Scientific Resources

Multiple myeloma is characterized by increased osteoclast activity that results in bone destruction and lytic lesions. With the prolonged overall patient survival achieved by new treatment modalities,additional drugs are required to inhibit bone destruction. We focused on a novel and more potent structural analog of the nonsteroidal anti-inflammatory drug etodolac,known as SDX-308,and its effects on osteoclastogenesis and multiple myeloma cells. SDX-101 is another structural analog of etodolac that is already used in clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Compared with SDX-101,a 10-fold lower concentration of SDX-308 induced potent (60%-80%) inhibition of osteoclast formation,and a 10- to 100-fold lower concentration inhibited multiple myeloma cell proliferation. Bone resorption was completely inhibited by SDX-308,as determined in dentin-based bone resorption assays. SDX-308 decreased constitutive and RANKL-stimulated NF-kappaB activation and osteoclast formation in an osteoclast cellular model,RAW 264.7. SDX-308 effectively suppressed TNF-alpha-induced IKK-gamma and IkappaB-alpha phosphorylation and degradation and subsequent NF-kappaB activation in human multiple myeloma cells. These results indicate that SDX-308 effectively inhibits multiple myeloma cell proliferation and osteoclast activity,potentially by controlling NF-kappaB activation signaling. We propose that SDX-308 is a promising therapeutic candidate to inhibit multiple myeloma growth and osteoclast activity and that it should receive attention for further study. View Publication

Overcoming imatinib mesylate (IM) resistance and disease persistence in patients with chronic myeloid leukemia (CML) is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL+ cells in the presence of IM and nilotinib (NI; AMN107),a novel,more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84 cell clones (R-CM) was found to substantially protect IM-naive LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony-stimulating factor (GM-CSF),which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the janus kinases 2 (JAK-2)/signal transducer and activator of transcription 5 (STAT-5) signaling pathway in GM-CSF receptor alpha receptor (CD116)-expressing cells,including primary CD34+/CD116+ GM progenitors (GMPs). Elevated mRNA and protein levels of GM-CSF were detected in IM-resistant patient samples,suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly,inhibition of JAK-2 with AG490 abrogated GM-CSF-mediated STAT-5 phosphorylation and NI resistance in vitro. Together,adaptive autocrine secretion of GM-CSF mediates BCR/ABL-independent IM and NI resistance via activation of the antiapoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF-induced IM and NI progenitor cell resistance,providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML. View Publication

Drug resistance resulting from emergence of imatinib-resistant BCR-ABL point mutations is a significant problem in advanced-stage chronic myelogenous leukemia (CML). The BCR-ABL inhibitor,nilotinib (AMN107),is significantly more potent against BCR-ABL than imatinib,and is active against many imatinib-resistant BCR-ABL mutants. Phase 1/2 clinical trials show that nilotinib can induce remissions in patients who have previously failed imatinib,indicating that sequential therapy with these 2 agents has clinical value. However,simultaneous,rather than sequential,administration of 2 BCR-ABL kinase inhibitors is attractive for many reasons,including the theoretical possibility that this could reduce emergence of drug-resistant clones. Here,we show that exposure of a variety of BCR-ABL+ cell lines to imatinib and nilotinib results in additive or synergistic cytotoxicity,including testing of a large panel of cells expressing BCR-ABL point mutations causing resistance to imatinib in patients. Further,using a highly quantifiable bioluminescent in vivo model,drug combinations were at least additive in antileukemic activity,compared with each drug alone. These results suggest that despite binding to the same site in the same target kinase,the combination of imatinib and nilotinib is highly efficacious in these models,indicating that clinical testing of combinations of BCR-ABL kinase inhibitors is warranted. View Publication

