Janus kinases (JAKs) mediate responses to cytokines, hormones and growth factors in haematopoietic cells1,2. The JAK gene JAK2 is frequently mutated in the ageing haematopoietic system3,4 and in haematopoietic cancers5. JAK2 mutations constitutively activate downstream signalling and are drivers of myeloproliferative neoplasm (MPN). In clinical use, JAK inhibitors have mixed effects on the overall disease burden of JAK2-mutated clones6,7, prompting us to investigate the mechanism underlying disease persistence. Here, by in-depth phosphoproteome profiling, we identify proteins involved in mRNA processing as targets of mutant JAK2. We found that inactivation of YBX1, a post-translationally modified target of JAK2, sensitizes cells that persist despite treatment with JAK inhibitors to apoptosis and results in RNA mis-splicing, enrichment for retained introns and disruption of the transcriptional control of extracellular signal-regulated kinase (ERK) signalling. In combination with pharmacological JAK inhibition, YBX1 inactivation induces apoptosis in JAK2-dependent mouse and primary human cells, causing regression of the malignant clones in vivo, and inducing molecular remission. This identifies and validates a cell-intrinsic mechanism whereby differential protein phosphorylation causes splicing-dependent alterations of JAK2–ERK signalling and the maintenance of JAK2V617F malignant clones. Therapeutic targeting of YBX1-dependent ERK signalling in combination with JAK2 inhibition could thus eradicate cells harbouring mutations in JAK2.
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The MS raw data files and Maxquant output files reported in this manuscript are available at ProteomeXchange Consortium with the data identifier PXD006921. RNA-seq data have been deposited in the Gene expression Omnibus database with accession number GSE123417. ChIP-Seq data has been deposited to the Gene expression Omnibus database with the accession code GSE154025 for mouse and GSE146717 for human. All other data supporting the findings of this study are available from corresponding authors upon reasonable request. Source data are provided with this paper.
Darnell, J. E., Jr, Kerr, I. M. & Stark, G. R. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264, 1415–1421 (1994).
Perner, F., Perner, C., Ernst, T. & Heidel, F. H. Roles of JAK2 in aging, inflammation, hematopoiesis and malignant transformation. Cells 8, 854 (2019).
Genovese, G. et al. Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. N. Engl. J. Med. 371, 2477–2487 (2014).
Jaiswal, S. et al. Age-related clonal hematopoiesis associated with adverse outcomes. N. Engl. J. Med. 371, 2488–2498 (2014).
Forbes, S. A. et al. COSMIC: somatic cancer genetics at high-resolution. Nucleic Acids Res. 45 (D1), D777–D783 (2017).
Deininger, M. et al. The effect of long-term ruxolitinib treatment on JAK2p.V617F allele burden in patients with myelofibrosis. Blood 126, 1551–1554 (2015).
Harrison, C. N. et al. Long-term findings from COMFORT-II, a phase 3 study of ruxolitinib vs best available therapy for myelofibrosis. Leukemia 30, 1701–1707 (2016).
Humphrey, S. J., Azimifar, S. B. & Mann, M. High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics. Nat. Biotechnol. 33, 990–995 (2015).
Hsu, T. Y. et al. The spliceosome is a therapeutic vulnerability in MYC-driven cancer. Nature 525, 384–388 (2015).
Climente-González, H., Porta-Pardo, E., Godzik, A. & Eyras, E. The functional impact of alternative splicing in cancer. Cell Rep. 20, 2215–2226 (2017).
Dolfini, D. & Mantovani, R. YB-1 (YBX1) does not bind to Y/CCAAT boxes in vivo. Oncogene 32, 4189–4190 (2013).
Stickeler, E. et al. The RNA binding protein YB-1 binds A/C-rich exon enhancers and stimulates splicing of the CD44 alternative exon v4. EMBO J. 20, 3821–3830 (2001).
Wei, W. J. et al. YB-1 binds to CAUC motifs and stimulates exon inclusion by enhancing the recruitment of U2AF to weak polypyrimidine tracts. Nucleic Acids Res. 40, 8622–8636 (2012).
Kaelin, W. G., Jr. The concept of synthetic lethality in the context of anticancer therapy. Nat. Rev. Cancer 5, 689–698 (2005).
McCubrey, J. A. et al. Involvement of p53 and Raf/MEK/ERK pathways in hematopoietic drug resistance. Leukemia 22, 2080–2090 (2008).
