Essential Role of the INK4/CDK4,6/Rb/E2F Signaling Pathway in AML with the Flt3 Internal Tandem Duplication.

Mutationally activated tyrosine kinases provide a critical survival signal to cancer cells, thus, making such kinases and their downstream effectors attractive targets for cancer therapy. To study signaling of mutated kinases we have chosen the receptor tyrosine kinase Flt3 that harbors an activating internal tandem duplication (ITD) in about 25% of AML patients. The use of a Flt3 inhibitor (THRX-165724, Theravance, Inc.) in two Flt3 ITD AML cell lines (MOLM13 and MV4-11) led to the inhibition of the INK4/CDK4,6/Rb/E2F pathway within three hours as reflected by the downregulation of D-cyclin gene expression followed by a decrease in D-cyclin protein. As a result of reduced D-cyclin levels, CDK4,6 activity was downregulated as revealed by the hypophosphorylation of the main substrate of CDK4,6, the Rb protein. THRX-165724 had no effect on D-cyclin levels or Rb hyperphosphorylation in THP-1 and U937 cells, two AML cell lines that express wildtype Flt3. Furthermore, THRX-165724 did not affect the proliferation or survival of these two cell lines. To investigate the role of the INK4/CDK4,6/Rb/E2F pathway as part of the proliferation and survival signal provided by the Flt3 ITD, we used PD-0332991, a highly selective CDK4,6 kinase inhibitor from Pfizer currently in phase I clinical trials for solid tumors. A dose-response experiment revealed that in cells PD-0332991 inhibits CDK4,6 activity with an IC50 of 30–40 nM as assayed by following the dephosphorylation of Rb. The compound does not inhibit Flt3 kinase activity even at the highest concentrations tested (500 nM). In MV4-11 and MOLM13 cells, PD-0332991 induced G1 specific cell cycle arrest within 24 hours and apoptosis after 3 days. Hence, in MV4-11 and MOLM13, PD-0332991 is cytostatic and cytotoxic. In contrast, PD-0332991 was neither cytotoxic nor cytostatic in THP-1 and U937 cells. In a clonogenic assay PD-0332991 reduced in a dose dependent manner the colony formation of MV4-11 and MOLM13 cells as well as primary patient blasts with Flt3 ITD. In contrast, THP-1 and U937 cells as well as CD34+ primary hematopoietic progenitor cells were not affected in their ability to form colonies. In MV4-11 and MOLM13 cells PD-0332991 induced the downregulation of the anti-apoptotic protein Bcl-2 and in MOLM13 it also induced the upregulation of the pro-apoptotic protein Bak. The deregulation of Bcl-2 and Bak may represent the underlying mechanism for the observed cytotoxicity of PD-0332991 in MV4-11 and MOLM13. In a mouse model of AML, NOD/SCID mice were inoculated with MOLM13 cells which caused leukemia within eight days. Mice treated with PD-0332991 starting at day seven after inoculation had a significant survival advantage demonstrating that PD-0332991 has anti-leukemic activity in vivo (median survival time 15.0 days versus 11.5 days, P=.0003). Our data suggest that the activation of the INK4/CDK4,6/Rb/E2F pathway by Flt3 ITD has an essential role for proliferation and survival in AML cells. Targeting the INK4/CDK4,6/Rb/E2F pathway in Flt3 ITD AML using CDK4,6 inhibitors like PD-0332991 should be explored for clinical efficacy in FLT3 ITD AML.