ALBERT

Description: Multiple Myeloma (MM) is an incurable disease that warrants novel treatments. MM survival is dependent on resistance conferred by interactions with the bone marrow (BM) microenvironment (stroma and/or extracellular matrix proteins): Environment Mediated-Drug Resistance (EM-DR). We have shown that adhesion to fibronectin inhibits CD95-induced caspase-8 activation resulting in apoptosis by regulating the cellular localization and availability of c- FLIPL (Shain K. et al. J Immunol. 2002;168(5):2544–53). TNF-related apoptosis inducing ligand/Apo-2L (TRAIL/Apo-2L) is a member of the TNF superfamily of death ligands that induce apoptosis of resistant MM cell lines and patient’s cells while cultured in suspension. TRAIL/Apo-2L also inhibits the growth of human plasmocytomas in xenografted NOD/SCID mice (Mitsiades C. et al. Blood2001; 98(3):795–804). To explore if the tumor microenvironment confers resistance to TRAIL/Apo-2L mediated apoptosis, we treated RPMI 8226 cells with recombinant human killer TRAIL (Alexis Biochemicals). In a series of 3 experiments, we observed a 50.8 ± 10 % specific apoptosis, measured by annexin/PI staining, when cells were treated with TRAIL (10ng/mL) in suspension (Su) media. In contrast, a dramatic resistance to TRAIL mediated apoptosis (6.9 ± 1.2 %) was observed when RPMI 8226 cells were adhered (Ad) to stroma cells (HS-5 and HS-5 transfected with green fluorescent protein-GFP) suggesting the EM-DR phenotype. We hypothesized that EM-DR can potentially be reversed by Bortezomib, a proteosome inhibitor, via NF-κB inhibition resulting in reduced transcription of cellular adhesion proteins genes (Mitsiades et al. Blood2002; 99(11): 4079–4086). Resistance to drug-induced apoptosis was also observed when RPMI 8226 cells were adhered to HS-5 and HS5-GFP and treated with Bortezomib alone at 5, 10 and 15nM (55.6 ± 11.6 % Su and 12.1 ± 1.6 % Ad). Microarray analysis of RPMI 8226 cells treated with Bortezomib showed that death receptor 4 (DR4) and DR5 expression was up-regulated suggesting a potential interaction between Bortezomib and TRAIL pathway. When RPMI 8226 cells were treated with Bortezomib (10nM) for 20 Hrs and subsequently treated with TRAIL for additional 4 Hrs, we observed an enhancement to 77.7 ± 3.64 % (Su) and 47.22 ± 13.6 % (Ad) specific apoptosis. In addition, in a series of 3 experiments using transwell inserts, RPMI 8226 cells (upper well) separated from HS-5 (lower well) were treated with Bortezomib (10nM), TRAIL (10ng/ML) or Bortezomib and TRAIL combined as described above. Results were comparable with direct contact experiments described, suggesting that cytokines and/or chemokines released by stroma may be responsible for EM-DR phenotype (Figure 1). Further mechanistic studies elucidating TRAIL and Bortezomib combination are currently under investigation. These preclinical studies provide the basis for clinical trial testing the combination of these agents. Figure Figure

Description: Here we demonstrate that treatment with SAHA (suberoylanilide hydroxamic acid), a known inhibitor of histone deacetylases (HDACs), alone induced p21 and/or p27 expressions but decreased the mRNA and protein levels of Bcr-Abl, which was associated with apoptosis of Bcr-Abl–expressing K562 and LAMA-84 cells. Cotreatment with SAHA and imatinib (Gleevec) caused more down-regulation of the levels and auto-tyrosine phosphorylation of Bcr-Abl and apoptosis of these cell types, as compared with treatment with either agent alone (P 

