CEBPA Mutations In Acute Myeloid Leukemia Research Articles

The Impact of Baseline Genetic Profile and Treatment on Hematological Toxicity in CEBPA-Mutated Acute Myeloid Leukemia

Background: CEBPA-mutated acute myeloid leukemia (AML) is a separate entity in the updated WHO and ICC classifications. Previously identified by double mutations, the clinical profile and favorable outcome were correlated with in-frame mutations in bZIP domain. Beyond mutations recurrent in myeloid neoplasms (involving TET2, FLT3, NRAS), CEBPA-mutated AML is enriched in gene mutations (i.e., GATA2, CSF3R, WT1) rarely observed in CEBPA-wild type AML. No conclusive findings have been drawn for the prognostic impact of additional mutations. Of note, somatic GATA2 mutations have been associated with invasive fungal infections in myeloid neoplasms. Although overall correlating with favorable prognosis, an unexpectedly high rate of treatment-related mortality (TRM) in CR has been described in a large multicenter study on intensively treated CEBPA-mutated patients (pts) (Schlenk et al Blood 2013;122(9):1576). Aims: The aim of our study was to correlate baseline and treatment characteristics with the outcome and hematological toxicity in an intensively treated cohort of CEBPA-bZIP mutated AML pts. Methods: Pts entering the study had a diagnosis of CEBPA-bZIP AML confirmed according to 2022 WHO and ICC criteria. Pts were characterized by next-Generation deep amplicon sequencing with Ion Torrent platform (ThermoFisher Scientific) covering a panel of 40 genes. Sequence alignment and filtering were performed by NextGENe version 2.4.2.1 (SoftGenetics). The first two chemotherapy cycles were categorized according to the delivery of an anthracycline (ANTHRAC) and cytarabine (ARA-C) at high dose (HDAC). The study was approved by the local institutional review board. Results: From 2004 to 2023, 49 pts with CEBPA-bZIP mutated AML met the inclusion criteria at project study sites (Firenze, Bergamo). Their features are detailed in Table 1. CR rate was achieved in 45 of 49 pts (91.8%), with a DFS and OS of 74.3% and 82.4% at 5 y, respectively. Based on NGS, at least one additional mutation was identified in 18 (36.7%) pts (NGS+), the majority of them involving GATA2 (n=14, 28.6%). As per treatment, 44 pts received at least two cycles, of which no (n=2, 4.6%), one (n=14, 31.8%), or two (n=28, 63.6%) ANTHRAC-containing cycles. As regarded HDAC, 19 (43.2%), 17 (38.6%) and 8 (18.2%) pts received no, one or two HDAC-containing cycles, respectively. No significant differences emerged for DFS or OS between NGS+ and NGS- groups (P=0.33 and 0.52, respectively). To gain insight into potential causes of toxicity during CR, we focused on hematopoietic recovery after induction and consolidation. NGS+ pts showed slower neutrophil (ANC) recovery (median, 22 days to >500/uL after induction and 25 days after consolidation) compared to NGS- group (19 days, P=0.018; 18 days P=0.058, respectively). Such effect was enhanced by treatment with ANTHRAC-containing cycles: NGS+ pts receiving two cycles required significantly longer time to recover from first consolidation cycle, both for ANC (30 days) and platelet count (34 days) vs 22 (P=0.012) and 27 (P=.010) days, respectively, of other categories (Figure 1). A proportion of 45.5% (5/11) NGS+/ANTHRAC-2 pts could not complete the planned chemotherapy program due to hematological toxicity compared to 19.3% (6/31, P=0.09) in other categories. No significant prolongation of recovery was observed for HDAC. Conclusions: CEBPA-bZIP mutated AML is featured by high response rate and favorable outcome. Delayed hematopoietic recovery was observed in pts bearing additional mutations (especially GATA2) when treated with anthracyclines in first two cycles. Our findings suggest the cumulative dosage of anthracyclines should be limited in CEBPA-mutated/NGS+ patients to spare hematological toxicity on turn impairing the therapeutic plan.

