In vitro activity of sorafenib against imatinib- and sunitinib-resistant kinase mutations associated with drug-resistant GI stromal tumors

10500 Background: Most gastrointestinal stromal tumors (GISTs) harbor mutant KIT or platelet-derived growth factor receptor alpha (PDGFRA) kinases, which are targets of imatinib (front-line therapy) or sunitinib (second-line therapy). Resistance to imatinib (IM) or sunitinib (SU) is commonly associated with the acquisition of secondary kinase mutations. In phase II studies, sorafenib (SOR), which targets KIT, PDGFRs, and several other kinases, has demonstrated efficacy in GIST patients after failure of IM and SU. We evaluated the in vitro activity of SOR against IM and/or SU resistant kinases. Methods: Kinase mutants were biochemically profiled for SOR, SU, and IM sensitivity by measuring inhibition of kinase phosphorylation after drug treatment in mutant-expressing CHO cells. We also tested the biochemical and cellular activity of SOR and IM against GIST cells derived from IM-resistant tumor clones. Results: SOR had superior potency to IM against IM-resistant KIT secondary mutations involving the ATP binding pocket (V654A, T670I) and the activation loop (D816H, D820A/G, N822K, Y823D). SOR and SU had similar potency against IM-resistant KIT secondary mutations involving the ATP binding pocket. In contrast, SOR had markedly increased potency, compared to SU, against IM-resistant KIT activation loop mutations. Both SOR and SU were more potent than IM against the isolated KIT exon 9 mutation. To confirm these observations in a GIST cellular context, we tested the relative potency of IM and SU against two previously described IM-resistant GIST cell lines (GIST430: exon 11 + V654A, GIST48: exon 11 + D820A). SOR was significantly more potent than IM against KIT in these cell lines. This also correlated with enhanced inhibition of downstream signaling pathways and cellular proliferation. Conclusions: SOR has superior in vitro potency compared with IM or SU against a panel of GIST-relevant mutant kinases. Notably, SOR, unlike SU, is active against most IM-resistant secondary mutations involving the KIT activation loop. Based on these results, we hypothesize that SOR might have superior clinical activity to SU in the setting of imatinib-resistant GIST and should be further evaluated for clinical efficacy in the second-line setting. [Table: see text]

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In vitro and in vivo activity of regorafenib (REGO) in drug-resistant gastrointestinal stromal tumors (GIST).

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.10510 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 10510-10510 ◽

Cited By ~ 6

Author(s):

Cesar Serrano-Garcia ◽

Michael C. Heinrich ◽

Meijun Zhu ◽

Chandrajit P. Raut ◽

Grant Eilers ◽

...

Keyword(s):

Kinase Inhibitors ◽

Initial Response ◽

Binding Pocket ◽

Atp Binding ◽

Activation Loop ◽

Line Therapy ◽

Exon 11 ◽

Clinical Trial Results

10510 Background: KIT and PDGFRA mutations (mut) are the crucial transforming events in most GISTs, and tyrosine kinase inhibitors (TKIs) with activity against KIT and PDGFRA, such as imatinib (IM) (front-line therapy) and sunitinib (SU) (second-line therapy), are effective treatments in GIST patients (pts). Resistance to IM and SU is commonly associated with evolution of secondary kinase mut. REGO is a multi-targeted TKI that inhibits KIT, PDGFR, and other oncologic targets and has recently shown benefit in pts with metastatic GIST after progression on standard treatments. We evaluated the in vitro and in vivo activity of REGO compared with IM, SU, and sorafenib (SOR) (a multi-TKI structurally related to REGO). Methods: REGO, IM, SU, and SOR inhibition of viability and KIT phosphorylation was assessed in human GIST cell lines and in Ba/F3 cells transformed by KIT oncoproteins with IM-resistant ATP binding pocket or activation-loop mut. KIT/PDGFRA genotyping was performed in GISTs responding or progressing on REGO in the academic phase II clinical trial. Results: In GISTs with KIT exon 11 mutant oncoproteins, REGO potently inhibited viability, KIT phosphorylation, and downstream effector phosphorylation (AKT, MAPK, S6). IM-resistant activation loop mut were more potently inhibited by REGO than SU, whereas the gatekeeper IM-resistant mut T670I was inhibited by both REGO and SU, and the common ATP-binding pocket mutant V654A was more potently inhibited by SU than REGO. Two GIST metastases progressing in one pt after initial response to REGO contained KIT V654A mut. SOR and REGO demonstrated comparable in vitro overall activity. Representative GIST cell line viability IC50s are shown in the Table (values in bold indicate expected clinical relevance). Conclusions: In vitro studies confirm REGO is a potent inhibitor of KIT exon 11 mut in GIST and appears to have stronger activity than SU against the most common KIT activation-loop mut observed in GIST. Ongoing clinical correlative analyses from REGO-treated study patients will be presented. [Table: see text]

