Neurofibromatosis
By Nilesh Jambhekar
What is Neurofibromatosis?
The basics:
 Autosomal-dominant
genetic disorder.
 Is a type of cancer of the nervous system that
can occur in all 3 germ layers and any organ
system.
 Causes small (usually benign) tumors to start
growing on neural crest cells (Schwann Cells,
oligodendrocytes, melanocytes, etc.)
 2 main types:
-Neurofibromatosis 1(NF1)
-Neurofibromatosis 2(NF2)
[1]
Neurofibromatosis 1:
 Caused
gene.



by a mutation on the NF1 (Neurofibromin1)
-is on chromosome 17 on the long arm (q) at
position 11.2
-has 60 exons and over 8500 base pairs
-encodes for Neurofibromin
NF1 is the more common form of the disease . Occurs
in ~1/3000 live births (in the U.S.). [1]
Is also called von Recklinghausen syndrome after
Friedrich Daniel von Recklinghausen who discovered
it in 1882. [2]
Causes tumors to develop in the peripheral nervous
system or along the optic nerve. [1]
Diagnosing Neurofibromatosis 1
 Must







have at least 2 of the following:
Six or more café-au-lait spots 1.5 cm or larger in postpubertal individuals, 0.5 cm or larger in pre-pubertal
individuals
Two or more neurofibromas of any type or one or
more plexiform neurofibroma
Freckling in the axilla or groin
Optic glioma (tumor of the optic pathway)
Two or more Lisch nodules (benign iris hamartomas)
A distinctive bony lesion: dysplasia of the sphenoid
bone or dysplasia or thinning of long bone cortex
A first-degree relative with NF1[1]
A young boy
café-au-lait spots
Source:
www.understandingnf1.org
Neurofibromin (NF1):
 Is
produced by many cells in the nervous system
(nerve cells as well Schwann cells and
oligodendrocytes).
 Acts as a GTPase activating protein (GAP) that
specifically regulates the activity of RAS, a family of
GTPases .
 Acts as a tumor suppressor.
 Any one of hundreds of possible mutations in NF1
incapacitate Neurofibromin causing nerve cells to
proliferate unchecked (tumors). Because the NF1
gene is so large, mutations are relatively frequent
but not always detrimental.
 Neurofibromin is a very large protein (2839 amino
acids).
 The cellular role of Neurofibromin is still not known.
[2][4]
RAS:
 RAS
is a family of genes that encode small
GTPases such as H-RAS.
 RAS has a molecular switch with 2 modes: on
and off.
 In its “on” mode, RAS is bound to GTP and
interacts with a growth activator called proto
oncogene c-RAF. RAS activates intrinsic
properties within c-RAF activating it. This
activates a pathway of kinases leading to cell
growth. [6][8]
Neurofibromin mechanism:
 Turns
off the growth-promoting function of the
RAS family by hydrolyzing the GTP bound to
them. Normally, RAS is active while it slowly
cleaves off a terminal phosphate using the time
to initiate various kinase pathways that lead to
cell growth and proliferation. Neurofibromin
essentially catalyzes this reaction.
 Neurofibromin is also known to have effects on
cAMP signaling in other pathways, but most of
these connections aren’t well understood.[7]
Fig 2
There are many known mutations of Neurofibromin but the most serious
involve the mutations of the above 3 residues (Glutamic acid, Lysine,
and especially Arganine).
The Arganine Finger
 The
Arganine finger (residue 1276) is the single
most important part of the GAP domain in
terms of catalyzing the removal of the
gamma (terminal) phosphate. Replacement
of this residue by any other will result in a
several fold decrease in complex stability and
up to a 8000 fold decrease in the catalytic
ability of Neurofibromin.
 Replacement of K1423 and E1437 will also
decrease the stability of the above complex
as well. [11]
General ( very simplified) NF1
Pathway:
Fig. 3:
Neurofibromatosis 2:


Caused by a mutation in the NF2 gene.
-Is on Chromosome 22 at location q 11-13.1.
-Encodes a protein called MERLIN
(Moesin-Ezrin-Radixin-Like Protein)
Causes tumors to develop in the CNS, especially in
cranial nerve VIII connecting the inner ear with the
brain.
