ALBERT

Description: Abstract 3772 Poster Board III-708 [Purpose] Bortezomib (BTZ), a proteasome inhibitor, has been introduced into the treatment of multiple myeloma (MM). It shows remarkable response against both relapsed/refractory and newly diagnosed MM. However, it is often encountered that BTZ treatment achieves very short duration of response and permits early drug resistance. Therefore, understanding the mechanisms underlying this drug resistance is necessary to develop novel treatments to overcome this problem. [Materials & Methods] We established two stable BTZ-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, whose IC50 values were respectively 24.7- and 16.6-fold higher than their parental cell lines, under continuous exposure to BTZ. Using these resistant cells, we investigated on their proteasome activity, the alteration of proteasome β5 subunit (PSMB5) gene, misfolded protein accumulation, endoplasmic reticulum (ER) stress, and apoptosis signals including BH3 only proteins in comparison with their parental cells at clinically achievable concentration of BTZ treatment. [Results & Discussion] No activation of caspase -3,-8, and -9 and BH3 only protein, Noxa, which were initially up-regulated in BTZ-treated cells, were noted in BTZ-resistant cells even in the presence of BTZ. These results indicate avoidance of fatal intracellular stress may block transcriptional activation of Noxa in resistant cells at an early phase after BTZ exposure. In gel shift assay detecting NF-kB-DNA complexes, BTZ-resistant cells maintained constitutive NF-kB activation, whereas their parental cells lost its activity in the presence of BTZ. In addition, cellular proteasome activities including chymotrypsin-like and caspase-like activity were markedly inhibited by BTZ treatment in parental cells, and moderately also in BTZ-resistant cells, when detected by fluorogenic substrates specific for each proteasome activity. While time-dependent accumulation of ubiquitinated proteins was prevented only in BTZ-resistant cells, but not in their parental cells after BTZ exposure. Resistance was partly explained by the presence of a unique point mutation, G322A, in the gene encoding PSMB5 in both BTZ-resistant cell lines, which substituted Thr for Ala at the codon 49 in amino acid level. This constitution has been reported to gives rise to the conformational change of BTZ-binding pocket in β5 subunit, which results in partial disruption of the contact between BTZ and chymotrypsin-like active site. Furthermore, BTZ-resistant and parental MM cells had nearly equal expression of cytoplasmic and ER chaperons, however, only BTZ-resistant cells could prevent misfolded protein accumulation and therefore avoid fatal ER stress represented as activation of CHOP and of caspase-4, -12 after BTZ treatment. [Conclusion] Two kinds of stable BTZ-resistant MM cell lines were established, which acquired the unique point mutation (G322A) in BTZ-binding pocket of PSMB5, prevented the accumulation of misfolded proteins probably via reduced affinity of 26S proteasome to BTZ and avoided the development of catastrophic ER stress unlike their parental cells. These cell lines will provide better understanding of the underlying mechanisms of the BTZ-resistance, and will lead to the development of novel treatment strategies for overcoming BTZ-resistance in the patients with MM. Disclosures: Iida: JANSSEN PHARMACEUTICAL: Honoraria; KYOWA KIRIN: Research Funding. Nakashima:KYOWA KIRIN: Employment. Miyazaki:KYOWA KIRIN: Employment. Shiotsu:KYOWA KIRIN: Employment.

