Dasatinib-induced Apoptosis Research Articles

Investigating the Role of JAK/STAT Pathway on Dasatinib-Induced Apoptosis for CML Cell Model K562.

We aimed to evaluate the cytotoxic and apoptotic effects of dasatinib (BMS-354825) on K562 chronic myeloid leukemia (CML) cells and to examine the roles of STAT genes on dasatinib-induced apoptosis. The results showed that dasatinib decreased proliferation and induced apoptosis in K562 cells in a dose- and time-dependent manner. mRNA and protein levels of STAT5A and STAT5B genes were significantly reduced in dasatinib-treated K562 cells. These data indicated that STAT inhibition by dasatinib might be therapeutic in JAK/STAT pathway-associated malignancies after confirmation with clinical studies.

Investigating The Role of JAK/STAT Pathway upon Dasatinib Induced Apoptosis for CML Cell Model K562

Investigating The Role of JAK/STAT Pathway upon Dasatinib Induced Apoptosis for CML Cell Model K562

Abstract A73: AMPK activation mediates dasatinib-induced EGFR degradation and apoptosis in head and neck cancer

Abstract Head and neck squamous cell carcinoma (HNSCC) is a worldwide disease with aggressive course and dismal outcome. Dasatinib, a Bcr-abl and Src in hibitor, has been approved clinically for CML and shown activities against solid tumors in vitro. In our recent work, degradation of epidermal growth factor receptor (EGFR) plays a role in dasatinib-induced apoptosis in HNSCC cells. We further explored the mechanism of this event. ER stress was induced by dasatinib and played a role in dasatinib-EGFR degradation. C-cbl activation was induced by ER stress and shown to mediate ER-stress induced EGFR degradation. In addition, AMPK activation was induced by dasatinib, which was proved to be involved in dasatinib-induced ER stress and EGFR degradation. Activation of AMPK with metformin not only enhanced dasatinib-induced growth inhibition and apoptosis but also sensitized dasatinib-resistant cells. Furthermore, xenograft model showed that metformin potentiated anti-cancer effect of dasatinib through activation of AMPK and ER stress. Our results disclose that AMPK-dependent ER stress plays a crucial role in the anti-cancer effect of dasatinib in HNSCC and further activation of AMPK by metformin might enhance dasatinib efficacy. Citation Format: Yu-Chin Lin Lin, Meng-Hsuan Wu, Tzu-Tang Wei, Yun-Chieh Lin, Ching-Chow Chen. AMPK activation mediates dasatinib-induced EGFR degradation and apoptosis in head and neck cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A73. doi:10.1158/1538-7445.CHTME14-A73

Transforming growth factor β signaling overcomes dasatinib resistance in lung cancer.

Lung cancer is the second most common cancer and the leading cause of cancer-related deaths. Despite recent advances in the development of targeted therapies, patients with advanced disease remain incurable, mostly because metastatic non-small cell lung carcinomas (NSCLC) eventually become resistant to tyrosine kinase inhibitors (TKIs). Kinase inhibitors have the potential for target promiscuity because the kinase super family is the largest family of druggable genes that binds to a common substrate (ATP). As a result, TKIs often developed for a specific purpose have been found to act on other targets. Drug affinity chromatography has been used to show that dasatinib interacts with the TGFβ type I receptor (TβR-I), a serine-threonine kinase. To determine the potential biological relevance of this association, we studied the combined effects of dasatinib and TGFβ on lung cancer cell lines. We found that dasatinib treatment alone had very little effect; however, when NSCLC cell lines were treated with a combination of TGFβ and dasatinib, apoptosis was induced. Combined TGFβ-1 + dasatinib treatment had no effect on the activity of Smad2 or other non-canonical TGFβ intracellular mediators. Interestingly, combined TGFβ and dasatinib treatment resulted in a transient increase in p-Smad3 (seen after 3 hours). In addition, when NSCLC cells were treated with this combination, the pro-apoptotic protein BIM was up-regulated. Knockdown of the expression of Smad3 using Smad3 siRNA also resulted in a decrease in BIM protein, suggesting that TGFβ-1 + dasatinib-induced apoptosis is mediated by Smad3 regulation of BIM. Dasatinib is only effective in killing EGFR mutant cells, which is shown in only 10% of NSCLCs. Therefore, the observation that wild-type EGFR lung cancers can be manipulated to render them sensitive to killing by dasatinib could have important implications for devising innovative and potentially more efficacious treatment strategies for this disease.

