Aidan G. Gilmartin

Antitumor activity of an allosteric inhibitor of centromere-associated protein-E

Centromere-associated protein-E (CENP-E) is a kinetochore-associated mitotic kinesin that is thought to function as the key receptor responsible for mitotic checkpoint signal transduction after interaction with spindle microtubules. We have identified GSK923295, an allosteric inhibitor of CENP-E kinesin motor ATPase activity, and mapped the inhibitor binding site to a region similar to that bound by loop-5 inhibitors of the kinesin KSP/Eg5. Unlike these KSP inhibitors, which block release of ADP and destabilize motor-microtubule interaction, GSK923295 inhibited release of inorganic phosphate and stabilized CENP-E motor domain interaction with microtubules. Inhibition of CENP-E motor activity in cultured cells and tumor xenografts caused failure of metaphase chromosome alignment and induced mitotic arrest, indicating that tight binding of CENP-E to microtubules is insufficient to satisfy the mitotic checkpoint. Consistent with genetic studies in mice suggesting that decreased CENP-E function can have a tumor-suppressive effect, inhibition of CENP-E induced tumor cell apoptosis and tumor regression.

Distinct Concentration-Dependent Effects of the Polo-like Kinase 1–Specific Inhibitor GSK461364A, Including Differential Effect on Apoptosis

Polo-like kinase 1 (Plk1) is a conserved serine/threonine kinase that plays an essential role in regulating the many processes involved in mitotic entry and progression. In humans, Plk1 is expressed primarily during late G(2) and M phases and, in conjunction with Cdk1/cyclin B1, acts as master regulatory kinases for the myriad protein substrates involved in mitosis. Plk1 overexpression is strongly associated with cancer and has been correlated with poor prognosis in a broad range of human tumor types. We have identified a potent, selective, reversible, ATP-competitive inhibitor of Plk1, GSK461364A, capable of inhibiting cell growth of most proliferating cancer cell lines tested. We observe distinct cell cycle effects of GSK461364A depending on the dose used. The predominant phenotype for cells treated with GSK461364A is prometaphase arrest with characteristic collapsed polar polo spindle. At high concentrations, GSK461364A delays mitotic entry in G(2) followed by gradual progression into terminal mitosis; in some cell lines, this correlates with decreased apoptosis. Cell culture growth inhibition by GSK461364A can be cytostatic or cytotoxic but leads to tumor regression in xenograft tumor models under proper dose scheduling. Finally, we describe pharmacodynamic biomarkers of GSK461364A activity (pHH3 and Plk1) that are currently being evaluated in human cancer clinical trials.

Comprehensive Predictive Biomarker Analysis for MEK Inhibitor GSK1120212

The MEK1 and MEK2 inhibitor GSK1120212 is currently in phase II/III clinical development. To identify predictive biomarkers, sensitivity to GSK1120212 was profiled for 218 solid tumor cell lines and 81 hematologic malignancy cell lines. For solid tumors, RAF/RAS mutation was a strong predictor of sensitivity. Among RAF/RAS mutant lines, co-occurring PIK3CA/PTEN mutations conferred a cytostatic response instead of a cytotoxic response for colon cancer cells that have the biggest representation of the comutations. Among KRAS mutant cell lines, transcriptomics analysis showed that cell lines with an expression pattern suggestive of epithelial-to-mesenchymal transition were less sensitive to GSK1120212. In addition, a proportion of cell lines from certain tissue types not known to carry frequent RAF/RAS mutations also seemed to be sensitive to GSK1120212. Among these were breast cancer cell lines, with triple negative breast cancer cell lines being more sensitive than cell lines from other breast cancer subtypes. We identified a single gene DUSP6, whose expression was associated with sensitivity to GSK1120212 and lack of expression associated with resistance irrelevant of RAF/RAS status. Among hematologic cell lines, acute myeloid leukemia and chronic myeloid leukemia cell lines were particularly sensitive. Overall, this comprehensive predictive biomarker analysis identified additional efficacy biomarkers for GSK1120212 in RAF/RAS mutant solid tumors and expanded the indication for GSK1120212 to patients who could benefit from this therapy despite the RAF/RAS wild-type status of their tumors. Mol Cancer Ther; 11(3); 720–9. ©2011 AACR.

