scholarly journals Kinome state is predictive of cell viability in pancreatic cancer tumor and stroma cell lines

2021 ◽  
Author(s):  
Matthew E. Berginski ◽  
Madison R. Jenner ◽  
Chinmaya U. Joisa ◽  
Silvia G. Herrera Loeza ◽  
Brian T. Golitz ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Information Transfer ◽  
Kinase Inhibitor ◽  
The State ◽  
Surrounding Tissue ◽  
Ductal Adenocarcinoma ◽  
Stroma Cell ◽  
Cancer Tumor ◽  
Insight Into

Numerous aspects of cellular signaling are regulated by the kinome - the network of over 500 protein kinases that guides and modulates information transfer throughout the cell. The key role played by both individual kinases and assemblies of kinases organized into functional subnetworks leads to kinome dysregulation being a key driver of many diseases, particularly cancer. In the case of pancreatic ductal adenocarcinoma (PDAC), a variety of kinases and associated signaling pathways have been identified for their key role in the establishment of disease as well as its progression. However, the identification of additional relevant therapeutic targets has been slow and is further confounded by interactions between the tumor and the surrounding tissue microenvironment. Fundamentally, it is an open question as to the degree to which knowledge of the state of the kinome at the protein level is able to provide insight into the downstream phenotype of the cell. In this work, we attempt to link the state of the kinome, or kinotype, with cell viability in representative PDAC tumor and stroma cell lines. Through the application of both regression and classification models to independent kinome perturbation and kinase inhibitor cell screen data, we find that the inferred kinotype of a cell has a significant and predictive relationship with cell viability. While regression models perform poorly, we find that classification approaches are able to predict drug viability effects. We further find that models are able to identify a set of kinases whose behavior in response to perturbation drive the majority of viability responses in these cell lines. These results suggest that characterizing the state of the protein kinome provides significant opportunity for better understanding signaling behavior and downstream cell phenotypes, as well as providing insight into the broader design of potential therapy design for PDAC.

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.

Compensatory Signaling From ROR1 and the Pre-B Cell Receptor Promote Survival of t(1;19) Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2466-2466
Author(s):  
Vincent T Bicocca ◽  
Bill H Chang ◽  
Markus Muschen ◽  
Brian J. Druker ◽  
Jeffrey W Tyner
Keyword(s):  
Cell Viability ◽  
B Cell ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Immunoblot Analysis ◽  
Cell Receptor ◽  
Cell Surface Receptor

