Current progress at TRI: Biology

1
Current progress at TRI Biology

• Biology Staff
• John C. Wheeler
• Rachel M. Squillace
• Suzanne G. Sobel
• Sara A. Takacs

2
Presentation Outline

• Introduction to TSC
• Cell-based Screening
• Rapamycin and AP023573
• Compound library screening
• Human TSC Cell lmmortalization
• Screening Workflow

3
Background on Tuberous Sclerosis

• Genetic disorder caused by a mutation in one of
  2 genes TSC1 and TSC2.

• Incidence in 1/6,000 live births prevalence in
  1/10,000 people in USA.

• gt80 will have neurological problems including
  seizures, mental retardation
• and autism. Rarely, a brain tumor will
  develop.

• gt70 will develop kidney problems including
  angiomyolipomas and cysts.

• gt90 will show skin lesions, most of which are
  cosmetic.

• The Rothberg Institute is working to develop
  drugs to treat tuberous
• sclerosis and other orphan childhood diseases.

4
Compound Screening and Cellular Assay Development

• Drug screening for compounds that differentially
  inhibit growth of TSC-/- cells (21,539 compounds
  screened)
• Rapamycin and AP023573
• ICCB collaboration
• NCI Diversity Set
• MicroSource Spectrum Collection
• NCI Open Compound Library
• Secondary assays
• Apoptosis
• Immunoblotting
• Migration
• Immunofluorescence
• Drosophila
• Human cell line development
• Subungual fibroma and normal patient-derived
  fibroblasts
• Angiomyolipoma (AML)
• Lymphangioleiomyoma (LAM)

5
The PI3K-Akt-Tuberin Pathway
Mitogenic Stimuli PI3K PIP2
PIP3 PTEN
Akt Tuberin-Hamartin
P
P
P
P
mTOR S6K1 4E-BP1 Protein
synthesis
6
TSC-relevant cell lines

• Primary Screening differential growth
  inhibition
• Mouse embryonic fibroblasts (Kwiatkowski)
• TSC2 PE118 (TSC2/ p53-/-) vs. PE119
  (TSC2-/- p53-/-)
• Secondary Screening differential growth
  inhibition between multiple cell lines
• Mouse embryonic fibroblasts (Kwiatkowski, ATCC)
• TSC2 PE109 (TSC2-/ p53-/-) PE110 (TSC2-/-
  p53-/-) 2645 (TSC2/ p53-/-)
• TSC1 H202 (TSC1/) H205, H207, H208 (TSC1-/-)
• Tertiary Screening differential growth
  inhibition between cell lines of multiple species
  cell type
• Eker rat cells (Knudson Howard, ATCC, Walker)
• ERC15, 18, 33A (TSC2-/-) RK3E (TSC2/ E1a)
• ELT3 (TSC2-/-)
• Human bladder cancer cells (Knowles)
• RT4 (TSC1-/-)

7
Validation of TSC2 MEF cell lines
Cell line genotypes
Immunoblotting
PE118 TSC1/, TSC2/ p53-/-
PE110
PE119
PE118
PE109
2645
Cell line
2645 TSC1/, TSC2/ p53-/-
TSC2
PE109 TSC1/, TSC2/- p53 -/-
PE119 TSC1/, TSC2-/- p53-/-
TSC1
PE110 TSC1/, TSC2-/- p53-/-
Tubulin
Serum - - - - -
PCR genotyping
S6-P
PE109
PE110
PE119
PE118
Tubulin
2645
Cell line
TSC2 -
Cell line
PE109
PE110
PE119
PE118
2645
TSC2
TSC2 / / -/- /- -/-
8
Validation of TSC1 MEF cell lines
Cell lines
Immunoblotting
H202 TSC1/, TSC2/ p53/
H208
H207
H202
H205
H207 TSC1-/-, TSC2/ p53/
Cell line
TSC2
H205 TSC1-/-, TSC2/ p53/
H208 TSC1-/-, TSC2/ p53/
TSC1
Tubulin
PCR genotyping
Serum - - - -
S6-P
H207
H208
H205
H202
Cell line
Tubulin
TSC1 -
TSC1
Cell line
H205
H207
H202
H208
TSC1 / -/- -/- -/-
9
Rapamycin and AP023573
Mitogenic Stimuli PI3K PIP2
PIP3 PTEN

• Rapamycin is an inhibitor of mTOR.
• AP023573 is a rapamycin analogue developed by
  Ariad Pharmaceuticals.

