment induced upregulation of most apoptosis-related molecules both in Tregs and CD4+ T cells, except for the decrease of Bcl-2 expression in CD4+ T cells. We found high basal cytosolic Ca2+ levels in untreated Treg cells, which further increased after DX treatment, while the specific TCR-induced Ca2+ signal was lower in Tregs than in CD4+ T cells. Our results suggest that in the background of the relative apoptosis resistance of Treg cells to GCs might be their high basal cytosolic Ca2+ level and upregulated Bcl-2 expression. In contrast, downregulation of Bcl-2 expression in CD4+ T cells can explain their higher, DX-induced apoptosis sensitivity.

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