Description: 17-allylamino-demethoxy geldanamycin (17-AAG) inhibits the chaperone function of heat shock protein—90 (Hsp-90) and promotes the proteasomal degradation of its misfolded client proteins. Here, we demonstrate that treatment of the human acute myeloid leukemia HL-60 cells with 17-AAG attenuates the intracellular levels of a number of Hsp-90 client proteins, including Akt, c-Raf-1, and c-Src. Also, 17-AAG induced the mitochondrial release and cytosolic accumulation of cytochrome c (cyt c) and second mitochondria-derived activator of caspases (Smac)/DIABLO, resulting in the activation of caspase-9 and caspase-3 and apoptosis. Treatment with 17-AAG triggered the B-cell lymphoma—2 (Bcl-2)—associated X protein (Bax) conformational change associated with apoptosis, while Bax-deficient cells were resistant to 17-AAG—induced apoptosis. In addition, in HL-60/Bcl-2 and HL-60/Bcl-xL cells, which ectopically express Bcl-2 and Bcl-xL respectively, 17-AAG—induced Bax conformational change, cytosolic accumulation of cyt c and Smac/DIABLO, and apoptosis were markedly inhibited. Although the rate of 17-AAG—mediated decline in Akt, c-Raf-1, and c-Src levels was blunted, the total decline was not compromised in HL-60/Bcl-2 and HL-60/Bcl-xL cells. Cotreatment with HA14-1, a nonpeptidic ligand that can bind and inhibit the antiapoptotic activity of Bcl-2, significantly overcame the resistance to 17-AAG—induced apoptosis in HL-60/Bcl-2 cells. Together, these findings indicate that although 17-AAG treatment causes the levels of a number of survival-signaling protein kinases to decline, the downstream engagement of the mitochondrial pathway of apoptosis is regulated by the activity of the Bcl-2 family of proteins. Also, neutralizing the antiapoptotic effect of Bcl-2 would further enhance the antileukemia activity of 17-AAG. (Blood. 2003;102:269-275)

Description: Purine nucleoside phosphorylase (PNP) deficiency in humans results in T lymphocytopenia. Forodesine, a potent inhibitor of PNP, was designed based on the transition-state structure stabilized by the enzyme. Previous studies established that forodesine in the presence of deoxyguanosine (dGuo) inhibits the proliferation of T lymphocytes. A phase 1 clinical trial of forodesine in T-cell malignancies demonstrated significant antileukemic activity with an increase in intracellular dGuo triphosphate (dGTP). High accumulation of dGTP in T cells may be dependent on the levels of deoxynucleoside kinases. Because B-cell chronic lymphocytic leukemia (B-CLL) cells have high activity of deoxycytidine kinase (dCK), we hypothesized that these lymphocytes would respond to forodesine. This postulate was tested in primary lymphocytes during in vitro investigations. Lymphocytes from 12 patients with CLL were incubated with forodesine and dGuo. These CLL cells showed a wide variation in the accumulation of intracellular dGTP without any effect on other deoxynucleotides. This was associated with DNA damage-induced p53 stabilization, phosphorylation of p53 at Ser15, and activation of p21. The dGTP accumulation was related to induction of apoptosis measured by caspase activation, changes in mitochondrial membrane potential, and PARP cleavage. Based on these data, a phase 2 clinical trial of forodesine has been initiated for CLL patients.

Description: Myeloma cells proliferate preferentially in the bone marrow and the latter has been shown to confer a survival advantage and drug resistance, (DR), through a process known as Cell Adhesion Mediated Drug Resistance or CAM-DR, a form of de-novo drug DR. Therefore, in studying the mechanisms of drug resistance in myeloma, the contribution of the microenvironment needs to be considered. Recently, we compared the genotypic and phenotypic profiles of CAM-DR melphalan resistance to acquired melphalan resistance. (Hazlehurst et al Cancer Res. 63: 7900–7906, 2003,)Briefly, we reported that de novo melphalan resistance associated with CAM-DR was significantly less complex, both phenotypically and genotypically, compared to acquired melphalan resistance in 8226 cells. GEP of adherent 8226 myeloma cells demonstrated fewer changes in gene expression compared to acquired melphalan resistance with no changes related to genes involved with DNA damage or cell cycle; however, changes were noted for genes involved in mitochondrial perturbation and caspase activation. Studies examining GEP profiles in myeloma patients, have been performed using isolated myeloma cells without considering the contribution of the tumor microenvironment and its influence on the phenotype or genotype of these cells. We hypothesize that the interactions of the myeloma cells with the microenvironment affects the myeloma GEP and needs to be considered when developing gene signatures that would ultimately predict level of response to treatment. In this study, we examined GEP of CD138+ myeloma cells isolated from 18 patients with relapsed, (n=8) or newly diagnosed (n=10), myeloma. The CAM-DR phenotype in CD-138+ cells was analyzed by exposing patient bone marrow specimens to Dexamethasone when cells were in suspension versus adhered to FN. We showed myeloma cells to be significantly more sensitive to Dexamethasone when kept in suspension vs adhered to FN in 5/6 specimens studied. For GEP studies, CD138+ were maintained in suspension or adhered to FN for 24 hours, then harvested for RNA extraction and hybridized to GeneChip arrays (Affymetrix HG-133A chip). Differential exression of genes was performed using a chi square test to compare observed vs expected. Only genes that were consistently differentially expressed in 〉50 % of patients with p value