AKT
P
P
Tuberin/Hamartin
Rapamycin/ AP023573
mTOR S6K1 4E-BP1 Protein
synthesis
10
MTS-based Cell Growth Assay Methods
PBS Compounds
PBS
Cells Cells
No cells
Murine Embryonic Fibroblasts
3 wells/compound
11
Rapamycin and AP023573 differentially inhibit
growth of TSC MEF cells
12
Rapamycin and AP023573 do not cause apoptosis

• Apoptosis was measured by Apo-One Caspase3/7
  assay (Promega).

13
Rapamycin and AP023573 inhibit phospho-S6(235/236)
in TSC cells
PE118 PE119
PE110
Serum - - - -
- - - - -
- - -
Rapamycin - 1nM 4nM 16nM -
1nM 4nM 16nM - 1nM 4nM 16nM

P-S6 tubulin
PE118 PE119
PE110
Serum - - - -
- - - - -
- - -
AP023573 - 1nM 4nM 16nM -
1nM 4nM 16nM - 1nM 4nM
16nM
P-S6 tubulin
14
Drosophila in vivo drug testing rapamycin and
AP023573

• Absence of TSC1 or TSC2 causes lethality during
  larval development.
• Can rapamycin and/or AP023573 rescue larval
  lethality?

TSC/TSC- x TSC/TSC-
Add Drug
TSC-/- pupae
TSC/TSC- pupae

• larval lethal

• TSC chromosome linked to gene
• with dominant pupal phenotype.

15
Screening compounds at the Harvard Medical School
- ICCB

• Multiple commercial libraries 101,003 compounds
• Screening Methods PE118 (TSC2/) vs. PE119
  (TSC2-/-)
• Plate at 600 cells/well in 384-well plates (2x
  density of TRI screening).
• Add 100nL compound to 20-60uM (10-30x greater
  than used at TRI).
• Incubate 24 hours.
• Perform MTS cell viability assay.

Cell plating
Pin transfer Assay
read-out
16
Hits from the ICCB screening

• Screened 17,563 compounds from 4 separate
  commercial libraries.
• 620 Hits across both cell lines (3.5)
• 27 Differential hits (0.15)
• ICCB will aliquot and ship 1-2uL of 0.3 of the
  compounds screened.
• Re-testing methods PE118 vs. PE119
• 24 hour assay same concentration as at ICCB
• 72 hour assay 110 compound dilution

17
ICCB repeated hits

• 27 hits re-tested
• 2 hits repeated in 24 hour assay
• ChemDiv 8001-1421 C31H28IN3O
• PE118 64 PE119 18
• Bionet 10N-024 C15H9F3N2O7
• PE118 38 PE119 16
• 2 hits repeated in 72 hour assay
• Maybridge AW 00945 C27H30N4O3S
• PE118 115 PE119 40
• Maybridge BTB 04272 C11H12N2O3S2
• PE118 68 PE119 32

18
TRI Cell-based Screening Methods
PBS Compounds (2 uM) PBS
Cells Cells
No cells
1 well/compound
19
NCI Diversity Set

• The Diversity Set comprises 1990 compounds from
  NCI Open Compound Library representing structural
  diversity.
• 2 Differential Hits
• NCI 131547 C29H37N3O3
• PE118 80 PE119 20
• NCI 259969 C40H48N6O9
• PE118 50 PE119 20

SAT
20
Diversity Set Hits
SAT
21
MicroSource Spectrum Collection

• The Spectrum Collection 2000 biologically
  active including marketed and experimental drugs
  as well as natural products. Purchased from
  Microsource Discovery Systems, Inc.,
  Gaylordsville, CT
• 2 differential hits puromycin, nigericin
• 65 hits in both cell lines
• Dose tested due to known bioactivity of compounds
  in this library.

• Screen Summary Data
• Mean Mode
• 92 19 103
• 94 20 104
• 110 92 17 106

22
Puromycin affects TSC2-/- cells, while nigericin
is a false-positive
cell growth
cell growth

• Puromycin inhibits cell growth of both TSC2 null
  lines (119,110), Nigericin does not.