Recognition of tumor-associated Ags (TAAs) on tumor cells by CTLs and the subsequent tumor cell death are assumed to be dependent on TAA protein expression and to correlate directly with the level of peptide displayed in the binding site of the HLA class I molecule. In this study we evaluated whether the levels of Her-2/neu protein expression on human tumor cell lines directly correlate with HLA-A*0201/Her2/neu peptide presentation and CTL recognition. We developed a TCR mimic (TCRm) mAb designated 1B8 that specifically recognizes the HLA-A2.1/Her2/neu peptide (369-377) (Her2(369)-A2) complex. TCRm mAb staining intensity varied for the five human tumor cell lines analyzed,suggesting quantitative differences in levels of the Her2(369)-A2 complex on these cells. Analysis of tumor cell lines pretreated with IFN-gamma and TNF-alpha for Her2/neu protein and HLA-A2 molecule expression did not reveal a direct correlation between the levels of Her2/neu Ag,HLA-A2 molecule,and Her2(369)-A2 complex expression. However,compared with untreated cells,cytokine-treated cell lines showed an increase in Her2(369)-A2 epitope density that directly correlated with enhanced tumor cell death (p = 0.05). Although a trend was observed between tumor cell lysis and the level of the Her2(369)-A2 complex for untreated cells,the association was not significant. These findings suggest that tumor cell susceptibility to CTL-mediated lysis may be predicted based on the level of specific peptide-MHC class I expression rather than on the total level of TAA expression. Further,these studies demonstrate the potential of the TCRm mAb for validation of endogenous HLA-peptide epitopes on tumor cells. View Publication

The complex hematopoietic effects of placental growth factor (PlGF) prompted us to test in mice and nonhuman primates the mobilization of peripheral blood progenitor cells (PBPCs) elicited by recombinant mouse PlGF-2 (rmPlGF-2) and recombinant human PlGF-1 (rhPlGF-1). PBPC mobilization was evaluated by assaying colony-forming cells (CFCs),high-proliferative potential-CFCs (HPP-CFCs),and long-term culture-initiating cells (LTC-ICs). In mice,both rmPlGF-2 and rhPlGF-1 used as single agents failed to mobilize PBPCs,whereas the combination of rhPlGF-1 and granulocyte colony-stimulating factor (rhG-CSF) increased CFCs and LTC-ICs per milliliter of blood by four- and eightfold,respectively,as compared with rhG-CSF alone. rhPlGF-1 plus rhG-CSF significantly increased matrix metalloproteinase-9 plasma levels over rhG-CSF alone,suggesting a mechanistic explanation for rhPlGF-1/rhG-CSF synergism. In rhesus monkeys,rhPlGF-1 alone had no mobilization effect,whereas rhPlGF-1 (260 microg/kg per day) plus rhG-CSF (100 microg/kg per day) increased rhG-CSF-elicited mobilization of CFCs,HPP-CFCs,and LTC-ICs per milliliter of blood by 5-,7-,and 15-fold,respectively. No specific toxicity was associated with the administration of rhPlGF-1 alone or in combination. In conclusion,our data demonstrate that rhPlGF-1 significantly increases rhG-CSF-elicited hematopoietic mobilization and provide a preclinical rationale for evaluating rhPlGF-1 in the clinical setting. View Publication

Therapeutic mAbs that target tumor-associated Ags on the surface of malignant cells have proven to be an effective and specific option for the treatment of certain cancers. However,many of these protein markers of carcinogenesis are not expressed on the cells' surface. Instead these tumor-associated Ags are processed into peptides that are presented at the cell surface,in the context of MHC class I molecules,where they become targets for T cells. To tap this vast source of tumor Ags,we generated a murine IgG2a mAb,3.2G1,endowed with TCR-like binding specificity for peptide-HLA-A*0201 (HLA-A2) complex and designated this class of Ab as TCR mimics (TCRm). The 3.2G1 TCRm recognizes the GVL peptide (GVLPALPQV) from human chorionic gonadotropin beta presented by the peptide-HLA-A*0201 complex. When used in immunofluorescent staining reactions using GVL peptide-loaded T2 cells,the 3.2G1 TCRm specifically stained the cells in a peptide and Ab concentration-dependent manner. Staining intensity correlated with the extent of cell lysis by complement-dependent cytotoxicity (CDC),and a peptide concentration-dependent threshold level existed for the CDC reaction. Staining of human tumor lines demonstrated that 3.2G1 TCRm was able to recognize endogenously processed peptide and that the breast cancer cell line MDA-MB-231 highly expressed the target epitope. The 3.2G1 TCRm-mediated CDC and Ab-dependent cellular cytotoxicity of a human breast carcinoma line in vitro and inhibited in vivo tumor implantation and growth in nude mice. These results provide validation for the development of novel TCRm therapeutic reagents that specifically target and kill tumors via recognition and binding to MHC-peptide epitopes. View Publication