Integrated Methylation and Transcriptional Landscape and Evolution of Pediatric AML

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy associated with poor outcomes despite intensive therapies. The genomic landscape of pediatric AML has been well characterized and has led to translational benefits, such as improved relapse predication and effective targeted therapies for a subset of individuals (e.g., FLT3-ITD AML). Despite these advances, the majority of patients continue to receive therapeutic interventions that have not changed in three decades. This underscores the need to advance our understanding of AML biology beyond genomic alterations (e.g., somatic mutations). Here, we present the largest (N = 858 patients) integrative analysis of pediatric AML paired transcriptomes and methylomes to date, performed at diagnosis for patients enrolled on Children's Oncology Group clinical trials AAML0531 (NCT00372593) or AAML1031 (NCT01371981). We also analyzed N = 400 and 253 corresponding transcriptomes and methylomes at relapse, respectively. Additionally, we performed comprehensive mutation and fusion calling from NGS data and integrated clinical covariates and outcomes. This robust dataset allowed us to uncover how pediatric AML epigenetics are perturbed from normal hematopoiesis, characterize the evolution of transcriptional and methylation changes from leukemia diagnosis to relapse, and identify more accurate relapse prediction models for pediatric AML. We initially performed unbiased hierarchical clustering on CpG sites that are differentially methylated between AML and normal bone marrow to identify distinct AML-restricted methylation (ARM) signatures. This analysis demonstrated that that these ARM clusters were enriched in specific cytomolecular subtypes of pediatric AML. Specifically, we identified methylation signatures that recapitulated their adult counterparts (e.g., IDH1/2, DNMT3A, WT1) and pediatric specific methylation signatures that clustered with RUNX1::RUNX1T1, CBFB::MYH11, KMT2A-rearrangement, NUP98::NSD1, NUP98::KDM5A, and CEBPA mutant AML. Next, we investigated the transcriptional and methylation signatures for the relapsed cases and compared them to diagnosis. We demonstrated that relapsed transcriptional and methylation signatures clustered within their cytomolecular subtype counterparts at diagnosis (i.e., AMLs at relapse more closely resemble AMLs at diagnosis with the same underlying gene fusion than other relapsed AMLs) ( Fig. 1A). However, analyzing our data in the context of these cytomolecular subtypes, we found that leukemias uniformly acquired a more stem-like state at relapse. We focused our biomarker discovery efforts on KMT2A-rearranged AMLs since (as a cohort) these patients have an intermediate risk of relapse and existing molecular biomarkers (e.g., KMT2A fusion partner and measurable residual disease detection) result in only modest improvements in relapse prediction. First, we found that CpG islands associated with CD34 and HOXA gene expression were frequently (but variably) hypermethylated, which was also associated with downregulation of CD34 and HOXA gene expression and favorable survival outcomes. Next, we performed non-negative matrix factorization on diagnosis and relapsed KMT2A-rearranged AMLs and identified a novel gene set associated with relapse and poor outcomes. These three distinct methylation-transcriptional findings (i.e., CD34, HOXA, and relapsed signatures) were subsequently transformed into a novel methylation-transcription biomarker score (MTBS) using machine learning algorithms (i.e., LASSO Cox regression). We used KMT2A-rearranged AML patients from AAML1031 as our training cohort (N = 129) to generate a MTBS that was able to distinguish KMT2A-rearranged cases with disparate outcomes. This algorithm was validated in an independent cohort from AAML0531 (N = 28) ( Fig. 1B). Additional methylation-transcriptional biomarker discovery efforts for pediatric AML are underway. Since pediatric AML is associated with a low mutational burden, understanding leukemia-specific transcriptional and epigenetic alterations is of utmost importance. Our study demonstrates the translational promise offered by deep functional genomic characterization of pediatric AML and we believe that further interrogation may provide additional novel insights into leukemia initiation, relapse prediction, and novel treatment approaches.

