Search

The aurora kinases facilitate transit from G2 through cytokinesis and, thus, are targets in cancer therapy. Multiple myeloma (MM) is a malignancy characterized by genetic instability, suggesting a disruption of checkpoints that arrest cells at G2M when injury to the mitotic machinery occurs. Since deficient checkpoints would prevent cell cycle arrest and may render cells susceptible to apoptosis in mitosis and since aurora kinases are intermediaries in checkpoint pathways, we tested antimyeloma effects of 2 agents that inhibit aurora kinases. Both inhibited growth of MM lines and primary myeloma samples at nanomolar concentrations while having less of an effect on proliferating lymphocytes and hematopoietic cells. MM cells were not protected by IL-6 or activating mutations of Ras. Antimyeloma effects included induction of tetraploidy followed by apoptosis. Apoptosis correlated with inhibition of aurora activity as shown by reduction of histone 3B phosphorylation. Ectopic expression of aurora A protected MM cells against aurora inhibitors but had no effect on apoptosis induced by bortezomib. As expression of RHAMM in MM contributes to genetic instability, we tested effects of RHAMM. RHAMM overexpression enhanced sensitivity to apoptosis and RHAMM silencing decreased sensitivity. These results suggest potential for aurora kinase inhibitors in MM especially in patients in whom RHAMM is overexpressed.

We have previously reported that an anti-human transferrin receptor IgG3-avidin fusion protein (anti-hTfR IgG3-Av) inhibits the proliferation of an erythroleukemia-cell line. We have now found that anti-hTfR IgG3-Av also inhibits the proliferation of additional human malignant B and plasma cells. Anti-hTfR IgG3-Av induces internalization and rapid degradation of the TfR. These events can be reproduced in cells treated with anti-hTfR IgG3 cross-linked with a secondary Ab, suggesting that they result from increased TfR cross-linking. Confocal microscopy of cells treated with anti-hTfR IgG3-Av shows that the TfR is directed to an intracellular compartment expressing the lysosomal marker LAMP-1. The degradation of TfR is partially blocked by cysteine protease inhibitors. Furthermore, cells treated with anti-hTfR IgG3-Av exhibit mitochondrial depolarization and activation of caspases 9, 8, and 3. The mitochondrial damage and cell death can be prevented by iron supplementation, but cannot be fully blocked by a pan-caspase inhibitor. These results suggest that anti-hTfR IgG3-Av induces lethal iron deprivation, but the resulting cell death does not solely depend on caspase activation. This report provides insights into the mechanism of cell death induced by anti-TfR Abs such as anti-hTfR IgG3-Av, a molecule that may be useful in the treatment of B-cell malignancies such as multiple myeloma.

Abstract 2892 Multiple myeloma (MM) is an incurable hematological malignancy. Thalidomide, bortezomib and lenalidomide have emerged as highly active agents in the treatment of MM. Although initial clinical responses to drug therapy are achieved, a significant number of MM patients relapses and no longer responds to further treatments. Hence, the mechanism of resistance of MM to current therapeutic regimens remains elusive. It will be useful to identify prognostic markers that can predict the poor response of patients to therapies and to develop novel therapeutics for these patients. Our studies and those of others have recently identified a gene product, Raf-1 kinase inhibitory protein (RKIP) that has been shown to be involved in the regulation of tumor sensitivity to drugs and that has been found to be poorly expressed in different histological resistant tumors studied. RKIP is a member of the phosphatidylethanolamine-binding protein (PEBP) that functions as a potent inhibitor of different kinases of the Raf/MAPK (Raf-1 kinase) and NF-k B (TAK-1, NIK) activation pathways, and, thereby, antagonizing both cell survival and the expression of anti-apoptotic gene products. RKIP activity undergoes a post translational modification involving PKC–meditated phosphorylation at Ser 153 that inhibits its association with RAF/MAPK and NF-k B. The objective of this study was to examine the expression and function of RKIP and phospho-RKIP (p-RKIP) in MM and examined their clinical significance. MM cell lines and bone marrow-derived MM tissues were analyzed by both immunohistochemistry (IHC) and western blot for the expression of RKIP and p-RKIP. The antibody to RKIP recognizes both non-phospho and p-RKIP whereas the antibody against p-RKIP is specific. We demonstrate the unusual overexpression of RKIP in MM cell lines and tissues and the majority was in its inactive