Animal studies.

For these studies, we used immunocompetent (C57BL/KaLwRijHsd) (26–28) and immunodeficient (C57BL/6 Prkdc^scid; Jackson’s Lab, Bar Harbor, ME, US; RRID: IMSR) murine models of established MM disease that closely resemble the human disease, as shown before (13, 29). Briefly, six to eight-week-old female and male mice were injected intratibially with 10⁵ murine 5TGM1 or human JJN3 MM cells, and control mice were injected with saline. Three weeks after MM cell inoculation, mice were randomized by tumor levels and bone lytic disease and assigned to subgroups receiving i.p. injections of either 1) vehicle (DMSO), 2) GSI (0.1mol/L (5 mg/kg), 3 times a week), 3) zoledronic acid (0.1 mg/kg, 2 times a week), 4) BT (0.1mol/L (5mg/kg), 0.05mol/L (2.5mg/kg) or 0.02mol/L (1mg/kg), 3 times a week) or 5) BT-GSI (0.1mol/L (10mg/kg), 0.05mol/L (5mg/kg) or 0.02mol/L (2mg/kg), 3 times a week). Treatments were administered in random order and given for a total of three weeks. For the gut toxicity studies, six to eight-week-old naïve C57BL/6 female mice were assigned to subgroups receiving i.p. injections of either 1) vehicle (DMSO), 2) GSI LY3039478 (Selleck Chemical LLC, Houston, TX 77014; 0.4mol/L (20 mg/kg), 2 times a day), 3) GSI (0.4mol/L (20/mg/kg), 2 times a day) or 4) BT-GSI (0.4mol/L (40 mg/kg), 2 times a day) for one week. Mice were fed a regular diet (Harlan, Indianapolis, IN, US), received water ad libitum, and maintained a 12-hour light/dark cycle. Animal studies were approved by the Institutional Animal Care and Use Committee of the Indiana University School of Medicine and the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences. Animal studies and data reporting follow ARRIVE guidelines.