23
Puromycin and PAN

• Puromycin
• A broad spectrum antibiotic with antitumor
  activity.
• Causes nephrotoxicity in animal studies.
• Inhibits protein synthesis by causing premature
  termination of peptidyl chain elongation.
• Induces S6K activity in the absence of amino
  acids (Iiboshi et al.,1999).
• PAN
• Structural analysis suggests it cannot inhibit
  protein synthesis.
• May be metabolite of puromycin in vivo.

24
Other protein synthesis inhibitors do not
differentially inhibit growth of TSC2 -/- MEF
cells
Puromycin - TSC2 cells
Emetine - TSC2 cells
118
118
118
118
119
119
119
119
100
100
109
109
110
109
110
110
109
110
80
80
cell growth
60
60
cell growth
cell growth
cell growth
40
40
20
20
0
0
0.01
0.1
1
10
0.01
0.1
1
10
Concentration (uM)
Concentration (uM)
Concentration (uM)
Concentration (uM)
Data not shown cycloheximide, paromomycin,
G418 protein synthesis inhibitors in TSC1 MEFs.

• Hygromycin, like puromycin, is an aminoglycoside
  that blocks at the translocation step of protein
  synthesis.
• Emetine inhibits translocation by binding to the
  40S ribosomal subunit.
• Blasticidin S blocks new peptidyl bond formation.

25
Puromycin and PAN inhibit cell growth of TSC2,
but not TSC1 cells
cell growth
cell growth
cell growth
cell growth
26
Puromycin is cytotoxic, PAN is cytostatic
Apoptosis ( of PBS control)
Apoptosis ( of PBS control)
27
Stimulated S6 and S6K phosphorylation by
puromycin and PAN
Puromycin
PAN
Cyclo (100uM)
Puro (100uM)
PAN (100uM)
Puro (10uM)
Puro (10uM)
PAN (30uM)
PAN (10uM)
Puro (1uM)
No Cmpd.
No Cmpd.
No Cmpd.
No Cmpd.
Compound
Compound
Serum - - - - -
Serum - - - - -

S6kinase - P
S6kinase - P
S6 - P
S6 - P
Tubulin
Tubulin
119
119

• 119 cells were serum-starved for 48 hours, then
  compound was added for 1 hour before harvest.

28
Summary of results

• Puromycin
• Inhibits growth of TSC2 null cells, but not TSC1
  lines.
• Induces apoptosis.
• Stimulates S6 phosphorylation.
• PAN
• Inhibits growth of TSC2 null cells (including
  2645) better than puromycin, but not TSC1 lines.
• Does not induce apoptosis.
• Stimulates S6K phosphorylation.

29
Immortalization of AML and LAM cells

• There are currently no cell lines derived from
  human-specific TSC lesions AML or LAM.
• TSC mutant mice do not get AMLs or LAM.
• Almost all TSC cell lines are rodent, not human.
• We will generate immortalized AML and LAM cell
  lines for drug discovery at TRI.

30
Immortalization strategy and validation

• Human primary TSC fibroma/fibroblast
  immortalization

• E6E7 Human papillomavirus type 16 genes.
• E6 activates hTERT, inhibits p53.
• E7 inhibits pRB, p21.
• E6E7 encoded by a single mRNA.

Immortalization protocol
24h _at_37oC
Fibroma
24h _at_32oC
48h _at_37oC
7d _at_37oC
Fibroblasts
neo
neo
neo
Wash
Grow in growth factors.
neo
neo
neo
neo
neo
neo
neo
neo
neo
Infect
Recover
Select
Genotype clones for mutant TSC1 and TSC2 status
at Athena Diagnostic.
3T3 cells expressing retrovirus containing E6E7
plasmid.
31
AML and LAM cells

• We will obtain genotyped AML cells from Lisa
  Henske (LOH TSC2), and LAM cells from Vera
  Krymskya and Joel Moss.
• There is no reliable biomarker for AML and LAM
  cells - the gold standard is sequencing.
• AML and LAM cells will be genotyped for TSC1/2
  before and after immortalization.
• A collaboration is in process with Athena
  Diagnostics to sequence TSC1/2 loci of starting
  samples and immortalized clones.
• Current progress retroviral producing lines and
  fibroma/fibroblast cells growing in culture.

