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T cells play a critical role in the control of cancer. The development of immune checkpoint blockers (ICB) aimed at enhancing antitumor T-cell responses has revolutionized cancer treatment. However, durable clinical benefit is observed in only a subset of patients, prompting research efforts to focus on strategies that target multiple inhibitory signals within the tumor microenvironment (TME) to limit tumor evasion and improve patient outcomes. Adenosine has emerged as a potent immune suppressant within the TME, and CD73 is the major enzyme responsible for its extracellular production. CD73 can be co-opted within the TME to impair T-cell–mediated antitumor immunity and promote tumor growth. To target this pathway and block the formation of adenosine, we designed a novel, selective, and potent class of small-molecule inhibitors of CD73, including AB680 (quemliclustat), which is currently being tested in patients with cancer. AB680 effectively restored T-cell proliferation, cytokine secretion, and cytotoxicity that were dampened by the formation of immunosuppressive adenosine by CD73. Furthermore, in an allogeneic mixed lymphocyte reaction where CD73-derived adenosine had a dominant suppressive effect in the presence of PD-1 blockade, AB680 restored T-cell activation and function. Finally, in a preclinical mouse model of melanoma, AB680 inhibited CD73 in the TME and increased the antitumor activity of PD-1 blockade. Collectively, these data provide a rationale for the inhibition of CD73 with AB680 in combination with ICB, such as anti–PD-1, to improve cancer patient outcomes.

Supplementary Data from Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity

Supplementary Figure from Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity

T cells play a critical role in the control of cancer. The development of immune checkpoint blockers (ICB) aimed at enhancing antitumor T-cell responses has revolutionized cancer treatment. However, durable clinical benefit is observed in only a subset of patients, prompting research efforts to focus on strategies that target multiple inhibitory signals within the tumor microenvironment (TME) to limit tumor evasion and improve patient outcomes. Adenosine has emerged as a potent immune suppressant within the TME, and CD73 is the major enzyme responsible for its extracellular production. CD73 can be co-opted within the TME to impair T-cell–mediated antitumor immunity and promote tumor growth. To target this pathway and block the formation of adenosine, we designed a novel, selective, and potent class of small-molecule inhibitors of CD73, including AB680 (quemliclustat), which is currently being tested in patients with cancer. AB680 effectively restored T-cell proliferation, cytokine secretion, and cytotoxicity that were dampened by the formation of immunosuppressive adenosine by CD73. Furthermore, in an allogeneic mixed lymphocyte reaction where CD73-derived adenosine had a dominant suppressive effect in the presence of PD-1 blockade, AB680 restored T-cell activation and function. Finally, in a preclinical mouse model of melanoma, AB680 inhibited CD73 in the TME and increased the antitumor activity of PD-1 blockade. Collectively, these data provide a rationale for the inhibition of CD73 with AB680 in combination with ICB, such as anti–PD-1, to improve cancer patient outcomes.

INTRODUCTION: Tumors employ many strategies to attenuate immune responses. High levels of extracellular adenosine generated in the tumor microenvironment engage A2a and A2b adenosine receptors on immune cells, resulting in immunosuppression. Expression of A2aR and A2bR can vary by cell type, with T cells predominantly expressing A2aR while myeloid cells express both A2aR and A2bR. We have previously shown that etrumadenant, a dual A2aR/A2bR antagonist, blocks the immunosuppressive effects of adenosine in immune cells and enhances anti-tumor immune responses in mouse syngeneic tumors. Using dual and selective adenosine receptor antagonists, we assessed the contribution of these receptors to adenosine-mediated phenotypes in immune and cancer cells. METHODS: Human CD8+ T cells were isolated from healthy human blood and activated using CD3/CD28/CD2 stimulation and cytokines were analyzed by cytokine bead array at 72 hours. Primary human dendritic cells (DC) were isolated from healthy blood and matured with LPS/IFN-γ for 24 hours. Cancer cell lines were purchased from ATCC. The adenosine analogue NECA was used to stimulate A2aR/A2bR-mediated signaling. RESULTS: Activated human CD8+ T cells stimulated in the presence of NECA showed suppression of activation markers (CD69) and cytokine production (IFN-γ, IL-2 and granzyme B). As expected, we observed similar rescue of this phenotype with both etrumadenant and an A2aR-specific antagonist owing to the sole expression of A2aR on T cells. In contrast, primary DC have comparable expression of A2aR and A2bR, suggesting that dual blockade may provide greater resistance to adenosine-mediated suppression than A2aR antagonism. Indeed, etrumadenant was able to attenuate the adenosine-mediated upregulation of IL-10 and enhance IL-12p70 production, whereas a comparable A2aR-specific antagonist showed no significant rescue versus NECA-stimulated controls. These observations may be extended to suppressive myeloid populations as well as tumor-resident macrophages and myeloid-derived suppressor cells isolated from mouse syngeneic tumors, which have very high expression of both A2aR and A2bR. RNA-sequencing identified a cassette of genes regulated by adenosine-signaling in these cells, which were largely reversed by etrumadenant. Finally, cultured human cancer cell lines have high expression of A2bR, which has been implicated in driving their tumorigenesis. Etrumadenant reversed adenosine-stimulated gene expression changes in non-small cell lung cancer cell lines, restoring enriched pathways driven by adenosine signaling. CONCLUSIONS: Taken together, these results show an important role for A2aR/A2bR in adenosine-mediated immunosuppression and provide a mechanistic rationale for stimulation of anti-tumor immune responses with the dual adenosine receptor antagonist etrumadenant. Citation Format: Sachie Marubayashi, Bindi Patel, Livia Yamashiro, Dana Piovesan, Sean Cho, Jenna Jeffrey, Manmohan Leleti, Jay Powers, Matt Walters, Daniel DiRenzo. Dual A2aR/A2bR antagonism with etrumadenant (AB928) eliminates the suppressive effects of adenosine on immune and cancer cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 256.