Meyer, S. C. Mechanisms of resistance to JAK2 inhibitors in myeloproliferative neoplasms. Hematol. Oncol. Clin. North Am. 31, 627–642 (2017).
Stivala, S. et al. Targeting compensatory MEK/ERK activation increases JAK inhibitor efficacy in myeloproliferative neoplasms. J. Clin. Invest. 129, 1596–1611 (2019).
Humphrey, S. J., Karayel, O., James, D. E. & Mann, M. High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform. Nat. Protocols 13, 1897–1916 (2018).
Kulak, N. A., Pichler, G., Paron, I., Nagaraj, N. & Mann, M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat. Methods 11, 319–324 (2014).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Tyanova, S. & Cox, J. Perseus: a bioinformatics platform for integrative analysis of proteomics data in cancer research. Methods Mol. Biol. 1711, 133–148 (2018).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).
Romero, J. P. et al. EventPointer: an effective identification of alternative splicing events using junction arrays. BMC Genomics 17, 467 (2016).
Schnöder, T. M. et al. Cell autonomous expression of CXCL-10 in JAK2V617F-mutated MPN. J. Cancer Res. Clin. Oncol. 143, 807–820 (2017).
Webb, C. H. & Hertel, K. J. Preparation of splicing competent nuclear extracts. Methods Mol. Biol. 1126, 117–121 (2014).
Uckelmann, H. J. et al. Therapeutic targeting of preleukemia cells in a mouse model of NPM1 mutant acute myeloid leukemia. Science 367, 586–590 (2020).
Lu, Z. H., Books, J. T. & Ley, T. J. YB-1 is important for late-stage embryonic development, optimal cellular stress responses, and the prevention of premature senescence. Mol. Cell. Biol. 25, 4625–4637 (2005).
Mullally, A. et al. Physiological Jak2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells. Cancer Cell 17, 584–596 (2010).
Cosgun, K. N. et al. Kit regulates HSC engraftment across the human-mouse species barrier. Cell Stem Cell 15, 227–238 (2014).
Schnöder, T. M. et al. Epo-induced erythroid maturation is dependent on Plcγ1 signaling. Cell Death Differ. 22, 974–985 (2015).
Till, J. E. & McCULLOCH, E. A. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14, 213–222 (1961).
Quintás-Cardama, A. et al. Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms. Blood 115, 3109–3117 (2010).
Heidel, F. H. et al. The cell fate determinant Llgl1 influences HSC fitness and prognosis in AML. J. Exp. Med. 210, 15–22 (2013).
Heidel, F. H. et al. Genetic and pharmacologic inhibition of β-catenin targets imatinib-resistant leukemia stem cells in CML. Cell Stem Cell 10, 412–424 (2012).
Arreba-Tutusaus, P. et al. Impact of FLT3-ITD location on sensitivity to TKI-therapy in vitro and in vivo. Leukemia 30, 1220–1225 (2016).
We thank A. Fenske (Central Animal Facility, OvGU Magdeburg) and M. van der Wall (Central Animal Facility, University Hospital Jena) for support with animal care; the Animal Facility of the Leibniz Institute on Aging Jena for providing NSGW41 animals; R. Hartig (FACS Core Facility, Medical Faculty, and CRC854, OvGU Magdeburg) and K. Schubert (FACS Core Facility, Leibniz-Institute on Aging, Jena) for support with cell sorting, M. Milsom (DKFZ, Heidelberg) for providing constructs; S. Frey, A. Sammt and C. Kathner-Schaffert for technical assistance; K. Mayr, I. Paron and G. Sowa for their assistance and support in the mass spectrometry analysis (MPI Biochemistry); M. Driessen for RNA sequencing and S.-H. Yu for ChIP sequencing analysis (Bioinformatics Core Facility, MPI Biochemistry); and Th. Fischer (Magdeburg), K.L. Rudolph (Jena) and C. Müller-Tidow (Heidelberg) for helpful comments and discussions.
F.H.H. has served as an advisory board member for and received research funding from Novartis, Celgene and CTI. S.W.L. has served on an advisory board for Novartis Australia. A.H. received research support by Novartis, BMS, Pfizer, Incyte. A.M. has received honoraria from Blueprint Medicines, Roche and Incyte and receives research support from Janssen and Actuate Therapeutics. No potential conflicts of interest were disclosed by the other authors.
