Preclinical Models Of Pancreatic Cancer Research Articles

Effect of stereotactic radiation (SBRT) on survival in a preclinical model of spontaneous pancreatic cancer with induction gemcitabine/nab-paclitaxel.

273 Background: There are no useful preclinical models that resemble relevant fractionated radiation, such as SBRT, with relevant chemotherapy. Most preclinical studies use a single fraction of radiation to large fields, causing significant toxicity and/or insufficient dose which leads to accelerated repopulation and worsened outcomes. Here, we employ mice with spontaneous pancreatic tumors, and treat them in a preclinical trial utilizing gemcitabine/nab-paclitaxel +/-SBRT. Methods: Mice with an activated KRas (G12D) allele and heterozygous for p53 expression only in the pancreas (KPC) were created and spontaneous tumors were diagnosed via weekly palpation and ultrasound. Mice with single tumors that were between 3-7mm at diagnosis were sequentially randomized to received either no treatment, gemcitabine/nab-paclitaxel x 2 weeks, or gemcitabine/nab-paclitaxel x 2 weeks followed by 8Gy x 5 daily fractions of SBRT to the pancreatic tumor. SBRT was delivered using X-RAD 225Cx (Precision X-Ray, Inc) small animal irradiator under image guidance by cone beam micro CT using AP/PA technique with a 10mm collimator. Every mouse was subjected to necropsy and a cause of death assigned. Tumor growth was monitored by small animal ultrasound. Kaplan-Meier survival analysis was performed to assess efficacy of treatments. Results: A total of 40 mice were enrolled with 12 receiving chemotherapy alone, 13 receiving chemotherapy +SBRT and 15 mice had no further treatment. The addition of induction chemotherapy improves lifespan compared to untreated mice. The addition of SBRT further improved survival. Causes of death in untreated animals of the gemcitabine/nab-paclitaxel group were largely from local progression, whereas the addition of SBRT provided local control causing most of the mice in this cohort to die from metastatic progression. Conclusions: SBRT improves survival when added to gem/nab-paclitaxel in a preclinical model, which may reflect favorable biology for this approach. Our novel system employs similar SBRT fields and doses, and may thus be useful for the evaluation of other chemotherapeutic regimens with SBRT.

Abstract LB-B29: Combined inhibition of MAPK and PI3K signaling augments standard chemotherapy response in preclinical models of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal cancer with mortality closely paralleling incidence despite substantial advances in biological understanding of the disease. Nab-paclitaxel (NPT) plus Gemcitabine (Gem) represents the current standard for systemic therapy of advanced PDAC. More than 90% of PDACs harbor an activating mutation in the KRAS oncogene. Presently, no therapeutics exist that effectively target this oncogene, but alternative strategies focus on inhibition of downstream effectors of KRAS signaling pathways. The RAF-MEK-ERK (MAPK) and the AKT-PI3K signaling pathways are well-described mediators of KRAS induced transformation and tumorigenesis in several cancers including PDAC and represent potential targets for combination therapy. We evaluated combination treatment benefits of NPT+Gem with the MEK inhibitor trametinib (Tra) and the AKT inhibitor MK-2206 (MK) in preclinical models to evaluate their therapeutic potential against PDAC. An animal survival study was performed in peritoneal dissemination model in NOD/SCID mice. Tumor growth inhibition studies were performed in subcutaneous PDAC cell-line-derived xenografts and PDAC patient-derived xenografts in mice. Intratumoral mechanism of action was determined by immunohistochemistry (IHC) and Immunoblot analysis. Median animal survival in peritoneal dissemination PDAC xenografts in mice revealed that the median survival was 21 days in controls, which was significantly improved by the NPT+Gem combination (35 days, a 67% increase over controls). Median survival was further increased by addition of trametinib or MK-2206 to the NPT+Gem chemotherapy regimen: NPT+Gem+Tra (43 days, a 105% increase over controls), NPT+Gem+MK (39 days, a 86% increase over controls) and NPT+Gem+MK+Tra (48 days, a 129% increase over controls). In human subcutaneous xenografts using AsPC-1 PDAC cells, trametinib and MK-2206 were also able to enhance NPT+Gem effects. Compared to controls (100±34.8), the percent net local tumor growth in different therapy groups was 21.8±5.9 for NPT+Gem, 7.1±11.2 for NPT+Gem+Tra, 17.2±4.8 for NPT+Gem+MK and 5.7±9.1 for NPT+Gem+MK+Tra. Effects of therapy on intratumoral proliferation and apoptosis corresponded with tumor growth inhibition data. Furthermore, in vivo effects of trametinib and MK-2206 correlated with reduced expression of phospho-ERK and phospho-AKT in tumor samples. More importantly, in a recently completed patient-derived xenograft study, we observed that the addition of trametinib or MK-2206 again augmented the NPT+Gem tumor growth inhibition response. In this experiment, compared to controls (350 mm3), the net local tumor growth in different therapy groups was 145.4 mm3 for NPT+Gem, 122 mm3 for NPT+Gem+Tra, 20.4 mm3 for NPT+Gem+MK and -43.7 mm3 (tumor regression) for NPT+Gem+MK+Tra. These findings from multiple preclinical model settings suggest that the effects of NPT+Gem can be enhanced through combined inhibition of MAPK and PI3K signaling, which supports the potential of these targets for clinical PDAC therapy. Citation Format: Niranjan Awasthi, Sheena Monahan, Alexis Stefaniak, Margaret A. Schwarz, Roderich E. Schwarz. Combined inhibition of MAPK and PI3K signaling augments standard chemotherapy response in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B29.

