Depletion of Jak2V617F myeloproliferative neoplasm-propagating stem cells by interferon-α in a murine model of polycythemia vera

Abstract

Interferon-α (IFNα) is an effective treatment of patients with myeloproliferative neoplasms (MPNs). In addition to inducing hematological responses in most MPN patients, IFNα reduces the JAK2V617F allelic burden and can render the JAK2V617F mutant clone undetectable in some patients. The precise mechanism underlying these responses is incompletely understood and whether the molecular responses that are seen occur due to the effects of IFNα on JAK2V617F mutant stem cells is debated. Using a murine model of Jak2V617F MPN, we investigated the effects of IFNα on Jak2V617F MPN-propagating stem cells in vivo. We report that IFNα treatment induces hematological responses in the model and causes depletion of Jak2V617F MPN-propagating cells over time, impairing disease transplantation. We demonstrate that IFNα treatment induces cell cycle activation of Jak2V617F mutant long-term hematopoietic stem cells and promotes a predetermined erythroid-lineage differentiation program. These findings provide insights into the differential effects of IFNα on Jak2V617F mutant and normal hematopoiesis and suggest that IFNα achieves molecular remissions in MPN patients through its effects on MPN stem cells. Furthermore, these results support combinatorial therapeutic approaches in MPN by concurrently depleting dormant JAK2V617F MPN-propagating stem cells with IFNα and targeting the proliferating downstream progeny with JAK2 inhibitors or cytotoxic chemotherapy.

Figure 1

Murine IFNα is effective treatment in Jak2^VF-induced MPN. (A) Schematic representation of generation of chimeric recipient mice. Purified HSC-enriched populations (lineage^negKit^highSca1⁺) were isolated from Jak2^VF (CD45.2) or Jak2^WT (CD45.1) and injected into lethally irradiated B6xPtprca.F1 (CD45.1/2 double positive) recipient mice. (B) Effects of 4 weeks of daily IFNα on Jak2^VF chimeric mice. Reduced HCT after IFNα (vehicle 76.0 ± 6.9% vs IFNα 65.5 ± 8.1%; P < .05; n = 9-10). (C) Reduced peripheral blood WCC after IFNα (vehicle 13.9 ± 3.7 × 10⁹/L vs IFNα 7.5 ± 3.5 × 10⁹/L; P < .01; n = 9-10). (D) Reduced peripheral blood RCC after IFNα (vehicle 17.8 ± 0.5 × 10¹²/L vs IFNα 14.7 ± 0.7 × 10⁹/L; P < .01; n = 9-10). (E) Reduced hemoglobin (Hb) after IFNα (vehicle 21.4 ± 0.6 g/L vs IFNα 19.4 ± 0.6 g/L; P < .05; n = 9-10). Each data point represents an individual mouse. Data shown are pooled results from 2 independent experiments. (F) Effects of 8 week daily IFNα on Jak2^VF chimeric mice. Reduced HCT after IFNα (vehicle 65.6 ± 5.6% vs IFNα 39.1 ± 0.8%; P < .01; n = 5). (G) Reduced peripheral blood WCC after IFNα (V = vehicle 11.4 ± 0.8 × 10⁹/L vs IFNα 6.7 ± 3.5 × 10⁹/L; P < .01; n = 5). (H) Reduced peripheral blood RCC after IFNα (vehicle 14.0 ± 1.0 × 10¹²/L vs IFNα 8.2 ± 0.2 × 10⁹/L; P < .01; n = 5). (I) Reduced Hb after IFNα (vehicle 20.9 ± 0.5 g/L vs IFNα 11.8 ± 0.3 g/L; P < .01; n = 5). Each data point represents an individual mouse. Results given are mean ± standard deviation. RCC, red cell count; WCC, white cell count.

