Discovery and Evaluation of Novel Angular Fused Pyridoquinazolinonecarboxamides as RNA Polymerase I Inhibitors.

RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) into the 47S ribosomal RNA (rRNA) precursor. Further processing produces the 28S, 5.8S, and 18S rRNAs that are assembled into mature ribosomes. Many cancers exhibit higher Pol I transcriptional activity, reflecting a need for increased ribosome biogenesis and protein synthesis and making the inhibition of this process an attractive therapeutic strategy. Lead molecule BMH-21 (1) has been established as a Pol I inhibitor by affecting the destruction of RPA194, the Pol I large catalytic subunit. A previous structure-activity relationship (SAR) study uncovered key pharmacophores, but activity was constrained within a tight chemical space. This work details further SAR efforts that have yielded new scaffolds and improved off-target activity while retaining the desired RPA194 degradation potency. Pharmacokinetic profiling was obtained and provides a starting point for further optimization. New compounds present additional opportunities for the development of Pol I inhibitory cancer therapies.

Widespread genetic heterogeneity of human ribosomal RNA genes.

Polymorphism drives survival under stress and provides adaptability. Genetic polymorphism of ribosomal RNA (rRNA) genes derives from internal repeat variation of this multicopy gene, and from interindividual variation. A considerable amount of rRNA sequence heterogeneity has been proposed but has been challenging to estimate given the scarcity of accurate reference sequences. We identified four rDNA copies on chromosome 21 (GRCh38) with 99% similarity to recently introduced reference sequence KY962518.1. We customized a GATK bioinformatics pipeline using the four rDNA loci, spanning a total 145 kb, for variant calling and used high-coverage whole-genome sequencing (WGS) data from the 1000 Genomes Project to analyze variants in 2504 individuals from 26 populations. We identified a total of 3791 variant positions. The variants positioned nonrandomly on the rRNA gene. Invariant regions included the promoter, early 5' ETS, most of 18S, 5.8S, ITS1, and large areas of the intragenic spacer. A total of 470 variant positions were observed on 28S rRNA. The majority of the 28S rRNA variants were located on highly flexible human-expanded rRNA helical folds ES7L and ES27L, suggesting that these represent positions of diversity and are potentially under continuous evolution. Several variants were validated based on RNA-seq analyses. Population analyses showed remarkable ancestry-linked genetic variance and the presence of both high penetrance and frequent variants in the 5' ETS, ITS2, and 28S regions segregating according to the continental populations. These findings provide a genetic view of rRNA gene array heterogeneity and raise the need to functionally assess how the 28S rRNA variants affect ribosome functions.

Phase 2 study of vismodegib, a hedgehog inhibitor, combined with gemcitabine and nab-paclitaxel in patients with untreated metastatic pancreatic adenocarcinoma.

BACKGROUND: The Hedgehog (Hh) signalling pathway is overexpressed in pancreatic ductal adenocarcinoma (PDA). Preclinical studies have shown that Hh inhibitors reduce pancreatic cancer stem cells (pCSC), stroma and Hh signalling. METHODS: Patients with previously untreated metastatic PDA were treated with gemcitabine and nab-paclitaxel. Vismodegib was added starting on the second cycle. The primary endpoint was progression-free survival (PFS) as compared with historical controls. Tumour biopsies to assess pCSC, stroma and Hh signalling were obtained before treatment and after cycle 1 (gemcitabine and nab-paclitaxel) or after cycle 2 (gemcitabine and nab-paclitaxel plus vismodegib). RESULTS: Seventy-one patients were enrolled. Median PFS and overall survival (OS) were 5.42 months (95% confidence interval [CI]: 4.37-6.97) and 9.79 months (95% CI: 7.85-10.97), respectively. Of the 67 patients evaluable for response, 27 (40%) had a response: 26 (38.8%) partial responses and 1 complete response. In the tumour samples, there were no significant changes in ALDH + pCSC following treatment. CONCLUSIONS: Adding vismodegib to chemotherapy did not improve efficacy as compared with historical rates observed with chemotherapy alone in patients with newly diagnosed metastatic pancreatic cancer. This study does not support the further evaluation of Hh inhibitors in this patient population. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01088815.

