Role of SHP2 for FLT3-dependent proliferation and transformation in 32D cells

Fms-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase, which plays a role in proliferation and differentiation of B-cell progenitors, myelomonocytic and dendritic cells, as well as in the maintenance of pluripotent hematopoietic stem cells (reviewed in Stirewalt and Radich,1and Schmidt-Arras et al.2). Recently, FLT3 has received much attention as an important oncoprotein. Mutations in FLT3 that lead to constitutive activation are among the most common molecular lesions found in acute myeloid leukemia.3 The most prevalent type of mutations result in internal tandem duplications (ITD) of amino-acid stretches in the juxtamembrane domain of FLT3. FLT3-ITD is constitutively and highly active, can transform myeloid cell lines in vitro4 and can induce a myeloproliferative syndrome. Wild-type FLT3 and FLT3-ITD exhibit qualitative differences in signal transduction. Notably, while FLT3-ITD strongly and directly activates STAT5, ligand-activated wild-type FLT3 is a comparatively weak STAT5 activator (reviewed in Choudhary et al.5). The mechanisms of differential signal transduction are incompletely understood. Signaling of receptor tyrosine kinases is modulated by protein-tyrosine phosphatases (PTPs), and aberrations in PTP function play a role in carcinogenesis.6 Some PTPs have been found to function in a positive manner in growth-stimulatory signaling pathways and gain-of-function of these PTPs can potentially be oncogenic. The oncogenic activity of a PTP is best characterized for the Src homology-2 (SH2) domain-containing phosphatase 2 (SHP2), in which activation mutations are found in hereditary and sporadic leukemias and, less frequently, in solid tumors.7 In most receptor tyrosine kinase signaling pathways, SHP2 is required for full activation of the Erk-MAP kinase pathway. Little is known about the control of FLT3 signaling by PTPs and the potential role of PTPs in FLT3-mediated transformation. We have recently shown that coexpression of FLT3 with the PTPs SHP1, PTP1B and PTP-PEST leads to FLT3 dephosphorylation, suggesting an inhibitory function of these PTPs for FLT3 signaling.8 Recent work by Heiss et al.9 has shown that SHP2 can directly interact with FLT3 in a phosphorylation-dependent manner through FLT3 phosphotyrosine 599. The possible role of SHP2 for FLT3-ITD-mediated transformation has not yet been addressed. To further elucidate the function of SHP2 in FLT3 signaling, we have downregulated SHP2 in human and murine cell systems using stable shRNA expression. A set of five different, but nonvalidated murine SHP2 targeting MISSION shRNA derived expression cassettes (designated LKO1-5) were packaged into lentiviral pseudotypes and used to transduce wild-type FLT3-expressing 32D cells (for details, see online supplement). Three out of five shRNA cassettes (LKO1, 4 and 5) resulted in a pronounced suppression of SHP2 levels as compared to cells transduced with particles encoding control shRNA (Figure 1a). Notably, it was not possible to stably knockdown SHP2 expression below a level of approximately 30%, whereas nearly complete SHP2 knockdown with the same targeting sequences was easily possible in transient transfection experiments in HEK293 cells (not shown). Also, prolonged cultivation of cells with SHP2 knockdown frequently resulted in acquisition of a cytokine-independent phenotype (not shown). These observations may indicate that a certain amount of SHP2 is essential for the viability of 32D cells. The effect of SHP2 knockdown on FLT3 signaling activity was initially assessed by analyzing FL-stimulated activation of Erk. All 32D SHP2 knockdown lines exhibited clearly reduced Erk phosphorylation compared to the control cell line (Figure 1b). Similar results were observed in the human acute monocytic cell line THP-1, in which SHP2 levels were also reduced by shRNA technology, and in 32D cell lines employing yet another targeting shRNA sequence and retroviral transduction with the pSUPER vector (Supplementary Figure S1, online supplement). The similar responses in various cell lines and with different SHP2-targeting shRNA sequences strongly suggest that this effect is specifically caused by the SHP2 knockdown. Next, the proliferation of the LKO-transduced cell lines was analyzed. All cell lines were cytokine dependent, as evident from their rapid death in absence of cytokine (Figure 1c, left panel). The downregulation of SHP2 affected cell growth in presence of 1 ng/ml IL3 only partially in one cell line, whereas growth in presence of 10 ng/ml FL was reduced to a variable extent in all three analyzed SHP2 shRNA-transduced cell lines compared to the control cells (Figure 1c, right and middle panel). Using an experimental approach of inducible expression of dominant negative SHP2 variants, it has previously been shown that SHP2 is involved in the antiapoptotic effects of IL3 in BaF/3 cells. However, these effects could only be observed at suboptimal concentrations.10 Saturating IL3 concentrations, as we have employed them to maintain the SHP2-depleted cells, can apparently over-ride the effects of SHP2 knockdown, in contrast to saturating FLT3 ligand (FL) concentrations. These findings highlight the qualitative differences in signaling patterns induced by the two cytokines and indicate