Journal: Cancer Discovery

Article Title: Jak2 V617F Reversible Activation Shows Its Essential Requirement in Myeloproliferative Neoplasms

doi: 10.1158/2159-8290.CD-22-0952

Figure Lengend Snippet: Jak2 V617F deletion abolishes JAK/STAT signaling and abrogates the MPN phenotype. A, Schematic representation of the dual-recombinase Jak2 V617F conditional knock-in/knock-out allele ( Jak2 RL ), the Jak2 RL knock-in allele following Dre recombination, and the null recombined allele following Cre-mediated deletion. Semicircles indicate Rox sequences; triangles indicate lox P sequences. B, Representative Western blot depicting phospho-STAT5 abundance of Dre-mediated Jak2 V617F knock-in (+Dre) vs. Jak2 V617F -deleted (+Dre +Cre) states from isolated splenocytes 7 days following tamoxifen (TAM) administration in comparison with unrecombined (Unrec.) Jak2 RL cells ( n = 2 biological replicates each; representative of n = 2 independent experiments). C, Peripheral blood count trends (weeks 0–24) of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated mice: WBCs (left), Hct (right; n ≥ 10 per arm; mean ± SEM). Gray bar represents duration of tamoxifen pulse/chow administration. Representative of n = 2 independent transplants. **, P ≤ 0.01; ****, P ≤ 0.0001. D, Kaplan–Meier survival analysis of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated mice ( n ≥ 12 per arm; log-rank test). Gray bar represents duration of tamoxifen pulse/chow administration. ****, P ≤ 0.0001. E, Spleen weights of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated mice at timed sacrifice (24 weeks) in comparison with WT control mice (mean ± SEM). Representative of n = 2 independent transplants. ****, P ≤ 0.0001. F, Heat map scaled using Z-scores of serum cytokine/chemokine concentrations of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated mice harvested at time of sacrifice 18–24 weeks posttransplant in comparison with WT control mice ( n = 4–7 biological replicates per arm pooled from n = 3 transplants). Asterisks denote cytokines with FDR ≤ 0.05. Kruskal–Wallis test with FDR correction. G, Representative hematoxylin and eosin (H&E) and reticulin stains of bone marrow of MPN (Control) vs. tamoxifen ( Jak2 V617F -deleted) treated mice from timed sacrifice at 24 weeks. Representative micrographs of n = 6 individual mouse replicates per arm. All images represented at 400× magnification. Scale bar, 20 μm.

Article Snippet: Single-mutant Tet2 −/− or double-mutant Jak2 RL / Tet2 −/− transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily ×4; purchased from MedChemExpress) 6–8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Knock-In, Knock-Out, Western Blot, Isolation, Comparison, Control

Journal: Cancer Discovery

Article Title: Jak2 V617F Reversible Activation Shows Its Essential Requirement in Myeloproliferative Neoplasms

