Anna N. Moraitis

The co-repressor hairless protects RORα orphan nuclear receptor from proteasome-mediated degradation

RORα is a constitutively active orphan nuclear receptor essential for cerebellar development and is previously shown to regulate genes involved in both myogenesis and adipogenesis. The transcriptional activity of RORα is dependent on the presence of a ubiquitous ligand and can be abolished by interaction with Hairless (Hr), a ligand-oblivious nuclear receptor co-repressor. In this study, we first demonstrate that RORα is a short-lived protein and that treatment with the MG-132 proteasome inhibitor results in the accumulation of ubiquitin-conjugated receptor and inhibition of transcription. These data show that RORα transcriptional activity and degradation are intrinsically linked. In addition, the introduction of inactivation mutations in the ligand-binding pocket and co-regulator-binding surface of RORα significantly increases protein stability, indicating that ligand and/or co-regulator binding perpetuates RORα degradation. Strikingly, expression of the co-repressor Hr results in the stabilization of RORα because of an inhibition of proteasome-mediated degradation of the receptor. Stabilization of RORα by Hr requires intact nuclear receptor recognition LXXLL motifs within Hr. Interestingly, the co-repressor nuclear receptor co-repressor (NCoR) has no effect on RORα protein turnover. This study shows that stabilization of RORα is an essential component of Hr-mediated repression and suggests a molecular mechanism to achieve transcriptional repression by a liganded receptor-co-repressor complex.

Mechanism and function of monoclonal antibodies targeting siglec-15 for therapeutic inhibition of osteoclastic bone resorption.

Background: Bone-resorbing osteoclasts express the Siglec-15 receptor on their plasma membrane. Results: Siglec-15 antibodies inhibit osteoclast differentiation and induce receptor endocytosis and degradation. Conclusion: These results establish that Siglec-15 antibodies target osteoclasts in vivo and reveal a likely mechanism of action. Significance: Siglec-15 could serve as a target of novel therapeutics for osteoporosis and cancer-associated bone loss. The use of monoclonal antibodies to target functionally important cell-surface proteins on bone-resorbing osteoclasts represents a promising approach for treatment of cancer-associated bone loss and other skeletal pathologies. Previously, we identified Siglec-15, a little studied sialic acid-binding receptor, as a candidate target that is highly up-regulated during osteoclast differentiation induced by the cytokine receptor activator of NF-κB ligand (RANKL). In this report, we confirm that Siglec-15 is localized to the plasma membrane where it can be targeted by monoclonal antibodies to inhibit differentiation of functional osteoclasts in vitro. Furthermore, we found that treatment of mice with these antibodies led to a marked increase in bone mineral density, consistent with inhibition of osteoclast activity. Interestingly, osteoblast numbers were maintained despite the anti-resorptive activity. At the molecular level, Siglec-15 interacts with the adapter protein DAP12 and can induce Akt activation when clustered on the osteoclast cell surface, which likely represents its normal signaling function. Importantly, we discovered that monoclonal antibodies induce rapid internalization, lysosomal targeting, and degradation of Siglec-15 by inducing receptor dimerization. This study defines a key regulatory node that controls osteoclast differentiation and activity downstream of RANKL and supports further development of Siglec-15 antibodies as a novel class of bone loss therapeutics.

Abstract LB-091: Inhibition of Siglec-15 prevents bone loss in a mouse model of multiple myeloma

