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.

Relevance of ATM Status in Driving Sensitivity to DNA Damage Response Inhibitors in Patient-Derived Xenograft Models.

Ataxia-telangiectasia mutated gene (ATM) is a key component of the DNA damage response (DDR) and double-strand break repair pathway. The functional loss of ATM (ATM deficiency) is hypothesised to enhance sensitivity to DDR inhibitors (DDRi). Whole-exome sequencing (WES), immunohistochemistry (IHC), and Western blotting (WB) were used to characterise the baseline ATM status across a panel of ATM mutated patient-derived xenograft (PDX) models from a range of tumour types. Antitumour efficacy was assessed with poly(ADP-ribose)polymerase (PARP, olaparib), ataxia- telangiectasia and rad3-related protein (ATR, AZD6738), and DNA-dependent protein kinase (DNA-PK, AZD7648) inhibitors as a monotherapy or in combination to associate responses with ATM status. Biallelic truncation/frameshift ATM mutations were linked to ATM protein loss while monoallelic or missense mutations, including the clinically relevant recurrent R3008H mutation, did not confer ATM protein loss by IHC. DDRi agents showed a mixed response across the PDX's but with a general trend toward greater activity, particularly in combination in models with biallelic ATM mutation and protein loss. A PDX with an ATM splice-site mutation, 2127T > C, with a high relative baseline ATM expression and KAP1 phosphorylation responded to all DDRi treatments. These data highlight the heterogeneity and complexity in describing targetable ATM-deficiencies and the fact that current patient selection biomarker methods remain imperfect; although, complete ATM loss was best able to enrich for DDRi sensitivity.

Identification of a Molecularly-Defined Subset of Breast and Ovarian Cancer Models that Respond to WEE1 or ATR Inhibition, Overcoming PARP Inhibitor Resistance.

PURPOSE: PARP inhibitors (PARPi) induce synthetic lethality in homologous recombination repair (HRR)-deficient tumors and are used to treat breast, ovarian, pancreatic, and prostate cancers. Multiple PARPi resistance mechanisms exist, most resulting in restoration of HRR and protection of stalled replication forks. ATR inhibition was highlighted as a unique approach to reverse both aspects of resistance. Recently, however, a PARPi/WEE1 inhibitor (WEE1i) combination demonstrated enhanced antitumor activity associated with the induction of replication stress, suggesting another approach to tackling PARPi resistance. EXPERIMENTAL DESIGN: We analyzed breast and ovarian patient-derived xenoimplant models resistant to PARPi to quantify WEE1i and ATR inhibitor (ATRi) responses as single agents and in combination with PARPi. Biomarker analysis was conducted at the genetic and protein level. Metabolite analysis by mass spectrometry and nucleoside rescue experiments ex vivo were also conducted in patient-derived models. RESULTS: Although WEE1i response was linked to markers of replication stress, including STK11/RB1 and phospho-RPA, ATRi response associated with ATM mutation. When combined with olaparib, WEE1i could be differentiated from the ATRi/olaparib combination, providing distinct therapeutic strategies to overcome PARPi resistance by targeting the replication stress response. Mechanistically, WEE1i sensitivity was associated with shortage of the dNTP pool and a concomitant increase in replication stress. CONCLUSIONS: Targeting the replication stress response is a valid therapeutic option to overcome PARPi resistance including tumors without an underlying HRR deficiency. These preclinical insights are now being tested in several clinical trials where the PARPi is administered with either the WEE1i or the ATRi.

Direct targeting of FOXP3 in Tregs with AZD8701, a novel antisense oligonucleotide to relieve immunosuppression in cancer.

