1. 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

2. 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 b treatment at 10 mg/kg weekly for 4 weeks in ovariectomized mice; and (5) Effectiveness of SM LP and LP treatments for 12 and 18 weeks, respectively, in ovariectomized rats.
NOTUM is an osteoporosis anabolic drug target stimulating new endocortical bone formation.

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

4. 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

5. 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

6. 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

7. Mineralization Studies in MC3T3 Cells Mineralization inhibited by NOTUM conditioned medium This inhibition is overcome with NOTUM small molecule inhibitor LP
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)

8. 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

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

10. 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 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)

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

12. 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:
Bone data have been presented at three conferences and are available on Brommage ResearchGate website
2015 ASBMR; 2016 Endocrine Society; 2016 WCO-IOF-ESCEO