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1360. Antimicrobial Activity of Cefepime in Combination with VNRX-5133 Against a Global Collection of Enterobacteriaceae Including Resistant Phenotypes

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy210.1191 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S416-S417 ◽

Cited By ~ 3

Author(s):

Meredith Hackel ◽

Dan Sahm

Keyword(s):

Vitro Activity ◽

In Vitro Activity ◽

First Line ◽

First Line Therapy ◽

Carbapenem Resistant ◽

Line Therapy ◽

Resistant Strains ◽

Further Development ◽

Dose Dependent

Abstract Background VNRX-5133 is a novel cyclic boronate-based broad-spectrum β-lactamase inhibitor with potent and selective direct inhibitory activity against both serine- and metallo-β-lactamases (Ambler Classes A, B, C, and D). In this analysis, we evaluated the activity of cefepime (FEP) in combination with VNRX-5133 and comparators against 1,120 recent Enterobacteriaceae clinical isolates, including carbapenem-resistant strains. Methods MICs of FEP with VNRX-5133 fixed at 4 µg/mL (FEP/VNRX-5133) were determined following CLSI M07-A10 guidelines against 1,120 Enterobacteriaceae from community and hospital infections collected globally in 2012–2013. Resistant phenotypes were based on 2017 CLSI breakpoints. As FEP/VNRX-5133 breakpoints have not yet been established, the FEP 2 g q8h susceptible dose-dependent (SDD) breakpoint of ≤8 µg/mL was considered for comparative purposes. Results FEP/VNRX-5133 showed potent in vitro activity against drug-resistant subsets of Enterobacteriaceae, with MIC90 values ranging from 1 µg/mL against ceftazidime-, levofloxacin-, or piperacillin–tazobactam-nonsusceptible isolates, to 8 µg/mL against meropenem-nonsusceptible isolates. FEP/VNRX-5133 inhibited >93% of all resistant subsets at ≤8 µg/mL. Conclusion Cefepime in combination with VNRX-5133 demonstrated potent in vitro activity against Enterobacteriaceae, including cephalosporin-, fluoroquinolone- and carbapenem-resistant (CRE) isolates. Because this drug combination exhibited substantial potential for the treatment of infections caused by isolates often resistant to first-line therapy, further development is warranted. Disclosures M. Hackel, IHMA, Inc.: Employee, Salary. VenatoRx: Consultant, Consulting fee. D. Sahm, IHMA, Inc.: Employee, Salary. VenatoRx: Consultant, Consulting fee.

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Flavopiridol Induces Apoptosis in Chronic Lymphocytic Leukemia Cells Via Activation of Caspase-3 Without Evidence of bcl-2 Modulation or Dependence on Functional p53

Blood ◽

10.1182/blood.v92.10.3804 ◽

1998 ◽

Vol 92 (10) ◽

pp. 3804-3816 ◽

Cited By ~ 197

Author(s):

