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ObjectiveIntrahepatic cholangiocarcinoma (ICC)—a rare liver malignancy with limited therapeutic options—is characterised by aggressive progression, desmoplasia and vascular abnormalities. The aim of this study was to determine the role of placental growth factor (PlGF) in ICC progression.DesignWe evaluated the expression of PlGF in specimens from ICC patients and assessed the therapeutic effect of genetic or pharmacologic inhibition of PlGF in orthotopically grafted ICC mouse models. We evaluated the impact of PlGF stimulation or blockade in ICC cells and cancer-associated fibroblasts (CAFs) using in vitro 3-D coculture systems.ResultsPlGF levels were elevated in human ICC stromal cells and circulating blood plasma and were associated with disease progression. Single-cell RNA sequencing showed that the major impact of PlGF blockade in mice was enrichment of quiescent CAFs, characterised by high gene transcription levels related to the Akt pathway, glycolysis and hypoxia signalling. PlGF blockade suppressed Akt phosphorylation and myofibroblast activation in ICC-derived CAFs. PlGF blockade also reduced desmoplasia and tissue stiffness, which resulted in reopening of collapsed tumour vessels and improved blood perfusion, while reducing ICC cell invasion. Moreover, PlGF blockade enhanced the efficacy of standard chemotherapy in mice-bearing ICC.ConclusionPlGF blockade leads to a reduction in intratumorous hypoxia and metastatic dissemination, enhanced chemotherapy sensitivity and increased survival in mice-bearing aggressive ICC.

Radiotherapy (RT) is a standard treatment for patients with advanced prostate cancer (PCa). Previous preclinical studies showed that SDF1α/CXCR4 axis could mediate PCa metastasis (most often to the bones) and cancer resistance to RT. We found high levels of expression for both SDF1α and its receptor CXCR4 in primary and metastatic PCa tissue samples. In vitro analyses using PCa cells revealed an important role of CXCR4 in cell invasion but not radiotolerance. Pharmacologic inhibition of CXCR4 using AMD3100 showed no efficacy in orthotopic primary and bone metastatic PCa models. However, when combined with RT, AMD3100 potentiated the effect of local single-dose RT (12 Gy) in both models. Moreover, CXCR4 inhibition also reduced lymph node metastasis from primary PCa. Notably, CXCR4 inhibition promoted the normalization of bone metastatic PCa vasculature and reduced tissue hypoxia. In conclusion, the SDF1α/CXCR4 axis is a potential therapeutic target in metastatic PCa patients treated with RT.