Abstract Abstract 2466 BACKGROUND: Aberrant tyrosine kinase activity is commonly implicated in the pathogenesis of leukemia and other cancers. Identification of these leukemogenic tyrosine kinases has proven invaluable for diagnostic and prognostic stratification of patients as well as for the development of novel strategies for therapeutic intervention. We previously demonstrated that siRNA screening of mononuclear cells from leukemia patients can determine sensitivity to individual tyrosine kinases. With the goal of uncovering novel viability-dependent tyrosine kinases in leukemia patients, we have employed an RNAi-assisted protein target identification (RAPID) assay to screen cytogenetic subtypes of acute lymphoblastic leukemia (ALL). ALL is the most common pediatric cancer, accounting for one-quarter of all childhood malignancies. Childhood ALL has a primarily B cell precursor phenotype and is characterized by chromosomal abnormalities, primarily translocations and duplications. One of the most common recurring translocations associated with pediatric ALL, t(1;19)(q23;p13.3), generates the E2A-PBX1 fusion product. Here we show unique viability-dependent expression of a receptor tyrosine kinase, ROR1, in the t(1;19) ALL background. In addition, we identify a kinase inhibitor, dasatinib, with significant activity against t(1;19) ALL cells due to its capacity to inhibit tyrosine kinases necessary for transduction of pre-B cell receptor (preBCR) signaling. Finally, we show that ROR1 and the preBCR activate mutually compensatory signaling pathways, suggesting that optimal therapeutic regimens would include agents targeting both pathways. METHODS: To identify targets required for viability of leukemic cells, we screened cell lines as well as primary cells from ALL patients with siRNAs and determined cell viability using an MTS assay. ROR1 expression levels were determined by RT-PCR, immunoblot analysis and flow cytometry. Kinase inhibitor screening was performed on both cells lines and primary ALL cells by treating samples with a library of small-molecule inhibitors and cell viability was assessed by MTS. Signaling pathways disrupted by inhibitor treatment or ROR1 knockdown were interrogated by phospho-protein arrays and confirmed by immunoblot analysis. RESULTS: The RAPID assay identified a unique sensitivity to the cell surface receptor ROR1 in a subject identified with t(1;19) pediatric ALL. Similar sensitivity was not observed in ALL patients of alternative cytogenetic subtypes. Examination of additional ALL patient samples revealed conserved overexpression of the ROR1 transcript in t(1;19)-positive specimens with absence of ROR1 expression in t(1;19)-negative samples. Cell lines and early passage xenograft cells confirmed overexpression and functional dependence of t(1;19)-positive cells on ROR1. A subsequent kinase inhibitor screen of t(1;19) ALL cell lines and patient samples revealed universal sensitivity to the FDA-approved drug dasatinib. Further examination revealed the dasatinib targets, BTK and LYN, which signal downstream of the preBCR as the viability dependent targets of dasatinib in t(1;19) ALL. Inhibition of the preBCR results in transient loss of AKT activity and, surprisingly, upregulation of ROR1. Analysis of signaling pathways after silencing of ROR1 or dasatinib treatment revealed compensatory signaling pathways emanating from ROR1 and the preBCR that both serve to regulate AKT activity. Consequently, combination of ROR1 knockdown and dasatinib treatment resulted in additive impairment of cell viability compared with either treatment alone. CONCLUSION: The cell surface receptor ROR1 is consistently overexpressed in t(1;19) ALL. RNAi mediated downregulation of ROR1 impairs the viability of these cells. Similarly, t(1;19) cells are sensitive to the kinase inhibitor dasatinib due to activity against the preBCR. Combined targeting of ROR1 and the preBCR with dasatinib yields an additive viability effect due to compensatory signaling pathways aimed at regulating AKT. These results demonstrate a novel mechanism of AKT regulation in t(1;19) ALL as well as a therapeutic strategy for treatment of t(1;19) ALL. Disclosures: Druker: MolecularMD: Equity Ownership, OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee and t; Ariad Pharmaceuticals: Consultancy; OHSU patent #843: Mutated ABL Kinase Domains: Patents & Royalties; Bristol-Myers Squibb: OHSU has clinical trial contracts with Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Novartis: OHSU has clinical trial contracts with Novartis to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Cylene: Consultancy.

scholarly journals A common phosphotyrosine signature for the Bcr-Abl kinase

Blood ◽  
2006 ◽  
Vol 107 (12) ◽  
pp. 4888-4897 ◽  
Author(s):  
Valerie L. Goss ◽  
Kimberly A. Lee ◽  
Albrecht Moritz ◽  
Julie Nardone ◽  
Erik J. Spek ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitor ◽  
Imatinib Treatment ◽  
Treatment Comparison ◽  
Abl Kinase ◽  
Abl Tyrosine Kinase ◽  
Abl Tyrosine Kinase Inhibitor ◽  
Disease Stages ◽  
Insight Into ◽  
Fusion Type

Abstract The Bcr-Abl fusion kinase drives oncogenesis in chronic myeloid leukemia (CML). CML patients are currently treated with the Abl tyrosine kinase inhibitor imatinib, which is effective in early stages of the disease. However, resistance to imatinib arises in later disease stages primarily because of a Bcr-Abl mutation. To gain deeper insight into Bcr-Abl signaling pathways, we generated phosphotyrosine profiles for 6 cell lines that represent 3 Bcr-Abl fusion types by using immunoaffinity purification of tyrosine phosphopeptides followed by tandem mass spectrometry. We identified 188 nonredundant tyrosine-phosphorylated sites, 77 of which are novel. By comparing the profiles, we found a number of phosphotyrosine sites common to the 6 cell lines regardless of cellular background and fusion type, several of which are decreased by imatinib treatment. Comparison of this Bcr-Abl signature with the profile of cells expressing an alternative imatinib-sensitive fusion kinase, FIP1L1-PDGFRα, revealed that these kinases signal through different pathways. This phosphoproteomic study of the Bcr-Abl fusion kinase highlights novel disease markers and potential drug-responsive biomarkers and adds novel insight into the oncogenic signals driven by the Bcr-Abl kinase.