John C. Byrd ◽

Charlotte Shinn ◽

Jamie K. Waselenko ◽

Ephraim J. Fuchs ◽

Teresa A. Lehman ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Lines ◽

Caspase 3 ◽

Mononuclear Cells ◽

P53 Protein ◽

Lymphocytic Leukemia ◽

Vitro Activity ◽

Interleukin 4 ◽

In Vitro Activity

Abstract Flavopiridol has been reported to induce apoptosis in lymphoid cell lines via downregulation of bcl-2. The in vitro activity of flavopiridol against human chronic lymphocytic leukemia (CLL) cells and potential mechanisms of action for inducing cytotoxicity were studied. The in vitro viability of mononuclear cells from CLL patients (n = 11) was reduced by 50% at 4 hours, 24 hours, and 4 days at a flavopiridol concentration of 1.15 μmol/L (95% confidence interval [CI] ±0.31), 0.18 μmol/L (95% CI ±0.04), and 0.16 μmol/L (95% CI ±0.04), respectively. Loss of viability in human CLL cells correlated with early induction of apoptosis. Exposure of CLL cells to 0.18 μmol/L of flavopiridol resulted in both decreased expression of p53 protein and cleavage of the caspase-3 zymogen 32-kD protein with the appearance of its 20-kD subunit. Contrasting observations of others in tumor cell lines, flavopiridol cytotoxicity in CLL cells did not correlate with changes in bcl-2 protein expression alterations. We evaluated flavopiridol’s dependence on intact p53 by exposing splenocytes from wild-type (p53+/+) and p53 null (p53−/−) mice that demonstrated no preferential cytotoxicity as compared with a marked differential with F-ara-a and radiation. Incubation of CLL cells with antiapoptotic cytokine interleukin-4 (IL-4) did not alter the LC50 of flavopiridol, as compared with a marked elevation noted with F-ara-a in the majority of patients tested. These data demonstrate that flavopiridol has significant in vitro activity against human CLL cells through activation of caspase-3, which appears to occur independently of bcl-2 modulation, the presence of IL-4, or p53 status. Such findings strongly support the early introduction of flavopiridol into clinical trials for patients with B-CLL.

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Evaluation of essential oils from 22 Guatemalan medicinal plants for in vitro activity against cancer and established cell lines

Journal of Medicinal Plants Research ◽

10.5897/jmpr2017.6528 ◽

2018 ◽

Vol 12 (3) ◽

pp. 42-49 ◽

Cited By ~ 1

Author(s):

Byron Miller Andrew ◽

Gordon Cates Rex ◽

O’Neill Kim ◽

Alfonso Fuentes Soria Juan ◽

Vicente Espinoza Luis ◽

...

Keyword(s):

Medicinal Plants ◽

Essential Oils ◽

Cell Lines ◽

Vitro Activity ◽

In Vitro Activity ◽

Established Cell Lines ◽

Established Cell

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Cyclin-Dependent Kinase Inhibitor Seliciclib Shows In Vitro Activity in Diffuse Large B Cell Lymphomas.

Blood ◽

10.1182/blood.v108.11.4738.4738 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4738-4738

Author(s):

Francesco Bertoni ◽

Katia Lacrima ◽

Andrea Rinaldi ◽

Sara Vignati ◽

Vittoria Martin ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Active Agent ◽

B Cell Lymphoma ◽

Lymphocytic Leukemia ◽

Vitro Activity ◽

In Vitro Activity ◽

Large B Cell

Abstract Background. Despite recent improvements in treatment, a significant fraction of patients with diffuse large B cell lymphoma (DLBCL) still fail therapy. Therefore, new therapeutic modalities are needed to advance the cure rate. Seliciclib (CYC202, R-roscovitine) is a purine analogue developed as an inhibitor of CDK2/cyclin E CDK7/cyclin H and CDK9/cyclin T. Seliciclib has been shown to be active in B cell neoplasms, such as mantle cell lymphoma, chronic lymphocytic leukemia and in multiple myeloma in vitro. The aim of this study was to assess the in vitro activity of seliciclib in DLBCL. Materials and methods. The anti-proliferative activity of seliciclib was tested in nine human DLBCL cell lines and six DLBCL primary cell cultures. The effects of seliciclib on the cell cycle and on apoptosis, as well as on transcription-related proteins were assessed. Results. The cell viability of all DLBCL cell lines and primary cells was reduced by seliciclib treatment. The IC50 for the cell lines ranged from 13 to 36 μM. The effect of seliciclib was independent of the genetic aberrations characterizing the cell lines. After seliciclib exposure cells accumulated in G2/M or in G1 phase, with most of the cells showing signs of apoptosis. Despite the clear cytotoxic effect and induction of apoptosis, we could not identify a unique mechanism of action. Conclusions. Our in vitro data suggest that seliciclib is an active agent in DLBCL. Its efficacy is apparently independent of the underlying chromosomal translocations characteristic of DLBCL. The drug might represent a new therapeutic agent in this lymphoma subtype.

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Significant in-Vitro Activity of a Novel JAK2 Inhibitor in Multiple Myeloma Associated with Preferential Cytotoxicity for CD45+ Myeloma Cells.