Preclinical evaluation of the maximum tolerated dose and toxicokinetics of enteric-coated lantibiotic OG253 capsules.

Clostridium difficile associated disease (CDAD) is the leading infectious cause of antibiotic-associated diarrhea and colitis in the United States. Both the incidence and severity of CDAD have been increased over the past two decades. We evaluated the maximum tolerated dose (MTD) and toxicokinetics of OG253, a novel lantibiotic in development for the treatment of CDAD. OG253 was orally administered to Wistar Han rats as enteric-coated capsules in a one-day dose escalation study, followed by a seven-day repeated dose toxicokinetics study. All three doses of OG253 (6.75, 27 and 108 mg/day) were generally well-tolerated with no treatment-related clinical signs, alterations in body weight or food consumption in both one-day acute tolerability and seven-days repeated dose tolerability and toxicokinetics study. OG253 capsule administration neither significantly alter the weight of organs nor affect the hematology, coagulation, clinical biochemistry parameters and urine pH compared to placebo capsule administered rats. LC-MS/MS analysis did not detect OG253 in the plasma, indicating that OG253 is not absorbed into the blood from the rat gastrointestinal tract. Glandular atrophy of the rectal mucosa was noticed in two out of six rats administered with a high dose of OG253. Surprisingly, we found that OG253 treatment significantly lowered both serum cholesterol and triglyceride levels in both sexes of rats. Overall, there was a 29.8 and 61.38% decrease in the serum cholesterol and triglyceride levels, respectively as compared to placebo-treated rats. The well-tolerated high dose of OG253 (425.7 mg/kg/day) is recommended as the MTD for safety and efficacy studies. Further preclinical study is needed to evaluate the safety profile of OG253 under longer exposure.

Direct Interactions With Cancer-Associated Fibroblasts Lead to Enhanced Pancreatic Cancer Stem Cell Function.

OBJECTIVE: Cancer-associated fibroblasts (CAFs) play an important role in the progression of pancreatic ductal adenocarcinoma (PDAC) by promoting tumor cell migration and drug resistance. We determined the impact of CAFs on PDAC cancer stem cells (CSCs). METHODS: Fibroblast cell lines from patients' tumors were cocultured with PDAC cells and examined for clonogenic growth and self-renewal using colony-forming assays and migration in vitro. Changes in the frequency of CSCs was determined by flow cytometry. The effect of integrin-focal adhesion kinase (FAK) signaling on CAF-mediated clonogenic growth was evaluated using short hairpin RNAs against ß1 integrin and FAK as well as a small-molecule FAK inhibitor. RESULTS: Cancer-associated fibroblasts enhanced PDAC clonogenic growth, self-renewal, and migration that was associated with an increase in the frequency of CSCs. These fibroblast cells were activated by PDAC cells and increased collagen synthesis resulting in FAK activation in PDAC cells. Knockdown of ß1-integrin and FAK or the inhibition of FAK kinase activity in PDAC cells abrogated the impact of CAFs on clonogenic growth. CONCLUSION: Therefore, CAFs enhance PDAC clonogenic growth, self-renewal, and the frequency of CSCs through type I collagen production that enhances integrin-FAK signaling in PDAC cells.

Small-Molecule Inhibition of Axl Targets Tumor Immune Suppression and Enhances Chemotherapy in Pancreatic Cancer.

Activation of the receptor tyrosine kinase Axl is associated with poor outcomes in pancreatic cancer (PDAC), where it coordinately mediates immune evasion and drug resistance. Here, we demonstrate that the selective Axl kinase inhibitor BGB324 targets the tumor-immune interface to blunt the aggressive traits of PDAC cells in vitro and enhance gemcitibine efficacy in vivo Axl signaling stimulates the TBK1-NFκB pathway and innate immune suppression in the tumor microenvironment. In tumor cells, BGB324 treatment drove epithelial differentiation, expression of nucleoside transporters affecting gemcitabine response, and an immune stimulatory microenvironment. Our results establish a preclinical mechanistic rationale for the clinical development of Axl inhibitors to improve the treatment of PDAC patients.Significance: These results establish a preclinical mechanistic rationale for the clinical development of AXL inhibitors to improve the treatment of PDAC patients. Cancer Res; 78(1); 246-55. ©2017 AACR.

