A noncanonical Flt3ITD/NF-κB signaling pathway represses DAPK1 in acute myeloid leukemia.

PURPOSE: Death-associated protein kinase 1 (DAPK1), a tumor suppressor, is a rate-limiting effector in an endoplasmic reticulum (ER) stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of acute myeloid leukemia (AML) with poor prognosis, which lacked ER stress-induced apoptosis. EXPERIMENTAL DESIGN: Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB-and c-Jun-responsive genes, was studied. RNA interference knockdown studies were carried out in an Flt3ITD(+) cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD-TAK1-DAPK1 repression, and chromatin immunoprecipitations were carried out to identify proximate effector proteins, including TAK1-activated p52NF-κB, at the DAPK1 locus. RESULTS: AMLs characterized by normal karyotype with Flt3ITD were found to have 10- to 100-fold lower DAPK1 transcripts normalized to the expression of c-Jun, a transcriptional activator of DAPK1, as compared with a heterogeneous cytogenetic category. In addition, Meis1, a c-Jun-responsive adverse AML prognostic gene signature was measured as control. These Flt3ITD(+) AMLs overexpress relB, a transcriptional repressor, which forms active heterodimers with p52NF-κB. Chromatin immunoprecipitation assays identified p52NF-κB binding to the DAPK1 promoter together with histone deacetylase 2 (HDAC2) and HDAC6 in the Flt3ITD(+) human AML cell line MV-4-11. Knockdown of p52NF-κB or its upstream regulator, NF-κB-inducing kinase (NIK), de-repressed DAPK1. DAPK1-repressed primary Flt3ITD(+) AMLs had selective nuclear activation of p52NF-κB. CONCLUSIONS: Flt3ITD promotes a noncanonical pathway via TAK1 and p52NF-κB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD(+) AML.

Constitutive c-jun N-terminal kinase activity in acute myeloid leukemia derives from Flt3 and affects survival and proliferation.

OBJECTIVE: c-jun N-terminal kinase (JNK) has been implicated in proliferation and survival downstream from the tyrosine kinase oncogene, p210 BCR-ABL, in chronic myeloid leukemia. We studied whether a similar relationship between JNK and FMS-like tyrosine kinase 3 (Flt3) describes acute myeloid leukemia (AML). METHODS: By immunoprecipitation, Flt3 was found to be activated and identified as the potential origin of JNK activity in a heavy majority of JNK+ve AML blasts tested. Often, Flt3 activity is associated with activating mutation of the gene locus. However, statistical linkage tied JNK activity with Flt3 expression levels rather than with mutation. An adaptor network to describe the signal cascade Flt3-to-JNK was uncovered. RESULTS: Active Flt3 was linked to p85 phosphoinositide-3 (PI-3) kinase, and p85 with cbl and CrkII/CrkL by co-immunoprecipitaton assays from lysates of model cell lines and primary AML blasts. JNK1 co-immunoprecipitated from such lysates with p85-cbl-crkII/L and bound to Crk species SH3 domain in pull-down assay. siRNA-mediated depletion of Flt3 or of cbl, the adaptor at the nexus of this signaling group, inhibited JNK activity on substrate c-jun. Within AML blast cells influenced by Flt3 signaling, selective inhibition of JNK by a small molecule inhibitor, led to proliferative inhibition, apoptosis, and sensitizing cells to the anthracycline, daunorubicin. These effects occurred upon JNK inhibition without off-target inhibition of extracellular signal-regulated kinase or AKT pathways, and p38-kinase activation, an effector in the p53/p14 arf tumor suppressor pathway, was also maintained or augmented. CONCLUSION: JNK is a bonafide signaling pathway from Flt3 in AML whose function for proliferation and survival is required in a significant AML cohort with active Flt3 signaling, by mutation or overexpression of Flt3.