Journal: Biology

Article Title: Nucleolar Protein 56 Deficiency in Zebrafish Leads to Developmental Abnormalities and Anemia via p53 and JAK2-STAT3 Signaling

doi: 10.3390/biology12040538

Figure Lengend Snippet: Knockdown of p53 and inhibition of jak2 partially rescue malformation and anemia in nop56 mutants. ( A ) WISH analysis of p53 in siblings and mutants. ( B ) qRT-PCR showed that p53 expression was increased significantly in nop56 mutants. qRT-PCR was performed with biological repeats and technical repeats in triplicate. n = 3, * p < 0.05. ( C ) Analyses of the phenotype and hemoglobin level after p53 knockdown and CEP-33779 treatment in nop56 mutants. Knockdown of p53 partially rescued the morphological malformations, but not anemia, at 48 hpf. However, CEP-33779 partially rescued the anemic phenotype, but not malformations, at 48 hpf. Arrowhead indicates erythroid cells. All scale bars represent 1 mm.

Article Snippet: The inhibitor of JAK2, CEP-33779, was purchased from TargetMol, Boston, MA, USA and dissolved in DMSO to a storage concentration of 10 mM.

Techniques: Knockdown, Inhibition, Quantitative RT-PCR, Expressing

Journal: Clinical Cancer Research

Article Title: Development of Resistance to Type II JAK2 Inhibitors in MPN Depends on AXL Kinase and Is Targetable

doi: 10.1158/1078-0432.CCR-23-0163

Figure Lengend Snippet: Resistance to type II JAK inhibition is overcome in vivo by combined JAK2/AXL inhibition. A, JAK2 inhibitor resistant SET2 cells (JAKi-R) were injected subcutaneously into the flank of NSG mice. Animals were treated orally with type II JAK2 inhibitor CHZ868 15 mg/kg every day, AXL inhibitor bemcentinib (bem) 50 mg/kg twice a day, combined CHZ868 15 mg/kg every day/bemcentinib 50 mg/kg twice a day or vehicle control. Treatment was initiated at 100 mm 3 tumor size and continued until maximal tumor size was reached in vehicle treated mice. B, Analysis of tumor weight at end of treatment confirmed significant reduction of tumor growth in vivo by AXL inhibition with bemcentinib or by combined JAK2/AXL inhibition with CHZ868/bemcentinib ( n = 6/group). C, Tumor size over time showed tumor growth in vehicle-treated mice and analogously in CHZ868-treated mice suggesting resistance to type II JAK2 inhibition in vivo . AXL inhibition by bemcentinib significantly reduced tumor growth, while combined JAK2/AXL inhibition almost suppressed tumor growth ( n = 6/group). D, Photographic image of isolated tumors at end of treatment. E, Tumor growth in individual mice is shown for each treatment group. Black lines indicate the average tumor size per group. Combined CHZ868/bemcentinib almost suppressed tumor growth in all the treated mice. Data are presented as mean ± SD and analyzed by one-way ANOVA. ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: JAK2 inhibitors CHZ868, BBT594 (type II), ruxolitinib (type I), and MEK inhibitor trametinib were from Novartis through Material Transfer Agreement; AXL inhibitors bemcentinib and gilteritinib were purchased from BioVision and Sellekchem.

Techniques: Inhibition, In Vivo, Injection, Control, Isolation

Journal: Clinical Cancer Research

Article Title: Development of Resistance to Type II JAK2 Inhibitors in MPN Depends on AXL Kinase and Is Targetable