phosphorylated form. The expression of both forms in normal bone marrow was very low. Analysis of the ratio of RKIP/p-RKIP suggested its clinical significance wereby a higher ratio correlated with poor progression and a low ratio correlated with progression. The overexpression of p-RKIP suggested that it maintains the survival and the resistance of MM to cytotoxic drugs. We hypothesized that the dephosphorylation of RKIP may render RKIP active and modify the resistance to cytotoxic drugs. This hypothesis was tested in MM cell lines. Tumor cells were treated with the PKC inhibitor bisindolylmalemide (BIM) (5–10 μg/ml) for different times and complete inhibition of p-RKIP as detected as early as 4 h, while non phospho-RKIP was preserved, as detected by IHC and western blot. Treatment with BIM and followed with bortezomib (2.5-nM) resulted in significant cytotoxicity of the bortezomib-resistant MM cells. Overall, the findings demonstrate the unusual overexpression of p-RKIP in MM cells that plays an important role in survival and maintenance of tumor cell-resistance to cytotoxic drugs. The reversal of the active non-phosphorylated form of RKIP by the use of the specific PKC inhibitor rendered the tumor cells sensitive to bortezomib-induced cytotoxicity. These findings support the clinical application of specific PKC inhibitors in combination with bortezomib or other drugs in the treatment of patients who are resistant to conventional therapies. Disclosures: No relevant conflicts of interest to declare.

Abstract 3991 The transcription factor Yin-Yang 1 (YY1) is a multi-functional DNA-binding protein which can activate, repress, or initiate transcription depending on the context in which it binds. We have previously reported that YY1 is a repressor for the death receptors Fas and DR5 as well as it regulates both chemo- and immuno- resistance in many tumors such as prostate carcinoma and lymphoma. YY1 has also been reported to be a prognostic factor for some cancers. Due to the inherent resistance of multiple myeloma (MM) cell lines to various cytotoxic drugs, we examined the expression levels of YY1 in both MM cell lines and bone marrow (BM)-derived MM tissues. Analysis for YY1 was performed by immunohistochemistry (IHC) and western blot. We found that, compared to normal peripheral blood mononuclear cells and normal bone marrow, the expression of YY1 was significantly upregulated in both the cell lines and patient-derived MM tissues. The expression of YY1 was distributed in both the cytoplasm and the nucleus. Further, the intensity and frequency of cells expressing YY1 in the nuclei of MM tissues were significantly higher among patients with progressive disease as compared to patients with stable disease. In order to ascertain the role of YY1 in the maintenance of drug resistance in MM, we hypothesized that, due to the overexpression of YY1 in MM, inhibition of YY1 expression and activity should lower the threshold of resistance and render the MM cells more sensitive to drug-induced apoptosis. This hypothesis was tested using the MM U266 cell line and the drugs bortezomib and melphalan as models. The inhibition of YY1 was done by knocking down YY1 following transfection with YY1 siRNA. Analysis of the transfected cells with YY1 siRNA, but not with control siRNA, inhibited YY1 expression as analyzed by IHC and western blot. The U266 cells were first transfected with YY1 siRNA or control siRNA for 48 hours and then treated with various concentrations of bortezomib (2.5 and 5.0 nM) or melphalan (10 and 20 μM) that were not toxic to the cells for 48 hours. The cells were analyzed for apoptosis by activation of the effector caspase 3. The findings demonstrate that YY1/siRNA-treated cells were sensitized to apoptosis by bortezomib or melphalan. Analysis of the underlying mechanism by which the inhibition of YY1 sensitizes the MM cells via regulation of the apoptotic pathways will be presented. We are also currently testing specific chemical inhibitors for YY1 that are being tested both in vitro and pre-clinically in mice. The present findings demonstrate that YY1 is a potential target for therapeutic intervention and the combination of YY1 inhibitors with non-toxic doses of bortezomib, melphalan or other cytotoxic drugs may be beneficial for the treatment of MM patients who are no longer responding to current treatments. Further, we propose that, based on our findings that the level and nuclear localization of YY1 dictates disease progression in a subset of patients, inhibition of YY1 with treatment may reduce the frequency of patients with progressive disease. *Contributed equally Disclosures: No relevant conflicts of interest to declare.

