Inhibition of Wnt/β-Catenin Signaling by AV65 Treatment Caused Cell Cycle Arrest and Induced Caspase-Independent or -Dependent Apoptosis in CML Cells.

Author: Kimura, Shinya ; Maekawa, Taira ; Yokota, Asumi ; Strovel, Jeffrey W ; Yao, Hisayuki ; Nagao, Rina ; Hirai, Hideyo ; Padia, Janak ; Cholody, Wieslaw M ; Ashihara, Eishi ; Takeuchi, Miki ; Tanaka, Ruriko

AbstractAbstract 2184Poster Board II-161Imatinib has dramatically improved the management of CML, but cases of imatinib resistance have been reported. The second-generation ABL tyrosine kinase inhibitors (TKIs) such as dasatinib and nilotinib overcome imatinib-resistant CML.These agents, however, are ineffective in CML cells harboring T315I mutation and in CML stem cells. Recently, loss of β-catenin has been reported to impair the renewal of CML stem cells (Chao et al, Cancer Cell 2007) and an in vivo study has showed that β-catenin is essential for survival of leukemic stem cells (Hu et al, Leukemia 2009). Thus, we hypothesized that the inhibition of β-catenin signaling may be efficacious in the treatment of CML. We have previously reported that a novel β-catenin inhibitor, AV65, suppresses the growth of imatinib resistant CML cell lines harboring Abl kinase domain mutations including T315I and hypoxia-adaptation (Nagao et al, ASH 2008). We herein examine the cell cycle and apoptotic effects of AV65 on CML cell lines and its therapeutic possibility for CML stem/progenitor cells.We observed that expression of β-catenin is increased 20 to 45-fold in K562, BV173, KT-1, and MYL CML cell lines compared with total bone marrow cells from healthy volunteers. We have previously demonstrated that AV65 induced apoptosis of CML cells. To investigate how AV65 inhibits β-catenin, we next analyzed the effects of AV65 using Western blotting and real time PCR. AV65 suppressed the expression of β-catenin in K562 in a time- and a dose-dependent manner in nuclear and cytosolic fractions as well as whole cell lysates. AV65 did not diminish the transcripts of β-catenin in K562 indicating the depletion of β-catenin due to an inhibition of its accumulation in CML cells. Next we examined the effects of AV65 on cell cycle. The fractions of G1 phase to S phase increased by AV65 treatment. TUNEL/PI staining showed that both K562 and BV173 began to be nicked by AV65 at 30 nM for 12 hours, resulting in the induciton of apoptosis from G1 phase to S phase 24 hours after AV65 treatment (Figure). In real-time PCR analysis, the transcripts of p21, p27, and p57 in CML cell lines increased by AV65 treatment, however, those of p53 were not altered. Taken together, it is suggested that CML cells first arrested from G1 phase to S phase and then induced apoptosis after AV65 treatment. Next we examined the mechanisms of apoptosis by AV65 treatment. AV65 treatment in the presence of Z-VAD did not induce cell death in BV173, indicating that AV65 induced caspase-dependent apoptosis in BV173. In K562 cells however, AV65 induced apoptosis with or without Z-VAD suggesting that AV65 induces apoptosis in CML cell lines in caspase-dependent or -independent pathways. Lastly, we investigated the effects on hypoxia-adapted CML cells. We established hypoxia-adapted K562 cell lines (K562/HA). This cell line shows characteristics of more primitive CML cells, including resistance to serial Abl TKIs and a higher transplantation efficacy compared to the parental K562 cells (Takeuchi, et al. ASH 2008). In Western blotting analysis, K562/HA cell line expressed more β-catenin than its parental K562 cell line. AV65 inhibited the growth of K562/HA at the similar concentration to K562. Taken together, AV65 is effective for primitive CML cells which overexpress β-catenin. This suggests that AV65 has a potential to eradicate CML stem/progenitor cells. In conclusion, AV65 inhibits the accumulation of β-catenin in CML cells and this causes cell cycle arrest from G1 to S phase, resulting in induction of caspase-independent or -dependent apoptosis in CML cells. The inhibition of Wnt/β-catenin signaling has great potential as a novel and attractive therapy for CML.Disclosures:No relevant conflicts of interest to declare.

16 Nov 2008·Blood

A Novel β-catenin Inhibitor, AV65 Suppresses the Growth of CML Cell Lines Which Acquire Imatinib-Resistance Because of Abl Kinase Domain Mutations Including T315I and Hypoxia-Adaptation.

Author: Kimura, Shinya ; Maekawa, Taira ; Tanaka, Ruriko ; Cholody, Wieslaw M ; Yokota, Asumi ; Takeuchi, Miki ; Nagao, Rina ; Yao, Hisayuki ; Strovel, Jeffrey W ; Ashihara, Eishi

