PTPN11 Plays Oncogenic Roles and Is a Therapeutic Target for BRAF Wild-Type Melanomas

Abstract

Melanoma is one of the most highly mutated cancer types. To identify functional drivers of melanoma, we searched for cross-species conserved mutations utilizing a mouse melanoma model driven by loss of PTEN and CDKN2A, and identified mutations in Kras, Erbb3, and Ptpn11. PTPN11 encodes the SHP2 protein tyrosine phosphatase that activates the RAS/RAF/MAPK pathway. Although PTPN11 is an oncogene in leukemia, lung, and breast cancers, its roles in melanoma are not clear. In this study, we found that PTPN11 is frequently activated in human melanoma specimens and cell lines and is required for full RAS/RAF/MAPK signaling activation in BRAF wild-type (either NRAS mutant or wild-type) melanoma cells. PTPN11 played oncogenic roles in melanoma by driving anchorage-independent colony formation and tumor growth. In Pten- and Cdkn2a-null mice, tet-inducible and melanocyte-specific PTPN11^E76K expression significantly enhanced melanoma tumorigenesis. Melanoma cells derived from this mouse model showed doxycycline-dependent tumor growth in nude mice. Silencing PTPN11^E76K expression by doxycycline withdrawal caused regression of established tumors by induction of apoptosis and senescence, and suppression of proliferation. Moreover, the PTPN11 inhibitor (SHP099) also caused regression of NRAS^Q61K -mutant melanoma. Using a quantitative tyrosine phosphoproteomics approach, we identified GSK3α/β as one of the key substrates that were differentially tyrosine-phosphorylated in these experiments modulating PTPN11. This study demonstrates that PTPN11 plays oncogenic roles in melanoma and regulates RAS and GSK3β signaling pathways. IMPLICATIONS: This study identifies PTPN11 as an oncogenic driver and a novel and actionable therapeutic target for BRAF wild-type melanoma.

Figure 1.. Identification of oncogenic mutations arising spontaneously in Cdkn2a (Ink4a/Arf) and Pten null (IP) tumors.

An illustration of the IP mouse model (A) where topical 4OHT treatment induces Cre activation within melanocytes and subsequent Ink4a/Arf and Pten deletion (B: PCR analysis of ear and tumor DNA). Gross and histological representations of melanomas arising in IP mice (C). Results of whole exome sequencing performed on melanoma and matched normal tissue samples from IP mice (D). KRAS A146T and Ptpn11 S506P mutation is confirmed by Sanger sequencing in melanoma, but not in matched normal DNA of PA543 and PA624 mice, respectively (E).

Figure 2.. PTPN11 is activated in human melanoma and regulates ERK phosphorylation in BRAF wt melanoma cell lines.

Mutations identified in human cancers (adapted from cBioPortal): SH2 and Y phosphatase domains; Gray: missense mutation; Black: in frame deletion (A). Western blot analysis of human metastatic melanoma specimens and WM793 cell lysates (“C”) as a control (B) and melanoma cell lines (C) with pPTPN11 (Y542), PTPN11, and β-actin antibodies. The effect of PTPN11 KD with siRNAs (A & B) on ERK activation in BRAF or NRAS mutant and BRAF/NRAS wt melanoma cells (D) and NRAS activation ((RAS-GTP pull down detected with NRAS antibody) in NRAS mutant cells (E).

Figure 3.. PTPN11 expression regulates growth of cells in soft agar and as xenografts.

PA624T and PA662T cells are murine melanomas cells from Cdkn2a(Ink4a/Arf) ^L/L; Pten^L/L; Tyr-CreER^T2 mice. Western analysis of PA624T with vector control or shRNAs (#4 and #9) targeting Ptpn11 (A top) and PA662T with vector control, wt, G503V, or E76K mutant PTPN11 expression (B top). Number of colonies grown in soft agar (A&B, bottom, seeded in triplicate) is shown graphically as mean +/− SD (p-value: two-tailed T-test). Subcutaneous tumor growth in nude mice implanted with PA662T cells expressing control, wt, or E76K PTPN11 over the time course (mean tumor volume +/− SEM, C). Significant difference (p<0.0001) is determined by two-way ANOVA comparing E76K with both control and wt on day 42 and comparing wt and control on day 52. Immunohistochemical (IHC) analysis of tumors for PTPN11 (flag Ab), pHistone H3 (pH3), pERK, and cleaved caspase 3 (D).

Figure 4.. PTPN11 E76K is required for growth and maintenance of tumors in mice.