Treatment of patients with relapsed or refractory B-NHL with rituximab alone or in combination with CHOP has resulted in significant clinical response. However, a subset of patients does not initially respond or develops resistance to such therapies. The mechanism underlying rituximab resistance is not fully understood. We have explored a possible mechanism by generating in the laboratory rituximab resistant clones (Ramos RR, Daudi RR, and 2F7 RR) and reported that the clones, unlike the parental wild type (wt), no longer respond to rituximab-induced cell signaling and chemo- immuno-sensitization. For instance, we have demonstrated that the resistant clones exhibit hyperactivation of cell survival signaling pathways, such as the NF-kB and Raf-1/MEK/ERK pathways, and overexpress several anti-apoptotic gene products that regulate apoptosis (Jazirehi et al., Cancer Research 67:1270–1281, 2007). To further characterize the molecular basis of rituximab-resistance, we analyzed the gene expression profile of Ramos and Ramos RR1 using oligonucleotide microarrays. There were only a few genes that were significantly modified and we have focused on such genes for analysis. First, we analyzed genes that were present in Ramos RR1 and silenced in wtRamos, namely, KIAA0738, DHTKDU1, PTPRO, REG3A, and ATXN10. Analysis of some of these gene products revealed their possible role in the regulation of resistance. For instance, REG3A, regenerating islet-derived 3α, a growth promoting lectin, has been reported to be involved in various biological functions including proliferation and resistance to apoptosis and silenced by hypermethylation. This suggests that REG3A expression in Ramos RR1 may be due to inhibition of hypermethylation. Another gene product silenced in wtRamos but expressed in Ramos RR1 is the protein tyrosine phosphatase receptor type O (PTPRO), which is known to be hypermethylated in response to rituximab therapy. Overexpression of PTPRO inhibits BCR-triggered syk tyrosyl phosphorylation and cell signaling. Hence, rituximab has been reported to trigger cells through the association of BCR with CD20 on the membrane. Second, we analyzed differentially expressed genes between Ramos RR1 and wtRamos. Two gene products, MAP3K14, and ACN9 were most overexpressed, whereas PAM, SDCCAG33, and NPAS4 were most downregulated in Ramos RR1. Overexpression of MAP3K14 (NIK) is in agreement with the hyperactivated state of Ramos RR1, and in addition to our findings of hyperactivation of the canonical NF-κB pathway, would suggest that the non-canonical pathway is also activated. Hyperactivation of NIK in Ramos RR1 may be due to its stabilization by NF-κB-dependent degradation of TRAF-3 and our findings are consistent with NIK overexpression in other blood malignancies. PAM (phosphatydyl-glycine α amidating monooxygenase) is an enzyme that has been reported to play a critical role in an autocrine pathway promoting proliferation. Its downregulation in Ramos RR1 suggests PAM-independence. Findings will be presented on the validation and roles of the above gene products in the regulation of rituximab resistance and approaches to target such gene products to reverse resistance.

