Gregor Werba

The necrosome promotes pancreatic oncogenesis via CXCL1 and Mincle-induced immune suppression

Neoplastic pancreatic epithelial cells are believed to die through caspase 8-dependent apoptotic cell death, and chemotherapy is thought to promote tumour apoptosis. Conversely, cancer cells often disrupt apoptosis to survive. Another type of programmed cell death is necroptosis (programmed necrosis), but its role in pancreatic ductal adenocarcinoma (PDA) is unclear. There are many potential inducers of necroptosis in PDA, including ligation of tumour necrosis factor receptor 1 (TNFR1), CD95, TNF-related apoptosis-inducing ligand (TRAIL) receptors, Toll-like receptors, reactive oxygen species, and chemotherapeutic drugs. Here we report that the principal components of the necrosome, receptor-interacting protein (RIP)1 and RIP3, are highly expressed in PDA and are further upregulated by the chemotherapy drug gemcitabine. Blockade of the necrosome in vitro promoted cancer cell proliferation and induced an aggressive oncogenic phenotype. By contrast, in vivo deletion of RIP3 or inhibition of RIP1 protected against oncogenic progression in mice and was associated with the development of a highly immunogenic myeloid and T cell infiltrate. The immune-suppressive tumour microenvironment associated with intact RIP1/RIP3 signalling depended in part on necroptosis-induced expression of the chemokine attractant CXCL1, and CXCL1 blockade protected against PDA. Moreover, cytoplasmic SAP130 (a subunit of the histone deacetylase complex) was expressed in PDA in a RIP1/RIP3-dependent manner, and Mincle—its cognate receptor—was upregulated in tumour-infiltrating myeloid cells. Ligation of Mincle by SAP130 promoted oncogenesis, whereas deletion of Mincle protected against oncogenesis and phenocopied the immunogenic reprogramming of the tumour microenvironment that was induced by RIP3 deletion. Cellular depletion suggested that whereas inhibitory macrophages promote tumorigenesis in PDA, they lose their immune-suppressive effects when RIP3 or Mincle is deleted. Accordingly, T cells, which are not protective against PDA progression in mice with intact RIP3 or Mincle signalling, are reprogrammed into indispensable mediators of anti-tumour immunity in the absence of RIP3 or Mincle. Our work describes parallel networks of necroptosis-induced CXCL1 and Mincle signalling that promote macrophage-induced adaptive immune suppression and thereby enable PDA progression.

Dectin 1 activation on macrophages by galectin 9 promotes pancreatic carcinoma and peritumoral immune tolerance

The progression of pancreatic oncogenesis requires immune-suppressive inflammation in cooperation with oncogenic mutations. However, the drivers of intratumoral immune tolerance are uncertain. Dectin 1 is an innate immune receptor crucial for anti-fungal immunity, but its role in sterile inflammation and oncogenesis has not been well defined. Furthermore, non-pathogen-derived ligands for dectin 1 have not been characterized. We found that dectin 1 is highly expressed on macrophages in pancreatic ductal adenocarcinoma (PDA). Dectin 1 ligation accelerated the progression of PDA in mice, whereas deletion of Clec7a—the gene encoding dectin 1—or blockade of dectin 1 downstream signaling was protective. We found that dectin 1 can ligate the lectin galectin 9 in mouse and human PDA, which results in tolerogenic macrophage programming and adaptive immune suppression. Upon disruption of the dectin 1–galectin 9 axis, CD4+ and CD8+ T cells, which are dispensable for PDA progression in hosts with an intact signaling axis, become reprogrammed into indispensable mediators of anti-tumor immunity. These data suggest that targeting dectin 1 signaling is an attractive strategy for developing an immunotherapy for PDA.

Dectin-1 Regulates Hepatic Fibrosis and Hepatocarcinogenesis by Suppressing TLR4 Signaling Pathways

Dectin-1 is a C-type lectin receptor critical in anti-fungal immunity, but Dectin-1 has not been linked to regulation of sterile inflammation or oncogenesis. We found that Dectin-1 expression is upregulated in hepatic fibrosis and liver cancer. However, Dectin-1 deletion exacerbates liver fibro-inflammatory disease and accelerates hepatocarcinogenesis. Mechanistically, we found that Dectin-1 protects against chronic liver disease by suppressing TLR4 signaling in hepatic inflammatory and stellate cells. Accordingly, Dectin-1(-/-) mice exhibited augmented cytokine production and reduced survival in lipopolysaccharide (LPS)-mediated sepsis, whereas Dectin-1 activation was protective. We showed that Dectin-1 inhibits TLR4 signaling by mitigating TLR4 and CD14 expression, which are regulated by Dectin-1-dependent macrophage colony stimulating factor (M-CSF) expression. Our study suggests that Dectin-1 is an attractive target for experimental therapeutics in hepatic fibrosis and neoplastic transformation. More broadly, our work deciphers critical cross-talk between pattern recognition receptors and implicates a role for Dectin-1 in suppression of sterile inflammation, inflammation-induced oncogenesis, and LPS-mediated sepsis.