Kaplan-Meier melanoma free survival curves of BT (PTPN11 ^E76K positive) and MT (rtTA only) mice on doxy (A). Representative images of a cutaneous melanoma developed in a BT mouse, H&E, and IHC staining for S100, Flag, pPTPN11 Y542 and pERK (B). Western blot analysis of W331 cells derived from a BT melanoma (Tyr-rtTA; tetO-PTPN11 ^E76K;Cdkn2a(Ink4a/Arf) ^L/L; Pten^L/L; Tyr-CreER^T2) (C). Weights of W331 allograft tumors (mean tumor weight (g) +/− SEM) grown in nude mice fed with (+Dox) or without (-Dox) doxy on day 11 following implantation (p=0.001; +Dox vs. -Dox group) or deinduced for 7 days following growth on doxy for 11 days (p=0.002; De-induction vs. +Dox group) (D). Repeated cycles of doxy induction/de-induction (PTPN11 E76K expression On/Off) drive growth and regression of s.c. tumors (E). Quantification of p-Histone H3 (F) and cleaved caspase 3 (G) by IHC analysis on tumor sections following PTPN11 de-induction on D0, D3, and D7. Senescence associated β-galactosidase staining (blue) of tumors on D0 and D3 (H). p-value is calculated using two-tailed t-test for D, F, and G.

Figure 5.. Phosphotyrosine proteomic analysis of PTPN11 expressing tumors identifies candidate substrates of PTPN11, including GSK3α/β.

A. Normalized scatter plot of the log₂ values of the median (E76K/ctrl) ratios for individual phosphosites. Peptides changed in PA662T (Cdkn2a^L/L; Pten^L/L;Tyr-CreER^T2) transduced with E76K vs. vector ctrl are shown along the y axis and ones altered in W331 (Tyr-rtTA;tetO-PTPN11^E76K; Cdkn2a^L/L; Pten^L/L;Tyr-CreER^T2) on doxy (D0, E76K+) vs. de-induced (D3, E76K-) are on the x axis. B. Western blot analysis of W331 cells with or without doxy with indicated antibodies. Immunoblotting with Ras antibody detected the level of total, activated (Ras-GTP pull-down), and tyr phosphorylated (pTyr pull down) Ras in W331. C. Co-immunoprecipitation using anti-FLAG beads in control or CSDA-PTPN11-Flag (trapping mutant) expressing 662T cells were immunoblotted with antibodies against pGab1, pY-GSK3, total GSK3 and Ras. D. IHC staining of pY-GSK3 in 662T control, E76K, and wt PTPN11 expressing tumors and in W331 tumors (D0 and D3). Western analysis of human melanoma cell lines MeWo (E, BRAF/NRAS wt) with PTPN11 (wt or E76K mutant) and WM1361A and 1366 (F, NRAS mutant) with PTPN11 KD (siRNAs A, B) compared to controls.

Figure 6.. Pharmacological inhibition of PTPN11 in mouse melanoma cells with NRAS ^Q61K induces tumor regression in part through regulation of GSK3 and cyclin D1.

Survival analysis of 2187 and 5037 (mouse melanoma cells with Tyr-rtTA; tetO-NRAS ^Q61K; Cdkn2a (Ink4a/Arf) ^−/−) following treatment with increasing concentrations of SHP099 at 72 hours (A). Decreased tumor volume following treatment of established subcutaneous allograft 5037 tumors with SHP099 (PTPN11 inhibitor, 100 mg/kg; po qd) compared to treatment with vehicle alone. Data shown as mean tumor volume ratio to day 0 +/− SEM (B) and final tumor weight at harvest (p=0.0003; two-tailed t-test) on day 8 (C). Waterfall plot demonstrating ratio of D8 to D0 tumor volume for tumors treated with vehicle or SHP099. SHP099 treated tumor segregated into (red) responders and partial-responders (blue) with and without regression, respectively (D). Western analysis of 5037 subcutaneous tumors with (+) or without (–) SHP099 treatment for 3 or 8 days (E). Western analysis of parental 5037 and a cell line established from a partial responding tumor (PR1; blue in panel D) treated with 0, 10, or 30 uM SHP099 for 3 hrs (F). Cell survival was assessed at 72 hrs of post-treatment as the mean +/−SD survival (relative to DMSO or GSK3β inhibitor CHIR-99021 alone) (G&H). The response of 5037 with vector control or GSK3β-Y216A mutant and PR1 cells to SHP099 (G) and that of 5037 parental cells to increasing concentrations of SHP099 alone or in combination with 1μM or 3μM CHIR-99021 (H) were shown.