Small Interfering RNA against Bcr-Abl Transcripts Sensitized Mutated T315I Cells to Nilotinib.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2188-2188
Author(s):  
Michael Koldehoff ◽  
Ahmet H Elmaagacli
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Interfering Rna ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
Cell Cycle Distribution ◽  
Multidrug Resistance Gene ◽  
Inhibition Of Proliferation ◽  
Interfering Rna

Abstract 2188 Poster Board II-165 Background: Selective inhibition of the BCR-ABL tyrosine kinase by RNA interference has been demonstrated in leukemic cells. Therefore, we evaluated the specific bcr-abl small interfering RNA (siRNA) silencing in BCR-ABL positive cell lines, including those resistant to imatinib (IM) and particularly those with the T315I mutation. Design and Methods: The factor-independent 32Dp210 bcr-abl oligoclonal cell lines and in human IM-resistant bcr-abl positive cells from different patients with leukemia disorders were investigated. The effects of bcr-abl siRNA or the combination of bcr-abl siRNA with both IM and nilotinib were compared with those of the ABL inhibitors IM and nilotinib. Results: Coadministration of bcr-abl siRNA with IM or nilotinib dramatically reduced the .{/MAIN;133}BCR-ABL expression in wild-type (wt) and mutated bcr-abl cells and increased the lethal capacity. The bcr-abl siRNA significantly induced apoptosis and inhibited proliferation in wt (p<0.0001) and mutated cells (H396P, T315I, p<0.0001) versus controls. Cotreatment of bcr-abl siRNA with IM or nilotinib resulted in an increased inhibition of proliferation and induction of apoptosis as compared to IM or nilotinib (p<0.0001) in T315I cells. Furthermore, the combination of bcr-abl siRNA with IM or nilotinib significantly (p<0.01) reversed multidrug resistance gene 1-dependent resistance of mutated cells. In T315I cells bcr-abl siRNA with nilotinib has shown powerful effect on the cell-cycle distribution. Conclusions: Our data suggest that silencing by bcr-abl siRNA with IM or nilotinib may be associated with an additive antileukemic activity against tyrosine kinase inhibitor-sensitive and –resistant BCR-ABL cells, and might be an alternative approaches to overcome BCR-ABL mutations. Disclosures: No relevant conflicts of interest to declare.

Combination of Crenolanib with Sorafenib Produces Synergistic Pro-Apoptotic Effects in FLT3-ITD-Inhibitor-Resistant Acute Myelogenous Leukemias with FLT3 Mutations

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3591-3591 ◽  
Author(s):  
Chen Gao ◽  
Weiguo Zhang ◽  
Rodrigo Jacamo ◽  
Abhijit Ramachandran ◽  
Donald Small ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Single Agent ◽  
Point Mutations ◽  
Type I ◽  
Type Ii ◽  
Apoptotic Effects ◽  
Resistant Cells

