TARGETING CHROMATIN REGULATORS INHIBITS LEUKEMOGENIC GENE EXPRESSION IN NPM1 MUTANT LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. Kühn M. 06/12/16; 135297; S803
Dr. Michael Kühn
Contributions
Contributions
Abstract
Abstract: S803
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:00 - 08:15
Location: Hall A3
Background
Homeobox (HOX) proteins and the receptor tyrosine kinase FLT3 are frequently highly expressed and mutated in acute myeloid leukemia (AML). Aberrant HOX expression is found in nearly all AMLs that harbor a mutation in the NPM1 gene (NPM1mut), and FLT3 is concomitantly mutated in ~50% of these cases. Little is known how these cells maintain aberrant gene expression. Since specific chromatin regulatory complexes control HOX expression in normal hematopoiesis and in leukemias expressing an oncogenic MLL-fusion protein, we hypothesized that they might also regulate HOX expression in other settings, and that inhibition of HOX and FLT3 expression would produce an antiproliferative effect in NPM1mut AML.
Aims
To investigate dependencies of NPM1mut AML on MLL and DOT1L complexes and to delineate their potential role in control of leukemogenic gene expression.
Methods
CRISPR-Cas9 genome editing across exons encoding specific protein domains was used in a negative selection screen to assess potential dependencies of NPM1mut AML on specific MLL protein domains, and this concept was extended with inhibitors of the menin-MLL interaction (MI2-2 and MI503). Dependencies on DOT1L were explored using the inhibitor EPZ4777. In vitro and in vivo target validation was performed in two conditional murine knock-in models (Npm1CA/+FLt3ITD/+; Npm1CA/+RosaSB/+) and in a disseminated human xenograft model of Npm1mut AML.
Results
Using a CRISPR-Cas9 negative selection screen, we discovered the menin binding site of MLL as a dependency in NPM1mut OCI-AML3 cells, a region critically involved in chromatin binding of the MLL complex. We next sought to assess treatment effects of MI2-2 and MI503 and observed dramatic suppression of HOX and MEIS1 expression, differentiation induction, and profound growth inhibition in human OCI-AML3 cells and murine Npm1mut AMLs. Of interest, ectopic expression of Meis1, Hoxb4, or Hoxa9-Meis1 rescued the antiproliferative effects of menin-MLL inhibition. In vivo MI503 treatment of leukemic OCI-AML3 xenografts resulted in profound HOX and MEIS1 suppression, significant reduction of leukemia burden, and in a significant survival advantage of both, human xenografts and secondarily transplanted Npm1CA/+RosaSB/+ mice compared to vehicle controls. As MEIS1 was most dramatically suppressed across all three models, we determined drug effects on FLT3 expression, a reported target gene of MEIS1 and found consistent suppression of FLT3 to almost undetectable levels.MI503 treatment depleted menin and H3K4me3 on HOX and MEIS1 loci. We also observed a significant decrease of H3K79me2 at those loci, pointing to a potential role for DOT1L, the sole H3K79 methyltransferase. Treatment with EPZ4777 resulted in HOX, MEIS1, and FLT3 down regulation, cell growth inhibition, and profound differentiation of NPM1mut AML blasts just as we found with inhibition of the menin-MLL interaction.Next, we investigated effects of combinatorial menin-MLL and DOT1L inhibition and found superior suppression of HOX and FLT3 expression, dramatically more pronounced differentiation, and synergistic growth inhibition. Combinatorial drug treatment further reduced leukemia initiating potential of NPM1mut AML cells dramatically compared to single drug treatment in vivo.
Conclusion
MLL and DOT1L are chromatin regulators that control HOX, MEIS1 and FLT3 expression in NPM1mut AML. Combinatorial small-molecule inhibition has synergistic on target activity and represents a potential therapeutic opportunity that should soon be ready for clinical assessment.
Session topic: AML Biology - Novel targeted therapies
Keyword(s): Acute myeloid leukemia, Epigenetic, MLL
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:00 - 08:15
Location: Hall A3
Background
Homeobox (HOX) proteins and the receptor tyrosine kinase FLT3 are frequently highly expressed and mutated in acute myeloid leukemia (AML). Aberrant HOX expression is found in nearly all AMLs that harbor a mutation in the NPM1 gene (NPM1mut), and FLT3 is concomitantly mutated in ~50% of these cases. Little is known how these cells maintain aberrant gene expression. Since specific chromatin regulatory complexes control HOX expression in normal hematopoiesis and in leukemias expressing an oncogenic MLL-fusion protein, we hypothesized that they might also regulate HOX expression in other settings, and that inhibition of HOX and FLT3 expression would produce an antiproliferative effect in NPM1mut AML.
Aims
To investigate dependencies of NPM1mut AML on MLL and DOT1L complexes and to delineate their potential role in control of leukemogenic gene expression.
