ALBERT

Description: Myeloma (MM) cells grow and expand almost exclusively in the bone marrow while creating a cellular microenvironment suitable for MM cell growth and survival (MM niche). In pursuing the molecular mechanisms whereby MM cells gain drug resistance in the “MM niche”, we have found that the serine/threonine kinase Pim-2 is constitutively over-expressed in MM cells, and further up-regulated by co-cultures with bone marrow stromal cells (BMSCs) as well as osteoclasts (Leukemia, 2011), and that Pim-2 is an important therapeutic target in MM for the progression of MM tumor and bone disease (Leukemia, 2014). The ABC transporter BCRP is preferentially expressed in drug resistant MM cells as well as in MM progenitors or stem cells. BCRP has been demonstrated to be phosphorylated by Pim kinases to trigger its dimerization and function; Pim inhibition may suppress the BCRP function to sensitize BCRP-expressing MM cells to chemotherapeutic agents. In the present study we therefore explored whether Pim inhibition is able to target and impair BCRP-expressing drug-resistant MM cells and MM progenitors. We analyzed an ABC transporter activity in BCRP-expressing RPMI8226 and KMS11 cells by intracellular accumulation and retention of BCRP substrates with auto-fluorescence emission, mitoxantrone and doxorubicin, in flow cytometry. Treatment with Pim inhibitors, SMI-16a or SMI-4a, increased the incorporation of these drugs into the MM cells and enhanced their subsequent intracellular retention after 6-hour incubation without these drugs, although BCRP expression on their surface was only marginally affected by the Pim inhibition. Interestingly, acidic conditions up-regulated Pim-2 expression while reducing the accumulation and retention of these drugs in BCRP-expressing RPMI8226 and KMS11 cells. However, the Pim inhibitors efficaciously restored the drug accumulation and retention reduced by extracellular acidification, and enhanced the cytotoxic activity of the BCRP substrate doxorubicin against RPMI8226 cells rather preferentially in acidic conditions. Furthermore, the Pim inhibition minimized the sizes of “side populations”, highly drug-resistant fractions with enhanced BCRP activity, and the ability of colony formation in RPMI8226 and KMS11 cells, which was more marked in acidic conditions. We previously demonstrated the in vivo effects of the Pim inhibitors in human INA-6 cell-bearing SCID-rab MM models and syngeneic mouse MM models with an intra-tibial inoculation of 5TGM1 MM cells (Leukemia, 2014). To further examine the acid-tropism of anti-tumorigenic activity of Pim inhibition, we pretreated murine 5TGM1 MM cells in vitro with or without SMI16a at pH6.8 for 24 hours, and transplanted to the tibiae in mice the same numbers of viable MM cells remaining in each treatment group. Treatment with SMI16a at pH6.8 almost completely abrogated in vivo tumorigenic capacity of 5TGM1 cells, while MM cells without the treatment rapidly grew and expanded in and outside of the tibiae, suggesting targeting clonogenic MM cells by Pim inhibition preferentially in acidic conditions. Taken together, Pim-2 may become an important therapeutic target of drug-resistant BCRP-expressing MM cells and their progenitors which appear to gain more drug resistance in acidic bone lesions. Combinatory treatment with Pim inhibitors warrants further study to overcome drug resistance in MM cells, including their tumorigenic cancer stem cells. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 454 Multiple myeloma (MM) develops and expands in the bone marrow, and causes devastating bone destruction by enhancing osteoclastic bone resorption in their close vicinity. In MM bone lesions, thus induced osteoclasts (OCs) in turn enhance MM cell growth and survival, thereby forming a vicious cycle between the progression of bone destruction and MM tumor expansion. Such cellular interactions create an acidic milieu not only through acids produced by OCs but also through a large amount of lactate by proliferating tumor cells (Warburg effect). Reveromycin A (RM-A), a small microbial metabolite, preferentially induces cellular apoptosis in an acidic milieu, and draws considerable attention as a novel anti-resorptive agent. In the present study, we explored whether an acidic condition induced by MM-OC interaction affects MM expansion and whether RM-A targets