Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer

Abstract

Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple-negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, and prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 inhibition, but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.

Figure 1. Kinome profiling of TNBC reveals elevated ERK signaling

(A) Experimental strategy for the rational design of kinase inhibitor combination therapies. To define kinome inhibitor response signatures, expression profiling is integrated with kinase affinity capture and MS quantitative assessment of the activation state of the kinome. RNAi is used to analyze kinase function in survival response to inhibitors. (B) Venn diagram shows number of expressed kinases defined by RNA-seq across patient TNBC and MDA-MB-231 and SUM159 cell lines. See Table S1 for normalized read count transcript expression profiles. (C) Venn diagram shows number of kinases captured and identified by MIB-based proteomics across patient-sample TNBC and MDA-MB-231 and SUM159 cell lines. MIB/MS captures 50-60% of the expressed kinome as defined by RNA-seq. See Table S2 for MS identifications of protein kinases. (D) Distribution and (E) overlap of expressed and MIB-bound kinases across patient-sample TNBC and MDA-MB-231 and SUM159 cell lines. (F) RAF-MEK-ERK pathway activated in patient TNBC tumors. Quantitative comparison of patient TNBC to matched uninvolved mammary tissue using MIB/MS. The line graphs show iTRAQ determined quantitative changes in MIB binding as a ratio of tumor/uninvolved. Ratio <1 denotes decreased MIB binding and >1 increased MIB binding of kinase in tumor versus control tissue. (G) Immunoblotting confirms an activated RAF-MEK-ERK pathway in TNBC cell lines and TNBC patient samples. (H) RTK array analysis of patient TNBC tumors reveals multiple Tyr phosphorylated RTKs, including EGFR and PDGFRβ. See also Figure S1 and Tables S1 and S2.

Figure 2. Reprogramming of the kinome in response to MEK inhibition

(A) Growth inhibition of SUM159 cells in response to AZD6244 or U0126. Triplicate experiments + SD. (B) Reactivation of MEK and ERK in the continued presence of 5 μM AZD6244 shown by western blot. (C) Loss of ERK regulated feedback of the RAF-MEK-ERK pathway and downstream signaling. SUM159 cells were treated with 5 μM AZD6244 for 12h and kinome phosphorylation analyzed by MIB/MS. (D) Activation and repression of the kinome in response to 5 μM AZD6244 in SUM159 cells. Line graphs show iTRAQ-determined quantitative changes in MIB binding as a ratio of AZD6244/DMSO. Ratio <1 denotes decreased and >1 denotes increased MIB binding of kinases in treated versus control cells. (E) MEK2 and ERK1 escape AZD6244 inhibition. MIB/MS binding profile of RAF-MEK-ERK from SUM159 cells treated with 5 μM AZD6244 for 4, 12 and 24h or 5 μM U0126 for 24h. (F) MEK2 and ERK1 promote survival following MEK inhibition. siRNA knockdown of MAPK signaling components in SUM159 cells shows loss of MEK2, but not MEK1, inhibits growth in the presence of U0126. (G) Kinome response signature for MEK inhibition in SUM159 cells. Triplicate MIB/MS runs of SILAC-labeled SUM159 cells ± 5 μM AZD6244 or U0126 relative to DMSO. Error bars represent mean + SD where kinases are significant at FDR of 0.05. (H) Kinome map of AZD6244 response (blue: inhibited, red: induced) as determined by MIB/MS and RTK arrays. (I) Increased Tyr phosphorylation of RTKs in response to MEK inhibition. SUM159 cells were treated with 5 μM AZD6244 for 24h and analyzed by RTK array. (J) Dose-dependent RTK reprogramming in response to AZD6244. Dose-dependent induction of RTK expression and activity in 24h-treated SUM159 cells was determined by western blot. See also Figure S2 and Table S3.

Figure 3. AZD6244-induced kinome reprogramming is target specific and involves rapid, stable transcriptional upregulation of RTKs and cytokines

(A) AZD6244 treatment initially causes ERK inhibition and accumulation of MKP3. With prolonged AZD6244 treatment, increased RTK expression and downstream survival signaling coincides with the reactivation of RAF-MEK-ERK signaling. (B) Time-dependent increase in RTK and (C) cytokine gene expression in SUM159 cells after AZD6244 treatment, determined by qRT-PCR. (D) Prolonged treatment of SUM159 cells with AZD6244 leads to stable upregulation of RTKs. SUM159 cells were treated with DMSO or AZD6244 for 4-72h or 30d and RTK tyrosine phosphorylation determined by RTK antibody arrays. (E) Treatment with AZD6244 enhances phosphorylation of PDGFRβ at multiple sites, including the activation loop. (F) Generation of AZD6244-resistant SUM159 cells following stable treatment with AZD6244. Increased cell growth of SUM159-R cells compared to SUM159 cells treated with AZD6244 for 72h determined by cell counts (*p-value<0.001). (G) Maintenance of RTK reprogramming in SUM159-R cells accompanied by increased survival signaling. Kinome reprogramming in SUM159 cells treated with DMSO or AZD6244 for 4h was compared to SUM159-R cells by western blot. (H) AZD6244 and BEZ235 are target-specific in their reprogramming of kinome response. BEZ235 induces a kinase response different from AZD6244 in SUM159 cells, despite similar growth inhibition. Error bars represent triplicate experiments ± S.D. See also Figure S3.