doi: 10.1158/2159-8290.CD-22-0952

Figure Lengend Snippet: Jak2 V617F reversal impairs the fitness of MPN cells, including MPN stem cells. A, Peripheral blood (PB) mutant Cd45.2 percent chimerism trend (weeks 0–24) of early (3 weeks posttransplant) tamoxifen (TAM; Jak2 V617F -deleted) treated (gold bar) and late (12 weeks posttransplant) tamoxifen-treated (maroon bar) mice ( n = 8 each) in comparison with MPN (dark gray bar; n = 6) mice (mean ± SEM). Gray bars represent duration of tamoxifen pulse/chow administration. Representative of n = 2 independent transplants. **, P ≤ 0.01; ***, P ≤ 0.001. B, Bone marrow–mutant cell fraction within LSK (Lineage − Sca1 + cKit + ), granulocytic-monocytic progenitor (GMP; Lineage − cKit + Sca1 − Cd34 + Fcg + ), and megakaryocytic-erythroid progenitor (MEP; Lineage − cKit + Sca1 − Cd34 − Fcg − ) compartments of early (3 weeks posttransplant) tamoxifen ( Jak2 V617F -deleted) treated and late (12 weeks posttransplant) tamoxifen-treated mice in comparison with MPN mice at timed sacrifice of 24 weeks ( n = 6–8 individual biological replicates per arm; mean ± SEM). Representative of n = 2 independent transplants. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. C, Gene-set enrichment analysis (GSEA) of significant Hallmark gene sets of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated LSKs isolated 7 days after initiation of tamoxifen ( n = 3–4 biological replicates per arm). D, Volcano plot demonstrating differential gene expression of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated LSKs 7 days following initiation of tamoxifen ( n = 3–4 biological replicates per arm). E, GMP and MEP stem cell frequencies of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated mice 7 days following initiation of tamoxifen ( n = 8 biological replicates per arm across two independent transplants; mean ± SEM). F, Row normalized heat map of RNA-seq data of key erythroid differentiation factor genes from harvested MEPs at baseline (MPN), day 3 (D3), and day 7 (D7) following initiation of tamoxifen ( Jak2 V617F deletion). G, HOMER motif analysis from ATAC-seq data demonstrating decreased accessibility of Gata motif signatures with concomitant increased accessibility of Cebp motif signatures of tamoxifen-treated ( Jak2 V617F -deleted) cKit + bone marrow cells isolated 7 days following initiation of treatment in comparison with MPN cells ( n = 3 biological replicates per arm). Non-Sig., nonsignificant.

Article Snippet: Single-mutant Tet2 −/− or double-mutant Jak2 RL / Tet2 −/− transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily ×4; purchased from MedChemExpress) 6–8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Mutagenesis, Comparison, Isolation, Gene Expression, RNA Sequencing

Journal: Cancer Discovery

Article Title: Jak2 V617F Reversible Activation Shows Its Essential Requirement in Myeloproliferative Neoplasms

