Design and Preclinical Evaluation of a Novel B7-H4-Directed Antibody-Drug Conjugate, AZD8205, Alone and in Combination with the PARP1-Selective Inhibitor AZD5305.

Purpose: We evaluated the activity of AZD8205, a B7-H4-directed antibody-drug conjugate (ADC) bearing a novel topoisomerase I inhibitor (TOP1i) payload, alone and in combination with the PARP1-selective inhibitor AZD5305, in preclinical models.

Experimental Design: IHC and deep-learning-based image analysis algorithms were used to assess prevalence and intratumoral heterogeneity of B7-H4 expression in human tumors. Several TOP1i-ADCs, prepared with Val-Ala or Gly-Gly-Phe-Gly peptide linkers, with or without a PEG8 spacer, were compared in biophysical, in vivo efficacy, and rat toxicology studies.

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

Unlabelled: 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.

Quantifying cell cycle-dependent drug sensitivities in cancer using a high throughput synchronisation and screening approach.

Background: Chemotherapy and targeted agent anti-cancer efficacy is largely dependent on the proliferative state of tumours, as exemplified by agents that target DNA synthesis/replication or mitosis. As a result, cell cycle specificities of a number of cancer drugs are well known. However, they are yet to be described in a quantifiable manner.

Cooperative Blockade of CK2 and ATM Kinases Drives Apoptosis in VHL-Deficient Renal Carcinoma Cells through ROS Overproduction.

Kinase-targeted agents demonstrate antitumor activity in advanced metastatic clear cell renal cell carcinoma (ccRCC), which remains largely incurable. Integration of genomic approaches through small-molecules and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. The 786-O cell line represents a model for most ccRCC that have a loss of functional pVHL (von Hippel-Lindau).

Complete loss of ATM function augments replication catastrophe induced by ATR inhibition and gemcitabine in pancreatic cancer models.

Background: Personalised medicine strategies may improve outcomes in pancreatic ductal adenocarcinoma (PDAC), but validation of predictive biomarkers is required. Having developed a clinical trial to assess the ATR inhibitor, AZD6738, in combination with gemcitabine (ATRi/gem), we investigated ATM loss as a predictive biomarker of response to ATRi/gem in PDAC.

Methods: Through kinase inhibition, siRNA depletion and CRISPR knockout of ATM, we assessed how ATM targeting affected the sensitivity of PDAC cells to ATRi/gem.

The ATR Inhibitor AZD6738 Synergizes with Gemcitabine and to Induce Pancreatic Ductal Adenocarcinoma Regression.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers, and overall survival rates have barely improved over the past five decades. The antimetabolite gemcitabine remains part of the standard of care but shows very limited antitumor efficacy. Ataxia telangiectasia and Rad3-related protein (ATR), the apical kinase of the intra-S-phase DNA damage response, plays a central role in safeguarding cells from replication stress and can therefore limit the efficacy of antimetabolite drug therapies.

Mechanistic Distinctions between CHK1 and WEE1 Inhibition Guide the Scheduling of Triple Therapy with Gemcitabine.

Combination of cytotoxic therapy with emerging DNA damage response inhibitors (DDRi) has been limited by tolerability issues. However, the goal of most combination trials has been to administer DDRi with standard-of-care doses of chemotherapy. We hypothesized that mechanism-guided treatment scheduling could reduce the incidence of dose-limiting toxicities and enable tolerable multitherapeutic regimens.

CLIQ-BID: A method to quantify bacteria-induced damage to eukaryotic cells by automated live-imaging of bright nuclei.

Pathogenic bacteria induce eukaryotic cell damage which range from discrete modifications of signalling pathways, to morphological alterations and even to cell death. Accurate quantitative detection of these events is necessary for studying host-pathogen interactions and for developing strategies to protect host organisms from bacterial infections. Investigation of morphological changes is cumbersome and not adapted to high-throughput and kinetics measurements.

Structure-Guided Strategy for the Development of Potent Bivalent ERK Inhibitors.

ERK is the effector kinase of the RAS-RAF-MEK-ERK signaling cascade, which promotes cell transformation and malignancy in many cancers and is thus a major drug target in oncology. Kinase inhibitors targeting RAF or MEK are already used for the treatment of certain cancers, such as melanoma. Although the initial response to these drugs can be dramatic, development of drug resistance is a major challenge, even with combination therapies targeting both RAF and MEK.

Protein kinase CK2 in breast cancer: the CK2β regulatory subunit takes center stage in epithelial plasticity.

Structurally, protein kinase CK2 consists of two catalytic subunits (α and α') and two regulatory subunits (β), which play a critical role in targeting specific CK2 substrates. Compelling evidence shows the complexity of the CK2 cellular signaling network and supports the view that this enzyme is a key component of regulatory protein kinase networks that are involved in several aspects of cancer. CK2 both activates and suppresses the expression of a number of essential oncogenes and tumor suppressors, and its expression and activity are upregulated in blood tumors and virtually all solid tumors.

Association of the breast cancer antiestrogen resistance protein 1 (BCAR1) and BCAR3 scaffolding proteins in cell signaling and antiestrogen resistance.