Abstract 5514: Metformin sensitizes anticancer effect of dasatinib through AMPK-dependent ER stress in head and neck squamous cell carcinoma cells

Abstract Purpose: Head and neck squamous cell carcinoma (HNSCC) is an important endemic disease in Taiwan with aggressive course and dismal outcome. Dasatinib is a Bcr-bl and Src kinase inhibitor that has potential against HNSCC. We recently disclosed that EGFR degradation is critical for dasatinib-induced apoptosis. Here, we further demonstrate that AMPK-dependent ER stress is responsible for this effect. Experimental design: Dasatinib-induced ER stress and AMPK activation was investigated in HNSCC cells including Ca9-22, HSC3, and SAS. The anti-cancer activity of dasatinib in combination with AMPK activator was examined in vitro and in vivo. EGFR expression and AMPK activation were correlated in human specimens. Results: Dasatinib-induced ER stress was involved in dasatinib-induced EGFR degradation and apoptosis, and ER stress mediated EGFR degradation in a c-cbl-dependent manner. In addition, dasatinib-induced AMPK activation led to dasatinib-induced ER stress and EGFR degradation, and activation of AMPK might be due to ATP decrease through the up-regulation of pyruvate dehydrogenase kinase 4 (PDK4) induced by dasatinib. Furthermore, activation of AMPK by metformin potentiated dasatinib-induced anti-cancer effect in vitro and in vivo. The correlation of AMPK activation and EGFR expression was seen in HNSCC cells and human tumor specimens. Conclusions: Our results disclose that AMPK-dependent ER stress plays a crucial role in anti-cancer effect of dasatinib on HNSCC and activation of AMPK might enhance dasatinib efficacy. Note: This abstract was not presented at the meeting. Citation Format: Yu-Chin Lin, Meng-Hsuan Wu, Tzu-Tang Wei, Yun-Chieh Lin, Ching-Chow Chen. Metformin sensitizes anticancer effect of dasatinib through AMPK-dependent ER stress in head and neck squamous cell carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5514. doi:10.1158/1538-7445.AM2014-5514

Abstract 347: Dasatinib accelerates valproic acid-induced acute myeloid leukemia cells death by regulation of differentiation capacity

Abstract Dastinib (BMS-354825) is a FDA-approved small compound that was developed for chronic myeloid leukemia as a multi-targeted kinase inhibitor of wild-type BCR-ABL and SRC family kinases. Valproic acid (VPA; Orfiril) has been a well-known anti-epileptic drug worldwidely, and belong to the class I histone deacetylase inhibitor. The aim of this research is to determine the anti-leukemic effects of combination of dasatinib and VPA or their mechanism in acute myeloid leukemia (AML) cells. In this study, dasatinib showed potent inhibitory effects on VPA-treated AML cells that were associated with induced G1 phase cell cycle arrest and also apoptosis including the cleavage of PARP, caspase-3, -7 and -9. Moreover, inhibitors of caspase-3 and -9 (Z-DEVD-FMK and LEHD-CHO) efficiently inhibited dasatinib-induced apoptosis in VPA-treated AML cells. Therefore, dasatinib/VPA-induced cell death occurred via caspase-dependent apoptosis. Also, inhibitors of MEK/ERK (U0126 and PD98059) and p38 MAPK (SB203580) efficiently inhibited dasatinib/VPA-induced apoptosis. The combination of dasatinib and VPA differentiation capacity of AML cells is more powerful than each drug alone. So the combination is enough to push AML cells to death strongly by G1 cell cycle arrest and caspase-dependent apoptosis. In addition, MEK/ERK and p38 MAPK controls dasatinib/VPA-induced apoptosis as an upstream regulator. Finally, co-treatment of dasatinib and VPA is contributed to AML cell death by regulation of differentiation capacity. Taken together, these results indicate that dasatinib and VPA together have a potential for anti-leukemic therapy. Citation Format: Sook-Kyoung Heo, Eui-Kyu Noh, Dong-Joon Yoon, Jae-Cheol Jo, Jae-Hoo Park, Hawk Kim. Dasatinib accelerates valproic acid-induced acute myeloid leukemia cells death by regulation of differentiation capacity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 347. doi:10.1158/1538-7445.AM2014-347

Metformin sensitizes anticancer effect of dasatinib in head and neck squamous cell carcinoma cells through AMPK-dependent ER stress

