Pharmacological IRS1/2 Inhibition Induces Apoptosis in BCR-ABL1T315I mutant Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1886-1886
Author(s):  
Renata Scopim-Ribeiro ◽  
João Agostinho Machado-Neto ◽  
Christopher A. Eide ◽  
Paula de Melo Campos ◽  
Priscila Santos Scheucher ◽  
...  

Abstract Introduction: Chronic myeloid leukemia (CML) is a hematological malignancy associated with the BCR-ABL1 oncoprotein, which results in deregulated kinase activity-driven leukemic phenotype. Most CML cases are successfully treated with tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1, but a significant percentage of patients develop drug resistance and/or progress to blast crisis, a disease stage that is refractory to therapy. Furthermore, recent studies have provided evidence that the CML leukemia stem cell is resistant to imatinib treatment. In this sense, the identification of other crucial proteins that cooperate with BCR-ABL1 signaling pathways may represent secondary therapeutic targets optimize treatment strategies. Increased IRS1 mRNA expression has been previously identified as positively correlated with worse overall survival in BCR-ABL1-positive adult acute lymphoblastic leukemia. In K562 cells, IRS1 has been identified as a binding partner of BCR-ABL1 and is capable of activating the PI3K/Akt/mTOR and MAPK pathways. Recently, a pharmacological IRS1/2 inhibitor (NT157) has been developed and has shown promising results in preclinical studies on solid tumors. We have previously shown that NT157 treatment reduces colony formation of primary CML cells but not in normal cells, decreases cell viability and proliferation of K562 cells, and increases apoptosis of K562 cells in a time- and dose-dependent manner (ASH 2015 - Abstract #83876). To further characterize the mechanism of the sensitivity of CML cells to IRS1/2 inhibition, here we performed PCR-based gene expression analyses in CML cells in response to NT157 treatment and investigated the effects of IRS1/2 inhibition on cell proliferation and apoptosis in Ba/F3 cells expressing wild-type or T315I mutant BCR-ABL1. Materials and Methods: K562 cells treated with vehicle or with the IRS1/2 pharmacological inhibitor NT157 (1.6 µM) for 48 hours were submitted to PCR array analysis using the PI3K-AKT Signaling Pathway and CancerPathwayFinder RT2 Profiler PCR Array kit (#PAHS-033A and #PAHS-058A, respectively; SA Biosciences, Frederick, MD, USA). Each cDNA sample was processed in a 96-well plate containing 156 signaling pathway-related genes and 5 endogenous control genes. Ba/F3 parental, BCR-ABL1WT and BCR-ABL1T315I cells were subjected to IRS1/2 pharmacological inhibition using NT157 (0.2, 0.4, 0.8, 1.6 and 3.2 µM) for 24, 48 and 72 hours and were evaluated for cell viability (MTT assay), apoptosis (annexin V/PI), and protein expression/activation (Western blot). Statistical analyses were performed by Student's t-test or Mann-Whitney test, as appropriate. Results: Sixteen genes were found to be differentially expressed (fold change ≥ 2); 5 upregulated genes (FOXO3A, CDKN1A, FOS, JUN, VEGF) and 1 downregulated gene (E2F1) were chosen for validation in a larger number of experiments. Notably, NT157 treatment also resulted in significant dose- and time-dependent decrease in viability in BCR-ABL1-expressing cells regardless of BCR-ABL1 mutation status, compared to parental Ba/F3 cells. NT157 treatment (1.6 µM) for 48 hours reduced cell viability of Ba/F3 BCR-ABL1WT and BCR-ABL1T315I cells to 64% and 76%, respectively (p<0.05). Identical treatment with NT157 also significantly induced apoptosis as noted by the percentage of cells positive for annexinV/PI staining compared to vehicle in Ba/F3 BCR-ABL1WT and BCR-ABL1T315I cells (84 vs. 13% and 92 vs. 22%, respectively; p<0.05). Similar results were observed after 24 and 72 hours of NT157 treatment for both cell lines. More importantly, NT157 treatment reduced phosphorylation of STAT3 at both serine (S)727 and tyrosine (Y)705 in Ba/F3 BCR-ABL1T315I in the absence of IL3. Conclusion: Pharmacological inhibition of IRS1/2 decreases cell viability, increases apoptosis, and reduces STAT3 activation in cells expressing wild-type or T315I-mutant BCR-ABL1 in a time- and dose-dependent manner. These findings suggest that targeting of IRS1/2 alone or in combination with ABL1 TKIs may offer further improved treatment strategies and outcomes for maximizing control of disease resistance and persistence in patients with CML. Disclosures Druker: Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy.