Atherosclerosis is the process underlying heart attack and stroke. Despite decades of research, its pathogenesis remains unclear. Dogma suggests that atherosclerotic plaques expand primarily via the accumulation of cholesterol and inflammatory cells. However, recent evidence suggests that a substantial portion of the plaque may arise from a subset of “dedifferentiated” vascular smooth muscle cells (SMCs) which proliferate in a clonal fashion. Herein we use multicolor lineage-tracing models to confirm that the mature SMC can give rise to a hyperproliferative cell which appears to promote inflammation via elaboration of complement-dependent anaphylatoxins. Despite being extensively opsonized with prophagocytic complement fragments, we find that this cell also escapes immune surveillance by neighboring macrophages, thereby exacerbating its relative survival advantage. Mechanistic studies indicate this phenomenon results from a generalized opsonin-sensing defect acquired by macrophages during polarization. This defect coincides with the noncanonical up-regulation of so-called don’t eat me molecules on inflamed phagocytes, which reduces their capacity for programmed cell removal (PrCR). Knockdown or knockout of the key antiphagocytic molecule CD47 restores the ability of macrophages to sense and clear opsonized targets in vitro, allowing for potent and targeted suppression of clonal SMC expansion in the plaque in vivo. Because integrated clinical and genomic analyses indicate that similar pathways are active in humans with cardiovascular disease, these studies suggest that the clonally expanding SMC may represent a translational target for treating atherosclerosis.

Much remains unknown regarding the regulatory networks formed by transcription factors in mature, differentiated mammalian cells in vivo, despite many studies of individual DNA-binding transcription factors. We report a constellation of feed-forward loops formed by the pancreatic transcription factors MIST1 and PTF1 that govern the differentiated phenotype of the adult pancreatic acinar cell. PTF1 is an atypical basic helix-loop-helix transcription factor complex of pancreatic acinar cells and is critical to acinar cell fate specification and differentiation. MIST1, also a basic helix-loop-helix transcription factor, enhances the formation and maintenance of the specialized phenotype of professional secretory cells. The MIST1 and PTF1 collaboration controls a wide range of specialized cellular processes, including secretory protein synthesis and processing, exocytosis, and homeostasis of the endoplasmic reticulum. PTF1 drives Mist1 transcription, and MIST1 and PTF1 bind and drive the transcription of over 100 downstream acinar genes. PTF1 binds two canonical bipartite sites within a 0.7-kb transcriptional enhancer upstream of Mist1 that are essential for the activity of the enhancer in vivo. MIST1 and PTF1 coregulate target genes synergistically or additively, depending on the target transcriptional enhancer. The frequent close binding proximity of PTF1 and MIST1 in pancreatic acinar cell chromatin implies extensive collaboration although the collaboration is not dependent on a stable physical interaction.

Many studies have noted similarities between atherosclerosis and cancer including pronounced cellular plasticity, clonal expansion of cellular subtypes, increased DNA mutations, defective efferocytosis pathways, and an important role for proto-oncogenes in disease development. Although it is clear that these 2 diseases have disparate causes, noting the parallels between atherosclerosis and cancer may help us identify unique, targeted therapeutic strategies. Early articles described atheromas as benign neoplasms of the blood vessel comprised primarily of fibrous smooth muscle cells (SMCs). However, additional studies soon noted the presence of macrophages and other cell types, thereby promoting controversy as to which cells actually give rise to the atherosclerotic plaque. Much of this controversy results from the promiscuity of the so-called lineage-specific markers.1 For example, bone marrow–derived cells can migrate into plaques and begin to express certain SMC markers,2 whereas SMCs can upregulate macrophage-specific markers such as galectin-3, CD11b, and F4/80 as they migrate into the plaque.3,4 In addition, recent lineage tracing studies suggest that endothelial cells can acquire mesenchymal cell characteristics, and some adventitial cells may have the capability of forming SMC-like cells. Collectively, these studies demonstrate the impressive capacity of many vascular cell types to dedifferentiate or transdifferentiate in response to the atherosclerotic environment and suggest that plaques may arise from multiple vascular and circulating cell types. Clearly, cellular plasticity is a major theme in atherosclerotic plaque development—as it is in cancer—and methods to reprogram proatherosclerotic cells could have tremendous therapeutic potential. The clonal evolution theory of cancer development posits that certain tumors arise from a single cell that expands in response to a series of acquired mutations. In 1973, Benditt and Benditt5 performed X-inactivation studies on human atherosclerotic lesions and found evidence that atherosclerotic plaques could be derived from a single cell that underwent monoclonal expansion, …

Background: The release of ATP from dying cancer cells in response to platinum-based chemotherapy increases extracellular immunosuppressive adenosine (A), which binds to and activates the A2a and A2b receptors on immune cells resulting in an ineffective anti-tumor immune response. Concomitant adenosine receptor blockade may therefore enhance the therapeutic efficacy of some chemotherapeutic agents. AB928, the first clinical-stage small molecule dual adenosine receptor antagonist, is highly potent, pharmacodynamically active, and has been well tolerated in dose escalation studies as a single agent or in combination with chemo/immunotherapy. Methods: ARC-3 (NCT03720678) is a Phase 1/1b, open-label study in participants (pts) with advanced CRC. Phase 1 escalation identified AB928 150 mg orally once daily as the recommended dose in combination with standard mFOLFOX-6. Phase 1b expansion is ongoing and includes at least 15 and up to 40 pts. Eligible pts must have unresectable or mCRC, ECOG performance status 0-1, and at least one RECIST measurable lesion. Phase 1 eligibility included up to 5 lines of prior therapy; Phase 1b is similarly scoped. Exploratory biomarker analyses include immunohistochemistry of the adenosine axis, tumoral next gene sequencing, and tumor/blood immune correlates. Results: As of 27Dec19, 21 pts received AB928 150 mg + mFOLFOX-6: 7 in Phase 1 and 14 in Phase 1b. All previously treated pts (n=12) were FOLFOX- and/or FOLFIRI-experienced. Prior metastatic therapies range from 3 to 5 in Phase 1 escalation and 0 to 3 in Phase 1b expansion. Adverse events (AEs) reported in >30% of pts included fatigue, diarrhea, and thrombocytopenia. AEs related to AB928 occurred in 13 pts and were mostly mild to moderate. AB928-related Grade 3 AEs reported by 3 pts were diarrhea, AST increase, and neutropenia; there were no Grade 4-5 AB928-related AEs. Out of 15 evaluable pts, by investigator assessment, the disease control rate was 100% with 2 partial responses (13%; 1 confirmed, 1 pending confirmation) and 13 stable disease (87%). Of pts with stable disease, 6/13 (46%) had tumor shrinkage >15%. Median time on treatment was 15.4 (range: 1.7 - 40.6+) and 11.9 (range: 2.7 - 15.7+) weeks for Phase 1 and Phase 1b, respectively, with initiation of Phase 1b dosing on 09Sep19. Enrollment up to 40 pts is proceeding based on early efficacy gates; 15 pts are currently receiving study treatment. Conclusions: AB928 with mFOLFOX-6 has been well tolerated without significant evidence of additive toxicity in pts with mCRC. Combination treatment was associated with disease control in all evaluable pts, including those with microsatellite stable and RAS/BRAF mutated mCRC. Additional updates on the safety, clinical activity, and correlative biomarker results for all escalation/expansion pts will be presented. Citation Format: Michael Cecchini, Manuel Modiano, Fadi Braiteh, Olivia S. Gardner, Houston N. Gilbert, Daniel DiRenzo, Lisa Seitz, Matt J. Walters, Fangfang Yin, Rachel Woloski, Melissa C. Paoloni, Ki Y. Chung. Efficacy and safety of AB928 plus modified FOLFOX-6 (mFOLFOX-6) in participants with metastatic colorectal cancer (mCRC): Initial results at the recommended dose for expansion (ARC-3) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-387.

