Contributions
Abstract: PB1676
Type: Publication Only
Background
FLT3-ITDs (internal tandem duplication) represent the most frequent molecular aberration in acute myeloid leukemia (AML) associated with an inferior prognosis. The pattern of downstream activation by this constitutively activated receptor tyrosine kinase is influenced by the localization of mutated FLT3 depending on its glycosylation status. Different pharmacological approaches can affect FLT3-driven oncogenic pathways by distinct compartmentalization of FLT3-ITD.
Aims
The objective of this study was to investigate the effects of either valproic acid (VPA) or tunicamycin on the localization of FLT3-ITD. We sought to investigate different susceptibility of mutated FLT3 towards combination treatment - e.g. with inhibitors of heat shock proteins (HSP) – dependent on both the localization of activated FLT3 and the subtype of FLT3-ITD variants.
Methods
Murine Ba/F3 leukemia cell lines were stably transfected with distinct FLT3-ITD variants resulting in IL-3-independent growth (Arreba-Tutusaus et al. Leukemia 2016). Signal transduction after exposing cells to VPA, tunicamycin and/ or the HSP inhibitor 17-AAG was characterized by Western blotting, MTS assay and flow cytometry analysis of apoptosis, cell cycle and surface expression of FLT3.
Results
Treatment of Ba/F3-FLT3-ITD cells with VPA is associated with a significant increase of FLT3-ITD surface expression that depends on FLT3 protein synthesis. In contrast, incubation with tunicamycin leads to intracellular retention of FLT3-ITD by inhibition of its glycosylation as an important mechanism of post-translational modification. Of note, allocation of FLT3 to different cellular compartments by VPA or tunicamycin is accompanied by distinct activation of oncogenic signaling pathways. In detail, VPA is associated with an increase of AKT and ERK phosphorylation while enhanced STAT5 activation can be observed following treatment with tunicamycin. Importantly, sequential combination of either VPA or tunicamycin with the HSP inhibitor 17-AAG demonstrates additive effects that are restricted to the treatment with tunicamycin and 17-AAG.
Conclusion
We can demonstrate the impact of VPA on cell surface stabilization of FLT3-ITD that results from FLT3 biosynthesis and is associated with an enhanced activation of the AKT and ERK pathway. Combination treatment suggests that the unglycosylated form of FLT3 is more susceptible to HSP inhibitor treatment. Thus, allocation of FLT3-ITD to different cellular compartments might represent a promising therapeutic strategy to overcome tyrosine kinase inhibitor resistance in FLT3-ITD-positive AML.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, Flt3-ITD, Signal transduction
Abstract: PB1676
Type: Publication Only
Background
FLT3-ITDs (internal tandem duplication) represent the most frequent molecular aberration in acute myeloid leukemia (AML) associated with an inferior prognosis. The pattern of downstream activation by this constitutively activated receptor tyrosine kinase is influenced by the localization of mutated FLT3 depending on its glycosylation status. Different pharmacological approaches can affect FLT3-driven oncogenic pathways by distinct compartmentalization of FLT3-ITD.
Aims
The objective of this study was to investigate the effects of either valproic acid (VPA) or tunicamycin on the localization of FLT3-ITD. We sought to investigate different susceptibility of mutated FLT3 towards combination treatment - e.g. with inhibitors of heat shock proteins (HSP) – dependent on both the localization of activated FLT3 and the subtype of FLT3-ITD variants.
Methods
Murine Ba/F3 leukemia cell lines were stably transfected with distinct FLT3-ITD variants resulting in IL-3-independent growth (Arreba-Tutusaus et al. Leukemia 2016). Signal transduction after exposing cells to VPA, tunicamycin and/ or the HSP inhibitor 17-AAG was characterized by Western blotting, MTS assay and flow cytometry analysis of apoptosis, cell cycle and surface expression of FLT3.
Results
Treatment of Ba/F3-FLT3-ITD cells with VPA is associated with a significant increase of FLT3-ITD surface expression that depends on FLT3 protein synthesis. In contrast, incubation with tunicamycin leads to intracellular retention of FLT3-ITD by inhibition of its glycosylation as an important mechanism of post-translational modification. Of note, allocation of FLT3 to different cellular compartments by VPA or tunicamycin is accompanied by distinct activation of oncogenic signaling pathways. In detail, VPA is associated with an increase of AKT and ERK phosphorylation while enhanced STAT5 activation can be observed following treatment with tunicamycin. Importantly, sequential combination of either VPA or tunicamycin with the HSP inhibitor 17-AAG demonstrates additive effects that are restricted to the treatment with tunicamycin and 17-AAG.
Conclusion
We can demonstrate the impact of VPA on cell surface stabilization of FLT3-ITD that results from FLT3 biosynthesis and is associated with an enhanced activation of the AKT and ERK pathway. Combination treatment suggests that the unglycosylated form of FLT3 is more susceptible to HSP inhibitor treatment. Thus, allocation of FLT3-ITD to different cellular compartments might represent a promising therapeutic strategy to overcome tyrosine kinase inhibitor resistance in FLT3-ITD-positive AML.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, Flt3-ITD, Signal transduction