BACKGROUND: The Regulatory T cell (Treg) lineage is defined by the transcription factor FOXP3, which controls immune-suppressive gene expression profiles. Tregs are often recruited in high frequencies to the tumor microenvironment where they can suppress antitumor immunity. We hypothesized that pharmacological inhibition of FOXP3 by systemically delivered, unformulated constrained ethyl-modified antisense oligonucleotides could modulate the activity of Tregs and augment antitumor immunity providing therapeutic benefit in cancer models and potentially in man. METHODS: We have identified murine Foxp3 antisense oligonucleotides (ASOs) and clinical candidate human FOXP3 ASO AZD8701. Pharmacology and biological effects of FOXP3 inhibitors on Treg function and antitumor immunity were tested in cultured Tregs and mouse syngeneic tumor models. Experiments were controlled by vehicle and non-targeting control ASO groups as well as by use of multiple independent FOXP3 ASOs. Statistical significance of biological effects was evaluated by one or two-way analysis of variance with multiple comparisons. RESULTS: AZD8701 demonstrated a dose-dependent knockdown of FOXP3 in primary Tregs, reduction of suppressive function and efficient target downregulation in humanized mice at clinically relevant doses. Surrogate murine FOXP3 ASO, which efficiently downregulated Foxp3 messenger RNA and protein levels in primary Tregs, reduced Treg suppressive function in immune suppression assays in vitro. FOXP3 ASO promoted more than 70% reduction in FOXP3 levels in Tregs in vitro and in vivo, strongly modulated Treg effector molecules (eg, ICOS, CTLA-4, CD25 and 4-1BB), and augmented CD8+ T cell activation and produced antitumor activity in syngeneic tumor models. The combination of FOXP3 ASOs with immune checkpoint blockade further enhanced antitumor efficacy. CONCLUSIONS: Antisense inhibitors of FOXP3 offer a promising novel cancer immunotherapy approach. AZD8701 is being developed clinically as a first-in-class FOXP3 inhibitor for the treatment of cancer currently in Ph1a/b clinical trial (NCT04504669).

ATR Inhibitor AZD6738 (Ceralasertib) Exerts Antitumor Activity as a Monotherapy and in Combination with Chemotherapy and the PARP Inhibitor Olaparib.

AZD6738 (ceralasertib) is a potent and selective orally bioavailable inhibitor of ataxia telangiectasia and Rad3-related (ATR) kinase. ATR is activated in response to stalled DNA replication forks to promote G2-M cell-cycle checkpoints and fork restart. Here, we found AZD6738 modulated CHK1 phosphorylation and induced ATM-dependent signaling (pRAD50) and the DNA damage marker γH2AX. AZD6738 inhibited break-induced replication and homologous recombination repair. In vitro sensitivity to AZD6738 was elevated in, but not exclusive to, cells with defects in the ATM pathway or that harbor putative drivers of replication stress such as CCNE1 amplification. This translated to in vivo antitumor activity, with tumor control requiring continuous dosing and free plasma exposures, which correlated with induction of pCHK1, pRAD50, and γH2AX. AZD6738 showed combinatorial efficacy with agents associated with replication fork stalling and collapse such as carboplatin and irinotecan and the PARP inhibitor olaparib. These combinations required optimization of dose and schedules in vivo and showed superior antitumor activity at lower doses compared with that required for monotherapy. Tumor regressions required at least 2 days of daily dosing of AZD6738 concurrent with carboplatin, while twice daily dosing was required following irinotecan. In a BRCA2-mutant patient-derived triple-negative breast cancer (TNBC) xenograft model, complete tumor regression was achieved with 3 to5 days of daily AZD6738 per week concurrent with olaparib. Increasing olaparib dosage or AZD6738 dosing to twice daily allowed complete tumor regression even in a BRCA wild-type TNBC xenograft model. These preclinical data provide rationale for clinical evaluation of AZD6738 as a monotherapy or combinatorial agent. SIGNIFICANCE: This detailed preclinical investigation, including pharmacokinetics/pharmacodynamics and dose-schedule optimizations, of AZD6738/ceralasertib alone and in combination with chemotherapy or PARP inhibitors can inform ongoing clinical efforts to treat cancer with ATR inhibitors.

Macrophage Activation Status Rather than Repolarization Is Associated with Enhanced Checkpoint Activity in Combination with PI3Kγ Inhibition.

