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Selective translational control by PABPC1 phase separation regulates blast crisis and therapy resistance in chronic myeloid leukaemia

Nature Cell Biology volume 27, pages 683–695 (2025)Cite this article

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Abstract

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion tyrosine kinase have revolutionized the treatment of chronic myeloid leukaemia (CML). However, the development of TKI resistance and the subsequent transition from the chronic phase (CP) to blast crisis (BC) threaten patients with CML. Accumulating evidence suggests that translational control is crucial for cancer progression. Our high-throughput CRISPR–Cas9 screening identified poly(A) binding protein cytoplasmic 1 (PABPC1) as a driver for CML progression in the BC stage. PABPC1 preferentially improved the translation efficiency of multiple leukaemogenic mRNAs with long and highly structured 5′ untranslated regions by forming biomolecular condensates. Inhibiting PABPC1 significantly suppressed CML cell proliferation and attenuated disease progression, with minimal effects on normal haematopoiesis. Moreover, we identified two PABPC1 inhibitors that inhibited BC progression and overcame TKI resistance in murine and human CML. Overall, our work identifies PABPC1 as a selective translation enhancing factor in CML-BC, with its genetic or pharmacological inhibition overcoming TKI resistance and suppressed BC progression.

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Fig. 1: CRISPR–Cas9 screening identifies PABPC1 as the key regulator of mRNA translation in CML-BC.
Fig. 2: Pabpc1 depletion suppresses CML disease progression and improves survival in vivo.
Fig. 3: PABPC1 promotes the translation of multiple leukaemogenic mRNAs, including BCR-ABL1 in CML-BC.
Fig. 4: PABPC1 inhibition suppresses TKI-resistant CML cells and improves survival.
Fig. 5: PABPC1 undergoes phase separation to form condensates to achieve robust translation in CML-BC.
Fig. 6: Small molecules 1,10-Phen and ML324 bind to PABPC1 and inhibit its RNA binding activity.
Fig. 7: Small molecules 1,10-Phen and ML324 suppress CML progression in the BC stage.

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Data availability

RNA-sequencing, RIP-seq and Polysome-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession codes GSE198142, GSE280877, GSE280878 and GSE280879. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the State Key Laboratory of Common Mechanism Research of Major Diseases Platform for consultation and the instrument availability that supported this work, as well as the High-performance Computing Platform at the Center for Bioinformatics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. This work was supported by the National Key Research and Development Program of China (2019YFA0802600 to Y.M., 2021YFA0805703 to Y.M., 2019YFA0801800 to J.Y.), the National Natural Science Foundation of China (92268205 to J.Y., 82122005 to Y.M., 82325002 to M.Z., 82270192 to X.W.), the CAMS Innovation Fund for Medical Sciences (2021-I2M-1-019 to J.Y.) and the Haihe Laboratory of Cell Ecosystem Innovation Fund (22HHXBSS00027 to Y.M.). This paper was edited by Life Science Editors.

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Author notes

  1. These authors contributed equally: Chenguang Sun, Xi Xu, Zhongyang Chen, Fanqi Zhou.

Authors and Affiliations

  1. State Key Laboratory of Common Mechanism Research for Major Diseases, Haihe Laboratory of Cell Ecosystem, Key Laboratory of RNA and Hematopoietic Regulation, Institute of Basic Medical Sciences, School of Basic Medicine Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China

    Chenguang Sun, Zhongyang Chen, Fanqi Zhou, Wen Wang, Mengyao Sun, Fang Wang, Siqi Liu, Jiayue Xu, Manman He, Bowei Su, Xiaoling Liu, Xiaoshuang Wang, Jia Yu & Yanni Ma

  2. Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, China

    Chenguang Sun, Fang Wang, Xiaoshuang Wang, Meng Zhao, Jia Yu & Yanni Ma

  3. Key Laboratory of Stem Cells and Tissue Engineering (Ministry of Education), Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China

    Xi Xu, Junzhu Chen & Meng Zhao

  4. Department of Hematology, Nanfang Hospital, Southern Medical University, Guangzhou, China

    Xi Xu

  5. Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China

    Linjia Jiang

  6. Institute of Materia Medica and Peking Union Medical College, Chinese Academy of Medical Sciences, Tianjin, China

    Ming Ji

  7. State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China

    Yingdai Gao & Hui Wei

  8. Department of Hematology, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China

    Jian Li

  9. State Key Laboratory for Complex, Severe, and Rare Diseases, Institute of Basic Medical Sciences, School of Basic Medicine Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China

