ALBERT

Description: Abstract 4147 Whole-body diffusion-weighted magnetic resonance imaging (DWI-MRI) provides functional information and is able to highlight oncological lesions, but the usefulness has not been established in malignant lymphoma especially for monitoring therapeutic response. Positron emission tomography with fluorine-18 fluorodeoxyglucose (FDG-PET) is a useful imaging producer for tumor staging and therapy monitoring that can visualize active tumor tissue including malignant lymphoma. The spatial resolution of FDG-PET is limited, and low accuracy rates in diabetic patients and those with low grade lymphoma have been reported. We prospectively studied the utility of DWI-MRI with T2 imaging and apparent diffusion coefficient (ADC) for staging and monitoring therapeutic responses in patients with malignant lymphoma compared with FDG-PET/CT. Twenty-eight patients with malignant lymphoma (16 patients with diffuse large B cell lymphoma: DLBCL, 7 with follicular Lymphoma: FL, 3 with aggressive T cell lymphoma: TCL and 2 with Hodgkin lymphoma: HL, including one diabetic patient) received both MRI and FDG-PET examination before (n=28), after 2 courses of chemotherapy (n=25) and one month after the end of chemotherapy (n=9). MRI examination was performed with a 3-Tesla MR system (Signa Excite, Generel Electrics). Whole-body DWI-MRI was performed with echo planar imaging sequence with short T1 inversion recovery (STIR) fat suppression. ADC measurement was performed based on the region of interest (ROI) method. ROI was placed on the lesion showing the highest standardized uptake value (SUV) on FDG-PET/CT scanner (Discovery LS, General Electrics) in each patient, and crucial parameters of the ADC and SUV were compared. Based on staging by PET/CT, 4 patients were clinical stage I, 8 were stage II, 7 were stage III and 9 were stage IV. DWI-MRI findings alone matched PET/CT in 22 patients (79%), whereas these findings combined with T2 imaging increased match in 26 patients (93%). Regarding the early response to chemotherapy, 19 of 25 patients (76%) were considered to show CR on PET/CT and the DWI findings matched PET/CT 23 patients (92%). To evaluate the final response after chemotherapy, 7 of 9 patients (78%) were considered to show CR on PET/CT and the DWI findings matched PET/CT in 8 of 9 patients (89%). Of these nine, one patient with DLBCL who did not show a match was a false positive on PET/CT. In all patients with TCL and HL, the DWI-MRI findings combined with T2 imaging matched PET/CT findings for staging and therapeutic response. Interestingly, the ADC values on DWI-MRI did not differ between DLBCL and FL (0.77 +/− 0.23 and 0.70 +/− 0.08, p=0.99, mean +/− SD respectively), whereas the SUV values of DLBCL on PET/CT were higher than those of FL (14.5 +/− 6.97 and 6.09 +/− 2.54, p 〈 0.0005, mean +/− SD respectively), suggesting the DWI-MRI could detect the lymphoma lesion more accurately than PET/CT in patients with indolent lymphoma such as FL. We conclude that whole-body DWI-MRI combined with T2 imaging and ADC analysis could be promising sensitive method for staging and therapeutic response evaluation in patients with malignant lymphoma. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3977 Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor prognosis, requiring novel anticancer strategies. Since p53 inactivating mutations occur primarily in the aggressive and refractory MCL variants, targeting p53-independent signaling pathways is of considerable interest. We previously reported the cytotoxic efficacy of a newly discovered tricyclic coumarin GUT-70 (5-methoxy-2,2-dimethyl-6-(2-methyl-1-oxo-2-butenyl) -10-propyl-2H,8H-benzo[1,2-b;3,4-b ]dipyran-8-one (C23H26O5) (synthesized at Nippon Shinyaku, Kyoto, Japan), originally derived from Calophyllum brasiliense, with more pronounced apoptotic effects in mutant-p53 MCL cells than in wild type-p53 cells (Jin et al., ASH abstract 2009). Some of the coumarin antibiotics are known to bind to chaperone molecule 90-kDa heat shock proteins (Hsp90) and induce degradation of Hsp90 client proteins including key components of multiple signaling pathways for cell proliferation and/or survival. In this study, the mechanisms of action of GUT-70 were investigated in MCL cell lines with known p53 mutation status (wt-p53: JVM-2, Granta-519, mt-p53: Jeko-1, MINO). GUT-70 demonstrated a dose-dependent binding affinity to Hsp90 (competitive binding assay using fluorescently labeled geldanamycin), increased ubiquitinated proteins accumulation, and further induced degradation of Hsp90 client proteins, including cyclin D1, Raf-1, Akt, and mt-p53, or increased Hsp70, a marker of Hsp90 inhibition. The downregulation of constitutively overexpressed cyclin D1 in MCL by GUT-70 was accompanied by p27 accumulation, decreased Rb phosphorylation, and impeded cell cycle progression in the wt-p53 JVM2 and Granta 519. However, GUT-70 induced apoptosis in mt-p53-bearing MINO and Jeko cells which was accompanied by only minimal cell cycle arrest. These findings suggest that apoptosis induction by GUT-70 in mt-p53 cells is in part independent from cell cycle arrest is known to protect cells from apoptosis. Moreover, the mt-p53 depletion by GUT-70 will further diminish its “gain of functions” for cell proliferation and anti-apoptosis. To determine if GUT-70 might potentiate the apoptotic effects of commonly used chemotherapeutic agents, we assessed the combination effects of GUT-70 with bortezomib (BTZ), a selective inhibitor of the 26S proteasome, and with doxoubicin (DOX), a conventional chemotherapeutic agent drug for MCL. Synergistic anti-proliferative effects of GUT-70 and BTZ or GUT-70 and DOX combinations were observed in both wt-p53 and mt-p53 MCL cells at 48 h post-exposure (combination index; GUT-70/Bortezomib; 0.59 for JVM2, 0.73 forMINO, GUT-70/Doxorubicin; 0.37 for JVM2, 0.35 for MINO). In conclusion, our results demonstrate that the novel anticancer agent tricyclic coumarin GUT-70, an Hsp90 inhibitor, has potential utility for mt-p53 bearing MCL cells, and in the combination therapy of MCL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5088 Aim: With the exception of CD5, the clinical implications of aberrant T-cell antigen expression on B cells in diffuse large B cell lymphoma (DLBCL) have not been well studied. The present retrospective study aimed to determine associations between T cell antigen expression and patients′ characteristics and prognosis Patients, Material and Methods: 249 cases with B-cell lymphoma (BCL) newly-diagnosed in our Institute between January 2002 and August 2009 were biopsied and also analyzed by flow cytometry. Biopsy specimens were fixed in formalin, stained with Hematoxylin-Eosin, and also immunostained. Histological subtypes were defined according to the World Health Organization Classification Version 3. Flow cytometric (F/C) analysis was performed following standard methods. The aberrant expression of one or more T cell surface antigens on B cells (CD2, 3, 4, 7, 8) was assessed. Statistical analysis was conducted to seek associations between aberrant positive and negative cases and patients′ background laboratory data, and prognosis. This study was approved by the Ethics Committee of Kitasato University School of Medicine. Results: 150 DLBCL, 68 Follicular lymphoma (FL), 17 Marginal zone lymphoma (MZL), 6 MCL and 4 CLL patients were tested. Of the DLBCL cases, 12 (8%) showed aberrant T cell antigen expression with 4, 1, 5, 1 and 1 patients′ B cells positive for CD2, CD4, CD7, CD8 and CD7 + 8 respectively. Aberrant expression among the FL, MCL, MZL and CLL cases was seen in 0, 1, 1 and 1 patient, respectively. Among the DLBCL patients, T antigen-negative cases tended to have higher WBC counts and greater CD20 expression by F/C analysis. No statistically significant associations with gender, age, IPI, clinical stage, laboratory data, expression of CD5 and other markers, ABC/GCB, or karyotype were observed between the positive and negative groups. 96 of the 150 DLBCL patients received rituximab-chemotherapy. Again, there were no statistically significant differences between the two groups in overall survival and response to treatment. Discussion: 1) CD2 and CD7 are relatively common among aberrant T cell antigen- positive DLBCL. 2) No statistically significant differences were observed between the two groups in terms of background and prognosis. 3) Both CD8-positive DLBCL patients experienced a very aggressive clinical course and rapidly succumbed to their disease. CD8-positive DLBCL cases may in general have poorer survival. 4) Further analysis will be necessary to confirm these results. Disclosures: No relevant conflicts of interest to declare.