not only OCs but also such an acidic microenvironment to regress tumor expansion in MM. INA6 and RPMI8226 MM cells potently enhanced osteoclastogenesis and osteoclastic pit formation when cocultured with rabbit bone marrow cells on bone slices. Notably, large multinucleated OCs were almost completely disappeared and pit formation on bone slices was abolished upon the treatment with RM-A at concentrations as low as 100nM. The cocultures with rabbit bone marrow cells stimulated INA6 MM cell growth; RM-A at 1microM was however able to substantially decrease the MM cell viability in the cocultures after 12 hours, although RM-A at this concentration did not affect MM cell growth when MM cells were cultured alone at pH7.4. The suppression of INA6 MM cell viability by RM-A was obviously more potent than that under bisphosphonate treatment in which mature OCs and pits on bone slices similarly decreased in number, suggesting that the anti-MM effects of RM-A is not merely due to depletion of mature OCs. Blockade of acid release by the proton pump inhibitor concanamycin A abolished such RM-A effects. Because an acidic microenvironment increases cell permeability of RM-A to cause apoptosis, it is plausible that a highly acidic milieu created by OC-MM interaction allows RM-A to act on nearby MM cells as well as OCs. In order to clarify a role of tumor acidity in RM-A-triggered cell death, we examined the effects of RM-A on MM cell growth upon acidification with lactic acid. When lactic acid was added to media to adjust their pH to be 7.0 and 6.75, the growth of INA6 and RPMI8226 MM cells was enhanced up to 150 and 120%, respectively, after 24 hours compared to that at pH7.4. However, RM-A at 1microM induced cell death in these MM cells at pH7.0 (60-70% reduction of alive MM cells compared to those at pH7.4) and at pH6.75 (〉90%), suggesting cytocidal effects of RM-A on lactate-producing MM cells densely proliferated in an acidic milieu. Because metoformin, anti-diabetic agent, up-regulates lactate production through stimulation of glycolysis, we next examined the effects of RM-A on MM cells in combination with metoformin. Metoformin dose-dependently enhanced lactate production by MM cells to decrease pH in their culture media over time; RM-A at 1microM showed potent cytotoxic effects on MM cells upon 24-hour preceded treatment with metoformin at 5 mM even when MM cells were started to be cultured at pH7.4, suggesting induction of anti-MM activity of RM-A with metoformin. Finally, in vivo RM-A effects were studied using INA6 MM cell-bearing SCID-rab mice. We injected RM-A sc at 4mg/kg twice daily for 18 days to the mice after confirming MM cell growth at 4 weeks after the MM cell inoculation. The RM-A treatment substantially decreased osteolytic lesions in X-ray and microCT images and MM tumor area in bone sections along with a reduction of INA6 cell-derived human soluble IL-6 receptor levels in mouse sera as a marker of MM tumor burden. These results collectively suggest that acidic microenvironment produced by MM-OC interaction enhances MM tumor progression but can trigger cytotoxic effects of RM-A on MM cells besides acid-producing OCs. RM-A may become a candidate for a novel therapeutic agent against MM with extensive bone resorption. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3025 Multiple myeloma (MM) remains incurable by the present therapeutic modalities, leading to the idea to develop various forms of immunotherapy. γδT cells are important effectors in the first-line defense against infections and tumors, and play a critical role in host defense and tumor surveillance. Aminobisphosphonates can effectively expand human γδT cells in combination with IL-2. Because expanded γδT cells have been demonstrated to exert potent anti-MM effects in vitro, γδT cell-based immunotherapy has attracted considerable attention as a novel treatment against MM. However, the clinical efficacy of γδT cell-based immunotherapy has been limited in patients with MM. We previously demonstrated that bone marrow stromal cells (BMSCs) blunt γδT cell activity and thus protect MM cells. To improve the therapeutic efficacy of γδT cells in vivo, therefore, we need to develop a maneuver to restore the anti-MM activity of γδT cells in the bone marrow microenvironment in which MM cells reside. Lenalidomide (LEN), a novel immunomodulatory anti-MM