By RT-qPCR analysis in primary AML cells, AML cell lines, and evaluation of Affymetrix data we have established a profile of PTP expression in AML. Highly expressed genes include three transmembrane classical PTPs and 6 non-receptor classical PTPs. Interestingly, several dual-specificity PTPs (DUSPs) were found abundantly expressed, suggesting important functions. This applies also to PTP4A1 and PTP4A2 (PRL-1 and −2), and ACP1 (LMW-PTP). The possible roles of some of these PTPs for cell biology in leukemia have been previously reviewed  but further functional investigation of these highly expressed PTPs appears warranted. PTP mRNA expression in different types of immune cells has been recently evaluated . This study has defined a set of PTPs commonly expressed in immune cell lineages. As one could expect, this set shows many overlaps with the PTP profile in AML cells described here, for example PTPRC/CD45 and PTPN6/SHP-1 are among the highly expressed genes in both analyses. However, certain of these “commonly expressed” PTP genes in immune cells were only weakly or not detected in our analysis, including for example PTPRF
PTPN9, or DUSP4. These PTPs may not be expressed in the early differentiation stages represented by the leukemic blasts, or may potentially be downregulated, another interesting topic for further investigation. Some obvious discrepancies in the expression levels of PTP genes in the patient samples either detected by RT-qPCR or by Affymetrix analysis were apparent. They may mostly relate to the different detection of splice versions by the two methods, and in part to the different sample sets. Still, of the 23 PTP genes detected among the upper 25% by expression level, 12 match for both detection methods. For the case of DUSP6, RT-qPCR analysis was clearly advantageous over the microarray technique to detect relevant and further validated changes.
We have focused our interest on the possible role of FLT3 ITD, a common oncogenic lesion in AML, on PTP expression. It appeared possible that changed PTP expression would contribute to FLT3 ITD mediated transformation. Only relatively few alterations of PTP expression were, however, observed. Notably, PTPN6 (SHP-1), a highly expressed PTP with a proven negative regulatory role for cytokine signaling and possible role in regulation of FLT3 was not altered in mRNA expression in our data sets, contrary to what has previously been proposed . Also, PTPRJ, a negative regulator of FLT3 autophosphorylation , was not altered in expression by FLT3 ITD (data not shown). As we have found recently, FLT3 ITD appears, however, to inactivate PTPRJ by production of high levels of reactive oxygen species . DUSP2 mRNA was downregulated in primary AML cells with FLT3 ITD, but this phenomenon could not be recapitulated in 32D or Ba/F3 cells. It is possible that further lesions in the primary AML cells contribute to DUSP2 regulation. Still, reduced DUSP2 levels may play a role in FLT3 ITD-transformed cells, which we have not yet explored.
Interestingly, DUSP6 was elevated in expression downstream of FLT3 ITD signaling, as found in our RT-qPCR analysis of primary AML cells, as well as in different model cell systems. DUSP6 induction at mRNA and protein level could be causally linked to FLT3 ITD signaling activity and ERK1/2 pathway activation. Upregulation of DUSP6 mRNA was, however, not detectable in the Affymetrix data set. It is possible that this method is not sufficiently sensitive to detect the FLT3 ITD-mediated alteration in DUSP6 expression. In patients with activating N-RAS mutations, an increase in DUSP6 expression could, however, be seen in the Affymetrix data set (not shown), possibly indicating that N-RAS mutations are more potent than FLT3 ITD in driving DUSP6 expression. Previous gene expression analyzes (also using Affymetrix expression arrays) in myeloma cells have identified DUSP6 as one of only three genes which were uniquely and strongly elevated in cells harboring activated N-RAS . Interestingly, other stimuli which only transiently activated ERK signaling such as interleukin-6 stimulation did not induce a sufficiently robust DUSP6 response to allow detection with this