1. Bone-fat reciprocity in progressive osseous heteroplasia (POH) Reciprocità tra osso e tessuto adiposo nella POH Robert J. Pignolo, M.D., Ph.D.

2. Bone-fat reciprocity in progressive osseous heteroplasia (POH)

3. Bone-fat phenotype in POH Osteoblast differentiation and HO Adipocyte differentiation and leaness Mechanisms of bone- fat reciprocity

4. Progressive osseous heteroplasia (POH; OMIM #166350) Rare genetic condition of progressive heterotopic bone formation Defined clinically by cutaneous ossification that progresses to involve deep connective tissues including skeletal muscle and fascia [Kaplan et al., 1994] Most cases are caused by heterozygous inactivating germline mutations in the GNAS gene that encodes the alpha subunit of the G- stimulatory protein of adenylyl cyclase [Shore et al., 2002]

5. Adegbite et al Am J Med Gen (2008) Clinical characteristics of POH and other GNAS-based disorders of HO All (+) or (no (-) patients within the diagnostic category displayed the indicated characteristic. a Deep HO refers to the extension of superficial (dermal) HO to deep tissues. b AHO features included: short statue, obesity, round face, brachydactaly, neurobehavioral abnormalities. The presence of heterotopic ossification was common to all presentations and excluded here. c An endocrine evaluation included a survey of calcium, phosphorus, TSH, and intact PTH blood levels. Two POH patients had endocrine abnormalities: one had a high TSH, and another had high calcium and phosphate levels. One POH/AHO patient had a high phosphate level. All POH/PHP 1a/1c patients had PTH resistance or PTH and thyroid hormone resistance. Differences found to be statistically significant when compared to POH are d p < 0.0001, e p = 0,0002.

6. Progression of HO in POH Kaplan, F. S. et al. J. Bone Joint Surg. 76, 425–436 (1994) 18 months30 months8 years

7. Spectrum of POH and other GNAS- based conditions of HO Adegbite et al Am J Med Gen (2008)

8. Low birth weight in patients with POH Shown are birth weights from 13 patients with POH. Three of 4 males and 7 of 9 females had measurements at or below the fifth percentile. Birth weights are plotted onto normative growth charts for sex-matched cohorts born in United States (Kuczmarski RJ, Ogden CL, Guo SS, et al. 2000 CDC growth charts for the United States: methods and development. Vital Health Stat 2002;246:1—190).

9. Adegbite et al Am J Med Gen (2008) Obesity and other AHO features among patients with GNAS-based disorders of HO DiagnosisAverage no. AHO features per patient (+/- SD) Short Stature (%) Obesity (%) Round face (%) Brachydactaly (%) Mental retardation (%) POH0.31 (0.61)7.70.03.815.43.8 POH/AHO2.7 (0.5)66.733.366.783.316.6 POH/PHP1 a/1c 2.2 (1.5)8040.020.040.0 Osteoma cutis 0.0 (0.0)0.0 AHO2.7 (1.9)50.040.050.080.020.0 PHP1a/1c2.6 (1.4)50.058.366.7 8.3

10. Bone-fat reciprocity in progressive osseous heteroplasia (POH)

11. GNAS encodes multiple transcripts Shore EM & Kaplan FS Nat Rev Rheumatol 6: 518-527 (2010)

12. Bone formation in POH Shore EM & Kaplan FS Nat Rev Rheumatol 6: 518-527 (2010)

13. Pignolo et al, Submitted (2011) Expression of Gnas protein products in bone and fat Bone Fat No 1 o AbXLαsGsαGsαNESP

14. * * * Expression of Gnas transcripts in adipocyte soft tissue stromal cells (STSCs) Pignolo et al, JBMR 25: 2647-55 (2011)

15. KO 754 WT 750 KO 764 WT 756 KO 765 WT 763 KO 752 WT 749 A  B Accelerated osteoblast differentiation in STSCs from Gnas +/- mice Pignolo et al, JBMR 25: 2647-55 (2011)

