Targeting integrin α5 in fibroblasts potentiates colorectal cancer response to PD-L1 blockade by affecting extracellular-matrix deposition.

BACKGROUND: One reason patients with cancer cannot benefit from immunotherapy is the lack of immune cell infiltration in tumor tissues. Cancer-associated fibroblasts (CAFs) are emerging as central players in immune regulation that shapes tumor microenvironment (TME). Earlier we reported that integrin α5 was enriched in CAFs in colorectal cancer (CRC), however, its role in TME and cancer immunotherapy remains unclear. Here, we aimed to investigate the role for integrin α5 in fibroblasts in modulating antitumor immunity and therapeutic efficacy combined with checkpoint blockade in CRC. METHODS: We analyzed the CRC single-cell RNA sequencing (scRNA-seq) database to define the expression of ITGA5 in CRC tumor stroma. Experimentally, we carried out in vivo mouse tumor xenograft models to confirm the targeting efficacy of combined α5ß1 inhibition and anti-Programmed death ligand 1 (PD-L1) blockade and in vitro cell-co-culture assay to investigate the role of α5 in fibroblasts in affecting T-cell activity. Clinically, we analyzed the association between α5 expression and infiltrating T cells and evaluated their correlation with patient survival and immunotherapy prognosis in CRC. RESULTS: We revealed that ITGA5 was enriched in FAP-CAFs. Both ITGA5 knockout fibroblasts and therapeutic targeting of α5 improved response to anti-PD-L1 treatment in mouse subcutaneous tumor models. Mechanistically, these treatments led to increased tumor-infiltrating CD8+ T cells. Furthermore, we found that α5 in fibroblasts correlated with extracellular matrix (ECM)-related genes and affected ECM deposition in CRC tumor stroma. Both in vivo analysis and in vitro culture and cell killing experiment showed that ECM proteins and α5 expression in fibroblasts influence T-cell infiltration and activity. Clinically, we confirmed that high α5 expression was associated with fewer CD3+ T and CD8+ T cells, and tissues with low α5 and high CD3+ T levels correlated with better patient survival and immunotherapy response in a CRC cohort with 29 patients. CONCLUSIONS: Our study identified a role for integrin α5 in fibroblasts in modulating antitumor immunity by affecting ECM deposition and showed therapeutic efficacy for combined α5ß1 inhibition and PD-L1 blockade in CRC.

Emerging and potential use of CRISPR in human liver disease.

CRISPR is a gene editing tool adapted from naturally occurring defense systems from bacteria. It is a technology that is revolutionizing the interrogation of gene functions in driving liver disease, especially through genetic screens and by facilitating animal knockout and knockin models. It is being used in models of liver disease to identify which genes are critical for liver pathology, especially in genetic liver disease, hepatitis, and in cancer initiation and progression. It holds tremendous promise in treating human diseases directly by editing DNA. It could disable gene function in the case of expression of a maladaptive protein, such as blocking transthyretin as a therapy for amyloidosis, or to correct gene defects, such as restoring the normal functions of liver enzymes fumarylacetoacetate hydrolase or alpha-1 antitrypsin. It is also being studied for treatment of hepatitis B infection. CRISPR is an exciting, evolving technology that is facilitating gene characterization and discovery in liver disease and holds the potential to treat liver diseases safely and permanently.

Lipid Nanoparticle Delivery of Small Proteins for Potent In Vivo RAS Inhibition.

Mutated RAS proteins are potent oncogenic drivers and have long been considered "undruggable". While RAS-targeting therapies have recently shown promise, there remains a clinical need for RAS inhibitors with more diverse targets. Small proteins represent a potential new therapeutic option, including K27, a designed ankyrin repeat protein (DARPin) engineered to inhibit RAS. However, K27 functions intracellularly and is incapable of entering the cytosol on its own, currently limiting its utility. To overcome this barrier, we have engineered a lipid nanoparticle (LNP) platform for potent delivery of functional K27-D30─a charge-modified version of the protein─intracellularly in vitro and in vivo. This system efficiently encapsulates charge-modified proteins, facilitates delivery in up to 90% of cells in vitro, and maintains potency after at least 45 days of storage. In vivo, these LNPs deliver K27-D30 to the cytosol of cancerous cells in the liver, inhibiting RAS-driven growth and ultimately reducing tumor load in an HTVI-induced mouse model of hepatocellular carcinoma. This work shows that K27 holds promise as a new cancer therapeutic when delivered using this LNP platform. Furthermore, this technology has the potential to broaden the use of LNPs to include new cargo types─beyond RNA─for diverse therapeutic applications.

A Therapeutically Targetable TAZ-TEAD2 Pathway Drives the Growth of Hepatocellular Carcinoma via ANLN and KIF23.

