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).

Optimization of an Imidazo[1,2-a]pyridine Series to Afford Highly Selective Type I1/2 Dual Mer/Axl Kinase Inhibitors with In Vivo Efficacy.

Inhibition of Mer and Axl kinases has been implicated as a potential way to improve the efficacy of current immuno-oncology therapeutics by restoring the innate immune response in the tumor microenvironment. Highly selective dual Mer/Axl kinase inhibitors are required to validate this hypothesis. Starting from hits from a DNA-encoded library screen, we optimized an imidazo[1,2-a]pyridine series using structure-based compound design to improve potency and reduce lipophilicity, resulting in a highly selective in vivo probe compound 32. We demonstrated dose-dependent in vivo efficacy and target engagement in Mer- and Axl-dependent efficacy models using two structurally differentiated and selective dual Mer/Axl inhibitors. Additionally, in vivo efficacy was observed in a preclinical MC38 immuno-oncology model in combination with anti-PD1 antibodies and ionizing radiation.

Cell-autonomous inflammation of BRCA1-deficient ovarian cancers drives both tumor-intrinsic immunoreactivity and immune resistance via STING.

In this study, we investigate mechanisms leading to inflammation and immunoreactivity in ovarian tumors with homologous recombination deficiency (HRD). BRCA1 loss is found to lead to transcriptional reprogramming in tumor cells and cell-intrinsic inflammation involving type I interferon (IFN) and stimulator of IFN genes (STING). BRCA1-mutated (BRCA1mut) tumors are thus T cell inflamed at baseline. Genetic deletion or methylation of DNA-sensing/IFN genes or CCL5 chemokine is identified as a potential mechanism to attenuate T cell inflammation. Alternatively, in BRCA1mut cancers retaining inflammation, STING upregulates VEGF-A, mediating immune resistance and tumor progression. Tumor-intrinsic STING elimination reduces neoangiogenesis, increases CD8+ T cell infiltration, and reverts therapeutic resistance to dual immune checkpoint blockade (ICB). VEGF-A blockade phenocopies genetic STING loss and synergizes with ICB and/or poly(ADP-ribose) polymerase (PARP) inhibitors to control the outgrowth of Trp53-/-Brca1-/- but not Brca1+/+ ovarian tumors in vivo, offering rational combinatorial therapies for HRD cancers.

Cancer-Specific Loss of p53 Leads to a Modulation of Myeloid and T Cell Responses.

Loss of p53 function contributes to the development of many cancers. While cell-autonomous consequences of p53 mutation have been studied extensively, the role of p53 in regulating the anti-tumor immune response is still poorly understood. Here, we show that loss of p53 in cancer cells modulates the tumor-immune landscape to circumvent immune destruction. Deletion of p53 promotes the recruitment and instruction of suppressive myeloid CD11b+ cells, in part through increased expression of CXCR3/CCR2-associated chemokines and macrophage colony-stimulating factor (M-CSF), and attenuates the CD4+ T helper 1 (Th1) and CD8+ T cell responses in vivo. p53-null tumors also show an accumulation of suppressive regulatory T (Treg) cells. Finally, we show that two key drivers of tumorigenesis, activation of KRAS and deletion of p53, cooperate to promote immune tolerance.

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.

Critical questions in ovarian cancer research and treatment: Report of an American Association for Cancer Research Special Conference.

Substantial progress has been made in understanding ovarian cancer at the molecular and cellular level. Significant improvement in 5-year survival has been achieved through cytoreductive surgery, combination platinum-based chemotherapy, and more effective treatment of recurrent cancer, and there are now more than 280,000 ovarian cancer survivors in the United States. Despite these advances, long-term survival in late-stage disease has improved little over the last 4 decades. Poor outcomes relate, in part, to late stage at initial diagnosis, intrinsic drug resistance, and the persistence of dormant drug-resistant cancer cells after primary surgery and chemotherapy. Our ability to accelerate progress in the clinic will depend on the ability to answer several critical questions regarding this disease. To assess current answers, an American Association for Cancer Research Special Conference on "Critical Questions in Ovarian Cancer Research and Treatment" was held in Pittsburgh, Pennsylvania, on October 1-3, 2017. Although clinical, translational, and basic investigators conducted much of the discussion, advocates participated in the meeting, and many presentations were directly relevant to patient care, including treatment with poly adenosine diphosphate ribose polymerase (PARP) inhibitors, attempts to improve immunotherapy by overcoming the immune suppressive effects of the microenvironment, and a better understanding of the heterogeneity of the disease.

Author Correction: CRISPR/Cas9-derived models of ovarian high grade serous carcinoma targeting Brca1, Pten and Nf1, and correlation with platinum sensitivity.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

CRISPR/Cas9-derived models of ovarian high grade serous carcinoma targeting Brca1, Pten and Nf1, and correlation with platinum sensitivity.

Transplantable murine models of ovarian high grade serous carcinoma (HGSC) remain an important research tool. We previously showed that ID8, a widely-used syngeneic model of ovarian cancer, lacked any of the frequent mutations in HGSC, and used CRISPR/Cas9 gene editing to generate derivatives with deletions in Trp53 and Brca2. Here we have used one ID8 Trp53 -/- clone to generate further mutants, with additional mutations in Brca1, Pten and Nf1, all of which are frequently mutated or deleted in HGSC. We have also generated clones with triple deletions in Trp53, Brca2 and Pten. We show that ID8 Trp53 -/-;Brca1 -/- and Trp53 -/-;Brca2 -/- cells have defective homologous recombination and increased sensitivity to both platinum and PARP inhibitor chemotherapy compared to Trp53 -/-. By contrast, loss of Pten or Nf1 increases growth rate in vivo, and reduces survival following cisplatin chemotherapy in vivo. Finally, we have also targeted Trp53 in cells isolated from a previous transgenic murine fallopian tube carcinoma model, and confirmed that loss of p53 expression in this second model accelerates intraperitoneal growth. Together, these CRISPR-generated models represent a new and simple tool to investigate the biology of HGSC, and the ID8 cell lines are freely available to researchers.

