Overcoming T-cell exhaustion in LCH: PD-1 blockade and targeted MAPK inhibition are synergistic in a mouse model of LCH.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with persistent MAPK pathway activation. Standard-of-care chemotherapies are inadequate for most patients with multisystem disease, and optimal strategies for relapsed and refractory disease are not defined. The mechanisms underlying development of inflammation in LCH lesions, the role of inflammation in pathogenesis, and the potential for immunotherapy are unknown. Analysis of the immune infiltrate in LCH lesions identified the most prominent immune cells as T lymphocytes. Both CD8+ and CD4+ T cells exhibited "exhausted" phenotypes with high expression of the immune checkpoint receptors. LCH DCs showed robust expression of ligands to checkpoint receptors. Intralesional CD8+ T cells showed blunted expression of Tc1/Tc2 cytokines and impaired effector function. In contrast, intralesional regulatory T cells demonstrated intact suppressive activity. Treatment of BRAFV600ECD11c LCH mice with anti-PD-1 or MAPK inhibitor reduced lesion size, but with distinct responses. Whereas MAPK inhibitor treatment resulted in reduction of the myeloid compartment, anti-PD-1 treatment was associated with reduction in the lymphoid compartment. Notably, combined treatment with MAPK inhibitor and anti-PD-1 significantly decreased both CD8+ T cells and myeloid LCH cells in a synergistic fashion. These results are consistent with a model that MAPK hyperactivation in myeloid LCH cells drives recruitment of functionally exhausted T cells within the LCH microenvironment, and they highlight combined MAPK and checkpoint inhibition as a potential therapeutic strategy.

CNS Langerhans cell histiocytosis: Common hematopoietic origin for LCH-associated neurodegeneration and mass lesions.

BACKGROUND: Central nervous system Langerhans cell histiocytosis (CNS-LCH) brain involvement may include mass lesions and/or a neurodegenerative disease (LCH-ND) of unknown etiology. The goal of this study was to define the mechanisms of pathogenesis that drive CNS-LCH. METHODS: Cerebrospinal fluid (CSF) biomarkers including CSF proteins and extracellular BRAFV600E DNA were analyzed in CSF from patients with CNS-LCH lesions compared with patients with brain tumors and other neurodegenerative conditions. Additionally, the presence of BRAFV600E was tested in peripheral mononuclear blood cells (PBMCs) as well as brain biopsies from LCH-ND patients, and the response to BRAF-V600E inhibitor was evaluated in 4 patients with progressive disease. RESULTS: Osteopontin was the only consistently elevated CSF protein in patients with CNS-LCH compared with patients with other brain pathologies. BRAFV600E DNA was detected in CSF of only 2/20 (10%) cases, both with LCH-ND and active lesions outside the CNS. However, BRAFV600E+ PBMCs were detected with significantly higher frequency at all stages of therapy in LCH patients who developed LCH-ND. Brain biopsies of patients with LCH-ND demonstrated diffuse perivascular infiltration by BRAFV600E+ cells with monocyte phenotype (CD14+ CD33+ CD163+ P2RY12- ) and associated osteopontin expression. Three of 4 patients with LCH-ND treated with BRAF-V600E inhibitor experienced significant clinical and radiologic improvement. CONCLUSION: In LCH-ND patients, BRAFV600E+ cells in PBMCs and infiltrating myeloid/monocytic cells in the brain is consistent with LCH-ND as an active demyelinating process arising from a mutated hematopoietic precursor from which LCH lesion CD207+ cells are also derived. Therapy directed against myeloid precursors with activated MAPK signaling may be effective for LCH-ND. Cancer 2018;124:2607-20. © 2018 American Cancer Society.

Loss of glutaredoxin 3 impedes mammary lobuloalveolar development during pregnancy and lactation.

