Combined Effects of novel Heat Shock Protein 90 Inhibitor NVP-AUY922 and Nilotinib in a Random Mutagenesis Screen.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3765-3765
Author(s):  
Tetsuzo Tauchi ◽  
Seiichi Okabe ◽  
Eishi Ashihara ◽  
Shinya Kimura ◽  
Taira Maekawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Heat Shock ◽  
Random Mutagenesis ◽  
Heat Shock Protein 90 ◽  
Kinase Domain ◽  
Hsp90 Inhibitor ◽  
Combined Effects ◽  
Endosteal Surface ◽  
Imatinib Resistant

Abstract Abstract 3765 Poster Board III-701 To overcome imatinib resistance, more potent ABL TKIs such as nilotinib and dasatinib have been developed, with demonstrable preclinical activity against most imatinib-resistant BCR-ABL kinase domain mutations, with the exception of T315I. However, imatinib-resistant patients already harbouring mutations have a higher likelihood of developing further mutations under the selective pressure of potent ABL TKIs. The challenge for development of an effective Ph-positive leukemia therapy is therefore to develop an alternative treatment strategy that does not rely solely on kinase domain inhibition but rather results in degradation of the offending BCR-ABL protein regardless of its mutation status. NVP-AUY922 (Novartis) is a novel 4,5-diaryloxazole ATP-binding site heat shock protein 90 (HSP90) inhibitor, which has been shown to inhibit the chaperone function of HSP90 and deplete the levels of HSP90 client protein. In the present study, we investigated the combined effects of AUY922 and Nilotinib on random mutagenesis for BCR-ABL mutation (Blood, 109; 5011, 2007). We performed a comprehensive drug combination experiment using a broader range of concentrations for AUY922 and Nilotinib. Compared with single agents, combination with AUY922 and Nilotinib was more effective at reducing the outgrowth of resistant cell clones. At the highest concentration of Nilotinib, the mutation spectrum narrowed to T315I and E344V by direct sequencing, whereas, at intermediate concentration of AUY922, the resistant clone was recovered by wild-type BCR-ABL only. No outgrowth was observed in the presence of 2 μM Nilotinib and 20 nM AUY922. To assess the in vivo efficacy of AUY922 and Nilotinib, athymic nude mice were injected s.c. with BaF3 cells expressing random mutagenesis for BCR-ABL mutation. 7 days after injection (average tumor volume, 100 mm3), the mice were divided four groups (5 mice per group), with each group receiving either vehicle, AUY922 (50mg/kg; i.p. weekly), Nilotinib (30mg/kg; p.o. once every day), AUY922 (50mg/kg; i.p. weekly) + Nilotinib (30mg/kg; p.o. once every day). Combination with AUY922 and Nilotinib effectively inhibited tumor growth in mice compared with vehicle- or Nilotinib- or AUY922-treated mice. Histopathologic analysis of tumor tissue from AUY922 plus Nilotinib-treated mice demonstrated an increased number of apoptotic cells detected by TUNEL stain. To investigate combined effects of AUY922 and Nilotinib on primary CML cells, NOD/SCID mice were injected i.v. with bone marrow mononuclear cells from a CML patients with F359V mutation. Treatment with AUY922 and Nilotinib demonstrated a marked segregation of apoptotic cells in both the central bone-marrow cavity and the endosteal surface. Furthermore, treatment with AUY922 and Nilotinib inhibited the phosphorylation of GSK-3β and Stat-3 in the endosteal surface. These results suggest that the combination with a HSP90 inhibitor and ABL TKIs may eliminate the CML initiating cells. Taken together, the present study shows that the combination of AUY922 and Nilotinib exhibits a desirable therapeutic index that can reduce the in vivo growth of mutant forms of BCR-ABL-expressing cells. Disclosures: No relevant conflicts of interest to declare.

