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BackgroundPancreatic ductal adenocarcinoma (PDAC) is resistant to many available therapies including immunotherapy because of its highly complex tumor microenvironment (TME). PDAC TME consists of a significant proportion of stromal cells, such as endothelial cells, perivascular cells, and cancer-associated fibroblasts (CAFs). Recent work indicates how CAFs can orchestrate the crosstalk cancer and immune cells, and contribute to many aspects of tumor progression, including angiogenesis, senescence, and inflammation. Recent studies based on scRNA-seq have increased understanding of CAF heterogeneity in PDAC in both human and genetically engineered mouse models (GEMMs) is of high interest. To understand the translatability of GEMMs in the setting of PDAC, we conducted a thorough scRNA-seq meta-analysis on CAFs across GEMMs and PDAC human samples. Hereafter, we characterized CAFs multi-dimensionally based on transcriptional, chromatin accessibility, and spatial profiles. Finally, we suggested certain transcription factors may be regulatory drivers of heterogeneous CAF phenotypes in both human and GEMMs.MethodsWe collected publicly available and internally generated scRNA-seq data of PDAC CAFs from human and mouse. After dataset alignment and label transfer, we conducted differential expression analysis across CAF subsets to characterize myofibroblasts (myCAFs) and other CAF subsets of interest. Bioinformatically, we further interrogated CAF heterogeneity in terms of regulatory potential of transcription factors, gene set enrichment, and functional state transition. Complemented by epigenomic assessment, we investigated chromatin accessibility and transcription factor binding availability on the single-cell level. Finally, to investigate the TME organization and spatial neighborhood of cell-to-cell interaction, we explored potential functional differences across location and transcriptional changes of CAF subsets by spatial transcriptomics.ResultsWe found that myofibroblasts (myCAFs) make up a substantial proportion of the CAF population, in both human and mouse TME. In a combination of transcriptional profiling, chromatin accessibility assessment, and spatial transcriptomics, we elucidated potential functional and phenotypic differences within myCAF population and compared to other CAF subsets in the TME. While myofibroblasts are traditionally described as matrix remodeling related, heterogeneity in myofibroblasts may suggest additional roles played by this specific subset. In addition, CAFs in human and mouse share similarities, in terms of transcriptional profiles and phenotypes. The use of GEMMs facilitates our understanding of CAF heterogenous behavior and phenotypes in the PDAC TME.ConclusionsHere, we presented a comprehensive overview of CAF heterogeneity in mouse PDAC models and human datasets. Our observations highlight molecular differences in CAFs, which facilitates our understanding on PDAC stromal microenvironment and translatability in GEMMs in imitating human TME.

KRAS is most frequently mutated in pancreatic adenocarcinomas (PDAC) with more than 90% of cases having a KRAS mutation. This leads to constitutive activation of the downstream signaling pathways, driving tumor cell proliferation whilst co-opting the tumor microenvironment (TME) to promote tumorigenesis and immune escape. The mutant specific KRASG12C inhibitor (AMG510) has been shown to suppress tumor growth and reconfigure the immune TME. However, covalent binding KRASG12C inhibitors have no efficacy on other KRAS alleles, such as G12D and V, the majority of KRAS mutations in PDAC (37% and 28%, respectively). Here, we use a SOS1 inhibitor (BI 1701963) in combination with a MEK inhibitor, blocking two non-redundant steps in the KRAS pathway, on a library of congenic tumor clones derived from an autochthonous mouse model of PDAC deemed the KPCY (Pdx-1-Cre, KRASG12D, p53-/-, YFP+). These tumor lines can generate both “hot” and “cold” TMEs regarding T cell infiltration. We observed that combined SOS1 and MEK inhibition suppressed tumor growth of all KPCY lines irrespective of their cold or hot TME status. In some KPCY lines, CD8 T-cells significantly increased in the TME following combination treatment. We found that the CD8 T-cell increase is restricted to the individual tumor site and resulted from both recruitment and intra-tumoral division. This was accompanied by a decrease of tumor associated macrophages and myeloid-derived suppressor cells. Single-cell RNAseq profiling of the TME was performed on three KPCY lines with distinct immune response to the treatment to characterize cellular composition change induced by the SOS1 and MEK inhibitor combination. To take advantage of the treatment-induced immune TME reconfiguration, we examined combination immune-oncology (IO) targeting T cells and antigen-presenting cells and found that the combination of SOS1i and MEKi with PD-1/CTLA4/CD40 enhanced and prolonged tumor growth inhibition mediated by anti-tumor immunity. Collectively, these results reveal that the combination of SOS1 and MEK inhibitors can be used to convert cold to hot tumors and highlights how we can rationalize combination of cancer-targeted therapy and IO therapy to prolong anti-tumor effects. Citation Format: Robert J Norgard, Pratha Budhani, Sarah O'Brian, Jessica Potts, Brianna Flynn, Xavier Salce, Joshua Tagore, Lucinda Thiede, Joseph Seco, Suzanne Segal, Mark Love, Ania Mikucka, Charlie Cote, Ruby Wasti, Mohanapriya Kamalakannan, Astrid Jeschko, Gabriela Melo-Zainzinger, Angela Mcnabola, Varenka Rodriguez Rodriguez DiBlasi, Joseph Tumang, Ulrike Tontsch-Grunt, Melanie Hinkel, Jeanine Pignatelli, Francesca Trapani, Sandra Martinez-Morilla, Kaja Kostyrko, Marco H Hofmann, Abhishek Kashyap, Emilio Flano, Kang Liu. Combination of SOS1::KRAS inhibitor with a MEK inhibitor reconfigures the immune tumor microenvironment of KRASG12D pancreatic ductal adenocarcinomas and sensitizes to immunotherapy [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B23.