- Causes a variety of symptoms related to the ear:
deafness, ear ringing, and difficulty keeping
balance.
- Less common form of NF1 with an
occurrence of ~1/(30000-40000) births in the U.S.[1]
 Is
MERLIN:
595 amino acids long: Residues 1-302->N-terminal
region, residues 303-478->alpha-helix region, residues
479-595-> C-terminal region.
 Is a membrane-cytoskeleton scaffolding protein
which binds actin filaments to the cell surface
glycoproteins.
 Resides in cellular (adherens) junctions and
membrane ruffles in isolated cells where it acts as a
tumor suppressor by means of contact-mediated
growth inhibition.
 Merlin has a closed form in which it is active and an
open form in which it is not.
 Without a functional MERLIN, cells cannot recognize
each other when the contact and continue to grow
even when they have no space.[3]
MERLIN’s Intramolecular
Associations:
 Like
other members of the ERM family (Ezrin, Radixin,
and Moesin), MERLIN’s interactions with itself are
crucial to its function.
 2 main types of interactions: N-terminus/N-terminus
and N-terminus/C-terminus.
 The N/N-terminus interactions are necessary for the
N/C-terminus interactions.
 The N/C-interactions also require residues 302-308
and an intact exon 17 ( residues 580-595). [5]
4
5
MERLIN’s Possible Mechanisms:
 The
full scope of Merlin’s f tumor suppression isn’t
known.
 Most of MERLIN’s tumors suppression abilities come
from its ability to inhibit the activity of RAS GTPases.
 RAS also inhibits MERLIN by initiating a pathway
ending with the phosphorylation of Serine 518 by
PAK-1 which causes MERLIN to become open and
switch off. There are other such feedback loops.
 Recent studies show that another possible
mechanism by which Merlin acts is binding to E3
ubiquitin ligase-CRL4DCAF1 complex(which is crucial in
ribosome genesis) in the cell nucleus and suppressing
its activity.[3][10]
The Activation of RAS
Fig 6 : Receptor
Tyrosine Kinases
and RAS. The
pathway involves
the GRB2, a key
target for MERLIN.
Source:
www.ncbi.nlm.nih.gov
Complete (simplified)
MERLIN-RAS pathway:
Fig. 7:
Fig : The
MERLIN-RAS
pathway is
filled with
chains of
Kinases and
feedback
loops.
Fig 8:
The all-important RAS:
• The RAS family is at the heart
of both forms of
Neurofibromatosis.
• In general, there seems to be
a trend in cancers involving
mutations upstream that lead
to protein kinases proliferating
cell growth. This chain of
kinases begins with a member
of the RAS family such as H-RAS
(right). As such, RAS is a topic
of interest for researchers trying
to cure all forms of cancer, not
just neurofibromatosis. [3][5][8]
Fig 9 : H-RAS
In complex
with GCP (an
analog of
GDP)
The Financial Costs of
Neurofibromatosis:
 The
costs associated with both forms of
neurofibromatosis depends greatly upon how
severe the disease is. Patients with more severe
forms may require expensive medical procedures
such as surgery.
 According to the British Journal of Dermatology,
the average patient with Neurofibromatosis (1)
has about £810 ($1215) in medical costs. Medical
costs are probably higher, though there seems to
be no such study performed in the US.
 The direct overall costs in the US are probably
around $200 million, given the above estimate
holds true for the U.S.
How Neurofibromatosis
Effects the Body:
Source: www.NFINC.org
Meet Yvonne Foong:
 When
Yvonne was 16 years old,
she was diagnosed with
Neurofibromatosis 2.
 The disease took an overwhelming
toll on the young athlete, who
used to practice karate, ballet,
figure skating, and even singing in
her school choir. Soon she lost her
ability to hear and walk properly.
 MRI scans showed a large brain
tumor growing in her inner ear
affecting her hearing and several
smaller ones in her spine affecting
motor function.
Source: yvonnefoong.com
Since Then:
 Yvonne
has undergone 1 spine
surgery and 3 separate brain
surgeries.
 To help defray the costs, Yvonne has
written 2 books outlining her struggles
with the disease and her experiences
through it all.
