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Supplementary figure legends for Figures S1-S8

Supplementary materials and methods

mTOR signaling controls several critical cellular functions and is deregulated in many cancers, including pancreatic cancer. To date, most efforts have focused on inhibiting the mTORC1 complex. However, clinical trials of mTORC1 inhibitors in pancreatic cancer have failed, raising questions about this therapeutic approach. We employed a genetic approach to delete the obligate mTORC2 subunit Rictor and identified the critical times during which tumorigenesis requires mTORC2 signaling. Rictor deletion resulted in profoundly delayed tumorigenesis. Whereas previous studies showed most pancreatic tumors were insensitive to rapamycin, treatment with a dual mTORC1/2 inhibitor strongly suppressed tumorigenesis. In late-stage tumor-bearing mice, combined mTORC1/2 and PI3K inhibition significantly increased survival. Thus, targeting mTOR may be a potential therapeutic strategy in pancreatic cancer. Cancer Res; 76(23); 6911–23. ©2016 AACR.

Supplementary Legends from Mist1-KrasG12D Knock-In Mice Develop Mixed Differentiation Metastatic Exocrine Pancreatic Carcinoma and Hepatocellular Carcinoma

Despite the prevalence of oncogenic Kras mutations in the earliest stages of pancreatic ductal adenocarcinoma, the cellular compartment in which oncogenic Kras initiates tumorigenesis remains unknown. To address this, we have gene targeted KrasG12D into the open reading frame of Mist1, a basic helix-loop-helix transcription factor that is expressed during pancreatic development and required for proper pancreatic acinar organization. Although the pancreata of Mist1KrasG12D/+ mutant mice predictably exhibited acinar metaplasia and dysplasia, the frequent death of these mice from invasive and metastatic pancreatic cancer with mixed histologic characteristics, including acinar, cystic, and ductal features, was unexpected and in contrast to previously described mutant mice that ectopically expressed the Kras oncogene in either acinar or ductal compartments. Interestingly, many of the mutant mice developed hepatocellular carcinoma, implicating Mist1KrasG12D/+ cells in both pancreatic and hepatic neoplasia. Concomitant Trp53+/− mutation cooperated with Mist1KrasG12D/+ to accelerate lethality and was associated with advanced histopathologic findings, including parenchymal liver metastasis. These findings suggest that Mist1-expressing cells represent a permissive compartment for transformation by oncogenic Kras in pancreatic tumorigenesis. (Cancer Res 2006; 66(1): 242-7)

Supplementary Figure 1 from Mist1-KrasG12D Knock-In Mice Develop Mixed Differentiation Metastatic Exocrine Pancreatic Carcinoma and Hepatocellular Carcinoma

Supplementary Table 1 from Mist1-KrasG12D Knock-In Mice Develop Mixed Differentiation Metastatic Exocrine Pancreatic Carcinoma and Hepatocellular Carcinoma

Gemcitabine (dFdC) is a common treatment for pancreatic cancer; however, it is thought that treatment may fail because tumor stroma prevents drug distribution to tumor cells. Gemcitabine is a pro-drug with active metabolites generated intracellularly; therefore, visualizing the distribution of parent drug as well as its metabolites is important. A multimodal imaging approach was developed using spatially coregistered mass spectrometry imaging (MSI), imaging mass cytometry (IMC), multiplex immunofluorescence microscopy (mIF), and hematoxylin and eosin (H&E) staining to assess the local distribution and metabolism of gemcitabine in tumors from a genetically engineered mouse model of pancreatic cancer (KPC) allowing for comparisons between effects in the tumor tissue and its microenvironment. Mass spectrometry imaging (MSI) enabled the visualization of the distribution of gemcitabine (100 mg/kg), its phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP, and the inactive metabolite dFdU. Distribution was compared to small-molecule ATR inhibitor AZD6738 (25 mg/kg), which was codosed. Gemcitabine metabolites showed heterogeneous distribution within the tumor, which was different from the parent compound. The highest abundance of dFdCMP, dFdCDP, and dFdCTP correlated with distribution of endogenous AMP, ADP, and ATP in viable tumor cell regions, showing that gemcitabine active metabolites are reaching the tumor cell compartment, while AZD6738 was located to nonviable tumor regions. The method revealed that the generation of active, phosphorylated dFdC metabolites as well as treatment-induced DNA damage primarily correlated with sites of high proliferation in KPC PDAC tumor tissue, rather than sites of high parent drug abundance.