To expand, analyze and extend published behavioral phenotypes relevant to autism spectrum disorder (ASD), we present a study of three ASD genetic mouse models: Feng's Shank3tm2Gfng model, hereafter Shank3/F, Jiang's Shank3tm1Yhj model, hereafter Shank3/J and the Cacna1c deletion model. The Shank3 models mimick gene mutations associated with Phelan-McDermid Syndrome and the Cacna1c model recapitulates the deletion underlying Timothy syndrome. This study utilizes both standard and novel behavioral tests with the same methodology used in our previously published companion report on the Cntnap2 null and 16p11.2 deletion models. We found that some but not all behaviors replicated published findings and those that did replicate, such as social behavior and overgrooming in Shank3 models, tended to be milder than reported elsewhere. The Shank3/F model, and to a much lesser extent, the Shank3/J and Cacna1c models, showed hypoactivity and a general anxiety-like behavior triggered by external stimuli which pervaded social interactions. We did not detect deficits in a cognitive procedural learning test nor did we observe perseverative behavior in these models. We did, however, find differences in exploratory patterns of Cacna1c mutant mice suggestive of a behavioral effect in a social setting. In addition, only Shank3/F showed differences in sensory-gating. Both positive and negative results from this study will be useful in identifying the most robust and replicable behavioral signatures within and across mouse models of autism. Understanding these phenotypes may shed light of which features to study when screening compounds for potential therapeutic interventions.

We report the synthesis and characterization of a series of enantiopure 5-cyclopropane-bearing pyridyldiazabicyclo[3.3.0]octanes that display low nanomolar binding affinities and act as functional agonists at α4β2-nicotinic acetylcholine receptor (nAChR) subtype. Structure-activity relationship studies revealed that incorporation of a cyclopropane-containing side chain at the 5-position of the pyridine ring provides ligands with improved subtype selectivity for nAChR β2 subunit-containing nAChR subtypes (β2*-nAChRs) over β4*-nAChRs compared to the parent compound 4. Compound 15 exhibited subnanomolar binding affinity for α4β2- and α4β2*-nAChRs with negligible interaction. Functional assays confirm selectivity for α4β2-nAChRs. Furthermore, using the SmartCube assay system, this ligand showed antidepressant, anxiolytic, and antipsychotic features, while mouse forced-swim assay further confirm the antidepressant-like property of 15.

To expand, analyze and extend published behavioral phenotypes relevant to autism spectrum disorder (ASD), we present a study of three ASD genetic mouse models: Feng’s Shank3tm2Gfng model, hereafter Shank3/F, Jiang’s Shank3tm1Yhj model, hereafter Shank3/J, and the Cacna1c deletion model. The Shank3/F and Shank3/J models mimick gene mutations associated with Phelan-Mcdermid syndrome and the Cacna1c model recapitulates the deletion underlying Timothy syndrome. The current study utilizes both standard and novel, computer-vision based behavioral tests, the same methdology used in our previously published companion report on the Cntnap2 null and 16p11.2 deletion models. Overall, some but not all behaviors replicated published findings. Those that replicated, such as social behavior and overgrooming in Shank3 models, also tended to be milder than previous reports. The Shank3/F model, and to a much lesser extent, the Shank3/J and Cacna1c models, showed hypoactivity and a general anxiety-like behavior triggered by external stimuli which pervaded social interactions. We did not detect deficits in a cognitive procedural learning test nor did we observe perseverative behavior in these models. We did, however, find differences in exploratory patterns of Cacna1c mutant mice suggestive of a behavioral effect in a social setting. In addition, Shank3/F but not Shank3/J KO or Cacna1c HET showed differences in sensory-gating. Discrepancies in our current results from previous reports may be dependent on subtle differences in testing conditions, housing enrichment, or background strain. Both positive and negative results from this study will be useful in identifying the most robust and replicable behavioral signatures within and across mouse models of autism. Understanding these phenotypes may shed light of which features to study when screening compounds for potential therapeutic interventions.

