Therapeutic targeting of cellular senescence in diabetic macular edema: preclinical and phase 1 trial results.

Compromised vascular endothelial barrier function is a salient feature of diabetic complications such as sight-threatening diabetic macular edema (DME). Current standards of care for DME manage aspects of the disease, but require frequent intravitreal administration and are poorly effective in large subsets of patients. Here we provide evidence that an elevated burden of senescent cells in the retina triggers cardinal features of DME pathology and conduct an initial test of senolytic therapy in patients with DME. In cell culture models, sustained hyperglycemia provoked cellular senescence in subsets of vascular endothelial cells displaying perturbed transendothelial junctions associated with poor barrier function and leading to micro-inflammation. Pharmacological elimination of senescent cells in a mouse model of DME reduces diabetes-induced retinal vascular leakage and preserves retinal function. We then conducted a phase 1 single ascending dose safety study of UBX1325 (foselutoclax), a senolytic small-molecule inhibitor of BCL-xL, in patients with advanced DME for whom anti-vascular endothelial growth factor therapy was no longer considered beneficial. The primary objective of assessment of safety and tolerability of UBX1325 was achieved. Collectively, our data suggest that therapeutic targeting of senescent cells in the diabetic retina with a BCL-xL inhibitor may provide a long-lasting, disease-modifying intervention for DME. This hypothesis will need to be verified in larger clinical trials. ClinicalTrials.gov identifier: NCT04537884 .

Reprogramming T cell differentiation and exhaustion in CAR-T cell therapy.

T cell differentiation is a highly regulated, multi-step process necessary for the progressive establishment of effector functions, immunological memory, and long-term control of pathogens. In response to strong stimulation, as seen in severe or chronic infections or cancer, T cells acquire a state of hypo-responsiveness known as exhaustion, limiting their effector function. Recent advances in autologous chimeric antigen receptor (CAR)-T cell therapies have revolutionized the treatment of hematologic malignancies by taking advantage of the basic principles of T cell biology to engineer products that promote long-lasting T cell response. However, many patients' malignancies remain unresponsive to treatment or are prone to recur. Discoveries in T cell biology, including the identification of key regulators of differentiation and exhaustion, offer novel opportunities to have a durable impact on the fate of CAR-T cells after infusion. Such next-generation CAR-T cell therapies and their clinical implementation may result in the next leap forward in cancer treatment for selected patients. In this context, this review summarizes the foundational principles of T cell differentiation and exhaustion and describes how they can be utilized and targeted to further improve the design and efficacy of CAR-T cell therapies.

PCA062, a P-cadherin Targeting Antibody-Drug Conjugate, Displays Potent Antitumor Activity Against P-cadherin-expressing Malignancies.

The cell surface glycoprotein P-cadherin is highly expressed in a number of malignancies, including those arising in the epithelium of the bladder, breast, esophagus, lung, and upper aerodigestive system. PCA062 is a P-cadherin specific antibody-drug conjugate that utilizes the clinically validated SMCC-DM1 linker payload to mediate potent cytotoxicity in cell lines expressing high levels of P-cadherin in vitro, while displaying no specific activity in P-cadherin-negative cell lines. High cell surface P-cadherin is necessary, but not sufficient, to mediate PCA062 cytotoxicity. In vivo, PCA062 demonstrated high serum stability and a potent ability to induce mitotic arrest. In addition, PCA062 was efficacious in clinically relevant models of P-cadherin-expressing cancers, including breast, esophageal, and head and neck. Preclinical non-human primate toxicology studies demonstrated a favorable safety profile that supports clinical development. Genome-wide CRISPR screens reveal that expression of the multidrug-resistant gene ABCC1 and the lysosomal transporter SLC46A3 differentially impact tumor cell sensitivity to PCA062. The preclinical data presented here suggest that PCA062 may have clinical value for treating patients with multiple cancer types including basal-like breast cancer.

Integrins as novel drug targets for overcoming innate drug resistance.

Acquired drug resistance continues to be one of the major obstacles hindering the successful treatment of many forms of cancer. Compounds utilized as antagonists of these cytoprotective mechanisms have, for the most part, proven to be ineffective at overcoming clinical resistance to cytotoxic drugs. Recently, the tumor cell microenvironment has been found to have a significant bearing on the survival of tumor cells following exposure to a wide variety of anti-neoplastic agents, prior to the acquisition of known drug resistance mechanisms. Specifically, interactions between cell surface integrins and extracellular matrix components have been shown to be responsible for this phenomenon of innate drug resistance, which we have termed Cell Adhesion Mediated Drug Resistance, or CAM-DR. Following its discovery using a multiple myeloma cell line model, evidence for CAM-DR has been found in a multitude of other human tumor cell types. In contrast to many other drug resistance mechanisms, integrin-mediated cell signaling is capable of protecting against death induced by an extremely wide variety of structurally and functionally diverse agents from traditional DNA damaging agents to the promising novel kinase inhibitor STI-571. This review examines the role of integrins in regard to their ability to protect tumor cells from drug- and radiation-induced apoptosis through numerous intracellular mechanisms. Current and future antagonists of specific integrin heterodimers may have the potential to sensitize tumor cells when used in combination with standard chemotherapy regimens. Specific signal transduction pathways initiated by integrin ligation will also be discussed as potential bridge points for inhibiting cell survival during cytotoxic drug exposure.

