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315 Forward Genetic Screen Implicates Drivers of Leukemic Progression in a Novel Model of Trp53R270H myelodysplastic Syndrome

Program: Oral and Poster Abstracts
Type: Oral
Session: 636. Myelodysplastic Syndromes – Basic and Translational: Molecular Drivers and Therapeutic Implications
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Diseases, Myeloid Malignancies
Saturday, December 9, 2023: 4:30 PM

Daniel Chang, BSc1*, Klara E Noble-Orcutt, BS, MS2*, Wendy Hudson1*, Aishwarya Iyer, BSc, MSc3*, Emily Pomeroy, MS1*, Craig E. Eckfeldt, MD, PhD4, Aaron Sarver, PhD1*, Nuri Alpay Temiz, PhD1*, Michael Linden, MD, PhD4, Chad L Myers, PhD5,6*, David A. Largaespada, PhD1* and Zohar Sachs, MD, PhD2,7

1University of Minnesota, Minneapolis, MN
2Division of Hematology, Oncology, and Transplantation, Department of Medicine, University of Minnesota, Minneapolis, MN
3University of Alberta, Edmonton, AB, CAN
4University of Minnesota Medical School, Minneapolis, MN
5Department of Computer Science and Engineering, University of Minnesota, Minneapolis, MN
6Bioinformatics and Computational Biology Program, University of Minnesota, Minneapolis, MN
7Masonic Cancer Center, University of Minnesota, Minneapolis, MN

Myelodysplastic syndrome (MDS) is characterized by bone marrow failure and a highly variable clinical course. The most catastrophic complication of MDS is transformation to secondary acute myeloid leukemia (sAML). Notably, mutations in TP53 confer the single highest risk of transformation to sAML and death. However, some patients with TP53 mutated MDS do not develop sAML, suggesting that additional genetic events cooperate with TP53 mutations to transform MDS to sAML. Understanding the mechanisms of transformation of MDS to sAML could provide targets for therapeutic intervention.

To model the genetics of MDS, we crossed mice bearing Trp53R270H (Trp53 is the murine TP53 gene) and deletion of genes syntenic with human chromosome 5q (del(5q)). To discover how additional mutations contribute to disease progression, we utilized Sleeping Beauty (SB) transposon mutagenesis in Trp53R270H/del(5q) mice. SB transposase mobilized SB mutagenic T2/Onc transposons which randomly insert within the genome. T2/Onc transposons are designed to induce gain or loss of function alterations depending on the site and orientation of insertion with respect to targeted genes. We used the Mx1-Cre transgene to activate SB transposase and T2/Onc transposition in hematopoietic progenitors. Trp53R270H and del(5q) (or cytogenetically normal, CN) mice were crossed to SB mice to generate donor mice of the following genotypes: Trp53R270H/del(5q)/SB, Trp53R270H/CN/SB, Trp53WT/del(5q)/SB, Trp53WT/CN/SB mice, and mice without SB transposition, (no transposition, NT: Trp53R270H/del(5q)/NT). Bone marrow cells were transplanted into recipients, and SB insertional mutagenesis was activated using pI-pC to activate Cre. Mice receiving Trp53WT/CN/SB bone marrow developed more frequent T-cell leukemia (n=3/10) than myeloid leukemia (n=1/10). In contrast, mice receiving Trp53R270H/del(5q)/SB and Trp53R270H/CN/SB bone marrow developed predominantly myeloid leukemia (n=14/28) more commonly than T-cell leukemia (1/28). Mixed phenotype leukemia was seen in 7/28 of these mice. Together, these data demonstrate a strong bias towards myeloid disease in SB-mutagenized Trp53R270H bone marrow.

To identify genes with SB insertions, we performed RNA sequencing to detect SB T2/Onc transposon–endogenous gene fusion transcripts. Among Trp53WT/CN/SB leukemias, the most common recurrent SB fusions involved Notch1 and Ikzf1, as has previously reported for SB-associated T-cell leukemias. Among Trp53R270H/del(5q)/SB and Trp53R270H/CN/SB leukemias, the most common recurrent SB-fusions involved Erg, Eras, and Il2rb with Erg fusions detected 85% of Trp53 R270H leukemias (n=17/20). SB inserted upstream of Erg promoter indicating that these fusions likely upregulate expression of Erg. Indeed, Erg levels are significantly higher in leukemias that express SB-Erg fusions relative to leukemias that do not (p<0.0023).

ERG is not recurrently mutated in human AML, but the ERG gene locus is commonly amplified, especially TP53 mutant AML. ERG is known to support normal hematopoietic stem cell self-renewal. Notably, Erg-insertions were also detected in a model of MDS expressing stabilized cyclin E with SB-mediated progression to erythroleukemia (Loeb 2019). Using gene set enrichment analysis, we found that hematopoietic stem cell and leukemic stem cell signatures are enriched in Erg-SB fusion leukemias. In our analyses of two independent data sets (TCGA and BEAT AML), stem cell signatures are also among the most highly enriched pathways in human AMLs expressing high ERG levels. Furthermore, in a human AML single cell RNA sequencing dataset (van Galen 2019), we found that ERG expression is highest in AML cells with the most immature stem and progenitor-like features. Together, these findings implicate a role for ERG as a driver of progression of MDS to AML by enhancing aberrant self-renewal.

In summary, we present a novel murine model of Trp53/del(5q) MDS. In this model, Erg upregulation is associated with progression to AML and upregulation of leukemia stem cell gene expression profiles. These data implicate ERG as a major contributor to progression of MDS to secondary AML in the setting of mutant p53. Understanding the mechanisms of disease progression and self-renewal in myeloid malignancies with p53 mutations is critical to define effective therapeutic strategies in these rapidly fatal, treatment resistant diseases.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH