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Neutrophil extracellular traps (NETs) occur when chromatin is decondensed and extruded from the cell, generating a web-like structure. NETs have been implicated in the pathogenesis of several sterile disease states and thus are a potential therapeutic target. Various pathways have been shown to induce NETs, including autophagy, with several key enzymes being activated like peptidyl arginine deiminase 4 (PAD4), an enzyme responsible for citrullination of histones, allowing for DNA unwinding and subsequent release from the cell. Pre-clinical studies have already demonstrated that chloroquine (CQ) and hydroxychloroquine (HCQ) are able to reduce NETs and slow disease progression. The exact mechanism as to how these drugs reduce NETs has yet to be elucidated. CQ and HCQ decrease NET formation from various NET activators, independent of their autophagy inhibitory function. CQ and HCQ were found to inhibit PAD4 exclusively, in a dose-dependent manner, confirmed with reduced CitH3+ NETs after CQ or HCQ treatment. Circulating CitH3 levels were reduced in pancreatic cancer patients after HCQ treatment. In silico screening of PAD4 protein structure identified a likely binding site interaction at Arg639 for CQ and Trp347, Ser468, and Glu580 for HCQ. SPR analysis confirmed the binding of HCQ and CQ with PAD4 with KD values of 54.1 µM (CQ) and 88.1 µM (HCQ). This data provide evidence of direct PAD4 inhibition as a mechanism for CQ/HCQ inhibition of NETs. We propose that these drugs likely reduce NET formation through multiple mechanisms; the previously established TLR9 and autophagy inhibitory mechanism and the novel PAD4 inhibitory mechanism.

The pancreas of adult prolactin deficient mice display no obvious abnormalities.

Supplementary Figure Legend

Ferroptosis is a form of regulated cell death driven by oxidative injury promoting lipid peroxidation, although detailed molecular regulators are largely unknown. Here, we show that heatshock 70-kDa protein 5 (HSPA5) negatively regulates ferroptosis in human pancreatic ductal adenocarcinoma (PDAC) cells. Mechanistically, activating transcription factor 4 (ATF4) resulted in the induction of HSPA5, which in turn bound glutathione peroxidase 4 (GPX4) and protected against GPX4 protein degradation and subsequent lipid peroxidation. Importantly, the HSPA5–GPX4 pathway mediated ferroptosis resistance, limiting the anticancer activity of gemcitabine. Genetic or pharmacologic inhibition of the HSPA5–GPX4 pathway enhanced gemcitabine sensitivity by disinhibiting ferroptosis in vitro and in both subcutaneous and orthotopic animal models of PDAC. Collectively, these findings identify a novel role of HSPA5 in ferroptosis and suggest a potential therapeutic strategy for overcoming gemcitabine resistance. Cancer Res; 77(8); 2064–77. ©2017 AACR.

Supplementary Data

Supplementary figure legends and methods.

Purpose:We hypothesized that autophagy inhibition would increase response to chemotherapy in the preoperative setting for patients with pancreatic adenocarcinoma. We performed a randomized controlled trial to assess the autophagy inhibitor hydroxychloroquine in combination with gemcitabine and nab-paclitaxel.Patients and Methods:Participants with potentially resectable tumors were randomized to two cycles of nab-paclitaxel and gemcitabine (PG) alone or with hydroxychloroquine (PGH), followed by resection. The primary endpoint was histopathologic response in the resected specimen. Secondary clinical endpoints included serum CA 19-9 biomarker response and margin negative R0 resection. Exploratory endpoints included markers of autophagy, immune infiltrate, and serum cytokines.Results:Thirty-four patients in the PGH arm and 30 in the PG arm were evaluable for the primary endpoint. The PGH arm demonstrated statistically improved Evans grade histopathologic responses (P = 0.00016), compared with control. In patients with elevated CA 19-9, a return to normal was associated with improved overall and recurrence-free survival (P < 0.0001). There were no differences in serious adverse events between arms and chemotherapy dose number was equivalent. The PGH arm had greater evidence of autophagy inhibition in their resected specimens (increased SQSTM1, P = 0.027, as well as increased immune cell tumor infiltration, P = 0.033). Overall survival (P = 0.59) and relapse-free survival (P = 0.55) did not differ between the two arms.Conclusions:The addition of hydroxychloroquine to preoperative gemcitabine and nab-paclitaxel chemotherapy in patients with resectable pancreatic adenocarcinoma resulted in greater pathologic tumor response, improved serum biomarker response, and evidence of autophagy inhibition and immune activity.