scholarly journals Lapatinib sensitivity in nasopharyngeal carcinoma is modulated by SIRT2-mediated FOXO3 deacetylation

2019 ◽  
Author(s):  
Sathid Aimjongjun ◽  
Zimam Mahmud ◽  
Yannasittha Jiramongkol ◽  
Glowi Alasiri ◽  
Shang Yao ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Cell Viability ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Protein Extraction ◽  
Treatment Strategies ◽  
Therapeutic Drug ◽  
Clonogenic Assays ◽  
High Doses ◽  
Novel Treatment Strategies

Abstract Background Chemoresistance is an obstacle to the successful treatment of nasopharyngeal carcinoma (NPC). Lapatinib is a targeted tyrosine kinase inhibitor therapeutic drug also used to treat NPC, but high doses are often required to achieve a result. To investigate the mechanism for the development of Lapatinib resistance, we characterised a number of NPC cell lines to determine the role of FOXO3 and sirtuins in regulating NPC resistance. Methods Sulforhodamine B (SRB) assays, Clonogenic assays, Protein extraction, quantification and western blotting, RT qPCR, Co-immunoprecipitation assay Results To explore novel treatment strategies, we first characterized the Lapatinib-sensitivity of a panel of NPC cell lines by SRB and clonogenic cytotoxic assays and found that the metastatic NPC (C666-1 and 5-8F) cells are highly resistant whereas the poorly metastatic lines (6-10B, TW01 and HK-1)are sensitive to Lapatinib. Western blot analysis of the Lapatinib-sensitive 6-10Band resistant 5-8FNPC cells showed that the expression of phosphorylated/inactive FOXO3 (P-FOXO3;T32), its target FOXM1 and its regulator SIRT2 correlate negatively with Lapatinib response and sensitivity, suggesting that SIRT2 mediates FOXO3 deacetylation to promote Lapatinib resistance. In agreement, clonogenic cytotoxic assays using wild-type and foxo1/3/4 −/− mouse embryonic fibroblasts (MEFs) showed that FOXO1/3/4-deletion significantly attenuates Lapatinib-induced cytotoxicity, confirming that FOXO proteins are essential for mediating Lapatinib response. SRB cell viability assays using chemical SIRT inhibitors (i.e. sirtinol, Ex527, AGK2 and AK1) revealed that all SIRT inhibitors can reduce NPC cell viability, but only the SIRT2-specific inhibitors AK1 and AGK2 further enhance the Lapatinib cytotoxicity. Consistently, clonogenic assays demonstrated that the SIRT2 inhibitors AK1 and AGK2 as well as SIRT2-knockdown increase Lapatinib cytotoxicity further in both the sensitive and resistant NPC cells. Co-immunoprecipitation studies showed that besides Lapatinib treatment, SIRT2-pharmaceutical inhibition and silencing also led to an increase in FOXO3 acetylation. Importantly, SIRT2 inhibition and depletion further enhanced Lapatinib-mediated FOXO3-acetylation in NPC cells. Conclusion Collectively, our results suggest the involvement of SIRT2-mediated FOXO3 deacetylation in Lapatinib response and sensitivity, and that SIRT2 can specifically antagonise the cytotoxicity of Lapatinib through mediating FOXO3 deacetylation in both sensitive and resistant NPC cells.

scholarly journals MCL-1 antagonism enhances the anti-invasive effects of dasatinib in pancreatic adenocarcinoma