Combined Effects of the Hedgehog Pathway Inhibitor LDE225 and Nilotinib In a Random Mutagenesis Screen

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 595-595
Author(s):  
Tetsuzo Tauchi ◽  
Seiichi Okabe ◽  
Eishi Ashihara ◽  
Shinya Kimura ◽  
Taira Maekawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Inhibition ◽  
Random Mutagenesis ◽  
Hedgehog Pathway ◽  
Receptor Protein ◽  
Parp Cleavage ◽  
Tunel Staining ◽  
Apoptotic Cells ◽  
Combined Effects

Abstract Abstract 595 The hedgehog signaling pathway is a developmental pathway that has been shown to play a role in primitive hematopoiesis. Recent studies have demonstrated that hedgehog pathway is activated in chronic myeloid leukemia (CML) stem cells via up-regulation of Smoothened (Smo), a seven transmembrane domain receptor protein. LDE225 is a small molecule Smo antagonist which has entered Phase I clinical evaluation in patients with solid tumors. In the present study, we investigated the combined effects of LDE225 and nilotinib in a random mutagenesis study for BCR-ABL mutation (Blood, 109; 5011, 2007). We performed a comprehensive drug combination experiment using a broader range of concentrations for LDE225 and nilotinib. Compared with single agents, the combination of LDE225 and nilotinib was more effective at reducing the outgrowth of resistant cell clones. No outgrowth was observed in the presence of 2 μM nilotinib plus 20 μM LDE225. Also co-treatment with LDE225 and nilotinib resulted in significantly more inhibition of growth than treatment with either agent alone in BaF3 cells expressing wt-BCR-ABL and BCR-ABL mutants (M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). The observed data from the isobologram indicated the synergistic effect of simultaneous exposure to LDE225 and nilotinib even in BaF3 cells expressing T315I. Co-treatment with 2μM of nilotinib significantly increased LDE225-induced apoptosis in BaF3 cells expressing T315I. Combined treatment with LDE225 and nilotinib in BaF3 T315I also associated with more PARP cleavage, resulting from increased activation of caspase-3 and -9 during apoptosis. To assess the mechanism of combination effect between LDE225 and nilotinib on T315I BCR-ABL-expressing cells, we used RNA interference to determine whether reduction of nilotinib off-target affected the growth inhibition. BaF3 cells expressing T315I BCR-ABL pretreated with DDR1 siRNA showed enhanced growth inhibition with LDE225. These results demonstrate that the enhanced growth inhibition by LDE225 and nilotinib in T315I-expressing cells may be mediated by DDR1. To assess the in vivo efficacy of LDE225 and nilotinib, athymic nude mice were injected s.c. with BaF3 cells expressing random mutagenesis for BCR-ABL mutation. 7 days after injection (average tumor volume, 100 mm3), the mice were randomised into four groups (5 mice per group), with each group receiving either vehicle, LDE225 (20mg/kg; p.o. once every day), nilotinib (30mg/kg; p.o. once every day), LDE225 (20mg/kg; p.o. once every day) + nilotinib (30mg/kg; p.o. once every day). The LDE225 and nilotinib combination more effectively inhibited tumor growth in mice compared to either vehicle- or nilotinib- or LDE225-treated mice. Histopathologic analysis of tumor tissue from LDE225 plus nilotinib-treated mice demonstrated an increased number of apoptotic cells detected by TUNEL staining. To investigate combined effects of LDE225 and nilotinib on primary Ph-positive acute lymphocytic leukemia (ALL) cells, NOD/SCID mice were injected i.v. with bone marrow mononuclear cells from a Ph positive ALL patient. Treatment with LDE225 and nilotinib demonstrated a marked segregation of apoptotic cells in both the central bone-marrow cavity and the endosteal surface. These results suggest that the combination with a Smo inhibitor and ABL TKIs may help to eliminate the Ph positive ALL cells. Taken together, the present study shows that the combination of LDE225 and nilotinib exhibits a desirable therapeutic index that can reduce the in vivo growth of mutant forms of BCR-ABL-expressing cells. Disclosures: Tauchi: Novartis Pharma KK: Research Funding.

