Jingda Xu

Th9 cells promote antitumor immune responses in vivo

Th9 cells are a subset of CD4+ Th cells that produce the pleiotropic cytokine IL-9. IL-9/Th9 can function as both positive and negative regulators of immune response, but the role of IL-9/Th9 in tumor immunity is unknown. We examined the role of IL-9/Th9 in a model of pulmonary melanoma in mice. Lack of IL-9 enhanced tumor growth, while tumor-specific Th9 cell treatment promoted stronger antitumor responses in both prophylactic and therapeutic models. Th9 cells also elicited strong host antitumor CD8+ CTL responses by promoting Ccl20/Ccr6-dependent recruitment of DCs to the tumor tissues. Subsequent tumor antigen delivery to the draining LN resulted in CD8+ T cell priming. In agreement with this model, Ccr6 deficiency abrogated the Th9 cell-mediated antitumor response. Our data suggest a distinct role for tumor-specific Th9 cells in provoking CD8+ CTL-mediated antitumor immunity and indicate that Th9 cell-based cancer immunotherapy may be a promising therapeutic approach.

Mature adipocytes in bone marrow protect myeloma cells against chemotherapy through autophagy activation

A major problem in patients with multiple myeloma is chemotherapy resistance, which develops in myeloma cells upon interaction with bone marrow stromal cells. However, few studies have determined the role of bone marrow adipocytes, a major component of stromal cells in the bone marrow, in myeloma chemotherapy resistance. We reveal that mature human adipocytes activate autophagy and upregulate the expression of autophagic proteins, thereby suppressing chemotherapy-induced caspase cleavage and apoptosis in myeloma cells. We found that adipocytes secreted known and novel adipokines, such as leptin and adipsin. The addition of these adipokines enhanced the expression of autophagic proteins and reduced apoptosis in myeloma cells. In vivo studies further demonstrated the importance of bone marrow-derived adipocytes in the reduced response of myeloma cells to chemotherapy. Our findings suggest that adipocytes, adipocyte-secreted adipokines, and adipocyte-activated autophagy are novel targets for combatting chemotherapy resistance and enhancing treatment efficacy in myeloma patients.

p38 MAPK in Myeloma Cells Regulates Osteoclast and Osteoblast Activity and Induces Bone Destruction

p38 mitogen-activated protein kinase (MAPK), which is constitutively activated in human myeloma, has been implicated in bone destruction by this cancer, but the processes it recruits are obscure. In this study, we show that p38 activity in myeloma inhibits osteoblast differentiation and bone formation, but also enhances osteoclast maturation and bone resorption. p38 regulated the expression and secretion of the Wnt pathway antagonist DKK-1 and the monocyte chemoattractant MCP-1. Attenuating p38, DKK-1, or MCP-1 were each sufficient to reduce bone lesions in vivo. Although it is well known that DKK-1 inhibits osteoblast differentiation, we found that together with MCP-1, it could also promote osteoclast differentiation and bone resorption. The latter effects were mediated by enhancing expression of RANK in osteoclast progenitor cells and by upregulating secretion of its ligand RANKL from stromal cells and mature osteoblasts. In summary, our study defined the mechanisms by which p38 signaling in myeloma cells regulates osteoblastogenesis, osteoclastogenesis, and bone destruction. Our findings, which may have implications for bone invasion by other cancers where p38 is elevated, strongly suggests that targeting p38 for inhibition may offer an effective therapeutic approach to treat osteolytic bone lesions in patients with myeloma.