Oncogene ◽  
2019 ◽  
Vol 39 (8) ◽  
pp. 1821-1829 ◽  
Author(s):  
Lesley Castillo ◽  
◽  
Adelaide I. J. Young ◽  
Amanda Mawson ◽  
Pia Schafranek ◽  
...  
Keyword(s):  
Cell Viability ◽  
Kinase Inhibitor ◽  
Additional Benefit ◽  
Ductal Adenocarcinoma ◽  
Murine Models ◽  
Primary Tumor Growth ◽  
Cytoskeletal Remodeling ◽  
Wide Range ◽  
Invasive Capacity ◽  
Tcga Dataset

AbstractPancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies. It is phenotypically heterogeneous with a highly unstable genome and provides few common therapeutic targets. We found that MCL1, Cofilin1 (CFL1) and SRC mRNA were highly expressed by a wide range of these cancers, suggesting that a strategy of dual MCL-1 and SRC inhibition might be efficacious for many patients. Immunohistochemistry revealed that MCL-1 protein was present at high levels in 94.7% of patients in a cohort of PDACs from Australian Pancreatic Genome Initiative (APGI). High MCL1 and Cofilin1 mRNA expression was also strongly predictive of poor outcome in the TCGA dataset and in the APGI cohort. In culture, MCL-1 antagonism reduced the level of the cytoskeletal remodeling protein Cofilin1 and phosphorylated SRC on the active Y416 residue, suggestive of reduced invasive capacity. The MCL-1 antagonist S63845 synergized with the SRC kinase inhibitor dasatinib to reduce cell viability and invasiveness through 3D-organotypic matrices. In preclinical murine models, this combination reduced primary tumor growth and liver metastasis of pancreatic cancer xenografts. These data suggest that MCL-1 antagonism, while reducing cell viability, may have an additional benefit in increasing the antimetastatic efficacy of dasatinib for the treatment of PDAC.

Synergistic Activity of the Aurora Kinase Inhibitor MK-0457 (VX-680) with Idarubicin, Ara-C, and Inhibitors of BCR-ABL.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1384-1384 ◽  
Author(s):  
Russell R. Hoover ◽  
Matthew W. Harding
Keyword(s):  
Clinical Trials ◽  
Cell Viability ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Single Agent ◽  
Leukemia Cell ◽  
Parp Cleavage ◽  
Synergistic Activity ◽  
Dependent Manner ◽  
Wild Type

Abstract MK-0457 (VX-680) is a reversible small molecule kinase inhibitor that targets Aurora A, B, and C with Ki values of 0.7, 18, and 4.6 nM, respectively. MK-0457 also inhibits Flt3 (Ki = 30 nM), and both the wild type and the T315I mutant of BCR-ABL (Ki = 30 and 40 nM, respectively). Clinical trials are ongoing in patients with solid tumors and hematological malignancies. Recent data show that MK-0457 is active in patients against subtypes of AML, BCR-ABL T315I mutant CML, and Philadelphia positive (Ph+) ALL. To support multi-agent clinical trials, the activity of MK-0457 in combination with idarubicin, Ara-C, and BCR-ABL inhibitors was investigated. The viability of a panel of AML, ALL, and CML cell lines was assessed following single agent and either simultaneous or sequential combinations of agents. Combination effects were evaluated using the Bliss Independence Model. MK-0457 as a single agent markedly inhibited leukemia cell viability (at 72 hrs) with an IC50 range of 20–300 nM for MV4-11, Molt-4, Molm-13, K562, LAMA-84, MEG-01, and KU812F cells. Additionally, MK-0457 inhibited the viability of BaF3 cells transformed by wild type, T315I, or Y253F mutants of BCR-ABL with similar IC50s (approximately 300 nM). The sequential combination of MK-0457 followed by either idarubicin or Ara-C showed greater synergy than simultaneous combinations in a cell line dependent manner. MK-0457 displayed strong synergy in simultaneous combination with Gleevec (imatinib mesylate) in a panel of human CML-derived cell lines and BaF3 cells expressing wild type BCR-ABL. MK-0457 enhanced the Gleevec-mediated cell death of K562 leukemia cells as evidenced by increased caspase activity, PARP cleavage, and induction of the sub-G1 population. At concentrations where synergy was observed by cell viability analysis, the MK-0457/Gleevec combination resulted predominantly in aneuploidy and G2/M arrest, consistent with inhibition of Aurora kinases by MK-0457. These results support the clinical evaluation of MK-0457 combined with idarubicin and Ara-C in AML and with BCR-ABL inhibitors in CML and Ph+ ALL.

