This document summarizes a study investigating the metabolic mechanisms underlying segmental overgrowth disorders and potential treatments. The study found that mutations in the PI3K-AKT pathway, such as in PIK3CA and AKT1, lead to increased cell proliferation and metabolism. Functional studies showed that cell lines with PIK3CA mutations had increased AKT phosphorylation and responded strongly to growth factors, while AKT1 mutant cell lines responded less. Treatment with PI3K or mTOR inhibitors normalized the metabolic profile in both AKT1 and PIK3CA mutant cell lines, suggesting these may be effective treatments. The study provides new insights into the molecular mechanisms and potential targeted therapies for segmental overgrowth disorders.

1. Metabolic investigation of
segmental overgrowth:
new insights in pathogenic
mechanisms and
treatments.
Luigi Boccuto, MD
Greenwood Genetic Center
October 26th, 2017

2. Insulin/IGF, FGF, EGF, PDGF,
VEGF, HGF/cMet
Cell survival and
proliferation,
increased
metabolism and
protein synthesis
mTOR
Caspase
p53
GSK3β
NF-κB
PI3K-AKT pathway

3. PIK3CA
(catalytic subunit, p110α)
PIK3R2
(regulatory subunit,
p85)
Proteus (AKT1, E17K)
Hemihypertrophy, lypodystrophy, hypoglycemia (AKT2, E17K)
HME and MPPH (AKT3, E17K)
MPPH
*PIK3CA-Related Overgrowth Spectrum (PROS)
CLOVES*
FAO* (FibroAdipose
Overgrowth)
MCAP* (Megalencephaly
CAPillary malformations)
MPPH* (Megalencephaly
Polymicrogiria Polydactyly
Hydrocephalus)
HHML* (HemiHyperplasia
Multiple Lipomatosis)
KTW (Klippel-Trenauney-
Weber)
HME (HemiMegalEncephaly)

4. (Cohen, AJMG-C, 2005)
(Lindhurst et al., NEJM, 2011)
Proteus syndrome

5. Congenital Lipomatous Overgrowth,
Vascular malformations, Epidermal nevi,
Scoliosis/skeletal and spinal (CLOVES)
syndrome
(Gucev et al.
AJMG-A,
2008)

6. (Keppler-Noreuil et al. AJMG 2015)

7. Proteus
(Modified from Keppler-Noreuil et al. AJMG 2015)
(and Proteus)

8. (Keppler-Noreuil et al. AJMG 2014)
23:
• 16 H1047R
• 7 H1047L
3 H1047R
1 E542K and
1 E545K
6:
• 3 E545K
• 2 E542K
• 1 C420R
Genotype-
phenotype
correlation in
PIK3CA
mutations

9. Case H
CLOVES/KTW
No mutation
Case B
CLOVES/KTW
PIK3CA p.E542K +
PDPK1 p.I294T

10. Case C
CLOVES
PIK3CA p.Q546K
Case E
Proteus
PIK3CA p.H1047R
(from Costa et al. Pediatrics 1985)

11. Functional studies

12. Biolog Phenotype MicroArray Mammalian (PM-M) plates
0.48
0.020
0.1
0.2
0.3
0.4
0.5
0.6
350 400 450 500 550 600 650 700 750
Absorbance (A590-750)
• 96-well plates; 50 μl/well of cell suspension (20,000
cells/well)
• Incubate 40-48 hours, add Redox Dye Mix (tetrazolium)
and incubate for 24 hours
• Plate substrates:
1. Carbon energy sources (sugars, polysaccharides,
nucleotides, and carboxylic acids)
2-4. Amino acids and dipeptides
5. Ions
6-8. Hormones and metabolic effectors

14. 5653 (PIK3CA
H1047R, <5%) vs.
control
CMS15740
(PIK3CA H1047R,
46%) vs. control
CMS25039 vs.
control
(PIK3CA E542K,
35-45%)

15. CMS17024 (AKT1
E17K, ~1%) vs.
control
CMS25037 vs.
control
CMS25038 vs.
control
(AKT1 p.E17K, 5-
15%)
(AKT1 p.E17K,
35-45%)