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to chemotherapy, partly due to the presence of a dense-fibrotic stroma and adaptive metabolism. Telmisartan is an angiotensin II type receptor 1 (AT1) antagonist with partial peroxisome proliferator-activated receptor gamma (PPARγ) agonistic activity used for treatment of hypertension. The aim of this study was to determine the effects of telmisartan on the viability of PDAC cells and tumor progression. In panels of 4 murine and 8 human PDAC cells, the telmisartan IC50 was lower in cells with a low steady-state expression of PPARγ and a mesenchymal cell morphology. In contrast, losartan—a selective AT1 inhibitor—did not affect the viability of PDAC cells. The siRNA knockdown of PPARγ enhanced the sensitivity of telmisartan and stimulated epithelial-mesenchymal transition, which was accompanied by an increase in Wnt signaling. PPARγ regulates glucose metabolism and autophagy. We thus assessed effects of telmisartan on bioenergetics and autophagy of PDAC cells. In PPARγ-knockdown and -scrambled cells telmisartan significantly reduced glucose uptake, without affecting ATP production, but increased respiratory capacity, which can maintain the production of ATP during hypoglycemia. Immunoblotting revealed that PPARγ knockdown compared to scramble cells had increased levels of phosphorylated-AMP-activated protein kinase (p-AMPK) and increased expression of LC3A/B—structural proteins of autophagosomal membranes—which implies higher levels of autophagy. We also compared effects of telmisartan treatment on LC3A/B expression to well-established autophagy modulators, chloroquine and verapamil. Under nutrient-rich conditions and as expected, chloroquine and verapamil treatment induced LC3A/B accumulation, consistent with active autophagic flux in these cells. Telmisartan treatment decreased the levels of LC3A/B in both scramble and PPARγ knockdown cells and decreased the formation of LC3A/B positive granules in other PDAC cell lines. Telmisartan can also induce the accumulation of the signal adaptor protein p62 (SQSTM1), even in the presence of verapamil, which is also consistent with autophagy inhibition. Telmisartan did not prevent the accumulation LC3A/B in the presence of chloroquine, implying that telmisartan acts after the autophagosome-lysosome fusion step. To assess the effects of telmisartan in vivo, we used an orthotopic PDAC model. Telmisartan monotherapy inhibited the growth of primary tumors, decreased the incidence of liver metastasis, and significantly improved the survival of mice. Hence, telmisartan can reduce autophagy and the viability of PDAC cells, and PDAC progression. Because telmisartan is an FDA-approved drug, our findings provide the scientific rationale for testing its efficacy in the prevention of PDAC progression. Citation Format: Jelena Grahovac, Shiwei Han, Hao Liu, Mark Duquette, Alba Luengo, Daniel Schanne, Andrew S. Liss, Matthew G. Vander Heiden, Rakesh K. Jain, Yves Boucher. The angiotensin receptor blocker and partial PPARγ agonist telmisartan inhibits the growth of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B06.

The resistance to therapy in pancreatic ductal adenocarcinoma (PDAC) is due in part to carcinoma-associated fibroblasts (CAFs) that produce a highly desmoplastic and proinflammatory tumor microenvironment (TME). Our laboratory has shown that the angiotensin receptor blocker (ARB) losartan reduces the CAF-density and desmoplasia, and improves vascular perfusion, and the uptake and efficacy of drugs in animal models of PDAC (Diop-Frimpong et al., PNAS 2011; Chauhan et al., Nature Comm 2013). These preclinical findings led to a phase II trial at Massachusetts General Hospital in locally advanced PDAC patients, which showed that losartan combined with neoadjuvant-FOLFIRINOX chemotherapy followed by chemoradiation more than doubled the resection rate (Murphy et al., JAMA Oncology 2019). Moreover, in a retrospective analysis, we found that the chronic use of angiotensin system inhibitors was independently associated with longer overall survival in nonmetastatic PDAC patients (Liu et al., Clin Cancer Res 2017). However, how ARBs affect CAFs and desmoplasia in human PDAC remains unknown. Here we performed immunohistochemistry in formalin-fixed, paraffin-embedded sections to examine the effect of chronic use of ARB on CAF phenotypes and the extracellular matrix (ECM) in resected PDAC samples from untreated patients and patients treated with ARBs to control their hypertension. We found that the chronic use of ARBs did not reduce the density of CAFs expressing the fibroblast activation protein (FAP), α-smooth muscle actin (α-SMA), or platelet-derived growth factor receptor-beta alone. However, ARBs increased the fraction of CAFs expressing both the angiotensin receptor 1 (AT1) and α-SMA and decreased the fraction of CAFs expressing both AT1 and FAP. ARBs also reduced the intratumoral levels of hyaluronan, suggesting that ARBs reduce desmoplasia in human PDAC. To reveal the underlying mechanisms, we performed a transcriptomic analysis of the bulk tumor as well as α-SMA+ cells isolated from mice bearing orthotopic PDAC tumors and treated with losartan. Gene expression profiling revealed that in both the tumor bulk and α-SMA+ cells, losartan “normalized” the ECM/cytoskeleton interaction, glycosaminoglycan metabolism and platelet-derived growth factor gene sets, and significantly reduced gene transcripts ofS100A8 and S100A9 . Gene products of S100A8 and S100A9 are S100 proteins that stimulate the development of myeloid-derived suppressor cells. In α-SMA+ cells—but not in tumor bulk—losartan reduced cell cycle and muscle contraction gene sets. These results suggest that ARBs can reprogram α-SMA+ cells and normalize the TME in PDAC. Citation Format: Hao Liu, William W. Ho, Kamila Naxerova, Jelena Grahovac, Hadi Nia, Ivy Chen, Jessica M. Posada, Daniel H. Schanne, Matthias Pinter, Jonathan Crain, Xialong Qi, Jeffrey W. Clark, Theodore S. Hong, David P. Ryan, Peigen Huang, Keith D. Lillemoe, Carlos Fernández-Del Castillo, Cristina R Ferrone, Michael Downes, Ronald M. Evans, Vikram Deshpande, Yves Boucher, Rakesh K. Jain. Angiotensin receptor blockers normalize the pancreatic ductal adenocarcinoma stroma by reprogramming carcinoma-associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A18.