Combination of the IGF-1 Receptor Inhibitor Picropodophylin and the BH3 Mimetic ABT-737 Has Synergistic Anti-Myeloma Activity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4010-4010
Author(s):  
Liesbeth Bieghs ◽  
Ken Maes ◽  
Els Van Valckenborgh ◽  
Eline Menu ◽  
Hans Erik Johnsen ◽  
...  
Keyword(s):  
Growth Factors ◽  
Cell Viability ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Differential Sensitivity ◽  
Expression Ratio ◽  
Therapy Strategy ◽  
Induced Apoptosis ◽  
Bh3 Mimetic

Abstract Abstract 4010 Despite intensive research multiple myeloma (MM) is still an incurable disease. MM cells are strongly dependent on the BM micro-environment where growth factors are secreted. IGF-1 is one of the most important growth factors in MM and thus forms an attractive target for anti-cancer therapy. Previously, we demonstrated that picropodophylin (PPP), an IGF-1R kinase inhibitor, indeed has potent anti-MM effects both on human cells and in the 5T2MM and 5T33MM murine models. However, mice eventually relapsed and showed signs of morbidity. Therefore it would be an attractive approach to combine PPP with other cytotoxic drugs. ABT-737 is a BH3 mimetic that binds with high affinity to Bcl-xL, Bcl-2 and Bcl-w, but not Mcl-1. In MM, elevated expression of the Bcl-2 pro-survival family of proteins, especially Mcl-1 and to a lesser extent Bcl-2, has extensively been shown to cause resistance to drug induced apoptosis in MM cells. Consequently, ABT-737 was shown to have potent anti-MM activity but only on a subset of human cell lines. Only very recently, it was shown that the ABT-737 sensitivity appears to be determined both by the Bcl-2/Mcl-1 expression ratio and the interaction of these anti-apoptotic proteins with Bim. Interestingly, we demonstrated that IGF-1R inhibition reduces the expression of Mcl-1 and Bcl-xL and that IGF-1 down-regulates the expression of Bim. In addition, a protective effect of IL-6 and BMSC against ABT-737 has been reported. Together, all the above suggests that it would be beneficial to combine ABT-737 with agents that target growth factors, like PPP. Here, we investigated the potential synergistic anti-MM effects of PPP and ABT-737 and studied the underlying mechanisms using two human myeloma cell lines (OPM-2 and RPMI-8226) and the murine 5T33MM model. Both PPP and ABT-737 (kindly provided by Abbott Laboratories) alone were found to significantly decrease cell viability and induce apoptosis dose and time dependently as evidenced by a decrease in ATP levels and an increase in the number of AnnexV/7'AAD positive cells. However, in agreement with previous reported data, we observed differential sensitivity to ABT-737 between the cell lines used. Nevertheless, treatment with PPP/ABT-737 synergistically decreased cell viability and induced apoptosis in all cell lines. In addition, by western blot analysis we could observe increased cleavage of caspase- 3,- 9 and PARP. Mechanistically, PPP was found to circumvent the adverse effect of ABT-737 by blocking the ABT-737 induced Mcl-1 expression and increasing the expression of Noxa. Interestingly, while CD138+ 5T33MM cells were more sensitive to PPP and the CD138- cells more sensitive to ABT-737, treatment with PPP/ABT-737 targeted both MM cell subpopulations to an equal extent. Finally, we tested the combination of PPP and ABT-737 in the 5T33MM model in a prophylactic setting. Whereas, vehicle and ABT-737 treated mice exhibited progressive MM growth, PPP, and to a significant greater extent, PPP/ABT-737 reduced the tumor burden and prolonged overall survival (p≤0.001). In conclusion, PPP combined with ABT-737 appears to have synergistic anti-MM activity and might thus be a novel and promising therapy strategy for MM. Disclosures: No relevant conflicts of interest to declare.

