Citations

Pancreatic cancer remains among the deadliest forms of cancer with a 5 year survival rate less than 10%. With increasing numbers being observed, there is an urgent need to elucidate the pathogenesis of pancreatic cancer. While both contribute to disease progression, neither genetic nor environmental factors completely explain susceptibility or pathogenesis. Defining the links between genetic and environmental events represents an opportunity to understand the pathogenesis of pancreatic cancer. Epigenetics, the study of mitotically heritable changes in genome function without a change in nucleotide sequence, is an emerging field of research in pancreatic cancer. The main epigenetic mechanisms include DNA methylation, histone modifications and RNA interference, all of which are altered by changes to the environment. Epigenetic mechanisms are being investigated to clarify the underlying pathogenesis of pancreatic cancer including an increasing number of studies examining the role as possible diagnostic and prognostic biomarkers. These mechanisms also provide targets for promising new therapeutic approaches for this devastating malignancy.

The Hedgehog-regulated transcription factors GLI1 and GLI2 play overlapping roles in development and disease; however, the mechanisms underlying their interplay remain elusive. We report for the first time that GLI1 and GLI2 physically and functionally interact in cancer cells. GLI1 and GLI2 were shown to co-immunoprecipitate in PANC1 pancreatic cancer cells and RMS13 rhabdomyosarcoma cells. Mapping analysis demonstrated that the zinc finger domains of both proteins are required for their heteromerization. RNAi knockdown of either GLI1 or GLI2 inhibited expression of many well-characterized GLI target genes (BCL2, MYCN, PTCH2, IL7 and CCND1) in PANC1 cells, whereas PTCH1 expression was only inhibited by GLI1 depletion. qPCR screening of a large set of putative canonical and non-canonical Hedgehog/GLI targets identified further genes (e.g. E2F1, BMP1, CDK2) strongly down-regulated by GLI1 and/or GLI2 depletion in PANC1 cells, and demonstrated that ANO1, AQP1 and SOCS1 are up-regulated by knockdown of either GLI1 or GLI2. Chromatin immunoprecipitation showed that GLI1 and GLI2 occupied the same regions at the BCL2, MYCN and CCND1 promoters. Furthermore, depletion of GLI1 inhibited GLI2 occupancy at these promoters, suggesting that GLI1/GLI2 interaction is required for the recruitment of GLI2 to these sites. Together, these findings indicate that GLI1 and GLI2 co-ordinately regulate the transcription of some genes, and provide mechanistic insight into the roles of GLI proteins in carcinogenesis.

Abstract Summary: In this issue, Hou and colleagues present their exciting work demonstrating that, through remodeling of the local tumor microenvironment (TME), pancreatic ductal adenocarcinoma forms a tumor-supportive niche capable of liberating cancer cells from dependence on oncogenic KRAS signaling. Through extensive experimentation both in vitro and in vivo, the authors reveal that the HDAC5–CCL2 axis drives the recruitment of tumor-associated macrophages to the TME to provide trophic signaling. See related article by Hou et al., p. 1058.

AbstractThe modulation of GLI2, an oncogenic transcription factor commonly upregulated in cancer, is in many cases not due to genetic defects, suggesting dysregulation through alternative mechanisms. The identity of these molecular events remains for the most part unknown. Here, we identified TFII-I as a novel repressor of GLI2 expression. Mapping experiments suggest that the INR region of the GLI2 promoter is necessary for GLI2 repression. ChIP studies showed that TFII-I binds to this INR. TFII-I knockdown decreased the binding of NELF-A, a component of the promoter–proximal pausing complex at this site, and enriched phosphorylated RNAPII serine 2 in the GLI2 gene body. Immunoprecipitation studies demonstrate TFII-I interaction with SPT5, another pausing complex component. TFII-I overexpression antagonized GLI2 induction by TGFβ, a known activator of GLI2 in cancer cells. TGFβ reduced endogenous TFII-I binding to the INR and increased RNAPII SerP2 in the gene body. We demonstrate that this regulatory mechanism is not exclusive of GLI2. TGFβ-induced genes CCR7, TGFβ1 and EGR3 showed similar decreased TFII-I and NELF-A INR binding and increased RNAPII SerP2 in the gene body post-TGFβ treatment. Together these results identify TFII-I as a novel repressor of a subset of TGFβ-responsive genes through the regulation of RNAPII pausing.

