alpha-chymotrypsin and Waldenstrom-Macroglobulinemia

Primary Waldenstrom's Macroglobulinemia (WM) cells present with a significantly higher level of the immunoproteasome compared with the constitutive proteasome. It has been demonstrated that selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive-(c20S) and immuno-(i20S) proteasome represents a valid strategy to induce antineoplastic effect in hematologic tumors. We therefore evaluated carfilzomib, a potent selective, irreversible inhibitor of the CT-L activity of the i20S and c20S in WM cells.. We tested the effect of carfilzomib on survival and proliferation of primary WM cells, as well as of other IgM-secreting lymphoma cell lines. Carfilzomib-dependent mechanisms of induced apoptosis in WM cells, and its effect on WM cells in the context of bone marrow (BM) microenvironment have been also evaluated. Moreover, the combinatory effect of carfilzomib and bortezomib has been investigated. In vivo studies have been performed.. We demonstrated that carfilzomib targeted the CT-L activity of both i20S and c20S, which led to the induction of toxicity in primary WM cells, as well as in other IgM-secreting lymphoma cells. Importantly, carfilzomib targeted WM cells even in the context of BM milieu. In addition, carfilzomib induced apoptosis through c-jun-N-terminal-kinase activation, caspase cleavage, and initiation of unfolded protein response. Importantly, the combination of carfilzomib and bortezomib synergistically inhibited CT-L activity, as well as caspase-, PARP-cleavage and GRP94 expression. Antitumor activity of carfilzomib has been validated in vivo.. These findings suggest that targeting i20S and c20S CT-L activity by carfilzomib represents a valid antitumor strategy in WM and other IgM-secreting lymphomas.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone Marrow Cells; Boronic Acids; Bortezomib; Caspases; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Survival; Chemokine CXCL12; Chymotrypsin; Coculture Techniques; DNA Fragmentation; Drug Synergism; Enzyme Activation; Female; Humans; JNK Mitogen-Activated Protein Kinases; Lymphoma; Mice; Mice, SCID; Oligopeptides; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteasome Inhibitors; Pyrazines; Serine Proteinase Inhibitors; Unfolded Protein Response; Waldenstrom Macroglobulinemia

Proteasome inhibition represents a valid antitumor approach and its use has been validated in Waldenström macroglobulinemia (WM), where bortezomib has been successfully tested in clinical trials. Nevertheless, a significant fraction of patients relapses, and many present toxicity due to its off-target effects. Selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive proteasome 20S (c20S) and immunoproteasome 20S (i20S) represents a sufficient and successful strategy to induce antineoplastic effect in hematologic tumors. We therefore studied ONX0912, a novel selective, irreversible inhibitor of the CT-L activity of i20S and c20S. Primary WM cells express higher level of i20S compared with c20S, and that ONX0912 inhibited the CT-L activity of both i20S and c20S, leading to induction of toxicity in primary WM cells, as well as of apoptosis through c-Jun N-terminal kinase activation, nuclear factor kappaB (NF-kappaB) inhibition, caspase cleavage, and initiation of the unfolded protein response. Importantly, ONX0912 exerted toxicity in WM cells, by reducing bone marrow (BM)-derived interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1) secretion, thus inhibiting BM-induced p-Akt and phosphorylated extracellular signal-related kinase (p-ERK) activation in WM cells. These findings suggest that targeting i20S and c20S CT-L activity by ONX0912 represents a valid antitumor therapy in WM.

Topics: Apoptosis; Chymotrypsin; Dipeptides; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; Immunoblotting; Insulin-Like Growth Factor I; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Lymphoma; NF-kappa B; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazoles; Waldenstrom Macroglobulinemia

Human J chain isolated from the plasma of a patient with Waldenstrom's macroglobulinemia was subjected to extended and limited digestion with trypsin and chymotrypsin. The digests were fractionated by combination of column chromatography and high voltage paper electrophoresis. Peptide purity was established by their amino acid analysis and a single amino terminal residue. All the necessary peptides which would provide the total primary structure of molecule were thus obtained.

Topics: Amino Acids; Chymotrypsin; Humans; Immunoglobulin J-Chains; Peptide Fragments; Trypsin; Waldenstrom Macroglobulinemia

The primary structure of the J chain from a human Waldenströms IgM protein has been determined using a combination of automated and conventional Edman degradative procedures. Eighty-five percent of the sequence was established with peptides isolated from tryptic digests of carboxyamidomethylated and citraconylated J chain, many of which were sequenced completely. Alignment of the tryptic fragments was achieved with peptides generated by chymotrypsin and limited acid hydrolyses. The j chain consits of 129 amino acids and a single oligosaccharide structure linked to asparagine at positon 43 of the sequence. The molecular weight, including 7.5% carbohydrate by weight, is 16 422. The location and arrangement of three half-cystines could be deduced from previous studies, whereas the pairing of the remaining five disulfide bonds still needs to be clarified.

Topics: Amino Acid Sequence; Amino Acids; Chemical Phenomena; Chemistry; Chymotrypsin; Humans; Hydrolysis; Immunoglobulin J-Chains; Peptide Fragments; Trypsin; Waldenstrom Macroglobulinemia

Topics: Animals; Antigens; Chemical Phenomena; Chemistry; Chromatography, Gel; Chymotrypsin; Complement Fixation Tests; Cysteine; Electrophoresis, Disc; Hexoses; Humans; Immunodiffusion; Immunoelectrophoresis; Immunoglobulin M; Macroglobulins; Molecular Weight; Oxidation-Reduction; Papain; Peptides; Rabbits; Ultracentrifugation; Waldenstrom Macroglobulinemia

Topics: Amino Acid Sequence; Autoanalysis; Chromatography, Gel; Chromatography, Ion Exchange; Chymotrypsin; Dialysis; Electrophoresis; Freeze Drying; gamma-Globulins; Humans; Hydrogen-Ion Concentration; Pepsin A; Peptides; Protein Hydrolysates; Waldenstrom Macroglobulinemia

Topics: Amino Acids; Carbohydrates; Chromatography, Gel; Chromatography, Ion Exchange; Chromatography, Thin Layer; Chymotrypsin; Hexosamines; Hexoses; Humans; Immunodiffusion; Macroglobulins; Neuraminic Acids; Ribose; Waldenstrom Macroglobulinemia