Preclinical evaluation of the dual specific Src/Abl kinase inhibitor AZD0530 in lung cancer

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 13108-13108 ◽  
Author(s):  
O. Gautschi ◽  
P. Purnell ◽  
C. P. Evans ◽  
J. C. Yang ◽  
W. S. Holland ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Growth ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Protein Levels ◽  
Abl Kinase ◽  
Potential Marker ◽  
Induced Apoptosis

13108 Background: AZD0530 is a highly selective, orally available, dual specific Src/Abl kinase inhibitor in clinical development. We tested this agent in multiple lung cancer cell lines in vitro. We hypothesized that activity of AZD0530 may depend on the level of activated (pY416-)Src, and that Src inhibition may decrease Bcl-xL protein levels and lower the barrier to apoptosis. Methods: NSCLC (A549, Calu-1, Calu-6) and SCLC (H69, H526) cells were incubated with 0.001–100 μM AZD0530 for 1–72hrs. Proliferation (MTT), DNA-content (flow cytometry), and protein levels of Src, pY416-Src, PARP, and Bcl-xL (Western blotting) were assessed. Results: Basal pY416-Src was detectable in most cell lines except H526. AZD0530 decreased pY416-Src levels at submicromolar concentrations in pY416-Src positive cells. In A549, Calu-1 and Calu-6, AZD0530 blocked cell growth in a time- and dose-dependent way (IC50 = 7–25 μM) by arrest in G1, retaining a cytostatic effect at submicromolar concentrations in A549 and Calu-1. AZD0530 induced apoptosis in 10–22% of these cells at micromolar concentrations, accompanied by a decrease of Bcl-xL protein in A549 and Calu-1. In H69 and H526, growth inhibition by AZD0530 was limited (IC50 >100 μM). Conclusions: 1) AZD0530 induced apoptosis at micromolar concentrations, and inhibited cell growth at micromolar to submicromolar concentrations in some cell lines. 2) pY416-Src is a potential marker for drug responsiveness, but other factors should also be tested. 3) Decrease of Bcl-xL by AZD0530 may render cancer cells more sensitive to chemotherapy. These data suggest that Src kinase inhibitors merit further testing in lung cancer, both alone and in combination with other agents. (Support: Swiss National Science Foundation, Swiss Cancer League, AstraZeneca). [Table: see text]

In Vitro and In Vivo Activity of the Src/Abl Kinase Inhibitor AP23464 and Analogs Against Activation Loop Mutants of KIT.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 793-793 ◽  
Author(s):  
Amie S. Corbin ◽  
Shadmehr Demehri ◽  
Ian J. Griswold ◽  
Chester A. Metcalf ◽  
William C. Shakespeare ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Myelogenous Leukemia ◽  
Activation Loop ◽  
Wild Type ◽  
Abl Kinase ◽  
Ic50 Values

