H2AX promotes replication fork degradation and chemosensitivity in BRCA-deficient tumours.

Histone H2AX plays a key role in DNA damage signalling in the surrounding regions of DNA double-strand breaks (DSBs). In response to DNA damage, H2AX becomes phosphorylated on serine residue 139 (known as γH2AX), resulting in the recruitment of the DNA repair effectors 53BP1 and BRCA1. Here, by studying resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient mammary tumours, we identify a function for γH2AX in orchestrating drug-induced replication fork degradation. Mechanistically, γH2AX-driven replication fork degradation is elicited by suppressing CtIP-mediated fork protection. As a result, H2AX loss restores replication fork stability and increases chemoresistance in BRCA1/2-deficient tumour cells without restoring homology-directed DNA repair, as highlighted by the lack of DNA damage-induced RAD51 foci. Furthermore, in the attempt to discover acquired genetic vulnerabilities, we find that ATM but not ATR inhibition overcomes PARP inhibitor (PARPi) resistance in H2AX-deficient tumours by interfering with CtIP-mediated fork protection. In summary, our results demonstrate a role for H2AX in replication fork biology in BRCA-deficient tumours and establish a function of H2AX separable from its classical role in DNA damage signalling and DSB repair.

Loss of the volume-regulated anion channel components LRRC8A and LRRC8D limits platinum drug efficacy.

In recent years platinum (Pt) drugs have been found to be especially efficient to treat patients with cancers that lack a proper DNA damage response, e.g. due to dysfunctional BRCA1. Despite this knowledge, we are still missing helpful markers to predict Pt response in the clinic. We have previously shown that volume-regulated anion channels, containing the subunits LRRC8A and LRRC8D, promote the uptake of cisplatin and carboplatin in BRCA1-proficient cell lines. Here, we show that the loss of LRRC8A or LRRC8D significantly reduces the uptake of cis- and carboplatin in BRCA1;p53-deficient mouse mammary tumor cells. This results in reduced DNA damage and in vivo drug resistance. In contrast to Lrrc8a, the deletion of the Lrrc8d gene does not affect the viability and fertility of mice. Interestingly, Lrrc8d-/- mice tolerate a two-fold cisplatin maximum-tolerable dose. This allowed us to establish a mouse model for intensified Pt-based chemotherapy, and we found that an increased cisplatin dose eradicates BRCA1;p53-deficient tumors, whereas eradication is not possible in WT mice. Moreover, we show that decreased expression of LRRC8A/D in head and neck squamous cell carcinoma patients, who are treated with a Pt-based chemoradiotherapy, leads to decreased overall survival of the patients. In particular, high cumulative cisplatin dose treatments lost their efficacy in patients with a low LRRC8A/D expression in their cancers. Our data therefore suggest that LRRC8A and LRRC8D should be included in a prospective trial to predict the success of intensified cis- or car-boplatin-based chemotherapy.

Foxp3+ T Regulatory Cells as a Potential Target for Immunotherapy against Primary Infection with Echinococcus multilocularis Eggs.

Alveolar echinococcosis (AE) is a lethal disease caused by infection with the metacestode stage of the helminth Echinococcus multilocularis, which develops into a tumorlike mass in susceptible intermediate hosts. The growth potential of this parasite stage is directly linked to the nature of the surrounding periparasitic immune-mediated processes. In a first step (experiment 1), mice were orally infected with E. multilocularis eggs, to be used for assessing the hepatic expression profiles of 15 selected cytokine and chemokine genes related to acquired immunity from 21 to 120 days postinfection. The early stage of infection in immunocompetent animals was marked by a mixed Th1/Th2 immune response, as characterized by the concomitant presence of gamma interferon (IFN-γ) and interleukin-4 (IL-4) and their related chemokines. At the late stage of AE, the profile extended to a combined tolerogenic mode including Foxp3, IL-10, and transforming growth factor beta (TGF-ß) as key components. In a second step (experiment 2), the effect of T regulatory cell (Treg) deficiency on metacestode growth was assessed in E. multilocularis-infected DEREG (depletion of regulatory T cells) mice upon induction of Treg deficiency with diphtheria toxin (DT). The parasite lesions were significantly smaller in the livers of treated mice than in corresponding control groups. Foxp3+ Tregs appear to be one of the key players in immune-regulatory processes favoring metacestode survival by affecting antigen presentation and suppressing Th1-type immune responses. For these reasons, we suggest that affecting Foxp3+ Tregs could offer an attractive target in the development of an immunotherapy against AE.

Depletion of FoxP3+ Tregs improves control of larval Echinococcus multilocularis infection by promoting co-stimulation and Th1/17 immunity.