16. Expression of osteoblast markers in adipose-derived stem cells Osteoblast differentiation (days) Relative OP mRNA expression 0 0.5 1 1.5 2 2.5 3 09 1720  Gnas +/+ Gnas +/- B Relative ALP mRNA expression Osteoblast differentiation (days) 0 0.5 1 1.5 2 2.5 3 3.5   0 9 1720 Gnas +/+ Gnas +/- A Osteoblast differentiation (days) Relative OC mRNA expression 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7  091720 Gnas +/+ Gnas +/- C Pignolo et al, JBMR 25: 2647-55 (2011)

17. A B D C B F F Gnas +/- mice develop HO Pignolo et al, JBMR 25: 2647-55 (2011)

18. Bone-fat reciprocity in progressive osseous heteroplasia (POH)

19. Paternally-inherited Gsα mutation impairs adipogenesis in vitro A WT Gsα +/p- Relative amounts of Oil Red O (day10) WTGsα +/p- Adipogenesis ** B Adipogenic differentiation (days) WTGsα +/p- Relative RNA expression C/EBPβ [ * C371 C/EBPα [ * C137 PPARγ [ * C137 aP2 [ * C137 C Lui et al, submitted (2012)

20. Adipogenic differentiation (days) WT Gsα +/p- A Relative Gsα RNA expression 0137 ** B Relative XLαs RNA expression 1370 * C Relative Nesp RNA expression 0137 * * Expression of Gnas transcripts during adipocyte differentiation Lui et al, submitted (2012)

22. Inactivating Gsα mutation reduces adipogenic tissues in vivo I Lui et al, submitted (2012)

23. WTGsα +/p- WTGsα +/p- Fat Pad (subcutaneous) WAT (abdominal) BAT (interscapular) Inactivating Gsα mutation reduces adipogenic tissues in vivo II Lui et al, submitted (2012)

25. Expression of adipose markers in adipose tissues WTGsα+/p- Relative RNA expression Brown fat tissue (BAT) Gsα αP2 PPARγ Leptin UCP1 ** Relative RNA expression White fat tissue (WAT) Gsα αP2 PPARγ Leptin **** Relative RNA expression Fat Pad Gsα αP2 PPARγ Leptin **** Lui et al, submitted (2012)

26. B s WTGsα +/p- ** Relative amounts Oil Red O (day 10) -- + day 1-2 -- + day 1-2 + day 5-6 Forskolin: -- -- + + + Adipogenic Induction: Differentiation day 1-2 forskolin A day 1day 10 day 5-6 Adenylyl cyclase activation rescues the adipogenic impairment of Gsα +/p- adipose-derived stem cells Lui et al, submitted (2012)

27. WTGsα +/p - Adipogenic differentiation (days) C * Relative Osteocalcin RNA expression 0 1 3 7 A Relative Msx2 RNA expression * * B Relative Runx2 RNA expression 0 1 3 7 * Paternally-inherited Gsα mutation potentiates osteogenesis during adipogenic induction Lui et al, submitted (2012)

28. Bone-fat reciprocity in progressive osseous heteroplasia (POH)

29. Gnas +/- (exon 1) ↓G s α XL αs ↑↑cAMP Regulation of bone-fat reciprocity in soft tissue by GNAS/Gnas Gnas +/+ (wild-type) GsαGsα XL αs ↑↑↑ cAMPHO POH ↓G s α ↓XLαs ↑ cAMP ? Superficial HO Progressive HO Leaness ↑ Osteogenic genes ↓ Adipogenic genes ↓ Osteogenic genes ↑ Adipogenic genes

30. Summary Unlike other GNAS-based disorders of HO, POH occurs in the absence of obesity and multiple other features of AHO or hormone resistance Impaired bone-fat reciprocity in soft tissue promotes osteogenesis and antagonizes adipogenesis in POH GNAS is a key factor which regulates cell fate decisions in adipose-derived stem cells

31. Acknowledgements Laboratory –Nichelle Adegbite –Jan-Jan Lui –Elizabeth Russell –Alec Richardson –Meiqi Xu –Josef Kaplan Collaborators –Eileen Shore –Fred Kaplan Funding –National Institutes of Health –John A. Hartford Foundation –International Progressive Osseous Heteroplasia Association –Italian Progressive Osseous Heteroplasia Association –Ian Cali Fund –University of Pennsylvania Center for Research in FOP & Related Disorders