Abstract Oncogenic mutations of the KIT receptor tyrosine kinase have been identified in several malignancies including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), seminomas/dysgerminomas and acute myelogenous leukemia (AML). Mutations in the regulatory juxtamembrane domain are common in GIST, while mutations in the activation loop of the kinase (most commonly D816V) occur predominantly in SM and at low frequency in AML. Several ATP-competitive kinase inhibitors, including imatinib, are effective against juxtamembrane KIT mutants, however, the D816V mutant is largely resistant to inhibition. We analyzed the sensitivities of cell lines expressing wild type KIT, juxtamembrane mutant KIT (V560G) and activation loop mutant KIT (D816V,F,Y and murine D814Y) to a potent Src/Abl kinase inhibitor, AP23464, and analogs. IC50 values for inhibition of cellular KIT phosphorylation by AP23464 were 5–11 nM for activation loop mutants, 70 nM for the juxtamembrane mutant and 85 nM for wild type KIT. Consistent with this, IC50 values in cell proliferation assays were 3–20 nM for activation loop mutants and 100 nM for wild type KIT and the juxtmembrane mutant. In activation loop mutant-expressing cell lines, AP23464, at concentrations ≤50 nM, induced apoptosis, arrested the cell cycle in G0/G1 and down-regulated phosphorylation of Akt and STAT3, signaling pathways critical for the transforming capacity of mutant KIT. In contrast, 500 nM AP23464 was required to induce equivalent effects in wild-type KIT and juxtamembrane mutant-expressing cell lines. These data demonstrate that activation loop KIT mutants are considerably more sensitive to inhibition by AP23464 than wild type or juxtamembrane mutant KIT. Non-specific toxicity in parental cells occurred only at concentrations above 2 μM. Additionally, at concentrations below 100 nM, AP23464 did not inhibit formation of granulocyte/macrophage and erythrocyte colonies from normal bone marrow, suggesting that therapeutic drug levels would not impact normal hematopoiesis. We also examined in vivo target inhibition in a mouse model. Mice were subcutaneously injected with D814Y-expressing (D816V homologous) murine mastocytoma cells. Once tumors were established, compound was administered three-times daily by oral gavage. One hour post treatment we observed >90% inhibition of KIT phosphorylation in tumor tissue. Following a three-day treatment regimen, there was a statistically significant difference in tumor size compared to controls. Thus, AP23464 analogs effectively target D816-mutant KIT both in vitro and in vivo and inhibit activation loop KIT mutants more potently than the wild type protein. These data provide evidence that this class of kinase inhibitors may have therapeutic potential for D816V-expressing malignancies such as SM or AML.

Bortezomib blocks Bax degradation in malignant B cells during treatment with TRAIL

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2797-2805 ◽  
Author(s):  
Feng-Ting Liu ◽  
Samir G. Agrawal ◽  
John G. Gribben ◽  
Hongtao Ye ◽  
Ming-Qing Du ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Resistant Cell ◽  
Protein Levels ◽  
Bax Protein ◽  
Degradation Activity ◽  
Potent Drug ◽  
Induced Apoptosis

Proapoptotic Bcl-2 family member Bax is a crucial protein in the induction of apoptosis, and its activation is required for this process. Here we report that Bax is a short-lived protein in malignant B cells and Bax protein levels decreased rapidly when protein synthesis was blocked. Malignant B cells were relatively resistant to tumor necrosis factor–related apoptosis inducing ligand (TRAIL)–induced apoptosis, and this correlated with low basal Bax protein levels. Furthermore, during treatment with TRAIL, the resistant cell lines showed prominent Bax degradation activity. This degradation activity was localized to mitochondrial Bax and could be prevented by truncated Bid, a BH3-only protein; in contrast, cytosolic Bax was relatively stable. The proteasome inhibitor bortezomib is a potent drug in inducing apoptosis in vitro in malignant B-cell lines and primary chronic lymphocytic leukemic (CLL) cells. In CLL cells, bortezomib induced Bax accumulation, translocation to mitochondria, conformational change, and oligomerization. Accumulation and stabilization of Bax protein by bortezomib-sensitized malignant B cells to TRAIL-induced apoptosis. This study reveals that Bax instability confers resistance to TRAIL, which can be reversed by Bax stabilization with a proteasome inhibitor.

scholarly journals Identification of Hsp90 inhibitors as potential drugs for the treatment of TSC1/TSC2 deficient cancer

2021 ◽  
Author(s):  
Evelyn M. Mrozek ◽  
Vineeta Bajaj ◽  
Yanan Guo ◽  
Izabela Malinowska ◽  
Jianming Zhang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Xenograft Model ◽  
Growth Delay ◽  
Hsp90 Inhibitors ◽  
Null Cell ◽  
Standard Dosage

