Antibody targeting of HER2/HER3 signaling overcomes heregulin-induced resistance to PI3K inhibition in prostate cancer.

Dysregulated expression and/or mutations of the various components of the phosphoinositide 3-kinase (PI3K)/Akt pathway occur with high frequency in prostate cancer and are associated with the development and progression of castration resistant tumors. However, small molecule kinase inhibitors that target this signaling pathway have limited efficacy in inhibiting tumor growth, primarily due to compensatory survival signals through receptor tyrosine kinases (RTKs). Although members of the epidermal growth factor receptor (EGFR), or HER, family of RTKs are strongly implicated in the development and progression of prostate cancer, targeting individual members of this family such as EGFR or HER2 has resulted in limited success in clinical trials. Multiple studies indicate a critical role for HER3 in the development of resistance against both HER-targeted therapies and PI3K/Akt pathway inhibitors. In this study, we found that the growth inhibitory effect of GDC-0941, a class I PI3K inhibitor, is markedly reduced in the presence of heregulin. Interestingly, this effect is more pronounced in cells lacking phosphatase and tensin homolog function. Heregulin-mediated resistance to GDC-0941 is associated with reactivation of Akt downstream of HER3 phosphorylation. Importantly, combined blockade of HER2 and HER3 signaling by an anti-HER2/HER3 bispecific antibody or a mixture of anti-HER2 and anti-HER3 antibodies restores sensitivity to GDC-0941 in heregulin-treated androgen-dependent and -independent prostate cancer cells. These studies indicate that the combination of PI3K inhibitors with HER2/HER3 targeting antibodies may constitute a promising therapeutic strategy for prostate cancer.

Type I IFN receptor and the B cell antigen receptor regulate TLR7 responses via distinct molecular mechanisms.

Toll-like receptor 7 (TLR7) signals to B cells are critically involved in the innate immune response to microbes, as well as pathogenesis of autoimmune diseases, but the molecular mechanisms that normally regulate these responses are incompletely understood. We previously reported that repeated stimulation through TLR7 induces a state of hyporesponsiveness (TLR tolerance) in both human and mouse B cells, characterized by marked inhibition of particular signaling pathways. BCR signals prevent and overcome TLR7 tolerance. Because optimal responses to TLR7 in B cells require type I IFN, we investigated whether BCR-mediated effects on TLR7 tolerance are mediated by type I IFN receptor (IFNAR) signals. Surprisingly, although BCR-mediated reversal of TLR7 tolerance was IFNAR independent, IFNAR signals alone also blocked TLR7 tolerance, despite enhancing TLR7 expression. Both BCR and IFNAR signals restored the phosphorylation of the transcriptional regulator c-Jun, but only BCR signals blocked the tolerance-mediated inhibition of JNK. Both BCR and IFNAR-mediated regulation was dependent on activation of the PI3K/Akt/mammalian target of rapamycin signaling pathway, indicating a central role for this axis in integrating TLR7, BCR, and IFNAR signals in B cells. These new findings reveal distinct and overlapping signaling mechanisms used by BCR and IFNAR in the regulation of TLR7 tolerance and activation.

Antigen receptor signals rescue B cells from TLR tolerance.

Interactions between innate and adaptive immune receptors are critical for an optimal immune response, but the role played by Ag receptors in modulating innate receptor functions is less clear. TLRs are a family of pattern recognition receptors that play crucial roles in detecting microbial pathogens and subsequent development of immune responses. However, chronic stimulation through TLRs renders immune cells hyporesponsive to subsequent stimulation with TLR ligands, a phenomenon known as TLR tolerance, well characterized in myeloid cells. However, it has not been studied in detail in B lymphocytes. In addition to the BCR, B cells express almost all known TLRs and respond robustly to many TLR ligands. Thus, B cells may receive signals through both TLRs and BCR during an infection and may respond differently to TLR stimulation than myeloid cells. We tested this possibility by stimulating repeatedly through either TLR alone or both TLR and BCR. Prestimulation through TLR7 resulted in reduced B cell proliferation, cytokine production, and IgM secretion upon subsequent TLR7 restimulation. The hyporesponsiveness to TLR7 restimulation was associated with reduced NF-kappaB and MAPK activation and defective c-Jun phosphorylation. However, simultaneous BCR signaling prevented or reversed TLR7 tolerance in both mouse and human B cells. Importantly, BCR signaling also rescued B cells from TLR7-mediated TLR9 tolerance. Additionally, the reversal of TLR7-mediated JNK activation was dependent on PI3K activation. Together these results present a novel mechanism to prevent and reverse TLR tolerance in B cells.

Malaria-induced murine pregnancy failure: distinct roles for IFN-gamma and TNF.