Combined Effects of Novel Heat Shock Protein 90 Inhibitor NVP-AUY922 and Nilotinib against Mutant Forms of BCR-ABL

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 724-724
Author(s):  
Tetsuzo Tauchi ◽  
Daigo Akahane ◽  
Seiichi Okabe ◽  
Eishi Ashihara ◽  
Shinya Kimura ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 90 ◽  
Parp Cleavage ◽  
Combined Effects ◽  
Wild Type ◽  
Abl Kinase ◽  
Histopathologic Analysis ◽  
Mutant Forms

Abstract NVP-AUY922 (Novartis) is a novel 4,5-diaryloxazole ATP-binding site heat shock protein 90 (HSP90) inhibitor, which has been shown to inhibit the chaperone function of HSP90 and deplete the levels of HSP90 client protein. Combining AUY922 with ABL kinase inhibitors may provide several advantages, such as enhanced efficacy and reducing the potential emergence of new resistant mutations. Treatment with AUY922 has been shown to exert greater potency against BCR-ABL mutants compared with wild type (wt) BCR-ABL. In the present study, we investigated the combined effects of AUY922 and Nilotinib on mutant forms of BCR-ABL-expressing cells. Co-treatment with AUY922 and Nilotinib resulted in significantly more inhibition of growth than treatment with either agent alone in BaF3 cells expressing wt-BCR-ABL and BCR-ABL mutants (M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). The observed data from the isobologram indicated the synergistic effect of simultaneous exposure to AUY922 and Nilotinib even in BaF3 cells expressing T315I. In contrast, we did not observe enhanced effects of AUY922 and imatinib in T315I BCR-ABL-expressing cells. Co-treatment with 500nM of Nilotinib significantly increased AUY922-induced apoptosis in BaF3 cells expressing T315I. Combined treatment with AUY922 and Nilotinib in BaF3 T315I also associated with more PARP cleavage, resulting from increased activation of caspase-3 and -9 during apoptosis. Following co-treatment of BaF3 T315I with AUY922 and Nilotinib caused more attenuation of phospho-T315I BCR-ABL levels and the downstream signal transducer, including phosho-CrkL, phospho-Stat5, and phospho-Akt. These results demonstrate that Nilotinib appears to inhibit T315I BCR-ABL kinase activity in BCR-ABL structurally compromised by loss of HSP90 chaperone activity. To assess the in vivo efficacy of AUY922 and Nilotinib, athymic nude mice were injected i.v. with mixture of BaF3 cells expressing wild type BCR-ABL and mutant forms of BCR-ABL(M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). 24 hrs after injection, the mice were divided four groups (5 mice per group), with each group receiving either vehicle, AUY922 (50mg/kg; i.p. two times per week), Nilotinib (30mg/kg; p.o. once every day), AUY922 (50mg/kg; i.p. two times per week) + Nilotinib (30mg/kg; p.o. once every day). Animals treated with either vehicle or Nilotinib-alone died of a condition resembling acute leukemia by 28 days; animals treated with AUY922 alone survived more than 40 days, and those treated with the combination of AUY922 + Nilotinib survived more than 60 days. Histopathologic analysis of vehicle or Nilotinib-alone treated mice revealed infiltration of the spleen and bone marrow with leukemic blasts. In contrast, histopathologic analysis of organs from AUY922 plus Nilotinib-treated mice demonstrated normal tissue architecture and no evidence of residual leukemia. Taken together, these preclinical studies show that the combination of AUY922 and Nilotinib exhibits a desirable therapeutic index that can reduce the in vivo growth of mutant forms of BCR-ABL-expressing cells, including T315I, in an efficacious manner.

scholarly journals Polymorphic glutathione S-transferase subunit 3 of rat liver exhibits different susceptibilities to carbon tetrachloride: differences in their interactions with heat-shock protein 90