Peer review information Nature thanks Fabienne Meier-Abt, Michael Yaffe and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Phosphoproteomic analysis uncovers differential regulation of splicing factors in Jak2-mutated cells.
a, Schematic of the phosphoproteome workflow. Following sample collection, phosphopeptides were enriched using EasyPhos work flow8 and analysed in single-run LC-MS/MS. Data were analysed in Maxquant and Perseus. b, Quantified phosphosite depth per sample. Samples were measured as biological quadruplicates. c, Heatmap of sample correlation matrix of all measured samples (in b) based on Pearson correlation values. The reproducibility between the phosphoproteome sample is highlighted. d, Summary of identified and quantified class-I phosphosites (localization probability of >0.75) corresponding to number of proteins of this experiment. e, Principal component analysis of the samples. f, Network map of significantly enriched GO terms (P value <0.05) of differentially phosphorylated proteins in JAK2V617F. Phosphorylated proteins significantly regulated in Jak2V617F were subsequently used as an input for Cytoscape to obtain the network. The highlighted sub-network was obtained with P value <0.01 and kappa score >0.6. Two-sided hypergeometric test, P value correction–Bonferroni stepdown (g, h), western blot validation of shRNA library targets. g, Pcbp1 protein and h, YBX1 protein in mouse JAK2VF cells. n = 3 with comparable results. i, Pearson correlation profile of the independent shRNA experiments.
a, In vitro shRNA validation of selected top 15 targets essential for JAK2VF cell survival and growth in the presence (green) and absence (grey) of JAK inhibitor (RUX, 0.5 μM) measured by proliferation assays (16 technical replicates measured in 2 plates independently, mean). b, Immunofluorescence analysis of YBX1 and JAK2 localization in JAKVF positive Ba/F3 cells (left) and mouse JAK2VF positive stem- and progenitor cells (lineage-negative cells) (right panel) (representative image of n = 3). c, Immunoprecipitation of JAK2 receptor from mouse JAK2VF cells showing binding of YBX1 to mutated JAK2 receptor (n = 3 with similar results). d, e, Percentages of cell growth in JAK2WT (d) and JAK2VF cells (e) following lentiviral infection with shRNAs targeting Ybx1 or non-targeting control (shNT) measured by MTS assay. f, Representative histogram and bar plot showing ROS levels measured in JAK2VF cells with shRNAs targeting Ybx1 or control. g, Representative histogram and bar plot showing proliferative marker PCNA levels in JAK2VF cells with shRNAs targeting Ybx1 or control. h, Cell cycle analysis after YBX1 inactivation in JAK2VF cells. d–h, n = 4 independent experiments, mean ± s.d., two-tailed t-test with equal variance. i, Representative confocal images of DNA damage marker yH2AX pS139 in JAK2VF cells with shRNAs targeting Ybx1 or non-targeting control. Cells exposed to UV light for 20 min were used as positive controls (n = 4 independent experiments). j, FACS plots showing percentage of apoptotic cells in Jak2+/+ and Jak2VF/+ mouse BM cells after JAK inhibitor treatment (RUX, 0.5 μM) after lentiviral knockdown of Ybx1 (sh1 and sh2) compared to non-targeting control (shNT).