The extracellular matrix and focal adhesion kinase signaling regulate cancer stem cell function in pancreatic ductal adenocarcinoma.

Cancer stem cells (CSCs) play an important role in the clonogenic growth and metastasis of pancreatic ductal adenocarcinoma (PDAC). A hallmark of PDAC is the desmoplastic reaction, but the impact of the tumor microenvironment (TME) on CSCs is unknown. In order to better understand the mechanisms, we examined the impact of extracellular matrix (ECM) proteins on PDAC CSCs. We quantified the effect of ECM proteins, ß1-integrin, and focal adhesion kinase (FAK) on clonogenic PDAC growth and migration in vitro and tumor initiation, growth, and metastasis in vivo in nude mice using shRNA and overexpression constructs as well as small molecule FAK inhibitors. Type I collagen increased PDAC tumor initiating potential, self-renewal, and the frequency of CSCs through the activation of FAK. FAK overexpression increased tumor initiation, whereas a dominant negative FAK mutant or FAK kinase inhibitors reduced clonogenic PDAC growth in vitro and in vivo. Moreover, the FAK inhibitor VS-4718 extended the anti-tumor response to gemcitabine and nab-paclitaxel in patient-derived PDAC xenografts, and the loss of FAK expression limited metastatic dissemination of orthotopic xenografts. Type I collagen enhances PDAC CSCs, and both kinase-dependent and independent activities of FAK impact PDAC tumor initiation, self-renewal, and metastasis. The anti-tumor impact of FAK inhibitors in combination with standard chemotherapy support the clinical testing of this combination.

Treatment of Pancreatic Cancer Patient-Derived Xenograft Panel with Metabolic Inhibitors Reveals Efficacy of Phenformin.

Purpose: To identify effective metabolic inhibitors to suppress the aggressive growth of pancreatic ductal adenocarcinoma (PDAC), we explored the in vivo antitumor efficacy of metabolic inhibitors, as single agents, in a panel of patient-derived PDAC xenograft models (PDX) and investigated whether genomic alterations of tumors correlate with the sensitivity to metabolic inhibitors.Experimental Design: Mice with established PDAC tumors from 6 to 13 individual PDXs were randomized and treated, once daily for 4 weeks, with either sterile PBS (vehicle) or the glutaminase inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), transaminase inhibitor aminooxyacetate (AOA), pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA), autophagy inhibitor chloroquine (CQ), and mitochondrial complex I inhibitor phenformin/metformin.Results: Among the agents tested, phenformin showed significant tumor growth inhibition (>30% compared with vehicle) in 5 of 12 individual PDXs. Metformin, at a fivefold higher dose, displayed significant tumor growth inhibition in 3 of 12 PDXs similar to BPTES (2/8 PDXs) and DCA (2/6 PDXs). AOA and CQ had the lowest response rates. Gene set enrichment analysis conducted using the baseline gene expression profile of pancreatic tumors identified a gene expression signature that inversely correlated with phenformin sensitivity, which is in agreement with the phenformin gene expression signature of NIH Library of Integrated Network-based Cellular Signatures (LINCS). The PDXs that were more sensitive to phenformin showed a baseline reduction in amino acids and elevation in oxidized glutathione. There was no correlation between phenformin response and genetic alterations in KRAS, TP53, SMAD4, or PTENConclusions: Phenformin treatment showed relatively higher antitumor efficacy against established PDAC tumors, compared with the efficacy of other metabolic inhibitors and metformin. Phenformin treatment significantly diminished PDAC tumor progression and prolonged tumor doubling time. Overall, our results serve as a foundation for further evaluation of phenformin as a therapeutic agent in pancreatic cancer. Clin Cancer Res; 23(18); 5639-47. ©2017 AACR.