doi: 10.1158/2159-8290.CD-22-0952

Figure Lengend Snippet: Differential efficacy of Jak2 V617F deletion compared with JAK inhibitor therapy. A, Scatter plot depicting −log 10 ( P adj )*sign(log 2 Fold Change) of ruxolitinib (RUX) treated vs. tamoxifen (TAM; Jak2 V617F -deleted) treated LSKs (Lineage − Sca1 + cKit + ) in comparison with MPN control LSKs isolated after 7 days of treatment ( n = 2–3 biological replicates per arm); differentially expressed genes as indicated by color (see Supplementary Tables S1 and S3). B, Gene-set enrichment analysis (GSEA) depicting a positive enrichment in heme metabolism in ruxolitinib-treated ( n = 3) vs. negative enrichment in tamoxifen ( Jak2 V617F -deleted) treated ( n = 3) LSKs isolated after 7 days of treatment. C, Box plot of the top leading edge genes in the Hallmark heme metabolism gene set of ruxolitinib-treated (blue) or tamoxifen ( Jak2 V617F -deleted) treated (red) megakaryocytic-erythroid progenitor (MEP; Lineage − cKit + Sca1 − Cd34 − Fcg − ) cells as compared with untreated MPN cohorts. D, Box plots of scATAC-seq motif accessibility for either NFKB1 or REL transcription factors for untreated human JAK2 WT ( n = 188 cells from 4 patients; gray), untreated JAK2 V617F -mutant ( n = 105 cells from 4 patients; gray), and ruxolitinib-treated JAK2 V617F -mutant ( n = 87 cells from 3 patients; blue) HSPCs . P values indicated are from linear mixture model explicitly modeling patient identity as random effect to account for patient-specific effects, followed by likelihood ratio test. ****, P ≤ 0.0001. E, Peripheral blood counts of vehicle (VEH), ruxolitinib (RUX), the type II JAK2 inhibitor CHZ868 (CHZ), or tamoxifen ( Jak2 V617F -deleted) treated mice at the conclusion of a 6-week in vivo trial: WBCs (left), Hct (right; n ≥ 4 each; mean ± SEM). **, P ≤ 0.01; ***, P ≤ 0.001; ****, p ≤ 0.0001. F, Peripheral blood (PB) mutant Cd45.2 percent chimerism trend (0–6 weeks) of vehicle, ruxolitinib, CHZ868, or tamoxifen ( Jak2 V617F -deleted) treated mice ( n ≥ 4 each; mean ± SEM). *, P ≤ 0.05. G, Bone marrow–mutant cell fraction of LSK (Lineage − Sca1 + cKit + ), granulocytic-monocytic progenitor (GMP; Lineage − cKit + Sca1 − Cd34 + Fcg + ), and megakaryocytic-erythroid progenitor (MEP; Lineage − cKit + Sca1 − Cd34 − Fcg − ) compartments of vehicle, ruxolitinib, CHZ868, or tamoxifen ( Jak2 V617F -deleted) treated mice at the conclusion of the 6-week in vivo trial ( n ≥ 4 each; mean ± SEM). *, P ≤ 0.05; ****, P ≤ 0.0001. H, GSEA depicting a negative enrichment in downregulation of KRAS signaling targets in ruxolitinib-treated ( n = 3) vs. positive enrichment in tamoxifen ( Jak2 V617F -deleted) treated ( n = 3) MEPs isolated after 7 days of respective treatment. I, IHC of phospho-ERK on sectioned bone marrow of vehicle, ruxolitinib, or tamoxifen ( Jak2 V617F -deleted) treated mice following 7 days of treatment ( n = 3 individual biological replicates per arm). All images represented at 400× magnification. Scale bar, 20 μm. J, Quantitative PCR demonstrating relative Ybx1 expression levels from isolated cKit + bone marrow of vehicle vs. ruxolitinib vs. tamoxifen ( Jak2 V617F -deleted) treated mice after 7 days of treatment ( n = 2–4 individual biological replicates per arm; mean ± SEM). *, P ≤ 0.05; **, P ≤ 0.01. E–G, Representative of n = 3 independent experiments.

Article Snippet: Single-mutant Tet2 −/− or double-mutant Jak2 RL / Tet2 −/− transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily ×4; purchased from MedChemExpress) 6–8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Comparison, Control, Isolation, Mutagenesis, In Vivo, Real-time Polymerase Chain Reaction, Expressing

Journal: Cancer Discovery

Article Title: Jak2 V617F Reversible Activation Shows Its Essential Requirement in Myeloproliferative Neoplasms

