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: Blood

Article Title: The sumoylation pathway is dysregulated in multiple myeloma and is associated with adverse patient outcome

doi: 10.1182/blood-2009-03-211045

Figure Lengend Snippet: Effect of UBE2I-dominant-negative mutant on growth, proliferation, and adhesion to BMSCs. (A) Myc-tagged fusion proteins were expressed in RPMI 8226 cells. Mo indicates mock transfected; Wt, UBE2I wild-type; and DN, UBE2I-DN (dominant negative). Lysates were probed with a Myc-tag specific mAb (top), Ube2I-specific mAb (middle), or Sumo-1 polyclonal Ab (bottom). (B) UBE2I-DN increased γ-radiation–induced apoptosis. Early apoptosis was detected by annexin V staining. Plots indicate percentage of RPMI 8226 cells in early apoptosis 24 hours after treatment with 10 Gy γ-radiation. Data are representative of 3 independent experiments. (C) UBE2I-wt and UBE2I-DN resulted in opposite effects on MM cell growth. Values represent the mean of triplicate measurements using the MTT assay. (D) Ube2I is necessary for MM adhesion to bone marrow stroma. Adhesion of GFP-labeled UBE2I transfectants to normal BMSCs is shown by fluorescence microscopy after 96 hours of incubation under standard conditions. Data are representative of 3 independent experiments. Images were viewed with a Zeiss Axiovert 200 inverted epifluorescence microscope using a 20× objective (37°C; cells were in PBS; FITC fluorescent filter). Images were acquired with a Zeiss AxioCam HRc 14-bit color CCD camera and were processed with Axio Vision software (Version 3.1). (E) UBE2I-DN decreased BMSCs-induced uptake of 3H-thymidine. Values represent the mean of triplicate measurements of 3H-thymidine after 96 hours of coculture.

Article Snippet: Rabbit polyclonal antibody to human NSE2 (BC100-2506) was obtained from Novus Biologicals and was used at a final concentration of 1:500 and rabbit polyclonal antibody to PIAS1 was from Abcam.

Techniques: Dominant Negative Mutation, Transfection, Staining, MTT Assay, Labeling, Fluorescence, Microscopy, Incubation, Inverted Epifluorescence, Software

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-6 promotes pancreatic cancer cell migration by rapidly activating the small GTPase CDC42

doi: 10.1074/jbc.RA118.003276

Figure Lengend Snippet: JAK/STAT signaling is required for IL-6 to activate CDC42. A, knockdown of JAK2 prevents IL-6 from activating CDC42. PANC-1 cells were transfected with a control nontargeting siRNA or an siRNA against JAK2 and then stimulated with IL-6 (50 ng/ml) for 5 min. Active CDC42 was precipitated using a GST-PBD biochemical pulldown and Western blotting for CDC42. B, knockdown of STAT3 prevents IL-6 from activating CDC42. PANC-1 cells were transfected with a control nontargeting siRNA or an siRNA against STAT3 and then treated as described in A. C, PANC-1 cells were treated with DMSO vehicle control or the STAT inhibitor S3I-201 (100 μm) for 1 h and then treated with IL-6 (50 ng/ml) for 5 min, and CDC42 activity was assessed as described above. D, a dominant-negative STAT3 mutant prevents activation of CDC42 by IL-6. PANC-1 cells were transfected to overexpress WT STAT3 or STAT3 Y705F and then treated with IL-6 (50 ng/ml; 5 min). CDC42 activation was assessed as described above. In B–D, IL-6 stimulation led to activation of CDC42 but not following inhibition of STAT3 using RNAi, a chemical inhibitor, or a phosphomutant. For A–D, active CDC42 was normalized to total CDC42, and the ratios were compared with t = 0 in the control cells. Data are represented as the mean of three to five independent biological replicates. Error bars represent S.E. *, p < 0.05; **, p < 0.01; ns, not a statistically significant difference. See also Fig. S3.

Article Snippet: Immunoprecipitation and immunoblotting For Western blotting, proteins were resolved by SDS-PAGE, transferred to PVDF (Millipore), blocked in 5% milk (Bio-Rad) or 5% BSA (Sigma), and incubated with primary antibodies overnight (CDC42, BD Transduction Laboratories, 610928; RAC1, clone 23A8, Millipore, 05-389; RhoA, Santa Cruz Biotechnology, sc-418; actin, Sigma, A2066; JAK2, clone D2E12, Cell Signaling Technology, 3230; STAT3, clone D3Z2G, Cell Signaling Technology, 12640; phospho-STAT3, clone D3A7, Cell Signaling Technology, 9145; phospho-ERK, clone 197G2, Cell Signaling Technology, 4377; IQGAP1, Santa Cruz Biotechnology, sc-376021).