Mammalian glutaredoxin 3 (Grx3) has been shown to be important for regulating cellular redox homeostasis in the cell. Our previous studies indicate that Grx3 is significantly overexpressed in various human cancers including breast cancer and demonstrate that Grx3 controls cancer cell growth and invasion by regulating reactive oxygen species (ROS) and NF-κB signaling pathways. However, it remains to be determined whether Grx3 is required for normal mammary gland development and how it contributes to epithelial cell proliferation and differentiation in vivo. In the present study, we examined Grx3 expression in different cell types within the developing mouse mammary gland (MG) and found enhanced expression of Grx3 at pregnancy and lactation stages. To assess the physiological role of Grx3 in MG, we generated the mutant mice in which Grx3 was deleted specifically in mammary epithelial cells (MECs). Although the reduction of Grx3 expression had only minimal effects on mammary ductal development in virgin mice, it did reduce alveolar density during pregnancy and lactation. The impairment of lobuloalveolar development was associated with high levels of ROS accumulation and reduced expression of milk protein genes. In addition, proliferative gene expression was significantly suppressed with proliferation defects occurring in knockout MECs during alveolar development compared with wild-type controls. Therefore, our findings suggest that Grx3 is a key regulator of ROS in vivo and is involved in pregnancy-dependent mammary gland development and secretory activation through modulating cellular ROS.

In-silico QTL mapping of postpubertal mammary ductal development in the mouse uncovers potential human breast cancer risk loci.

Genetic background plays a dominant role in mammary gland development and breast cancer (BrCa). Despite this, the role of genetics is only partially understood. This study used strain-dependent variation in an inbred mouse mapping panel, to identify quantitative trait loci (QTL) underlying structural variation in mammary ductal development, and determined if these QTL correlated with genomic intervals conferring BrCa susceptibility in humans. For about half of the traits, developmental variation among the complete set of strains in this study was greater (P < 0.05) than that of previously studied strains, or strains in current common use for mammary gland biology. Correlations were also detected with previously reported variation in mammary tumor latency and metastasis. In-silico genome-wide association identified 20 mammary development QTL (Mdq). Of these, five were syntenic with previously reported human BrCa loci. The most significant (P = 1 × 10(-11)) association of the study was on MMU6 and contained the genes Plxna4, Plxna4os1, and Chchd3. On MMU5, a QTL was detected (P = 8 × 10(-7)) that was syntenic to a human BrCa locus on h12q24.5 containing the genes Tbx3 and Tbx5. Intersection of linked SNP (r(2) > 0.8) with genomic and epigenomic features, and intersection of candidate genes with gene expression and survival data from human BrCa highlighted several for further study. These results support the conclusion that mammary tumorigenesis and normal ductal development are influenced by common genetic factors and that further studies of genetically diverse mice can improve our understanding of BrCa in humans.

Developmental regulation of mitochondrial biogenesis and function in the mouse mammary gland during a prolonged lactation cycle.

The regulation of mitochondrial biogenesis and function in the lactating mammary cell is poorly understood. The goal of this study was to use proteomics to relate temporal changes in mammary cell mitochondrial function during lactation to changes in the proteins that make up this organelle. The hypothesis tested was that changes in mammary cell mitochondrial biogenesis and function during lactation would be accounted for by coordinated changes in the proteins of the electron transport chain and that some of these proteins might be linked by their expression patterns to PPARGC1α and AMP kinase. The mitochondrial proteome was studied along with markers of mitochondrial biogenesis and function in mammary tissue collected from mice over the course of a single prolonged lactation cycle. Mammary tissue concentrations of AMP and ADP were increased (P < 0.05) during early lactation and then declined with prolonged lactation. Similar changes were also observed for mitochondrial ATP synthesis activity, mitochondrial mass and DNA copy number. Analysis of the mammary cell mitochondrial proteome identified 244 unique proteins. Of these, only two proteins of the electron transport chain were found to increase during early lactation. In contrast, coordinated changes in numerous electron transport chain proteins were observed both during mid- and late lactation. There were six proteins that could be directly linked to PPARGC1α through network analysis. Abundance of PPARGC-1α and phosphorylation of AMP kinase was highest on day 2 postpartum. The results suggest that the increases in mammary mitochondria ATP synthesis activity during early lactation results from changes in only a limited number proteins. In addition, decreases in a handful of proteins linked to lipid oxidation could be temporally linked to decreases in PPARGC1α and phospho-AMP kinase suggesting potential roles for these proteins in coordinating mammary gland metabolism during early lactation.