a, Schematic representation of the wild-type Ybx1 allele (Genomic Locus), the targeting vector (Targeting Vector), the desired targeted allele (Targeted Allele), the desired conditional allele flanked by LoxP sequences (Conditional Allele) and the null recombined allele (Recombined Allele) after Cre-mediated recombination of the conditional allele. Triangles indicate loxP sequences. b, Excision control by genomic PCR on whole bone marrow cells at week 16 following genetic inactivation of Ybx1 in conditional knockout mice. Representative micrograph of n = 5 animals from a total of n = 9 controls (+/+) and n = 9 knockout (−/−) replicates. c, Schematic as in Fig. 2j. FACS plots showing percentage of Jak2V617F (CD45.2) cells of Ybx1+/+ or Ybx1−/− recipients. d, Histology of liver, spleen and lung of Jak2V617F-Ybx1+/+ and Jak2V617F-Ybx1−/− recipient mice at week 20 after BMT. Representative micrographs of n = 9 individual mouse replicates. Haematoxylin and eosin stain (H&E) at ×10 magnification. Focal leukocyte infiltration (arrows) and haemorrhage (stars) of liver, spleen and lung, respectively. Scale bar: 200 μm. e, Peripheral blood chimerism of lethally irradiated (12 Gy) recipient mice. FACS plots showing abundance of CD45.2 myeloid cells in Jak2V617F-Ybx1+/+ and Jak2V617F-Ybx1−/− recipient mice at week 20 after BMT. f, Design for assessment of steady state haematopoiesis. g, White blood count (WBC), Gr-1 positive cells (Gr1+), haemoglobin (HGB) and platelets (PLT) following genetic inactivation of Ybx1 (Ybx1−/− mice, n = 10) compared to Ybx1+/+ controls (n = 10). Data represented as mean ± s.e.m. h, FACS plots showing comparable percentages of LSK cells and HSCs (SLAM+CD34–L–S+K+ cells) following genetic inactivation of Ybx1 in conditional knockout mice (compared to wildtype littermate controls). i, stem- and progenitor cell numbers per 1 × 106 whole bone marrow cells at week 16 after genetic inactivation of Ybx1 (n = 6; mean ± s.d.). j, FACS plot showing comparable abundance of mature myeloid and erythroid cells following genetic deletion of Ybx1. k, Total numbers of mature blood cells of the myeloid (Gr1+), erythroid (TER119+), B-lymphoid (CD19) and T-lymphoid (CD3) lineages at week 16 after genetic inactivation of Ybx1 (n = 6; mean ± s.d.). l, Experimental protocol for investigation of haematopoietic progenitor cell function. m, Colony numbers of Ybx1+/+ versus Ybx1−/− mouse stem/progenitor cells. Colonies were counted at day 8 after plating (each sample plated in duplicate, n = 3 independent experiments, mean ± s.d.). n, Spleen colony numbers counted on day 12 after injection of Ybx1+/+ or Ybx1−/− LSK cells into lethally irradiated (12Gy) recipient mice (CFU-S12) (n = 12 Ybx1+/+; n = 12 Ybx1−/− independent biological mouse replicates in n = 3 independent cohorts). o, BM chimerism of primary recipient mice (n = 10 individual biological replicates) at week 20 after BMT. Whole bone marrow chimerism (WBMC) and chimerism of myeloid cells (Gr1+ BMC) (left panel). HSPC chimerism (LSK) and HSC chimerism (CD34– LSK) (right panel). Data shown as mean ± s.d. p, BM chimerism of secondary recipient mice (n = 5 individual biological replicates) at week 20 after BMT. Whole BM chimerism (WBMC) and chimerism of myeloid cells (Gr1+ BMC) (left panel). HSPC chimerism (LSK) and HSC chimerism (CD34– LSK) (right panel). Data shown as mean ± s.d.
a, Profile plot showing significantly regulated individual phosphorylated residues of YBX1 in JAK2WT and JAK2VF. Each data point is the averaged median of biological quadruplicate and significance was tested using two-sample test. b, Profile plot showing individual phosphorylated residues of YBX1 that are significantly regulated in JAK2WT when unstimulated (control) or stimulated with erythropoietin or erythropoietin + JAK inhibitor. c, Profile plot showing significantly regulated individual phosphorylated residues of YBX1 in JAK2VF when unstimulated or stimulated with EPO or EPO + JAK inhibitor. b, c, Each data point is the averaged median of biological quadruplicate, z-scored (log2 phosphosite intensity), and significance was tested using multiple sample test. d, Experimental design for phosphoproteome analysis of short-term JAK2 downstream effector kinase inhibitor treatment in JAK2VF cells. n = 4 per group, phosphopeptides were enriched using EasyPhos workflow and analysed in single-run LC-MS/MS. e, Dot plot showing the successful inhibition of respective targets of the corresponding kinase inhibitor used in this study (ANOVA test with permutation-based FDR < 0.01). f, Dot plot showing changes in quantified YBX1 phosphosites after various kinase inhibitor treatment (ANOVA test, permutation-based FDR < 0.01). The highlighted YBX1 pS30 phosphosite is the only site highly significantly downregulated upon MEK/ERK inhibitor treatment compared to controls. e, f, Size and colour of the dots are proportional to the phosphosite intensity, z-scored (log2 intensity). g, Amino acid sequence alignment of YBX1 across different species shows that mouse YBX1 Ser30 and Ser34 are conserved. Source data
Extended Data Fig. 5 YBX1 interaction with spliceosome components and validation of YBX1-MAPK1 partnership.
