ALBERT

Description: Abstract 1839 The implementation of hematopoietic stem cell transplantation and new agents such as bortezomib, thalidomide, and lenalidomide (Len) has dramatically improved survival in patients with multiple myeloma (MM). However, most MM patients eventually relapse after achieving of complete response. The existence of cancer stem cells is suggested to cause the relapse and considered as an important therapeutic target. We have demonstrated that HM1.24 (CD317) is selectively over-expressed on neoplastic plasma cells and that a defucosylated humanized monoclonal antibody (mAb) specific to HM1.24 (YB-AHM) is able to induce potent antibody-dependent cellular cytotoxicity (ADCC) against human MM cells in the presence of human effector cells. On the other hand, Len, an immunomodulatory drug, has been shown to activate NK cells to enhance their ADCC activity. Recently, we have reported that “side population (SP)” cells expelling a Hoechst 33342 dye represent a fraction with cancer stem cell-like property in MM cells. Moreover, we have found that MM cancer stem-like cells over-express pluripotency-associated transcription factors such as Sox2 and Nanog, and that these factors are useful for evaluating the potential of MM cancer stem-like cells. Here, we evaluated the efficacy of the combinatory therapy of YB-AHM and Len on MM cancer stem-like cells. We first examined the expression levels of the target molecule, HM1.24 on SP fraction of MM cells. Although SP cells expressed higher levels of ABC transporter ABCG2 compared with main population (MP) cells, HM1.24 was highly expressed in both SP and MP fractions in MM cells. We next examined the inhibitory effect of YB-AHM and Len on clonogenic activity of MM cell lines. RPMI 8226, U266, and OPM-2 cells were pre-incubated with YB-AHM (0.1 μg/ml) and Len (3 μM)-stimulated peripheral blood mononuclear cells (PBMCs) at an effector/target (E/T) ratio of 10 for 4 hours, and then were cultured into H4034 methylcellulose medium. Colony formation of MM cells was examined after 14 days. Treatment with YB-AHM and Len-stimulated PBMCs significantly suppressed the colony formation of MM cell lines compared with the no-treatment group (4±5 vs 62±2 colonies/well in RPMI 8226, p

Description: Abstract 2799 Poster Board II-775 Multiple myeloma (MM) remains an incurable disease despite high overall response rates induced by combination therapy of chemotherapeutic drugs and new agents such as thalidomide, lenalidomide, and bortezomib. Recently, the existence of cancer stem cells is proposed for several tumors including MM, and such cells are considered as an important target for curative therapy. The side population (SP) cells are identified by their ability to efflux Hoechst 33342 dye, which represent a small fraction with stem cell properties. Our previous studies have demonstrated that MM cells expressed HLA class I at high levels than normal hematopoietic cells, and that antibodies against HLA class I specifically induced MM cell death by Rho-mediated actin aggregation. In this study, we characterized SP fraction in MM cell lines (RPMI 8226, U266, and MM.1S) and primary MM cells (n=3) by flow cytometry, and investigated the efficacy of chemotherapeutic drugs as well as a recombinant single-chain Fv diabody specific to HLA class I (C3B3-DB, Chugai Pharmaceutical Co., Ltd, Tokyo, Japan). MM cell lines and primary MM cells contained a distinct fraction of SP cells ranging from 0.01% to 0.6% of the gated cells, which was confirmed by disappearance after treatment with verapamil. Treatment with melphalan (10 μM, 48 hours) decreased the percentage of non-SP cells (16.7% to 3.9%) but not of SP cells (0.6% to 0.7%) in RPMI 8226. In contrast, treatment with C3B3-DB (1 μg/ml, 48 hours) caused a significant reduction in both non-SP cells (16.7% to 4.0%) and SP cells (0.6% to 0.3%) in RPMI 8226. Similar results were observed in primary MM cells enriched from patient bone marrow cells by negative selection with antibody cocktail. Next, we isolated SP cells and non-SP cells in RPMI 8226 using a cell sorter, and characterized in detail. SP cells exhibited elevated levels of ABCG2 and low levels of CD138 compared with non-SP cells, but HLA class I was expressed at high levels in both SP and non-SP cells by flow cytometry. Annexin/PI assay showed that SP cells were 1.5- and 2.0-fold more resistant to melphalan and bortezomib than non-SP cells. Whereas both SP cells and non-SP cells showed similar sensitivity to C3B3-DB. Methylcellulose colony-forming assay showed that SP cells have a higher potential for colony formation (numbers of colonies, 13.0±1.0) than non-SP cells (1.3±0.6), and the colony formation of SP cells was significantly inhibited by C3B3-DB (0.7±0.6, p