Radiation Therapy Induces Macrophages to Suppress T-Cell Responses Against Pancreatic Tumors in Mice.

BACKGROUND & AIMS The role of radiation therapy in the treatment of patients with pancreatic ductal adenocarcinoma (PDA) is controversial. Randomized controlled trials investigating the efficacy of radiation therapy in patients with locally advanced unresectable PDA have reported mixed results, with effects ranging from modest benefit to worse outcomes compared with control therapies. We investigated whether radiation causes inflammatory cells to acquire an immune-suppressive phenotype that limits the therapeutic effects of radiation on invasive PDAs and accelerates progression of preinvasive foci. METHODS We investigated the effects of radiation therapy in p48(Cre);LSL-Kras(G12D) (KC) and p48(Cre);LSLKras(G12D);LSL-Trp53(R172H) (KPC) mice, as well as in C57BL/6 mice with orthotopic tumors grown from FC1242 cells derived from KPC mice. Some mice were given neutralizing antibodies against macrophage colony-stimulating factor 1 (CSF1 or MCSF) or F4/80. Pancreata were exposed to doses of radiation ranging from 2 to 12 Gy and analyzed by flow cytometry. RESULTS Pancreata of KC mice exposed to radiation had a higher frequency of advanced pancreatic intraepithelial lesions and more foci of invasive cancer than pancreata of unexposed mice (controls); radiation reduced survival time by more than 6 months. A greater proportion of macrophages from radiation treated invasive and preinvasive pancreatic tumors had an immune-suppressive, M2-like phenotype compared with control mice. Pancreata from mice exposed to radiation had fewer CD8(+) T cells than controls, and greater numbers of CD4(+) T cells of T-helper 2 and T-regulatory cell phenotypes. Adoptive transfer of T cells from irradiated PDA to tumors of control mice accelerated tumor growth. Radiation induced production of MCSF by PDA cells. A neutralizing antibody against MCSF prevented radiation from altering the phenotype of macrophages in tumors, increasing the anti-tumor T-cell response and slowing tumor growth. CONCLUSIONS Radiation treatment causes macrophages murine PDA to acquire an immune-suppressive phenotype and disabled T-cell-mediated anti-tumor responses. MCSF blockade negates this effect, allowing radiation to have increased efficacy in slowing tumor growth.

The Pancreatic Cancer Microbiome Promotes Oncogenesis by Induction of Innate and Adaptive Immune Suppression

We found that the cancerous pancreas harbors a markedly more abundant microbiome compared with normal pancreas in both mice and humans, and select bacteria are differentially increased in the tumorous pancreas compared with gut. Ablation of the microbiome protects against preinvasive and invasive pancreatic ductal adenocarcinoma (PDA), whereas transfer of bacteria from PDA-bearing hosts, but not controls, reverses tumor protection. Bacterial ablation was associated with immunogenic reprogramming of the PDA tumor microenvironment, including a reduction in myeloid-derived suppressor cells and an increase in M1 macrophage differentiation, promoting TH1 differentiation of CD4+ T cells and CD8+ T-cell activation. Bacterial ablation also enabled efficacy for checkpoint-targeted immunotherapy by upregulating PD-1 expression. Mechanistically, the PDA microbiome generated a tolerogenic immune program by differentially activating select Toll-like receptors in monocytic cells. These data suggest that endogenous microbiota promote the crippling immune-suppression characteristic of PDA and that the microbiome has potential as a therapeutic target in the modulation of disease progression.Significance: We found that a distinct and abundant microbiome drives suppressive monocytic cellular differentiation in pancreatic cancer via selective Toll-like receptor ligation leading to T-cell anergy. Targeting the microbiome protects against oncogenesis, reverses intratumoral immune tolerance, and enables efficacy for checkpoint-based immunotherapy. These data have implications for understanding immune suppression in pancreatic cancer and its reversal in the clinic. Cancer Discov; 8(4); 403-16. ©2018 AACR.See related commentary by Riquelme et al., p. 386This article is highlighted in the In This Issue feature, p. 371.