Homozygous CEBPA Mutations Predict Favorable Outcomes in AML Patients

Background: Loss of heterozygosity (LOH) of CEBPA has been sporadically reported in acute myeloid leukemia (AML), resulting in a complete or nearly complete homozygosity of CEBPA mutations ( CEBPA homo). However, its clinical significance has been underappreciated. Methods: We retrospectively analyzed the prognostic impact of CEBPA homo in 1223 adult AML patients, where CEBPA mutations were detected using whole exome sequencing (n=296) or targeted sequencing (n=927) technology. CEBPA homo is defined as the variant allele frequency (VAF) of a CEBPA mutation over 50%, and CEBPA heter is the opposite. Propensity score matching was used to reduce selection bias when comparing the survival difference between two groups. Results: CEBPA mutations were identified in 106 patients (8.7%), 47(44.3%) of which carried the C-terminal DNA-binding or basic leucine zipper region ( CEBPA bZIP). Compared to patients with other CEBPA mutations ( CEBPA others), patients carrying CEBPA bZIP were significantly younger (40.38±14.80 vs. 52.05±17.44 years, P<0.001) and had higher hemoglobin [89(65.5, 98) vs 70(58, 81) g/dL, P=0.001], lower platelet counts [35(17.5, 43) vs. 44(24, 78.5) 10 9/L, P=0.019) and higher proportion of peripheral blast cells [80(52.5, 90.5) vs. 51(14, 86.5) %, P=0.001] at diagnosis. CEBPA bZIP was associated with a higher complete remission rate (CR; 85% vs. 61%, P=0.012) in AML patients but did not reduce the relapse rate (32% vs. 39%, P=0.582). CEBPA bZIP significantly improved 4-year overall survival (OS; 0.70±0.09 vs. 0.30±0.13, P=0.013), 2-year event-free survival (EFS; 0.47±0.09 vs. 0.2±0.09, P=0.01), and 3-year pre-transplantation survival (PTS; 0.54±0.09 vs. 0.38±0.09, P=0.003) in AML patients compared to those with CEBPA others. These data are consistent with previous reports, suggesting the representative of this cohort. We further evaluated the clinical significance of CEBPA homo in this cohort and found that patients with CEBPA homo had higher white blood cell counts [29.37 (9.16, 76.11) vs. 10.37 (3.38, 43.43) 10 9/L, P=0.048] than those with CEBPA heter. Although CEBPA homo was not associated with CR rate (65% vs. 80%, P=0.145), it did reduce relapse rate (26% vs. 47%, P=0.045) in AML patients compared to those with CEBPA heter. CEBPA homo was significantly associated with improved 5-year OS (0.68±0.10 vs. 0.29±0.10, P=0.006), 5-year EFS (0.30±0.09 vs. 0.10±0.08, P=0.018), and 5-year PTS (0.40±0.10 vs. 0.14±0.07, P=0.046) compared to CEBPA heter. There is no difference in CEBPA VAF between patients carrying CEBPA bZIP and CEBPA other, suggesting similar distribution of CEBPA homo between these two groups. Conclusions: The clinical significance of CEBPA homo is different from CEBPA bZIP, which suggests a distinct biological meaning. Our study would add one new layer to the current stratification of CEBPA-mutated AML based on the prognosis. More cases are needed to further examine the clinical significance of the concurrency of CEBPA homo and CEBPA bZIP.

TET2 lesions enhance the aggressiveness of CEBPA-mutant acute myeloid leukemia by rebalancing GATA2 expression

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. The most frequently co-mutated genes in CEBPADM AML are GATA2 and TET2, however the molecular mechanisms underlying 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. Elevated CEBPA levels, driven by CEBPANT, mediate recruitment of TET2 to the Gata2 distal hematopoietic enhancer thereby increasing Gata2 expression. Concurrent loss of TET2 in CEBPADM AML induces a competitive advantage by increasing Gata2 promoter methylation, thereby rebalancing GATA2 levels. Of clinical relevance, demethylating treatment of Cebpa-Tet2 co-mutated AML restores Gata2 levels and prolongs disease latency.

The histone demethylase KDM5C functions as a tumor suppressor in AML by repression of bivalently marked immature genes

Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). Thus, the identification and characterization of novel epigenetic drivers affecting AML biology holds potential to improve our basic understanding of AML and to uncover novel options for therapeutic intervention. To identify novel tumor suppressive epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of CEBPA mutant AML. This identified the Histone 3 Lysine 4 (H3K4) demethylase KDM5C as a tumor suppressor, and we show that reduced Kdm5c/KDM5C expression results in accelerated growth both in human and murine AML cell lines, as well as in vivo in Cebpa mutant and inv(16) AML mouse models. Mechanistically, we show that KDM5C act as a transcriptional repressor through its demethylase activity at promoters. Specifically, KDM5C knockdown results in globally increased H3K4me3 levels associated with up-regulation of bivalently marked immature genes. This is accompanied by a de-differentiation phenotype that could be reversed by modulating levels of several direct and indirect downstream mediators. Finally, the association of KDM5C levels with long-term disease-free survival of female AML patients emphasizes the clinical relevance of our findings and identifies KDM5C as a novel female-biased tumor suppressor in AML.

Differential Engraftment of CEBPA Mutated Acute Myeloid Leukemia Cells in NSGS Mice

Differential Engraftment of CEBPA Mutated Acute Myeloid Leukemia Cells in NSGS Mice

Frequency and clinical impact of WT1 mutations in the context of CEBPA-mutated acute myeloid leukemia