Abstract LB-B19: Re-sensitizing Pancreatic Cancer, targeted siRNA inhibition of HuR

Abstract We have demonstrated that 3DNA nanocarriers can specifically, effectively, and safely deliver siRNA oligos targeted against a pro-survival hub, HuR (the ELAVL1 gene) in an in vivo, pre-clinical model of pancreatic cancer. Pancreatic cancer remains one of the deadliest cancers with an overall 5 year survival rate of 9%; and is on pace to become the second leading cause of cancer related deaths in the U.S. by 2020. Pancreatic cancer is so deadly, in part, because it is highly refractory to standard chemotherapeutics. In fact, new standard of care regiments such as FOLFIRINOX have only improved survival by a few months. Our previous work has established HuR as a target in pancreatic cancer. HuR is an RNA binding protein that is primarily retained in the nucleus, but upon exposure to cancer associated stressors translocates to the cytoplasm. In the cytoplasm, HuR functions to bind, stabilize, and up-regulate expression of pro-survival target mRNAs. CRISPR-mediated deletion of HuR causes a xenograft lethal phenotype in vivo and increases sensitivity to a variety of drugs, particularly PARP inhibitor therapy, enhancing Olaparib therapy over 20-fold in vitro. In an independent doxycycline inducible knockdown model, HuR depletion combined with Olaparib treatment resulted in a 9.3 fold (p<.001) decrease in tumor volume in vivo. Based on these findings and our previous published work, we have launched into a line of investigation attempting to target HuR in pancreatic cancer cells. We have delivered siHuR oligos tethered to 3DNA nanostructures (Genisphere, LLC) in a targeted fashion using either a folic acid targeting moiety, a transferrin receptor targeted antibody, or an EGFR targeted antibody (i.e., siHuR nanotherapy). Additionally, we are working to combine siHuR nanotherapy with conventional therapeutics. 3DNA nanocarriers are constructed using stable DNA strands assembled and crosslinked in a stepwise fashion, to create a layered nanoscaffold capable of attaching multiple different targeting moieties and deliverables. The siRNA oligos hybridized to the 3DNA were modified in order to withstand systemic RNases during treatment. We first validated the modified siHuR nanotherapy in vitro by evaluating protein and RNA levels of HuR. Second, we validated the expression of our intended targeting moieties in both human cell lines and a tissue microarray consisting of patient derived pancreatic cancer tumors. For the mouse studies, athymic female nude mice were injected with 4 million MIA PaCa-2 cells per flank, and randomized to treatment arms when tumors reached 100mm3. Mice were then treated intraperitoneally twice weekly with either siHuR or siControl nanotherapies (3ug siRNA per treatment) with one of the three targeting moieties. We demonstrated efficacy of systemic targeted delivery of siHuR nanotherapy which improved average overall survival by 35% (p=.012) compared to the control arm, with an increase in survival using a Kaplan-Meier survival analysis (p=.01). We are currently setting up treatments with siHuR nanotherapy in conjunction with a targeted treatment strategy, Olaparib, which has previously been shown to work in conjunction with dox-inducible HuR silencing (i.e.,shRNA targeting) in vivo. Herein, we describe the development of siHuR nanotherapy that, as a monotherapy or most likely with clinically available drugs, may improve outcomes for patients with pancreatic cancer. Citation Format: Chris W. Schultz, Kevin O'Hayer, Kathryn M. Bormes, Teena Dhir, Samantha Brown, Avinoam Nevlar, Saswati Chand, Henry Thomsett, Wei Jiang, Jessica Bowers, Kelly Rhodes, Michael Pishvaian, Robert Getts, Jonathan Brody. Re-sensitizing Pancreatic Cancer, targeted siRNA inhibition of HuR [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B19.

Inhibition of the MEK/ERK pathway augments nab-paclitaxel-based chemotherapy effects in preclinical models of pancreatic cancer.

Nab-paclitaxel (NPT) combination with gemcitabine (Gem) represents the standard chemotherapy for pancreatic ductal adenocarcinoma (PDAC). Genetic alterations of the RAS/RAF/MEK/ERK (MAPK) signaling pathway yielding constitutive activation of the ERK cascade have been implicated as drivers of PDAC. Inhibition of downstream targets in the RAS-MAPK cascade such as MEK remains a promising therapeutic strategy. The efficacy of trametinib (Tra), a small molecule inhibitor of MEK1/2 kinase activity, in combination with nab-paclitaxel-based chemotherapy was evaluated in preclinical models of PDAC. The addition of trametinib to chemotherapy regimens showed a trend for an additive effect on tumor growth inhibition in subcutaneous AsPC-1 and Panc-1 PDAC xenografts. In a peritoneal dissemination model, median animal survival compared to controls (20 days) was increased after therapy with NPT (33 days, a 65% increase), Tra (31 days, a 55% increase), NPT+Tra (37 days, a 85% increase), NPT+Gem (39 days, a 95% increase) and NPT+Gem+Tra (49 days, a 145% increase). Effects of therapy on intratumoral proliferation and apoptosis corresponded with tumor growth inhibition. Trametinib effects were specifically accompanied by a decrease in phospho-ERK and an increase in cleaved caspase-3 and cleaved PARP-1 proteins. These findings suggest that the effects of nab-paclitaxel-based chemotherapy can be enhanced through specific inhibition of MEK1/2 kinase activity, and supports the clinical application of trametinib in combination with standard nab-paclitaxel-based chemotherapy in PDAC patients.

