Targeting the IL1ß Pathway for Cancer Immunotherapy Remodels the Tumor Microenvironment and Enhances Antitumor Immune Responses.

High levels of IL1ß can result in chronic inflammation, which in turn can promote tumor growth and metastasis. Inhibition of IL1ß could therefore be a promising therapeutic option in the treatment of cancer. Here, the effects of IL1ß blockade induced by the mAbs canakinumab and gevokizumab were evaluated alone or in combination with docetaxel, anti-programmed cell death protein 1 (anti-PD-1), anti-VEGFα, and anti-TGFß treatment in syngeneic and humanized mouse models of cancers of different origin. Canakinumab and gevokizumab did not show notable efficacy as single-agent therapies; however, IL1ß blockade enhanced the effectiveness of docetaxel and anti-PD-1. Accompanying these effects, blockade of IL1ß alone or in combination induced significant remodeling of the tumor microenvironment (TME), with decreased numbers of immune suppressive cells and increased tumor infiltration by dendritic cells (DC) and effector T cells. Further investigation revealed that cancer-associated fibroblasts (CAF) were the cell type most affected by treatment with canakinumab or gevokizumab in terms of change in gene expression. IL1ß inhibition drove phenotypic changes in CAF populations, particularly those with the ability to influence immune cell recruitment. These results suggest that the observed remodeling of the TME following IL1ß blockade may stem from changes in CAF populations. Overall, the results presented here support the potential use of IL1ß inhibition in cancer treatment. Further exploration in ongoing clinical studies will help identify the best combination partners for different cancer types, cancer stages, and lines of treatment.

Inhibition of IL1ß by Canakinumab May Be Effective against Diverse Molecular Subtypes of Lung Cancer: An Exploratory Analysis of the CANTOS Trial.

In the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS), inhibition of the IL1ß inflammatory pathway by canakinumab has been shown to significantly reduce lung cancer incidence and mortality. Here we performed molecular characterization of CANTOS patients who developed lung cancer during the study, including circulating tumor DNA (ctDNA) and soluble inflammatory biomarker analysis. Catalogue of Somatic Mutations in Cancer (COSMIC) database ctDNA mutations were detected in 65% (46/71) of the CANTOS patients with lung cancer, with 51% (36/71) having detectable ctDNA at the time point closest to lung cancer diagnosis and 43% (29/67) having detectable ctDNA at trial randomization. Mutations commonly found in lung cancer were observed with no evidence of enrichment in any mutation following canakinumab treatment. Median time to lung cancer diagnosis in patients with (n = 29) versus without (n = 38) detectable COSMIC ctDNA mutations at baseline was 407 days versus 837 days (P = 0.011). For serum inflammatory biomarker analysis, circulating levels of C-reactive protein (CRP), IL6, IL18, IL1 receptor antagonist, TNFα, leptin, adiponectin, fibrinogen, and plasminogen activator inhibitor-1 were determined. Patients with the highest level of baseline CRP or IL6, both downstream of IL1ß signaling, trended toward a shorter time to lung cancer diagnosis. Other inflammation markers outside of the IL1ß pathway at baseline did not trend with time to lung cancer diagnosis. These results provide further evidence for the importance of IL1ß-mediated protumor inflammation in lung cancer and suggest canakinumab's effect may be mediated in part by delaying disease progression of diverse molecular subtypes of lung cancer. SIGNIFICANCE: These findings suggest that targeting the IL1ß inflammatory pathway might be critical in reducing tumor-promoting inflammation and lung cancer incidence.

A community-based lung cancer rapid tissue donation protocol provides high-quality drug-resistant specimens for proteogenomic analyses.

BACKGROUND: For the advancement of cancer research, the collection of tissue specimens from drug-resistant tumors after targeted therapy is crucial. Although patients with lung cancer are often provided targeted therapy, post-therapy specimens are not routinely collected due to the risks of collection, limiting the study of targeted therapy resistance mechanisms. Posthumous rapid tissue donation (RTD) is an expedient collection process that provides an opportunity to understand treatment-resistant lung cancers. METHODS: Consent to participate in the thoracic RTD protocol was obtained during patient care. When death occurred, tumor and paired non-tumor, cytology, and blood specimens were collected within 48 hours and preserved as formalin-fixed and frozen specimens. Tissue sections were evaluated with hematoxylin and eosin staining and immunohistochemistry (IHC) against multiple biomarkers, including various programmed death ligand 1 (PD-L1) clones. Next-generation sequencing was performed on 13 specimens from 5 patients. RESULTS: Postmortem specimens (N = 180) were well preserved from 9 patients with lung cancer. PD-L1 IHC revealed heterogeneity within and between tumors. An AGK-BRAF fusion was newly identified in tumor from a donor with a known echinoderm microtubule-associated protein-like 4 to anaplastic lymphoma kinase (EML4-ALK) fusion and history of anaplastic lymphoma kinase (ALK) inhibitor therapy. RNA expression analysis revealed a clonal genetic origin of metastatic cancer cells. CONCLUSIONS: Post-therapy specimens demonstrated PD-L1 heterogeneity and an acyl glycerol kinase to B-rapidly accelerated fibrosarcoma (AGK-BRAF) fusion in a patient with an EML4-ALK-positive lung adenocarcinoma as a potential resistance mechanism to ALK inhibitor therapy. Rapid tissue donation collection of postmortem tissue from lung cancer patients is a novel approach to cancer research that enables studies of molecular evolution and drug resistance.

Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage.

We report studies of preimplantation human embryo development that correlate time-lapse image analysis and gene expression profiling. By examining a large set of zygotes from in vitro fertilization (IVF), we find that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA). These parameters can be reliably monitored by automated image analysis, confirming that successful development follows a set of carefully orchestrated and predictable events. Moreover, we show that imaging phenotypes reflect molecular programs of the embryo and of individual blastomeres. Single-cell gene expression analysis reveals that blastomeres develop cell autonomously, with some cells advancing to EGA and others arresting. These studies indicate that success and failure in human embryo development is largely determined before EGA. Our methods and algorithms may provide an approach for early diagnosis of embryo potential in assisted reproduction.

High-efficiency stem cell fusion-mediated assay reveals Sall4 as an enhancer of reprogramming.

Several methods allow reprogramming of differentiated somatic cells to embryonic stem cell-like cells. However, the process of reprogramming remains inefficient and the underlying molecular mechanisms are poorly understood. Here, we report the optimization of somatic cell fusion with embryonic stem cells in order to provide an efficient, quantitative assay to screen for factors that facilitate reprogramming. Following optimization, we achieved a reprogramming efficiency 15-590 fold higher than previous protocols. This allowed observation of cellular events during the reprogramming process. Moreover, we demonstrate that overexpression of the Spalt transcription factor, Sall4, which was previously identified as a regulator of embryonic stem cell pluripotency and early mouse development, can enhance reprogramming. The reprogramming activity of Sall4 is independent of an N-terminal domain implicated in recruiting the nucleosome remodeling and deacetylase corepressor complex, a global transcriptional repressor. These results indicate that improvements in reprogramming assays, including fusion assays, may allow the systematic identification and molecular characterization of enhancers of somatic cell reprogramming.

A novel and critical role for Oct4 as a regulator of the maternal-embryonic transition.

BACKGROUND: Compared to the emerging embryonic stem cell (ESC) gene network, little is known about the dynamic gene network that directs reprogramming in the early embryo. We hypothesized that Oct4, an ESC pluripotency regulator that is also highly expressed at the 1- to 2-cell stages in embryos, may be a critical regulator of the earliest gene network in the embryo. METHODOLOGY/PRINCIPAL FINDINGS: Using antisense morpholino oligonucleotide (MO)-mediated gene knockdown, we show that Oct4 is required for development prior to the blastocyst stage. Specifically, Oct4 has a novel and critical role in regulating genes that encode transcriptional and post-transcriptional regulators as early as the 2-cell stage. Our data suggest that the key function of Oct4 may be to switch the developmental program from one that is predominantly regulated by post-transcriptional control to one that depends on the transcriptional network. Further, we propose to rank candidate genes quantitatively based on the inter-embryo variation in their differential expression in response to Oct4 knockdown. Of over 30 genes analyzed according to this proposed paradigm, Rest and Mta2, both of which have established pluripotency functions in ESCs, were found to be the most tightly regulated by Oct4 at the 2-cell stage. CONCLUSIONS/SIGNIFICANCE: We show that the Oct4-regulated gene set at the 1- to 2-cell stages of early embryo development is large and distinct from its established network in ESCs. Further, our experimental approach can be applied to dissect the gene regulatory network of Oct4 and other pluripotency regulators to deconstruct the dynamic developmental program in the early embryo.

The proximal cis-acting elements Sp1, Sp3 and E2F regulate mouse mer gene transcription in Sertoli cells.

Mer belongs to the Tyro 3 family of receptor tyrosine kinases (RTKs). Together with Axl and Rse, the three RTKs are believed to play important functional roles in the male gonads because gene knockout male mice lacking all of these receptors are infertile. In the present study, postnatal expression of Axl and Rse in mouse testes decreased during maturation while expression of Mer increased age-dependently during testicular development. To investigate the transcriptional regulation of gene expression in the testis, a approximately 1.5 kb fragment of the 5' flanking sequence of Mer was isolated. The sequence lacks a typical TATA or CAAT box. 5' RACE revealed that the putative major transcriptional start site of Mer is located at +102 bp upstream of the translation initiation site. Using transient transfections of luciferase reporter constructs driven by various lengths of the 5' flanking sequence, the gene segment -321/+126 showed the highest transcriptional activity in a mouse Sertoli cell line (TM4). DNAase I footprinting experiments revealed four footprints within the region from -321 to -26, including three binding sites for the transcriptional factor Specificity protein 1 (Sp1) and one for an unknown transcriptional factor. Electrophoretic mobility shift assay (EMSA), supershift assay, mutation studies and cotransfection demonstrated that those Sp1 cis-acting motifs interacted either with Sp1 or Sp1/Sp3, depending on location and the nearby nucleotide sequences. An E2F binding site which down-regulates Mer transcription, as revealed by EMSA, deletion and mutation studies, was identified downstream in the proximity of the promoter. Taking all of these data together, the study has demonstrated that Sp1, Sp3, E2F and probably another unknown transcriptional factor play a critical role in regulating the proximal promoter activities of Mer.