Mosaic loss of chromosome Y (LOY) is a highly prevalent somatic event in many cancers (~30% of male tumors) and is frequently observed in males with age-associated clonal hematopoiesis of indeterminate potential (CHIP; ~50% by age 70-80 years). However, the mechanistic role of LOY in cancer remains poorly understood. Some studies suggest LOY is, in part, a manifestation of inherent genomic instability and a marker of biological aging, but mounting evidence supports an alternative hypothesis — that LOY is itself a cancer driver.

We analyzed >1500 male patients with acute myeloid leukemia (AML) and found that ~10% harbored somatic LOY. Notably, LOY was significantly enriched (p<0.01) in specific subtypes — most strikingly in 54% of male patients with RUNX1::RUNX1T1 (AML1-ETO) AML — including pediatric cases. These data suggest that LOY may actively contribute to the pathogenesis of certain AML subtypes rather than being a mere passenger event or age-related artifact. Supporting clinical relevance, Schlenk et al. (2004) reported that LOY in AML1-ETO AML predicts shorter remission and inferior overall survival.

Several candidate Y-encoded genes — including UTY/KDM6C, KDM5D, CSF2RA, TMSB4Y, and CRLF2 — have been implicated as drivers of LOY's oncogenic effects across different malignancies. However, loss of multiple genes, rather than any single gene, may underlie the phenotypic consequences of LOY, highlighting the need for robust, lineage-specific, and otherwise isogenic models to study pathogenic mechanisms.

To investigate this, we immortalized primary murine hematopoietic progenitors in an ex vivo culture system using a retrovirally-expressed estrogen receptor-HoxB8 fusion protein, which maintains hematopoietic cells as immature, multipotent progenitors in the presence of estradiol and allows synchronized differentiation upon estradiol removal. Using two CRISPR/Cas9 strategies targeting ChrY repeat elements, we generated isogenic progenitors with or without ChrY deletion, confirmed by qRT-PCR and DNA PCR. In short-term culture of undifferentiated myeloid progenitors, LOY had no significant effects on growth kinetics, confirmed by flow cytometry cell tracing and cell cycle analysis. However, in longer-term competitive co-culture assays, LOY cells showed significantly reduced growth versus wildtype ChrY (WTY) cells, evident by day 7. Similar paradoxical reduced fitness has been seen previously with single gene models of clonal hematopoiesis-associated mutations. Transcriptomic profiling revealed 195 upregulated and 198 downregulated genes in LOY versus WTY cells (padj<0.05). Downregulated ChrY-encoded genes including Uty, Ddx3y, and Kdm5d validated LOY, and pathway enrichment of non-ChrY genes revealed significant downregulation (p=0.008) of oxidative phosphorylation genes in LOY cells, suggesting metabolic reprogramming.

Following estradiol withdrawal, which inactivates the HoxB8 stem cell transcription factor and induces terminal differentiation, both WTY and LOY Hoxb8 cells lost the progenitor marker Cd117 (c-Kit) and upregulated mature myeloid markers Gr-1 and Cd11b within four days. However, LOY cells consistently showed accelerated myeloid maturation, confirmed by flow cytometry and morphology.

In contrast to cells with LOY alone, in cells co-expressing the oncogene AML-ETO9a (AE9a), LOY significantly impaired myeloid differentiation compared to WTY. RNA-seq of AE9a cells with and without LOY revealed 638 up- and 540 down-regulated genes (padj<0.05), with significant reprogramming of translation, stress response, and biosynthetic pathways. Functionally via in vitrotransformation assays, both AE9a WTY and LOY cells proliferated and survived two-fold longer than empty vector controls (6 vs 12 days); however, only AE9a LOY cells sustained long-term viability beyond 21 days, while AE9a WTY cells progressively died off. In methylcellulose colony formation assays, AE9a LOY cells were the only genotype that produced large rapidly growing colonies (>200 cells), while other groups (AE9a WTY, and LOY or WTY without AE9a) formed only microcolonies (<20 cells).Together, our findings suggest that LOY impedes myeloid differentiation and enhances self-renewal in concert with specific leukemia oncogenes, revealing a context-dependent effect to promote clonal expansion and leukemic progression. These results challenge the paradigm that LOY is merely a bystander in leukemia and support further mechanistic study of its role in AML.

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2025
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