Inactivating mutations in either TSC1 or TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder, characterized by multi-system tumor and hamartoma development. Mutation and loss of function of TSC1 and/or TSC2 also occur in a variety of sporadic cancers, and rapamycin and related drugs show highly variable treatment benefit in patients with such cancers. The TSC1 and TSC2 proteins function in a complex that inhibits mTORC1, a key regulator of cell growth, which acts to enhance anabolic biosynthetic pathways. In this study, we identified and validated five cancer cell lines with TSC1 or TSC2 mutations and performed a kinase inhibitor drug screen with 197 compounds. The five cell lines were sensitive to several mTOR inhibitors, and cell cycle kinase and HSP90 kinase inhibitors. The IC50 for Torin1 and INK128, both mTOR kinase inhibitors, was significantly increased in three TSC2 null cell lines in which TSC2 expression was restored.  Rapamycin was significantly more effective than either INK128 or ganetespib (an HSP90 inhibitor) in reducing the growth of TSC2 null SNU-398 cells in a xenograft model. Combination ganetespib-rapamycin showed no significant enhancement of growth suppression over rapamycin. Hence, although HSP90 inhibitors show strong inhibition of TSC1/TSC2 null cell line growth in vitro, ganetespib showed little benefit at standard dosage in vivo. In contrast, rapamycin which showed very modest growth inhibition in vitro was the best agent for in vivo treatment, but did not cause tumor regression, only growth delay.

The Src-Abl Kinase Inhibitor Dasatinib (BMS-354825) Shows Anti-Proliferative and Anti-Apoptotic Effects in Chronic Lymphocytic Leukemia (CLL) Cells In Vitro.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3101-3101
Author(s):  
Aditya Veldurthy ◽  
Michaela Patz ◽  
Christian P. Pallasch ◽  
Clemens M. Wendtner ◽  
Michael Hallek ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Parp Cleavage ◽  
Apoptosis Induction ◽  
Abl Kinase ◽  
Two Samples ◽  
Apoptotic Effects ◽  
Dasatinib Treatment

Abstract Introduction: Dasatinib is an ATP competitive, dual-specific Src-Abl kinase inhibitor used for the treatment of imatinib-resistant Bcr-Abl-positive leukemias. Originally it was developed from the aminothiazole scaffold as an inhibitor of Src family kinases (SFKs). Since the aberrant expression and activity of the SFK Lyn seems to enhance the survival of CLL cells, dasatinib is currently tested in clinical studies for CLL. In order to explore the anti-leukemic potential of dasatinib we tested its effects on freshly isolated CLL cells and on CLL cell lines. Methods and Results: In freshly isolated CLL cells, dasatinib showed a dose and time dependent reduction of global tyrosine phosphorylation which was paralleled by a decreased phosphorylation of the activating tyrosine residue of SFKs. The comparison of the inhibitory effect of several protein tyrosine kinase inhibitors on overall tyrosine phosphorylation in cellular lysates of freshly isolated CLL cells showed that 0.1 μM dasatinib had a more pronounced effect than 5 μM of nilotinib, or 10 μM of PP1 or imatinib. In the CLL cell lines, Mec1 and JVM-3, 0.1 μM of dasatinib appeared to interfere with survival signaling, since decreased levels of the activated phosphorylated forms of Akt, Erk1/2 and p38 were observed after exposure to dasatinib for 2 hours. In these cell lines, dasatinib treatment increased p53 protein levels and induced PARP cleavage and caspase activity. Pro-apoptotic effects of dasatinib were observed by an increase of annexin V-positive cells and a decrease of metabolic activity measured as XTT reduction, alone and in combination with fludarabine. Among six patient samples, two clones were particularly sensitive to dasatinib and fludarabine-resistant. While in these two samples the apoptosis induction by 5 μM dasatinib surpassed that by 5 μM fludarabine, the same dose of the two drugs led to similar apoptosis induction in the fludarabine-sensitive samples. Dasatinib treatment sensitized CLL cells for fludarabine-induced apoptosis. This enhancement of apoptosis induction was more pronounced in fludarabine-resistant patient samples and JVM-3 cells than in Mec1 and fludarabine-sensitive CLL cells. Conclusion: The Src-Abl kinase inhibitor dasatinib shows potent inhibitory effects on the survival of CLL cells in vitro.