Rationale: Genetic variation at the chromosome 9p21 cardiovascular risk locus has been associated with peripheral artery disease, but its mechanism remains unknown. Objective: To determine whether this association is secondary to an increase in atherosclerosis, or it is the result of a separate angiogenesis-related mechanism. Methods and Results: Quantitative evaluation of human vascular samples revealed that carriers of the 9p21 risk allele possess a significantly higher burden of immature intraplaque microvessels than carriers of the ancestral allele, irrespective of lesion size or patient comorbidity. To determine whether aberrant angiogenesis also occurs under nonatherosclerotic conditions, we performed femoral artery ligation surgery in mice lacking the 9p21 candidate gene, Cdkn2b . These animals developed advanced hindlimb ischemia and digital autoamputation, secondary to a defect in the capacity of the Cdkn2b -deficient smooth muscle cell to support the developing neovessel. Microarray studies identified impaired transforming growth factor β ( TGF β) signaling in cultured cyclin-dependent kinase inhibitor 2B ( CDKN2B )–deficient cells, as well as TGF β 1 upregulation in the vasculature of 9p21 risk allele carriers. Molecular signaling studies indicated that loss of CDKN2B impairs the expression of the inhibitory factor, SMAD-7 , which promotes downstream TGF β activation. Ultimately, this manifests in the upregulation of a poorly studied effector molecule, TGF β 1-induced-1 , which is a TGF β-rheostat known to have antagonistic effects on the endothelial cell and smooth muscle cell. Dual knockdown studies confirmed the reversibility of the proposed mechanism, in vitro. Conclusions: These results suggest that loss of CDKN2B may not only promote cardiovascular disease through the development of atherosclerosis but may also impair TGF β signaling and hypoxic neovessel maturation.

To date, there is no periadventitial drug delivery method available in the clinic to prevent restenotic failure of open vascular reconstructions. Resveratrol is a promising anti-restenotic natural drug but subject to low bioavailability when systemically administered. In order to reconcile these two prominent issues, we tested effects of periadventitial delivery of resveratrol on all three major pro-restenotic pathologies including intimal hyperplasia (IH), endothelium impairment, and vessel shrinkage. In a rat carotid injury model, periadventitial delivery of resveratrol either via Pluronic gel (2-week), or polymer sheath (3-month), effectively reduced IH without causing endothelium impairment and vessel shrinkage. In an in vitro model, primary smooth muscle cells (SMCs) were stimulated with elevated transforming growth factor (TGFβ) and its signaling protein Smad3, known contributors to IH. TGFβ/Smad3 up-regulated Kruppel-like factor (KLF5) protein, and SMC de-differentiation which was reversed by KLF5 siRNA. Furthermore, TGFβ/Smad3-stimulated KLF5 production and SMC de-differentiation were blocked by resveratrol via its inhibition of the Akt-mTOR pathway. Concordantly, resveratrol attenuated Akt phosphorylation in injured arteries. Taken together, periadventitial delivery of resveratrol produces durable inhibition of all three pro-restenotic pathologies — a rare feat among existing anti-restenotic methods. Our study suggests a potential anti-restenotic modality of resveratrol application suitable for open surgery.

Background High levels of adenosine present in the tumor microenvironment have direct immunosuppressive impact on T-cell function and activation. AB928, a selective, small-molecule dual A2aR/A2bR antagonist, potently blocks the effects of adenosine and, in combination with chemotherapy or anti-PD-1, may have a more profound effect on reactivating anti-tumor immunity. Methods Four phase I, open-label, disease-specific platform studies assessing the safety, pharmacokinetics (PK), pharmacodynamics (PD), and clinical activity of AB928 in combination with chemotherapy (chemo) or AB122. In dose escalation (3 + 3 design), AB928 (cohort 1: 75 mg, cohort 2: 150 mg, cohort 3: 200 mg orally once daily) and chemo (pegylated liposomal doxorubicin (PLD), FOLFOX or carboplatin/pemetrexed plus pembrolizumab) or AB122 (240 mg IV every 2 weeks) were evaluated in eligible pts with advanced malignancies. The recommended phase II dose of each AB928 combination will advance into tumor-specific dose-expansion cohorts. Adverse events (AEs) were graded according to NCI CTCAE 5.0 and antitumor activity assessed using RECIST v1.1. Results As of 29APR2019, 26 pts have been treated across 4 studies (cohort 1, n = 12; cohort 2, n = 12; cohort 3, n = 2) with time on treatment ranging 9-268 days. In dose escalation, AB928 combination therapy was well tolerated with 1 dose limiting toxicity (Gr 2 rash, Conclusions Early results show a favorable safety profile of AB928 combination therapy and predictable PK/PD correlation. Further evaluation of AB928 + chemotherapy and AB928 + AB122 will continue in cohorts of triple-negative breast, ovarian, colorectal, gastro-esophageal, non-small cell lung, renal cell and castration-resistant prostate cancer. Clinical trial identification NCT03719326, NCT03720678, NCT03846310, NCT03629756. Legal entity responsible for the study Arcus Biosciences, Inc. Funding Arcus Biosciences, Inc. Disclosure J. Powderly: Research grant / Funding (institution): Arcus Biosciences; Speaker Bureau / Expert testimony, Research grant / Funding (institution): BMS; Speaker Bureau / Expert testimony, Research grant / Funding (institution), Full / Part-time employment: Genentech; Speaker Bureau / Expert testimony: Merck; Leadership role, Shareholder / Stockholder / Stock options, Officer / Board of Directors: Carolina BioOncology Institute; Leadership role, Shareholder / Stockholder / Stock options, Officer / Board of Directors: BioCytics; Shareholder / Stockholder / Stock options: Iovance; Shareholder / Stockholder / Stock options: BlueBird; Shareholder / Stockholder / Stock options: Juno; Shareholder / Stockholder / Stock options: KitePharma; Research grant / Funding (institution): EMD Serono; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Incyte; Research grant / Funding (institution): FLX Biosciences; Research grant / Funding (institution): Alkermes; Research grant / Funding (institution): Tempest; Research grant / Funding (institution): Curis; Research grant / Funding (institution): Corvus; Research grant / Funding (institution): AbbVie; Research grant / Funding (institution): Sequenom. A. Spira: Research grant / Funding (institution): Arcus Biosciences; Honoraria (self), Research grant / Funding (institution): Cytomx; Honoraria (self), Research grant / Funding (institution): AstraZeneca; Honoraria (self), Research grant / Funding (institution): BMS; Honoraria (self), Research grant / Funding (institution): Roche; Honoraria (self), Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Astellas; Research grant / Funding (institution): Takeda; Research grant / Funding (institution): Arch Oncology; Research grant / Funding (institution): Lam; Research grant / Funding (institution): Tesaro; Research grant / Funding (institution): Nektar; Shareholder / Stockholder / Stock options: Lilly; Honoraria (self): Ariad; Leadership role: ASCO; Leadership role: US Oncology; Leadership role: Longevity. R. Gutierrez: Research grant / Funding (institution): Arcus Biosciences. D. DiRenzo: Shareholder / Stockholder / Stock options, Full / Part-time employment: Arcus Biosciences. A. Udyavar: Shareholder / Stockholder / Stock options, Full / Part-time employment: Arcus Biosciences. J.J. Karakunnel: Shareholder / Stockholder / Stock options, Full / Part-time employment: Arcus Biosciences; Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. A. Rieger: Shareholder / Stockholder / Stock options, Full / Part-time employment: Arcus Biosciences; Shareholder / Stockholder / Stock options: AstraZeneca. J. Colabella: Shareholder / Stockholder / Stock options, Full / Part-time employment: Arcus Biosciences. D.W. Lai: Shareholder / Stockholder / Stock options, Full / Part-time employment: Arcus Biosciences; Shareholder / Stockholder / Stock options: Primevax. P. de Souza: Research grant / Funding (institution): Arcus Biosciences.