Description: 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.

Description: Abstract 5081 Background: Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is an uncommon tumor with unfavorable prognosis. The purpose of this retrospective study was to investigate the cytological features and treatment outcomes in patients with PTCL-NOS and to obtain new information on the therapeutic implications of PTCL-NOS. Method: A total of 6 institutions in Japan and Asia submitted clinical and pathologic information on PTCL-NOS diagnosed and treated at their respective centers. Among these cases, 55 (71%) were from Japan, 8 (10%) were from Hong Kong, 8 (10%) were from Thailand, and 7 (9%) were from Korea. From 1992 to 2006, 78 cases of PTCL-NOS were examined for cytological features, focusing on morphometric image analysis. The average nuclear area of lymphoma cells, as well as the median, the minimum, the maximum and the standard deviation, was estimated in 50 cells for each case. In addition, we performed an immunohistochemical study of CCR4 expression in all cases. Univariate analyses were performed using Kaplan-Meier survival estimates, and data were compared using the log-rank test. Cox proportional-hazard regression test was used for multivariate analysis. P-values of less than 0.05 were considered significant. Results: Clinical information was available for review in 78 cases. There was a 1.52:1 male/female ratio, and median age was 62 years (range, 3 to 87 years). Sixty-two percent of patients had advanced clinical stages. Lactic dehydrogenase (LDH) was elevated in 53% of cases, and most patients were treated with combination chemotherapy. Median survival was 693 days. On the morphometric image analysis, median nuclear area was 41.7 μ m2 (range, 22 to 119 μ m2). Thirty-three of the cases (42%) showed positive CCR4 immunostaining. Statistical analysis confirmed that nuclear area of lymphoma cells in PTCL-NOS is correlated with overall survival, whereas immunohistochemical expression of CCR4 is not correlated with overall survival or nuclear area of lymphoma cells. Multivariate analysis confirmed that the nuclear area of lymphoma cells in PTCL-NOS is an independent prognostic factor. Conclusion: These results indicate that nuclear area of lymphoma cells in PTCL-NOS is an independent prognostic factor that may predict overall survival. Because PTCL-NOS is a heterogeneous disease with regard to histological type and pathological state, nuclear area of lymphoma cells could be used to stratify patients with PTCL-NOS for therapies, although we are continuing to accumulate data. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1871 Introduction: PI3K/AKT pathway is involved in cell growth, proliferation and apoptosis. A key downstream effector is the phosphorylated serine-threonine Akt (p-AKT). Constitutive activation of PI3K/AKT has been observed in solid tumours and leukemic cells. Inhibition of PI3K/AKT activity, results in apoptosis in cell lines (CL) after treatment with different compounds, e.g. deguelin, a natural product from the leguminous Mundulea sericea, with antitumour effects. Aims: To evaluate PI3K/AKT activation in MDS patients and its therapeutic potential in MDS. Methods: PI3K/AKT activation was evaluated by flow cytometry (FC) using an alexa-fluor 488-antibody Ser 473 p-AKT (Cell Signalling Technology). A triple immunostaining procedure using CD45-PerCP and CD34-PE was used for p-AKT expression in CD34+ primary samples. The p-AKT activity was determined using Kolmogorov-Smirnov test (D). CD34+ cells from healthy donors and Jurkat cells were used as negative and positive controls respectively. Apoptosis (determined by Annexin V and PI/7AAD) and cell cycle arrest (using RNAse and PI) were determined following treatments with LY294002 (50uM), and deguelin (100-500nM) in P-39 myeloid leukemia cell line, with constitutive PI3K/AKT activation. Apoptosis was determined in bone marrow mononuclear cells and CD34+ cells from MDS patients with the same treatments. To evaluate in vivo activity of deguelin, we used a xenotransplant model. Briefly, NODSCID mice were injected intrafemurally with P-39 CL and 12 days post transplant a three week-course of treatment, every other day, was started (deguelin 4mg/Kg, n=3 vs vehicle, n=3). Results: P-39 CL showed constitutive PI3K/AKT activation with levels significantly higher than in CD34+cells from controls (median±SD= 0.73. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3240 Introduction: Treatment of adult acute lymphoblastic leukemia (ALL) has shown only modest improvements over the last 2 decades, with overall survival of 15% to 40%. The mitogen-activated protein kinase (MAPK) signaling cascade and the phosphoinosytol-3 phosphate/AKT (PI3K/AKT) pathways are involved in proliferation and differentiation of hematopoietic cells. It has been reported that those pathways are frequently activated in solid tumors and acute myeloid leukemia. However, their role in adult ALL is still uncertain. Better understanding of such pathways is necessary for development of novel therapeutic strategies. Aims: To evaluate the phopho-ERK and phospho-AKT protein expression in ALL at diagnosis and to correlate with biological and clinical parameters. Material and Methods: Twenty eight patients (median age 33y, 14–69y) with ALL at diagnosis were studied. Bone marrow and/or peripheral blood mononuclear cells (PBMC) (10 fresh and 18 cryopreserved cells at diagnosis) were stained using phospho-ERK and phospho-AKT/alexafluor 488 monoclonal antibodies (Cell Signaling Technology, Beverly, MA) and their expression was evaluated by flow cytometry (FACScalibur cytometer and CELL Quest program - BDB, San Jose, CA). The monoclonal antibodies CD19, CD10, CD3, CD45, IgM, CD34, CD7, CD2 were used for leukemic cells characterization by four-colour staining. Healthy donor PBMC and Jurkat cell line were used as controls: normal T lymphocytes are negative for p-ERK and Jurkat cell line express p-ERK and p-AKT in low levels. Samples were analyzed for constitutive expression of p-ERK and p-AKT and also after cell activation by phorbol-myristate-acetate (PMA). The expression of these proteins was evaluated by Kolmogorov-Smirnov test using fluorescence ratio between control isotype and phospho-protein (D). p-ERK and p-AKT expression was also evaluated in fresh and frozen samples of the same patients (2 cases) and similar results were obtained. In addition, patients were evaluated for multidrug resistance (MDR) through p-glycoprotein (PGP) expression and Rhodamine (Rh) efflux test and minimal residual disease (MRD) detection at the end of induction by flow cytometry. Results: Twenty cases were B-ALL (EGIL B-I 3, B-II 9, B-III 8, B-IV 5) and 3 T-ALL. Median WBC count was 25.3×109/L (2.3-373×109/L). The expression of p-ERK and p-AKT varied and the median value of p-ERK expression was D = 0.16 (0.01-0.80) and p-AKT median D = 0.08 (0.00-0.63). Considering these values as cutoff there was no difference regarding the patients` age and WBC count at the diagnosis between the positive and negative groups. In regards to EGIL subtypes, p-ERK expression was higher in T-ALL [median 0.50 (0.18-0.54)] than in B-precursor ALL [median 0.14 (0.01-0.80)] (p=0.03). Conversely p-AKT expression was similar in all cases, although high levels were observed among BIII cases. The frequency of Rh efflux was 88% in pERK negative cases and 66% in positive group but there was no difference on PGP expression. On the contrary, PGP expression and Rh efflux were more frequently seen in p-AKT positive cases (88% and 100%, respectively) than p-AKT negative ones (63% and 60%). MRD analysis was performed in seven patients. Two cases presented detectable MRD (〉0.01%) and both were p-ERK positive and p-AKT negative. Interestingly all five MRD (-) cases were p-ERK negative and p-AKT positive. In addition, PMA test showed p-ERK activation of normal T lymphocytes and the expression was increased in 52% of ALL cases upon treatment. Conclusion: MAPK and PI3/AKT activation varied among ALL patients. MAPK pathway showed to be more activated in T-ALL than in precursor-B ALL. The functional analysis of these pathways can address the role in ALL pathogenesis. Both pathways may be potential therapeutic targets for novel therapies. (Support: FAPESP proc.09/51002-8). Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3613 BMI-1 is essential for the self-renewal and proliferation of leukemic and hematopoietic stem and progenitor cells. Increased expression of BMI-1 is known to be an indicator for a poor prognosis in cancer patients. Analysis of the expression of BMI-1 and survivin in 6 patients with B-cell lymphoma (3 drug-resistant and 3 sensitive cases) showed that in the drug-resistant patients, high levels of BMI-1 and survivin were maintained even after drug administration in vitro. However, there observed was a down-regulation of both BMI-1 and survivin expression in the drug-sensitive patients. BMI-1 transduction induced the drug-resistance of two B-cell lymphoma cell lines, HT and RL, to the anti-cancer