Figure 2

Effects on extramedullary hematopoiesis of 4 weeks of daily IFNα on Jak2^VF chimeric mice. (A) Reduced spleen WCC after IFNα (vehicle 149.6 ± 44.1 × 10⁶ vs IFNα 100 ± 27.1 × 10⁶; P < .01; n = 9-10). (B) Reduced spleen weight after IFNα (vehicle 262 ± 51.2 mg vs IFNα 192 ± 40.6 mg; P ≤ .01; n = 9-10) (C) Representative flow cytometry plots showing maturation of erythropoiesis in WT and vehicle- and IFNα-treated mice. Early erythropoiesis is CD71^high, late erythropoiesis is CD71^mid-lowTer119⁺. (D) Representative histogram plots demonstrating annexin V staining on early erythroid cells. (E) Total early erythropoiesis reduced in IFNα-treated mice (vehicle 87.2 ± 8.0 × 10⁶/spleen vs IFNα 27.6 ± 5.0 × 10⁶/spleen; P < .01; n = 9-10) (F) No change in total late erythropoiesis (vehicle 44.3 ± 5.7 × 10⁶/spleen vs IFNα 54.4 ± 6.0 × 10⁶/spleen; P = .24; n = 9-10). Each data point represents an individual mouse. Data shown are pooled results from 2 independent experiments. (G) Apoptosis in early erythroid precursors increased after IFNα treatment (vehicle 12.3 ± 2.4% vs IFNα 42.4 ± 1.7; P < .01; n = 5). Apoptosis in late erythroid precursors increased after IFNα treatment (vehicle 3.7 ± 0.6% vs IFNα 16.4 ± 1.3; P < .01; n = 5). Results given are mean ± standard deviation.

Figure 3

Effect on HSPC populations of 4 weeks of daily IFNα on Jak2^VF chimeric mice. (A) Representative flow cytometry plots showing reduction in lineage^lowKit^highSca1⁺CD150⁺CD48⁻ LT-HSCs from the Jak2^VF IFNα-treated cells (expressed as percentage of parent LKS⁺ gate). (B) Reduction in LT-HSCs expressed as absolute number per lower limb cellularity (vehicle-treated WT: 8896 ± 6612 vs Jak2^VF 6930 ± 6128; P = .34; n = 14; IFNα-treated WT: 6503 ± 8885 vs Jak2^VF 2020 ± 3007; P = .01; n = 15; vehicle vs IFNα: Jak2^VF; P = .01; vehicle vs IFNα WT: P = .78). Experimental replicates are shown as circles, squares, and diamonds (experiments 1, 2, and 3, respectively). (C) Representative flow cytometry plots showing that IFNα treatment in WT cells causes a relative reduction in MEPs (lineage^lowKit^highSca1⁻CD34⁻FcGR⁻), with relative expansion of GMP (lineage^lowKit^highSca1⁻CD34⁺FcGR⁺). Conversely, IFNα treatment in Jak2^VF cells causes further expansion in MEP and relative reduction in GMP. (Percentage of parent lineage^lowKit^high, LK+ gate). (D) Two weeks of IFNα treatment in WT cells increases GMP differentiation (WT vehicle: 60.8 ± 2.7% vs IFNα: 80.5 ± 3.3%; P < .01; n = 4-5) and reduces GMP differentiation in Jak2^VF cells (Jak2^VF vehicle: 36.8 ± 8.1% vs IFNα: 19.6 ± 2.7%; P < .01; n = 4-5). (E) Two weeks of IFNα treatment in WT cells reduces MEP differentiation (WT vehicle: 19.8 ± 2.8% vs IFNα: 12.9 ± 3.2%; P < .01; n = 4-5) and increases MEP differentiation in Jak2^VF cells (Jak2^VF vehicle: 50.7 ± 9.6% vs IFNα: 76.3 ± 1.9%; P < .01; n = 4-5). Each data point represents an individual mouse. Data shown are representative of at least 2 independent experiments. Similar results were found in each experiment. Results given are mean ± standard deviation.