The transcription factor "nuclear factor erythroid 2" (NFE2) is overexpressed in the majority of patients with myeloproliferative neoplasms (MPNs). In murine models, elevated NFE2 levels cause an MPN phenotype with spontaneous leukemic transformation. However, both the molecular mechanisms leading to NFE2 overexpression and its downstream targets remain incompletely understood. Here, we show that the histone demethylase JMJD1C constitutes a novel NFE2 target gene. JMJD1C levels are significantly elevated in polycythemia vera (PV) and primary myelofibrosis patients; concomitantly, global H3K9me1 and H3K9me2 levels are significantly decreased. JMJD1C binding to the NFE2 promoter is increased in PV patients, decreasing both H3K9me2 levels and binding of the repressive heterochromatin protein-1α (HP1α). Hence, JMJD1C and NFE2 participate in a novel autoregulatory loop. Depleting JMJD1C expression significantly reduced cytokine-independent growth of an MPN cell line. Independently, NFE2 is regulated through the epigenetic JAK2 pathway by phosphorylation of H3Y41. This likewise inhibits HP1α binding. Treatment with decitabine lowered H3Y41ph and augmented H3K9me2 levels at the NFE2 locus in HEL cells, thereby increasing HP1α binding, which normalized NFE2 expression selectively in JAK2V617F-positive cell lines.

The transcription factor Nuclear Factor-Erythroid 2 (NF-E2) is overexpressed in the vast majority of patients with polycythaemia vera (PV). In murine models, NF-E2 overexpression increases proliferation and promotes cellular viability in the absence of erythropoietin (EPO). EPO-independent growth is a hallmark of PV. We therefore hypothesized that NF-E2 overexpression contributes to erythrocytosis, the pathognomonic feature of PV. Consequently, we investigated the effect of NF-E2 overexpression in healthy CD34+ cells. NF-E2 overexpression led to a delay in erythroid maturation, manifested by a belated appearance of glycophorin A-positive erythroid precursors. Maturation delay was similarly observed in primary PV patient erythroid cultures compared to healthy controls. Protracted maturation led to a significant increase in the accumulated number of erythroid cells both in PV cultures and in CD34+ cells overexpressing NF-E2. Similarly, NF-E2 overexpression altered erythroid colony formation, leading to an increase in erythroid burst-forming unit formation. These data indicate that NF-E2 overexpression delays the early phase of erythroid maturation, resulting in an expansion of erythroid progenitors, thereby increasing the number of erythrocytes derived from one CD34+ cell. These data propose a role for NF-E2 in mediating the erythrocytosis of PV.

