Contributions
Abstract: EP387
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
The unfolded protein response (UPR) is a stress sensing signaling network that allows cells to deal with endoplasmic reticulum (ER) stress, a condition characterized by an accumulation of mis- and unfolded proteins in the ER. Acute myeloid leukemia (AML) stem cells reside in the bone marrow niche, where they face stressful conditions, such as hypoxia, low pH and nutrient fluctuations. Together with their oncogene-driven metabolism, they are prone to develop ER stress. Elevated UPR gene expression levels, particularly of IRE1α and XBP1, were found in FLT3-ITD+ AML patient cells. Patients with high XBP1 mRNA expression showed an inferior overall survival compared to those with low XBP1 mRNA expression.
Aims
In this study, we aimed to uncover the potential of pharmacological IRE1α-XBP1 inhibition as a new therapeutic strategy in FLT3-ITD+ AML in combination with a 2nd generation FLT3 tyrosine kinase inhibitor (TKI; quizartinib, AC220). We further wanted to elucidate whether this approach is efficient under hypoxia, the physiological condition in the bone marrow niche that potentially counteracts the TKI response.
Methods
Human FLT3-ITD+ MV4-11, FLT3 wildtype (WT) RS4-11 and murine 32D cells transduced with FLT3-ITD or FLT3 WT were analyzed via Western blot and qRT-PCR. Cell viability was assessed by MTT assay, cell death and apoptosis were measured with propidium iodide (PI) or AnnexinV staining using FACS analysis. The clonogenic potential was determined in CFU assays, using primary patient-derived cells. Hypoxic cell culture conditions were applied (2-3% O2). Analysis of phosphoproteomics was performed by mass spectrometry.
Results
Pharmacological inhibition of the IRE1α RNase activity using STF-083010 blocked XBP1 splicing and reduced the cell viability of human AML cell lines in a dose dependent way. Interestingly, FLT3 inhibition strongly enhanced the IRE1α mRNA level in 32D FLT3-ITD cells (9.3-fold p<0.05) but only weak in 32D FLT3 WT cells (1.5-fold p<0.01). 32D FLT3-ITD also showed a 3-fold higher number of PI+ cells after IRE1α inhibition compared to 32D FLT3 WT cells (p<0.01). While single treatment of human FLT3-ITD+ MV4-11 cells with either the IRE1α inhibitor or AC220 significantly induced apoptosis, the combinatorial treatment strongly enhanced this effect (by 3-fold p<0.001). This potent induction of cell death was only observed in FLT3-ITD+ MV4-11 cells, but not in FLT3 WT expressing RS4-11 cells. Likewise, the combination significantly decreased the clonogenic potential of FLT3-ITD+ AML cell lines and AML mononuclear patient cells, but not of healthy donor cells. Cultivation of MV4-11 cells under hypoxia further induced IRE1α signaling and revealed that the effect of AC220 was largely abolished under hypoxia (normoxia: 58.4-fold induction of dead cells p<0.01 hypoxia: 2.2-fold induction p>0.05). Western blot and phosphoproteomic analysis revealed that FLT3 signaling was reduced under hypoxia. Intriguingly, combined targeting of IRE1α and FLT3 overcame this hypoxia-induced AC220 resistance and strongly induced cell death.
Conclusion
IRE1α-XBP1 signaling is activated in FLT3-ITD+ AML and further enhanced by hypoxic conditions in the bone marrow niche. Targeting IRE1α in FLT3-ITD+ cells is effective to decrease proliferation, clonogenic potential and induces apoptosis. Our data demonstrate that hypoxia-conferred resistance against AC220 can be overcome by simultaneous IRE1α inhibition, making this pathway an interesting novel therapeutic target.
Keyword(s): Acute myeloid leukemia, Flt3-ITD
Abstract: EP387
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
The unfolded protein response (UPR) is a stress sensing signaling network that allows cells to deal with endoplasmic reticulum (ER) stress, a condition characterized by an accumulation of mis- and unfolded proteins in the ER. Acute myeloid leukemia (AML) stem cells reside in the bone marrow niche, where they face stressful conditions, such as hypoxia, low pH and nutrient fluctuations. Together with their oncogene-driven metabolism, they are prone to develop ER stress. Elevated UPR gene expression levels, particularly of IRE1α and XBP1, were found in FLT3-ITD+ AML patient cells. Patients with high XBP1 mRNA expression showed an inferior overall survival compared to those with low XBP1 mRNA expression.
Aims
In this study, we aimed to uncover the potential of pharmacological IRE1α-XBP1 inhibition as a new therapeutic strategy in FLT3-ITD+ AML in combination with a 2nd generation FLT3 tyrosine kinase inhibitor (TKI; quizartinib, AC220). We further wanted to elucidate whether this approach is efficient under hypoxia, the physiological condition in the bone marrow niche that potentially counteracts the TKI response.
Methods
Human FLT3-ITD+ MV4-11, FLT3 wildtype (WT) RS4-11 and murine 32D cells transduced with FLT3-ITD or FLT3 WT were analyzed via Western blot and qRT-PCR. Cell viability was assessed by MTT assay, cell death and apoptosis were measured with propidium iodide (PI) or AnnexinV staining using FACS analysis. The clonogenic potential was determined in CFU assays, using primary patient-derived cells. Hypoxic cell culture conditions were applied (2-3% O2). Analysis of phosphoproteomics was performed by mass spectrometry.
Results
Pharmacological inhibition of the IRE1α RNase activity using STF-083010 blocked XBP1 splicing and reduced the cell viability of human AML cell lines in a dose dependent way. Interestingly, FLT3 inhibition strongly enhanced the IRE1α mRNA level in 32D FLT3-ITD cells (9.3-fold p<0.05) but only weak in 32D FLT3 WT cells (1.5-fold p<0.01). 32D FLT3-ITD also showed a 3-fold higher number of PI+ cells after IRE1α inhibition compared to 32D FLT3 WT cells (p<0.01). While single treatment of human FLT3-ITD+ MV4-11 cells with either the IRE1α inhibitor or AC220 significantly induced apoptosis, the combinatorial treatment strongly enhanced this effect (by 3-fold p<0.001). This potent induction of cell death was only observed in FLT3-ITD+ MV4-11 cells, but not in FLT3 WT expressing RS4-11 cells. Likewise, the combination significantly decreased the clonogenic potential of FLT3-ITD+ AML cell lines and AML mononuclear patient cells, but not of healthy donor cells. Cultivation of MV4-11 cells under hypoxia further induced IRE1α signaling and revealed that the effect of AC220 was largely abolished under hypoxia (normoxia: 58.4-fold induction of dead cells p<0.01 hypoxia: 2.2-fold induction p>0.05). Western blot and phosphoproteomic analysis revealed that FLT3 signaling was reduced under hypoxia. Intriguingly, combined targeting of IRE1α and FLT3 overcame this hypoxia-induced AC220 resistance and strongly induced cell death.
Conclusion
IRE1α-XBP1 signaling is activated in FLT3-ITD+ AML and further enhanced by hypoxic conditions in the bone marrow niche. Targeting IRE1α in FLT3-ITD+ cells is effective to decrease proliferation, clonogenic potential and induces apoptosis. Our data demonstrate that hypoxia-conferred resistance against AC220 can be overcome by simultaneous IRE1α inhibition, making this pathway an interesting novel therapeutic target.
Keyword(s): Acute myeloid leukemia, Flt3-ITD