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Dual Blockade of Lipid and Cyclin-Dependent Kinases Induces Synthetic Lethality in Malignant Glioma

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Dual Blockade of Lipid and Cyclin-Dependent Kinases Induces Synthetic Lethality in Malignant Glioma Dual blockade of lipid and cyclin-dependent kinases induces synthetic lethality in malignant glioma Christine K. Chenga,b,c,d, W. Clay Gustafsonb,d, Elizabeth Charrona,b,c,d, Benjamin T. Housemane,f, Eli Zundere,f, Andrei Gogad,g, Nathanael S. Grayh, Brian Polloki, Scott A. Oakesd,j, C. David Jamesc,d, Kevan M. Shokatd,e,f,k, William A. Weissa,b,c,d,1, and Qi-Wen Fana,b,c,d,1 Departments of aNeurology, bPediatrics, cNeurological Surgery and Brain Tumor Research Center, fCellular and Molecular Pharmacology, and gMedicine and dHelen Diller Family Comprehensive Cancer Center, eProgram in Chemistry and Chemical Biology, and hDepartment of Cancer Biology, Dana Farber Cancer Institute, Boston, MA 02115; iResearch and Development, Invitrogen Corp., Carlsbad, CA 92008; and jDepartment of Pathology and kHoward Hughes Medical Institute, University of California, San Francisco, CA 94158 Edited by Eric C. Holland, Sloan-Kettering Institute, New York, NY, and accepted by the Editorial Board June 15, 2012 (received for review February 18, 2012) Malignant glioma, the most common primary brain tumor, is inhibitor, PIK-75, as uniquely able to induce apoptosis rather generally incurable. Although phosphatidylinositol-3-kinase (PI3K) than growth arrest in glioma. Lacking stability in solution and signaling features prominently in glioma, inhibitors generally block in vivo (8, 9), the broad target profile and overall toxicity of PIK- proliferation rather than induce apoptosis. Starting with an in- 75 precluded clinical development. We hypothesized that the hibitor of both lipid and protein kinases that induced prominent efficacy of PIK-75 in inducing apoptosis resulted from a syn- apoptosis and that failed early clinical development because of its thetic-lethal interaction between inhibition of PI3K and in- broad target profile and overall toxicity, we identified protein hibition of one or more protein kinase targets of PIK-75. kinase targets, the blockade of which showed selective synthetic lethality when combined with PI3K inhibitors. Prioritizing protein Results kinase targets for which there are clinical inhibitors, we demon- PI3K Inhibitor PIK-75 Induces Apoptosis in Glioma. We screened six strate that cyclin-dependent kinase (CDK)1/2 inhibitors, siRNAs glioma cell lines using a panel of PI3K inhibitors including the against CDK1/2, and the clinical CDK1/2 inhibitor roscovitine all PI3Kα-specific compound PIK-90 (Bayer) and the PI3K/mTOR cooperated with the PI3K inhibitor PIK-90, blocking the antiapop- inhibitors PI-103 (Piramed) and LY294002 (Lilly). In each cell totic protein Survivin and driving cell death. In addition, over- line, flow cytometric analyses demonstrated cell cycle arrest with expression of CDKs partially blocked some of the apoptosis caused no appreciable apoptosis (Fig. 1A). In testing additional PI3K by PIK-75. Roscovitine and PIK-90, in combination, were well inhibitors, we demonstrated that the Piramed p110α/δ inhibitor tolerated in vivo and acted in a synthetic-lethal manner to induce PIK-75 arrested cells and induced apoptosis (Fig. 1A and apoptosis in human glioblastoma xenografts. We also tested Fig. S1A). Like PIK-90, PI-103, and LY294002, PIK-75 could clinical Akt and CDK inhibitors, demonstrating induction of apo- block phosphorylation of Akt (Fig. 1B and Fig. S1B). The PTEN ptosis in vitro and providing a preclinical rationale to test this wild type (WT) cell line LN229 showed increased levels of apo- combination therapy in patients. ptosis in comparison with the PTEN mutant (MT) line U87, using doses of PIK-75 that led to equivalent reductions in p-Akt (Fig. 1C liomas are the most common primary brain tumors. Glio- and Fig. S1 A and B). In comparing biochemical activity of blastoma multiforme represents the most frequently occur- LY294002 with PIK-75, LY294002 more potently blocked both p- G B ring, highest grade, and most lethal glioma. Amplification of the Akt and the mTOR target p-rpS6 (Fig. S1 ), and yet LY294002, epidermal growth factor receptor (EGFR) occurs in 40% of as- in contrast to PIK-75, was unable to promote apoptosis. trocytomas and correlates with advanced disease (1, 2). EGFR signals, in part, through the lipid kinase phosphatidylinositol-3′- Apoptosis Induced by PIK-75 Requires Inhibition of PI3K and Is Mitochondrial-Dependent. Data in Fig. 1 A and B demonstrated kinase (PI3K). Activation of PI3K in glioma also occurs that PIK-75 was unique among PI3K inhibitors in inducing ap- independently of upstream EGFR, through gain-of-function optosis, suggesting an off-target effect. In contrast, however, data mutations in the structural gene for PI3Kα (PIK3CA) and