Abstract
Juvenile myelomonocytic leukemia (JMML) is a rare but aggressive pediatric myeloproliferative neoplasm driven by hyperactivation of the Ras signaling pathway. Elevated fetal hemoglobin, thrombocytopenia, and PTPN11 mutations are recognized poor prognostic factors in JMML, although the underlying mechanisms remain unclear. SHP2, encoded by PTPN11, functions as a critical positive regulator of Ras signaling, and pathogenic gain-of-function mutations in PTPN11 are the leading driver in approximately 35% of JMML patients. However, knock-in mice with the most common SHP2-E76K mutation display typical MPN-like phenotypes but do not fully reproduce critical JMML features such as onset during infancy, erythroid dysplasia, and thrombocytopenia (Dan Xu, et al. Journal of Experimental Medicine, 2011). These findings suggest that SHP2 mutation alone may be insufficient to fully drive JMML pathogenesis, and that additional genetic alterations, such as PTPN11 gene copy number variation (CNV), may be required to recapitulate the full disease phenotype.
In this study, we investigated the role of PTPN11 copy number gain in JMML pathogenesis and explored its underlying mechanisms. Quantitative PCR were performed on bone marrow samples from JMML patients to assess PTPN11 CNV. Copy number gain of PTPN11 was detected in approximately 68.7% of CFU-derived colonies from JMML patients carrying PTPN11 mutations. These mutant colonies exhibited markedly elevated PTPN11 copy numbers (mean 6.4 ± 3.2) compared to non-mutant colonies (mean 2.0 ± 0.2), suggesting that clonal expansion is associated with both mutational activation and gene dosage amplification of PTPN11. Colony-forming unit (CFU) assays revealed that hematopoietic progenitor cells carrying PTPN11 mutations exhibited higher PTPN11 copy number compared to wild-type cells from the same patients, suggesting a selective advantage for mutant clones with gene dosage gain. These mutant clones demonstrated marked clonal expansion and lineage skewing, as evidenced by increased formation of BFU-E, CFU-E, and CFU-GM colonies, consistent with enhanced self-renewal and impaired differentiation. Mechanistically, PTPN11 copy number gain resulted in elevated expression of mutant SHP2 protein, leading to hyperactivation of the Ras/ERK signaling pathway and promoting the proliferative advantage of mutant progenitors.
To experimentally evaluate the pathogenic impact of mutant PTPN11 gene dosage in vivo, we established transgenic mice expressing human SHP2-E76K mutant under the hematopoietic-specific Vav promoter, with defined transgene copy numbers. Three independent lines were generated, carrying approximately 1, 2, or 6 copies of the SHP2-E76K transgene haploid genome. The transgenic mice exhibited copy number–dependent hematopoietic abnormalities. Notably, mice from the 6-copy line developed significant thrombocytopenia and anemia as early as 3 weeks of age, consistent with early-onset disease of JMML, and these abnormalities persisted into adulthood. These mice also showed the most pronounced leukocytosis, with marked elevations in circulating granulocytes and monocytes. In contrast, the 1-copy and 2-copy lines exhibited only mild increases in leukocytosis. Although transient thrombocytopenia and anemia were observed in these lower-copy lines at 3 weeks of age, both parameters gradually returned to near-normal levels by adulthood. Flow cytometric analysis revealed a marked increase in the frequency of Lineage⁻Sca-1⁺c-Kit⁺ (LSK) cells, enriched for hematopoietic stem and progenitor cells, in both the bone marrow and spleen of 6-copy mice, relative to 1- and 2-copy lines. This expansion reflects increased hematopoietic output and stem/progenitor activity driven by high transgene dosage. The substantial accumulation of LSK cells in the spleen also indicates robust extramedullary hematopoiesis (EMH), a hallmark of JMML. In contrast, 1- and 2-copy mice exhibited only modest LSK expansion and minimal EMH. Histological analysis further confirmed EMH and myeloid infiltration in the spleen and liver of high-copy animals.
Together, these findings define PTPN11 copy number gain as a novel leukemogenic mechanism that cooperates with activating mutations to drive JMML pathogenesis. These results also highlight the clinical relevance of assessing PTPN11 copy number as a potential biomarker for risk stratification and prognosis in JMML.