drugs etoposide and oxaliplatin, but not to irinotecan. The expression of survivin was clearly augmented in the cells transduced with BMI-1. Moreover, we detected sustained expression of survivin level in the presence of etoposide in the BMI-1-overexpressing cells. By contrast, the mock-transduced cells succumbed in the medium with anti-cancer drugs with an accompanying decrease in the expression of survivin as well as BMI-1. Survivin has been reportedly implicated in resistance to chemotherapeutic agents. Intriguingly, survivin mRNA levels in BMI-1-overexpressing cells were consistent with those in controls. Also, the level of survivin was enhanced by treatment with a proteasomal inhibitor, MG132, suggesting that overexpression of BMI-1 stabilized survivin expression post-translationally. We further showed that sh RNA-mediated knockdown of BMI-1 or survivin restored sensitivity to etoposide in the HT cells overexpressing BMI-1. Our findings suggest survivin as a potential target for BMI-1. Thus BMI-1, by acting as an upstream regulator, may control the expression of survivin, facilitating drug resistance in B-cell lymphoma. Next, we examined whether B-cell lymphoma cells overexpressing BMI-1 are abrogated by immunotherapy with T cells containing anti-CD38 chimeric receptor in vitro. Interestingly, these B-cell lymphoma cells were effectively eliminated by specific T cells against B-cell lymphoma cells bearing CD38. These results suggest that the immunotherapy is useful for treatment of patients with B-cell lymphoma cells overexpressing BMI-1, which are refractory to chemotherapeutic reagents. BMI-1 may be an important prognostic marker as well as a future therapeutic target in the treatment of drug-resistant lymphomas. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2766 γδT cells, which control the innate immune system, are classified into three subtypes on the basis of Vγ chain. Of these subtypes, Vγ2Vδ9 T cells display anti-tumor immunity. We have demonstrated that nitrogen-containing bisphosphonate (N-BP) treatment expands Vγ2Vδ9 T cells ex vivo and that these expanded cells can kill tumor cells in a major histocompatibility complex-unrestricted manner (Sato, Int J Cancer, 2005; Uchida, Biochem Biophys Res Commun, 2007; Sato, Cancer Immunol Immunother, 2008.). N-BP inhibits farnesyl pyrophosphate synthase in the mevalonate pathway, resulting in the accumulation of isopentenyl pyrophosphate (IPP), which is a stimulatory antigen for Vγ2Vδ9T cells. In the present study, we investigated the chemotactic factors for Vγ2Vδ9T cells by using a micro total analysis system-based microfluidic cellular analysis device (Kanai, Sens Actuators A, 2004; Munaka, Analyst, 2007.). This microchip possesses a minute-volume (240 nL) chamber integrated with a micro-sample injector that permits the injection of a small amount (several nL) of a solute (Figure 1). Because of the minute size of this chamber, a concentration gradient can be maintained free from the influence of fluid convection and stirring, and the solute can consequently spread in a diffusion-dependent manner. Therefore, administration of a humoral factor via the sample injector mimics its release from the cell surface. We first investigated whether the supernatant of RPMI8226 multiple myeloma (MM) cells treated with zoledronic acid (ZOL) induced chemotaxis of γδT cells. We treated RPMI8226 MM cells with ZOL (1 mM) overnight and collected the supernatant. Human γδT cells were obtained by the culture of peripheral blood mononuclear cells as previously reported (Uchida, Biochem Biophys Res Commun, 2007.), and these cells were cultured in the microchip. After the injection of supernatant, γδT cells migration was observed under a microscope and continuous time-lapse recording was performed for 30 min. γδT cells migrated toward the injector, indicating that the supernatant of ZOL-treated RPMI8226 cells includes a chemoattractant factor for γδT cells. We next applied soluble MICA (sMICA), sICAM-1, sVCAM-1, and IPP and examined the migration of γδT cells. Among them, sMICA and IPP were chemoattractive for γδT cells, and the velocity of γδT cell migration was increased by the injection of IPP compared to the solvent control (Figure 2). These observations indicate that IPP, a metabolite of the mevalonate pathway in MM cells, or sMICA is a chemotactic factor for γδT cells when the target MM ells are treated with ZOL. Disclosures: Munaka: Shimadzu Corporation: Employment. Kanai:Shimadzu Corporation: Employment. Abe:Shimadzu Corporation: Employment.

