Essential requirement for PP2A inhibition by the oncogenic receptor c-KIT suggests PP2A reactivation as a strategy to treat c-KIT+ cancers

Abstract

Oncogenic mutations of the receptor tyrosine kinase c-KIT play an important role in the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis, and some acute myeloid leukemias (AML). Although juxtamembrane mutations commonly detected in gastrointestinal stromal tumor are sensitive to tyrosine kinase inhibitors, the kinase domain mutations frequently encountered in systemic mastocytosis and AML confer resistance and are largely unresponsive to targeted inhibition by the existing agent imatinib. In this study, we show that myeloid cells expressing activated c-KIT mutants that are imatinib sensitive (V560G) or imatinib resistant (D816V) can inhibit the tumor suppressor activity of protein phosphatase 2A (PP2A). This effect was associated with the reduced expression of PP2A structural (A) and regulatory subunits (B55alpha, B56alpha, B56gamma, and B56delta). Overexpression of PP2A-Aalpha in D816V c-KIT cells induced apoptosis and inhibited proliferation. In addition, pharmacologic activation of PP2A by FTY720 reduced proliferation, inhibited clonogenic potential, and induced apoptosis of mutant c-KIT(+) cells, while having no effect on wild-type c-KIT cells or empty vector controls. FTY720 treatment caused the dephosphorylation of the D816V c-KIT receptor and its downstream signaling targets pAkt, pSTAT5, and pERK1/2. Additionally, in vivo administration of FTY720 delayed the growth of V560G and D816V c-KIT tumors, inhibited splenic and bone marrow infiltration, and prolonged survival. Our findings show that PP2A inhibition is essential for c-KIT-mediated tumorigenesis, and that reactivating PP2A may offer an attractive strategy to treat drug-resistant c-KIT(+) cancers.

Figure 1. Expression of PP2A subunits in FDC-P1 c-KIT⁺ cell lines

Immunoblot of FDC-P1 cell lysates showing expression of PP2A-A, PP2Ac, PP2Ac pY³⁰⁷, PP2A-B55α, -B56α, -B56γ, -B56δ, -B56ε, and SET. Actin was used as a loading control. Blots are a representative of three independent experiments.

Figure 2. Over-expression of PP2A-Aα induces apoptosis and inhibits proliferation of FDC-P1 D816V c-KIT cells

The pMIG EV and PP2A-Aα expression vectors were retrovirally transduced into FDC-P1 D816V c-KIT cells. A, Immunofluorescent staining of cytopsins detecting PP2A-Aα 24 h post-infection (day 0). B, The induction of apoptosis at day 0 was assessed by annexin-V binding (left) and cleaved caspase-3 immunofluorescent staining of cytopsins (right). C, Total cell numbers were determined by trypan blue exclusion starting at day 0. Scale bars, 50 μm or 20 μm (600× magnification). UTF, untransfected.

Figure 3. Reactivation of PP2A induces apoptosis and suppresses the clonogenic potential of mutant c-KIT cells

A, FDC-P1 cells were treated with FTY720 or FTY720-P (2.5 μM; 6 h). PP2A activity was determined by incubating the isolated PP2Ac complex with a PP2A-specific phosphopeptide and measuring free phosphate release using a colorimetric assay. PP2A activity is normalised to untreated EV controls. B, FDC-P1 cells were treated with FTY720 (2.5 μM, 24 h) and assessed for annexin-V by flow cytometry. C, FDC-P1 cells were treated with FTY720 (2.5 μM; 36 h) and stained with PI to analyse DNA content. Plots are a representative of four independent experiments. D, E, FDC-P1 cells were grown in methylcellulose for 7days in the presence of indicated drugs. Columns, mean colony number (n=4); bars, SEM. *, p<0.05; **, p<0.01, Student’s t-test compared to untreated.

Figure 4. Reactivation of PP2A dephosphorylates the c-KIT receptor and its downstream signaling targets

A, c-KIT was immunoprecipitated from FDC-P1 cells treated with 2.5 μM FTY720 (0, 6, 12, 24 h). Levels of tyrosine phosphorylated (pY) and total c-KIT were determined by immunoblotting (top). Quantitation of three independent experiments (bottom). B, Immunoblots detecting phosphorylated or total Akt, STAT5, ERK1/2, p38MAPK, MEK and BAD after FTY720 treatment (2.5μM; 12h).

Figure 5. FTY720 delays mutant c-KIT tumor growth in vivo

FDC-P1 cells expressing either V560G (left) or D816V c-KIT (right) were s.c. inoculated into a separate group of mice. Treatment started day 5 post-tumor injection with daily i.p. injections of saline, 100 mg/Kg imatinib or 10 mg/Kg FTY720. A, The estimated probabilities for survival were calculated using the Kaplan-Meier method, and log-rank test was used to determine the survival difference compared to saline-treated mice. B, On day 14, mice were sacrificed and tumors weighed. Columns, mean of individual tumor weights; bars, SEM. *, p<0.05, n=6. **, p<0.01, n=12, Student’s t-test compared to saline. C, Apoptosis within the V560G and D816V c-KIT tumors assessed by TUNEL staining. Images were acquired at 400x on DAPI (blue) and FITC (green) channels with an Axioplan 2 imaging system. Scale bars, 20 μm. D, Splenic weight (mg) recorded from mice harboring D816V c-KIT tumors on day 14 (top). Columns, mean; bars, SEM. *, p<0.05; **, p<0.01. Immunohistochemical staining of human c-KIT on spleen and bone marrow harvested at day 14 (bottom). Representative pictures were taken with a ColorView II camera and analysis software at 100× and 400× magnification. Scale bars, 100 μm (100×) and 20 μm (400×). The % TUNEL (C) or human c-KIT⁺ (D) cells were determined by counting the number of positive cells divided by total cells seen in 10 individual fields from 3 different mice. Columns, mean percentage of positive cells; bars, SEM. **, p<0.01, n=3, Student’s t-test compared to saline.