Abstract Abstract 3591 Activating mutations in the FLT3 gene, including internal tandem duplications (ITDs) and missense point mutations of the tyrosine kinase domain (TKD), are frequently observed in AML patients and confer poor prognosis (1). Targeting FLT3 ITD mutations using the multi-kinase inhibitor Sorafenib (a type II kinase inhibitor, which binds to inactive conformation of a kinase ATP pocket)(2) showed impressive anti-leukemia effects in FLT3-ITD mutated AML in Phase I/II clinical trials (3) However, resistance/relapse develops regularly during prolonged Sorafenib therapy (4), in part through acquired point mutations of TKD domains. We postulated that the conformational change of FLT3 protein resulting from acquired point mutations limits the accessibility of sorafenib and leads to resistance (5, 6). Recently, Crenolanib, a novel PDGFRβ tyrosine kinase inhibitor, showed impressive anti-tumor effects by targeting the active conformation of a kinase ATP pocket of FLT3 protein (a type I kinase inhibitor). Therefore, we hypothesize that targeting different sites of FLT3 protein simultaneously using different types of kinase inhbitors may be effective in overcoming sorafenib resistance. We here report that Crenolanib has anti-leukemic activity in Sorafenib-resistant cells which harbor both ITD and acquired TKD point mutations i, and that its combination with Sorafenib in Sorafenib-resistant cells exerts synergistic pro-apoptotic effects. The anti-leukemic activity of Crenolanib was assessed by measuring cell viability (trypan Blue exclusion) and apoptosis induction (annexin V/propidium iodide staining) in isogenic murine Ba/F3 AML cell lines with stable transfection of human FLT3-ITD mutations, in Sorafenib resistant Ba/F3-ITD-Res cells derived from long-term, low-dose exposure of Ba/F3-ITD to Sorafenib in vitro, which harbor N676D and Y842C mutations, and Sorafenib-resistant cell lines Ba/F3-ITD+676, Ba/F3-ITD+842 and Ba/F3-ITD+676/842 which carry ITD and TKD point mutations (N676D, Y842C and N676D/Y842C mutations, respectively). Effects of combinatorial regimen employing Crenolanib and Sorafenib were analyzed using CalcuSyn software (combination index (CI) : CI<1 = synergistic, CI>1 = antagonistic effects). Results show that single agent Crenolanib induced cell growth arrest in leukemia cells Ba/F3-ITD, Ba/F3-ITD+676, Ba/F3-ITD+842 and Ba/f3-ITD+842/676, at IC50s of 0.012, 0.012 0.037 and 0.038uM, respectively, and induced apoptosis (EC50s) at 0.17, 0.23, 0.19, and 0.22uM, respectively, after 72 hours of treatment. Western Blot showed that Crenolanib profoundly suppressed phosphorylation levels of FLT3 protein and its downstream targets ERK and AKT and induced cleavage of caspase 3. Sorafenib-resistant cells Baf3-ITD+Res and Baf3-ITD+842/676 (EC50s for Sorafenib were 4.2 ± 1.50 and 6.6 ± 0.53 μM, respectively) were exposed to submicromolar concentrations of Crenolanib and Sorafenib concomitantly for 48 h, resulting in impressive synergistic pro-apoptotic effects (CIs were 0.56 ± 0.12 and 0.36 ± 0.04, respectively), implying high synergistic potency of Type I and Type II FLT3 kinase inhibitors, when given concomitantly. In vivo experiments are in progress. Our findings provide therapeutic rationale for a combinatorial treatment strategy with Crenolanib and Sorafenib of FLT-ITD inhibitor-refractory AML. Disclosures: No relevant conflicts of interest to declare.

USP47 Is a New Target in Chronic Myelogenous Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1572-1572
Author(s):  
Hu Lei ◽  
Chunmin Ma ◽  
Zhen Liu ◽  
Shenmeng Gao ◽  
Li Zhou ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Cell Lines ◽  
Chronic Myelogenous Leukemia ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Myelogenous Leukemia ◽  
Great Success

Abstract Despite of the great success of tyrosine kinase inhibitor in the therapy of chronic myelogenous leukemia (CML), CML is still not curable. Further investigation on the molecular mechanism of CML pathogenesis and identification of novel therapeutic target are required. In this study, we found that overexpression of CML characterized Bcr/Abl oncoprotein could significantly upregulate the expression of ubiquitin-specific protease Usp47. Also, increased expression of Usp47 was observed in primary CML cells and its expression was associated with the treatment response of imatinib, the first generation of tyrosine kinase inhibitor. Furthermore, Usp47 knockdown could inhibit the proliferation of K562 cells in vitro and in vivo. P22077, a Usp47 inhibitor, could inhibit the proliferation of CML cell lines, which comprise BCR/ABL mutation, including the T315I mutation. P22077 inhibits the proliferation of primary CML cells sensitive or resistant to imatinib. The colony forming activity of CD34+ from CML primary cells but not from normal cord blood cells were also inhibited by P22077. We further demonstrated that inactivation of STAT5 significantly reduce the expression of Usp47, indicating that BCR/ABL-induced upregulation of Usp47 is mediated by STAT5 activition. Interestingly, Usp47 could interact with Sirt1, another STAT5 target gene. Inactivation of Usp47 reduces the expression of Sirt1 in CML cell lines and primary CML cells. Taken together, we demonstrated that Usp47 plays an critical role in CML, revealing a novel BCR/ABL/STAT5/Usp47/Sirt1 signaling pathway in CML, providing a potential target to overcome tyrosine kinase inhibitor resistance. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibiting the Core Autophagy Enzyme ATG4B with Novel Drugs Sensitizes Resistant Leukemic Stem/Progenitor Cells to Standard Targeted Therapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 933-933 ◽  
Author(s):  
Katharina Rothe ◽  
Akie Watanabe ◽  
Donna L. Forrest ◽  
Sharon Gorski ◽  
Robert Young ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Interaction Analysis ◽  
Leukemic Cells ◽  
In Vivo Model ◽  
Autophagic Flux ◽  
Proof Of Concept