Head and neck squamous cell carcinoma (HNSCC) is an important endemic disease in Taiwan with aggressive course and dismal outcome. Dasatinib is a Bcr-bl and Src kinase inhibitor that has potential against HNSCC. We recently disclosed that EGFR degradation is critical for dasatinib-induced apoptosis. Here, we further demonstrate that AMPK-dependent ER stress is responsible for this event. Dasatinib induced ER stress which mediated EGFR degradation in a c-cbl-dependent manner. AMPK activation induced by dasatinib might be due to ATP decrease through the up-regulation of pyruvate dehydrogenase kinase 4 (PDK4). Furthermore, activation of AMPK by metformin sensitized dasatinib-induced in vitro and in vivo anti-cancer effect. The correlation of AMPK activation and EGFR expression was seen in HNSCC cells and human tumor specimens. Our results disclose that AMPK-dependent ER stress plays a crucial role in the anti-cancer effect of dasatinib in HNSCC and further activation of AMPK by metformin might enhance dasatinib efficacy.

Abstract 5404: Metabolic stress played a role in dasatinib-induced EGFR degradation and apoptosis in head and neck cancer.

Abstract Head and neck squamous cell carcinoma (HNSCC) is a worldwide disease with aggressive course and dismal outcome. Dasatinib, a Bcr-abl and Src in hibitor, has been approved clinically for CML and shown activities against solid tumors in vitro. In our recent work, degradation of epidermal growth factor receptor (EGFR) plays a role in dasatinib-induced apoptosis in HNSCC cells. We further revealed the role of metabolic stress in dasatinib-induced EGFR degradation and apoptosis. Dasatinib decreased cellular ATP, and activated AMPK activation and induced endoplasmic reticulum stress (ER stress) as well. Inhibition or knockdown of AMPK reversed dasatinib-induced EGFR degradation, and pharmacological activation of AMPK led to ER stress and EGFR degradation. Addition of ER stress inhibitor to dasatinib reversed EGFR degradation and apoptosis, and pharmacologic activation of ER stress resulted in EGFR degradation. In conclusion, metabolic stress may play a role in dasatinib-induced EGFR degradation and apoptosis in HNSCC cells. Citation Format: Yu-Chin Lin, Meng-Hsuan Wu, Tzu-Tang Wei, Kuen-Feng Chen, Ann-Lii Cheng, Ching-Chow Chen. Metabolic stress played a role in dasatinib-induced EGFR degradation and apoptosis in head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5404. doi:10.1158/1538-7445.AM2013-5404

Degradation of Epidermal Growth Factor Receptor Mediates Dasatinib-Induced Apoptosis in Head and Neck Squamous Cell Carcinoma Cells

Epidermal growth factor receptor (EGFR) is an important oncoprotein that promotes cell growth and proliferation. Dasatinib, a bcr-abl inhibitor, has been approved clinically for the treatment of chronic myeloid leukemia and demonstrated to be effective against solid tumors in vitro through Src inhibition. Here, we disclose that EGFR degradation mediated dasatinib-induced apoptosis in head and neck squamous cell carcinoma (HNSCC) cells. HNSCC cells, including Ca9-22, FaDu, HSC3, SAS, SCC-25, and UMSCC1, were treated with dasatinib, and cell viability, apoptosis, and underlying signal transduction were evaluated. Dasatinib exhibited differential sensitivities against HNSCC cells. Growth inhibition and apoptosis were correlated with its inhibition on Akt, Erk, and Bcl-2, irrespective of Src inhibition. Accordingly, we found that down-regulation of EGFR was a determinant of dasatinib sensitivity. Lysosome inhibitor reversed dasatinib-induced EGFR down-regulation, and c-cbl activity was increased by dasatinib, indicating that dasatinib-induced EGFR down-regulation might be through c-cbl-mediated lysosome degradation. Increased EGFR activation by ligand administration rescued cells from dasatinib-induced apoptosis, whereas inhibition of EGFR enhanced its apoptotic effect. Estrogen receptor α (ERα) was demonstrated to play a role in Bcl-2 expression, and dasatinib inhibited ERα at the pretranslational level. ERα was associated with EGFR in dasatinib-treated HNSCC cells. Furthermore, the xenograft model showed that dasatinib inhibited HSC3 tumor growth through in vivo down-regulation of EGFR and ERα. In conclusion, degradation of EGFR is a novel mechanism responsible for dasatinib-induced apoptosis in HNSCC cells.

