p53-deficiency is associated with MET inhibitor resistance in MPNSTs
Previously, we compared response to MET and MEK inhibition in tumor xenografts derived from MPNSTs of genetically engineered mouse models of NF1, including a Met-amplified, Trp53-wildtype model (NF1-MET; genotype: Nf1fl/ko;lox-stop-loxMETtg/+;Plp-creERTtg/+) and a Trp53 deficient model (NF1-P53; genotype: Nf1ko/+;p53R172H;Plp-creERTtg/+). NF1-P53 MPNSTs were less sensitive to both MET and MEK inhibition in vivo (33). To further investigate the molecular mechanisms of drug resistance, we generated tumor cell line isolates from these murine MPNSTs. Cell viability analysis confirmed that the MPNST cell lines maintained their drug sensitivity phenotypes in vitro, as the NF1-P53 cell line was resistant to single agent MET (capmatinib) and MEK (trametinib) inhibition as well as combination therapy (Fig. 1A). A targeted analysis of the RAS/ERK and PI3K/AKT pathways with reverse phase protein arrays (RPPA) was used to identify both immediate (2 hours) and adaptive (48 hours) kinome responses to MET and MEK inhibition. After controlling for drug treatment and exposure time, several PI3K effectors (including AKT and S6) were significantly activated in the NF1-P53 cells, suggesting PI3K/AKT pathway activation may promote resistance to MET and MEK inhibition (Fig. 1B, Supplementary Fig. 1).
To determine if p53 also regulates sensitivity to MET inhibition in human MPNSTs, we screened a panel of 6 NF1-related and 1 sporadic (STS26T) MPNST cell lines for functional p53 based on sensitivity to the p53 stabilizing drugs, MI-773 and nutlin-3a. These p53 stabilizers inhibit MDM2-mediated p53 degradation and are selectively active in p53 wild-type cells (45). Based on the IC50 of these drugs, we classified four cell lines as being “p53-functional” and three cell lines as being “p53-deficient” (Fig. 1C), which was consistent with a recent genomic analysis of MPNST cell lines (46). In addition, we screened the cell lines for sensitivity to of a panel of MET inhibitors. All but one of the p53-intact cell lines were sensitive to MET inhibition while, the p53-deficient cells were profoundly resistant to MET inhibition (Fig. 1C). The IC50 between several of the p53 stabilizers and MET inhibitors significantly correlated, similar to drugs within the same class (Fig. 1D), indicating a critical role for p53 in regulating MET-dependency.
p53 loss drives MET inhibitor resistance in MPNSTs
To evaluate the impact of p53 loss-of-function in the context of MET-addiction, we used CRISPR-Cas9-mediated knockout of p53 in murine NF1-MET cells to create the NF1‑MET;sgP53 line. p53 protein levels as well as the p53 target gene, p21, were diminished in NF1-MET;sgP53 cells (Supplemental Fig. 2A-B). Cell viability analysis demonstrated that NF1-MET;sgP53 cells were significantly less sensitive to MET inhibition than NF1-MET cells; however, combined MET-MEK inhibition restored drug sensitivity (Fig. 2A). We evaluated the impact of p53 on HGF-induced MET signaling and observed augmented MET signaling in NF1-MET;sgP53 cells, yet capmatinib inhibited HGF-dependent ERK and AKT activation regardless of p53 status (Fig. 2B). Stimulation with FBS (which contains other growth factors as well as HGF) also induced pERK and pAKT. Interestingly, the effect of capmatinib was quite modest in NF1-MET;sgP53 cells, suggesting capmatinib resistance may be partially mediated by parallel pathway activation that converges on ERK and AKT (Fig. 2C).
Resistant clonal populations within heterogeneous MPNSTs may explain the clinical failures of targeted kinase inhibitors in MPNSTs (47). To determine if p53 is a key driver of clonal selection and drug resistance in MPNSTs, we performed a clonal competition assay using labeled isogenic NF1-MET-GFP and NF1-MET;sgP53-RFP cell lines. Notably, when cultured separately, NF1‑MET;sgP53 cells do not have a significant proliferative advantage compared to NF1‑MET cells (Supplementary Fig. 2C). In contrast, when cocultured NF1-MET;sgP53-RFP cells had a strong growth advantage, which was significantly enhanced by both MET or MEK inhibition (Fig. 2D-E). After 5 days of capmatinib treatment, NF1-MET;sgP53-RFP cells comprised 83% of the culture (Fig. 2D-E), indicating that p53 loss drives strong clonal selection with MET inhibition.
To evaluate p53 loss and capmatinib sensitivity in vivo, we treated orthotopic MPNST xenografts with capmatinib. Tumor growth rate was significantly increased in the NF1-MET;sgP53 tumors compared to the parental tumors (p-value = 4.7e-4) (Fig. 2F; Supplementary Fig. 2D-E). Capmatinib strongly inhibited tumor growth in both the NF1-MET and NF1-MET;sgP53 models (Fig. 2F; Supplementary Fig. 2D), however only the NF1‑MET tumors regressed on treatment (p-value = 0.0083), while the NF-MET;sgP53 tumors remained stable throughout treatment, with a subset of tumors actually showing increased growth (Fig. 2F-G; Supplementary Fig. 2D). These data confirm our in vitro findings and further demonstrate that p53 loss promotes MET inhibitor resistance.
