Loss of Tyrosine Kinase 2 Does Not Affect the Severity of Jak2V617F-induced Murine Myeloproliferative Neoplasm

Results

Loss of TYK2 did not affect disease severity of JAK2VF-induced murine MPN. The expression levels of Jak1, Jak2, Jak3, and Tyk2 in the BM cells of WT, Jak2VF, and Jak2VF/Tyk2KO mice were measured by qPCR (n=3–5), and the results are shown in Figure 1A. Tyk2 expression was not detected in Jak2VF/Tyk2KO mice, and was 0.7-fold lower in Jak2VF mice than in WT mice (p<0.01). Due to transgene expression, Jak2 expression was 1.7-fold and 1.4-fold higher in Jak2VF and Jak2VF/Tyk2KO mice, respectively, than in WT mice. Jak1 expression was 0.8-fold and 0.7-fold lower in Jak2VF and Jak2VF/Tyk2KO mice, respectively, than in WT mice. The expression of Jak3 did not differ among the three groups. All JAK kinases, with the exception of TYK2, were equally expressed in Jak2VF and Jak2VF/Tyk2KO mice.

Next, we compared the phenotype of WT, Jak2VF, and Jak2VF/Tyk2KO mice. Both Jak2VF mice and Jak2VF/Tyk2KO mice showed increased white blood cell count compared with WT mice (Figure 1B), and also showed progressive anemia. The platelet count of Jak2VF and Jak2VF/Tyk2KO mice was markedly higher than that of WT mice at 8 weeks of age, but decreased thereafter. There was no difference in PB cell count between Jak2VF and Jak2VF/Tyk2KO mice. FACS analysis of PB cells showed that macrophage-antigen-1-positive/granulocyte-differentiation antigen-1-positive (Mac1⁺/Gr1⁺) myeloid cells were increased and B-cell isoform of 220-kDa-positive (B220⁺) B-cells were decreased in both Jak2VF and Jak2VF/Tyk2KO mice compared to WT mice, but there were no differences between Jak2VF and Jak2VF/Tyk2KO mice (data not shown).

Regarding organ weight, significant hepatomegaly and splenomegaly were observed in Jak2VF and Jak2VF/Tyk2KO mice compared to WT mice (p<0.01), but no difference was observed between Jak2VF and Jak2VF/Tyk2KO mice, even after 9 months of observation (Figure 1C). The survival of Jak2VF and Jak2VF/Tyk2KO mice was worse than that of WT mice (p<0.01), and as with leukocytosis and organomegaly, there was no difference between these two groups of mice (Figure 1D).

Loss of TYK2 did not affect the therapeutic efficacy of a JAK2 inhibitor. We determined whether the loss of TYK2 affected the therapeutic efficacy of a JAK2 inhibitor. At 12 weeks of age, all of the Jak2VF and Jak2VF/Tyk2KO mice developed MPN, exhibiting leukocytosis with an average white blood cell count of 7-8×10¹⁰/l (Figure 2A). Jak2VF and Jak2VF/Tyk2KO mice were divided into two groups: one group received an oral gavage dose of 50 mg/kg JAK2 inhibitor twice daily (Jak2VF-inh; n=16, Jak2VF/Tyk2KO-inh; n=16) and the other group received vehicle twice daily (Jak2VF-veh; n=16, Jak2VF/Tyk2KO-veh; n=16) for 16 weeks. As a control for vehicle-treated mice, WT mice were treated with vehicle (WT-veh; n=16) were used. vehicle-treated Jak2VF and Jak2VF/Tyk2KO mice showed marked leukocytosis compared with vehicle-treated WT mice. Significant suppression of leukocytosis was observed by administration of JAK2 inhibitor in both treatment groups (Jak2VF: p<0.05; Jak2VF/Tyk2KO: p<0.01) (Figure 2A). Inhibitor-treated Jak2VF and Jak2VF/Tyk2KO mice showed no difference in leukocyte count, and there was also no difference in the therapeutic effects of JAK2 inhibitor between the two groups. In the PB cells of vehicle-treated Jak2VF and Jak2VF/Tyk2KO mice, Mac1⁺/Gr1⁺ myeloid cells increased and B220⁺ B-cells and CD3⁺ T-cells decreased compared with vehicle-treated WT mice, but the proliferation of myeloid cells was suppressed and the B- and T-lymphocyte fractions were restored by treatment with JAK2 inhibitor (Figure 2B). There were no differences in leukocyte fractions between inhibitor-treated Jak2VF and Jak2VF/Tyk2KO mice. In the vehicle-treated groups of transgenic mice, progressive anemia was observed, but in the JAK2 inhibitor-treated groups, anemia progression stopped, although the difference was not significant (Figure 2A). No significant differences in hemoglobin levels were observed between JAK2 inhibitor-treated groups. Vehicle-treated Jak2VF and Jak2VF/Tyk2KO mice showed thrombocytosis compared to WT mice treated with vehicle in the early stages, but the platelet count declined with time. JAK2 inhibitor treatment resulted in sustained thrombocytosis (Jak2VF: p<0.05; Jak2VF/Tyk2KO: p=0.370) (Figure 2A) compared to the vehicle-treated counterparts. No difference in platelet count was observed between inhibitor-treated Jak2VF and Jak2VF/Tyk2KO mice.