32
Paclitaxel and colchicine hits from the
MicroSource Spectrum Collection

• 1 original differential hit from this library
  (puromycin).
• 65 compounds strongly inhibited growth in all
  cell lines and were dose reduced.
• 3 of these compounds were differential hits in
  the TSC2 MEFs
• Paclitaxel (taxol) and 2 variants of colchicine
  were found as hits.
• These are microtubule inhibitor drugs.
• Both have been used medicinally, but are known
  for their high toxicities.

2 colchicine variants
Paclitaxel
33
Paclitaxel and colchicine differentially inhibit
growth of TSC2-/- MEF cells
cell growth
cell growth
cell growth

• Paclitaxel differentially inhibits growth of
  all strains except PE118 (/).
• Colchicine and AP023573 differentially inhibit
  all strains except PE118, PE109 (/-).

34
Paclitaxel and colchicine do not differentially
inhibit growth of TSC1-/- MEF cells
Colchicine - TSC1 cells
Paclitaxel - TSC1 cells
100
100
202
202
205
205
207
207
208
208
80
80
60
60
cell growth
cell growth
cell growth
40
40
20
20
0
0
10
100
1000
10
100
1000
Concentration (nM)
Concentration (nM)

• Only AP023573 differentially inhibits the -/-
  strains.

35
Distinguishing the red herrings from the caviar

• Identifying cell line specific (and TSC loci
  independent) effects
• Puromycin, paclitaxel, and both Diversity Set
  hits are PE118 cell line specific.
• AP023573 and rapamycin differentially inhibit
  multiple MEF cell lines.
• True hit compounds should have differential
  effects across on multiple sets of cell lines.

36
Distinguishing the red herrings from the caviar

• Identifying cell line specific (and TSC loci
  independent) effects
• Puromycin, paclitaxel, and both Diversity Set
  hits are PE118 cell line specific.
• AP023573 and rapamycin differentially inhibit
  multiple MEF cell lines.
• True hit compounds should have differential
  effects across on multiple sets of cell lines.
• mTOR pathway dependence
• Rapamycin (and AP023573) directly inhibits mTOR.
• Paclitaxel has been found to induce inactivation
  of S6K via phosphorylation (Oncogene 22484,
  2003).
• Since modulation of the mTOR pathway by a
  compound does not indicate whether the affect is
  direct, this may not be a useful early step
  within our general workflow plan.

37
Proposed Workflow
Primary Screen
Confirm hits and dose reduction
Determine cell line specificity
Determine species cell type specificity
(non-MEFs)
To proceed differential response in 2/3 sets
of cell lines
38
Combinatorial dose response

• Combinations of compounds allow for modulation of
  multiple targets.
• Combination therapies for many diseases (HIV,
  cancer, hepatitis C, etc.) are already part of
  FDA approved regimens.
• We will assay all TSC-specific true hits in
  combination with rapamycin and AP023573 to
  identify any synergistic effects.

APO23573 75nM Paclitaxel
APO23573 Alone
cell growth
cell growth
118 75 nM Taxol
118 0 nM Taxol
119 75 nM Taxol
119 0 nM Taxol
0
0
1
1
Concentration of APO23573 (nM)
Concentration of APO23573 (nM)
39
Screening throughput at TRI

• Current throughput in 96 well plates
• 3-4K compounds/week.
• All 4 FTEs fully occupied.
• Screening in 384 well plates
• 7K compounds/week.
• Most time of all 4 FTEs occupied.
• Primary screen of NCI library (re-formatted to
  100,000) complete in 15 weeks.
• Cost of reformatting library
• 1FTE/week 3/5 Wolfgang, 2/5 other FTE.
• Cost of disposables 6000.
• Screening in 384 well plates with an automatic
  stacker (16,000)
• 14K compounds/week.
• 1 FTE fully occupied.
• NCI library (not re-formatted) primary screen
  complete in 13 weeks.
• 3 FTEs available for follow-up on hits!
• Saves cost of reformatting library!
• More Libraries !!

40
An Environment for Creativity
41
Proposed screening workflow
Primary screen
Select all hits reformat
Confirm hits dose reduction
2 mM
0.2 mM
Dose response determine species and cell type
specificity
True positive differential response in 2/3
sets of cell lines