Phase I clinical trial repurposing all-trans retinoic acid as a stromal targeting agent for pancreatic cancer

Abstract: Pre-clinical models have shown that targeting pancreatic stellate cells with all-trans-retinoic-acid (ATRA) reprograms pancreatic stroma to suppress pancreatic ductal adenocarcinoma (PDAC) growth. Here, in a phase Ib, dose escalation and expansion, trial for patients with advanced, unresectable PDAC (n = 27), ATRA is re-purposed as a stromal-targeting agent in combination with gemcitabine-nab-paclitaxel chemotherapy using a two-step adaptive continual re-assessment method trial design. The maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D, primary outcome) is the FDA/EMEA approved dose of gemcitabine-nab-paclitaxel along-with ATRA (45 mg/m2 orally, days 1–15/cycle). Dose limiting toxicity (DLT) is grade 4 thrombocytopenia (n = 2). Secondary outcomes show no detriment to ATRA pharmacokinetics.. Median overall survival for RP2D treated evaluable population, is 11.7 months (95%CI 8.6–15.7 m, n = 15, locally advanced (2) and metastatic (13)). Exploratory pharmacodynamics studies including changes in diffusion-weighted (DW)-MRI measured apparent diffusion coefficient after one cycle, and, modulation of cycle-specific serum pentraxin 3 levels over various cycles indicate stromal modulation. Baseline stromal-specific retinoid transport protein (FABP5, CRABP2) expression may be predicitve of response. Re-purposing ATRA as a stromal-targeting agent with gemcitabine-nab-paclitaxel is safe and tolerable. This combination will be evaluated in a phase II randomized controlled trial for locally advanced PDAC. Clinical trial numbers: EudraCT: 2015-002662-23; NCT03307148. Trial acronym: STARPAC

HNF4A and GATA6 loss reveals therapeutically actionable subtypes in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) can be divided into transcriptomic subtypes with two broad lineages referred to as classical (pancreatic) and squamous. We find that these two subtypes are driven by distinct metabolic phenotypes. Loss of genes that drive endodermal lineage specification, HNF4A and GATA6, switch metabolic profiles from classical (pancreatic) to predominantly squamous, with glycogen synthase kinase 3 beta (GSK3β) a key regulator of glycolysis. Pharmacological inhibition of GSK3β results in selective sensitivity in the squamous subtype; however, a subset of these squamous patient-derived cell lines (PDCLs) acquires rapid drug tolerance. Using chromatin accessibility maps, we demonstrate that the squamous subtype can be further classified using chromatin accessibility to predict responsiveness and tolerance to GSK3β inhibitors. Our findings demonstrate that distinct patterns of chromatin accessibility can be used to identify patient subgroups that are indistinguishable by gene expression profiles, highlighting the utility of chromatin-based biomarkers for patient selection in the treatment of PDAC

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer

Background and aims: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress and novel therapeutic response in PC to develop a biomarker driven therapeutic strategy targeting DDR and replication stress in PC. Methods: We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient derived xenografts and human PC organoids. Results: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum (P < 0.001) and PARP inhibitor therapy (P < 0.001) in vitro and in vivo. We generated a novel signature of replication stress with which predicts response to ATR (P < 0.018) and WEE1 inhibitor (P < 0.029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < 0.001) but not associated with DDR deficiency. Conclusions: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy

Retinoic Acid--Induced Pancreatic Stellate Cell Quiescence Reduces Paracrine Wnt-β-Catenin Signaling to Slow Tumor Progression

