Journal: Science Advances

Article Title: Therapeutic inhibition of the SRC-kinase HCK facilitates T cell tumor infiltration and improves response to immunotherapy

doi: 10.1126/sciadv.abl7882

Figure Lengend Snippet: ( A ) Mass of individual subcutaneous MC38 tumors from WT and Hck KO hosts. Where indicated, mice were treated with αPD1 (200 μg, once every 3 days) or an isotype-matched IgG control for 10 days. Representative tumors are depicted above the graphs. Scale bar, 1 cm. Each symbol represents an individual mouse. ( B ) Flow cytometry quantification of the indicated myeloid cell populations from subcutaneous MC38 tumors of WT and Hck KO hosts treated as described in (A). Each symbol represents an individual mouse. ( C ) Quantitative polymerase chain reaction (qPCR) analysis on fluorescence-activated cell sorter (FACS)–purified CD45 + CD11b + F4/80 High Ly6C − Ly6G − TAMs and CD45 + CD11c + F4/80 − MHCII + dendritic cells for markers associated with immune cell activation. Cells were isolated from subcutaneous MC38 tumors of WT and Hck KO hosts treated as described in (A). n = 4 mice per group. ( D ) Mass of individual subcutaneous MC38 tumors from WT and Hck KO hosts. Where indicated, mice were treated with αIL-12, αCXCR3, or an isotype-matched IgG control once every 3 days (total of three treatments) before subcutaneous MC38 tumor cell injection and continued until the experimental end point. Once palpable tumors formed, mice were treated with αPD1 for 10 days. Each symbol represents an individual mouse. ( E ) Mass of individual subcutaneous MC38 tumors from WT bone marrow chimeras reconstituted with cDC1-deficient (cDC1 KO ) or cDC1-proficient (WT) bone marrow. To deplete TAMs, half of each cohort were treated with αCSF1R before MC38 tumor cell injection and continued until the experimental end point. Once palpable tumors formed, all hosts were treated with αPD1 in the presence or absence of the small-molecule HCK inhibitor RK20449 for 20 days. Each symbol represents an individual mouse. Data represent mean ± SEM; * P < 0.05, ** P < 0.01, and *** P < 0.001, with statistical significance determined by one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: Where indicated, mice were treated with 12% Captisol [vehicle for RK20449 and dasatinib, intraperitoneally (i.p.)], an isotype-matched IgG control (JPP Biologics, 100 to 200 μg once every 3 days i.p.), RK20449 (synthesized by Reagency, 30 mg/kg, diluted in 12% Captisol, twice daily i.p.), dasatinib (Selleckchem, #S1021, 30 mg/kg, diluted in 12% Captisol, twice daily i.p.), IPI549 [Selleckchem, #S8330, 15 mg/kg, diluted in 5% dimethyl sulfoxide and 30% PEG300 in ddH 2 O, once daily orally (p.o.)], BLZ945 (Selleckchem, #953769-46-5, 200 mg/kg, diluted in 12% Captisol, once daily p.o.), αCD40 (JPP Biologics, clone FGK45, 100 μg once every 3 days i.p.), αPD1 (JPP Biologics, clone RMP1-14, 200 μg once every 3 days i.p.), or αCTLA4 (JPP Biologics, clone 4F10, 200 μg once every 3 days i.p.) for a minimum of 10 days.

Techniques: Control, Flow Cytometry, Real-time Polymerase Chain Reaction, Fluorescence, Purification, Activation Assay, Isolation, Injection, Comparison

Journal: Science Advances

Article Title: Therapeutic inhibition of the SRC-kinase HCK facilitates T cell tumor infiltration and improves response to immunotherapy

