Distinct effects of concomitant Jak2V617F expression and Tet2 loss in mice promote disease progression in myeloproliferative neoplasms

Abstract

Signaling mutations (eg, JAK2V617F) and mutations in genes involved in epigenetic regulation (eg, TET2) are the most common cooccurring classes of mutations in myeloproliferative neoplasms (MPNs). Clinical correlative studies have demonstrated that TET2 mutations are enriched in more advanced phases of MPNs such as myelofibrosis and leukemic transformation, suggesting that they may cooperate with JAK2V617F to promote disease progression. To dissect the effects of concomitant Jak2V617F expression and Tet2 loss within distinct hematopoietic compartments in vivo, we generated Jak2V617F/Tet2 compound mutant genetic mice. We found that the combination of Jak2V617F expression and Tet2 loss resulted in a more florid MPN phenotype than that seen with either allele alone. Concordant with this, we found that Tet2 deletion conferred a strong functional competitive advantage to Jak2V617F-mutant hematopoietic stem cells (HSCs). Transcriptional profiling revealed that both Jak2V617F expression and Tet2 loss were associated with distinct and nonoverlapping gene expression signatures within the HSC compartment. In aggregate, our findings indicate that Tet2 loss drives clonal dominance in HSCs, and Jak2V617F expression causes expansion of downstream precursor cell populations, resulting in disease progression through combinatorial effects. This work provides insight into the functional consequences of JAK2V617F-TET2 comutation in MPNs, particularly as it pertains to HSCs.

Figure 1

Tet2 loss accelerates the MPN phenotype of Jak2V617F mice. (A) Spleen weights of age-matched WT, Tet2^null, Jak2^VF, and Jak2^VF/Tet2^null mice 24 to 30 weeks old (mean ± standard error of the mean [SEM]; n = 4 in each group). (B) Photograph of spleens from Jak2^VF and Jak2^VF/Tet2^null mice. (C) Histopathologic sections of spleen from representative WT, Tet2^null, Jak2^VF, and Jak2^VF/Tet2^null mice (original magnification ×4; H&E stain). (D) Frequency of CD71⁺ Ter119⁺ erythroid precursor cells in spleen from age-matched WT, Tet2^null, Jak2^VF, and Jak2^VF/Tet2^null mice (mean ± SEM; n = 4 in each group). (E) Frequency of Mac1⁺ Gr1⁺ myeloid precursor cells in spleen from age-matched WT, Tet2^null, Jak2^VF, and Jak2^VF/Tet2^null mice (mean ± SEM; n = 4 in each group). (F) Frequency of CD150⁺ CD48⁻ LSK cells (LT-HSC), CD150⁻ CD48⁻ LSK cells (ST-HSC), and CD48⁺ LSK cells (MPP) in spleen from age-matched WT, Tet2^null, Jak2^VF, and Jak2^VF/Tet2^null mice (mean ± SEM; n = 4 in each group). *P < .05; **P < .005; ***P < .001.

Figure 2

Tet2 loss influences hematopoietic differentiation within the myeloid progenitor compartment of Jak2V617F mice but is insufficient to induce leukemic transformation. (A-C) White blood cell (WBC) count, hematocrit, and platelet (PLT) count of age-matched WT, Tet2^null, Jak2^VF, and Jak2^VF/Tet2^null mice 24 to 30 weeks old (mean ± SEM; n = 4 in each group). (D-F) Relative frequency of common myeloid progenitor (CMP), granulocyte macrophage progenitor (GMP), and megakaryocyte erythroid progenitor (MEP) cells in bone marrow from age-matched WT, Tet2^null, Jak2^VF, and Jak2^VF/Tet2^null mice (mean ± SEM; n = 4 in each group). (G-I) Histopathologic sections of bone marrow from representative WT, Tet2^null, Jak2^VF, and Jak2^VF/Tet2^null mice (original magnifications ×10 for G1-G4, ×40 for H1-H4, and ×100 for I1-I4; H&E stain).

Figure 3

Tet2 loss confers enhanced self-renewal to Jak2V617F HSPCs in vitro and in vivo. (A) Colony-forming unit assays from unfractionated bone marrow derived from WT, Jak2^VF, Tet2^null, and Jak2^VF/Tet2^null mice. Results represent the average of triplicate assays (mean ± SEM). The percentage of 45.2 donor chimerism assessed in peripheral blood (PB) total WBCs (B), PB Gr1⁺ cells (C), and PB B220⁺ cells (D) from lethally irradiated secondary recipients of Jak2^VF, Tet2^null, or Jak2^VF/Tet2^null LSK cells competed against an approximately equal number of 45.1 WT LSK cells, measured 4 to 17 weeks posttransplantation (mean ± SEM; n = 5 in each group). The percentage of 45.2 donor chimerism assessed in whole bone marrow (BM) cells (E), whole spleen (SPL) (F), and LSK BM cells (G) from lethally irradiated secondary recipients of Jak2^VF, Tet2^null, or Jak2^VF/Tet2^null LSK cells competed against an approximately equal number of 45.1 WT LSK cells, measured 18 weeks posttransplantation (mean ± SEM; n = 5 in each group). P values for each of the comparisons are indicated in the figure.

Figure 4

Jak2V617F expression and Tet2 loss cause distinct and nonoverlapping gene expression changes. (A) Dendrogram constructed from unsupervised hierarchical clustering of all 13 data sets from WT (n = 4), Tet2^null (n = 2), Jak2^VF (n = 3), and Jak2^VF/Tet2^null (n = 4) LSK cells using Pearson correlation. (B) Venn diagram depicting differentially expressed genes in LSK cells from Jak2^VF, Tet2^null, and Jak2^VF/Tet2^null mice (false discovery rate = 10%; minimum fold change relative to WT samples = 1.3). (C) Hierarchical clustering of expression profiles of all 12 data sets according to the 17 genes differentially expressed in either Jak2^VF, Tet2^null, or Jak2^VF/Tet2^null mice relative to WT controls. A red/blue color scale depicts normalized gene expression levels (red: high; blue: low). Dendrograms were constructed using Pearson correlation. (D) GSEA demonstrating enrichment for STAT5A target genes in Jak2^VF and Jak2^VF/Tet2^null LSK cells but not in Tet2^null LSK cells (top row: STAT5A targets UP; bottom row: STAT5A targets DOWN). (E) GSEA demonstrating enrichment of an HSC self-renewal signature in Tet2^null and Jak2^VF/Tet2^null LSK cells but not in Jak2^VF LSK cells. P values for each of the comparisons are indicated in the figure. NES, net enrichment score; n.s., not significant.