A critical role of autocrine sonic hedgehog signaling in human CD138+myeloma cell survival and drug resistance

Hedgehog (Hh) signaling plays an important role in the oncogenesis of B-cell malignancies such as multiple myeloma (MM). However, the source of Hh ligand sonic hedgehog (SHH) and its target cells remains controversial. Previous studies showed that stromally induced Hh signaling is essential for the tumor cells and that CD19(+)CD138(-) MM stem cells are the target cells of Hh signaling. Here we demonstrate that SHH was mainly secreted by human myeloma cells but not by stromal cells in MM bone marrow. Autocrine SHH enhanced CD138(+) myeloma cell proliferation and protected myeloma cells from spontaneous and stress-induced apoptosis. More importantly, autocrine SHH protected myeloma cells against chemotherapy-induced apoptosis in vitro and in vivo. Combinational treatment with chemotherapy and SHH-neutralizing antibody displayed synergistic antimyeloma effects. Mechanistic studies showed that SHH signaling activated the SHH/GLI1/BCL-2 axis, leading to the inhibition of myeloma cell apoptosis. Thus, this study identifies the myeloma autocrine Hh signaling pathway as a potential target for the treatment of MM. Targeting this pathway may improve the efficacy of chemotherapy in MM patients.

Tonic B-cell receptor signaling in diffuse large B-cell lymphoma

We used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtypes exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO-induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL.

Human osteoclasts are inducible immunosuppressive cells in response to T cell-derived IFN-γ and CD40 ligand in vitro

Osteoclasts (OCs) are bone resorbing cells whose activity can be regulated by activated T cells and their cytokines. However, the immune function of OCs is largely unknown. In this study, we found that as bystanders, human OCs effectively suppressed T‐cell proliferation induced by allogeneic, microbial antigenic, and T‐cell receptor stimuli in vitro. Mechanism studies revealed that T cell–derived IFN‐γ and CD40 ligand (CD40L) induced the expression of indoleamine 2,3‐dioxygenase (IDO) in OCs, which mediated the immunosuppressive function on T‐cell proliferation through depleting tryptophan. Neutralizing IFN‐γ and blocking CD40L, or silencing or inhibiting IDO in OCs restored T‐cell proliferation in the presence of OCs. Our data reveal a novel function of human OCs as inducible immunosuppressive cells, and a feedback loop between OCs and activated T cells. Thus, this study provides new insight into the mechanism of the immunosuppressive function of OCs, and may be helpful for developing novel therapeutic strategies for human diseases involving both the bone and immune systems.

MAPK11 in breast cancer cells enhances osteoclastogenesis and bone resorption.

Breast cancer cells frequently metastasize to bone and induce osteolytic bone destruction in patients. These metastases cause severe bone pain, high risk of fractures and hypercalcemia, and are essentially incurable and fatal. Recent studies show that breast cancer cells in bone activate osteoclastogenesis and bone resorption. However the underlying mechanism is poorly understood. This study shows that the p38 MAPK (p38) isoform MAPK11 (p38β) is expressed in breast cancer cells. By using specific small hairpin RNAs for MAPK11, we demonstrated that p38β-mediated p38 activity in breast cancer cells is responsible for breast cancer-induced osteolytic bone destruction. The addition of conditioned media from breast cancer cell lines MDA-MB-231 and MDA-MB-468, which have high expression of p38β, induced osteoclast differentiation and bone resorption. In contrast, knockdown of p38β in breast cancer cells reduced osteoclast differentiation in vitro and reduced bone destruction in severe combined immunodeficiency (SCID) mouse models. The knockdown of p38β did not affect tumor growth or survival or the ability of cancer cells to home to bone. Furthermore, our results showed that p38β upregulated the expression and secretion of monocyte chemotactic protein-1 (MCP-1) in breast cancer cells, and upregulated MCP-1 activates osteoclast differentiation and activity. This study elucidates a novel molecular mechanism of breast cancer cell-induced osteolytic bone destruction. This study also indicates that targeting breast cancer cell p38β and its product MCP-1 may be a viable approach to treat or prevent bone destruction in patients with bone-metastatic breast cancer.