Suppressive myeloid cells mediate resistance to immune checkpoint blockade. PI3Kγ inhibition can target suppressive macrophages, and enhance efficacy of immune checkpoint inhibitors. However, how PI3Kγ inhibitors function in different tumor microenvironments (TME) to activate specific immune cells is underexplored. The effect of the novel PI3Kγ inhibitor AZD3458 was assessed in preclinical models. AZD3458 enhanced antitumor activity of immune checkpoint inhibitors in 4T1, CT26, and MC38 syngeneic models, increasing CD8+ T-cell activation status. Immune and TME biomarker analysis of MC38 tumors revealed that AZD3458 monotherapy or combination treatment did not repolarize the phenotype of tumor-associated macrophage cells but induced gene signatures associated with LPS and type II INF activation. The activation biomarkers were present across tumor macrophages that appear phenotypically heterogenous. AZD3458 alone or in combination with PD-1-blocking antibodies promoted an increase in antigen-presenting (MHCII+) and cytotoxic (iNOS+)-activated macrophages, as well as dendritic cell activation. AZD3458 reduced IL-10 secretion and signaling in primary human macrophages and murine tumor-associated macrophages, but did not strongly regulate IL-12 as observed in other studies. Therefore, rather than polarizing tumor macrophages, PI3Kγ inhibition with AZD3458 promotes a cytotoxic switch of macrophages into antigen-presenting activated macrophages, resulting in CD8 T-cell-mediated antitumor activity with immune checkpoint inhibitors associated with tumor and peripheral immune activation.

STAT3 Antisense Oligonucleotide Remodels the Suppressive Tumor Microenvironment to Enhance Immune Activation in Combination with Anti-PD-L1.

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.

Pharmacology of the ATM Inhibitor AZD0156: Potentiation of Irradiation and Olaparib Responses Preclinically.

AZD0156 is a potent and selective, bioavailable inhibitor of ataxia-telangiectasia mutated (ATM) protein, a signaling kinase involved in the DNA damage response. We present preclinical data demonstrating abrogation of irradiation-induced ATM signaling by low doses of AZD0156, as measured by phosphorylation of ATM substrates. AZD0156 is a strong radiosensitizer in vitro, and using a lung xenograft model, we show that systemic delivery of AZD0156 enhances the tumor growth inhibitory effects of radiation treatment in vivo Because ATM deficiency contributes to PARP inhibitor sensitivity, preclinically, we evaluated the effect of combining AZD0156 with the PARP inhibitor olaparib. Using ATM isogenic FaDu cells, we demonstrate that AZD0156 impedes the repair of olaparib-induced DNA damage, resulting in elevated DNA double-strand break signaling, cell-cycle arrest, and apoptosis. Preclinically, AZD0156 potentiated the effects of olaparib across a panel of lung, gastric, and breast cancer cell lines in vitro, and improved the efficacy of olaparib in two patient-derived triple-negative breast cancer xenograft models. AZD0156 is currently being evaluated in phase I studies (NCT02588105).

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.

Rapid activation of epithelial-mesenchymal transition drives PARP inhibitor resistance in Brca2-mutant mammary tumours.

Tumours defective in the DNA homologous recombination repair pathway can be effectively treated with poly (ADP-ribose) polymerase (PARP) inhibitors; these have proven effective in clinical trials in patients with BRCA gene function-defective cancers. However, resistance observed in both pre-clinical and clinical studies is likely to impact on this treatment strategy. Over-expression of phosphoglycoprotein (P-gp) has been previously suggested as a mechanism of resistance to the PARP inhibitor olaparib in mouse models of Brca1/2-mutant breast cancer. Here, we report that in a Brca2 model treated with olaparib, P-gp upregulation is observed but is not sufficient to confer resistance. Furthermore, resistant/relapsed tumours do not show substantial changes in PK/PD of olaparib, do not downregulate PARP1 or re-establish double stranded DNA break repair by homologous recombination, all previously suggested as mechanisms of resistance. However, resistance is strongly associated with epithelial-mesenchymal transition (EMT) and treatment-naïve tumours given a single dose of olaparib upregulate EMT markers within one hour. Therefore, in this model, olaparib resistance is likely a product of an as-yet unidentified mechanism associated with rapid transition to the mesenchymal phenotype.

Modeling Dose and Schedule Effects of AZD2811 Nanoparticles Targeting Aurora B Kinase for Treatment of Diffuse Large B-cell Lymphoma.