BACKGROUND & AIMS: Patients with pancreatic ductal adenocarcinoma are deficient in vitamin A, resulting in activation of pancreatic stellate cells (PSCs). We investigated whether restoration of retinol to PSCs restores their quiescence and affects adjacent cancer cells. METHODS: PSCs and cancer cell lines (AsPc1 and Capan1) were exposed to doses and isoforms of retinoic acid (RA) in 2-dimensional and 3-dimensional culture conditions (physiomimetic organotypic culture). The effects of all-trans retinoic acid (ATRA) were studied in LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mice, a model of human pancreatic ductal adenocarcinoma. RESULTS: After incubation with ATRA, PSCs were quiescent and had altered expression of genes that regulate proliferation, morphology, and motility; genes that encode cytoskeletal proteins and cytokines; and genes that control other functions, irrespective of culture conditions or dosage. In the organotypic model, and in mice, ATRA induced quiescence of PSCs and thereby reduced cancer cell proliferation and translocation of beta-catenin to the nucleus, increased cancer cell apoptosis, and altered tumor morphology. ATRA reduced the motility of PSCs, so these cells created a "wall" at the junction between the tumor and the matrix that prevented cancer cell invasion. Restoring secreted frizzled-related protein 4 (sFRP4) secretion to quiescent PSCs reduced Wnt-beta-catenin signaling in cancer cells and their invasive ability. Human primary and metastatic pancreatic tumor tissues stained strongly for cancer cell nuclear beta-catenin but had low levels of sFRP4 (in cancer cells and PSCs). CONCLUSIONS: RA induces quiescence and reduces motility of PSCs, leading to reduced proliferation and increased apoptosis of surrounding pancreatic cancer cells. RA isoforms might be developed as therapeutic reagents for pancreatic cancer. [KEYWORDS: Animals, Antineoplastic Agents/ pharmacology, Apoptosis/drug effects, Carcinoma, Pancreatic Ductal/ drug therapy/genetics/metabolism/pathology, Cell Aging/ drug effects, Cell Line, Tumor, Cell Movement/drug effects, Cell Proliferation/drug effects, Disease Models, Animal, Disease Progression, Dose-Response Relationship, Drug, Gene Expression Regulation, Neoplastic/drug effects, Humans, Isotretinoin/pharmacology, Mice, Mice, Mutant Strains, Pancreatic Neoplasms/ drug therapy/genetics/

Polyethnic-1000: Advancing cancer genomics by studying ethnically diverse, underserved patient populations in New York

Background and aims Recent advances in DNA sequencing technologies have revolutionized approaches to the prevention, risk assessment, early detection, diagnosis, and treatment of cancers. However, many ethnic groups, especially non-European populations, have been significantly under-represented in cancer research, including clinical trials, and have not received equal benefits in clinical practice. As a result, our current knowledge about tumor biology, cancer risk, and response to treatment has primarily been derived from patients of European descent. These inequities limit our understanding of the many types of cancer and may exacerbate health disparities in the United States. This multi-institutional study, named Polyethnic-1000, aims to address both the scientific and social issues by creating a dynamic research platform within the ethnically diverse greater New York City area. Methods and results Polyethnic-1000 is a collaborative effort organized by the New York Genome Center (NYGC), involving staff and patients at academic centers and partnering hospitals in the New York City region. The genomics and informatics capabilities of the NYGC will be used to determine how inherited and somatically acquired genetic variations affect the behavior of cancers occurring in ethnically diverse populations. In a first, retrospective stage we are establishing the necessary infrastructure and workflow from sample acquisition to whole-exome and RNA sequencing, data analysis and data sharing within the consortium. Then we will start a prospective study enabling the formation of cohorts of interest for particular cancer types and particular ethnicities, with uniform consent allowing germline and somatic sequencing with broad data sharing of the somatic variants identified. Conclusion By establishing a collaborative network, Polyethnic-1000 will deepen our understanding of the contributions that ethnicities make to the incidence and biology of cancers, potentially improving outcomes for patients who currently lack access to the most recent advances in medical science

RhoC Interacts with Integrin alpha 5 beta 1 and Enhances Its Trafficking in Migrating Pancreatic Carcinoma Cells

This work was funded by an NIHR Clinician Scientist Award to HMK. DAT acknowledges the support of the University of Cambridge, Cancer Research UK and Hutchinson Whampoa Limited

Src activation is downstream of RhoC-enhanced integrin α5β1 trafficking, partially contributing to increased cell migration.

<div><p>(A)Total RhoA and RhoB were unaltered upon transfection with various RhoC constructs in Capan1 cells. Rotekin immunoprecipitation binding assays revealed an increase of active RhoC-GTP in nRhoC cells (RhoA-GTP, RhoB-GTP levels were unaltered). Densitometric analysis for various RhoC-harboring constructs’ cell lines are normalized to parental Capan1 levels from triplicate experiments. </p> <p>(B)Upon plating on fibronectin, there was an increased level of phospho-Src (Tyr416) in nRhoC cells only. This increase was abrogated upon treatment with integrin α5β1-neutralizing antibody. Both cRhoC and nDCT cells showed significantly lower phospho-Src than parental Capan1 cells after treatment (IgG controls: no difference from the non-treated cells). </p> <p>(C)Functionally, both nRhoC and cRhoC cells showed enhanced migration in the IgG control conditions as compared with the parental, nEV, or nDCT cells. Blocking cells with 8μg/ml of the integrin α5β1-neutralizing antibody significantly diminished cell migration of both the nRhoC and cRhoC cells but did not alter basal migratory capacity of the nDCT cells. However, in nRhoC cells this reduction in migration capacity did not return to the level of parental or nEV cells. </p> <p>(D)Similarly in the endogenously high-expressing Panc0403 cells (also for HPAF (data not shown)) and the vector control transfected (pSilencer) cell line there was an abrogation of the enhanced migratory capacity upon blockade with integrin α5β1-neutralizing antibody compared with the lack of effect on the shRhoC cell line. </p> <p>*p<0.05, **p<0.001, Student’s t-test, error bars: SEM. </p></div

RhoC enhanced integrin α5β1 internalization and recycling upon fibronectin adherence.