Bone Marrow Stromal Cells Derived MCP-1 Reverses the Inhibitory Effects of Multiple Myeloma Cells on Osteoclastogenesis by Upregulating the RANK Expression

Multiple myeloma (MM) cells are responsible for aberrant osteoclast (OC) activation. However, when cocultured monocytes, but not OC precursors, with MM cells, we made a novel observation that MM cells inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced increase of OC differentiation, OC gene expression, signaling pathways and bone resorption activity. Our results showed that MM cells produced multiple inhibitory cytokines of osteoclastogenesis, such as IL-10, which activated STAT3 signaling and induce OC inhibition. However, cocultures of bone marrow stromal cells (BMSCs) reversed MM-induced OC inhibition. We found that MM cells increased production of MCP-1 from BMSCs and BMSC-derived MCP-1 enhanced OC formation. Mechanistic studies showed that IL-10 downregulated RANK expression in monocytes and thus, inhibited RANKL-induced OC formation. In contrast, MCP-1 upregulated RANK expression and thus, enhanced OC formation. Overall, our studies for the first time demonstrated that MM cell have inhibitory effects on osteoclastogenesis by producing inhibitory cytokines. Our results further indicate that activation of osteoclastogenesis in bone marrow requests the crosstalk of MM cells, BMSCs and their produced cytokines. Thus, our studies provide evidences that targeting bone marrow microenvironmental cells and/or cytokines may be a new approach to treating MM bone destruction.

Abstract 5606: Fibrinogen-like protein 2 drives malignant tumor progression in glioma

Gliomas are the most common type of brain tumor in both children and adults. Several low-grade gliomas (LGG) have the ability to progress into more aggressive tumors -high-grade gliomas (HGG) including glioblastoma (GB). Although patients harboring a LGG may survive for years, after the tumor transforms to HGG, life expectancy rapidly declines to 12 to 15 months in adults and 40 months in children. Thus, inhibiting this process of malignant transformation (MT) is an attractive therapeutic strategy because of the more indolent course associated with LGGs. Immune response plays a critical role in surveillance against malignant transformation. Our previous study shows that fibrinogen-like protein 2 (FGL2) is a key hub of tumor-mediated immune suppression. Hence, we investigated the role of FGL2 in promoting tumor progression from LGG to HGG in glioma. Analysis of TCGA expression data showed that increased FGL2 expression is associated with poorer survival in LGG and GB patients. And there is a positive correlation of expression level between FGL2 and mesenchymal glioma marker CD44, and a negative correlation between FGL2 and proneural glioma marker OLIG2. Engineered expression of FGL2 in a PDGFB-dependent mouse model of oligodendroglioma, a common glioma subtype, yielded a significantly higher rate of HGGs (72% vs 29%, p=0.034) and poorer-symptom free survival (63 vs 90 days, p=0.003) than PDGFB expression alone. And HGGs from FGL2 + PDGFB expressing mice exhibited a distinct mesenchymal phenotype validating TCGA data. Further, FGL2 induced high numbers of CD4+FoxP3+ cells from an early time point of tumor formation underscoring its role in tumor progression. And FGL2 overexpression educated M2 skew in the tumors characterized by high expression of Iba1 and Arginase1 in macrophages. Finally, treatment with anti-FGL2 antibody significantly improves survival in mice, shifts the phenotype from mesenchymal HGG to proneural LGG, and rescues M2 macrophage skewing. Our results show that FGL2 is critical for malignant progression of glioma by inducing immunosuppression in tumor microenvironment, and raise the potential of FGL2 to be a promising target to suppress/reverse glioma progression and provide survival benefit in clinical. Citation Format: Khatri Latha, Jun Yan, Yuhui Yang, Loyola V. Gressot, Lingyuan Kong, Ganiraju Manyam, Ravesanker Ezhilarasan, Qianghu Wang, Erik P. Sulman, Jingda Xu, Richard E. Davis, Suyun Huang, Gregory N. Fuller, Arvind Rao, Amy B. Heimberger, Shulin Li, Ganesh Rao. Fibrinogen-like protein 2 drives malignant tumor progression in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5606. doi:10.1158/1538-7445.AM2017-5606