Evidence for Allosteric Interactions of Antagonist Binding to the Smoothened Receptor

The Smoothened receptor (Smo) mediates hedgehog (Hh) signaling critical for development, cell growth, and migration, as well as stem cell maintenance. Aberrant Hh signaling pathway activation has been implicated in a variety of cancers, and small-molecule antagonists of Smo have entered human clinical trials for the treatment of cancer. Here, we report the biochemical characterization of allosteric interactions of agonists and antagonists for Smo. Binding of two radioligands, [3H]3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)-phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.3) (agonist) and [3H]cyclopamine (antagonist), was characterized using human Smo expressed in human embryonic kidney 293F membranes. We observed full displacement of [3H]cyclopamine by all Smo agonist and antagonist ligands examined. N-[(1E)-(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-piperazinamine (SANT-1), an antagonist, did not fully inhibit the binding of [3H]SAG-1.3. In a functional cell-based β-lactamase reporter gene assay, SANT-1 and N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)-benzamide (SANT-2) fully inhibited 3-chloro-4,7-difluoro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.5)-induced Hh pathway activation. Detailed “Schild-type” radioligand binding analysis with [3H]SAG-1.3 revealed that two structurally distinct Smoothened receptor antagonists, SANT-1 and SANT-2, bound in a manner consistent with that of allosteric modulation. Our mechanism of action characterization of radioligand binding to Smo combined with functional data provides a better understanding of small-molecule interactions with Smo and their influence on the Hh pathway.

Sensitivity of Cancer Cells to Plk1 Inhibitor GSK461364A Is Associated with Loss of p53 Function and Chromosome Instability

Polo-like kinases are a family of serine threonine kinases that are critical regulators of cell cycle progression and DNA damage response. Predictive biomarkers for the Plk1-selective inhibitor GSK461364A were identified by comparing the genomics and genetics of a panel of human cancer cell lines with their response to a drug washout followed by an outgrowth assay. In this assay, cell lines that have lost p53 expression or carry mutations in the TP53 gene tended to be more sensitive to GSK461364A. These more sensitive cell lines also had increased levels of chromosome instability, a characteristic associated with loss of p53 function. Further mechanistic studies showed that p53 wild-type (WT) and not mutant cells can activate a postmitotic tetraploidy checkpoint and arrest at pseudo-G1 state after GSK461364A treatment. RNA silencing of WT p53 increased the antiproliferative activity of GSK461364A. Furthermore, silencing of p53 or p21/CDKN1A weakened the tetraploidy checkpoint in cells that survived mitotic arrest and mitotic slippage. As many cancer therapies tend to be more effective in p53 WT patients, the higher sensitivity of p53-deficient tumors toward GSK461364A could potentially offer an opportunity to treat tumors that are refractory to other chemotherapies as well as early line therapy for these genotypes. Mol Cancer Ther; 9(7); 2079–89. ©2010 AACR.

Discovery of a Highly Potent and Selective MEK Inhibitor: GSK1120212 (JTP-74057 DMSO Solvate).

Inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) represents a promising strategy for the discovery of a new generation of anticancer chemotherapeutics. Our synthetic efforts, beginning from the lead compound 2, were directed at improving antiproliferative activity against cancer cells as well as various drug properties. These efforts led to the discovery of N-{3-[3-cyclopropyl-5-(2-fluoro-4-iodophenylamino)-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamide dimethylsulfoxide solvate (GSK1120212, JTP-74057 DMSO solvate; 1), a selective and highly potent MEK inhibitor with improved drug properties. We further confirmed that the antiproliferative activity correlates with cellular MEK inhibition and observed significant antitumor activity with daily oral dosing of 1 in a tumor xenograft model. These qualities led to the selection of 1 for clinical development.