BACKGROUND & AIMS: Despite recent progress, long-term survival remains low for hepatocellular carcinoma (HCC). The most effective HCC therapies target the tumor immune microenvironment (TIME), and there are almost no therapies that directly target tumor cells. Here, we investigated the regulation and function of tumor cell-expressed Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in HCC. METHODS: HCC was induced in mice by Sleeping Beauty-mediated expression of MET, CTNNB1-S45Y, or TAZ-S89A, or by diethylnitrosamine plus CCl4. Hepatocellular TAZ and YAP were deleted in floxed mice via adeno-associated virus serotype 8-mediated expression of Cre. TAZ target genes were identified from RNA sequencing, confirmed by chromatin immunoprecipitation, and evaluated in a clustered regularly interspaced short palindromic repeats interference (CRISPRi) screen. TEA domain transcription factors (TEADs), anillin (ANLN), Kif23, and programmed cell death protein ligand 1 were knocked down by guide RNAs in dead clustered regularly interspaced short palindromic repeats-associated protein 9 (dCas9) knock-in mice. RESULTS: YAP and TAZ were up-regulated in murine and human HCC, but only deletion of TAZ consistently decreased HCC growth and mortality. Conversely, overexpression of activated TAZ was sufficient to trigger HCC. TAZ expression in HCC was regulated by cholesterol synthesis, as demonstrated by pharmacologic or genetic inhibition of 3-hydroxy-3-methylglutaryl- coenzyme A reductase (HMGCR), farnesyl pyrophosphate synthase, farnesyl-diphosphate farnesyltransferase 1 (FDFT1), or sterol regulatory element-binding protein 2 (SREBP2). TAZ- and MET/CTNNB1-S45Y-driven HCC required the expression of TEAD2 and, to a lesser extent, TEAD4. Accordingly, TEAD2 displayed the most profound effect on survival in patients with HCC. TAZ and TEAD2 promoted HCC via increased tumor cell proliferation, mediated by TAZ target genes ANLN and kinesin family member 23 (KIF23). Therapeutic targeting of HCC, using pan-TEAD inhibitors or the combination of a statin with sorafenib or anti-programmed cell death protein 1, decreased tumor growth. CONCLUSIONS: Our results suggest the cholesterol-TAZ-TEAD2-ANLN/KIF23 pathway as a mediator of HCC proliferation and tumor cell-intrinsic therapeutic target that could be synergistically combined with TIME-targeted therapies.

Integrin α5 subunit is required for the tumor supportive role of fibroblasts in colorectal adenocarcinoma and serves as a potential stroma prognostic marker.

The tumorigenesis of colorectal cancer (CRC) is a complicated process, involving interactions between cancer cells and the microenvironment. The role of α5 integrin subunit in CRC remains controversial, and previous studies mainly focused on cancer cells. Herein, we report an important role of α5 in stroma fibroblasts in the tumorigenesis of CRC. The expression of α5 was found to be located in colorectal tumor stroma rather than in epithelia cancer cells. Immunofluorescence colocalization and gene correlation analysis confirmed that α5 was mainly expressed in cancer-associated fibroblasts (CAFs). Moreover, experimental evidence showed that α5 expression was required for the tumor-promoting effect of fibroblast cells. In an in vivo xenograft nude mice model, α5 depletion in fibroblasts dramatically suppressed fibroblast-induced tumor growth. In an in vitro cell coculture assay, α5 depletion or knockdown reduced the ability of fibroblasts to promote cancer cell migration and invasion compared with wild-type fibroblasts; moreover, we observed that the expression and assembly of fibronectin were downregulated after α5 depletion or knockdown in fibroblasts. Analysis of the RNA-Seq data of the Cancer Genome Atlas cohort revealed that high expression of ITGA5 (α5 integrin subunit) was correlated with poor overall survival in colorectal adenocarcinoma, which was further confirmed by immunohistochemistry in an independent cohort of 355 patients. Thus, our study identifies α5 integrin subunit as a novel stroma molecular marker for colorectal adenocarcinoma, offers a fresh insight into colorectal adenocarcinoma progression, and shows that α5 expression in stroma fibroblasts underlies its ability to promote the tumorigenesis of colorectal adenocarcinoma.

Tankyrase Mediates K63-Linked Ubiquitination of JNK to Confer Stress Tolerance and Influence Lifespan in Drosophila.

Tankyrase (Tnks) transfers poly(ADP-ribose) on substrates. Whereas studies have highlighted the pivotal roles of Tnks in cancer, cherubism, systemic sclerosis, and viral infection, the requirement for Tnks under physiological contexts remains unclear. Here, we report that the loss of Tnks or its muscle-specific knockdown impairs lifespan, stress tolerance, and energy homeostasis in adult Drosophila. We find that Tnks is a positive regulator in the JNK signaling pathway, and modest alterations in the activity of JNK signaling can strengthen or suppress the Tnks mutant phenotypes. We further identify JNK as a direct substrate of Tnks. Although Tnks-dependent poly-ADP-ribosylation is tightly coupled to proteolysis in the proteasome, we demonstrate that Tnks initiates degradation-independent ubiquitination on two lysine residues of JNK to promote its kinase activity and in vivo functions. Our study uncovers a type of posttranslational modification of Tnks substrates and provides insights into Tnks-mediated physiological roles.

Systematic screening of a Drosophila ORF library in vivo uncovers Wnt/Wg pathway components.

We created a site-directed UAS-ORF library of 655 growth-regulating genes in Drosophila. This library represents a large collection of genes regulating cell cycle, cell size, and proliferation and will be a valuable resource for studying growth regulation in vivo. By using misexpression of genes, we prevent problems arising from genetic redundancy and can uncover novel gene functions. To validate the usefulness of this library, we screened for Wingless (Wg) pathway components. We used a combination of experimental and bioinformatic approaches to predict candidates and identified three serine/threonine kinases as regulators of Wg signaling. We show that one of these, Nek2, optimizes pathway response by direct phosphorylation of Dishevelled. In addition, we describe functional relations for roughly 5% of all Drosophila genes and identify a large number of genes that regulate cell size, proliferation, and final organ size upon misexpression.