2003 ◽  
Vol 372 (2) ◽  
pp. 611-616 ◽  
Author(s):  
Jun MAYAMA ◽  
Takayuki KUMANO ◽  
Makoto HAYAKARI ◽  
Takehiko YAMAZAKI ◽  
Shu AIZAWA ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 90 ◽  
Hsp90 Inhibitor ◽  
Glutathione S Transferase ◽  
Ccl4 Administration ◽  
A Subunit ◽  
Rat Livers

Rat glutathione S-transferase (GST) subunit 3 gene has polymorphism, one type encoding Asn198-Cys199 (NC type) and another encoding Lys198-Ser199 (KS type). To examine whether the two types of GST 3-3 exhibit different susceptibilities to oxidative stress in vivo, rats were administered with CCl4, a hepatotoxin causing severe oxidative stress, and its effect on liver GST 3-3 was compared. Decrease in GST activities in liver due to CCl4 administration was more evident in NC type rats than in KS type rats, and most GST activities of KS type rats were confined to S-hexylglutathione–Sepharose, whereas those of NC type rats were not. Decreases in GST subunits 1 and 3 were more marked in NC type rats and glutathiolated NC type GST 3-3 was also detected. These results indicated that KS and NC type GST 3-3 of rat livers exhibited different susceptibilities to CCl4in vivo. A protein consisting of a subunit with molecular mass of 90 kDa was shown to bind to KS type GST 3-3 but not to NC type. This protein was identified as heat-shock protein (HSP) 90β by N-terminal amino acid sequencing and immunoblotting. A specific HSP90 inhibitor geldanamycin released their binding. There was no difference in the binding of apoptosis signal-regulating kinase 1 to GST 3-3 between NC and KS type rats. These findings suggest that HSP90 interacts with KS type GST 3-3 and thereby protects it from inactivation due to CCl4.

Potential role of the heat shock protein 90 (hsp90) in buffering mutations to favour cyclical parthenogenesis in the peach potato aphid Myzus persicae (Aphididae, Hemiptera)

2018 ◽  
Vol 109 (4) ◽  
pp. 426-434 ◽  
Author(s):  
M. Mandrioli ◽  
E. Zanetti ◽  
A. Nardelli ◽  
G.C. Manicardi
Keyword(s):  
Heat Shock ◽  
Myzus Persicae ◽  
Heat Shock Protein 90 ◽  
Hsp90 Inhibitor ◽  
Cyclical Parthenogenesis ◽  
Potato Aphid ◽  
Wide Range ◽  
Peach Potato Aphid

AbstractHeat-shock proteins 90 (hsp90s) are a class of molecules able to stabilize a network of ‘client’ proteins that are involved in several processes. Furthermore, recent studies indicated that mutations in the hsp90-encoding gene induce a wide range of phenotypic abnormalities, which have been interpreted as an increased sensitivity of different developmental pathways to hidden/cryptic mutations. In order to verify the role of hsp90 in aphids, we amplified and sequenced the hsp90 gene in 17 lineages of the peach potato aphid Myzus persicae (Sulzer, 1776) looking for the presence of mutations. In particular, we compared lineages with different reproductive modes (obligate vs. cyclical parthenogenesis), propensity to develop winged females and karyotype stability. Differently from the cyclical parthenogenetic lineages that possessed functional hsp90 genes, the seven analysed asexual lineages showed severe mutations (including frameshift and non-sense mutations). In vivo functional assays with the hsp90-inhibitor geldanamycin showed that some lineages with cyclical parthenogenesis may lose their ability to induce sexuales in the absence of active hsp90 revealing the presence of cryptic mutations in their genomes. As a whole, our data suggest that hsp90 could play in aphids a role in buffering hidden/cryptic mutations that disrupt cyclical parthenogenesis.

scholarly journals Combined effects of novel heat shock protein 90 inhibitor NVP-AUY922 and nilotinib in a random mutagenesis screen

Oncogene ◽  
2011 ◽  
Vol 30 (24) ◽  
pp. 2789-2797 ◽  
Author(s):  
T Tauchi ◽  
S Okabe ◽  
E Ashihara ◽  
S Kimura ◽  
T Maekawa ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Random Mutagenesis ◽  
Heat Shock Protein 90 ◽  
Combined Effects ◽  
Mutagenesis Screen

Effect of inhibition of heat-shock protein 90 on mutant c-Kit protein as modulated by mRNA expression and protein degradation through both proteasome and autophagy-mediated pathways.

