The online version of this article has a Supplementary Appendix. BackgroundMultiple myeloma is characterized by the accumulation of tumor plasma cells in the bone marrow. Despite therapeutic improvements brought by proteasome inhibitors such as bortezomib, myeloma remains an incurable disease. In a variety of human cancers, human immunodeficiency virus protease inhibitors (e.g. nelfinavir) effectively inhibit tumor progression, but their impact on myeloma is unknown. We assessed the in vitro and in vivo effects of nelfinavir on multiple myeloma. Design and MethodsThe effects of nelfinavir (1-10 mM) on proteasome activity, proliferation and viability of myeloma cell lines and plasma cells from patients were assessed by measuring PERK, AKT, STAT3 and ERK1/2 phosphorylation and CHOP expression with immunoblotting or flow cytometry. The in vivo effect was assessed in NOD/SCID mice injected with luciferase expressing human myeloma cell lines and treated with nelfinavir at a dose of 75 mg/kg/day. Tumor progression was evaluated using a bioluminescent system. ResultsNelfinavir inhibited 26S chymotrypsin-like proteasome activity, impaired proliferation and triggered apoptosis of the myeloma cell lines and fresh plasma cells. It activated the pro-apoptotic unfolded protein response pathway by inducing PERK phosphorylation and CHOP expression. Cell death triggered by nelfinavir treatment correlated with decreased phosphorylation of AKT, STAT3 and ERK1/2. Nelfinavir enhanced the anti-proliferative activity of bortezomib, dexamethasone and histone deacetylase inhibitors and delayed tumor growth in a myeloma mouse model. ConclusionsThese results suggest that nelfinavir, used at a pharmacological dosage, alone or in combination, may be useful in the treatment of myeloma. Our data provide a preclinical basis for clinical trials using nelfinavir in patients with myeloma.Key words: HIV protease inhibitor; nelfinavir; proteasome; multiple myeloma; apoptosis; PI3K/AKT. Haematologica 2012;97(7):1101-1109. doi:10.3324/haematol.2011 This is an open-access paper. Citation: Bono C, Karlin L, Harel S, Mouly E, Labaume S, Galicier L, Apcher S, Sauvageon H, Fermand J-P, Bories J-C and Arnulf B. The human immunodeficiency virus-1 protease inhibitor nelfinavir impairs proteasome activity and inhibits the proliferation of multiple myeloma cells in vitro andin vivo. The human immunodeficiency virus-1 protease inhibitor nelfinavir impairs proteasome activity and inhibits the proliferation of multiple myeloma cells in vitro and in vivo ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n

We previously identified IgM autoantibodies in the sera of patients with Wiskott-Aldrich syndrome (WAS) that react with a subset of normal human B lymphocytes and induce B cell differentiation in vitro. From splenocytes of a patient with WAS we generated heterohybridomas (HY18 and HY21) and a lymphoblastoid cell line (LWA10) that produce human IgM lambda or IgM kappa anti-B lymphocyte autoantibodies, respectively. Immunohistochemical and multiparameter flow cytometric analyses demonstrate that these autoantibodies are specific for lymphocytes of the B lineage and preferentially stain B cells that reside in the mantle zone of secondary follicles and that constitutively co-express the CD5 surface antigen and most major autoantibody-associated cross-reactive idiotypes; in addition, these antibodies stain most pre-B cells in adult bone marrow. Molecular studies show that these anti-B lymphocyte autoantibodies are encoded by a highly conserved VH4 gene, designated VH4.21. The gene encodes a number of autoantibodies, especially anti-i and anti-I IgM cold agglutinins. Hemagglutination and surface labeling studies reveal that HY18 and LWA10 recognize the "i" carbohydrate antigenic determinant(s) which is classically found on human cord red blood cells and, as shown now by this study, on a subpopulation of human B cells which expresses it early in B cell development. These studies raise the possibility that the gene product encoded by this highly conserved germ-line VH4 gene may play a physiological role in B cell development and/or differentiation.

Present study confirms in vivo the in vitro effects of ATO on myeloma cells. Delayed relapses were observed suggesting that prolonged or maintenance therapy has to be considered in future clinical trials. Whether or not this will translate into clinically relevant effect of the drug in myeloma patients deserves further consideration.

Idiotype-bearing and antigen-binding receptors produced by blood T lymphocytes in a case of human myeloma"Twenty-five t o 30 % of blood lymphocytes from a myeloma patient with an IgGl(K) protein with antibody activity to horse a2-macroglobulin (a2M) reacted with purified IgG from a rabbit antiserum t o the idiotypic determinants of this protein. A small proportion of these idiotype-bearing lymphccytes were IgG-bearing B cells, but most of them were T cells. Several experiments demonstrated the actual synthesis of the idiotypic structures carried by the T lymphocytes. All idiotype-bearing lymphocytes bound horse a2M, and the structures that bore the idiotype and bound the antigen were shown t o completely cocap. After biosynthetic labeling with I4C-labeled amino acids, immunoprecipitation and sodium dodecyl sulfate gel electrophoresis, the antiidiotypic serum and the a2M antigen were found t o react with the same molecules. Preliminary data & the size of these antigen-binding T lymphocytederived molecules are in accord with those of Binz and Wigzell ( S c u d . ; I . Immunol. 1976. 5: 559) in the rat. The finding in this patient with multiple myeloma of a homogeneous and possibly malignant population of T lymphocytes, synthesizing identical antigen receptors which share idiotypic (but not isotypic) determinants of the immunoglobulin molecule produced by the malignant B cell clone, therefore confirms in man the results of several recent studies in other mammalian species. Abbreviations: PBL: Peripheral blood lymphocytes IBL: Idiotypebearidg lymphocytes; lymphocytes bearing (and synthesizing) the same idiotypic determinants as those of the serum myeloma protein SIg: Surface immunoglobulins a2M: ~~2-Macroglobulin RFC: Rosette-forming cells SDS: Sodium dodecyl sulfateThe presence, in human and murine myelomas, of peripheral blood lymphocytes (PBL) bearing on their membrane molecules which react with antisera specific for the individual myeloma protein idiotypic determinants, has been reported by several groups [l-41. The fact that these molecules were actual cell products was demonstrated by their regrowth after stripping of the membrane by proteolytic enzymes [3-51.Blood lymphocytes which carry the idiotypic determinants of the myeloma protein, here referred t o as "idiotype-bearing lymphocytes" (IBL), were considered t o be B cells. This interpretation is supported by our finding, in about one third of untreated myeloma patients, of "monoclonal" populations