Mariko Ishibashi

Marrow stromal cells induce B7-H1 expression on myeloma cells, generating aggressive characteristics in multiple myeloma.

Tumor-associated B7-H1 molecules inhibit antitumor immunity in some malignancies. We found that B7-H1 expression on patient myeloma cells and human myeloma cell lines (HMCLs) was upregulated by cultivating the cells with autologous stromal cells and the human stromal cell line HS-5. Among major cytokines produced by HS-5 cells, interleukin (IL)-6-induced B7-H1 expression on HMCLs. Moreover, HS-5 cell-mediated B7-H1 expression was downregulated by inhibiting IL-6. B7-H1+ HMCLs were more proliferative and less susceptible to antimyeloma chemotherapy compared with B7-H1− HMCLs. Moreover, the former cells showed higher levels of Bcl-2 and FasL expression than the latter. Finally, B7-H1 molecules on HMCLs induced T-cell apoptosis and anergy of tumor-specific T cells. Consistent with these in vitro observations, patients whose myeloma cells expressed high levels of B7-H1 had higher myeloma cell percentages in the bone marrow (BM) and higher serum lactate dehydrogenase levels compared with other myeloma patients. In addition, B7-H1 expression levels were often upregulated after myeloma patients relapsed or became refractory to therapy. Our data indicate that the BM microenvironment upregulates B7-H1 expression on myeloma cells, which links to the two biological actions of inducing T-cell downregulation and enhancing aggressive myeloma-cell characteristics. Modulating the B7-H1 pathway may be worthwhile in myeloma.

Myeloma drug resistance induced by binding of myeloma B7-H1 (PD-L1) to PD-1

B7-H1 suppresses T cells by binding to PD-1, but it is unclear how binding to B7-H1 on cancer cells affects tumors. “Reverse signaling” of B7-H1 on myeloma cells was found to induce drug resistance through the Akt-signaling pathway. B7 homolog 1 (B7-H1)–expressing myeloma cells not only inhibit myeloma-specific cytotoxic T lymphocytes (CTL), but also confer a proliferative advantage: resistance to antimyeloma chemotherapy. However, it remains unknown whether B7-H1 expressed on myeloma cells induces cellular responses associated with aggressive myeloma behaviors. To address this question, we analyzed the proliferation and drug sensitivity of B7-H1–expressing myeloma cells transfected with B7-H1–specific short-hairpin RNA or treated with programmed cell death (PD)-1-Fc–coupled beads. Knockdown of B7-H1 expression in myeloma cells significantly inhibited cell proliferation and increased apoptosis induced by the chemotherapeutic alkylating agent melphalan, with downregulation of the expression of cell cycle–related genes (CCND3 and CDK6) and antiapoptotic genes (BCL2 and MCL1). B7-H1 molecules thus contributed to myeloma cell-cycle progression and suppression of drug-induced apoptosis. B7-H1–expressing myeloma cells had a higher affinity for PD-1 than for CD80. PD-1-Fc bead–treated myeloma cells also became resistant to apoptosis that was induced by melphalan and the proteasome inhibitor bortezomib. Apoptosis resistance was associated with the PI3K/AKT pathway. Both myeloma cell drug resistance and antiapoptotic responses occurred through the PI3K/AKT signaling pathway, initiated from “reverse” stimulation of B7-H1 by PD-1. Therefore, B7-H1 itself may function as an oncogenic protein in myeloma cells. The interaction between B7-H1 on myeloma cells and PD-1 molecules not only inhibits tumor-specific CTLs but also induces drug resistance in myeloma cells through the PI3K/AKT signaling pathway. These observations provide mechanistic insights into potential immunotherapeutic benefits of blocking the B7-H1–PD-1 pathway. Cancer Immunol Res; 4(9); 779–88. ©2016 AACR.

Disease progression mechanism in myelodysplastic syndromes: insight into the role of the microenvironment.

The somatic mutation theory proposing that a sequential accumulation of genetic abnormalities plays a major role in cancer pathogenesis has not yet been confirmed for myelodysplastic syndromes (MDS). Meanwhile, recent data in some cancers has underscored the role of the microenvironment in tumor growth. MDS CD34+CD38- cells usually fail to repopulate after transplantation in mice, suggesting the importance of the microenvironment for MDS cells. Our recent data have provided a disease-progression model in which overproduction of interferon-γ and tumor necrosis factor-α in the microenvironment is the primary event. This causes B7-H1 molecule expression on MDS blasts, which generates a bifunctional signal inducing T-cell apoptosis and enhancing blast proliferation. The latter may provide more opportunity for developing secondary genetic changes.

