Recent Advances and Opportunities in Targeting Cancer Stem Cells

Recent Advances and Opportunities in
Targeting Cancer Stem Cells
Mattia Mori
Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia
m.mattia79@gmail.com
mattia.mori@iit.it
Puerto de la Cruz, October 14th-15th, 2014
Center for Life Nano Science@SAPIENZA
 Brain tumors, the most life-threatening diseases of adulthood
and childhood
 Understanding the interplay between Cancer Stem Cells (CSCs)
and neoangiogenesis and the dynamics of the CSCs population.
Research at CLNS
Hedgehog
Signaling Pathways
Notch
Molecular Modeling
Virtual Screening
Molecular Dynamics
Quantum Mechanics
Organic Synthesis
Muta-synthesis
Total synthesis of natural products
Extraction/isolation/characterization of
natural products
Biology/Pharmacology
In vitro/in vivo facilities
Biochemistry
Recombinant and mutant proteins
The collaborative network
Center for Life Nano Science IIT
Mattia Mori, PhD
Paola Infante, PhD
Rocco Palermo, PhD
Cinzia Ingallina
University of Siena
Prof. Maurizio Botta
University of Rome – ITALY
Dept. Pharmaceutical Sciences
Bruno Botta, Prof.
Sara Toscano
Francesca Ghirga, PhD
Ilaria D’acquarica, PhD
Simone Berardozzi
Patrizio Ghirga
Dept. Molecular Medicine
Alberto Gulino, Prof.
Lucia Di Marcotullio, Prof.
Isabella Screpanti, Prof.
Romina Alfonsi
View-point presentation - layout
1. Critical survey covering the year 2014
Statistical analysis
General considerations
2. STAT3 – state of the art and opportunities
3. Hedgehog – state of the art and opportunities
Critical survey covering the year 2014
Method:
• SciFinder (https://scifinder.cas.org/)
• Primary search: cancer stem cells
• Refine #1: topic “targeting”
• Refine #2: year (i.e. 2009, 2010, … 2014)
• Analysis #1: document type
Reviews
Research papers
Patents
Signaling
pathways/targets
Small molecules
Trend for “targeting CSCs”
Average # documents/month
Research papers/reviews/patents
70
60
50
40
30
20
10
0
2005
2009
2010
2011
2012
2013
2014
What in 2014?
Research papers
4%
Reviews
17%
Patents
79%
Searching “cancer stem cells” AND “targeting” on SciFinder, September 2014
Reviews 2014
As expected, reviews address most targets and most small
molecules published in the field of “targeting CSCs”
A significant number of reviews address the NOTCH signaling
pathway modulation, most often by curcumin
Particular interest in pancreatic cancer
Reviews 2014
Drug Discov Today. 2014, 19(10):1547-1562
Patents 2014
Most patents focus on small molecule modulators
of CSCs, biomarkers, detection methods, …
PKC/FRAP
STAT
MAPK
Hedgehog
PCT EP 2014063449
Botta B. et al, 2014, PCT EP 2014063449
Research papers 2014
23%
36%
18%
7%
STAT
WNT
NOTCH
HEDGEHOG
OTHER
16%
STAT is the most addressed target, followed by the canonical
signaling pathways that regulate CSCs proliferation/differentiation
(Hh, Wnt, Notch)
Small Molecules 2014 – CAS number
Frequency of small molecules usage in research papers (targeting CSCs)
5-FU
Cisplatin
Paclitaxel
Salinomycin
Doxorubicin
Gemcitabine
Eposin
Curcumin
Metformin
Temozolomide
Consideration #1
OLD DRUGS are still very useful in
biological/pharmacological investigations
Mostly used to understand the interplay between CSCs and
tumor initiation/progression in several cancer types
Extended dataset
Drug Repositioning
Combination Therapies
Mori et al, ChemMedChem 2014, 9, 973 - 983 – CM1106 partnership
An overview of Clinical Trials 2014
Most CSCs-oriented clinical trials monitor the efficacy & safety of
small molecule inhibitors (or vaccines) of HEDGEHOG, WNT or
NOTCH pathways.
 evaluation of New Molecular Entities efficacy in combination
with old drugs
 Significant interest in pancreatic cancer
Consideration #2
Lack of NMEs in advanced stages of development
Most biological investigations are performed with old drugs or, at
least, with a combination of old drugs + NMEs
However, NMEs are still quite far from advanced stages (clinical)
 big effort is required to MedChem & Pharma Companies.
2014 summary
Other Targets
HITs
LEADs
STAT 
Hh
Wnt
Notch
PRE-CLINICAL
CLINICAL
View-point presentation - layout
1. Critical survey covering the year 2014
Statistical analysis
General considerations
2. STAT3 – state of the art and opportunities
3. Hedgehog – state of the art and opportunities
STAT3
STAT3 is a transcription factor
constitutively activated in many tumors
Head & Neck squamous cell carcinoma
Non-small cell lung cancer
Breast cancer
2014 CD44+ cells isolated from 17
hepatocellular carcinoma (HCC)
positive patients have CSCs activity in
vitro.
