Inositol Phospholipid Signaling in Physiology and Disease

Abstracts

Day 1: Tuesday, June 26, 2012

Keynote Lecture 1

Phosphoinositol Signaling and Disease
Lewis C. Cantley, PhD, Harvard University Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA

Phosphoinositide 3-Kinase (PI3K) is a central enzyme in a signaling pathway that mediates cellular responses to growth factors. This enzyme phosphorylates the 3 position of phosphatidylinositol-4,5-bisphosphate to produce phosphatidylinositol-3,4,5-trisphosphate (PIP3) at the plasma membrane. A number of signaling proteins, including the Ser/Thr protein kinases, AKT and PDK1, contain pleckstrin homology domains that bind specifically to PIP3. Thus, the generation of PIP3 at the plasma membrane in response to activation of PI3K by growth factors results in the initiation of downstream Ser/Thr phosphorylation cascades that control a variety of cellular responses. The signaling pathway downstream of PI3K is highly conserved from worms and flies to humans and genetic analysis of the pathway has revealed a conserved role in regulating glucose metabolism and cell growth. Based on deletion of genes encoding the catalytic or regulatory subunits of PI3K in the mouse, PI3K mediates insulin dependent regulation of glucose metabolism, and defects in activation of this pathway result in insulin resistance. In contrast, mutational events that lead to hyperactivation of the PI3K pathway result in cancers. Activating mutations in PIK3CA, encoding the p110alpha catalytic subunit of PI3K or inactivating mutations in PTEN, a phosphoinositide 3-phosphatases that reverses the effects of PI3K, are among the most common events in solid tumors. We have generated mouse models in which a mutated form of the PIK3CA gene is expressed in a tissue specific and reversibly inducible manner. These mice develop cancers that are dependent on continuous expression of the mutant PIK3CA gene. The PIK3CA driven tumors are FDG-PET positive and turning off PI 3-Kinase with PI3K inhibitors that are in human clinical trials results in an acute decline in FDG-PET signal that precedes tumor shrinkage. These results suggest that the ability of PI3K to stimulate high rates of glucose uptake and metabolism may be critical for the survival of PIK3CA mutant tumors. The role of PI3K inhibitors for treating cancers in mouse models and in human trials will be discussed.
 

Session I: Contribution of Inositol Phospholipid Signaling to Oncogenesis

Characterisation of INPP4B Tumor Suppressor Activity
Christina Mitchell, PhD, Monash University

Phosphoinositide 3-kinase generates PtdIns(3,4,5)P₃ at the plasma membrane, which is rapidly dephosphorylated either by PTEN (phosphatase and tensin homologue deleted on chromosome 10) to PtdIns(4,5)P2, or by the inositol polyphosphate 5-phosphatases, generating PtdIns(3,4)P2.The inositol polyphosphate 4-phosphatases, INPP4A and INPP4B degrade PtdIns(3,4)P₂ to PtdIns(3)P and respectively regulate neuroexcitatory cell death, or act as a tumour suppressor in breast, prostate and other cancers. INPP4B loss of heterozygosity (LOH) and loss of expression of INPP4B protein has been identified in specific breast cancer subtypes, suggesting INPP4B acts as a tumour suppressor. INPP4B is expressed in nonproliferative estrogen receptor (ER)-positive cells in the normal breast and INPP4B knockdown in ER-positive breast cancer cells increases Akt activation, cell proliferation, and xenograft tumour growth. INPP4B protein expression is lost most commonly in aggressive basal-like breast carcinomas and its loss is frequently associated with PTEN-null tumours. INPP4B is expressed in benign prostatic tissue and in prostate cancers INPP4B protein expression is often reduced or lost. We have examined the molecular regulation of INPP4B enzyme activity and have identified INPP4B acts as an allosteric enzyme in its degradation of PtdIns(3,4)P₂. These studies reveal INPP4B enzyme activity is regulated by both protein:protein interactions and intramolecular interactions with other phosphoinositides.
 

