Both JAK2V617F and TET2 knockdown are required to sustain MPNs over long periods. (A) Schematic depiction of competitive and serial transplantation assay. FLs (CD45.2, 1 × 106 cells) and competitor WT BM cells (CD45.1, 5 × 106 cells) were cotransplanted into lethally irradiated recipients (CD45.1), and serial BM transplantation was performed using 1 × 106 recipient BM cells after 16 weeks posttransplantation. (B) Assessment of donor chimerism in the peripheral blood (PB; B1), spleen weight (B2), and histologic analysis (B3) of the first recipients. (B1) Chimerism at 20 weeks was significantly higher in all JAK2V617F, TET2KD, and double-mutant mice compared with WT mice (n = 10, each group). (B2) Spleens in first recipient mice after 12 weeks transplanted with JAK2V617F and double-mutant cells were much heavier than those receiving WT cells (n = 8-10, each group). (B3) Compared with WT mice, TET2KD mice show very mild extramedullary hematopoiesis; JAK2V617F mice show moderate extramedullary hematopoiesis, abnormal megakaryocytes, and fibrosis in BM and spleen; double-mutant mice show severe extramedullary hematopoiesis, abnormal megakaryocytes, and fibrosis. Compared with JAK2V617F mice, double-mutant mice exhibit more severe splenomegaly and extramedullary hematopoiesis. Hematoxylin and eosin (HE) stain and Gomori silver stain. Error bars represent standard deviation. *P < .05 and **P < .01 vs WT; †P < .05 and ††P < .01 vs JAK2V617F. (C) Assessment of donor chimerism in the PB (C1), spleen weight (C2), and histologic analysis (C3) of the second recipients. (C1) Chimerism at 16 weeks was significantly higher in TET2KD and lower in JAK2V617F mice compared with WT mice (n = 4, each group). Double-mutant mice showed sustained chimerism. (C2) Spleens in second recipient mice 12 weeks after transplantation with TET2KD and double-mutant cells were much heavier, and those in JAK2V617F were much lighter than those in WT cells (n = 6-8, each group). (C3) Compared with WT mice, TET2KD mice show mild extramedullary hematopoiesis; JAK2V617F mice show almost normal histologic findings; double-mutant mice exhibit severe extramedullary hematopoiesis, abnormal megakaryocytes, and fibrosis in BM and spleen. HE stain and Gomori silver stain. Error bars represent standard deviation. *P < .05 and **P < .01 vs WT; †P < .05 and ††P < .01 vs JAK2V617F.