A phase 2 trial of personalized cytotoxic therapy based on tumor immunohistochemistry in previously treated metastatic pancreatic cancer patients.

BACKGROUND: The choice of a regimen in metastatic pancreatic cancer patients following progression on 1st line therapy is empiric and outcomes are unsatisfactory. This phase II study was performed to evaluate the efficacy of therapy selected by immunohistochemistry (IHC) in these patients following progression after one or more therapies. METHODS: Eligible patients underwent a percutaneous biopsy of a metastatic lesion and treatment selection was determined by IHC. The study required 35 evaluable patients (power of 86%) for detecting a true 1-year survival rate of >20%. RESULTS: A tumor biopsy was performed in 48 of 49 accrued patients. Study therapy was not given (n=13) either due to insufficient tumor on biopsy (n=8) or due to worsening cancer related symptoms after biopsy (n=5). The demographics of evaluable patients (n=35) are male/female (59%/41%), with age range 34-78 years (median 63 years). Patients had 1-6 prior regimens (median of 2). The most common IHC targets were topoisomerase 1 or 2, thymidylate synthase, excision repair cross-complementation group 1 protein (ERCC1), and osteonectin secreted protein acidic and rich in cysteine (SPARC). Commercially available treatment regimens prescribed included FOLFIRI, FOLFOX, irinotecan, and doxorubicin. The response (RECIST) was 9%, the median survival was 5.6 months (94% CI, 3.8-8.2), and the 1-year survival was 20% (95% CI, 7-33%). CONCLUSIONS: In all patients, IHC assays resulted in identification of at least two targets for therapy and a non-cross resistant regimen could be prescribed for therapy with evidence of some benefit. An IHC based treatment strategy is feasible and needs validation in larger studies.

Superior therapeutic efficacy of nab-paclitaxel over cremophor-based paclitaxel in locally advanced and metastatic models of human pancreatic cancer.

BACKGROUND: Albumin-bound paclitaxel (nab-paclitaxel, nab-PTX) plus gemcitabine (GEM) combination has demonstrated efficient antitumour activity and statistically significant overall survival of patients with metastatic pancreatic ductal adenocarcinoma (PDAC) compared with GEM monotherapy. This regimen is currently approved as a standard of care treatment option for patients with metastatic PDAC. It is unclear whether cremophor-based PTX combined with GEM provide a similar level of therapeutic efficacy in PDAC. METHODS: We comprehensively explored the antitumour efficacy, effect on metastatic dissemination, tumour stroma and survival advantage following GEM, PTX and nab-PTX as monotherapy or in combination with GEM in a locally advanced, and a highly metastatic orthotopic model of human PDAC. RESULTS: Nab-PTX treatment resulted in significantly higher paclitaxel tumour plasma ratio (1.98-fold), robust stromal depletion, antitumour efficacy (3.79-fold) and survival benefit compared with PTX treatment. PTX plus GEM treatment showed no survival gain over GEM monotherapy. However, nab-PTX in combination with GEM decreased primary tumour burden, metastatic dissemination and significantly increased median survival of animals compared with either agents alone. These therapeutic effects were accompanied by depletion of dense fibrotic tumour stroma and decreased proliferation of carcinoma cells. Notably, nab-PTX monotherapy was equivalent to nab-PTX plus GEM in providing survival advantage to mice in a highly aggressive metastatic PDAC model, indicating that nab-PTX could potentially stop the progression of late-stage pancreatic cancer. CONCLUSIONS: Our data confirmed that therapeutic efficacy of PTX and nab-PTX vary widely, and the contention that these agents elicit similar antitumour response was not supported. The addition of PTX to GEM showed no survival advantage, concluding that a clinical combination of PTX and GEM may unlikely to provide significant survival advantage over GEM monotherapy and may not be a viable alternative to the current standard-of-care nab-PTX plus GEM regimen for the treatment of PDAC patients.

Therapeutic Targeting of the Warburg Effect in Pancreatic Cancer Relies on an Absence of p53 Function.

