Have lessons from past failures brought us closer to the success of immunotherapy in metastatic pancreatic cancer?

Pancreatic cancer is extremely resistant to chemo- and radiation-therapies due to its inherent genetic instability, the local immunosuppressive microenvironment and the remarkable desmoplastic stromal changes which characterize this cancer. Therefore, there is an urgent need for improvement on standard current therapeutic options. Immunotherapies aimed at harnessing endogenous antitumor immunity have shown promise in multiple tumor types. In this review, we give an overview of new immune-related therapeutic strategies currently being tested in clinical trials in pancreatic cancer. We propose that immunotherapeutic strategies in combination with current therapies may offer new hopes in this most deadly disease.

Loss of P53 Function Activates JAK2-STAT3 Signaling to Promote Pancreatic Tumor Growth, Stroma Modification, and Gemcitabine Resistance in Mice and Is Associated With Patient Survival.

BACKGROUND & AIMS: One treatment strategy for pancreatic ductal adenocarcinoma is to modify, rather than deplete, the tumor stroma. Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) is associated with progression of pancreatic and other solid tumors. We investigated whether loss of P53 function contributes to persistent activation of STAT3 and modification of the pancreatic tumor stroma in patients and mice. METHODS: Stat3, Il6st (encodes gp130), or Trp53 were disrupted, or a mutant form of P53 (P53R172H) or transgenic sgp130 were expressed, in mice that developed pancreatic tumors resulting from expression of activated KRAS (KrasG12D, KC mice). Pancreata were collected and analyzed by immunohistochemistry, in situ hybridization, quantitative reverse-transcription polymerase chain reaction (qPCR), or immunoblot assays; fluorescence-activated cell sorting was performed to identify immune cells. We obtained frozen pancreatic tumor specimens from patients and measured levels of phosphorylated STAT3 and P53 by immunohistochemistry; protein levels were associated with survival using Kaplan-Meier analyses. We measured levels of STAT3, P53, ligands for gp130, interleukin 6, cytokines, sonic hedgehog signaling, STAT3 phosphorylation (activation), and accumulation of reactive oxygen species in primary pancreatic cells from mice. Mice with pancreatic tumors were given gemcitabine and a Janus kinase 2 (JAK2) inhibitor; tumor growth was monitored by 3-dimensional ultrasound. RESULTS: STAT3 was phosphorylated constitutively in pancreatic tumor cells from KC mice with loss or mutation of P53. Tumor cells of these mice accumulated reactive oxygen species and had lower activity of the phosphatase SHP2 and prolonged phosphorylation of JAK2 compared with tumors from KC mice with functional P53. These processes did not require the gp130 receptor. Genetic disruption of Stat3 in mice, or pharmacologic inhibitors of JAK2 or STAT3 activation, reduced fibrosis and the numbers of pancreatic stellate cells in the tumor stroma and altered the types of immune cells that infiltrated tumors. Mice given a combination of gemcitabine and a JAK2 inhibitor formed smaller tumors and survived longer than mice given control agents; the tumor stroma had fewer activated pancreatic stellate cells, lower levels of periostin, and alterations in collagen production and organization. Phosphorylation of STAT3 correlated with P53 mutation and features of infiltrating immune cells in human pancreatic tumors. Patients whose tumors had lower levels of phosphorylated STAT3 and functional P53 had significantly longer survival times than patients with high levels of phosphorylated STAT3 and P53 mutation. CONCLUSIONS: In pancreatic tumors of mice, loss of P53 function activates JAK2-STAT3 signaling, which promotes modification of the tumor stroma and tumor growth and resistance to gemcitabine. In human pancreatic tumors, STAT3 phosphorylation correlated with P53 mutation and patient survival time. Inhibitors of this pathway slow tumor growth and stroma formation, alter immune cell infiltration, and prolong survival of mice. Transcript profiling: ArrayExpress accession number: E-MTAB-3278.

The MEK1/2 Inhibitor Pimasertib Enhances Gemcitabine Efficacy in Pancreatic Cancer Models by Altering Ribonucleotide Reductase Subunit-1 (RRM1).