Figure 4. AZD6244–mediated loss of ERK1/2 causes rapid degradation of c-Myc, destabilization of Myc-Max complexes and promotion of RTK expression

(A) Loss of ERK1/2 activity following AZD6244 treatment promotes c-Myc degradation. SUM159 cells were treated with AZD6244 and monitored by western blot. (B) Stable suppression of c-Myc RNA levels following AZD6244 treatment. MDA-MB-231 and SUM159 cells were treated with AZD6244 and c-Myc gene expression determined by qRT-PCR. (C) Disruption of Myc-Max complexes following AZD6244 treatment. SUM159 cells were treated with AZD6244 for 0, 4 and 72h. Nuclear extracts were immunoprecipitated with anti-Max antibodies and immunoblotted for Myc and Max. (D) RNAi knockdown of c-Myc using pooled siRNA for 72h in SUM159 cells upregulates gene expression of PDGFRβ, VEGFR2 and PDGFB as determined by qRT-PCR. (E) RNAi knockdown of c-Myc upregulates PDGFRβ, VEGFR2 and DDR1 expression. SUM159 cells were transfected with deconvolved c-Myc or GAPDH siRNA for 48h and kinome reprogramming determined by western blot. (F) Retroviral expression of non-degradable c-Myc(T58A) in SUM159 cells suppresses RTK reprogramming after 24h treatment with 5 μM AZD6244, as shown by western blot and quantified by densitometry. (G) Transcript levels of DDR1, PDGFRβ and VEGFR2 are reduced by the expression of c-Myc(T58A) in the presence and absence of 5 μM AZD6244. SUM159 cells were treated for 24h and analyzed by qRT-PCR. (H) Stabilization of c-Myc protein levels by bortezomib prevents AZD6244-mediated kinome reprogramming. SUM159 cells were treated with AZD6244 (5 μM) or bortezomib alone or in combination for 24h. Bortezomib blocked the AZD6244-dependent induction of RTKs as shown by western blot. (I) Removal of AZD6244 results in stabilization of c-Myc protein and reversal of RTK reprogramming. AZD6244 was removed from the media of SUM159 cells after 24h of treatment and cells were grown without AZD6244 for a further 24h. (J) c-Myc protein levels partially return in SUM159-R cells, while AZD6244-mediated RTK reprogramming is reduced but still maintained. SUM159 cells were treated with AZD6244 and RTK and c-Myc levels compared to SUM159-R cells by western blot. (K) Increased c-Myc RNA levels in SUM159-R cells relative to AZD6244 treated SUM159 cells. SUM159 cells were treated with DMSO or 5 μM AZD6244 for 4h and c-Myc gene expression compared to SUM159-R cells using qRT-PCR (*p-value<0.001). (L) AZD6244-induced RTK expression is maintained at reduced levels in SUM159-R cells. qRT-PCR was used to compare gene expression in SUM159 cells treated with AZD6244 for 24h or SUM159-R cells relative to DMSO-treated cells. (M) c-Myc stabilized by RTK-mediated ERK activation in SUM159-R cells. RTK reprogramming and c-Myc levels were determined by western blot comparing SUM159-R treated with AZD6244 for 24h. (N) RTK-mediated reactivation of ERK is incomplete in the continued presence of AZD6244. AZD6244 (5 μM) was removed from media of SUM159-R cells for 1h and ERK1/2 phosphorylation of c-Myc and RSK1 determined by western blot. Error bars represent triplicate experiments ± S.D. See also Figure S4.

