Alex W. Tong

CD40 ligand-induced apoptosis is Fas-independent in human multiple myeloma cells.

We and others previously demonstrated that human multiple myeloma (MM) cells express CD40 and have an active CD40-growth regulatory pathway. This study characterizes the growth outcome of soluble (gp39) or membrane-bound recombinant human CD40-ligand (rCD4()L) and its relationship with Fas-dependent apoptosis. Contrary to the moderate growth-stimulatory effect of the CD40-MAb G28.5, gp39 inhibited 3H-thymidine uptake of the plasma dyscrasia lines ARH-77, U266, and HS-Sultan in a dose-dependent fashion by up to 82%. By comparison, RPMI 8226 cells were resistant to CD40L-growth modulation, which may be attributable to a single base substitution (TCA→TTA, serine→leucine) at the 3rd cysteine-rich extramembrane region of CD40. Gp39 similarly reduced myeloma clono-genic colony (MCC) formation in patient primary bone marrow cultures by 50% (40-76%; n=6). Studies using transfectant L cells that constitutively expressed CD40L showed that membrane-bound CD40L inhibited the growth of ARH-77, U266, and HS-Sultan cells (66%, 63%, and 32%. respectively), whereas untransfected L cells did not. Growth inhibition by gp39 or CD40L+ L cells was neutralized by coincubation with the CD40L antibodies 5c8 or LL48. CD40L-treatment increased apoptotic activity of MM cells, as defined by oligonucleo-somal DNA fragmentation and an increased binding to annexin V (16-28%). All three untreated CD40-responsive MM lines expressed the Fas/Apo-1/CD95 antigen (65-92% CD95+). However, only ARH-77 cells responded to the growth inhibitory effect of the CD95-agonistic antibody CH-II. CD95 expression was not affected significantly by gp39 treatment, and growth inhibition by CH-11 was additive to gp39 (from 42% to 64% decrease in H-thmidine uptake). Conversely, the CD95 antagonist antibody ZB4 reversed the Fas-dependent growth inhibitory process but did not significantly alter gp39-mediated growth outcome. Gp39 treatment lowered the expression of TNFR-associated factors TRAF4 and TRAF6 by 38% and 32%, respectively, whereas detectable levels of TRAF1,2,3, and 5 levels remained unchanged. Our observations indicate that the CD40L-binding inhibits human MM cell growth and increases its apoptotic activity. This growth inhibitory effect corresponds to lower levels of cytoplasmic TRAF signaling elements, and appears independent of the Fas-signaling pathway. CD40 receptor mutation may lead to unresponsiveness to CD40 growth modulation in multiple myeloma cells.

CD40 and the Effect of Anti-CD40-Binding on Human Multiple Myeloma Clonogenicity

CD40 is a 48 kDa glycosylated phospoprotein that is a member of the tumor necrosis factor receptor (TNF-R) superfamily. CD40 was originally identified in B lymphocytes, and is found on monocytes, dendritic cells, some carcinoma cell lines, and the thymic epithelium. CD40 is expressed on normal pre-B through mature B stages of differentiation. For normal B cells, the cross-linking of CD40 induces cell cycle progression, long-term proliferation in vitro, IgE secretion, increased adhesion molecule (LFA-1) expression, and low level IL-6 secretion. The natural ligand of CD40 (CD40L, gp39, or T-BAM, for T-B cell activating molecule) was recently identified as an inducible molecule expressed transitionally on activated T cells. Although originally believed to be absent in normal and malignant plasma cells, CD40 has been demonstrated on the majority of myeloma cell lines and myeloma cells from plasma cell dyscrasia (PCD) patient specimens tested. CD40 activation modulated myeloma cell proliferation and clonogenicity in vitro, suggesting that the CD40 pathway is active in myeloma cell growth. For the IL-6 dependent cell line ANBL-6, CD40 activation was associated with autocrine IL-6 production. However, the IL-6 pathway does not appear to play a predominant role in CD40 activation of non-IL-6-dependent MM cell lines and patient primary bone marrow cultures. The possible pathophysiologic role of the CD40 receptor in human multiple myeloma is discussed.

