1. Background & Introduction
Each year approximately 220,000 people are diagnosed with non-small cell lung cancer (NSCLC) in the US alone. The LKB1 (also
known as STK11) tumor suppressor is mutationally inactivated in ~30% of sporadic non-small cell lung adenocarcinomas, however
there are no effective therapies for lung tumors harboring these mutations. LKB1 functions as a master regulator of cell growth,
metabolism and mitochondrial homeostasis through the adenosine monophosphate activated kinase (AMPK) and the Unc-51 like
kinase 1 (ULK1) pathways. Work in the Shackelford lab has identified the LKB1 signaling pathway to be critical for maintaining
cellular energy balance and mitochondrial turnover known as mitophagy in both normal and lung tumor cells. Importantly, LKB1
mutant cells unable to appropriately sense metabolic stress and are selectively sensitive to energy stress inducing compounds
such as phenformin. Phenformin is a mitochondrial inhibitor that induces energy stress and cell death in LKB1 mutant lung tumor
cells therefore we wanted to explore the possibility that other highly potent mitochondrial inhibitors may selectively kill LKB1
mutant lung tumors. In an effort to identify selective and highly effective new therapies for the treatment of LKB1 mutant lung
cancer, we investigated FDA approved mitochondrial inhibitors to be repurposed for use as anti-cancer agents.
Conclusion & Discussion:
Targeted therapeutics have made significant advances in subsets of NSCLC bearing activated oncogenic targets. Currently there are limited
options for LKB1-mutant tumors, but here we illustrate the hypersensitivity of LKB1-defective cells to metabolic and mitochondrial stress
and test the therapeutic use of a mitochondrial inhibitor MI007 on LKB1 mutant human and mouse lung tumor lines and MEFs. MI007
selective and efficiently killed LKB1 mutant lung tumor cell lines at low, clinically relevant doses. Our results suggest that MI007 and future
mitochondrial inhibitors identified in our screen may have clinical benefit in treating LKB1 and KRAS mutant lung tumors in patients. and
that Grp78 may have potential be developed as a clinical biomarker to measure of mitochondrial based therapies.
Future Directions:
We plan to assess MI007 on a larger panel of human NSCLC tumor cell lines and begin performing preclinical studies using GEMMs of lung
cancer. Development of biomarkers P-AMPK and Grp78 analyzed in this study will help to guide future in vivo pre-clinical studies.
Figure 1. Compound MI007 significantly decreases membrane potential and cell viability at low doses and in an LKB1 specific manner. (A)
Measurement of MMP (ΔΨ) following treatment with mitochondrial inhibitors (MI’s) or ROS inducers (RI’s) as indicated. IC50’s of MI007 determined using KrasMUT
or KrasMUT;Lkb1-/- mouse lung tumors lines in (B) and human A549 isogenic NSCLC lines in (C). IC50’s were calculated after treatment with varying doses of
MI007. Viability measured using CellTiter-Glo® Luminescent Cell Viability Assay (Promega). Viability normalized as a percentage of no treatment (NT) control. (D).
KW634 and KW821 cell lines were plated in clear-bottom 96-well plates and Red:Green ratio of JC-1 Dye was measured as described in Figure 1.B. (E). Viability
was measured using the Caspase-Glo 3/7 Assay (Promega) following 6hrs of treatment with 0.5% DMSO, MI007 or phenformin. Samples were ran in triplicate.
Significance was calculated using student’s t-test.
Figure 2. Biomarker analysis of K-Ras mutant, Lkb1-/- MEFs and Kras
mutant lung tumors following treatment with MI007 or phenformin.
Immunoblots from whole cell lysates Kras mutant (KrasMUT), Kras mutant + Lkb1
null (Lkb1-/-;KrasMUT) wild MEFs in (A) and KrasMUT lung tumors in (B). Cells
were not treated (NT) or treated with MI007 for 24 hours. Lysates were blotted
and probed with antibodies against caspase 3 (CC3), a marker of apoptosis,
Grp78, a marker of mitochondrial stress, phospho-AMPK-threonine 172 (p-AMPK
thr172) a marker of cellular energy stress or B-actin as a loading control.
LKB1 is the 3rd most frequently mutated gene in lung adenocarcinoma
Objectives: 1) To use a high throughput screen measuring tumor cell mitochondrial membrane potential (MMP) and
apoptosis to identify mitochondria inhibitors with anti-cancer properties; 2) perform functional assays on lead candidate
compounds and 3) assess potential biomarkers to guide pre-clinical studies.
Results:
Summary of Results:
1) JC-1 dye accurately measures loss of ΔΨ following treatment with MI’s.
2) MI007 found to have an IC50 of 500nM in human and mouse LKB1 mutant NSCLC tumor cell lines.
3) MI007 selectively induces both loss of ΔΨ and apoptosis in LKB1 mutant NSCLC tumor cell lines.
