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2755 Identifying PTPRJ As a Novel Mediator of CEBPA-Mutated AML

Program: Oral and Poster Abstracts
Session: 602. Myeloid Oncogenesis: Basic: Poster II
Hematology Disease Topics & Pathways:
Research, Diseases, Biological Processes
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Alexandra Lubin, PhD1, Catherine Hockings, FRCPath, MBBChir, MRCP, MA1*, Yvette Hoade, BSc1*, Lucy Copper, MSci1*, Phoebe Dace, BSc1*, Catherine Zhu, MB BChir1*, Helen Brown, PhD1*, Ellen Nuttall Musson1*, Katie-Jo Miller, BSc1* and Elspeth Payne, MD, PhD, FRCPath1,2

1Haematology, Cancer Institute, University College London, London, United Kingdom
2Department of Haematology, University College London Hospital, London, United Kingdom

10-20% of acute myeloid leukaemia (AML) cases in children and adults carry mutations in the transcription factor CCAAT/enhancer binding protein alpha (CEBPA). CEBPA is transcribed from a single exon and produces two isoforms, p30 and p42, with characteristic mutations, either frameshift mutations in the N-terminal portion (CEBPANterm) leading to production of only the p30 isoform, or in frame mutations in the C-terminal portion (CEBPACterm), disrupting DNA binding.

The most frequent secondary driver mutation in CEBPA-mutated AML, is the second allele of CEBPA. Additionally, in rare germline CEBPA cases, the founding mutation is usually CEBPANterm, with all individuals showing acquisition of a second hit mutation in the CEBPACterm on the alternate CEBPA allele at the time of AML diagnosis, which has near 100% penetrance in these families. This highlights a strong selective pressure for this very specific biallelic mutation pattern.

We have developed zebrafish models with germline cebpaNterm and cebpaCterm mutations that faithfully recapitulates the disease, with all homozygous and double heterozygous mutants showing loss of mature myeloid cells, and impaired survival. All double or compound mutants die by 10 weeks of age, with haematopoietic stem and progenitor cell (HSPC) expansion, and leukaemia in around 30% of these animals.

Our model also demonstrates distinct phenotypes for cebpaNterm and cebpaCterm mutations during developmental haematopoiesis prior to leukaemia onset. We observed that cebpaNterm and cebpaCterm mutations have opposite effects on the expression of c-myb, a transcription factor expressed by proliferating HSPC, from 3 days post fertilization. cebpaNterm/Nterm mutants show an increase in c-myb while cebpaCterm/Cterm show reduced c-myb expression during development.

To uncover the mechanisms by which cebpaNterm and cebpaCterm effect HSPCs we undertook RNASeq of sorted Tg(cd41:eGFP)lo HSPCs from fish of all possible genotype combinations. We identified both shared and unique expression profiles between genotypes. We also identified 4 genes that that were differentially regulated in both cebpaNterm/Nterm and cebpaCterm/Cterm HSPC, but in opposing vectors (upregulated in cebpaCterm/Cterm and downregulated in cebpaNterm/Nterm) (Figure 1). We hypothesised that these 4 genes are candidate drivers of selective pressure for the common CEBPACterm/Nterm mutation combination in human AML, and thus the malignant phenotype it confers. One of these candidate genes, PTPRJ, has also recently been identified as a frequently mutated gene in CEBPA-mutated AML in children thereby validating the relevance of our dataset and our approach.

To define the effects of these 4 candidate genes we then conducted an F0 CRISPR knock-out screen in all cebpa mutant combinations in our zebrafish model, using c-myb expression as a phenotypic readout. We identified ptprja as our primary candidate gene and showed that loss of ptprja alone and in combination with cebpaCterm/+ reduced expression of c-myb but had no effect on expression of c-myb in cebpaCterm/Cterm, indicating that ptprja loss reduces c-myb-expressing HSPC and that this is dependent on functional cebpa DNA binding.

We then examined the impact of ptprja knockout in juvenile zebrafish. Homozygous and double heterozygous mutants develop a pre-leukaemia expansion of HSPC before they succumb to death. We showed that that loss of ptprja in cebpaCterm/Cterm fish accelerates this pre-leukaemic expansion of Tg(cd41:eGFP)lo HSPCs in 4 week fish (Figure 2). We also showed an opposing effect in the cebpaNterm/Nterm mutants at 2 weeks, where loss of ptprja resulted in decreased pre-leukemic expansion of HSPC.

These data indicate a role for PTPRJ in mediating HSPC expansion in CEBPA-mutated AML and suggest that PTPRJ may mediate the mechanism of clonal selection in those with a CEBPA mutation that drives the acquisition of a second CEBPA mutation on the opposing allele, leading to the common CEBPACterm/Nterm in patients with CEBPA-mutated AML.

Disclosures: Payne: Jazz: Honoraria; Novartis: Honoraria; clovis: Honoraria.

*signifies non-member of ASH