technique. Consistent with our findings, DUSP6 has previously been identified as one of the genes which are most effectively downregulated in FLT3 ITD expressing AML cells treated with the FLT3/broad spectrum kinase inhibitor CEP701 (Lestaurtinib) . Based on its capacity for dephosphorylating the pThr-X-pTyr motif in ERK1/2 , DUSP6 can negatively regulate pERK1/2 levels in multiple cell types. Since the ERK1/2 pathway mediates mitogenic signaling, among other responses, DUSP6 has been proposed as a tumor suppressor . Notably, in pancreatic cancer, DUSP6 levels are downregulated by gene deletion or promoter hypermethylation, consistent with such a function [30–32]. Recently, DUSP6 was shown to inhibit growth, migration and epithelial-to-mesenchymal transition (EMT) of esophagal squamous cell carcinoma and nasopharyngeal carcinoma cells . In non-small cell lung cancer, however, high DUSP6 levels have been found to predict poor prognosis, and evidence for a tumor-promoting role of DUSP6 in human glioblastoma has recently been provided [34, 35]. Moreover, DUSP6 has recently been found as a predictor of invasiveness in papillary thyroid cancer. Silencing of DUSP6 expression decreased the cell viability and migration rate of a corresponding cell line . As shown in the present study, elevated DUSP6 levels correlate with the presence of FLT3 ITD, a negative predictor of prognosis  in AML cells. This observation, taken together with the functional findings discussed below, indicates that DUSP6 may play a pro-oncogenic role in FLT3 ITD-positive AML. Obviouosly, depending on the context, DUSP6 affects tumor biology in very different ways.
The function of high DUSP6 levels in FLT3 ITD expressing cells was addressed by RNAi experiments. Consistent with its function as ERK1/2-PTP, downregulation of DUSP6 caused elevated constitutive ERK1/2 activation in FLT3 ITD expressing cells. Moreover, ERK1/2 phosphorylation was also elevated in FL-stimulated cells, albeit only moderately. We observed that FL-stimulation caused downregulation of DUSP6 protein levels, similar as it has been described earlier for the stimulation of cells with other mitogens. In these studies the authors linked the reduction of DUSP6 levels to proteasomal degradation, which was prompted by phosphorylation at serine residues [22, 37]. Surprisingly, 32D cell pools with attenuated Dusp6 expression were, however, moderately impaired in proliferation, indicating that high DUSP6 levels may not play a negative but rather some positive role in FLT3 ITD dependent cell growth. Forced overexpression of exogenous DUSP6 in stable transfectants reduced ERK1/2 phosphorylation, but did not inhibit cell proliferation (data not shown), further supporting the notion that ERK1/2 activity and cell growth in FLT3 ITD-expressing cells are not simply positively correlated. It is well known that the biological outcome of ERK1/2 activation depends both on its magnitude and on its kinetics, which are determined by several feedback mechanisms . For example, sustained high ERK1/2 activation by the phorbol ester TPA in MCF7 cells causes growth arrest . Similarly, nerve growth factor (NGF) stimulation of PC-12 pheochromocytoma cells as opposed to epidermal growth factor (EGF) stimulation leads to sustained high-level ERK1/2 activation and not a proliferative, but a differentiation response [39, 40]. The alternate cellular responses to different kinetics and magnitude of ERK1/2 activation seem based on differential transcriptional sensing , which is likely to depend on the specific cell type and the simultaneous activation of other signaling pathways. In the context of FLT3 ITD, high constitutive expression of DUSP6 appears associated with a relatively low but constitutive level of ERK1/2 activity, which is compatible with efficient cell growth. ShRNA-mediated DUSP6 downregulation causes a higher level of constitutive ERK1/2 activation (see Figure 4), which, similar as in the case of MCF7 or PC12 cells, may ultimately result in diminished cell proliferation by yet unclear downstream mechanisms. Alternatively or in addition, potential other substrates of DUSP6 may play a role. These possibilities need to be further explored.