ABSTRACT Introduction Several studies have confirmed that mutations in the Wilms tumor 1 (WT1) gene occur in adult acute myeloid leukemia (AML). However, few data are available regarding the incidence of WT1 mutations in CEBPAmut AML and their impact. Methods We retrospectively analyzed the frequency and clinical impact of WT1 mutations in 220 newly diagnosed AML patients with CEBPA mutations(CEBPAmut). Chromosome karyotype analysis was performed by R or G banding method and further confirmed either by fluorescence in situ hybridization (FISH) and/or by multiple reverse transcription polymerase chain reaction (multiple RT–PCR). Mutations were detected with a panel of 112mutational genes using next-generation sequencing (NGS). Results Overall, 30 WT1 mutations were detected in 29 of the 220 CEBPAmut AML patients (13.18%) screened. These mutations clustered overwhelmingly in exon 7 (n=16). WT1 mutations were found to be significantly more frequent in AML patients with double-mutated CEBPA (CEBPAdm) than in AML patients with single-mutated CEBPA (17.36%vs. 8.08%, P = 0.043). Among WT1-mutated patients, the most common co-mutation was FLT3-ITD (n = 7, 24.14%), followed by NRAS (n = 5, 17.24%), CSF3R (n = 4, 13.79%), GATA2 (n = 4, 13.79%), and KIT (n = 4, 13.79%). The most frequent functional pathway was signaling pathways inas many as 62.07% of cases. Notably,the concomitant mutations in epigenetic regulatorswere inversely correlated with WT1 mutations(P = 0.003). CEBPAdm AML patients with WT1 mutations had inferior relapse-free survival, event-free survival and overall survival compared with patients CEBPAdm AML without WT1 mutations (P = 0.002, 0.004, and 0.010, respectively). Conclusion Our data showed that WT1 mutations are frequently identified in CEBPAmut AML, especially in CEBPAdm AML. CEBPAmut AML patients with WT1 mutations show distinct spectrum of comutations. In the context of CEBPAdm AML, WT1 mutations predict a poor prognosis.

Acute Myeloid Leukemia With CEBPA Mutations: Current Progress and Future Directions.

Mutations in CCAAT enhancer binding protein A gene (CEBPA) are one of the common genetic alterations in acute myeloid leukemia (AML). Recently, the emergence of new evidence makes it necessary to reconsider the subsets and treatment of AML patients with CEBPA mutations. This review will summarize the history of research progress of CEBPA mutations in AML, the heterogeneities of AML with CEBPA double mutations (CEBPAdm), and two special subtypes of CEBPA mutated AML. We will discuss the treatment of AML with CEBPA mutations as well, and finally propose a new algorithm for the treatment of these patients, including both familial and sporadic CEBPA mutated AML patients. This review may be beneficial for further investigation and optimizing clinical management of AML patients with CEBPA mutations.

CEBPA mutants down-regulate AML cell susceptibility to NK-mediated lysis by disruption of the expression of NKG2D ligands, which can be restored by LSD1 inhibition

ABSTRACT NK group 2, member D (NKG2D) is one of the most critical activating receptors expressed by natural killer (NK) cells. There is growing evidence that acute myeloid leukemia (AML) cells may evade NK cell-mediated cell lysis by expressing low or no ligands for NKG2D (NKG2D-Ls). We hypothesized that CCAAT/enhancer-binding protein α (C/EBPα), one of the most studied lineage-specific transcription factors in hematopoiesis, might influence the expression of NKG2D-Ls. To test this hypothesis, we first examined the endogenous expression of wild-type C/EBPα (C/EBPα-p42) in human AML cell lines and demonstrated that its expression level was highly relevant to the sensitivity of AML cells to NK cell cytotoxicity. Induction of C/EBPα-p42 in the low endogenous CEBPA-expressing AML cell line increased the sensitivity to NK-induced lysis. Moreover, decreased expression of C/EBPα-p42 by RNA interference in AML cells abrogated NK-mediated cytotoxicity. We further showed that the increase in NK susceptibility caused by C/EBPα-p42 occurred through up-regulation of the NKG2D-Ls ULBP2/5/6 in AML cells. More importantly, chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing captured C/EBPα motif signatures at the enhancer regions of the ULBP 2/5/6 genes. Whilst, the AML-associated C/EBPα C-terminal mutant and N-terminal truncated mutant (C/EBPα-p30) diminished ULBP2/5/6 transcription. Finally, we identified that histone demethylase lysine-specific demethylase 1 (LSD1) inhibition can restore the expression of ULBPs via induction of CEBPA expression in AML cells, which may represent a novel therapeutic strategy for CEBPA-mutated AML. Abbreviations: C/EBPα: CCAAT/enhancer-binding protein α; TF: Transcription factor; AML: Acute myeloid leukemia; TAD: Transactivation domain; FS: Frameshift; NK: Natural killer; NKG2D: NK group 2, member D; NKG2D-Ls: Ligands for NKG2D; MHC: Major histocompatibility complex; MICA: MHC class I-related chain A; ULBP: UL16-binding protein; STAT3: Signal transducer and activator of transcription 3; LSD1: Lysine-specific demethylase 1; Ab: Antibody; PBMC: Peripheral blood mononuclear cell; PBS: Phosphate-buffered saline; CFSE: Carboxyfluorescein diacetate succinimidyl ester; PI: Propidium iodide; shRNA: Short hairpin RNA; ChIP: Chromatin immunoprecipitation; BM: Binding motif; HCNE: Highly conserved noncoding element; TSS: Transcription start site; HMA: Hypomethylating agent; AZA: Azacitidine/5-azacytidine; DAC: Decitabine/5-aza-29-deoxycytidine; 2-PCPA: Tranylcypromine; RBP: RNA-binding protein; MSI2: MUSASHI-2; HDACi: Inhibitor of histone deacetylases; VPA: Valproate; DNMTi: DNA methyl transferase inhibitor; SCLC: Small cell lung cancer