by loss in Fig. 1C demonstrated that the degree of apoptosis induced in of phosphatase and tensin homolog (PTEN), a lipid phosphatase response to PIK-75 varied as a function of PTEN status, sug- and negative regulator of PI3K signaling (3). gesting that apoptosis induced by PIK-75 resulted, at least in Given the prominence of signaling through EGFR and PI3K part, from PI3K inhibition. WT in glioma, small-molecule inhibitors of these pathways have To clarify this issue, we treated LN229 PTEN cells with the tremendous potential as therapeutics. To date, clinical com- PTEN inhibitor bisperoxovanadium (bpv) (10). Addition of bpv pounds directed against this axis have been tested against EGFR led to increased levels of p-Akt (Fig. 2 A and B) and partially and, to a more limited extent, against mammalian target of attenuated cell death induced by PIK-75 (Fig. 2C), without af- rapamycin (mTOR), a kinase that signals downstream of PI3K fecting induction of G2M arrest. This result was also validated in (4). Clinical responses to these agents in glioma have been dis- appointing, likely, in part, because these therapies are primarily cytostatic rather than apoptotic. In preclinical systems, whereas Author contributions: C.K.C., B.T.H., E.Z., N.S.G., S.A.O., C.D.J., K.M.S., W.A.W., and inhibitors of PI3K effectively block survival signaling by Akt, Q.-W.F. designed research; C.K.C. and Q.-W.F. performed research; W.C.G., E.C., B.T.H., inhibitors of PI3K alone and in combination with inhibitors of E.Z., A.G., N.S.G., B.P., S.A.O., and C.D.J. contributed new reagents/analytic tools; C.K.C., EGFR and of the downstream kinase mTOR failed to induce B.T.H., E.Z., A.G., N.S.G., K.M.S., W.A.W., and Q.-W.F. analyzed data; and C.K.C., W.A.W., and Q.-W.F. wrote the paper. cell death in vitro or in vivo (5, 6). The authors declare no conflict of interest. Our knowledge of the PI3K signaling pathway stems in large This article is a PNAS Direct Submission. E.C.H. is a guest editor invited by the Editorial part from the development of inhibitors that are either pan- Board. selective or that block specific isoforms within the PI3K family. 1To whom correspondence may be addressed. E-mail: [email protected] or qiwen.fan@ In screening isoform-selective inhibitors of PI3K, including ucsf.edu. chemotypes representative of most pharmaceutical drugs enter- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ing clinical trials (5, 7), we identified the imidazopyridine 1073/pnas.1202492109/-/DCSupplemental. 12722–12727 | PNAS | July 31, 2012 | vol. 109 | no. 31 www.pnas.org/cgi/doi/10.1073/pnas.1202492109 we identified 21 kinases that were inhibited over 95% in response to PIK-75 (Table S1). From this list of serine–threonine kinases, we tested the cyclin- dependent kinases 1 and 2 (CDK1 and CDK2) because these were strongly inhibited by PIK-75, had well-described roles in regulating the G2M transition, and inhibitors of these kinases are now in clinical trials (14, 15). Because G2 arrest was unique to PIK-75, blockade of CDK1 and/or CDK2 could conceivably contribute to the abilities of PIK-75 to induce apoptosis over other G1-arresting PI3K inhibitors. PIK-75 also induced p-Erk (Fig. 1B and Fig. S4 A and B). We, therefore, asked whether combination therapy using inhibitors of PI3Kα with inhibitors of CDK1 and/or CDK2 could induce apoptosis, with accompanying biomarkers demonstrating G2 arrest and induction of p-Erk. WT We treated LN229 PTEN cells with small-molecule inhibitors of CDK1 [3-(2-chloro-3-indolylmethylene)-1,3-dihydroindol-2-one] or CDK2 [4-(6-cyclohexylmethoxy-9H-purin-2-ylamino)-N,N-dieth- ylbenzamide]. Flow cytometric analysis at 24 h showed that both inhibitors induced arrest at G2M(Fig.3A), and the CDK2 inhibitor demonstrated a dose-dependent increase in p-Erk by immunoblot (Fig. 3B). Combining the CDK1 inhibitor with the PI3Kα inhibitor PIK-90 did not result in appreciable apoptosis (Fig. 3C). Combining the CDK2 inhibitor with PIK-90 did demonstrate an increase in Fig. 1. The PI3K inhibitor PIK-75 induces apoptosis and G2M arrest in WT MT WT apoptosis in comparison with monotherapy. Levels of apoptosis in PTEN and PTEN glioma cell lines. (A) PTEN cell line LN229 was treated response to combination therapy were much lower than those ob- with DMSO, LY294002 (10 μM), PIK-90 (0.5 μM), PI-103 (0.5 μM), or PIK-75 D PTENWT μ served using PIK-75 (Fig. 3 ) and were limited to LN229 , (0.5 M) for 24 h. Cells were stained with propidium iodide and analyzed by fi PTENMT flow cytometry. In comparison with PIK-90 and PI-103, PIK-75 induced arrest with no signi cant apoptosis seen in cell lines U87 and C D at G2M and apoptosis (increase in sub-G1 fraction). (B) LN229 cells were U373 (Fig. S1 and ) over monotherapy. These data suggest that treated for 6 h with agents and doses used in A and analyzed by immuno- blockade of CDK2 cooperates with inhibition of PI3K to drive blot.
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