Barasertib (AZD1152), a pro-drug of the highly potent and selective Aurora B kinase inhibitor AZD2811, showed promising clinical activity in relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients administered as a 4-day infusion. To improve potential therapeutic benefit of Aurora B kinase inhibition, a nanoparticle formulation of AZD2811 has been developed to address limitations of repeated intravenous infusion. One of the challenges with the use of nanoparticles for chronic treatment of tumors is optimizing dose and schedule required to enable repeat administration to sustain tumor growth inhibition. AZD2811 gives potent cell growth inhibition across a range of DLBCL cells lines in vitro In vivo, repeat administration of the AZD2811 nanoparticle gave antitumor activity at half the dose intensity of AZD1152. Compared with AZD1152, a single dose of AZD2811 nanoparticle gave less reduction in pHH3, but increased apoptosis and reduction of cells in G1 and G2-M, albeit at later time points, suggesting that duration and depth of target inhibition influence the nature of the tumor cell response to drug. Further exploration of the influence of dose and schedule on efficacy revealed that AZD2811 nanoparticle can be used flexibly with repeat administration of 25 mg/kg administered up to 7 days apart being sufficient to maintain equivalent tumor control. Timing of repeat administration could be varied with 50 mg/kg every 2 weeks controlling tumor control as effectively as 25 mg/kg every week. AZD2811 nanoparticle can be administered with very different doses and schedules to inhibit DLBCL tumor growth, although maximal tumor growth inhibition was achieved with the highest dose intensities.

PI3Kα/δ inhibition promotes anti-tumor immunity through direct enhancement of effector CD8+ T-cell activity.

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.

Combination of dual mTORC1/2 inhibition and immune-checkpoint blockade potentiates anti-tumour immunity.

mTOR inhibition can promote or inhibit immune responses in a context dependent manner, but whether this will represent a net benefit or be contraindicated in the context of immunooncology therapies is less understood. Here, we report that the mTORC1/2 dual kinase inhibitor vistusertib (AZD2014) potentiates anti-tumour immunity in combination with anti-CTLA-4 (αCTLA-4), αPD-1 or αPD-L1 immune checkpoint blockade. Combination of vistusertib and immune checkpoint blocking antibodies led to tumour growth inhibition and improved survival of MC-38 or CT-26 pre-clinical syngeneic tumour models, whereas monotherapies were less effective. Underlying these combinatorial effects, vistusertib/immune checkpoint combinations reduced the occurrence of exhausted phenotype tumour infiltrating lymphocytes (TILs), whilst increasing frequencies of activated Th1 polarized T-cells in tumours. Vistusertib alone was shown to promote a Th1 polarizing proinflammatory cytokine profile by innate primary immune cells. Moreover, vistusertib directly enhanced activation of effector T-cell and survival, an effect that was critically dependent on inhibitor dose. Therefore, these data highlight direct, tumour-relevant immune potentiating benefits of mTOR inhibition that complement immune checkpoint blockade. Together, these data provide a clear rationale to investigate such combinations in the clinic.

The Combination of the PARP Inhibitor Olaparib and the WEE1 Inhibitor AZD1775 as a New Therapeutic Option for Small Cell Lung Cancer.

Purpose: 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.Experimental Design: 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 in vivo and/or ex vivo These CDX represent chemosensitive and chemorefractory disease including the first reported paired CDX generated longitudinally before treatment and upon disease progression.Results: 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.Conclusions: 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. Clin Cancer Res; 24(20); 5153-64. ©2018 AACR.

Structure-activity relationship of a series of non peptidic RGD integrin antagonists targeting α5ß1: part 1.

Potent antagonists of the integrin α(5)ß(1), which are RGD mimetics built from tyrosine are described. This letter describes the optimization of in vitro potency obtained by variation of two parts of the molecule, the basic group and the linker between the basic group and the phenyl central core.

Structure-activity relationship of a series of non peptidic RGD integrin antagonists targeting α5ß1: part 2.

Potent antagonists of the integrin α(5)ß(1), which are RGD mimetics built from tyrosine are described. This paper describes the optimization of in vitro potency obtained by variation of two parts of the molecule, the central aromatic core and the amide moiety.