Prognostic impact of CEBPA bZIP domain mutation in acute myeloid leukemia

AbstractMutations of CCAAT/enhancer–binding protein alpha (CEBPAmu) are found in 10% to 15% of de novo acute myeloid leukemia (AML) cases. Double-mutated CEBPA (CEBPAdm) is associated with a favorable prognosis; however, single-mutated CEBPA (CEBPAsm) does not seem to improve prognosis. We investigated CEBPAmu for prognosis in 1028 patients with AML, registered in the Multi-center Collaborative Program for Gene Sequencing of Japanese AML. It was found that CEBPAmu in the basic leucine zipper domain (bZIP) was strongly associated with a favorable prognosis, but CEBPAmu out of the bZIP domain was not. The presence of CEBPAmu in bZIP was a strong indicator of a higher chance of achieving complete remission (P < .001), better overall survival (OS; P < .001) and a lower risk of relapse (P < .001). The prognostic significance of CEBPAmu in bZIP was also observed in the subgroup with CEBPAsm (all patients: OS, P = .008; the cumulative incidence of relapse, P = .063; patients aged ≤70 years and with intermediate-risk karyotype: OS, P = .008; cumulative incidence of relapse, P = .026). Multivariate analysis of 744 patients aged ≤70 years showed that CEBPAmu in bZIP was the most potent predictor of OS (hazard ratio, 0.3287; P < .001). CEBPAdm was validated as a cofounding factor, which was overlapping with CEBPAmu in bZIP. In summary, these findings indicate that CEBPAmu in bZIP is a potent marker for AML prognosis. It holds potential in the refinement of treatment stratification and the development of targeted therapeutic approaches in CEBPA-mutated AML.

Identification of gene targets of mutant C/EBPα reveals a critical role for MSI2 in CEBPA-mutated AML.

Mutations in the gene encoding the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) occur in 10-15% of acute myeloid leukemia (AML). Frameshifts in the CEBPA N-terminus resulting in exclusive expression of a truncated p30 isoform represent the most prevalent type of CEBPA mutations in AML. C/EBPα p30 interacts with the epigenetic machinery, but it is incompletely understood how p30-induced changes cause leukemogenesis. We hypothesized that critical effector genes in CEBPA-mutated AML are dependent on p30-mediated dysregulation of the epigenome. We mapped p30-associated regulatory elements (REs) by ATAC-seq and ChIP-seq in a myeloid progenitor cell model for p30-driven AML that enables inducible RNAi-mediated knockdown of p30. Concomitant p30-dependent changes in gene expression were measured by RNA-seq. Integrative analysis identified 117 p30-dependent REs associated with 33 strongly down-regulated genes upon p30-knockdown. CRISPR/Cas9-mediated mutational disruption of these genes revealed the RNA-binding protein MSI2 as a critical p30-target. MSI2 knockout in p30-driven murine AML cells and in the CEBPA-mutated human AML cell line KO-52 caused proliferation arrest and terminal myeloid differentiation, and delayed leukemia onset in vivo. In summary, this work presents a comprehensive dataset of p30-dependent effects on epigenetic regulation and gene expression and identifies MSI2 as an effector of the C/EBPα p30 oncoprotein.

Combined inhibition of JAK/STAT pathway and lysine-specific demethylase 1 as a therapeutic strategy in CSF3R/CEBPA mutant acute myeloid leukemia

Acute myeloid leukemia (AML) is a deadly hematologic malignancy with poor prognosis, particularly in the elderly. Even among individuals with favorable-risk disease, approximately half will relapse with conventional therapy. In this clinical circumstance, the determinants of relapse are unclear, and there are no therapeutic interventions that can prevent recurrent disease. Mutations in the transcription factor CEBPA are associated with favorable risk in AML. However, mutations in the growth factor receptor CSF3R are commonly co-occurrent in CEBPA mutant AML and are associated with an increased risk of relapse. To develop therapeutic strategies for this disease subset, we performed medium-throughput drug screening on CEBPA/CSF3R mutant leukemia cells and identified sensitivity to inhibitors of lysine-specific demethylase 1 (LSD1). Treatment of CSF3R/CEBPA mutant leukemia cells with LSD1 inhibitors reactivates differentiation-associated enhancers driving immunophenotypic and morphologic differentiation. LSD1 inhibition is ineffective as monotherapy but demonstrates synergy with inhibitors of JAK/STAT signaling, doubling median survival in vivo. These results demonstrate that combined inhibition of JAK/STAT signaling and LSD1 is a promising therapeutic strategy for CEBPA/CSF3R mutant AML.