This file contains supplemental figures of clonogenic cell survival assay.

Related Article from The Power of Negative Thinking: Which Cells Limit Tumor Immunity?

Why human tumors grow infiltrated by specific antitumor T cells has been a mystery attributed to negative factors released directly by the tumor or indirectly through immune intermediaries. The frequency and phenotype of myeloid-derived suppressor cells in the peripheral blood of melanoma patients and healthy donors are surprisingly similar. Clin Cancer Res; 18(19); 5157–9. ©2012 AACR.

Supplementary Figure S1. Effects of combination treatment of both bortezomib and oHSV on cancer cell killing. Supplementary Figure S2. Necrostatin-1 (Nec-1) treatment and RIPK1 knock down did not reduce the bortezomib-induced increase in virus replication. Supplementary Figure S3. Single treatment with bortezomib or oHSV enhances NK cell mediated glioma killing. Supplementary Figure S4. Combination treatment with bortezomib and oHSV enhance mice survival in vivo.

Pancreatic ductal adenocarcinoma (PDAC) is associated with significant fibrosis. Recent findings have highlighted the profibrotic activity of tissue-resident macrophages in the pancreatic cancer microenvironment. Here, we show that neoplastic pancreatic epithelium, as well as a subset of tissue-resident macrophages, expresses the prolactin-receptor (PRLR). High mobility group box 1–induced prolactin expression in the pancreas maintained FAK1 and STAT3 phosphorylation within the epithelium and stroma. Gain-of-function and loss-of-function experiments demonstrated the essential role of prolactin in promoting collagen deposition and fibrosis. Finally, the signaling cascade downstream of prolactin/PRLR activated STAT3 rather than STAT5 in PDAC. These findings suggest that targeting prolactin together with IL6, a known major activator of STAT3, could represent a novel therapeutic strategy for treating pancreatic cancer.Significance:Prolactin is a key factor in the cross-talk between the stroma and neoplastic epithelium, functioning to promote fibrosis and PDAC progression.

Purpose: Both the proteasome inhibitor bortezomib and an oncolytic herpes simplex virus-1 (oHSV)–expressing GM-CSF are currently FDA approved. Although proteasome blockade can increase oHSV replication, immunologic consequences, and consequent immunotherapy potential are unknown. In this study, we investigated the impact of bortezomib combined with oHSV on tumor cell death and sensitivity to natural killer (NK) cell immunotherapy.Experimental Design: Western blot, flow cytometry, and caspase 3/7 activity assays were used to evaluate the induction of apoptosis/autophagy and/or necroptotic cell death. Cellular and mitochondrial reactive oxygen species (ROS) production was measured using CellROX and MitoSOX. Inhibitors/shRNA–targeting ROS, JNK and RIP1 kinase (RIPK1) were used to investigate the mechanism of cell killing. The synergistic interaction between oHSV and bortezomib was calculated using a Chou–Talalay analysis. NK cells isolated from normal human blood were co-cultured with tumor cells to evaluate cellular interactions. Q-PCR, ELISA, and FACS analysis were used to evaluate NK cell activation. Intracranial tumor xenografts were used to evaluate antitumor efficacy.Results: Combination treatment with bortezomib- and oHSV-induced necroptotic cell death and increased the production of mitochondrial ROS and JNK phosphorylation. Inhibitors/shRNA of RIPK1 and JNK rescued synergistic cell killing. Combination treatment also significantly enhanced NK cell activation and adjuvant NK cell therapy of mice treated with bortezomib and oHSV improved antitumor efficacy.Conclusions: This study provides a significant rationale for triple combination therapy with bortezomib, oHSV, and NK cells to improve efficacy, in glioblastoma patients. Clin Cancer Res; 22(21); 5265–76. ©2016 AACR.See related commentary by Suryadevara et al., p. 5164