Background Nab-paclitaxel plus gemcitabine (nabP+gemcitabine) offers modest survival gains for patients with metastatic pancreatic ductal adenocarcinoma (PDAC). Sequential scheduling of nabP+gemcitabine in a PDAC mouse model improved efficacy; this hypothesis was tested in a clinical trial. Methods Patients with previously untreated metastatic PDAC were randomised to receive nabP+gemcitabine administered either concomitantly on the same day, or sequentially, with gemcitabine administered 24 h after nabP. The primary outcome measure was progression-free survival (PFS). Secondary outcome measures were objective response rate (ORR), overall survival (OS), safety, quality of life (QoL) and predictive biomarkers. Results In total, 71 patients received sequential (SEQ) and 75 concomitant (CON) treatment. Six-month PFS was 46% with SEQ and 32% with CON scheduling. Median PFS (5.6 versus 4.0 months, hazard ratio [HR] 0.67, 95% confidence interval [95% CI] 0.47–0.95, p = 0.022) and ORR (52% versus 31%, p = 0.023) favoured the SEQ arm; median OS was 10.2 versus 8.2 months (HR 0.93, 95% CI 0.65–1.33, p = 0.70). CTCAE Grade ≥3 neutropaenia incidence doubled with SEQ therapy but was not detrimental to QoL. Strongly positive tumour epithelial cytidine deaminase (CDA) expression favoured benefit from SEQ therapy (PFS HR 0.31, 95% CI 0.13–0.70). Conclusions SEQ delivery of nabP+gemcitabine improved PFS and ORR, with manageable toxicity, but did not significantly improve OS. Clinical trial registration ISRCTN71070888; ClinialTrials.gov (NCT03529175).

Significance Patients with microsatellite stable (MSS) pancreatic (PDA) or colorectal cancer (CRC) do not respond to immunotherapy with inhibitors of T cell checkpoints. A possible explanation is suggested by finding that cancer cells in these tumors are coated with the chemokine, CXCL12, and that stimulation of CXCR4, the CXCL12 receptor on immune cells, suppresses directed migration mediated by other chemokine receptors on these cells. We assessed the relevance of these findings by treating patients for seven days with continuous infusion of AMD3100/Plerixafor, a CXCR4 inhibitor. Comparison of pre- and end-of-treatment paired biopsies of metastatic lesions by transcriptomic analysis revealed that AMD3100 induced an integrated immune response that is predictive of a clinical response to T cell checkpoint inhibition., Inhibition of the chemokine receptor CXCR4 in combination with blockade of the PD-1/PD-L1 T cell checkpoint induces T cell infiltration and anticancer responses in murine and human pancreatic cancer. Here we elucidate the mechanism by which CXCR4 inhibition affects the tumor immune microenvironment. In human immune cell-based chemotaxis assays, we find that CXCL12-stimulated CXCR4 inhibits the directed migration mediated by CXCR1, CXCR3, CXCR5, CXCR6, and CCR2, respectively, chemokine receptors expressed by all of the immune cell types that participate in an integrated immune response. Inhibiting CXCR4 in an experimental cancer medicine study by 1-wk continuous infusion of the small-molecule inhibitor AMD3100 (plerixafor) induces an integrated immune response that is detected by transcriptional analysis of paired biopsies of metastases from patients with microsatellite stable colorectal and pancreatic cancer. This integrated immune response occurs in three other examples of immune-mediated damage to noninfected tissues: Rejecting renal allografts, melanomas clinically responding to anti-PD1 antibody therapy, and microsatellite instable colorectal cancers. Thus, signaling by CXCR4 causes immune suppression in human pancreatic ductal adenocarcinoma and colorectal cancer by impairing the function of the chemokine receptors that mediate the intratumoral accumulation of immune cells.

