Abstract NG05: Depletion of a putatively druggable class of phosphatidylinositol kinases inhibits growth of p53 null tumors
Authors: Emerling Brooke M. , Hurov Jonathan B. , Poulogiannis George , Choo-Wing Rayman , Wulf Gerburg M. , Shim Hye-Seok , Lamia Katja A. , Rameh Lucia E. , Yuan Xin , Bullock Andrea , DeNicola Gina M. , Song Jiaxi , Signoretti Sabina , Cantley Lewis C. 
Published: May 2015

Abstract
The phosphoinositide family of lipids includes seven derivatives of phosphatidylinositol (PI) that are formed through the phosphorylation of the 3-, 4-, and 5-positions on the inositol ring. Phosphoinositides have distinct biological roles and regulate many cellular processes, including proliferation, survival, glucose uptake, and migration. Phosphoinositide kinases, phosphatases, and phospholipases spatially and temporally regulate the generation of the different phosphoinositide species, which localize to different subcellular compartments. Phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3) is synthesized by phosphoinositide 3-kinase (PI3K) and serves as the plasma membrane docking site for a subset of proteins that have pleckstrin-homology (PH) domains that bind this lipid, including the serine/threonine protein kinase AKT (also known as protein kinase B or PKB). AKT is a proto-oncogene that has critical regulatory roles in insulin signaling and cancer progression. Phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) is the major substrate for Class I PI3Ks and has a significant role itself in mediating the localization of proteins to the plasma membrane and in nucleating cortical actin polymerization (1).
Until 1997 it was thought that PI-4,5-P2 was produced exclusively by phosphorylation of phosphatidylinositol-4-phosphate (PI-4-P) at the 5 position of the inositol ring, a reaction catalyzed by the Type 1 PI-4-P 5-kinases (encoded by the genes PIP5K1A, B and C). Unexpectedly, a second highly-related family of PIP kinases (called Type 2) was found to produce PI-4,5-P2 by phosphorylating the 4 position of phosphatidylinositol-5-phosphate (PI-5-P), a lipid that had been previously overlooked due to its co-migration with the much more abundant PI-4-P (2-3). The Type 2 PIP kinases are not present in yeast but are conserved in higher eukaryotes from worms and flies to mammals. Humans and mice have three distinct genes, PIP4K2A, B and C encoding enzymes called PI5P4Kα, β, and γ, respectively. The bulk of PI-4,5-P2 in most tissues is almost certainly derived from the Type 1 PIP5Ks, yet recent quantitative proteomic studies on cell lines have revealed a higher abundance of PI5P4Ks than PI4P5Ks (4). This high abundance of the Type 2 enzymes may, in part, explain why the substrate, PI-5-P is present at very low levels. While the Type 1 PIP kinases generate PI-4,5-P2 at the plasma membrane, the Type 2 kinases are located at internal membranes, including the ER, Golgi and nucleus and probably generate PI-4,5-P2 at those locations (5-8). The vast majority of PI-4,5-P2 is located at the plasma membrane and it is not clear whether the critical function of the Type 2 PIP kinases is to generate PI-4,5-P2 at intracellular sites or to maintain low levels of PI-5-P (or both).
In a previous study we generated mice in which one of the Type 2 PIP kinase genes (PIP4K2B) was deleted in the germline. These mice were viable, exhibited enhanced insulin sensitivity and enhanced insulin-dependent activation of AKT in skeletal muscle (9). Paradoxically, despite increased AKT activation the mice were smaller and had decreased adiposity on a high fat diet. Cell based assays revealed that PI5P4Kβ (encoded by PIP4K2B) becomes phosphorylated by p38 at Ser326 in response to cellular stresses, such as UV and H2O2, and that this causes inhibition of the PI5P 4-kinase activity and results in increased cellular PI-5-P levels (10). These studies suggest that the Type 2 PIP kinases mediate cellular stress responses downstream of p38 (presumably by altering the PI-5-P/PI-4,5-P2 ratio at intracellular locations) and that under conditions of low stress, these enzymes suppress the PI3K/AKT signaling pathway. It should be pointed out that the Type 2 PIP kinases are unlikely to supply PI-4,5-P2 as a substrate for PI3K since activation of AKT correlates with loss of PI5P4K activity rather than gain.
In this study we have interrogated the potential role of Type 2 PIP kinases in cancers. We found high levels of either PI5P4Kα or PI5P4Kβ enzymes or both in a number of breast cancer cell lines, and more importantly, found amplification of the PIP4K2B gene and high levels of both the PI5P4Kα and PI5P4Kβ proteins in a subset of human breast tumors. We found that knocking down the levels of both PI5P4Kα and PI5P4Kβ in a TP53 deficient breast cancer cell line blocked growth on plastic and in xenografts. This impaired growth correlated with impaired glucose metabolism and enhanced levels of reactive oxygen species (ROS) leading to senescence. The impaired glucose metabolism, despite activation of the PI3K-AKT pathway (which typically enhances glucose metabolism) was paradoxical. The results indicate that PI3K activation is not driving the ROS production, but may be an inadequate feedback attempt to restore glucose uptake and metabolism.
