Introduced in 1987, platinum-based chemotherapy remains standard of care for small cell lung cancer (SCLC), a most aggressive, recalcitrant tumor. Prominent barriers to progress are paucity of tumor tissue to identify drug targets and patient-relevant models to interrogate novel therapies. Following our development of circulating tumor cell patient-derived explants (CDX) as models that faithfully mirror patient disease, here we exploit CDX to examine new therapeutic options for SCLC. We investigated the efficacy of the PARP inhibitor olaparib alone or in combination with the WEE1 kinase inhibitor AZD1775 in 10 phenotypically distinct SCLC CDX and/or These CDX represent chemosensitive and chemorefractory disease including the first reported paired CDX generated longitudinally before treatment and upon disease progression. There was a heterogeneous depth and duration of response to olaparib/AZD1775 that diminished when tested at disease progression. However, efficacy of this combination consistently exceeded that of cisplatin/etoposide, with cures in one CDX model. Genomic and protein analyses revealed defects in homologous recombination repair genes and oncogenes that induce replication stress (such as MYC family members), predisposed CDX to combined olaparib/AZD1775 sensitivity, although universal predictors of response were not noted. These preclinical data provide a strong rationale to trial this combination in the clinic informed by prevalent, readily accessed circulating tumor cell-based biomarkers. New therapies will be evaluated in SCLC patients after first-line chemotherapy, and our data suggest that the combination of olaparib/AZD1775 should be used as early as possible and before disease relapse. .

BackgroundThe ability to modulate immune-inhibitory pathways using checkpoint blockade antibodies such as αPD-1, αPD-L1, and αCTLA-4 represents a significant breakthrough in cancer therapy in recent years. This has driven interest in identifying small-molecule-immunotherapy combinations to increase the proportion of responses. Murine syngeneic models, which have a functional immune system, represent an essential tool for pre-clinical evaluation of new immunotherapies. However, immune response varies widely between models and the translational relevance of each model is not fully understood, making selection of an appropriate pre-clinical model for drug target validation challenging.MethodsUsing flow cytometry, O-link protein analysis, RT-PCR, and RNAseq we have characterized kinetic changes in immune-cell populations over the course of tumor development in commonly used syngeneic models.ResultsThis longitudinal profiling of syngeneic models enables pharmacodynamic time point selection within each model, dependent on the immune population of interest. Additionally, we have characterized the changes in immune populations in each of these models after treatment with the combination of α-PD-L1 and α-CTLA-4 antibodies, enabling benchmarking to known immune modulating treatments within each model.ConclusionsTaken together, this dataset will provide a framework for characterization and enable the selection of the optimal models for immunotherapy combinations and generate potential biomarkers for clinical evaluation in identifying responders and non-responders to immunotherapy combinations.

PI3K inhibitors with differential selectivity to distinct PI3K isoforms have been tested extensively in clinical trials, largely to target tumor epithelial cells. PI3K signaling also regulates the immune system and inhibition of PI3Kδ modulate the tumor immune microenvironment of pre-clinical mouse tumor models by relieving T-regs-mediated immunosuppression. PI3K inhibitors as a class and PI3Kδ specifically are associated with immune-related side effects. However, the impact of mixed PI3K inhibitors in tumor immunology is under-explored. Here we examine the differential effects of AZD8835, a dual PI3Kα/δ inhibitor, specifically on the tumor immune microenvironment using syngeneic models. Continuous suppression of PI3Kα/δ was not required for anti-tumor activity, as tumor growth inhibition was potentiated by an intermittent dosing/schedule in vivo. Moreover, PI3Kα/δ inhibition delivered strong single agent anti-tumor activity, which was associated with dynamic suppression of T-regs, improved CD8+ T-cell activation and memory in mouse syngeneic tumor models. Strikingly, AZD8835 promoted robust CD8+ T-cell activation dissociated from its effect on T-regs. This was associated with enhancing effector cell viability/function. Together these data reveal novel mechanisms by which PI3Kα/δ inhibitors interact with the immune system and validate the clinical compound AZD8835 as a novel immunoncology drug, independent of effects on tumor cells. These data support further clinical investigation of PI3K pathway inhibitors as immuno-oncology agents.Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0457-0) contains supplementary material, which is available to authorized users.

