Nuclear PFKP promotes CXCR4-dependent infiltration by T cell acute lymphoblastic leukemia
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
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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Research Article Cell biology Oncology

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 2

Nuclear localization of PFKP depends on its interaction with cyclin D3/CDK6.

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Nuclear localization of PFKP depends on its interaction with cyclin D3/C...
(A) More dimeric PFKP was detected in the nuclear fraction than in the cytoplasmic fraction. Quantification (ImageJ) was performed by ultrafiltration of nuclear extract (NE) or cytoplasmic extract (CE) from leukemia cells followed by immunoblotting (IB) using an anti-PFKP antibody from Supplemental Figure 2A. (B) CDK6 inhibition decreases nuclear accumulation of PFKP. Cells were treated with 1 μM palbociclib (Palbo) or DMSO (Con) for 24 hours. WCL, whole cell lysate. (C) Decreased nuclear PFKP in KOPTK1 cells in which either cyclin D3 or CDK6 was knocked down. The knockdown efficiency was examined with IB. (D) Pull-down assay using anti-FLAG M2 agarose beads shows direct interaction between FLAG-PFKP and recombinant cyclin D3/CDK6. FLAG peptide served as control. PFKP-RXL is an RXL motif mutant of PFKP. (E and F) Immunoprecipitation using anti-PFKP antibody–conjugated beads followed by IB showed that the interaction between PFKP and importin-9 was disrupted when CDK6 kinase activity was inhibited (1 μM palbociclib, 24 hours) (E) or when CDK6 was knocked down (F), while the interaction between PFKP and CRM1 was not affected. (G) Immunoprecipitation using anti-FLAG beads followed by IB shows that higher levels of importin 9 co-immunoprecipitated with mutant S679E-PFKP than WT. HC is the heavy chain of the FLAG antibody stained with Ponceau S (PS: HC). (H) IB of ectopic PFKP expression in NE or CE of cells expressing S679E- or WT-PFKP (left). FLAG-PFKP expression was normalized to nuclear FLAG from cells expressing WT-PFKP (right). (I) IB shows decreased nuclear accumulation of PFKP in importin-9–knockdown cells. IB of WCL shows no significant alteration of PFKP expression. Lamin A/C and actin were used as loading controls. n = 4 (A) and 3 (B–I). Data represent mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-tailed Student’s t test (A) or 1-way ANOVA (H).