doi: 10.1126/sciadv.abl7882

Figure Lengend Snippet: ( A ) Mass of individual subcutaneous MC38 tumors from WT hosts treated with RK20449 (30 mg/kg, twice daily) and/or αPD1 (200 μg, once every 3 days) for a total of 10 days. Vehicle control mice were treated with 12% Captisol and an isotype-matched IgG control. Representative tumors are depicted above graphs. Scale bar, 1 cm. Each symbol represents an individual mouse. ( B ) Tumor volume of subcutaneous MC38 tumors established in Hck KO hosts treated with dasatinib (30 mg/kg, twice daily) and/or αPD1 (200 μg, once every 3 days) until the experimental end point when tumors reached ≥600 mm 3 . n ≥ 9 mice per group. ( C ) t-distributed stochastic neighbor embedding (tSNE) plot depicting Csf1r , Hck , and Pik3cg gene expression in subcutaneous MC38 tumors analyzed from Zhang et al. . ( D ) Heatmap showing the relative expression of CSF1R , PIK3CG , HCK , and other SFK family members in human colon cancer samples analyzed from Zhang et al. . ( E ) Mass of individual subcutaneous MC38 tumors from Hck KO hosts treated with IPI549 (15 mg/kg, once daily) or BLZ945 (200 mg/kg, once daily) in the presence or absence of αPD1 (200 μg, once every 3 days) for 20 days. Each symbol represents an individual mouse. Data represent mean ± SEM; *** P < 0.001; n.s., not significant, with statistical significance determined by one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: Where indicated, mice were treated with 12% Captisol [vehicle for RK20449 and dasatinib, intraperitoneally (i.p.)], an isotype-matched IgG control (JPP Biologics, 100 to 200 μg once every 3 days i.p.), RK20449 (synthesized by Reagency, 30 mg/kg, diluted in 12% Captisol, twice daily i.p.), dasatinib (Selleckchem, #S1021, 30 mg/kg, diluted in 12% Captisol, twice daily i.p.), IPI549 [Selleckchem, #S8330, 15 mg/kg, diluted in 5% dimethyl sulfoxide and 30% PEG300 in ddH 2 O, once daily orally (p.o.)], BLZ945 (Selleckchem, #953769-46-5, 200 mg/kg, diluted in 12% Captisol, once daily p.o.), αCD40 (JPP Biologics, clone FGK45, 100 μg once every 3 days i.p.), αPD1 (JPP Biologics, clone RMP1-14, 200 μg once every 3 days i.p.), or αCTLA4 (JPP Biologics, clone 4F10, 200 μg once every 3 days i.p.) for a minimum of 10 days.

Techniques: Control, Gene Expression, Expressing, Comparison

Journal: Science Advances

Article Title: Therapeutic inhibition of the SRC-kinase HCK facilitates T cell tumor infiltration and improves response to immunotherapy

doi: 10.1126/sciadv.abl7882

Figure Lengend Snippet: ( A and B ) Tumor volume and mass of individual subcutaneous PDXs from humanized NSG-SGM3 hosts following treatment with vehicle (12% Captisol) or RK20449 (30 mg/kg, twice daily) for 25 days. Each symbol represents an individual mouse. ( C ) qPCR analysis on whole PDX tumors from humanized NSG-SGM3 hosts treated as described in (A) for genes associated with human immune cells. n = 4 mice per group. ( D ) qPCR analysis on whole PDX tumors from humanized NSG-SGM3 hosts treated as described in (A) for genes associated with immune cell activation (i.e., IL12 , IFNG , IL1B , TNF , CXCL9 , and CXCL10 ) and immune suppression (i.e., CD163 , MRC1 , TGFB , IL10 , and CXCL12 ). n = 4 mice per group. Data represent mean ± SEM; * P < 0.05, ** P < 0.01, and *** P < 0.001, with statistical significance determined by an unpaired Student’s t test.

Article Snippet: Where indicated, mice were treated with 12% Captisol [vehicle for RK20449 and dasatinib, intraperitoneally (i.p.)], an isotype-matched IgG control (JPP Biologics, 100 to 200 μg once every 3 days i.p.), RK20449 (synthesized by Reagency, 30 mg/kg, diluted in 12% Captisol, twice daily i.p.), dasatinib (Selleckchem, #S1021, 30 mg/kg, diluted in 12% Captisol, twice daily i.p.), IPI549 [Selleckchem, #S8330, 15 mg/kg, diluted in 5% dimethyl sulfoxide and 30% PEG300 in ddH 2 O, once daily orally (p.o.)], BLZ945 (Selleckchem, #953769-46-5, 200 mg/kg, diluted in 12% Captisol, once daily p.o.), αCD40 (JPP Biologics, clone FGK45, 100 μg once every 3 days i.p.), αPD1 (JPP Biologics, clone RMP1-14, 200 μg once every 3 days i.p.), or αCTLA4 (JPP Biologics, clone 4F10, 200 μg once every 3 days i.p.) for a minimum of 10 days.

Techniques: Activation Assay

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: HSP90-CDC37 functions as a chaperone for the oncogenic FGFR3-TACC3 fusion.

doi: 10.1016/j.ymthe.2022.02.009

Figure Lengend Snippet: Figure 3. F3-T3 is a strong HSP90 client (A and B) Time course of 200-nM HSP90 inhibitor treatments in U-251 MG cells stably expressing F3-T3 or FGFR3. Whole-cell lysates were harvested and immunoblotted with the indicated antibodies. (C–F) Upon treatment with DMSO vehicle (Vec) or 100 nM onalespib (C and D) or with (E and F) DMSO Vec or 100 nM 17-AAG, U-251 MG cells stably expressing FGFR3 or F3-T3 were treated with 50 mg/mL CHX, harvested at the indicated times, and lysates were immunoblotted with anti-FGFR3 (a representative WB is shown). WB band densities were quantified, normalized to b-actin, and the mean percent density levels relative to 0 h ± SEMs from 3 independent experiments are plotted. p values were calculated using a paired, 2-tailed Student’s t test.

Article Snippet: TMZ (5 mg/mL in 20% DMSO) was administered by oral gavage and onalespib (6 mg/mL in 15% SelleckChem Captisol-S4592 in water) by tail vein injection given on the days and in the concentrations indicated in the Figures 7A and S6B) schematic overview.

Techniques: Stable Transfection, Expressing

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: HSP90-CDC37 functions as a chaperone for the oncogenic FGFR3-TACC3 fusion.

doi: 10.1016/j.ymthe.2022.02.009

Figure Lengend Snippet: Figure 4. Targeting the HSP90-cdc37 system inhibits F3-T3 mediated signaling and oncogenic potency (A) U-251 MG cells stably expressing F3-T3 were exposed for 6 h to 200 nM of HSP90 inhibitors (onalespib and 17-AAG) or FGFR inhibitors (BGJ398 and PD173074) and lysates were immunoblotted with the indicated antibodies. (B) U-251 MG cells stably expressing F3-T3 were exposed for 6 h to 200 nM onalespib, 5 mg/mL brefeldin A (BFA) or 10 mg/mL of tunicamycin and lysates were immunoblotted with the indicated antibodies. (C) U-251 MG cells stably expressing F3-T3 were cultured in the absence or presence of FGF9 with or without 200 nM onalespib or 5 mg/mL BFA for 6 h and lysates were immunoblotted with the indicated antibodies. (D) U-251 MG cells stably expressing EV or F3-T3 were exposed to increasing concentrations of onalespib, 17-AAG, BGJ398, PD173074, or Vec control (DMSO) for 72 h, after which relative cell numbers were estimated using the CCK8 assay. A representative plot from 1 of 3 experiments is shown; means ± SEMs. (E) Mean IC50 of onalespib, 17-AAG, BGJ398, and PD173074 in U-251 MG cells stably expressing EV or F3-T3 from 3 independent experiments as illustrated in (D) and analyzed by 1-way ANOVA with Tukey’s post-test; *p < 0.05, ***p < 0.001 (selectively

Article Snippet: TMZ (5 mg/mL in 20% DMSO) was administered by oral gavage and onalespib (6 mg/mL in 15% SelleckChem Captisol-S4592 in water) by tail vein injection given on the days and in the concentrations indicated in the Figures 7A and S6B) schematic overview.

Techniques: Stable Transfection, Expressing, Cell Culture, Control, CCK-8 Assay

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: HSP90-CDC37 functions as a chaperone for the oncogenic FGFR3-TACC3 fusion.

doi: 10.1016/j.ymthe.2022.02.009

Figure Lengend Snippet: Figure 6. Hsp90 inhibitor chemosensitizes F3-T3 glioma cells to TMZ (A) Combination drug matrix for estimates of interactions using CalcuSyn software. LN-229 cells stably expressing F3-T3 were treated (+/) onalespib and TMZ as shown and relative CCK8 viability was measured after 72 h. Percentages are the means from 3 independent experiments and indicate cell death relative to the untreated controls. (B) U-251 MG cells or LN-229 cells stably expressing F3-T3 treated with onalespib or 17-AAG in combination with DMSO or TMZ and assayed for colony formation after 8 days. Mean colonies (treatment/DMSO ±SD (n = 3); ***p < 0.001 each drug alone to each combination by 1-way ANOVA with Tukey’s post-test. (C) U-251 MG cells (legend continued on next page)

Article Snippet: TMZ (5 mg/mL in 20% DMSO) was administered by oral gavage and onalespib (6 mg/mL in 15% SelleckChem Captisol-S4592 in water) by tail vein injection given on the days and in the concentrations indicated in the Figures 7A and S6B) schematic overview.

