Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia

Abstract

The protein tyrosine phosphatase PTPN11 is implicated in the pathogenesis of juvenile myelomonocytic leukemia (JMML), acute myeloid leukemia (AML), and other malignancies. Activating mutations in PTPN11 increase downstream proliferative signaling and cell survival. We investigated the signaling upstream of PTPN11 in JMML and AML cells and found that PTPN11 was activated by the nonreceptor tyrosine/serine/threonine kinase TNK2 and that PTPN11-mutant JMML and AML cells were sensitive to TNK2 inhibition. In cultured human cell-based assays, PTPN11 and TNK2 interacted directly, enabling TNK2 to phosphorylate PTPN11, which subsequently dephosphorylated TNK2 in a negative feedback loop. Mutations in PTPN11 did not affect this physical interaction but increased the basal activity of PTPN11 such that TNK2-mediated activation was additive. Consequently, coexpression of TNK2 and mutant PTPN11 synergistically increased mitogen-activated protein kinase (MAPK) signaling and enhanced colony formation in bone marrow cells from mice. Chemical inhibition of TNK2 blocked MAPK signaling and colony formation in vitro and decreased disease burden in a patient with PTPN11-mutant JMML who was treated with the multikinase (including TNK2) inhibitor dasatinib. Together, these data suggest that TNK2 is a promising therapeutic target for PTPN11-mutant leukemias.

Fig. 1.. A primary patient sample containing a PTPN11 mutation demonstrates dasatinib sensitivity and over-reliance on TNK2.

(A) Sanger sequencing confirming that the PTPN11 mutation G60R was first identified in a patient with recurrent JMML by whole-exome sequencing. (B) Peripheral blood mononuclear cells from this patient were incubated with an siRNA library, and viability was assessed by MTS assay. Each bar represents cell viability after silencing of an individual kinase (table S2). (C) Peripheral blood mononuclear cells from the same JMML patient were incubated with graded concentrations of each of 66 small-molecule kinase inhibitors for 3 days. Cell viability was determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, and the IC₅₀ for each drug was calculated with respect to cells incubated in the absence of drug. These IC₅₀ values were compared to the median IC₅₀ for each drug across 151 patient samples. Each bar represents the percentage of median IC₅₀ for an individual kinase inhibitor (table S1). (D) Mouse bone marrow cells were transduced to express PTPN11, PTPN11 G60R, or PTPN11 E76K and plated in a methylcellulose GM-CSF sensitivity colony formation assay. Colonies were counted at 14 days [GM-CSF] = 0.05 nM (0.71 ng/ml). **P < 0.005 and ***P < 0.0005. (E) 293T17 cells were transiently transfected with expression constructs containing PTPN11, PTPN11 G60R, PTPN11 E76K, or empty vector, and lysates were subjected to immunoblot. Blots are representative of four biological replicates. Data (B to D) are means ± SEM of four experiments.

Fig. 2.. TNK2 increases signaling through PTPN11/RAS/MAPK in cells overexpressing mutant PTPN11.

(A) 293T17 cells were cotransfected with expression constructs containing PTPN11, PTPN11 E76K, TNK2, or empty vector controls. Lysates were collected at 48 hours and subjected to immunoblot. (B) Relative phospho-p44/42 MAPK (phospho-ERK1/2), relative phospho-TNK2 (Y284), and relative phospho-PTPN11 values were calculated with GAPDH as a loading control. Data are means ± SEM of four experiments. ***P = 0.0005 and **P < 0.005 by one-way analysis of variance (ANOVA). (C) 293T17 cells were cotransfected with expression constructs containing PTPN11, PTPN11 E76K, TNK2, or empty vector controls. Lysates were collected 48 hours after and subjected to immunoblot. (D) 293T17 cells were cotransfected with expression constructs containing PTPN11 WT FLAG or PTPN11 G60R FLAG constructs, TNK2, or empty vector controls. Lysates were collected 48 hours after and subjected to TNK2 immunoprecipitation, followed by Western blot. Blots (A, C, and D) are representative of four biological replicates.

Fig. 3.. Inhibition of TNK2 reduces signaling through PTPN11/RAS/MAPK.

