Work Performed at Lexicon Pharmaceuticals, Texas, USA Poster Presentation at 2016 EMBO WNT Congress, Brno, Czech Republic Stimulation of Endocortical Bone Formation by Inhibiting WNT-Inactivating Lipase NOTUM Robert Brommage, Ph.D. Work Performed at Lexicon Pharmaceuticals, Texas, USA

NOTUM is a secreted lipase that inactivates WNTs by removing the palmitoleic acid essential for Frizzled binding and WNT signalling. Lexicon’s high-throughput gene knockout (KO) mouse phenotyping campaign, examining ≈4650 genes, confirmed skeletal phenotypes in Lrp5, Sost and Wnt10b KO mice and first identified Sfrp4 and Wnt16 as key WNT-signalling bone genes. Notum KO mice had elevated cortical bone thickness and strength. Bone length and trabecular bone parameters were normal. Except for dentin dysplasia, KO mice were healthy. The osteoporosis drug teriparatide stimulated cortical bone Notum expression 10-fold. As a secreted enzyme, NOTUM is amenable to inhibition by both neutralizing antibodies (Ab) and small molecule (SM) active-site inhibitors. Conditioned media containing mouse and human NOTUM proteins were employed to develop both enzymatic and cell-based assays to screen chemical libraries and Ab hybridomas for NOTUM inhibition. Standard drug development procedures yielded potent Abs and orally active SMs for testing in mice and rats. Rodent pharmacology studies showed dose-dependent anabolic actions of NOTUM inhibition at multiple skeletal sites, including (1) Increased cortical bone thickness and strength with reduced marrow cavity diameter; (2) New bone formation on endocortical bone surfaces adjacent to the marrow cavity by quantitative histomorphometry; (3) Elevated serum concentrations of bone formation markers ALP and PINP; (4) Effectiveness of Ab 2.78.33b treatment at 10 mg/kg weekly for 4 weeks in ovariectomized mice; and (5) Effectiveness of SM LP-922056 and LP-935001 treatments for 12 and 18 weeks, respectively, in ovariectomized rats. NOTUM is an osteoporosis anabolic drug target stimulating new endocortical bone formation.

Endocortical Bone Loss during Aging Contributes to Fragility Endocortex Periosteum New Bone Formed Between Red and Yellow Fluorochromes with Notum Inhibition in Adult Rats

Overview of WNT Signaling in Bone Genetic variations or mutations in WNT signaling genes affect human bone development (AMER1, LRP4, PORCN, WNT1, WNT5A, WNT7A) and/or architecture (LRP5, LRP6, SOST, WNT16) Mouse genes affecting skeletal WNT signaling include these genes and also Dkk1, Fzd9, Sfrp2, Wls, Wnt7b and Wnt10b Osteoporosis is defined as reduced amounts of normal bone, resulting in increased fragility Most current osteoporosis drugs inhibit bone loss Teriparatide, given by daily injections, is the only osteoporosis drug that stimulates new bone formation An orally active bone anabolic drug is desirable

WNT Signaling Mediates Anabolic Bone Responses to Exercise and Teriparatide (TPTD) Treatment Unstimulated Bone Exercise Teriparatide Treatment (Sclerostin Inhibition) (PTHrP ↓Sclerostin) (TPTD Mimics PTHrP) BONE BONE New Bone BONE New Bone ↓ WNT Signaling ↑ WNT Signaling ↑ WNT Signaling Quiescent Osteoblast Active Osteoblast Osteocyte Fluid Flow Secreted Sclerostin PTHrP TPTD

High-Throughput Bone Screening Data (Brommage et al High-Throughput Bone Screening Data (Brommage et al., Bone Research 2:14034, 2014) Consistent Observations No trabecular bone changes All cortical bones are affected, including vertebral body shell, femoral neck and pelvis Elevated femoral cortical bone thickness, averaging 21%, was observed in 12 separate cohorts of male and female Notum KO mice, with a total of 228 WT and 210 KO mice examined from 2004 through 2011 KOs show 32% reduction in viability (931 WT mice, 1825 heterozygous mice and 635 homozygous KO mice) and 26% of survivors (23 of 89 examined) have a single kidney Lexicon Pharmaceuticals NOTUM Studies

Mineralization Studies in MC3T3 Cells Mineralization inhibited by NOTUM conditioned medium This inhibition is overcome with NOTUM small molecule inhibitor LP-922056 Both studies lasted 21 days with CM present throughout the study NOTUM concentrations were  0.4 and  1.2 μg/ml in mouse and human conditioned media (CM), respectively, by Western blot analysis (data not shown)

Femur Cortical Thickness NOTUM Antibody Treatment Increases Bone Mass in Both Intact and OVX Mice Mice treated once weekly for 4 weeks starting 4 weeks after OVX at 16 weeks of age Dose = 10 mg/kg; Enzyme IC50 = 37 nM; Cell-based EC50 = 4 nM Treatment increased midshaft femur cortical thickness, cortical (but not trabecular) bone mass in LV5 vertebral body, and total BV/TV in femoral neck (Scanco μCT40) Two-Factor ANOVA Femur Cortical Thickness LV5 Cortical Bone BV/TV Femoral Neck BV/TV Effect of OVX P < 0.001 P = 0.002 Effect of NOTUM Ab Interaction P = 0.37 P = 0.32 P = 0.29

Transient Stimulation of Midshaft Femur Endocortical Bone Formation OVX Control OVX Treated Modeling-dependent bone formation

Treatment with Orally-Active Small Molecule NOTUM Inhibitor Increases Bone Mass in OVX Rats OVX Fischer 344 rats treated daily by oral gavage for 12 weeks starting at 80 weeks of age OVX at 17 weeks of age LP-935001 examined at doses of 1 and 5 mg/kg Plus untreated OVX and Sham-surgery groups Two courses of double fluorochrome labeling Alizarin at 1 and 3 weeks Calcein at 9 and 11 weeks Multiple bones examined by standard DEXA, microCT, dynamic histomorphometry and breaking strength analyses OVX reduced trabecular bone mass by 88% in proximal tibia metaphysis and 50% in spine, with no effects of treatment (P > 0.9)

Treatment with Orally-Active Small Molecule NOTUM inhibitor Increases Bone Strength and Formation in OVX Rats

Contributors Jennifer P. Bardenhagen, Dawn Bright, Deon D. Doree, Jie Cui, Gwenn M. Hansen, Sabrina Jeter-Jones, Jeff Liu, Qingyun (Jim) Liu, Faika Mseeh, David G. Potter, David R. Powell, James E. Tarver, Andrea Y. Thompson, Peter Vogel, Brian Zambrowicz Dentin dysplasia data published (Peter Vogel) Veterinary Pathology 2016; 53:853-862. Bone data have been presented at three conferences and are available on Brommage ResearchGate website 2015 ASBMR; 2016 Endocrine Society; 2016 WCO-IOF-ESCEO