Met inhibition induces p53-dependent lineage plasticity
To understand how loss of p53 drives resistance we used RNA-seq to examine capmatinib-induced transcriptional changes in NF1-MET and NF1-MET;sgP53 cells. Unsupervised hierarchical clustering identified strong clustering by treatment followed by genotype, with p53 status modifying expression of some gene subsets (Fig. 3A). GO term enrichment analysis of genes upregulated by MET inhibition identified biological processes related to positive regulation of actin organization, cell adhesion, collagen deposition/ossification, and muscle differentiation (Fig. 3B, Supplementary Fig. 3A). We next examined genes that were downregulated in capmatinib treated NF1-MET;sgP53 cells and identified biological processes related to bone and kidney development (Fig. 3C, Supplementary Fig. 3B). Together, these data suggest that response to MET inhibition may be partially mediated through the induction of linage plasticity pathways in MPNSTs and that p53 loss disrupts this process to promote drug resistance. As differentiation and cell cycle arrest are coupled, we next examined whether p53 loss altered expression of known p53 target genes that promote cell cycle arrest. Indeed, expression of the cell cycle regulators Cdkn1a and Zmat3 were lost in the NF1-MET;sgP53 cells, while expression of genes involved in senescence or apoptosis were unchanged (Supplementary Fig. 3C).
p53 regulates MET stability and localization
RTK expression, activation, and recycling are tightly regulated processes that ensure RTK regulation in normal physiological conditions. Deregulation of this cycle of RTK activation and recycling is often observed in cancers, such as the MET exon 14 deletions observed in lung cancers (48). To determine whether RTK spaciotemporal regulation promoted the enhanced MET signaling observed in p53-deficient MPNST cells (Figs. 1B & 2B-C), we measured the kinetics of MET activation and turnover to increase effector activation. In NF1‑MET cells an immediate, and expected, increase in MET activation was observed within 5 minutes of HGF treatment that quickly diminished along with total MET levels over time (Fig. 4A). In contrast, HGF-treatment induced a drastic pMET increase that remained elevated for 60 minutes in NF1‑MET;sgP53 cells. Both phosphorylated and total MET were persistently elevated in the NF1-MET;sgP53 cells, which corresponded to increased and prolonged activation of both ERK and AKT (Fig. 4A). Activation of the mTOR effector S6 was similar between the two cell lines.
To examine whether p53 loss also alters MET subcellular localization, we performed immunostaining of MET after HGF-treatment. In normal conditions, HGF treatment results in MET localization and activation at the plasma membrane; however MET can also be internalized to the nucleus, where it’s function is incompletely understood (49). At baseline, pMET was localized to the cytoplasm in the NF1-MET cells, whereas we observed both nuclear and cytoplasmic staining in NF1‑MET;sgP53 cells. Interestingly, HGF dramatically increased nuclear MET localization in the p53-deficient NF1‑MET;sgP53 cells, while treatment induced nuclear MET localization only in a small percentage of NF1-MET cells (Fig. 4B). In several cancers, nuclear MET is associated with drug resistance and poor prognosis (50–54)). These results indicate that p53 loss induces nuclear MET localization, promoting tumor aggressive phenotypes in MPNST cells.
To understand how loss of p53 promotes increased stability and nuclear localization of MET, we used RNA-seq to examine the expression of genes involved in MET activation and turnover. Capmatinib treatment induced sweeping compensatory expression changes in both cell lines (Fig. 4C). In NF1-MET;sgP53 cells, expression of genes critical for MET degradation, Sh3kpb1 and Cblb, (55–57) were significantly downregulated compared to the parental cell line. Conversely, Prkce expression, which is required for nuclear MET translocation (58), was significantly upregulated in the NF1-MET;sgP53 cells in the presence of capmatinib. GO cellular compartment enrichment analysis revealed that p53 loss promoted significant downregulation in plasma membrane and receptor organization pathways (Fig. 4D, Supplementary Fig. 3D).
MET and the RTK EGFR share much of the same recycling machinery (57,59), and EGFR is also frequently amplified in MPNST (60). To determine if p53 loss also enhanced EGFR activation, we treated the NF1-MET and NF1-MET;sgP53 cells with EGF in a time course of 5 minutes to 1 day. Strikingly, EGF induced much stronger EGFR phosphorylation in the NF1-MET;sgP53 cells than the parental cell line (Supplementary Fig. 4), similar to the increased MET activation upon HGF treatment. This corresponded with increased and prolonged activation of pERK, pAKT, and pS6. These results suggest an important role for p53 in regulating both MET and EGFR signaling in MPNSTs and may partially explain resistance to EGFR inhibition in MPNSTs (61).
