Purpose: There are several agents in early clinical trials targeting components of the adenosine pathway including A2AR and CD73. The identification of cancers with a significant adenosine drive is critical to understand the potential for these molecules. However, it is challenging to measure tumor adenosine levels at scale, thus novel, clinically tractable biomarkers are needed.Experimental Design: We generated a gene expression signature for the adenosine signaling using regulatory networks derived from the literature and validated this in patients. We applied the signature to large cohorts of disease from The Cancer Genome Atlas (TCGA) and cohorts of immune checkpoint inhibitor-treated patients.Results: The signature captures baseline adenosine levels in vivo (r 2 ¼ 0.92, P ¼ 0.018), is reduced after small-molecule inhibition of A2AR in mice (r 2 ¼ À0.62, P ¼ 0.001) and humans (reduction in 5 of 7 patients, 70%), and is abrogated after A2AR knockout. Analysis of TCGA confirms a negative association between adenosine and overall survival (OS, HR ¼ 0.6, P < 2.2e -16 ) as well as progressionfree survival (PFS, HR ¼ 0.77, P ¼ 0.0000006). Further, adenosine signaling is associated with reduced OS (HR ¼ 0.47, P < 2.2e -16 ) and PFS (HR ¼ 0.65, P ¼ 0.0000002) in CD8 þ T-cell-infiltrated tumors. Mutation of TGFb superfamily members is associated with enhanced adenosine signaling and worse OS (HR ¼ 0.43, P < 2.2e -16 ). Finally, adenosine signaling is associated with reduced efficacy of anti-PD1 therapy in published cohorts (HR ¼ 0.29, P ¼ 0.00012).Conclusions: These data support the adenosine pathway as a mediator of a successful antitumor immune response, demonstrate the prognostic potential of the signature for immunotherapy, and inform patient selection strategies for adenosine pathway modulators currently in development.

The objective of this study was to determine if the material properties of bone at the lamellar level are related to the predominant mode and magnitude of mechanical strain experienced in sku. The tibia and first metatarsal bones of five unpaired cadaveric lower extremities were instrumented with strain gauge rosettes and subjected to repeated loading trials in an apparatus that replicates the muscle forces and external loads experienced by the foot and shank while walking. The spatial distributions of axial strain within diaphyseal cross-sections taken from each bone were subsequently determined. Nanoindentation measurements were then performed on the same cross-sections to determine the compressive elastic inoduli of individual lamellae located within osteonal, interstitial, and outer circumferential microstructures. Twenty percent of the variance in interstitial elastic modulus within ct-osssectioiis of diaphyseal bone was explained by local strain magnitude. Lainellae residing in regions of compressive strain displayed significantly higher compressive elastic modulus values than those located in predominantly tensile regions (19.9 i 1.6 GPa compared to 17.9 & 1.7 GPa, p < 0.05). Elastic mod~ili of interstitial lamellae were 11% greater than those of osteonal or outer circumferential lamellae, irrespective of strain or anatomical location 0, < 0.001). Differences exist in the material properties of individual bone lamellae located within different anatomical regions and different microstructures, and these differences are related to the distribution of axial strain. These findings suggest that mechanical strain, or another closely related variable, may influence the design and ultimate mechanical behavior of the extra-cellular matrix found in lamellar bone. This tissue heterogeneity is of potential importance in bone fragility and adaptation.

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Adenosine signaling through the high affinity adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which can promote tumor growth and limit the efficacy of immune checkpoint inhibitors. AZD4635 (HTL-1071) is a potent and selective oral A2AR antagonist, currently in a Phase 1 clinical trial as a single agent and in combination with durvalumab (anti-PD-L1 Ab) in patients with solid malignancies. In functional in vitro assays, the IC50 of AZD4635 for inhibition of A2AR signaling is dependent on adenosine concentrations and ranges from 1, 10.0, 143 nM in the presence of 0.1, 1, 10 μM adenosine, respectively. However, full understanding of the impact of adenosine on AZD4635 mediated anti-tumor responses requires characterization of intratumoral adenosine concentration and spatial heterogeneity. Using a novel LC/MS based method, measurement of intratumoral adenosine concentrations in a panel of syngeneic tumor models demonstrated a wide range of adenosine levels (5-122 μM). Additionally, measurement of intratumoral adenosine by desorption electrospray ionisation - mass spectroscopy (DESI-MS) demonstrated that adenosine levels are spatially heterogeneous, with levels varying up to 50-fold among regions of a single tumor. High intratumoral adenosine concentrations were correlated with decreased intratumoral CD8 infiltration in vivo. The therapeutic benefit of A2AR blockade alone and in combination with anti-PD-L1 was evaluated in a panel of syngeneic mouse tumor models with varying adenosine concentrations. In sensitive models, inhibition of A2AR signaling with AZD4635 led to a dose dependent reduction in tumor growth alone and in combination with checkpoint inhibitors. Tumors harvested from the treated mice exhibited increases in the functional activity of T cell and myeloid subsets. These results demonstrate that AZD4635 is a potent and selective A2AR inhibitor, that blockade of A2AR signaling with an inhibitor such as AZD4635 can reduce tumor burden and enhance antitumor immunity. Citation Format: Alexandra Borodovsky, Yanjun Wang, Minwei Ye, Joseph C. Shaw, Kris Sachsenmeier, Nanhua Deng, Kelly Goodwin, James D. Clarke, Richard Goodwin, Nicole Strittmatter, Carl Hay, Vasu Sah, Lawson Deborah, Corinne Reimer, Miles Congreve, Jonathan Mason, Fiona Marshall, Paul Lyne, Richard Woessner. Inhibition of A2AR by AZD4635 induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical models [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 3751.