HUMAN PFKFB3 IN COMPLEX WITH KAN0438241

The glycolytic PFKFB3 enzyme is widely overexpressed in cancer cells and an emerging anti-cancer target. Here, we identify PFKFB3 as a critical factor in homologous recombination (HR) repair of DNA double-strand breaks. PFKFB3 rapidly relocates into ionizing radiation (IR)-induced nuclear foci in an MRN-ATM-γH2AX-MDC1-dependent manner and co-localizes with DNA damage and HR repair proteins. PFKFB3 relocalization is critical for recruitment of HR proteins, HR activity, and cell survival upon IR. We develop KAN0438757, a small molecule inhibitor that potently targets PFKFB3. Pharmacological PFKFB3 inhibition impairs recruitment of ribonucleotide reductase M2 and deoxynucleotide incorporation upon DNA repair, and reduces dNTP levels. Importantly, KAN0438757 induces radiosensitization in transformed cells while leaving non-transformed cells unaffected. In summary, we identify a key role for PFKFB3 enzymatic activity in HR repair and present KAN0438757, a selective PFKFB3 inhibitor that could potentially be used as a strategy for the treatment of cancer.

[1]  C. Lindskog,et al.  A pathology atlas of the human cancer transcriptome , 2017, Science.

[2]  S. Sivanand,et al.  Nuclear Acetyl-CoA Production by ACLY Promotes Homologous Recombination. , 2017, Molecular cell.

[3]  Min Huang,et al.  Phosphoglycerate mutase 1 regulates dNTP pool and promotes homologous recombination repair in cancer cells , 2017, The Journal of cell biology.

[4]  Yanping Zhang,et al.  p53 coordinates DNA repair with nucleotide synthesis by suppressing PFKFB3 expression and promoting the pentose phosphate pathway , 2016, Scientific Reports.

[5]  J. Chesney,et al.  Inhibition of 6-phosphofructo-2-kinase (PFKFB3) suppresses glucose metabolism and the growth of HER2+ breast cancer , 2016, Breast Cancer Research and Treatment.

[6]  E. Sonnhammer,et al.  A genome-wide IR-induced RAD51 foci RNAi screen identifies CDC73 involved in chromatin remodeling for DNA repair , 2015, Cell Discovery.

[7]  Piotr Malecki,et al.  The macromolecular crystallography beamlines at BESSY II of the Helmholtz-Zentrum Berlin: Current status and perspectives , 2015 .

[8]  J. Debreczeni,et al.  Structure-Based Design of Potent and Selective Inhibitors of the Metabolic Kinase PFKFB3. , 2015, Journal of medicinal chemistry.

[9]  P. Nordlund,et al.  The cellular thermal shift assay for evaluating drug target interactions in cells , 2014, Nature Protocols.

[10]  W. Denny,et al.  Targeting the Warburg Effect in cancer; relationships for 2-arylpyridazinones as inhibitors of the key glycolytic enzyme 6-phosphofructo-2-kinase/2,6-bisphosphatase 3 (PFKFB3). , 2014, Bioorganic & medicinal chemistry.

[11]  P. Nordlund,et al.  Monitoring Drug Target Engagement in Cells and Tissues Using the Cellular Thermal Shift Assay , 2013, Science.

[12]  J. Trent,et al.  Targeting 6-Phosphofructo-2-Kinase (PFKFB3) as a Therapeutic Strategy against Cancer , 2013, Molecular Cancer Therapeutics.

[13]  Y. Shiloh,et al.  The ATM protein kinase: regulating the cellular response to genotoxic stress, and more , 2013, Nature Reviews Molecular Cell Biology.

[14]  A. Schulze,et al.  Balancing glycolytic flux: the role of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatases in cancer metabolism , 2013, Cancer & metabolism.

[15]  K. Dahlman-Wright,et al.  Estrogen receptor-α, RBCK1, and protein kinase C β 1 cooperate to regulate estrogen receptor-α gene expression. , 2012, Journal of molecular endocrinology.

[16]  A. Urbani,et al.  Proteomic profiling of ATM kinase proficient and deficient cell lines upon blockage of proteasome activity , 2012, Journal of proteomics.

[17]  R. Muschel,et al.  The novel ATR inhibitor VE-821 increases sensitivity of pancreatic cancer cells to radiation and chemotherapy , 2012, Cancer biology & therapy.

[18]  M. Pagano,et al.  Cyclin F-Mediated Degradation of Ribonucleotide Reductase M2 Controls Genome Integrity and DNA Repair , 2012, Cell.

[19]  Philippe Pasero,et al.  dNTP pools determine fork progression and origin usage under replication stress , 2012, The EMBO journal.

[20]  Stephen J. Elledge,et al.  A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA damage response , 2012, Nature Cell Biology.

[21]  N. Pannu,et al.  REFMAC5 for the refinement of macromolecular crystal structures , 2011, Acta crystallographica. Section D, Biological crystallography.

[22]  Randy J. Read,et al.  Overview of the CCP4 suite and current developments , 2011, Acta crystallographica. Section D, Biological crystallography.

[23]  H. Niida,et al.  Mechanisms of dNTP supply that play an essential role in maintaining genome integrity in eukaryotic cells , 2010, Cancer science.

[24]  T. Helleday,et al.  Methylated DNA causes a physical block to replication forks independently of damage signalling, O(6)-methylguanine or DNA single-strand breaks and results in DNA damage. , 2010, Journal of molecular biology.

[25]  N. Lowndes,et al.  MRN and the race to the break , 2010, Chromosoma.

[26]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[27]  T. Helleday,et al.  Hydroxyurea-Stalled Replication Forks Become Progressively Inactivated and Require Two Different RAD51-Mediated Pathways for Restart and Repair , 2010, Molecular cell.