    Xiaoshuang Wang

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Contributions

J.Y., Y.M., M.Z. and X.W. conceived and directed the study. C.S. designed and performed the molecular and mechanistic experiments, assisted by M.S., S.L., B.S. and X.L. X.X. designed and performed the animal experiments, assisted by C.S., J.C. and M.J. Z.C. performed the bioinformatics analysis, assisted by W.W. and J.X. F.Z. performed the small-molecule screening experiments, assisted by C.S. X.W., F.W. and M.H. designed the CRISPR library. Y.G. provided TKI-resistant cells and technical assistance. L.J., H.W. and J.L. provided the primary CML-patient samples. J.Y., Y.M., M.Z. and C.S. wrote the paper.

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Correspondence to Xiaoshuang Wang, Meng Zhao, Jia Yu or Yanni Ma.

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Extended data

Extended Data Fig. 1 CRISPR–Cas9 screening of RBPs in CML-BC.

a, Differentially expressed genes (DEGs) between CML-CP (n = 42) and CML-BC (n = 28) patients from GSE4170. b, Functional enrichment analysis of DEGs in a; the top eight enriched terms of downregulated genes are labelled. c, Relative expression of genes enriched in four representative GO terms from Fig. 1a. d, Cell proliferation of primary leukemia cells from CML-CP (1#, 2#) and CML-BC (1#, 2#) patients (n = 3). e, Differential expression of all known RBPs between CML-CP (n = 42) and CML-BC (n = 28) patients from GSE4170. f, Western blots showing Cas9 protein levels in Tet-on/Tet-off edited K562 cells post-Dox induction. g, Flow cytometry gating strategy for sorting HPGHigh and HPGLow cell populations following CRISPR/Cas9 screening. h-i, sgRNA distribution of seven known translational regulators between HPGHigh and HPGLow cells (h), or between Day 0 and Day 20 cells post-Dox induction (left of i) with sgRNA abundance in cells on Day 0, 5, 10, 15, 20 was shown (right). j-k, Representative confocal microscopy images and corresponding flow cytometry histograms showing nascent peptides synthesis (j), and cell proliferation (k) in K562 cells following inhibition of each of the seven RBPs (n = 3). l, Normalized expression of PABPC1 in CML patients before (n = 9) and after (n = 9) TKI treatment (GSE33075). Data are shown as the mean ± SEM. P-values were determined by two-way repeated measures ANOVA (d, k), and paired two-tailed Student’s t-test (l), **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 2 PABPC1 inhibition suppresses CML progression in BC stage and improves survival.

a, Schematic outline of conditional Pabpc1 knockout mouse model construction mediated by CRISPR/Cas9. b, Genomic iditification of mice with Pabpc1 deletion (n = 5). c, Representative FACS plots of GFP+ CD11b+ leukemia cells from PB of CML mice generated in Fig. 2a (n = 6 mice). d-e, Cell cycle distribution (d) and apoptosis rate (e) of LSK and LSC (LT-HSC) populations from primary recipients at day 21 after engraftment (in Fig. 2b) (n = 5 mice) f, Western blots showing protein levels in BMCs from Pabpc1 cKO mice (n = 3 mice). g, Apoptosis rate of BMCs in Pabpc1 cKO mice (n = 5 mice). h-i, Colony formation ability (h) and cell proliferation (i) of primary leukemia cells from (1#, 2#, 3#) CML-CP patients following PABPC1 inhibition (n = 3). j-k, Colony formation ability (j) and cell proliferation (k) of normal HSPCs from healthy donors following PABPC1 inhibition (n = 3). l, The generation of cell-derived xenograft (CDX) CML mouse model using K562 cells. m, Quantification of CD33+ CML cells in PB (left) and survival curves (right) of recipients (n = 6 mice). Data are shown as the mean ± SEM. P-values were determined by Student’s unpaired two-tailed t-test (d, e, g, h-k), one-way ANOVA with Bonferroni correction (m), and Kaplan–Meier survival analysis (m), ns P > 0.05, **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 3 PABPC1 inhibition represses the translation of BCR-ABL1 and other leukemogenic transcripts in CML-BC.