Abstract Waldenström macroglobulinemia (WM) is a B-cell malignancy characterized by the aberrant production of a monoclonal IgM protein that may lead to hyperviscosity. Although this is a major factor causing significant morbidity in patients, little is known about the mechanisms that regulate monoclonal protein synthesis. Cytokines are protein mediators that are known to be involved in many biological processes, and can profoundly affect tumor cells and the tumor microenvironment. Many cytokines have been shown to have potent therapeutic efficacy in preclinical cancer models; however, the role of cytokine networks in WM is not fully understood and few studies have described the precise functional roles of cytokines in WM. To address this issue we performed a multiplex ELISA analysis to test cytokine levels in sera from patients with WM. We found that Rantes/CCL5 is significantly elevated in WM patients and correlates with disease activity. Elevated expression of RANTES in the serum was confirmed by ELISA and was also detected in the bone marrow of WM patients by ELISA and immunohistochemistry. RANTES expression serves as a marker for recruitment of immune cells and is associated with a wide range of immune-mediated diseases. However, the impact of RANTES in WM is not known. We analyzed the expression of receptors for Rantes by flow cytometry and found that WM B cells and stromal cells express CCR1 and CCR3, but not CCR5. Using a standard chemotaxis assay, we determined that Rantes had no effect on B cell or stromal cell recruitment. Rantes also had no effect on B cell or stromal cell survival, however it did promote stromal cell proliferation (p<0.04). The interaction between Rantes and IL-6 has been described in an autoimmune disease model. Since stromal cells secrete significantly more IL-6 than WM B cells, we treated stromal cells with Rantes for 24 hr and found that Rantes increases IL-6 secretion (p<0.03). To characterize the mechanism of Rantes-mediated IL-6 secretion, we transfected stromal cells with an IL-6 promoter construct and treated with Rantes for 12 hr and found a significant increase in IL-6 promoter activity (p<0.0162) indicating Rantes can regulate IL-6 transcription. Bioinformatics analysis of the IL-6 promoter indicates the presence of multiple candidate binding sites for transcription factors that have been previously shown to play a role in the biology of B cells, including NFkB, AP1, and GLI. Co-transfection of stromal cells with an IL-6 reporter construct and a plasmid expressing GLI1, GLI2 or GLI3 demonstrates that GLI2 and GLI3 proteins can regulate the IL-6 promoter. We then transfected stromal cells with a reporter construct containing 8X-GLI binding sites and demonstrate that Rantes can regulate GLI transcription further supporting a role for the interaction between Rantes and IL-6 through GLI transcription factors. IL-6 rich tumor microenvironment supports malignant cells. Elevated IL-6 levels have no effect on survival of BCWM.1 cells or CD19+ 138+ cells from WM patients, but leads to upregulation of IgM secretion by BCWM.1 cells and CD19+ CD138+ cells from WM patients, and increases their proliferation (p<0.0039). IL-6 activates Erk1/2 and Jak/ Stat in WM and stimulation of the IgM secreting cells BCWM.1 with IL-6 in the presence of PD98059 MAPK inhibitor had no effect on IgM secretion. However, stimulation of BCWM.1 cells with IL-6 in the presence of JakI inhibitor abolished the IL-6 mediated IgM secretion suggesting IL-6 mediated increase in IgM secretion occurs through Jak/ Stat signaling pathway. Analysis of Rantes levels in other B cell malignancies including follicular lymphoma, chronic lymphocytic leukemia, monoclonal gammopathy of undetermined significance and multiple myeloma indicates that Rantes is elevated in other B cell lymphoproliferative disorders and suggest Rantes may play a similar role in other malignancies. In summary, our data identifies a novel Rantes-GLI-IL-6 interplay in the stromal microenvironment that promotes IgM production by malignant B cells. This therefore provides multiple new potential therapeutic avenues, targeting both malignant cells and the microenvironment to control malignant cell growth, and immunoglobulin secretion in WM and Ig-mediated diseases.