Techniques: Knockdown, Transfection, Control, Western Blot, Activity Assay, Dominant Negative Mutation, Mutagenesis, Activation Assay, Inhibition

Journal: Scientific Reports

Article Title: Activated FXR Inhibits Leptin Signaling and Counteracts Tumor-promoting Activities of Cancer-Associated Fibroblasts in Breast Malignancy

doi: 10.1038/srep21782

Figure Lengend Snippet: ( a ) MCF-7 and MDA-MB-231 cells were pretreated for 12 h with GW4064 and then treated or not for 10 min with Lep. Levels of phosphorylated (p) JAK2, STAT3, Akt, and MAPK, and total non-phosphorylated proteins were evaluated in cellular extracts by immunoblot analysis. β-Actin was used as loading control. ( b ) Total RNA was extracted from MCF-7 and MDA-MB-231 cells, pretreated for 12 h with GW4064 and then treated or not for 24 h with Lep, reverse transcribed and cDNAs were subjected to PCR using primers specific for Ob or 36B4 (internal control). NC: negative control. ( c ) Immunoblot analysis for Cyclin D1 and Survivin expression in MCF-7 and MDA-MB-231 cells pretreated for 12 h with GW4064 and then treated or not for 24 h with Lep. (d) MCF-7 cells were transiently transfected with either empty vector (e.v.) or FXR dominant negative plasmid (FXR-DN), pretreated for 12 h with GW4064 and then treated or not for 24 h with Lep. Cyclin D1 and Survivin expression levels were evaluated by immunoblotting. β-Actin was used as loading control. The histograms represent the mean ± SD of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary units (OD) and expressed as percentage of vehicle-treated samples which were assumed to be 100%. n.s. = non significant, *p < 0.05.

Article Snippet: Antibodies against total non-phosphorylated and Phosphorylated (p) JAK2 (Tyr 1007/1008 ), STAT3 (Tyr 705 ), Akt (Ser 473 ), and MAPK (Thr 202 /Tyr 204 ) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Western Blot, Control, Reverse Transcription, Negative Control, Expressing, Transfection, Plasmid Preparation, Dominant Negative Mutation

Journal: Experimental Hematology & Oncology

Article Title: The SMAC mimetic LCL-161 selectively targets JAK2 V617F mutant cells

doi: 10.1186/s40164-019-0157-6

Figure Lengend Snippet: JAK2 V617F mutant cells lines are more sensitive to killing by LCL-161 under certain circumstances. a – c . L929 cells expressing JAK2 WT , JAK2 V617F or empty vector were incubated with increasing concentrations of a LCL-161 alone, b LCL-161 with the addition of 0.25 ng/ml mTNFα, c LCL-161 with the addition of 400 ng/ml mTNFa neutralizing antibody for 48 h and then analyzed with a resazurin based viability assay. **** P < 0.0001, 2way ANOVA. d Western blot of L929 cell lines harvested 24 h after exposure to combinations of LCL-161 and mTNFα. e – g HEL and K562 cells were incubated with e LCL-161 alone, f LCL-161 + 1 ng/ml hTNFα, or g LCL-161 + 10 ng/ml hTNFα for 48 h. Apoptosis was measured with Annexin V and PI staining. ** P < 0.01, **** P < 0.0001 unpaired t test

Article Snippet: We found that JAK2 V617F -mutated cells are hypersensitive to the SMAC mimetic LCL-161 in the absence of exogenous TNFα.

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Incubation, Viability Assay, Western Blot, Staining

Journal: Experimental Hematology & Oncology

Article Title: The SMAC mimetic LCL-161 selectively targets JAK2 V617F mutant cells

doi: 10.1186/s40164-019-0157-6

Figure Lengend Snippet: JAK inhibitors rescue hypersensitivity of JAK2 V617F mutant cells to LCL-161. L929 cells expressing JAK2 WT , JAK2 V617F or empty vector were incubated with increasing concentrations of a LCL-161 alone, b LCL-161 with the addition of 1 µM ruxolitinib, or c LCL-161 with the addition of 1 µM pacritinib. After 48 h in culture a resazurin-based cell viability assay was performed. **** P < 0.0001, 2way ANOVA

Article Snippet: We found that JAK2 V617F -mutated cells are hypersensitive to the SMAC mimetic LCL-161 in the absence of exogenous TNFα.