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2178
Author(s):  
Fabio Morandi ◽  
Veronica Bensa ◽  
Enzo Calarco ◽  
Fabio Pastorino ◽  
Patrizia Perri ◽  
...  

Neuroblastoma (NB) is the most common extra-cranial solid tumor of pediatric age. The prognosis for high-risk NB patients remains poor, and new treatment strategies are desirable. The olive leaf extract (OLE) is constituted by phenolic compounds, whose health beneficial effects were reported. Here, the anti-tumor effects of OLE were investigated in vitro on a panel of NB cell lines in terms of (i) reduction of cell viability; (ii) inhibition of cell proliferation through cell cycle arrest; (iii) induction of apoptosis; and (iv) inhibition of cell migration. Furthermore, cytotoxicity experiments, by combining OLE with the chemotherapeutic topotecan, were also performed. OLE reduced the cell viability of NB cells in a time- and dose-dependent manner in 2D and 3D models. NB cells exposed to OLE underwent inhibition of cell proliferation, which was characterized by an arrest of the cell cycle progression in G0/G1 phase and by the accumulation of cells in the sub-G0 phase, which is peculiar of apoptotic death. This was confirmed by a dose-dependent increase of Annexin V+ cells (peculiar of apoptosis) and upregulation of caspases 3 and 7 protein levels. Moreover, OLE inhibited the migration of NB cells. Finally, the anti-tumor efficacy of the chemotherapeutic topotecan, in terms of cell viability reduction, was greatly enhanced by its combination with OLE. In conclusion, OLE has anti-tumor activity against NB by inhibiting cell proliferation and migration and by inducing apoptosis.


Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2937-2937
Author(s):  
Manujendra N Saha ◽  
Hua Jiang ◽  
Yijun Yang ◽  
Donna Reece ◽  
Hong Chang

Abstract Abstract 2937 Mutation of p53, a tumor suppressor protein, is relatively rare (∼10% in newly diagnosed patients) in multiple myeloma (MM). However, p53 mutations/deletions are important risk factors for predicting the resistant to chemotherapy and no treatment is currently available for this subgroup of patients. MIRA-1, a novel class of small molecules with the ability to restore wild type conformation and function to mutant p53, induces apoptosis in different types of solid tumors harboring mutant p53. However, its effect on MM cells is not known. In this study we examined the ability of MIRA-1 to induce cytotoxic and apoptotic response in MM cells and inhibit tumor growth in MM mouse xenograft model. In addition, we explored the molecular mechanisms of MIRA-1-induced apoptosis in MM cells. Treatment of MM cells with MIRA-1 resulted in a time- and dose-dependent decrease in survival and increase in apoptosis of MM cells harboring either wild type (MM.1S, H929) or mutant (U266, 8226, and LP1) p53 suggesting that MIRA-induced apoptosis in MM cells is independent of p53 status. The IC50 of MIRA-1 observed in these cells was ranged between 10 and 15 μM. In addition, MIRA-1 elicited a dose-dependent inhibition of myeloma cell growth in seven primary MM samples with an average IC50of 10 μM. Two of the seven patient samples harbors p53 mutations/deletions. In contrast, MIRA-1 did not have a significant inhibitory effect on the survival of bone marrow or peripheral blood mononuclear cells obtained from three healthy donors at the concentrations (10–20 μM) that induced apoptosis of MM cells, indicating a preferential killing of myeloma cells by this drug. Apoptosis induced by MIRA-1 in MM cells harbouring either wild type or mutant p53 was associated with time- and dose-dependent activation of caspas-8, caspase-3 and PARP with subsequent up-regulation of a pro-apoptotic protein, Noxa and down-regulation of an anti-apoptotic protein, Mcl-1. Interestingly, MIRA-1 did not significantly modulate the level of p53 expression, although immunoprecipitation studies confirmed the restoration of wild type conformation of mutant p53 in LP1 and 8226 cells. Importantly, genetic knockdown of p53 using siRNA against wild type or mutant p53 had only a little effect on apoptosis induction by MIRA-1 in MM.1S or LP1 cells, respectively, confirming that apoptosis induction by MIRA-1 in MM cells is independent of p53. Furthermore, the combination of MIRA-1 with current anti-myeloma agents, dexamethasone or doxorubicin displayed synergistic cytotoxic response in MM.1S or LP1 cells (CI<1; p<0.05). To delineate the molecular mechanisms of apoptosis in MM cells induced by MIRA-1, we performed RT2 profiler PCR array analysis for the differential expression of 84 genes related to mitogen activated protein kinase (MAPK) signaling pathway. A significant number of genes of the MAPK family including MAP3K: MAP3K2 (MEKK2), MAP3K4 (MEKK4), PAK1; MAP2K: MAP2K5 (MEK5); and MAPK: MAPK11 (p38bMAPK) as well as transcription factors such as c-Jun, c-FOS, EGR1, and MKNK1, whose expression is induced by MAPK signaling, were up-regulated by more than 2-fold in MIRA-1-treated 8226 cells. On the other hand, expression of the scaffolding/anchoring genes, MAPK8IP2 (JIP-1) was down-regulated by ∼2-fold. Up-regulations of c-Jun, c-Fos, and EGR1 at their protein levels were further confirmed by Western blot analysis of MM.1S and 8226 cells treated with MIRA-1. Importantly, Western blot analysis revealed that treatment of MIRA-1 resulted in a time- and dose-dependent increase of phosphorylated p38 MAPK level in both MM.1S and 8226 cells. Taken together, our data indicates that activation of the MAPK signaling pathway is, at least in part, associated with MIRA-1-induced apoptosis of MM cells. Finally, we evaluated anti-tumorigenic potential of MIRA-1 in MM xenograft SCID mouse models. 8266 cells were inoculated into SCID mice and the mice received i.p. injections of either 100 μL PBS (control) or 10 mg/kg MIRA-1 once daily for 18 days after tumor formation was evident. Administration of MIRA-1 resulted in significant inhibition of tumor growth (p<0.05) and increase in survival (p=0.007) of the mice with no apparent toxicity. Our study for the first time demonstrates potent in vitro and in vivo anti-myeloma activity of MIRA-1 and thus providing a framework for clinical evaluation of MIRA-1 either alone or in combination with current anti-myeloma agents. Disclosures: Reece: Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Millennium Pharmaceuticals: Research Funding.


Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5428-5428 ◽  
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Yuko Tanaka ◽  
Kazuma Ohyashiki

Abstract Introduction: Chronic myeloid leukemia (CML) is characterized by the t(9:22) translocation known as the Philadelphia chromosome (Ph). Although ABL tyrosine kinase inhibitors (ABL TKI) such as imatinib, dasatinib and nilotinib have improved CML treatment, such therapies cannot cure patients with Philadelphia chromosome (Ph)-positive leukemia because of leukemia stem cells. Moreover, some patients develop BCR-ABL point mutations including T315I and become resistant to ABL TKI therapy. These leukemia stem cells are contained within a niche in the bone marrow and are often impervious to current treatments. Therefore, new approach against BCR-ABL mutant cells and LSCs may improve the outcome of Ph-positive leukemia patients. B cell lymphoma 2 (BCL-2) protein families are key regulator of apoptosis and highly promising targets for the development of anti-cancer treatment. Venetoclax, also known as ABT-199 is a selective, orally bioavailable BCL-2 inhibitor. Venetoclax is investigated in a pivotal phase 3 clinical trial against hematological malignancies such as chronic lymphocytic leukemia (CLL) and approved for the treatment of patients with CLL. Materials and methods: In this study, we investigated whether venetoclax could suppress Ph-positive leukemia cells including T315I mutation and primary samples. Results: BCL-2 expression was found in Ph-positive leukemia cells including primary samples, however, BCL-2 expression was reduced in K562 cells. We found 72 h venetoclax treatment inhibited the growth of Ba/F3 BCR-ABL and KCL-22 cells in a dose dependent manner. However, venetoclax activity was reduced in K562 cells. We examined the intracellular signaling after treatment of venetoclax. Phosphorylation of BCR-ABL and Crk-L was not reduced. However, activity of caspase 3, poly (ADP-ribose) polymerase (PARP) was increased. We next investigated the efficacy between ABL TKI and venetoclax by using these cell line. Combined treatment of Ba/F3 BCR-ABL cells with imatinib and venetoclax caused significantly more cytotoxicity than each drug alone. Apoptotic cells were also increased. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved caspase 3 and PARP activity was increased after imatinib and venetoclax treatment. We investigated the venetoclax activity against T315I positive cells. Venetoclax potently induced cell growth inhibition of Ba/F3 T315I mutant cells in a dose dependent manner. Combined treatment of Ba/F3 T315I mutant cells with ponatinib and venetoclax caused significantly more cytotoxicity than each drug alone. Apoptotic cells were also increased. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved PARP was increased after ponatinib and venetoclax treatment. To assess the activity of ponatinib and venetoclax, we examined tumor formation in mice model. We injected subcutaneously 1×107 Ba/F3 T315I mutant cells in nude mice. A dose of 20 mg/kg/day p.o of ponatinib and 50 mg/kg/day p.o of venetoclax inhibited tumor growth and reduced tumor volume compared with control mice. In the immunohistochemical analysis, we found that tumors in mice treated with ponatinib and venetoclax exhibited an increase in apoptotic cells. We also found that co-treatment with ponatinib and venetoclax increased mouse survival. The treatments were well tolerated with no animal health concerns observed. We also found that the treatment of venetoclax exhibits cell growth inhibition against CD34 positive CML samples. Conclusion: The results of our study indicate that the BCL-2 inhibitor venetoclax may be a powerful strategy against ABL TKI resistant cells including T315I mutation and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. Disclosures Tauchi: Pfizer Inc.: Research Funding. Ohyashiki:Novartis International AG,: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding.


Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2772-2772
Author(s):  
Renata Scopim-Ribeiro ◽  
João Agostinho Machado-Neto ◽  
Paula de Melo Campos ◽  
Adriana da Silva Santos Duarte ◽  
Priscila Santos Scheucher ◽  
...  

Abstract Introduction: Chronic myeloid leukemia (CML) is a hematological malignancy associated with the BCR-ABL1 fusion gene, which drives the proliferative disease phenotype by activating multiple signaling pathways. Most CML cases are successfully treated with tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1. However, in some cases, drug resistance limits TKIs efficacy, and the identification of other crucial proteins in the BCR-ABL1 signaling pathways may contribute to optimize anti-CML approaches. IRS1 mRNA expression has been previously identified as positively correlated with overall survival in BCR-ABL1-positive adult acute lymphoblastic leukemia. In K562 cells, IRS1 has been identified as a binding partner of BCR-ABL1 protein and was capable of activating PI3K/Akt/mTOR and MAPK pathways. Recently, a pharmacological IRS1/2 inhibitor (NT157) has been developed and has shown promising results in preclinical studies on solid tumors. We herein aimed to investigate IRS1 and IRS2 expression and the effects of IRS1/2 inhibition on cell proliferation, apoptosis and clonogenicity in BCR-ABL1 positive and normal hematopoietic cells. Materials and Methods: Total bone marrow cells from healthy donors (n=11) and CML patients at the time of diagnosis (n=24) were submitted to gene expression analysis by quantitative PCR with specific primers for IRS1, IRS2 and β-actin. All subjects provided informed written consent and the study was approved by the ethics committee of the Institution. K562 cells were submitted to IRS1/2 pharmacological inhibition using NT157 (0.2, 0.4, 0.8, 1.6, 3.2 and/or 6.4 µM) for 24, 48 and 72 hours and were evaluated for cell viability (MTT assay), proliferation (Ki-67), apoptosis (Annexin V/PI), and protein expression/activation (Western blot). Alternatively, cells were submitted to IRS1 and IRS2 gene silencing using specific shRNA lentiviral delivery, and submitted to functional studies. NT157 effects were analyzed by in vitro hematopoietic colony formation of bone marrow cells from two patients with CML at diagnosis, and of normal cord blood cells from one individual. Cells were seeded at 4.5x104 per well in a culture system for 14 days. Statistical analyses were performed by Student's t-test or Mann-Whitney test, as appropriate. Results: IRS1 and IRS2 mRNA expression was similar between normal donors and CML samples (p ≥.05). NT157 treatment reduced K562 cell viability in a time and dose-dependent manner; using a nonlinear regression analysis, IC50 for cytotoxicity was 9.8, 0.6 and 0.68 µM for 24, 48 and 72 hours, respectively. NT157 0.8 and 3.2 µM reduced cell viability in 14% and 19% at 24 hours, 50% and 61% at 48 hours and in 59% and 68% at 72 hours of treatment (all p <.05). After 48 hours of NT157 exposure, Ki-67 staining revealed a decrease in cell proliferation by 10% at 0.8 µM, 40% at 1.6 µM, and 75% at 3.2 µM. Pharmacological IRS1/2 inhibition significantly induced apoptosis as noted by increased cleaved caspase 3 and 9 by Western blotting analysis, and AnnexinV/PI staining. The percentage of apoptotic (AnnexinV+) cells for control, NT157 0.8 and 3.2 µM at 48 hours of treatment were 15%, 38% and 61%, respectively (p<.05). Upon NT157 0.8, 3.2 and 6.4 µM, colony formation of CML primary cells was inhibited by 7%, 36% and 78% for patient #1, and by 29%, 19% and 62% for patient #2, with a reduction predominance in granulomonocytic colonies for both patients. Interestingly, NT157 treatment did not inhibit colony formation of committed normal cord blood cells; the number of colonies for control, NT157 0.8, 3.2 and 6.4 µM were 88, 84, 97, and 92, respectively. Of note, lentiviral-mediated silencing of IRS1, but not of IRS2, significantly decreased K562 cell viability. Conclusion: Although IRS gene expression analysis did not differ between CML patients and normal controls, the functional studies indicate that IRS protein activation status is implicated in the biology of CML cells. NT157 pharmacological IRS1/2 inhibition (i) reduces colony formation in primary CML, but not in normal cells, (ii) decreases cell viability and proliferation, and (iii) increases apoptosis of K562 cells in a time and dose-dependent manner. Since IRS2 lentiviral-mediated silencing did not reduce K562 cell viability, IRS1 inhibition may be the main mechanism by which NT157 exerts its effects on BCR-ABL1 positive cells. NT157 IRS1/2-targeting may optimize the anti-CML approaches. Disclosures No relevant conflicts of interest to declare.


Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 245-245
Author(s):  
Stephen M Ansell ◽  
Lucy S. Hodge ◽  
Frank Secreto ◽  
Michelle Manske ◽  
Esteban Braggio ◽  
...  

Abstract Massively parallel sequencing analyses have revealed a common mutation within the MYD88 gene (MYD88L265P) occurring at high frequencies in many non-Hodgkin lymphomas (NHL) including the rare lymphoplasmacytic lymphoma, Waldenström’s macroglobulinemia (WM). Using whole exome sequencing, Sanger sequencing and allele-specific PCR, we validate the initial studies and detect the MYD88L265P mutation in the tumor genome of 97% of WM patients analyzed (n=39). MYD88L265P was detected at lower frequencies in other indolent lymphomas including LPL (0%), MALT (4%), nodal MZL (5%) and splenic MZL (8%); all but one MYD88L265P was heterozygous. Due to the high frequency of MYD88 mutation in WM and other NHL, and its known effects on malignant B cell survival, therapeutic targeting of MYD88 signaling pathways may be useful clinically. However, while the effects of MYD88L265P on the activity of IRAK1/4 and NF-κB are have been studied previously, we are lacking a thorough characterization of the role of intermediary signaling proteins such as TRAF6 and TAK1 on the biology of MYD88L265P-expressing B cells. A better understanding of the proteins involved in MYD88L265P signaling may lead to the development of more targeted and effective therapeutic approaches. In an attempt to identify MYD88L265P –specific therapeutic targets we first wanted to characterize the role of intermediary signaling proteins that facilitate the downstream activation of NF-κB. Upon activation of TLRs or IL-1b receptors, MYD88 forms a homodimer and recruits IRAK1/4 and TRAF6 into a complex resulting in association and phosphorylation of TAK1 followed by activation of NF-κB. We monitored the formation of a complex comprised of MYD88, IRAK1, IRAK4 and TRAF6 and immunoprecipitation of either endogenous IRAK4 or IRAK1 revealed constitutive association of IRAK with TRAF6 and MYD88L265P. To assess if the formation of a MYD88L265P/IRAK/TRAF6 complex results in downstream activation of TAK1, constitutive TAK1 phosphorylation was measured and detected in all three cell lines that express MYD88L265P. An association between TAK1 and TRAF6, another measure of TAK1 activation, was also detectable. When a similar analysis of TAK1 was performed in DLBCL cells expressing wild-type MYD88, no phosphorylation of TAK1 was detected, nor was TAK1 associated with TRAF6. IRAK1, IRAK4, TAK1, TRAF6, and MYD88 were expressed at similar levels in all cell lines studied and therefore did not contribute the differences in MYD88 complex formation observed between cell lines. These studies were further confirmed using HEK 293T cells that were transduced with either a vector control plasmid or HA-tagged MYD88WT or MYD88L265P expression plasmids. Together, these studies suggest that MYD88L265P forms a complex with IRAK and TRAF6 resulting in constitutive activation of TAK1 and NF-κB. To confirm the significance of TAK1-mediated MYD88L265P signaling on lymphoma cell growth, the effect of the selective TAK1 inhibitor, (5Z)-7-Oxozeaenol, on cell proliferation was tested. All MYD88L265P-expressing cell lines were sensitive to TAK1 inhibition in a dose-dependent manner (0-10 μM). In contrast, NHL cells expressing MYD88WT were found to be insensitive to TAK1 inhibition. We next tested the impact of the TAK1 inhibitor on a MYD88L265P positive WM patient sample. Similar to what was seen in the WM cell lines, the TAK1 inhibitor inhibited WM cell growth and survival in a dose dependent manner. Additionally, the TAK1 inhibitor significantly reduced the level of IL-10 secreted by each of the cell lines. Together, these data suggest that MYD88L265P drives cell proliferation and cytokine secretion through a TAK1-dependent mechanism. In conclusion, we are the first to validate by NGS in a large patient cohort the high prevalence and specificity of MYD88L265P in WM. Cells harboring the L265P mutation but not wild-type MYD88 exhibit constitutive signaling leading to the hyperactivation of NF-κB. We have established the role of TAK1 as an integral component of MYD88L265P signaling in both WM and DLBCL cell. Our data suggest that targeting TAK1 clinically may be an effective strategy for the treatment of WM and other lymphomas driven by MYD88L265P signaling. Disclosures: Fonseca: millennium: Consultancy; amgen: Consultancy; Binding site: Consultancy; onyx: Consultancy; medtronic: Consultancy; Genzyme: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; lilly: Consultancy; Onyx: Research Funding; cylene: Research Funding.


Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4222-4222
Author(s):  
Koji Yada ◽  
Keiji Nogami ◽  
Tomoko Matsumoto ◽  
Takehisa Kitazawa ◽  
Kunihiro Hattori ◽  
...  

Abstract Activated protein C (APC) directly inactivates factor (F)Va and FVIIIa dependently of protein S (PS), and furthermore inactivates FVIIIa through cofactor function of FV. Any impairment in these inactivation pathways may increase thrombotic risk. A previous report described that the APC resistance (APCR) associated with FV-Leiden was mainly due to the impaired cofactor activity of FV (Castoldi et al. Blood 2004;103, 4173). We have recently reported that FV-Nara carrying W1920R mutation exhibited APCR caused by a loss of FVa susceptibility to APC and APC cofactor activity resulting in more serious thrombotic potential than FV-Leiden (Nogami et al. Blood 2014;123, 2420). The contribution of each inactivation pathway associated with FVIII(a) and/or FV(a) for the hemostatic regulation remains unclear, however. An ACE910, bispecific antibody (Ab) to FIXa and FX mimicking the functions of FVIII, exerts tenase activities without FVIII(a) (Kitazawa et al. Nature Medicine. 2012;18, 1570). In this study, to elucidate the contribution of each pathway, we investigated the regulatory mechanism(s) by APC utilizing ACE910. FXa generation with ACE910 (10μg/ml) or with FVIIIa (0.3 nM) in FVIII-deficient plasma (ΔFVIII) was evaluated by the addition of APC (0-0.5 nM), PS (5 nM), and FV (1 nM). FXa generation with FVIIIa was reduced by ~60% maximally in an APC dose-dependent manner, whilst that with ACE910 was unaffected, indicating that ACE910 itself was not susceptible to inactivation by APC through the cofactor activity of FV. Thrombin generation in ΔFVIII with ACE910 (0-30μg/ml) was enhanced in an ACE910 dose-dependent fashion and ~10-fold of peak thrombin (PeakTh) (442 ± 7.0 nM), equivalent to 77.5% of PeakTh in pool normal plasma (PNP), was observed with ACE910 (30μg/ml) compared to that without ACE910 (44.6 ± 5.0 nM). To investigate the effect of APC upon the hemostatic enhancement by ACE910 in ΔFVIII, thrombin generation in ΔFVIII mixed with ACE910 (30μg/ml) and APC (0-16 nM) was examined. PeakTh showed the APC-dependent reduction to 156 ± 22nM and the %inhibition was 64.7%. Subsequently, we evaluated the effects of APC on thrombin generation in PNP or in PNP with an anti-FVIII Ab (FVIIIAb) (16BU/ml) supplemented by ACE910. Of note, the %inhibition of PeakTh by APC in latter with ACE910 and FVIIIAb (64%) was greater than that in PNP alone (35%) by ~1.9-fold. To elucidate the contribution of APC-catalyzed inactivation of FV(a) to the hemostatic regulation, we evaluated the thrombin generation in FV-diluted plasma (FV-dil) consisting of FV-deficient plasma and FV (8nM) corresponded to ~25% of its physiological concentration mixed with or without ACE910 and FVIIIAb in the presence or absence of APC. The %inhibition of PeakTh by APC in FV-dil with ACE910 and FVIIIAb (46.5%), being reduced in comparison with that of PNP with both Abs, was greater than that in FV-dil alone (18.3%) by ~2.5-fold, indicating that the inhibition of thrombin generation by APC-catalyzed FV(a) inactivation was FV(a) dose-dependent but enhanced in the absence of FVIII(a). Furthermore, the effects on thrombin generation with APC (0-16 nM) in plasmas carrying FV-Leiden without or with FVIIIAb and ACE910 (30μg/ml) were examined. It was of surprise that PeakTh in latter with FVIIIAb and ACE910 reduced exponentially by ~40% in an APC dose-dependent manner, whilst APC showed any little effects upon thrombin generation in FV-Leiden plasma alone indicating APCR. The susceptibility index to APC calculated by the rate of inhibition with FVIIIAb and ACE910 was 0.174, greater than that with FV-Leiden plasma alone (0.085) by ~2-fold. These results strongly suggested that APC could regulate the hemostatic enhancement by inactivation of FV(a) even FV-Leiden in the absence of another substrate, FVIII(a). We conclude that APC potentiates its catalytic activity to FVa more in the absence of FVIII(a) than in the presence of FVIII(a) and this mechanism would play significant roles in the downregulation of coagulation as one of the breaking systems. Disclosures Yada: Chugai Pharmaceutica Co., Ltd: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.


Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1837-1837 ◽  
Author(s):  
Shengjian Huang ◽  
Loretta J. Nastoupil ◽  
Hui Guo ◽  
Taylor Bell ◽  
Makhdum Ahmed ◽  
...  

Abstract Background: Mantle cell lymphoma (MCL) accounts for 6% of all non-Hodgkin lymphoma and is a therapeutic challenge. Phosphoinositide-3 kinase (PI3K) has been shown to be an alternative survival pathway in relapsed/refractory MCL. KA2237 (designed by Karus Therapeutics Ltd, Oxfordshire, United Kingdom) is a dual inhibitor of the class I beta and delta isoforms of the 110 kDa catalytic subunit of PI3K. By selectively targeting PI3K-beta and -delta isoforms and preventing their activation, KA2237 may decrease proliferation and induce cell death in susceptible tumor cells. Methods: We assessed the effects of KA2237 on the in vitro cell proliferation of both ibrutinib-sensitive (Mino, Jeko-1, and Rec-1) and primary ibrutinib-resistant (Z-138 and Maver-1) cell lines, and acquired ibrutinib-resistant MCL cell line, Jeko-R. We also tested the viability of patient-derived xenograft (PDX) tumor cells to KA2237. We compared the efficacy of KA2237 with two other commercial PI3K inhibitors, duvelisib (IPI-145, Selleck) and idelalisib (Cal-101, Selleck). Also, we paired these three inhibitors (KA2237, duvelisib and idelalisib) each with ibrutinib to evaluate the potential synergistic effects of these combinations. Lastly, we also tested in vivo efficacy of KA2237 and its combination with ibrutinib in PDX tumor cells. Results: KA2237 inhibited cell proliferation in both ibrutinib-sensitive and ibrutinib-resistant cell lines in a dose-dependent and time-dependent manner. For Mino and Jeko-1, the IC50 was 4.8 uM and 2.9 uM and for Z-138 and Maver-1 cell lines, the IC50 was 0.6 uM and 0.1 uM, respectively. KA2237 also decreased cell viability of ibrutinib-sensitive and ibrutinib-resistant MCL PDX tumor cells. However, KA2237 did not decrease the cell viability of normal human peripheral blood mono-nuclear cells. KA2237 arrested phase G0/G1 in Rec-1 and Jeko-R cell lines. We detected the expression of PI3K isoforms in MCL, finding higher expression of PI3K β and δ in MCL-resistant cell lines as compared with sensitive cell lines. We found that KA2237 induced MCL cell apoptosis in a time-dependent and dose-dependent manner. In comparison with duvelisib and idelalisib, KA2237 achieved greater inhibition of cell viability, cell apoptosis and cell cycle arrest. Furthermore, we found synergistic effects of KA2237 and ibrutinib combination in several MCL cell lines and in PDX models. In an ibrutinib-resistant PDX model, KA2237 treated mice reduced tumor burden significantly compared with vehicle control, and higher tumor growth inhibition was achieved as compared with ibrutinib. Conclusion: The novel PI3K inhibitor, KA2237 may be a potential candidate for MCL therapy, especially in the ibrutinib-resistant cases. Disclosures Wang: Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Research Funding; BeiGene: Research Funding; Asana BioSciences: Research Funding; Kite Pharma: Research Funding; Celgene: Research Funding.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ryota Ko ◽  
Masahiko Hayashi ◽  
Miho Tanaka ◽  
Tomoaki Okuda ◽  
Chiharu Nishita-Hara ◽  
...  