Abstract Background: A wide range of cancers, including pancreatic cancer, display altered expression of amino acid transporter LAT-1. LAT-1 overexpression has been shown to be an important prognostic factor and predicts chemo resistance in pancreatic cancer (Kaira K, 2012; Altan B, 2018). Radiolabeled amino acids for PET imaging, including 3-18F-l-α-methyl-tyrosine ([18F] FAMT), have successfully leveraged cancer’s aberrant LAT1 expression and amino acid uptake for decades. SM-88 is a denatured D/L racemic form of α-methyl-tyrosine designed for selective uptake and disruption of protein synthesis in cancer cells, as well as disruption of catecholamine production via inhibition of tyrosine hydroxylase. Catecholamines have been reported to potentially have a role in driving pancreatic cancer progression and changes in the tumor immune status (Renz B, Cancer Cell 2018; Calvani M, Oncotarget 2014). Initial clinical and preclinical results with SM-88 indicate that this agent could pose a novel approach in the treatment of pancreatic cancer. Methods and Results: Initial xenograft models of human gastrointestinal cancers using monotherapy SM-88 showed a >50% decrease in tumor growth at three weeks of treatment (p<0.05). Ongoing preclinical experiments are testing the full pharmacodynamic effects of SM-88 on pancreatic cancer cells, as well as potential effects on the tumor microenvironment due to catecholamine interruption. In results from the ongoing phase 2 trial of SM-88 in heavily pretreated metastatic pancreatic cancer patients, SM-88 has shown monotherapy antitumor activity based on RECIST 1.1 criteria. Additionally, 16/24 patients demonstrated a reduction in circulating tumor cells, with the median nadir decline of 63%. LAT1 has been reported to be important in EMT transition and mesenchymal cell survival (Holldorsson S, Cancer letters 2017); examination of the impact of SM-88 on this important emerging biomarker is ongoing. Conclusion: Altered amino acid (LAT1) metabolism has been shown to be a negative prognosticator in pancreatic cancer outcomes and progression. Targeting LAT1 and amino acid pathways has been identified as a therapeutic approach in multiple cancers (Fuchs BC, Semin Ca Biol 2005). Initial and ongoing studies of SM-88 may clarify the potential utility of this approach in the treatment of pancreatic cancer. Citation Format: Martin Fernandez-Zapico, Dae Won Kim, Philip Philip, Alexander Vandell, Jonathan Eckard, Ron Korn, Giuseppe Del Priore, Diane Simeone. Therapeutic potential of targeting amino acid metabolism in pancreatic cancer [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 B15.

TPS594 Background: Effective treatment options are very limited for patients with advanced refractory biliary tract cancer (BTC). Fluoropyrimidine-based chemotherapy regimen such as 5-fluorouracil and irinotecan are frequently utilized for these patients after first-line therapy despite lack of FDA approval. Trifluridine/tipiracil (FTD/TPI) is a novel oral nucleoside with antitumor activity in both fluoropyrimidine sensitive and resistant tumors due to its unique mechanisms of action. Given early toxicity and efficacy data from our previous study on single-agent trifluridine/tipiracil (FTD/TPI) in advanced BTC, the clinical evaluation of its combination with irinotecan represents a rational approach for the treatment of advanced refractory BTC. Methods: This is a single-arm phase II trial with a two-stage design to assess the efficacy of trifluridine/tipiracil (FTD/TPI) and irinotecan in advanced refractory BTC. Key eligibility criteria include histologically confirmed advanced, unresectable BTC who have progressed on at least one line of systemic therapy and have measurable disease per RECIST v1.1. Target accrual is 25. Treatment includes trifluridine/tipiracil (FTD/TPI) 25 mg/m2 on days 1-5 and irinotecan 180 mg/m2 on day 1 in 14-day cycles. Patients will be evaluated for response every 4 cycles and in the absence of disease progression, therapy may be given up to 2 years. The primary end point is the progression-free survival rate at 16 weeks. Secondary endpoints include overall response rate, disease control rate, progression-free survival, overall survival, and incidence of adverse events. Correlative biomarker studies include evaluations of circulating tumor DNA and circulating tumor cells at baseline, after 4 cycles and at progression; and development of patient-derived tumor organoids from pre-treatment biopsies for parallel treatments. This study was approved and funded in part by the National Comprehensive Cancer Network (NCCN) Oncology Research Program from general research support provided by Taiho Oncology, Inc. Clinical trial information: NCT 04072445.