To assess the role of Type 2 PIP kinases in tumor formation, we generated mice with germline deletions of PIP4K2A and PIP4K2B and crossed these with TP53-/- mice and evaluated tumor formation in all the viable genotypes. We found that mice with homozygous deletion of both TP53 and PIP4K2B were not viable, indicating a synthetic lethality for loss of these two genes. Importantly, mice with the genotype PIP4K2A-/-, PIP4KB+/-, TP53-/- were viable and had a dramatic reduction in tumor formation compared to siblings that were TP53-/- and wild type for PIP4K2B and/or PIP4K2A genes. The decreased tumor incidence in the background of PIP4K2A-/-, PIP4K2B+/-, TP53-/- compared to TP53-/- alone is particularly interesting in respect to Li Fraumeni Syndrome (germline TP53 mutations). Our results indicate that expression of PI5P4Kα and/or β is critical for the growth of tumors with TP53 mutations or deletions. Thus, co-amplification of PIP4K2B with ERBB2 might explain why breast cancers in patients with Li Fraumeni syndrome show ERBB2 amplifications (HER2-positive) in over 83% of cases as opposed to 16% of age-matched patients with wild type TP53 (11).
The results that we present here suggest that PI5P4Kα and β play a critical role in mediating changes in metabolism in response to stress, and in particular ROS stress that occurs in the absence of p53. Germ-line deletion of either PIP4K2A or PIP4K2B alone resulted in mice with normal lifespans, and germ-line deletion of both PIP4K2A and PIP4K2B resulted in full-term embryos of normal size and appearance at birth, indicating that these genes do not play a major role in normal embryonic growth and development. Yet the PIP4K2A-/-, PIP4K2B-/- pups die shortly after birth, consistent with these genes having a role in mediating stress responses known to occur following birth. Importantly, germ-line deletion of both PIP4K2B and TP53 resulted in lethality while germ-line deletion of either gene alone resulted in Mendelian ratios of viable pups. Thus, the genetic studies suggest that TP53 and PIP4K2B have overlapping roles in mediating cellular responses to stress and that, while neither gene alone is essential, loss of both genes is not tolerated.
The most exciting observation from these studies in regard to potentially new therapies for p53 mutant tumors is that germ-line deletion of both alleles of PIP4K2A and one allele of PIP4K2B in the context of TP53-/- results in a viable mouse with a dramatic reduction in tumor-dependent death compared to TP53-/- mice that are wild type for PIP4K2A and B. These results (and studies of the PIP4K2A-/-, PIP4K2B+/- or PIP4K2A+/-, PIP4K2B-/- mice in the context of wild type TP53) indicate that normal tissues tolerate well the loss of three out of four alleles of the PIP4K2A and PIP4K2B genes, but that tumors are not viable in this context. PI5P4Kα and β are kinases and pharmaceutical companies have shown that it is possible to develop highly specific inhibitors of both protein kinases and lipid kinases. The synthetic lethality that we observe between TP53 loss and loss of these kinases indicates that drugs that target either the enzyme PI5P4Kβ alone or that target both PI5P4Kα and β are likely to be well-tolerated and very effective on tumors that have loss of function mutations or deletions of TP53. Our observations with BT474 cells suggest that HER2 positive tumors that have amplifications of PIP4K2B and mutations in TP53 may be particularly sensitive to PI5P4Kα,β inhibitors. The ERBB2 (Her2) amplicon on chromosome 17 is variable in size and can contain a number of cancer-related genes in addition to the ERBB2 locus. Clinically, patients who have tumors with small amplicons confined to the ERBB2 locus have the greatest benefit from ERBB2-directed therapies such as Trastuzumab, while tumors with wider ERBB2 amplicons have poor responses, suggesting co-amplification of genes that contribute to Trastuzumab resistance (11). PIP4K2B, which is located in a chromosomal region (17q12) close to ERBB2, may be a candidate for an adjacent co-amplified gene that confers Trastuzumab resistance, and, conversely, concomitant inhibition of ERBB2 and PIP4K2B could be a highly effective treatment option for ERBB2 (Her2) positive tumors that are p53-mutant and PIP4K2B-amplified.
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Citation Format: Brooke M. Emerling, Jonathan B. Hurov, George Poulogiannis, Rayman Choo-Wing, Gerburg M. Wulf, Hye-Seok Shim, Katja A. Lamia, Lucia E. Rameh, Xin Yuan, Andrea Bullock, Gina M. DeNicola, Jiaxi Song, Sabina Signoretti, Lewis C. Cantley. Depletion of a putatively druggable class of phosphatidylinositol kinases inhibits growth of p53 null tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr NG05. doi:10.1158/1538-7445.AM2014-NG05