2011 ◽  
Vol 29 (4_suppl) ◽  
pp. 54-54
Author(s):  
L. Chen ◽  
Y. Hsueh ◽  
N. Chiang ◽  
C. Yen ◽  
N. Shih
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Protein Degradation ◽  
Cell Model ◽  
Mrna Level ◽  
Heat Shock Protein 90 ◽  
Hsp90 Inhibitor ◽  
Cmv Promoter ◽  
Proteasome Degradation ◽  
Imatinib Resistant

54 Background: c-Kit is a known client protein of heat-shock protein 90 (Hsp90). Inhibition of Hsp90 can reduce the expression of c-Kit protein in human mastocytoma and gastrointestinal stromal tumor (GIST) cells, which is conceived through the proteasome degradation pathway. Methods: COS-1 cells transfected with vectors containing CMV promoter-driven various c-Kit mutants (imatinib- sensitive or imatinib-resistant) and human GIST882 cells (with endogenous exon 13 mutant c-Kit) were used to investigate the efficacy NVP-AUY922, a new class of Hsp90 inhibitor, on suppressing the constitutive activation of mutated c-Kit. Detail mechanisms of protein regulation, as RNA transcription, RNA stability, and protein degradation were studied. Results: NVP-AUY922 is more potent than 17-AAG to inhibit the proliferation of imatinib-sensitive GIST882 cells. Further study showed that NVP-AUY922 down-regulated both total and phosphorylated c-Kit proteins in dose- and time-dependent manners in both GIST882 cells and mutant c-Kit (including imatinib-resistant exon11/17 double mutants) expressed in COS-1 cells. Surprisingly, the NVP-AUY922-induced c-Kit degradation in both cell models could be reversed by proteasome-degradation inhibitor and autophagy inhibitor. The involvement of autophagy in NVP-AUY922-induced c-Kit protein degradation was further confirmed by co-localization of c-Kit and autophagosome by immuno-staining. In addition, NVP-AUY922 treatment resulted in a reduction of c-Kit mRNA level in GIST882 cells but not c-Kit expressing COS-1 cells, which could be explained by the use of CMV promoter in artificial COS-1 cell model. Conclusions: Taken together, these findings provide the first evidence that down-regulation of mutant c-Kit protein by Hsp90 inhibitor involved both transcription and post-translation levels. Of the latter, autophagy- as well as proteasome-mediated degradation pathways were involved. These observations highlight the use of NVP-AUY922 either alone or in combination with autophagy modulators in imatinib-refractory, c-Kit-expressing GIST. Supported by 99D1-TC-CADOH03. [Table: see text]

scholarly journals Targeting BCL6-Mediated Resistance to BCR-ABL Targeted Tyrosine Kinase Inhibitors (TKIs) in Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL) through the Addition of Histone Deacetylase (HDAC) Inhibitors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1277-1277
Author(s):  
Gordon Cohen ◽  
Colleen E. Annesley ◽  
Rachel E. Rau ◽  
Shannon Kelley ◽  
Daniel Magoon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Lymphoblastic Leukemia ◽  
Survival Rates ◽  
Kinase Domain ◽  
Western Blots ◽  
Imatinib Resistant ◽  
Ph Chromosome ◽  
Kinase Domain Mutations