Oxidative stress is involved in Dasatinib-induced apoptosis in rat primary hepatocytes

Dasatinib, a multitargeted inhibitor of BCR–ABL and SRC kinases, exhibits antitumor activity and extends the survival of patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL). However, some patients suffer from hepatotoxicity, which occurs through an unknown mechanism. In the present study, we found that Dasatinib could induce hepatotoxicity both in vitro and in vivo. Dasatinib reduced the cell viability of rat primary hepatocytes, induced the release of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in vitro, and triggered the ballooning degeneration of hepatocytes in Sprague–Dawley rats in vivo. Apoptotic markers (chromatin condensation, cleaved caspase-3 and cleaved PARP) were detected to indicate that the injury induced by Dasatinib in hepatocytes in vitro was mediated by apoptosis. This result was further validated in vivo using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Here we found that Dasatinib dramatically increased the level of reactive oxygen species (ROS) in hepatocytes, reduced the intracellular glutathione (GSH) content, attenuated the activity of superoxide dismutase (SOD), generated malondialdehyde (MDA), a product of lipid peroxidation, decreased the mitochondrial membrane potential, and activated nuclear factor erythroid 2-related factor 2 (Nrf2) and mitogen-activated protein kinases (MAPK) related to oxidative stress and survival. These results confirm that oxidative stress plays a pivotal role in Dasatinib-mediated hepatotoxicity. N-acetylcysteine (NAC), a typical antioxidant, can scavenge free radicals, attenuate oxidative stress, and protect hepatocytes against Dasatinib-induced injury. Thus, relieving oxidative stress is a viable strategy for reducing Dasatinib-induced hepatotoxicity.

Abstract 2880: Dasatinib induces apoptosis in head and neck squamous cell carcinoma cells through down-regulation of epidermal growth factor receptor (EGFR) in vitro and in vivo

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is a worldwide disease with aggressive course and dismal outcome. Dasatinib, a bcr-abl inhibitor, has been approved clinically for the treatment of chronic myeloid leukemia and demonstrated to be effective against solid tumors in vitro through Src inhibition. Here, we disclose that dasatinib induced apoptosis of HNSCC cells in vitro and in vivo through down-regulation of EGFR. Methods: HNSCC cells, including Ca9-22, FaDu, HSC3, SAS, SCC-25, and UMSCC1 were treated with dasatinib. Cell viability, apoptosis, and underlying signal transduction were evaluated. Results: Dasatinib exhibited differential sensitivities against HNSCC cells. The growth inhibition and apoptosis were correlated with its inhibition on Akt, Erk, and bcl-2, but irrespective of Src inhibition. Accordingly, we found that down-regulation of epidermal growth factor receptor (EGFR) was a determinant of dasatinib sensitivity. Dasatinib inhibited EGFR through lysosome degradation. Increased EGFR activation by ligand administration rescued cells from dasatinib-induced apoptosis, whereas inhibition of EGFR enhanced its apoptotic effect. Estrogen receptor (ER) was demonstrated to play a role in bcl-2 expression, and dasatinib inhibited ER at pre-translational level. ER worked in concert with EGFR to regulate dasatinib-induced apoptosis. Furthermore, xenograft model showed that dasatinib inhibited HSC3 tumor growth through in vivo down-regulation of EGFR and ER. Conclusion: Down-regulation of EGFR is a novel mechanism responsible for dasatinib-induced apoptosis in HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2880. doi:1538-7445.AM2012-2880

Abstract 1027: Bcl-2 protected HNSCC cells from dasatinib-induced apoptosis

Abstract Introduction: Head and neck squamous cell carcinoma (HNSCC) is a worldwide prevalent cancer with aggressive clinical courses and dismal outcomes. Incorporation of cetuximab, an antibody against epidermal growth factor receptor, had led to advance of treatment, but showed a modest efficacy. Therefore, new treatment for HNSCC is needed. Dasatinib, a Bcr-Abl and Src inhibitor, approved for treatment of chronic myeloid leukemia, was demonstrated to have activities for solid tumors. Here, we report the differential sensitivity of dasatinib for HNSCC cells and identify the possible molecular events. Material and method: HNSCC cells including Ca9-22, HSC3, UMSCC1, FaDu, and SAS were treated with dasatinib. Cell viability, apoptosis, and underlined signal transduction were evaluated by MTT assay, flow cytometry and Western blotting, respectively. Results: Five HNSCC cells treated with dasatinib displayed differential sensitivities. Ca9-22 and HSC3 cells are the most sensitive cells in response to dasatinib and showed significant growth inhibition and apoptosis. In contrast, FaDu and SAS cells are resistant to dasatinib-induced growth inhibition and apoptosis. UMSCC1 cells showed intermediate sensitivity to dasatinib; their growth was inhibited without induction of apoptosis. Src inhibition (Y416 dephosphorylation) was observed in all 5 cells treated with dasatinib. The growth inhibition of Ca9-22, HSC3, and UMSCC1 cells was correlated with inhibition of Akt and Erk activities, which is not observed in SAS and FaDu cells. In addition, anti-apoptotic protein bcl-2 was increased in UMSCC1, FaDu, and SAS cells, but decreased in Ca9-22 and HSC3 cells. Therefore, the increases of bcl-2 in UMSCC1 may protect cells from dasatinib-induced apoptosis. Conclusion: Bcl-2 plays a crucial role in dasatinib-induced apoptosis in HNSCC cells, providing a new therapeutic approach to target bcl-2 in HNSCC treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1027. doi:10.1158/1538-7445.AM2011-1027