Partially or Fully BCR-ABL Independent Mechanisms of in Vitro Resistance to Ponatinib

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2481-2481 ◽  
Author(s):  
Qian Yu ◽  
Anna M Eiring ◽  
Matthew S. Zabriskie ◽  
Jamshid Khorashad ◽  
David J Anderson ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Synthetic Lethality ◽  
Immunoblot Analysis ◽  
Kinase Domain ◽  
Resistant Cell ◽  
Abl Kinase ◽  
Kinase Domain Mutation

Abstract Abstract 2481 Ponatinib (AP24534) is a pan-BCR-ABL inhibitor developed for treatment-refractory chronic myeloid leukemia (CML) and has significant activity in patients who fail second-line dasatinib and/or nilotinib tyrosine kinase inhibitor (TKI) therapy. A pivotal phase II trial (clinicaltrials.gov NCT01207440) is underway. BCR-ABL kinase domain mutation-mediated ponatinib resistance has been investigated in vitro (Cancer Cell 16, 2009, 401). Here, we developed ponatinib-resistant, BCR-ABL+ cell lines lacking a kinase domain mutation and investigated mechanisms of resistance to ponatinib and other TKIs. Methods: Four BCR-ABL+ CML cell lines (K562, AR230, BV173, and 32D(BCR-ABL)) were maintained in liquid culture containing ponatinib (0.1 nM) for 10 days. The ponatinib concentration was increased in small increments for a minimum of 90 days, yielding corresponding ponatinib-resistant cell lines. BCR-ABL kinase domain sequencing of sensitive and resistant cells confirmed BCR-ABL to be unmutated. Real-time qPCR was used to compare the expression of BCR-ABL in ponatinib-sensitive and -resistant cell lines. Immunoblot analysis (total and tyrosine-phosphorylated BCR-ABL) was used to the compare levels of BCR-ABL protein and to determine whether resistance to ponatinib corresponded with reduced (partially BCR-ABL-independent) or complete inhibition of BCR-ABL tyrosine phosphorylation (fully BCR-ABL-independent). Cell proliferation assays were performed on resistant and sensitive cell lines in the presence of ponatinib, nilotinib, and dasatinib. A small-molecule inhibitor screen composed of >90 cell-permeable inhibitors that collectively target the majority of the tyrosine kinome as well as other kinases (Blood 116, 2010, abstract 2754) is currently being applied to the 32D(BCR-ABL)R cell line in the presence of 24 nM ponatinib to assess synthetic lethality, with results analyzed using a companion drug sensitivity algorithm. As a second strategy to generate resistant lines, N-ethyl-N-nitrosourea (ENU) mutagenesis was done to investigate BCR-ABL kinase domain-mediated resistance in myeloid K562, AR230, BV173, and 32D(BCR-ABL) cells. After ENU exposure, cells were washed and cultured in 96-well plates with escalating ponatinib. Results: The