Coauthors: N.K. Rynkiewicz¹, H.J. Liu¹, L.M. Ooms¹, C.G. Fedele¹, C.A. McLean², C. A Mitchell^1
1 Department of Biochemistry, Monash University, Melbourne, Australia
²Department of Anatomical Pathology, Alfred Hospital, Melbourne, Australia

 

High-Frequency Mutation of the PI3K Signaling Suite in T-ALL
Thomas Look, MD, Dana Farber Cancer Center

To more comprehensively assess the pathogenic contribution of the PTEN-PI3K-AKT pathway to T-cell acute lymphoblastic leukemia (T-ALL), we examined diagnostic DNA samples from children with T-ALL using array CGH and sequence analysis. Alterations of PTEN, PI3K or AKT were identified in 47.7% of 44 cases. There was a striking clustering of PTEN mutations in exon 7 in 12 cases, all of which were predicted to truncate the C2 domain without disrupting the phosphatase domain of PTEN. Induction chemotherapy failed to induce remission in 3 of the 4 patients whose lymphoblasts harbored PTEN deletions at the time of diagnosis, compared with none of the 12 patients with mutations of PTEN exon 7 (P = 0.007), suggesting that PTEN deletion has more adverse therapeutic consequences than mutational disruptions that preserve the phosphatase domain. To establish the role of PI3K signaling in T-ALL, we generated a 4-hydroxytamoxifen inducible zebrafish model of MYC-induced T-ALL, in which most tumors demonstrate dependence on the MYC oncoprotein. To test whether MYC addiction requires pten in vivo, MYC-ER transgenic zebrafish were mated to zebrafish harboring loss-of-function mutations in their pten genes, and offspring were raised in 4-hydroxytamoxifen. Pten haploinsufficiency impaired T-ALL regression upon MYC transgene downregulation, an effect that was phenocopied by expression of constitutively active Akt. Our findings suggest that Akt pathway activation is sufficient to maintain the transformed phenotype in T-ALL lymphoblasts, even after MYC transgene downregulation. These findings add significantly to the rationale for the development of therapies targeting the PTEN-PI3K-AKT pathway in T-ALL.
 

Coauthors: Alejandro Gutierrez,^1,5 Takaomi Sanda,¹ Donna Neuberg,² Stuart S. Winter,⁷ Richard Larson,⁸ Jianhua Zhang,³ Alexei Protopopov,³ Lynda Chin,^3,4 Pier Paolo Pandolfi,⁶ Lewis B. Silverman,^1,5 Stephen P. Hunger,⁹ Stephen E. Sallan,^1,5 and A. Thomas Look^1,5
1Department of Pediatric Oncology
²Department of Biostatistics and Computational Biology
³Center for Applied Cancer Science of the Belfer Institute for Innovative Cancer Science
⁴Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
⁵Division of Hematology/Oncology, Children’s Hospital, Boston, MA
⁶Cancer Genetics Program, Beth Israel Deaconess Cancer Center, Boston, MA
⁷Department of Pediatrics
⁸Department of Pathology, The University of New Mexico Health Sciences Center, Albuquerque, NM
⁹Center for Cancer and Blood Disorders, The Children’s Hospital and University of Colorado School of Medicine, Aurora, CO

 

Glioma Formation, Cancer Stem Cells and PI3K/Akt Signaling
Eric C. Holland, MD, PhD, Memorial Sloan-Kettering Cancer Center

Cells exist in brain tumors that have stem-cell characteristics including expression of markers, ability to grow as neurospheres, and enhanced ability to form tumors upon transplantation. One marker of stem-ness is the activity of the transmembrane ATP binding cassette protein ABCG2 which pumps small molecules out of the cytoplasm and can be identified by its ability to eflux florescent dye from the cell. This phenotype can be detected on FACS as cells excluding Hoechst dye and is called the side population (SP). SP analysis enriches for stem-like cells in many tumor types including gliomas. Stem-like cells are relatively resistant to therapy and maintenance of this character is driven by complex interactions of several pathways including PI3K, notch, and nitric oxide (NO).Notch signaling elevates ABCG2 mRNA while Akt activity translocates ABCG2 to the plasma membrane to be active. In medulloblastomas, radiation therapy spares cells in the perivascular niche (PVN). These PVN cells acquire increased stem-cell characteristics and elevated Akt activity in response to radiation, and Aktblockade reduces their survival. Cells with stem-cell character also occupy the glioma PVN. In these tumors, NO is produced from endothelial cellsvia eNOS. NO activates cGMP, PKG and notch in the cells surrounding blood vessels leading to the SP phenotype and other stem-cell characteristics. Finally, SP appears to be more than just a marker for stem-like cells. The activity of ABCG2 actively drives stem-cell characteristics although the substrate that it pumps to achieve this effect is currently unknown.
 

Coordinate Tumor Suppressor Activities of Inositol Phosphatases PTEN and SHIP1 in B Cells
Robert C. Rickert, PhD, Sanford-Burnham Medical Research Institute

PTEN and SHIP antagonize PI3K signaling by dephosphorylating PtdIns(3,4,5)P₃. While PTEN is widely expressed and constitutively active, SHIP is hematopoietically-restricted and needs to be recruited to phosphorylated target receptors via an SH2-domain interaction. Both PTEN and SHIP are also targets of downregulation by microRNAs. We have shown that in the absence of PTEN, B cell transformation does not occur due to the retained function of SHIP. Here I will present new findings on the distinct roles of SHIP and PTEN in modulating PI3-kinase signaling in response to antigen receptor signaling as well as microenvironmental cues. These studies comment on the contributions of inflammation and homeostatic signaling to B lymphomagenesis.
 