PURPOSE: Gemcitabine, a nucleoside analogue, is an important treatment for locally advanced and metastatic pancreatic ductal adenocarcinoma (PDAC) but provides only modest survival benefit. Targeting downstream effectors of the RAS/ERK signaling pathway by direct inhibition of MEK1/2 proteins is a promising therapeutic strategy, as aberrant activation of this pathway occurs frequently in PDAC. In this study, the ability of pimasertib, a selective allosteric MEK1/2 inhibitor, to enhance gemcitabine efficacy was tested and the molecular mechanism of their interaction was investigated. EXPERIMENTAL DESIGN: Cell survival and apoptosis were assessed by MTT and Caspase 3/7 Glo assays in human pancreatic cancer cell lines. Protein expression was detected by immunoblotting. The in vivo sensitivity of gemcitabine with pimasertib was evaluated in an orthotopic model of pancreatic tumor. RESULTS: Synergistic activity was observed when gemcitabine was combined sequentially with pimasertib, in human pancreatic cancer cells. In particular, pimasertib reduced ribonucleotide reductase subunit 1 (RRM1) protein, and this was associated with sensitivity to gemcitabine. Pretreatment with MG132 impaired reduction of RRM1 protein induced by pimasertib, suggesting that RRM1 is degraded posttranslationally. Immunoprecipitation indicated enhanced MDM2-mediated polyubiquitination of RRM1 through Lys-48-mediated linkage following pimasertib treatment, an effect mediated, in part, by AKT. Finally, the combination treatment with pimasertib and gemcitabine caused significant tumor growth delays in an orthotopic pancreatic cancer model, with RRM1 downregulation in pimasertib-treated mice. CONCLUSIONS: These results confirm an important role of RRM1 in gemcitabine response and indicate MEK as a potential target to sensitize gemcitabine therapy for PDAC. Clin Cancer Res; 21(24); 5563-77. ©2015 AACR.

PD-L1 expression in small cell neuroendocrine carcinomas.

Small cell lung cancer and extrapulmonary small cell carcinomas are the most aggressive type of neuroendocrine carcinomas. Clinical treatment relies on conventional chemotherapy and radiotherapy; relapses are frequent. The PD-1/PD-L1/PD-L2 pathway is a major target of anti-tumour immunotherapy. Aberrant PD-L1 or PD-L2 expression may cause local immune-suppression. Here we investigated expression of PD-1 and its ligands by immunohistochemistry and RNA-seq in small cell carcinomas. PD-L1 and PD-1 protein expression were analysed in 94 clinical cases of small cell carcinomas (61 pulmonary, 33 extrapulmonary) by immunohistochemistry using two different monoclonal antibodies (5H1, E1L3N). RNA expression was profiled by RNA-seq in 43 clinical cases. None of the small cell carcinomas showed PD-L1 protein expression in tumour cells. PD-L1 and PD-1 expression was noticed in the stroma: Using immunohistochemistry, 18.5% of cases (17/92) showed PD-L1 expression in tumour-infiltrating macrophages and 48% showed PD-1 positive lymphocytes (45/94). RNA-seq showed moderate PD-L1 gene expression in 37.2% (16/43). PD-L1 was correlated with macrophage and T-cell markers. The second PD-1 ligand PD-L2 was expressed in 27.9% (12/43) and showed similar correlations. Thus, the PD-1/PD-L1 pathway seems activated in a fraction of small cell carcinomas. The carcinoma cells were negative in all cases, PD-L1 was expressed in tumour-infiltrating macrophages and was correlated with tumour-infiltrating lymphocytes. Patients with stromal PD-L1/PD-L2 expression may respond to anti-PD-1 treatment. Thus, evaluation of the composition of the tumour microenvironment should be included in clinical trials. Besides conventional immunohistochemistry, RNA-seq seems suitable for detection of PD-L1/PD-L2 expression and might prove to be more sensitive.