Methods
CRISPR-Cas9 genome editing across exons encoding specific protein domains was used in a negative selection screen to assess potential dependencies of NPM1mut AML on specific MLL protein domains, and this concept was extended with inhibitors of the menin-MLL interaction (MI2-2 and MI503). Dependencies on DOT1L were explored using the inhibitor EPZ4777. In vitro and in vivo target validation was performed in two conditional murine knock-in models (Npm1CA/+FLt3ITD/+; Npm1CA/+RosaSB/+) and in a disseminated human xenograft model of Npm1mut AML.
Results
Using a CRISPR-Cas9 negative selection screen, we discovered the menin binding site of MLL as a dependency in NPM1mut OCI-AML3 cells, a region critically involved in chromatin binding of the MLL complex. We next sought to assess treatment effects of MI2-2 and MI503 and observed dramatic suppression of HOX and MEIS1 expression, differentiation induction, and profound growth inhibition in human OCI-AML3 cells and murine Npm1mut AMLs. Of interest, ectopic expression of Meis1, Hoxb4, or Hoxa9-Meis1 rescued the antiproliferative effects of menin-MLL inhibition. In vivo MI503 treatment of leukemic OCI-AML3 xenografts resulted in profound HOX and MEIS1 suppression, significant reduction of leukemia burden, and in a significant survival advantage of both, human xenografts and secondarily transplanted Npm1CA/+RosaSB/+ mice compared to vehicle controls. As MEIS1 was most dramatically suppressed across all three models, we determined drug effects on FLT3 expression, a reported target gene of MEIS1 and found consistent suppression of FLT3 to almost undetectable levels.MI503 treatment depleted menin and H3K4me3 on HOX and MEIS1 loci. We also observed a significant decrease of H3K79me2 at those loci, pointing to a potential role for DOT1L, the sole H3K79 methyltransferase. Treatment with EPZ4777 resulted in HOX, MEIS1, and FLT3 down regulation, cell growth inhibition, and profound differentiation of NPM1mut AML blasts just as we found with inhibition of the menin-MLL interaction.Next, we investigated effects of combinatorial menin-MLL and DOT1L inhibition and found superior suppression of HOX and FLT3 expression, dramatically more pronounced differentiation, and synergistic growth inhibition. Combinatorial drug treatment further reduced leukemia initiating potential of NPM1mut AML cells dramatically compared to single drug treatment in vivo.
Conclusion
MLL and DOT1L are chromatin regulators that control HOX, MEIS1 and FLT3 expression in NPM1mut AML. Combinatorial small-molecule inhibition has synergistic on target activity and represents a potential therapeutic opportunity that should soon be ready for clinical assessment.
Session topic: AML Biology - Novel targeted therapies
Keyword(s): Acute myeloid leukemia, Epigenetic, MLL
Abstract: S803
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:00 - 08:15
Location: Hall A3
Background
Homeobox (HOX) proteins and the receptor tyrosine kinase FLT3 are frequently highly expressed and mutated in acute myeloid leukemia (AML). Aberrant HOX expression is found in nearly all AMLs that harbor a mutation in the NPM1 gene (NPM1mut), and FLT3 is concomitantly mutated in ~50% of these cases. Little is known how these cells maintain aberrant gene expression. Since specific chromatin regulatory complexes control HOX expression in normal hematopoiesis and in leukemias expressing an oncogenic MLL-fusion protein, we hypothesized that they might also regulate HOX expression in other settings, and that inhibition of HOX and FLT3 expression would produce an antiproliferative effect in NPM1mut AML.
Aims
To investigate dependencies of NPM1mut AML on MLL and DOT1L complexes and to delineate their potential role in control of leukemogenic gene expression.
Methods
CRISPR-Cas9 genome editing across exons encoding specific protein domains was used in a negative selection screen to assess potential dependencies of NPM1mut AML on specific MLL protein domains, and this concept was extended with inhibitors of the menin-MLL interaction (MI2-2 and MI503). Dependencies on DOT1L were explored using the inhibitor EPZ4777. In vitro and in vivo target validation was performed in two conditional murine knock-in models (Npm1CA/+FLt3ITD/+; Npm1CA/+RosaSB/+) and in a disseminated human xenograft model of Npm1mut AML.
