Ubiquitin conjugase UBCH8 targets active FMS-like tyrosine kinase 3 for proteasomal degradation

Abstract

The class III receptor tyrosine kinase FMS-like tyrosine kinase 3 (FLT3) regulates normal hematopoiesis and immunological functions. Nonetheless, constitutively active mutant FLT3 (FLT3-ITD) causally contributes to transformation and is associated with poor prognosis of acute myeloid leukemia (AML) patients. Histone deacetylase inhibitors (HDACi) can counteract deregulated gene expression profiles and decrease oncoprotein stability, which renders them candidate drugs for AML treatment. However, these drugs have pleiotropic effects and it is often unclear how they correct oncogenic transcriptomes and proteomes. We report here that treatment of AML cells with the HDACi LBH589 induces the ubiquitin-conjugating enzyme UBCH8 and degradation of FLT3-ITD. Gain-and loss-offunction approaches show that UBCH8 and the ubiquitin-ligase SIAH1 physically interact with and target FLT3-ITD for proteasomal degradation. These ubiquitinylating enzymes though have a significantly lesser effect on wild-type FLT3. Furthermore, physiological and pharmacological stimulation of FLT3 phosphorylation, inhibition of FLT3-ITD autophosphorylation and analysis of kinase-inactive FLT3-ITD revealed that tyrosine phosphorylation determines degradation of FLT3 and FLT3-ITD by the proteasome. These results provide novel insights into antileukemic activities of HDACi and position UBCH8, which have been implicated primarily in processes in the nucleus, as a previously unrecognized important modulator of FLT3-ITD stability and leukemic cell survival.

Breakdown of the FLT3-ITD/STAT5 Axis and Synergistic Apoptosis Induction by the Histone Deacetylase Inhibitor Panobinostat and FLT3-Specific Inhibitors

“…Compared with single-inhibitor treatment, we also found pronounced protein degradation of FLT3-ITD and STAT5 upon combinatorial treatment. Complementary to previous findings (18,20,27), caspases contribute to the decay of these prosurvival factors.…”

“…This treatment modality frequently resulted in the development of inhibitor-resistant FLT3 mutations (6,9,16). Therefore, combinatorial approaches with FLT3 inhibitors and other chemotherapeutic agents may be a valuable strategy for the treatment of AML (17)(18)(19)(20).…”

“…Of note, LBH589 is one of the most potent HDACi tested in clinical trials. While LBH589 shows favorable activity against various hematologic malignancies (25,26), it became apparent that HDACi produce best results when they are applied in combination with other drugs (20). Bali and colleagues showed a superior activity of the HDACi LAQ824/ PKC412 combinations in FLT3-ITD-positive cells and found that STAT5 DNA-binding activity is decreased by this treatment (18).…”

“…In a previous study, we showed that LBH589 upregulates the ubiquitin conjugase UBCH8 mediating poly-ubiquitinylation of FLT3-ITD. Consequently, FLT3-ITD undergoes proteasomal degradation in AML cells (20). FLT3-ITD degradation was also reported to occur via HDACi-induced inhibition of the chaperone HSP90 (18,27).…”

Proteasome inhibitors induce FLT3-ITD degradation through autophagy in AML cells

“…Indeed, histone deacetylase inhibitors has been shown recently to induce proteasomal degradation of FLT3-ITD catalyzed by the E2 ubiquitin conjugase UBCH8 and the E3 ubiquitin ligase SIAH1 in MV4-11 cells. 37 As UPS and autophagy are functionally interrelated catabolic processes sharing certain substrates, enhanced degradation by autophagy may have become critical for FLT3-ITD expression under conditions in which UPS was compromised by proteasome inhibitors. 38,39 Further studies are needed to clarify which molecular players are involved in this proteasome-to-autophagy switch in the context of FLT3-ITD AML cells.…”