Dual HDAC and PI3K Inhibitor, CUDC-907 Alone or in Combination with ABL Tyrosine Kinase Inhibitor Against ABL Tyrosine Kinase Inhibitor Resistant Leukemia Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3693-3693
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Yuko Tanaka ◽  
Juri Sakuta ◽  
Kazuma Ohyashiki
Keyword(s):  
Cell Growth ◽  
Growth Inhibition ◽  
Tyrosine Kinase Inhibitor ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Cell Growth Inhibition ◽  
Abl Tyrosine Kinase ◽  
Abl Tyrosine Kinase Inhibitor ◽  
Dose Dependent

Abstract Introduction: Chronic myeloid leukemia (CML) is characterized by the t(9:22) translocation known as the Philadelphia chromosome (Ph). ABL tyrosine kinase inhibitor (TKI), imatinib and second-generation ABL TKIs, nilotinib and dasatinib have demonstrated the potency against CML patients. However, resistance to ABL TKI can develop in CML patients due to BCR-ABL point mutations. Moreover, ABL TKIs do not eliminate the leukemia stem cells (LSCs). Therefore, new approach against BCR-ABL mutant cells and LSCs may improve the outcome of Ph-positive leukemia patients. In eukaryotic cells, histone acetylation/deacetylation is important in transcriptional regulation. Chromatin acetylation is controlled by the opposing effects of two families of enzymes: histone acetyltransferases (HAT) and histone deacetylases (HDACs). Deregulation of HDAC activity may be a cause of malignant disease in humans. Phosphoinositide 3-kinase (PI3K) pathway also regulates cell metabolism, proliferation and survival. Furthermore, aberrant activation of PI3K signaling pathway has been shown to be important in initiation maintenance of human cancers. CUDC-907 is an oral inhibitor of class I PI3K as well as class I and II HDAC enzymes. CUDC-907 is currently being investigated in a pivotal phase 1 clinical trial against hematological malignancies such as malignant lymphoma. We suggested that CUDC-907 mediated inhibition PI3K and HDAC activity and in combination with ABL TKIs may abrogate the proliferation and survival of Ph-positive leukemia cells including T315I mutation and ABL TKI resistant. Materials and methods: In this study, we investigated the combination therapy with a CUDC-907 and an ABL TKIs (imatinib, nilotinib and ponatinib) by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line which was transfected with T315I mutant, nilotinib resistant K562 and ponatinib resistant Ba/F3 cells and primary samples. Results: The treatment of imatinib, nilotinib and ponatinib exhibits cell growth inhibition partially against K562 cells in the presence of feeder cell (HS-5). We found that mRNA of PI3K subunit is significantly increased after a co-culture with HS-5 in K562 and primary CD34 positive CML samples. 72 h treatment of CUDC-907 exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. We examined the intracellular signaling after treatment of CUDC-907. Phosphorylation of JNK, histone acetylation and activity of caspase 3, poly (ADP-ribose) polymerase (PARP) was increased. Anti-apoptotic protein, Mcl-1 was decreased in a dose dependent. We next investigated the efficacy between imatinib and CUDC-907 by using these cell line. Combined treatment of K562 cells with imatinib and CUDC-907 caused significantly more cytotoxicity than each drug alone. Caspase activity was increased and Akt activity was reduced. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved PARP was increased after imatinib and CUDC-907 treatment. We investigated the CUDC-907 activity against T315I positive cells. CUDC-907 potently induced cell growth inhibition of Ba/F3 T315I cells in a dose dependent manner. Combined treatment of Ba/F3 T315I cells with ponatinib and CUDC-907 caused significantly more cytotoxicity than each drug alone. Caspase activity was increased and Akt activity was reduced after ponatinib and CUDC-907 treatment. To assess the activity of ponatinib and CUDC-907, we performed to test on tumor formation in mice. We injected nude mice subcutaneously with Ba/F3 T315I mutant cells. A dose of 20 mg/kg/day p.o of ponatinib and 30 mg/kg/day p.o of CUDC-907 inhibited tumor growth and reduced tumor volume compared with control mice. The treatments were well tolerated with no animal health concerns observed. We also found that the treatment of CUDC-907 exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells, K562 nilotinib resistant cells, T315I mutant primary samples and CD34 positive CML samples. Conclusion: These results indicated that administration of the dual PI3K and HDAC inhibitor, CUDC-907 may be a powerful strategy against ABL TKI resistant cells including T315I mutation and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. Disclosures No relevant conflicts of interest to declare.

