MDM2 and MDMX function as key regulators of p53 by binding to its N terminus, inhibiting its transcriptional activity, and promoting degradation. MDM2 and MDMX overexpression or hyperactivation directly contributes to the loss of p53 function during the development of nearly 50% of human cancers. Recent studies showed that disrupting p53-MDM2 and p53-MDMX interactions can lead to robust activation of p53 but also revealed a need to develop novel dual specific or MDMXspecific inhibitors. Using phage display we identified a 12-residue peptide (pDI) with inhibitory activity against MDM2 and MDMX. The co-crystal structures of the pDI and a single mutant derivative (pDI6W) liganded with the N-terminal domains of human MDMX and MDM2 served as the basis for the design of 11 distinct pDI-derivative peptides that were tested for inhibitory potential. The best derivative (termed pDIQ) contained four amino acid substitutions and exhibited a 5-fold increase in potency over the parent peptide against both MDM2 (IC 50 ‫؍‬ 8 nM) and MDMX (IC 50 ‫؍‬ 110 nM). Further structural studies revealed key molecular features enabling the high affinity binding of the pDIQ to these proteins. These include large conformational changes of the pDIQ to reach into a hydrophobic site unique to MDMX. The findings suggest new strategies toward the rational design of small molecule inhibitors efficiently targeting MDMX.The p53 tumor suppressor is a potent inducer of cell cycle arrest, apoptosis, cellular senescence, and innate immunity. It is activated in response to oncogenic transformation, extrinsic stress, and viral infection to protect higher organisms from cancer (1-3). p53 also facilitates maternal reproduction through induction of the growth factor leukemia inhibitory factor (LIF) that promotes embryo implantation (4). p53 activity is kept at minimal levels in unstressed cells by interactions with MDM2 and MDMX. MDM2 is an ubiquitin E3 ligase for p53 and an important regulator of p53 stability by forming a negative feedback loop (5, 6). The MDM2 homolog MDMX also binds to p53 and inhibits p53-dependent transcription (7). Loss of MDM2 or MDMX leads to embryonic lethality (8 -10). Therefore, the expression of MDM2 and the expression of MDMX are both necessary for regulation of p53 during normal development.Genetic or functional inactivation of p53 is an obligatory step during cancer development. In human tumors that retain wild type p53, amplification of MDM2 or MDMX serves as an alternative mechanism of p53 inactivation in a subset of tumors (11,12). Furthermore, MDM2 activity is controlled by the tumor suppressor ARF (alternative reading frame) encoded by the INK4a locus (2). Deletion/epigenetic silencing of ARF occur in most tumors expressing wild type p53, resulting in hyperactive MDM2 and lack of p53 response to oncogenic stress in malignant tumors (13,14). ARF has also been shown to promote MDMX degradation by MDM2 (15). Loss of ARF expression may result in MDMX stabilization that further inactivates p53. Therefore, MDM2 and MDMX are d...

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BackgroundIntraductal papillary mucinous neoplasms (IPMNs) are pancreatic ductal adenocarcinoma (PDAC) precursors. Differentiating between high-risk IPMNs that warrant surgical resection and low-risk IPMNs that can be monitored is a significant clinical problem, and we sought to discover a panel of mi(cro)RNAs that accurately classify IPMN risk status.Methodology/Principal FindingsIn a discovery phase, genome-wide miRNA expression profiling was performed on 28 surgically-resected, pathologically-confirmed IPMNs (19 high-risk, 9 low-risk) using Taqman MicroRNA Arrays. A validation phase was performed in 21 independent IPMNs (13 high-risk, 8 low-risk). We also explored associations between miRNA expression level and various clinical and pathological factors and examined genes and pathways regulated by the identified miRNAs by integrating data from bioinformatic analyses and microarray analysis of miRNA gene targets. Six miRNAs (miR-100, miR-99b, miR-99a, miR-342-3p, miR-126, miR-130a) were down-regulated in high-risk versus low-risk IPMNs and distinguished between groups (P<10−3, area underneath the curve (AUC) = 87%). The same trend was observed in the validation phase (AUC = 74%). Low miR-99b expression was associated with main pancreatic duct involvement (P = 0.021), and serum albumin levels were positively correlated with miR-99a (r = 0.52, P = 0.004) and miR-100 expression (r = 0.49, P = 0.008). Literature, validated miRNA:target gene interactions, and pathway enrichment analysis supported the candidate miRNAs as tumor suppressors and regulators of PDAC development. Microarray analysis revealed that oncogenic targets of miR-130a (ATG2B, MEOX2), miR-342-3p (DNMT1), and miR-126 (IRS-1) were up-regulated in high- versus low-risk IPMNs (P<0.10).ConclusionsThis pilot study highlights miRNAs that may aid in preoperative risk stratification of IPMNs and provides novel insights into miRNA-mediated progression to pancreatic malignancy. The miRNAs identified here and in other recent investigations warrant evaluation in biofluids in a well-powered prospective cohort of individuals newly-diagnosed with IPMNs and other pancreatic cysts and those at increased genetic risk for these lesions.