A novel mechanism of dasatinib-induced apoptosis in chronic myeloid leukemia; ceramide synthase and ceramide clearance genes

Sphingolipids are bioeffector molecules that control various aspects of cell growth, proliferation, apoptosis, and drug resistance. Ceramides, the central molecule of sphingolipid metabolism, are inducer of apoptosis and inhibitors of proliferation. Sphingosine-1-phosphate (S1P) and glucosyleceramide, converted from ceramides by sphingosine kinase-1 (SK-1) and glucosyleceramide synthase (GCS) enzymes, respectively, inhibit apoptosis and develop resistance to chemotherapeutic drugs. In this study, we examined the therapeutic potentials of bioactive sphingolipids in chronic myeloid leukemia (CML) alone and in combination with dasatinib in addition to investigate the roles of ceramide-metabolizing genes in dasatinib-induced apoptosis. Cytotoxic effects of dasatinib, C8:ceramide, PDMP, and SK-1 inhibitor were determined by XTT cell proliferation assay. Changes in caspase-3 enzyme activity and mitochondrial membrane potential (MMP) were measured using caspase-3 colorimetric assay and JC-1 MMP detection kit. Expression levels of ceramide-metabolizing genes were examined by qRT-PCR. Application of ceramide analogs and inhibitors of ceramide clearance genes decreased cell proliferation and induced apoptosis. Targeting bioactive sphingolipids towards generation/accumulation of ceramides increased apoptotic effects of dasatinib, synergistically. It was shown for the first time that dasatinib induces apoptosis through downregulating expression levels of antiapoptotic SK-1 but not GCS, and upregulating expression levels of ceramide synthase (CerS) genes, especially CerS1, in K562 cells. On the other hand, dasatinib downregulates expression levels of both GCS and SK-1 and upregulate apoptotic CerS2, -5 and -6 genes in Meg-01 cells. Increasing endogenous ceramide levels and decreasing prosurvival lipids, S1P, and GC, can open the way of more effective treatment of CML.

Co-administration of NVP-AEW541 and dasatinib induces mitochondrial-mediated apoptosis through Bax activation in malignant human glioma cell lines

Glioblastoma multiforme represents a largely incurable tumor for which novel therapeutic strategies are required. We report the effect of NVP-AEW541, an inhibitor of insulin-like growth factor-I receptor (IGF-IR) kinase activity on growth and signaling in a panel of glioma cell lines. NVP-AEW541 blocked phosphorylation of IGF-IR in a dose- and time-dependent manner and inhibited proliferation and clonogenicity with median effective concentrations of 2.5-10 microM. NVP-AEW541 also induced loss of mitochondrial membrane potential and release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria. Because concentrations of NVP-AEW541 required to significantly inhibit glioma cell viability and downstream signaling also inhibited non-neoplastic astrocytes, we questioned whether differential efficacy could be enhanced by combination with inhibition of other tyrosine kinases dysregulated in gliomas. Dasatinib was selected as a combination agent based on its distinct inhibitory profile for other relevant signaling targets. Combined treatment with NVP-AEW541 and dasatinib induced significantly more apoptosis than either agent alone in glioma cells, but not non-neoplastic astrocytes, and synergistically inhibited clonogenic survival. Mechanistic studies indicated that combination of NVP-AEW541 and dasatinib significantly reduced pERK and pAkt and markedly increased AIF release, Bax oligomerization and loss of mitochondrial potential compared to each agent alone. Overexpression of Bcl-2 and Akt significantly attenuated NVP-AEW541 and dasatinib-induced Bax activation and cell death. Our data indicate that activation of Bax plays a critical role in mediating NVP-AEW541 and dasatinib-induced apoptosis, and suggest the potential value of combining IGFR inhibition with other classes of tyrosine kinase inhibitors to potentiate therapeutic efficacy.