Results
Using a CRISPR-Cas9 negative selection screen, we discovered the menin binding site of MLL as a dependency in NPM1mut OCI-AML3 cells, a region critically involved in chromatin binding of the MLL complex. We next sought to assess treatment effects of MI2-2 and MI503 and observed dramatic suppression of HOX and MEIS1 expression, differentiation induction, and profound growth inhibition in human OCI-AML3 cells and murine Npm1mut AMLs. Of interest, ectopic expression of Meis1, Hoxb4, or Hoxa9-Meis1 rescued the antiproliferative effects of menin-MLL inhibition. In vivo MI503 treatment of leukemic OCI-AML3 xenografts resulted in profound HOX and MEIS1 suppression, significant reduction of leukemia burden, and in a significant survival advantage of both, human xenografts and secondarily transplanted Npm1CA/+RosaSB/+ mice compared to vehicle controls. As MEIS1 was most dramatically suppressed across all three models, we determined drug effects on FLT3 expression, a reported target gene of MEIS1 and found consistent suppression of FLT3 to almost undetectable levels.MI503 treatment depleted menin and H3K4me3 on HOX and MEIS1 loci. We also observed a significant decrease of H3K79me2 at those loci, pointing to a potential role for DOT1L, the sole H3K79 methyltransferase. Treatment with EPZ4777 resulted in HOX, MEIS1, and FLT3 down regulation, cell growth inhibition, and profound differentiation of NPM1mut AML blasts just as we found with inhibition of the menin-MLL interaction.Next, we investigated effects of combinatorial menin-MLL and DOT1L inhibition and found superior suppression of HOX and FLT3 expression, dramatically more pronounced differentiation, and synergistic growth inhibition. Combinatorial drug treatment further reduced leukemia initiating potential of NPM1mut AML cells dramatically compared to single drug treatment in vivo.
Conclusion
MLL and DOT1L are chromatin regulators that control HOX, MEIS1 and FLT3 expression in NPM1mut AML. Combinatorial small-molecule inhibition has synergistic on target activity and represents a potential therapeutic opportunity that should soon be ready for clinical assessment.
Session topic: AML Biology - Novel targeted therapies
Keyword(s): Acute myeloid leukemia, Epigenetic, MLL
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:00 - 08:15
Location: Hall A3
Background
Homeobox (HOX) proteins and the receptor tyrosine kinase FLT3 are frequently highly expressed and mutated in acute myeloid leukemia (AML). Aberrant HOX expression is found in nearly all AMLs that harbor a mutation in the NPM1 gene (NPM1mut), and FLT3 is concomitantly mutated in ~50% of these cases. Little is known how these cells maintain aberrant gene expression. Since specific chromatin regulatory complexes control HOX expression in normal hematopoiesis and in leukemias expressing an oncogenic MLL-fusion protein, we hypothesized that they might also regulate HOX expression in other settings, and that inhibition of HOX and FLT3 expression would produce an antiproliferative effect in NPM1mut AML.
Aims
To investigate dependencies of NPM1mut AML on MLL and DOT1L complexes and to delineate their potential role in control of leukemogenic gene expression.
Methods
CRISPR-Cas9 genome editing across exons encoding specific protein domains was used in a negative selection screen to assess potential dependencies of NPM1mut AML on specific MLL protein domains, and this concept was extended with inhibitors of the menin-MLL interaction (MI2-2 and MI503). Dependencies on DOT1L were explored using the inhibitor EPZ4777. In vitro and in vivo target validation was performed in two conditional murine knock-in models (Npm1CA/+FLt3ITD/+; Npm1CA/+RosaSB/+) and in a disseminated human xenograft model of Npm1mut AML.
Results
Using a CRISPR-Cas9 negative selection screen, we discovered the menin binding site of MLL as a dependency in NPM1mut OCI-AML3 cells, a region critically involved in chromatin binding of the MLL complex. We next sought to assess treatment effects of MI2-2 and MI503 and observed dramatic suppression of HOX and MEIS1 expression, differentiation induction, and profound growth inhibition in human OCI-AML3 cells and murine Npm1mut AMLs. Of interest, ectopic expression of Meis1, Hoxb4, or Hoxa9-Meis1 rescued the antiproliferative effects of menin-MLL inhibition. In vivo MI503 treatment of leukemic OCI-AML3 xenografts resulted in profound HOX and MEIS1 suppression, significant reduction of leukemia burden, and in a significant survival advantage of both, human xenografts and secondarily transplanted Npm1CA/+RosaSB/+ mice compared to vehicle controls. As MEIS1 was most dramatically suppressed across all three models, we determined drug effects on FLT3 expression, a reported target gene of MEIS1 and found consistent suppression of FLT3 to almost undetectable levels.MI503 treatment depleted menin and H3K4me3 on HOX and MEIS1 loci. We also observed a significant decrease of H3K79me2 at those loci, pointing to a potential role for DOT1L, the sole H3K79 methyltransferase. Treatment with EPZ4777 resulted in HOX, MEIS1, and FLT3 down regulation, cell growth inhibition, and profound differentiation of NPM1mut AML blasts just as we found with inhibition of the menin-MLL interaction.Next, we investigated effects of combinatorial menin-MLL and DOT1L inhibition and found superior suppression of HOX and FLT3 expression, dramatically more pronounced differentiation, and synergistic growth inhibition. Combinatorial drug treatment further reduced leukemia initiating potential of NPM1mut AML cells dramatically compared to single drug treatment in vivo.
Conclusion
MLL and DOT1L are chromatin regulators that control HOX, MEIS1 and FLT3 expression in NPM1mut AML. Combinatorial small-molecule inhibition has synergistic on target activity and represents a potential therapeutic opportunity that should soon be ready for clinical assessment.
Session topic: AML Biology - Novel targeted therapies
Keyword(s): Acute myeloid leukemia, Epigenetic, MLL
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