doi: 10.1158/1078-0432.CCR-23-0163

Figure Lengend Snippet: Targeting MAPK pathway abrogates acquired resistance to type II JAK2 inhibition in vivo . A, Schema of in vivo model with subcutaneous engraftment. JAK2 inhibitor resistant SET2 cells (JAK2i-R) were injected into the flank of NSG mice. Animals were treated orally with type II JAK2 inhibitor CHZ868 15 mg/kg every day, MAPK inhibitor trametinib (tram) 0.3 mg/kg every day, combined CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day, or vehicle control. Treatment was initiated at 100 mm 3 tumor size and continued until maximal tumor size was reached in vehicle-treated mice. B, Tumor size over time showed tumor growth in vehicle-treated mice similarly to CHZ868-treated mice. Combined JAK2/MAPK inhibition effectively reduced tumor size ( n = 13/group). C, Analysis of tumor weight at end of treatment confirmed significant reduction of tumor growth in vivo by combined JAK2/MAPK inhibition with CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day ( n = 13/group). D, Expression of MAPK pathway target DUSP6 was significantly reduced by combined JAK2/MAPK inhibition with CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day ( n = 4–5/group). E, Expression of MAPK pathway target ETV5 was significantly reduced by combined JAK2/MAPK inhibition with CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day ( n = 4–5/group). F, Schema of in vivo model with intravenous engraftment. JAK2 inhibitor resistant SET2 cells (JAK2i-R) stably expressing luciferase were injected intravenously into NSG mice and engraftment documented by bioluminescent imaging. Animals were treated orally with type II JAK2 inhibitor CHZ868 15 mg/kg every day, MAPK inhibitor trametinib 0.3 mg/kg every day, combined CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day, or vehicle control. G, Bioluminescent signal was significantly reduced in mice treated with combined CHZ868/trametinib at 7 days of treatment ( n = 7–9/group). H, Representative bioluminescence images are shown. I, BM infiltration of JAK2i-R cells was determined as human CD45 positivity by IHC on BM sections. A significant reduction of BM infiltration was observed with combined CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day ( n = 5–6/group). J, Representative images of BM sections stained with H&E (top) and IHC for hCD45 (lower panel) showed reduced infiltration with combined CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day. Original magnification x400. Data are presented as mean ± SD and analyzed by one-way ANOVA. ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: JAK2 inhibitors CHZ868, BBT594 (type II), ruxolitinib (type I), and MEK inhibitor trametinib were from Novartis through Material Transfer Agreement; AXL inhibitors bemcentinib and gilteritinib were purchased from BioVision and Sellekchem.

Techniques: Inhibition, In Vivo, Injection, Control, Expressing, Stable Transfection, Luciferase, Imaging, Staining

Journal: Clinical Cancer Research

Article Title: Development of Resistance to Type II JAK2 Inhibitors in MPN Depends on AXL Kinase and Is Targetable

doi: 10.1158/1078-0432.CCR-23-0163

Figure Lengend Snippet: Acquired resistance to type II JAK2 inhibition occurs via MAPK pathway activation in MPN cells. A, JAK2 V617F mutant SET2 cells upon continuous exposure to the type II JAK2 inhibitor CHZ868 at 0.3 μmol/L (JAKi-r) or at 0.5 μmol/L (JAKi-R) developed significantly increased IC 50 for CHZ868 as compared with JAK2 inhibitor sensitive SET2 cells (JAKi-S) reflecting acquired resistance ( n = 3–5). B, Susceptibility for apoptosis induction in JAKi-r and JAKi-R cells was significantly reduced as compared with JAK2 inhibitor sensitive SET2 cells (JAKi-S) as reflected by caspase 3 activation upon exposure to CHZ868 for 48 hours ( n = 3–4). C, Phosphoproteomic mass spectrometry analysis revealed differentially phosphorylated residues in JAKi-R cells as compared with JAKi-S cells as indicated by Volcano plot (blue: significant upregulation in JAKi-R cells, black: significant upregulation in JAKi-S cells, grey: nonsignificant changes). D, Pathway analysis by PathfindR of differentially phosphorylated sites in JAKi-R as compared with JAKi-S cells highlighted the MAPK pathway as most differentially phosphorylated pathway based on Biocarta gene sets. E and F, Immunoblotting confirmed activated MAPK pathway signaling in JAKi-r ( E ) and JAKi-R cells ( F ) in comparison with JAKi-S cells as reflected by pERK1/2 and pMEK1/2 despite exposure to increasing concentrations of type II JAK2 inhibitor CHZ868. JAK2, STAT3, and STAT5 remained inhibited in presence of CHZ868 in JAKi-r and JAKi-R cells similarly to JAKi-S cells. G, Densitometry of phosphoproteins shown in immunoblots confirmed increased ERK1/2 phosphorylation in JAKi-r and JAKi-R cells exposed to type II JAK2 inhibition with CHZ868 whereas JAKi-S cells showed dose-dependent inhibition of pERK (right panel). Densitometry also confirmed dose-dependent suppression of JAK2 and STAT3 phosphorylation upon type II JAK2 inhibition with CHZ868 in JAKi-r and JAKi-R cells similarly to JAKi-S cells (left and middle, n = 7–9). Data are presented as mean ± SD. Comparisons between 2 groups were performed by unpaired Student t test; multiple comparisons were performed by one-way ANOVA test with Tukey correction. P value ≤ 0.05 was considered significant (ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001).

Article Snippet: JAK2 inhibitors CHZ868, BBT594 (type II), ruxolitinib (type I), and MEK inhibitor trametinib were from Novartis through Material Transfer Agreement; AXL inhibitors bemcentinib and gilteritinib were purchased from BioVision and Sellekchem.