Transferrin receptor 1 (TfR1 or CD71) is the major gateway for iron entry into cells and is overexpressed on a variety of cancer cells. Antibodies targeting TfR1 have shown efficacy in vitro and in vivo against different malignancies by affecting internalization of iron into target cells; however, in some cases their response rate is limited despite the high expression of TfR1 on the target cells. The cause of this limited response is still not well understood. We have developed a mouse/human chimeric IgG3-avidin fusion protein (ch128.1Av) that binds specifically to human TfR1 and exhibits a significant intrinsic anti-proliferative and pro-apoptotic activity against hematopoietic malignant cells by inducing TfR1 degradation and iron starvation. We have previously found a wide range of responses to treatment with ch128.1Av, which can be categorized as high, intermediate, or low sensitivity to the drug. Among the cells tested, the human B-lymphoblastoid cell line IM-9 was one of the most sensitive to ch128.1Av, while the human myeloma cell line U266 was tolerant. To identify the mechanisms responsible for mediating sensitivity or tolerance to the treatment with ch128.1Av, we conducted confocal microscopy studies and a whole genome time-course microarray analysis of IM-9 and U266 cells treated with this drug. In the current work we describe the molecular pathways associated with sensitivity or resistance to this therapy in vitro. We demonstrate that IM-9 cells treated with ch128.1Av can re-route TfR into the lysosomal compartment and exhibit a cytotoxic response that is predicted to be mediated in part by p53 and its target genes based on a transcription factor binding site analysis of the promoter region of genes affected by treatment with ch128.1Av. We show that a siRNA mediated reduction of wild type p53 expression rescues in part the inhibition of proliferation of IM-9 cells treated with ch128.1Av, which validates the in silico promoter analysis. In contrast, we show that U266 cells are tolerant to the cytotoxic response induced by ch128.1Av, probably due to a combination of factors including their failure to redirect trafficking of TfR targeted by ch128.1Av into the lysosomal compartment, lack of a wild type copy of the p53 protein, an early pro-proliferative response to treatment, and a high expression of the cyclin D1 gene. This work has important implications in the clinical use of TfR targeted therapies and is broadly applicable to the advancement and development of novel therapeutics targeting iron metabolism in hematopoietic malignancies. Figure Figure Hierarchical clustering of expression changes (p

Objective and Rationale Raf-1 kinase inhibitor protein (RKIP) is a modulator of cell signaling and survival that functions as an endogenous inhibitor of multiple kinases, including kinases involved in the Raf-1/MEK/ERK and NF-kB pathways. RKIP has been identified as a metastasis suppressor gene and an immune surveillance cancer gene, since loss of RKIP protein expression has been associated with tumor progression, metastasis and escape from immune surveillance. Further, RKIP expression has been associated with prognostic significance in many cancers. Recently, we have demonstrated that induction of RKIP expression in tumors with low RKIP levels results in increased tumor cell sensitivity to immuno- or chemo-therapy via inhibition of the above pathways. However, multiple myeloma (MM) cells have been shown to express high RKIP levels compared to other tumors and still remain highly resistant to conventional cytotoxic therapies. These findings were unexpected and thus, it was plausible that the high level of RKIP expression was not functionally active. It has been reported that phosphorylation of RKIP at Ser-153 renders the cells inactive (Corbit et al., 2003, J Biol Chem 278:13061-8). Thus, we examined the expression and the phosphorylation status of the RKIP protein in several multiple myeloma cell lines and tissues and compared them with other cell lines with low RKIP expression. Hypothesis We hypothesized that MM tumor cells express high levels of the inactive phosphorylated RKIP protein which antagonizes the active non-phosphorylated RKIP form in the inhibition of the survival signaling pathways. Experimental Designs and Methods Multiple myeloma (IM-9, RPMI 8226, MM1S and U266 cell lines and fresh bone marry samples from MM patients), PC-3 prostatic carcinoma and Ramos B-NHL cell lines were examined for total and phosphorylated RKIP expression by IHC and Western Blot analyses. The total RKIP protein was significantly elevated in multiple myeloma cell lines compared to the prostate and B-NHL lines. The predominant RKIP form in patients’ multiple myeloma tumors was the phosphorylated RKIP protein with high nuclear localization, as assessed by IHC, while the phosphorylated RKIP levels in the non-myeloma tumors were relatively low. It has been reported that the phosphorylation of RKIP is mediated by protein kinase C (Corbit et al., 2003, J Biol Chem 278:13061–8). Additional studies in MM cell lines also revealed high expression of the zeta isoform of PKC (PKCζ), known to phosphorylate and inactivate RKIP. Conclusions and Implications The present findings demonstrate that the aberrant RKIP phosphorylation in MM tumors may result in the inhibition of the suppressive effect of RKIP on tumor survival signaling pathways. We postulate that the high expression of RKIP may be due to inhibition of proteasome degradation. The present findings also suggest that screening of RKIP levels and RKIP phosphorylation status in MM may be useful as prognostic factors of tumor cell response to anti-tumor therapies. (Baritaki and Huerta-Yepez contributed equally.)

