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The PI3K catalytic subunit p110δ is a crucial mediator of leukemia-associated mutant PTPN11 -induced GM-CSF hypersensitivity. Charles B Goodwin 1 , Rachel L. Gearinger 2 , Raghuveer Mali 2 , Donna Cerabona 2 , Gordon Chan 3 , Benjamin G. Neel 3 , Reuben Kapur 2 , Rebecca J. Chan 1, 2
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The PI3K catalytic subunit p110δ is a crucial mediator of leukemia-associated mutant PTPN11-induced GM-CSF hypersensitivity Charles B Goodwin1, Rachel L. Gearinger2, Raghuveer Mali2, Donna Cerabona2, Gordon Chan3, Benjamin G. Neel3, Reuben Kapur2, Rebecca J. Chan1, 2 1Department of Medical and Molecular Genetics and 2Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202, 3University Health Network, Toronto, Ontario 6th International Symposium on MDS and Bone Marrow Failure Syndromes in Childhood Prague, Czech Republic November 7, 2012
Juvenile Myelomonocytic Leukemia (JMML) • Rare Myelodysplastic Syndrome/Myelproliferative Neoplasm (MDS/MPN) of infancy and early childhood • Patients usually 4 or younger (average age is 2) • 25-50 cases/year in US (2% of childhood leukemias) • Responds poorly to chemotherapy • Distinguishing characteristics: • Hypersensitivity to Granulocyte Macrophage-Colony-Stimulating Factor (GM-CSF) stimulation • Clonal hyperproliferationof maturing myeloid cells, particularly monocyte/macrophage lineage
GM-CSF Receptor Shp2 PI3K Ras Ras
Class IA Phosphoinositol-3-Kinase (PI3K) Regulatory Subunits: -p85α [p55α, p50α] (Pik3r1) -p85β (Pik3r2) -p55γ (Pik3r3) Catalytic Subunits: -p110α (Pik3ca) -p110β (Pik3cb) -p110δ (Pik3cd) • Catalytic Subunit Function: • Lipid Kinase: • PIP2 PIP3 • PIP3 recruits Plekstrin Homology (PH) domain-containing proteins (e.g., PDK1 and Akt) • Catalytic Subunit Expression: • p110α: ubiquitous • p110β: ubiquitous • p110δ: hematopoietic cells p110 p85 PI3K
Hypothesis Ras
Genetic Disruption of p85α Regulatory Subunit Partially Normalizes GM-CSF Hypersensitivity C Goodwin, et al 2012
Hypothesis Ras
Inhibitors with High Specificity for p110δ Reduce GM-CSF Hypersensitivity in the Presence of Endogenously Expressed Mutant Shp2 D61Y
Inhibitors with High Specificity for p110δ Significantly Increase Apoptosis in Cells Expressing Mutant Shp2 E76K
Experimental Strategy to Evaluate the Effect of Genetic Disruption of p110δ on Gain-of-function Mutant Shp2 D61Y-Induced GM-CSF Hypersensitivity in vitro and in vivo
Genetic Disruption of p110δ Reduces GM-CSF Hypersensitivity in vitro
Genetic Disruption of p110δ Results in Significantly Smaller Spleens in vivo
Functional Domains of Shp2 E76K • Scaffolding function: • Bind Tyr577 or Tyr612 of βC subunit of the GM-CSF Receptor • Shp2 forms complex with Grb2 or Sos • Activation of Ras • Shp2 forms complex with Grb2 or Gab2 and p85 • Activation of PI3K • Catalytic function • Shown to be necessary for full activation of Ras • Molecular target of enzymatic activity is still not clear PTPase C-SH2 N-SH2 Shp2 R138K C463A R32K Scaffolding Function Enzymatic Function Rachel Gearinger
Genetic Disruption of Mutant Shp2’s SH2 and Phosphatase Domains Significantly Reduces GM-CSF Hypersensitivity and Akt Hyper-phosphorylation
Summary • Genetic disruption of the PI3K regulatory subunit, p85α, results in significant reduction in gain-of-function mutant Shp2-induced GM-CSF-stimulated hyperproliferation and Akt and Erk hyper-phosphorylation • Inhibitors with high specificity for the PI3K catalytic subunit, p110δ, significantly reduce activating mutant Shp2-induced GM-CSF-stimulated hyperproliferation, Akt and Erkhyperphosphorylation, and survival • Genetic disruption of p110δ significantly reduces Shp2 D61Y-induced GM-CSF-stimulated hyperproliferation and Akt and Erkhyperphosphorylation in vitro, and significantly reduces spleen size in vivo • Genetic disruption of mutant Shp2’s SH2 and phosphatase domains significantly reduces GM-CSF-stimulated hyperproliferation and Akt and Erkhyperphosphorylation • The PI3K catalytic subunit, p110δ, represents a potential therapeutic target for gain-of-function mutant Shp2-induced Myeloproliferative Disease
Thank You! • Kapur Lab (Indiana University): • Reuben Kapur, PhD • Raghuveer Mali, PhD • Neel Lab (University Health Network, Toronto): • Benjamin G. Neel, MD, PhD • Gordon Chan, PhD • IUSM Medical Scientist Training Program • Chan Lab: • Rebecca J. Chan, MD, PhD • Sarah Nabinger, PhD • Xingjun Li, MD, PhD • Rachel Gearinger • Donna Cerabona • Tirajeh Saadatzadeh • Briana Richine • Zhenyun Yang, PhD • Fuqin Yin • Support: • F30 HL104867 (NIH/NHLBI) • Merilyn Hester Scholarship (IU Simon Cancer Center) Questions?
Cre-Inducible Shp2 D61Y Knock-in Mice PolyI:PolyC IFNγ Mx1 Cre Chan, et al. Blood, 2009 D61Y loxP loxP Shp2 Mutant Floxed Allele Exon 2 Stop Exon 3 Hemizygous WT Shp2 Allele Exon 2 Exon 3 Heterozygous
Cre-inducible Shp2 D61Y Knock-in mice display splenomegaly and GM-CSF Hypersensitivity Vevo 770 Ultrasound