-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

839 CEBPA-Driven Expression of the Transcriptionally Inactive deltaTP73 Isoform Phenocopies TP53mutated Poor Risk and Drug-Resistant Acute Myeloid Leukemia

Program: Oral and Poster Abstracts
Type: Oral
Session: 617. Acute Myeloid Leukemias: Biomarkers, Molecular Markers and Minimal Residual Disease in Diagnosis and Prognosis: Deciphering Risk by Genetic and Epigenetic Features
Hematology Disease Topics & Pathways:
Research, Acute Myeloid Malignancies, AML, apoptosis, Translational Research, Combination therapy, bioinformatics, Diseases, Therapies, therapy sequence, Myeloid Malignancies, Biological Processes, molecular biology, Technology and Procedures, gene editing, multi-systemic interactions, molecular testing, omics technologies
Monday, December 11, 2023: 3:45 PM

Diego Pereira-Martins, PhD1*, César Alexander Ortiz Rojas, Ph.D.2*, Isabel Weinhaeuser, PhD1*, Bart-Jan Wierenga, PhD1*, Vincent Van Den Boom, Ph.D.1*, Fatemeh Mojallali, MSc1*, Dominique Sternadt, MSc1*, Thiago Mantello Bianco, Ph.D.3*, Nisha van der Meer, MD, MSc1*, Douglas RA Silveira, MD, PhD4*, Lynn Quek, MD, PhD5*, Emanuele Ammatuna, MD, PhD1*, Antonio R Lucena-Araujo, Prof.6*, Gerwin A. Huls, Prof.7, Eduardo Magalhães Rego, MD, PhD8,9 and Jan Jacob Schuringa, PhD10

1Department of Experimental Hematology, University Medical Centre Groningen, Groningen, Netherlands
2Department of Internal Medicine, Medical School of Ribeirao Preto and the Center for Cell Based Therapy, University of São Paulo, Sao Paulo, Brazil
3Department of Medical Images, Haematology, and Clinical Oncology, Ribeirão Preto Medical School, University of Sao Paulo, Ribeirão Preto, Brazil
4School of Cancer and Pharmaceutical Sciences, King's College London, London, ENG, United Kingdom
5Myeloid Leukaemia Genomics and Biology Group, School of Cancer and Pharmaceutical Sciences, King's College London, London, United Kingdom
6Department of Genetics, Federal University of Pernambuco, Recife, Brazil
7Department of Hematology, University Medical Center Groningen, Groningen, Netherlands
8Hematology Service, LIM-31 and State of Sao Paulo Cancer Institute, University of São Paulo, Faculdade de Medicina, São Paulo, São Paulo, Brazil
9Instituto do Cancer do Estado de Sao Paulo, Faculdade de Medicina da Universidade de Sao Paulo, Sao Paulo, BRA
10Department of Experimental Hematology, University Medical Center - Groningen, Groningen, Netherlands

Mutations in TP53 (TP53mut) are present in roughly 10% of acute myeloid leukemia (AML) patients and represent a unique subgroup of patients with very poor prognosis. While the exact mechanisms of TP53mut-driven leukemogenesis remain elusive, transcriptional changes associated with suppression of the TP53 signaling pathway are considered to confer a dismal prognosis on TP53mut patients. Using molecular profiling of a large cohort of AML patients (n=823) we identify a new poor prognostic subgroup in AML that is TP53WT but shares strong similarities with TP53mut patients. Patients included in this new group present increased expression of the oncogenic TP73 gene isoform ΔNp73, which lacks the transactivation domain but still binds to chromatin. We find that the transcriptional and metabolic program of ΔNp73high AMLs strongly resembles that of TP53mut AML, being enriched for stemness signatures like “17LSC” and “LSC UP”. In line with TP53mut AML, ΔNp73high/TP53WT frequently co-occurs with U2AF1, SRSF2, TET2, and RUNX1 mutations. Lentiviral overexpression (OE) of ΔNp73 in healthy CD34+ cells increased cell proliferation and stemness. ΔNp73-OE in TP53WT AML models, imposed drug resistance against several standard-of-care cytotoxic therapies in AML (e.g., AraC, venetoclax (VEN) ± azacytidine (AZA), and FLT3 inhibitors), while drug resistance was not further enhanced upon ΔNp73-OE in TP53mut AML models. These results were validated using ex vivo treated primary AML samples (n=80), whereby ΔNp73high samples were more resistant to several cytotoxic therapies, including VEN+AZA. Lentiviral ΔNp73-OE in primary APL samples (n=10) allowed efficient engraftment in vivo using xenograft mouse models with colonization of secondary organs and splenomegaly, which was not observed in the empty vector controls.

To unravel mechanisms via which ΔNp73 controls oncogenic transformation we performed RNA-seq and ChIP-seq to identify direct transcriptional targets of TP53, ΔNp73, and the transcriptionally active isoform TAp73. These studies revealed that ΔNp73 and TP53 compete for the same target genes, whereby ΔNp73-OE resulted in complete loss of TP53 binding and strong downregulation of these loci, including downregulation of known TP53 target genes CDKN1A, BBC3, and GAS6, but also of TP53 itself. Gene set enrichment analyses of molecular programs in ΔNp73-OE cells revealed that these were associated with the terms “LSC UP”, “CEBPA targets”, and “SIGNAL TRANSDUCTION BY TP53”, indicating that ΔNp73 phenocopies TP53mut AML.

Chromatin accessibility data on AML patients revealed a differentially accessible region in intron 2 (+24 Kb after the transcription start site, referred to as peak B) of the TP73 gene, with unknown regulatory functions on TP73 expression in AML. To access the potential enhancer functions of this region driving ΔNp73 expression we generated CRISPR/Cas9-KO MOLM13 cells lacking the peak B region (MOLM13-KO). MOLM13-KO cells exhibited reduced ΔNp73 expression, and the ability to up-regulate ΔNp73 expression upon high dose AraC-induced stress was abrogated. Since CEBPA was a transcription factor predicted to bind to Peak B, we investigated whether CEBPA could regulate the expression of the different TP73 isoforms. CEBPA-KD in AML cells resulted in decreased ΔNp73 levels and restored the TAp73 expression, suggesting that CEBPA controls the balance of the distinct TP73 isoforms. Pharmacological inhibition of CEBPA using the FDA-approved drug Guanfacine (GFC), diminished ΔNp73 levels and restored drug sensitivity against several cytotoxic agents in ΔNp73-OE AML cells. Moreover, the combination of GFC+VEN displayed strong synergistic effects in a cohort of ex vivo treated AML blasts (n=20), including patients with TP53mut, which are often resistant to VEN.

Here, we describe a new AML patient subgroup that is TP53WT but behaves similarly to TP53mut AML due to the expression of the ΔNp73 isoform that impairs TP53 signaling. This patient subgroup displays a very poor prognosis and is resistant to standard-of-care drugs used for AML patients. We identify a novel enhancer region that drives the expression of oncogenic TP73 isoforms, which is controlled by CEBPA, and inhibition of CEBPA by GFC enhances sensitivity to cytotoxic therapies in TP53mut-like AMLs. Our insights further refine patient stratification and provide alternatives for treatment for the most dismal group of AML patients.

Disclosures: Rego: Astellas: Research Funding, Speakers Bureau; Abbvie: Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding. Schuringa: Byondis BV: Research Funding.

*signifies non-member of ASH