Nuclear PFKP promotes CXCR4-dependent infiltration by T cell acute lymphoblastic leukemia
Xueliang Gao, … , Xue-Zhong Yu, Haizhen Wang
Xueliang Gao, … , Xue-Zhong Yu, Haizhen Wang
Published July 13, 2021
Citation Information: J Clin Invest. 2021;131(16):e143119. https://doi.org/10.1172/JCI143119.
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Nuclear PFKP promotes CXCR4-dependent infiltration by T cell acute lymphoblastic leukemia

Abstract

PFKP (phosphofructokinase, platelet), the major isoform of PFK1 expressed in T cell acute lymphoblastic leukemia (T-ALL), is predominantly expressed in the cytoplasm to carry out its glycolytic function. Our study showed that PFKP is a nucleocytoplasmic shuttling protein with functional nuclear export and nuclear localization sequences (NLSs). Cyclin D3/CDK6 facilitated PFKP nuclear translocation by dimerization and by exposing the NLS of PFKP to induce the interaction between PFKP and importin 9. Nuclear PFKP stimulated the expression of C-X-C chemokine receptor type 4 (CXCR4), a chemokine receptor regulating leukemia homing/infiltration, to promote T-ALL cell invasion, which depended on the activity of c-Myc. In vivo experiments showed that nuclear PFKP promoted leukemia homing/infiltration into the bone marrow, spleen, and liver, which could be blocked with CXCR4 antagonists. Immunohistochemical staining of tissues from a clinically well-annotated cohort of T cell lymphoma/leukemia patients showed nuclear PFKP localization in invasive cancers, but not in nonmalignant T lymph node or reactive hyperplasia. The presence of nuclear PFKP in these specimens correlated with poor survival in patients with T cell malignancy, suggesting the potential utility of nuclear PFKP as a diagnostic marker.

Authors

Xueliang Gao, Shenghui Qin, Yongxia Wu, Chen Chu, Baishan Jiang, Roger H. Johnson, Dong Kuang, Jie Zhang, Xi Wang, Anand Mehta, Kenneth D. Tew, Gustavo W. Leone, Xue-Zhong Yu, Haizhen Wang

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Figure 5

Nuclear PFKP interacts with c-Myc to regulate CXCR4 transcription.

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Nuclear PFKP interacts with c-Myc to regulate CXCR4 transcription. (A) I...
(A) Immunoblots show that PFKP interacts with c-Myc (upper). Exogenous PFKP was immunoprecipitated from lysates of DND41 cells expressing WT-, NLS-, or S679E-PFKP using an anti-FLAG antibody. The presence of c-Myc was determined by immunoblotting (IB). Phosphorylation of c-Myc at S62, but not T58, increases in cells expressing nuclear PFKP (NLS- or S679E-PFKP) (lower). c-Myc was immunoprecipitated from lysates of DND41 cells expressing WT-, NLS-, or S679E-PFKP. Phosphorylation of c-Myc was determined by IB. c-Myc IB shows the loading of c-Myc proteins. (B) ChIP-qPCR shows c-Myc and PFKP binding to the same promoter region of CXCR4. c-Myc and PFKP antibodies were applied to ChIP in formaldehyde-fixed DND41 cells, in which c-Myc was either intact or knocked down with shRNA. Mouse IgG served as a control. (C) c-Myc knockdown decreases CXCR4 expression as measured by flow cytometry (upper) but does not affect PFKP expression and its nuclear distribution (lower). c-Myc expression was knocked down in DND41 cells expressing WT-, NLS-, or S679E-PFKP. Expression of nuclear PFKP and c-Myc were examined by IB (lower). (D) c-Myc knockdown with independent shRNAs (shMyc-1 or shMyc-2) significantly decreases the invasive capability of DND41 cells expressing WT-, NLS-, or S679E-PFKP. shCon is nontargeting control shRNA. (E) Luciferase reporter assay shows c-Myc inhibition decreases CXCR4 promoter activity. DND41 cells expressing WT-, NLS-, S679E-PFKP, or empty vector were treated with JQ1 (0.5 or 1.0 μM) for 48 hours. (F) c-Myc inhibitor (50 μM 10058-F4) treatment decreases the invasiveness of DND41 cells expressing WT-, NLS-, or S679E-PFKP. Con is a vehicle control. n = 3 (A–C, E, and F) and 4 (D). Data represent mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001 by 2-tailed Student’s t test (C) or 1-way ANOVA (B and D–F).