Activity of the Histone Deacetylase Inhibitor, Vorinostat, Against BCR-ABL Positive Leukemia Cells with Random Mutagenesis and In Combination with Nilotinib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4460-4460
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Eishi Ashihara ◽  
Shinya Kimura ◽  
Taira Maekawa ◽  
...  
Keyword(s):  
Cell Growth ◽  
Tyrosine Kinase ◽  
Growth Inhibition ◽  
Tyrosine Kinase Inhibitor ◽  
Hdac Inhibitor ◽  
Kinase Inhibitor ◽  
Random Mutagenesis ◽  
Cell Growth Inhibition ◽  
Abl Tyrosine Kinase ◽  
Abl Tyrosine Kinase Inhibitor

Abstract Abstract 4460 The clinical use of imatinib, a specific BCR-ABL tyrosine kinase inhibitor (TKI) is effective in inducing a complete hematological and cytogenetic remission in a high percentage of chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph) positive acute lymphoblastic leukemia (ALL) patients. However, imatinib does not efficiently kill leukemic stem cells and is limited by the emergence of resistance due to the point mutations in the BCR-ABL kinase domain. Histone acetyltransferases (HAT) and histone deacetylases (HDAC) control the acetylation of histones and intracellular proteins, and regulate the transcription and function of the proteins. HDAC inhibitor is a structurally diverse class of targeted anti-cancer agent. One of the pan-HDAC inhibitor, vorinostat (suberoylanilide hydroxamic acid: SAHA) is a small-molecule inhibitor of most human class I and class II HDAC, and is reported the efficacy of malignant cells including lymphomas and myeloid malignancies.Therefore, combination therapy using a BCR-ABL tyrosine kinase inhibitor and an HDAC inhibitor, vorinostat may help prevent CML relapse due to BCR-ABL point mutation and may improve their long-term outcome. In this study, we investigated the efficacy of vorinostat by using the Ph-positive leukemia cell line, K562 and Ba/F3 BCR-ABL cell in a random mutagenesis study for BCR-ABL mutation. We first performed a comprehensive drug combination experiment using vorinostat and BCR-ABL tyrosine kinase inhibitor, imatinib or nilotinib. The treatment of imatinib or nilotinib exhibits cell growth inhibition partially against Ba/F3 BCR-ABL cell in a random mutagenesis. We also found the BCR-ABL point mutation such as T315I or M344V after 2 weeks nilotinib treatment by direct sequence analysis. We show that vorinostat potently induced cell growth inhibition of K562 and Ba/F3 BCR-ABL cells in a random mutagenesis in a dose dependent manner. Combined treatment of Ba/F3 BCR-ABL cell in a random mutagenesis with vorinostat and nilotinib or imatinib caused significantly more cytotoxicity than each drug alone by colony assay. We investigated the intracellular signaling of vorinostat. Phosphorylation of BCR-ABL, Crk-L were reduced after vorinostat treatment for 24 hours in a dose dependent manner. Caspase 3 and poly (ADP-ribose) polymerase (PARP) activation were increased after vorinostat treatment. Vorinostat potently enhanced cell growth inhibition of Ba/F3 BCR-ABL point mutants (G250E, Q252H, Y253F, E255K, M294V, T315I, T315A, F317L, F317V, M351T and H396P) compared with Ba/F3 expressing Wt BCR-ABL cells. The protein level of BCR-ABL was reduced after vorinostat treatment. BCR-ABL degradations in BCR-ABL mutant cells were significantly enhanced compared with Ba/F3 Wt BCR-ABL cells. Although long term culture of Ba/F3 BCR-ABL cell in a random mutagenesis with 2μ M vorinostat significantly decreased cell growth, the cells were increased after removal of vorinostat. We found these cells were wild type BCR-ABL by direct sequence analysis. Data from this study suggested that administration of the vorinostat may mediate its effects on BCR-ABL positive cells included BCR-ABL point mutation and enhance cytotoxic effects of nilotinib or imatinib in BCR-ABL mutant cells, and provide information of potential therapeutic relevance. Disclosures: Ohyashiki: Nippon Shinyaku Co., Ltd.: Research Funding.