Figure 4

Effect of IFNα treatment on LT-HSC cell cycle in chimeric transplant recipients and primary mice. (A) Representative flow cytometry plots showing cell cycle in lineage^lowKit^highSca1⁺CD150⁺ WT (CD45.1) or Jak2^VF (CD45.2) LT-HSCs from chimeric recipient mice. G0 (quiescent) cells are Ki-67^lowH33342²ⁿ, G1 cells are Ki-67⁺H33342²ⁿ, and SG2M cells are Ki67⁺H33342⁴ⁿ. (B) Reduction in quiescent HSCs after IFNα treatment in WT (gated on CD45.1 WT vehicle: 40.3 ± 6.6% vs IFNα: 22.4 ± 6.0%; P < .01; n = 4) and reduction in quiescent HSCs after IFNα treatment in Jak2^VF (gated on CD45.2 Jak2^VF, vehicle: 28.0 ± 2.3% vs IFNα: 5.4 ± 0.7%; P < .0001; n = 5). There were significantly fewer quiescent Jak2^VF compared with WT HSCs (1.4-fold; P = .02). More profound depletion of quiescent Jak2^VF HSC compared with WT HSCs (4.2 fold; P = .0003). (C) Increase in actively cycling (Ki67⁺H33342⁴ⁿ) HSCs after IFNα treatment in Jak2^VF cells (gated on CD45.1 WT vehicle: 19.6 ± 0.4% vs WT IFNα: 24.2 ± 6.5%; P = .21; gated on CD45.2 Jak2^VF vehicle: 24.1 ± 0.9% vs Jak2^VF IFNα: 35.9 ± 9.6%; P < .05; n = 4-5). There were significantly more Jak2^VF than WT HSCs in active cell cycle in vehicle-treated recipients (1.2-fold; P < .01) and also in IFNα-treated recipients (1.5-fold; P = .05). Each data point represents an individual mouse. Data shown are representative of at least 2 independent experiments using chimeric recipient mice. Similar results were found in each experiment. Results given are mean ± standard deviation. (D) GSEA performed on lineage^lowKit^highSca1⁺CD150⁺ WT (CD45.1) or Jak2^VF (CD45.2) cells from chimeric recipient mice revealed that genes regulating cell cycle were enriched in Jak2^VF HSCs compared with WT HSCs. (E) Genes regulating quiescence were relatively enriched in WT HSCs compared with Jak2^VF HSCs. (F) Reduction in quiescent (Ki67^lowH33342²ⁿ) HSCs in WT and Jak2^VF primary mice after IFNα treatment (WT vehicle: 31.0 ± 3.2% vs WT IFNα: 21.7 ± 4.9%; P = .16; Jak2^VF vehicle: 22.0 ± 3.6% vs Jak2^VF IFNα: 11.1 ± 0.9%; P = .05; n = 3-4/group). (G) Increase in actively cycling (Ki67⁺H33342⁴ⁿ) HSCs in WT and Jak2^VF primary mice after IFNα treatment (WT vehicle: 20.0 ± 1.1% vs WT IFNα: 31.7 ± 2.6%; P < .01; Jak2^VF vehicle: 30.1 ± 1.6% vs Jak2^VF IFNα: 41.3 ± 1.4%; P < .01; n = 3-4/group). There were significantly more Jak2^VF than WT HSCs in active cell cycle in vehicle-treated recipients (P < .01) and also in IFNα-treated recipients (P = .03).

Figure 5

Functional depletion of Jak2^VF disease-initiating stem cells after IFNα treatment. (A) Normal HCT in recipients from IFNα-treated but not vehicle-treated donor Jak2^VF chimeric mice bone marrow 4 weeks after transplantation (vehicle: 60.5 ± 9.2% vs IFNα: 42.6 ± 0.8%: P < .01; n = 5). (B) Reduced Jak2^VF chimerism in recipients 4 weeks after transplantation from IFNα-treated mice but not from vehicle-treated donor mice (vehicle: 42.6 ± 18.8% vs IFNα: 8.2 ± 6.7%; P < .01; n = 5). (C) Absolute engraftment (total CD45.2+ cells in peripheral blood) 4 weeks after transplantation in secondary transplant recipient (WT vehicle: 3.5 ± 1.3% vs IFNα: 1.5 ± 0.3%; P < .05; n = 5; Jak2^VF vehicle: 2.9 ± 2.3% vs IFNα: 0.2 ± 0.2%; P < .05; n = 5; vehicle WT vs Jak2^VF: 3.5% vs 2.9%; P = .64; IFNα-treated WT vs Jak2^VF: 1.5% vs 0.2%; P < .01). (D) Sixteen weeks of chimerism demonstrating sustained reduction in Jak2^VF chimerism in recipients of bone marrow from IFNα-treated mice but not vehicle-treated donor mice (vehicle: 30.6 ± 20.2% vs IFNα: 6.1 ± 4.2%; P < .05; n = 5). (E) Engraftment in secondary transplant recipients 16 weeks after transplantation (WT vehicle: 1.5 ± 0.7% vs IFNα: 1.6 ± 2.4%; P = .90; n = 5; Jak2^VF vehicle: 0.6 ± 0.5% vs IFNα: 0.04 ± 0.02%; P < .05; n = 5). Each data point represents an individual mouse. Data shown are representative of at least 2 independent experiments. Similar results were found in each experiment. Results given are mean ± standard deviation.