Blood ◽

10.1182/blood.v114.22.2848.2848 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2848-2848

Author(s):

Vijay Ramakrishnan ◽

Jessica Haug ◽

Teresa Kimlinger ◽

Timothy Halling ◽

Linda Wellik ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Lines ◽

Research Funding ◽

Myeloma Cell ◽

Vitro Activity ◽

In Vitro Activity ◽

Myeloma Cells ◽

Stat Pathway

Abstract Abstract 2848 Poster Board II-824 Background: Multiple myeloma remains incurable with current therapies and novel approaches based on disease biology are needed. IL-6 is a critical cytokine involved in myeloma cell proliferation and survival and exerts its activity primarily through the JAK/STAT pathway. In addition to IL6, other cytokines are also believed to cross talk with the JAK/STAT pathway, making it a crucial interface for survival signals. It has been implicated in myeloma cell interaction with the microenvironment and resistance to apoptotic stimuli from different drugs, and represents a potential therapeutic target. We examined the pre-clinical activity of a novel JAK2 tyrosine kinase inhibitor TG101209. Methods: TG101209 (N-tert-butyl-3-(5-methyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-ylamino)-benzenesulfonamide) was synthesized by TargeGen Inc. (San Diego, CA, USA). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and using Apo2.7 in primary patient cells. CD45 expression was estimated using flow cytometry and cells were gated by their CD45 expression to assess differential effects of the drug. Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. Results: TG101209 resulted in a dose and time dependent inhibition of cell growth in the MM cell lines tested. Most of the cytotoxicity was evident by 48 hours, with minimal increase seen up to 96 hours of incubation. At 48 hours of incubation, the median inhibitory concentration was between 2 and 4uM with similar IC50 seen for myeloma cell lines sensitive or resistant to conventional therapies. The IC50s were maintained when the cells were treated in co-culture with stromal cells or in the presence of IL6, IGF or VEGF. Increasing doses of IL6 was not able to rescue the cells from the drug. Dose dependent decrease in proliferation of the cell lines was evidenced by decreased thymidine incorporation. Apoptotic changes in cells following drug treatment was confirmed by flow cytometry for Annexin and PI. Cleavage of caspases 3, 8 and 9 were confirmed on flow cytometry. Addition of the pan-caspase inhibitor zvad-fmk did not prevent drug-induced apoptosis confirming non-caspase mediated mechanisms of cell death as well. Primary myeloma cells from several patients were treated with increasing doses of the drug and IC50 similar to cell lines were seen in 8/10 patient samples tested. Interestingly, evaluation of U266 cell lines, which have a mix of CD45+ and negative cells as well as primary patient cells demonstrated more profound cytotoxicity and anti-proliferative activity of the drug on the CD45+ population relative to the CD45- cells. Immunoblotting studies demonstrated significant down regulation of IL-6 induced pSTAT3 with minor effects on the pERK and pAkt. The effect on pSAT3 was sustained compared to that on pERK and pAkt. This was accompanied by significant down regulation of Bcl-xL. Studies in a mouse model of myeloma are planned. Conclusion: These studies demonstrate significant in-vitro activity of JAK2 inhibition in multiple myeloma. In particular, the preferential targeting of CD45 cells, considered to reflect the proliferative compartment in myeloma holds out the promise for more sustained impact on the disease from a therapeutic standpoint. This is likely explained by the increased sensitivity of the CD45 cells to cytokines as a result of higher expression of different cytokine receptors as has been previously shown. This leads to increased activity of and dependence of the cells on the JAK-STAT pathway and likely explains the increased effect of the pathway inhibition. These studies form the framework for clinical evaluation of the drug in the setting of myeloma. Disclosures: Kumar: CELGENE: Research Funding; MILLENNIUM: Research Funding; BAYER: Research Funding; GENZYME: Research Funding; NOVARTIS: Research Funding.

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Resveratrol Plus Simvastatin Are Effective in Decreasing IgM Secretion in Asymptomatic Waldenstrom Macroglobulinemia. One Year Follow-up.

Blood ◽

10.1182/blood.v114.22.4401.4401 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4401-4401

Author(s):

Francesco Iuliano ◽

Stefania Infusino ◽

Alessia Perricelli ◽

Massimo Di Maio ◽

Angelo Pomillo ◽

...

Keyword(s):