Anti-Leukemic Activity of Phosphoinositide 3-Kinase Inhibitor, Copanlisib in ABL Tyrosine Kinase Resistant Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3127-3127
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Yuko Tanaka ◽  
Seiichiro Katagiri ◽  
Kazuma Ohyashiki
Keyword(s):  
Cell Growth ◽  
Growth Inhibition ◽  
Cell Line ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Cell Growth Inhibition ◽  
Abl Tyrosine Kinase ◽  
Primary Sample ◽  
Resistant Cells

Abstract Introduction: Chronic myelogenous leukemia (CML) is characterized by cytogenetic aberration (Philadelphia chromosome: Ph) and chimeric oncoprotein BCR-ABL. ABL tyrosine kinase inhibitor (TKI) therapy such as imatinib, nilotinib and dasatinib has improved the survival of Ph-positive leukemia patients. Although impressive clinical responses are obtained in the majority of CML patients, increasing evidence of acquired resistance to TKIs have been documented in clinically. Moreover, ABL TKIs cannot eradicate leukemia stem cells, thus, TKIs do not appear to lead to a cure the diseases. Therefore, new approach against BCR-ABL mutant cells and leukemia stem cells may improve the outcome of Ph-positive leukemia patients. Phosphoinositide 3-kinase (PI3K) pathway regulates cell metabolism, proliferation and survival. Furthermore, aberrant activation of PI3K signaling pathway has been shown to be important in initiation maintenance of human cancers. Copanlisib, also known as BAY80-6946 is a PI3K inhibitor with potential antineoplastic activity. Copanlisib is being investigated in a pivotal phase 2 clinical trial against hematological malignancies such as malignant lymphoma. We hypothesized that targeting PI3K, in combination with ABL TKI, would result in enhanced therapeutic activity in Ph-positive leukemia cells including T315I mutation and ABL TKI resistant. Materials and methods: We investigated the combination therapy with a copanlisib and an ABL TKIs (imatinib, nilotinib and ponatinib) by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line which was transfected with T315I mutant, nilotinib resistant K562, ponatinib resistant Ba/F3 cells and primary sample. Results: 72 h treatment of copanlisib exhibits cell growth inhibition against K562 cells in a dose dependent manner. The treatment of imatinib, nilotinib and ponatinib exhibits cell growth inhibition partially against K562 cells co-cultured with feeder cell line, HS-5. We found that the treatment of copanlisib abrogated the protective effects of HS-5 in K562 cells. We examined the intracellular signaling after treatment of copanlisib. High concentration of copanlisib reduced the phosphorylation of BCR-ABL and Crk-L. Activity of caspase 3 and poly (ADP-ribose) polymerase (PARP) was increased. We next investigated the efficacy between ABL TKI and copanlisib by using these cell line. Phosphorylation of BCR-ABL, Crk-L and Akt was reduced after imatinib and copanlisib treatment. We investigated the copanlisib activity against T315I positive cells. Copanlisib potently induced cell growth inhibition of Ba/F3 T315I cells. Combined treatment of Ba/F3 T315I cells with ponatinib and copanlisib caused significantly more cytotoxicity than each drug alone. Phosphorylation of BCR-ABL and Crk-L was reduced and cleaved PARP was increased after ponatinib and copanlisib treatment. To assess the activity of copanlisib and ponatinib, we performed to test on CML tumor formation in mice. We injected nude mice subcutaneously with Ba/F3 T315I mutant cells. A dose of 20 mg/kg/day p.o of ponatinib and 6 mg/kg/three times per week i.p of copanlisib inhibited tumor growth and reduced tumor volume compared with control mice. The treatments were well tolerated with no animal health concerns observed. We also found that the treatment of copanlisib exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells, K562 nilotinib resistant cells and primary sample. Conclusion: These results indicated that administration of the PI3K inhibitor, copanlisib may be a powerful strategy against ABL TKI resistant cells including T315I mutation and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. Disclosures Ohyashiki: Bristol: Honoraria, Research Funding.

