TET2 lesions enhance the aggressiveness of CEBPA-mutant AML by rebalancing GATA2 expression [RNA-Seq]
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ABSTRACT: The myeloid transcription factor CEBPA is recurrently biallelically mutated (i.e., double mutated; CEBPADM) in acute myeloid leukemia (AML) with a combination of hypermorphic N-terminal mutations (CEBPANT), promoting expression of the leukemia-associated p30 isoform, and amorphic C-terminal mutations. CEBPADM AML features recurrent co-occurring mutations including GATA2 lesions, however insights into mechanisms governing this co-mutational spectrum are incomplete. By combining transcriptomic and epigenomic analyses of CEBPA-TET2 co-mutated patients with models thereof, we identify GATA2 as a conserved target of the CEBPA-TET2 mutational axis, providing a rationale for the mutational spectra in CEBPADM AML. Mechanistically, we suggest that elevated CEBPA levels, driven by CEBPANT, mediate recruitment of TET2 to the Gata2 distal hematopoietic enhancer thereby increasing Gata2 expression. Conversely, CEBPADM AML gains a competitive advantage through TET2 loss, by decreasing Gata2 promoter demethylation thereby rebalancing GATA2 levels. Of clinical relevance, demethylating treatment of Cebpa-Tet2 co-mutated AML restores Gata2 levels and prolongs disease latency.
Project description:The myeloid transcription factor CEBPA is recurrently biallelically mutated (i.e., double mutated; CEBPADM) in acute myeloid leukemia (AML) with a combination of hypermorphic N-terminal mutations (CEBPANT), promoting expression of the leukemia-associated p30 isoform, and amorphic C-terminal mutations. CEBPADM AML features recurrent co-occurring mutations including GATA2 lesions, however insights into mechanisms governing this co-mutational spectrum are incomplete. By combining transcriptomic and epigenomic analyses of CEBPA-TET2 co-mutated patients with models thereof, we identify GATA2 as a conserved target of the CEBPA-TET2 mutational axis, providing a rationale for the mutational spectra in CEBPADM AML. Mechanistically, we suggest that elevated CEBPA levels, driven by CEBPANT, mediate recruitment of TET2 to the Gata2 distal hematopoietic enhancer thereby increasing Gata2 expression. Conversely, CEBPADM AML gains a competitive advantage through TET2 loss, by decreasing Gata2 promoter demethylation thereby rebalancing GATA2 levels. Of clinical relevance, demethylating treatment of Cebpa-Tet2 co-mutated AML restores Gata2 levels and prolongs disease latency.
Project description:The myeloid transcription factor CEBPA is recurrently biallelically mutated (i.e., double mutated; CEBPADM) in acute myeloid leukemia (AML); with a combination of hypermorphic N-terminal mutations (CEBPANT), promoting expression of the leukemia-associated p30 isoform, and amorphic C-terminal mutations. CEBPADM AML features recurrent co‑occurring mutations; however, insight into the underlying mechanisms for the co-mutational spectra is incomplete. By combining transcriptomic and epigenomic analyses of data from CEBPA-TET2-co-mutant patients with experimental models thereof, we identify GATA2 as a conserved target of the CEBPA-TET2 mutational axis, providing a rationale for the mutational spectra in CEBPADM AML. Mechanistically, we suggest that elevated CEBPA levels, driven by the CEBPANT, mediate recruitment of TET2 to the GATA2 distal hematopoietic enhancer and thereby increase GATA2 expression. Conversely, CEBPADM AML gains a competitive advantage by loss of TET2; decreasing GATA2 promoter demethylation and re-balancing GATA2 levels. Further, demethylating treatment of CEBPA-TET2-co-mutant AML restores GATA2 levels, and prolongs disease latency.
Project description:Acute myeloid leukemia (AML) with CEBPA mutations is determined as provisional entity in the current WHO. A difference in clinical outcome between single- (sm) and double-mutated (dm) cases has been reported, whereupon dm cases were shown to be associated with longer overall survival (OS). The occurrence and prognostic impact of concomitant molecular mutations in addition to CEBPAdm has not been assessed until now. Here, we investigated a cohort of 95 AML CEBPAdm cases for concomitant mutations. TET2 was found to be the most frequent mutation (32/94, 34.0%), followed by GATA2 (20/95, 21.0%), WT1 (13/95, 13.7%), DNMT3A (9/94, 9.6%), ASXL1 (9/95, 9.5%), NRAS (8/95, 8.4%), KRAS (3/94, 3.2%), IDH1/2 (6/95, 6.3%), FLT3-ITD (6/95, 6.3%), FLT3-TKD (2/95, 2.1%), NPM1 (2/95, 2.1%), and RUNX1 (1/94). No mutation was detected in MLL-PTD and TP53. With respect to prognostic impact, we observed that those cases harboring additional mutations in TET2 showed significant worse survival than wild-type cases (P=0.035), whereas GATA2 mutated cases showed improved survival (P=0.032). Further, using gene expression microarray analysis we identified no clear different clustering within the CEBPAdm cases with the distinct concomitant mutated genes. In conclusion, we demonstrated that 76.8% of CEBPAdm cases harbored additional alterations in other molecular markers and that CEBPA is a suitable MRD marker to control therapy. Total cohort contains 95 cases, but expression data available for 38 cases which were analyzed on Affymetrix HG-U133 Plus 2.0 arrays: 8 CEBPAdm and GATA2 mutated cases, 12 CEBPAdm and TET2 mutated cases, 7 CEBPAdm and ASXL1 mutated cases (n=3 showed an additional TET2 mutation) and 11 CEBPAdm without mutations in GATA2, TET2, and ASXL1.