Description: Abstract 1342 In mature lymphoid malignancies including T-cell lymphoma and multiple myeloma (MM), aberrant acetylation status has been strongly linked to their tumorigenesis. Thus, the modulation of acetylation through targeting histone deacetylases (HDACs) is considered to be a viable therapeutic strategy. Vorinostat (SAHA) is the first HDAC inhibitor approved by the FDA for the patients with cutaneous T-cell lymphoma (CTCL). Anti-tumor activity of SAHA against CTCL, adult T-cell leukemia/lymphoma (ATLL), and MM cell lines was examined. Most of these cells were found to be sensitive to this drug at IC50 of less than 1–2uM. For further clarification of its mechanism of action, we established five SAHA-resistant cell lines consisting of 3 CTCL and 2 MM using dose stepwise increase method over six months. IC50 of the SAHA-resistant cells was 4-to 14-fold higher than that of their parental cells. These cell lines also showed cross-resistance of 2.8- to 17.5-fold against another pan-HDAC inhibitor, panobinostat (LBH589). Regarding HDAC activity, it was greatly inhibited by SAHA in parental cells, whereas it was only partly inhibited in SAHA-resistant cells. That is, SAHA-resistant cells have lost apart of the HDAC inhibiting function caused by SAHA. Moreover, SAHA-resistant cells showed higher anti-apoptosis ability when exposed with SAHA than their parental cells with acetylation status of histone H3 being remained low. Next, we performed microarray analysis to compare expression levels of various HDACs and other related genes between parental and SAHA-resistant cells. Results indicated that the expression level of HDAC3 being obviously low in resistant cells among various HDACs, which was also confirmed by real-time PCR. In line with mRNA analysis, protein level of HDAC3 was also decreased in SAHA-resistant cells compared with their parental cells, while other HDAC expression remained unchanged. We assumed that HDAC3 could be a main target of SAHA. To examine this possibility, we established both HDAC3 knocked-down and over-expressing cell lines, and examined the sensitivity of these cells to SAHA. HDAC3 knocked-down cells showed obviously SAHA-resistant feature in MTS assay, however, HDAC3 over-expressing cells showed higher sensitivity to SAHA. Knocking out other HDACs (1, 2 and 8) in parental cell lines did not change the sensitivity to SAHA. Thus, our results suggest that SAHA induced apoptosis depends on the inhibition of HDAC3. To search for other possible mechanisms, we screened for the mutations in HDAC2, 3, 4 and 8, but did not find them. Finally, we supposed that HDAC3 expression was epigenetically silenced by promoter methylation in SAHA resistant cells, and attempted to restore HDAC3 expression in the presence of 5-azacytidine, a DNA demethylase. We incubated SAHA-resistant cells with non-toxic levels of 5-azacytidine (4uM) for 9 days, and confirmed that HDAC3 expression was restored during 6–9 days after exposure. When HDAC3 expression being restored, the resistant cells showed higher sensitivity to SAHA. It suggests that hyper-methylation of promoter sequences of HDAC3 contributed to the mechanism of SAHA-resistance. From these results, we conclude that anti-tumor effect of HDAC inhibitors depends on the expression level of HDCA3 in mature lymphoid malignancies, and HDAC3 might provide a useful biomarker for identifying favorable response to HDAC inhibitors, and the overcoming the resistance of HDAC inhibitors. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5034 Introduction: The IRE1α-XBP1 pathway, a key component of the endoplasmic reticulum (ER) stress response, is considered to be a critical regulator for survival of multiple myeloma (MM) cells. Because of the production of abundant immunoglobulins and cytokines, MM cells need to survive under chronic ER stress. In addition, MM cells are located in the bone marrow milieu, which is usually considered hypoxic compared to other organs. Therefore, MM cells need to possess mechanisms to protect against ER stress. Among the unfolded protein responses in MM cells, the IRE1α-XBP1 pathway has been implicated in the proliferation and survival of MM cells to a greater extent than in those of monoclonal gammopathy of undetermined significance or normal plasma cells. It has been reported to be a prognostic factor and could be a target for immunotherapy or chemotherapy. Based on previous reports, it is proposed that an inhibitor of IRE1α-XBP1 activation should be a potent therapeutic agent for MM. Therefore, the availability of small molecule inhibitors targeting this pathway would offer a new therapeutic strategy for MM. Here, we screened small molecule inhibitors of ER stress-induced XBP1 activation, and identified toyocamycin from a culture broth of an Actinomycete strain. Materials & Methods: First, we evaluated the mechanism of toyocamycin-induced inhibition of IREα activity, with focused on its kinase activity, endonuclease activity, and other unfolded protein responses. Next, the activity of toyocamycin was evaluated on MM cell lines and other tumor cells about IRE1α activity and cytotoxicity. Similarly, 9 primary MM cells were tested. Finally, the in vivo efficacy of toyocamycin was evaluated in a human MM xenograft model. Results & Discussion: Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting ATF6 and PERK activation. Furthermore, although toyocamycin was unable to inhibit IRE1 a phosphorylation, it prevented IRE1α-induced XBP1 mRNA cleavage in vitro. Thus, toyocamycin is an inhibitor of IRE1α-induced XBP1 mRNA cleavage. Next, we examined the effect of toyocamycin on MM cells. Most MM cell lines have activated XBP1 protein expression, represented as the overexpression of spliced XBP1 isoform, whereas non-MM cells including other hematological and solid tumor cells have little activation of XBP1. Toyocamycin inhibited constitutive activation of XBP1 in MM cell lines without affecting IRE1α phosphorylation. This inhibition occurred within 6 hours after exposure to 30 nM toyocamycin. We then evaluated the growth inhibitory effect of toyocamycin on 7 MM cell lines with high spliced-XBP1 expression, 3 MM cell lines with low spliced-XBP1 expression, and 4 non-MM cell lines as assessed by MTS assay. All MM cells with high spliced-XBP1 expression showed remarkable decline in cellular viability at 30 nM or higher concentrations of toyocamycin than other MM cells with low spliced-XBP1 expression, and non-MM cell lines showed little reduction in cellular viability. MM cell lines expressing high spliced-XBP1 showed robust dose-dependent apoptosis after exposure to various concentrations of toyocamycin for 24 hours, as assessed by the number of Annexin V-positive cells. Toyocamycin also induces marked apoptosis on two bortezomib (BTZ)-resistant MM cells at nM concentration. It also inhibited constitutive activation of XBP1 expression in primary MM cells derived from patients, showing dose-dependent reduced viability without any cytotoxicity to PBMCs from healthy donors. Toyocamycin also showed synergistic effects with bortezomib, and induced apoptosis of primary MM cells from patients including bortezomib-resistant cases at nano-molar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human MM, and showed enhanced growth inhibition when combined with bortezomib. Taken together, we found that adenosine analog toyocamycin has a potent IRE1α-XBP1 inhibitory effect on MM cells with excessive ER-stress. It triggers dose-dependent apoptosis in MM cells. These results suggest toyocamycin can be a lead compound for developing novel anti-MM therapy, and also provide a preclinical rationale for conducting clinical trials using toyocamycin or other adenosine analog alone or in combination with BTZ for treating MM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 423 Purpose: Engineering the Fc region of monoclonal antibodies (mAb) to enhance effector functions is likely to be a promising approach for next-generation mAb therapy. 113F, a complement-dependent cytotoxicity-(CDC)-enhancing variant of rituximab is such an antibody (Cancer Res 2008;68:3863-72). The first of the three major aims of the present study was to identify tumor-associated factors influencing tumor susceptibility to 113F-induced CDC, especially focusing on complement regulatory proteins (CRPs). The second aim of the present study was to compare 113F-induced CDC against primary lymphoma cells with rituximab in vitro. A current crucial problem in the field of human immunotherapy research, including antibody therapy, is the lack of suitable small animal models for in vivo preclinical testing. With respect to antibody-dependent cellular cytotoxicity (ADCC), we have established a human tumor-bearing mouse model, using NOD/Shi-scid, IL-2Rγnull (NOG) mice as recipients, in which human immune cells are engrafted and mediate ADCC (Cancer Immunol Immunother 2009;58:1195-206, J Immunol. 2009;183:4782-91). On the other hand, there is no mouse model in which human CDC can be evaluated. Thus, the third and final aim of the present study was to establish a mouse model in which it is human complement that mediates CDC against human tumor cells.Using this model, we assessed the therapeutic potential of 113F in comparison with rituximab. Experimental Design: Rituximab- and 113F-induced human CDC was compared in vitro, and in vivo using NOG mice with human complement. Result: First, we determined that tumor-associated factors influencing tumor susceptibility to 113F-induced CDC included the quantity of CRPs such as CD55 and CD59 on the cell surface, as observed in rituximab-induced CDC. Second, we found that 113F mediated