Background: The Notch pathway is frequently activated in cancer. Pathway inhibition by g-secretase inhibitors has been shown to be effective in pre-clinical models of pancreatic cancer, in combination with gemcitabine. Methods: A multi-centre, non-randomised Bayesian adaptive design study of MK-0752, administered per os weekly, in combination with gemcitabine administered intravenously on days 1, 8 and 15 (28 day cycle) at 800 or 1000mgm-2, was performed to determine the safety of combination treatment and the recommended phase 2 dose (RP2D). Secondary and tertiary objectives included tumour response, plasma and tumour MK-0752 concentration, and inhibition of the Notch pathway in hair follicles and tumour. Results: Overall, 44 eligible patients (performance status 0 or 1 with adequate organ function) received gemcitabine and MK-0752 as first or second line treatment for pancreatic cancer. RP2Ds of MK-0752 and gemcitabine as single agents could be combined safely. The Bayesian algorithm allowed further dose escalation, but pharmacokinetic analysis showed no increase in MK-0752 AUC (area under the curve) beyond 1800mg once weekly. Tumour response evaluation was available in 19 patients; 13 achieved stable disease and 1 patient achieved a confirmed partial response. Conclusions: Gemcitabine and a g-secretase inhibitor (MK-0752) can be combined at their full, single-agent RP2Ds.

mTOR signaling controls several critical cellular functions and is deregulated in many cancers, including pancreatic cancer. To date, most efforts have focused on inhibiting the mTORC1 complex. However, clinical trials of mTORC1 inhibitors in pancreatic cancer have failed, raising questions about this therapeutic approach. We employed a genetic approach to delete the obligate mTORC2 subunit Rictor and identified the critical times during which tumorigenesis requires mTORC2 signaling. Rictor deletion resulted in profoundly delayed tumorigenesis. Whereas previous studies showed most pancreatic tumors were insensitive to rapamycin, treatment with a dual mTORC1/2 inhibitor strongly suppressed tumorigenesis. In late-stage tumor-bearing mice, combined mTORC1/2 and PI3K inhibition significantly increased survival. Thus, targeting mTOR may be a potential therapeutic strategy in pancreatic cancer. Cancer Res; 76(23); 6911–23. ©2016 AACR.

Pancreatic ductal adenocarcinoma is the most common form of pancreatic tumour, with a very limited survival rate and currently no available disease-modifying treatments. Despite recent advances in the production of genetically engineered mouse models (GEMMs), the development of new therapies for pancreatic cancer is still hampered by a lack of reliable and predictive preclinical animal models for this disease. Preclinical models are vitally important for assessing therapies in the first stages of the drug development pipeline, prior to their transition to the clinical arena. GEMMs carry mutations in genes that are associated with specific human diseases and they can thus accurately mimic the genetic, phenotypic and physiological aspects of human pathologies. Here, we discuss different GEMMs of human pancreatic cancer, with a focus on the Lox-Stop-Lox (LSL)-KrasG12D; LSL-Trp53R172H; Pdx1-cre (KPC) model, one of the most widely used preclinical models for this disease. We describe its application in preclinical research, highlighting its advantages and disadvantages, its potential for predicting clinical outcomes in humans and the factors that can affect such outcomes, and, finally, future developments that could advance the discovery of new therapies for pancreatic cancer., Drug Discovery Collection: This Review discusses GEMMs of human pancreatic cancer, particularly focusing on the KPC model, its use in preclinical research, advantages and disadvantages, and its potential for predicting clinical outcomes.