four BCR-ABL+ cell lines initially grew in the presence of 0.1 nM but not 0.5 nM ponatinib. Upon gradual exposure to escalating ponatinib, each of the cell lines exhibited a degree of adaptation to growth in the presence of the inhibitor (range: 10 to 240-fold). Real-time qPCR showed a modest two-fold increase in BCR-ABL expression level in K562R, AR230R and BV173R cell lines relative to the respective parental lines. Based on immunoblot analysis, cell lines segregated into two categories of ponatinib resistance: partially (K562R and AR230R) or fully BCR-ABL-independent (BV173R and 32D(BCR-ABL)R). Cell proliferation assays showed that ponatinib resistant cell lines also exhibited resistance to nilotinib and dasatinib. The 32D(BCR-ABL)R cell line exhibited a level of ponatinib resistance comparable to that of the Ba/F3 BCR-ABLE255V cell line, which carries the most ponatinib-resistant BCR-ABL mutation. BCR-ABL tyrosine phosphorylation was efficiently blocked by low concentrations of ponatinib (<5 nM) in the 32D(BCR-ABL)R cell line, yet these cells remained viable in the presence of up to 24 nM ponatinib. The effects of providing a second kinase inhibitor along with ponatinib (24 nM) in order to probe for synthetic lethality are under study. Possible involvement of a second, moderately ponatinib-sensitive target is suggested by the sharp ponatinib maximum at 24 nM; even 26 nM ponatinib is toxic to 32D(BCR-ABL)R cells. Thus far, ENU mutagenesis screens in human CML cell lines failed to yield resistant clones and only a few were recovered from the murine 32D(BCR-ABL)R cell line (3/1440 wells; the only BCR-ABL mutant recovered was BCR-ABLL387F). Conclusions: The ponatinib resistant, BCR-ABL+ cell lines described here exhibit either a partially or fully BCR-ABL independent mechanism of resistance. The molecular details of both processes will be reported, with an emphasis on the striking level of resistance (240-fold over starting conditions) exhibited by the 32D(BCR-ABL)R cell line. Our in vitro results indicate that BCR-ABL independent mechanisms may contribute to ponatinib resistance in myeloid CML cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Pim Kinase Inhibitor AZD1208 Enhances Apoptosis Induction By Clinically Active FLT3 Inhibitors in FLT3-ITD Acute Myeloid Leukemia Cells in Vitro and in Vivo through Synergistic Downregulation of Mcl-1 and of Bcl-xL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3601-3601 ◽  
Author(s):  
Karthika Natarajan ◽  
Trevor J Mathias ◽  
Kshama A Doshi ◽  
Adriana E Tron ◽  
Manfred Kraus ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Protein Levels ◽  
Flt3 Inhibitors ◽  
Synergistic Induction ◽  
Induction Of Apoptosis