that IL3 signaling either does not absolutely require SHP2 or can function with only low levels of the PTP. To further validate the specificity of the knockdown phenotype, we carried out rescue experiments by re-expressing a version of SHP2, which is refractory to the shRNA. We chose the wild-type FLT3 expressing LKO5 cells for such rescue experiments since knockdown of endogenous murine SHP2 had the most pronounced effects in this line. The cells were transduced with retroviruses encoding wild-type human SHP2, or the gain-of-function D61Y SHP2, and stable cell pools were selected. The LKO5-encoded murine-targeting sequence differs in three positions from the corresponding sequence in human SHP2. As shown in Supplementary Figure S2a (online supplement), restoration of SHP2 levels was possible with this strategy and re-expression of human wild-type SHP2 resulted in partial, and that of the SHP2 D61Y mutant in complete compensation of the impaired cell proliferation of the SHP2 knockdown cell line (Supplementary Figure S2b, online supplement). Still, cells expressing human SHP2 D61Y remained fully cytokine dependent, suggesting that at least in 32D cells, SHP2 D61Y can function only in the context of upstream receptor activation and cannot bypass FLT3-mediated signaling. Taken together, our analysis revealed that SHP2 is an important mediator of wild-type FLT3-mediated Erk activation and cell proliferation. The possible role of SHP2 for cell transformation by oncogenic FLT3 versions has not yet been investigated. We, therefore, used the efficient lentiviral SHP2 knockdown strategy described above, to address this issue. Lentivirus-mediated transduction of FLT3-ITD-expressing 32D cells with SHP2-targeting shRNAs resulted in effective SHP2 knockdown with the LKO variants LKO1, 4 and 5 (Figure 2). FLT3-ITD expression in 32D cells causes the constitutive activation of several signaling events, including the Erk pathway.5 As shown in Figure 2b, SHP2 knockdown in FLT3-ITD-expressing 32D cells resulted in a significant reduction of Erk phosphorylation. FLT3-ITD also confers factor-independent growth and transformation to 32D cells and to other IL3-dependent hematopoietic cells.11 Interestingly, the SHP2 knockdown in FLT3-ITD-expressing 32D cells did not significantly affect cytokine-independent growth (Figure 2c). To further investigate the role of SHP2 for FLT3-ITD-mediated transformation, we also examined the ability of the SHP2 knockdown cells lines to confer clonogenic growth, as a marker of transformation. All cell lines readily formed colonies in semisolid media regardless of the cellular SHP2 expression level, which did not differ in number or size (Figure 2d). Finally, the capacity of FLT3-ITD-expressing 32D cells with downregulated SHP2 levels to generate a leukemia-like disease in syngeneic C3H/HeJ mice was analyzed. Disease development occurred with similar features as in mice inoculated with FLT3-ITD-expressing 32D cells, with massive enlargement of spleen (Supplementary Figure S3, online supplement) and liver and infiltration of the bone marrow (Supplementary Table S1, online supplement). Diseased animals were moribund within 4 weeks after injection (Figure 2e). In contrast, animals injected with parental 32D cells did not develop an obvious disease up to 3 months after injection. Taken together, although constitutive Erk activation by FLT3-ITD was reduced by SHP2 knockdown, FLT3-ITD-dependent cell proliferation, colony formation and tumor formation in syngeneic C3H/HeJ mice were not affected. Signal transduction of FLT3-ITD is qualitatively different from signal transduction of ligand-activated wild-type FLT3. FLT3-ITD also exhibits an altered cellular localization, partially existing as an immature, intracellular form, which may contribute to the different signaling quality.8 It is possible that the FLT3-ITD evoked alternate signaling pathways can bypass SHP2 in their effects on cell proliferation and survival. Alternatively or in addition, low levels of SHP2, as they still exist in the shRNA-expressing cells, may be sufficient to mediate essential effects of FLT3-ITD. It has recently been observed that FLT3-ITD mutations and SHP2 gain-of-function mutations are mutually exclusive in acute myeloid leukemia patients, indicating that both lesions drive leukemogensis by similar mechanisms.12 It remains to be elucidated if FLT3-ITD can transform cells in the complete absence of SHP2. Clearly, our findings indicate that alternate signaling downstream of FLT3-ITD is associated with a reduced requirement of SHP2 for promoting cell proliferation and transformation, as compared to the case of FL-stimulated wild-type FLT3. Reduced requirement for SHP2 is a previously unrecognized aspect of FLT3-ITD signaling quality and FLT3-ITD dependent transformation. We thank Dr Justus Duyster and Dr Rebekka Grundler (Munich), and Dr M Golam Mohi and Dr Benjamin G Neel (Boston) for provision of different reagents; Dr Chunaram Choudhary, Dr Bülent Sargin and Dr Lara Tickenbrock (University of Münster) for help with establishing several techniques, and Ulla Bergholz (Hamburg) for expert technical assistance. This work was supported by the collaborative Grant 106291 from Deutsche Krebshilfe and by the EC Grant MRTN-CT-2006-035830 PTP-NET. Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)