The Ellipticine Derivative NSC 338258 Has Anti-Myeloma Activity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3379-3379
Author(s):  
Erming Tian ◽  
Owen Stephens ◽  
Susan Holbeck ◽  
Bart Barlogie ◽  
John Shaughnessy
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Topoisomerase Ii ◽  
Kinase Inhibitor ◽  
Myeloma Cell ◽  
The Other ◽  
Response Analysis ◽  
Time Points ◽  
Cell Kill

Abstract We recently discovered that amplification and overexpression of the CKS1B gene, which regulates the ubiquitination and proteasomal degradation of the cyclin-dependent kinase inhibitor p27Kip1, is linked to a poor prognosis in myeloma. We used the COMPARE algorithm (NCI, Developmental Therapeutics Program) to identify correlations between the expression of CKS1B in the NCI 60 cell line panel and the GI50 of nearly 50,000 anticancer compounds. This analysis revealed a strong correlation between CKS1B expression and the anticancer activity of Ellipticine and multiple derivatives of it. Ellipticine (5,11-Dimethyl-6H-Pyrido[4,3]Carbazole, MW=246.3), an alkaloid isolated from Apocyanaceae, is a topoisomerase II poison that induces topoisomerase II-dependent DNA cleavages. Previous studies have shown that the anti-neoplastic mechanism of Ellipticine is to form covalent DNA adducts mediated by human cytochromes P450. We obtained Ellipticine and twelve other CKS1B-correlated antineoplastic compounds from the NCI-DTP. In vitro analyses were carried out with 12 myeloma cell lines. Cell viability was detected using CellTiter-Glo Luminescent Assay (Promega, Co.). We found that one of the Ellipticine derivatives, NSC 338258 (EPED3) showed significant cell kill activity not observed with Ellipticine or the other compounds in all cell lines tested. In a dose response analysis of cell kill using 0.2, 0.02, and 0.002 uM EPED3, 12 out of 12 myeloma cell lines showed at least 50% reduction in cell viability within 24 hrs at 0.2 uM compared to no treatment controls. By 48 hours essentially all cell growth was inhibited. Only weak to moderate cell kill was noted at 0.02 and 0.002 uM at all time points tested. We next tested the activity of the topoisomerase II inhibitor VP-16-213 at the same concentrations and time periods and found only modest reduction of cell viability at day 5 at 0.2uM. Essentially no effect on cell viability was observed at 0.02 and 0.002 uM at any of the time points tested. We next tested whether EPED3 had a comparable anti-myeloma activity as Adriamycin at the same concentrations. Twelve myeloma cell lines were exposed to EPED3, VP-16-213, and Adriamycin at 0.2mM for a total of 5 days. Results of the mean relative luminescence showed that EPED3 promoted an immediate cell proliferation arrest within the first 24 hours of culture, with almost complete cell kill within 3 days. The other drugs were less effective with Adriamycin inducing a > 90% reduction only after 4 days and VP-16 a > 70% reduction at day 5. These data suggests that EPED3 may represent a potential new therapeutic for myeloma. Studies are currently underway to test the efficacy of EPED3 in our in-vivo SCID-Hu mouse model of primary myeloma and results of these studies will be presented.