The "Warburg effect" describes a peculiar metabolic feature of many solid tumors, namely their increased glucose uptake and high glycolytic rates, which allow cancer cells to accumulate building blocks for the biosynthesis of macromolecules. During aerobic glycolysis, pyruvate is preferentially metabolized to lactate by the enzyme lactate dehydrogenase-A (LDH-A), suggesting a possible vulnerability at this target for small-molecule inhibition in cancer cells. In this study, we used FX11, a small-molecule inhibitor of LDH-A, to investigate this possible vulnerability in a panel of 15 patient-derived mouse xenograft (PDX) models of pancreatic cancer. Unexpectedly, the p53 status of the PDX tumor determined the response to FX11. Tumors harboring wild-type (WT) TP53 were resistant to FX11. In contrast, tumors harboring mutant TP53 exhibited increased apoptosis, reduced proliferation indices, and attenuated tumor growth when exposed to FX11. [18F]-FDG PET-CT scans revealed a relative increase in glucose uptake in mutant TP53 versus WT TP53 tumors, with FX11 administration downregulating metabolic activity only in mutant TP53 tumors. Through a noninvasive quantitative assessment of lactate production, as determined by 13C magnetic resonance spectroscopy (MRS) of hyperpolarized pyruvate, we confirmed that FX11 administration inhibited pyruvate-to-lactate conversion only in mutant TP53 tumors, a feature associated with reduced expression of the TP53 target gene TIGAR, which is known to regulate glycolysis. Taken together, our findings highlight p53 status in pancreatic cancer as a biomarker to predict sensitivity to LDH-A inhibition, with regard to both real-time noninvasive imaging by 13C MRS as well as therapeutic response.

Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1) redox function negatively regulates NRF2.

Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1) (henceforth referred to as Ref-1) is a multifunctional protein that in addition to its base excision DNA repair activity exerts redox control of multiple transcription factors, including nuclear factor κ-light chain enhancer of activated B cells (NF-κB), STAT3, activator protein-1 (AP-1), hypoxia-inducible factor-1 (HIF-1), and tumor protein 53 (p53). In recent years, Ref-1 has emerged as a promising therapeutic target in cancer, particularly in pancreatic ductal carcinoma. Although a significant amount of research has centered on Ref-1, no wide-ranging approach had been performed on the effects of Ref-1 inhibition and transcription factor activity perturbation. Starting with a broader approach, we identified a previously unsuspected effect on the nuclear factor erythroid-related factor 2 (NRF2), a critical regulator of cellular defenses against oxidative stress. Based on genetic and small molecule inhibitor-based methodologies, we demonstrated that repression of Ref-1 potently activates NRF2 and its downstream targets in a dose-dependent fashion, and that the redox, rather than the DNA repair function of Ref-1 is critical for this effect. Intriguingly, our results also indicate that this pathway does not involve reactive oxygen species. The link between Ref-1 and NRF2 appears to be present in all cells tested in vitro, noncancerous and cancerous, including patient-derived tumor samples. In particular, we focused on understanding the implications of the novel interaction between these two pathways in primary pancreatic ductal adenocarcinoma tumor cells and provide the first evidence that this mechanism has implications for overcoming the resistance against experimental drugs targeting Ref-1 activity, with clear translational implications.

Heterogeneity of pancreatic cancer metastases in a single patient revealed by quantitative proteomics.