[28]  M. Nakanishi,et al.  Essential role of Tip60-dependent recruitment of ribonucleotide reductase at DNA damage sites in DNA repair during G1 phase. , 2010, Genes & development.

[29]  Vincent B. Chen,et al.  MolProbity: all-atom structure validation for macromolecular crystallography , 2009, Acta crystallographica. Section D, Biological crystallography.

[30]  A. Yalçin,et al.  Regulation of glucose metabolism by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases in cancer. , 2009, Experimental and molecular pathology.

[31]  A. Lane,et al.  Nuclear Targeting of 6-Phosphofructo-2-kinase (PFKFB3) Increases Proliferation via Cyclin-dependent Kinases* , 2009, The Journal of Biological Chemistry.

[32]  Yves Pommier,et al.  Implication of Checkpoint Kinase-dependent Up-regulation of Ribonucleotide Reductase R2 in DNA Damage Response* , 2009, The Journal of Biological Chemistry.

[33]  Yves Pommier,et al.  γH2AX and cancer , 2008, Nature Reviews Cancer.

[34]  Ricky A. Sharma,et al.  DNA repair pathways as targets for cancer therapy , 2008, Nature Reviews Cancer.

[35]  L. Kopelovich,et al.  A forward chemical genetic screen reveals an inhibitor of the Mre11-Rad50-Nbs1 complex. , 2008, Nature chemical biology.

[36]  John O Trent,et al.  Small-molecule inhibition of 6-phosphofructo-2-kinase activity suppresses glycolytic flux and tumor growth , 2008, Molecular Cancer Therapeutics.

[37]  A. Lane,et al.  Ras transformation requires metabolic control by 6-phosphofructo-2-kinase , 2006, Oncogene.

[38]  J. Schneider,et al.  ATM-dependent suppression of stress signaling reduces vascular disease in metabolic syndrome. , 2006, Cell metabolism.

[39]  R. Bartrons,et al.  PFKFB3 gene silencing decreases glycolysis, induces cell‐cycle delay and inhibits anchorage‐independent growth in HeLa cells , 2006, FEBS letters.

[40]  M. Cuadrado,et al.  "ATR activation in response to ionizing radiation: still ATM territory" , 2006, Cell Division.

[41]  A. Hofer,et al.  Regulation of Mammalian Ribonucleotide Reduction and dNTP Pools after DNA Damage and in Resting Cells* , 2006, Journal of Biological Chemistry.

[42]  R. Bucala,et al.  Phosphorylation of the 6-Phosphofructo-2-Kinase/Fructose 2,6-Bisphosphatase/PFKFB3 Family of Glycolytic Regulators in Human Cancer , 2005, Clinical Cancer Research.

[43]  L. Wodicka,et al.  A small molecule–kinase interaction map for clinical kinase inhibitors , 2005, Nature Biotechnology.

[44]  J. L. Rosa,et al.  6-Phosphofructo-2-kinase (pfkfb3) Gene Promoter Contains Hypoxia-inducible Factor-1 Binding Sites Necessary for Transactivation in Response to Hypoxia* , 2004, Journal of Biological Chemistry.

[45]  N. Curtin,et al.  Identification and Characterization of a Novel and Specific Inhibitor of the Ataxia-Telangiectasia Mutated Kinase ATM , 2004, Cancer Research.

[46]  R. Bambara,et al.  Macrophage Tropism of HIV-1 Depends on Efficient Cellular dNTP Utilization by Reverse Transcriptase* , 2004, Journal of Biological Chemistry.

[47]  D. Vertommen,et al.  6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis. , 2004, The Biochemical journal.

[48]  J. Caro,et al.  Hypoxic regulation of the 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase gene family (PFKFB‐1–4) expression in vivo , 2003, FEBS letters.

[49]  Yair Andegeko,et al.  Requirement of the MRN complex for ATM activation by DNA damage , 2003, The EMBO journal.

[50]  Stephen J. Elledge,et al.  MDC1 is a mediator of the mammalian DNA damage checkpoint , 2003, Nature.

[51]  L. Leng,et al.  High expression of inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (iPFK-2; PFKFB3) in human cancers. , 2002, Cancer research.

[52]  K. Khanna,et al.  DNA double-strand breaks: signaling, repair and the cancer connection , 2001, Nature Genetics.

[53]  J. Petrini,et al.  DNA Damage-Dependent Nuclear Dynamics of the Mre11 Complex , 2001, Molecular and Cellular Biology.

[54]  L. Thompson,et al.  XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. , 1999, Genes & development.

[55]  R. Bucala,et al.  An inducible gene product for 6-phosphofructo-2-kinase with an AU-rich instability element: role in tumor cell glycolysis and the Warburg effect. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[56]  P. Baumann,et al.  Human Rad51 Protein Promotes ATP-Dependent Homologous Pairing and Strand Transfer Reactions In Vitro , 1996, Cell.

[57]  E. Schaftingen,et al.  A kinetic study of pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers. Application to a microassay of fructose 2,6-bisphosphate. , 1982, European journal of biochemistry.

[58]  N. Curtin,et al.  DNA-PK inhibition by NU7441 sensitizes breast cancer cells to ionizing radiation and doxorubicin , 2013, Breast Cancer Research and Treatment.

[59]  K. Vousden,et al.  Control of glycolysis through regulation of PFK1: old friends and recent additions. , 2011, Cold Spring Harbor symposia on quantitative biology.

[60]  P. Huertas,et al.  DNA resection in eukaryotes: deciding how to fix the break , 2010, Nature Structural &Molecular Biology.

[61]  K. Sameith,et al.  Stratification of pediatric ALL by in vitro cellular responses to DNA double-strand breaks provides insight into the molecular mechanisms underlying clinical response. , 2009, Blood.