Enhancement of maternal lactation performance during prolonged lactation in the mouse by mouse GH and long-R3-IGF-I is linked to changes in mammary signaling and gene expression.

GH, prolactin (PRL), and IGF-I stimulate lactation-related metabolic processes in mammary epithelial cells. However, the ability of these factors to stimulate milk production in animals varies depending on species and experimental variables. Previous work in our laboratory demonstrated that transgenic overexpression of des(1-3)IGF-I within the mammary glands of lactating mouse dams increased lactation capacity during prolonged lactation. This work also suggested that some of the effects of the overexpressed IGF-I may have been mediated through elevated concentrations of IGF-I or PRL in the systemic circulation. In the present study, murine GH and PRL, and a human IGF-I analog, long-R3-IGF-I (LR3), were administered as s.c. injections to compare their ability to enhance milk production, and alter mammary gland signaling and gene expression. Lactation capacity, as measured by litter gain, was increased (P<0.05) by GH, but not by PRL. LR3 increased (P<0.05) mammary phospho-Akt and suppressors of cytokines signaling 3 (SOCS3) gene expression, and had a modest ability to increase (P<0.05) lactation capacity. GH both increased (P<0.05) mammary SOCS1 expression and decreased (P<0.05) mammary expression of tryptophan hydroxylase 1, the rate-limiting enzyme in the synthesis of serotonin and a potential feedback inhibitor of lactation. These results suggest that while both GH and IGF-I stimulate milk production in the lactating mouse, the effect of GH may be additionally mediated through IGF-I-independent effects associated with repression of mammary serotonin synthesis.

Decreased lactation capacity and altered milk composition in insulin receptor substrate null mice is associated with decreased maternal body mass and reduced insulin-dependent phosphorylation of mammary Akt.

Expression of insulin receptor substrates (IRS)-1 and -2 within the mammary gland was found to be high at mid-lactation and dramatically decreased with mammary involution. This observation supports the hypothesis that these proteins are induced in the mammary gland with lactogenesis and involved in normal milk synthesis. To test this hypothesis, lactation capacity, along with indices of mammary secretory cell glucose metabolism and cell signaling were compared in normal mice and mice carrying targeted mutations in either the Irs1 or Irs2 genes. Mammary IRS-1 and IRS-2 protein levels were increased within 1 day of parturition and reached maximal levels by 5 days post partum. Dams carrying germline mutations of Irs1 or Irs2 displayed reduced lactation capacity as assessed by weight gain of pup litters. The reduction was more dramatic in Irs1(-/-) versus Irs2(-/-) dams. Maternal body weight was also reduced in Irs1(-/-) dams as well as in Irs1(+/-) Irs2(+/-) dams. The loss of IRS-1 had little impact on mammary gland expression of milk protein mRNAs, glucose transport, or on the abundance and subcellular localization of hexokinases I and II. The loss of IRS-1 was associated with a compensatory increase in insulin-induced IRS-2 phosphorylation; however, the loss of IRS-1 did also cause a reduction in insulin-dependent mammary gland-specific activation of Akt phosphorylation. These results support the conclusion that IRS-1 is important for insulin-dependent activation of Akt signaling within the lactating mammary gland, but that loss of this protein has only modest impact on normal milk synthesis, since related signaling proteins such as IRS-2 may act in compensatory fashion.