AbstractImatinib and the second-generation ABL tyrosine kinase inhibitors (TKIs) such as dasatinib and nilotinib have dramatically changed the management of CML. However, these agents are effective neither for CML cells harboring T315I mutation nor for CML stem cells. Genetic analysis of blasts from CML patients in blast crisis has identified numerous members of the Wnt/b-catenin pathway as being activated (Radich et al, PNAS 2006) and loss of b-catenin has been reported to impair the renewal of CML stem cells (Chao et al, Cancer Cell 2007). Thus, b-catenin signaling inhibition may be useful for CML treatment. We herein describe the effects of a novel b-catenin inhibitor, AV65 on various imatinib-sensitive and –resistant CML cell lines (Table 1). Eight imatinib-sensitive human CML cell lines and one normal cell line derived from hepatocyte were exposed to AV65 for 72 hours and its anti-proliferative effects were examined by MTT assay. AV65 induced apoptosis in these CML cell lines time- and does-dependent manners and inhibited the growth of all eight CML cell lines with IC50 at ranging from 9.8 to 33.1nM. While that for hepatocyte was 204.8nM. Interestingly, AV65 induced polyploidies in K562 but not in BV173. In addition, AV65 augmented the anti-proliferative effects of imatinib additively against K562 and BV173. These findings suggested that AV65 alone or the combination with imatinib was effective for imatinib-sensitive CML cells. To investigated the effects of AV65 against imatinib-resistant CML cells, four imatinib-resistant CML cell lines such as K562/IMR (bcr-abl amplification), MYL-R1 (LYN overexpression), KBM5/ STIR (harboring T315I) and K562/D1-9 (P-glycoprotein overexpression), and BaF3 cells expressing the wild type BCR-ABL or its ten different mutant BCR-ABL forms including T315I, were used. There was little difference on the induction of apoptosis and anti-proliferative effects of AV65 except K562/D1-9 between each parental cell line and its resistant clone and AV65 inhibited the growth of all examined BaF3 cells harboring various mutations including T315I with IC50 at ranging from 21.6 to 46.5nM. IC50 values of AV65 for K562 and K562/D1-9 were 11.0 and 60.1nM, respectively. These findings suggested that the effects of AV65 were independent either to BCR-ABL expression level, ABL mutations or LYN overexpression, but might be affected by P-glycoprotein. Next, the effects of AV65 against the hypoxia-adapted CML cell lines such as K562/ HA and KCL22/HA were also investigated. These cell lines were resistant to imatinib, dasatinib, INNO-406 (another second-generation ABL TKI; Kimura et al, Blood 2005) and alkylating agents via the up-regulation of glyoxalase-I, a detoxification enzyme for the cytotoxic byproducts of glycolysis. Intriguingly, AV65 inhibited the growth of hypoxiaadapted CML cell lines at almost same concentration compared with their parental cell lines. Although CML stem cell niche has not been definitely identified, it might located in hypoxia because the inoculated human leukemia cells to immunodeficient mice preferably localized on the surface of osteoblasts in the epiphysis (Ninomiya et al, Leukemia 2007) where could be hypoxic. Taken together, AV65 may be effective for CML stem cells in hypoxia. In conclusion, AV65 inhibited the growth of CML cell lines which acquire imatinib-resistance because of Abl kinase domain mutations including T315I and hypoxiaadaptation. Therefore, AV65 may become a promising agent for CML treatment including both imatinib and the second-generation Abl TKIs-resistant patients.Figure

16 Nov 2007·Blood

In Vitro Characterization of Wnt/β-Catenin Pathway Inhibition in Multiple Myeloma Cell Lines Using a Novel Class of Small Molecules.

Author: Strovel, Jeffrey W. ; Horrigan, Stephen K. ; Weaver, Zoe ; Watthey, Jeffrey W. ; Padia, Janka ; Cholody, Wieslaw M. ; Hamilton, J. Michael ; Lonskaya, Irina ; Bol, David K. ; Natarajan, Pachai ; Lawrence, Tammy ; Glanowski, Stephen

AbstractMultiple Myeloma (MM) is a fatal form of hematologic cancer characterized by the clonal expansion of plasma cells in the bone marrow. Recent finding by several research laboratories have shown that Wnt/β-Catenin signaling pathway is activated in MM leading to the translocation of β-Catenin to the nucleus where it binds to T cell factor (TCF) and drives transcription of genes involved in the progression of cancer (Derksen et. Al., PNAS 2004). The mechanism by which Wnt/β-Catenin signaling is activated in MM is not well understood, however, β-Catenin is expressed in the majority of MM cell lines and inhibition of the pathway with a dominant negative form of TCF4 or the small molecule PKF115-584 have shown anti-proliferative effects in in vitro and in vivo cell line and xenograft models making inhibition of Wnt/β-Catenin signaling a promising modality for the treatment of MM (Sukhdeo et al., PNAS 2007). To this end, Avalon Pharmaceuticals has developed a series of small molecule compounds, lead candidate series-363 (LC-363), with potent inhibitory effects on Wnt/β-Catenin signaling and anti-proliferative effects in colon cancer cell lines and in vivo models (EORTC, 2007). LC-363 compounds down regulate expression of the noted β-Catenin/TCF transcriptional targets c-jun, fra-1, and PPARδ as well as increase expression of the Dickkopf family of secreted proteins 1 and 3 which inhibit Wnt signaling. In this study, we report on the in vitro characteristics of LC-363 in MM cell line models. We show that LC-363 compounds have broad and potent growth inhibitory and cytotoxic effects on MM cell lines. Additionally, these effects are mediated through a G1 phase cell cycle block, apoptosis, and decrease in cytoplasmic levels of β-Catenin. Importantly, we show the relationship between expression level of β-Catenin protein and sensitivity to LC-363 compounds in terms of growth inhibition and apoptosis. Lastly, we describe the gene expression signatures induced by LC-363 in MM cell models across multiple time and dose studies to detail their dynamic effects on expression of genes within the Wnt/β-Catenin signaling pathway. In conclusion, we believe that inhibition of Wnt/β-Catenin signaling is a viable therapeutic alternative for treatment of MM patients with activated Wnt/β-Catenin signaling and to that end; LC-363 compounds are promising candidates currently in pre-clinical development for treatment of cancers with activated Wnt/β-Catenin signaling including MM.

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