HM1.24 (CD317) is a novel target against lung cancer for immunotherapy using anti-HM1.24 antibody

HM1.24 antigen (CD317) was originally identified as a cell surface protein that is preferentially overexpressed on multiple myeloma cells. Immunotherapy using anti-HM1.24 antibody has been performed in patients with multiple myeloma as a phase I study. We examined the expression of HM1.24 antigen in lung cancer cells and the possibility of immunotherapy with anti-HM1.24 antibody which can induce antibody-dependent cellular cytotoxicity (ADCC). The expression of HM1.24 antigen in lung cancer cells was examined by flow cytometry as well as immunohistochemistry using anti-HM1.24 antibody. ADCC was evaluated using a 6-h ⁵¹Cr release assay. Effects of various cytokines on the expression of HM1.24 and the ADCC were examined. The antitumor activity of anti-HM1.24 antibody in vivo was examined in SCID mice. HM1.24 antigen was detected in 11 of 26 non-small cell lung cancer cell lines (42%) and four of seven (57%) of small cell lung cancer cells, and also expressed in the tissues of lung cancer. Anti-HM1.24 antibody effectively induced ADCC in HM1.24-positive lung cancer cells. Interferon-β and -γ increased the levels of HM1.24 antigen and the susceptibility of lung cancer cells to ADCC. Treatment with anti-HM1.24 antibody inhibited the growth of lung cancer cells expressing HM1.24 antigen in SCID mice. The combined therapy with IFN-β and anti-HM1.24 antibody showed the enhanced antitumor effects even in the delayed treatment schedule. HM1.24 antigen is a novel immunological target for the treatment of lung cancer with anti-HM1.24 antibody.     

Bst-2/HM1.24 is a raft-associated apical membrane protein with an unusual topology

An expression screen of a rat cDNA library for sequences encoding Golgi-localized integral membrane proteins identified a protein with an apparent novel topology, i.e. with both an N-terminal transmembrane domain and a C-terminal glycosyl-phosphatidylinositol (GPI) anchor. Our data are consistent with this. Thus, the protein would have a topology that, in mammalian cells, is shared only by a minor, but pathologically important, topological isoform of the prion protein (PrP). The human orthologue of this protein has been described previously (BST-2 or HM1.24 antigen) as a cell surface molecule that appears to be involved in early pre-B-cell development and which is present at elevated levels at the surface of myeloma cells. We show that rat BST-2/HM1.24 has both a cell surface and an intracellular (juxtanuclear) location and is efficiently internalized from the cell surface. We also show that the cell surface pool of BST-2/HM1.24 is predominantly present in the apical plasma membrane of polarized cells. The fact that rat BST-2/HM1.24 apparently possesses a GPI anchor led us to speculate that it might exist in cholesterol-rich lipid microdomains (lipid rafts) at the plasma membrane. Data from several experiments are consistent with this localization. We present a model in which BST-2/HM1.24 serves to link adjacent lipid rafts within the plasma membrane.     

IL-6-induced Bcl6 variant 2 supports IL-6-dependent myeloma cell proliferation and survival through STAT3

IL-6 is a growth and survival factor for myeloma cells, although the mechanism by which it induces myeloma cell proliferation through gene expression is largely unknown. Microarray analysis showed that some B-cell lymphoma-associated oncogenes such as Bcl6, which is absent in normal plasma cells, were upregulated by IL-6 in IL-6-dependent myeloma cell lines. We found that Bcl6 variant 2 was upregulated by STAT3. ChIP assay and EMSA showed that STAT3 bound to the upstream region of variant 2 DNA. Expression of p53, a direct target gene of Bcl6, was downregulated in the IL-6-stimulated cells, and this process was impaired by an HDAC inhibitor. Bcl6 was knocked down by introducing small hairpin RNA, resulting in decreased proliferation and increased sensitivity to a DNA damaging agent. Thus, STAT3-inducible Bcl6 variant 2 appears to generate an important IL-6 signal that supports proliferation and survival of IL-6-dependent myeloma cells.     

Epigenetic silencing of the human nucleotide excision repair gene, hHR23B, in interleukin-6-responsive multiple myeloma KAS-6/1 cells

During tumorigenesis, selective proliferative advantage in certain cell subsets is associated with accumulation of multiple genetic alterations. For instance, multiple myeloma is characterized by frequent karyotypic instability at the earliest stage, progressing to extreme genetic abnormalities as the disease progresses. These successive genetic alterations can be attributed, in part, to defects in DNA repair pathways, perhaps based on epigenetic gene silencing of proteins involved in DNA damage repair. Here we report epigenetic hypermethylation of the hHR23B gene, a key component of the nucleotide excision repair in response to DNA damage, in interleukin-6 (IL-6)-responsive myeloma KAS-6/1 cells. This hypermethylation was significantly abated by Zebularine, a potent demethylating agent, with a consequent increase in the hHR23B mRNA level. Subsequent removal of this drug and supplementation with IL-6 in the culture medium re-established DNA hypermethylation of the hHR23B gene and silencing of mRNA expression levels. The inclination of DNA to be remethylated, at least within the hHR23B gene promoter region, reflects an epigenetic driving force by the cytogenetic/tumorigenic status of KAS-6/1 myeloma. The IL-6 response of KAS-6/1 myeloma also raises a question of whether the proneoplastic growth factor, such as IL-6, supports the epigenetic silencing of important DNA repair genes via promoter hypermethylation during the development of multiple myeloma.     

Suppression of IL-2-induced T cell proliferation and phosphorylation of STAT3 and STAT5 by tumor-derived TGF beta is reversed by IL-15.

IL-2 responses are susceptible to suppression by TGFbeta, a cytokine widely implicated in suppression of inflammatory responses and secreted by many different tumor cell types. There have been conflicting reports regarding inhibition of IL-2-induced STAT3 and STAT5 phosphorylation by TGFbeta and subsequent suppression of immune responses. Using TGFbeta-producing multiple myeloma tumor cells we demonstrate that tumor-derived TGFbeta can block IL-2-induced proliferation and STAT3 and STAT5 phosphorylation in T cells. High affinity IL-2R expression was required for the suppression of IL-2 responses as a novel CD25(-) T cell line proliferated and phosphorylated STAT3 when cultured with tumor cells or rTGFbeta1. Activating T cells with IL-15, which does not use the high affinity IL-2R, completely restored the ability of T cells to phosphorylate STAT3 and STAT5 when cultured with tumor cells. IL-15-treated T cells proliferated normally when cocultured with tumor cells or rTGFbeta1, whereas IL-2 responses were consistently inhibited. Preincubation with IL-15 also restored the ability of T cells to respond to IL-2 by phosphorylating STAT3 and STAT5, and proliferating normally in the presence of tumor cells. IL-2 pretreatment did not restore T cell function. IL-15 also restored T cell responses by T cells from multiple myeloma patients, and against freshly isolated bone marrow tumor samples. Thus, activation of T cells by IL-15 renders T cells resistant to suppression by TGFbeta1-producing tumor cells and rTGFbeta1. This finding may be exploited in the design of new immunotherapy approaches that will rely on T cells avoiding tumor-induced suppression.