Research ArticleLEUKEMIA

Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia

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Science Signaling  17 Jul 2018:
Vol. 11, Issue 539, eaao5617
DOI: 10.1126/scisignal.aao5617
  • 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).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/539/eaao5617/DC1

    Fig. S1. Increase in PTPN11 activity is TNK2 kinase–dependent.

    Fig. S2. TNK2 inhibition reduces signaling through RAS/MAPK in 293T17 lysates.

    Fig. S3. PTPN11 inhibition by SHP099 increases phospho-Tyr284 TNK2 and phospho-Tyr580 PTPN11.

    Fig. S4. Mouse bone marrow colony formation assays with TNK2-specific inhibitors.

    Fig. S5. Signaling effects of PTPN11 S502P mimic those seen with other PTPN11-activating mutations.

    Fig. S6. Relative mRNA expression of dasatinib targets in AML patient samples from Fig. 5A.

    Table S1. Inhibitor IC50 values in the JMML patient sample.

    Table S2. siRNA panel data from the JMML patient sample.

    Table S3. Mutations identified in PTPN11-mutant patient samples from Fig. 5A.

    Table S4. Kd (dissociation constant) values of top dasatinib targets.

  • This PDF file includes:

    • Fig. S1. Increase in PTPN11 activity is TNK2 kinase–dependent.
    • Fig. S2. TNK2 inhibition reduces signaling through RAS/MAPK in 293T17 lysates.
    • Fig. S3. PTPN11 inhibition by SHP099 increases phospho-Tyr284 TNK2 and phospho-Tyr580 PTPN11.
    • Fig. S4. Mouse bone marrow colony formation assays with TNK2-specific inhibitors.
    • Fig. S5. Signaling effects of PTPN11 S502P mimic those seen with other PTPN11-activating mutations.
    • Fig. S6. Relative mRNA expression of dasatinib targets in AML patient samples from Fig. 5A.
    • Table S1. Inhibitor IC50 values in the JMML patient sample.
    • Table S2. siRNA panel data from the JMML patient sample.
    • Table S3. Mutations identified in PTPN11-mutant patient samples from Fig. 5A.
    • Table S4. Kd (dissociation constant) values of top dasatinib targets.

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