Abstract 103: Disruption of Notch Signaling targeted to the myeloma bone marrow microenvironment simultaneously inhibits tumor growth and prevents bone loss without inducing gut toxicity
Authors: Ferrari Adam J. , McAndrews Kevin , Nelson Jessica H. , Bell James T. , Srinivasan Venkatesan , Ebetino Frank H. , Boeckman Robert K. , Roodman G. David , Bellido Teresita , Delgado-Calle Jesus 
Published: August 2019

Abstract
Communication between myeloma (MM) cells and cells of the bone marrow via Notch signaling promotes tumor growth/survival and stimulates bone resorption. Systemic inhibition of Notch, using γ-secretase inhibitors (GSIs), decreases MM growth and reduces bone destruction, but the clinical use of GSIs is limited due to dose-limiting severe gut toxicity.
To circumvent GSI side effects, we generated a bone specific Notch inhibitor (BT-GSI) by conjugating GSI-XII to a targeting molecule (BT) with high bone affinity using an acid hydrolyzable linker. In vitro, BT-GSI was inactive unless pre-incubated at low pH, and exhibited equal inhibition of Notch target genes in MM cells as unconjugated GSI. Ex vivo, BT-GSI decreased Notch expression and reduced MM growth in bone organ cultures that reproduce acidic conditions in the MM-bone microenvironment. In vivo, treatment with BT-GSI (5mg/kg/3x/wk, i.p.) for 2 wks decreased Notch signaling in bone more efficiently than unconjugated GSI (10mg/kg/5x/wk, i.p.) in naïve mice. In addition, BT-GSI increased cancellous bone mass (30%) and decreased bone resorption by 40%, without affecting bone formation. In contrast, these parameters remained unchanged by GSI. Next, we examined in vivo the impact of BT-GSI on MM growth and bone disease in a preclinical model of established MM. 8-wk-old immunodeficient mice were injected intratibially with 105 JJN3 human MM (hMM) cells or saline. hMM injected mice exhibited detectable serum levels of the tumor biomarker human K-light chain (40 ng/mL) and visible osteolytic disease (osteolytic area 1.7 mm2) 3 wks after hMM inoculation. Then, hMM-injected mice were randomized based on tumor levels to two subgroups to receive either BT-GSI (10mg/kg/3x/wk) or vehicle (DMSO) for 3 wks. Saline-injected mice received vehicle injections. BT-GSI selectively decreased Notch gene expression in bone, but had no effect in the brain or gut. Further, BT-GSI did not increase the expression of Adipsin in the gut, a biomarker of gut toxicity, nor showed evidence of gut toxicity at necropsy. Mice treated with BT-GSI exhibited a 45% decrease in tumor burden (168 vs 254 ng/mL human K-light chain) and 50% less osteolytic area compared to vehicle treated mice bearing hMM (4.4 vs 10.2 mm2). Moreover, BT-GSI decreased serum CTX by 30%, but did not affect serum P1NP. Importantly, equimolar administration of the unconjugated BT molecule did not alter MM growth nor prevented bone loss in mice with established MM.
In conclusion, these results show that bone-targeted Notch inhibition reduces MM growth and preserves bone mass in mice with established MM. Because BT-GSI shows bone specific Notch inhibition and lacks gut toxicity, it should circumvent the deleterious side effects that limit GSI use in patients. Thus, BT-GSI is a promising approach to inhibit MM growth and to prevent bone loss in MM patients.
Citation Format: Adam J. Ferrari, Kevin McAndrews, Jessica H. Nelson, James T. Bell, Venkatesan Srinivasan, Frank H. Ebetino, Robert K. Boeckman Jr, G. David Roodman, Teresita Bellido, Jesus Delgado-Calle. Disruption of Notch Signaling targeted to the myeloma bone marrow microenvironment simultaneously inhibits tumor growth and prevents bone loss without inducing gut toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 103.