ROR1 as a Therapeutic Target In E2A-PBX1-Positive Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 539-539
Author(s):  
Vincent Bicocca ◽  
Bill H Chang ◽  
Markus Muschen ◽  
Brian J Druker ◽  
Jeffrey W Tyner
Keyword(s):  
Tyrosine Kinase ◽  
Cell Viability ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Mononuclear Cells ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Fusion Product ◽  
Rapid Assay ◽  
Pediatric All

Abstract Abstract 539 Background: Aberrant tyrosine kinase activity is commonly implicated in the pathogenesis of leukemia and other cancers. Identification of these leukemogenic tyrosine kinases has proven invaluable for diagnostic and prognostic stratification of patients as well as for the development of novel strategies for therapeutic intervention. We previously demonstrated that siRNA screening of mononuclear cells from leukemia patients can determine sensitivity to individual tyrosine kinases. With the goal of uncovering novel viability-dependent tyrosine kinases in leukemia patients, we have employed an RNAi-assisted protein target identification (RAPID) assay to screen cytogenetic subtypes of acute lymphoblastic leukemia (ALL). ALL is the most common pediatric cancer, accounting for one-quarter of all childhood malignancies. Childhood ALL has a primarily B cell precursor phenotype and is characterized by chromosomal abnormalities, primarily translocations and duplications. These lesions can result in aberrant tyrosine kinase expression and activity required for leukemogenesis. One of the most common recurring translocations associated with pediatric ALL, t(1;19)(q23;p13.3), generates the E2A-PBX1 fusion product. The role of E2A-PBX1 in the development of acute leukemia remains unclear. Here we show unique viability-dependent expression of a receptor tyrosine kinase, ROR1, in the E2A-PBX1 ALL background. In addition, we identify a kinase inhibitor, dasatinib, with significant activity against E2A-PBX1-positive ALL cells. Methods: To identify targets required for viability of leukemic cells, we screened cell lines as well as primary cells from ALL patients by electroporating siRNAs individually targeting each member of the tyrosine kinase gene family. Four days later, we determined the cell viability and tabulated sensitivity of the cells to any individual tyrosine kinase. ROR1 expression levels were determined by RT-PCR, immunoblot analysis and flow cytometry. Kinase inhibitor screening was performed on both cell lines and primary ALL cells by treating samples with a library of small-molecule inhibitors consisting of 90 cell-permeable inhibitor compounds. Inhibitors are plated at four serial dilutions to allow IC50 calculations. The effect of each drug on cell viability is determined at day three by an MTS cell viability assay. Results: The RAPID assay identified a unique sensitivity to the receptor tyrosine kinase-like orphan receptor 1 (ROR1) in a subject identified with E2A-PBX1-positive B cell precursor pediatric ALL. Similar sensitivity was not observed in patients of other leukemic backgrounds or ALL patients of alternative cytogenetic subtypes. Examination of mononuclear cells from this patient by reverse-transcriptase-PCR revealed overexpression of the ROR1 transcript compared with ALL patients lacking the E2A-PBX1 fusion product. Examination of 12 additional E2A-PBX1-positive ALL patient samples revealed universal overexpression of ROR1 within the E2A-PBX1 background. Analysis of E2A-PBX1-positive cell lines and early passage xenograft cells showed both overexpression of ROR1 and sensitivity to siRNA-mediated ROR1 silencing, confirming the ROR1 dependent survival observed in primary cells with the RAPID assay. Finally, since ROR1 is defined as a tyrosine kinase, we performed a kinase inhibitor screen and identified universal sensitivity of E2A-PBX1-positive cell lines and patient samples to the FDA-approved drug dasatinib. Hence, dasatinib is suggested as a potential therapeutic for E2A-PBX1-positive ALL patients. Conclusion: The cell surface receptor ROR1 is consistently overexpressed in E2A-PBX1-positive ALL. RNAi mediated downregulation of ROR1 impairs the viability of these cells. Finally, the kinase inhibitor dasatinib is suggested as a novel therapeutic tool for treatment of E2A-PBX1-positive ALL based on universal sensitivity of E2A-PBX1 samples to this kinase inhibitor. Disclosures: Druker: Molecular MD: Equity Ownership.