doi: 10.1158/2159-8290.CD-22-0952

Figure Lengend Snippet: Jak2 V617F dependency with cooperative Tet2 loss. A, Schematic of the experimental setup for the double-mutant Jak2 RL / Tet2 f/f competitive transplants. Downward arrows represent initial pulse tamoxifen (TAM) administration to genetically inactivate Tet2 . B, WBC counts of primary Jak2 RL vs. Tet2 −/− vs. Jak2 RL / Tet2 −/− transplanted mice at 16 weeks posttransplant ( n = 5–6 each; mean ± SEM). Representative of n = 2 independent transplants. *, P ≤ 0.05; ***, P ≤ 0.001. C, Spleen weights of primary Jak2 RL vs. Tet2 −/− vs. Jak2 RL /Tet2 −/− transplanted mice at time of sacrifice ( n = 5–6 each; mean ± SEM). Representative of n = 2 independent transplants. *, P ≤ 0.05; **, P ≤ 0.01. D, Peripheral blood Cd45.2-mutant percent chimerism of Jak2 RL vs. Tet2 −/− vs. Jak2 RL / Tet2 −/− secondary competitive transplant mice at 9 weeks posttransplant ( n ≥ 10 per arm; mean ± SEM). Representative of n = 2 independent transplants. *, P ≤ 0.05. E, Peripheral blood count trends (weeks 0–21) of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated Jak2 RL vs. Jak2 RL / Tet2 −/− competitive transplant mice: WBCs (left), hematocrit (Hct; right; n = 3–4 per arm; mean ± SEM). Gray bars represent duration of tamoxifen pulse/chow administration. Representative of n = 2 independent transplants. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. F, Fold change from baseline (pretreatment) to posttreatment of Cd45.2-mutant peripheral blood chimerism of Jak2 RL vs. Tet2 −/− vs. Jak2 RL / Tet2 −/− transplanted mice treated for 6 weeks with either vehicle, ruxolitinib (RUX; 60 mg/kg twice daily), or tamoxifen ( Jak2 VF deletion; n = 4–5 per arm; mean ± SEM). *, P ≤ 0.05. G, Reticulin stains of bone marrow from MPN vs. tamoxifen ( Jak2 V617F -deleted) treated Jak2 RL vs. Jak2 RL / Tet2 −/− mice at timed sacrifice (21 weeks). Representative micrographs of n = 3 individual mouse replicates per arm. All images represented at 400× magnification. Scale bar, 20 μm. H, Bone marrow–mutant Cd45.2 percent chimerism within the LSK (Lineage − Sca1 + cKit + ) compartment of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated Jak2 RL vs. Jak2 RL / Tet2 −/− mice at timed sacrifice (21 weeks; n ≥ 7 biological replicates per arm across two independent transplants; mean ± SEM). *, P ≤ 0.05; ***, P ≤ 0.001. I, Serial replating assay of plated MPN vs. tamoxifen ( Jak2 V617F -deleted) treated Jak2 RL vs. Jak2 RL / Tet2 −/− bone marrow cells harvested at timed sacrifice 21 weeks and scored at day 8 after each plating (each sample plated in triplicate, representative of n = 2 independent experiments, mean ± SD). cGy, centigray; KI, knock-in; KO, knock-out; Lin-neg BM, lineage-negative bone marrow; trx, transplant.

Article Snippet: Single-mutant Tet2 −/− or double-mutant Jak2 RL / Tet2 −/− transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily ×4; purchased from MedChemExpress) 6–8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Mutagenesis, Knock-In, Knock-Out

Journal: Communications Biology

Article Title: Whole genome sequencing identifies pathogenic genetic variants in Han Chinese patients with familial venous thromboembolism

doi: 10.1038/s42003-025-07935-x

Figure Lengend Snippet: A Two pedigrees (F11 and F34) shared by TET2: c.G3451T:p.E1151X variant and pedigrees of F03, F32 and F32, F33 shared by GP6 : c.G1094A:p.R365H and JAK2 : c.G380A:p.G127D variant co-segregating with VTE, respectively. The proband is indicated by an arrowhead. A triangle symbol indicates individuals who underwent whole genome sequencing. Genotypes and phenotypes of each family member are shown in tables. B Sanger sequencing of TET2, GP6 and JAK2 variants are listed. C TET2 mutant reduced itself expression while GP6 mutant enhanced itself expression compared to wild-type by qPCR and Western-blot experiments. Data are presented as mean ± SE. Error bars indicate the standard error of the mean. WT: wild-type; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: GFP-tagged full-length wild-type (WT) and mutant (MT) plasmids for TET2 , JAK2 and GP6 was synthesized and verified by GenePharma (GenePharma, Shanghai, China).

Techniques: Variant Assay, Sequencing, Mutagenesis, Expressing, Western Blot

Journal: Journal of blood medicine

Article Title: Emerging treatments for essential thrombocythemia

doi: 10.2147/JBM.S19053

Figure Lengend Snippet: Diagnostic criteria for essential thrombocythemia (ET) 37

Article Snippet: However, in a recent update, this group did show that whilst the JAK2 V617F clone was reduced by Pegasys, the TET-2 mutant clones were apparently not affected.

Techniques: Diagnostic Assay, Biomarker Discovery, Mutagenesis