Description: TNF-related apoptosis inducing ligand/Apo-2L (TRAIL), a member of the TNF superfamily of death ligands, is preferentially cytotoxic against neoplastic cells, while sparing normal tissue. TRAIL has been implicated in tumor immune-surveillance and mediates murine allogeneic graft-versus-tumor responses. The bone marrow microenvironment provides tumor protection from chemotherapy and Fas-death receptor mediated apoptosis through a process known as environmental mediated death resistance (EM-DR). We tested whether the tumor microenvironment inhibits TRAIL-mediated killing of myeloma cells, as this process may contribute to multiple myeloma escape from immune-surveillance. Three drug-sensitive myeloma cell lines (RPMI-8226, U266 and MM1s) exhibit apoptosis resistance to recombinant human (rh) TRAIL while adhered to HS5 stromal cells that expresses green-fluorescence-protein (HS5-GFP). Apoptosis resistance to TRAIL was time- and dose- dependent. Evaluation of TRAIL apoptosis in a transwell (TW) assay, with HS5-GFP cells on the bottom and RPMI-8226 cells in the upper well (TW+HS5) revealed that HS5-GFP stromal cells blocked TRAIL-induced apotosis through soluble factors. We then assessed the modifications in the TRAIL signaling pathway induced by TW+HS5 and responsible for the observed phenotype. RPMI-8226 treated in TW+HS5 exhibited attenuated pro-caspase-8, pro-caspase-3, PARP, and BID cleavage, with diminished mitochondrial membrane potential changes, but without alterations on TRAIL receptors and other Bcl-2 family members. We found increased levels of Fas-associated death domain like IL-1 converting enzyme-like inhibitory protein (FLIP), a regulatory factor that competes with caspase-8 inhibiting apoptosis. Subcellular fractionation of RPMI-8226 cells showed that FLIPL is maintained in or associated with organelle membranes and is released to the cytosol when exposed to soluble factor (TW+HS5) suggesting that that soluble factor signaling may influence FLIPL localization and availability. Furthermore, FLIP reduction by FLIP-RNA interference increases TRAIL sensitivity of RPMI-8226 treated in TW+HS5. Bortezomib is a proteosome inhibitor that inhibits NF-KB and reduces FLIP levels. To this end, pretreatment of RPMI-8226 cells with bortezomib for 20 hours followed by TRAIL for 4 hours overcame TRAIL EM-DR through inhibition of FLIP, thus providing rationale for testing this drug combination in a clinical trial. In addition, rh-IL-6 induced FLIP levels in RPMI-8226 cells conferring resistance to TRAIL mediated apoptosis. Our results suggest that IL-6 and other soluble factors produced by marrow stromal cells promote myeloma cell survival by upregulating FLIP, thereby mitigating the influence of the microenvironment on TRAIL-induced apoptosis. We conclude that the cytotoxic effect of TRAIL may be enhanced by FLIP inhibitors.

Description: An increasing body of evidence suggests that interactions between myeloma cells and the microenvironment are critical for tumor cell survival, proliferation and drug resistance. These pro-survival interactions include binding of soluble factors, and direct contact with stromal cells, or extra cellular matrix (ECM) components such as fibronectin (FN). More specifically, adhesion of multiple myeloma (MM) to FN via ν1 integrins has been demonstrated to confer resistance to a host of chemotherapeutic agents (Shain et al., 2000). This anti-apoptotic phenomenon, termed cell adhesion-mediated drug resistance (CAM-DR), is an intrinsic pathway for evading the therapy-induced apoptosis (Damiano et al., 2000). Bortezomib (PS-341), a specific inhibitor of proteosome, has been shown to cause apoptosis of several tumor cell lines including multiple myeloma. In contrast to conventional cytotoxics, Bortezomib, showed more apoptosis in 24 hours for 8226 myeloma cells adhered to FN (25 % 10 nM) compared to cells in suspension (10 % 10 nM) in a dose -dependent manner. To investigate the signal transduction mechanism responsible for the reversal of CAM-DR phenotype by Bortezomib, we conducted Affymetrix GeneChipÒ Expression of 8226 cells cultured either in suspension, or adhered to FN in the presence and absence of Bortezomib (50 nM) for 4 and 24 hours using the Affymetrix U133A chips. A total of 197 genes were upregulated on FN compared to suspension, among which only 12 genes were increased with Bortezomib and 105 genes were decreased with PS-341. A total of 870 genes were downregulated on FN adhesion, among which the majority of them were increased with Bortezomib. Similar patterns of gene expression profiles were observed after 24 hrs of treatment with Bortezomib. To examine how Bortezomib is reversing CAMDR phenotype, we chose to examine the genes that were changed with FN adhesion and reversed when treated with Bortezomib. The adhesion of 8226 cells to FN enhanced both ERK1/2 and AKT signaling pathways, and both pathways were down regulated when treated with Bortezomib. The main indicators of the AKT signaling on FN are upregulation of PYK2, AKT2, MDM2 and downregulation of p21 and BAD. In conformation with our micro-array data, PYK2 phosphorylation and ERK1/2 phosphorylation were enhanced when 8226 cells were adhered to FN. The mechanisms to understand role of PYK2 phosphorylation and downstream AKT signaling in Bortezomib-induced reversal of CAMDR are under investigation.

