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This file contains the additional information for Material and method, including method for cell transduction, cell treatment and WB, RT-qPCR analysis, viablity assays, proliferation assays, DEVD cleavage assay, flow cytometry, soft agar assay, organoid and soft agar assay quantification and ImageJ plugin, scriot for analaysis of unpaired heavy/light phosphopeptides, method for SILAC labelling, SILAC sample preparation, data-dependent acquisition LC-MS/MS, phosphoproteomic data analysis, mice housing, details on statistics, as well as Legends of supplementary figures and tables, table with sequences of shRNAs, used antibodies, RT-qPCR primers.

Supplementary Figures, Legends and Methods

Supplementary Table 3 contains the location of isoform selective phosphorylations in the protein.

Supplementary Table 2 contains the list of isoform selective peptides in each condition, with quantification and global pathway analysis.

Supplementary Figures file contains merged Figure S1 to S8 including description of cell lines, Human samples, PI3K inhibitor stability, SILAC setup and controls, STRING analysis, quantifications of WB and cellular assays, prediction of kinase motifs found regulated, effects of combination of treatment in vitro, correlation between cell survival and phosphopeptide intensity, effects of combination treatment in Human samples and in vivo.

Supplementary Table 1 contains the list of files deposited on PRIDE Proteome X Change under the number PXD008410.

The PI3K pathway is highly active in human cancers. The four class I isoforms of PI3K are activated by distinct mechanisms leading to a common downstream signaling. Their downstream redundancy is thought to be responsible for treatment failures of PI3K inhibitors. We challenged this concept, by mapping the differential phosphoproteome evolution in response to PI3K inhibitors with different isoform selectivity patterns in pancreatic cancer, a disease currently without effective therapy. In this cancer, the PI3K signal was shown to control cell proliferation. We compared the effects of LY294002 that inhibit with equal potency all class I isoenzymes and downstream mTOR with the action of inhibitors with higher isoform-selectivity towards PI3Kα, PI3Kβ or PI3Kγ (namely A66, TGX-221 and AS-252424). A bioinformatics global pathway analysis of phosphoproteomics data allowed us to identify common and specific signals activated by PI3K inhibitors supported by the biological data. AS-252424 was the most effective treatment and induced apoptotic pathway activation as well as the highest changes in global phosphorylation-regulated cell signal. However, AS-252424 treatment induced re-activation of Akt, therefore decreasing the treatment outcome on cell survival. Reversely, AS-252424 and A66 combination treatment prevented p-Akt reactivation and led to synergistic action in cell lines and patient organoids. The combination of clinically approved α-selective BYL-719 with γ-selective IPI-549 was more efficient than single molecule treatment on xenograft growth. Mapping unique adaptive signaling responses to isoform-selective PI3K inhibition will help to design better combinative treatments that prevent the induction of selective compensatory signals.Graphical abstractSignificanceHalf of all human cancers show increased PI3K activity but the mutational pattern of the PI3K pathway is not sufficient to predict sensitivity to PI3K inhibitors. By identifying for the first time specific signaling induced by PI3K inhibitors with different isoform-selectivity patterns, we provide insight in how to handle heterogeneity of PI3K expression in tumor samples for the choice of available PI3K-targetting drugs. Our work provides a roadmap to target the PI3K pathway, balancing the use of isoform-selective PI3K inhibitors with personalized information extending beyond the specific mutational status.

By accentuating drug efficacy and impeding resistance mechanisms, combinatorial, multi‐agent therapies have emerged as key approaches in the treatment of complex diseases, most notably cancer. Using high‐throughput drug screens, we uncovered distinct metabolic vulnerabilities and thereby identified drug combinations synergistically causing a starvation‐like lethal catabolic response in tumor cells from different cancer entities. Domperidone, a dopamine receptor antagonist, as well as several tricyclic antidepressants (TCAs), including imipramine, induced cancer cell death in combination with the mitochondrial uncoupler niclosamide ethanolamine (NEN) through activation of the integrated stress response pathway and the catabolic CLEAR network. Using transcriptome and metabolome analyses, we characterized a combinatorial response, mainly driven by the transcription factors CHOP and TFE3, which resulted in cell death through enhanced pyrimidine catabolism as well as reduced pyrimidine synthesis. Remarkably, the drug combinations sensitized human organoid cultures to the standard‐of‐care chemotherapy paclitaxel. Thus, our combinatorial approach could be clinically implemented into established treatment regimen, which would be further facilitated by the advantages of drug repurposing., This study identifies novel combinatorial drug treatments to induce death of different tumor cells, and defines the mechanisms of synergism between a mitochondrial uncoupler and antidepressants or dopamine receptor antagonists.