WIP1 Phosphatase as a Potential Therapeutic Target in Neuroblastoma

The wild-type p53-induced phosphatase 1 (WIP1) is a serine/threonine phosphatase that negatively regulates multiple proteins involved in DNA damage response including p53, CHK2, Histone H2AX, and ATM, and it has been shown to be overexpressed or amplified in human cancers including breast and ovarian cancers. We examined WIP1 mRNA levels across multiple tumor types and found the highest levels in breast cancer, leukemia, medulloblastoma and neuroblastoma. Neuroblastoma is an exclusively TP53 wild type tumor at diagnosis and inhibition of p53 is required for tumorigenesis. Neuroblastomas in particular have previously been shown to have 17q amplification, harboring the WIP1 (PPM1D) gene and associated with poor clinical outcome. We therefore sought to determine whether inhibiting WIP1 with a selective antagonist, GSK2830371, can attenuate neuroblastoma cell growth through reactivation of p53 mediated tumor suppression. Neuroblastoma cell lines with wild-type TP53 alleles were highly sensitive to GSK2830371 treatment, while cell lines with mutant TP53 were resistant to GSK2830371. The majority of tested neuroblastoma cell lines with copy number gains of the PPM1D locus were also TP53 wild-type and sensitive to GSK2830371A; in contrast cell lines with no copy gain of PPM1D were mixed in their sensitivity to WIP1 inhibition, with the primary determinant being TP53 mutational status. Since WIP1 is involved in the cellular response to DNA damage and drugs used in neuroblastoma treatment induce apoptosis through DNA damage, we sought to determine whether GSK2830371 could act synergistically with standard of care chemotherapeutics. Treatment of wild-type TP53 neuroblastoma cell lines with both GSK2830371 and either doxorubicin or carboplatin resulted in enhanced cell death, mediated through caspase 3/7 induction, as compared to either agent alone. Our data suggests that WIP1 inhibition represents a novel therapeutic approach to neuroblastoma that could be integrated with current chemotherapeutic approaches.

Abstract 961: GSK1120212 inhibits both MEK kinase activity and activation

Negative feedback loops are commonplace in signal transduction pathways, restoring homeostatic equilibrium. Inhibitory drugs to these pathways, by suppressing the negative feedback, can result in hyperactivation of upstream pathway components. MEK inhibitors have been reported to suppress ERK1/2 mediated activation of DUSPs and Sprouty, resulting in increased phosphorylation and activation of both Raf and Mek. This feedback response may negatively impact an inhibitor9s efficacy both by increasing pathway activation and by weakening the compound binding site. Here we report on studies characterizing the effect of the MEK1/2 inhibitor GSK1120212 in preventing activation of MEK by Raf kinases, a complementary effect to its inhibition of MEK kinase activity. We conducted in vitro MEK activation assays with Raf1 or BRAF and U-MEK in the presence or absence of GSK1120212, and analyzed MEK phosphorylation by ftMSMS. We observed that GSK1120212 completely prevented Raf-dependent phosphorylation of S217 on MEK1, resulting in mono-phosphorylated (S221) MEK1 (p-MEK1). We then showed that although p-MEK1(pS221) is more active than U-MEK, it is 83-fold less active than the fully-activated diphospho-MEK1 (pp-MEK1). Our inhibition study indicated that the affinity of GSK1120212 for MEK1 was reduced by S217 phosphorylation since the IC50 for pp-MEK1 was 15.3 nM, but We then assessed whether the effect of GSK1120212 on MEK1 activating phosphorylation occurs in cells. In Sk-MEL-28, A375P, and HCT116 cells, treatment with GSK1120212 results in a transient decrease in MEK phosphorylation detected by immunoblotting; however the levels increase over time for both HCT116 and A375P. Since the phospho-MEK antibodies may not distinguish the mono from diphospho MEK, we immunoprecipitated MEK1 from lysates and analyzed MEK phosphorylation by MS. As with the in vitro reaction, GSK1120212 prevented S217 phosphorylation, but not phosphorylation of S221. As suggested by immunblotting, S221 phosphorylation increased over time, presumably reflecting the block of negative feedback mechanisms. Since GSK1120212 does not directly inhibit either RAF1 or BRAF kinase activity, we believe that GSK1120212 binds to MEK in a way that specifically blocks the accessibility of S217 to Raf kinases. This is further supported by the observation that once S217 is phosphorylated, as in pp-MEK, the affinity for GSK1120212 is reduced. S217 phosphorylation likely alters the adjacent activation loop that partially defines the compound binding site. These results suggest that in Ras and Raf mutant tumors, GSK1120212 may suppress both MEK kinase activity and partially abrogate the activating effects due to negative feedback. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 961. doi:10.1158/1538-7445.AM2011-961