Abstract 3735: Dasatinib induces dephosphorylation of tyrosine proteins and apoptosis in both B-cell lymphoma lines and primary B-cell Non-Hodgkin Lymphoma cells in vitro for a subgroup of patients with phospho-Src (Y416) expression

Abstract The Non-Hodgkin Lymphomas (NHLs) are a heterogeneous group of disease with differing clinical behavior and responses to treatment. Approximately 85% of NHLs belonging to the B-cell lineage. Src family kinases (SFKs) are the predominant tyrosine kinase expressed in B-cell lines and are important in regulating multiple signaling pathways. We attempted to correlate the efficacy of SFK inhibitors with the expression of p-SFKs in lymphoma cells, to examine p-Src (Y416) expression in NHL tissues, and study dasatinib-induced apoptosis in primary lymphoma cells. In screening for the effects of different kinases inhibitors on B-cell lymphoma lines, we observed that SFK inhibitors, PP2 and dasatinib, inhibited proliferation and induced apoptosis at 24 hrs (PP2: 31% at 10 μM; dasatinib: 39% at 100 nM) in Burkitt's lymphoma (BL) line Raji. Apoptosis was associated with cleavage of caspases-3 & 8. SFK inhibitor-induced apoptosis in Raji cells correlated with high level expression of p-Src. A similar effect was observed in a follicular lymphoma (FL) line DOHH2 despite expression of BCL2 in both. Raji and DOHH2 cells. In contrast, dasatinib showed no apoptotic induction in B-cell lymphoma lines Ramos (BL line) & Sud-HL4 [GC type diffuse large B-cell lymphoma (DLBCL) line] which have no detectable expression of p-Src. Dasatinib caused complete de-phosphorylation of tyrosine proteins (around 160 kDa, 110 kDa and 60 kDa) in Raji cells as early as 15 mins. We are currently trying to identify these proteins. IHC staining of B-cell NHLs by tissue microarray indicated 26/55 (47.3%) of NHL tissues are positive for p-Src, which including DLBCL, FL, BL, small lymphocytic lymphoma (SLL), mantle cell lymphoma, marginal zone lymphoma (MZL) and lymphoblastic lymphoma. Fresh lymphoma cells from 12 cases [DLBCL (3), FL (3), SLL (2), MZL (1), NK/T cell lymphoma (1) and two reactive cases] were studied for in vitro effects of dasatinib. 6 cases (3 FL, 2 DLBCL and one reactive case) had clear expression of p-Src and underwent dasatinib-induced apoptosis (100 nM, 24h) of B-cells gated by CD45+/CD19+ co-expression. De-phosphorylation of p-Src and cleavage of caspase-3 were observed in the two DLBCL cases. The other 6 cases had undetectable p-Src and showed no apoptotic induction upon expose to dasatinib. Purified B-cells from 3 healthy donors have no detectable p-Src and no dasatinib induced apoptosis. These data suggest that 1) rational application of molecularly targeted therapy for aggressive NHL may be possible by directly examining key signaling nodes promoting survival and proliferation. 2) dasatinib is a potential therapeutic candidate for a subgroup of aggressive NHL which express p-Src (Y416). 3) profiling patients’ lymphoma cells for phospho-SFK might be a strategy selection of such therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3735.

Dasatinib-Induced Apoptosis in Chronic Lymphocytic Leukaemia Is Abrogated by Selective Micro-Environmental Signals.