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Figure 1.

Tyrosine kinase 2 (TYK2) deficiency does not affect the severity of myeloproliferative neoplasms in mice. A: The mRNA expression levels of Janus kinases (JAK) in bone marrow cells from wild-type (WT) mice (n=3), JAK2 V617F transgenic (Jak2VF) mice (n=5), and Jak2VF/tyrosine kinase 2 gene knockout (Tyk2KO) mice (n=4). The expression level in WT mice was set at 1. Tukey's HSD test with one-way ANOVA was used. B: Differential blood counts in WT mice (n=28), Jak2VF mice (n=34), and Jak2VF/Tyk2KO mice (n=35) every 8 weeks after birth. ANOVA with repeated measures was used. Significant differences for all counts vs. WT: Jak2VF, p<0.01; Jak2VF/Tyk2KO, p<0.01. C: Liver and spleen weights at 3, 6, and 9 months after birth (n=5). Tukey's HSD test with one-way ANOVA was used. D: Kaplan–Meier survival curves of WT mice (n=33), Jak2VF mice (n=62), and Jak2VF/Tyk2KO mice (n=63) mice. Log-rank test was used. WBCs, White blood cells; Hb, hemoglobin; Plt, platelets. Data are presented as the mean±SEM. Significantly different at *p<0.05 and **p<0.01.

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Figure 2.

Loss of tyrosine kinase 2 (TYK2) does not affect the therapeutic efficacy of a Janus kinase 2 (JAK2) inhibitor. A: Differential blood cell count every 4 weeks after drug administration (n=16 in each group). ANOVA with repeated measures was used. B: Hematopoietic compartment of peripheral blood assessed by flow cytometry 16 weeks after drug administration (n=10 in each group). Tukey's HSD test with one-way ANOVA was used. C: Spleen and liver weights 16 weeks after drug administration (n=10 in each group). Tukey's HSD test with one-way ANOVA was used. D: Histological analysis 16 weeks after drug administration. WBCs, White blood cells; Hb, hemoglobin; Plt, platelets. Data are presented as the mean±SEM. Significantly different at *p<0.05 and **p<0.01.

The spleen weight was markedly increased by 10.4-fold in vehicle-treated Jak2VF mice and 10.2-fold in Jak2VF/Tyk2KO vehicle-treated mice compared to vehicle-treated WT mice. A significant reduction of spleen weight by 4.1-fold in inhibitor-treated Jak2VF mice (p<0.01) and by 3.4-fold in Jak2VF/Tyk2KO inhibitor-treated mice (p<0.01) compared to vehicle-treated WT mice. There was no difference in improvement of splenomegaly between Jak2VF and Jak2VF/Tyk2KO mice upon administration of JAK2 inhibitor (Figure 2C), and there was also no difference in liver weight between those groups of mice (Figure 2C). Because the observation period was short, no difference was observed among groups regarding survival (data not shown).

Histological examination showed that myeloid cells markedly infiltrated the spleen, and the follicular structure had disappeared in vehicle-treated Jak2VF and Jak2VF/Tyk2KO mice (Figure 2D), but JAK2 inhibitor therapy improved cell invasion and restored follicular structure in both groups of mice (Figure 2D). In the liver and lung, cell infiltration was observed in the vehicle-treated groups (Figure 2D), but cell infiltration completely disappeared in the JAK2 inhibitor-treated groups (Figure 2D). In both inhibitor-treated Jak2VF and Jak2VF/Tyk2KO mice, histological improvement upon JAK2 inhibitor therapy was observed equally. In contrast to the drastic pathological improvement of these organs, JAK2 inhibitor had little effect on fibrosis in the BM, and TYK2 loss did not affect these result (data not shown). Constitution of hematopoietic cells in the BM and spleen was examined by FACS analysis (Table I). The number of BM cells in vehicle-treated Jak2VF and Jak2VF/Tyk2KO mice decreased compared with the corresponding WT mice due to fibrotic changes, and consistent with pathological findings, cellularity did not recover with JAK2 inhibitor therapy. The number of cells in the spleen of vehicle-treated Jak2VF and Jak2VF/Tyk2KO mice increased because of extramedullary hematopoiesis, which was reduced with JAK2 inhibitor, although not with statistical significance. TYK2 loss did not affect cellularity in the BM or spleen. In Jak2VF and Jak2VF/Tyk2KO mice, JAK2 inhibitor therapy significantly increased common myeloid progenitors in the BM and significantly reduced cluster of differentiation 71-positive/lymphocyte antigen 76 (Ly76, also known as Ter119) -positive (CD71⁺/Ter119⁺) erythroid progenitors in the BM and spleen, although the biological significance of this is unknown. Both inhibitor-treated Jak2VF and Jak2VF/Tyk2KO mice showed similar changes upon JAK2 inhibitor administration compared with vehicle-treated groups, and there was no significant difference observed between inhibitor-treated Jak2VF and Jak2VF/Tyk2KO mice (Table I).