Abstract Internal tandem duplication (ITD) mutations of the receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) are present in acute myeloid leukemia (AML) cells in 30% of cases and are associated with high relapse rate and short disease-free survival. FLT3 inhibitors have clinical activity, but their activity is limited and transient. New therapeutic approaches combining FLT3 inhibitors and inhibitors of downstream or parallel signaling pathways may increase depth and duration of responses. The Pim-1 serine/threonine kinase is transcriptionally upregulated by FLT3-ITD. We previously demonstrated that Pim-1 phosphorylates and stabilizes FLT3 and thereby promotes its signaling in a positive feedback loop. Pim kinase inhibitors are in clinical trials. Here we studied the effect of combinations of the Pim kinase inhibitor AZD1208 and clinically active FLT3 inhibitors on AML with FLT3-ITD in vitro and in vivo. Ba/F3-ITD cells, with FLT3-ITD, were grown in medium with the Pim kinase inhibitor AZD1208 at 1 μM and/or the FLT3 inhibitors quizartinib (Q), sorafenib (S) or crenolanib (C) at their IC50values of 1, 2.5 and 20 nM, respectively, and viable cells were measured at serial time points. While Q, S, C or AZD1208 treatments reduced cell numbers, compared to DMSO control, combined AZD1208 and Q, S or C treatments abrogated proliferation. Because FLT3-ITD cells remain responsive to FLT3 ligand (FLT3L) despite constitutive FLT3 activation and increased FLT3L levels following chemotherapy have been hypothesized to contribute to relapse, we repeated the proliferation experiments in the presence of 0, 1, 3 and 10 ng/ml FLT3L. FLT3L produced a concentration-dependent increase in proliferation and, while Q, S, C or AZD1208 treatments individually reduced cell numbers, combined AZD1208 and Q, S or C abrogated proliferation at all FLT3L concentrations tested, suggesting that these combinations overcome growth stimulation by FLT3L. To understand the anti-proliferative effect of combined Pim-1 and FLT3 inhibitors, we first studied cell cycle effects of AZD1208 and Q, S or C in Ba/F3-ITD cells and of AZD1208 and Q in the additional FLT3-ITD cell lines 32D-ITD, MV4-11 and MOLM14. We found a progressive increase in sub-G1 phase cells at 24, 48 and 72 hours, consistent with induction of apoptosis. Synergistic induction of apoptosis was confirmed by Annexin V/propidium iodide labeling of Ba/F3-ITD and 32D-ITD cells treated for 48 hours with AZD1208 combined with Q (p<0.0001), S (p<0.0001) or C (p<0.001), and of MV4-11 (p<0.0001) and MOLM14 (p<0.05) cells treated with AZD1208 combined with Q, in relation to each drug alone. Apoptosis was additionally confirmed by loss of mitochondrial membrane potential. Synergistic induction of apoptosis was not seen in Ba/F3-WT or 32D-WT cells, with wild-type FLT3, indicating a FLT3-ITD-specific effect. Synergistic (p<0.01) induction of apoptosis was seen in three FLT3-ITD AML patient samples treated in vitro with AZD1208 combined with Q. In an in vivo model, synergistic decrease in tumor volume was seen with combined AZD1208 and Q therapy in mice with subcutaneously implanted MV4-11 cells, with FLT3-ITD, but not with KG1a cells, with wild-type FLT3. Mechanistically, combined AZD1208 and Q treatment in vitro did not increase reactive oxygen species, compared to each drug alone, but increased both cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) levels, and caspase 3 cleavage was reduced by co-incubation with the pan-caspase inhibitor Z-VAD. Moreover, combined AZD1208 and Q treatment caused a synergistic decrease in expression of the anti-apoptotic Mcl-1 and of Bcl-xL proteins, but did not significantly alter Bim-1, p-Bad, Bad, Bax, Bak or Bcl-2, pro- and anti-apoptotic protein levels. Bcl-xL mRNA expression decreased along with protein levels, but Mcl-1 mRNA levels remain unchanged, indicating post-transcriptional down-regulation of Mcl-1 by the combination treatment. In summary, synergistic cytotoxicity of AZD1208 and clinically active FLT3 inhibitors was demonstrated in FLT3-ITD cell lines and patient samples in vitro and in cell lines in vivo, via caspase-mediated apoptosis, associated with a synergistic decrease in Mcl-1 and Bcl-xL expression. Our data suggest clinical promise for combination therapy with Pim kinase and FLT3 inhibitors in patients with AML with FLT3-ITD. Disclosures No relevant conflicts of interest to declare.