Abstract 1264Poster Board I-286 BackgroundRecent advances in chronic lymphocytic leukaemia (CLL) biology indicate that a plethora of micro-environmental factors are important in promoting the pathogenesis of the disease. As B cell antigen receptor (BCR) signalling plays a critical role in CLL progression, we chose to study the effect of dasatinib, a dual src/abl tyrosine kinase inhibitor, on CLL cells, thus targeting the key, BCR-proximal tyrosine kinases Lyn and c-Abl that are over-expressed in CLL. We were interested to determine the impact of BCR-signalling directed therapy on CLL cell viability in the context of the leukaemic micro-environment. ResultsClinically relevant concentrations of dasatinib (100 nM) both inhibited ERK-MAPK and Akt phosphorylation, and prevented calcium mobilisation following BCR crosslinking. Furthermore, dasatinib prevented up-regulation of Mcl-1 observed on prolonged BCR stimulation, abrogating the BCR-driven increase in CLL cell survival. Dasatinib induced apoptosis of CLL cells, with a mean reduction in viability of 35.2% ± 3.8% following 48 hr exposure (n=26). Neither ZAP-70 expression nor cytogenetic abnormalities were predictive of response. In addition, dasatinib exhibited synergy with both fludarabine and chlorambucil, with mean ED50 combination indices of 0.29 and 0.62 respectively. Additional micro-environmental elements responsible for the maintenance and progression of CLL include stromal or ‘nurse-like' cells and activated T lymphocytes, the latter of which express CD40 ligand and secrete IL-4. CLL cell co-culture with the murine bone marrow stromal cell line NT-L significantly inhibited both spontaneous and dasatinib induced apoptosis. As we observed that stromal co-culture significantly increased phosphorylation of both ERK-MAPK and the Akt/mTOR target p70 S6 kinase in CLL cells, we investigated whether pharmacological inhibition these signalling pathways may re-sensitise CLL cells to dasatinib in co-culture. Both the MEK inhibitor PD98059 and PI3K inhibitor LY294002 significantly increased the level of apoptosis observed on dasatinib treatment of CLL cells in stromal co-culture. Dasatinib also retained the ability to potentiate the effects of fludarabine and chlorambucil in stromal co-culture, however, CLL cells co-cultured with NT-L cells stably transfected with CD40 ligand (CD154L cells) were resistant to all drug combinations. Furthermore, on CD154L/IL-4 co-culture, dasatinib failed to prevent up-regulation of Bcl-xL and Mcl-1, or inhibit CLL cell proliferation. Conclusions:While dasatinib was capable of reversing BCR-mediated survival signals, it was unable to overcome stromal-derived survival signals, indicating that combination of dasatinib with agents targeting antigen-independent micro-environmental pathways are required to effectively target CLL cells in tissue niches. DisclosuresNo relevant conflicts of interest to declare.

The Broad Spectrum Bcl-2 Inhibitor Apogossypol Induces Apoptosis and Differentiation of Blast Crisis Chronic Myeloid Leukemia Stem Cells.

Abstract 3275Poster Board III-1▪▪This icon denotes an abstract that is clinically relevant. Introduction:Leukemia stem cells (LSC) are rare, self-renewing cells capable of proliferation and differentiation into the bulk of cells that make up a leukemia. LSC, like normal tissue-specific stem cells, can be highly quiescent and resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. While traditional chemotherapy may successfully eradicate the bulk of tumor cells, it often fails to kill LSC resulting in their reactivation and eventual relapse of disease.Apoptosis resistance in cancer often involves deregulation of Bcl-2 family proteins. Bcl-2 family expression also seems to have an important role in normal stem cell function as expression changes of Mcl-1, Bcl-2, and Bcl-XL in mouse hematopoietic stem and progenitors characterize the differentiation of hematopoietic cells to different lineages.Several studies have linked expression of Bcl-2 family members to the development of blast crisis chronic myeloid leukemia (BC CML) in vitro, however there has been little data on the role of Bcl-2 expression in apoptosis resistance in human LSC. Also, various data has shown that CML cells become increasingly resistant to BCR-ABL inhibition with progression to blast crisis. In BC CML, LSC are enriched in the progenitor population and can be serially transplanted in immunodeficient mice leading to BC CML in vivo. We hypothesize that human BC CML LSC may resist chemotherapy by overexpression of Bcl-2 family proteins and maintenance of quiescence. We analyzed Bcl-2 family protein expression in BC CML LSC and analyzed whether these cells were sensitive to chemotherapy treatment in vitro. We further analyzed whether BC CML LSC could maintain quiescence in vivo. Finally, we tested the efficacy of the broad spectrum Bcl-2 antagonist apogossypol on BC CML LSC in vitro and in vivo. Methods:Bcl-2 and Mcl-1 protein expression was measured in 1° BC CML LSC by intracellular FACS analysis and compared to expression in normal and chronic phase CML cells. CD34+ cells isolated from serially transplanted BC CML LSC were used for all subsequent studies. To assess whether BC CML cells maintained quiescence in vivo, they were stained with DiR, an infrared fluorescent cell membrane dye, transplanted into neonatal mice, and analyzed for DiR fluorescence 18 weeks later. Dividing cells will distribute DiR to all daughter cells leading to dilution of the dye and a decrease in fluorescent signal per cell. LSC drug resistance was tested in vitro by culturing the cells with etoposide, dasatinib, and apogossypol and by measuring apoptosis by FACS using annexin-V/7-AAD staining. Finally, apogossypol efficacy was tested in vivo in LSC transplanted mice. At 8 weeks post-transplantation, mice were treated for 3 weeks with apogossypol or vehicle and then analyzed for human hematopoietic cell engraftment and apoptosis by FACS. Results:1° BC CML progenitors expressed significantly higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. LSC cultured in vitro were also resistant to etoposide and dasatinib-induced apoptosis. Apogossypol treatment in vitro however led to a dose-dependent increase in cell death and apoptosis and resulted in a significant increase in the frequency of lin+ staining cells along with a significant shift in the frequency of the common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) populations compared to vehicle treated controls.Mice transplanted with BC CML LSC developed diffuse myeloid sarcomas and had high levels of human engraftment in the liver, spleen, and bone marrow. Human cells in tumors and the liver were uniformly DiR- while cells engrafted in the spleen and bone marrow retained DiR fluorescence. In vivo treatment with apogossypol led to a significant reduction in human cell engraftment in mouse bone marrow compared to vehicle controls. Consistent with the results in vitro, there was also a significant increase in the frequency of lin+ staining cells in engrafted mouse spleens as well as a significant decrease in engrafted GMP in the mouse bone marrow. Conclusions:Our results demonstrate that BC CML LSC are highly resistant to conventional chemotherapy but are sensitive to apogossypol in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC by inducing apoptosis as well as by inducing differentiation. Disclosures:Goff:Coronado Biosciences: Research Funding. Tesi:Coronado Biosciences: President and CEO. Jamieson:Coronado Biosciences: Research Funding. Jamieson:Coronado Biosciences: Research Funding.