Multiple myeloma (MM) is a B-cell malignancy associated with infiltration and growth of plasma cells in the bone marrow resulting in end-organ damage including devastating osteolytic bone disease. We examined the ability of 25B2, an antibody targeting Siglec-15, to prevent bone loss in the 5T2MM syngeneic model of MM. C57BlKaLwRij male mice (n = 10/group) were injected with 5T2MM cells and 24h later treated for 12 weeks with the PBS, 25B2 (10 mg/kg, q3d), bortezomib (0.7 mg/kg, q3d) or a combination of 25B2 and bortezomib. 25B2 treatment caused a 3.7-, 5.4- and 1.2-fold increase in bone volume (%BV/TV) compared to PBS-treated mice in the tibia, femur and lumbar vertebra (LV), respectively. Increases in bone volume were also seen with bortezomib with 3.1-, 4.1- and 1.4-fold increases in the tibia, femur and LV, respectively. Combination of the two agents further increased%BV/TV by 6.9-, 10.5- and 1.5-fold in the tibia, femur and LV, respectively, when compared to the PBS treated mice. All of the changes were statistically significant with the exception of those in the LV. Unlike bortezomib treatment, which inhibits the growth of 5T2MM cells in this model, there was no direct effect of 25B2 on tumor burden in 5T2MM mice when the percentage of plasmacytosis and the serum paraprotein levels were measured. However, 25B2 caused a significant decrease in the number of bone lesions by 49.5%. The number of lesions in the bortezomib group decreased by 71.7%, but there was no significant difference between the 25B2 and bortezomib groups. The number of lesions decreased by 89.4% in the combination group. Consistent with the mechanism of action of Siglec-15 antibodies, osteoclasts were rendered inactive, however cross-talk with bone-forming osteoblasts was likely maintained. This unique mechanism of action would be particularly appropriate for MM, where osteolytic disease is not only the result of increased osteoclast activity but also decreased osteoblast-mediated bone formation. The 5T2MM model is one of the most representative of the human pathology and therefore, the effect observed in response to the Siglec-15 antibody holds the promise that such a treatment in MM patients could provide benefit and further delay skeletal related events. Citation Format: Gilles B. Tremblay, Anna Moraitis, Karin Vanderkerken, Mario Filion. Inhibition of Siglec-15 prevents bone loss in a mouse model of multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-091.

Abstract 666: AB-3A4: A novel KAAG1-targeting antibody-drug conjugate is active in models of ovarian carcinoma, triple-negative breast cancer and castration-resistant prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Starting from total RNA prepared from late-stage serous ovarian tumors and low-malignancy potential tumors, a sensitive subtractive cloning technology called STAR was used to identify differentially expressed sequences that were up-regulated in the malignant tumors. This analysis led to the identification of KAAG1, an antigen that is specifically expressed in the vast majority of the ovarian tumors. Monoclonal antibodies (mAbs) that bind to human KAAG1 were generated and one of these, AB-3A4, interacted with KAAG1 expressed on the surface of several ovarian cancer, triple-negative breast cancer (TNBC) and prostate cancer cell lines. Further studies indicated that the AB-3A4/KAAG1 complex was rapidly internalized and co-localized with LAMP1, a marker of lysosomes, indicating that AB-3A4 has potential as an antibody-drug conjugate (ADC). Here we describe preclinical results of AB-3A4 conjugated to the cytotoxic agent monomethyl auristatin E (AB-3A4-vcMMAE). Binding studies with KAAG1-positive cell lines, such as the SKOV-3 (ovarian), MDA-MB-231 (TNBC) and PC-3 (prostate) showed that AB-3A4-vcMMAE interacted with its antigen in a similar manner to the unconjugated AB-3A4. In cytotoxicity assays, AB-3A4-vcMMAE killed cancer cell lines expressing KAAG1 with an IC50 value of 1 - 2 nM, whereas it displayed no specific potency in KAAG1-low or -negative cells. SKOV-3 cells were subcutaneously implanted in SCID mice and allowed to grow to a volume of 150 - 200 mm3 after which four injections of AB-3A4-vcMMAE at 5 mg/kg were administered over two weeks. Rapid and sustained tumor regression was observed for several weeks post-injection. In PC-3 xenografts, a single injection of AB-3A4-vcMMAE at 3 mg/kg was sufficient to cause significant tumor regression and this efficacy was found to be dose-dependent between doses of 1 and 10 mg/kg. In parallel treatment groups, no significant tumor shrinkage was observed with either a control IgG-vcMMAE or a mixture of unconjugated AB-3A4 and free MMAE at a drug-antibody ratio corresponding to that of AB-3A4-vcMMAE. Furthermore, xenografts of TNBC MDA-MB-231 cells were significantly regressed following a single injection of AB-3A4-vcMMAE with no recurrence after several weeks post-treatment. Finally, using an intraperitoneal orthotopic model of ovarian cancer, treatment with AB-3A4-vcMMAE significantly increased the overall survival of mice compared to mice treated with the control ADC. Collectively, our results demonstrate the potent efficacy of AB-3A4-vcMMAE in several disease models and underscore the potential of this novel agent in ovarian cancer, TNBC and castration-resistant prostate cancer, three areas for which very few targeted therapies exist. Citation Format: Gilles B. Tremblay, Anna Moraitis, Dominique Bedard, Adriana Orimoto, Martine Page, Anne-Marie Mes-Masson, Mario Filion. AB-3A4: A novel KAAG1-targeting antibody-drug conjugate is active in models of ovarian carcinoma, triple-negative breast cancer and castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 666. doi:10.1158/1538-7445.AM2014-666