Contributions
Abstract: EP404
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Children with Down syndrome are predisposed to develop acute megakaryoblastic leukemia (ML-DS). Thereby, ML-DS leukemogenesis is a stepwise process evolving from pre-leukemic transient abnormal myelopoiesis (TAM). Previously, we showed that the transition from TAM to ML-DS is characterized by the activation of target genes of the histone demethylase LSD1 and acquisition of mutations in signaling pathway genes, e.g. JAK-STAT signaling. As children with Down syndrome often suffer from severe toxicities upon intensive chemotherapy, targeted approaches for the treatment of ML-DS are desirable.
Aims
Here we set out to investigate pharmacological LSD1 inhibition (LSD1i) – also in combination with the JAK1/2 inhibitor Ruxolitinib – as therapeutic strategy in ML-DS.
Methods
In vitro drug efficacy screens for LSD1 inhibitors as monotherapy or in combination with Ruxolitinib were performed in pediatric AML cell lines as well as different patient-derived xenografts (PDX) calculating EC50 and Bliss synergy score. This drug combination of LSD1i and Ruxolitinib was further investigated in vivo in a PDX-based ML-DS mouse model. To identify molecular mechanisms of drug synergy between LSD1i and inhibition of JAK-STAT signaling we performed RNAseq in two different PDX.
Results
In line with the activation of genes positively regulated by LSD1, we observed particularly high LSD1 protein expression in ML-DS primary samples. LSD1i in the ML-DS cell line CMK as well as in five different PDX induced myeloid differentiation displayed by increased expression of CD86 and CD11b. However, LSD1i only led to proliferation arrest without increasing the rate of apoptotic cells. Thus, we tried various drugs in combination with LSD1i (e.g. Azacytidine, Venetoclax) to enhance the antileukemic effect, and identified the JAK1/2 inhibitor Ruxolitinib as the most effective. The combination of LSD1i and Ruxolitinib caused a synergistic as opposed to a merely additive antileukemic effect in ML-DS samples measured by the Bliss synergy score. Consequently, we tested LSD1i and Ruxolitinib in a PDX-based murine ML-DS model, where the combination led to a significant reduction in blast count compared to monotherapies and control group. Concerning molecular mechanisms of drug synergy, RNAseq data revealed that LSD1i and Ruxolitinib synergistically inhibited pathways involved in cell division, such as DNA replication or transition from G1 to S phase during cell cycle. On the contrary, LSD1i induced interleukin signaling in mono- and combination therapy, which was repressed by Ruxolitinib. Western blot analysis confirmed increased STAT phosphorylation upon LSD1i that was abrogated in combination with Ruxolitinib. Hence, our data point towards increased interleukin signaling after LSD1i as potential mechanism to sensitize cells to additional Ruxolitinib treatment.
Conclusion
We identified LSD1i in combination with disruption of JAK-STAT signaling by Ruxolitinib as potential new treatment strategy in ML-DS with efficacy in vitro and in vivo. Thereby, both drugs synergized in inducing proliferation arrest and differentiation. The regulation of interleukin signaling by LSD1i might be a potential mechanism of sensitizing ML‑DS blasts to Ruxolitinib.
Keyword(s): Acute myeloid leukemia, AML, Down Syndrome, Drug interaction
Abstract: EP404
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Children with Down syndrome are predisposed to develop acute megakaryoblastic leukemia (ML-DS). Thereby, ML-DS leukemogenesis is a stepwise process evolving from pre-leukemic transient abnormal myelopoiesis (TAM). Previously, we showed that the transition from TAM to ML-DS is characterized by the activation of target genes of the histone demethylase LSD1 and acquisition of mutations in signaling pathway genes, e.g. JAK-STAT signaling. As children with Down syndrome often suffer from severe toxicities upon intensive chemotherapy, targeted approaches for the treatment of ML-DS are desirable.
Aims
Here we set out to investigate pharmacological LSD1 inhibition (LSD1i) – also in combination with the JAK1/2 inhibitor Ruxolitinib – as therapeutic strategy in ML-DS.
Methods
In vitro drug efficacy screens for LSD1 inhibitors as monotherapy or in combination with Ruxolitinib were performed in pediatric AML cell lines as well as different patient-derived xenografts (PDX) calculating EC50 and Bliss synergy score. This drug combination of LSD1i and Ruxolitinib was further investigated in vivo in a PDX-based ML-DS mouse model. To identify molecular mechanisms of drug synergy between LSD1i and inhibition of JAK-STAT signaling we performed RNAseq in two different PDX.
Results
In line with the activation of genes positively regulated by LSD1, we observed particularly high LSD1 protein expression in ML-DS primary samples. LSD1i in the ML-DS cell line CMK as well as in five different PDX induced myeloid differentiation displayed by increased expression of CD86 and CD11b. However, LSD1i only led to proliferation arrest without increasing the rate of apoptotic cells. Thus, we tried various drugs in combination with LSD1i (e.g. Azacytidine, Venetoclax) to enhance the antileukemic effect, and identified the JAK1/2 inhibitor Ruxolitinib as the most effective. The combination of LSD1i and Ruxolitinib caused a synergistic as opposed to a merely additive antileukemic effect in ML-DS samples measured by the Bliss synergy score. Consequently, we tested LSD1i and Ruxolitinib in a PDX-based murine ML-DS model, where the combination led to a significant reduction in blast count compared to monotherapies and control group. Concerning molecular mechanisms of drug synergy, RNAseq data revealed that LSD1i and Ruxolitinib synergistically inhibited pathways involved in cell division, such as DNA replication or transition from G1 to S phase during cell cycle. On the contrary, LSD1i induced interleukin signaling in mono- and combination therapy, which was repressed by Ruxolitinib. Western blot analysis confirmed increased STAT phosphorylation upon LSD1i that was abrogated in combination with Ruxolitinib. Hence, our data point towards increased interleukin signaling after LSD1i as potential mechanism to sensitize cells to additional Ruxolitinib treatment.
Conclusion
We identified LSD1i in combination with disruption of JAK-STAT signaling by Ruxolitinib as potential new treatment strategy in ML-DS with efficacy in vitro and in vivo. Thereby, both drugs synergized in inducing proliferation arrest and differentiation. The regulation of interleukin signaling by LSD1i might be a potential mechanism of sensitizing ML‑DS blasts to Ruxolitinib.
Keyword(s): Acute myeloid leukemia, AML, Down Syndrome, Drug interaction