Hypoxia promotes angiogenesis, proliferation, invasion, and metastasis of pancreatic cancer. Essentially, all studies of the hypoxia pathway in pancreatic cancer research to date have focused on fully malignant tumors or cancer cell lines, but the potential role of hypoxia inducible factors (HIF) in the progression of premalignant lesions has not been critically examined. Here, we show that HIF2α is expressed early in pancreatic lesions both in human and in a mouse model of pancreatic cancer. HIF2α is a potent oncogenic stimulus, but its role in Kras-induced pancreatic neoplasia has not been discerned. We used the Ptf1aCre transgene to activate KrasG12D and delete Hif2α solely within the pancreas. Surprisingly, loss of Hif2α in this model led to markedly higher, rather than reduced, number of low-grade pancreatic intraepithelial neoplasia (mPanIN) lesions. These lesions, however, failed to progress to high-grade mPanINs, and displayed exclusive loss of β-catenin and SMAD4. The relationship among HIF2α, β-catenin, and Smad4 was further confirmed in vitro, where silencing of Hif2α resulted in reduced β-catenin and Smad4 transcript levels. Thus, with oncogenic Ras expressed in the pancreas, HIF2α modulates Wnt-signaling during mPanIN progression by maintaining appropriate levels of both Smad4 and β-catenin. Cancer Res; 73(15); 4781–90. ©2013 AACR.

Supplementary Tables S1-S2. Primer pairs for qRT-PCR (S1); Oligonucleotides used for cloning RECK/TIMP3 3'UTR to pMIR-REPORT (S2).

Supplementary Figure S4. The HMGB1-IL6-STAT3-miR-21 axis promotes HCC tumor progression.

Legend for Supplementary Figures S1-S4.

PDF file - 324K

PDF file - 86K, Transmission electron microscopy (TEM), ATP Quantification, and XF Bioenergetic Assay.

Supplementry Figure 4 - PDF file 812K, HIF2� promotes B-catenin and Smad4 expression

Supplementary Figure 2 - PDF file 2100K, Normal pancreas development in Ptf1Cre;Hif2�f/f mice

Supplementary Figure 5 - PDF file 5613 K, Loss of Smad4 in PKH2 mPanINs

Supplementary Materials and Methods- PDF file 100K, Additional experimental procedures including Generation of Hif2�-floxed mouse line, Tissue processing and Immunostaining, Western Blot, and Small Interfering RNA transfection

PDF file - 428K, Supplemental Figure 1. Administration of rIL-2 inhibits tumor growth in murine hepatic metastasis model in a dose dependent fashion. Supplemental Figure 2. HDIL-2 alone or combined with CQ significantly inhibits Panc02 tumor growth, prolonging survival time in a hepatic metastasis model. Supplemental Figure 3. HDIL-2 combined with CQ inhibits Renca tumor growth in an hepatic metastasis model. Supplemental Figure 4. CQ treatment results in mitochondrial morphologic changes in MC38 cells. Supplemental Figure 5. Murine tumor cells are dependent on glycolysis. Supplemental Figure 6. Ethyl pyruvate alters tumor cell metabolism. Supplemental Figure 7. Proposed mechanisms of autophagy inhibition during HDIL-2 immunotherapy promoting long term tumor regression.

PDF file - 56K, HDIL-2 administration does not result in liver and idney dysfunction in mice.

Supplementary Figure 1 - PDF file 2170K, Generation of the Hif2�-floxed strain, and validation of Hif2� inactivation in the pancreas

Supplementary Tables S1-S2 - PDF file 73K, List of primary antibodies, along with the sources and working dilutions (S1); primers used in RT-PCR analyses (S2)

Supplementary Figure and Table Legends�- PDF file 93K, Legend for Supplemental Tables S1-S2 and Supplemental Figures S1-S5

PDF file - 1.2MB, 1) cell population discrimination and lysis, 2) CD4 T-cell C-MA, 3) ATG5 KO, 4) Prolonged survival, 5) Caspase and Granzyme inhibitors, 6) Bafilomycin and LAMP1, 7) Imaging starvation and HCQ, 8) TGF-beta, 9)NKG2D ligands, 10) KIR inhibition, 11) STAT1,2 translocation and IFNGR1, 12) IFN alpha combinations, 13) Chemotherapy.