Summary Pancreatic ductal adenocarcinoma (PDAC) is resistant to most therapies including single-agent immunotherapy and has a dense desmoplastic stroma, and most patients present with advanced metastatic disease. We reveal that macrophages are the dominant leukocyte population both in human PDAC stroma and autochthonous models, with an important functional contribution to the squamous subtype of human PDAC. We targeted macrophages in a genetic PDAC model using AZD7507, a potent selective inhibitor of CSF1R. AZD7507 caused shrinkage of established tumors and increased mouse survival in this difficult-to-treat model. Malignant cell proliferation diminished, with increased cell death and an enhanced T cell immune response. Loss of macrophages rewired other features of the TME, with global changes in gene expression akin to switching PDAC subtypes. These changes were markedly different to those elicited when neutrophils were targeted via CXCR2. These results suggest targeting the myeloid cell axis may be particularly efficacious in PDAC, especially with CSF1R inhibitors., Graphical Abstract, Highlights • Macrophages functionally contribute to the squamous subtype of human PDAC • Inhibition of CSF1R alters the TME and results in an enhanced T cell immune response • Loss of macrophages rewires PDAC gene expression and switches subtype • Macrophage inhibition induces changes markedly different to neutrophil targeting, Candido et al. find that CSF-1R+ macrophages play a vital role in pancreatic tumor maintenance, suppression of T cells, and tumor gene networks based on transcriptome analysis. In an autochthonous model, CSF-1R inhibition alters the gene expression programs that influence subtype specification of PDAC and extends survival.

The use of oligosaccharides as active pharmaceutical ingredients lags behind that of other biological molecules such as proteins and nucleic acids. However, there is now a growing understanding of the complex biological functions of saccharide structures. This has resulted in an increased interest in exploring the pharmaceutical applications of both oligosaccharides and glycoconjugates. Despite recent advances in the treatment of pancreatic adenocarcinoma (PDAC), the median survival remains Here we report a novel oligosaccharide drug candidate, RiXOVA (G-blocks). The term “G-blocks” refers to highly defined and specialized short oligomers comprising α-L-guluronate residues derived from alginates extracted from brown seaweed. The G-block oligomers are well tolerated at i.v./i.p. doses >100mg/kg in mice. These oligomers have previously demonstrated the ability to alter matrices of biological macromolecules including mucins, giving rise to the hypothesis that they may have similar effects on the dense desmoplastic tissue within tumours. We here describe the anti-tumour properties of RiXOVA in different PDAC mouse models. Initial efficacy studies were performed in a xenograft model using the CAPAN-2 cells. RiXOVA was found to be non-toxic and non-immunogenic (I.P, 25mg/kg Q3D10 ) in mice. Relative to the start of dosing, the tumour growth in RiXOVA treated mice was 160% +/-30 (n=10) compared to 240% +/- 61 (n=10, p value=0.001) in the vehicle treated mice. Further testing was carried out in a Genetically Engineered Mouse-Derived Allograft (GEDA). The GEDA model recapitulates the dense desmoplastic stroma of the original donor (KPC mouse), unlike cell line-based allografts. KPC (LSL-KrasG12D; LSL-Trp53R172H; Pdx1-cre) mouse tumour fragments (2 donors) were implanted subcutaneously in the flank of recipient LSL-Trp53R172H; Pdx1-cre (PC), immunocompetent mice. Relative to the start of dosing, the tumour growth in the RiXOVA treated group (25 mg/kg TIW) was 260% +/- 160 (n=10), compared to 540% +/- 210 (n=8) for the Vehicle (p = 0.001) and 130 % +/- 57 (n=9) for the combination of RiXOVA + gemcitabine (100 mg/kg BIW p results indicate RiXOVA to be a non-toxic oligosaccharide, that acts at the level of the tumour microenviroment to inhibit tumour growth. Whilst these results are in a preliminary stage they indicate G-block oligomers may represent a novel treatment modality in PDAC. Citation Format: Shalini V. Rao, Tonje S. Steigedal, Aarthi Gopinathan, Synnøve N. Magnussen, Sabina S. Strand, Duncan Jodrell, Fran Richards, Kurt I. Draget, Catherine T. Nordgård. Antitumor effect of an oligosaccharide API in a genetically engineered mouse-derived allograft (GEDA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 627.