ADAM8 as a drug target in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a grim prognosis with <5% survivors after 5 years. High expression levels of ADAM8, a metalloprotease disintegrin, are correlated with poor clinical outcome. We show that ADAM8 expression is associated with increased migration and invasiveness of PDAC cells caused by activation of ERK1/2 and higher MMP activities. For biological function, ADAM8 requires multimerization and associates with ß1 integrin on the cell surface. A peptidomimetic ADAM8 inhibitor, BK-1361, designed by structural modelling of the disintegrin domain, prevents ADAM8 multimerization. In PDAC cells, BK-1361 affects ADAM8 function leading to reduced invasiveness, and less ERK1/2 and MMP activation. BK-1361 application in mice decreased tumour burden and metastasis of implanted pancreatic tumour cells and provides improved metrics of clinical symptoms and survival in a Kras(G12D)-driven mouse model of PDAC. Thus, our data integrate ADAM8 in pancreatic cancer signalling and validate ADAM8 as a target for PDAC therapy.

Dual-action combination therapy enhances angiogenesis while reducing tumor growth and spread.

Increasing chemotherapy delivery to tumors, while enhancing drug uptake and reducing side effects, is a primary goal of cancer research. In mouse and human cancer models in vivo, we show that coadministration of low-dose Cilengitide and Verapamil increases tumor angiogenesis, leakiness, blood flow, and Gemcitabine delivery. This approach reduces tumor growth, metastasis, and minimizes side effects while extending survival. At a molecular level, this strategy alters Gemcitabine transporter and metabolizing enzyme expression levels, enhancing the potency of Gemcitabine within tumor cells in vivo and in vitro. Thus, the dual action of low-dose Cilengitide, in vessels and tumor cells, improves chemotherapy efficacy. Overall, our data demonstrate that vascular promotion therapy is a means to improve cancer treatment.

Tumour Microenvironment: Overview with an Emphasis on the Colorectal Liver Metastasis Pathway.

The tumour microenvironment (TME) represents a dynamic network that plays an important role in tumour initiation, proliferation, growth, and metastasis. Cell behaviour may be regulated by interplay of molecular interactions involving positive and negative reinforcement as well as a high level of cross-talk, which determines this system. Additionally, cancer involves cell proliferation, its malignancy defined by the tumour's ability to break down normal tissue architecture and by a dynamic process of invasion and metastasis. The metastatic cascade is regulated by a chain of molecular steps which triggers the progression of the developing cancer cell in the primary tumour into a number of transformations, leading to invasion and proceeding to metastases. Tumour-associated macrophages (TAMs) play a key-role in the progression from inflammatory conditions to cancer; TAMs are also capable of infiltrating the tumour microenvironment. Furthermore, myeloid-derived suppressor cells (MDSCs), a population of inhibitory immune cells, have been reported to increase in various cancer types, although characterising human MDSCs remains difficult, as their phenotype is quite variable. The future of cancer treatment is likely to involve creating more drugs that target these elements as well as others. An overview of the tumour's microenvironment is, therefore, presented in this paper, focusing on the metastatic pathways of primary colorectal cancer to the liver.

A vaccinia virus armed with interleukin-10 is a promising therapeutic agent for treatment of murine pancreatic cancer.

PURPOSE: Vaccinia virus has strong potential as a novel therapeutic agent for treatment of pancreatic cancer. We investigated whether arming vaccinia virus with interleukin-10 (IL10) could enhance the antitumor efficacy with the view that IL10 might dampen the host immunity to the virus, increasing viral persistence, thus maximizing the oncolytic effect and antitumor immunity associated with vaccinia virus. EXPERIMENTAL DESIGN: The antitumor efficacy of IL10-armed vaccinia virus (VVLΔTK-IL10) and control VVΔTK was assessed in pancreatic cancer cell lines, mice bearing subcutaneous pancreatic cancer tumors and a pancreatic cancer transgenic mouse model. Viral persistence within the tumors was examined and immune depletion experiments as well as immunophenotyping of splenocytes were carried out to dissect the functional mechanisms associated with the viral efficacy. RESULTS: Compared with unarmed VVLΔTK, VVLΔTK-IL10 had a similar level of cytotoxicity and replication in vitro in murine pancreatic cancer cell lines, but rendered a superior antitumor efficacy in the subcutaneous pancreatic cancer model and a K-ras-p53 mutant-transgenic pancreatic cancer model after systemic delivery, with induction of long-term antitumor immunity. The antitumor efficacy of VVLΔTK-IL10 was dependent on CD4(+) and CD8(+), but not NK cells. Clearance of VVLΔTK-IL10 was reduced at early time points compared with the control virus. Treatment with VVLΔTK-IL10 resulted in a reduction in virus-specific, but not tumor-specific CD8(+) cells compared with VVLΔTK. CONCLUSIONS: These results suggest that VVLΔTK-IL10 has strong potential as an antitumor therapeutic for pancreatic cancer.

