Order Matters: Sequence Of Mutation Acquisition In Myeloproliferative Neoplasms Impacts Disease Pathogenesis and Stem Cell Potency

Cancers evolve as a consequence of the stepwise accumulation of somatic lesions, with competition between subclones and sequential subclonal evolution. Some driver mutations arise either early or late in the evolution of different individual tumors, indicating that the final malignant properties of a subclone reflect the sum of mutations acquired rather than the order in which they arose. However virtually nothing is known about the cellular consequences of altering the order in which mutations are acquired.

The myeloproliferative neoplasms (MPNs) readily permit clonal analysis and are chronic malignancies, thereby facilitating the dissection of disease evolution and intra-tumoral clonal architecture. In this study, we genotyped >7000 colonies from 24 MPN patients who harbored mutations in both JAK2 and TET2. We found that mutation of JAK2 and TET2 each occurred first in 12/24 patients, with TET2-first and JAK2-first patients observed in all 3 MPN subtypes. Patients who acquired a TET2 mutation first presented on average 12.3 years later than JAK2-first patients (p=0.0043) and had a lower proportion of JAK2V617F homozygous erythroid colonies (p=0.0001) suggesting that the order of acquisition affected both cellular composition and disease evolution. Moreover, compared to TET2-first patients, JAK2-first patients had an increased frequency of megakaryocyte/erythrocyte progenitors (p=0.0001) and decreased frequency of common myeloid progenitors (p=0.001). In order to determine whether disease evolution was also affected at the stem cell level, we isolated individual HSCs (lin-CD34+CD38+CD90-CD45RA+) and found that JAK2 single-mutant HSCs were significantly less prevalent than their JAK2/TET2 double-mutant counterparts. In marked contrast, TET2 single-mutant HSCs were significantly more prevalent than JAK2/TET2 double-mutant HSCs, suggesting that TET2 single-mutant HSCs exhibit a substantial self-renewal advantage and were not out-competed by their double-mutant progeny. Consistent with this interpretation, functional progenitor expansion from single cell cultures of individual HSCs (as assessed by secondary colony formation) was differentially altered by the second mutation acquired. Whereas the mean number of secondary colonies produced by double-mutant HSCs was increased when TET2 was the second mutation (p=0.012), the mean number of colonies was decreased when JAK2 was the second mutation (p=0.002) despite both HSCs bearing the same set of genetic lesions.

Together our data indicate that acquisition of a JAK2 mutation reduces the competitiveness of TET2 single mutant HSCs, whereas acquisition of a TET2 mutation enhances that of JAK2 single mutant HSCs. These observations represent the first demonstration that the order of mutation acquisition influences stem and progenitor cell behaviour and clonal evolution in cancer.