INTRODUCTION: The growth potential of the tumor-like Echinococcus multilocularis metacestode (causing alveolar echinococcosis, AE) is directly linked to the nature/function of the periparasitic host immune-mediated processes. Previous studies had shown that regulatory T cells (Tregs) become gradually up-regulated in the course of both chronic human and murine AE. Thus we now tackled the role of FoxP3+ Tregs and FoxP3+ -Treg-regulated immune response in contributing to the control of this helminthic infection. METHODS: The infection outcome in E. multilocularis-infected DEREG mice was measured upon determining parasite load (wet weight of parasitic metacestode tissue). Flow cytometry and qRT-PCR were used to assess Treg, Th17-, Th1-, Th2-type immune responses and antigen presenting cell activation. RESULTS: We showed that E. multilocularis-infected DEREG-mice treated with DT (as compared to infected control DEREG-mice without DT application) exhibited a significantly lower parasite load, associated with a persisting capacity of co-stimulation, and an increased Th1/Th17-polarization. CONCLUSIONS: FoxP3+ Tregs appear as one of the key players in immune regulatory processes favoring (i) metacestode survival by inhibiting the maturation potential of co-stimulatory activity and (ii) T cell exhaustion (suppressing Th1/Th17-type immune responses). We showed as well that prospectively, targeting FoxP3+ Tregs could be an option to develop an immunotherapy against AE.

Repeated Long-Term DT Application in the DEREG Mouse Induces a Neutralizing Anti-DT Antibody Response.

Regulatory T (Tregs) cells play an important role in mediating tolerance to self-antigens but can also mediate detrimental tolerance to tumours and pathogens in a Foxp3-dependent manner. Genetic tools exploiting the foxp3 locus including bacterial artificial chromosome- (BAC-) transgenic DEpletion of REGulatory T cells (DEREG) mice have provided essential information on Treg biology and the potential therapeutic modulation of tolerance. In DEREG mice, Foxp3+ Tregs selectively express enhanced green fluorescent protein (eGFP) and diphtheria toxin (DT) receptor, allowing for the specific depletion of Tregs through DT administration. We here provide a detailed overview about an important consideration that long-term administration of DT induces a humoral immune response with an appropriate production of anti-DT antibodies that can inactivate DT and thus abrogate its effect in the DEREG mouse. Additionally, we showed that anti-DT mouse serum partially neutralized DT-induced Foxp3 inhibition.

EGFR inhibitors erlotinib and lapatinib ameliorate epidermal blistering in pemphigus vulgaris in a non-linear, V-shaped relationship.

Novel insights into intra-cellular signalling involved in pemphigus vulgaris (PV), an autoimmune blistering disease of skin and mucous membranes, are now revealing new therapeutic approaches such as the chemical inhibition of PV-associated signals in conjunction with standard immunosuppressive therapy. However, extensive inhibition of signalling molecules that are required for normal tissue function and integrity may hamper this approach. Using a neonatal PV mouse model, we demonstrate that epidermal blistering can be prevented in a dose-dependent manner by clinically approved EGFR inhibitors erlotinib and lapatinib, but only up to approximately 50% of normal EGFR activity. At lower EGFR activity, blisters again aggravated and were highly exacerbated in mice with a conditional deletion of EGFR. Statistical analysis of the relation between EGFR activity and the extent of skin blistering revealed the best fit with a non-linear, V-shaped curve with a median break point at 52% EGFR activity (P = 0.0005). Moreover, lapatinib (a dual EGFR/ErbB2 inhibitor) but not erlotinib significantly reduced blistering in the oral cavity, suggesting that signalling mechanisms differ between PV predilection sites. Our results demonstrate that future clinical trials evaluating EGFR/ErbB2 inhibitors in PV patients must select treatment doses that retain a specific level of signal molecule activity. These findings may also be of relevance for cancer patients treated with EGFR inhibitors, for whom skin lesions due to extensive EGFR inhibition represent a major threat.

An adult passive transfer mouse model to study desmoglein 3 signaling in pemphigus vulgaris.

Evidence has accumulated that changes in intracellular signaling downstream of desmoglein 3 (Dsg3) may have a significant role in epithelial blistering in the autoimmune disease pemphigus vulgaris (PV). Currently, most studies on PV involve passive transfer of pathogenic antibodies into neonatal mice that have not finalized epidermal morphogenesis, and do not permit analysis of mature hair follicles (HFs) and stem cell niches. To investigate Dsg3 antibody-induced signaling in the adult epidermis at defined stages of the HF cycle, we developed a model with passive transfer of AK23 (a mouse monoclonal pathogenic anti-Dsg3 antibody) into adult 8-week-old C57Bl/6J mice. Validated using histopathological and molecular methods, we found that this model faithfully recapitulates major features described in PV patients and PV models. Two hours after AK23 transfer, we observed widening of intercellular spaces between desmosomes and EGFR activation, followed by increased Myc expression and epidermal hyperproliferation, desmosomal Dsg3 depletion, and predominant blistering in HFs and oral mucosa. These data confirm that the adult passive transfer mouse model is ideally suited for detailed studies of Dsg3 antibody-mediated signaling in adult skin, providing the basis for investigations on novel keratinocyte-specific therapeutic strategies.