AUTHORS:L.A. Hazlehurst, M. Emmons, W.S. Dalton
Moffitt Cancer Center, Tampa Florida, USA
Introduction. Multiple myeloma (MM) is a disease that will initiallyrespond to chemotherapy. However, the disease is not curable with standardchemotherapy, indicating that standard therapy fails to eliminateminimal residual disease. Myeloma homes to the bone marrow an environmentthat is considered a rich source of extracellular matrixes. Wepropose that MM-matrix interactions, contributes to failure ofchemotherapy to eliminate minimal residual disease. In support of theimportance of MM-matrix interactions in evaluating drug response ourlaboratory previously demonstrated that adhesion of MM cell lines andprimary patients specimens via beta 1 integrins is sufficient to confer amulti-drug resistant phenotype. We have referred to this phenotype ascell adhesion mediated drug resistance or CAM-DR. Beta 1 integrin signalinginduces a network of signaling pathways that could contribute todrug resistance which include activation of Src family kinases, AKT anda reduction in the levels of the pro-apoptotic Bcl-2 family member Bim.Because of the multiplicity of integrin signaling we proposed that targetingthe integrin receptor may be an effective strategy to block integrinmediated survival signals. The goal of this study was to determinewhether inhibition of beta 1 integrin mediated adhesion with aninhibitory peptide referred to HYD1 is a viable strategy for increasing theefficacy of standard chemotherapy. Material and Methods. Peptide: TheD-amino acid peptide referred to as HYD1 was discovered using combinatorialchemistry. HYD1 was identified as the lead peptide based onthe potency of blocking beta 1 integrin mediated adhesion. Co-culturemodel: The bone marrow stroma cell line HS-5 was used for co-culturestudies. Myeloma cells were treated with 50 ug/mL HYD1 for 45 minutesprior to co-incubating the target myeloma cell line with HS-5 cells.Following 24 hrs of drug treatment apoptotic cells were measured byAnnexin V positivity by FACS analysis. SCID-Hu in vivo model: SCIDmice that were 4 to 6 weeks old received two fetal human bones subcutaneously(humerus, femur or tibia). Six weeks after implantation ofhuman bone, 5X104 RMPI8226 myeloma cells in PBS were injecteddirectly into the bone. Following four weeks of tumor engraftment, micewere randomized into drug treatment groups and tumor burden wasmeasured by circulating lambda levels as detected by ELISA. On day 28appropriate mice, were treated with either, vehicle control, HYD1 peptideor the scrambled peptide HYD1S which was administered I.P. dailyfor 14 days. For mice randomized to receive melphalan treatment, 1.5mg/Kg melphalan was administered I.P. on day 29 and day 32. Results.In this report we demonstrate that the beta 1 integrin D-amino acid inhibitory peptide HYD1, induces apoptosis as single agent in 8226 andH929 multiple myeloma cells lines. In addition, we show that HYD1treatment significantly enhanced melphalan induced cell death in suspensioncultures, as well as in the bone marrow stroma HS-5 co-culturemodel of drug resistance (p<0.05 students t-test). Finally in this reportwe show that HYD1 has activity as a single agent, and enhances melphalanactivity in the SCID-Hu in vivo model. HYD1 was injected I.P.indicating that the peptide is likely to have good bioavailability. Furthermore,our finding that HYD1 has activity as a single agent in vitro andin vivo indicates that multiple myeloma cells are dependent on beta 1integrin mediated signaling for cell survival. Further studies are warrantedto determine whether MM cells are more dependent on beta 1 integrinsignaling for cell survival compared to non-malignant plasma cells.Conclusions. Together these data provide essential proof of principle thatbeta 1 integrins represent a novel target for drug development in thetreatment of multiple myeloma. In addition, our data demonstrate thatHYD1 represents a potential novel therapeutic agent for enhancing theefficacy of standard chemotherapy in the treatment of multiple myeloma.These data provide the rationale for further pre-clinical studies ofHYD1, which may ultimately provide support for designing clinical trialswith HYD1 for the treatment of multiple myeloma.