Gain-of-Function Effects of N-Terminal CEBPA Mutations in Acute Myeloid Leukemia.

Mutations in the CEBPA gene are present in 10–15% of acute myeloid leukemia (AML) patients. The most frequent type of mutations leads to the expression of an N-terminally truncated variant of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα), termed p30. While initial reports proposed that p30 represents a dominant-negative version of the wild-type C/EBPα protein, other studies show that p30 retains the capacity to actively regulate gene expression. Recent global transcriptomic and epigenomic analyses have advanced the understanding of the distinct roles of the p30 isoform in leukemogenesis. This review outlines direct and indirect effects of the C/EBPα p30 variant on oncogenic transformation of hematopoietic progenitor cells and discusses how studies of N-terminal CEBPA mutations in AML can be extrapolated to identify novel gain-of-function features in oncoproteins that arise from recurrent truncating mutations in transcription factors.

PS966 FUNCTIONAL COOPERATION OF CEBPA AND TET2 MUTATIONS IN ACUTE MYELOID LEUKEMIA

Background:The transcription factor CCAAT‐enhancer‐binding protein alpha (C/EBPα) is a master regulator of granulopoiesis and regulates the switch between proliferating, uncommitted progenitors and cell‐cycle‐arrested, differentiated myeloid cells. Usage of two alternative translation initiation sites in the CEBPA mRNA results in expression of a full‐length C/EBPα protein p42 (42 kDa) and a shorter p30 isoform (30 kDa). CEBPA mutations are found in 9–15% of Acute Myeloid Leukemia (AML) patients. N‐terminal frameshift mutations in the CEBPA gene lead to selective ablation of p42 expression, while C‐terminal mutations disrupt the dimerization and DNA‐binding ability of C/EBPα. AML patients harbor either mono‐ or biallelic CEBPA mutations (CEBPAmo or CEBPAbi) and both genotypes are frequently associated with concurrent mutations in other genes. The most commonly co‐occurring mutations in both groups are loss‐of‐function mutations in the methylcytosine dioxygenase TET2 (44.4% in CEBPAmo / 34.8% in CEBPAbi). While CEBPA mutations typically confer good prognosis, we found that the presence of mutated TET2 resulted in significantly reduced overall survival. Thus, we hypothesize that combinatorial effects of CEBPA mutations with TET2 loss specifically rewire transcriptional and epigenetic circuitries in AML cells, thereby strongly influencing disease outcome.Aims:We aim to elucidate the molecular mechanisms that underlie the cooperative effects of CEBPA and TET2 mutations by combining novel in vitro and in vivo AML models with integrated transcriptomic and epigenetic analyses.Methods:Cebpa‐mutated AML mouse models were used to generate cell line models for N‐terminal CEBPA mutations (Cebpa p30/p30 ) as well as mimicking biallelic CEBPA mutations (Cebpa p30/C−mut. ). We used the CRISPR‐Cas9 technology to introduce Tet2 mutations into Cebpa p30/p30 and Cebpa p30/C−mut. cell lines to study the functional cooperation of these mutations. RNA‐ and ATAC‐Seq analysis of &gt;30 clones representing the spectrum of CEBPA and/or TET2 mutations yielded a comprehensive dataset for in‐depth comparative analysis and correlation with patient data. In parallel, we establish fetal liver transplant models harboring Cebpa and Tet2 mutations to study their cooperative effect during leukemogenesis in vivo.Results:Introduction of Tet2 mutations into Cebpa p30/p30 and Cebpa p30/C−mut. cell lines conveyed a strong selective advantage on Tet2‐targeted cells over Tet2‐wild‐type cells. ATAC‐Seq analysis revealed that Tet2 loss caused a significant decrease in the accessibility of chromatin regions that are associated with cellular differentiation. Integration of these results with RNA‐Seq data showed that Tet2 deletion led to a strong deregulation of genes involved in RNA binding and splicing. Furthermore, Cebpa‐Tet2 co‐mutation induced significant down‐regulation of components of the p53 pathway (including Rprm and Zmat3).Summary/Conclusion:Our results from these novel models provide deeper insights into the global epigenetic and transcriptomic changes that depend on CEBPA and TET2 aberrations in a physiologically relevant mutational context. The data imply that loss of TET2 reinforces the differentiation block imposed by CEBPA mutations in AML. In addition, TET2‐deficiency interferes with components of the p53 pathway, thereby possibly compromising its activity. Integration of these findings with in vivo models and available patient data will enhance our understanding of gene cooperativity in AML and will provide entry points for the development of novel patient management strategies for patients with CEBPA and TET2 mutations.