New cholecystokinin-1 receptor (CCK1R) agonist 'triggers' were identified using iterative library synthesis. Structural activity relationship studies led to the discovery of compound 10e, a potent CCK1R agonist that demonstrated robust weight loss in a diet-induced obese rat model with very low systemic exposure. Pharmacokinetic data suggest that efficacy is primarily driven through activation of CCK1R's located within the intestinal wall.

Autism spectrum disorder comprises several neurodevelopmental conditions presenting symptoms in social communication and restricted, repetitive behaviors. A major roadblock for drug development for autism is the lack of robust behavioral signatures predictive of clinical efficacy. To address this issue, we further characterized, in a uniform and rigorous way, mouse models of autism that are of interest because of their construct validity and wide availability to the scientific community. We implemented a broad behavioral battery that included but was not restricted to core autism domains, with the goal of identifying robust, reliable phenotypes amenable for further testing. Here we describe comprehensive findings from two known mouse models of autism, obtained at different developmental stages, using a systematic behavioral test battery combining standard tests as well as novel, quantitative, computer-vision based systems. The first mouse model recapitulates a deletion in human chromosome 16p11.2, found in 1% of individuals with autism. The second mouse model harbors homozygous null mutations in Cntnap2, associated with autism and Pitt-Hopkins-like syndrome. Consistent with previous results, 16p11.2 heterozygous null mice, also known as Del(7Slx1b-Sept1)4Aam weighed less than wild type littermates displayed hyperactivity and no social deficits. Cntnap2 homozygous null mice were also hyperactive, froze less during testing, showed a mild gait phenotype and deficits in the three-chamber social preference test, although less robust than previously published. In the open field test with exposure to urine of an estrous female, however, the Cntnap2 null mice showed reduced vocalizations. In addition, Cntnap2 null mice performed slightly better in a cognitive procedural learning test. Although finding and replicating robust behavioral phenotypes in animal models is a challenging task, such functional readouts remain important in the development of therapeutics and we anticipate both our positive and negative findings will be utilized as a resource for the broader scientific community.

Modifying the intestinal microbiome has been shown to influence behavior and brain development in selected rodent models. This study assessed the impact of consuming dietary prebiotics polydextrose...

Background Cannabinoid 1 (CB1) receptor antagonists exhibit pharmacological properties favorable for the treatment of obesity and other related metabolic disorders. CE-178253 (1-[7-(2-Chlorophenyl)-8-(4-chlorophenyl)-2-methylpyrazolo[1,5-a]-[1,3,5]triazin-4-yl]-3-ethylaminoazetidine-3-carboxylic acid hydrochloride) is a recently discovered selective centrally-acting CB1 receptor antagonist. Despite a large body of knowledge on cannabinoid receptor antagonists little data exist on the quantitative pharmacology of this therapeutic class of drugs. The purpose of the current studies was to evaluate the quantitative pharmacology and concentration/effect relationships of CE-178253 based on unbound plasma concentration and in vitro pharmacology data in different in vivo preclinical models of FI and energy expenditure. Results In vitro, CE-178253 exhibits sub-nanomolar potency at human CB1 receptors in both binding (Ki = 0.33 nM) and functional assays (Ki = 0.07 nM). CE-178253 has low affinity (Ki > 10,000 nM) for human CB2 receptors. In vivo, CE-178253 exhibits concentration-dependent anorectic activity in both fast-induced re-feeding and spontaneous nocturnal feeding FI models. As measured by indirect calorimetry, CE-178253 acutely stimulates energy expenditure by greater than 30% in rats and shifts substrate oxidation from carbohydrate to fat as indicated by a decrease the respiratory quotient from 0.85 to 0.75. Determination of the concentration-effect relationships and ex vivo receptor occupancy in efficacy models of energy intake and expenditure suggest that a greater than a 2-fold coverage of the Ki (50-75% receptor occupancy) is required for maximum efficacy. Finally, in two preclinical models of obesity, CE-178253 dose-dependently promotes weight loss in diet-induced obese rats and mice. Conclusions We have combined quantitative pharmacology and ex vivo CB1 receptor occupancy data to assess concentration/effect relationships in food intake, energy expenditure and weight loss studies. Quantitative pharmacology studies provide a strong a foundation for establishing and improving confidence in mechanism as well as aiding in the progression of compounds from preclinical pharmacology to clinical development.