<div><p>The well-established Biotin-labeling assay (labeling integrin α5β1 with Biotin and allowing internalization and recycling (separate assays: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s001" target="_blank">Methods S1</a>) followed by cleavage of Biotin and measurement of integrin by ELISA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#B18" target="_blank">18</a>]) to compare the internalization and recycling rates of integrin α5β1. Graphs represent summary data from three representative individual experiments. The trend-line shown is second-order polynomial regression fit for the data, as previously used [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#B46" target="_blank">46</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#B47" target="_blank">47</a>]. Thus, compared with parental Capan1 cells, nRhoC cells demonstrated significantly increased internalization (A) and recycling (B) of integrin α5β1 on fibronectin-coated surface. The masking of RhoC CAAX in cRhoC cells resulted in a significant increase in internalization, but not recycling, of integrin α5β1 (compared with parental Capan1 cells). However, deletion of the C-terminal of RhoC in nDCT cells resulted in significant reduction in internalization and recycling of integrin α5β1. </p> <p>Compared with the parental HPAF and pSilencer (empty vector) cells, shRhoC (stable RhoC knockdown) cells showed a significant decline of integrin α5β1 internalization (C) and recycling (D) on a fibronectin-coated surface. The dramatic reduction in the HPAF-shRhoC cells was not due to a vector artifact, since HPAF-pSilencer cells actually showed a significant enhancement of the recycling rate. Similar data were obtained after knock-down of endogenous RhoC in Panc0403 (not shown). In addition, there was no change in Transferrin receptor recycling after manipulation of RhoC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s013" target="_blank">Figure S8</a>). *** p<0.0001, ** p<0.001, * p<0.01, ANOVA. Error bars: SEM. </p></div

RhoC overexpression causes rapid spreading and movement of Capan1 cells.

<div><p>Initial screen of PDAC cell lines revealed low expression levels of RhoC protein in Capan1 pancreatic cancer cells which were then transfected with RhoC constructs (nRhoC, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s006" target="_blank">Figures S1, S2</a>). </p> <p>(A)Cells were plated on the fibronectin-coated surface and incubated at 37°C till the indicated time-point, then fixed and stained with Crystal Violet before imaging with Zeiss Axiovert 200M microscope. nRhoC cells (Capan1 cells with N-terminus tagged RhoC overexpression) spread more within the first two hours as compared to the rounded parental Capan1 cells. </p> <p>(B)A selection of images from time-lapse videos (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s006" target="_blank">videos S1, S2</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s008" target="_blank">S3</a> for detailed information) of cell movement during two hours after plating on fibronectin-coated surface. These images demonstrate that nRhoC transfected cells had accelerated cell spreading and contraction, associated with rapid movement, as compared to Capan1 cells (arrows). Time of capture is indicated at the top-left corner of each image (m= minutes). </p> <p>Scale bar: 20µm.</p></div

RhoC knockdown decrease cellular migration and is rescued by ectopic RhoC overexpression.

<div><p>Initial screen of PDAC cell lines revealed high expression levels of RhoC protein in HPAF and Panc0403 pancreatic cancer cells. Endogenous RhoC was silenced using shRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s006" target="_blank">Figures S1, S2</a>). </p> <p>shRhoC (stable RhoC knockdown: two different constructs were used: shRhoC1 and shRhoC2) resulted in a significant reduction of cell migration compared with the parental or pSilencer (empty vector control) cell lines towards 10% FBS (A) or through Fibronectin (B). Transient over-expression of RhoC in shRhoC cells (pTag2BRhoC) restored cell migration significantly and comparable to parental cell line. pTag2B is the empty vector control cell line. Similarly, another cell line (HPAF) demonstrated RhoC-dependent migration (C). Here the summary data of both ShRhoC are presented for migration towards 10% FBS. ** p<0.001, * p<0.01, ANOVA. Individual data points represent technical/biological repeats with summary statistics represented by mean ± SEM.</p></div