Figure 5. RTK inhibition synergizes to enhance AZD6244-induced growth arrest

(A) RNAi knockdown of PDGFRβ enhances AZD6244-induced growth arrest. PDGFRβ knockdown was performed in the presence or absence of 1.25 μM AZD6244 in MDA-MB-231 and SUM159 for 96h and cell growth monitored using Cell-Titer Glo. (B) RNAi knockdown of MEK inhibitor-responsive RTKs in SUM159 cells synergizes with U0126 to inhibit growth. Cell proliferation was determined at 96h of treatment by Cell-Titer Glo. (C) Cotreatment with AZD6244 and sorafenib synergizes in cell growth inhibition of SUM159 cells, as determined by Cell-Titer Glo. (D) Cotreatment of SUM159 cells with AZD6244 and foretinib enhances growth inhibition, as determined by Cell-Titer Glo. (E) Cell counting confirms synergistic growth inhibition of combined AZD6244 and sorafenib treatment in SUM159 cells. (F) Sorafenib inhibits AZD6244-mediated activation of RTKs. SUM159 cells were treated as indicated for 72h and RTK Tyr phosphorylation determined by RTK antibody arrays. (G) Cotreatment of SUM159 cells with AZD6244 and sorafenib enhances inhibition of ERK activity and primes cells for apoptosis. SUM159 cells were treated with for 72h and BIM, cyclin D1 expression and ERK activity determined by western blot. (H) AZD6244 activation of ERK1/2 requires RTK and MEK activity. Inhibition of ERK activity in SUM159-R cells occurs after treatment with 50 μM AZD6244 or cotreatment of 5 μM AZD6244 with 250 nM sorafenib. (I) SUM159-R cells require AZD6244-induced RTK activity for drug resistance. SUM159-R cells treated with 250 nM sorafenib are growth arrested over 72h, as determined by cell counts. *p-value<0.001; Error bars represent triplicate experiments ± S.D. See also Figure S5 and Table S4.

Figure 6. AZD6244-mediated kinome reprogramming in C3Tag mouse model of TNBC

(A) PDGFRβ is induced in C3Tag tumors after 2d AZD6244 treatment, as shown by anti-PDGFRβ immunofluorescence. (B) AZD6244 treatment of C3Tag mice for 2 and 7d causes c-Myc degradation and induced PDGFRβ expression, as shown by western blot. (C) Tumor-derived C3Tag cell line shows AZD6244-mediated c-Myc loss and induction of RTKs. T2-C3Tag cells were treated with AZD6244 and RTK reprogramming determined by western blot. (D) Expression of c-Myc(T58A) in T2-C3Tag cells suppresses AZD6244-mediated RTK reprogramming. T2-C3Tag cells stably expressing vector or human c-Myc(T58A) were treated with AZD6244 for 24h and analyzed by western blot. (E) MIB/MS profile of C3Tag tumors in response to AZD6244 for 28d, sorafenib for 26d, or combined AZD6244 and sorafenib for 26d show distinct kinome responses. The line graphs show iTRAQ-determined quantitative changes in MIB binding as a ratio of inhibitor/untreated. Ratio <1 denotes decreased MIB binding and >1 increased MIB binding of kinases in treated versus control tumors. (F) AZD6244 plus sorafenib inhibits AZD6244-induced kinome response in C3Tag tumors, as identified by MIB/MS. Changes in MIB binding (>1.5 fold) of AZD6244-responsive kinases following cotreatment with sorafenib are shown in yellow. (G) Cotreatment of C3Tag mice with AZD6244 and sorafenib prevents upregulation of kinases observed by AZD6244 treatment alone, including CDKs and PDGFRβ. These kinases were selected from (E) with a >1.5-fold reduction in MIB binding between AZD6244 treatment alone and cotreatment with AZD6244 and sorafenib. See also Figure S6.

Figure 7. Combination of AZD6244 and sorafenib causes apoptosis and tumor regression in C3Tag TNBC mouse model

(A) AZD6244 (20 mg/kg) or sorafenib (30 mg/kg) fed in chow results in ERK activation after 2d of treatment in C3Tag GEMM, while cotreatment with AZD6244 and sorafenib inhibits RTK-mediated ERK activation. RTK reprogramming was monitored in tumors treated with AZD6244 and/or sorafenib relative to untreated tumors by western blot. (B) Sorafenib inhibits AZD6244-dependent reactivation of ERK, promoting c-Myc degradation and loss of cyclin B1 expression in T2-C3Tag cells. T2-C3Tag cells were treated for 24h and analyzed by western blot. (C) AZD6244 and sorafenib synergize to inhibit cell growth in C3Tag cell line. T2-C3Tag cells were treated with AZD6244 and sorafenib, alone or in combination, and cell growth determined by cell counting (*p-value<0.001; quadruplicate experiments). (D) Cotreatment of C3Tag mice with AZD6244 and sorafenib for 21d causes significant tumor regression compared to AZD6244 alone. C3Tag mice were treated with AZD6244 (20 mg/kg), sorafenib (30 mg/kg) or the combination of AZD6244 and sorafenib and compared to untreated tumors. Percent change in tumor volume of drug treated relative to untreated is shown (* Wilcoxon p-value=0.007). (E) Increased apoptosis of C3Tag mouse tumors following cotreatment with AZD6244 and sorafenib. Apoptosis in C3Tag tumors treated for 2d was determined by TUNEL staining (shown in red; DAPI is grayscale). See also Figure S7.