Description: Abstract 4930 Tyrosine kinase inhibitors (TKIs) against Bcr-Abl fusion oncoprotein, such as imatinib mesylate (IM), nilotinib, or dasatinib, are the first-line molecular targeted therapeutics for chronic myelogenous leukemia (CML). However, the resistance to Bcr-Abl TKIs is induced in leukemic cells not only by loss of sensitivity to TKIs through Bcr-Abl-related molecular mechanisms, such as the acquisition of Abl mutation or the overexpression of Bcr-Abl, but also by loss of addiction to Bcr-Abl TK activity by acquiring Bcr-Abl-unrelated additional oncogenic mutations. Therefore, a new treatment approach that induces an anti-leukemic effect via Bcr-Abl-unrelated molecular pathways is urgently needed for achievement of a complete cure and to overcome TKI resistance. Galectins are a family of animal lectins that show specific affinity for beta-galactosides. Among fourteen mammalian galectins, galectin-9 (Gal9) has been shown to possess the anti-cancer properties by regulating various cellular functions, such as cell adhesion, cell proliferation, or apoptosis. These prompted us to investigate whether Gal9 can have an anti-CML effect via signaling cascades distinct from the pathway utilized by Bcr-Abl TKIs or by other commonly utilized anti-cancer agents. Modified human Galectin-9 (hGal9) inhibits the proliferation of six CML-derived cell lines, BV173, KT-1, KCL22, K562, KBM5 and MYL, by inducing apoptosis at their IC50s from 17.5 to 164.9 nM, with the activation of caspases-3, -4, - 8 and -9. The addition of 25 mM lactose prevented the growth inhibitory effect by hGal9 in K562, indicating the essential role of beta-galactoside binding activity in the anti-CML activity of hGal9. Because hGal9 treatment caused upregulation of Noxa, a pro-apoptotic BH3-only protein of Bcl-2 family proteins, and Mcl-1, a member of anti-apoptotic Bcl-2 proteins, in CML cell lines, we next investigated the involvement of Bcl-2-regulated apoptosis pathway in cell death by hGal9. K562 sublines overexpressing Bcl-2, Bcl-XL, or Mcl-1, showed resistance to cell death induced by IM, but were as sensitive to hGal9-induced cell death as the parental cells, suggesting the involvement of a pathway which is independent of Bcl-2 family proteins. These results also indicate that the accumulation of Mcl-1 following hGal9 treatment does not hamper apoptotic induction by hGal9. Besides, the expression of dominant-negative FADD protein did not hamper the effect of hGal9, also indicating that the death receptor pathway was not responsible for apoptosis induced by hGal9. In contrast, our study revealed that hGal9 caused the upregulation of activating transcription factor 3 (ATF3), a member of the ATF/CREB family transcription factors, within 3 hour treatment, and the gene knockdown experiments using RNA interference (RNAi) technique revealed that ATF3 is the critical mediator for cell killing by hGal9. Moreover, RNAi experiments indicated that Noxa is one of the downstream effector molecules of ATF3, and that Noxa partly mediates cell death induction by hGal9. Bim, on the other hand, the BH3-only protein essential for apoptosis by Bcr-Abl TKIs, was not associated with hGal9-induced cell death. Considering that the activation of caspase-4 and caspase-8 is involved in ER stress-induced apoptosis, and that Noxa induction by ATF3 has been shown to be crucial in the cell death induced by inhibitors for ER-associated protein degradation, we suggest that hGal9-induced cell death may at least partly involve ER stress. ATF3-mediated cell death by hGal9 was not hampered by the absence of p53, the presence of mutant AblT315I, or by P-glycoprotein overexpression. In addition, hGal9 showed the additive growth inhibitory effect with IM on CML cell lines. Collectively, hGal9 is a candidate agent that may overcome various kinds of resistance to treatment for CML, and suggest that ATF3 may be a new target molecule for the development of new treatment modalities that can overcome resistance to currently available chemotherapeutics. Disclosures: No relevant conflicts of interest to declare.