Project description:Acute myeloid leukemia (AML) with CEBPA mutations is determined as provisional entity in the current WHO. A difference in clinical outcome between single- (sm) and double-mutated (dm) cases has been reported, whereupon dm cases were shown to be associated with longer overall survival (OS). The occurrence and prognostic impact of concomitant molecular mutations in addition to CEBPAdm has not been assessed until now. Here, we investigated a cohort of 95 AML CEBPAdm cases for concomitant mutations. TET2 was found to be the most frequent mutation (32/94, 34.0%), followed by GATA2 (20/95, 21.0%), WT1 (13/95, 13.7%), DNMT3A (9/94, 9.6%), ASXL1 (9/95, 9.5%), NRAS (8/95, 8.4%), KRAS (3/94, 3.2%), IDH1/2 (6/95, 6.3%), FLT3-ITD (6/95, 6.3%), FLT3-TKD (2/95, 2.1%), NPM1 (2/95, 2.1%), and RUNX1 (1/94). No mutation was detected in MLL-PTD and TP53. With respect to prognostic impact, we observed that those cases harboring additional mutations in TET2 showed significant worse survival than wild-type cases (P=0.035), whereas GATA2 mutated cases showed improved survival (P=0.032). Further, using gene expression microarray analysis we identified no clear different clustering within the CEBPAdm cases with the distinct concomitant mutated genes. In conclusion, we demonstrated that 76.8% of CEBPAdm cases harbored additional alterations in other molecular markers and that CEBPA is a suitable MRD marker to control therapy.
Project description:The gene encoding the transcription factor C/EBPα is mutated in 10-15% of all patients with de novo acute myeloid leukemia (AML). N-terminal CEBPA mutations cause selective ablation of full-length C/EBPα without affecting the expression of a shorter oncogenic isoform, termed p30. The mechanistic basis of p30-induced leukemogenesis is not well understood. Here, we show that the SET/MLL histone methyltransferase complex represents a critical actionable vulnerability in CEBPA-mutated AML. The oncogenic C/EBPα p30 isoform and MLL show global co-localization on chromatin and core SET/MLL complex components exhibit robust physical interaction with p30. We also show that C/EBPα p30-expressing cells require the expression of an intact MLL protein, as targeted CRISPR/Cas9-mediated mutagenesis results in proliferation arrest and myeloid differentiation. In line with this, CEBPA-mutated hematopoietic progenitor cells are hypersensitive to pharmacological inhibition of the SET/MLL complex. SET/MLL complex inhibition impairs proliferation, induces cell cycle arrest and causes apoptosis in mouse and human AML cells with CEBPA mutations. Global analysis of gene expression changes shows that inhibitor treatment restores myeloid differentiation potential in CEBPA-mutated cells. Finally, we identify the transcription factor GATA2 as a direct critical target of the cooperative gene regulation between p30 and MLL in CEBPA-mutated AML. Taken together, we show that C/EBPα p30 requires the SET/MLL complex to regulate oncogenic gene expression and that CEBPA-mutated AML is hypersensitive to perturbation of the SET/MLL complex. These findings expand our understanding of CEBPA-mutated AML and identify the SET/MLL complex as a potential therapeutic target in this disease.
Project description:A previously predictive CEBPA double mutant (CEBPAdm) signature was hampered by the recently reported CEBPA silenced AML cases that carry a similar gene expression profile (GEP). Two independent AML cohorts were used to train and evaluate the predictive value of the CEBPAdm signature in terms of sensitivity and specificity. A predictive signature was created, containing 25-probe sets by using a logistic regression model with Lasso regularization, which selects discriminative probe sets between the classes, CEBPAdm and all other AML cases, CEBPA wild type (CEBPAwt) and CEBPA single mutant (CEBPAsm). Subsequently, a classifier was trained on the entire HOVON-SAKK cohort based on a two-class approach; CEBPAdm versus all other cases (CEBPAwt and CEBPAsm). This trained classifier subsequently classified 16 candidate CEBPAdm cases in the AMLSG-cohort out of 154 AML cases. This approach showed perfect sensitivity and specificity (both 100%). In addition, we have performed a classification between CEBPAdm ,CEBPAsm, and CEBPAwt to infer if we were able to accurately classify CEBPAsm cases. We observed that all CEBPAsm cases were classified as CEBPAwt, thus CEBPAsm cases do not have a consistent gene expression pattern and are different from the CEBPAdm group.
Project description:A previously predictive CEBPA double mutant (CEBPAdm) signature was hampered by the recently reported CEBPA silenced AML cases that carry a similar gene expression profile (GEP). Two independent AML cohorts were used to train and evaluate the predictive value of the CEBPAdm signature in terms of sensitivity and specificity. A predictive signature was created, containing 25-probe sets by using a logistic regression model with Lasso regularization, which selects discriminative probe sets between the classes, CEBPAdm and all other AML cases, CEBPA wild type (CEBPAwt) and CEBPA single mutant (CEBPAsm). Subsequently, a classifier was trained on the entire HOVON-SAKK cohort based on a two-class approach; CEBPAdm versus all other cases (CEBPAwt and CEBPAsm). This trained classifier subsequently classified 16 candidate CEBPAdm cases in the AMLSG-cohort out of 154 AML cases. This approach showed perfect sensitivity and specificity (both 100%). In addition, we have performed a classification between CEBPAdm ,CEBPAsm, and CEBPAwt to infer if we were able to accurately classify CEBPAsm cases. We observed that all CEBPAsm cases were classified as CEBPAwt, thus CEBPAsm cases do not have a consistent gene expression pattern and are different from the CEBPAdm group. All samples were obtained from untreated patients at the time of diagnosis. Cells used for microarray analysis were collected from the purified fraction of mononuclear cells after Ficoll density centrifugation. Routine diagnostic algorithms, including the characterization of molecular markers are performed.