Contributions
Abstract: EP419
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
The pivotal role of hypoxic bone marrow microenvironment (BMM) has been demonstrated in acute myeloid leukemia (AML) development and maintenance, however whether it influences the fitness of leukemic stem cells (LSCs) and their susceptibility to drug treatment still remains to be clarified. Increasing evidences suggest that hypoxic BMM represents a key regulator of transcriptomic and metabolomic profiles of LSCs, influencing their proliferation capacity and leading resistance to chemotherapeutic treatments. Aurora kinases A and B are crucial actors during mitosis and their over-expression has been described in AML, making them a promising therapeutic target. Phase II studies of Aurora kinase inhibitors combined with induction chemotherapy have demonstrated good tolerability and efficacy in patients with high-risk AML (Brunner, Lancet Haematol 2020).
Aims
The present study aim to explore the effects of Aurora kinases inhibitor Tozasertib in AML cell lines cultured either under normoxic or hypoxic conditions.
Methods
AML cell lines (NOMO-1: t(9;11); MOLM-13: FLT3-ITD; MV-4-11: t(4;11), +8, +19; KASUMI-1 t(8;21), c-KIT and p53 mutated) were treated under either normoxia or hypoxia (1%O2) with the pan Aurora inhibitor Tozasertib (100 and 2500 nM). After 24 and 48h of treatment, Annexin V and PI staining were performed to evaluate the induction of apoptosis and proliferation capacity. Protein level alterations were analyzed by Western blotting (WB).
Results
Tozasertib strongly inhibited growth and proliferation in AML models, but it was less effective in a low oxygen environment. In particular, all tested AML cell lines showed increased cell death in a dose-dependent manner, however under hypoxic conditions the apoptotic cell death was significantly reduced compared to the normoxic counterpart (AnnexinV+ cells were 32-60% under hypoxia vs 47-70% under normoxia after 48h treatment; p<0.05). Of note, inhibition of Aurora kinases induced mitotic arrest; therefore, we evaluated the effect of tozasertib on cellular proliferation demonstrating a reduced ability of the drug to induce the accumulation of cells in G2/M phase under hypoxic culture conditions. The reduced anti-leukemic activity of tozasertib in hypoxic AML cell lines was confirmed also at the protein level. WB analysis demonstrated that inhibition of Aurora kinases was associated with activation of the TP53 pathway, inducing the expression of p-p53(ser15), p21 and γH2AX as well as the cleavage of CASPASE 3 and PARP in all tested cell lines. However, the upregulation of the above proteins was less pronounced under hypoxic conditions. Interestingly, the drug treatment correlated, in an O2 independent manner, with a strong increase of trimethylation levels at lysine 36 of histone H3 (H3K36me3), that is involved in regulation of the DNA damage response (DDR) network.
Conclusion
Cytostatic and cytotoxic effects after treatment with Tozasertib were observed in MLL-AF9 translocated, FLT3-ITD, and both wt and mutant TP53 AML models, which represent an important goal in AML therapy. Nevertheless, the hypoxic microenvironment strongly influenced the efficacy of Aurora kinase inhibition in AML cell lines, highlighting the crucial role of environment in AML treatment. In conclusion, our study confirmed the pivotal role of low oxygen environment in regulating cellular response to Aurora kinase inhibition and sets the rationale for further investigations aimed to identify new combination therapies against AML LSCs.
This study was supported in part by AILBologna.
Keyword(s): Acute myeloid leukemia, Hypoxia-sensing, Kinase inhibitor, Microenvironment
Abstract: EP419
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
The pivotal role of hypoxic bone marrow microenvironment (BMM) has been demonstrated in acute myeloid leukemia (AML) development and maintenance, however whether it influences the fitness of leukemic stem cells (LSCs) and their susceptibility to drug treatment still remains to be clarified. Increasing evidences suggest that hypoxic BMM represents a key regulator of transcriptomic and metabolomic profiles of LSCs, influencing their proliferation capacity and leading resistance to chemotherapeutic treatments. Aurora kinases A and B are crucial actors during mitosis and their over-expression has been described in AML, making them a promising therapeutic target. Phase II studies of Aurora kinase inhibitors combined with induction chemotherapy have demonstrated good tolerability and efficacy in patients with high-risk AML (Brunner, Lancet Haematol 2020).
Aims
The present study aim to explore the effects of Aurora kinases inhibitor Tozasertib in AML cell lines cultured either under normoxic or hypoxic conditions.
Methods
AML cell lines (NOMO-1: t(9;11); MOLM-13: FLT3-ITD; MV-4-11: t(4;11), +8, +19; KASUMI-1 t(8;21), c-KIT and p53 mutated) were treated under either normoxia or hypoxia (1%O2) with the pan Aurora inhibitor Tozasertib (100 and 2500 nM). After 24 and 48h of treatment, Annexin V and PI staining were performed to evaluate the induction of apoptosis and proliferation capacity. Protein level alterations were analyzed by Western blotting (WB).
Results
Tozasertib strongly inhibited growth and proliferation in AML models, but it was less effective in a low oxygen environment. In particular, all tested AML cell lines showed increased cell death in a dose-dependent manner, however under hypoxic conditions the apoptotic cell death was significantly reduced compared to the normoxic counterpart (AnnexinV+ cells were 32-60% under hypoxia vs 47-70% under normoxia after 48h treatment; p<0.05). Of note, inhibition of Aurora kinases induced mitotic arrest; therefore, we evaluated the effect of tozasertib on cellular proliferation demonstrating a reduced ability of the drug to induce the accumulation of cells in G2/M phase under hypoxic culture conditions. The reduced anti-leukemic activity of tozasertib in hypoxic AML cell lines was confirmed also at the protein level. WB analysis demonstrated that inhibition of Aurora kinases was associated with activation of the TP53 pathway, inducing the expression of p-p53(ser15), p21 and γH2AX as well as the cleavage of CASPASE 3 and PARP in all tested cell lines. However, the upregulation of the above proteins was less pronounced under hypoxic conditions. Interestingly, the drug treatment correlated, in an O2 independent manner, with a strong increase of trimethylation levels at lysine 36 of histone H3 (H3K36me3), that is involved in regulation of the DNA damage response (DDR) network.
Conclusion
Cytostatic and cytotoxic effects after treatment with Tozasertib were observed in MLL-AF9 translocated, FLT3-ITD, and both wt and mutant TP53 AML models, which represent an important goal in AML therapy. Nevertheless, the hypoxic microenvironment strongly influenced the efficacy of Aurora kinase inhibition in AML cell lines, highlighting the crucial role of environment in AML treatment. In conclusion, our study confirmed the pivotal role of low oxygen environment in regulating cellular response to Aurora kinase inhibition and sets the rationale for further investigations aimed to identify new combination therapies against AML LSCs.
This study was supported in part by AILBologna.
Keyword(s): Acute myeloid leukemia, Hypoxia-sensing, Kinase inhibitor, Microenvironment