The Novel, Orally Available Multi-Kinase Inhibitor BAY 73-4506 Targets Myeloma Cells and Their Bone Marrow Microenvironment.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2525-2525
Author(s):  
Marc S. Raab ◽  
Iris Breitkreutz ◽  
Podar Klaus ◽  
Jing Zhang ◽  
Simona Blotta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cell ◽  
Cell Growth ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Dependent Manner ◽  
The Novel ◽  
Phase I Clinical Trials ◽  
Potent Inducer

Abstract Multitargeted treatment approaches have been shown to be more effective than single agent therapy in multiple myeloma (MM). In addition, agents targeting not only the MM cells directly but also their microenvironment, like bone marrow stromal cells (BMSCs), endothelial cells, and osteoclasts (OCLs) causing enhancement of tumor cell growth, angiogenesis, and MM bone disease, respectively, are promising new treatment modalities for this still non-curable disease.Here we investigated the novel, orally available multi-kinase inhibitor BAY 73-4506, currently in phase I clinical trials, for its therapeutic effect in MM. BAY is a potent inhibitor of angiogenic (VEGFR 1-3, PDGFR-b), as well as oncogenic, kinases (cKIT, RET, FGFR, Raf). We first tested the ability of BAY to suppress MM cell proliferation and survival in a wide array of MM cell lines (MM.1S, RPMI 8226, NCI H929, OPM2, KMS11, KMS 18, INA6, U266, KMS12BM, S6B45), including those resistant to conventional chemotherapeutics (MM.1R, Dox40, LR5). Our data show that BAY is active in all cell lines tested in a low micromolar range equivalent to concentrations achieved in patient plasma during the first clinical trial in solid tumors. Importantly, BAY also overcomes the growth advantage conferred in a BMSC-MM, as well as an endothelial cell-MM, coculture system. BAY treatment abrogates MEK, ERK and AKT phosphorylation in a time and dose dependent manner, followed by induction of apoptosis, evidenced by Annexin staining and DNA fragmentation. Since VEGF signaling pathway is a potent inducer of angiogenesis and BAY targets VEGFR 1-3, we examined anti-angiogenic properties of BAY. This compound inhibits endothelial cell growth and endothelial cell tubuli formation in vitro at concentrations less than 1mM; moreover, BAY markedly inhibits the VEGF-induced cell migration on fibronectin. Activation of MAP kinase is a critical event during OCL differentiation, activation, and survival; BAY inhibits osteoclastogenesis, evidenced by blockade of M-CSF/RANKL-triggered differentiation of mononuclear cells to TRAP-positive osteoclasts, an important marker of osteoclastogenesis. Finally, combination treatment of BAY with dexamethasone shows synergistic effects on MM cell growth and survival. These in vitro experiments on the effects of BAY on MM tumor cells directly, in co-culture with endothelial or BMSCs, as well as on osteoclast differentiation, provides the basis for its evaluation in a murine model of human MM to confirm these promising in vitro effects of this novel multi-kinase inhibitor, finally leading to clinical evaluation to improve patient outcome.

scholarly journals Evaluation of Hsp90 and mTOR inhibitors as potential drugs for the treatment of TSC1/TSC2 deficient cancer

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0248380
Author(s):  
Evelyn M. Mrozek ◽  
Vineeta Bajaj ◽  
Yanan Guo ◽  
Izabela A. Malinowska ◽  
Jianming Zhang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Xenograft Model ◽  
Mtor Inhibitors ◽  
Growth Delay ◽  
Null Cell ◽  
Standard Dosage

Inactivating mutations in either TSC1 or TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder, characterized by multi-system tumor and hamartoma development. Mutation and loss of function of TSC1 and/or TSC2 also occur in a variety of sporadic cancers, and rapamycin and related drugs show highly variable treatment benefit in patients with such cancers. The TSC1 and TSC2 proteins function in a complex that inhibits mTORC1, a key regulator of cell growth, which acts to enhance anabolic biosynthetic pathways. In this study, we identified and validated five cancer cell lines with TSC1 or TSC2 mutations and performed a kinase inhibitor drug screen with 197 compounds. The five cell lines were sensitive to several mTOR inhibitors, and cell cycle kinase and HSP90 kinase inhibitors. The IC50 for Torin1 and INK128, both mTOR kinase inhibitors, was significantly increased in three TSC2 null cell lines in which TSC2 expression was restored. Rapamycin was significantly more effective than either INK128 or ganetespib (an HSP90 inhibitor) in reducing the growth of TSC2 null SNU-398 cells in a xenograft model. Combination ganetespib-rapamycin showed no significant enhancement of growth suppression over rapamycin. Hence, although HSP90 inhibitors show strong inhibition of TSC1/TSC2 null cell line growth in vitro, ganetespib showed little benefit at standard dosage in vivo. In contrast, rapamycin which showed very modest growth inhibition in vitro was the best agent for in vivo treatment, but did not cause tumor regression, only growth delay.

The effects of lapatinib and neratinib on HER2 protein levels in breast cancer cell lines.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 637-637 ◽  
Author(s):  
Denis Collins ◽  
Norma O'Donovan ◽  
Naomi Walsh ◽  
Kathy Gately ◽  
Connla Edwards ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Breast Cancer Cell Lines ◽  
Breast Cancers ◽  
Her2 Gene ◽  
Her2 Protein ◽  
Protein Levels