A non-controlled, single arm, open label, phase II study of intravenous and intratumoral administration of ParvOryx in patients with metastatic, inoperable pancreatic cancer: ParvOryx02 protocol

BackgroundMetastatic pancreatic cancer has a dismal prognosis, with a mean six-month progression-free survival of approximately 50% and a median survival of about 11 months. Despite intensive research, only slight improvements of clinical outcome could be achieved over the last decades. Hence, new and innovative therapeutic strategies are urgently required. ParvOryx is a drug product containing native parvovirus H-1 (H-1PV). Since H-1PV was shown to exert pronounced anti-neoplastic effects in pre-clinical models of pancreatic cancer, the drug appears to be a promising candidate for treatment of this malignancy.MethodsParvOryx02 is a non-controlled, single arm, open label, dose-escalating, single center trial. In total seven patients with pancreatic cancer showing at least one hepatic metastasis are to be treated with escalating doses of ParvOryx according to the following schedule: i) 40% of the total dose infused intravenously in equal fractions on four consecutive days, ii) 60% of the total dose injected on a single occasion directly into the hepatic metastasis at varying intervals after intravenous infusions. The main eligibility criteria are: age ≥ 18 years, disease progression despite first-line chemotherapy, and at least one hepatic metastasis. Since it is the second trial within the drug development program, the study primarily explores safety and tolerability after further dose escalation of ParvOryx. The secondary objectives are related to the evaluation of certain aspects of anti-tumor activity and clinical efficacy of the drug.DiscussionThis trial strongly contributes to the clinical development program of ParvOryx. The individual hazards for patients included in the current study and the environmental risks are addressed and counteracted adequately. Besides information on safety and tolerability of the treatment after further dose escalation, thorough evaluations of pharmacokinetics and intratumoral spread as well as proof-of-concept (PoC) in pancreatic cancer will be gained in the course of the trial.Trial registrationClinicalTrials.gov-ID: NCT02653313, Registration date: Dec. 4th, 2015.

A Combination of Radiation and the Hypoxia-Activated Prodrug Evofosfamide (TH-302) is Efficacious against a Human Orthotopic Pancreatic Tumor Model

This study was designed to investigate the effect of single-dose radiation therapy (RT) in combination with evofosfamide (TH-302), a hypoxia-activated prodrug, in a pre-clinical model of pancreatic cancer. AsPC1 tumors were implanted orthotopically in the pancreas of nude mice. Tumors were treated with 15 Gy of RT, using a 1 cm diameter field, and delivered as a continuous arc. Image-guidance to center the field on the tumor was based on CT imaging with intraperitoneal contrast. Evofosfamide (100 mg/kg, i.p.) was administered 3 hours before RT. Tumor volumes were measured using ultrasound, and regrowth curves were plotted. Tumor hypoxia and cell proliferation were measured using pimonidazole and the thymidine analog EdU, respectively. In vitro clonogenic assays were performed. Tumors were shown to contain substantial areas of hypoxia, as calculated by percent pimonidazole staining. Evofosfamide was active in these tumors, as demonstrated by a significant reduction in uptake of the thymidine analog EdU. This effect was visible in oxygenated tissue, consistent with the previously reported bystander effects of evofosfamide. RT produced significant regrowth delay, as did evofosfamide. The combination of both agents produced a growth delay that was at least equal to the sum of the two treatments given separately. The improvement in tumor response when evofosfamide is combined with RT supports the hypothesis that hypoxia is a cause of radioresistance in high dose RT for pancreatic cancer. Assessing the efficacy and safety of stereotactic radiation treatment and evofosfamide is warranted in patients with locally advanced pancreatic cancer.

Abstract 4202: Trametinib, a MEK inhibitor, augments nab-paclitaxel based chemotherapy response in preclinical models of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer related deaths in the USA with a 5-year survival less than 6%. Nanoparticle albumin-bound paclitaxel (nab-paclitaxel, NPT) has demonstrated 8.5 months median survival in combination with gemcitabine, and now represents a standard of care for PDAC patients. Genetic alterations of the RAS/RAF/MEK/ERK (MAPK) signaling pathway yielding constitutive activation of the ERK cascade have been implicated in many human cancers. In PDAC, activating K-ras mutations occur at a frequency of 90%, rendering this a potential therapeutic target. Efforts to develop drugs that directly target mutant KRAS protein remain challenging due to target specificity issues. Alternative strategies therefore focus towards inhibition of downstream targets in the RAS-MAPK cascade such as MEK. Trametinib (Tra) is a potent and highly selective small molecule inhibitor of MEK1/2 kinase activity. We evaluated efficacy of trametinib to enhance antitumor response of nab-paclitaxel based chemotherapy regimens in preclinical models of PDAC using K-ras mutant cell lines. In subcutaneous PDAC xenografts using AsPC-1 cells, net tumor growth in different therapy groups was 432.6 mm3 in controls, 105.3 mm3 after NPT (p=0.0023), 184 mm3 after Tra (p=0.0018), 81 mm3 after NPT+Tra (p=0.0003), 37.3 mm3 after NPT+Gem (p=0.0025) and -8.1 mm3 (tumor regression) after NPT+Gem+Tra (p<0.0001). In another subcutaneous PDAC xenografts using Panc-1 cells, net tumor growth in different therapy groups was: 274.1 mm3 in controls, 80.8 mm3 after NPT (p=0.0002), 150.6 mm3 after Tra (p=0.0047), 75.1 mm3 after NPT+Tra (p=0.0002), 48.4 mm3 after NPT+Gem (p=0.0004) and 3.8 mm3 after NPT+Gem+Tra (p<0.0001). In PDAC peritoneal dissemination model using AsPC-1 cells, median animal survival compared to controls (20 days) was increased after therapy with NPT (33 days, a 65% increase, p=0.0004), Tra (31 days, a 55% increase, p=0.0004), NPT+Tra (37 days, a 85% increase, p=0.0001), NPT+Gem (39 days, a 95% increase, p=0.0001) and NPT+Gem+Tra (49 days, a 145% increase, p<0.0001). Effects of therapy on intratumoral proliferation and apoptosis corresponded with tumor growth inhibition data. In vitro studies demonstrated inhibition in PDAC cell (AsPC-1, Panc-1, Mia PaCa-2, CFPAC) proliferation by NPT+Gem, Tra, and combination. Immunoblot analysis revealed that trametinib effects were specifically accompanied by decrease in phospho-ERK expression and increase in the expression of apoptosis-related cleaved caspase-3 and cleaved PARP-1 proteins. These findings suggest that the effects of nab-paclitaxel based chemotherapy regimens can be enhanced through specific inhibition of MEK1/2 kinase activity, which clinically could lead to improved PDAC therapy effects. Citation Format: Niranjan Awasthi, Sheena Monahan, Alexis Stefaniak, Margaret A. Schwarz, Roderich E. Schwarz. Trametinib, a MEK inhibitor, augments nab-paclitaxel based chemotherapy response in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4202. doi:10.1158/1538-7445.AM2017-4202