16. Patients Phenotype
Gene
(Mutations)
AKT phospho
Starvation
AKT phospho
Stimulation
Metabolic
response to
growth
factors
5 Proteus
AKT1
(E17K)
Increased Increased Increased
19
CLOVES,
KTW, FAO,
HHML
PIK3CA
(C420R,
E542K,
E545K,
Q546K,
H1047R,
H1047L)
Increased Decreased Decreased
Identification of gene-specific
metabolic patterns

17. -
Insulin/IGF, FGF, EGF, PDGF,
VEGF, HGF/cMet
Cell survival and
proliferation,
increased
metabolism and
protein synthesis
mTOR
Caspase
p53
GSK3β
NF-κB

18. Functional studies – Conclusions
Combining AKT phosphorylation test and Biolog PM
arrays can be helpful in:
• distinguishing AKT1 vs. PIK3CA mutations;
• understanding the response of a mutation to
growth factors (insulin, FGF-1, IGF-1, PDGF >> hGH);
• suggesting treatment approaches;
• following up the response of patients to
pharmaceutical protocols.

19. Treatment
• BYL719: selective inhibitor of the p110α subunit of Pi3K (encoded
by PIK3CA), at 40 and 80 nM.
• Wortmannin: inhibitor of the Pi3K complex, at 0.1 and 1 μM.
• Rapamycin: inhibitor of the mTOR pathway, at 0.1 and 1 μM.
mTOR

20. BYL719
CMS15740 BYL719
80 nM vs. untreated
control
CMS15740 BYL719 40
nM vs. untreated
control
CMS15740 (PIK3CA
H1047R, 46%) vs.
control

21. Wortmannin
5653 (PIK3CA
H1047R, <5%) vs.
control
5653 Wort 1 μM vs.
untreated control
5653 Wort 0.1 μM vs.
untreated control

22. Wortmannin
CMS25038 Wort 1 μM
vs. untreated control
CMS25038 Wort 0.1
μM vs. untreated
control
CMS25038 (AKT1
p.E17K, 35-45%)
vs. control

23. Rapamycin
CMS15740 (PIK3CA
H1047R, 46%) vs.
control
CMS15740 Rapa 1 μM
vs. untreated control
CMS15740 Rapa 0.1
μM vs. untreated
control

24. Rapamycin
CMS17024 (AKT1
E17K, ~1%) vs.
control
CMS17024 Rapa 1 μM
vs. untreated control
CMS17024 Rapa 0.1
μM vs. untreated
control

25. 0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
Empty
Empty
Empty
Empty
Empty
Empty
Insulin
Insulin
Insulin
Insulin
Insulin
Insulin
hGH
hGH
hGH
hGH
hGH
hGH
IGF-I
IGF-I
IGF-I
IGF-I
IGF-I
IGF-I
FGF-1
FGF-1
FGF-1
FGF-1
FGF-1
FGF-1
PDGF
PDGF
PDGF
PDGF
PDGF
PDGF
EndpointAbsorbance
Control AKT1 AKT1-Rapa1
Metabolic profiles (4 AKT1+, 8 controls)

26. 0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
Empty
Empty
Empty
Empty
Empty
Empty
Insulin
Insulin
Insulin
Insulin
Insulin
Insulin
hGH
hGH
hGH
hGH
hGH
hGH
IGF-I
IGF-I
IGF-I
IGF-I
IGF-I
IGF-I
FGF-1
FGF-1
FGF-1
FGF-1
FGF-1
FGF-1
PDGF
PDGF
PDGF
PDGF
PDGF
PDGF
EndpointAbsorbance
Control PIK3CA PIK3CA-Rapa1
Metabolic profiles (10 PIK3CA+, 8 controls)

27. Treatment – Conclusions
• Wortmannin normalized the metabolic
response in Proteus cell lines.
• BYL719 reduced the metabolic response in all
cell lines but is probably toxic (especially at 80
nM).
• Rapamycin normalized metabolic response in
both AKT1+ and PIK3CA+.

28. Acknowledgments
Lauren Cascio
Sujata Srikanth
Kelly Jones
Chin-Fu Chen
Rini Pauly
Cindy Skinner
Roger Stevenson
Charles Schwartz