Many patients with pancreatic cancer have metastases to distant organs at the time of initial presentation. Recent studies examining the evolution of pancreatic cancer at the genetic level have shown that clonal complexity of metastatic pancreatic cancer is already initiated within primary tumors, and organ-specific metastases are derived from different subclones. However, we do not yet understand to what extent the evolution of pancreatic cancer contributes to proteomic and signaling alterations. We hypothesized that genetic heterogeneity of metastatic pancreatic cancer results in heterogeneity at the proteome level. To address this, we employed a model system in which cells isolated from three sites of metastasis (liver, lung, and peritoneum) from a single patient were compared. We used a SILAC-based accurate quantitative proteomic strategy combined with high-resolution mass spectrometry to analyze the total proteome and tyrosine phosphoproteome of each of the distal metastases. Our data revealed distinct patterns of both overall proteome expression and tyrosine kinase activities across the three different metastatic lesions. This heterogeneity was significant because it led to differential sensitivity of the neoplastic cells to small molecule inhibitors targeting various kinases and other pathways. For example, R428, a tyrosine kinase inhibitor that targets Axl receptor tyrosine kinase, was able to inhibit cells derived from lung and liver metastases much more effectively than cells from the peritoneal metastasis. Finally, we confirmed that administration of R428 in mice bearing xenografts of cells derived from the three different metastatic sites significantly diminished tumors formed from liver- and lung-metastasis-derived cell lines as compared with tumors derived from the peritoneal metastasis cell line. Overall, our data provide proof-of-principle support that personalized therapy of multiple organ metastases in a single patient should involve the administration of a combination of agents, with each agent targeted to the features of different subclones.

Autophagy is critical for pancreatic tumor growth and progression in tumors with p53 alterations.

UNLABELLED: Pancreatic ductal adenocarcinoma is refractory to available therapies. We have previously shown that these tumors have elevated autophagy and that inhibition of autophagy leads to decreased tumor growth. Using an autochthonous model of pancreatic cancer driven by oncogenic Kras and the stochastic LOH of Trp53, we demonstrate that although genetic ablation of autophagy in the pancreas leads to increased tumor initiation, these premalignant lesions are impaired in their ability to progress to invasive cancer, leading to prolonged survival. In addition, mouse pancreatic cancer cell lines with differing p53 status are all sensitive to pharmacologic and genetic inhibition of autophagy. Finally, a mouse preclinical trial using cohorts of genetically characterized patient-derived xenografts treated with hydroxychloroquine showed responses across the collection of tumors. Together, our data support the critical role of autophagy in pancreatic cancer and show that inhibition of autophagy may have clinical utility in the treatment of these cancers, independent of p53 status. SIGNIFICANCE: Recently, a mouse model with embryonic homozygous Trp53 deletion showed paradoxical effects of autophagy inhibition. We used a mouse model with Trp53 LOH (similar to human tumors), tumor cell lines, and patient-derived xenografts to show that p53 status does not affect response to autophagy inhibition. These findings have important implications on ongoing clinical trials.

Notch signaling pathway targeted therapy suppresses tumor progression and metastatic spread in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) remains a lethal human malignancy with historically limited success in treatment. The role of aberrant Notch signaling, which requires the constitutive activation of γ-secretase, in the initiation and progression of PDA is well defined and inhibitors of this pathway are currently in clinical trials. Here we investigated the in vivo therapeutic effect of PF-03084014, a selective γ-secretase inhibitor, alone and in combination with gemcitabine in pancreatic cancer xenografts. PF-03084014 treatment inhibited the cleavage of nuclear Notch 1 intracellular domain and Notch targets Hes-1 and Hey-1. Gemcitabine treatment showed good response but not capable of inducing tumor regressions and targeting the tumor-resident cancer stem cells (CD24(+)CD44(+) and ALDH(+) tumor cells). A combination of PF-03084014 and gemcitabine treatment resulted tumor regression in 3 of 4 subcutaneously implanted xenograft models. PF-03084014, and in combination with gemcitabine reduced putative cancer stem cells, indicating that PF-03084014 target the especially dangerous and resilient cancer stem cells within pancreatic tumors. Tumor re-growth curves plotted after drug treatments demonstrated that the effect of the combination therapy was sustainable than that of gemcitabine. Notably, in a highly aggressive orthotopic model, PF-03084014 and gemcitabine combination was effective in inducing apoptosis, inhibition of tumor cell proliferation and angiogenesis, resulting in the attenuation of primary tumor growth as well as controlling metastatic dissemination, compared to gemcitabine treatment. In summary, our preclinical data suggest that PF-03084014 has greater anti-tumor activity in combination with gemcitabine in PDA and provides rationale for further investigation of this combination in PDA.