13-cis-retinoic acid (RA) and thalidomide have shown a synergistic anti-proliferative effect on U343 glioblastoma (GBM) cells. In the present study, we test if their combined treatment might enhance the radiosensitivity of these cells.The radiosensitivity of U343 GBM cells was determined by the colony formation assay. Fibroblast growth factor-2 (FGF2) gene expression was determined by a quantitative polymerase chain reaction (qPCR).RA up-regulated FGF2 gene expression, which was abrogated by thalidomide. No radiosensitisation by RA was observed under standard culture conditions with 10% serum, but enhanced radiosensitivity was observed under 1% serum during irradiation. However, a synergistic effect with thalidomide was not observed.Growth factors in the culture medium may mask radiosensitization by RA while autocrine expression of FGF2 did not seem to be protective. Importantly, the anti-proliferative effect of RA in combination with thalidomide would not compromise the radiosensitivity of these GBM cells.

Expression of the transcription factor and epigenetic modulator “nuclear factor erythroid-2” (NF-E2) is aberrantly elevated in patients with Myeloproliferative Neoplasms (MPN). We have recently shown that NF-E2 overexpression in a murine model causes a phenotype similar to MPN in humans. This includes thrombocytosis, leukocytosis, expansion of the stem- and progenitor cell compartments as well as spontaneous transformation to acute leukemia. However, both the downstream pathways by which this transcription factor exerts its effects as well as the mechanisms leading to its overexpression in MPN patients remain incompletely characterized. We show here that the histone demethylases JMJD1C and JMJD2C constitute novel NF-E2 target genes. Using chromatin immunoprecipitation (ChIP) we demonstrate NF-E2 binding to the JMJD1C and JMJD2C loci. Both JMJD1C and JMJD2C protein levels are statistically significantly elevated in patients with polycythemia vera (PV) compared to healthy controls. JMJD1C and 2C proteins mediate the demethylation of histone H3K9, converting H3K9me2 via H3K9me to unmethylated H3K9. Consistent with increased demethylase activity, we observed statistically significantly decreased levels of global H3K9me and H3K9me2 in PV patients compared to healthy controls. We subsequently investigated whether the NF-E2 gene is regulated by epigenetic mechanisms and whether these are altered in PV patients. Indeed, using ChIP we demonstrate presence of the repressive H3K9me2 mark at three sites within the NF-E2 locus in healthy control granulocytes, where NF-E2 is expressed at low levels. These repressive histone marks are significantly reduced or completely absent in PV patients, which overexpress NF-E2. At the same time, binding of the repressive heterochromatin protein 1-alpha (HP1a) to the NF-E2 gene, while present in healthy controls, is significantly reduced in PV patients. HP1a chromatin binding is dependent on the presence of the H3K9me2 modification. Decreased levels of H3K9me2 and decreased HP1a binding are therefore both consistent with elevated NF-E2 expression in PV. We hypothesized that increased levels of the H3K9me2 demethylase JMJD1C in PV patients are responsible for the loss of this histone mark in the NF-E2 gene. Consistent with this model, we demonstrate significantly increased binding of JMJD1C to the NF-E2 locus in purified primary PV granulocytes. Our data demonstrate that the NF-E2 gene is regulated by epigenetic mechanisms and that these histone modifications are perturbed in PV patients. Moreover, NF-E2 participates in an auto-regulatory loop, by directly regulating transcription of the histone demethylase JMJD1C. Increased levels of NF-E2 thus cause increased JMJD1C expression. The JMJD1C protein, in turn, demethylates H3K9me2 residues in the NF-E2 locus, thereby further augmenting NF-E2 expression. Besides sustaining this novel autoregulatory loop, increased JMJD1C activity may contribute to MPN pathology by altering gene expression at additional loci. We sought to determine whether elevated NF-E2 levels could be normalized by pharmacological intervention. Treatment with the nucleoside analogue Decitabine reduced NF-E2 expression, and restored both the H3K9me2 marks and HP1a binding to the NF-E2 locus. These data provide a molecular rational for pre-clinical investigation of the effects of histone demethylase inhibitors and Decitabine on MPN cell biology and, subsequently, for a phase I trial investigating a combination of Decitabine and histone demethylase inhibitors in MPN patients. Disclosures: No relevant conflicts of interest to declare.