Abstract 5659: Local treatment with novel TLR9 agonist IMO-2125 demonstrates antitumor activity in preclinical models of pancreatic cancer

Abstract Pancreatic cancer remains a serious unmet medical need. The majority of pancreatic tumors are composed of stromal cells that make tumors resistant to conventional therapies. We are developing an approach in which a TLR9 agonist, IMO-2125, is administered intratumorally, which induces local Th1 responses and modulates the tumor microenvironment. This approach has shown promising antitumor activity in preclinical models of multiple tumor types in combination with checkpoint inhibitors. We are employing a similar approach to the treatment of pancreatic cancer. We evaluated the local therapeutic effect of IMO-2125 in a murine syngeneic pancreatic ductal adenocarcinoma (Panc02) cancer models. In the first study, C57BL/6 mice were intraperitoneally (i.p.) implanted with Panc02 cells to generate peritoneal metastatic cancer. Tumor-bearing mice (n=10 per group) were then treated with 2.5 mg/kg IMO-2125 either by i.p. or subcutaneous (s.c.) injections for six times in two weeks. In the second study, to establish dual solid tumors in C57BL/6 mice, Panc02 cells were subcutaneously implanted in both the right and left flanks of the animals. When tumors reached to 50-150 mm3, we started the intratumoral (i.t.) administration of IMO-2125 in right tumors at doses of either 0.25 mg/kg, 1 mg/kg or 2.5 mg/kg, or PBS (n=10 per group), 3x/week for three weeks. In the peritoneal metastatic pancreatic tumor model, 80% of IMO-2125 intraperitoneally treated mice survived for more than 88 days, while 90% of the control group died within 29 days. Compared to i.p. treatment, the s.c. treated group survived for only 36 days. Tumor-free mice from the IMO-2125 i.p treatment group (n=7) rejected the same tumor rechallenge, suggesting establishment of durable tumor-specific immune response. In the dual solid pancreatic tumor model, intratumoral IMO-2125 treatment led to a dose dependent antitumor activity in both injected and distant tumors. Tumors treated with 0.25, 1 and 2.5 mg/kg doses of IMO-2125 showed reductions in tumor volume by 66% (p = 0.004), 74% (p = 0.002) and 95% (p = 0.003), respectively. Moreover, the distant tumors exhibited a reduction of 0% (p = 0.65), 64% (p = 0.006) and 69% (p = 0.001), respectively. Antitumor activity was associated with increased T-cell infiltration and upregulation of the gene expression of various checkpoints in both treated and distant tumors. In an independent study, treatment with a control IMO showed minimal antitumor activity. All treatments were well tolerated. In summary, local treatment with IMO-2125 exerts antitumor activity in preclinical pancreatic cancer models, associated with an increase in T-cell infiltration in the tumor microenvironment, providing a novel approach to the treatment of pancreatic cancer. The clinical trial of intratumoral IMO-2125 is in progress in patients with melanoma. Citation Format: Daqing Wang, Evren Kocabas Argon, Fugang Zhu, Sudhir Agarwal. Local treatment with novel TLR9 agonist IMO-2125 demonstrates antitumor activity in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5659. doi:10.1158/1538-7445.AM2017-5659

Abstract 5082: The selective bromodomain inhibitor, INCB054329 targets both cancer cells and the tumor microenvironment in the KC inflammatory preclinical model of ductal pancreatic cancer

Abstract Pancreatic cancer is expected to become the second most deadly cancer by 2030, with few effective therapeutic options available to improve patient survival. The Kras gene is mutated in over 90% of pancreatic cancers, but the Kras protein is considered to be an undruggable target. p-Erk is a downstream effector of Kras and has been shown to be essential for the progression and maintenance of pancreatic cancer. The LSL-KrasG12D/+; Pdx-1-Cre (KC) mouse model is used to study pancreatic cancer, especially cancer progression, after stimulation with an inflammatory agent, such as caerulein or LPS. Bromodomain inhibitors are a new generation of drugs that target BET (bromodomain and extra-terminal) proteins, impairing their ability to bind to acetylated lysines and therefore interfering with transcriptional initiation and elongation. BET proteins regulate several genes responsible for cell growth, apoptosis and inflammation. INCB54329 is a novel, orally bioavailable BET inhibitor that is currently being investigated in Phase 1 clinical trials.INCB054329 inhibits cell growth in murine pancreatic cancer cells harboring Kras mutations, derived from a KrasG12D/+; Trp53R172H/+; Pdx-1-Cre (KPC) mouse with an IC50 values less than 100 nM. Moreover, INCB054329 reduces the levels of p-ERK 1/2 in these cells lines. When KC mice are injected with LPS to induce inflammation and pancreatitis, p-Stat3 protein levels are significantly increased, but this pro-survival protein is reduced by more than 50%, in mice that were pre-treated with INCB054329. Tumor cells can use epigenetic modulation to evade immune recognition and to shape the TME (tumor microenvironment) toward an immunosuppressive phenotype. Since epigenetic modulation is a mechanism used by tumors to regulate the TME, using small molecules as epigenetic modulators to activate immune recognition is a therapeutically desirable approach. Within the TME, several cytokines and chemokines play crucial roles, recruiting and regulating inflammatory cells [macrophages, T cells and Myeloid derived suppressor cells]. KC mice stimulated with LPS have higher levels of CCL2 (1947 ± 591 ng/mL) and IL-6 (2459 ± 577 pg/mL) than unstimulated mice; these increased levels of inflammatory cytokines and the subsequent immune cell infiltration can be modulated by treatment with INCB054329, with INCB054329 demonstrating >50% inhibition of both cytokines (IL-6: 1063 ± 502 pg/mL; CCL2: 713 ± 134 ng/mL). These data suggest that INCB054329 has a dual activity, targeting cancer cells and modulating the TME; both activities may prove beneficial for the treatment of pancreatic cancer. Moreover, this work suggests that additional mechanisms may underlie the beneficial effects of bromodomain inhibitors. Citation Format: Ana Sofia Leal, Karen T. Liby, Phillip Liu, Bruce Ruggeri. The selective bromodomain inhibitor, INCB054329 targets both cancer cells and the tumor microenvironment in the KC inflammatory preclinical model of ductal pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5082. doi:10.1158/1538-7445.AM2017-5082