Abstract Leukemic stem cell (LSC) persistence is a major cause of therapy failure and relapse in patients, thus warranting further investigations into the cells' molecular properties to aid in meaningful clinical decisions. Chronic myeloid leukemia (CML) is a clear example and excellent study model with rare, propagating LSCs that are not eradicated by BCR-ABL1-directed tyrosine kinase inhibitor (TKI) monotherapy such as Imatinib Mesylate (IM), since the LSCs do not exclusively rely on BCR-ABL1 for their survival. We and others have shown that the persistence of primitive leukemic cells is mediated by macroautophagy (autophagy), a catabolic cellular recycling process. In particular, we discovered that transcript and protein expressions of all ATG4 family members, including ATG4A, B, C, and D, are significantly increased in CD34+ CML cells compared to CD34+ normal bone marrow (BM) cells. ATG4B expression is also significantly higher in pre-treatment CD34+ CML cells from IM-nonresponders vs. IM-responders, including LSCs. Moreover, we revealed that stable suppression of ATG4B significantly suppressed autophagy, impaired survival of IM-nonresponder stem/progenitor cells and sensitized leukemic cells to TKI treatment. Thus, we identified ATG4B as a critical therapeutic target in CML (Rothe et al., Blood, 2014). To further explore whether targeting of ATG4B would be a viable strategy in the successful treatment of various leukemia, two lead ATG4B inhibitors were recently developed for pre-clinical proof-of-concept studies. Compound 4-28, a styrylquinoline, was identified by in silico screening and high content cell-based screening. Its structure-activity relationship (SAR)-based optimization led to a more stable and potent compound, LV-320. LV-320 was further evaluated by Microscale Thermophoresis and showed consistent Kd values for binding to the ATG4B enzyme (Kd=16±1 μM). LV-320 can inhibit autophagic flux, shows excellent tolerability and a good PK profile. It is also characterised as a non-competitive inhibitor of ATG4B and displays similar potency against ATG4A. Interestingly, inhibition of ATG4B with compound 4-28 decreased viability by 40-60% and increased apoptosis by 30-40% in different BCR-ABL1+ leukemic cell lines upon serum-deprivation as compared to the same cells without treatment of 4-28 (p<0.05). 4-28 treatment also efficiently inhibited autophagic flux in these cells as shown by Western blot analysis of LC3-II/I and p62 accumulation. In addition, compound 4-28 was able to inhibit the clonal growth of patient-derived CML stem/progenitor cells from IM-nonresponders, in particular when combined with various TKIs compared to single agents (20 vs. 50%, p<0.05). However, this combination approach also showed slight toxic effects on healthy BM cells. We then tested the more stable and potent compound LV-320 with superior results. Treatment of several drug-resistant and mutated CML and aggressive BCR-ABL1+ B-ALL cell lines with LV-320 increased apoptosis up to 90% compared to controls and reached almost 100% when combined with various TKIs (p<0.05). Moreover, LV-320 sensitized IM-nonresponder stem and progenitor cells to TKIs in colony-forming short-term and long-term cell assays as compared to single agents (10 % vs. 40 %, p<0.05), but was not toxic to primitive BM cells from healthy donors (up to 10µM). Mechanistically, we found that LV-320 effectively inhibited autophagic flux in leukemic cells: Confocal microscopy demonstrated an increase in LC3-II-positive punctae (autophagosomes) and the presence of yellow punctae (blocked autophagosome-lysosome fusion) when leukemic cells where transduced with a mRFP-GFP-LC3 construct and treated with LV-320 or a combination of LV-320 and TKIs. Drug interaction analysis further indicated synergy between LV-320 plus IM (CI value ≤ 0.9). A novel in vivo model is currently being investigated to validate our proof-of-concept. Together, our results suggest that targeting of ATG4B with novel autophagy inhibitors in combination with TKIs may be able to circumvent drug resistance in CML and possibly other aggressive leukemia. Disclosures No relevant conflicts of interest to declare.

