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. 2010 Jun 15;17(6):584-96.
doi: 10.1016/j.ccr.2010.05.015.

Physiological Jak2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells

Ann Mullally  1 , Steven W Lane, Brian Ball, Christine Megerdichian, Rachel Okabe, Fatima Al-Shahrour, Mahnaz Paktinat, J Erika Haydu, Elizabeth Housman, Allegra M Lord, Gerlinde Wernig, Michael G Kharas, Thomas Mercher, Jeffery L Kutok, D Gary Gilliland, Benjamin L Ebert
Affiliations

Physiological Jak2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells

Ann Mullally et al. Cancer Cell. .

Abstract

We report a Jak2V617F knockin mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem cell (HSC) compartment has the unique capacity for disease initiation but does not have a significant selective competitive advantage over wild-type HSCs. In contrast, myeloid progenitor populations are expanded and skewed toward the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to JAK2V617F-positive MPN.

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Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Physiological Jak2V617F expression causes a lethal MPN resembling PV
(A) Targeting vector schema for the Jak2V617F conditional KI mouse. (B) PCR analysis of tail DNA from Jak2+/+, Jak2+/Fl and Jak2+/VF mice for genotyping (Geno), detection of floxed band (Flox) and detection of excised band (Exc);(Fl = Floxed). (C) Sequencing analysis of unfractionated BM cells from Jak2+/+ and Jak2+/VF mice. (D) Kaplan-Meier analysis of a cohort of Jak2+/VF mice (n=17) and littermate controls (n=13). (E) Composite data from age-matched littermates of Jak2+/+ and Jak2+/VF mice (unpaired two-tailed t test; mean +/− SEM; n=4 in each group) demonstrating increased in CD71+, Ter119+ erythroid precursors in Jak2+/VF spleens. (F) Hematocrits of age-matched littermate Jak2+/+ and Jak2+/VF mice aged 4–14 weeks (mean +/− SEM; n=16 in each group). (G) Photograph of spleens from Jak2+/+ (upper; weight = 0.08G) and Jak2+/VF mice (lower; weight = 3.1G) demonstrating marked splenomegaly in Jak2+/VF mouse. (H) Representative flow-cytometric analysis of single cell suspensions of spleen from age-matched littermate Jak2+/+ and Jak2+/VF mice, indicating expansion of CD71+ Ter119+ cells (subpopulations I and II) in Jak2+/VF mice. (I) Histopathologic H & E sections of bone marrow (BM) and spleen (SPL) from representative Jak2+/+ and Jak2+/VF mice demonstrating megakaryocyte hyperplasia with clustering and emperipolesis in Jak2+/VF BM and marked erythroid hyperplasia with complete effacement of white pulp in Jak2+/VF SPL when compared with unremarkable BM and SPL from Jak2+/+ controls (BM: first column [scale bars 250µM], second column [scale bars 50µM]; SPL: third column [scale bars 500µM], fourth column [scale bars 125µM]. (J) Histopathologic reticulin sections of bone marrow (BM) and spleen (SPL) from representative Jak2+/+ and Jak2+/VF mice demonstrating absence of reticulin fibrosis (BM: first and second panels [scale bars 250µM]; SPL: third and fourth panels [scale bars 250µM]. See also Figure S1.
Figure 2
Figure 2. Erythroid skewing in the myeloid progenitor compartment of Jak2V617F mice
(A – B) Multiparameter flow cytometry of Jak2+/+ and Jak2+/VF BM. Representative contour plots indicating LK subsets (MkPs, GMPs, Pre Meg-Es and Pre CFU-Es) are shown. All values are mean frequency of total BM cells. Representative data from one of 2 independent experiments (n=4 in each group). (C) Composite data from age-matched littermates of Jak2+/+ and Jak2+/VF mice demonstrating significantly increased numbers of MEP cells in Jak2+/VF BM over Jak2+/+ BM (unpaired two-tailed t test; mean +/−SEM; n=4 in each group). (D) Composite data from age-matched littermates of Jak2+/+ and Jak2+/VF mice demonstrating significantly increased numbers of Pre CFU-E cells in Jak2+/VF BM over Jak2+/+ BM (unpaired two-tailed t test; mean +/−SEM; n=4 in each group). (E – F) Unfractionated BM and MEP cells from Jak2+/+ and Jak2+/VF mice were plated and scored for BFU-E colony formation 7–10 days later. Results are the average of two independent experiments performed in duplicate (unpaired two-tailed t test; mean +/−SEM). See also Figure S2.
Figure 3
Figure 3. The Jak2V617F MPN-initiating population is contained within the LSK population
(A) HCT of lethally (Lethal) or sub-lethally (Sublethal) irradiated littermate secondary recipients of unfractionated BM or SPL cells from diseased Jak2+/VF mice measured 4–14 weeks post transplantation (mean +/−SEM; n = 3 in each group). (B) HCT of lethally irradiated secondary (2°) recipients of purified Jak2+/VF BM sub-populations (LSKs, MEPs and GMPs) and corresponding Jak2+/+ BM sub-populations; and of lethally irradiated tertiary (3°) recipients of unfractionated BM, measured 2–21 weeks post transplantation (mean +/−SEM; n = 5 in each group). (C) Composite data from secondary recipients of purified Jak2+/VF BM sub-populations (LSKs, MEPs and GMPs), demonstrating increased CD71+, Ter119+ erythroid precursors in spleens of secondary recipients of purified Jak2+/VF LSK cells (unpaired two-tailed t test; mean +/− SEM; n=3 in each group). (D – G) Histopathologic H & E sections of BM from representative secondary recipients of purified Jak2+/+ and Jak2+/VF LSK cells, Jak2+/VF MEP cells and Jak2+/VF GMP cells, demonstrating erythroid hyperplasia, increased megakaryocytes in clusters with large forms and emperipoliesis in Jak2+/VF LSK; slight increase in megakaryocytes in Jak2+/VF MEP and mild myeloid hyperplasia in Jak2+/VF GMP as compared with unremarkable BM from Jak2+/+ LSK (BM: [scale bars 250µM]). See also Figure S3.
Figure 4
Figure 4. Jak2V617F has modest effects in the LSK compartment and does not confer a significant competitive advantage to LSK cells
(A) Representative multiparameter flow cytometry of LSK populations in Jak2+/+ and Jak2+/VF mice demonstrating no significant differences in cell numbers in different phases of cell cycle. All values are mean frequency of total LSK cells. Representative data from one of two independent experiments (n=4 in each group). (B) Representative flow cytometric analysis of levels of phospho-STAT5 (p-STAT5) in LSK cells from Jak2+/+ and Jak2+/VF littermate controls, following serum starvation (starve) or stimulation with EPO (3U/mL) and IL3 (10ng/mL) for 10 mins, in the absence (EPO/IL3) or presence of the JAK2 inhibitor, TG101348 (EPO/IL3/JAK2i). Representative data from one of three independent experiments. (C) Composite data from Jak2+/+ and Jak2+/VF LSK cells purified from primary mice (unpaired two-tailed t test; mean +/− SEM), demonstrating no significant difference in phosphoSTAT5 activation after stimulation with EPO/IL3 (n=4 in each group). (D) Jak2+/VF(VF) to Jak2+/+(WT) chimerism ratios assessed in BM LSK compartment from lethally irradiated secondary recipients of Jak2+/VF and Jak2+/+ LSK cells in 75:25, 50:50 and 25:75 ratios respectively, measured 16 weeks post transplantation (mean +/− SEM; n=4 in each group). (E) Jak2+/VF(VF) to Jak2+/+(WT) myeloid chimerism ratios assessed in peripheral blood (PB) from lethally irradiated secondary recipients of Jak2+/VF and Jak2+/+ LSK cells in 75:25, 50:50 and 25:75 ratios respectively, measured 5–16 weeks post transplantation (mean +/− SEM; n=6 in each group). See also Figure S4 and Table S1.
Figure 5
Figure 5. JAK2 kinase inhibition does not eradicate the MPN-initiating population
(A) Composite data from primary Jak2+/VF age-matched littermates treated with vehicle or the JAK2 inhibitor, TG101348 for 6 weeks, demonstrating reduced spleen weight in mice treated with TG101348 as compared with those treated with vehicle (unpaired two-tailed t test; mean +/− SEM; n=3 in each group). (B – C) Histopathologic H & E sections of spleen from representative primary Jak2+/VF mice treated with vehicle or JAK2 inhibitor, TG101348 for 6 weeks, demonstrating complete effacement of normal splenic architecture in vehicle treated mice and improvement towards normal in TG101348 treated mice (SPL: [scale bars 250µM]). (D) Composite data from tertiary Jak2+/VF recipients treated with vehicle or TG101348 for 6 weeks and from untreated Jak2+/+ controls, demonstrating reduced splenic cellularity and reduced number of CD71+ Ter119+ erythroid precursor cells in mice treated with TG101348 (mean +/−SEM; n=4 in each group; Tot = Total; Ery = Erythroid). (E) HCTs of lethally irradiated secondary recipients of purified Jak2+/VF LSKs from mice treated with vehicle or JAK2 inhibitor, TG101348 for 6 weeks pre-transplant and resumed 3 weeks post transplantation. HCTs of lethally irradiated secondary recipients of purified Jak2+/+ control LSKs, measured 3–10 weeks post transplantation also shown. HCTs of tertiary recipients of unfractionated Jak2+/VF BM from treated secondary recipients measured 3 weeks post transplantation are also shown (mean +/−SEM; n=3 in each group for secondary recipients; n=6 for tertiary recipients). (F) Schema outlining schedule for primary, secondary and tertiary Jak2+/VF mice with treated with TG101348 or vehicle.
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References

