Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe reversal of immune tolerance represents one central goal in cancer immune therapies and serves as a rationale for developing [3,5]. Furthermore, these drugs can enhance the immunogenicity of the tumor epithelium, and as well change the immunosuppressive cytokine milieu produced by the tumor and its microenvironment, thereby facilitating the maturation and function of effector cells in innate and adaptive immunity [6]. The immunomodulatory effects of established anticancer drugs are also exploited to improve tumor vaccination protocols. An important animal model used in these studies is FVB/ N-MMTV-neu transgenic mice developing mammary cancer due to overexpression of neu, the normal rat homologue of HER2/erbB2 in the mammary gland [7]. These mice are immunotolerant to neu [8,9], but can be vaccinated with neu-directed vaccines to prevent tumor formation in combination with appropriate adjuvants such as GM-CSF, IL-12, and cyclophosphamide [10,11]. The IFN-induced transcription factor Stat1 has been described as important mediator of the antitumor response [12]. As a key regulator of innate as well as adaptive immunity, Stat1 is involved in immune surveillance [13] but has also been postulated to act as a tumor suppressor by tumor epithelium intrinsic mechanisms. As has been shown recently, Stat1-deficient mice spontaneously develop mammary tumors [14,15]. In MMTV-neu mammary tumor mice, deletion of Stat1 in the tumor epithelium as well as in the tumor stroma was shown to contribute to accelerated tumorigenesis [16,17]. The aim of the present study was to investigate this issue further in MMTV-neu mice as an animal model for erbB2-positive breast cancer, treated in vivo with two different types of drugs, the dual tyrosine kinase inhibitor lapatini which targets HER2/erbB2/neu and EGFR/erbB1, and the genotoxic anthracycline drug doxorubicin.Here, we show that in MMTV-neu mice the in vivo efficacy of lapatinib and/or doxorubicin treatment is dependent on CD8 . Both Stat1-deficient and -proficient mice developed mammary tumors with no significant differences in expression levels for erbB2 and in tumor histology ( Fig. 1A and Supporting Information Fig. 1).As described previously for other mouse models of erbB2-positive breast cancer [16,17], Stat1 deficiency resulted in a slight acceleration of the development of palpable tumors. Furthermore, we observed an increase in tumor multiplicity, with lowered levels of caspase 3 cleavage in the tumor and a slight increase of the fraction of proliferating cells in the tumor (Supporting Information Fig. 1). To assess the role of Stat1 in the response to chemotherapy, mice were treated with the erbB1/erbB2 targeting drug lapatinib and/or the genotoxic agent doxorubicin as soon as tumors were palpable. The response to therapy was monitored for 6 weeks. Treatment of Stat1-proficient mice either with lapatinib alone, doxorubicin or drug combination resu...

Infiltration of a neoplasm with tumor-associated macrophages (TAMs) is considered an important negative prognostic factor and is functionally associated with tumor vascularization, accelerated growth, and dissemination. However, the ontogeny and differentiation pathways of TAMs are only incompletely characterized. Here, we report that intense local proliferation of fully differentiated macrophages rather than low-pace recruitment of blood-borne precursors drives TAM accumulation in a mouse model of spontaneous mammary carcinogenesis, the MMTVneu strain. TAM differentiation and expansion is regulated by CSF1, whose expression is directly controlled by STAT1 at the gene promoter level. These findings appear to be also relevant for human breast cancer, in which an interrelationship between STAT1, CSF1, and macrophage marker expression was identified. We propose that, akin to various MU subtypes in nonmalignant tissues, local proliferation and CSF1 play a vital role in the homeostasis of TAMs. Additional supporting information may be found in the online version of this article at the publisher's web-site

The ability of B-1 cells to become positively selected into the mature B cell pool, despite being weakly self-reactive, has puzzled the field since its initial discovery. Here, we explore changes in B cell positive selection as a function of developmental time by exploiting a link between CD5 surface levels and the natural occurrence of self-reactive B cell receptors (BCRs) in BCR wild-type mice. We show that the heterochronic RNA binding protein Lin28b potentiates a neonatal mode of B cell selection characterized by enhanced overall positive selection in general and the developmental progression of CD5+ immature B cells in particular. Lin28b achieves this by amplifying the CD19/PI3K/c-Myc positive feedback loop, and ectopic Lin28b expression restores both positive selection and mature B cell numbers in CD19−/− adult mice. Thus, the temporally restricted expression of Lin28b relaxes the rules for B cell selection during ontogeny by modulating tonic signaling. We propose that this neonatal mode of B cell selection represents a cell-intrinsic cue to accelerate the de novo establishment of the adaptive immune system and incorporate a layer of natural antibody-mediated immunity throughout life.