agent, shows pivotal anti-MM activity by targeting immune cells as well as MM cells and their surrounding cells in the bone marrow microenvironment. The present study was undertaken to further clarify the role of the bone marrow microenvironment in MM in the cytotoxic activity of γδT cells against MM cells and to determine whether LEN is able to modulate γδT cell activity and restore its anti-MM effects in the bone marrow. When γδT cells expanded with zoledronic acid (Zol) and IL-2 were cocultured with peripheral blood mononuclear cells (PBMC)-derived OCs or MM cell lines, γδT cells almost completely destroyed both types of cells, suggesting the susceptibility of OCs as well as MM cells to γδT cells. However, the cytotoxic activity of γδT cells against MM cells was markedly attenuated in the presence of BMSCs but not OCs. γδT cells expanded with Zol and IL-2 up-regulated their surface expression of LFA-1, CD26, and the molecules associated with NK or cytotoxic T cell activation including NKG2D and DNAX accessory molecule-1 (DNAM-1; CD226). Among these molecules, stromal cells markedly down-regulated DNAM-1 expression along with a decrease in interferon-γ production by γδT cells. DNAM-1 is regarded as an essential factor in NK cell-mediated cytotoxicity against various cancers. MM cells constitutively expressed DNAM-1 ligands, poliovirus receptor (CD155) and nectin-2; treatment with a blocking antibody against DNAM-1 substantially reduced the cytotoxic activity of γδT cells against MM cells, suggesting a critical role of DNAM-1 in the anti-MM effects of γδT cells. Interestingly, when γδT cells were cocultured with BMSCs in the presence of clinically relevant doses of LEN (1 microM) and Zol (0.1-1 microM), the suppression of interferon-γ production and DNAM-1 expression of γδT cells by BMSCs were substantially reduced. Moreover, such γδT cells exerted potent anti-MM effects, suggesting antagonism of the suppressive effects of BMSCs on γδT cell activity and its anti-MM effects. LEN (0.1-10 microM) and Zol (0.1-5 microM) in combination were able to substantially expand γδT cells from PBMCs in the absence of IL-2. These expanded γδT cells expressed the activation markers LFA-1, CD26, NKG2D and DNAM-1 at levels similar to those in γδT cells expanded by Zol and IL-2. However, LEN alone did not show any significant effects on γδT cell expansion and activation, suggesting a costimulatory role of LEN on Zol-primed γδT cells. These results collectively suggest that MM cells evade attack by γδT cells in the bone marrow through attenuation of γδT cell activity by BMSCs, and that LEN and Zol in combination may restore γδT cell activity and its anti-MM effects to improve the efficacy of γδT cell-based immunotherapy against MM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5104 Bortezomib (BTZ) is widely used in the treatment of myeloma (MM) with marked response rates in both relapsed/refractory as well as newly diagnosed MM. However, significant numbers of patients still remain outside benefit of the BTZ treatment; and various combinatory treatments with BTZ have been implemented to improve BTZ's anti-MM effects. On the other hand, immunotherapies seem attractive for yet incurable malignancies by chemotherapeutic agents such as MM and their clinical application has been studied. One such approach is a TNF-related apoptosis-inducing ligand (TRAIL)-mediated immunotherapy. In the present study, we therefore explored the role of BTZ on TRAIL receptor editing and its downstream signaling with special reference to endoplasmic reticulum (ER) stress and the cytotoxic effects of BTZ and anti-TRAIL receptor agonistic antibody in combination on MM cells. Most MM cells expressed DR4 but weakly DR5, while normal peripheral blood mononuclear cells expressed neither DR4 nor DR5. BTZ at 10 nM markedly up-regulated the surface levels of DR5 and its mRNA expression but not those of DR4 in MM cell lines and primary MM cells. Furthermore, BTZ decreased the levels of c-FLIP, an inhibitor of DISC, along with activation of caspase-8 and caspase-3, suggesting potentiation of the DR-mediated extrinsic apoptotic pathway. Consistently, BTZ and anti-DR5 agonistic antibody cooperatively enhanced the cytotoxicity against MM cells. BTZ induced phosphorylation of eIF2alpha, ATF4 and CHOP, along with disappearance of anti-apoptotic proteins including Mcl-1 in MM