NLRP3 signaling drives macrophage-induced adaptive immune suppression in pancreatic carcinoma.

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDA) is characterized by immune tolerance, which enables disease to progress unabated by adaptive immunity. However, the drivers of this tolerogenic program are incompletely defined. In this study, we found that NLRP3 promotes expansion of immune-suppressive macrophages in PDA. NLRP3 signaling in macrophages drives the differentiation of CD4+ T cells into tumor-promoting T helper type 2 cell (Th2 cell), Th17 cell, and regulatory T cell populations while suppressing Th1 cell polarization and cytotoxic CD8+ T cell activation. The suppressive effects of NLRP3 signaling were IL-10 dependent. Pharmacological inhibition or deletion of NLRP3, ASC (apoptosis-associated speck-like protein containing a CARD complex), or caspase-1 protected against PDA and was associated with immunogenic reprogramming of innate and adaptive immunity within the TME. Similarly, transfer of PDA-entrained macrophages or T cells from NLRP3−/− hosts was protective. These data suggest that targeting NLRP3 holds the promise for the immunotherapy of PDA.

Necroptotic cell death – An unexpected driver of pancreatic oncogenesis

Cell growth and death are tightly regulated mechanisms that, in concert, facilitate tissue formation and integrity. Disruption in either pathway is a well-described hallmark that cancer cells exploit to survive noxious stimuli such as DNA damage, Reactive Oxygen Species (ROS), Fas-Fas-Ligand interaction or loss of cell-cell adhesion. Thus, many chemotherapeutics aim to disrupt survival and drive cells down a Caspase 8 mediated apoptotic pathway. Necroptosis is a more recently described mechanism of cell death that denotes coordinated cellular necrosis, leading to pore formation in the cell membrane and the release of cellular components. Evolutionarily, necroptosis can be considered a second line of defense against viruses, which enter cells and inhibit cellular demise via Caspase 8mediated apoptosis. In the presence of dysfunctional Caspase 8, receptor interacting kinase (RIP) 1 and 3 associate and assemble the necrosome, which in turn recruits mixed lineage kinase domain-like protein (MLKL) leading to necroptosis and the release of danger signals. Our recent work shows that the components of the necrosome are highly expressed in human and murine pancreatic ductal adenocarcinoma (PDA). Expression was further inducible by the chemotherapeutic agent Gemcitabine. To investigate the role of necroptosis in PDA progression, we deleted RIP3 in pancreatic cancer cells. In accordance with our hypothesis, RIP3 deletion resulted in a more aggressive oncogenic phenotype in vitro. However, contrary to these observations RIP3 deletion in vivo resulted in marked protection against PDA including a significant survival benefit. RIP1 inhibition was similarly protective. We found that targeting the necrosome led to a more immunogenic inflammatory infiltrate as evidenced by an increase in tumor infiltrating CD8C T cells and Th1-polarized CD4C T cells, B cells, as well as a reduction in myeloid derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs). Furthermore, TAMs exhibited a shift toward an M1-like immunogenic phenotype. Interestingly, pro-tumorigenic effects of necroptosis seemed to be specific to the pancreatic tumor microenvironment (TME), as neither subcutaneously implanted PDA nor B16 melanoma cells exhibited altered tumor growth in the context of RIP3 deletion. We discovered that CXCL1, a potent chemoattractant for myeloid cells, was highly expressed in a RIP3 dependent manner. We postulated that CXCL1 could mediate the protumorigenic immune suppression associated with RIP3 signaling by mobilizing myeloid cells. CXCL1 blockade protected against PDA, reduced myeloid cell infiltration, and increased peri-tumoral T cells. However, CXCL1 overexpression alone could not account for the entire immunosuppressive phenotype associated with intact necroptosis signaling in PDA. Since necroptosis produces danger signals to the surrounding environment, we postulated that the release of damage associated molecular patterns (DAMPs) by necroptotic cells within the TME triggers immune-suppressive inflammation. Mincle is a C type lectin receptor involved in fungal immunity and has recently been implicated in promoting sterile inflammation by ligating SAP130, a subunit of the histone deacetylase complex, which is released by necrotic cells. We found that Mincle and its associated signaling intermediates were highly expressed on antigen presenting cells (APCs) and Mincle coassociated with SAP130 in the PDA TME. Further emphasizing the role of necroptosis, SAP130 levels and Mincle signaling were reduced in RIP3¡/¡ tumors. Moreover, Mincle deletion was protective against PDA, extended survival, and directly phenocopied the immunogenic infiltrate associated with RIP3 deletion. In contrast, Mincle ligation led to tumor progression, promoted MDSC infiltration and M2-polarization of TAMs and induced adaptive immune suppression. Cellular depletion experiments revealed that TAMs promote tumorigenesis in PDA; however, they lose their immune-suppressive effects when RIP3 or Mincle are deleted. As such, T cells, which do not protect against PDA progression in hosts with intact RIP3 or Mincle signaling, are reprogrammed into potent mediators of anti-tumor immunity in the absence of RIP3 or Mincle. Our work describes parallel axes of necroptosis-induced CXCL1 and Mincle signaling that promote macrophage-induced adaptive immune suppression and thereby enable pancreatic cancer progression (Fig. 1). Each of these axes represents a novel target for experimental therapeutics.

