Serum erythropoietin level as a selective pressure in clonal hematopoiesis: Insights into the mutational landscape of myeloid neoplasias Free

Outgrowth of a founder mutant clone is an initial event in leukemogenesis. The corresponding somatic hit likely conveys a fitness advantage over a polyclonal background. Several factors may modulate the competitiveness of hematopoietic stem or progenitor cells, including aging, genotoxic stress, repeated cycles of stress hematopoiesis, and inherited genetic fitness deficiency. The nature of these conditions provides a particularly fertile environment in which only certain mutant clones thrive. For instance, chemotherapy facilitates expansion of PPMD1^MT or TP53^MT clones, aging-related loss of fitness predisposes to outgrowth of TET2^MT clones, while many inherited bone marrow failure (BMF) syndromes are associated with specific clonal acquisitions due to somatic gene rescue. Identification of factors enabling the expansion of clones with specific mutations may help to develop targeted preventive, screening, or therapeutic strategies.

Recently, high levels of erythropoietin (EPO) induced by frequent blood donation were shown to favor the emergence of DNMT3A^R882 clones due to their increased EPO sensitivity¹. We theorized that this may be a more general phenomenon operating in other diseases characterized by high EPO levels. Thus, we selected 644 patients in whom endogenous serum EPO levels had been assessed and who underwent mutational NGS analysis. Given that this cohort consisted of patients with hematologic neoplasms, EPO levels were broad with the majority falling outside the normal clinical reference range. For this study we used EPO levels ≥100mU/mL as the threshold to define high and low EPO groups. This threshold laid above the 75^th percentile for our cohort (M=31.1; IQR=109.7) to capture outliers more likely to reflect biologically significant erythropoietic stress. DNMT3A^MT were present in 14% of high EPO and in 12% of low EPO groups (p=0.5). However, in the high EPO group, R882 constituted 32% of DNMT3A^MT while in the low EPO group R882 was found in only 11% of DNMT3A^MT cases (p=.02). In a reverse fashion, the mean level of EPO in DNMT3A^R882 was 441.7mU/mL while it was 215.3mU/mL in other DNMT3A^MT carriers (p=0.03) and 186.2mU/mL in DNMT3A^WT cases (p<0.001). To assess whether the effect on DNMT3A was dependent on EPO levels rather than anemia, we determined mean hemoglobin levels in R882 (µ=10.2g/dL), other DNMT3A^MT (µ=10.4g/dL), and wild type cases (µ=10.9g/dL) and found no significant differences (p=0.33). To assess whether R882-EPO sensitivity was related to EPO-R expression levels, we compared EPO-R expression in R882 (µ=2.7), non-R882 (µ=2.6) mutations, and WT cases (µ=2.8) and found no significant difference.

To assess whether the impact of high EPO states was specific to DNMT3A^MT we next analyzed the distribution of other mutations. We observed that frameshift mutations in BCOR (p = 0.013) and BCORL1 (p=0.036) were significantly more frequent in high EPO patients. BCOR^MT was also overall more prevalent in the high EPO group (p=.001), while BCORL1 frequency was similar across EPO strata (p=0.77) showing a similar trend to DNMT3A^MT. Beyond these genes, other patterns emerged, particularly within CH-associated genes. Individuals with U2AF1^MT had elevated EPO (p=.0087), with variant-specific enrichment: p.S34F was more common in high EPO (p=.038) whereas p.Q157P predominated in low EPO patients (p=0.038). No significant EPO associations were observed for other CH-related genes such as ASXL1, CBL, and TET2.

The limitations of this study notwithstanding, these findings support a biologically meaningful relationship between elevated EPO levels and the acquisition or enrichment of specific CH-associated mutations in patients with hematologic malignancies. The association of high EPO with DNMT3A^R882, frameshift mutations in BCOR/L1, and variant-specific enrichment in U2AF1 suggests that EPO-driven stress may contribute to clonal selection or expansion.

Our study of high EPO levels and DNMT3A^MT analysis illustrates that external conditions may influence selection pressure for specific mutations. However, selection pressure may be a complex combination of factors. Non-malignant conditions with high EPO, such as hypoxia or intrinsic BMF, may also create conditions facilitating the acquisition of specific somatic hits. Recognition of evolutionary pressures and their associations with specific mutations may improve our understanding of CH pathogenesis, and conceptualization of therapeutic or preventative measures.