Abstract Introduction: Patients who harbor the Philadelphia (Ph+) chromosome t(9;22) translocation account for approximately 20-30% of adult ALL and 2-5% of pediatric ALL. Prior to approval and use of imatinib, a small molecule TKI which targets the Ph+ chromosome BCR-ABL1, these patients had poor survival & EFS - with long term survival rates in the 20% range. With the addition of imatinib and later generation TKIs to chemotherapy backbones and bone marrow transplant, EFS & survival rates have substantially improved - surpassing 50% in studies in adults and even higher in children. However, resistance to imatinib and other TKIs has become a significant problem in Ph+ ALL, especially in adults. ABL1 kinase domain mutations are the dominant form of TKI resistance, however other resistance mechanisms include upregulation of parallel pathways such as SRC family kinases, MAPK and BCL6 pathways. BCL6 is an oncogene that suppresses transcription of tumor suppressor genes such as p53 and CDNK1A. Interestingly, BCL6 has been shown to be upregulated and activated through deacetylation following imatinib treatment in Ph+ ALL, likely leading to its role in resistance. Histone deacetylase inhibitors (HDACi) have been shown to act synergistically with TKIs in imatinib sensitive and resistant Ph+ leukemia though multiple mechanisms including attenuation of BCR-ABL1 levels and other downstream proliferation promoting pathways. We have shown that HDACi treatment acetylates (and thus inactivates) BCL6 in Ph+ ALL, and that the combination of HDACis and TKIs leads to synergistic effects in vitro and in vivo (using xenograft models). Methods: In vitro WST-1 cell viability assays were carried out on TOM1 cells (non-ABL1 mutant, imatinib sensitive Ph+ ALL) and NALM1 cells (non-ABL1 mutant, imatinib resistant CML lymphoid blast crisis) with imatinib and entinostat (a HDACi). Synergy was assessed using Calcusyn software. Western blots were performed assessing BCL6 expression and acetylation, and expression of downstream effectors of apoptosis. Two separate in vivo xenograft mouse experiments were performed transplanting TOM1 and NALM1 cells into Nod SCID Gamma (NSG) mice. Cohorts of TOM1 mice were treated with imatinib 50mg/kg BID, entinostat 15mg/kg QD, imatinib plus entinostat combination, or vehicle control. In the NALM1 mice we added a higher dose imatinib cohort (100 mg/kg BID) due to known imatinib resistance. Results: In vitro, there was substantially more synergy of the imatinib/entinostat combination in imatinib-resistant NALM1 cells vs. the imatinib-sensitive TOM1 cells. Average Combination Index (CI) values in TOM1 cells across multiple entinostat and imatinib doses was 1.2 (CI: =1 suggest additive effect, <1 = synergy, >1 = antagonism), while the CI in NALM1 cells at the same dose combinations was 0.53. We noted BCL6 upregulation and decreased BCL6 acetylation - signs correlating with resistance - in Western blots of NALM1 and TOM1 cells treated with imatinib, while exposure to entinostat caused increased acetylation of BCL6 and increased expression of downstream tumor suppressors. In the imatinib-sensitive TOM1 xenograft trial, the combination displayed a significant reduction in bone marrow leukemic blast involvement versus control following 6 weeks of dosing as measured by flow cytometry (36.9% mean decrease, p=0.001). There was a trend toward decreased bone marrow involvement between the combination treatment and other active treatment arms. There was no difference in peripheral blood blast percentage between arms. In the imatinib-resistant NALM1 xenograft trial, the combination showed a significant decrease in peripheral blood blast percentage in the combination arms versus all other arms after only two weeks of therapy (p=0.0008). Conclusions: Upregulation of activated BCL6 is a known mechanism of resistance in Ph+ ALL that may be abrogated by acetylation of BCL6 with HDACi, as our in-vitro data suggests. Further, we have shown in xenograft models of Ph+ acute lymphoblastic leukemia that combination therapy with HDACi + imatinib, even in imatinib-resistant leukemia, has significant activity. Interestingly, the combination appears more active in resistant disease than in imatinib-sensitive disease. This combination could prove a viable strategy to attenuate imatinib- (and perhaps other TKI-) resistance in Ph+ ALL relapse, particularly in cases not driven by ABL1 kinase domain mutations. Disclosures No relevant conflicts of interest to declare.

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