JAK2V617F myeloproliferative neoplasm eradication by a novel interferon/arsenic therapy involves PML

Abstract

Interferon α (IFNα) is used to treat JAK2V617F-driven myeloproliferative neoplasms (MPNs) but rarely clears the disease. We investigated the IFNα mechanism of action focusing on PML, an interferon target and key senescence gene whose targeting by arsenic trioxide (ATO) drives eradication of acute promyelocytic leukemia. ATO sharply potentiated IFNα-induced growth suppression of JAK2V617F patient or mouse hematopoietic progenitors, which required PML and was associated with features of senescence. In a mouse MPN model, combining ATO with IFNα enhanced and accelerated responses, eradicating MPN in most mice by targeting disease-initiating cells. These results predict potent clinical efficacy of the IFNα+ATO combination in patients and identify PML as a major effector of therapy, even in malignancies with an intact PML gene.

Conflict of interest statement

Disclosures: B. Cassinat reported a pending patent to WO2018134260. W. Vainchenker reported a patent to JAK2V617F licensed "Quiagen." V. Lallemand-Breitenbach reported a pending patent to WO2018134260. J. Kiladjian reported personal fees from AOP Orphan during the conduct of the study and personal fees from Novartis outside the submitted work. In addition, J. Kiladjian had a pending patent to WO2018134260 naming Hugues de Thé, Bruno Cassinat, Valérie Lallemand-Breitenbach, Isabelle Plo, Jean-Luc Villeval, and Jean-Jacques Kiladjian as inventors. H. de Thé reported personal fees from Vectorlab outside the submitted work; in addition, H. de Thé had a pending patent to WO2018134260. No other disclosures were reported.

© 2020 Dagher et al.

Figures

Graphical abstract

Figure 1.

IFNα+ATO enforces potent NB formation in JAK2V617F cells.(a) Representative immunofluorescence images (upper panel) and quantification of PML-NB numbers and intensities (lower panel) in CD34+ cells from a healthy donor (control) and a JAK2V617F MF patient, not treated (NT) or treated in vitro with ATO (1 µM) for 1 h, IFNα (1,000 IU/ml) for 24 h, or both (lower panel represents the pooled results of three patients and controls). Two-tailed unpaired Student’s t test. Scale bars = 5 µm. (b) Same as panel a in LK cells from Jak2V617F KI mice (VF) or Jak2WT littermates (WT) not treated or treated in vitro with ATO (0.3 µM) for 1 h, IFNα (1,000 IU/ml) for 22 h, or both (n = 6). Scale bars = 10 µm. (c) Western blot analysis of PML in LK cells from Jak2V617F KI, Jak2WT littermate, or Pml−/− mice not treated or treated with ATO for 1 h, IFNα for 22 h, or both (n = 3). The values shown are mean ± SEM; **P< 0.01; ***P< 0.001; ****P< 0.0001.

Figure S1.

Arsenic potently enhances NB formation and IFN-driven growth suppression in JAK2V617F-expressing cells. (a) Representative immunofluorescence images (upper panel) and quantification of PML-NB numbers and intensities per cell (lower panel) in JAK2WT (WT) and JAK2V617F (VF) UT-7 cells untreated (NT) or treated with ATO (0.5 µM) for 2 h, IFNα (1,000 IU/ml) for 24 h, or the combination of both drugs in these conditions. Three independent experiments with at least 20 cells analyzed per condition. Two-tailed unpaired Student’s t test. Scale bars = 5 µm. (b) Proliferation of JAK2WT (WT) or JAK2V617F (VF) UT-7 cells after 72 h of treatment with increasing doses of ATO alone or combined with IFNα 200 IU/ml. Three independent experiments. (c) Top panels: example of cell cycle analysis (BrdU/DAPI staining) of either JAK2WT (WT) or JAK2V617F (VF) UT-7 cells 1 d after being untreated or treated with ATO (0.25 µM), IFNα (1,000 IU/ml), or the combination of both. Bottom plot: summary and statistical analysis of three independent experiments. (d) Absolute numbers of JAK2WT/WT (WT), JAK2WT/V617F (WT/VF), or JAK2V617F/V617F (VF) colonies counted after 14 d of culture of CD34+ cells from seven PV patients. The effect of increasing doses of IFNα (200 IU/ml, 500 IU/ml, and 1,000 IU/ml) is shown on the upper panel, and the effect of increasing doses of IFNα (0 IU/ml, 200 IU/ml, 500 IU/ml, and 1,000 IU/ml) in combination with a constant dose of ATO (0.1 µM) is shown on the lower panel. Seven patient samples in duplicate cultures. Two-tailed unpaired Student’s t test. (e) Percentages of erythroid colonies relative to untreated controls after 14 d of culture of CD34+ cells from five healthy donors untreated or treated with ATO (0.1 µM), IFNα (200 IU/ml), or both drugs in triplicate cultures. Two-tailed unpaired Student’s t test. The values shown are mean ± SEM. *P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001; ns, not significant; CFC, colony-forming cell.

Figure 2.

Arsenic enhances IFNα-driven growth suppression in JAK2V617F cells in vitro.(a) Relative proliferation of JAK2WT (WT) UT-7 cells or UT-7 cells expressing ectopic JAK2V617F (VF) treated with ATO (0.25 µM), IFNα (1,000 IU/ml), or both for 3 d (n = 3). (b) Numbers of JAK2WT (WT, light shade) and JAK2V617F-positive (VF, dark shade) erythroid colonies from CD34+ patient cells cultured for 14 d. Triplicate cultures from 10 JAK2V617F MF patients untreated (NT) or treated with ATO (0.1 µM), IFNα (200 IU/ml), or both. (c) Numbers of JAK2WT/WT (WT; light shade), JAK2WT/V617F (WT/VF; patterned), and JAK2V617F/V617F (VF; dark shade) erythroid and myeloid colonies counted from CD34+ patient cells cultured for 14 d. Results represent duplicate cultures from seven PV patients untreated or treated with ATO (0.1 µM), IFNα (1,000 IU/ml), or both. (d) Erythroid colonies formed from CD34+ cells isolated from a PV patient taken after 14 d of culture: untreated or treated with ATO (0.1 µM), IFNα (200 or 1,000 IU/ml), or both. Scale bars = 50 µm. (e) Numbers of erythroid and myeloid colonies after 7 d of culture of 5 × 104 BM cells collected from Jak2V617F; UbiGFP KI mice (left), from Jak2WT littermate mice (right), or a 1:1 mixture from both, mimicking the situation found in patients (bottom). Cultures were untreated or treated with ATO (0.1 µM), IFNα (200 or 1,000 IU/ml), or both. Three independent experiments (except for ATO n = 1) treated with ATO (0.2 µM), IFNα (200 or 1,000 IU/ml as indicated) with or without ATO. Two-way ANOVA. Error bars represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant; CFC, colony-forming cell.

Figure S2.

Experimental design of the preclinical MPN mouse model. Recipient mice were lethally irradiated and transplanted with 3 × 106 40:60 mix of Jak2V617F/UbiGFP and Jak2WT BM cells. 4 wk after BM transplantation (BMT), mice were treated with ATO (5 mg/kg/2 d), IFNα (3 × 104 IU/d), or both for 8 wk. At completion of treatment, some mice were sacrificed, and their BM cells were used to assess disease-initiating cells in secondary transplantations, also with 3 × 106 BM cells; the remaining mice were kept alive to assess disease relapse around 9 wk without further treatment.

Figure 3.

The IFNα+ATO combination efficiently targets Jak2V617F disease in vivo.(a) Hematological parameters of mice bearing Jak2V617F disease were obtained from four independent experiments (untreated, n = 25; ATO, n = 19; IFNα, n = 28; and IFNα+ATO, n = 29; Fig. S2 for protocol details). One-way ANOVA with multiple comparisons. (b) Percentage of GFP-positive Jak2V617F Gr-1+/CD11b+ cells (granulocytes), CD42+ cells (platelets), and Ter119+ cells (RBCs) in chimeric recipients during treatment. (c) Spleen weight after treatment discontinuation obtained from four independent experiments (untreated [NT], n = 12; ATO, n = 8; IFNα, n = 11; and IFN+ATO, n = 19). Unpaired two-sided Student’s t test. (d) Percentage of Jak2V617F cells (untreated, n = 5; ATO, n = 4; IFNα, n = 4; and IFNα+ATO, n = 9). Unpaired two-sided Student’s t test. (e) Percentage of healthy secondary recipients evaluated at 8–15 wk after BM transplantation (four independent experiments: untreated, n = 17; ATO, n = 12; IFNα, n = 22; and IFNα + ATO, n = 18). Mice were considered healthy based on the hematocrit (<50%) and the absence of Jak2V617F cells (GFP labeled) in granulocytes (Gr-1+/CD11b+) and platelets (CD42+). One-way ANOVA with multiple comparisons. (f) Percentage of relapses 9 wk after treatment completion in primary recipients. Results from four independent experiments (untreated, n = 11; ATO, n = 10; IFNα, n = 15; and IFNα + ATO, n = 15). One-way ANOVA with multiple comparisons. The values shown are mean ± SEM. *P< 0.05; **P< 0.01; ***P< 0.001.

Figure 4.

PML drives IFNα+ATO response.(a) Absolute numbers of JAK2WT (WT) and JAK2V617F (VF) erythroid colonies after 14 d of culture of shSCR–treated (PML expressing) or shPML-treated (PML down-regulated) CD34+ cells from four JAK2V617F MF patients untreated (NT) or treated with the combination of IFNα (200 IU/ml) and ATO (0.1 µM). Unpaired two-sided Student’s t test. (b) Lethally irradiated mice were grafted with 50% BM cells from Jak2V617F KI/UbiGFP mice and 50% BM cells from Jak2V617F KI Pml−/− mice. Mice were treated with IFNα alone or IFNα+ATO 4 wk after transplantation. (c) Blood parameters (WBCs, platelets, and hematocrit) every 2 wk during the treatment. n = 5 for each treatment; one-way ANOVA with multiple comparisons. (d) Percentage of Pml−/− Gr-1+ cells (granulocytes), Pml−/− CD42+ cells (platelets), and Pml−/− Ter119+ cells (RBCs). n = 5 for each treatment; one-way ANOVA with multiple comparisons. Color code as in panel c. The values shown are mean ± SEM. **P< 0.01; ***P< 0.001; ****P< 0.0001. BMT, BM transplantation.

Figure S3.

IFN/ATO drives senescence more efficiently in the JAK2V617F context.(a) Percentages of JAK2WT (WT) or JAK2V617F (VF) UT-7 cells untreated (NT) or treated with ATO (0.25 µM), IFNα (1,000 IU/ml), or both drugs for 7 d stained positively for SA-β-Gal. (b) Relative expression to untreated control of the senescence-associated genes PML, PAI1, and CDKN1A in UT-7 cells. Color code as in panel a. Error bars represent mean ± SEM of three independent experiments; unpaired two-sided Student’s t test. (c) SA-β-Gal staining assay of CD34+-derived cells from three MF patients or three healthy controls after 10 d of megakaryocytic differentiation and untreated or treated with the combination of IFNα (1,000 IU/ml) and ATO (0.25 µM). The values shown are mean ± SEM. *P< 0.05; **P< 0.01; ***P< 0.001.

Figure 5.

IFNα+ATO drives PML-dependent senescence.(a) Percentages of SA-β-Gal+ cells after CD34+ cells from three healthy donors’ (control) and three JAK2V617F MF patients’ samples (patient) were treated with ATO (0.25 µM), IFNα (1,000 IU/ml), or both for 10 d. Unpaired two-sided Student’s t test. (b) Relative expression to untreated control of senescence-associated genes PML, PAI1, CDKN1A, and GDF15 of patient samples described in panel a. Color code as in panel a. Unpaired two-sided Student’s t test. (c) SA-β-Gal staining of shSCR–treated (PML expressing) or shPML-treated (PML down-regulated) CD34+-derived cells from two MF patients after 10 d of megakaryocytic differentiation and untreated (NT) or treated with the combination IFNα (1,000 IU/ml) and ATO (0.25 µM). (d) Percentages of SA-β-Gal+ cells of shSCR–treated (PML-expressing) or shPML-treated (PML down-regulated) CD34+-derived cells from three MF patient samples after 10 d of megakaryocytic differentiation untreated or treated with the combination IFNα (1,000 IU/ml) and ATO (0.25 µM). (e) Relative expression of PML, PAI1, CDKN1A, and GDF15 of samples described in panel d. Unpaired two-sided Student’s t test. (f) Expression of Cdkn2a, Pai1, and Gdf15 in mouse Lin− BM cells derived from Jak2V617F KI (VF) or littermate Jak2WT (WT) mice deficient (hatched bars) or not (not hatched bars) in Pml treated in vitro with IFNα (1,000 IU/ml)+ATO (0.3 µM) for 24 h. The values shown are mean ± SEM. *P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001.