The ATR inhibitor ceralasertib potentiates cancer checkpoint immunotherapy by regulating the tumor microenvironment.

The Ataxia telangiectasia and Rad3-related (ATR) inhibitor ceralasertib in combination with the PD-L1 antibody durvalumab demonstrated encouraging clinical benefit in melanoma and lung cancer patients who progressed on immunotherapy. Here we show that modelling of intermittent ceralasertib treatment in mouse tumor models reveals CD8+ T-cell dependent antitumor activity, which is separate from the effects on tumor cells. Ceralasertib suppresses proliferating CD8+ T-cells on treatment which is rapidly reversed off-treatment. Ceralasertib causes up-regulation of type I interferon (IFNI) pathway in cancer patients and in tumor-bearing mice. IFNI is experimentally found to be a major mediator of antitumor activity of ceralasertib in combination with PD-L1 antibody. Improvement of T-cell function after ceralasertib treatment is linked to changes in myeloid cells in the tumor microenvironment. IFNI also promotes anti-proliferative effects of ceralasertib on tumor cells. Here, we report that broad immunomodulatory changes following intermittent ATR inhibition underpins the clinical therapeutic benefit and indicates its wider impact on antitumor immunity.

Longitudinal immune characterization of syngeneic tumor models to enable model selection for immune oncology drug discovery.

BACKGROUND: The 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. METHODS: Using 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. RESULTS: This 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. CONCLUSIONS: Taken 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.

Estrogen receptor beta: expression profile and possible anti-inflammatory role in disease.

Estrogen receptor (ER) beta agonists have been demonstrated to possess anti-inflammatory properties in inflammatory disease models. The objective of this study was to determine whether ERbeta agonists affect in vitro and in vivo preclinical models of asthma. mRNA expression assays were validated in human and rodent tissue panels. These assays were then used to measure expression in human cells and our characterized rat model of allergic asthma. ERB-041 [7-ethenyl-2-(3-fluoro-4-hydroxyphenyl)-1,3-benzoxazol-5-ol], an ERbeta agonist, was profiled on cytokine release from interleukin-1beta-stimulated human airway smooth muscle (HASM) cells and in the rodent asthma model. Although ERbeta expression was demonstrated at the gene and protein level in HASM cells, the agonist failed to have an impact on the inflammatory response. Similarly, in vivo, we observed temporal modulation of ERbeta expression after antigen challenge. However, the agonist failed to have an impact on the model endpoints such as airway inflammation, even though plasma levels reflected linear compound exposure and was associated with an increase in receptor activation after drug administration. In these modeling systems of airway inflammation, an ERbeta agonist was ineffective. Although ERbeta agonists are anti-inflammatory in certain models, this novel study would suggest that they would not be clinically useful in the treatment of asthma.

Functional significance of co-occurring mutations in PIK3CA and MAP3K1 in breast cancer.

The PI3Kα signaling pathway is frequently hyper-activated in breast cancer (BrCa), as a result of mutations/amplifications in oncogenes (e.g. HER2), decreased function in tumor suppressors (e.g. PTEN) or activating mutations in key components of the pathway. In particular, activating mutations of PIK3CA (~45%) are frequently found in luminal A BrCa samples. Genomic studies have uncovered inactivating mutations in MAP3K1 (13-20%) and MAP2K4 (~8%), two upstream kinases of the JNK apoptotic pathway in luminal A BrCa samples. Further, simultaneous mutation of PIK3CA and MAP3K1 are found in ~11% of mutant PIK3CA tumors. How these two alterations may cooperate to elicit tumorigenesis and impact the sensitivity to PI3K and AKT inhibitors is currently unknown. Using CRISPR gene editing we have genetically disrupted MAP3K1 expression in mutant PIK3CA cell lines to specifically create in vitro models reflecting the mutational status of PIK3CA and MAP3K1 in BrCa patients. MAP3K1 deficient cell lines exhibited ~2.4-fold increased proliferation rate and decreased sensitivity to PI3Kα/δ(AZD8835) and AKT (AZD5363) inhibitors (~2.61 and ~5.23-fold IC50 increases, respectively) compared with parental control cell lines. In addition, mechanistic analysis revealed that MAP3K1 disruption enhances AKT phosphorylation and downstream signaling and reduces sensitivity to AZD5363-mediated pathway inhibition. This appears to be a consequence of deficient MAP3K1-JNK signaling increasing IRS1 stability and therefore promoting IRS1 binding to p85, resulting in enhanced PI3Kα activity. Using 3D-MCF10A-PI3KαH1047R models, we found that MAP3K1 depletion increased overall acinar volume and counteracted AZD5363-mediated reduction of acinar growth due to enhanced proliferation and reduced apoptosis. Furthermore, in vivo efficacy studies revealed that MAP3K1-deficient MCF7 tumors were less sensitive to AKT inhibitor treatment, compared with parental MCF7 tumors. Our study provides mechanistic and in vivo evidence indicating a role for MAP3K1 as a tumor suppressor gene at least in the context of PIK3CA-mutant backgrounds. Further, our work predicts that MAP3K1 mutational status may be considered as a predictive biomarker for efficacy in PI3K pathway inhibitor trials.