BACKGROUND: AB154 is a humanized antibody that blocks human TIGIT (T-cell immunoreceptor with Ig and ITIM domains), an inhibitory receptor expressed on natural killer (NK) cells, CD8+ T cells, CD4+ T cells and regulatory T cells (Treg). DNAM-1 (DNAX Accessory Molecule-1) is an activating receptor found on NK cells, monocytes and a subset of T cells that competes with TIGIT for shared ligands CD155 (PVR) and CD112 (Nectin-2), expressed by cancer and antigen-presenting cells. TIGIT blockade prevents binding to its ligands and shifts the immune balance towards a more favorable DNAM-1 interaction. AB154 has the potential to promote sustained immune activation and tumor clearance, particularly in combination with other immunotherapies such as AB122 (α-PD1). METHODS: Binding affinity of AB154 was determined in CHO cells over-expressing human TIGIT and in human T cells. TIGIT blockade was quantified using a TIGIT-expressing reporter gene cell line. TIGIT and PD-1 expression in cancer patient PBMCs and dissociated tumor cells (DTCs) were assessed by flow cytometry. Gene expression of these markers were also derived from TCGA (The Cancer Genome Atlas), GTEX (Genotype-Tissue Expression Project), RNAseq, and by immunohistochemistry in various tumor types and normal tissues. A receptor occupancy (RO) assay was developed using a competing α-TIGIT antibody and validated ex vivo in whole blood leukocytes from healthy donors and cancer patients. RESULTS: AB154 binds to and blocks human TIGIT with sub-nanomolar affinity. Data assembled from TCGA identified tumor types in which expression of TIGIT is greater than PD-1, equivalent to PD-1, or less than PD-1. Expression of TIGIT and CD155 at the protein level was confirmed by IHC. Immunophenotyping performed on dissociated human tumor cells demonstrated strong correlation between TIGIT and PD-1 expression on immune cells. The intensity of TIGIT staining was lowest on conventional CD4+ T cells while its intensity in Treg and CD8+ T cells was 1.5 to 3-fold higher on average. Using flow cytometry, we profiled lymphocyte populations in peripheral whole blood and demonstrated target engagement by AB154 on T cells and NK cells in the low nanomolar range in both healthy and cancer patients (ex vivo). This receptor occupancy assay is being used to monitor target engagement in AB154 dosed patient samples in an ongoing Phase 1 dose escalation study in oncology patients. CONCLUSION: Blockade of multiple immune checkpoint proteins can confer effective and durable responses in the treatment of cancer. The data presented will provide: 1) the basis for selection of tumor types by TIGIT RNA and protein expression profiles, 2) rationale for combining AB154 with AB122 (α-PD-1) in upcoming clinical trials, 3) methodology to evaluate TIGIT receptor occupancy and 4) preliminary PK/PD data from the AB154 Phase 1 dose escalation study in oncology subjects. Citation Format: Amy E. Anderson, Daniel DiRenzo, Susan Lee, Akshata Udyavar, Kimberline Gerrick, Hema Singh, Xiaoning Zhao, Lixia Jin, Lisa Seitz, Nigel P. Walker, Matthew J. Walters, Joanne B. Tan. Characterization of AB154, a humanized anti-TIGIT antibody, for use in combination studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1557.