Inhibition of Proliferation and Induction of Apoptosis by Thymoquinone via Modulation of TGF Family, p53, p21 and Bcl-2α in Leukemic Cells

2018 ◽  
Vol 18 (2) ◽  
pp. 210-215 ◽  
Author(s):  
Mona Diab-Assaf ◽  
Josiane Semaan ◽  
Marwan El-Sabban ◽  
Soad K. Al Jaouni ◽  
Rania Azar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
T Cell ◽  
Flow Cytometric Analysis ◽  
Leukemic Cells ◽  
Cell Cycle Distribution ◽  
Dependent Manner ◽  
Apoptosis Induction ◽  
Inhibition Of Proliferation ◽  
Human T Cell

Introduction: Adult T-cell leukemia (ATL) is an aggressive form of malignancy caused by human T- cell lymphotropic virus 1 (HTLV-1). Currently, there is no effective treatment for ATL. Thymoquinone has been reported to have anti-cancer properties. Objective: The aim of this study is to investigatthe effects of TQ on proliferation, apoptosis induction and the underlying mechanism of action in both HTLV-1 positive (C91-PL and HuT-102) and HTLV-1 negative (CEM and Jurkat) malignant T-lymphocytes. Materials and Methods: Cells were incubated with different thymoquinone concentrations for 24h. Cell cytotoxicity was assayed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit. Cell proliferation was determined using CellTiter 96® Non-Radioactive Cell Proliferation. Cell cycle analysis was performed by staining with propidium iodide. Apoptosis was assessed using cell death ELISA kit. The effect of TQ on p53, p21, Bcl-2 protein expression was determined using Western blot analysis while TGF mRNA expression was determined by RT-PCR. Results: At non-cytotoxic concentrations of TQ, it resulted in the inhibition of proliferation in a dose dependent manner. Flow cytometric analysis revealed a shift in the cell cycle distribution to the PreG1 phase which is a marker of apoptosis. Also TQ increase DNA fragmentation. TQ mediated its anti-proliferative effect and apoptosis induction by an up-regulation of TGFβ1, p53 and p21 and a down-regulation of TGF-α and Bcl-2α. Conclusion: Thymoquinone presents antiproliferative and proapoptotic effects in ATL cells. For this reason, further research is required to investigate its possible application in the treatment of ATL.

scholarly journals Advantages of Tyrosine Kinase Anti-Angiogenic Cediranib over Bevacizumab: Cell Cycle Abrogation and Synergy with Chemotherapy

2021 ◽  
Vol 14 (7) ◽  
pp. 682
Author(s):  
Jianling Bi ◽  
Garima Dixit ◽  
Yuping Zhang ◽  
Eric J. Devor ◽  
Haley A. Losh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Endometrial Cancer ◽  
Tyrosine Kinase ◽  
Cell Viability ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Kinase Inhibitor ◽  
Cell Cycle Checkpoint ◽  
Mitotic Catastrophe ◽  
M Cell