Personalized invitro models for dysplasia and carcinogenesis in the pancreas have been constrained by insufficient differentiation of human pluripotent stem cells (hPSCs) into the exocrine pancreatic lineage. Here, we differentiate hPSCs into pancreatic duct-like organoids (PDLOs) with morphological, transcriptional, proteomic, and functional characteristics of human pancreatic ducts, further maturing upon transplantation into mice. PDLOs are generated from hPSCs inducibly expressing oncogenic GNAS, KRAS, or KRAS with genetic covariance of lost CDKN2A and from induced hPSCs derived from a McCune-Albright patient. Each oncogene causes a specific growth, structural, and molecular phenotype invitro. While transplanted PDLOs with oncogenic KRAS alone form heterogenous dysplastic lesions or cancer, KRAS with CDKN2A loss develop dedifferentiated pancreatic ductal adenocarcinomas. In contrast, transplanted PDLOs with mutant GNAS lead to intraductal papillary mucinous neoplasia-like structures. Conclusively, PDLOs enable invitro and invivo studies of pancreatic plasticity, dysplasia, and cancer formation from a genetically defined background.

Several studies have suggested an oncogenic role for the neural Wiskott-Aldrich syndrome protein (N-WASP, encoded by the Wasl gene), but thus far, little is known about its function in pancreatic ductal adenocarcinoma (PDAC). In this study, we performed in silico analysis of WASL expression in PDAC patients and found a correlation between low WASL expression and prolonged survival. To clarify the role of Wasl in pancreatic carcinogenesis, we used 2 oncogenic Kras-based PDAC mouse models with pancreas-specific Wasl deletion. In line with human data, both mouse models had an increased survival benefit due to either impaired tumor development in the presence of the tumor suppressor Trp53 or the delayed tumor progression and senescent phenotype upon genetic ablation of Trp53. Mechanistically, loss of Wasl resulted in cell-autonomous senescence through displacement of the N-WASP binding partners WASP-interacting protein (WIP) and p120ctn; vesicular accumulation of GSK3β, as well as YAP1 and phosphorylated β-catenin, which are components of the destruction complex; and upregulation of Cdkn1a(p21), a master regulator of senescence. Our findings, thus, indicate that Wasl functions in an oncogenic manner in PDAC by promoting the deregulation of the p120-catenin/β-catenin/p21 pathway. Therefore, strategies to reduce N-WASP activity might improve the survival outcomes of PDAC patients. CA extern

ObjectivePancreatic ductal adenocarcinoma (PDAC) still carries a dismal prognosis with an overall 5-year survival rate of 9%. Conventional combination chemotherapies are a clear advance in the treatment of PDAC; however, subtypes of the disease exist, which exhibit extensive resistance to such therapies. Genomic MYC amplifications represent a distinct subset of PDAC with an aggressive tumour biology. It is clear that hyperactivation of MYC generates dependencies that can be exploited therapeutically. The aim of the study was to find and to target MYC-associated dependencies.DesignWe analysed human PDAC gene expression datasets. Results were corroborated by the analysis of the small ubiquitin-like modifier (SUMO) pathway in a large PDAC cohort using immunohistochemistry. A SUMO inhibitor was used and characterised using human and murine two-dimensional, organoid and in vivo models of PDAC.ResultsWe observed that MYC is connected to the SUMOylation machinery in PDAC. Components of the SUMO pathway characterise a PDAC subtype with a dismal prognosis and we provide evidence that hyperactivation of MYC is connected to an increased sensitivity to pharmacological SUMO inhibition.ConclusionSUMO inhibitor-based therapies should be further developed for an aggressive PDAC subtype.

Pancreatic ductal adenocarcinoma is one of the most aggressive forms of cancer, and the third leading cause of cancer-related mortality in the United States. Although important advances have been made in the last decade, the mortality rate of pancreatic ductal adenocarcinoma has not changed appreciably. This review summarizes a rapidly emerging model of pancreatic cancer research, focusing on 3-dimensional organoids as a powerful tool for several applications, but above all, representing a step toward personalized medicine.