Abstract A08: Dectin-1 signaling drives pancreatic oncogenesis by promoting adaptive immune suppression

Progression of pancreatic oncogenesis requires immune-suppressive inflammation in cooperation with oncogenic mutations. However, the drivers of intra-tumoral immune tolerance are uncertain. Dectin-1 is an innate immune receptor critical in anti-fungal immunity, but its role in sterile inflammation and oncogenesis is not well-defined. Further, non-pathogen-derived ligands for Dectin-1 have not been characterized. We found that Dectin-1 is highly expressed on myeloid cells in pancreatic ductal adenocarcinoma (PDA). Moreover, Dectin-1 ligation accelerates PDA, whereas Dectin-1 deletion, or blockade of its downstream signaling, was highly protective. We show that Dectin-1 ligates the lectin Galectin-9 in the PDA tumor microenvironment (TME) leading to macrophage-induced adaptive immune suppression. Upon interruption of the Dectin-1–Galectin-9 axis, CD4+ and CD8+ T cells – which are dispensable to PDA progression in hosts with an intact signaling axis – become reprogrammed into indispensable mediators of anti-tumor immunity. These data suggest that Dectin-1 and Galectin-9 are novel and attractive targets for PDA immunotherapy. Citation Format: Donnele Daley, Neha Akkad, Navyatha Mohan, Atsuo Ochi, Gregor Werba, Vishnu Mani, Rocky Barilla, Constantinos Zambirinis, Mautin Hundeyin, Ki Buom Lee, Steven Chang, Ding Wang, Lawrence Gardener, Beatrix Ueberheide, George Miller. Dectin-1 signaling drives pancreatic oncogenesis by promoting adaptive immune suppression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A08.

Abstract A145: Radiation therapy induces tumor-promoting immune suppression in the microenvironment of pancreatic carcinoma

Radiation therapy (RT) has shown marginal efficacy in patients with pancreatic ductal adenocarcinoma (PDA). Two of the past three randomized controlled trials investigating the efficacy of RT for patients with locally advanced unresectable PDA have shown a statistically significant worse survival by 40% or more for patients receiving RT. We postulated that RT reprograms inflammatory cells within the tumor microenvironment to an immune suppressive phenotype limiting the efficacy of RT in invasive PDA and accelerating disease progression in surrounding pre-invasive foci. We found that RT markedly accelerates the progression of pre-invasive PDA in a dose-dependent manner and reduces animal survival by more than 6 months. In both invasive and pre-invasive PDA, RT reprograms immunogenic macrophages towards an immune-suppressive M2 phenotype resulting in CD8 T cell scarcity and Th2 and Treg differentiation of CD4 T cells. Moreover, adoptive transfer of T cells harvested from RT-treated tumors accelerates tumor growth in recipient hosts. We show that M-CSF blockade concurrent with RT prevents immune-suppressive macrophage and T cell reprogramming and markedly enhances the efficacy of RT in PDA. These data suggest that targeting macrophage reprogramming can unleash the utility of RT for PDA. Citation Format: Lena Tomkotter, Gregor Werba, Susanna Nguy, Sara Alothman, Dalia Alqunaibit, Shaun Tiwari, Nancy Ngoc Giao Ly, Donnele Daley, Atsuo Ochi, Rocky Barilla, Alejandro Torres-Hernandez, Ilenia Pellicciotta, Kevin Du, George Miller. Radiation therapy induces tumor-promoting immune suppression in the microenvironment of pancreatic carcinoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A145.