AbstractWe evaluated the effects of ambient particulate matter (PM) on the corneal epithelium using a reconstructed human corneal epithelium (HCE) model. We collected two PM size fractions [aerodynamic diameter smaller than 2.4 µm: PM0.3–2.4 and larger than 2.4 µm: PM>2.4] and exposed these tissues to PM concentrations of 1, 10, and 100 µg/mL for 24 h. After exposure, cell viability and interleukin (IL) IL-6 and IL-8 levels were determined, and haematoxylin and eosin and immunofluorescence staining of the zonula occludens-1 (ZO-1) were performed on tissue sections. In addition, the effects of a certified reference material of urban aerosols (UA; 100 µg/mL) were also examined as a reference. The viability of cells exposed to 100 μg/mL UA and PM>2.4 decreased to 76.2% ± 7.4 and 75.4% ± 16.1, respectively, whereas PM0.3–2.4 exposure had a limited effect on cell viability. These particles did not increase IL-6 and IL-8 levels significantly even though cell viability was decreased in 100 μg/mL UA and PM>2.4. ZO-1 expression was reduced in a dose-dependent manner in all groups. Reconstructed HCE could be used as an in vitro model to study the effects of environmental PM exposure on ocular surface cell viability and inflammation.


2020 ◽  
Vol 63 (1) ◽  
Author(s):  
Yunjeong Gwon ◽  
Jisun Oh ◽  
Jong-Sang Kim

AbstractSulforaphane is a well-known phytochemical that stimulates nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant cellular response. In this study, we found that sulforaphane promoted cell proliferation in HCT116 human colon cancer cells expressing a normal p53 gene in a dose-dependent but biphasic manner. Since p53 has been reported to contribute to cell survival by regulating various metabolic pathways to adapt to mild stress, we further examined cellular responses in both p53-wild-type (WT) and p53-knockout (KO) HCT116 cells exposed to sulforaphane in vitro and in vivo. Results demonstrated that sulforaphane treatment activated Nrf2-mediated antioxidant enzymes in both p53-WT and p53-KO cells, decreased apoptotic protein expression in WT cells but increased in KO cells in a dose-dependent manner, and increased the expression of a mitochondrial biogenesis marker PGC1α in WT cells but decreased in KO cells. Moreover, a low dose of sulforaphane promoted tumor growth, upregulated the Nrf2 signaling pathway, and decreased apoptotic cell death in p53-WT HCT116 xenografts compared to that in p53-KO HCT116 xenografts in BALB/c nude mice. These findings suggest that sulforaphane can influence colon cancer cell proliferation and mitochondrial function through a crosstalk between the Nrf2 signaling pathway and p53 axis.


2020 ◽  
Vol 11 (2) ◽  
pp. 148-155
Author(s):  
Pinjari Hameeda ◽  
Sandeep Katti ◽  
Rajkishore Jammalamadugu ◽  
Kishore Bhatt ◽  
Malleswara Rao Peram ◽  
...  

Aim: To evaluate and compare the effect of curcumin (CUR) and Nano-curcumin (N-CUR) on human-derived mesenchymal stem cells (MSCs) in a dose-dependent manner. Materials and Methods: An experimental study performed with putative MSCs from a total of five systemically healthy subjects with chronic periodontitis. These putative MSCs were isolated by cell culture and were further characterized and identified by colony-forming unit assay and immunocytochemical analysis using cell surface markers CD105, CD146, CD45 and CD73. The identified MSCs were treated with different doses of CUR and N-CUR, and compared with α-minimum essential medium (α -MEM) for its cell viability by performing MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay for 48 and 72 hr. The statistically analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test and Bonferroni’s post hoc test. Results: Compared to the α-MEM group, both CUR and N-CUR treated cells have shown significantly ( P = .029) higher survival rate at lower concentration (0.1 and 0.5 µM/L), at 48 hr incubation. However, there was no statistically significant difference between the CUR and N-CUR groups on cell survival rate at both 48 and 72 hr incubation. When compared between the concentrations of the same group, significantly higher cell viability ( P = .001) was observed at lower concentrations (0.1, 0.5 µM/L) in both test groups after incubation for 48 and 72 hr. Conclusion: Both CUR and N-CUR have a dose-dependent effect on human derived MSCs survival when incubated for 48 hr, whereas N-CUR shows increased cell survival rate even at 72 hr of incubation. Although, the cautious use of CUR and N-CUR at higher concentrations is recommended.


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