4) Low dose MI007 induces apoptosis, energy stress and mitochondrial stress in Lkb1-/-;KrasMUT ;Lkb1-/- MEFs and mouse lung tumor cell
lines. Additionally, MI007 induces mitochondrial stress in KrasMUT and wild type MEFs represented by elevated Grp78 protein levels.
Summary of the Experimental Design:
1) Performed a literature search of studies assessing FDA approved compounds to disrupt mitochondrial function and MMP.
2) Performed IC50’s on lead compounds using the cell titer glo assay.
3) Performed a 96 well formatted H.T.S screen on MI’s that measured both MMP and apoptosis. MMP was measure using the
voltage sensitive dye JC-1 and cellular apoptosis was measure by the caspase 3/7 glo assay.
4) Performed functional assays in cell culture by immunoblotting for markers of: i) apoptosis, ii) energy stress and iii)
mitochondrial stress.
5) We will perform PK and PD analysis of lung tissue and lung tumors in mice followed by pre-clinical studies assessing MI’s as
anti-cancer agents targeting LKB1 mutant lung cancer.
Developing a H.T.S of mitochondrial inhibitors to be repurposed for the treatment of LKB1 mutant NSCLC
Mitochondria Disrupters / ROS Inducers
N
T
2
m
M
Phen10
uM
PL20
uM
PL50
uM
PL
500
nM
O
ligom
ycin
1
uM
O
ligom
ycin
5
uM
O
ligom
ycin
25
uM
Antim
ycin
A
50
uM
Antim
ycin
A
100
uM
Antim
ycin
A
5
uM
B-lapachone
10
uM
B-lapachone
20
uM
B-lapachone
10
uM
A
-D
H
C
20
uM
A
-D
H
C
40
uM
A
-D
H
C
0
1
2
3
4
Red:GreenRatio
KW634
KW821
KrasMUT
KrasMUT ;Lkb1-/-
N
T
10
uM
PL
10
uM
A
-D
H
C
0
1
2
3
4
Red:GreenRatio
KW634
KW821
**
*** ***
**
Red:GreenRatio
Mitochondria Disruptors/ROS inducers
NT
Phen(2mM)
RI001(10µM)
OligoA(0.5µM)
OligoA(1µM)
OligoA(1µM)
AntA(1µM)
RI001(20µM)
RI001(50µM)
AntA(1µM)
AntA(1µM)
RI002(5µM)
RI002(10µM)
RI002(20µM)
MI007(10µM)
MI007(20µM)
MI007(40µM)
A
NT
10
uM
PL
10
uM
A-DHC
0
1
2
3
4
Red:GreenRatio
KW634
KW821
**
*** ***
**
KrasMUT
KrasMUT ;Lkb1-/-
N
T
10
uM
PL
10
uM
A
-D
H
C
0
1
2
3
4
Red:GreenRatio
KW634
KW821
**
*** ***
**
ΔΨ
MI007PhenNT
N
T
5
uM
A
-D
H
C
2
m
M
Phen
0.0
0.2
0.4
0.6
0.8
1.0
1.2
CellViability(%NTControl)
***
***
Viability
MI007PhenNT
96 well format
Literature Search of
FDA approved MI’s
Multiplex: Measure both
MMP (ΔΨ) and apoptosis
IC50’s on lead
compounds
Functional assays
in cell culture
PK and PD analysis
Preclinical trials
LKB1+/+'
AMPK'
P
AMP
Cell'Growth'
AMPAMP
ADPAMP
AMP
AMP
Energy'Stress'
ADP
LKB1:/:'
AMPK'
Cell$Death$
Cell'Growth'mTORC1'
Ding%et%al.,%Nature%2008%LKB1 = STKII
LKB1 mutant cells are unable to restore energy balance and
undergo apoptosis in response to metabolic stress
AMPKα
β-actin
Veh Phen
K-RasG12D
℗-AMPKα
Grp78
Mouse lung
tumors
β-actin
KrasMUT
KrasMUT;
Lkb1-/-
Grp78
CC3
MI007
NT
MI007
NT
MEFs
℗-AMPKα
2 4 6
-0.5
0.0
0.5
1.0
1.5
log[ADHC]nM
Viability(%Control)
KW634
KW821
IC50 = 500nM
Viability
MI007 (nM)
!KrasMUT
!KrasMUT ;Lkb1-/-
IC50 = 500nM
log[ADHC]nM
Viability(%Control)
0 2 4 6
0.0
0.5
1.0
1.5
A549B
A549WT
A549KD
IC50
520nM
430nM
570nM
A549B
A549WT
A549KD
IC50 = 500nM
Viability
MI007 (nM)
A
B C
D E
A B
MEFs Mouse lung tumors
Alex Yang1, Robert J. McMickle1, David B. Shackelford1
1Pulmonary and Critical Care Medicine, David Geffen UCLA School of Medicine, Los Angeles, CA 90095 *Correspondence: DShackelford@mednet.ucla.edu,
Identification of personalized therapies for LKB1 mutant lung cancer using a
high throughput screen of FDA approved compounds