ALBERT

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.