Human metapneumovirus (hMPV) is a recently described member of the Paramyxoviridae family/Pneumovirinae subfamily and shares many common features with respiratory syncytial virus (RSV),another member of the same subfamily. hMPV causes respiratory tract illnesses that,similar to human RSV,occur predominantly during the winter months and have symptoms that range from mild to severe cough,bronchiolitis,and pneumonia. Like RSV,the hMPV virus can be subdivided into two genetic subgroups,A and B. With RSV,a single monoclonal antibody directed at the fusion (F) protein can prevent severe lower respiratory tract RSV infection. Because of the high level of sequence conservation of the F protein across all the hMPV subgroups,this protein is likely to be the preferred antigenic target for the generation of cross-subgroup neutralizing antibodies. Here we describe the generation of a panel of neutralizing monoclonal antibodies that bind to the hMPV F protein. A subset of these antibodies has the ability to neutralize prototypic strains of both the A and B hMPV subgroups in vitro. Two of these antibodies exhibited high-affinity binding to the F protein and were shown to protect hamsters against infection with hMPV. The data suggest that a monoclonal antibody could be used prophylactically to prevent lower respiratory tract disease caused by hMPV. View Publication

BACKGROUND: Resistance to imatinib monotherapy frequently emerges in advanced stages of chronic myelogenous leukemia (CML),supporting the rationale for combination drug therapy. In the present study,the activities of the farnesyltransferase inhibitors (FTIs) L744,832 and LB42918,as single agents and in combination with imatinib,were investigated in different imatinib-sensitive and -resistant BCR-ABL-positive CML cells. MATERIALS AND METHODS: Growth inhibition of the cell lines and primary patient cells was assessed by MTT assays and colony-forming cell assays,respectively. Drug interactions were analyzed according to the median-effect method of Chou and Talalay. The determination of apoptotic cell death was performed by annexin V/propidium iodide staining. RESULTS: Combinations of both FTIs with imatinib displayed synergism or sensitization (potentiation) in all the cell lines tested. In primary chronic phase CML cells,additive and synergistic effects were discernible for the combination of imatinib plus L744,832 and imatinib plus LB42918,respectively. Annexin V/propidium iodide staining showed enhancement of imatinib-induced apoptosis with either drug combination,both in imatinib-sensitive and -resistant cells. CONCLUSION: The results indicated the potential of L744,832 and LB42918 as combination agents for CML patients on imatinib treatment. View Publication

Current therapies for delayed- or nonunion bone fractures are still largely ineffective. Previous studies indicated that the VEGF homolog placental growth factor (PlGF) has a more significant role in disease than in health. Therefore we investigated the role of PlGF in a model of semi-stabilized bone fracture healing. Fracture repair in mice lacking PlGF was impaired and characterized by a massive accumulation of cartilage in the callus,reminiscent of delayed- or nonunion fractures. PlGF was required for the early recruitment of inflammatory cells and the vascularization of the fracture wound. Interestingly,however,PlGF also played a role in the subsequent stages of the repair process. Indeed in vivo and in vitro findings indicated that PlGF induced the proliferation and osteogenic differentiation of mesenchymal progenitors and stimulated cartilage turnover by particular MMPs. Later in the process,PlGF was required for the remodeling of the newly formed bone by stimulating osteoclast differentiation. As PlGF expression was increased throughout the process of bone repair and all the important cell types involved expressed its receptor VEGFR-1,the present data suggest that PlGF is required for mediating and coordinating the key aspects of fracture repair. Therefore PlGF may potentially offer therapeutic advantages for fracture repair. View Publication