RIPK3 promotes adenovirus type 5 activity.

Oncolytic adenoviral mutants infect human malignant cells and replicate selectively within them. This induces direct cytotoxicity that can also trigger profound innate and adaptive immune responses. However, the mechanism by which adenoviruses produce cell death remains uncertain. We previously suggested that type 5 adenoviruses, including the E1A CR2 deletion mutant dl922-947, might induce a novel form of programmed death resembling necroptosis. Here we have investigated the roles of core necrosis proteins RIPK1, RIPK3 and MLKL in the cytotoxicity of dl922-947 and other adenovirus serotypes. By electron microscopy, we show that dl922-947 induces similar necrotic morphology as TSZ treatment (TNF-α, Smac mimetic, zVAD.fmk). However, dl922-947-mediated death is independent of TNF-α signalling, does not require RIPK1 and does not rely upon the presence of MLKL. However, inhibition of caspases, specifically caspase-8, induces necroptosis that is RIPK3 dependent and significantly enhances dl922-947 cytotoxicity. Moreover, using CRISPR/Cas9 gene editing, we demonstrate that the increase in cytotoxicity seen upon caspase inhibition is also MLKL dependent. Even in the absence of caspase inhibition, RIPK3 expression promotes dl922-947 and wild-type adenovirus type 5 efficacy both in vitro and in vivo. Together, these results suggest that adenovirus induces a form of programmed necrosis that differs from classical TSZ necroptosis.

CRISPR/Cas9-Mediated Trp53 and Brca2 Knockout to Generate Improved Murine Models of Ovarian High-Grade Serous Carcinoma.

There is a need for transplantable murine models of ovarian high-grade serous carcinoma (HGSC) with regard to mutations in the human disease to assist investigations of the relationships between tumor genotype, chemotherapy response, and immune microenvironment. In addressing this need, we performed whole-exome sequencing of ID8, the most widely used transplantable model of ovarian cancer, covering 194,000 exomes at a mean depth of 400× with 90% exons sequenced >50×. We found no functional mutations in genes characteristic of HGSC (Trp53, Brca1, Brca2, Nf1, and Rb1), and p53 remained transcriptionally active. Homologous recombination in ID8 remained intact in functional assays. Further, we found no mutations typical of clear cell carcinoma (Arid1a, Pik3ca), low-grade serous carcinoma (Braf), endometrioid (Ctnnb1), or mucinous (Kras) carcinomas. Using CRISPR/Cas9 gene editing, we modeled HGSC by generating novel ID8 derivatives that harbored single (Trp53-/-) or double (Trp53-/-;Brca2-/-) suppressor gene deletions. In these mutants, loss of p53 alone was sufficient to increase the growth rate of orthotopic tumors with significant effects observed on the immune microenvironment. Specifically, p53 loss increased expression of the myeloid attractant CCL2 and promoted the infiltration of immunosuppressive myeloid cell populations into primary tumors and their ascites. In Trp53-/-;Brca2-/- mutant cells, we documented a relative increase in sensitivity to the PARP inhibitor rucaparib and slower orthotopic tumor growth compared with Trp53-/- cells, with an appearance of intratumoral tertiary lymphoid structures rich in CD3+ T cells. This work validates new CRISPR-generated models of HGSC to investigate its biology and promote mechanism-based therapeutics discovery. Cancer Res; 76(20); 6118-29. ©2016 AACR.

Systematic review of the body of evidence for the use of biomarkers in the diagnosis of dementia.

BACKGROUND: Although recent diagnostic criteria for Alzheimer's disease propose the use of biomarkers, validation of these biomarkers by diagnostic test accuracy studies is a necessary first step, followed by the synthesis of the evidence from these studies in systematic reviews and meta-analyses. The quality of the resulting evidence depends on the number and size of the primary studies, their quality, and the adequacy of their reporting. This systematic review assesses the weight and quality of the evidence available from primary diagnostic test accuracy studies. METHODS: A MEDLINE search was performed in August 2011 to identify all potentially relevant publications relating to the biomarkers ß-amyloid, tau, positron emission tomography ((18)F-fluorodeoxyglucose or ligands for amyloid), or magnetic resonance imaging (MRI). The reporting and methodology were assessed using the Standards for Reporting of Diagnostic Accuracy and Quality Assessment of Diagnostic Accuracy Studies assessment tools, respectively. Because clinical progression to dementia is the most commonly used reference standard, this review focuses on participants with objective cognitive impairment but no dementia at baseline. RESULTS: Of the 19,104 published references identified by the search, 142 longitudinal studies relating to the biomarkers of interest were identified, which included subjects who had objective cognitive impairment but no dementia at baseline. The highest number of studies (n = 70) and of participants (n = 4722) related to structural MRI. MRI also yielded the highest number of studies with extractable data for meta-analysis (n = 32 [46% of all structural MRI studies]), followed by cerebrospinal fluid tau (n = 24 [73%]). There were few studies on positron emission tomography ligands for amyloid having suitable data for meta-analysis (n = 4). There was considerable variation across studies in reporting outcomes, methods of blinding and selection, means of accounting for indeterminate or missing values, the interval between the test and assessments, and the determination of test thresholds. CONCLUSIONS: The body of evidence for biomarkers is not large and is variable across the different types of biomarkers. Important information is missing from many study reports, highlighting the need for standardization of methodology and reporting to improve the rigor of biomarker validation.