a, Study design of mouse YBX1 interactome. n = 4 biological replicates and YBX1 interactome analysed in LC-MS/MS. Using Perseus, samples were filtered for a minimum of three valid values in at least one group. In total 614 high confident interactors of YBX1 were identified (t-test with permutation-based FDR < 0.05) with 260 JAK2VF-specific interactors. b, Network representation of YBX1 interacting spliceosomal proteins in JAK2VF cells. The size and colour of the node indicates the abundance of the corresponding proteins (z-scored protein intensity) and the edges are connected by STRING database interactions. c, List of significant YBX1-interacting spliceosomal proteins presented according to their spliceosome complex. d, Spliceosome proteins interacting with YBX1 participate in spliceosome assembly reaction in a stepwise manner to excise intronic sequences from immature mRNA to form a mature mRNA. e, Network representation of YBX1 interactome (regulated in both JAK2WT and JAK2VF) based on annotation keywords. The keywords are highlighted in colours according to the protein function. n = 4 biological replicates, t-test with permutation-based FDR < 0.05. f, Scatter plot of YBX1 interactome in JAK2VF vs control. YBX1 interactome is enriched for GO term mRNA splicing factor (green) and Ribonucleoproteins (blue) assessed by Fisher’s exact test. Fold enrichment of YBX1 and MAPK1 in JAK2VF cells compared to IgG control plotted against –log10 Student t-test P value. g, Scatter plot of YBX1 interactome in DMSO vs JAK inhibitor (RUX 0.5 μM, 4 h)-treated JAK2VF cells. Fold enrichment of MAPK1 in DMSO vs JAK inhibitor plotted against –log10 Student’s t-test P value. h, Immunoprecipitation of YBX1 from mouse JAK2VF cells ± RUX, 0.5 –μM for 4 h and analysed for interaction with MAPK1 by western blot analysis using ERK1/2 antibody. Representative images from n = 3 biological experiments. Source data
Extended Data Fig. 6 YBX1 phospho-null mutants display impaired nuclear localization and increased sensitivity to JAK inhibitor.
a, Western blot showing the expression of YBX1 phosphomutants in Ba/F3 JAK2VF cells as indicated. b, Confocal images of YBX1 localization (Red) in YBX1 phosphomutants expressing Ba/F3 JAK2VF cells. Cells were counterstained with DAPI and GFP confirms the phosphomutant expression. a b, Representative images from n = 3 biological experiments. c, Bar plot showing quantification of nuclear YBX1 expression in YBX1 phosphomutants expressing Ba/F3 JAK2VF cells; P values were determined by two-tailed t-test. Control (n = 67): min = 15.39; max = 33.01; whisker = [15.39–33.01]; median = [24.74], S1(n = 70): min = 4.14; max = 9.75; whisker = [4.14–9.75]; median = [7.0053], S2(n = 64): min = 5.32; max = 14.11; whisker = [5.32–14.11]; median = [7.9], S3(n = 57):min = 4.44; max = 17.6; whisker = [4.44–17.6]; median = [11.4], S4(n = 72): min = 13.32; max = 23.82; whisker = [13.32–23.82]; median = [19.3], S5(n = 61): min = 13.08; max = 27.97; whisker = [13.08–27.97]; median = [22.05], S6(n = 57):min = 18.89; max = 30.04; whisker = [18.89–30.04]; median = [22.92], S7(n = 60): min = 16.69; max = 32.53; whisker = [16.69–32.53]; median = [24.65]. d, Cell growth curve of YBX1 phosphomutants expressing Ba/F3 JAK2VF cells following treatment with increase doses of JAK inhibitor (1 nM–10 μM RUX) measured by MTS assay. n = 4 independent experiments each with 8 technical replicates. e, FACS plots showing induction of apoptosis in Ba/F3 JAK2VF cells expressing YBX1 phosphomutants following JAK inhibitor treatment (RUX, 0.5 μM) compared to untreated YBX1 wild-type Ba/F3 JAK2VF cells. f, Bar plot shows quantification of the percentages of apoptotic (annexin V- and 7-AAD-positive) cells. n = 6 independent experiments since YBX1 phosphomutants endogenously express GFP, n = 3 for annexin V–APC staining and n = 3 for 7-AAD–APC staining (error bars represent mean ± s.d.).
