Anti-tumor activity of a novel LAIR1 antagonist in combination with anti-PD-1 to treat collagen-rich solid tumors.

We recently reported that resistance to PD-1-blockade in a refractory lung cancer-derived model involved increased collagen deposition and the collagen-binding inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), and thus we hypothesized that LAIR1 and collagen cooperated to suppress therapeutic response. Here, we report LAIR1 is associated with tumor stroma and is highly expressed by intratumoral myeloid cells in both human tumors and mouse models of cancer. Stroma-associated myeloid cells exhibit a suppressive phenotype and correlate with LAIR1 expression in human cancer. NGM438, a novel humanized LAIR1 antagonist monoclonal antibody, elicits myeloid inflammation and allogeneic T cell responses by binding to LAIR1 and blocking collagen engagement. Further, a mouse-reactive NGM438 surrogate antibody sensitized refractory KP mouse lung tumors to anti-PD-1 therapy and resulted in increased intratumoral CD8+ T cell content and inflammatory gene expression. These data place LAIR1 at the intersection of stroma and suppressive myeloid cells and support the notion that blockade of the LAIR1/collagen axis can potentially address resistance to checkpoint inhibitor therapy in the clinic.

ILT2 and ILT4 Drive Myeloid Suppression via Both Overlapping and Distinct Mechanisms.

Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.

Forskolin Stimulates Estrogen Receptor (ER) α Transcriptional Activity and Protects ER from Degradation by Distinct Mechanisms.

Estradiol action is mediated by estrogen receptors (ERs), a and ß. Estradiol binding initiates ER-mediated transcription and ER degradation, the latter of which occurs via the ubiquitin-proteasome pathway. Inhibition of proteasome activity prevents estradiol-induced ERα degradation and transactivation. In ER-positive GH3 cells (a rat pituitary prolactinoma cell line), forskolin, acting via protein kinase A (PKA), stimulates ERα transcriptional activity without causing degradation, and proteasome inhibition does not block forskolin-stimulated transcription. Forskolin also protects liganded ERα from degradation. In the current study, we first examined ERα and ERß transcriptional activity in ER-negative HT22 cells and found that forskolin stimulated ERα-, but not ERß-dependent transcription, through the ligand-binding domain (LBD). We also identified four mutations (L396R, D431Y, Y542F, and K534E/M548V) on the ERα LBD that selectively obliterated the response to forskolin. In GH3 cells, transfected ERα mutants and ERß were protected from degradation by forskolin. Ubiquitination of ERα and ERß was increased by forskolin or estradiol. ERα ubiquitination was diminished by a mutated ubiquitin (K48R) that prevents elongation of polyubiquitin chains for targeting the proteasome. Increased ERα ubiquitination was not affected by the deletion of the A/B domain but significantly diminished in the F domain deletion mutant. Our results indicate distinct and novel mechanisms for forskolin stimulation of ERα transcriptional activity and protection from ligand-induced degradation. It also suggests a unique mechanism by which forskolin increases unliganded and liganded ERα and ERß ubiquitination but uncouples them from proteasome-mediated degradation regardless of their transcriptional responses to forskolin.

The Fibronectin-ILT3 Interaction Functions as a Stromal Checkpoint that Suppresses Myeloid Cells.

Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, ex vivo treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3-fibronectin interaction represents a "stromal checkpoint" through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.

FGF21 contributes to neuroendocrine control of female reproduction.

Preventing reproduction during nutritional deprivation is an adaptive process that is conserved and essential for the survival of species. In mammals, the mechanisms that inhibit fertility during starvation are complex and incompletely understood. Here we show that exposure of female mice to fibroblast growth factor 21 (FGF21), a fasting-induced hepatokine, mimics infertility secondary to starvation. Mechanistically, FGF21 acts on the suprachiasmatic nucleus (SCN) in the hypothalamus to suppress the vasopressin-kisspeptin signaling cascade, thereby inhibiting the proestrus surge in luteinizing hormone. Mice lacking the FGF21 co-receptor, ß-Klotho, in the SCN are refractory to the inhibitory effect of FGF21 on female fertility. Thus, FGF21 defines an important liver-neuroendocrine axis that modulates female reproduction in response to nutritional challenge.

Inhibition of growth hormone signaling by the fasting-induced hormone FGF21.

Starvation blocks the actions of growth hormone (GH) and inhibits growth through mechanisms that are not well understood. In this report, we demonstrate that fibroblast growth factor 21 (FGF21), a hormone induced by fasting, causes GH resistance. In liver, FGF21 reduces concentrations of the active form of signal transducer and activator of transcription 5 (STAT5), a major mediator of GH actions, and causes corresponding decreases in the expression of its target genes, including insulin-like growth factor 1 (IGF-1). FGF21 also induces hepatic expression of IGF-1 binding protein 1 and suppressor of cytokine signaling 2, which blunt GH signaling. Chronic exposure to FGF21 markedly inhibits growth in mice. These data suggest a central role for FGF21 in inhibiting growth as part of its broader role in inducing the adaptive response to starvation.

Truncated estrogen receptor product-1 stimulates estrogen receptor alpha transcriptional activity by titration of repressor proteins.

The truncated estrogen receptor product-1 (TERP-1, or TERP) is a pituitary-specific isoform of estrogen receptor alpha (ERalpha), and its expression is regulated by estrogen. TERP modulates the transcriptional activity of ERalpha but has no independent effect on transcription of estrogen-response element-containing promoters. At low concentrations, TERP stimulates ERalpha transcriptional activity in transient transfection assays. At TERP concentrations equal to or greater than full-length ERalpha, TERP forms dimers with ERalpha and reduces both ligand-dependent and -independent transcription. A dimerization mutant of TERP, TERP L509R, stimulated ERalpha transcription at all concentrations. We hypothesized that TERP stimulates ERalpha transcriptional activity by titrating suppressors of ERalpha activity. We found that repressor of estrogen receptor activity (REA), originally isolated from human breast cancer cells, is present in mouse pituitary gonadotrope cell lines. Levels of REA vary slightly throughout the rat reproductive cycle, but TERP mRNA and protein vary much more dramatically. In transfection experiments, REA suppressed ERalpha transcriptional activity, and TERP L509R was able to alleviate transcriptional suppression by REA. In glutathione S-transferase pull-down assays, TERP bound to REA more efficiently than did ERalpha at equivalent concentrations, suggesting that REA will preferentially bind to TERP. Our findings suggest that the stimulation of pituitary ERalpha activity by low concentrations of TERP can occur by titration of corepressors such as REA.