Haematopoietic focal adhesion kinase deficiency alters haematopoietic homeostasis to drive tumour metastasis.

Metastasis is the main cause of cancer-related death and thus understanding the molecular and cellular mechanisms underlying this process is critical. Here, our data demonstrate, contrary to established dogma, that loss of haematopoietic-derived focal adhesion kinase (FAK) is sufficient to enhance tumour metastasis. Using both experimental and spontaneous metastasis models, we show that genetic ablation of haematopoietic FAK does not affect primary tumour growth but enhances the incidence of metastasis significantly. At a molecular level, haematopoietic FAK deletion results in an increase in PU-1 levels and decrease in GATA-1 levels causing a shift of hematopoietic homeostasis towards a myeloid commitment. The subsequent increase in circulating granulocyte number, with an increase in serum CXCL12 and granulocyte CXCR4 levels, was required for augmented metastasis in mice lacking haematopoietic FAK. Overall our findings provide a mechanism by which haematopoietic FAK controls cancer metastasis.

Inactivation of PI(3)K p110δ breaks regulatory T-cell-mediated immune tolerance to cancer.

Inhibitors against the p110δ isoform of phosphoinositide-3-OH kinase (PI(3)K) have shown remarkable therapeutic efficacy in some human leukaemias. As p110δ is primarily expressed in leukocytes, drugs against p110δ have not been considered for the treatment of solid tumours. Here we report that p110δ inactivation in mice protects against a broad range of cancers, including non-haematological solid tumours. We demonstrate that p110δ inactivation in regulatory T cells unleashes CD8(+) cytotoxic T cells and induces tumour regression. Thus, p110δ inhibitors can break tumour-induced immune tolerance and should be considered for wider use in oncology.

Inflammatory cytokines in human pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal types of cancer with poor prognosis. Despite extensive efforts, the current treatment methods have limited success. Therefore, novel therapeutic approaches are required. The pancreatic tumor microenvironment is rich in growth factors and inflammatory cytokines that support tumor growth, and it is highly immunosuppressive. Up-regulation of cytokine pathways has been shown to modulate PDAC progression and immune evasion; therefore targeting cytokines may have therapeutic benefits. In this review we provide an overview of current understanding of pro- and anti-inflammatory cytokines in pancreatic cancer and their potential as therapeutic targets.

A multi-gene signature predicts outcome in patients with pancreatic ductal adenocarcinoma.

BACKGROUND: Improved usage of the repertoires of pancreatic ductal adenocarcinoma (PDAC) profiles is crucially needed to guide the development of predictive and prognostic tools that could inform the selection of treatment options. METHODS: Using publicly available mRNA abundance datasets, we performed a large retrospective meta-analysis on 466 PDAC patients to discover prognostic gene signatures. These signatures were trained on two clinical cohorts (n = 70), and validated on four independent clinical cohorts (n = 246). Further validation of the identified gene signature was performed using quantitative real-time RT-PCR. RESULTS: We identified 225 candidate prognostic genes. Using these, a 36-gene signature was discovered and validated on fully independent clinical cohorts (hazard ratio (HR) = 2.06, 95% confidence interval (CI) = 1.51 to 2.81, P = 3.62 × 10(-6), n = 246). This signature serves as a good alternative prognostic stratification marker compared to tumour grade (HR = 2.05, 95% CI = 1.45 to 2.88, P = 3.18 × 10(-5)) and tumour node metastasis (TNM) stage (HR = 1.13, 95% CI = 0.66 to 1.94, P = 0.67). Upon multivariate analysis with adjustment for TNM stage and tumour grade, the 36-gene signature remained an independent prognostic predictor of clinical outcome (HR = 2.21, 95% CI = 1.17 to 4.16, P = 0.01). Univariate assessment revealed higher expression of ITGA5, SEMA3A, KIF4A, IL20RB, SLC20A1, CDC45, PXN, SSX3 and TMEM26 was correlated with shorter survival while B3GNT1, NOSTRIN and CADPS down-regulation was associated with poor outcome. CONCLUSIONS: Our 36-gene classifier is able to prognosticate PDAC independent of patient cohort and microarray platforms. Further work on the functional roles, downstream events and interactions of the signature genes is likely to reveal true molecular candidates for PDAC therapeutics.