cells, suggesting the enhancement of ER stress and subsequent suppression of protein translation by BTZ. However, such induction of ER stress by BTZ was not observed in BTZ-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, with a point mutation in BTZ-binding proteasome beta5 subunit (Ri et al. Leukemia 2010). In KMS-11/BTZ and OPM-2/BTZ, surface protein as well as mRNA levels of DR5 were not up-regulated by BTZ, suggesting a critical role of ER stress in up-regulation of DR5 expression by BTZ. Because DR5 expression has been shown to be transcriptionally up-regulated by CHOP, the up-regulation of DR5 mRNA and protein in MM cells by BTZ is suggested to be at least in part due to CHOP induced by BTZ-mediated ER stress. Although BTZ exerts its anti-MM effects through induction of ER stress, the present study demonstrates that induction of ER stress by BTZ is also able to sensitize MM cells to TRAIL-mediated immunotherapy. Therefore, the combination of BTZ and TRAIL-mediated immunotherapy is warranted for further study. Disclosures: No relevant conflicts of interest to declare.

Description: Devastating bone destruction in multiple myeloma (MM) still remains a significant clinical problem. In pursuing factors responsible for MM tumor expansion and bone destruction, we found that the serine/threonine kinase Pim-2 is constitutively over-expressed as an anti-apoptotic mediator, and further up-regulated in MM cells when cocultured with bone marrow stromal cells or osteoclasts (OCs) (Leukemia, 2011). We also demonstrated that Pim inhibition is able to induce bone formation while suppressing MM tumor growth (Leukemia, 2015). However, the impact of Pim inhibition on MM-induced bone resorption remains unknown. Therefore, the present study was undertaken to clarify the role of Pim-2 in osteoclastogenesis enhanced in MM and the therapeutic effects of Pim inhibition on mutual interaction between MM cells and OCs. Pim-2 was highly expressed almost exclusively in cathepsin K-positive mature OCs on the surface of bone but not in other bone marrow cells in normal mouse bone tissues. RANK ligand and TNF-α induced the expression of Pim-2 in monocytes and RAW264.7 preosteoclastic cells at mRNA and protein levels. Inhibitors of the classical NF-κB pathway, SN50 or IMG2001, abolished Pim-2 up-regulation in RAW264.7 cells by RANK ligand or TNF-α, while Pim inhibition marginally affected the nuclear translocation of NF-κB subunits, p50 and p65, as well as the promoter activity of NF-κB, suggesting Pim-2 up-regulation downstream of the NF-κB pathway. Pim-2 appeared to be up-regulated along with c-fos, NFATc1 and cathepsin K during osteoclastogenesis. The Pim inhibitor SMI-16a potently suppressed the RANK ligand-induced expression of c-fos, NFATc1 and cathepsin K in RAW264.7 cells, and abolished osteoclastogenesis and bone resorption enhanced by MM cell conditioned media on hydroxyapatite-coated dishes. Furthermore, the Pim inhibition was found to suppress [Ca2+ ]i oscillation and thereby nuclear translocation of NFATc1, a critical transcription factor for osteoclastogenesis. MM cells and acid-producing OCs are mutually interacted in bone lesions to enhance MM tumor growth and bone destruction while creating an acidic milieu, thereby forming a progressive vicious cycle. Pim-2 was also up-regulated in MM cells when cocultured with OCs as well as bone marrow stromal cells, and to lesser extent merely by acidic conditions. Interestingly, acidic conditions rather preferentially enhanced the cytotoxic effects of the Pim inhibitor SMI-16a on MM cells even in cocultures with OCs or bone marrow stromal cells. Finally, treatment with SMI-16a reduced OC numbers in bone lesions together with tumor reduction and the restoration of bone formation in mouse MM models with intra-tibial injection of murine 5TGM1 MM cells. These results collectively demonstrated that Pim-2 play a critical role in osteoclastogenesis and tumor growth in acidic bone lesions in MM, and further corroborated that Pim-2 is a pivotal therapeutic target for MM bone disease and tumor progression. Disclosures Abe: Novartis Pharma K.K.: Speakers Bureau; Takeda Pharmaceutical Company Limited: Research Funding; Kyowa Hakko Kirin Company, Limited: Research Funding; Astellas Pharma Inc.: Research Funding; Ono Pharmaceutical Co.,Ltd.: Research Funding; GlaxoSmithKline plc: Research Funding.