Generation of a ribozyme-adenoviral vector against K-ras mutant human lung cancer cells.

Abstractras mutations represent one of the most common oncogenetic lesions in human non-small cell lung cancer (NSCLC) and adversely affect the survival of patients afflicted with this disease. ras-directed gene therapy in the past employed primarily antisense oligonucleotides (AS-ODN) or expression vectors (such as a viral vector construct) that deliver the antisense sequence to inactivate the mutant oncogene message. These approaches produced minimal toxicity, and yet were limited in efficacy. Ribozymes present a viable alternative in antisense therapy by virtue of their renewable catalytic capability for site-specific RNA cleavage. We recently produced an adenoviral vector with a hammerhead ribozyme transgene (KRbz) that is specific for the K-ras codon 12 mutant sequence GUU, given the considerations that (a) in the United States, approx 30% of human NSCLCs express K-ras oncogene mutations, nearly all of which reside in codon 12; (b) anti-K-ras, anti-H, as well as anti-N-ras hammerhead ribozymes are potent growth inhibitors in various human cancers tested; and (c) in vitro and animal model studies suggest that ribozymes directed at oncogene (K- and H-ras C-fos, BCR-ABL) or human immunodeficiency viral gene messages are more effective than their antisense counterpart. This article describes the techniques involved in the production of the KRbz-adenoviral vector that is specific for the K-ras mutation GTT, and summarizes its in vivo antitumor effect against NSCLC xenografts expressing the relevant K-ras mutation in athymic mice.

Preclinical Assessment of wt GNE Gene Plasmid for Management of Hereditary Inclusion Body Myopathy 2 (HIBM2)

Hereditary Inclusion Body Myopathy (HIBM2) is a chronic progressive skeletal muscle wasting disorder which generally leads to complete disability before the age of 50 years. There is currently no effective therapeutic treatment for HIBM2. Development of this disease is related to expression in family members of an autosomal recessive mutation of the GNE gene, which encodes the bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE/MNK). This is the rate limiting bifunctional enzyme that catalyzes the first 2 steps of sialic acid biosynthesis. Decreased sialic acid production, consequently leads to decreased sialyation of a variety of glycoproteins including the critical muscle protein alpha-dystroglycan (α-DG). This in turn severely cripples muscle function and leads to the onset of the syndrome. We hypothesize that replacing the mutated GNE gene with the wildtype gene may restore functional capacity of GNE/MNK and therefore production of sialic acid, allowing for improvement in muscle function and/or delay in rate of muscle deterioration. We have constructed three GNE gene/CMV promoter plasmids (encoding the wildtype, HIBM2, and Sialuria forms of GNE) and demonstrated enhanced GNE gene activity following delivery to GNE-deficient CHO-Lec3 cells. GNE/MNK enzyme function was significantly increased and subsequent induction of sialic acid production was demonstrated after transfection into Lec3 cells with the wild type or R266Q mutant GNE vector. These data form the foundation for future preclinical and clinical studies for GNE gene transfer to treat HIBM2 patients.

Prognostic role of K-ras in patients with progressive colon cancer who received treatment with Marimastat (BB2516).

Abstract We determined the prognostic role of K-ras mutation in tumor tissue of patients with refractory colon cancer who received Marimastat (BB2516). DNA was extracted from paraffin-stored tumor tissue of 27 patients who previously failed 5-fluorouracil and were treated with BB2516. The presence of K-ras mutation was characterized by Polymerase Chain Reaction using ras- and p53-specific primers, ras and p53 oncoprotein expression was analyzed by an automated biotin-avidin immunoproxidase technique. Seventeen patients had a normal K-ras sequence and 10 patients had a K-ras mutation. Median survival of patients with a normal ras sequence was 330 days from the time of BB2516 treatment compared with 160 days for patients with a K-ras mutation (p = 0.0442, Wilcoxon; 0.0130 Log-Rank). No differences in age, sex, cancer stage, surgical treatment, or chemotherapy treatment were observed. Abnormalities involving ras expression did not affect survival. By comparison, median survival for patients with p53 mutation or p53 overexpression was both 158 days after BB2516 treatment. Patients having both K-ras and p53 mutations had the poorest median survival of 113 days (p = 0.035). There is a suggestion by univariate analysis that the presence of a K-ras mutation may predict survival in patients with progressive colon cancer. Further assessment with larger patient numbers and multivariate analysis is indicated.