Angiogenesis plays a crucial role in tumor development and metastasis. Both bevacizumab and cediranib have demonstrated activity as single anti-angiogenic agents in endometrial cancer, though subsequent studies of bevacizumab combined with chemotherapy failed to improve outcomes compared to chemotherapy alone. Our objective was to compare the efficacy of cediranib and bevacizumab in endometrial cancer models. The cellular effects of bevacizumab and cediranib were examined in endometrial cancer cell lines using extracellular signal-related kinase (ERK) phosphorylation, ligand shedding, cell viability, and cell cycle progression as readouts. Cellular viability was also tested in eight patient-derived organoid models of endometrial cancer. Finally, we performed a phosphoproteomic array of 875 phosphoproteins to define the signaling changes related to bevacizumab versus cediranib. Cediranib but not bevacizumab blocked ligand-mediated ERK activation in endometrial cancer cells. In both cell lines and patient-derived organoids, neither bevacizumab nor cediranib alone had a notable effect on cell viability. Cediranib but not bevacizumab promoted marked cell death when combined with chemotherapy. Cell cycle analysis demonstrated an accumulation in mitosis after treatment with cediranib + chemotherapy, consistent with the abrogation of the G2/M checkpoint and subsequent mitotic catastrophe. Molecular analysis of key controllers of the G2/M cell cycle checkpoint confirmed its abrogation. Phosphoproteomic analysis revealed that bevacizumab and cediranib had both similar and unique effects on cell signaling that underlie their shared versus individual actions as anti-angiogenic agents. An anti-angiogenic tyrosine kinase inhibitor such as cediranib has the potential to be superior to bevacizumab in combination with chemotherapy.

scholarly journals Protein arginine N-methyltransferase 1 promotes the proliferation and metastasis of hepatocellular carcinoma cells

Tumor Biology ◽  
2017 ◽  
Vol 39 (2) ◽  
pp. 101042831769141 ◽  
Author(s):  
Qing Gou ◽  
ShuJiao He ◽  
ZeJian Zhou
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Lines ◽  
Small Interfering Rna ◽  
Carcinoma Cell ◽  
Carcinoma Cells ◽  
Hepatocellular Carcinoma Cells ◽  
Carcinoma Cell Lines ◽  
Hepatocellular Carcinoma Cell ◽  
Interfering Rna ◽  
Methyltransferase 1

Hepatocellular carcinoma is the most common subtype of liver cancer. Protein arginine N-methyltransferase 1 was shown to be upregulated in various cancers. However, the role of protein arginine N-methyltransferase 1 in hepatocellular carcinoma progression remains incompletely understood. We investigated the clinical and functional significance of protein arginine N-methyltransferase 1 in a series of clinical hepatocellular carcinoma samples and a panel of hepatocellular carcinoma cell lines. We performed suppression analysis of protein arginine N-methyltransferase 1 using small interfering RNA to determine the biological roles of protein arginine N-methyltransferase 1 in hepatocellular carcinoma. In addition, the expression of epithelial-mesenchymal transition indicators was verified by western blotting in hepatocellular carcinoma cell lines after small interfering RNA treatment. Protein arginine N-methyltransferase 1 expression was found to be significantly upregulated in hepatocellular carcinoma cell lines and clinical tissues. Moreover, downregulation of protein arginine N-methyltransferase 1 in hepatocellular carcinoma cells by small interfering RNA could inhibit cell proliferation, migration, and invasion in vitro. These results indicate that protein arginine N-methyltransferase 1 may contribute to hepatocellular carcinoma progression and serves as a promising target for the treatment of hepatocellular carcinoma patients.

Tyrosine kinase inhibitor resistance in chronic myeloid leukemia cell lines: investigating resistance pathways

2011 ◽  
Vol 52 (11) ◽  
pp. 2139-2147 ◽  
Author(s):  
Carine Tang ◽  
Lisa Schafranek ◽  
Dale B. Watkins ◽  
Wendy T. Parker ◽  
Sarah Moore ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Leukemia Cell ◽  
Leukemia Cell Lines ◽  
Inhibitor Resistance ◽  
Myeloid Leukemia Cell
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