BACKGROUND: The tumor microenvironment (TME) contains high levels of immunosuppressive adenosine (ADO), which activates the A2aR and A2bR receptors on immune cells, leading to an ineffective anti-tumor response. Ecto-5’-nucleotidase (CD73) and tissue non-specific alkaline phosphatase (TNAP) are primarily responsible for the conversion of extracellular adenosine monophosphate (AMP) to ADO and exhibit both membrane-bound and secreted forms. We have previously shown that AB928, a dual A2aR/A2bR antagonist, rescues the immunosuppressive effects of ADO in experimental tumor models. Herein, we describe the development of assays to measure the expression and activity of adenosine-generating enzymes in human tumor samples and peripheral blood. These assays are being used to define an “adenosine fingerprint” that identifies tumor types and patients most sensitive to adenosine inhibition by AB928. METHODS: CD73 and TNAP immuno-histochemistry (IHC) and mRNA analysis were performed on sections of formalin fixed paraffin embedded (FFPE) tumor tissue. Circulating levels of CD73 were quantified with an in-house developed ELISA, and AMP-ase enzymatic activity in serum was determined using an AMP-GloTM(Promega) assay. Gene expression data were extracted from The Cancer Genome Atlas (TCGA) and expressed as a ratio of log2 counts per million per sample. RESULTS: TCGA data identified non-small cell lung (NSCLC), renal clear cell, triple-negative breast, ovarian, colorectal, and gastro-esophageal cancers as tumors that highly express the adenosine producing enzymes CD73 or TNAP. To confirm these gene expression patterns, IHC assays for both CD73 and TNAP were developed using normal and tumor human tissue. IHC for CD73 was strongest in NSCLC (54.3 +/- 11.2 µm2) and colorectal (22.5 +/- 8.1 µm2) adenocarcinomas, whereas prostate (1.0 +/- 0.3 µm2) cancer exhibited the weakest staining. In contrast, TNAP staining was strongest in ovarian cancer and NSCLC adenocarcinoma, whereas gastric and colorectal adenocarcinomas showed very little TNAP staining. Therefore, CD73 and TNAP IHC broadly recapitulate the gene expression patterns found in TCGA. A CD73-specific ELISA was developed using human cancer patient blood which established a range of circulating CD73 protein levels (2-8 ng/mL) and showed a strong correlation between plasma and serum levels (r2 = 0.94). To measure adenosine-generating enzyme activity in peripheral blood, the AMP-Glo biochemical assay was performed and showed strong concordance with the CD73 ELISA in cancer patient serum (r2 = 0.72). CONCLUSIONS: These assays provide a detailed picture of the adenosine-generating capacity in the local TME as well as the peripheral activity and levels of CD73/TNAP to better identify patients that may benefit from adenosine inhibition. Citation Format: Daniel DiRenzo, Devika Ashok, Amy E. Anderson, Akshata Udyavar, Joanne B. Tan, Irene M. Luu, Kristen Zhang, Jenna L. Jeffrey, Lisa Seitz, Manmohan R. Leleti, Stephen W. Young, Jay P. Powers, Matthew J. Walters. Methods for assessment of the “adenosine fingerprint” in clinical trials of AB928 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3168.

Background: Adenosine production mediated by CD73 and/or TNAP is a potential mechanism of immune suppression across many cancer types. We have previously shown that AB928, a dual A2aR/A2bR antagonist, in combination with anti-PD-1 or chemotherapy, rescues the immunosuppressive effects of adenosine in experimental tumor models. Oncogene-driven cancers are targeted with specific tyrosine kinase inhibitors, typically leading to the development of several resistance mechanisms that bypass the kinase signaling. These oncogene-driven cancers tend to be non-responsive to PD(L)-1 inhibition. We hypothesized that oncogenic drivers might be regulating adenosine production machinery as a mechanism to suppress and evade the immune system. Here we show that pan-RAS, BRAF and EGFR alterations drive the expression of CD73, which may contribute to suppressed anti-tumor immunity. Methods and Results: We used linear models to evaluate the ability of 299 pan-cancer consensus oncogenic drivers (Bailey et al, 2018) to predict CD73 expression independent of tumor type in PanCanAtlas TCGA dataset. We defined a given gene as either wild type or altered if it exhibited a SNV/copy number/fusion event in a given patient. Out of the 299 oncogenes, we identified 20 oncogenes that upregulated and 26 that downregulated CD73 expression (FDR < 0.05). Alterations in KRAS, BRAF and EGFR were the top 3 oncogenes upregulating CD73 expression, followed by other kinases such as RASA1, MET, RET, SMAD4 and CDK4. In KRAS/BRAF/EGFR altered tumors, CD73 expression was significantly higher compared to respective wild-type patients in all cancers combined, as well as in individual tumor types (BRCA, LUAD, LUSC, CRC, PAAD, HNSCC, UBC, CESC, ESCA, STAD, UCEC and LGG). Furthermore, the pan-cancer Ras activation classifier score (Way et.al 2018) was highly predictive of CD73 expression independent of tumor type (p-value < 2e-16). KRAS, HRAS, BRAF, EGFR as well as pan-Ras mutant cancer cell lines exhibited a significantly higher level of CD73 expression than wild-type cell lines, independent of cancer type. Conclusions: Activating mutations in KRAS and HRAS that result in deregulation of the Ras pathway serve as oncogenic drivers in a variety of cancer types such as CRC, NSCLC, gastric-esophageal, cervical, bladder and HNSCC and upregulate CD73. BRAF mutations are enriched in breast, CRC, NSCLC, and low-grade gliomas that exhibit higher CD73 expression. Finally, EGFR mutations are prominent in RCC, low-grade gliomas and NSCLC, which drive CD73 expression. Oncogene-driven cancers are largely unresponsive to PD-(L)1 inhibition and are targeted with kinase inhibitors resulting in significant but not durable clinical responses. Our data strongly suggests that CD73 inhibition with AB680 and/or A2R antagonism with AB928 might be effective in Ras pathway mutant and EGFR mutant cancers, either in TKI-naïve or relapsed settings. Citation Format: Akshata R. Udyavar, Daniel DiRenzo, Devika Ashok, Amy E. Anderson, Stephen W. Young, Matt J. Walters, Joanne Bl Tan. Altered pan-Ras pathway and activating mutations in EGFR result in elevated CD73 in multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4980.

Coronary artery disease (or coronary heart disease), is the leading cause of mortality in many of the developing as well as the developed countries of the world. Cholesterol-enriched plaques in the heart's blood vessels combined with inflammation lead to the lesion expansion, narrowing of blood vessels, reduced blood flow, and may subsequently cause lesion rupture and a heart attack. Even though several environmental risk factors have been established, such as high LDL-cholesterol, diabetes, and high blood pressure, the underlying genetic composition may substantially modify the disease risk; hence, genome composition and gene-environment interactions may be critical for disease progression. Ongoing scientific efforts have seen substantial advancements related to the fields of genetics and genomics, with the major breakthroughs yet to come. As genomics is the most rapidly advancing field in the life sciences, it is important to present a comprehensive overview of current efforts. Here, we present a summary of various genetic and genomics assays and approaches applied to coronary artery disease research.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers owing to a number of characteristics including difficulty in establishing early diagnosis and the absence of effective therapeutic regimens. A large number of genetic alterations have been ascribed to PDAC with mutations in the KRAS2 proto-oncogene thought to be an early event in the progression of disease. Recent lineage-tracing studies have shown that acinar cells expressing mutant Kras(G12D) are induced to transdifferentiate, generating duct-like cells through a process known as acinar-ductal metaplasia (ADM). ADM lesions then convert to precancerous pancreatic intraepithelial neoplasia (PanIN) that progresses to PDAC over time. Thus, understanding the earliest events involved in ADM/PanIN formation would provide much needed information on the molecular pathways that are instrumental in initiating this disease. As studying the transition of acinar cells to metaplastic ductal cells in vivo is complicated by analysis of the entire organ, an in vitro three dimensional (3D) culture system was used to model ADM outside the animal. Kras(G12D)-expressing acinar cells rapidly underwent ADM in 3D culture, forming ductal cysts that silenced acinar genes and activated duct genes, characteristics associated with in vivo ADM/PanIN lesions. Analysis of downstream KRAS signaling events established a critical importance for the Raf/MEK/ERK pathway in ADM induction. In addition, forced expression of the acinar-restricted transcription factor Mist1, which is critical to acinar cell organization, significantly attenuated Kras(G12D)-induced ADM/PanIN formation. These results suggest that maintaining MIST1 activity in Kras(G12D)-expressing acinar cells can partially mitigate the transformation activity of oncogenic KRAS. Future therapeutics that target both the MAPK pathway and Mist1 transcriptional networks may show promising efficacy in combating this deadly disease.