Regulation of TNF-alpha- and IFN-gamma-induced CXCL10 expression: participation of the airway smooth muscle in the pulmonary inflammatory response in chronic obstructive pulmonary disease.

The chemokine CXCL10 is produced by many inflammatory cells found in the diseased lung and has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). The present study demonstrates elevated CXCL10 protein in the lungs of COPD patients, which appears histologically in airway smooth muscle (hASM). In primary cultured hASM cells taken from normal donors, CXCL10 protein expression was induced by IFN-gamma and TNF-alpha, cytokines reported as elevated in COPD, and a synergistic response was obtained when they were combined. TNF-alpha stimulation of hASM enhanced accumulation of CXCL10 mRNA, indicating regulation at the transcriptional level, while IFN-gamma stimulation resulted in a smaller accumulation of CXCL10 mRNA. When these cytokines were applied simultaneously, an additive effect was obtained. TNF-alpha-induced CXCL10 expression in hASM was dependent on NFkappaB activation, and a salicylanilide NFkappaB inhibitor blocked the CXCL10 expression. In contrast, IFN-gamma stimulation resulted in transient NFkappaB activation, and the inhibitor had little effect on CXCL10 expression. When these cytokines were added simultaneously, NFkappaB was activated earlier and lasted longer, and the effect was blocked by the inhibitor. These data demonstrate a potential active role for hASM in pulmonary inflammatory diseases such as COPD by producing CXCL10.

Second-generation inhibitors demonstrate the involvement of p38 mitogen-activated protein kinase in post-transcriptional modulation of inflammatory mediator production in human and rodent airways.

The exact role of p38 mitogen-activated protein kinase (MAPK) in the expression of inflammatory cytokines is not clear; it may regulate transcriptionally, post-transcriptionally, translationally, or post-translationally. The involvement of one or more of these mechanisms has been suggested to depend on the particular cytokine, the cell type studied, and the specific stimulus used. Interpretation of some of the published data is further complicated by the use of inhibitors such as 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580) used at single, high concentrations. The aim of this study was to determine the impact of two second-generation p38 MAPK inhibitors on the expression of a range of inflammatory cytokines at the gene and protein levels in human cultured cells. Similar assessment of the impact of these compounds on inflammatory cytokine expression in a preclinical in vivo model of airway inflammation was performed. The results in THP-1 cells and primary airway macrophages clearly show that protein expression is inhibited at much lower concentrations of inhibitor than are needed to impact on gene expression. In the rodent model, both compounds, at doses that cause maximal inhibition of cellular recruitment, inhibit tumor necrosis factor alpha (TNFalpha) protein production without impacting on nuclear factor kappaB pathway activation or TNFalpha gene expression. In summary, the data shown here demonstrate that, although at high compound concentrations there is some level of transcriptional regulation, the predominant role of p38 MAPK in cytokine production is at the translational level. These data question whether the effect of p38 inhibitors on gene transcription is related to their potential therapeutic role as anti-inflammatory compounds.

IkappaB kinase-2-independent and -dependent inflammation in airway disease models: relevance of IKK-2 inhibition to the clinic.

Nuclear factor kappaB (NF-kappaB) is a transcription factor believed to be central in the expression of numerous inflammatory genes and the pathogenesis of many respiratory diseases. We have previously demonstrated increased NF-kappaB pathway activation in a steroid-sensitive animal model of lipopolysaccharide (LPS)-driven airway inflammation. It is noteworthy that this phenomenon was not observed in a steroid-insensitive model of elastase-induced inflammation in the rat. The aim of this study was to gather further evidence to suggest that these similar profiles of neutrophilic inflammation can be NF-kappaB-dependent or -independent by determining the impact of an IkappaB kinase-2 (IKK-2) inhibitor, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1). In the LPS model, TPCA-1 blocked the increase in NF-kappaB DNA binding, a marker of NF-kappaB pathway activation. This inhibition was associated with a reduction in inflammatory mediator release [tumor necrosis factor alpha (TNFalpha)/interleukin-1beta (IL-1beta)/matrix metalloproteinase-9 (MMP-9)] and lung inflammatory cell burden (neutrophilia/eosinophilia). These data were paralleled with a steroid and in human cell based assays. In the elastase-driven inflammation model, in which our group has previously failed to measure an increase in NF-kappaB DNA binding, neither TPCA-1 nor the steroid, affected mediator release (IL-1beta/MMP-9) or cellular burden (neutrophilia/lymphomononuclear cells). This is the first study to examine the effect of an IKK-2 inhibitor in well validated models that mimic aspects of the inflammatory lesion evident in diseases such as COPD. In conclusion, we have demonstrated that animal models with similar profiles of airway inflammation can be IKK-2 inhibitor/steroid-sensitive or -insensitive. If both profiles of inflammation exist in the clinic, then this finding is extremely exciting and may lead to greater understanding of disease pathology and the discovery of novel anti-inflammatory targets.