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Objective The mainstay of treatment for patients with malignant pleural disease is fluid drainage and systemic therapy. A tumor-specific oncolytic virus or T-cell–activating interleukin-2 immunotherapy may provide an opportunity for local control. We previously developed a vaccinia virus–expressing interleukin-2, an oncolytic virus that mediated tumor regression in preclinical peritoneal tumor models with expansion of tumor-infiltrating lymphocytes. We evaluated the antitumor efficacy and immune modulatory effects of vaccinia virus–expressing interleukin-2 in malignant pleural disease. Methods A murine model of malignant pleural disease was established with percutaneous intrapleural deposition of the Lewis lung carcinoma cell line and monitored with bioluminescent imaging. After intrapleural or systemic administration of vaccinia viruses (vaccinia virus yellow fluorescent protein control, vaccinia virus–expressing interleukin-2), systemic anti–programmed cell death-1 antibody, or combination therapy (vaccinia virus–expressing interleukin-2 and anti–programmed cell death-1), tumor mass, immune cell infiltration, T-cell receptor diversity, and survival were assessed. Results Intrapleural vaccinia virus resulted in significant tumor regression compared with phosphate-buffered saline control (P Conclusions Intrapleural vaccinia virus–expressing interleukin-2 reduced tumor burden and enhanced survival in a murine malignant pleural disease model. Increased CD8+ tumor-infiltrating lymphocytes and αβ T-cell receptor diversity are associated with enhanced response. Clinical trials will enable assessment of intrapleural vaccinia virus–expressing interleukin-2 therapy in patients with malignant pleural disease.

Introduction Preoperative autophagy inhibition with hydroxychloroquine (HCQ) in combination with gemcitabine in pancreatic adenocarcinoma (PDAC) has been shown to be safe and effective in inducing a serum biomarker response and increase resection rates in a previous phase I/II clinical trial. We aimed to analyze the long‐term outcomes of preoperative HCQ with gemcitabine for this cohort. Methods A review of patients enrolled between July 2010 and February 2013 in the completed phase I/II single arm (two doses of fixed‐dose gemcitabine (1500 mg/m2) in combination with oral hydroxychloroquine administered for 31 consecutive days until the day of surgery for high‐risk pancreatic cancer) was undertaken. Progression‐free survival (PFS) and overall survival analysis (OS) using Kaplan–Meier estimates were performed. Results Of 35 patients initially enrolled, 29 patients underwent surgical resection (median age at diagnosis: 62 years, 45% females). Median duration of follow‐up was 7.5 years. There was a median 15% decrease in the serum CA19‐9 levels following completion of neoadjuvant therapy and 83% of the cohort underwent a pancreaticoduodenectomy, 7 (24%) patients had a concomitant venous resection. On histopathology, 14 (48%) patients had at least a partial treatment response. The median PFS and OS were 11 months (95% Confidence interval [CI]: 7–28) and 31 months (95% CI: 13–47), respectively, while 9 (31%) patients survived beyond 5 years from diagnosis; a rate that compares very favorably with contemporaneous series. Conclusion Compared to historical data, neoadjuvant autophagy inhibition with HCQ plus gemcitabine is associated with encouraging long‐term survival for patients with PDAC., Preoperative autophagy inhibition with hydroxychloroquine enhances the therapeutic efficacy of neoadjuvant chemotherapy leading to a sustained and improved survival benefit in high‐risk pancreatic cancer.

SMAD4, a tumor suppressor gene, is lost in up to 60%–90% of pancreatic adenocarcinomas (PDAs). Loss of SMAD4 allows tumor progression by upregulating autophagy, a cell survival mechanism that counteracts apoptosis and allows intracellular recycling of macromolecules. Hydroxychloroquine (HCQ) is an autophagy inhibitor. We studied whether HCQ treatment in SMAD4 deficient PDA may prevent therapeutic resistance induced by autophagy upregulation. We retrospectively analyzed the SMAD4 status of patients with PDA enrolled in two prospective clinical trials evaluating pre‐operative HCQ. The first dose escalation trial demonstrated the safety of preoperative gemcitabine with HCQ (NCT01128296). More recently, a randomized trial of gemcitabine/nab‐paclitaxel +/− HCQ evaluated Evans Grade histopathologic response (NCT01978184). The effect of SMAD4 loss on response to HCQ and chemotherapy was studied for association with clinical outcome. Fisher’s exact test and log‐rank test were used to assess response and survival. Fifty‐two patients receiving HCQ with neoadjuvant chemotherapy were studied. Twenty‐five patients had SMAD4 loss (48%). 76% of HCQ‐treated patients with SMAD4 loss obtained a histopathologic response greater than or equal to 2A, compared with only 37% with SMAD4 intact (p = 0.006). Although loss of SMAD4 has been associated with worse outcomes, in the current study, loss of SMAD4 was not associated with a detriment in median overall survival in HCQ‐treated patients (34.43 months in SMAD4 loss vs. 27.27 months in SMAD4 intact, p = 0.18). The addition of HCQ to neoadjuvant chemotherapy in patients with PDA may improve treatment response in those with SMAD4 loss. Further study of the relationship among SMAD4, autophagy, and treatment outcomes in PDA is warranted.

Recent studies defined a potentially important role of the microbiome in modulating pancreatic ductal adenocarcinoma (PDAC) and responses to therapies. We hypothesized that antibiotic usage may predict outcomes in patients with PDAC. We retrospectively analyzed clinical data of patients with resectable or metastatic PDAC seen at MD Anderson Cancer from 2003 to 2017. Demographic, chemotherapy regimen and antibiotic use, duration, type, and reason for indication were recorded. A total of 580 patients with PDAC were studied, 342 resected and 238 metastatic patients, selected retrospectively from our database. Antibiotic use, for longer than 48 hrs, was detected in 209 resected patients (61%) and 195 metastatic ones (62%). On resectable patients, we did not find differences in overall survival (OS) or progression‐free survival (PFS), based on antibiotic intake. However, in the metastatic cohort, antibiotic consumption was associated with a significantly longer OS (13.3 months vs. 9.0 months, HR 0.48, 95% CI 0.34–0.7, p = 0.0001) and PFS (4.4 months vs. 2 months, HR 0.48, 95% CI 0.34–0.68, p =, We have analyzed the effect of antibiotics’ intake on two cohorts of patients with pancreatic adenocarcinoma, resectable, and metastatic. We have found that on the metastatic cohort, antibiotics use was significantly associated with better outcomes, particularly, on patients that received gemcitabine based‐chemotherapy as the first line.

Despite its increased application in pancreatic ductal adenocarcinoma (PDAC), complete response to neoadjuvant therapy (NAT) is rare. Given the critical role of host immunity in regulating cancer, we sought to correlate baseline inflammatory profiles to significant response to NAT. PDAC patients receiving NAT were classified as responders (R) or nonresponders (NR) by carbohydrate antigen 19-9 response, pathologic tumor size, and lymph node status in the resected specimen. Baseline (treatment-naive) plasma was analyzed to determine levels of 27 inflammatory mediators. Logistic regression was used to correlate individual mediators with response. Network analysis and Pearson correlation maps were derived to determine baseline inflammatory mediator profiles. Forty patients (20R and 20NR) met study criteria. The R showed significantly higher overall survival (59.4 vs. 21.25 mo, P=0.002) and disease-free survival (50.97 vs. 10.60 mo, P=0.005), compared with NR. soluble interleukin-2 receptor alpha was a significant predictor of no response to NAT (P=0.045). Analysis of inflammatory profiles using the Pearson heat map analysis followed by network analysis depicted increased inflammatory network complexity in NR compared with R (1.69 vs. 1), signifying a more robust baseline inflammatory status of NR. A panel of inflammatory mediators identified by logistic regression and Fischer score analysis was used to create a potential decision tree to predict NAT response. We demonstrate that baseline inflammatory profiles are associated with response to NAT in PDAC, and that an upregulated inflammatory status is associated with a poor response to NAT. Further analysis into the role of inflammatory mediators as predictors of chemotherapy response is warranted.