a, Differentially expressed genes in K562 cells identified by RNA-seq. b, The top GO terms enriched in DEGs identified in (a). c, Relative BCR-ABL1 mRNA abundance per component to that in total RNA in MEG cells following PABPC1 inhibition (n = 3). d-e, Western blots showing BCR-ABL1 protein levels in MEG-01 cells following PABPC1 inhibition (d), or in BMCs of CML recipients in Fig. 2b (e) (n = 3 mice). f, Summary and definition of BCR-ABL1 downstream genes. g, Gene set enrichment analysis (GSEA) of BCR-ABL1 downstream genes using RNA-seq data (a). h, Differential expression of BCR-ABL1 downstream genes in leukemia cells derived from CML patients at different stages of disease, CML-CP (n = 42) and CML-BC (n = 28) patients from the GSE4170; CML patients (n = 9) before/after TKI treatment from the GSE33075; IM-resistant (n = 13) and remission (n = 83) CML patients from the GSE130404. i, Western blots showing BCR-ABL1 protein levels in K562 cells with PABPC1 inhibition rescued by BCR-ABL1 overexpression. j-k, Cell cycle distributions (j), early (Annexin V+-633-) and late (Annexin V+-633+) apoptosis (k) rates in K562 cells with PABPC1 inhibition rescued by BCR-ABL1 overexpression (n = 3). Data are shown as the mean ± SEM. P-values were determined by Student’s unpaired two-tailed t-test (a, h), and one-way ANOVA with Bonferroni correction (c, j, k), ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 4 PABPC1 inhibition represses TKI-resistant CML cell proliferation.

a-b, Colony formation ability of TKI-resistant K562 cells with PABPC1 inhibition or imatinib treatment (n = 3). c-d, Cell cycle distributions of TKI-resistant K562 cells with PABPC1 inhibition or imatinib treatment (n = 3). e-f, Early (Annexin V+-633-) and late (Annexin V+-633+) apoptosis rates in TKI-resistant K562 cells with PABPC1 inhibition or imatinib treatment (n = 3). g, The degree of PABPC1 inhibition on the proliferation of K562, K562/G01, and K562-r cells. Data are shown as the mean ± SEM. P-values were determined by one-way ANOVA with Bonferroni correction (a-f); ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

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Extended Data Fig. 5 PABPC1 undergoes LLPS to form condensates in CML-BC.

a, The colocalization ratio (colocalized condensates/individual protein condensates) per cell according to Fig. 5a (n = 10 per sample). b, Co-immunofluorescence staining of PABPC1 (red) and eIF4E/RPL7A (green) in bone marrow cells from CML-BC (1#, 2#, 3#) patients, and nuclei counterstained with DAPI (blue). Scale bar, 2 μm. c, The colocalization ratio (colocalized condensates/individual protein condensates) per cell (b) (n = 10 per sample). d, PLA assay of PABPC1-eIF4G/eIF4E colocalization in bone marrow cells from CML-BC (1#, 2#, 3#) patients, and nuclei counterstained with DAPI (blue). Scale bar, 2 μm. e-f, Immunofluorescence staining showing the effects of PABPC1 inhibition on formation (e) or quantity (f) of eIF4G/eIF4E/RPL7A condensates (green) in bone marrow cells from CML-BC (1#, 2#, 3#) patients, and nuclei counterstained with DAPI (blue), (n = 10 per patient). Scale bar, 2 μm. g, PONDR predicted intrinsically disordered regions in PABPC1. h, Summary of phase-separation behavior of PABPC1 (Fig. 5d). i, FRAP analysis of the PABPC1/ PolyA+ mRNA condensates with 488 nm laser in vitro. j, Schematic diagram of truncation and chimeric mutation in PABPC1 protein. k, Fluorescence area per droplet at 0.8 μM protein concentration (Fig. 5h). l, FRAP analysis of EGFP-PABPC1 (IDR3_FUS) in transfected K562 cells. m, R-EMSA assay showing purified recombined EGFP-PABPC1 binding to poly(A) RNA sequences in vitro. n, Pull-down assay showing purified recombined EGFP-PABPC1 binding to eIF4G protein in vitro. o, Schematic diagram of in vitro translation from Fig. 5j. p, Western blot showing the protein expression levels of K562 cells from Fig. 5k-l. Data are shown as the mean ± SEM. Adjusted p-values were calculated by Wilcoxon rank sum test (a, f) and one-way ANOVA with Bonferroni correction (k); ns P > 0.05, *P < 0.05, ***P < 0.001.