INTRODUCTION: Extracellular adenosine triphosphate (ATP) is efficiently hydrolyzed to adenosine by ecto-nucleotidases CD39 and CD73, which converts adenosine-monophosphate (AMP) into adenosine (ADO). ADO suppresses immune responses including those of T cells, natural killer (NK) cells, and dendritic cells (DC) through activation of A2aR and A2bR receptors. Treatment of cancer cells with platinum-based and anthracycline chemotherapy has been shown to induce immunogenic cell death (ICD), characterized by increased extracellular ATP levels, and upregulation of CD39 and CD73, leading to the enhanced generation of adenosine. METHODS: The ability of AB928 to inhibit adenosine-mediated suppression of immune cell function was assessed using human CD4 and CD8 cells. BALB/c mice were inoculated with AT-3-OVA tumors, which express the model antigen ovalbumin, and subsequently treated with chemotherapy or AB928 alone, or a combination of both. The growth rate and immune composition of the tumors were assessed (flow cytometry). RESULTS: Consistent with the ability of adenosine to suppress immune function, AB928 inhibited the ability of adenosine to suppress CD4 or CD8 T cell activation. Tumor-bearing mice that have been treated with either doxorubicin (DOX, an anthracycline) or oxaliplatin (OX) exhibit increased expression of CD39 and CD73. Established AT-3-OVA tumors treated with AB928 alone had a small but significant decrease in their growth rate; similarly, tumors treated with chemotherapy exhibited a reduction in growth rate. Consistent with the ICD hypothesis, treatment of AT-3-OVA-bearing mice with DOX or OX resulted in an increase in OVA-specific CD8 T cells in the tumor. Concurrent treatment with AB928 and chemotherapy resulted in significantly reduced tumor growth rates, when compared to chemotherapy alone. Analysis of the tumor-infiltrating cell populations showed a significant increase in OVA-specific CD8 T cells, relative to those treated with chemotherapy alone. CONCLUSIONS: Treatment of tumor-bearing mice with a combination of AB928, a dual A2aR and A2bR antagonist, and ICD-inducing chemotherapy results in increased tumor antigen-specific CD8 T cell responses and significantly reduced tumor growth. AB928 is currently undergoing clinical evaluation. Citation Format: Matt J. Walters, Dana Piovesan, Joanne Tan, Daniel DiRenzo, Fangfang Yin, Dillon Miles, Manmohan R. Leleti, Tim Park, Ferdie Soriano, Eshan Sharif, Ulrike Schindler, Jay P. Powers. Combining adenosine receptor inhibition with AB928 and chemotherapy results in greater immune activation and tumor control [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 5556.

INTRODUCTION: CD73 catalyzes the extracellular generation of adenosine (ADO) from adenosine monophosphate (AMP). ADO suppresses immune responses, including those of T cells, NK cells and dendritic cells through activation of A2aR and A2bR receptors. Exhausted T cells and NK cells express high levels of several immune checkpoint proteins, including PD-1 and TIGIT. We present here preclinical data on the ability of CD73i to reverse effector cell suppression from exposure to ADO even in the presence of ICI. METHODS: CD73i effects in a monotherapeutic setting were assessed by CD3/CD28/CD2 T cell stimulation and cytolytic assays. Combinatorial settings were assessed using mixed lymphocyte reactions (MLRs). In vivo effects of CD73i + ICI were determined using syngeneic tumor models. RESULTS: CD73 is expressed across a wide range of tumor types, including those with limited response to anti-PD-1 therapy. CD73i completely rescued AMP-mediated inhibition of T cell proliferation and effector function as well as NK cell cytolytic function. AMP abrogated the enhanced allogeneic CD4+ T cell activation and IFN-γ production mediated by blocking PD-1/PD-L1 and TIGIT, an effect that was reversed by CD73i. Mechanistically, addition of AMP in MLRs repressed expression of activation markers and immune checkpoint proteins. Thus, activation of the adenosinergic pathway may limit the efficacy of ICI. TCGA data from anti-PD-1-treated melanoma patients identified CD73 expression as a negative prognostic factor. Finally, co-administration of a CD73i with an anti-PD-1 mAb resulted in significant reduction of tumor volume associated with increases in immune cell infiltration. CONCLUSIONS: CD73 inhibition, alone or in combination with anti-PD-1 and anti-TIGIT antibodies, translates into potent enhancement of immune cell activation in a variety of studies. These data provide a rationale for CD73i + ICI combinations. Citation Format: Annette Becker, Nell Narasappa, Fangfang Yin, Kristen Zhang, Daniel DiRenzo, Timothy Park, Jaroslaw Kalisiak, Ken Lawson, Jenna Jeffrey, Jay P. Powers, Ulrike Schindler, Matthew J. Walters, Joanne B. Tan. CD73 inhibitors (CD73i) reverse the AMP/adenosine-mediated impairment of immune effector cell activation by immune checkpoint inhibitors (ICI) [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 710.