Combining ABL1 Kinase Inhibitor, Imatinib and the Jak Kinase Inhibitor TG101348: A Potential Treatment for Residual BCR-ABL Positive Leukemia Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3737-3737
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Seiichiro Katagiri ◽  
Yuko Tanaka ◽  
Kazuma Ohyashiki
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Intracellular Signaling ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Cell Growth Inhibition

Abstract Abstract 3737 ABL kinase inhibitor, imatinib is highly effective therapy against chronic myeloid leukemia (CML) patients and eliminates disease progression and transformation. However, imatinib is not curative for most CML patients. Residual CML cells are present in bone marrow microenvironment. Bone marrow microenvironment is a source of soluble factors and regulates the proliferation of leukemia cells. These leukemia cells are contained within a niche in the bone marrow and are often impervious to current treatments, thus maintaining their proliferative activity when the treatment is ceased, suggests that the new therapeutic strategies designed to override stroma-associated drug resistance are required to treat against Philadelphia (Ph)-positive leukemia patients. The hematopoietic cytokine receptor signaling is mediated by tyrosine kinases termed Janus kinases (Jaks) and downstream transcription factors, signal transducers and activators of transcription (STATs). Jak-STAT signaling is also activated in CML cells. One of the Jak kinase inhibitor, TG101348 (SAR302503) is an orally available inhibitor of Jak2 and developed for the treatment of patients with myeloproliferative diseases. Therefore, combination therapy using a BCR-ABL tyrosine kinase inhibitors and a Jak inhibitor, TG101348 may help prevent stroma-associated drug resistance and these approaches may be expected to improve the outcomes of CML patients. In this study, we investigated the ABL tyrosine kinase inhibitor, imatinib and TG101348 efficacy by using the BCR-ABL positive cell lines, K562 and primary CML samples when leukemic cells were protected by the feeder cell lines (HS-5 and S9). 72 hours treatment of imatinib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. However, the treatment of imatinib exhibits cell growth inhibition partially against K562 cells in the presence of HS-5 conditioned media. We found that the treatment of TG101348 did not exhibit cell growth inhibition against K562 cells directly, but the combination treatment with imatinib and TG101348 abrogated the protective effects of HS-5 conditioned media in K562 cells. We next investigated the intracellular signaling of imatinib and TG101348. Phosphorylation of BCR-ABL, Crk-L was not reduced after TG101348 treatment. However, phosphorylation of BCR-ABL, Crk-L was significantly reduced and increased apoptosis after combination treatment with imatinib and TG101348. We next investigated the efficacy between imatinib and TG101348 by using CD34 positive primary CML samples. The treatment of imatinib exhibits cell growth inhibition partially against CD34 positive CML samples in the presence of feeder cells. Combined treatment of CD34 positive primary samples with imatinib and TG101348 caused significantly more cytotoxicity and induced apoptosis. We also found that mitogen-activated protein kinase (MAPK) was also inhibited by imatinib and TG101348 treatment. We next investigated the intracellular signaling of imatinib and TG101348 by using the CD34 positive primary samples. Phosphorylation of BCR-ABL, Crk-L was significantly reduced and increased apoptosis after treatment with imatinib and TG101348. Moreover, combination of imatinib and TG101348 inhibited the colony growth of Ph-positive primary samples. We also investigated the TG101348 activity against feeder cell. Phosphorylation of STAT5 was reduced by TG101348 in a dose dependent manner. The cytokine production was analyzed by using cytokine array systems. The cytokine production such as granulocyte macrophage colony-stimulating factor (GM-CSF) from HS-5 was also reduced by TG101348 treatment. Data from this study suggested that administration of the imatinib and Jak inhibitor, TG101348 may be a powerful strategy against stroma-associated drug resistance of Ph-positive cells and enhance cytotoxic effects of imatinib in those residual CML cells. Disclosures: No relevant conflicts of interest to declare.