Description: Multiple myeloma is an incurable disease where new treatment strategies are required to improve on current treatment standards. Based on this knowledge we have performed a phase II clinical trial testing the farnesyltransferase inhibitor R115777 in patients with relapsed myeloma (Alsina M. et al, Blood 2004). R115777 was found to have a favorable toxicity profile and 64% of patients achieved disease stabilization. RAS mutation and inhibition of farnesyltransferase did not correlate with clinical efficacy; findings consistent with our prior observation that R115777 induces apoptosis via a Ras independent mechanism (Beaupre D.M. et al, Mol. Cancer Ther. 2004). In order to further characterize the mechanisms by which R115777 induces apoptosis in myeloma cells and to investigate drug resistance, we have identified and characterized a R115777 resistant human myeloma cell line. 8226/S cells were cultured continuously in increasing concentrations of R115777 for over 6 months. 8226/R5 cells were found to be nearly 50 times more resistant to R115777 compared to the parent cell line. K-Ras remained prenylated in both resistant and sensitive cells after R115777 treatment; however, HDJ-2 farnesylation was inhibited in both lines implying that farnesyltranseferase (the drug target) is not modified. 8226/R5 cells were also resistant to a diverse group of anti-tumor agents including PS-341. Many 8226 lines that acquire drug resistance have elevated expression of P-glycoprotein. We found that P-glycoprotein expression is not increased in the 8226/R5 line and furthermore influx and efflux of R115777 was similar in both parent and resistant cells. Expression of the drug resistance proteins hsp27, 70, and 90 were also not increased. Comparison of 8226/S and 8226/R5 gene expression profiles revealed increased expression of JAK2 in resistant cells. Interestingly, STAT3 phosphorylation was significantly increased in the 8226/R5 line consistent with its reported role in myeloma drug resistance. Experiments are underway to delineate the contribution of JAK2 to the multidrug resistant phenotype. Characterization of aberrant JAK2 activation is relevant since constitutive STAT3 activity is frequently observed in primary myeloma isolates.

Description: Multiple myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells that accumulate preferentially in the bone marrow. In spite of high dose chemotherapy and novel targeted therapies this disease remains incurable with a median survival of 3–6 years mainly because of the emergence of drug resistance. Improved survival requires new strategies to prevent relapse. Heat shock proteins (HSPs) are a super family of highly conserved proteins, which are induced in plant, yeast, bacterial and mammalian cells in response to an array of physiological and environmental stress cues. Among heat shock protein families, HSP70 is one of the most highly conserved and is the only protein expressed in response to cellular stress. Exogenous HSP70 has been demonstrated to act as a cytokine to human monocytes by stimulating rapid calcium influx, activating nuclear factor (NF)-kB and up-regulating the expression of IL-1b, IL-6 and tumor necrosis factor alpha (TNF-a) (Asea A et al., 2000). Adhesion of myeloma cells to bone marrow stromal cells mediates IL-6 secretion and tumor cell proliferation in part mediated by STAT-3 activation (Cheung WC et al., 2001). We have shown that adhesion of myeloma cells to bone marrow stromal cells enhances IL-6 secretion by stromal cells and HSP70 secretion by myeloma cells. When we inhibited the HSP70 expression using either KNK437 (HSF-1 inhibitor) or RNAi to HSP70, IL-6 secretion by stromal cells as well as activation of STAT-3 in myeloma cells was inhibited in dose-dependent manner. These results suggest that HSP70 released from myeloma cells is enhancing IL-6 secretion from stromal cells. Incubation of stromal cells with recombinant HSP70 did not enhance IL-6 secretion in stromal cells suggesting that some other soluble factor released from myeloma cells cooperates with HSP70 to enhance IL-6 secretion by stromal cells, We examined whether HSP70 can modulate IL-6 mediated STAT-3 activation by stimulating 8226 cells with IL-6 in the presence or absence of KNK437 and RNAi to HSP70 and measuring phospho-STAT-3 by western analysis. HSP70 inhibition attenuated IL-6 induced STAT-3 activity, but not ERK1/2 activity, indicating that HSP70 mediated IL-6 signaling is very specific to STAT-3. The signal transduction cascade by which HSP70 induces IL-6 secretion and the mechanism by which HSP70 mediates IL-6 induced STAT-3 activity are currently under investigation.