In the field of pathology it is clear that molecular genomics and digital imaging represent two promising future directions, and both are as relevant to the tumor microenvironment as they are to the tumor itself (Beck AH et al. Sci Transl Med 3(108):108ra113-08ra113, 2011). Digital imaging, or whole slide imaging (WSI), of glass histology slides facilitates a number of value-added competencies which were not previously possible with the traditional analog review of these slides under a microscope by a pathologist. As an important tool for investigational research, digital pathology can leverage the quantification and reproducibility offered by image analysis to add value to the pathology field. This chapter will focus on the application of image analysis to investigate the tumor microenvironment and how quantitative investigation can provide deeper insight into our understanding of the tumor to tumor microenvironment relationship.

BackgroundT cell immunoglobulin and mucin domain containing−3 (TIM-3) blocking antibodies are currently being evaluated in clinical trials for solid and hematological malignancies. Despite its identification on T cells, TIM-3 is predominantly expressed by myeloid cells, including XCR1+ type I conventional dendritic cells (cDC1s). We have recently shown that TIM-3 blockade promotes expression of CXCR3 chemokine ligands by tumor cDCs, but how this drives a CD8+ T cell-dependent response to therapy is unclear.MethodsT cell infiltration, effector function, and spatial localization in relation to XCR1+ cDC1s were evaluated in a murine orthotopic mammary carcinoma model during response to TIM-3 blockade and paclitaxel chemotherapy. Mixed bone marrow chimeras and diphtheria toxin depletion were used to determine the role of specific genes in cDC1s during therapeutic responses.ResultsTIM-3 blockade increased interferon-γ expression by CD8+ T cells without altering immune infiltration. cDC1 expression of CXCL9, but not CXCL10, was required for response to TIM-3 blockade. CXCL9 was also necessary for the increased proximity observed between CD8+ T cells and XCR1+ cDC1s during therapy. Tumor responses were dependent on cDC1 expression of interleukin-12, but not MHCI.ConclusionsTIM-3 blockade increases exposure of intratumoral CD8+ T cells to cDC1-derived cytokines, with implications for the design of therapeutic strategies using antibodies against TIM-3.

This study proposes lipid nanocapsules (LNCs)-based Trojan particles made by entrapping 59±3 nm LNCs in carbohydrate-based microparticles via spray drying. This work focused on optimisation of maintenance of the nanocarrier colloidal properties, solid state properties and suitability of Trojan particles for pulmonary deposition. The properties of Trojan particles were evaluated as a function of the nature of carbohydrate, the feed concentration and LNC loading. The presence of nanocapsules had a significant impact on the size and morphology of microparticles and their solid state properties. Spray drying did not destroy the LNCs and their size after reconstitution varied between 78±1 and 121±1 nm. The lactose, trehalose and raffinose-based Trojan particles were readily dispersed as aerosols with mass median aerodynamic diameters between 5.3±0.1 and 6.2 ±0.1 µm using a dry powder inhaler. In conclusion, LNC-Trojan particles were shown to successfully not only to encapsulate the lipid nanocapsules but also to release them.