Growth Inhibitory Function Of a Novel T-LAK Cell-Originated Protein Kinase Inhibitor On FLT3-ITD Positive Acute Myeloid Leukemia (AML) Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1459-1459
Author(s):  
Houda Alachkar ◽  
Jae-Hyun Park ◽  
Makoto Nakakido ◽  
Alex Wood ◽  
Gordana Raca ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Viability ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
U937 Cells ◽  
Transfected Cells ◽  
Lak Cell ◽  
Acute Myeloid

Abstract T-LAK cell-originated protein kinase (TOPK/PBK), a serine-threonine mitogen-activated protein kinase kinase is highly expressed and correlated with more aggressive disease in several types of cancer, but is undetectable in normal tissues except the testis and fetal tissues. TOPK is up-regulated in a variety of hematologic malignancies including acute myeloid leukemia (AML) and may be involved in disease pathogenesis. To investigate the role of TOPK in AML and to develop the rationale for therapeutic targeting, we initially examined the expression level of TOPK protein in AML cell lines by western blot analysis. TOPK expression was detected at high levels in 8 out of 11 AML cell lines. Importantly, TOPK was not detected in mobilized, peripheral blood-derived stem cells from healthy donors. To determine the role of TOPK in AML, we utilized a loss of function approach in two AML cell lines (MV4-11 and U937 cells) and assessed cell viability and apoptosis by MTS and Annexin/PI staining assays, respectively. Cells transfected with TOPK-siRNA showed significant decrease in cell viability (∼70%, P<0.001, both cell lines) and significant increase in apoptosis 48 hours following transfection compared to cells transfected with control-siRNA. A small molecule compound that inhibits TOPK kinase is currently undergoing pharmaceutical development for cancer treatment. We treated nine AML cell lines with the compound for 48 hours and assessed cells viability by MTS assay. Decrease in cell viability following treatment with increasing doses of the compound was noted with variable sensitivities among AML cell lines. In addition, the compound showed enhancement of cell differentiation assessed by CD11b staining in U937 cells. Cell lines with FLT3 mutations (MV4-11, MOLM13 and KOCL-48) were significantly more sensitive to the treatment compared to cell lines with unmutated FLT3. We next treated cells with increasing doses of the compound and assessed cells viability and apoptosis by MTS and flow cytometry assays. Following the treatment with the compound, IC50 was <10nM for MV4-11 and MOLM13 cells (FLT3-ITD positive cell lines), and >20nM for U937 and KG1 (FLT3-ITD negative cell lines). 40 nM of the compound induced apoptosis by ∼80% in MV4-11 and MOLM13 cells compared to 49% in U937 and 9% in KG1 cells, at 48 hours following treatment. We also validated the anti-leukemia activity in primary blasts from patients with AML (n=2). FLT3-ITD positive blasts had an IC50 of ∼15nM and showed 40% increase in apoptosis following treatment with 20nM of the compound. To gain a mechanistic insight into why FLT3-ITD mutant cells are preferentially sensitive to this TOPK inhibitor, we performed a gene expression profile microarray analysis on MV4-11 cells treated with 20nM of the compound or transfected with TOPK siRNA in comparison with untreated cells and cells transfected with control-siRNA. We observed significant downregulation in genes involved in cell cycle control pathways in the signatures associated with the compound-treated and TOPK siRNA-transfected cells. Interestingly, FLT3 was among the significantly downregulated genes in the compound treated cells (∼80%) and in TOPK-siRNA transfected cells (∼30%). We then examined the activity and the expression levels of FLT3 protein following the treatment. Consistently, we found that both the phospho-FLT3 and the total FLT3 protein levels were completely depleted in cells treated with 10, 20 and 40 nM of the compound as early as 16 hours following treatment. In conclusion, TOPK is highly expressed in AML and may act as novel therapeutic target. A novel TOPK kinase inhibitor exhibits preferential cytotoxicity to FLT3-ITD mutated AML cells, possibly through inhibition of FLT3 protein expression. Although further research is needed to determine the mechanism by which the compound inhibits FLT3 protein expression, this novel compound may represent a new targeted therapy for this adverse risk subset of patients with AML. Disclosures: Matsuo: OncoTherapy Science, Inc.: Employment. Nakamura:Oncotherapy Science. Ltd.: share holder Other.

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