Techniques: Software, Stable Transfection, Expressing

Journal: Molecular Cancer Research

Article Title: METTL3-Mediated m 6 A Modification Regulates the Polycomb Repressive Complex 1 Components BMI1 and RNF2 in Hepatocellular Carcinoma Cells

doi: 10.1158/1541-7786.MCR-24-0362

Figure Lengend Snippet: METTL3 inhibitor, STM2457, significantly inhibited HCC cell growth, in vitro and in vivo . A, Huh7 and PLC/PRF/5 cells were treated with METTL3 inhibitor STM2457 at the indicated concentrations for 72 and 96 hours. The cell proliferation was analyzed by WST-1 assay. B, Colony formation analysis of Huh7 and PLC/PRF/5 cells after treatment of METTL3 inhibitor STM2457 at the indicated concentrations for 14 days (left). Quantification of the colony formation assay results are shown in the right. C, Western blot analysis of the protein levels of BMI1, RNF2, and HA2K119ub in Huh7 and PLC/PRF/5 cells after treatment with the METTL3 inhibitor STM2457 (20 µmol/L) for 48 hours. β-Actin was used as loading control. D, Western blot for BMI1 and RNF2 in Huh7 and PLC/PRF/5 cells transfected with BMI1 or RNF2 expression vector. E, WST-1 cell proliferation assay in BMI1 or RNF2 transfected Huh7 and PLC/PRF/5 cells treated with STM2457 (20 µmol/L) for 48h . F, Huh7 cells (1 × 10 6 ) were mixed in Matrigel solution and inoculated directly into the livers of SCID mice ( n = 15). Three weeks after intrahepatic inoculation, the mice were treated with STM2457 (50 mg/kg; n = 6) or vehicle (10% DMSO, 20% captisol in 1× PBS; n = 9) via i.p. injection daily. The mice were sacrificed at 7 weeks after intrahepatic inoculation. Representative gross images of the livers are shown in left. The tumor volumes are shown in right. G, IHC staining of Ki67. The images were analyzed using Motic Digital Slide Assistant. Scale bar, 200 μm. H, The levels of BMI1 and RNF2 proteins in liver tumor tissues from mice treated with vehicle or STM2457 were analyzed by Western blot. β-Actin was used as loading control. Data are expressed as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Three weeks after intrahepatic inoculation, the mice were randomly divided into two groups and treated with STM2457 (MedChemExpress; 50 mg/kg, 10% DMSO, 20% captisol in 1× PBS; n = 6) or vehicle (10% DMSO, 20% captisol in 1× PBS; n = 9) via i.p. injection daily for 4 weeks.

Techniques: In Vitro, In Vivo, WST-1 Assay, Colony Assay, Western Blot, Control, Transfection, Expressing, Plasmid Preparation, Proliferation Assay, Injection, Immunohistochemistry

Journal: International Journal of Pharmaceutics: X

Article Title: In vivo pharmacokinetic study of remdesivir dry powder for inhalation in hamsters

doi: 10.1016/j.ijpx.2021.100073

Figure Lengend Snippet: Lung concentration-time profiles of REM-CAP (remdesivir-Captisol®; 80/20 w/w) and REM-LEU (remdesivir-leucine; 80/20 w/w) after a single inhalation administration in hamsters; (A) remdesivir; (B) GS-441524.

Article Snippet: Interestingly, our current study demonstrated that the complexation of Captisol® and remdesivir appeared only to have an effect on the absorption rate, but not on the extent of drug absorption into the systemic circulation.

Techniques: Concentration Assay

Journal: International Journal of Pharmaceutics: X

Article Title: In vivo pharmacokinetic study of remdesivir dry powder for inhalation in hamsters

doi: 10.1016/j.ijpx.2021.100073

Figure Lengend Snippet: Plasma concentration-time profiles of REM-CAP (remdesivir-Captisol®; 80/20 w/w) and REM-LEU (remdesivir-leucine; 80/20 w/w) after a single inhalation administration in hamsters; (A) remdesivir; (B) GS-441524. Dash line and dot line represent EC 50 of remdesivir and GS-441524 in human epithelial cells (HAE) , and continuous human lung epithelial cell line (Calu-3) , respectively.