Efficacy Of The Polo-Like Kinase Inhibitor, Rigosertib Alone Or In Combination With ABL Tyrosine Kinase Inhibitor Against BCR-ABL-Positive Leukemia Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3985-3985
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Yuko Tanaka ◽  
Toshihiko Kitahara ◽  
Kazuma Ohyashiki
Keyword(s):  
Cell Growth ◽  
Growth Inhibition ◽  
Cell Line ◽  
Kinase Inhibitor ◽  
Combined Treatment ◽  
Point Mutations ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Cell Growth Inhibition ◽  
Resistant Cells

Abstract Chronic myeloid leukemia (CML) is characterized by the t(9:22) translocation known as the Philadelphia chromosome (Ph). ABL tyrosine kinase inhibitor (TKI), imatinib and second-generation ABL TKIs, nilotinib and dasatinib have demonstrated the potency against CML patients. However, resistance to ABL TKI can develop in CML patients due to BCR-ABL point mutations. Moreover, ABL TKIs do not eliminate the leukemia stem cells (LSCs). These leukemia cells are contained within a niche in the bone marrow and are often impervious to current treatments. Therefore, new approach against BCR-ABL mutant cells and LSCs may improve the outcome of Ph-positive leukemia patients. Polo-like kinases (PLK) are the family of serine threonine kinases and essential for mitosis. PLK is also critical regulator of cell cycle progression and DNA damage response. One of the PLK and phosphoinositide 3-kinase (PI3K) inhibitor, rigosertib (ON 01910.Na) is a novel synthetic benzyl styryl sulfone that is cytotoxic against a variety of human tumor cell lines. Rigosertib is currently being investigated in a pivotal phase 3 clinical trial against hematological malignancies such as myelodysplastic syndromes (MDS). We suggested that rigosertib mediated inhibition PLK and PI3K activity and in combination with ABL TKIs may abrogate the proliferation and survival of Ph-positive leukemia cells including T315I mutation and ABL TKI resistant. In this study, we investigated the combination therapy with a rigosertib and an ABL TKIs (imatinib, nilotinib and ponatinib) by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line with T315I mutant, nilotinib resistant K562 and ponatinib resistant Ba/F3 cells. 72 h treatment of rigosertib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. The treatment of imatinib, nilotinib and ponatinib exhibits cell growth inhibition partially against K562 cells in the presence of feeder cell (HS-5) conditioned media. We found that the treatment of rigosertib abrogated the protective effects of HS-5 conditioned media in K562 cells. We examined the intracellular signaling after treatment of rigosertib. Phosphorylation of BCR-ABL, Crk-L was not reduced. However, activity of caspase 3 was increased. We next investigated the efficacy between imatinib and rigosertib by using these cell line. Combined treatment of K562 cells with imatinib and rigosertib caused significantly more cytotoxicity than each drug alone. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved PARP and γH2A.X phosphorylation was increased after imatinib and rigosertib treatment. Anti-apoptotic protein, Mcl-1 was also decreased. We also found the phosphorylation of histone H3 was increased after rigosertib treatment suggested that the cells arrested in G2/M phase. We investigated the rigosertib activity against T315I positive cells. Rigosertib potently induced cell growth inhibition of Ba/F3 T315I cells. Combined treatment of Ba/F3 T315I cells with ponatinib and rigosertib caused significantly more cytotoxicity than each drug alone.We next investigated by using ponatinib resistant Ba/F3 cells and nilotinib resistant K562 cells. In the ponatinib resistant cell lines, IC50 of ponatinib was up to 200 nM. BCR-ABL triple point mutations (T315I, E255K and Y253H) were detected by direct sequence analysis. The treatment of rigosertib exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells and K562 nilotinib resistant cells. These results indicated that administration of the PLK and PI3K inhibitor, rigosertib may be a powerful strategy against ABL TKI resistant cells and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. Disclosures: Ohyashiki: Novartis: Honoraria, Research Funding.