CEBPA-mutated leukemia is sensitive to genetic and pharmacological targeting of the MLL1 complex.

The gene encoding the transcription factor C/EBPα is mutated in 10-15% of acute myeloid leukemia (AML) patients. N-terminal CEBPA mutations cause 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 incompletely understood. Here, we demonstrate that the MLL1 histone-methyltransferase complex represents a critical actionable vulnerability in CEBPA-mutated AML. Oncogenic C/EBPα p30 and MLL1 show global co-localization on chromatin and p30 exhibits robust physical interaction with the MLL1 complex. CRISPR/Cas9-mediated mutagenesis of MLL1 results in proliferation arrest and myeloid differentiation in C/EBPα p30-expressing cells. In line, CEBPA-mutated hematopoietic progenitor cells are hypersensitive to pharmacological targeting of the MLL1 complex. Inhibitor treatment impairs proliferation and restores myeloid differentiation potential in mouse and human AML cells with CEBPA mutations. Finally, we identify the transcription factor GATA2 as a direct critical target of the p30-MLL1 interaction. Altogether, we show that C/EBPα p30 requires the MLL1 complex to regulate oncogenic gene expression and that CEBPA-mutated AML is hypersensitive to perturbation of the MLL1 complex. These findings identify the MLL1 complex as a potential therapeutic target in AML with CEBPA mutations.

The splicing factor RBM25 controls MYC activity in acute myeloid leukemia

Cancer sequencing studies have implicated regulators of pre-mRNA splicing as important disease determinants in acute myeloid leukemia (AML), but the underlying mechanisms have remained elusive. We hypothesized that “non-mutated” splicing regulators may also play a role in AML biology and therefore conducted an in vivo shRNA screen in a mouse model of CEBPA mutant AML. This has led to the identification of the splicing regulator RBM25 as a novel tumor suppressor. In multiple human leukemic cell lines, knockdown of RBM25 promotes proliferation and decreases apoptosis. Mechanistically, we show that RBM25 controls the splicing of key genes, including those encoding the apoptotic regulator BCL-X and the MYC inhibitor BIN1. This mechanism is also operative in human AML patients where low RBM25 levels are associated with high MYC activity and poor outcome. Thus, we demonstrate that RBM25 acts as a regulator of MYC activity and sensitizes cells to increased MYC levels.

3159 - CEBPA-Mutant Acute Myeloid Leukemia is Sensitive to Small-Molecule-Mediated Inhibition of the Menin-MLL Interaction

3159 - CEBPA-Mutant Acute Myeloid Leukemia is Sensitive to Small-Molecule-Mediated Inhibition of the Menin-MLL Interaction

Differences Between CEBPA bZIP and TAD Mutations and Their Effect on Outcome-an Analysis in 4578 Patients with Acute Myeloid Leukemia