a, Confocal images of YBX1 localization (Red) in Ba/F3 JAK2VF cells after treatment for 2 h with RUX (0.5 μM), MEK inhibitor (2 μM), the ERK inhibitor trametinib (Tram; 100 and 200 nM), RUX + Tram or DMSO (control). Cells were counterstained with DAPI. b, Bar plot shows quantification of nuclear YBX1 expression in Ba/F3 JAK2VF cells (n ≥ 3 independent imaging experiments, P value determined by Student’s t-test). DMSO (n = 67): min = 7.05; max = 27.27; whisker = [7.05–27.27]; median = [15.01], RUX (n = 77): min = 6.6; max = 35.8; whisker = [6.6-35.8]; median = [16.39], CGP57380 (n = 89): min = 3.2; max = 16.35; whisker = [3.2–16.35]; median = [8.35], Tram (n = 67): min = 2.44; max = 14.18; whisker = [2.44–14.18]; median = [12.01], Tram (n = 62): min = 2.5; max = 17.96; whisker = [2.5–17.96]; median = [12.01], Tram + RUX (n = 64): min = 3.44; max = 19.23; whisker = [3.44–19.23]; median = [13.04], MEK inhibitor + RUX (n = 68): min = 4.06 max = 14.68; whisker = [4.06–14.68]; median = [13.01]. c, Bubble plot showing the regulation of human YBX1 pS32 and pS36 phosphorylation in HEL cells treated with RUX (0.5 μM), CGP57380 (10 μM), Tram (500 nM and 2 μM) or DMSO (control) for 4 h in vitro. Phosphorylation status of MAPK and JAK2 is shown as successful inhibition of respective targets of the corresponding kinase inhibitors. n = 4 biological samples per group. Size and colour of bubbles are proportional to the z-scored log2 phosphosite intensity; significance assessed using multiple sample test. d, Confocal images of YBX1 localization (red) in HEL cells treated with inhibitors or DMSO for 2 h. Cells were counterstained with DAPI. n = 3 biological experiments. e, Bar plot shows quantification of nuclear YBX1 expression in HEL cells; P values were determined by Student’s t-test. DMSO (n = 44): min = 17.51; max = 34.56; whisker = [17.51–34.56];median = [24.30], RUX (n = 44): min = 13.04; max = 36.92; whisker = [13.04–36.92]; median = [23.9], MEK inhibitor (n = 41): min = 7.62; max = 15.36; whisker = [7.62-15.36]; median = [11.89], Tram (n = 48): min = 6.38; max = 19.03; whisker = [6.38-19.03]; median = [12.01], Tram + RUX (n = 55): min = 3.31; max = 19.19; whisker = [3.31-19.19]; median = [13.04], MEK inhibitor + RUX (n = 49): min = 4.32; max = 19.46; whisker = [4.32-19.46]; median = [13.01]. f, Immunoprecipitation of YBX1 from Ba/F3 JAK2VF cells expressing YBX1 phosphomutants. n = 3 independent experiments. Source data
a, Experimental design of RNA sequencing and data analysis. b, Bars represent number of retained intron events significantly upregulated in YBX1-depleted cells with two-tailed test P value < 0.05 (1,064 RI events), then filtered for P value < 0.01 and ΔPSI 0.1, reducing the number to 472 highly significant RI events. c, Network map displaying enrichment of gene sets in the 472 highly significant RI events. Each node represents significantly enriched gene sets. Clusters of functionally related gene sets are circled, and labels are highlighted. Two-sided hypergeometric test, P value correction–Bonferroni step down. d, Visualization of spliced Mknk1 mRNA product after in vitro splicing assay. Nuclear extracts with and without YBX1 knockdown and with and without ATP were incubated for 2 h with biotin-labelled Mknk1 pre-mRNA (exon 12–exon 13). mRNA was isolated and reverse transcribed, PCR was performed using the primers at the indicated arrow, and products were agarose gel resolved and visualized using gel-red stain. Representative images from n = 3 biological experiments. e, f, Flow cytometric analysis of MKNK1 protein expression rescue experiment in CRISPR–Cas9-induced YBX1 knockout mouse JAK2VF cells upon nonsense-mediated decay (NMD) inhibition. Representative flow cytometry histogram and violin plot showing quantification of MKNK1–DyLight 649 mean fluorescence intensity (MFI) upon e, compound C treatment (NMD inhibitor (dorsomorphin), termed CC, 10 μM, for 24 h), and f, VG-1 treatment (5 μM and 10 μM, for 20 h) (n = 3, two-sided t-test; mean ± s.d.). g, Genomic track profile of human MKNK1, ARAF and BRAF loci in HEL cells from YBX1 ChIP sequence data set (n = 2). h, Genomic track profile of mouse Mknk1, Araf and Braf loci in Ba/F3 JAK2VF cells from YBX1 ChIP sequence data set. n = 3 biological experiments. Genomic track profile of human and mouse Prkcb shown as positive control from the YBX1 ChIP-seq data set compared to IgG controls. i, Western blot showing the regulation of MKNK1 protein abundance in YBX1 phosphomutants expressing Ba/F3 JAK2VF cells as indicated. n = 3 independent experiments. Source data
a, Unsupervised hierarchical clustering of significantly down regulated phosphosites (n = 2,390 sites) in human HEL cells following inactivation of YBX1 by two independent shRNAs compared to non-targeting control. n = 4 biological replicates. Heatmap represents z-scored and averaged log2 phosphosite intensity, significance by ANOVA test with permutation-based FDR < 0.01. b, Kinase-substrate motifs significantly downregulated in YBX1-targeted HEL cells are shown including Benjamini–Hochberg FDR value (–log10). c, ERK substrate motifs significantly downregulated and shared between Ybx1-targeted mouse and human JAK2VF cells. d, Western blot analysis of total protein abundance and phosphorylation of JAK2 downstream targets upon treatment with JAK inhibitor and/or genetic inactivation of YBX1 by RNAi. GAPDH used as loading control. Representative images from n = 3 independent experiments. e, f, Bar plots show the mean fluorescence intensity of pERK levels measured in human HEL (e; n = 3) and in patient JAK2-mutated cells (f; n = 4 independent biological replicates from 4 individual patients) following genetic inactivation of YBX1 by RNAi with or without drug treatment as indicated. Representative FACS plots shown in Fig. 4d. Data shown as mean ± s.d. and P value determined by two-tailed Student’s t-test. g, Western blot validation of Mknk1-targeting shRNAs in mouse Ba/F3 JAK2VF cells. h, Representative western blot analysis of pERK upon genetic inactivation of Mknk1 in Ba/F3 JAK2VF cells. n = 4 with comparable results. i, Growth curve of JAK2VF cells following lentiviral infection with shRNAs targeting Mknk1 or non-targeting control and treatment with increase doses of JAK inhibitor (1nM–10 μM RUX) measured by MTS assay. n = 4, each with 8 technical replicates. j, Percentage of apoptotic JAK2VF cells following lentiviral knockdown of Mknk1 (sh2, sh3 ± RUX 0.5 μM) or infection with non-targeting control (shSCR) (n = 4, two-tailed Student t-test, mean ± s.d.). Source data
Extended Data Fig. 10 MCL-1 rescue of Ybx1-targeted cells and phosphoproteome analysis of primary Jak2-mutated cells upon JAK inhibitor treatment.
a, Proteome analysis of mouse Jak2-mutated cells following inactivation of Ybx1 by two shRNAs compared to non-targeting control. Heatmap representation of significantly enriched GO term biological processes in YBX1-depleted JAK2VF cells assessed by Fisher’s exact test (P (–log10) shown). b, Quantification of MCL-1 phosphosite pT144 in sh1Ybx1, sh2Ybx1 and shNT control in mouse JAK2VF cells. The y axis is the log2 intensity of the phosphopeptide (n = 4 biological replicates, two-tailed Student t-test). shNT: min = 23.8; max = 24.7; whisker = [23.8–24.7]; box = [24–24.2], sh1Ybx1: min = 19.8; max = 22; whisker = [19.8–22]; box = [21.3–21.6], sh2Ybx1: min = 19.2; max = 22.4; whisker = [19.2–22.4]; box = [21.8–22.4]. c, Scatter dot plot of MCL-1 phosphosite pT144 after respective kinase inhibitor treatment (n = 3, biological replicates, P values using two-tailed Student t-test, error bars represent ± s.d.). The y axis is the z-scored, log2 phosphopeptide intensity. DMSO: min = –0.09; max = 1.02; whisker = [–0.09 