PTEN Signaling in Prostate Cancer
Loyd Trotman, PhD, Cold Spring Harbor Laboratories

Session II: Pharmaceutical Modulation of Inositol Phospholipid Signaling in Cancer

Session Chair: Langdon M. Miller, MD, Gilead Sciences

Targeting PI3Kdelta in Lymphoid Malignancies
Langdon L. Miller, MD, Gilead Sciences, Inc.

The PI3K signaling pathway has emerged as a promising therapeutic target in cancer and inflammatory disorders. Among several forms of the PI3K enzyme is PI3K-delta, which is expressed in lymphocytes, other immune cells, and in various tumor types. Genetic and pharmacological approaches have established an important role for PI3K-delta in immune cell function, mast cell degranulation, and leukocyte migration. PI3K-delta has been shown to integrate signaling from cell surface receptors that promote malignant B-cell proliferation, migration, and survival. GS-1101 (also known as CAL-101) is a novel drug that selectively inhibits PI3K-delta activity. In preclinical efficacy pharmacology studies evaluating lymphoid tumors, GS-1101 has reduced PI3K-delta signaling, interrupted cell cycling, and enhanced apoptosis. GS-1101 has been evaluated in clinical studies in patients with lymphoid malignancies, including indolent NHL, mantle cell lymphoma, and chronic lymphocytic leukemia. Among patients with these cancers, substantial antineoplastic activity has been observed; GS-1101 has induced durable tumor regressions in the majority of patients. Thus, initial characterization of PI3K delta has allowed development of GS-1101 as the first drug to therapeutically modulate this important enzyme. In turn, the early success of GS-1101 is setting the stage for further examination of the role of PI3K-delta in health and disease.
 

Challenges in the Clinical Development of PI3 Kinase Inhibitors for the Treatment of Solid Tumors
Cristian Massacesi, MD, Novartis Pharmaceuticals, France

Multiple components of the PI3K pathway are often dysregulated in cancer cells and over-activation of PI3K signaling is implicated in many aspects of tumor growth and survival. Activation of this pathway can be the result of: i) Amplification and/or overexpression of the p110α catalytic subunit; ii) Presence of activating mutations in the PIK3CA gene encoding the p110α catalytic subunit; iii) Constitutively active mutants or overexpression of receptor tyrosine kinases (e.g. EGFR, ErbB2) leading to constitutive recruitment and activation of PI3K; iv) Constitutive recruitment and activation by mutant forms of the Ras oncogene; v) Loss or inactivating mutation of the tumor suppressor gene PTEN, a endogenous negative regulator of the PI3K pathway; or vi) Overexpression of the downstream kinase Akt.
 
Preclinical work suggests that inhibition of the PI3K signaling pathway might provide benefit for the treatment of many solid tumors such as breast cancer, prostate cancer, glioblastoma multiforme, colon cancer, lung cancer, etc. Therefore, therapeutic interventions on PI3K signaling could represent a rationale approach for the treatment of many tumors and could, in addition, increase the efficacy of already established antineoplastic treatments like cytotoxic agents, endocrine agents, targeted agents, etc.
 
Currently a large number of PI3K and Akt inhibitors are being investigated in clinical trials. Despite strong scientific rationale and supportive preclinical data, a consistent marginal single agent clinical activity has been reported to date for most of these compounds, across different tumor types. Nevertheless, interesting preliminary clinical data are emerging when this class of compounds are combined with other antineoplastic agents (eg, with hormonal agents in breast cancer or with chemotherapy in NSCLC), These emerging data suggest that targeting the overactivated PI3K pathway may be critical to overcome drug-resistance and may provide clinical benefit.
 
With regards to biomarker strategy, the complexity of PI3K/Akt pathway and the high number of key players in the pathway has thus far limited the profiling of biomarkers which can guide the pre-identification of patients most likely to benefit from PI3K inhibition.
 
An overview on the challenges faced in the clinical development of PI3K inhibitors will be discussed using Novartis compounds as example.
 

Mechanisms of PI3K Inhibition in Cancer
Neal Rosen, MD, PhD, Memorial Sloan-Kettering Cancer Center

Interrupting the PI3K-AKT-mTOR Pathway in Cancer Therapy
David B. Solit, MD, Memorial Sloan-Kettering Cancer Center