Introduction: Endovascular interventions are the first line treatment for patients with atherosclerotic disease. However, a significant number of these fail due to neointimal hyperplasia. The Wnt4/β-catenin pathway has been reported to play an important role in this process, but the mechanism underlying the up-regulation of this pathway after injury remains unclear. Our lab has previously demonstrated that following vascular injury, elevated TGF-β and its signaling protein, Smad3, exacerbate intimal hyperplasia in Smooth Muscle Cells (SMCs). Moreover, our Affymetrix array data revealed an upregulation (>3 fold) of several members of the Wnt family in response to TGF-β/Smad3 treatment of SMCs. Therefore, we hypothesize that the TGF-β/Smad3 axis is responsible for the activation of the Wnt/β-catenin signaling pathway that promotes neointimal hyperplasia. Methods and Results: To mimic the injury induced up-regulation of TGF-β/Smad3 in vitro, SMCs were infected with an adenovirus to increase Smad3 and then treated with recombinant TGF-β for 48 hrs. Real time PCR confirmed that Wnt expression increased substantially in response to TGF-β/Smad3 stimulation. Using western blotting, we found that SMCs treated with TGF-β/Smad3 had a significantly increased stabilization of β-catenin compared to SMCs treated with TGF-β alone. This effect was abolished by Niclosamide, a broad-spectrum Wnt inhibitor. Conversely, β-catenin stabilization increased after stimulation by a Wnt agonist and recombinant Wnts 2b, 4, 5a, and 9a, but not Wnt11. Importantly, we found that these Wnt ligands stimulated the expression of SMC de-differentiation markers including CXCR4, CD34, Sox18, and FGF1 to varying levels as measured by RT-PCR. Wnt4 and Wnt5a were also found to significantly promote SMC proliferation (21% and 27%, respectively, p Conclusions: These results suggest that the neointima-producing Wnt/β-catenin pathway may be activated due to elevated TGF-β/Smad3 signaling and promote SMC de-differentiation and proliferation. Thus, the cross talk of these two pathways may provide a potential therapeutic target for preventing failure of endovascular interventions.

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

Background— Three major processes, constrictive vessel remodeling, intimal hyperplasia (IH), and retarded re-endothelialization, contribute to restenosis after vascular reconstructions. Clinically used drugs inhibit IH but delay re-endothelialization and also cause constrictive remodeling. Here we have examined halofuginone, an herbal derivative, for its beneficial effects on vessel remodeling and differential inhibition of IH versus re-endothelialization. Methods and Results— Two weeks after perivascular application to balloon-injured rat common carotid arteries, halofuginone versus vehicle (n=6 animals) enlarged luminal area 2.14-fold by increasing vessel size (adaptive remodeling; 123%), reducing IH (74.3%) without inhibiting re-endothelialization. Consistent with its positive effect on vessel expansion, halofuginone reduced collagen type 1 (but not type 3) production in injured arteries as well as that from adventitial fibroblasts in vitro. In support of its differential effects on IH versus re-endothelialization, halofuginone produced greater inhibition of vascular smooth muscle cell versus endothelial cell proliferation at concentrations ≈50 nmol/L. Furthermore, halofuginone at 50 nmol/L effectively blocked Smad3 phosphorylation in smooth muscle cells, which is known to promote smooth muscle cell proliferation, migration, and IH, but halofuginone had no effect on phospho-Smad3 in endothelial cells. Conclusions— Periadventitial delivery of halofuginone dramatically increased lumen patency via adaptive remodeling and selective inhibition of IH without affecting endothelium recovery. Halofuginone is the first reported small molecule that has favorable effects on all 3 major processes involved in restenosis.

Atherosclerotic-associated diseases are the leading cause of death in the United States. Despite recent progress, interventional treatments for atherosclerosis can be complicated by restenosis resulting from neo-intimal hyperplasia. We have previously demonstrated that TGF-β and its downstream signaling protein Smad3 ∶ 1) are up-regulated following vascular injury, 2) together drive smooth muscle cell (SMC) proliferation and migration and 3) enhance the development of intimal hyperplasia. In order to determine a mechanism through which TGF-β/Smad3 promote these effects, Affymetrix gene expression arrays were performed on primary rat SMCs infected with Smad3 and stimulated with TGF-β or infected with GFP alone. More than 200 genes were differentially expressed (>2.0 fold change, p

Background & Aims Early embryogenesis involves cell fate decisions that define the body axes and establish pools of progenitor cells. Development does not stop once lineages are specified; cells continue to undergo specific maturation events, and changes in gene expression patterns lead to their unique physiological functions. Secretory pancreatic acinar cells mature postnatally to synthesize large amounts of protein, polarize, and communicate with other cells. The transcription factor MIST1 is expressed by only secretory cells and regulates maturation events. MIST1-deficient acinar cells in mice do not establish apical-basal polarity, properly position zymogen granules, or communicate with adjacent cells, disrupting pancreatic function. We investigated whether MIST1 directly induces and maintains the mature phenotype of acinar cells. Methods We analyzed the effects of Cre-mediated expression of Mist1 in adult Mist1 –deficient ( Mist1 KO ) mice. Pancreatic tissues were collected and analyzed by light and electron microscopy, immunohistochemistry, real-time polymerase chain reaction analysis, and chromatin immunoprecipitation. Primary acini were isolated from mice and analyzed in amylase secretion assays. Results Induced expression of Mist1 in adult Mist1 KO mice restored wild-type gene expression patterns in acinar cells. The acinar cells changed phenotypes, establishing apical-basal polarity, increasing the size of zymogen granules, reorganizing the cytoskeletal network, communicating intercellularly (by synthesizing gap junctions), and undergoing exocytosis. Conclusions The exocrine pancreas of adult mice can be remodeled by re-expression of the transcription factor MIST1. MIST1 regulates acinar cell maturation and might be used to repair damaged pancreata in patients with pancreatic disorders.

MIST1 is a transcription factor expressed in pancreatic acinar cells and other serous exocrine cells. Mice harboring a targeted deletion of the Mist1 gene (Mist1(-/-)) exhibit alterations in acinar regulated exocytosis and aberrant Ca(2+) signaling that are normally controlled by acinar cell Ca(2+)-ATPases. Previous studies indicated that total sarcoendoplasmic reticulum Ca(2+)-ATPases (SERCA) and plasma membrane Ca(2+)-ATPases (PMCA) remained unaffected in Mist1(-/-) acinar cultures. Therefore, we have assessed the expression of Atp2c2, the gene that encodes the secretory pathway Ca(2+)-ATPase 2 (SPCA2). We revealed a dramatic decrease in pancreatic expression of Atp2a2 mRNA and SPCA2 protein in Mist1(-/-) mice. Surprisingly, this analysis indicated that the acinar-specific Atp2c2 mRNA is a novel transcript, consisting of only the 3' end of the gene and the protein and localizes to the endoplasmic reticulum. Expression of SPCA2 was also lost in Mist1(-/-) secretory cells of the salivary glands and seminal vesicles, suggesting that Atp2c2 transcription is regulated by MIST1. Indeed, inducible MIST1 expression in Mist1(-/-) pancreatic acinar cells restored normal Atp2c2 expression, supporting a role for MIST1 in regulating the Atp2c2 gene. Based on these results, we have identified a new Atp2c2 transcript, the loss of which may be linked to the Mist1(-/-) phenotype.