Figure 6

The effects of IFNα are cell autonomous and specific to type 1 interferon signaling. (A) Peripheral blood WCC was reduced in IFNα-treated Jak2^VF chimeric mice compared with vehicle controls (12.5 ± 2.9 × 10⁶/L vs 8.3 ± 2.0 × 10⁶/L, respectively; P = .05) but not in IFNα-treated Ifnar1^−/−Jak2^VF chimeric mice compared with vehicle controls (16.5 ± 4.6 × 10⁶/L vs 15.1 ± 2.8 × 10⁶/L, respectively; P = .62). (B) Peripheral blood RCC was reduced in IFNα-treated Jak2^VF chimeric mice compared with vehicle controls (20.2 ± 0.8 × 10⁹/L vs 16.6 ± 0.7 × 10⁹/L, respectively; P = .02) but not in IFNα-treated Ifnar1^−/−Jak2^VF chimeric mice compared with vehicle controls (18.9 ± 1.2 × 10⁹/L vs 19.4 ± 1.2 × 10⁹/L, respectively; P = .81). (C) Spleen WCC was reduced in IFNα-treated Jak2^VF chimeric mice compared with vehicle controls (165 ± 8 × 10⁶ vs 215 ± 10 × 10⁶, respectively; P < .01) but not in IFNα-treated Ifnar1^−/−Jak2^VF chimeric mice compared with vehicle controls (181 ± 29 × 10⁶ vs 174 ± 8 × 10⁶, respectively; P = .83). (D) Spleen weight was reduced in IFNα-treated Jak2^VF chimeric mice compared with vehicle controls (298 ± 30 mg vs 410 ± 17 mg, respectively; P = .02); however, there was no difference in IFNα-treated Ifnar1^−/−Jak2^VF chimeric mice compared with vehicle controls (321 ± 41 mg vs 364 ± 12 mg, respectively; P = .36). (E) MEP differentiation was potentiated by IFNα treatment in Jak2^VF chimeric mice compared with vehicle controls (76.4 ± 1.7% vs 56.9 ± 4.4%, respectively; P < .01), but there was no effect in Ifnar1^−/−Jak2^VF chimeric mice compared with vehicle controls (53.4 ± 2.7% vs 51.3 ± 4.4%, respectively; P = .69). (F) GMP differentiation was impaired in IFNα-treated Jak2^VF chimeric mice compared with vehicle controls (17.1 ± 1.5% vs 31.7 ± 3.3%, respectively; P < .01), but there was no effect in IFNα-treated Ifnar1^−/−Jak2^VF chimeric mice compared with vehicle controls (30.4 ± 1.6% vs 29.6 ± 3.4%, respectively; P = .85). (G) Reduced total LT-HSC numbers in IFNα-treated Jak2^VF chimeric mice compared with vehicle controls (5040 ± 1502 vs 13 430 ± 2026, respectively; P = .01) but no effect in IFNα-treated Ifnar1^−/−Jak2^VF chimeric mice compared with vehicle controls (7662 ± 1843 vs 7849 ± 1237, respectively; P = .94). (H) Representative flow cytometry plots showing cell cycle in lineage^lowKit^highSca1⁺CD150⁺ Jak2^VF or Ifnar1^−/−Jak2^VF LT-HSCs from chimeric recipient mice. G0 (quiescent) cells are Ki-67^lowDNA²ⁿ, G1 cells are Ki-67⁺DNA²ⁿ, and SG2M cells are Ki67⁺DNA⁴ⁿ. (I) Quiescence in Jak2^VF HSCs was reduced after IFNα treatment compared with vehicle control (6.0 ± 1.2% vs 15.4 ± 3.4%, respectively; P = .04), but there was no difference in Ifnar1^−/−Jak2^VF HSCs with IFNα treatment or vehicle control (27.8 ± 4.5% vs 30.6 ± 7.3%, respectively; P = .75). (J) Actively cycling Jak2^VF HSCs increased after IFNα treatment compared with vehicle control (47.0 ± 2.1% vs 29.8 ± 2.9%, respectively; P < .01), but there was no difference in Ifnar1^−/−Jak2^VF HSCs after IFNα treatment compared with vehicle control (24.2 ± 3.4% vs 24.0 ± 2.4%, respectively; P = .96) (n = 4/group for each experiment, mean +/− standard deviation).