Heterotypic interactions among NACHT domains: implications for regulation of innate immune responses.

Proteins of the NACHT [NAIP (neuronal apoptosis inhibitory protein), CIITA (MHC class II transcription activator), HET-E (incompatibility locus protein from Podospora anserina) and TP1 (telomerase-associated protein)] family may serve as critical pathogen-sensing and signal-transducing molecules within the innate immune system. In the present paper, we show that CLAN [CARD (caspase-recruitment domain), LRR (leucine-rich repeat) and NACHT domain-containing protein], a NACHT-containing protein originally demonstrated to bind and activate pro-caspase 1, is also capable of influencing the functions of other members of the NACHT family. Through heterotypic NACHT-domain interactions, CLAN was found to associate with Nod1, Nod2 and NAC [nucleotide-binding domain and CARD-containing protein; NALP1 (NACHT, LRR and PYRIN protein 1)] when co-expressed in HEK-293T (human embryonic kidney) cells. NF-kappaB (nuclear factor kappaB) reporter assays demonstrated that co-expression of either full-length CLAN or the NACHT domain of CLAN significantly inhibited NF-kappaB activation induced by Nod1 or Nod2 overexpression. In addition, co-expression of CLAN or the NACHT domain of CLAN with Nod1 or Nod2 inhibited the ability of these proteins to generate active IL-1beta (interleukin 1beta) through their association with pro-caspase 1. The NACHT domain of CLAN was demonstrated by co-immunoprecipitation experiments to bind all NACHT domains that were tested, including the NACHT domains from CLAN itself, Nod1, Nod2, cryopyrin, NAC, PAN2 [PAAD [pyrin, AIM (absent-in-melanoma), ASC (apoptosis-associated speck-like protein containing a CARD) and death-domain-like]- and NACHT-containing protein] and NAIP (neuronal apoptosis inhibitory protein). Finally, monocyte-expressed CLAN was found to associate with Nod2 following exposure to bacterial peptidoglycan, implying a regulatory role for interaction of these NACHT proteins in the innate immune response. These studies suggest that by mediating hetero-oligomerization, NACHT domains provide a means by which various NACHT-containing proteins may interact, creating protein-interaction networks that potentially modulate immune responses to invading pathogens.

CARD proteins as therapeutic targets in cancer.

Proteins containing a caspase-associated recruitment domain (CARD) have been established as key regulators of cell death and, more recently, cytokine production. During the last several years, the number of proteins identified within this family has grown immensely and many aspects of their function point to their potential utility as novel drug targets in the treatment of cancer. Several CARD family proteins are critical components of the conserved cell death machinery which, when dysregulated, promotes oncogenesis and contributes prominently to tumor resistance to chemotherapy. The pro-apoptotic protein Apaf1, which is inactivated in some cancers, is a CARD protein that is indispensable for mitochondria-induced apoptosis. Other anti-apoptotic CARD proteins, such as TUCAN/CARDINAL/CARD8, have been shown to protect tumors from cell death stimuli and to be over-expressed in certain forms of cancer. Therapeutics that activate or inhibit CARD proteins may therefore be potentially utilized as novel chemo-sensitizing agents when used in conjunction with conventional chemotherapy. Other CARD proteins influence cellular processes through the regulation of NF-kappaB or caspase-1, which governs the levels of interleukin-1beta (IL-1beta). In addition to its pro-inflammatory properties, this cytokine also contributes to neoplastic progression by promoting angiogenesis, proliferation, and the metastasis of many tumors. Many of the IL-1beta-regulating CARD proteins also contain a nucleotide binding/oligomerization domain known as a NACHT and may therefore be amenable to targeting by small molecule compounds. This review examines the role of CARD proteins in cytoprotection and cytokine processing in the context of neoplasia and presents strategies for using this information in devising potential novel anticancer agents.

Molecular pathways: blockade of the PRLR signaling pathway as a novel antihormonal approach for the treatment of breast and prostate cancer.

