Contributions
Abstract: EP388
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Acute myeloid leukemia (AML) is a heterogenous disease with multiple morphological, immunophenotypic and genetic features. The t(8;21), which results in the expression of RUNX1-ETO (aka AML1-ETO), is accountable for 12% of AML cases. Even though several studies have determined the transcriptomic changes observed in cells expressing RUNX1-ETO, there is a paucity of studies quantitating proteomic changes.
Aims
To analyze the proteomic profile of RUNX1-ETO expressing cord blood-derived human hematopoietic stem progenitor cells (HSPC) using mass spectrometry techniques.
Methods
We performed quantitative proteomics by SWATH-MS to determine the impact of RUNX1-ETO expression on the proteome using HSPC. Further, to determine changes in subcellular localization induced by RUNX1-ETO, cytosolic or nuclear fractions from these cells were separately analyzed.
Results
We quantified 4,635 proteins of which 2,787 were detected in the cytoplasm, and 1,848 in the nucleus. Statistical analysis identified 257 significantly differentially expressed proteins in RUNX1‑ETO HSPC compared to controls, of which 71% were detected in cytoplasm and 29% in the nucleus. We also identified 29 proteins that were seen only in the cytosol and 24 that were seen only in nuclear preparations of RUNX1-ETO cells. Pathway analysis demonstrated significant changes to ‘myeloid differentiation’ (p=2.5x10-6) pathway. Thus, we focused our analysis on identifying the transcription factors potentially responsible for differential expression in RUNX1-ETO cells. Using Metacore™’s transcription factor overconnectivity algorithm we identified C/EBPβ as a novel significant regulator (p=1.4x10-4) of the observed 257 proteomic changes. C/EBPβ is a transcription factor belonging to the C/EBP family and related to C/EBPB⍺, which is mutated in approximately 5-10% of AML patients. In our data, RUNX1‑ETO significantly downregulated the expression of C/EBPβ protein vs control (2-fold; p=0.01). Analysis of parallel transcriptomic data from the same model showed a 1.4-fold reduction in C/EBPβ mRNA level compared to controls suggesting transcriptional suppression by RUNX1‑ETO. Further, C/EBPβ mRNA is significantly downregulated in t(8;21) patients compared to non-t(8;21) AML FAB M2 patients (2.7-fold; p<0.05). Since C/EBPβ is normally upregulated during myeloid development, these data suggest that RUNX1‑ETO may inhibit myeloid differentiation by suppressing C/EBPβ expression. In support of this, C/EBPβ knockdown (KD) in the RUNX1-ETO-expressing cell line, SKNO-1, completely suppressed myeloid cell surface marker expression (CD13, CD33 and CD38) and gave a concomitant 4-fold increase in cell proliferation (p<0.001; n=3). Interestingly, in non-t(8;21) cells lines (HEL and U937) KD of C/EBPβ ablated cell growth (p<0.001) and increased the frequency of apoptotic cells (Annexin V+ 7-AAD- cells increased by 6-fold in HEL, p<0.01; n=3 and 2-fold in U937 cells, p<0.01; n=3), suggesting that the effects of C/EBPβ KD are context dependent.
Conclusion
In conclusion, proteomics identified hundreds of significantly differentially expressed proteins arising from RUNX1-ETO expression in the cytoplasmic and nuclear compartments. Importantly, we identified C/EBPβ as significantly downregulated mimicking the reduced expression of this transcription factor seen in t(8;21) patients compared AML FAB M2 patients without t(8;21). Functional studies suggest that reduced C/EBPβ expression inhibits myeloid differentiation in a RUNX1-ETO-dependant manner. Currently, we are determining the effect of C/EBPβ KD on normal HSPC growth and differentiation.
Keyword(s): Acute myeloid leukemia, AML1-ETO, C/EBP, Proteomics
Abstract: EP388
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Acute myeloid leukemia (AML) is a heterogenous disease with multiple morphological, immunophenotypic and genetic features. The t(8;21), which results in the expression of RUNX1-ETO (aka AML1-ETO), is accountable for 12% of AML cases. Even though several studies have determined the transcriptomic changes observed in cells expressing RUNX1-ETO, there is a paucity of studies quantitating proteomic changes.
Aims
To analyze the proteomic profile of RUNX1-ETO expressing cord blood-derived human hematopoietic stem progenitor cells (HSPC) using mass spectrometry techniques.
Methods
We performed quantitative proteomics by SWATH-MS to determine the impact of RUNX1-ETO expression on the proteome using HSPC. Further, to determine changes in subcellular localization induced by RUNX1-ETO, cytosolic or nuclear fractions from these cells were separately analyzed.
Results
We quantified 4,635 proteins of which 2,787 were detected in the cytoplasm, and 1,848 in the nucleus. Statistical analysis identified 257 significantly differentially expressed proteins in RUNX1‑ETO HSPC compared to controls, of which 71% were detected in cytoplasm and 29% in the nucleus. We also identified 29 proteins that were seen only in the cytosol and 24 that were seen only in nuclear preparations of RUNX1-ETO cells. Pathway analysis demonstrated significant changes to ‘myeloid differentiation’ (p=2.5x10-6) pathway. Thus, we focused our analysis on identifying the transcription factors potentially responsible for differential expression in RUNX1-ETO cells. Using Metacore™’s transcription factor overconnectivity algorithm we identified C/EBPβ as a novel significant regulator (p=1.4x10-4) of the observed 257 proteomic changes. C/EBPβ is a transcription factor belonging to the C/EBP family and related to C/EBPB⍺, which is mutated in approximately 5-10% of AML patients. In our data, RUNX1‑ETO significantly downregulated the expression of C/EBPβ protein vs control (2-fold; p=0.01). Analysis of parallel transcriptomic data from the same model showed a 1.4-fold reduction in C/EBPβ mRNA level compared to controls suggesting transcriptional suppression by RUNX1‑ETO. Further, C/EBPβ mRNA is significantly downregulated in t(8;21) patients compared to non-t(8;21) AML FAB M2 patients (2.7-fold; p<0.05). Since C/EBPβ is normally upregulated during myeloid development, these data suggest that RUNX1‑ETO may inhibit myeloid differentiation by suppressing C/EBPβ expression. In support of this, C/EBPβ knockdown (KD) in the RUNX1-ETO-expressing cell line, SKNO-1, completely suppressed myeloid cell surface marker expression (CD13, CD33 and CD38) and gave a concomitant 4-fold increase in cell proliferation (p<0.001; n=3). Interestingly, in non-t(8;21) cells lines (HEL and U937) KD of C/EBPβ ablated cell growth (p<0.001) and increased the frequency of apoptotic cells (Annexin V+ 7-AAD- cells increased by 6-fold in HEL, p<0.01; n=3 and 2-fold in U937 cells, p<0.01; n=3), suggesting that the effects of C/EBPβ KD are context dependent.
Conclusion
In conclusion, proteomics identified hundreds of significantly differentially expressed proteins arising from RUNX1-ETO expression in the cytoplasmic and nuclear compartments. Importantly, we identified C/EBPβ as significantly downregulated mimicking the reduced expression of this transcription factor seen in t(8;21) patients compared AML FAB M2 patients without t(8;21). Functional studies suggest that reduced C/EBPβ expression inhibits myeloid differentiation in a RUNX1-ETO-dependant manner. Currently, we are determining the effect of C/EBPβ KD on normal HSPC growth and differentiation.
Keyword(s): Acute myeloid leukemia, AML1-ETO, C/EBP, Proteomics