STAT1 serves as an important regulator in the response to pathogens, oncogenic transformation, and genotoxic insults. It exerts these effects by shaping the innate and adaptive immune response and by participating in genotoxic stress pathways, leading to apoptosis and inhibition of cell proliferation. We have investigated the role of STAT1 in hematopoietic toxicity induced by doxorubicin in STAT1-proficient and -deficient mice. Whereas the early genotoxic effect of doxorubicin did not depend on STAT1, expression of STAT1 was required for efficient B lymphocyte repopulation in the recovery phase. We found a lower abundance of lymphocyte precursors in the BM of STAT1-deficient animals, which was particularly evident after doxorubicin-induced hematopoietic toxicity. In accordance, colony-forming assays with STAT1-deficient BM cells revealed a decreased number of pre-B colonies. Differentiation from the pro-B to the pre-B stage was not affected, as demonstrated by unaltered differentiation of purified B cell precursors from BM in the presence of IL-7. With the exception of Sca-1, expression of genes implicated in early lymphocyte development in pro-B cells did not depend on STAT1. Our findings indicate a specific requirement for STAT1 in lymphoid development before differentiation to pre-B cells, which becomes particularly apparent in the recovery phase from doxorubicin-induced hematopoietic toxicity.

Inhibition of extracellular secreted enzyme autotaxin (ATX) represents an attractive strategy for the development of new therapeutics to treat various diseases and a few inhibitors entered in clinical trials. We herein describe structure-based design, synthesis, and biological investigations revealing a potent and orally bioavailable ATX inhibitor 1. During the molecular docking and scoring studies within the ATX enzyme (PDB-ID: 4ZGA), the S-enantiomer (Gscore = −13.168 kcal/mol) of the bound ligand PAT-494 scored better than its R-enantiomer (Gscore = −9.562 kcal/mol) which corroborated with the reported observation and analysis of the results suggested the scope of manipulation of the hydantoin substructure in PAT-494. Accordingly, the docking-based screening of a focused library of 10 compounds resulted in compound 1 as a better candidate for pharmacological studies. Compound 1 was synthesized from L-tryptophan and evaluated against ATX enzymatic activities with an IC 50 of 7.6 and 24.6 nM in biochemical and functional assays, respectively. Further, ADME-PK studies divulged compound 1 as non-cytotoxic (19.02% cell growth inhibition at 20 μM in human embryonic kidney cells), metabolically stable against human liver microsomes (CL int = 15.6 μl/min/mg; T 1/2 = 113.2 min) with solubility of 4.82 μM and orally bioavailable, demonstrating its potential to be used for in vivo experiments.

Signal Transducer and Activator of Transcription 1 (STAT1) is known to play a role in sensitizing cells to chemotherapy induced apoptosis. Doxorubicin (Dox), a commonly used chemotherapeutic drug is known to exert serious side effects, most remarkably cardiotoxicity and myelosuppression. In this study we analyzed the role of STAT1 in Dox induced toxicity and recovery using STAT1 knock out (KO) mice. Dox injected STAT1 wild type (WT) mice showed an early onset of acute toxicity whereas toxicity in KOmice was later but prolonged. A strong effect of Dox on the organs related to immune function such as shrinkage of spleen and thymus were only observed in KO mice on day 16 after Dox injection. Flow‐cytometry analysis of peripheral blood cells from KO mice showed an increase in the granulocyte population but a decrease in B lymphocytes, while no change was noted in WT mice. A severe depletion of bone marrow cellularity was observed on day 3 in both WT and KO mice. However, after 16 days WT mice were able to partially restore the depleted bone marrow, while KO mice were recovering more slowly. On day 30, WT mice showed significant recovery on the population of bone marrow progenitors and B lymphocytes compared to a little recovery in the KO mice. In summary, STAT1 KO mice are differentially sensitive to Dox induced lympho‐toxicity, thus STAT1 has an important role in the recovery from Dox induced immuno‐suppression. Supported by IFTZ grant (WD).