Histone deacetylase inhibitor panobinostat induces calcineurin degradation in multiple myeloma

Multiple myeloma (MM) is a relapsed and refractory disease, one that highlights the need for developing new molecular therapies for overcoming of drug resistance. Addition of panobinostat, a histone deacetylase (HDAC) inhibitor, to bortezomib and dexamethasone improved progression-free survival (PFS) in relapsed and refractory MM patients. Here, we demonstrate how calcineurin, when inhibited by immunosuppressive drugs like FK506, is involved in myeloma cell growth and targeted by panobinostat. mRNA expression of PPP3CA, a catalytic subunit of calcineurin, was high in advanced patients. Panobinostat degraded PPP3CA, a degradation that should have been induced by inhibition of the chaperone function of heat shock protein 90 (HSP90). Cotreatment with HDAC inhibitors and FK506 led to an enhanced antimyeloma effect with a greater PPP3CA reduction compared with HDAC inhibitors alone both in vitro and in vivo. In addition, this combination treatment efficiently blocked osteoclast formation, which results in osteolytic lesions. The poor response and short PFS duration observed in the bortezomib-containing therapies of patients with high PPP3CA suggested its relevance to bortezomib resistance. Moreover, bortezomib and HDAC inhibitors synergistically suppressed MM cell viability through PPP3CA inhibition. Our findings underscore the usefulness of calcineurin-targeted therapy in MM patients, including patients who are resistant to bortezomib.

Functional expression of Tim-3 on blasts and clinical impact of its ligand galectin-9 in myelodysplastic syndromes

T-cell immunoglobulin mucin-3 (Tim-3), an inhibitory immune checkpoint receptor, is highly expressed on acute myeloid leukemia cells and its ligand galectin-9 is reported to drive leukemic progression by binding with Tim-3. However, it remains unclear whether the Tim-3–galectin-9 pathway is associated with the pathophysiology of myelodysplastic syndromes (MDS). Thus, we investigated the expression and function of Tim-3 and the clinical impact of its ligand galectin-9 in MDS. Tim-3 expression levels on MDS blasts by CD45/side-scatter or CD34/CD45 gating were increased as MDS progressed to the advanced stage. Tim-3 expression in the MDS blasts was upregulated in the presence of the cell culture supernatant of human stromal cells or the MDS-related cytokine transforming growth factor-β1. The proliferation of Tim-3+ MDS blasts was inhibited by the blockade of anti-Tim-3 antibody. Furthermore, plasma levels of galectin-9 were elevated as MDS progressed to the advanced stage in 70 MDS/acute leukemia transformed from MDS patients and was a prognostic factor in 40 MDS patients. Our data demonstrated that the Tim-3-galectin-9 pathway is associated with the pathogenesis and disease progression of MDS. These findings provide new insight into potential immunotherapy targeting the galectin-9–Tim-3 pathway in MDS.

Clinical impact of serum soluble SLAMF7 in multiple myeloma

The signaling lymphocytic activation molecule family (SLAMF7; also known as CS1 or CD319) is highly expressed on plasma cells from multiple myeloma (MM) as well as natural killer (NK) cells and is a well-known therapeutic target of elotuzumab. The objective of this study was to evaluate the clinical significance of serum soluble SLAMF7 (sSLAMF7) levels in patients with MM (n=103) and furthermore the impact of sSLMF7 on the antitumor activity of anti-SLAMF7 antibody. Thirty-one percent of MM patients, but not patients with monoclonal gammopathy of undetermined significance and healthy controls, had detectable levels of serum sSLAMF7, which were significantly increased in advanced MM patients. Further, MM in sSLAMF7-postive patients exhibited aggressive clinical characteristics with shorter progression-free survival times in comparison with sSLAMF7-negative patients. In responders to MM therapy, the levels of sSLAMF7 were undetectable or decreased compared with those before treatment. In addition, the anti-SLAMF7 antibody-mediated antibody-dependent cellular cytotoxicity of NK cells against MM cell lines was inhibited by recombinant SLAMF7 protein. Thus, our findings suggest that high concentrations of sSLAMF7, which could transiently suppress the therapeutic effects of elotuzumab, may be a useful indicator of disease progression in MM patients.