Abstract LB-197: First-in-class TREM-1 inhibitors attenuate tumor growth and angiogenesis by suppressing intratumoral macrophage infiltration and activation in preclinical models of lung and pancreatic cancer

Abstract Non-small cell lung cancer (NSCLC) and pancreatic cancer (PC) are among the most lethal cancer types. Despite significant recent advances, effective treatments are still lacking and novel therapies are needed. High intratumoral macrophage content promotes tumor aggressiveness and metastasis, ultimately leading to poor patient prognosis. In cancer patients, high expression levels of triggering receptor expressed on myeloid cells 1 (TREM-1) on intratumoral macrophages have been linked with cancer recurrence and poor patient survival. Thus, blockade of TREM-1 could be a promising therapeutic strategy. To test this hypothesis, we employed our novel ligand-independent strategy of TREM-1 inhibition that includes the use of the signaling chain homooligomerization (SCHOOL) approach to rationally design TREM-1 inhibitory peptide sequences. These SCHOOL TREM-1 inhibitors are non-toxic, non-immunogenic and can be deployed in either free form or incorporated into our lipopeptide complexes for targeted delivery to macrophages. This incorporation significantly increased peptide half-life and dose efficacy in our prior studies. In this study, to determine whether SCHOOL TREM-1 inhibitors have an anticancer effect in vivo, xenograft mouse models of NSCLC (H292 and A549) and PC (AsPC-1, BxPC-3, and CAPAN-1) were used. Administration of these inhibitors in free and lipopeptide complex-bound form was found to significantly inhibit tumor growth in NSCLC and PC models achieving an optimal T/C value of 23% depending on the xenograft and formulation used. This suggests that the therapeutic effect of our TREM-1-targeted treatment is cancer type-independent. In addition, SCHOOL TREM-1 inhibitors did not alter body weight, which indicates that it is well-tolerated. Interestingly, the growth delay in NSCLC and PC tumors persisted even after treatment was halted, thus highlighting the long lasting anticancer effects. Importantly, the tumor growth inhibition in select xenograft models correlated significantly with: 1) increased survival from 36 to 54 days (P<0.001) 2) up to 60% reduced intratumoral macrophage infiltration as assessed by a F4/80 macrophage marker (P<0.001 vs. vehicle), and 3) up to 50% reduced angiogenesis as assessed by an CD31 epithelial marker (P<0.001 vs. vehicle). Finally, treatment with SCHOOL TREM-1 inhibitors significantly reduced serum levels of interleukin (IL)-1α, IL-6 and macrophage colony-stimulating factor (M-CSF, also known as CSF-1), but unexpectedly not vascular endothelial growth factor (VEGF), suggesting the inhibitory effect on angiogenesis could be M-CSF-dependent as previously reported for osteosarcoma and retinopathy. Taken together, these results clearly demonstrate that SCHOOL TREM-1 inhibitors can have beneficial anticancer activity by inhibiting intratumoral macrophage infiltration and activation and tumor angiogenesis. Alleviation of intratumoral macrophage activity by SCHOOL TREM-1 inhibitors would be ideal in combinatorial approaches with chemotherapy. Citation Format: Zu T. Shen, Alexander B. Sigalov. First-in-class TREM-1 inhibitors attenuate tumor growth and angiogenesis by suppressing intratumoral macrophage infiltration and activation in preclinical models of lung and pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-197. doi:10.1158/1538-7445.AM2017-LB-197

P-200 - PANOVA-2: A phase II study of TTFields (150 kHz) concomitant with standard chemotherapy for front-line therapy of advanced pancreatic adenocarcinoma – Updated efficacy results

Introduction: Background TTFields are a non-invasive, regional antimitotic treatment modality, which has been approved for the treatment of glioblastoma by the FDA. TTFields predominantly act by disrupting the formation of the mitotic spindle during metaphase. TTFields were effective in multiple preclinical models of pancreatic cancer. PANOVA was the first trial testing TTFields in pancreatic cancer patients. Results from the first cohort of the study testing TTFields in combination with gemcitabine, have demonstrated superiority in efficacy compared to historical controls (Rivera F. et al, J Clin Oncol 34, 2016 (suppl 4S; abstr 269). Methods: Twenty advanced pancreatic cancer patients enrolled in the second cohort, PANOVA-2, were treated with TTFields in combination with gemcitabine concomitant to nab-paclitaxel. All patients had unresectable tumors, an ECOG performance score of 0-1 and no prior therapy. The primary endpoint was the incidence and severity of adverse events. Results: The median age was 68.2 (range – 58-81) and most patients (65%) had an ECOG score of 1. Twelve patients (60%) had distant metastases. Ten patients (50%) had serious AEs during the study period. Eleven patients (55%) had treatment-related skin toxicity, of which 5 had grade 3 toxicity. No TTFields-related serious AEs were reported. The median PFS was 12.7 months (95% CI 5.4, NA): 9.3 months in patients with metastatic disease, and not reached in locally-advanced patients. PFS rate at 6 months was 65%: 50% in metastatic disease and 87.5% in locally advanced patients. Of the evaluable tumors, 40% had partial response and another 47% stable disease. The median OS was not reached, and the 1-year survival rate was 72% (62.5% in metastatic disease and 87.5% in locally advanced disease). Conclusion: TTFields concomitant to gemcitabine and nab-paclitaxel are safe for advanced pancreatic cancer patients, with promising clinical outcome which doubled historical data. A phase III trial (PANOVA-3) is planned to test the efficacy of TTFields combined with gemcitabine and nab-paclitaxel in locally-advanced pancreatic cancer patients.