Forced expression of C-X-C chemokine receptor type 6 in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer.The efficacy of adoptive cell therapy for solid tumours is hampered by the poor accumulation of the transferred T cells in tumour tissue. Here, we show that forced expression of C-X-C chemokine receptor type 6 (whose ligand is highly expressed by human and murine pancreatic cancer cells and tumour-infiltrating immune cells) in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer. In mice with subcutaneous pancreatic tumours treated with T cells with either a transgenic T-cell receptor or a murine chimeric antigen receptor targeting the tumour-associated antigen epithelial cell adhesion molecule, and in mice with orthotopic pancreatic tumours or patient-derived xenografts treated with T cells expressing a chimeric antigen receptor targeting mesothelin, the T cells exhibited enhanced intratumoral accumulation, exerted sustained anti-tumoral activity and prolonged animal survival only when co-expressing C-X-C chemokine receptor type 6. Arming tumour-specific T cells with tumour-specific chemokine receptors may represent a promising strategy for the realization of adoptive cell therapy for solid tumours.

e21014 Background: ALK rearrangements are key targets in non-small-cell lung cancer (NSCLC). Unfortunately, the optimal sequential strategy of ALK-tyrosine kinase inhibitors (TKI) remains to be defined. Testing drug sensitivity in patient derived organoids (PDOs) could support a rational drug selection in this setting. Methods: We designed an observational study assessing the correlation between drug sensitivity of PDOs established in Invitrocue and the clinical outcome in our institution. To date, forty cases have been included, of which nine were lung cancers. Results: PDO was sucessfully established in 7/9 cases (77%). Three patients did not receive any of the drugs tested in vitro due to clinical deterioration. One patient was deemed as sensitive to carboplatin but tumor showed to be resistant. A meaningful correlation was observed in an oncogenic addicted tumor. A 54-year-old never-smoker man who had been diagnosed with lung adenocarcinoma stage IVa (T3N1M1a). He received standard first-line therapy with platinum-based chemotherapy, immunotherapy and antiangiogenics achieving tumor progression. A next-generation sequencing (NGS) panel revealed the presence of the EML4-ALK fusion variant 3a/b. The patient started alectinib but showed progression after 12 months. A second NGS panel did not identify any new ALK resistance mutation but acquiring TP53. Treatment was switched to brigatinib with no response. Finally, fresh tumor tissue was obtained from a liver biopsy to establish PDOs and drug sensitivity to 8 compounds was tested. The In vitro results demonstrated no activity of lorlatinib but a strong response to crizotinib. The patient started Crizotinib 250mg BID achieving partial response after 8 weeks and treatment duration of 6 months. A third liver biopsy for PDOs and NGS revealed acquired ALK C1156Y and I1171T mutations that confer resistance to crizotinib but sensitive to ceritinib, respectively. Conclusions: We present clinical evidence that PDOs are an alternative tool in oncogenic addicted tumors helping to guide treatment decisions and increasing a more personalized sequential treatment approach.

In many solid tumors, parasympathetic input is provided by the vagus nerve, which has been shown to modulate tumor growth. However, whether cholinergic signaling directly regulates progression of pancreatic ductal adenocarcinoma (PDAC) has not been defined. Here, we found that subdiaphragmatic vagotomy in LSL-Kras+/G12D;Pdx1-Cre (KC) mice accelerated PDAC development, whereas treatment with the systemic muscarinic agonist bethanechol restored the normal KC phenotype, thereby suppressing the accelerated tumorigenesis caused by vagotomy. In LSL-Kras+/G12D;LSL-Trp53+/R172H;Pdx1-Cre mice with established PDAC, bethanechol significantly extended survival. These effects were mediated in part through CHRM1, which inhibited downstream MAPK/EGFR and PI3K/AKT pathways in PDAC cells. Enhanced cholinergic signaling led to a suppression of the cancer stem cell (CSC) compartment, CD11b+ myeloid cells, TNFα levels, and metastatic growth in the liver. Therefore, these data suggest that cholinergic signaling directly and indirectly suppresses growth of PDAC cells, and therapies that stimulate muscarinic receptors may be useful in the treatment of PDAC. Significance: Subdiaphragmatic vagotomy or Chrm1 knockout accelerates pancreatic tumorigenesis, in part via expansion of the CSC compartment. Systemic administration of a muscarinic agonist suppresses tumorigenesis through MAPK and PI3K/AKT signaling, in early stages of tumor growth and in more advanced, metastatic disease. Therefore, CHRM1 may represent a potentially attractive therapeutic target. Cancer Discov; 8(11); 1458–73. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333