4100 Background: NabP+GEM chemotherapy improves survival compared with GEM monotherapy as treatment for mPDAC. A PDAC mouse model suggested that nabP potentiates GEM activity by reducing cytidine deaminase levels and scheduling may be critical to optimise clinical benefit. Methods: Patients (pts) were randomised to receive standard concomitant (CON) nabP+GEM or sequential (SEQ) administration, with nabP given 24 hours before GEM. After 6 cycles, pts benefiting from treatment could continue the same regimen until disease progression. The primary endpoint was progression-free survival (PFS) by RECIST v1.1; secondary endpoints included safety, objective response rate (ORR), overall survival (OS) and quality of life (QoL). Serial blood and baseline tumour samples were collected for exploratory biomarkers. Results: Between March 2014 and 2016, 146 pts (71 SEQ, 75 CON) were recruited. Median age (range) was 66 (45-82) years; Karnofsky performance status was 70 (in 12% pts), 80 (27%), 90 (38%) or 100 (24%); 47% had pancreatic head primaries; 84% had liver metastases. Median no. cycles received was 4 SEQ, 3 CON; 51 pts (35%) received ≥6 cycles of treatment (42% SEQ, 28% CON). A 24+2hr interval was achieved in > 90% SEQ admin. Grade ≥3 adverse events experienced by ≥10% pts (SEQ, CON) were neutropaenia (54%, 30%; p = 0.003), febrile neutropaenia (12%, 12%), fatigue (22%, 15%), vomiting (7%, 11%) and anaemia (10%, 5%). G-CSF was administered at local investigator's discretion to 35 pts (23 SEQ, 12 CON; p = 0.015). To date, 112 pts have died. 6 month (m) PFS by SEQ and CON arms were 47% and 33%; median PFS were 5.8 and 4.0m; hazard ratio (HR) = 0.66, 95% CI = 0.46-0.95; 12m OS by SEQ and CON arms were 29% and 26%; median OS were 10.1 and 7.9m; HR = 0.88, 95% CI = 0.61-1.29. ORR was 50% SEQ and 33% CON (p = 0.065). Mean baseline QoL Global health status score was 60.6 SEQ and 63.4 CON. The mean change in QoL score from baseline at 24 weeks was -2.1 SEQ and -12.1 CON. Conclusions: Sequential delivery of nabP combined with GEM trended towards improving all clinically relevant efficacy end points: PFS, OS, and ORR. Translational correlates will be reported in due course. Clinical trial information: ISRCTN71070888.

