1. Integrated genomic and proteomic analysis identifies PTEN loss and AKT/MTOR as drivers of resistance to MEK inhibitors in NSCLC cells
Dianren Xia1, Lauren Averett Byers1, Lixia Diao2, Jing Wang2, Steven H. Lin3, Don Lynn Gibbons1, Kathryn Ann Gold1, Juliane Paul4, Ningshu Liu4, and John V. Heymach1
Departments of Thoracic, Head and Neck Medical Oncology1, Bioinformatics2, and Radiation Oncology3, The University of Texas MD Anderson Cancer Center. Bayer HealthCare Pharmaceuticals, Mueller ST 178, Berlin 13342, Germany4.
Introduction
Lung cancer is the leading cause of cancer death worldwide.
Non-small cell lung cancer (NSCLC) is diagnosed in 85% of lung cancer patients.
NSCLC therapy has been largely limited to chemotherapy and radiotherapy with un-resectable tumors.
Recent development in targeted therapy, such as EGFR inhibitors erlotinib, gefitinib, and afatinib, provides new hope for NSCLC patients.
However, their therapeutic efficacy is impeded by the emergence of drug resistance.
MEK inhibitors (MEKi) is a group of small molecule inhibitors targeting MEK1/2 of the RAS/RAF/MEK/ERK pathway.
MEKi have shown promising clinical outcome in NSCLC in clinical development.
Clinical and pre-clinical studies have demonstrated that MEKi is only effective in a sub-population of NSCLC patients, indicating the existence of intrinsic drug resistance.
Thus, it is urgent to identify predictive biomarkers of MEKi to assist the patient selection and design combination treatment to overcome the resistance in future clinical application.
Results
Figure 2: PTEN mutation is associated with MEKi resistance in NSCLC cells
Figure 4: PTEN knockdown renders sensitive cells more resistant to MEKi in vivo
Figure 6. Decitabine sensitizes part of NSCLC cells to MEKi
Figure 1: Diminished basal PTEN expression and elevated AKT activation are associated with MEKi resistance in NSCLC cells A A A
No. of cell lines with mutations *
P=0.0322
P=0.9808
P=0.0194
P=0.9475
Tumor volume (mm3)
Trametinib (days)
H661
H661 A
AKT pT308
AKT pS473
GSK3 pS21.9
PDK1 pS211
p53BP1
4EBP1 pS65
PTEN
Cyclin D1
SMAD3
Resistant
Sensitive
Sensitivity:
RPPA expression
Relative cell viability
Relative cell viability
Refametinib (μM)
Trametinib (μM) B B
IC50 to refametinib (μM)
PTEN wild-type
H838
H838 B
PTEN del or lost
Survival rate (%)
Trametinib (days)
Relative cell viability
Relative cell viability B
AKT pS473 (log2 ratio)
Wilcox rank
P=0.003 -1 1 -2 2 -3
Sensitive
AKT pT308 (log2 ratio)
PTEN (log2 ratio)
Resistant
P: 0.017 3
Refametinib (μM)
Trametinib (μM)
Figure 3: Decrease in PTEN expression causes NSCLC cells resistance to MEKi
Conclusions
Basal low PTEN expression is correlated with the activation of PI3K pathway and MEKi resistance.
Genetic PTEN loss is associated with MEKi resistance in NSCLC cells.
Alteration of PTEN expression through knockdown or forced expression affects MEKi resistance of NSCLC cells.
Knockdown of PTEN increases the resistance to MEKi in vivo.
Low PTEN expression in a subset of MEKi resistant cells is associated with higher DNA methylation. Pre-treatment with decitabine re-sensitizes these cells to MEKi. A
H2009
H2009
Figure 5: PTEN promoter is hypermethylated in part of NSCLC cells resistant to MEKi
PTEN
HCC44
A549
H1666
H2108
H2405
H2887
HCC4006
H4017
H28
HCC193
H838
H1793
H1819
H2170
H2882
HCC3051
Sensitive
Resistant
H23
H1437
H2291
H2347
Calu1
H157
H441
H520
ACTB
Relative cell viability
Relative cell viability A C
Methods
We have employed an integrative, multi-dimensional approach to study the mechanisms of MEKi resistance of NSCLC cells, which include:
Reverse Phase Protein Array (RPPA) for protein expression.
cDNA microarray for mRNA expression.
Genome-wide DNA methylation analysis (Infinium HumanMethylation27 BeadChip).
Publicly available datasets for genomic alterations (COSMIC, CCLE).
Xenograft validation and clinical confirmation.
Cells with PTEN decreased with decitabine
Cells with PTEN increased with decitabine
H522
H661
H1793
H441
H838
H1975
Decitabine - + - + - + - + - + - +
Refametinib (μM)
Trametinib (μM)
PTEN B
ACTB B
Relative cell viability
Relative cell viability
Cells with PTEN decreased with decitabine
Cells with PTEN increased with decitabine M U
PTEN
H441
H838
H522
H1793
H661
H1975
Refametinib (μM)
Trametinib (μM)