Article Snippet: Interestingly, our current study demonstrated that the complexation of Captisol® and remdesivir appeared only to have an effect on the absorption rate, but not on the extent of drug absorption into the systemic circulation.

Techniques: Concentration Assay

Journal: Science Advances

Article Title: Therapeutic inhibition of the SRC-kinase HCK facilitates T cell tumor infiltration and improves response to immunotherapy

doi: 10.1126/sciadv.abl7882

Figure Lengend Snippet: ( A ) Mass of individual subcutaneous MC38 tumors from WT hosts treated with RK20449 (30 mg/kg, twice daily) and/or αPD1 (200 μg, once every 3 days) for a total of 10 days. Vehicle control mice were treated with 12% Captisol and an isotype-matched IgG control. Representative tumors are depicted above graphs. Scale bar, 1 cm. Each symbol represents an individual mouse. ( B ) Tumor volume of subcutaneous MC38 tumors established in Hck KO hosts treated with dasatinib (30 mg/kg, twice daily) and/or αPD1 (200 μg, once every 3 days) until the experimental end point when tumors reached ≥600 mm 3 . n ≥ 9 mice per group. ( C ) t-distributed stochastic neighbor embedding (tSNE) plot depicting Csf1r , Hck , and Pik3cg gene expression in subcutaneous MC38 tumors analyzed from Zhang et al. . ( D ) Heatmap showing the relative expression of CSF1R , PIK3CG , HCK , and other SFK family members in human colon cancer samples analyzed from Zhang et al. . ( E ) Mass of individual subcutaneous MC38 tumors from Hck KO hosts treated with IPI549 (15 mg/kg, once daily) or BLZ945 (200 mg/kg, once daily) in the presence or absence of αPD1 (200 μg, once every 3 days) for 20 days. Each symbol represents an individual mouse. Data represent mean ± SEM; *** P < 0.001; n.s., not significant, with statistical significance determined by one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: Where indicated, mice were treated with 12% Captisol [vehicle for RK20449 and dasatinib, intraperitoneally (i.p.)], an isotype-matched IgG control (JPP Biologics, 100 to 200 μg once every 3 days i.p.), RK20449 (synthesized by Reagency, 30 mg/kg, diluted in 12% Captisol, twice daily i.p.), dasatinib (Selleckchem, #S1021, 30 mg/kg, diluted in 12% Captisol, twice daily i.p.), IPI549 [Selleckchem, #S8330, 15 mg/kg, diluted in 5% dimethyl sulfoxide and 30% PEG300 in ddH 2 O, once daily orally (p.o.)], BLZ945 (Selleckchem, #953769-46-5, 200 mg/kg, diluted in 12% Captisol, once daily p.o.), αCD40 (JPP Biologics, clone FGK45, 100 μg once every 3 days i.p.), αPD1 (JPP Biologics, clone RMP1-14, 200 μg once every 3 days i.p.), or αCTLA4 (JPP Biologics, clone 4F10, 200 μg once every 3 days i.p.) for a minimum of 10 days.

Techniques: Control, Gene Expression, Expressing, Comparison

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: Mice were then treated with ruxolitinib (60 mg/kg orally twice daily; dissolved in 20% Captisol in PBS; purchased from MedChemExpress), CHZ868 (30 mg/kg orally daily; dissolved in 0.5% methylcellulose + 0.5% Tween-80 in dH 2 O; purchased from MedChemExpress), tamoxifen to delete Jak2 V617F (as above; purchased from MedChemExpress), or vehicle.

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: Mice were then treated with ruxolitinib (60 mg/kg orally twice daily; dissolved in 20% Captisol in PBS; purchased from MedChemExpress), CHZ868 (30 mg/kg orally daily; dissolved in 0.5% methylcellulose + 0.5% Tween-80 in dH 2 O; purchased from MedChemExpress), tamoxifen to delete Jak2 V617F (as above; purchased from MedChemExpress), or vehicle.

Techniques: Mutagenesis, Knock-In, Knock-Out