342 Background: NabP+GEM chemotherapy improves survival compared with standard GEM monotherapy as treatment for advanced PDAC. A PDAC mouse model showed that delivery of nabP prior to GEM generated a higher intratumoural dFdC:dFdU ratio, which correlated with improved outcome. Therefore, scheduling may be critical to optimise clinical benefit. Methods: Patients were randomised to receive nabP+GEM, either concomitantly (CON) or sequentially (SEQ), 24 hours apart. After 6 cycles, patients benefiting from treatment could continue the same regimen until disease progression. The primary endpoint was progression-free survival (PFS) assessed by 8-weekly CT scanning; secondary endpoints included safety, response rate, overall survival (OS) and quality of life (QoL). Results: Between March 2014 and 2016, 146 patients (71 SEQ, 75 CON) from 19 UK sites were randomly assigned. Median age (range) was 66 (45-82) years; Karnofsky performance status was 70 (in 12% of patients), 80 (27%), 90 (38%) or 100 (24%); 47% of primary tumours were situated in the pancreatic head; 84% had liver metastases. Median number of cycles was 4 SEQ, 3 CON; 50 patients (34%) received ≥ 6 cycles of treatment (41% SEQ, 28% CON). A 24+2hr interval was achieved in over 90% SEQ administrations. Most patients stopped treatment prior to 6 cycles due to disease progression/death (42%) or toxicity (41%). Grade ≥ 3 adverse events experienced by ≥ 10% patients (SEQ, CON) were neutropaenia (54%, 30%), febrile neutropaenia (12%, 12%), fatigue (22%, 16%), vomiting (7%, 11%) and anaemia (10%, 5%). G-CSF was administered to 35 patients (23 SEQ, 12 CON). To date, 109 patients have died. 6-month PFS by SEQ and CON arms was 46.7% and 33.8%; median PFS was 5.8 and 4.3 months. 12-month OS by SEQ and CON arms was 29.1% and 20.1%; median OS was 10.1 and 7.9 months. The objective response rate was 44% SEQ and 30% CON. Mean baseline QoL Global health status was 60.6 SEQ and 63.4 CON. The change from baseline (mean) at 24 weeks was -2.1 SEQ and -12.1 CON. Conclusions: Sequential delivery of nabP and GEM improves progression-free and overall survival. Although more myelosuppressive, QoL was maintained. Clinical trial information: ISRCTN71070888.

Despite recent advances in the treatment of patients with pancreatic adenocarcinoma (PDAC), survival remains low. Pre-clinical models of human malignancies often fail to capture the complexity of tumors and as such lack clinical predictive power. Patient-derived tumor xenografts (PDXs) are an emerging pre-clinical platform, capable of retaining the molecular heterogeneity of their originating sample and able to predict the response to therapies in some cases. However, the lack of immune system of the mouse PDX host is a major drawback for a preclinical model, especially with the advent of promising cancer immunotherapies (e.g. anti-PD-1/PD-L1, CXCR4 antagonist). Genetically Engineered Mouse models (GEMMs) carry mutations in genes that are associated with the human disease and can thus mimic the genetic, phenotypic and physiological aspect of the human disease, in an immunocompetent background. The Lox-Stop-Lox (LSL)-Kras(G12D); LSL-Trp53(R172H); Pdx1-cre (KPC) mouse is a favoured model of PDA, recapitulating the desmoplastic tumor histology, immune profile and drug resistance of human PDAC. However, KPC tumor development speed is variable and tumor monitoring requires ultrasound imaging, so therapeutic studies are long, complex and expensive. We describe here a new model, Genetically Engineered mouse-Derived Allograft (GEDA), that applies the methods developed for PDXs to KPC tumors. Pieces of KPC tumor were implanted subcutaneously in the flank of recipient LSL-Trp53 (R172H); Pdx1-cre (PC), immunocompetent mice. At primary passage, 10 mice were implanted from a single primary tumor, and the engraftment rate is 90%. Tumors are palpable 2 weeks after the tumor implantation and can be monitored by calliper measurement for up to 10 weeks, with a tumor doubling time of 14.4 ± 1.5 days. The incidence of metastases was similar to KPC mice, but organ distribution was different, with 57% of 45 recipient mice developing lung metastases, and 0% liver metastases. Histologically the GEDAs appeared more like primary KPC tumors than subcutaneous allograft of KPC cell lines, with intratumoral heterogeneity including desmoplastic regions identified by the presence of collagen fibres and αSMA-expressing cells. Implantation of GEDAs onto GFP-mice revealed that the original donor stroma disappeared within 2 weeks and was replaced by host stroma, but the stromal composition remained comparable even over 5 passages in vivo, with ±15% of variation in αSMA-expressing area between donor and recipients. Typically, around 20% of the GEDA cells were positive for Ki67, remaining stable over 5 passages, suggesting that aggressiveness of the tumor was stable. Qualitatively, by CD3 IHC, the T cell distribution is similar to that reported in KPC tumors (Feig et al, PNAS 2013) with T cell exclusion from tumor cell nests. A therapeutic study was performed with gemcitabine. Tumor growth was not inhibited by gemcitabine (100 mg/kg IP, twice per week); tumor volume change at day 60 (%): 1544 ± 263 (treated) vs. 1350 ± 156 (control), n= 6 per group, p=0.25 ). Few KPC primary tumors show significant response to gemcitabine, so the PDA GEDA was more like KPC than subcutaneous allografts of KPC cell lines which tend to be gemcitabine sensitive. A biobank of KPC GEDAs is now being generated, which will be tested to determine the heterogeneity of therapeutic response to gemcitabine and other agents.In conclusion, we have generated PDAC GEDAs, which combine advantages of both GEMMs and PDXs, cost-effective generation of cohorts of mice for therapeutic studies. The stromal content and intact immune system should enable the evaluation of immunomodulatory therapies and also agents targeting the stromal components of PDAC. Citation Format: Séverine Mollard, Kristopher K. Frese, Aarthi Gopinathan, Frances M. Richards, Duncan I. Jodrell.{Authors}. Genetically engineered mouse-derived allografts (GEDAs): an immunocompetent mouse model of PDAC for the evaluation of novel therapeutic strategies. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B54.