Most breast cancers are estrogen receptor-positive and treated with antiestrogens, but aberrant signaling networks can induce drug resistance. One of these networks involves the scaffolding protein BCAR1/p130CAS, which regulates cell growth and migration/invasion. A less investigated scaffolding protein that also confers antiestrogen resistance is the SH2 domain-containing protein BCAR3.

Structure of ERK2 bound to PEA-15 reveals a mechanism for rapid release of activated MAPK.

ERK1/2 kinases are the principal effectors of a central signalling cascade that converts extracellular stimuli into cell proliferation and migration responses and, when deregulated, can promote cell oncogenic transformation. The scaffolding protein PEA-15 is a death effector domain protein that directly interacts with ERK1/2 and affects ERK1/2 subcellular localization and phosphorylation. Here, to understand this ERK1/2 signalling complex, we have solved the crystal structures of PEA-15 bound to three different ERK2 phospho-conformers.

NSP-CAS Protein Complexes: Emerging Signaling Modules in Cancer.

The CAS (CRK-associated substrate) family of adaptor proteins comprises 4 members, which share a conserved modular domain structure that enables multiple protein-protein interactions, leading to the assembly of intracellular signaling platforms. Besides their physiological role in signal transduction downstream of a variety of cell surface receptors, CAS proteins are also critical for oncogenic transformation and cancer cell malignancy through associations with a variety of regulatory proteins and downstream effectors. Among the regulatory partners, the 3 recently identified adaptor proteins constituting the NSP (novel SH2-containing protein) family avidly bind to the conserved carboxy-terminal focal adhesion-targeting (FAT) domain of CAS proteins.

NSP-Cas protein structures reveal a promiscuous interaction module in cell signaling.

Members of the novel SH2-containing protein (NSP) and Crk-associated substrate (Cas) protein families form multidomain signaling platforms that mediate cell migration and invasion through a collection of distinct signaling motifs. Members of each family interact via their respective C-terminal domains, but the mechanism of this association has remained enigmatic. Here we present the crystal structures of the C-terminal domain from the NSP protein BCAR3 and the complex of NSP3 with p130Cas.

SHEP1 partners with CasL to promote marginal zone B-cell maturation.

The marginal zone is a cellular niche bordering the marginal sinus of the spleen that contains specialized B-cell and macrophage subsets poised to capture bloodborne antigens. Marginal zone B cells are retained in this niche by integrin-mediated signaling induced by G protein-coupled receptors (GPCRs) and, likely, the B-cell receptor (BCR). Sphingosine-1-phosphate (S1P) signaling via the S1P family of GPCRs is known to be essential for B-cell localization in the marginal zone, but little is known about the downstream signaling events involved.

The SRC homology 2 domain protein Shep1 plays an important role in the penetration of olfactory sensory axons into the forebrain.

Shep1 is a multidomain signaling protein that forms a complex with Cas, a key scaffolding component of integrin signaling pathways, to promote the migration of non-neuronal cells. However, the physiological function of Shep1 in the nervous system remains unknown. Interestingly, we found that Shep1 and Cas are both concentrated in the axons of developing olfactory sensory neurons (OSNs).

The SH2 domain protein Shep1 regulates the in vivo signaling function of the scaffolding protein Cas.

The members of the p130Cas (Cas) family are important scaffolding proteins that orchestrate cell adhesion, migration and invasiveness downstream of integrin adhesion receptors and receptor tyrosine kinases by recruiting enzymes and structural molecules. Shep1, BCAR3/AND-34 and NSP1 define a recently identified family of SH2 domain-containing proteins that constitutively bind Cas proteins through a Cdc25-type nucleotide exchange factor-like domain. To gain insight into the functional interplay between Shep1 and Cas in vivo, we have inactivated the Shep1 gene in the mouse through Cre-mediated deletion of the exon encoding the SH2 domain.

Endothelial adherens and tight junctions in vascular homeostasis, inflammation and angiogenesis.

Endothelial cells lining the vessel wall are connected by adherens, tight and gap junctions. These junctional complexes are related to those found at epithelial junctions but with notable changes in terms of specific molecules and organization. Endothelial junctional proteins play important roles in tissue integrity but also in vascular permeability, leukocyte extravasation and angiogenesis.

Angiogenesis: the VE-cadherin switch.

Because angiogenesis is a key step in a number of pathologic processes, including tumor growth and atherosclerosis, many research studies have investigated the regulatory signals active at various stages of vascular invasion. The differential activities of the endothelial junction protein vascular endothelial (VE)-cadherin reflect the versatile behavior of endothelial cells between vascular quiescence and angiogenesis. VE-cadherin function and signaling are deeply modified in proliferating cells, and this conversion is accompanied by phosphorylation of the protein on tyrosine residues and enhanced transcription of its gene.

Vascular endothelial-cadherin tyrosine phosphorylation in angiogenic and quiescent adult tissues.

Vascular endothelial-cadherin (VE-cadherin) plays a key role in angiogenesis and in vascular permeability. The regulation of its biological activity may be a central mechanism in normal or pathological angiogenesis. VE-cadherin has been shown to be phosphorylated on tyrosine in vitro under various conditions, including stimulation by VEGF.