Dasatinib Induces Apoptosis in Chronic Lymphocytic Leukemia and Enhances the Activity of Rituximab and Fludarabine.

Chronic lymphocytic leukemia (CLL) is a disease characterized by the monoclonal accumulation of well-differentiated CD5 B cells. Previously, we reported that CLL cells that use unmutated immunoglobulin VH (IgVH) genes and/or express ZAP-70 are more responsive to signaling induced by ligation of surface IgM compared to cells that use mutated IgVH and lack expression of ZAP-70 (Chen et al. Blood 2005) Because signaling through the Ig receptor appears to play a role in the pathogenesis or progression of CLL, targeting the Ig signal transduction pathway in CLL might have therapeutic utility, particularly for those patients with high-risk disease. Dasatinib (Sprycel) is a tyrosine kinase inhibitor (TKI) with antiproliferative activity against hematological and solid tumor cell lines and it is FDA approved for the treatment of patients with CML and Ph+ ALL. Contrary to Imatinib (Gleevec), Dasatinib is a potent TKI not only of the Abl family of kinases but also of Src kinases, which regulate Ig-receptor signal transduction and govern the early events following Ig receptor ligation. Because Dasatinib TKI profile and its potential role inhibiting BCR signaling we studied its in vitro activity in CLL. Primary leukemia cells from 40 different CLL patients were evaluated. We found that Dasatinib, but not Imatinib, induced apoptosis in CLL cells at doses that were pharmacologically achievable. The IC50 for Dasatinib was in the 30–100 nM range. Interestingly, the pro-apoptotic activity of Dasatinib in CLL cells was not observed in normal B, T cells or blood mononuclear cells of healthy donors, suggesting that Dasatinib has a specific effect on CLL cells. Dasatinib induced apoptosis in CLL cells in a time and concentration dependent manner. Peak apoptosis occurred after 2 hours of in vitro exposure. Leukemia cells that expressed ZAP-70 were significantly more sensitive to Dasatinib-induced apoptosis than CLL cells lacking expression of ZAP-70. In addition, Dasatinib enhanced in vitro the pro-apoptotic activity of Rituximab and Fludarabine in CLL cells. Dasatinib treatment of CLL cells induced changes in the expression profile of apoptosis related genes as well as changes in apoptosis related proteins such as cleavage of PARP-1 and Caspases. Moreover, CLL cells treated in vitro with Dasatinib showed reduced tyrosine kinase activity measured by ELISA and also by immunoblots of CLL-cell lysates using specific phospho-TK antibodies. In addition, Ig receptor signaling following surface IgM ligation was decreased in CLL cells that were pre-treated with Dasatinib relative to that of untreated CLL cells. In conclusion, Dasatinib induces apoptosis of CLL cells at low nanomolar concentrations that do not appear to affect the viability of B cells, T cells, or blood mononuclear cells of healthy adults. CLL cells that expressed ZAP-70 were significantly more sensitive to Dasatinib than CLL cells that lacked expression of ZAP-70. This process was associated with impairment of BCR signaling, decrease TK activity and regulation of genes and proteins related to apoptosis. In addition, treatment of CLL cells with Dasatinib enhanced the in vitro activity of Rituximab and Fludarabine. These results reveal that Dasatinib is potentially active in CLL, providing a rationale for clinical trials evaluating its clinical activity in the treatment of patients with this disease.