highly enhanced CDC against primary CD20-expressing lymphoma cells from patients, greater than rituximab. Finally, a novel human tumor-bearing mouse model has been developed in which human complement functions in CDC. NOG mouse serum is defective in its capacity to induce CDC against human cells, and endogenous immune cells from NOG mice are unable to mediate ADCC of therapeutic antibodies with an Fc region consisting of human IgG. Thus, we were able to evaluate purely human CDC without interference from endogenous mouse immune cells or complement-mediated mAb induced antitumor effects in this NOG mouse model. The present observation of significant therapeutic efficacy of rituximab together with pooled human serum (PHS) compared to rituximab with inactivated PHS indicated that human complement does function in rituximab-induced CDC in these mice in vivo. The finding of specific localization of human C1q on CD20-expressing tumor cell membranes indicated that human CDC indeed contributed to the antitumor effect in this model. In addition, enhanced therapeutic efficacy of 113F together with PHS compared to rituximab with PHS in this mouse model was observed in vivo. In the cell proliferation assay, viability of the target cell line was not affected by rituximab or 113F alone, and thus no significant difference between these mAbs was observed in vitro. Therefore, the present in vivo observation emphasizes the concept of this type of CDC-enhancing antibody. Furthermore, the more abundant, denser signals of C1q at the tumor cell membrane in these human serum-bearing mice receiving 113F compared with rituximab, are consistent with the Fc region of the former having a much higher C1q binding affinity than the latter. These findings are concordant with the present observation of the greater therapeutic efficacy of 113F compared to rituximab in vivo. Conclusion: This animal model overcomes the limitations of preclinical in vivo investigations of CDC caused by species incompatibilities between humans and mice. This model also makes it possible to reconstitute the human complement system during mAb-based immunotherapy and to perform more appropriate preclinical evaluations of novel therapeutic mAb which mediate CDC. In the present study, highly enhanced human CDC mediated by this type of CDC-enhancing mAb was demonstrated both in vitro and in a humanized mouse model in vivo. In the near future, the efficacy of the type of CDC-enhancing antibody described here will be established in planned clinical trials in humans. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Thymus and activation-regulated chemokine (TARC/CCL17) and macrophage derived chemokine (MDC/CCL22) are specific ligands of CC chemokine receptor 4 (CCR4). We have previously reported that tumor cells obtained from a large majority of patients with adult T-cell leukemia/lymphoma (ATLL) express CCR4 and that the extent of CCR4 expression is significantly associated with unfavorable prognosis and with skin involvement. We also reveal TARC/CCL17 and MDC/CCL22 secreted by tumor cells create a favorable environment for tumor escape from host immune defense withattract CCR4+ regulatory T cells to the tumor. Collectively, it is assumed that the interactions between CCR4 and its ligands play an important role for pathogenesis of ATLL. The goal of the present study was to clarify the clinical roles of individual CCR4 ligands in ATLL patients. Material and Method: We compared the serum levels of CCR4 ligands (TARC/CCL17 and MDC/CCL22) and helper T-cell associated cytokines (IFN-g, TNF-a, IL-2, IL-4, IL- 5, IL-6, IL-10), in 103 ATLL patients, 40 asymptomatic HTLV-1 carriers, and 50 healthy adult volunteers. We also analyzed the associations between the serum concentration of individual CCR4 ligands and the clinical characteristics such as survival time among the ATLL patients. Result: Serum levels of TARC/CCL17 and MDC/CCL22 were significantly lower both in ATLL patients and asymptomatic HTLV-1 carriers than in healthy controls. The overall survivals were significantly shorter in ATLL patients with a high TARC/CCL17 level than in those with a low TARC/CCL17 level. Univariate cox proportional hazard analysis identified the 12 unfavorable prognostic factors including a high TARC/CCL17 level as well as a high IL-5 level, a high IL-6 level, and a high IL-10 level(Table). Multivariate analysis confirmed that a high TARC/CCL17 level was an independent and significant unfavorable prognostic factor among the ATLL patients (Table). Although a high MDC/CCL22 level was significantly associated with a high TARC/CCL17 level, serum MDC/CCL22 levels did not have prognostic significance in ATLL patients. Conclusion: Measurement of serum TARC/CCL17 levels is useful for predicting the prognosis and for determining a suitable treatment strategy for