Multiple Myeloma (MM) is an incurable disease of monoclonal malignant plasma cells. Treatment of MM with conventional chemotherapeutic drugs has resulted in improved response rates, however, with no sufficient improvement in overall survival. Bortezomib has been recently used and results in significant clinical responses in refractory MM. However, many patients relapse and become refractory to cytotoxic therapies and, hence, the need for new therapies. We have generated an Anti-TfR-IgG3-Avidin Fusion Protein (Anti-TfR-IgG3-Av) that can bind MM which express high levels of transferrin receptor and can deliver biotinylated molecules into cancer cells (Ng et al PNAS2002; 79:10706). We have reported that treatment of MM with Anti-TfR-IgG3-Av results in inhibition of cell proliferation and direct cytotoxicity in few cell lines. Further, we have also found that Anti-TfR-IgG3-Av can sensitize resistant MM cells to drugs (eg. CDDP)-induced apoptosis. Sensitization by Anti-TfR-IgG3-Av resulted in the inhibition of several anti-apoptotic gene products like XIAP, Bid, Bcl-2 and BclXL. Since these gene products are regulated by the NF-κB pathway, we hypothesized that Anti-TfR-IgG3-Av may inhibit the AKT pathway in MM cell lines. The AKT signaling inactivates several pro-apoptotic factors, such as Bad, which is phosphorylated and inhibits its binding and inactivation of BclXL. AKT also activates IκB kinase (IKK) to phosphorylate IκB (inhibitor of NF-κB ) and leading to its proteasomal degradation and NF-κB nuclear localization. The AKT and NF-κB pathways result in the transcription of many anti-apoptotic gene products like XIAP, Bcl-2, survivin and BclXL. Treatment of MM cell lines with Anti-TfR-IgG3-Av resulted in inhibition of phospho-AKT and inhibition of NF-κB activity and downstream inhibition of above anti-apoptotic gene products. We then examined the roles of AKT and NF-κB in Anti-TfR-IgG3-Av-induced sensitization of MM to CDDP-apoptosis. Treatment of IM-9 cells with siRNA AKT, not control siRNA, resulted in inhibition of AKT concomitantly with inhibition of Bcl-2 and survive in. The cells treated with si-RNA AKT were sensitized to CDDP-induced apoptosis. These findings suggested that Anti-Anti-TfR-IgG3-Av-induced sensitization to CDDP may be due, in part, to inhibition of AKT. Likewise, the role of NF-κB inhibition by Anti-TfR-IgG3-Av in the sensitization to CDDP was demonstrated by the use of the specific NF-κB inhibitor, DHMEQ. Thus, both inhibition of AKT and NF-κB pathways by Anti-TfR-IgG3-Av play a major role in Anti-TfR-IgG3-Av-induced sensitization to CDDP. The apoptosis achieved by the combination of Anti-TfR-IgG3-Av and CDDP resulted from the complementation of several gene products modified by each agent alone and resulting in the activation of caspases 9, 8 and 3 and apoptosis. The above findings provide an underlying mechanism of Anti-TfR-IgG3-Av-induced cell signaling modification that renders drug-resistant MM cells sensitive to apoptosis by drugs.