Recombinant human erythropoietin (rhEPO) is receiving increasing attention as a potential therapy for prevention of injury and restoration of function in nonhematopoietic tissues. However,the minimum effective dose required to mimic and augment these normal paracrine functions of erythropoietin (EPO) in some organs (e.g.,the brain) is higher than for treatment of anemia. Notably,a dose-dependent risk of adverse effects has been associated with rhEPO administration,especially in high-risk groups,including polycythemia-hyperviscosity syndrome,hypertension,and vascular thrombosis. Of note,several clinical trials employing relatively high dosages of rhEPO in oncology patients were recently halted after an increase in mortality and morbidity,primarily because of thrombotic events. We recently identified a heteromeric EPO receptor complex that mediates tissue protection and is distinct from the homodimeric receptor responsible for the support of erythropoiesis. Moreover,we developed receptor-selective ligands that provide tools to assess which receptor isoform mediates which biological consequence of rhEPO therapy. Here,we demonstrate that rhEPO administration in the rat increases systemic blood pressure,reduces regional renal blood flow,and increases platelet counts and procoagulant activities. In contrast,carbamylated rhEPO,a heteromeric receptor-specific ligand that is fully tissue protective,increases renal blood flow,promotes sodium excretion,reduces injury-induced elevation in procoagulant activity,and does not effect platelet production. These preclinical findings suggest that nonerythropoietic tissue-protective ligands,which appear to elicit fewer adverse effects,may be especially useful in clinical settings for tissue protection. View Publication

Deletions and/or mutations of p53 are relatively rare and late events in the natural history of B-cell chronic lymphocytic leukemia (B-CLL). However,it is unknown whether p53 signaling is functional in B-CLL and if targeted nongenotoxic activation of the p53 pathway by using nutlin-3,a small molecule inhibitor of the p53/MDM2 interaction,is sufficient to kill B-CLL cells. In vitro treatment with nutlin-3 induced a significant cytotoxicity on primary CD19(+) B-CLL cells,but not on normal CD19(+) B lymphocytes,peripheral-blood mononuclear cells,or bone marrow hematopoietic progenitors. Among 29 B-CLL samples examined,only one was resistant to nutlin-3-mediated cytotoxicity. The induction of p53 by nutlin-3 in B-CLL samples was accompanied by alterations of the mitochondrial potential and activation of the caspase-dependent apoptotic pathway. Among several genes related to the p53 pathway,nutlin-3 up-regulated the steady-state mRNA levels of PCNA,CDKN1A/p21,GDF15,TNFRSF10B/TRAIL-R2,TP53I3/PIG3,and GADD45. This profile of gene activation showed a partial overlapping with that induced by the genotoxic drug fludarabine. Moreover,nutlin-3 synergized with both fludarabine and chlorambucil in inducing B-CLL apoptosis. Our data strongly suggest that nutlin-3 should be further investigated for clinical applications in the treatment of B-CLL. View Publication

SPD754 (AVX754) is a deoxycytidine analogue nucleotide reverse transcriptase inhibitor (NRTI) in clinical development. These studies characterized the in vitro activity of SPD754 against NRTI-resistant human immunodeficiency virus type 1 (HIV-1) and non-clade B HIV-1 isolates,its activity in combination with other antiretrovirals,and its potential myelotoxicity and mitochondrial toxicity. SPD754 was tested against 50 clinical HIV-1 isolates (5 wild-type isolates and 45 NRTI-resistant isolates) in MT-4 cells using the Antivirogram assay. SPD754 susceptibility was reduced 1.2- to 2.2-fold against isolates resistant to zidovudine (M41L,T215Y/F,plus a median of three additional nucleoside analogue mutations [NAMs]) and/or lamivudine (M184V) and was reduced 1.3- to 2.8-fold against isolates resistant to abacavir (L74V,Y115F,and M184V plus one other NAM) or stavudine (V75T/M,M41L,T215F/Y,and four other NAMs). Insertions at amino acid position 69 and Q151M mutations (with or without M184V) reduced SPD754 susceptibility 5.2-fold and 14- to 16-fold,respectively (these changes gave values comparable to or less than the corresponding values for zidovudine,lamivudine,abacavir,and didanosine). SPD754 showed similar activity against isolates of group M HIV-1 clades,including A/G,B,C,D,A(E),D/F,F,and H. SPD754 showed additive effects in combination with other NRTIs,tenofovir,nevirapine,or saquinavir. SPD754 had no significant effects on cell viability or mitochondrial DNA in HepG2 or MT-4 cells during 28-day exposure at concentrations up to 200 microM. SPD754 showed a low potential for myelotoxicity against human bone marrow. In vitro,SPD754 retained activity against most NRTI-resistant HIV-1 clinical isolates and showed a low propensity to cause myelotoxicity and mitochondrial toxicity. View Publication