Nf1-Mutant Tumors Undergo Transcriptome and Kinome Remodeling after Inhibition of either mTOR or MEK

Abstract

Loss of the tumor suppressor NF1 leads to activation of RAS effector pathways, which are therapeutically targeted by inhibition of mTOR (mTORi) or MEK (MEKi). However, therapeutic inhibition of RAS effectors leads to the development of drug resistance and ultimately disease progression. To investigate molecular signatures in the context of NF1 loss and subsequent acquired drug resistance, we analyzed the exomes, transcriptomes, and kinomes of Nf1-mutant mouse tumor cell lines and derivatives of these lines that acquired resistance to either MEKi or mTORi. Biochemical comparisons of this unique panel of tumor cells, all of which arose in Nf1^+/- mice, indicate that loss of heterozygosity of Nf1 as an initial genetic event does not confer a common biochemical signature or response to kinase inhibition. Although acquired drug resistance by Nf1-mutant tumor cells was accompanied by altered kinomes and irreversibly altered transcriptomes, functionally in multiple Nf1-mutant tumor cell lines, MEKi resistance was a stable phenotype, in contrast to mTORi resistance, which was reversible. Collectively, these findings demonstrate that Nf1-mutant tumors represent a heterogeneous group biochemically and undergo broader remodeling of kinome activity and gene expression in response to targeted kinase inhibition.

Fig. 1

Ras signaling among Nf1 null tumor cell lines. A. Basal Ras signaling was assessed by Western blot analysis. Phosphorylated and total AKT (S473), S6 Ribosomal protein (Ser235/236) and p44/42 MAPK (ERK1/2) (Thr202/Tyr204) in untreated Nf1 null tumor cell lines shown. β actin was used as loading control. B. RAS activation varies among serum-stimulated Nf1 null cell lines. Nf1 null tumor cell lines were serum starved for 24 hours, then stimulated with media containing 10% FBS. Whole cell lysates were collected at 0, 5, 30 and 60 minutes after stimulation and RAS-GTP pull-down was performed. Shown in right panel: Corresponding immunoblotting for phospho-specific antibodies against AKT (S473), S6 (Ser235/236) and p44/42 MAPK (ERK1/2) (Thr202/Tyr204) was assessed. β actin was used as loading control. C. Heat map of transcriptome profiling data shows the unsupervised clustering of analyzed parental cell lines (881 and 989 cell lines, two biological replicates analyzed for both. D. Scatter plots depicting relative expression differences between 989 and 881 cell lines against corresponding relative differences in kinase binding to MIB.

Fig. 2

Drug resistant 989 Nf1 mutant cells. A. Two sets of resistant cell lines were derived from 989 parental cells after chronic drug exposure; these were resistant to either 100 nM (989 PD_R1, 989 RAD_R1) or resistant to 500 nM (989 PD_R, 989 RAD_R). Acquired resistance was confirmed by comparing growth by parental and resistant cell lines to increasing concentrations of drug after 72 hours of exposure (*P<0.05; **P<0.01; ns, not significant) by MTS assay. B. Basal Ras signaling in parental and drug resistant derivatives was assessed by Western blotting. Total and phosphorylated AKT (S473), S6 Ribosomal Protein (Ser235/236) and p44/42 MAPK (ERK1/2) (Thr202/Tyr204) were assessed, with β actin used as loading control. C. After 24 hours of serum starvation, subconfluent cells were stimulated for 0, 5, 30 or 60 minutes with media containing 10% FBS, and protein lysates collected. RAS-GTP levels were assessed by GTPase pull-down. Corresponding immunoblotting for total RAS and phospho-RAS effectors AKT (S473), S6 (Ser235/236) and p44/42 MAPK (ERK1/2) (Thr202/Tyr204) was performed. D. Growth by parental and drug resistant cell lines after 72 hours of exposure to 0.5 μM PD0325901 alone, 0.5 μM RAD001 alone, or the combination was assessed by an MTS assay. Cell viability was assessed after 72 hours of drug exposure (*P<0.05; **P<0.01; ns, not significant). E. Total and phosphorylated AKT (S473), S6 Ribosomal Protein (Ser235/236) and p44/42 MAPK (ERK1/2) (Thr202/Tyr204) in control and drug treated cells were assessed by western blotting.