Activity of the Aurora Kinase Inhibitor, MLN8237 (alisertib) Alone or in Combination with Ponatinib Against Imatinib-Resistant BCR-ABL-Positive Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1333-1333
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Seiichiro Katagiri ◽  
Yuko Tanaka ◽  
Kazuma Ohyashiki
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Aurora Kinase ◽  
Dependent Manner ◽  
Cell Growth Inhibition ◽  
Cycle Arrest ◽  
T315i Mutation

Abstract Abstract 1333 Chronic myeloid leukemia (CML) is characterized by cytogenetic aberration (Philadelphia chromosome: Ph) and chimeric tyrosine kinase BCR-ABL. ABL tyrosine kinase inhibitor, imatinib has demonstrated the potency against CML patients. However, resistance to imatinib can develop in CML patients due to BCR-ABL point mutations. One of T315I mutation is resistant to currently available ABL tyrosine kinase inhibitors. Therefore, new approach against T315I mutant may improve the outcome of Ph-positive leukemia patients. Aurora kinases are serine/threonine kinases and upregulated in many malignancies including leukemia, and play an important role in cell cycle control and tumor proliferations. Because Aurora kinases are overexpressed in leukemia cells, Aurora kinases may present attractive targets for leukemia treatment. One of Aurora kinase inhibitor, MLN8237 (alisertib) is an oral and selective Aurora kinase A inhibitor and is currently being investigated in a pivotal phase 3 clinical trial against hematological malignancies. We suggested that alisertib mediated inhibition Aurora kinase activity and in combination with ponatinib, also known as AP24534 may abrogate the proliferation and survival of Ph-positive cells including T315I mutation. In this study, we investigated the combination therapy with a ponatinib and an alisertib by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line which was transfected with T315I mutant, ponatinib resistant Ba/F3 cells and T315I primary sample. Protein expression of Aurora A and B were increased in Ph-positive leukemia cells. 72 hours treatment of alisertib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. Alisertib also induced cell cycle arrest. The treatment of ponatinib exhibits cell growth inhibition partially against K562 cells in the presence of feeder cell (HS-5) conditioned media. We found that the treatment of alisertib abrogated the protective effects of HS-5 conditioned media in K562 cells. We investigated the alisertib activity against T315I positive cells. Alisertib potently induced cell growth inhibition of Ba/F3 cells ectopically expressing T315I mutation and induced cell cycle arrest. We investigated the efficacy between ponatinib and alisertib by using these cell lines. Combined treatment of Ba/F3 T315I cells with ponatinib and alisertib caused significantly more cytotoxicity than each drug alone. Ponatinib and alisertib were also effective against T315I primary samples. We examined the intracellular signaling of alisertib. Phosphorylation of Aurora A was inhibited in a time dependent manner. We also found the phosphorylation of histone H3 was also reduced in a dose dependent manner suggested that high concentration of alisertib also inhibits Aurora B activity. We next investigated by using ponatinib resistant Ba/F3 cells. In the ponatinib resistant cell lines, IC50 of ponatinib was up to 200 nM. BCR-ABL triple point mutations (T315I, E255K and Y253H) were detected by direct sequence analysis. The treatment of alisertib exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells in the dose dependent manner. Alisertib induced cell cycle arrest in ponatinib resistant cells. Combined treatment of Ba/F3 ponatinib resistant cells with ponatinib and alisertib caused significantly more cytotoxicity. To assess the activity of alisertib and ponatinib, we performed to test on CML tumor formation in mice. We injected nude mice subcutaneously with 1×107 Ba/F3 T315I cells. A dose of 30 mg/kg/day p.o of ponatinib and 30 mg/kg/day p.o of alisertib inhibited tumor growth and reduced tumor volume compared with control mice. The treatments were well tolerated with no animal health concerns observed indicating the feasibility of alisertib combination strategies in the clinic. Data from this study suggested that administration of the ponatinib and Aurora inhibitor, alisertib may be a powerful strategy against BCR-ABL mutant cells including T315I. Disclosures: No relevant conflicts of interest to declare.