to 1.02], RUX: min = -0.554; max = 0.135; whisker = [–0.55 to 0.135], AKT inhibitor: min = 1.071; max = 1.263; whisker = [1.07–1.2634], PI3Ki: min = –1.008; max = –0.89; whisker = [–1.008 to 0.89], MEK inhibitor: min = –1.266; max = –0.699; whisker = [–1.266 to 0.699]. d, Western blot analysis of MCL-1, YBX1, BIM and BCL-XL following genetic inactivation of Ybx1 with four different shRNA constructs, compared to non-targeting control. n = 3 independent experiments. e, Measurement of apoptosis (annexin V- and Sytox-positive cells) after genetic inactivation of Ybx1 and concomitant JAK inhibitor treatment (RUX, 100 nM, 500 nM). Rescue by ectopic overexpression of MCL-1 (n = 4 independent experiments, two-tailed t-test). f, Western blot analysis of Ba/F3 JAK2VF cells showing downregulation of MKNK1 protein abundance following overnight combination treatment with RUX and Tram. n = 3 biological independent experiments. g, Peripheral blood cell analysis of human cell chimerism in NSGW41 humanized mice at week 4 and 20 (n = 5 per cohort). h, Heatmap shows unsupervised hierarchical clustering of significantly regulated (t-test with permutation-based FDR < 0.01) phosphosites with (n = 24) and without (n = 24) JAK inhibitor treatment in Jak2-mutated primary patient samples. Phosphoproteome analysis of Jak2-mutated primary patient samples (total n = 48) samples following in vitro (n = 18, JAK inhibitor treatment for 2 h) or in vivo (n = 6, 2 h post dosing of RUX samples) exposure to ruxolitinib. i, Network map of significantly enriched GO terms (P value <0.01) of dephosphorylated proteins upon JAK inhibitor treatment. Two-sided hypergeometric test, P value correction–Bonferroni step down. j, Box plot shows no significant changes in the MAPK1 and MAPK3 phosphorylation in control (n = 24) vs JAK inhibitor treated (n = 24) patient samples. MAPK1pT185 (DMSO): min = 19.61; max = 24.82; whisker = [19.61–24.82]; box = [21.34–23.11], MAPK1pT185 (RUX): min = 19.5; max = 24.81; whisker = [19.5–24.81]; box = [21.64–23.72], MAPK1pY187 (DMSO): min = 21.04; max = 26.45; whisker = [21.04–26.45];box = [23.22-25.46], MAPK1pY187(RUX): min = 21.09; max = 25.89; whisker = [21.09–25.89]; box = [22.63–24.77], MAPK3pT202(DMSO): min = 19.72; max = 26.06; whisker = [19.72–26.06]; box = [21.46–24.78], MAPK3pT202 (RUX): min = 20.41; max = 26.93; whisker = [20.41–26.93]; box = [22.05–23.88], MAPK3pY204 (DMSO): min = 20.70; max = 26.51; whisker = [20.70–26.51]; box = [22.02–24.99], MAPK3pY204 (RUX): min = 20.31; max = 25.76; whisker = [20.31–25.76]; box = [22.72–24.42]. k, Box plot shows significant changes in IKBKB, STAT3 and STAT5 phosphorylation in control (n = 24) vs JAK inhibitor treated (n = 24) patient samples. P values as determined by Mann–Whitney test. IKBKBpS697 (DMSO): min = 20.66; max = 25.79; whisker = [20.66–25.79]; box = [22.89–24.40], IKBKBpS697 (RUX): min = 20.66; max = 25.04; whisker = [20.66–25.04]; box = [21.88–23.88], IKBKBpS672 (DMSO): min = 20.71; max = 26.30; whisker = [20.71–26.30]; box = [22.5–23.86], IKBKBpS672 (RUX): min = 18.85; max = 24.73; whisker = [18.85–24.73]; box = [21.44–23.24], STAT5pY699 (DMSO): min = 22.02; max = 25.33; whisker = [22.02–25.33]; box = [22.73–23.63], STAT5pY699 (RUX): min = 19.90; max = 24.83; whisker = [19.90–24.83]; box = [21.8–23.2], STAT3pY705 (DMSO): min = 21.87; max = 26.66; whisker = [21.87–26.66]; box = [22.41–25.62], STAT3pY705 (RUX): min = 20.07; max = 26.17; whisker = [20.07–26.17]; box = [21.22–23.26]. l, Schematic depicting the mechanism of YBX1-mediated JAK inhibitor persistence. Source data
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Jayavelu, A.K., Schnöder, T.M., Perner, F. et al. Splicing factor YBX1 mediates persistence of JAK2-mutated neoplasms. Nature 588, 157–163 (2020). https://doi.org/10.1038/s41586-020-2968-3
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