M Geugien

Regulation of constitutive STAT5 phosphorylation in acute myeloid leukemia blasts

In the present study, we examined the underlying mechanism, which causes the constitutive tyrosine phosphorylation of signal transducer and activator of transcription 5 (STAT5) in acute myeloid leukemia (AML) blasts. Constitutive STAT5 phosphorylation was observed in 18 of 26 (69%) patients with AML. The constitutive STAT5 phosphorylation was caused by different mechanisms. In the majority of the investigated cases (71% (12 of 17)) constitutive STAT5 phosphorylation was associated with autophosphorylation of the type III receptor tyrosine kinase Flt3. In 47% (eight of 17) of these cases autophosphorylation of Flt3 coincided with tandem duplications of the Flt3 gene, resulting in constitutive phosphorylation of the receptor, while 24% (four of 17) of the cases demonstrated STAT5 phosphorylation and Flt3 autophosphorylation without mutations. In addition, a subset of AML cases (29% (five of 17)) had no autophosphorylation of the Flt3 receptor, but demonstrated constitutive STAT5 phosphorylation, which was partly due to autocrine growth factor production. All AML cases with high STAT5 and Flt3 phosphorylation demonstrated, in general, a lower percentage of spontaneous apoptosis, compared to AML blasts with no spontaneous STAT5 phosphorylation. Addition of the receptor tyrosine III kinase inhibitor AG1296 strongly inhibited STAT5 phosphorylation and enhanced the percentage of apoptotic cells without modulating the Bcl-xl protein levels. These data indicate that in the majority of AML cases the constitutive STAT5 phosphorylation is caused by Flt3 phosphorylation mostly due to mutations in the receptors and associated with a low degree of spontaneous apoptosis.

Constitutive NF-kappa B DNA-binding activity in AML is frequently mediated by a Ras/PI3-K/PKB-dependent pathway

In the present study, we aimed to elucidate the mechanism responsible for constitutive NF-κB DNA-binding activity in AML cells. Intervening in aberrant signaling pathway provides a rational approach for in vivo targeting of AML cells. Constitutive NF-κB DNA-binding activity was observed in 16 of 22 (73%) investigated AML cases and was, in general, associated with resistance to spontaneous apoptosis. Indeed, inhibition of NF-κB activity by the NF-κB inhibitor SN-50 peptide resulted in enhanced chemotherapy-induced apoptosis. In the majority of cases, constitutive NF-κB activity was mediated by a Ras/PI3 kinase (PI3-K)/protein kinase B (PKB)-mediated pathway. The PI3-K inhibitor Ly294002 and the Ras inhibitor L-744832 both inhibited PKB phosphorylation and NF-κB DNA-binding activity. The constitutive activation of Ras GTP-ase was caused by mutations in the gene encoding for N-Ras in 29% of the cases. The constitutive NF-κB activity could so far not be ascribed to the autocrine production of growth factors or to mutations in the Flt3 receptor, since anti-GM-CSF, -IL-1, -IL6, -TNFα or the tyrosine kinase inhibitor AG1296 did not affect the NF-κB DNA-binding activity. The present study demonstrates that Ras activation is an important pathway for triggering the NF-κB pathway in AML cells.

Constitutive cytoplasmic localization of p21(Waf1/Cip1) affects the apoptotic process in monocytic leukaemia

In the present study, we analysed the expression and localization of p21Waf1/Cip1 in normal and malignant haematopoietic cells. We demonstrate that in normal monocytic cells, protein kinase C (PKC)-induced p21 gene activation, which is nuclear factor-κB (NF-κB) independent, results in predominantly cytoplasmic localized p21 protein. In acute monocytic leukaemia (M4, M5), monocytic blasts (N=12) show constitutive cytoplasmic p21 expression in 75% of the cases, while in myeloid leukaemic blasts (N=10), low nuclear and cytoplasmic localization of p21 could be detected, which is also PKC dependent. Constitutive p21 expression in monocytic leukaemia might have important antiapoptotic functions. This is supported by the finding that in U937 cells overexpressing p21, VP16-induced apoptosis is significantly reduced (20.0±0.9 vs 55.8±3.8%, P<0.01, N=5), reflected by a reduced phosphorylation of p38 and JNK. Similarly, AML blasts with high cytoplasmic p21 were less sensitive to VP16-induced apoptosis as compared to AML cases with low or undetectable p21 expression (42.25 vs 12.3%, P<0.01). Moreover, complex formation between p21 and ASK1 could be demonstrated in AML cells, by means of coimmunoprecipitation. In summary, these results indicate that p21 has an antiapoptotic role in monocytic leukaemia, and that p21 expression is regulated in a PKC-dependent and NF-κB-independent manner.

CITED2 affects leukemic cell survival by interfering with p53 activation

CITED2 (CBP/p300-interacting-transactivator-with-an-ED-rich-tail 2) is a regulator of the acetyltransferase CBP/p300 and elevated CITED2 levels are shown in a number of acute myeloid leukemia (AML). To study the in vivo role of CITED2 in AML maintenance, AML cells were transduced with a lentiviral construct for RNAi-mediated knockdown of CITED2. Mice transplanted with CITED2-knockdown AML cells (n=4) had a significantly longer survival compared to mice transplanted with control AML cells (P<0.02). In vitro, the reduction of CITED2 resulted in increased p53-mediated apoptosis and CDKN1A expression, whereas BCL2 levels were reduced. The activation of p53 upon CITED2 knockdown is not a direct consequence of increased CBP/p300-activity towards p53, since no increased formation of CBP/p300/p53 complexes was demonstrated and inhibition of CBP/p300-activity could not rescue the phenotype of CITED2-deficient cells. Instead, loss of CITED2 had an inhibitory effect on the AKT-signaling pathway, which was indicated by decreased levels of phosphorylated AKT and altered expression of the AKT-pathway regulators PHLDA3 and SOX4. Notably, simultaneous upregulation of BCL2 or downregulation of the p53-target gene PHLDA3 rescued the apoptotic phenotype in CITED2-knockdown cells. Furthermore, knockdown of CITED2 led to a decreased interaction of p53 with its inhibitor MDM2, which results in increased amounts of total p53 protein. In summary, our data indicate that CITED2 functions in pathways regulating p53 activity and therefore represents an interesting target for AML therapy, since de novo AML cases are characterized by an inactivation of the p53 pathway or deregulation of apoptosis-related genes.