The Analysis of HDAC Inhibitor, Vorinostat Efficacy against Wild Type and BCR-ABL Mutant Positive Leukemia Cells in Monotherapy and in Combination with a Pan-Aurora Kinase Inhibitor, MK-0457

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5025-5025 ◽  
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Shinya Kimura ◽  
Taira Maekawa ◽  
Kazuma Ohyashiki
Keyword(s):  
Cell Growth ◽  
Growth Inhibition ◽  
Kinase Inhibitor ◽  
Aurora Kinase ◽  
Dependent Manner ◽  
Wild Type ◽  
Cell Growth Inhibition ◽  
Imatinib Resistant ◽  
T315i Mutation ◽  
Dose Dependent

Abstract The use of imatinib, an ABL tyrosine kinase inhibitor, has led to a dramatic change in the management of BCR-ABL positive leukemia patients. However, the resistance to imatinib mediated by mutations in the BCR-ABL domain has become a major problem in the treatment. Histone deacetylase (HDAC) inhibitors have been shown to mediate the regulation of gene expression, induce cell growth, cell differentiation and apoptosis of tumor cells. Vorinostat (suberoylamide hydroxamic acid:SAHA) is a hydroxamic acid based polar HDAC inhibitor. Vorinostat have shown efficacy in a wide range of cancers such as cutaneous T-cell lymphoma (CTCL). However, efficacy of vorinostat against the BCR-ABL mutants has fully not known. Here we report on the studies performed against murine Ba/F3 cell line which was transfected wild type (Wt) p210 and p185 BCR-ABL or imatinib resistant BCR-ABL mutants such as G250E, Q252H, Y253F, E255K, M294V, T315I, T315A, F317L, F317V, M351T, H396P and T315I(p185). 48 hours treatment of vorinostat exhibits cell growth inhibition and proapoptotic activity murine Ba/F3 cells ectopically expressing Wt and imatinib resistant BCR-ABL mutants including T315I mutation in a dose dependent manner. IC50 of these cell lines are Wt(720nM), G250E(625nM), Q252H(220nM), Y253F(525nM), E255K(685nM), M294V(785nM), T315I(500nM), T315A(715nM), F317L(560nM), F317V(565nM), M351T(375nM) and H396P(485nM). Aurora kinases play a pivotal role in the regulator of mitotic processes during cell division. MK-0457 is a small molecule inhibitor of the Aurora kinase family and was found to be active against the cells from BCR-ABL positive patients with T315I mutation in clinical trial. Because vorinostat also depleted BCR-ABL, as well as induced apoptosis and sensitized BCR-ABL-expressing leukemia cells, we examined whether vorinostat and MK-0457 enhances the apoptosis in imatinib resistant BCR-ABL-expressing cells. 48 hours treatment of MK-0457 exhibits cell growth inhibition of Ba/F3 cells ectopically expressing Wt and imatinib resistant BCR-ABL mutants including T315I mutation. IC50 of MK-0457 is Wt(215nM), G250E(205nM), Q252H(185nM), Y253F(245nM), E255K(185nM), M294V(238nM), T315I(205nM), T315A(165nM), F317L(200nM), F317V(200nM), M351T(225nM) and H396P(195nM). We examined the intracellular signaling by using these cell lines. We found that caspase 3, and poly (ADPribose) polymerase (PARP) were activated after MK-0457 treatment in a dose dependent manner. Phosphorylation of BCR-ABL and Crk-L which is downstream target of BCR-ABL was reduced after MK-0457 treatment. We found that combination of vorinostat and MK-0457 synergistically cell growth inhibition of Wt and BCR-ABL mutants Ba/F3 cells in 48 hours treatment. Phosphorylation of Crk-L was reduced after vorinostat and MK-0457 treatment. Caspase 3 and PARP activation were also synergistically increased after vorinostat and MK-0457 treatment. We evaluated the activity of MK-0457 and vorinostat in primary BCR-ABL positive acute lymphoblastic leukemia (ALL) cells with the T315I mutation. We found that MK-0457 potently induced cell growth inhibition of primary T315I cells in 48 hours treatment. Moreover, combination of vorinostat and MK-0457 synergistically increased the cell growth inhibition in primary T315I cells. This study demonstrate monotherapy of vorinostat and the combination of vorinostat and MK-0457 are more potent efficacy not only wild type BCR-ABL but also imatinib resistant BCR-ABL mutants cells and represents a promising new strategy for treatment of imatinib resistant BCR-ABL positive leukemias, including those harboring the T315I mutation.