Bone Marrow ◽

Cell Lines ◽

Vitro Activity ◽

Bone Marrow Infiltration ◽

In Vitro Activity ◽

Waldenström Macroglobulinemia ◽

Waldenstrom Macroglobulinemia ◽

Asymptomatic Patients

Abstract Abstract 4401 Background Waldenström macroglobulinemia (WM) is a distinct B-cell lymphoproliferative disorder characterized by lymphoplasmacytic bone marrow infiltration along with an immunoglobulin M (IgM) monoclonal gammopathy. Asymptomatic patients with monoclonal IgM and without morphologic evidence of bone marrow infiltration < 10% clonal marrow cells) are classified as having IgM-MGUS.Therapy is postponed for asymptomatic patients, and progressive anemia is the most common indication for initiation of treatment. Resveratrol (3,4',5-tri-hydroxy-trans-stilbene) is an antioxidant constituent of a wide variety of plant species including grapes. It has gained considerable attention because of its anticancer properties, as shown in solid and hematologic malignancies. Published data show that resveratrol has significant antitumor activity in WM cells line. Moreover, simvastatin, a 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor, induced inhibition of proliferation, cytotoxic effect and apoptosis in IgM secreting cell lines as well as in primary CD19(+) WM cells. With this background we have treated 4 patients with asymptomatic WM with an association of simvastatin and resveratrol to test the efficacy of such of drugs in asymptomatic Waldenstrom macroglobulinemia Methods 4 pts (3 males and 1 female), median age 42,3 yrs (range, 42–73) and asymptomatic WM were treated with a schedule containing resveratrol 40 mg/die and simvastatin 20 mg/die for at least 90 days. At enrollment patients characteristic were hemoglobin level median, 12.1 g/dL,serum beta(2)-microglobulin level median, 2.4 mg/L, and IgM peaks median, 1.8 g/dL. All patients have taken regularly the drugs and there have been no adverse events.CK and LDH serum levels were kept in the normal range. Results In all IgM-MGUS patients a reduction of more than 50% of the IgM peak was observed after 3 months of therapy and it was still maintained at 12 months of follow-up. In SWM patient the reduction was about 25% and it was manteined over time. Striking, another patient with Waldentrom disease resistant to the previous therapy with EDX and anti-CD20 MoAb achieved a CR only after adding resveratrol and simvastatin. Conclusions Our data demonstrate clearly that the association between resveratrol with simvastatin decreases IgM secretion in Waldenstrom macroglobulinaemia and can be useful in asymptomatic or low risk patients not having any adverse effects. Disclosures: Off Label Use: Simvastatin showed in vitro activity on waldentrom cell lines Resveratrol showed in vitro activity on waldenstro cell lines.

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Flavopiridol Induces Apoptosis in Chronic Lymphocytic Leukemia Cells Via Activation of Caspase-3 Without Evidence of bcl-2 Modulation or Dependence on Functional p53

Blood ◽

10.1182/blood.v92.10.3804.422k36_3804_3816 ◽

1998 ◽

Vol 92 (10) ◽

pp. 3804-3816 ◽

Cited By ~ 7

Author(s):

John C. Byrd ◽

Charlotte Shinn ◽

Jamie K. Waselenko ◽

Ephraim J. Fuchs ◽

Teresa A. Lehman ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Lines ◽

Caspase 3 ◽

Mononuclear Cells ◽

P53 Protein ◽

Lymphocytic Leukemia ◽

Vitro Activity ◽

Interleukin 4 ◽

In Vitro Activity

Flavopiridol has been reported to induce apoptosis in lymphoid cell lines via downregulation of bcl-2. The in vitro activity of flavopiridol against human chronic lymphocytic leukemia (CLL) cells and potential mechanisms of action for inducing cytotoxicity were studied. The in vitro viability of mononuclear cells from CLL patients (n = 11) was reduced by 50% at 4 hours, 24 hours, and 4 days at a flavopiridol concentration of 1.15 μmol/L (95% confidence interval [CI] ±0.31), 0.18 μmol/L (95% CI ±0.04), and 0.16 μmol/L (95% CI ±0.04), respectively. Loss of viability in human CLL cells correlated with early induction of apoptosis. Exposure of CLL cells to 0.18 μmol/L of flavopiridol resulted in both decreased expression of p53 protein and cleavage of the caspase-3 zymogen 32-kD protein with the appearance of its 20-kD subunit. Contrasting observations of others in tumor cell lines, flavopiridol cytotoxicity in CLL cells did not correlate with changes in bcl-2 protein expression alterations. We evaluated flavopiridol’s dependence on intact p53 by exposing splenocytes from wild-type (p53+/+) and p53 null (p53−/−) mice that demonstrated no preferential cytotoxicity as compared with a marked differential with F-ara-a and radiation. Incubation of CLL cells with antiapoptotic cytokine interleukin-4 (IL-4) did not alter the LC50 of flavopiridol, as compared with a marked elevation noted with F-ara-a in the majority of patients tested. These data demonstrate that flavopiridol has significant in vitro activity against human CLL cells through activation of caspase-3, which appears to occur independently of bcl-2 modulation, the presence of IL-4, or p53 status. Such findings strongly support the early introduction of flavopiridol into clinical trials for patients with B-CLL.

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