The human transferrin receptor (hTfR) is a cell surface glycoprotein responsible for iron uptake, which is required for several cellular reactions including respiration and DNA synthesis. In cancer cells, the hTfR is over-expressed due to the high amount of iron required to maintain the uncontrolled cell proliferation. This makes the cancer cells highly sensitive to death induced by iron starvation. In addition to iron uptake, the TfR may also play a role in cell growth signaling. We have developed a mouse/human chimeric anti-human transferrin receptor IgG3-avidin fusion protein (anti-hTfR IgG3-Av) that induces cell death in human malignant B-cells such as IM9 and ARH-77 (B lymphoblastoid cell lines). The TfR is constitutively internalized and recycled back to cell surface. Anti-hTfR IgG3-Av binding to the hTfR disrupts this cycling and redirects the hTfR to lysosomal compartments where hTfR is presumably degraded. This promotes a state of iron starvation that induces cancer cell death by apoptosis, which can be blocked by iron supplementation. However, the cytotoxic effect induced by anti-hTfR IgG3-Av was minor or absent in U266 (multiple myeloma cell line). Gambogic acid (GA), a natural compound from traditional Chinese medicine, was previously shown to bind the TfR, blocking its internalization and inducing apoptosis in an iron independent manner in Jurkat (acute T cell leukemia) and HL-60 (acute promyelocytic leukemia) cell lines. The goal of the present work is to explore the cytotoxic effect of anti-hTfR IgG3-Av and GA alone or in combination in a panel of hematopoietic malignant cell lines and understand the relevance of iron in the cytotoxicity. We demonstrate that GA inhibits the proliferation of Ramos, HS-sultan, Raji (Burkitt’s lymphomas), IM9, U266, Jurkat and HL-60 cell lines. Our results showed that 0.3 μM GA at 24 h decreases cell proliferation by 70–90%, and 95–99.9% at 48 h, in all cell lines tested. We also observed morphological changes in the cells treated with GA consistent with cell death. Importantly, using ferric ammonium citrate as an iron supplement, we confirmed that the cytotoxic effect of GA in the cell lines tested is iron independent. This work also demonstrates, for the first time, that anti-hTfR IgG3-Av has a potent cytotoxic effect in the panel of leukemia and lymphoma cell lines tested above. Additionally, we confirmed that anti-hTfR IgG3-Av induces cell death in an iron dependent manner. Since both anti-hTfR IgG3-Av and GA target the TfR, we tested the effect of the combination of these components. Despite a potential antagonistic effect due to the fact that anti-hTfR IgG3-Av decreases the cell surface levels of the hTfR and GA blocks its internalization, the combination of anti-hTfR IgG3-Av plus GA enhanced the cytotoxicity, including cases of synergy, of either one alone in the cell lines tested. Studies are currently underway to understand the role of iron in the enhancement of toxicity. Our results suggest that anti-hTfR IgG3-Av and GA, alone or in combination, could be an effective treatment option for various hematopoietic malignancies.