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Extended Data Fig. 6 Small molecules 1,10-Phen and ML324 target PABPC1 to inhibit the robust translation in CML-BC.

a, R-EMSA assay showing PABPC1 binding to poly(A) RNA sequences. b, AlphaScreen signals showing optimum concentrations of PABPC1 protein (purple) and poly(A) RNA (orange). c, Scatter plots showing percentage inhibition of compounds in the second round of screening. d, Binding constant (Kd) values detected by SPR assay between four compounds (Fig. 6c) and PABPC1 protein molecules. e-f, Cell proliferation (e), early (Annexin V+-633-) and late (Annexin V+-633+) apoptosis rates (f) of K562 or MEG-01 cells with 1,10-Phen (purple) or ML324 (orange) treatment (n = 3). g, Cell proliferation of TKI-resistant K562 cells with 1,10-Phen or ML324 treatment (n = 3). h-i, Representative confocal microscopy imaging and corresponding flow histogram showing the fluorescence intensity of nascent peptides in K562 (h) and MEG-01 (i) (n = 3). Scale bar, 10 μM. j, Relative mutated BCR-ABL1 mRNA abundance per component to that in total RNA in K562 cells with 1,10-Phen or ML324 treatment (n = 3). k, Correlation of PABPC1-binding genes in K562 cells between PABPC1 inhibition and 1,10-Phen, ML324 treatment or two random genes (LARP7 and NONO) inhibition. l-o, IC50 values of 1,10-Phen or ML324 treatment on K562/MEG-01 cells with PABPC1 inhibition or not (n = 3). Data are shown as the mean ± SEM. P-values were determined by two-way repeated measures ANOVA (e, g, l-o), and one-way ANOVA with Bonferroni correction (f, h-j), ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

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Extended Data Fig. 7 Small molecules 1,10-Phen and ML324 suppress CML-BC progression.

a, Acute toxicity (above) with intraperitoneal administration, pharmacokinetics (below) with intravenous administration of 1,10-Phen or ML324 in vivo (n = 3 mice). b, The strategy analyzing the safety of 1,10-Phen or ML324 treatment in vivo. qd.ip, intraperitoneal injection once a day. c-d, The weights (c) and H&E staining (d) of heart, liver, spleen, lung, and kidney of mice treated with 1,10-Phen or ML324 (n = 5 mice). e, Spleen weights of CML recipients post 1,10-Phen, ML324, IM, 1,10-Phen+IM, or ML324 + IM treatment (n = 5 mice). f-g, Representative confocal microscopy images and corresponding flow cytometry histograms showing nascent peptides synthesis (f), and western blots showing BCR-ABL1 protein levels (g) in leukemic cells from CML recipients post 1,10-Phen, ML324, IM treatment (n = 3 mice). Scale bar, 20 μM. h, Quantification of HPCs (CD34+ CD38+), HSCs (CD38- CD90-) and LT-HSCs (CD90+) in the bone marrow of PDX CML-BC mice after 1,10-Phen, ML324, IM, 1,10-Phen+IM, or ML324 + IM treatment (n = 3 mice). i-j, Representative confocal microscopy images and corresponding flow cytometry histograms showing nascent peptides synthesis (i), and western blots showing BCR-ABL1 protein levels (j) in leukemic cells from PDX CML-BC mice after 1,10-Phen, ML324, IM treatment (n = 3 mice). Scale bar, 20 μM. k, The inhibition of shPABPC1, 1,10-Phen and ML324 on the proliferation of normal HSPCs, leukemia cells from CML-CP and CML-BC patients (n = 3). Data are shown as the mean ± SEM. P-values were determined by one-way ANOVA with Bonferroni correction (e-f, h-i), and Student’s unpaired two-tailed t-test (k), ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

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Sun, C., Xu, X., Chen, Z. et al. Selective translational control by PABPC1 phase separation regulates blast crisis and therapy resistance in chronic myeloid leukaemia. Nat Cell Biol 27, 683–695 (2025). https://doi.org/10.1038/s41556-024-01607-4

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