Wan S et al, Gastroenterology, doi: 10.1053/j.gastro.2014.08.039
STAT3
Indirect targeting
(upstream STAT effectors)
Problems connected with kinase
inhibitors promiscuity, cross-talk
in signaling pathways, low
specificity/selectivity
Direct STAT targeting
- SH2 dimerization site
- STAT3/DNA interaction
Not trivial design
Thought to be specific
Recent progresses
#1 STAT3 dimerization inhibitors - 2013
R
Organic synthesis
In vitro assays
O
OO
S
N
Ar
R1
N
HO
O
HO
R
O
N
HO
O
HO
Low activity
Poor drug-like
properties
O
R
H
N
HO
HO
O
Ar
R
N
Ar
O
O
#1 STAT3 dimerization inhibitors - 2014
FP-based assay for STAT3 dimerization:
-full length STAT3
-5FAM-GpTLPQTV (from IL6 receptor)
-SH2 binding
13g
S31-1757
O
O
HO
HO
N
IC50 = 15 +/- 0.44 µM
O
Reference STAT3-STAT3 inhibitor
Cell permeable; not-cytotoxic
200 µM
pSTAT3 and STAT3 Immunoblotting
MDA-MB-469 (breast cancer)
100 µM
Breast
Lung
Zhang H et al, Cancer Res; 73(6) March 15, 2013
#2 STAT3 dimerization inhibitors - 2014
Tetrahydro-pyridine-pyrazoles as new
chemotypes for STAT3 inhibition
Breast & Hepatocellular cancer
HCC  connected to CSCs activity
#2 STAT3 dimerization inhibitors - 2014
STAT3 phosphorylation and DNA binding (10 µM)
12g
NH
O
NH
N
N
Cl
pSTAT3
N
O
F F
Cl
STAT3
Not cytotoxic
HCC cells proliferation
STAT3 dimerization inhibitors - 2014
Good examples of medicinal chemistry
13g
S31-1757
12g
NH
O
O
O
N
N
HO
HO
NH
N
Cl
N
O
O
F F
Cl
Selective inhibitors of STAT3 signaling in vitro, safe,
clear mechanism of action
Possible pre-clinical candidates
STAT3/DNA interaction inhibitors - 2014
192000 compounds
• Water soluble
• Resistant to proteolysis
High-throughput screening in
vitro against STAT3
Antiproliferative effect in cells, not affecting STAT3 SH2 phosphorylation
100 µM
Selective inhibitor of
STAT3/DNA interaction
New tool for STAT3 modulation
New opportunity for drug design
View-point presentation - layout
1. Critical survey covering the year 2014
Statistical analysis
General considerations
2. STAT3 – state of the art and opportunities
3. Hedgehog – state of the art and opportunities
Hedgehog - pitfalls
IC50 = 0.005 – 0.02 μM
(Gli1-reporter gene assay)
Smo antagonist
Metastatic BCC – FDA 2012
 Drug resistant Smo
mutant (D473H)
 Hh activation
downstream of Smo
Yauch et al. Science (2009) 326, 572.
Hedgehog - pitfalls
IC50 = 0.005 – 0.02 μM
(Gli1-reporter gene assay)
NEW STRATEGIES ARE NEEDED TO
TARGET THE HEDGEHOG PATHWAY
NEW TARGETSMetastatic BCC – FDA 2012
NEW SMALL MOLECULES
Drug resistant Smo
mutant (D473H) isolated
from a patient treated
with Vismodegib
Yauch et al. Science (2009) 326, 572.
Hedgehog – new opportunities - 2014
1.6M mols
HTS
Non-Smo
Tox
Dose
dependent
Smo
Gli-luc
Pharma
filtering
trash
TARGET
identification
cyclohexyl-methyl aminopyrimidines (CMAPs)
CMAPs
Mechanism of investigation
Smo or non-Smo binders?
Non-Smo binders do not
change their activity in
presence of increasing
concentration of a Smo
agonist
Bodipy-cyclopamine
Weierstall U, et al. Nat Commun 2014, 5: 3309-3309
Non-Smo binders do not compete
with bodipy-cyclopamine from Smoexpressing cells or membranes
isolated from these cells.
Target identification  GPR39
Main hypothesis: GPCR
1) Previous experience with this class of molecules
2) Chemical similarity with Neuropeptide Y5 receptor (GPCR) antagonists
GPCR binders - experimental proof:
1) cAMP, calcium and inositol triphosphate (IP3) production measurement
(markers of GPCR-mediated signaling)
absence of calcium signaling
massive IP3 production
Non-canonical GPCR
signaling
Target identification  GPR39
GPCR type - experimental proof:
2) GPCR mRNA expression was measured in TM3 (CMAP-responsive)
and MC3T3 (CMAP-non-responsive) cells by qPCR
GPR39 was highly expressed in TM3 cells
IP3 production
Hh inhibition
HEK293 cells stably expressing GPR39
HEK 293
GPR39 is the target receptor of Hh inhibitor CMAPs
Summary – GPR39
GRP39 acts downstream of Smo
and modulates Gli signaling
through a yet un-elucidated
mechanism
GRP39 is a potential therapeutic
target for Hedgehog pathway–
driven tumors, particularly those
with acquired SMO inhibitor
resistance
New opportunity for drug design
PLoSONE 2009, 4(8): e6709.
Viewpoint 2014 – Summary
1) Targeting CSCs is an emerging and promising anti-cancer
strategy
2) Many biological studies, few NMEs…
3) Biology/biochemistry is highlighting new promising strategies
or targets to impact selectively on CSCs proliferation
4) Much effort from medicinal chemists is required to design
and develop NMEs up to preclinical or clinical candidates