Objective and Rationale Raf-1 kinase inhibitory protein (RKIP) is a modulator of cell signaling and survival that functions as an endogenous inhibitor of multiple kinases, including kinases involved in the Raf/MEK/ERK and NF-κB pathways. RKIP has been identified as a metastasis suppressor gene and an immune surveillance cancer gene, since loss of RKIP protein expression has been associated with tumor progression, metastasis and escape from immune surveillance. Further, RKIP expression has been associated with prognostic significance in many cancers. Recently, we have demonstrated that induction of RKIP expression in tumors with low RKIP levels results in increased tumor cell sensitivity to immuno- or chemo-therapy via inhibition of the above pathways. However, multiple myeloma (MM) cells have been shown to express high RKIP levels compared to other tumors and still remain highly resistant to conventional cytotoxic therapies. These findings were unexpected and thus, it was plausible that the high level of RKIP expression was not functionally active. It has been reported that phosphorylation of RKIP at Ser-153 renders the cells inactive (Rosner et al., 2003, J Biol Chem 278:13061–8). Thus, we examined the expression and the phosphorylation status of the RKIP protein in several multiple myeloma cell lines and tissues and compared them with other cell lines with low RKIP expression. Hypothesis We hypothesized that MM tumor cells express high levels of the inactive phoshorylated RKIP protein which antagonizes the active non-phoshorylated RKIP form in the inhibition of the survival signaling pathways. Experimental Designs and Methods Multiple myeloma (IM-9, RPMI 8226, MM1S, U266 cell lines and fresh bone marrow samples from MM patients), PC-3 prostatic carcinoma and Ramos B-NHL cell lines were examined for total and phosphorylated RKIP expression by IHC and Western Blot analyses. The total RKIP protein was significantly elevated in multiple myeloma cell lines compared to the prostate and B-NHL lines. The predominant RKIP form in multiple myeloma tumors was the phosphorylated RKIP protein with high nuclear localization, as assessed by IHC, while the phosphorylated RKIP levels in the non-myeloma tumors were relatively low. It has been reported that the phosphorylation of RKIP is mediated by protein kinase C (Rosner et al., 2003, J Biol Chem 278:13061–8). Additional studies in multiple myeloma cell lines also revealed high expression of the zeta isoform of PKC (PKCζ), known to phosphorylate and inactivate RKIP. Conclusions and Implications The present findings demonstrate that the aberrant RKIP phosphorylation in multiple myeloma tumors may result in the inhibition of the suppressive effect of RKIP on tumor survival signaling pathways. We postulate that the high expression of RKIP may be due to inhibition of proteasome degradation. The present findings also suggest that screening of RKIP levels and RKIP phosphorylation status in MM may be useful as prognostic factors of tumor cell response to anti-tumor therapies.

Multiple myeloma (MM) is an incurable proliferative disorder of monoclonal malignant plasma cells. Multiple agents conventional dose chemotherapy regimens have resulted in improved response rates, however, there have been no significant improvements in overall survival. Recently, Bortezomib has been introduced for the treatment of MM and has resulted in significant response rates. Nevertheless, additional strategies for direct treatment of MM patients are urgently needed. In general, cancer cells have been shown to express significantly higher levels of the transferrin receptor (TfR) and thus, this receptor is an attractive target for cancer therapy. We have generated an anti-TfR IgG3-avidin fusion protein (anti-TfR-IgG3-Av) that can deliver biotinylated molecules into cancer cells and exhibits intrinsic pro-apoptotic activity against hematopoietic malignant cells including myeloma (Ng P. et al., 2002, PNAS 99:10706–11). We have also reported that certain monoclonal antibodies (e.g., rituximab) can sensitize tumor cells to various chemotherapeutic drug-induced apoptosis (Jazirehi and Bonavida, 2005, Oncogene 65:264–76). Thus, this study investigated whether anti-TfR IgG3-Av sensitizes drug-resistant MM cell lines to drug-induced apoptosis. We initially examined the CDDP-resistant lymphoblastoid IM-9 and ARH-77 cell lines for chemosensitization using a suboptimal dose of anti-TfR IgG3-Av. Several methods were used to assess the cytotoxic activity, namely viable cell recovery and apoptosis by both PI and caspase-3 activation. The findings demonstrate that treatment of tumor cells with anti-TfR IgG3-Av for 18 h following treatment with CDDP for an additional 18 h resulted in significant cytotoxicity and apoptosis and synergy was achieved. The combined treatment also resulted in significant mitochondrial membrane depolarization, suggesting that apoptosis was achieved through a type-II pathway. In comparison with tumor cell treatment with anti-TfR-IgG3-Av, the anti-TfR IgG3 was a more potent chemosensitizer, based on both the concentration used and the extent of apoptosis achieved. These findings demonstrate that anti-TfRIgG3-Av is a potential therapeutic antibody that will reverse MM drug resistance when used in combination with chemotherapeutic drugs.