Abstract 6500: Application of human inducible pluripotent stem cells (hiPSCs) with CRISPR/Cas9-mediated deletion of <i>CUX1</i> as a model for high-risk myeloid neoplasms

Abstract Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with cytogenetic abnormalities of chromosome 7 have dismal survival outcomes. Monosomy 7 or deletion of 7q (-7/del7q) are adverse-risk cytogenetic features found in up to 12% of MDS/AML patients and are further enriched in therapy-related MDS/AML. Despite significant scientific effort, no targeted therapy is available for patients with -7/del7q MDS/AML. Several genes are associated with -7/del7q leukemogenesis. Cut-like homeobox 1 (CUX1) is a gene encoding a context-dependent transcription factor required for several molecular pathways, including epigenetic signaling. CUX1 is deleted in -7/del7q MDS/AML and somatically mutated in 2-4% of MDS/AML. CUX1 mutations and CUX1 deletions are associated with a poor prognosis, and are known to co-occur with adverse risk TP53 mutations and a complex cytogenetic background. Thus, there is a need for novel human models of MDS/AML to isolate the effect of CUX1 alterations. To address the need for high-fidelity models of MDS/AML with high-risk genetic changes, we transfected human inducible pluripotent stem cells (hiPSCs) with CUX1 targeting gRNA and CRISPR/Cas9 ribonucleotide-protein (RNP) complexes to generate isogenic hiPSC lines bearing CUX1+/- and CUX1-/- genotypes alongside parent hiPSCs and sham transfection lines with a CUX1+/+ background. These derivative cell lines underwent rigorous validation by karyotypic G-banding analysis, sequencing, immunoblotting, trilineage germ layer differentiation, and analysis of hiPSC transcriptional and immunophenotypic markers. We hypothesized that the loss of one or both alleles of CUX1 would result in transcriptional changes similar to CUX1 mutated and -7/del7q MDS/AML. This model can be further leveraged to identify potential treatment targets. We obtained bulk RNA sequencing of undifferentiated hiPSCs and hiPSC-derived hematopoietic stem and progenitor cells (i-HSPCs) of each genotype (CUX1+/+, CUX1+/-, and CUX1-/-). We identified several differentially expressed genes and novel long non-coding RNAs in hiPSCs with Cas9-mediated CUX1 deletions, including downregulation of distinct subsets of genes in CUX1-/- and CUX1+/- hiPSCs. Bulk RNA sequencing of CUX1-/- and CUX1+/- i-HSPCs identified an association between reduced expression of thioredoxin-interacting protein (TXNIP), a known regulator of autophagy. This study supports the use of hiPSC-based CRISPR/Cas9 gene editing as a novel platform to identify gene-specific alterations in genotypically and cytogenetically complex myeloid neoplasms. Citation Format: Joseph Michael Cannova, Kaina A. Millan, Yuqing Xue, Aubrianna S. Ramsland, Zara Khosravi, Adesewa Ogunsusi, Matthew Jotte, Megan McNerney, Michael Drazer. Application of human inducible pluripotent stem cells (hiPSCs) with CRISPR/Cas9-mediated deletion of CUX1 as a model for high-risk myeloid neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6500.