    1. Abdel-Wahab O, Manshouri T, Patel J, Harris K, Yao J, Hedvat C, Heguy A, Bueso-Ramos C, Kantarjian H, Levine RL, Verstovsek S. Genetic analysis of transforming events that convert chronic myeloproliferative neoplasms to leukemias. Cancer Res. 70:447–452. - PMC - PubMed
    1. Akada H, Yan D, Zou H, Fiering S, Hutchison RE, Mohi MG. Conditional expression of heterozygous or homozygous Jak2V617F from its endogenous promoter induces a polycythemia vera-like disease. Blood - PMC - PubMed
    1. Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, Vassiliou GS, Bench AJ, Boyd EM, Curtin N, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005;365:1054–1061. - PubMed
    1. Braun BS, Tuveson DA, Kong N, Le DT, Kogan SC, Rozmus J, Le Beau MM, Jacks TE, Shannon KM. Somatic activation of oncogenic Kras in hematopoietic cells initiates a rapidly fatal myeloproliferative disorder. Proc Natl Acad Sci U S A. 2004;101:597–602. - PMC - PubMed
    1. Campbell PJ, Baxter EJ, Beer PA, Scott LM, Bench AJ, Huntly BJ, Erber WN, Kusec R, Larsen TS, Giraudier S, et al. Mutation of JAK2 in the myeloproliferative disorders: timing, clonality studies, cytogenetic associations, and role in leukemic transformation. Blood. 2006;108:3548–3555. - PubMed

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