Anti-K- ras 1 Ribozyme Adenoviral Vector for Gene Therapy of Non-Small Cell Lung Cancer

Lung cancer is the leading cause of cancer death for men and women in the United States. Several factors affect survival in nonsmall-cell lung cancer [NSCLC; i.e., stage, age, Karnofsky Performance Status (KPS)]. The 5-yr survival rate is<15% for newly diagnosed cases following conventional treatments. Tumor oncogenetic defects appear to be associated with adverse survival (1,2). The presence of a ras mutation or p21(ras) oncoprotein overexpression correlates with an unfavorable prognosis for patients with nonsmall-cell lung cancer (3). These findings suggest that abnormal ras function contributes to pathophysiology, and raise the possibility that reversal of aberrant ras activity may serve as a viable therapeutic approach.

Quantification of Non-Light Chain Amyloid Precursors and Acute Phase-Related Proteins in AL-Amyloid Sera.

This study examined the serum concentration of various non-light chain amyloid-related proteins and enzymes in patients (pts) with plasma cell dyscrasia+amyloid (AL, n=13), plasma cell dyscrasia without amyloid (PCD, n=28), pts with non-PCD non-AL disorders (NPCD, n=13), and normal controls (n=30). Compared with normals, AL pts had significantly lower serum levels of prealbumin and plasminogen. s2-microglobulin and protease inhibitors α1-antichymotrypsin, α1-antitrypsin and α2-macroglobulin were significantly elevated. Thirty-eight percent of AL sera (vs. 46% of normal controls) contained ≥0.03 mg/ml of amyloid P-component (SAP). No serum amyloid A (SAA) was detectable in AL pts, despite significant elevation of C-reactive protein (CRP) levels. PCD pts had below normal prealbumin, and elevated values of s2-microglobulin, α1-antichymotrypsin, α2-macroglobulin and CRP. Plasminogen and αl-antitrypsin levels in PCD pts did not differ from normals. NPCD sera contained elevated levels of all three protease inhibitors, whereas the other proteins were within normal limits. Serum levels of interleukin-6 (IL6) were increased in AL and NPCD groups. These observations suggest that AL is accompanied by quantitative changes in serum non-light chain amyloid precursors and acute phase-related proteins; these altered levels may be relevant to amyloid deposition.

Expression of Plasma Cell-Associated Non-Light Chain Antigens in Patients with Plasma Cell Dyscrasia and Amyloidosis

In this study, we examined reactivity of the plasma cell-reactive monoclonal antibodies MM4 and PCA-1 with bone marrow and other tissue biopsies from patients with documented amyloidosis. Thirty-six biopsies (bone marrow, GI tract, gall bladder, rectum, peripheral nerve, heart, lung, kidney, liver, spleen, tongue, skin, muscle, and adrenal gland) from 17 patients were evaluated independently by standard histopathology. The presence of tissue amyloid was established by the finding of green birefrigence under polarized light after Congo red staining and/or characteristic fibrillar structure by electron microscopy. Cases studied included 13 patients with plasma cell dyscrasia (PCD) and 4 patients without evidence of an M-protein (non-PCD). Reactivity with MM4 or PCA-1 was established by the immunoperoxidase technique, based on comparison with a negative control (no primary monoclonal antibody) section from the same biopsy. MM4 and PCA-1 reacted with marrow plasma cells from the majority of these amyloid patients. In addition, a number of solid organ biopsies demonstrated positive staining with MMA or both monoclonal antibodies in areas of tissue amyloid deposits. Reactivity of either antibody with plasma cells and/or tissue amyloid was independent of light chain isotype. These observations suggest that plasma cells from patients with amyloidosis commonly express MM4-and PCA-1-reactive antigens, which also may be detected in certain tissue amyloid deposits. Further studies with these or similar reagents may help in understanding the pathogenesis of immunocytic amyloidosis.