 Her story came out in the October
08’ issue of Marie Claire magazine.
 Through all this Yvonne is optimistic:
“fear not. I am excited and
anticipating my next surgery and the
wonders it would lead to.”
Source: yvonnefoong.com
Hope for a cure?
 As
of now, the methods to deal with
any form of neurofibromatosis
target the symptom and not the
disease mechanism itself.
 BAY 43-9006 (Sorafenib), a new
drug being developed by Onyx
Pharmaceuticals in conjunction
with Bayer Pharmaceutical Group
targets cRAF and could potentially
treat both forms of
neurofibromatosis.
 So far the drug is in Phase I of
clinical trials, so it won’t be
available any time soon. [12]
Source:
Clinicaltrials.gov
How BAY 43-9006 (Sorafenib)
works:
 Sorafenib
seems to cause the
phosphorylation of serines on a variety of
kinases (particularly RAF-1) inhibiting their
activity.
 It also dephosphorylates serines 75 & 99
on an apoptotic protein called BAD
turning it on. Similarly it activates BAK and
BAX. Together these cells increase the
rate of cellular apoptosis.
 Sorafenib also causes a reduction in the
mitochondrial membrane potential
inhibiting respiration. [12]
References:
1.
2.
3.
4.
U.S. National Library of Medicine. “Neurofibromatosis.
“Genetics Home Reference. National Institute of
Health, March 2007. Web. 5 April 2010.
Davis, Ronald. “Neurobiology: Neurofibromin Progress
on the Fly.”Nature 403 (2000): 846-847. 1 April 2010.
JY Lim, Kim H, Jeun SS, Kang SG, Lee KJ. “Merlin
inhibits growth hormone-regulated Raf-ERKs pathways
by binding to Grb2 protein.” Biochemical and
Biophysical Research Communications 340.4 (2006):
1151-1157. 3 April 2010.
Scheffzek K, Ahmadian MR, Wiesmüller L, Kabsch
W, Stege P, Schmitz F, Wittinghofer A. “Structural
analysis of the GAP-related domain from
neurofibromin and its implications.” EMBO J.17.15
More References:
5.) Li, Wei, Liru You. “Merlin/NF2 Suppresses Tumorigenesis by
Inhibiting the E3 Ubiquitin Ligase CRL4DCAF1 in the
Nucleus.”Cell 140.4 (2010): 477-90. 29 March 2010.
6.) Klose, Anja, M. Rheza Ahmadian. “Selective disactivation
of neurofibromin GAP activity in neurofibromatosis type 1
(NF1).” Oxford University Press 7.8 (1998): 1261-1268. 29
March 2010.
7.) Dasgupta, Bislab, Laura Dugan. “The Neurofibromatosis 1
Gene Product Neurofibromin Regulates Pituitary Adenylate
Cyclase-Activating Polypeptide-Mediated Signaling in
Astrocytes.”The Journal of Neuroscience 23.26 (2003): 89498954. 29 March 2010.
8.) Guha, A, N. Lau. “Ras-GTP levels are elevated in human
NF1 peripheral nerve tumors.”Oncogene 12.3 (1996): 507-13.
30 March 2010.
Even More References:
9.) Fraz A. Ismat, Junwang Xu, Min Min Lu and Jonathan A. Epstein.
”The Neurofibromin GAP-related Domain Rescues Endothelial But
Not Neural Crest Development in Nf1–/– mice.”
J. Clin. Invest. 116.9 (2006): 2378-2384. 12 April 2010.
10.) NCBI. Molecular Cell Biology. Baltimore: W. H. Freeman and
Company, 2000. Print.
11.) Rob te Biesebeke,‡ Ivo M. Krab,§ and Andrea Parmeggian.
“The Arginine Finger Loop of Yeast and Human GAP Is a
Determinant for the Specificity toward Ras GTPase.”
Biochemistry.40.25 (2001): 7474–7479. 3 April 2010.
12.) David J. Panka1, Wei Wang2, Michael B. Atkins1 and James W.
Mier1. “The Raf Inhibitor BAY 43-9006 (Sorafenib) Induces CaspaseIndependent Apoptosis in Melanoma Cells.” Cancer Research 66
(2006) 1611-19. 13 April 2010.