GDC-0449, the Small Molecule Inhibitor of Hedgehog-Gli Pathway for the Treatment of BCR-ABL Positive Leukemia Cells and In Combination with Dasatinib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2136-2136
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Kazuma Ohyashiki
Keyword(s):  
Cell Growth ◽  
Tyrosine Kinase ◽  
Growth Inhibition ◽  
Cell Line ◽  
Kinase Inhibitors ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Feeder Cell ◽  
Cell Growth Inhibition ◽  
Abl Tyrosine Kinase

Abstract Abstract 2136 Imatinib has shown clinical efficacy against Philadelphia chromosome (Ph) positive leukemia cells and it is now the standard care for initial therapy. However, recent studies reported imatinib are not effective in quiescent primitive chronic myeloid leukemia (CML) stem cells. Moreover, many Ph-positive leukemia patients develop resistance or fail to respond to imatinib by mutation in the ABL kinase domain in clinically. These results indicated that alternative combination therapy such as BCR-ABL targeting tyrosine kinase inhibitors (TKIs) and nontoxic agents are required to cure the Ph positive leukemia patients. Hedgehog (Hh)- Glioma-associated oncogene homolog (Gli) signaling regulates self-renewal of stem cells and implicates in a large number of human cancers. One of the Hh inhibitor, GDC-0449 is a potent small molecule inhibitor of Hedgehog-Gli pathway. It has been reported GDC-0449 showed high target specificity and demonstrated antiproliferative activity against tumors and it is now in clinical trial. Therefore, combination therapy using a BCR-ABL tyrosine kinase inhibitors and a Hedgehog-Gli inhibitor, GDC-0449 may help prevent CML relapse and these approaches may be expected to improve the outcomes of Ph-positive leukemia patients. In this study, we investigated the GDC-0449 efficacy by using the BCR-ABL positive cell lines, OM9;22, K562 and primary samples when leukemic cells were protected by the feeder cell line, S9 cells. We examined a comprehensive drug combination experiment using GDC-0449 and dual Src/ABL tyrosine kinase inhibitor, dasatinib. Gli proteins (Gli1, Gli2 and Gli3) were existed in Ph-positive cell lines. We found the cell numbers of OM9;22 were significantly increased with the feeder cell line, S9 cells compared to without S9 cells. The treatment of dasatinib exhibits cell growth inhibition partially against OM9;22 cells in the presence of feeder cell line, S9 cells. Caspase-3 activity by 100 nM dasatinib treatment was also reduced in the presence of S9 cells. 72 h of combined treatment of Ph-positive leukemia cells with 10 μM of GDC-0449 and 100 nM of dasatinib in the presence of feeder cell line, caused significantly more cytotoxicity than each drug alone. We next investigated the efficacy and intracellular signaling of GDC-0449. The treatment of GDC-0449 exhibits cell growth inhibition and induced apoptosis against OM9;22 cells in a dose and time dependent manner. Expression of Gli1 and Gli2 proteins were reduced after GDC-0449 treatment. 10 μM of GDC-0449 also inhibited the growth of Ph-positive primary samples by colony assay. Another Hh inhibitor, SANT-2 also exhibits cell growth inhibition against OM9;22 cells in a dose dependent manner. Data from this study suggested that administration of the Hh inhibitor, GDC-0449 may be a powerful strategy against Ph-positive leukemia cells and enhance cytotoxic effects of dasatinib in the presence of feeder cell. Disclosures: Ohyashiki: Nippon Shinyaku Co., Ltd.: Research Funding.

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