Fig. 3

Acquired drug resistance in Nf1 mutant tumor cell lines is associated with remodeled transcriptomes and kinomes. A. Schema describing samples and molecular analysis of parental, acute treated (989+PD901, 989+RAD001) and drug resistant derivatives (989 PD-R5, 989 RAD-R5). B. Heat map depicts unsupervised clustering analysis of transcriptomes of parental and resistant 989 tumor cell lines. Parental and resistant cell lines were grown to a confluence of 70%. Acutely treated parental cell lines (989 + RAD, 989 + PD) were treated with 500 nM PD0325901 or RAD001 for 24 hours. Resistant cells (989 RAD_R, 989 PD_R) were maintained in drug (500 nM) continuously. Drug resistant cells (989 PD_R and 989 RAD_R) cluster together and separately from parental drug-sensitive counterparts (two biological replicates for analyzed for each). C. The upper two panels show log-fold changes in mRNA of acutely drug treated cells relative to parental 989 cells plotted against p-value on the y-axis. Acute (24 hour) exposure to MEKi or mTORi resulted in 774 or 619 genes respectively showing significant expression change compared to parental 989 cells (significantly altered genes are indicated by blue crosses). In contrast, PD resistant and RAD resistant 989 cell lines (relative to parental cells) demonstrate both more genes undergoing expression changes (5133 and 1702 respectively) and greater magnitudes of expression change for significantly altered genes. D. The log2 of LFQ (label free quantification) intensity of each kinase (194 total) was computed and plotted in a heatmap plotting the differences (as compared to control untreated parental cells) in the binding of kinases to MIB by acutely treated (989 + PD, 989 + RAD), PD901- or RAD001-resistant 989 cells (each sample compared to control parental cells). For each experimental condition (acutely treated or drug-resistant), kinase activation or repression in response to 24h treatment with 500 nM PD901 or RAD001 was quantified by subtracting the average Log2LFQs of two biological control replicates (parental cell lines) from the average Log2LFQs of two biological replicates of each experimental group. Red indicates increased MIB binding, blue indicates decreased MIB binding (Kinase families: AGC, Atypical, CAMK, CK1, CMGC, Metabolic, NEK, other, STE, TK, TKL).

Fig. 4

Kinome profiles in drug resistant Nf1 mutant cell lines. A. Scatter plots displaying relative expression differences between drug resistant and parental cells against corresponding relative differences in kinase binding to MIB. B-E. Log2LFQ (label free quantification) intensity of each kinase in parental cells was subtracted from the corresponding Log2LFQ of kinases in drug resistant derivatives. Kinases with Log2LFQ values of >1.5 or <−1.5 were identified, then organized into protein interaction networks using Cytoscape software. Shown in panels B and D are kinases demonstrating increased MIB binding in both 989 PD_R and 989 RAD_R (B) and 989 PD_R and 881 PD_R (D). Shown in panels C and E are kinases demonstrating decreased MIB binding in both 989 PD_R and 989 RAD_R (C) and 989 PD_R and 881 PD_R (E).

Fig. 5

Somatic mutations in Nf1 mutant parental and drug resistant cells. A. The Venn diagram depicts the number of shared and unique somatic SNVs identified in 989, 989 PD_R and 989 RAD_R cell lines. B. The stacked bar graphs depict the private and shared somatic SNVs as percentages of each cell line’s total somatic SNVs (normalized, total summing to 100%). Private mutations in 989 PD_R and 989 RAD_R comprised 54.33% and 33.95% of the total somatic SNVs, respectively. C. Variant allele frequency plots comparing Nf1 mutant 989 parental and drug resistant cell lines. Cell-line specific genes involved by SNVs were highlighted in blue and green. The Nfrkb gene was mutated in both resistant 989 PD_R and 989 RAD_R cell lines.

Fig. 6

Effect of drug withdrawal on drug resistant Nf1 mutant tumor cells. A-B. Resistant cell lines were washed twice with PBS and then grown drug-free for at least 12 days. Viability of parental, resistant and resistant washout cell lines treated with 0.5 μM PD0325901 or 0.5 μM RAD001 for 72 hours was assessed by MTS proliferation assay. Washout resistant cell lines were normalized to each untreated resistant counterpart (*P<0.05; **P<0.01; ns, not significant). C-D. Western blot analyses were performed to evaluate Ras pathway effector activation in parental, resistant and resistant washout cell lines in response to 24 hours of exposure to either PD0325901 or RAD001. E. Heat map depicts unsupervised clustering analysis of kinase expression in parental and drug resistant 989 and 881 tumor cells (resistant and washout). Drug resistant and washout cells each segregate separately from parental cells but also from each other. F. Schema describing the drug response-based relationship of drug resistant Nf1 cell lines to parental cells. Cells chronically treated with PD901 and RAD001 differentiate into new cell lines resistant to the two drugs, 989 PD_R and 989 RAD_R. In response to drug withdrawal, 989 PD_R WO cells progress in the evolution and diverge further from 989 PD_R, whereas 989 RAD_R WO re-sensitize to the drug and revert to the parental state.