A preclinical and clinical study of mycophenolate mofetil in pancreatic cancer.

A high throughput screening for anticancer activity of FDA approved drugs identified mycophenolic acid (MPA), an inhibitor of inositol monophosphate dehydrogenase (IMPDH) as an active agent with an antiangiogenesis mode of action. Exposure of pancreatic cancer cell lines to MPA resulted in growth inhibition and reduced the expression of VEGF that was reversed by supplementing the media with guanosine supporting and IMPDH-dependant mechanism. In preclinical in vivo study, MPA showed a moderate inhibition of tumor growth in a panel of 6 human derived pancreatic cancer xenografts but reduced the expression of VEGF. To investigate the effects of MPA in human pancreatic cancer, a total of 12 patients with resectable pancreatic cancer (PDA) received increasing doses of mycophenolate mofetil (MMF) in cohorts of 6 patients each from 5-15 days prior to surgical resection. Treatment was well tolerated with one episode of grade 1 muscle pain, one episode of grade 2 lymphopenia (2 gr/day dose) and one episode of grade 2 elevantion in LFT (all in the 2 gr./day dose). Patients recovered from surgery uneventfully with no increased post-operative complications. Assessment of CD31, VEGF, and TUNEL in resected specimens compared to a non treated control of 6 patients showed no significant variations in any of the study endpoints. In conclusion, this study shows the feasibility of translating a preclinical observation to the clinical setting and to explore a drug mechanism of action in patients. MPA, however, did not show any hints of antiangiogenesis of anticancer clinical activity questioning if this agent should be further developed in PDA.

Conceptual framework for cutting the pancreatic cancer fuel supply.

Pancreatic ductal adenocarcinoma (a.k.a. pancreatic cancer) remains one of the most feared and clinically challenging diseases to treat despite continual improvements in therapies. The genetic landscape of pancreatic cancer shows near ubiquitous activating mutations of KRAS, and recurrent inactivating mutations of CDKN2A, SMAD4, and TP53. To date, attempts to develop agents to target KRAS to specifically kill cancer cells have been disappointing. In this regard, an understanding of cellular metabolic derangements in pancreatic cancer could lead to novel therapeutic approaches. Like other cancers, pancreatic cancer cells rely on fuel sources for homeostasis and proliferation; as such, interrupting the use of two major nutrients, glucose and glutamine, may provide new therapeutic avenues. In addition, KRAS-mutant pancreatic cancers have been documented to depend on autophagy, and the inhibition of autophagy in the preclinical setting has shown promise. Herein, the conceptual framework for blocking the pancreatic fuel supply is reviewed.

The gamma secretase inhibitor MRK-003 attenuates pancreatic cancer growth in preclinical models.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy, with most patients facing an adverse clinical outcome. Aberrant Notch pathway activation has been implicated in the initiation and progression of PDAC, specifically the aggressive phenotype of the disease. We used a panel of human PDAC cell lines as well as patient-derived PDAC xenografts to determine whether pharmacologic targeting of Notch pathway could inhibit PDAC growth and potentiate gemcitabine sensitivity. MRK-003, a potent and selective γ-secretase inhibitor, treatment resulted in the downregulation of nuclear Notch1 intracellular domain, inhibition of anchorage-independent growth, and reduction of tumor-initiating cells capable of extensive self-renewal. Pretreatment of PDAC cells with MRK-003 in cell culture significantly inhibited the subsequent engraftment in immunocompromised mice. MRK-003 monotherapy significantly blocked tumor growth in 5 of 9 (56%) PDAC xenografts. A combination of MRK-003 and gemcitabine showed enhanced antitumor effects compared with gemcitabine in 4 of 9 (44%) PDAC xenografts, reduced tumor cell proliferation, and induced both apoptosis and intratumoral necrosis. Gene expression analysis of untreated tumors indicated that upregulation of NF-κB pathway components was predictive of sensitivity to MRK-003, whereas upregulation in B-cell receptor signaling and nuclear factor erythroid-derived 2-like 2 pathway correlated with response to the combination of MRK-003 with gemcitabine. Our findings strengthen the rationale for small-molecule inhibition of Notch signaling as a therapeutic strategy in PDAC.