PANOVA: A phase II study of TTFields (150 kHz) concomitant with standard chemotherapy for front-line therapy of advanced pancreatic adenocarcinoma—Updated efficacy results.

e15790 Background: TTFields are a non-invasive, regional antimitotic treatment modality, which has been approved for the treatment of glioblastoma by the FDA. TTFields predominantly act by disrupting the formation of the mitotic spindle during metaphase. TTFields were effective in multiple preclinical models of pancreatic cancer. PANOVA was the first trial testing TTFields in pancreatic cancer patients. Results from the first arm of the study, testing TTFields in combination with gemcitabine, have demonstrated superiority in efficacy compared to historical controls (Rivera F. et al, J Clin Oncol 34, 2016 (suppl 4S; abstr 269). Methods: Twenty advanced pancreatic cancer patients were enrolled in the second arm of PANOVA and treated with TTFields in combination with gemcitabine concomitant to nab-paclitaxel. All patients had unresectable tumors, an ECOG performance score of 0-1 and no prior therapy. The primary endpoint was the incidence and severity of adverse events. Results: The median age was 68.2 (range – 58-81) and most patients (65%) had an ECOG score of 1. Twelve patients (60%) had distant metastases. Ten patients (50%) had serious AEs during the study period. Eleven patients (55%) had treatment-related skin toxicity, of which 5 had grade 3 toxicity. No TTFields-related serious AEs were reported. The median PFS was 12.7 months (95% CI 5.4, NA): 9.3 months in patients with metastatic disease, and not reached in locally-advanced patients. PFS rate at 6 months was 65%: 50% in metastatic disease and 87.5% in locally advanced patients. Of the evaluable tumors, 40% had partial response and another 47% stable disease. The median OS was not reached, and the 1-year survival rate was 72% (62.5% in metastatic disease and 87.5% in locally advanced disease). Conclusions: TTFields concomitant to gemcitabine and nab-paclitaxel are safe for advanced pancreatic cancer patients, with promising clinical outcome which doubled historical data. A phase III trial is planned, testing the efficacy of TTFields combined with gemcitabine and nab-paclitaxel in locally-advanced pancreatic cancer patients. Clinical trial information: NCT01971281.

Targeting ERK enhances the cytotoxic effect of the novel PI3K and mTOR dual inhibitor VS-5584 in preclinical models of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease in urgent need of newer therapeutic modalities. Majority of patients with PDAC have mutations in KRAS, which unfortunately remains an ineffectual target. Our strategy here is to target KRAS downstream effectors PI3K and mTOR. In this study, we investigated the antitumor efficacy of the novel PI3K and mTOR dual inhibitor VS-5584 in PDAC. Our data shows that PI3K/mTOR dual inhibition causes ERK activation in all tested PDAC cell lines. Although the MEK inhibitor GSK1120212 could abrogate VS-5584-induced ERK activation, it did not substantially enhance cell death in all the cell lines tested. However, combination with ERK inhibitor SCH772984 not only mitigated VS-5584-induced ERK activation but also enhanced VS-5584-induced cell death. In a xenograft model of PDAC, we observed 28% and 44% tumor inhibition for individual treatment with VS-5584 and SCH772984, respectively, while the combined treatment showed superior tumor inhibition (80%) compared to vehicle control treatment. Our findings support the clinical development of VS-5584 and ERK inhibitor combination for PDAC treatment.

Correction: Pulsed High-Intensity Focused Ultrasound Enhances Delivery of Doxorubicin in a Preclinical Model of Pancreatic Cancer.

Correction: Pulsed High-Intensity Focused Ultrasound Enhances Delivery of Doxorubicin in a Preclinical Model of Pancreatic Cancer.

Establishment of the In Vivo Efficacy of Pretargeted Radioimmunotherapy Utilizing Inverse Electron Demand Diels-Alder Click Chemistry.

The pretargeting system based on the inverse electron demand Diels-Alder reaction (IEDDA) between trans-cyclooctene (TCO) and tetrazine (Tz) combines the favorable pharmacokinetic properties of radiolabeled small molecules with the affinity and specificity of antibodies. This strategy has proven to be an efficient method for the molecularly targeted delivery of pharmaceuticals, including isotopes for radiological imaging. Despite encouraging results from in vivo PET imaging studies, this promising system has yet to be thoroughly evaluated for pretargeted radioimmunotherapy (PRIT). Toward that end, we synthesized two novel 177Lu-labeled tetrazine-bearing radioligands. Next, we compared the usefulness of our ligands for PRIT when paired with TCO-modified 5B1-a human, anti-CA19.9 mAb-in preclinical murine models of pancreatic cancer. The exemplary ligand, 177Lu-DOTA-PEG7-Tz, showed rapid (4.6 ± 0.8% ID/g at 4 hours) and persistent (16.8 ± 3.9% ID/g at 120 hours) uptake in tumors while concurrently clearing from blood and nontarget tissues. Single-dose therapy studies using 5B1-TCO and varying amounts of 177Lu-DOTA-PEG7-Tz (400, 800, and 1,200 μCi) showed that our system elicits a dose-dependent therapeutic response in mice bearing human xenografts. Furthermore, dosimetry calculations suggest that our approach is amenable to clinical applications with its excellent dosimetric profile in organs of clearance (i.e., liver and kidneys) as well as in dose-limiting tissues, such as red marrow. This study established that a pretargeted methodology utilizing the IEDDA reaction can rapidly and specifically deliver a radiotherapeutic payload to tumor tissue, thus illustrating its excellent potential for clinical translation. Mol Cancer Ther; 16(1); 124-33. ©2016 AACR.