Purpose: Danvatirsen is a therapeutic antisense oligonucleotide (ASO) that selectively targets STAT3 and has shown clinical activity in two phase I clinical studies. We interrogated the clinical mechanism of action using danvatirsen-treated patient samples and conducted back-translational studies to further elucidate its immunomodulatory mechanism of action. Experimental Design: Paired biopsies and blood samples from danvatirsen-treated patients were evaluated using immunohistochemistry and gene-expression analysis. To gain mechanistic insight, we used mass cytometry, flow cytometry, and immunofluorescence analysis of CT26 tumors treated with a mouse surrogate STAT3 ASO, and human immune cells were treated in vitro with danvatirsen. Results: Within the tumors of treated patients, danvatirsen uptake was observed mainly in cells of the tumor microenvironment (TME). Gene expression analysis comparing baseline and on-treatment tumor samples showed increased expression of proinflammatory genes. In mouse models, STAT3 ASO demonstrated partial tumor growth inhibition and enhanced the antitumor activity when combined with anti–PD-L1. Immune profiling revealed reduced STAT3 protein in immune and stromal cells, and decreased suppressive cytokines correlating with increased proinflammatory macrophages and cytokine production. These changes led to enhanced T-cell abundance and function in combination with anti–PD-L1. Conclusions: STAT3 ASO treatment reverses a suppressive TME and promotes proinflammatory gene expression changes in patients' tumors and mouse models. Preclinical data provide evidence that ASO-mediated inhibition of STAT3 in the immune compartment is sufficient to remodel the TME and enhance the activity of checkpoint blockade without direct STAT3 inhibition in tumor cells. Collectively, these data provide a rationale for testing this combination in the clinic.

AZD6738 is a potent and selective orally bioavailable kinase inhibitor of ataxia telangiectasia and rad3 related (ATR). Here we report the pre-clinical in vitro and in vivo and biological profile of AZD6738. ATR is a serine/threonine protein kinase involved in DNA damage response signalling caused by DNA replication associated stress. Activation of ATR at stalled replication forks leads to suppression of replication fork origin firing, promotes repair and S/G2-cell cycle checkpoints to prevent premature mitosis and maintain genomic integrity. Failure to resolve damage leads to genomic instability and if sufficiently high, cell death. Stalled replication forks may collapse leading to formation of DNA double stranded breaks and activation of the ataxia telangiectasia mutated (ATM) kinase. ATM works in conjunction with ATR to efficiently resolve replication associated DNA damage creating a co-dependency with loss of one leading to a greater reliance on the other to maintain genomic stability. ATM is frequently inactivated across B-cell malignancies, head and neck, breast and lung cancers through chromosomal deletion, promoter hypermethylation or mutation. ATM-deficient tumours are hypothesised to be more reliant on ATR for survival and specific inhibition of ATR may lead enhanced anti-tumour activity while minimizing normal tissue toxicity. AZD6738 demonstrates this preclinical profile. AZD6738 inhibits the phosphorylation of direct downstream substrate CHK1 while increasing the phosphorylation of ATM-dependent substrate CHK2 and DNA damage marker γH2AX. This is associated with impaired S-phase cell cycle progression with prolonged inhibition causing cell death, indicative of replication fork stalling, collapse and irreversible damage. AZD6738 is active as a single agent across cancer cell line panels but shows enhanced sensitivity in cell lines with ATM-pathway defects. AZD6738 when used in combination with DNA damaging inducing agents’ gemcitabine, cisplatin or ionising radiation (IR) shows enhanced synergistic cell killing activity. In vivo, AZD6738 monotherapy treatment leads to significant anti-tumour activity in ATM-deficient but not ATM-proficient xenograft models at equivalent, tolerated doses. When AZD6738 is used in combination with carboplatin or IR anti-tumour growth inhibitory activity or regression is observed. AZD6738 in vivo activity is also associated with a persistent increased γH2AX staining in tumour tissue but only a transient increase in normal bone marrow or gut tissue suggesting a favourable therapeutic index can be achieved. Early pre-clinical safety studies support these findings with target related bone marrow suppression, neutropenia and GI tract impact only at high-doses. These data merit further investigation of AZD6738 as a monotherapy or in combination with chemo or radiotherapy. Citation Format: Sylvie M. Guichard, Elaine Brown, Rajesh Odedra, Adina Hughes, Dan Heathcote, Jen Barnes, Alan Lau, Steve Powell, Clifford D. Jones, Willem Nissink, Kevin M. Foote, Philip J. Jewsbury, Martin Pass. The pre-clinical in vitro and in vivo activity of AZD6738: A potent and selective inhibitor of ATR kinase. [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 3343. doi:10.1158/1538-7445.AM2013-3343