Integrated preclinical and clinical development of S-trans, trans-Farnesylthiosalicylic Acid (FTS, Salirasib) in pancreatic cancer.

PURPOSE: S-trans,trans-Farnesylthiosalicylic Acid (FTS, salirasib) inhibits Ras-dependent cell growth by dislodging all isoforms of Ras, including mutant Ras, from the plasma membrane. This study evaluated the activity, safety, and toxicity of salirasib in preclinical models and patients with metastatic pancreatic adenocarcinoma (PDA). PATIENTS AND METHODS: In the preclinical study, salirasib was tested, alone and in combination with gemcitabine, in patient derived xenografts (PDX) of PDA. In the clinical study, treatment-naïve patients with advanced, metastatic PDA were treated with a standard dose schedule of gemcitabine and salirasib 200-800 mg orally (PO) twice daily (bid) for 21 days every 28 days. Tissue from preclinical models and patients' biopsies were collected pre-treatment and on Cycle (C) 1, Day (D) 9 to characterize the effect of gemcitabine and salirasib on activated Ras protein levels. Plasma samples for pharmacokinetics were collected for salirasib administered alone and in combination. RESULTS: Salirasib inhibited the growth of 2/14 PDX models of PDA and modulated Ras signaling in these tumors. Nineteen patients were enrolled. No DLTs occurred. Common adverse events included hematologic and gastrointestinal toxicities and fatigue. The median overall survival was 6.2 months and the 1 year survival 37 %. In 2 patients in whom paired tissue biopsies were available, Ras and KRas protein levels were decreased on C1D9. Salirasib exposure was not altered by gemcitabine and did not correlate with PD outcomes. CONCLUSION: The combination of gemcitabine and salirasib appears well-tolerated, with no alteration of salirasib exposure, and exerted clinical and PD activity in PDA.

Gemcitabine plus nab-paclitaxel is an active regimen in patients with advanced pancreatic cancer: a phase I/II trial.

PURPOSE: The trial objectives were to identify the maximum-tolerated dose (MTD) of first-line gemcitabine plus nab-paclitaxel in metastatic pancreatic adenocarcinoma and to provide efficacy and safety data. Additional objectives were to evaluate positron emission tomography (PET) scan response, secreted protein acidic and rich in cysteine (SPARC), and CA19-9 levels in relation to efficacy. Subsequent preclinical studies investigated the changes involving the pancreatic stroma and drug uptake. PATIENTS AND METHODS: Patients with previously untreated advanced pancreatic cancer were treated with 100, 125, or 150 mg/m(2) nab-paclitaxel followed by gemcitabine 1,000 mg/m(2) on days 1, 8, and 15 every 28 days. In the preclinical study, mice were implanted with human pancreatic cancers and treated with study agents. RESULTS: A total of 20, 44, and three patients received nab-paclitaxel at 100, 125, and 150 mg/m(2), respectively. The MTD was 1,000 mg/m(2) of gemcitabine plus 125 mg/m(2) of nab-paclitaxel once a week for 3 weeks, every 28 days. Dose-limiting toxicities were sepsis and neutropenia. At the MTD, the response rate was 48%, with 12.2 median months of overall survival (OS) and 48% 1-year survival. Improved OS was observed in patients who had a complete metabolic response on [(18)F]fluorodeoxyglucose PET. Decreases in CA19-9 levels were correlated with increased response rate, progression-free survival, and OS. SPARC in the stroma, but not in the tumor, was correlated with improved survival. In mice with human pancreatic cancer xenografts, nab-paclitaxel alone and in combination with gemcitabine depleted the desmoplastic stroma. The intratumoral concentration of gemcitabine was increased by 2.8-fold in mice receiving nab-paclitaxel plus gemcitabine versus those receiving gemcitabine alone. CONCLUSION: The regimen of nab-paclitaxel plus gemcitabine has tolerable adverse effects with substantial antitumor activity, warranting phase III evaluation.