ATLL patients. Prognostic factors affecting overall survival in all ATLL patients. (n=103) Variable Unfavorable Unlvarlate Multivariate Hazard ratio (95%*CI P value Hazard ratio (95%*CI) P value *CI, confidence Interval TARC/CCL17 〉 285 pg/mL 1.638 (1.022–2 626) .0405 1.814 (1.109–2.969) .0177 IL-5 〉 1.7 pg/mL 2.307 (1.357–3.922) .0020 1.767 (1.008–3.096) .0468 IL-6 〉 0.7 pg/mL 1.956 (1.183–3.233) .0089 - IL-10 〉 2.6 pg/mL 2.192 (1.254–3.830) .0059 - Clinical subtype Acute, lymphoma 3.539(1.851–6.767) .0001 - PS 2–4 2 187 (1.372–3.488) .0010 - B symptoms Present 2.104 (1.279–3.461) .0034 1,729 (1.040–2.872) .0346 Hemoglobin 〈 100 g/dL 1.923 (1.030–3.592) .0401 - Albumin 〈 3.0 g/dL 1.854(1.029–3.338) .0397 - LDH 〉 2N 2.683(1.683–4.279) 〈 .0001 1.876(1.086–3.242) .0241 sIL-2R 〉 10,000 U/mL 3.232(1.896–5.507) 〈 .0001 1.956(1.024–3.738) .0423 Extranodal involvement ≥ 2 2.152 (1.352–3.426) .0012 -

Description: Purpose: ADCC is a major antitumor mechanism for the action of therapeutic monoclonal antibodies (mAbs) such as rituximab, trastuzumab and cetuximab. Therefore, a better understanding of ADCC will allow the development of novel, more effective treatment strategies, and may help overcome the resistance which can develop against the effects of the therapeutic mAbs. However, the tumor-associated factors which determine susceptibility to rituximab-induced ADCC have not been identified. In the present study, we focused on this issue, especially focused on the molecules expressed by the tumor cells that interact with NK cells, such as NKG2D ligands, because of the importance of NK cells for rituximab-induced ADCC. The aim of this study was to identify tumor associated factors which determine susceptibility to rituximab-induced ADCC. Experimental Design: 30 different CD20+ non-Hodgkin lymphoma (B-NHL) cell lines were phenotyped for characteristics of cell surface protein: expression levels of CD20, MHC class I, NKG2D ligands (ULBP1-3, MICA, and MICB), CD48 (2B4 ligands), HLA-G, cathepsin B, and complement inhibitors (CD46, CD55, and CD59), and the influence thereof on susceptibility to rituximab-induced ADCC was established. Result: The degree of rituximab-induced ADCC were correlate with the expression levels of CD20 and ULBP1-3, and inversely correlate with the expression levels of MHC class I among 30 different CD20+ B-NHL cell lines. The importance of the ULBPs was confirmed using antibody blockade. In the presence of blocking mAb to ULBP1, 2 or 3, a decrease of rituximab-induced ADCCs against B-NHL cell lines were observed. In addition, the present study clearly identified the key mechanism of rituximab-induced ADCC as antibody-dependent target-specific cytotoxicity mediated by highly activated NK cells. Strong NK cells activation was due to the combination of Fc„dR stimulation via the Fc portion of rituximab, together with stimulation of activating NK cell receptors via their ligands expressed on the tumor cells, particularly ULBPs, which occurred in a robustly synergistic manner. Conclusions: Tumor cell susceptibility to rituximab-induced ADCC was determined by three major tumor-associated factors: the amount of the target molecule, CD20; the amount of the ligands for inhibitory killer Ig-like receptors, MHC class I; and the amounts of some of the NKG2D ligands, especially ULBP1-3. This is the first report to show the importance of ULBPs on tumor cells for rituximab-induced ADCC. The ULBPs could be valuable diagnostic biological makers and significant targets for immunotherapy to improve efficacy not only of rituximab but also of other therapeutic mAbs. Figure. Correlations between the expression of the CD20, MHC class I, and NKG2D ligands ULBP1-3, and rituximab-induced ADCC in B-NHL cell lines. ADCC in the presence of 10μg/mL rituximab against 30 different CD20+ B-NHL cell lines determined by 51Cr release assays. Y-axis: % lysis. X-axis: MFI ratio of CD20 (upper left panel), MHC class I (upper right panel), ULBP-1 (lower left panel), ULBP-2 (lower middle panel), and ULBP-3 (lower right panel). Each dot plot in each panel represents one cell line. The coefficients and P values assessed by Spearman rank correlation coefficient testing are indicated in each panel. Figure. Correlations between the expression of the CD20, MHC class I, and NKG2D ligands ULBP1-3, and rituximab-induced ADCC in B-NHL cell lines. ADCC in the presence of 10μg/mL rituximab against 30 different CD20+ B-NHL cell lines determined by 51Cr release assays. Y-axis: % lysis. X-axis: MFI ratio of CD20 (upper left panel), MHC class I (upper right panel), ULBP-1 (lower left panel), ULBP-2 (lower middle panel), and ULBP-3 (lower right panel). Each dot plot in each panel represents one cell line. The coefficients and P values assessed by Spearman rank correlation coefficient testing are indicated in each panel.