Background: Intraductal papillary mucinous neoplasms (IPMNs) are cystic pancreatic cancer (PC) precursors that are increasingly being detected incidentally by cross-sectional imaging. IPMNs harbor potential for invasive malignancy depending on the degree of histologic dysplasia which ranges from low to high grade, yet the only way to determine disease severity is through surgical resection and histopathologic investigation. Mi(cro)RNAs, non-coding RNAs that regulate one-third of protein coding genes, are attractive candidate biomarkers of early pancreatic malignancy because they are detectable in tissue and blood in a stable form, making them amenable to reliable measurement, and they have been implicated in the development and progression of PC. The goal of our pilot project was to discover and characterize a miRNA signature that accurately differentiates between low-grade IPMNs that merit continued surveillance and high-grade IPMNs that warrant immediate surgical resection. Methods: In our discovery phase, we microdissected formalin-fixed paraffin-embedded tissue (FFPE) and isolated total RNA from 28 pathologically-confirmed IPMNs (9 low-grade and 19 high-grade) surgically resected at our institution between 1999 and 2011, and evaluated the expression of 378 mature miRNAs using high-throughput Taqman Low Density Arrays. After normalization using the endogenous control RNU44, a rank-sum test was performed to compare the expression between the two groups for each miRNA. Results: Using a false discovery rate of 10%, there were 13 differentially expressed miRNAs with statistical significance. The top candidates include miR-100 (P=1.6 x10−3), miR-99b and miR-99a (P=2.7 x10−3), miR-342-3p and miR-126 (P = 3.7 x10−3), and miR-130a (P = 3.7 x10−3). Several of these miRNAs were highlighted in a recent investigation of FFPE tissue and cyst fluid from patients with IPMNs and other pancreatic cysts, demonstrating consistency of findings. Furthermore, the expression level of the top candidate miRNAs was down-regulated in high-grade compared to low-grade IPMN tissue, in line with recent data supporting a role for these miRNAs as tumor suppressors and regulators of key genes that contribute to cell proliferation and invasion in pancreatic and other malignancies. Analysis is underway to evaluate the expression of the top candidate miRNAs in an independent set of high- and low-grade IPMN tissue specimens, and to correlate miRNA expression with other possible clinical predictors of malignant potential. Conclusions: Although preliminary, our findings suggest that miRNAs may serve as a diagnostic adjunct for stratifying patients with IPMNs for continued surveillance or surgical resection. Citation Format: Jennifer Permuth Wey, Susan McCarthy, Y. Ann Chen, Kate Fisher, Agnieszka Kasprzak, Mark Lloyd, Xiaotao Qu, Timothy Yeatman, Jason Klapman, Domenico Coppola, Mokenge Malafa. Tackling a clinical challenge: Using microRNAs to differentiate between low-and high-risk Pancreatic Cysts . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-70. doi:10.1158/1538-7445.AM2013-LB-70

Progression from a ductal carcinoma in situ (DCIS) to an invasive tumor is a major step initiating a devastating and often lethal metastatic cascade. One sentinel event that initiate this process is the development of ductal microinvasions, i.e., small cohorts of tumor cells that breach the basement membrane surrounding the duct, and migrate through the extracellular matrix (ECM). This process is difficult to reproduce experimentally and investigate in a fully controlled way. However, mathematical modeling based on physical principles and medical imaging can shed light on interactions between tumor cells and the surrounding stroma during the spread of microinvasions. We used a combination of advanced image analysis techniques applied to patients' histology data, that include both tissue regions and single cells segmentation, with multi-factorial classification to extract feature data which identifies specific properties of individual tumor cells inside the duct and on the invasive front. These histology-based quantitative data were then used to calibrate a hybrid agent-based mathematical model of DCIS-ECM interactions that takes into account both individual tumor cells and the underlying ECM fibril structure. The model was employed in a computational simulation study to delineate physical properties of the matrix fibers that can facilitate and/or prevent formation and progression of ductal microinvasions. Our integrated approach incorporating medical imaging and computational modeling, allowed us to predict the dynamics of emerging ductal microinvasions that are consistent with the observed clinical data. We showed how changes in the local microenvironmental niche near the DCIS edge leads to initiation of ductal microinvasions. We identified physical properties of the matrix that can facilitate or prevent the progression of such microinvasions. Our methods also provide a tool for quantifying morphological and immunohistochemical properties of individual cells within the mammary ducts and ductal microinvasion, as well as a tool for testing biomechanical hypotheses of tumor cell-tumor matrix interactions. These findings can be directly compared to the patient histology samples and can lead to development of new prognostic methods and therapeutic interventions by targeting the tumor preinvasive niche. Citation Format: Katarzyna A. Rejniak, Mark C. Lloyd, Agnieszka Kasprzak, Marilyn Bui. Tumor-matrix tnteractions in early ductal invasions: Integrating histology imaging with computational modeling. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A32.