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Incubation, Viability Assay

Journal: Experimental Hematology & Oncology

Article Title: The SMAC mimetic LCL-161 selectively targets JAK2 V617F mutant cells

doi: 10.1186/s40164-019-0157-6

Figure Lengend Snippet: LCL-161 preferentially decreases MPN colony formation. Methylcellulose colony formation of a , b whole bone marrow from JAK2 V617F or wild-type mice and c , d peripheral blood mononuclear cells from JAK2 V617F mutated MPN patients or normal controls with increasing concentrations of LCL-161 alone ( a , c ) or LCL-161 + 0.25 ng/ml TNFα ( b , d ). * P < 0.05, *** P < 0.001, unpaired t test

Article Snippet: We found that JAK2 V617F -mutated cells are hypersensitive to the SMAC mimetic LCL-161 in the absence of exogenous TNFα.

Techniques:

Journal: Experimental Hematology & Oncology

Article Title: The SMAC mimetic LCL-161 selectively targets JAK2 V617F mutant cells

doi: 10.1186/s40164-019-0157-6

Figure Lengend Snippet: Impact of LCL-161 treatment in a transduction–transplantation model of JAK2V617F mutated MPN. a Percentage of GFP+ (empty vector or JAK2 V617F ) cells in peripheral blood. b White blood cell (WBC), c hematocrit (HCT), d platelet (PLT) and e hemoglobin (HGB) counts in wild-type (empty vector) or JAK2 V617F mice treated with LCL-161. f Spleen weights in JAK2 V617F mice, P < 0.05, unpaired t test. g Percentage of GFP+ (JAK2 V617F ) cells in the spleen and bone marrow. h Snook’s reticulin staining of paraffin-embedded bone marrows in wildtype (empty vector) and JAK2 V617F mice treated with LCL-161. n = 3–4 mice per group

Article Snippet: We found that JAK2 V617F -mutated cells are hypersensitive to the SMAC mimetic LCL-161 in the absence of exogenous TNFα.

Techniques: Transduction, Transplantation Assay, Plasmid Preparation, Staining

Journal: PLoS ONE

Article Title: JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms

doi: 10.1371/journal.pone.0012165

Figure Lengend Snippet: Top, schematic diagram of the JAK2 protein showing JAK homology domains 1 through 7 (JH1-JH7) with the JH2 pseudokinase domain highlighted in black. The corresponding exon regions of the mRNA is shown with the exons 13, 14, and 15. Because exon 14 is consists of 88 bp, its deletion leads to frameshift and early termination of translation after coding for seven new amino acids and elimination of the V617 codon of JAK2 (lower panel). The resulting truncated JAK2 protein is shown on the bottom.

Article Snippet: A patent has been filed on finding by Quest Diagnostics and testing for JAK2 various mutations is offered at Quest Diagnostics.

Techniques:

Journal: PLoS ONE

Article Title: JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms

doi: 10.1371/journal.pone.0012165

Figure Lengend Snippet: Upper panels: Detection of Δexon14 is relatively easy when the transcript is present at high levels (eg, 29% of total JAK2 transcript). Detection is more difficult when the Δexon14 transcript makes up a small proportion of total JAK2 transcript (eg, 6.9%). Normal control is shown on the bottom.

Article Snippet: A patent has been filed on finding by Quest Diagnostics and testing for JAK2 various mutations is offered at Quest Diagnostics.

Techniques: Control

Journal: PLoS ONE

Article Title: JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms

doi: 10.1371/journal.pone.0012165

Figure Lengend Snippet: Prevalence and Relative Level of the ΔExon14 JAK2 Transcript in Patients with Suspected or Confirmed Myeloproliferative Neoplasms (MPNs)

Article Snippet: A patent has been filed on finding by Quest Diagnostics and testing for JAK2 various mutations is offered at Quest Diagnostics.

Techniques:

Journal: PLoS ONE

Article Title: JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms

doi: 10.1371/journal.pone.0012165

Figure Lengend Snippet: Lysates were prepared from the indicated human CML K562 cell line (Lane1), a patient with chronic myelogenous leukemia (lane 2), and 5 patients with chronic MPNs (Lanes 3–7). Patient 1: non-CML CMPD, JAK2 V617F positive; Patient 2: non-CML CMPD, JAK2 V617F negative; Patient 3: non-CML CMPD, JAK2 Δexon14 positive; Patient 4: non-CML CMPD, JAK2 Δexon14 positive; Patient 5: non-CML CMPD, JAK2 Δexon14 positive. Top Panel: Probing with an anti-JAK2 N-terminal clone yielded a wild-type JAK2 band at 130 kDa in the K562 and other negative control lanes, and an additional band at 75 kDa only in patients with expression of Δexon14 transcript. Bottom Panel: An anti-JAK2 clone directed against the carboxyl-terminus of JAK2 yielded only a single band at 130 kDa.

Article Snippet: A patent has been filed on finding by Quest Diagnostics and testing for JAK2 various mutations is offered at Quest Diagnostics.

Techniques: Negative Control, Expressing