c-Cbl and Cbl-b Ligases Mediate 17-Allylaminodemethoxygeldanamycin-induced Degradation of Autophosphorylated Flt3 Kinase with Internal Tandem Duplication through the Ubiquitin Proteasome Pathway

Abstract

Fms-like tyrosine kinase 3 (Flt3) 2 also known as fetal liver kinase-2 (Flk-2) is a receptor-tyrosine kinase expressed on hematopoietic progenitors and regulates early steps of hematopoietic progenitor cell proliferation, survival, and differentiation (1, 2). Wild-type Flt3 (Flt3-WT) has been shown to be highly expressed in several hematopoietic malignancies including 70 -100% cases of acute myeloid leukemia (AML). Oncogenic internal tandem duplication (ITD) mutations in the juxtamembrane domain of Flt3 (Flt3-ITD) and point mutations within the tyrosine kinase domain, such as the most predominant D835Y mutation, are the most frequent kinase mutations in AML, occurring in 25-30 and 5-10% of cases, respectively, and are associated with poor prognosis (1-3). Flt3-ITD results in ligand-independent autophosphorylation and activation of the receptor with subsequent activation of multiple downstream targets, including the signal transducer and activator of transcription 5 (STAT5), mitogen-activated protein kinase, and Akt pathways. Flt3-ITD has been shown to induce a myeloproliferative disorder in murine models. Flt3-ITD exists partially in an immature, underglycosylated, constitutively phosphorylated form (4). Aberrant intracellular localization and activity of Flt3-ITD generate oncogenic phosphorylation patterns and aberrantly activate signaling cascades (5). Because Flt3 or Flt3-ligand (FL) knock-out mice have only a subtle hematopoietic stem/progenitor cell deficit and show no significant disadvantage in viability, interference with deregulated Flt3 functions appears as a promising treatment option for AML (1-3). Thus, several Flt3 inhibitors are currently under evaluation for their efficacy in AML patients with Flt3-ITD.Recent studies showed that both activated Flt3 and mutant Flt3 are degraded through the proteasome (6 -11). The proteasomal degradation of targeted proteins, including protein kinases, requires polyubiquitination catalyzed by a series of enzymes containing ubiquitin activating enzymes (E1), ubiquitin conjugases (E2), and ubiquitin ligases (E3) (12, 13). E3 ubiquitin ligases confer substrate specificity and are responsible for mediating the transfer of ubiquitin from E2s to the substrates. The Cbl proteins are a highly conserved family of RING finger E3 ubiquitin ligases that regulate signaling by receptor and non-receptor-tyrosine kinases, including EGF receptor, PDGF receptor ␣, and FMS (14). There are three mammalian Cbl proteins encoded by separate genes: c-Cbl, Cbl-b, and Cbl-c. These proteins contain an N-terminal phosphotyrosine binding domain that allows direct interaction with activated receptortyrosine kinases and a RING finger domain that classifies Cbl

Adaptation to TKI Treatment Reactivates ERK Signaling in Tyrosine Kinase–Driven Leukemias and Other Malignancies

“…A minimal rebound in AKT phosphorylation was observed in the Molm14 cell line starting 8 hours after treatment, but was reduced by 24 hours. An increase in total FLT3 protein and, in some cases, FLT3 phosphorylation was also observed, consistent with previous reports which demonstrate that FLT3 inhibition significantly decreases the rate of its proteasomal degradation (25). …”

E3 ubiquitin ligase Cbl-b activates the p53 pathway by targeting Siva1, a negative regulator of ARF, in FLT3 inhibitor-resistant acute myeloid leukemia

“…9 In fact, mice with a loss-of-function mutation of Cbl-b (Cbl-b-C373A) have been shown to exhibit augmented FLT3 signaling and develop a myeloproliferative disease that progresses to leukemia. 10 Cbl-b was expressed in MOLM13-R-PKC412 (which was sensitive to Nutlin-3) but not in MV4-11R-PKC412 (which was resistant to Nutlin-3) (Figure 2a). Furthermore, Cbl-b expression was up-regulated in FLT3 inhibitor-resistant MOLM13-R-PKC412 when compared with its parental, FLT3 inhibitor-sensitive MOLM13 (Supplementary Figure S1).…”

Down regulation of Chk1 by p53 plays a role in synergistic induction of apoptosis by chemotherapeutics and inhibitors for Jak2 or BCR/ABL in hematopoietic cells

“…Ton.B210 cells were cultured in 10% fetal calf serum (FCS)-containing RPMI 1640 medium supplemented either with 5 U/ml murine IL-3 or with 1 μg/ml doxycycline, which induces the expression of BCR/ABL. Ton.B210/T315I cells, which inducibly express BCR/ABL harboring the T315I mutation, murine IL-3-dependent 32Dcl3 cells, Ton.32D cells, and Ton.32D210 cells expressing BCR/ABL were described previously [21, 38, 39]. …”

“…Transfection of the retroviral vector pRevTRE-p53-DD into PLAT-A cells and infection of Ton.32D or Ton.B210 cells was performed as described previously [39]. After selection with hygromycin, infected cells were used as Ton.32D/TRE-p53-DD or Ton.B210/TRE-p53-DD cells.…”

“…After selection with hygromycin, infected cells were used as Ton.32D/TRE-p53-DD or Ton.B210/TRE-p53-DD cells. Transfection of pTRE-Tight-p53-DD into Ton.32D210 cells by electroporation with pMAM2-BSD (Funakoshi, Tokyo, Japan), followed by selection and limiting dilution, was performed as described previously [39]. Selected clones were found to express p53-DD with or without doxycycline by Western blot analysis.…”