We have previously shown that in the presence of elevated Smad3, transforming growth factor-β (TGF-β) transforms from an inhibitor to a stimulant of vascular smooth muscle cell (SMC) proliferation and intimal hyperplasia (IH). Here we identify a novel mechanism through which TGF-β/Smad3 also exacerbates IH by inhibiting SMC apoptosis. We found that TGF-β treatment led to inhibition of apoptosis in rat SMCs following viral expression of Smad3. Conditioned media from these cells when applied to naive SMCs recapitulated this effect, suggesting an autocrine pathway through a secreted factor. Gene array of TGF-β/Smad3-treated cells revealed enhanced expression of vascular endothelial growth factor (VEGF), a known inhibitor of endothelial cell apoptosis. We then evaluated whether VEGF is the secreted mediator responsible for TGF-β/Smad3 inhibition of SMC apoptosis. In TGF-β/Smad3-treated cells, VEGF mRNA and protein as well as VEGF secretion were increased. Moreover, recombinant VEGF-A inhibited SMC apoptosis and a VEGF-A-neutralizing antibody reversed the inhibitory effect of conditioned media on SMC apoptosis. Stimulation of SMCs with TGF-β led to the formation of a complex of Smad3 and hypoxia-inducible factor-1α (HIF-1α) that in turn activated the VEGF-A promoter and transcription. In rat carotid arteries following arterial injury, Smad3 and VEGF-A expression were upregulated. Moreover, Smad3 gene transfer further enhanced VEGF expression as well as inhibited SMC apoptosis. Finally, blocking either the VEGF receptor or Smad3 signaling in injured carotid arteries abrogated the inhibitory effect of Smad3 on vascular SMC apoptosis. Taken together, our study reveals that following angioplasty, elevation of both TGF-β and Smad3 leads to SMC secretion of VEGF-A that functions as an autocrine inhibitor of SMC apoptosis. This novel pathway provides further insights into the role of TGF-β in the development of IH.

Our studies are focused on the identification and prevention of the initial transformation events associated with pancreatic ductal adenocarcinoma (PDA) development, an extremely lethal malignancy with poor prognosis and ineffective treatments. PDA initiates through activating mutations in the KRAS proto-oncogene (KrasG12D) and recent studies from our laboratory have determined that targeted expression of an endogenous KrasG12D allele in pancreatic acinar cells is sufficient to produce PDA precursor lesions (PanINs). These results suggest that PDA arises via acinar cells undergoing a process of acinar-to-ductal metaplasia (ADM) which is characterized by a loss of acinar cell properties with the acquisition of duct-like characteristics. Several studies indicate that the process of ADM requires inactivation of the acinar-specific transcription factor Mist1, since Mist1 null (Mist1KO) mice display accelerated ADM and PanIN formation upon KrasG12D expression. Additionally, acinar cell polarity is disrupted in Mist1KO mice with diffuse zymogen granule organization and mis-localization of nuclei. These results suggest that Mist1 has a key role in suppressing PDA formation by maintaining the acinar cell differentiation program. To identify the downstream genes affected by the absence of Mist1, we have generated an inducible Mist1 transgenic mouse line (LSL-Mist1myc) in which constitutive Mist1 expression is activated through Cre-mediated recombination. When LSL-Mist1myc mice are crossed to Mist1Cre-ER mice, Mist1myc expression can be induced in acinar cells upon Tamoxifen (TM) addition. Analysis of Mist1Cre-ER/Cre-ER; LSL-Mist1myc mice confirmed that exogenous Mist1myc expression is detected as early as 6 hours post-TM. Time course studies in a Mist1KO background revealed that nuclear and zymogen granule organization can be restored within 24 hours of Cre activation. We conclude that Mist1 actively controls pancreatic acinar cell organization and that induction of Mist1 in Mist1KO mice leads to complete reorganization of pancreatic tissues. Our current efforts are focused on establishing how Mist1 negatively regulates ADM and PanIN formation and to identify Mist1 targets that negatively regulate Kras-induced pancreas metaplasia and transformation. Citation Information: Cancer Res 2009;69(23 Suppl):A76.

Significance: Pancreatic ductal adenocarcinoma (PDA) is one of the most fatal malignancies, partially due to the lack of early detection methods. Background: Efforts to understand the early stages of PDA progression have led to identification of Kras mutations as the initiating event and pancreatic intraepithelial neoplasias (PanINs) as one of the cellular precursors of PDA. Recently, several labs have demonstrated that PanINs can be generated from pancreatic acinar cells upon expression of Kras mutants in mouse models. This dramatic shift from differentiated acinar cells to duct-like PanIN cells, generally termed acinar-ductal metaplasia (ADM), is accelerated by loss of Mist1, an acinar-restricted basic helix-loop-helix transcription factor. Despite this recent progress, the transcription and signaling networks that regulate ADM remain largely unknown. Results: To study these networks, we employed a collagen-based 3-dimensional (3D) culture model of ADM, where primary acinar cells can be transformed into duct-like cysts when treated with transforming growth factor-alpha (TGF-α). A similar acinar-to-ductal conversion was observed with primary acinar cells that express the KrasG12D mutant. Initial characterization of the ADM process revealed that the MAPK pathway is required in both settings. However, EGFR activity is required for TGF-α-induced conversion but not for KrasG12D-induced conversion. Consistent with previous data generated from mouse models, loss of Mist1 accelerates KrasG12D-induced conversion to ductal cysts in vitro. This accelerated ADM formation can be reversed by inhibition of EGFR, without affecting the basal level conversion induced by KrasG12D, suggesting that loss of Mist1 promotes transformation through an EGFR-dependent pathway. Further investigation has revealed that Mist1 directly activates transcription of the epidermal growth factor (EGF) gene, which encodes a classic ligand of EGFR. Conclusion: These data suggest that EGFR has a complex role in this ADM model. EGFR activity either promotes maintenance of acinar differentiation, or cooperates with KrasG12D-induced transformation, possibly depending on the functional specificity of different ligands. Citation Information: Cancer Res 2009;69(23 Suppl):A71.