637 Background: HER2, a member of the c-erbB receptor tyrosine kinase family, is over-expressed (HER2 gene amplification/IHC 3+, >30% cells) in approx. 25% of breast cancers. Pre-clinical studies have shown that the HER2-targeted small molecule tyrosine kinase inhibitor (TKI) lapatinib (LAP) can increase HER2 levels in cell line models and can potentiate trastuzumab-mediated antibody dependent cell-mediated cytotoxicity. To assess the potential effects of the next generation TKI neratinib (NER) on expression of HER2 in breast cancer, this study compares the effects of LAP and NER on HER2 protein levels in the HER2-amplified SKBR3 and HER2-non-amplified T47D cell lines in vitro. Methods: SKBR3 and T47D were treated with LAP or NER (0.2, 1 and 2 µM) for 12, 24 and 48 hours.HER2 protein levels were determined by ELISA, immunoblotting and high content analysis (HCA). HER2 protein was examined using two antibodies targeting the extracellular domain (ECD) and the intracellular domain (ICD) of HER2. pHER2, EGFR/pEGFR, MAPK/pMAPK and AKT/pAKT levels were determined by immunoblotting. Proliferation studies utilized an acid phosphatase-based assay. Results: ELISA analysis confirmedsignificantly lower HER2 expression in T47D (44 +/- 17 pg/µg) compared to SKBR3 (748 +/- 296 pg/µg), p = 0.015. NER proved a more potent inhibitor of pHER2 and pEGFR as analyzed by immunoblotting and in proliferation studies (NER IC50 - SKBR3 0.03 +/- 0.01 nM, T47D 199 +/- 70 nM, LAP IC50 - SKBR3 20 +/- 1 nM, T47D 1.2 +/- 0.2 µM). LAP induced an increase in both ECD and ICD-containing HER2 protein levels in SKBR3 and T47D cells. No increase in HER-2 levels was observed with NER treatment, as determined by HCA and immunoblotting. EGFR protein levels increased in response to lapatinib in both cell lines. Conclusions: Our results suggest that LAP and NER have differing effects on HER2 protein levels in the models examined. NER provides greater inhibition of HER2 signaling activity but does not increase HER2 protein levels as was observed with LAP. Further pre-clinical assessment of combinations of LAP and NER with HER2 and EGFR monoclonal antibody therapies is warranted in HER2-amplified, HER2-non-amplified and EGFR-expressing breast cancer models.

scholarly journals The Impact of Cand1 in Prostate Cancer

Cancers ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 428
Author(s):  
Andrea Eigentler ◽  
Piotr Tymoszuk ◽  
Johanna Zwick ◽  
Arndt A. Schmitz ◽  
Andreas Pircher ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Clinical Findings ◽  
Protein Levels ◽  
Bioinformatic Tools ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Genes Encoding ◽  
The Impact

Evidence has accumulated asserting the importance of cullin-RING (really interesting new gene) ubiquitin ligases (CRLs) and their regulator Cullin-associated neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) dissociated protein 1 (Cand1) in various cancer entities. However, the role of Cand1 in prostate cancer (PCa) has not been intensively investigated so far. Thus, in the present study, we aimed to assess the relevance of Cand1 in the clinical and preclinical setting. Immunohistochemical analyses of radical prostatectomy specimens of PCa patients showed that Cand1 protein levels are elevated in PCa compared to benign areas. In addition, high Cand1 levels were associated with higher Gleason Scores, as well as higher tumor recurrence and decreased overall survival. In line with clinical findings, in vitro experiments in different PCa cell lines revealed that knockdown of Cand1 reduced cell viability and proliferation and increased apoptosis, therefore underlining its role in tumor progression. We also found that the cyclin-dependent kinase inhibitor p21 is significantly upregulated upon downregulation of Cand1. Using bioinformatic tools, we detected genes encoding for proteins linked to mRNA turnover, protein polyubiquitination, and proteasomal degradation to be significantly upregulated in Cand1high tumors. Next generation sequencing of PCa cell lines resistant to the anti-androgen enzalutamide revealed that Cand1 is mutated in enzalutamide-resistant cells, however, with little functional and clinically relevant impact in the process of resistance development. To summarize the present study, we found that high Cand1 levels correlate with PCa aggressiveness.

Sign in / Sign up

Export Citation Format

Share Document