Description: Abstract 5011 Myeloma (MM) cells and osteoclasts (OCs) mutually interact in MM bone lesions to confer aggressiveness and drug resistance in MM cells along with the progression of bone destruction. The MM-OC interaction appears to create a highly acidic milieu in bone lesions by proton produced from activated OCs and lactate from proliferating MM cells (the Warburg effect). Tumor acidity is known to cause drug resistance in cancers. As suggested, acidic conditions or cocultures with OCs blunt the cytotoxic effects of anti-MM agents such as doxorubicin. Therefore, the development of innovative modalities is strongly needed to overcome the drug resistance in an acidic microenvironment in MM. We have found that the serine/threonine kinase Pim-2 is over-expressed in MM cells as an anti-apoptotic mediator, and further up-regulated to cause their aggressiveness and drug resistance when cocultured with bone marrow stromal cells or OCs (Leukemia, 2011). In the present study, we aim to clarify the role of Pim-2 in MM cell growth and survival in acidic conditions and the therapeutic impact of Pim inhibition on drug-resistant MM cells. The phosphorylation of 4E-BP1, a substrate of Pim-2, as well as Pim-2 expression were up-regulated in MM cells in media acidified by lactic acid or HCl; the Pim inhibitor SMI-16a preferentially induced MM cell death at pH 6. 8 or 6. 4 rather than at pH7. 4, suggesting Pim-2-dependent MM cell survival in an acidic milieu. In contrast to their quick death at pH 7. 4, substantial numbers of MM cells remained intact at pH6. 8 or lower upon treatment with doxorubicin. Interestingly, Pim inhibition enhanced the cytotoxic activity of doxorubicin against MM cells, which was more marked in acidic conditions. The ABC transporter BCRP is aberrantly over-expressed in drug-resistant MM cells. The intracellular levels of auto-fluorescence emitting doxorubicin and mitoxantron were reduced over time after their passive incorporation into BCRP-expressing RPMI8226 cells. However, the Pim inhibitor SMI-16a at 50 microM substantially restored the intracellular levels of these drugs; the intracellular retention of these drugs by the Pim inhibition was more prominent in acidic conditions, suggesting the correlation of BCRP function with Pim-2 up-regulation by acid. BCRP phosphorylation has been demonstrated to be essential for its functionality. Treatment with SMI-16a reduced BCRP phosphorylation in BCRP-expressing INA6 cells, suggesting Pim-dependent activation of BCRP. The reduction was more obvious at pH6. 8 than at pH7. 4. “Side population (SP)” is regarded as a highly drug-resistant fraction with enhanced BCRP activity, which is considered to contain clonogenic or tumor-initiating cells. The Pim inhibitor SMI-16a minimized the size of SP fractions in RPMI8226 and KMS11 cells; the reduction of SP fractions by the Pim inhibition was also more marked in acidic conditions. Collectively, these results suggest that Pim-2 plays an important role in MM cell survival and drug resistance in an acidic milieu, and that Pim-2 may become an important therapeutic target of MM cells which preferentially gain drug resistance in acidic bone lesions. Disclosures: Nakamura: Janssen Pharmaceutical K. K.: Honoraria. Abe:Janssen Pharmaceutical K. K.: Honoraria, Research Funding.