To further examine the effect of p53 expression on MET signaling, we used RPPA to evaluate the response of 98 protein phosphosites to short and extended HGF stimulation. After 2 hours of HGF treatment, numerous phosphosites were significantly upregulated in both the NF1‑MET and NF1-P53 cell lines, yet the number of upregulated sites as well as the magnitude of change was higher in the p53-deficient cells (Fig. 4E-F, Table S1). Additionally, phosphorylation of several proteins, including STAT3, JAK2, and B-RAF, were significantly decreased in the NF1-MET cell line after just 2 hours HGF treatment, (Fig. 4E, Table S1) confirming that rapid negative feedback signaling in response to MET activation is p53‑dependent. Remarkably, after 48 hours of HGF treatment, only 3/98 phospho-sites were significantly changed in the NF1-MET cells compared to 27/98 sites in the NF1-P53 cells (Fig. 4E-F, Table S1). Notably, STAT3 Y705 phosphorylation, which is sustained by perinuclear MET (62), was among the most significantly increased phosphosites in the p53-deficient cells after 48 hours of HGF exposure (Fig. 4F, Table S1). Collectively, these data suggest that p53 loss moderates MET-addiction by modulating the location, timing, and magnitude of MET effector signaling.
p53 loss drives mTOR Dependency in MPNSTs
AKT activation was consistently elevated and sustained in p53-deficient MPNST cell lines and is targetable therapeutically, either directly or via its downstream effectors. To determine the scope of AKT/mTOR pathway activation in vivo, we assessed the phosphorylation status of mTORC1 and mTORC2 pathway effectors in MPNST tumorgrafts by RPPA. Globally, phosphorylation was significantly increased in NF1-P53 tumors compared to NF1-MET tumors (p-value = 0.0034) (Fig. 5A), with increased activation of 7/12 phosphosites in NF1-P53 tumors (Fig. 5B). AKT and S6RP phosphorylation were significantly increased in the NF1-P53 tumors (Fig. 5B), suggesting increased dependency on the AKT/mTOR pathway. Regardless of p53 status, treatment with the AKT inhibitor, afuresertib, had no effect on the MPNST cell lines, either as a single agent or in combination with trametinib (Supplementary Fig. 5A-B). However, treatment with the mTOR inhibitor, everolimus, significantly decreased viability in the p53-deficient cell lines compared to the p53-intact cells (Fig. 5C). This enhanced inhibition of the p53-deficient cells was observed even though everolimus strongly inhibited downstream pS6RP regardless of p53 status (Fig. 5D). Further, combination therapy of mTOR (everolimus) and MEK (trametinib) inhibition reversed clonal selection for p53 loss (Fig. 5E-F).
As combination mTOR and MEK inhibition was so effective in inhibiting MPNST cell growth, we next asked whether treatment reversed the nuclear localization of MET leading to global downregulation of MAPK and AKT/mTOR signaling. Unexpectedly, combination everolimus and trametinib actually induced ligand-independent MET localization specifically in the NF1-MET;sgP53 cells compared to the parental line (Supplementary Fig. 6A). Moreover, treatment with everolimus or the dual PI3K/mTOR inhibitor BEZ235 induced stronger compensatory ERK and AKT activation in NF1-P53 cells, consistent with increased MET activation (Supplementary Fig. 6B). These data reinforce the role of p53 in regulating MET localization and effector signaling in response to diverse stimuli.
We next tested whether the excessive AKT activation induced by everolimus resulted in p53-independent oncogene induced apoptosis or senescence in p53 null MPNST cells (63,64). Combination everolimus and trametinib treatment did induce apoptosis in the NF1-MET;sgP53 cells compared to the parental cell line, however, the difference was minor (Supplementary Fig. 6C). We also did not observe senescence based on the senescence marker beta-galactosidase after 7 days of drug treatment (Supplementary Fig. 5D).
We evaluated whether differentiation of MPNST cells was impacted by treatment by combined everolimus and trametinib treatment. We observed that mTOR and MEK inhibition in NF1-MET;sgP53 cells significantly increased expression of the Schwann cell markers S100B and FABP7, yet this increased Schwann cell differentiation was not observed in NF1-MET cells (Fig. 5G). We also examined differentiation marker expression of neuron, chondrocyte, adipocyte, bone, kidney, endothelial, and muscle cells, as well as the multipotency marker SOX9, which is expressed in neural crest cells. Combined MEK and mTOR inhibition resulted in increase of the neuronal marker MAP2 in NF1-MET;sgP53 cells, while chondrocyte and adipocyte differentiation was strongly induced in NF1-MET cells (Fig. 5G). Interestingly, both kidney and muscle markers were strongly induced by treatment in both NF1-MET and NF1-MET;sgP53 line (Supplementary Fig. 6E). Consistent with the induction of multiple differentiation pathways upon mTOR and MEK inhibition, the multipotency marker SOX9 was decreased in both cell lines (Fig. 5G). These results indicate that lineage differentiation is altered by mTOR and MEK inhibition, with p53-deficient MPNST cells most sensitive to shifts back towards differentiated Schwann cell states.