The ubiquitously expressed non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, is involved in signal transduction downstream of multiple growth factor, cytokine and integrin receptors1. Its requirement for complete RAS-MAPK activation and its role as a negative regulator of JAK-STAT signaling have established SHP2 as an essential player in oncogenic signaling pathways1-7. Recently, a novel potent allosteric SHP2 inhibitor was presented as a viable therapeutic option for receptor tyrosine kinase-driven cancers, but was shown to be ineffective in KRAS-mutant tumor cell lines in vitro8. Here, we report a central and indispensable role for SHP2 in oncogenic KRAS-driven tumors. Genetic deletion of Ptpn11 profoundly inhibited tumor development in mutant KRAS-driven murine models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. We provide evidence for a critical dependence of mutant KRAS on SHP2 during carcinogenesis. Deletion or inhibition of SHP2 in established tumors delayed tumor progression but was not sufficient to achieve tumor regression. However, SHP2 was necessary for resistance mechanisms upon blockade of MEK. Synergy was observed when both SHP2 and MEK were targeted, resulting in sustained tumor growth control in murine and human patient-derived organoids and xenograft models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. Our data indicate the clinical utility of dual SHP2/MEK inhibition as a targeted therapy approach for KRAS-mutant cancers., This work was supported by grants from Deutsche Forschungsgemeinschaft (DFG AL1174/5-1 to H.A. and LE3222/1-1 to M.L.), Deutsche Krebshilfe (no. 111646 and no. 111464 to H.A.; Max Eder Program no. 111273 to M.R.), the Wilhelm Sander Stiftung (2014.052.1 to H.A.) and the Fundación Asociación Española Contra el Cáncer (to B.S.).

Background: Although the mechanistic target of rapamycin (MTOR) kinase, included in the mTORC1 and mTORC2 signalling hubs, has been demonstrated to be active in a significant fraction of patients with pancreatic ductal adenocarcinoma (PDAC), the value of the kinase as a therapeutic target needs further clarification. Methods: We used Mtor floxed mice to analyse the function of the kinase in context of the pancreas at the genetic level. Using a dual-recombinase system, which is based on the flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies, we generated a novel cellular model, allowing the genetic analysis of MTOR functions in tumour maintenance. Cross-species validation and pharmacological intervention studies were used to recapitulate genetic data in human models, including primary human 3D PDAC cultures. Results: Genetic deletion of the Mtor gene in the pancreas results in exocrine and endocrine insufficiency. In established murine PDAC cells, MTOR is linked to metabolic pathways and maintains the glucose uptake and growth. Importantly, blocking MTOR genetically as well as pharmacologically results in adaptive rewiring of oncogenic signalling with activation of canonical extracellular signal-regulated kinase and phosphoinositide 3-kinase-AKT pathways. We provide evidence that interfering with such adaptive signalling in murine and human PDAC models is important in a subgroup. Conclusions: Our data suggest developing dual MTORC1/TORC2 inhibitor-based therapies for subtype-specific intervention.

Catecholamines stimulate epithelial proliferation, but the role of sympathetic nerve signaling in pancreatic ductal adenocarcinoma (PDAC) is poorly understood. Catecholamines promoted ADRB2-dependent PDAC development, nerve growth factor (NGF) secretion, and pancreatic nerve density. Pancreatic Ngf overexpression accelerated tumor development in LSL-Kras+/G12D;Pdx1-Cre (KC) mice. ADRB2 blockade together with gemcitabine reduced NGF expression and nerve density, and increased survival of LSL-Kras+/G12D;LSL-Trp53+/R172H;Pdx1-Cre (KPC) mice. Therapy with a Trk inhibitor together with gemcitabine also increased survival of KPC mice. Analysis of PDAC patient cohorts revealed a correlation between brain-derived neurotrophic factor (BDNF) expression, nerve density, and increased survival of patients on nonselective β-blockers. These findings suggest that catecholamines drive a feedforward loop, whereby upregulation of neurotrophins increases sympathetic innervation and local norepinephrine accumulation. Renz et al. show that catecholamines promote ADRB2-dependent pancreatic ductal adenocarcinoma development and secretion of neurotrophins (NT), which in turn promote tumor innervation leading to increased NE and tumor growth. Blockade of ADRB2 or NT receptors improves gemcitabine's therapeutic effect.

One of the major challenges in using pancreatic cancer patient–derived organoids (PDOs) in precision oncology is the time from biopsy to functional characterization. This is particularly true for endoscopic ultrasound-guided fine-needle aspiration biopsies, typically resulting in specimens with limited tumor cell yield. Here, we tested conditioned media of individual PDOs for cell-free DNA to detect driver mutations already early on during the expansion process to accelerate the genetic characterization of PDOs as well as subsequent functional testing. Importantly, genetic alterations detected in the PDO supernatant, collected as early as 72 hours after biopsy, recapitulate the mutational profile of the primary tumor, indicating suitability of this approach to subject PDOs to drug testing in a reduced time frame. In addition, we demonstrated that this workflow was practicable, even in patients for whom the amount of tumor material was not sufficient for molecular characterization by established means. Together, our findings demonstrate that generating PDOs from very limited biopsy material permits molecular profiling and drug testing. With our approach, this can be achieved in a rapid and feasible fashion with broad implications in clinical practice. Kein CA