Abstract B12: CLPTM1L and GRP78 interact and promote pancreatic tumor survival under ER-stress and chemoresistance through cell-surface translocation and interaction with PI3K

Abstract Altered regulation of endoplasmic reticulum (ER) homeostasis has been implicated in many cancers and has recently become a therapeutic and chemosensitization target of interest. Tumor cells must endure ER stress caused by microenvironmental, oxidative, energetic, and therapeutic stresses to survive. However, the role of ER stress survival signaling in pancreatic tumorigenesis has not been elucidated. We have identified Cleft Lip and Palate Transmembrane 1-Like (CLPTM1L)/Cisplatin Resistance Related Protein 9 (CRR9) as an ER stress related target in pancreatic cancer. We recently demonstrated that CRR9/CLPTM1L is overexpressed in 90% of pancreatic ductal adenocarcinomas and associated with poor outcome, while it is not expressed in normal pancreatic ductal epithelium. Furthermore, we have shown that CLPTM1L can confer resistance to chemotherapeutic killing, apoptosis, and anoikis, and is critical for KRas transformation and tumorigenesis. Here, we demonstrate that CLPTM1L interacts with phosphoinositol-3-kinase (PI3K) and the ER stress protein Glucose Related Protein 78 (GRP78) at the tumor cell surface and causes up-regulation of Bcl-xL and pAkt survival signaling. Surface accumulation of both CLPTM1L and GRP78 are induced by ER stress and by chemotherapy in vitro. Subsequent to our recent discovery of the presence of CLPTM1L at the cell surface, we have developed antibody inhibitors of CLPTM1L, which deplete chemotherapy-inducible surface accumulation of CLPTM1L and GRP78, as well as downstream survival signaling through the Akt pathway and Bcl-xL expression. These agents have shown striking anti-tumorigenic efficacy in preclinical models of pancreatic cancer and present a promising therapeutic and chemosensitization approach. Citation Format: Michael James.{Authors}. CLPTM1L and GRP78 interact and promote pancreatic tumor survival under ER-stress and chemoresistance through cell-surface translocation and interaction with PI3K. [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 B12.

Abstract B36: Preclinical development of a multikinase targeting molecule with activity against the cancer stem cell phenotype in pancreatic adenocarcinoma

Abstract Background: Pancreatic cancer is highly resistant to existing chemotherapy regimens, with only a minority of patients exhibiting a response to standard therapy. One aspect of treatment resistance is based on the cancer stem cell (CSC) hypothesis, which holds that a small population of highly chemoresistant cells is responsible for initiating and sustaining tumor growth. Compounds with activity against this population are highly desired in the adjuvant setting, where failure to sterilize micrometastases not detected at time of operation results in recurrence in nearly all patients. Methods: A high-throughput drug screen (HTS) was performed in an in vitro model of pancreatic cancer stem-like cells (spheroids). Molecules with high potency against both the cancer stem cell-like spheroid and monolayer cell formats were evaluated for efficacy and mechanism of action in a variety of preclinical models of pancreatic cancer. Unbiased global in situ phosphoproteomic profiling using KiNativ technology was used to identify kinase targets impacted by the top hit. Stemness phenotype was interrogated by qRT-PCR of stemness genes, flow cytometry, colony formation assays, and tumor initiation in immunocompromised mice. Results: The top hit from the HTS was a 1H-benzo[d]imidazol-2-amine-based inhibitor of interleukin-2-inducible T-cell kinase (ITK) (NCGC00188382, 1). 1 effectively mediated growth inhibition, blocked migration and invasion, and reduced clonogenicity of cancer cells with an associated decrease in the expression of several CSC-associated stemness markers at non-lethal concentrations (Sox2, Notch1) compared to gemcitabine used at >10-fold concentration. Metastatic burden in both the liver and lung was significantly reduced in mice orthotopically injected with pancreatic CSCs in the treatment group after 6 weeks. Target deconvolution of 1 demonstrated inhibition of multiple select kinase targets. There were 16 targets identified from an initial screening panel of >400 kinases. The targets were further evaluated for the ability to phenocopy the effect of treatment with 1 via shRNA knockdown. Inhibition of the thousand-and-one amino acid kinase 3 (TAOK3), cyclin-dependent kinase 7 (CDK7), aurora B kinase (AURKB), and ephrin B2 receptor (EPHB2) effectively phenocopied the effect of treatment with 1. Concurrent silencing of two or more targets increased apoptosis induced by 1, suggesting intrinsic additivity of the multikinase agent. Enforced overexpression of TAOK3 increased expression of stemness markers, clonogenicity, and tumor initiation in vivo. Conclusion: A potent inhibitor of ITK with a unique target profile and anti-stemness activities was derived from a differential HTS in a pancreatic spheroid CSC model. The 1H-benzo[d]imidazol-2-amine-derivative targets select characteristics attributed to pancreatic CSCs, including cell migration, colony formation, and metastasis formation with concomitant suppression of stemness traits without inducing cell death at the concentrations used. Target deconvolution of the compound identified multiple kinase targets, including TAOK3, a kinase not previously known to be involved in pancreatic tumor biology. Overexpression of TAOK3 results in an enforced CSC phenotype including increased colony formation and tumor initiation in vivo. The observed inhibitory effect of 1 on stemness phenotype at nanomolar concentrations without affecting cell viability or showing toxicity in vivo supports further preclinical development. Citation Format: Taylor Aiken, Yansong Bian, Yaroslav Teper, Lesley Mathews Griner, Rajarshi Guha, Paul Shinn, Hong-Wu Xin, Holger Pflicke, Jian-kang Jiang, Paresma Patel, Steven Rogers, Jeffery Aube, Marc Ferrer, Craig J. Thomas, Udo Rudloff.{Authors}. Preclinical development of a multikinase targeting molecule with activity against the cancer stem cell phenotype in pancreatic adenocarcinoma. [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 B36.