Although an important role for excessive proinflammatory cytokines in compromise of pregnancy has been established, an immunological basis for malaria-induced fetal loss remains to be demonstrated. In this study, the roles of IFN-gamma and TNF in Plasmodium chabaudi AS-induced fetal loss in mice were directly investigated. Pregnant IFN-gamma(-/-) mice experienced a more severe course of infection compared with intact C57BL/6 mice, characterized by high parasitemia, severe anemia, and marked weight loss. However, fetal loss was delayed in these mice relative to intact controls. Because IFN-gamma(-/-) mice exhibited sustained levels of plasma TNF, the role of this cytokine was examined. Whereas splenic tnf expression in C57BL/6 mice was highest 3 days before peak parasitemia, increased placental expression relative to uninfected mice was sustained, indicating that locally produced TNF may be important in malaria-induced pregnancy failure. Indeed, Ab neutralization of TNF resulted in preservation of embryos until day 12 of gestation, at which point all embryos were lost in untreated mice. Histological analysis revealed that TNF ablation preserved placental architecture whereas placentae from untreated infected mice had widespread hemorrhage and placental disruption, with fibrin thrombi in some maternal blood sinusoids. Consistent with a role for cytokine-driven thrombosis in fetal loss, expression of procoagulant tissue factor was significantly increased in the placentae of infected C57BL/6 mice but was reduced in mice treated with anti-TNF Ab. Together, these results suggest that IFN-gamma contributes to malaria-induced fetal loss and TNF is a critical factor that acts by inducing placental coagulopathy.

Engineering multivalent antibodies to target heregulin-induced HER3 signaling in breast cancer cells.

The use of antibodies in therapy and diagnosis has undergone an unprecedented expansion during the past two decades. This is due in part to innovations in antibody engineering that now offer opportunities for the production of "second generation" antibodies with multiple specificities or altered valencies. The targeting of individual components of the human epidermal growth factor receptor (HER)3-PI3K signaling axis, including the preferred heterodimerization partner HER2, is known to have limited anti-tumor effects. The efficacy of antibodies or small molecule tyrosine kinase inhibitors (TKIs) in targeting this axis is further reduced by the presence of the HER3 ligand, heregulin. To address these shortcomings, we performed a comparative analysis of two distinct approaches toward reducing the proliferation and signaling in HER2 overexpressing tumor cells in the presence of heregulin. These strategies both involve the use of engineered antibodies in combination with the epidermal growth factor receptor (EGFR)/HER2 specific TKI, lapatinib. In the first approach, we generated a bispecific anti-HER2/HER3 antibody that, in the presence of lapatinib, is designed to sequester HER3 into inactive HER2-HER3 dimers that restrain HER3 interactions with other possible dimerization partners. The second approach involves the use of a tetravalent anti-HER3 antibody with the goal of inducing efficient HER3 internalization and degradation. In combination with lapatinib, we demonstrate that although the multivalent HER3 antibody is more effective than its bivalent counterpart in reducing heregulin-mediated signaling and growth, the bispecific HER2/HER3 antibody has increased inhibitory activity. Collectively, these observations provide support for the therapeutic use of bispecifics in combination with TKIs to recruit HER3 into complexes that are functionally inert.

Maternal malaria induces a procoagulant and antifibrinolytic state that is embryotoxic but responsive to anticoagulant therapy.

Low birth weight and fetal loss are commonly attributed to malaria in endemic areas, but the cellular and molecular mechanisms that underlie these poor birth outcomes are incompletely understood. Increasing evidence suggests that dysregulated hemostasis is important in malaria pathogenesis, but its role in placental malaria (PM), characterized by intervillous sequestration of Plasmodium falciparum, proinflammatory responses, and excessive fibrin deposition is not known. To address this question, markers of coagulation and fibrinolysis were assessed in placentae from malaria-exposed primigravid women. PM was associated with significantly elevated placental monocyte and proinflammatory marker levels, enhanced perivillous fibrin deposition, and increased markers of activated coagulation and suppressed fibrinolysis in placental plasma. Submicroscopic PM was not proinflammatory but tended to be procoagulant and antifibrinolytic. Birth weight trended downward in association with placental parasitemia and high fibrin score. To directly assess the importance of coagulation in malaria-induced compromise of pregnancy, Plasmodium chabaudi AS-infected pregnant C57BL/6 mice were treated with the anticoagulant, low molecular weight heparin. Treatment rescued pregnancy at midgestation, with substantially decreased rates of active abortion and reduced placental and embryonic hemorrhage and necrosis relative to untreated animals. Together, the results suggest that dysregulated hemostasis may represent a novel therapeutic target in malaria-compromised pregnancies.