(A) Phospho-p44/42 MAPK (phospho-ERK1/2) in 293T17 cells that were cotransfected with expression constructs containing PTPN11, PTPN11 E76K, TNK2, TNK2 T205I, or empty vector controls and treated 48 hours later with dasatinib (100 nM), AIM-100 (500 nM), or 0.05% dimethyl sulfoxide (DMSO) vehicle control for 2 hours. Lysates were subjected to immunoblot. (B and C) Quantification of Western blots represented in (A). Relative phospho-MAPK p44/42 (phospho-MAPK1/2), values were calculated with GAPDH as a loading control. ***P < 0.0005 by one way ANOVA. (D) As described in (A), phospho-PTPN11 (Y542) in 293T17 cells treated with TNK2 inhibitors. (E and F) Quantification of Western blots represented in (A). Relative phospho-p44/42 MAPK values were calculated with GAPDH as a loading control. No significance, as determined by one-way ANOVA. (G and H) Quantification of Western blots represented in (D). Relative phospho-PTPN11 values were calculated with GAPDH as a loading control. (I) Inhibition of PTPN11 in cells cotransfected with PTPN11 WT and TNK2 vectors. Cells were treated with SHP099 or vehicle control in increasing doses for 2 and 48 hours after transfection. Lysates were then immunoblotted. Blots (A and I) are representative of four biological replicates. Data (B to G) are means ± SEM of four experiments.

Fig. 4.. Functional assays show increased transformation potential and sensitivity to TNK2 inhibition.

(A) Total colony formation in mouse bone marrow colony formation assay. Mouse bone marrow cells were cotransduced to express PTPN11, PTPN11 E76K, TNK2, or empty vector controls. Cells were selected for GFP⁺ (green fluorescent protein-positive) and puromycin resistance and plated in a methylcellulose GM-CSF sensitivity colony formation assay. Colonies were counted at 14 days [GM-CSF] = 0.05 nM (0.71 ng/ml). ****P < 0.0001 by one-way ANOVA. (B) Total colony formation in mouse bone marrow colony formation assay in cells transduced with PTPN11, PTPN11 E76K, or PTPN11 G60R. Cells were sorted for GFP⁺. Cells were plated with increasing concentrations of dasatinib. ***P < 0.005 and ****P < 0.0005 by one-way ANOVA. (C) Total colony formation and percent total colony formation in mouse bone marrow colony formation assay. Mouse bone marrow cells were cotransduced to express PTPN11 E76K and TNK2 or TNK2 T205I gatekeeper mutant (fig. S4). Cells were selected for GFP+ and puromycin resistance and plated in a methylcellulose GM-CSF sensitivity colony formation assay. Colonies were counted at 7 days [GM-CSF] = 0.05 nM (0.71 ng/ml). ***P < 0.005 and **P < 0.005 by one-way ANOVA. (D) Total colony formation in mouse bone marrow colony formation assay. Mouse bone marrow cells were cotransduced to express PTPN11 E76K and TNK2 (fig. S4). Cells were selected for GFP⁺ and puromycin resistance and plated in a methylcellulose GM-CSF sensitivity colony formation assay. Colonies were counted at 7 days [GM-CSF] = 0.05 nM (0.71 ng/ml). No significance, as determined by one-way ANOVA. Data (A to D) are means ± SEM of three experiments.

Fig. 5.. PTPN11 mutations in AML confer dasatinib sensitivity.

(A) Mean dasatinib IC₅₀ of AML samples from patients, by PTPN11, NRAS, or KRAS mutation status (table S4). If no IC₅₀ was reached here, IC₅₀ was set to 1 μM. n = 128 samples. P values are determined by two-tailed Student’s t tests. *P < 0.05 or no significance, as determined by one-way ANOVA. (B) Sanger sequencing confirming a PTPN11 S502P mutation, identified by GeneTrail analysis, in a patient’s AML sample. Asterisk indicates the mutation. (C) Immunoblotting on lysates from 293T17 cells were transiently cotransfected with expression constructs containing PTPN11, PTPN11 S502P, TNK2, or empty vector controls. Blot is representative of five biological replicates. (D) Total colony formation in mouse bone marrow colony formation assay. Mouse bone marrow cells were cotransduced to express PTPN11, PTPN11 E76K, PTPN11 S502P, TNK2, or empty vector controls and plated in a methylcellulose GM-CSF sensitivity colony formation assay. Colonies were counted at 14 days [GM-CSF] = 0.05 nM (0.71 ng/ml). Data are means ± SEM of three experiments. (E) Peripheral blood counts for JMML patient at the time of recurrence after second bone marrow transplant. Dasatinib therapy is shown over a 3-month period. The patient was diagnosed with Klebsiella bacteremia (denoted by asterisk), which is resolved with antibiotic therapy.

Fig. 6.. Working model: Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia.

PTPN11 signaling is necessary for sustaining RAS/MAPK activation, with activating mutations of PTPN11 leading to increased RAS/MAPK signaling and cell proliferation (left). Our findings suggest a new paradigm in which TNK2 activates PTPN11, especially mutant PTPN11, leading to even more RAS/MAPK signaling and leukemogenesis (middle). Inhibition of TNK2 with dasatinib abolishes this RAS/MAPK signaling (right).