Inhibition of the GAS6/AXL pathway augments the efficacy of chemotherapies.

The AXL receptor and its activating ligand, growth arrest-specific 6 (GAS6), are important drivers of metastasis and therapeutic resistance in human cancers. Given the critical roles that GAS6 and AXL play in refractory disease, this signaling axis represents an attractive target for therapeutic intervention. However, the strong picomolar binding affinity between GAS6 and AXL and the promiscuity of small molecule inhibitors represent important challenges faced by current anti-AXL therapeutics. Here, we have addressed these obstacles by engineering a second-generation, high-affinity AXL decoy receptor with an apparent affinity of 93 femtomolar to GAS6. Our decoy receptor, MYD1-72, profoundly inhibited disease progression in aggressive preclinical models of human cancers and induced cell killing in leukemia cells. When directly compared with the most advanced anti-AXL small molecules in the clinic, MYD1-72 achieved superior antitumor efficacy while displaying no toxicity. Moreover, we uncovered a relationship between AXL and the cellular response to DNA damage whereby abrogation of AXL signaling leads to accumulation of the DNA-damage markers γH2AX, 53BP1, and RAD51. MYD1-72 exploited this relationship, leading to improvements upon the therapeutic index of current standard-of-care chemotherapies in preclinical models of advanced pancreatic and ovarian cancer.

Widely metastatic atypical pituitary adenoma with mTOR pathway STK11(F298L) mutation treated with everolimus therapy

Pituitary adenomas are the commonest intracranial tumor, but metastases are rare (0.2% yearly incidence) and portend poor prognosis. CAPecitabine and TEMozolomide improved outcomes for neuroendocrine tumors. However, no chemotherapy is approved for refractory pituitary carcinomas. Next-generation sequencing revealed an actionable mTOR pathway STK11 mutation in a woman with adrenocorticotropic hormone-secreting pituitary carcinoma refractory to six resections, radiation and CAPecitabine and TEMozolomide. Given efficacy in preclinical pancreatic cancer models with STK11 mutations, she received radiation and everolimus leading to clinical improvement and stability on MRI and PET for >6 months. She ultimately expired from widely metastatic disease. Next-generation sequencing can identify actionable mutations in rare or treatment refractory tumors. Earlier targeted therapy may improve outcomes.

Abstract 644: Utilizing a novel luciferase labeling technique to establish and validate preclinical models of pancreatic cancer

Abstract Bioluminescent-labeling imaging (BLI) allows sensitive non-invasive sequential imaging of tumor development and early metastasis. Current methods for the genetic modification of cells typically use integrating genotoxic viruses that can disrupt the molecular behavior of cancer cell lines due to their random nature of integration. A primary aim of the study was to utilize a non-integrating DNA vector that comprises an S/MAR (Scaffold/Matrix Attachment Region) element to stably genetically modify pancreatic cancer cells to persistently express the reporter gene luciferase without altering the molecular behavior of the cell or altering its sensitivity to therapeutic drug treatments. Once a novel isogenic cell line is generated the cells can subsequently be used in xenograft studies. A second aim was to validate these established models with gemcitabine and test the efficacy of VAL401, formulation of risperidone in rumenic acid. Human BxPC3, Capan-1, MiaPaCa-2 and Panc-1 pancreatic cancer cells were stably transfected with a pSMARt-UBC-Luc DNA vector and cultured for 4 weeks under selection. Colonies that formed after this period were isolated and expanded in normal medium and evaluated for luciferase expression and the molecular integrity of the DNA vector. Efficacy of gemcitabine was tested in these new luciferase expressing cell lines and VAL401 was tested in Capan-1-luc cells. For in vivo studies, BxPC3-luc cells were inoculated orthotopically into the pancreas of athymic nude mice and stratified into groups: control, gemcitabine, VAL401 (1mg/kg, p.o. daily) and VAL401 (2mg/kg, p.o. daily). In vitro validation results indicated that the luciferase transfected cells maintained their original properties with stable expression. Gemcitabine inhibited cell proliferation in all established cell lines. VAL401 inhibited cell proliferation of Capan-1-luc cells at 50 μM concentration. BxPC3-luc cells inoculated orthotopically into the pancreas were followed for 5 weeks with BLI by IVIS, and the results demonstrated high-quality follow-up of tumor growth. BxPC3-luc cells induced growth of pancreatic tumors with high take rate in all groups. Gemcitabine and both studied doses of VAL401 decreased tumor volume, and the same trend was seen in tumor weight and the BLI during the study. In conclusion, both gemcitabine and VAL401 decreased tumor volume and the same trend was observed using BLI. Our results demonstrated that S/MAR DNA vectors are able to produce genetically modified cells without the limitations of random genomic integration, whilst providing extra-chromosomal mitotic stability and high levels of sustained transgene expression. When utilized in orthotopic xenograft studies, these luciferase expressing cells formed a reliable and essentially non-invasive imaging platform that substantially improves the efficacy of testing anticancer drug candidates. Citation Format: Jenni Bernoulli, Matthias Bozza, Katja M. Fagerlund, Johanna Tuomela, Mari I. Suominen, Suzanne Dilly, George Morris, Jussi M. Halleen, Richard Harbottle. Utilizing a novel luciferase labeling technique to establish and validate preclinical models of pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 644.