JAK2V617F Impairs Lymphoid Differentiation in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) arise from the clonal acquisition of a driver mutation in a hematopoietic stem cell (HSC), often decades before the emergence of clinical disease. A defining characteristic of an HSC is its potential to give rise to both myeloid and lymphoid hematopoietic lineages. Yet the most common MPN driver mutation, JAK2^V617F, has only rarely been observed in lymphocytes. We conducted this study to determine why the JAK2^V617F clone does not contribute to mature lymphocytes.

We directly measured myeloid and lymphoid lineage contribution of normal and MPN HSCs in 133 patients with JAK2^V617F MPN. Peripheral blood subpopulations were fractionated via fluorescence-activated cell sorting (FACS) and JAK2^V617F mutation allele frequency (MAF) was measured in each subpopulation by droplet digital PCR. JAK2^V617F was enriched in neutrophils and maintained in erythroid progenitors (EPs) relative to HSCs. In contrast, JAK2^V617F alleles were largely absent in T and B cells relative to HSCs.

Lymphopoiesis declines with age, potentially explaining the absence of JAK2^V617F in lymphocytes from older patients with MPN even if JAK2^V617F has no effect on lymphopoiesis. This “JAK2^V617F neutral” hypothesis predicts that patients who acquired the mutation at a younger age, and those who harbor the mutation for a longer duration, have a higher likelihood of accumulating JAK2 mutated lymphocytes. However, we found that JAK2^V617F MAF in T and B cells across our cohort does not correlate with either patient age at diagnosis or duration of clinical disease. Thus, predictions of the “JAK2^V617F neutral” hypothesis are not supported by patient data.

The alternative explanation is that JAK2^V617F impairs lymphocyte differentiation from mutated progenitors. We tested this “JAK2^V617F adverse” hypothesis by tracking JAK2^V617F during various stages of lymphopoiesis. To assess the relative bias of JAK2^V617F HSCs during the early stages of lymphoid and myeloid commitment, we measured JAK2^V617F MAF in common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs) and HSCs isolated by FACS from the peripheral blood of 79 patients with JAK2^V617F MPN. We found that while myeloid commitment from HSC to CMP was associated with an increase in mean JAK2^V617F MAF, there was no statistically significant change in mean MAF between CLPs and the parent HSCs across the cohort. Therefore, JAK2^V617F HSCs are not completely excluded from the earliest stages of lymphoid differentiation.

To further define the extent to which JAK2^V617F affects lymphopoiesis, we used FACS to isolate CLPs and CMPs from 17 patients with JAK2^V617F polycythemia vera (PV) and differentiated each subpopulation in vitro into T cell progenitors (pro- and pre-T cells) and erythroblasts (EBs), respectively. We found that all CLP samples could be differentiated into T cell progenitors. However, we observed a marked depletion of JAK2^V617F alleles in progressively mature T cell progenitors (Figure 1). In contrast, JAK2^V617F alleles were further enriched in progressively mature erythroblasts. The in vitro differentiation potential of MPN progenitors thus supports the “JAK2V617F adverse” hypothesis.

To determine the effect of JAK2^V617F on lymphopoiesis in vivo, we performed competitive transplantations of lethally irradiated CD45.1 mice with congenic CD45.2 donor whole bone marrow (WBM) cells harboring JAK2^V617F or wild type JAK2 (JAK2^WT). Measuring CD45.2 chimerism by FACS in lymphocytes and neutrophils from engrafted mice revealed that only CD45.2 cells harboring JAK2^V617F were preferentially depleted in lymphocytes (Figure 2). Therefore, JAK2^V617F HSCs displayed a competitive disadvantage relative to their JAK2^WT counterparts during lymphopoiesis in vivo.

Myeloid proliferation in MPN driven by JAK2^V617F is well recognized but does not fully explain certain MPN complications such as increased risk of infections and second malignancies. Our findings demonstrate impaired lymphoid differentiation from JAK2^V617F stem and progenitor cells that may also contribute to clinical MPN phenotypes. Further study is ongoing to define actionable mechanisms through which JAK2^V617F interferes with T cell differentiation.