Abstract Background : The 2016 revision to the World Health Organization classification of myeloid neoplasms has recommended distinction between "proliferative" (WBC ≥ 13 x 10(9)/L) and "dysplastic" (WBC < 13 X 10(9)/L) subtypes of chronic myelomonocytic leukemia (CMML). In addition, CMML is characterized by a spectrum of cytogenetic and molecular abnormalities (Patnaik Leukemia 2014). In the current study, we looked for correlations between gene expression profiles (GEP) and morphologic or molecular categories of previously untreated patients with CMML. Methods : 35 patients with CMML were included in the study. Targeted capture assays were carried out on BM DNA specimens obtained at diagnosis for 28 myeloid-relevant genes (Patnaik Blood C. J.). RNA sequencing (Stranded Total RNA- Illumina, San Diego, CA), was performed on 9 normal BM controls, and 35 CMML samples (25 peripheral blood (PB) and 10 BM). The RNA-Seq data was analyzed using MAPRSeq v.2.0. Normalization and differential expression analysis was performed using edgeR 2.6.2. We selected genes that were differentially expressed with a False Discovery Rate (FDR) <0.1 and a fold change bigger than 2. Pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA). Results: Among the 35 study patients, 75% were males and median age was 71 years (range, 55-87). 21 (60%), 9 (25%) and 5 (15%) patients were classified as CMML-0, 1 and 2, respectively. 16 (45%) had a dysplastic while 19 (55%) had a proliferative phenotype. At a median follow-up of 16 months, 5 (14%) deaths and 4 (14%) leukemic transformations were documented. Mutational frequencies included; TET2 45%, ASXL1 45%, SRSF2 40%, NRAS 14%, SETBP1 13%, CBL 10%, JAK2 7%, RUNX1 6%, DNMT3A 6%, U2AF1 6%, SF3B1 5%, ZRSR2 4%, Tp53 4%, and IDH2 4%. Abnormal cytogenetics (n=9, 35%) included; -Y 8%, trisomy 8 6%, monosomy 7 3%, del(13q) 3% and monosomal karyotype 3%. i) Differential GEP in PB samples: 25 PB CMML samples were analyzed. Unsupervised GEP demonstrated two unique clusters (figure one); cluster one was enriched with dysplastic CMML (90%) and cluster two with proliferative CMML (64%). Consistent with the difference in the distribution of morphologic categories, in comparison to cluster one, patients in cluster two had a higher white blood count (p=0.003), higher monocyte count (p=0.02) and higher risk prognostication by the Mayo Model (p=0.016); there was no significant difference between the two clusters in terms of age and gender distribution, hemoglobin level, neutrophil and platelet counts, PB or BM blast content, cytogenetic risk stratification, ASXL1, TET2, RAS, CBL and SETBP1 mutational status. Genes significantly upregulated (FDR<1e-5 and fold change ≥ 16x); in cluster two (proliferative CMML) in comparison to cluster one included; PRG2, CTSG, BEX1, CD34, CRISP2, while those significantly down-regulated included (FDR<1e-15 and fold change ≥ 5x); IL2RB, PRF1, GNLY, PDGFRB. IPA analysis identified the following pathways preferentially expressed in cluster two (proliferative CMML) 1) mitotic roles of polo-like kinase (KIF23, WEE-1, PLK1, PLK4), 2) Cell cycle: G2/M checkpoint regulation (CDC25C, WEE-1, AURKA, CDK1), 3) Cell cycle: chromosomal replication (CDC45, RPA3, MCM2, CDC7), and 4) BRCA1 in DNA damage response (BARD1, RBBP8, PLK1, RAD51). Whereas in the cluster one (dysplastic CMML), preferentially expressed pathways included; 1) T-cell receptor signalling (CD247 PRKCQ, CD4, PLCG1, CD28) and 2) STAT3 signalling (SOCS3, MAP3K9). ii) Differential GEP in BM samples: 10 BM CMML samples were analyzed along with 10 normal controls. Samples and controls clustered separately and within the CMML samples two clusters were identified (figure two). These clusters had equal representations of dysplastic (n=5) and proliferative (n=5) CMML sub-types. Additional samples are being processed. Conclusions: Differential gene expression profiling in patients with CMML identifies two unique clusters, clinically demarcated, at least in PB samples, by "proliferative" and "dysplastic" CMML subtypes. The proliferative cluster is enriched in genes involved in cell cycle regulation and DNA damage response; whereas the dysplastic cluster is enriched in T-cell receptor and STAT3 signalling. The prognostic and therapeutic impact of these gene clusters remains to be assessed in a larger group of informative patients. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Al-Kali: Novartis: Research Funding; Celgene: Research Funding.