IL-17 mediates resistance to anti-VEGF therapy.

Interleukin-17 (IL-17) released in the tumor microenvironment in response to drugs blocking vascular endothelial growth factor (VEGF) triggers stromal-derived inflammatory and VEGF-independent angiogenic programs that induce the drug refractoriness found in cancers resistant to anti-angiogenic therapy.

An apolipoprotein A-I mimetic targets scavenger receptor A on tumor-associated macrophages: A prospective anticancer treatment?

Tumor-associated macrophages polarize toward an M2 phenotype and express scavenger receptor A (SRA), hence promoting tumor progression. We demonstrated that SRA can be therapeutically targeted in vivo with the small peptide inhibitor 4F to prevent metastatic spread. Beyond our study, 4F is emerging as a promising anticancer agent.

Targeting immunosuppression for cancer therapy.

Failing immunity has been acknowledged for its contribution to cancer development and progression. Recent clinical findings have provided payoffs for significant preclinical evaluation and refinement over the last 20 years, but many questions remain to be answered. In this issue of the JCI, Marabelle et al. describe a novel method for targeting the Tregs that infiltrate tumors, demonstrating that dampening the tumor immunosuppressive environment while activating innate antitumor immunity may be an effective approach to cancer treatment.

Tumour-associated macrophages and cancer.

Our understanding of the complex roles and functions of tumour-associated myeloid cells has improved vastly over the last few years. Alternatively activated macrophages, TAMs, are an abundant part of solid and haematological malignancies and have been linked with progression, metastasis and resistance to therapy. Still, characterisation and TAM targeting is hindered by a lack of TAM specific markers, but advances in next generation technologies are rapidly increasing our understanding of the sheer diversity of myeloid differentiation and phenotypic regulation. These technologies help to shed light on the heterogeneous phenotypic states of myeloid cells within the tumour. Alternative approaches to influence the myeloid compartment within cancers surround inhibition of myeloid recruitment or 're-education' of the plastic TAM phenotype. Our knowledge continuously grows on how even 'established' therapies might influence the myeloid compartment within tumours. Now the promising results from elegant pre-clinical studies at first translate into the clinic and use combination therapies with myeloid inhibitors and standard chemotherapy.

Current clinical immunotherapy targets in advanced nonsmall cell lung cancer (NSCLC).

NSCLC remains one of the most challenging malignancies to treat. Despite the introduction of innovative therapies over the last decade, the 5-year survival of NSCLC is still <20%. Clearly, novel, therapeutic approaches are required. Targeting the immune system to derive meaningful clinical benefit has proved successful in various malignancies in recent years. As a result, there is renewed focus on the use of immunotherapy in lung cancer. In this review, we provide an overview of current immune-modulatory approaches in the treatment of NSCLC.

Macrophage scavenger receptor a promotes tumor progression in murine models of ovarian and pancreatic cancer.

Alternatively activated macrophages express the pattern recognition receptor scavenger receptor A (SR-A). We demonstrated previously that coculture of macrophages with tumor cells upregulates macrophage SR-A expression. We show in this study that macrophage SR-A deficiency inhibits tumor cell migration in a coculture assay. We further demonstrate that coculture of tumor-associated macrophages and tumor cells induces secretion of factors that are recognized by SR-A on tumor-associated macrophages. We tentatively identified several potential ligands for the SR-A receptor in tumor cell-macrophage cocultures by mass spectrometry. Competing with the coculture-induced ligand in our invasion assay recapitulates SR-A deficiency and leads to similar inhibition of tumor cell invasion. In line with our in vitro findings, tumor progression and metastasis are inhibited in SR-A(-/-) mice in two in vivo models of ovarian and pancreatic cancer. Finally, treatment of tumor-bearing mice with 4F, a small peptide SR-A ligand able to compete with physiological SR-A ligands in vitro, recapitulates the inhibition of tumor progression and metastasis observed in SR-A(-/-) mice. Our observations suggest that SR-A may be a potential drug target in the prevention of metastatic cancer progression.