Abstract C74: Reassessing IKKε as a novel oncology target

Recent years have seen increasing efforts to employ integrative genomic approaches to identify novel oncogenes. In this manner, IKBKE coding for IKKe was recently proposed as a novel oncogene, aberrantly activating the NF‐κB signaling pathway in breast cancers (Boehm et al. Cell. 2007). We pursued a series of experiments that delve more deeply into the hypothesis of IKKe as a novel oncology target; the resulting evidence challenges the proposed oncogenic mechanism. We conducted RNA interference studies on mutiple breast cancer cell lines with multiple IKBKE siRNA motifs to define a general on‐target phenotype. Consistent with published findings, we confirmed that although 4 IKBKE siRNA motifs tested significantly reduced IKKe protein expression (78–90% silencing) only 2 of these motifs significantly inhibited cell growth. We then engineered ‘non‐silenceable’ IKBKE constructs packaged in BacMam virus and were able to simultaneously silence the endogenous IKBKE and exogenously express the non‐silenceable IKKe at protein levels below, equivalent to, and significantly above the endogenous levels. We demonstrated that the antiproliferative effects of these 2 siRNAs could not be rescued by functional, exogenously expressed IKBKE, indicating likely off‐target toxicity. We further explored the correlations between IKBKE gene amplification, IKKe protein overexpression, and NF‐κB pathway activation using RNAi in conjunction with an NF‐κB ‐luciferase reporter assay. We observed that IKBKE amplification correlated poorly with both IKKe protein expression and NF‐κB pathway activation. Furthermore, silencing downstream components of the NF‐κB signaling pathway had little impact on the IKBKE‐amplified cells; and conversely, silencing IKBKE had no impact on NF‐κB ‐reporter activity in the IKBKE‐amplified cells. In addition, we over‐expressed the kinase‐dead IKKe (K38A), and observed no effect on tumor cell growth. Finally, using BacMam virus to serially titrate IKK , we confirmed that the level of exogenous IKKe needed to achieve a modest pathway activation (2‐fold increase in NF‐κB ‐luciferase reporter assay) was >10 times the endogenous protein level found in an IKBKE‐amplified breast cancer line. We also undertook a limited medicinal chemistry effort to arrive at several potent small molecule tool inhibitors of both IKK and TBK1 including GSK2292978A (8nM & 1nM respectively). GSK2292978A confirmably inhibited IKKe activity in a cellular mechanistic assay (IC50∼170nM). Using a 3‐day proliferation assay, we observed no evident selectivity of GSK2292978A for IKBKE‐amplified or IKKe overexpressing cell lines compared to non‐amplified cell lines. These findings resulted in our conclusion that IKKe likely does not represent a true oncogene in breast cancer and suggest the vital need for well‐designed controls in cases where RNA interference significantly defines target validation. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C74.