Techniques: Inhibition, Activation Assay, Mutagenesis, Mass Spectrometry, Western Blot, Comparison, Phospho-proteomics

Journal: Clinical Cancer Research

Article Title: Development of Resistance to Type II JAK2 Inhibitors in MPN Depends on AXL Kinase and Is Targetable

doi: 10.1158/1078-0432.CCR-23-0163

Figure Lengend Snippet: Transcriptional programs and histone occupancy in acquired resistance to type II JAK2 inhibition. A, Differential expression analysis of JAK inhibitor resistant (JAKi-r and JAKi-R) vs. sensitive (JAKi-S) SET2 cells showed JAKi-r and JAKi-R cells clustered together as opposed to JAKi-S cells. The top 500 differentially expressed genes are displayed and genes with relation to MAPK signaling or epigenetic and transcriptional changes are highlighted (red box: upregulation, blue box: downregulation) including RAS and AXL tyrosine kinase. B, Differentially expressed genes between JAK2 inhibition resistant and sensitive cells were assessed by GSEA. C, RNA-seq and ATAC-seq analysis of paired samples revealed a high number of differentially expressed genes and differentially accessible chromatin regions, respectively, in JAK2 inhibition resistant as compared with sensitive cells ( n = 3). Log 2 (FoldChange) of expression (y-axis) and accessibility (x-axis) is given for JAKi-R versus JAKi-S cells. Number of genes in each quadrant is indicated in brackets. A subset of genes relating to MAPK pathway signaling are highlighted (FDR-adjusted P value < 0.01, Fold Change > 1.2). D and E, Accessible chromatin regions were assessed for the presence of TF binding motifs by HOMER analysis in JAK2 inhibition sensitive (JAKi-S; upper panels) and resistant (JAKi-R; lower panels) cells, highlighting GATA and AP1 complex motifs in JAKi-R cells.

Article Snippet: JAK2 inhibitors CHZ868, BBT594 (type II), ruxolitinib (type I), and MEK inhibitor trametinib were from Novartis through Material Transfer Agreement; AXL inhibitors bemcentinib and gilteritinib were purchased from BioVision and Sellekchem.

Techniques: Inhibition, Quantitative Proteomics, RNA Sequencing, Expressing, Binding Assay

Journal: Clinical Cancer Research

Article Title: Development of Resistance to Type II JAK2 Inhibitors in MPN Depends on AXL Kinase and Is Targetable

doi: 10.1158/1078-0432.CCR-23-0163

Figure Lengend Snippet: Acquired resistance to type II JAK2 inhibition is dependent on AXL tyrosine kinase. A, AXL mRNA expression was significantly increased in JAK inhibitor resistant (JAKi-r and JAKi-R) vs. sensitive cells (JAKi-S) relative to GAPDH ( n = 3) consistent with RNA-seq . B and C, Increased AXL expression in JAKi resistant cells (JAKi-r and JAKi-R) was confirmed on protein level as indicated by immunoblotting ( B ) and respective densitometry ( C ; n = 4). D, Reduced AXL protein expression shown by immunoblotting upon shRNA-induced AXL knock-down with shAXL-1 and shAXL-2 hairpins vs. shSCR control in JAKi-R SET2 cells 48 hours after doxycycline induction. E, Significantly reduced AXL mRNA expression shown by qRT-PCR upon shRNA-induced AXL knock-down with shAXL-1 and shAXL-2 hairpins vs. shSCR control in JAKi-R SET2 cells 48 hours after doxycycline induction ( n = 3). F, Significantly increased susceptibility of JAKi-R SET2 cells to type II JAK2 inhibition with CHZ868 as shown by reduced IC 50 upon AXL depletion by two different shRNAs (shAXL-1, shAXL-2) upon doxycycline induction (+dox) as compared with shSCR control. Non-induced control (-dox), n = 3. G, Significantly reduced IC 50 values in JAKi-R SET2 cells were achieved upon combined exposure with the AXL inhibitor bemcentinib and type II JAK2 inhibition with CHZ868 ( n = 3). H, Representative graph showing reduced proliferation of JAKi-R SET2 cells exposed to the AXL inhibitor bemcentinib or combined JAK2 / AXL inhibition with CHZ868 / bemcentinib as compared with CHZ868 as a single agent. I, Significantly reduced IC 50 values in JAKi-R SET2 cells were achieved upon combined exposure with the AXL/FLT3 inhibitor gilteritinib and type II JAK2 inhibition with CHZ868 ( n = 4). J, Representative graph showing reduced proliferation of JAKi-R SET2 cells exposed to the AXL/FLT3 inhibitor gilteritinib or combined JAK2 / AXL inhibition with CHZ868 / gilteritinib as compared with CHZ868 as a single agent. K, Synergy analysis of type II JAK2 inhibitor CHZ868 with the AXL inhibitor bemcentinib in JAKi-S (upper panel) and JAKi-R (lower panel) SET2 cells showed positive synergy in JAKi-R cells, but not in JAKi-S cells ( n = 3, mean values are shown for synergy scores along with representative graphs). Data are presented as mean ± SD and analyzed by one-way ANOVA (panels A, G, I ) or two-tailed Student t test ( F ). ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: JAK2 inhibitors CHZ868, BBT594 (type II), ruxolitinib (type I), and MEK inhibitor trametinib were from Novartis through Material Transfer Agreement; AXL inhibitors bemcentinib and gilteritinib were purchased from BioVision and Sellekchem.