Description: Abstract 135 Malignant cells aberrantly up-regulate ATP-binding cassette (ABC) transporters and ATP-dependent drug efflux pumps, which causes drug resistance. Because the activity of TCA cycle in mitochondria is suppressed through oncogenic alterations including the mutation of p53, ATP is largely produced by aerobic glycolytic metabolism enhanced in malignant cells (the Warburg effect). Hexokinase II (HKII), a key enzyme of glycolysis, is widely over-expressed in cancer cells. However, HKII levels and its roles in ATP production and ATP-dependent cellular process have not been elucidated in hematopoietic malignant cells including myeloma (MM) cells. In the present study, we therefore explored the expression levels of HKII and the effect of HKII inhibition on ABC transporter activity as well as the susceptibility to chemotherapeutic agents in MM cells. HKII protein was constitutively expressed at higher level in MM cells than in normal peripheral blood mononuclear cells (PBMCs). The expression level of HKII in MM cells was further up-regulated when cocultured with osteoclasts. 3-bromopyruvate (3BrPA), an inhibitor of HKII promptly inhibited glycolysis and substantially suppressed ATP production in MM cells but not in normal PBMCs. 3BrPA preferentially induced cell death in MM cells but not in normal hematopoietic cells in bone marrow samples from patients with MM, suggesting that HKII is a potential target for treatment of MM cells. We next examined the effects of 3BrPA on ABC transporter activity in RPMI 8226 (MM) and KG-1 (acute myeloid leukemia) cells which are aberrantly over-expressed breast cancer resistance protein (ABCG2) and P-glycoprotein (ABCB1), respectively. After passive incorporation of auto-fluorescence emitting daunorubicin, these cells were washed and incubated for 2 hours without daunorubicin in the absence or presence of 3BrPA, and then the intracellular daunorubicin levels were measured by flow cytometry. Treatment with 3BrPA markedly enhanced the accumulation and retention of daunorubicin in both cells. Therefore, inhibition of HKII by 3BrPA appears to be able to effectively deplete intracellular ATP production and suppress ABC transporter activity. Importantly, 3BrPA restored cytotoxic effects of doxorubicin and daunorubicin on RPMI 8226 and KG-1 cells. We next focused on “Side population (SP)” which is regarded as a highly drug-resistant fraction with enhanced ABC transporter activity, and contains clonogenic or tumor-initiating cells. SP cells isolated from RPMI 8226 cells exhibited higher levels of glycolysis with an increased expression of genes involved in the glycolytic pathway. Treatment with 3BrPA abolished Hoechst 33342 exclusion in the SP cells, and clonogenic capacity in RPMI 8226 and KG-1 cells. Furthermore, 3BrPA cooperatively suppressed subcutaneous tumor growth in combination with doxorubicin in RPMI 8226-implanted mice. These results demonstrate that HKII is a tumor specific target for treatment of MM and that inhibition of HKII effectively depletes ATP and inactivate ABC transporters to overcome drug resistance. ABC transporter-expressing SP cells with enhanced glycolysis and clonogenic cells with high proliferative potential are suggested to be a good target of the inhibition of glycolysis. These findings highlight a novel role of enhanced glycolysis in malignant cells in tumor growth and drug resistance, and relevance to anti-cancer strategies attempting to target unique metabolic pathway of cancer cells. Disclosures: No relevant conflicts of interest to declare.