Mutations of the key myeloid transcription factor CCAAT/enhancer binding protein alpha (C/EBPa) are found in 5-10% of patients with acute myeloid leukemia (AML). Two mutational clusters exist, in the aminoterminal transcription activation domains (TAD1 or 2) and in the basic leucine zipper domain (bZIP) located at the carboxyterminal-part of the protein. Biallelic mutations (biCEBPA) have been found to be associated with improved outcome and are now included as an independent entity in the WHO-classification. In contrast, monoallelic CEBPA-mutations (moCEBPA) do not appear to provide prognostic information. We characterized a large cohort of AML patients for CEBPA mutations and further analyzed the mutational spectrum of mono- and biallelic CEBPA-mutant AML patients to better understand potential differences in the biology of these groups. Patients and Methods: Patients (including all age groups) analyzed had a newly diagnosed AML and were registered in clinical protocols of the Study Alliance Leukemia (SAL)(AML96, AML2003 or AML60+, SORAML) or the SAL-register. Screening for CEBPA mutations was done using PCR and capillary electrophoresis. All identified CEBPA mutations were confirmed using conventional Sanger sequencing and the samples were further analyzed using next generation sequencing (Trusight Myeloid Panel, Illumina) for the presence of associated alterations. Results: In the 4578 patients analyzed, 228 (5%) with CEBPA-mutations were identified. An initial analysis revealed substantial clinical differences between the different mutation subtypes. Patients with biCEBPA (n=111) were significantly younger (median age 46 yrs) than wt-CEBPA patients (median 57 yrs; p<.001). Interestingly, single bZIP mutant patients (n=64) had a similar median age (50 yrs.) as biCEBPA, whereas single TAD mutant patients (n=53) were significantly older (median 63 yrs.). In addition, WBC counts, CD34 positivity as well as the history of prior MDS differed between the subgroups (single TAD mutant had significantly lower WBC counts, lower rate of CD34 positivity and had a higher rate of prior MDS than biCEBPA and single bZIP mutant patients). Along with this, the distribution of co-mutations differed significantly between the subgroups, especially GATA2 mutations were more common in biCEBPA and single bZIP mutant patients (37% and 34%, respectively) compared to only 3% (single TAD)(p=.001). A similar pattern was seen for mutations in DNMT3A (8% biCEBPA, 20% single bZIP vs. 36% single TAD; p=.001), and NPM1 (3% biCEBPA, 8% single bZIP, 32% single TAD; p<.001). In 2897 patients, the different CEBPA mutations were correlated with outcome. This analysis indicated a differential effect of the individual mutations on outcome, with an improved rate of complete remission (CR), overall survival (OS) and event free survival (EFS) for biCEBPA and single bZIP mutations in univariate and multivariate analyses (shown for OS in Figure 1a). Given the similarity of single bZIP and biCEBPA mutations, it appears reasonable to speculate on a common mechanistical background, since most of the biCEBPA mutants include a bZIP alteration. Recent experimental evidence generated by several groups indeed supports a specific role of these bZIP missense mutations. To address this in the clinical context, we regrouped patients with mutant CEBPA into patients with (n=157) or without bZIP mutations (n=71), irrespective of the biallelic status. As illustrated in Figure 1b, the bZIP mutant group had a significantly better OS, similar results were obtained for EFS and CR. In multivariate analysis, the presence of a bZIP mutation was the strongest indicator for achievement of CR (HR 7.5, 95% CI: 3-19; p<.001), and together with favorable cytogenetics the factor associated with best OS (HR: .48; 95% CI .36-.64; p<.001). In conclusion, our results obtained in one of the largest cohorts of AML patients analyzed for CEBPA mutations indicate that especially the presence of a missense bZIP mutation is associated with a favorable outcome in AML patients. These data point to substantial differences in prognostic implications of individual CEBPA mutations and support the major functional divergence of these alterations. If confirmed, these results might necessitate further refinement of their use in AML-classification. [Display omitted] DisclosuresMiddeke:Sanofi: Honoraria. Platzbecker:Janssen-Cilag: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; TEVA Pharmaceutical Industries: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Thiede:AgenDix: Employment, Other: Ownership.

Disease evolution and outcomes in familial AML with germline CEBPA mutations

AbstractIn-depth molecular investigation of familial leukemia has been limited by the rarity of recognized cases. This study examines the genetic events initiating leukemia and details the clinical progression of disease across multiple families harboring germ-line CEBPA mutations. Clinical data were collected from 10 CEBPA-mutated families, representing 24 members with acute myeloid leukemia (AML). Whole-exome (WES) and deep sequencing were performed to genetically profile tumors and define patterns of clonal evolution. Germline CEBPA mutations clustered within the N-terminal and were highly penetrant, with AML presenting at a median age of 24.5 years (range, 1.75-46 years). In all diagnostic tumors tested (n = 18), double CEBPA mutations (CEBPAdm) were detected, with acquired (somatic) mutations preferentially targeting the C-terminal. Somatic CEBPA mutations were unstable throughout the disease course, with different mutations identified at recurrence. Deep sequencing of diagnostic and relapse paired samples confirmed that relapse-associated CEBPA mutations were absent at diagnosis, suggesting recurrence was triggered by novel, independent clones. Integrated WES and deep sequencing subsequently revealed an entirely new complement of mutations at relapse, verifying the presentation of a de novo leukemic episode. The cumulative incidence of relapse in familial AML was 56% at 10 years (n = 11), and 3 patients experienced ≥3 disease episodes over a period of 17 to 20 years. Durable responses to secondary therapies were observed, with prolonged median survival after relapse (8 years) and long-term overall survival (10-year overall survival, 67%). Our data reveal that familial CEBPA-mutated AML exhibits a unique model of disease progression, associated with favorable long-term outcomes.

Pharmacological targeting of the Wdr5-MLL interaction in C/EBPα N-terminal leukemia.

The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short 30 kDa C/EBPα translational isoform, termed p30, represents the most common type of CEBPA mutations in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPα p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as its down-regulation inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a novel small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30-cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML.