Faculty Bibliography

The basis for persistence of leukemic stem cells in the bone marrow microenvironment remains poorly understood. We present evidence that signaling cross-talk between alpha4 integrin and Abelson interactor-1 (Abi-1) is involved in the acquisition of an anchorage-dependent phenotype and drug resistance in Bcr-Abl-positive leukemia cells. Comparison of Abi-1 (ABI-1) and alpha4 integrin (ITGA4) gene expression in relapsing Bcr-Abl-positive CD34+progenitor cells demonstrated a reduction in Abi-1 and an increase in alpha4 integrin mRNA in the absence of Bcr-Abl mutations. This inverse correlation between Abi-1 and alpha4 integrin expression, as well as linkage to elevated phospho-Akt and phospho-Erk signaling, was confirmed in imatinib mesylate -resistant leukemic cells. These results indicate that the alpha4-Abi-1 signaling pathway may mediate acquisition of the drug-resistant phenotype of leukemic cells.

Impairing dendritic cell (DC) function to prevent graft versus host disease (GvHD) is an appealing concept. DC antigen presentation is NF-kappaB pathway-dependent and bortezomib might therefore play a role in preventing alloreactivity. We obtained DC from the blood of patients enrolled in a phase I study using post-transplant cyclophosphamide and bortezomib for prevention of GvHD. Control samples were obtained from patients receiving standard GvHD prevention regimen. Pre-treatment samples were also collected from enrolled patients. DC isolated on days +1, +4, and +7 showed progressive decrease in the expression of maturation markers in comparison to control. In a DC-CD4+ mixed lymphocyte reaction (MLR) where DC isolated from the recipient blood before graft infusion were the stimulator cells, T cell proliferation measured by bromodeoxyuridine (BrdU) integration was decreased in samples obtained on days +14 and +21 in comparison to control group. Finally, measured by real-time PCR, the expression of IkappaB progressively increased while the expression of NF-kappaB decreased in DC on days +1, +4, and +7, in comparison to pre-treatment paired controls. We conclude that our data further justify exploring the role of bortezomib in GvHD prevention and propose a novel mechanism of action of bortezomib in DC.

Dendritic cells (DC) play a central role in the pathophysiology of graft versus host disease (GvHD). Their antigen presenting capacity is nuclear factor kappaB- (NF-kappaB) dependent. Consequently, DC have emerged as a potential target for the prevention of GvHD and clinical trials with bortezomib are underway. We explored the activity of novel proteasome and immunoproteasome inhibitors on healthy volunteer peripheral blood DC. After incubation with the drug or drug combination, DC were stimulated with lipopolysaccharide, stained for maturation surface markers and then analyzed by flow cytometry. We found that the different molecule(s) inhibited DC maturation marker expression to variable degrees, with the constitutive proteasome-selective agent being the least active. In a DC and allogeneic CD4+ mixed lymphocyte reaction, DC incubation with the studied proteasome and immunoproteasome inhibitor(s), impeded T cell proliferation as measured by BrDU incorporation. Finally, we found that DC incubation with the drug(s) enhanced IkappaB expression and that oprozomib inhibited NF-kappaB expression. We concluded that based on its activity and oral bioavailability, oprozomib merits further investigation in an animal GvHD prevention model. We also suggest that altering IkappaB and NF-kappaB expressions may, in DC, represent a new mechanism of action of proteasome and immunoproteasome inhibitors.

BACKGROUND: A significant barrier to islet transplantation is the rapid loss of human islet function in vivo. The present study evaluates whether bone marrow (BM) could be used to support human islet survival and function in vivo. METHODS: We cocultured human islets and BM for 3 weeks before transplantation into the left subrenal capsule of diabetic severe combined immunodeficient mice. RESULTS: The cocultured human islets before transplantation demonstrated improved viability, increased size, and migration capacity in vitro. After 4 months, animals transplanted with precultured BM/islets exhibited euglycemia and detectable human insulin levels (157 muU/mL), whereas no human insulin was detected in the islet-only transplantation group. Furthermore, the removal of the transplants on day 126 resulted in hyperglycemia, indicating that the reduction of blood glucose was dependent on the transplants. Diabetic mice transplanted with BM/islets demonstrated the longest survival period (130 vs. 40 days for those with islet-only transplants). The transplanted BM/islets showed signs of vascularization and migration from the renal capsule into medulla. CONCLUSIONS: Our results suggest that BM precultured with human islets may enhance the survival and function of transplanted islets, thus significantly improving the therapeutic efficacy of islet transplantation for type 1 diabetes.