The prolactin (PRL)-prolactin receptor (PRLR) signaling complex has been implicated in the pathology of breast and prostate carcinoma. A multitude of pro-oncogenic intracellular signaling pathways are activated by PRL in breast and prostate epithelial cells, leading to enhanced cellular proliferation, survival, and tumorigenesis in numerous model systems. Emerging evidence suggests that targeting the PRL-PRLR axis in human cancer may represent an unexploited avenue for therapeutic intervention and, given the extensive cross-talk between PRLR and other signal transduction pathways, a potential means through which other anticancer agents could be rendered more efficacious in the clinic. LFA102 is a potent anti-PRLR neutralizing antibody that efficiently abrogates the function of this receptor in vivo, mediating significant antitumor effects in preclinical models. The clean safety profile of this antibody in animals and in the clinical experiences to date suggests that blocking the PRLR signaling pathway in human tumors may have few significant toxicologic consequences and may be a promising approach to treating cancer. A phase I trial in patients with breast and prostate cancer is underway to better understand the clinical utility of LFA102 and the contribution of PRL to the maintenance and progression of human cancer.

Neutralization of prolactin receptor function by monoclonal antibody LFA102, a novel potential therapeutic for the treatment of breast cancer.

Numerous lines of evidence suggest that the polypeptide hormone prolactin (PRL) may contribute to breast and prostate tumorigenesis through its interactions with the prolactin receptor (PRLR). Here, we describe the biologic properties of LFA102, a humanized neutralizing monoclonal antibody directed against the extracellular domain of PRLR. This antibody was found to effectively antagonize PRL-induced signaling in breast cancer cells in vitro and in vivo and to block PRL-induced proliferation in numerous cell line models, including examples of autocrine/paracrine PRL activity. A single administration of LFA102 resulted in regression of PRL-dependent Nb2-11 tumor xenografts and significantly prolonged time to progression. Finally, LFA102 treatment significantly inhibited PRLR signaling as well as tumor growth in a carcinogen-induced, estrogen receptor-positive rat mammary cancer model as a monotherapy and enhanced the efficacy of the aromatase inhibitor letrozole when administered in combination. The biologic properties of LFA102, elucidated by the preclinical studies presented here, suggest that this antibody has the potential to be a first-in-class, effective therapeutic for the treatment of PRL-dependent cancers.

Multiple roles of CLAN (caspase-associated recruitment domain, leucine-rich repeat, and NAIP CIIA HET-E, and TP1-containing protein) in the mammalian innate immune response.

NAIP CIIA HET-E and TP1 (NACHT) family proteins are involved in sensing intracellular pathogens or pathogen-derived molecules, triggering host defense responses resulting in caspase-mediated processing of proinflammatory cytokines and NF-kappaB activation. Caspase-associated recruitment domain, leucine-rich repeat, and NACHT-containing protein (CLAN), also known as ICE protease-activating factor, belongs to a branch of the NACHT family that contains proteins carrying caspase-associated recruitment domains (CARDs) and leucine-rich repeats (LRRs). By using gene transfer and RNA-interference approaches, we demonstrate in this study that CLAN modulates endogenous caspase-1 activation and subsequent IL-1beta secretion from human macrophages after exposure to LPS, peptidoglycan, and pathogenic bacteria. CLAN was also found to mediate a direct antibacterial effect within macrophages after Salmonella infection and to sensitize host cells to Salmonella-induced cell death through a caspase-1-independent mechanism. These results indicate that CLAN contributes to several biological processes central to host defense, suggesting a prominent role for this NACHT family member in innate immunity.

Comparative analysis of apoptosis and inflammation genes of mice and humans.

Apoptosis (programmed cell death) plays important roles in many facets of normal mammalian physiology. Host-pathogen interactions have provided evolutionary pressure for apoptosis as a defense mechanism against viruses and microbes, sometimes linking apoptosis mechanisms with inflammatory responses through NFkappaB induction. Proteins involved in apoptosis and NFkappaB induction commonly contain evolutionarily conserved domains that can serve as signatures for identification by bioinformatics methods. Using a combination of public (NCBI) and private (RIKEN) databases, we compared the repertoire of apoptosis and NFkappaB-inducing genes in humans and mice from cDNA/EST/genomic data, focusing on the following domain families: (1) Caspase proteases; (2) Caspase recruitment domains (CARD); (3) Death Domains (DD); (4) Death Effector Domains (DED); (5) BIR domains of Inhibitor of Apoptosis Proteins (IAPs); (6) Bcl-2 homology (BH) domains of Bcl-2 family proteins; (7) Tumor Necrosis Factor (TNF)-family ligands; (8) TNF receptors (TNFR); (9) TIR domains; (10) PAAD (PYRIN; PYD, DAPIN); (11) nucleotide-binding NACHT domains; (12) TRAFs; (13) Hsp70-binding BAG domains; (14) endonuclease-associated CIDE domains; and (15) miscellaneous additional proteins. After excluding redundancy due to alternative splice forms, sequencing errors, and other considerations, we identified cDNAs derived from a total of 227 human genes among these domain families. Orthologous murine genes were found for 219 (96%); in addition, several unique murine genes were found, which appear not to have human orthologs. This mismatch may be due to the still fragmentary information about the mouse genome or genuine differences between mouse and human repertoires of apoptotic genes. With this caveat, we discuss similarities and differences in human and murine genes from these domain families.