Techniques: Inhibition, Expressing, RNA Sequencing, Western Blot, shRNA, Knockdown, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Clinical Cancer Research

Article Title: Development of Resistance to Type II JAK2 Inhibitors in MPN Depends on AXL Kinase and Is Targetable

doi: 10.1158/1078-0432.CCR-23-0163

Figure Lengend Snippet: Targeting MAPK pathway abrogates acquired resistance to type II JAK2 inhibition in MPN cells. A, Proliferation of JAKi-r SET2 cells was effectively inhibited by combined JAK2/MAPK pathway inhibition with CHZ868/trametinib as shown by a representative graph of proliferation capacity upon 48 hours exposure of inhibitor exposure. B, IC 50 was significantly reduced by combined CHZ868/trametinib in JAKi-r cells ( n = 3). C, Synergy analysis of type II JAK2 inhibition with CHZ868 and MAPK pathway inhibition with trametinib showed positive synergy in JAKi-r cells. Mean of n = 3 experiments is shown for synergy score along with a representative graph D . Proliferation of JAKi-R SET2 cells was effectively inhibited by combined JAK2/MAPK pathway inhibition with CHZ868/trametinib as shown by a representative graph of proliferation capacity upon 48 hours of inhibitor exposure. E, IC 50 was significantly reduced by combined CHZ868/trametinib in JAKi-R cells ( n = 3). F, Synergy analysis of type II JAK2 inhibition with CHZ868 and MAPK pathway inhibition with trametinib showed positive synergy in JAKi-R cells. Mean of n = 3 experiments is shown for synergy score along with a representative graph G . Apoptotic cell death reflected by positivity for caspase-3 was induced by combined JAK2 / MAPK pathway inhibition by CHZ868/trametinib in JAKi-R cells upon 48 hours exposure to inhibitors ( n = 3–4). H, Representative histograms of caspase-3 positivity in JAKi-R cells are shown. I, Immunoblotting showed dose-dependent suppression of MAPK pathway signaling reflected by pERK1/2 (arrowhead) in JAKi-r and JAKi-R SET2 cells upon exposure to increasing concentrations of trametinib combined with CHZ868. Data are presented as mean ± SD and analyzed by one-way ANOVA. ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: JAK2 inhibitors CHZ868, BBT594 (type II), ruxolitinib (type I), and MEK inhibitor trametinib were from Novartis through Material Transfer Agreement; AXL inhibitors bemcentinib and gilteritinib were purchased from BioVision and Sellekchem.

Techniques: Inhibition, Western Blot

Journal: Leukemia research

Article Title: Recent advances in the genomics and therapy of BCR/ABL1 -positive and -negative chronic myeloproliferative neoplasms

doi: 10.1016/j.leukres.2018.02.008

Figure Lengend Snippet: Mutationsin IDH1, IDH2, EZH2, ASXL1, and SRSF2 have all been shown to predict risk for leukemic transformation of patients with myelofibrosis. Moreover, mutations and deletions in TP53 have been shown to predict leukemic transformation of all classic BCR- ABL1 negative MPNs and promote transformation to acute myeloid leukemia in an experimental model with JAK2V617F expression. Interestingly, mutations in ASXL1, EZH2, and SRSF2 all appear to impinge on methylation of histone H3 lysine 27 methylation. (B) The genetic alterations shown in (A) may occur in a cell with a pre-existing JAK2/CALR/MPL mutation to result in AML or could occur in a cell prior to acquisition of the JAK2/CALR/MPL mutation (in which case the patient may have both CALR/JAK2/MPL mutant MPN and CALR/JAK2/MPL wildtype AML). (C) As a final possibility, occasionally AML may be generated from a hematopoietic stem cell (HSC) unrelated to the HSC which gave rise to MPN.

Article Snippet: Other JAK inhibitors in development for MF include the JAK2 inhibitor NS018 (NS Pharma, Japan), which has demonstrated modest activity and safety in phase 2 setting [ 83 ].

Techniques: Transformation Assay, Expressing, Methylation, Mutagenesis, Generated