The PI3K/Akt/mTOR pathway is frequently activated in human pancreatic ductal adenocarcinoma (PDAC) and in mouse models of Kras-driven pancreatic cancer. In addition, 20-30% of pancreatic cancers may have “unstable” genomes, associated with somatic or germline BRCA mutations, mutations in genes involved in DNA maintenance (eg. PALB2, ATM) or genes not previously associated with DNA maintenance. We therefore evaluated the efficacy of the mTORC1/2 inhibitor AZD2014 and olaparib, as single agents and in combination in vitro in human (MIA PaCa-2 and Capan-1) and murine (K8484: KRASG12D; p53R172H; Pdx1-cre (KPC)) PDAC cell lines. We also assessed the effect of AZD2014 in combination with gemcitabine, and the triple agent combination. Drug-induced cytotoxicity and cell proliferation were evaluated in 96-well plates, using the high-throughput sulforhodamine B (SRB) assay. The efficacy of 2-drug combinations was assessed by treating cells with serial dilutions of the compounds in an 8X8 grid format. Synergy was assessed using the Bliss Independence model, as implemented in Combenefit software (http://www.cruk.cam.ac.uk/research-groups/jodrell-group/combenefit). Western blotting showed that phosphorylation of Akt, NDRG1, S6 ribosomal protein and 4EBP1 were suppressed by AZD2014. AZD2014 inhibited the growth of all 3 cell lines, with GI50s in the nanomolar range. At the highest AZD2014 concentration, the growth of all 3 cell lines was inhibited at least 70%. Olaparib was ineffective as a single agent in K8484 and MIA PaCa-2 cells, but reassuringly inhibited the growth of Brca2 deficient Capan-1 cells. Synergy was observed in K8484 cells, and mild synergy in MIA PaCa-2 cells, when AZD2014 was combined with gemcitabine. No synergy was observed with the combination of AZD2014 and olaparib in any of the cell lines, although some additivity was observed in Capan-1 cells. When the 3 drugs were combined, higher olaparib concentrations increased cytotoxicity at lower concentrations of AZD2014, however this did not provide any additional benefit, compared to the combination of AZD2014 and gemcitabine at higher concentrations. Although the combination of AZD2014 and olaparib did not exhibit synergistic activity, AZD2014 showed efficacy as a single agent, and with gemcitabine is a promising combination that warrants further investigation in patients with pancreatic cancer. Further work is required to understand the mechanism of action of this combination, which may also reveal other promising targets to combine with inhibition of mTORC1/2. This study was funded by the Cancer Research UK New Agents Committee, with compounds supplied by AstraZeneca. Citation Format: Aarthi Gopinathan, Frances M. Richards, Sabina Cosulich, Bristi Basu, Duncan I. Jodrell.{Authors}. Evaluation of the combination of AZD2014 and olaparib in pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B40.