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This review describes our recent efforts in the development of novel therapies for cancer. Our primary approach is to design synthetic agents that antagonize the function of growth factors that are critically involved in oncogenesis and angiogenesis. We achieve this by designing synthetic molecules that can recognize the exterior surface of the growth factor and so block the interaction with its receptor tyrosine kinase. A key step is the construction of synthetic agents that contain a large (> 400A2) and functionalized surface area to recognize a complementary surface on the target growth factor. In the course of this work we have discovered a molecule, GFB-111, that binds to PDGF, prevents it from binding to its receptor tyrosine kinase, blocks PDGF-induced receptor autophosphorylation, activation of Erk1 and Erk2 kinases and DNA synthesis. The binding affinity for PDGF is high (IC50=250 nM) and selective over EGF, IGF-1, aFGF, bFGF and HRGbeta. In nude mouse models GFB-111 also shows significant inhibition of tumor growth and angiogenesis.

In 1990, more than 10 years after the discovery that the low molecular weight GTPase Ras is a major contributor to human cancer, farnesylation, a lipid posttranslational modification required for the cancer-causing activity of Ras, emerged as a major target for the development of novel anticancer agents. However, it took only 5 years from 1993, when the first farnesyltransferase inhibitors (FTIs) were reported, to 1998 when results from the first phase I clinical trials were described. This rapid progress was due to the demonstration of outstanding antitumor activity and lack of toxicity of FTIs in preclinical models. Although, many FTIs are currently in phase H and at least one is in phase III clinical trial, the mechanism of FTI antitumor activity is not known. In this review a brief summary of the development of FTIs as antitumor agents will be given. The focus of the review will be on important mechanistic and bench-to-bedside translational issues. Among the issues that will be addressed are: evidence for and against inhibition of the prenylation of Ras and RhoB proteins in the mechanism of action of FTIs; implications of the alternative prenylation of K-Ras by geranylgeranyl-transferase I (when FTase is inhibited) in cancer therapy; GGTase I inhibitors (GGTIs) as antitumor agents; effects of FTIs and GGTIs on cell cycle machinery and progression and potential mechanisms by which FTIs and GGTIs induce apoptosis in human cancer cells. A thorough discussion about bench-to-bedside issues relating to hypothesis-driven clinical trials with proof-of-principle in man will also be included. This section will cover issues relating to whether the biochemical target (FTase) is inhibited and the level of inhibition of FTase required for clinical response; are signaling pathways such as H-Ras/PI3K/Akt and/or K-Ras/Raf/MEK/Erk relevant biological readouts?; is Ras (particularly N-Ras and H-Ras) mutation status a good predictor of clinical response?; in phase I trials should effective biological dose, not maximally tolerated dose, be used to determine phase II dose?; and finally, in phase II/III trials what are the most appropriate clinical end points for anti-signaling molecules such as FTIs? Parts of this topic have been recently reviewed (Sebti and Hamilton, 2000c).

While both nitric oxide synthase-2 (NOS-2) and low molecular weight GTPases, such as Ras and Rho, have been implicated in malignant transformation, the cross talk between these important proteins is ill understood. In this study we examined the ability of H-Ras, RhoA, RhoB and Rac1 to modulate cytokine-induced NOS2. In the normal human liver AKN-1 cell line and in the human non-small cell lung carcinoma cell line, A-549, the ability of the cytokines (INF-gamma, IL-1beta and TNF-alpha) to activate NOS-2 was blocked by activated L61-H-Ras whereas dominant negative N17-H-Ras enhanced NOS-2 activation. Consistent with this dominant negative Erk2 as well as a MEK inhibitor also enhanced cytokine activation of NOS-2. Furthermore, activated L63-RhoA blocked whereas activated V14-RhoB enhanced cytokine NOS-2 activation. Activated I115-Racl did not affect NOS-2 activation. These results demonstrate that the Ras/Erk and the Ras/RhoA pathways negatively regulate whereas RhoB enhances cytokine-induced NOS-2. This is the first demonstration that genes that promote malignant transformation such as Ras and RhoA inhibit, whereas genes with tumor suppressor activity such as RhoB enhance NOS2 induction.

Several small GTPases of the Ras superfamily have been shown to antagonize TGFbeta signaling in human tumor cell lines. Some of these GTPases are post-translationally modified by farnesylation, a lipid modification catalyzed by farnesyltransferase and required for the proteins to attach to membranes and to function. In this study, we investigated the effect of the farnesyltransferase inhibitor FTI-277 on TGFbeta-regulated cell growth and transcription. Treatment of the human pancreatic tumor cell line, Panc-1, with FTI-277 enhanced the ability of TGFbeta to inhibit both anchorage-dependent and -independent tumor cell growth. FTI-277 also enhanced the ability of TGFbeta to induce transcription, as measured by p3TP-lux reporter activity and collagen synthesis. The enhancement of TGFbeta responses by FTI-277 correlated with the stimulation of transcription and protein expression of type II TGFbeta receptor (TbetaRII). Consequently, FTI-277-treated cells exhibited a higher level of TGFbeta binding to its receptor. Thus, inhibition of protein farnesylation stimulates TbetaRII expression, which leads to increased TGFbeta receptor binding and signaling as well as inhibition of tumor cell growth and transformation.

The fact that proteins such as Ras, Rac and RhoA require farnesylation or geranylgeranylation to induce malignant transformation prompted many investigators to develop farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) inhibitors (FTIs and GGTIs, respectively) as novel anticancer drugs. Although FTIs have been shown to antagonise oncogenic signalling, reverse malignant transformation, inhibit human tumour growth in nude mice and induce tumour regression in transgenic mice without any signs of toxicity, their mechanism of action is not known. This review will focus on important mechanistic issues as well as bench to bedside translational issues. These will include the relevance to cancer therapy of the alternative geranylgeranylation of K-Ras when FTase is inhibited; a thorough discussion about evidence for and against the involvement of inhibition of prenylation of Ras and RhoB in the mechanism of FTIs' antitumour activity as well as effects of FTIs and GGTIs on the cell cycle machinery and the dynamics of bipolar spindle formation and chromosome alignment during mitosis. Bench to bedside issues relating to the design of hypothesis-driven clinical trials with biochemical correlates for proof-of-concept in man will also be discussed. This will include Phase I issues such as determining maximally tolerated dose (MTD) versus effective biological dose (EBD), as well as whether Phase II trials are still needed for clinical evaluations of anti-signalling agents. Other questions that will be addressed include: what levels of inhibition of FTase activity are required for tumour response in Phase II clinical evaluations? What FTase substrates are most relevant as biochemical correlates? Are signalling pathways such as H-Ras/PI3K/Akt and K-Ras/Raf/MEK/Erk significant biological readouts? Does Ras mutation status predict response? What are appropriate clinical end-points for FTI Phase II trials? For this latter important question, time to tumour progression, median survival, percentage of patients that progress, clinical benefits and improvement in quality of life will all be discussed.

Colony-stimulating factor-1 (CSF-1) induces expression of immediate early gene, such as c-myc and c-fos and delayed early genes such as D-type cyclins (D1 and D2), whose products play essential roles in the G1 to S phase transition of the cell cycle. Little is known, however, about the cytoplasmic signal transduction pathways that connect the surface CSF-1 receptor to these genes in the nucleus. We have investigated the signaling mechanism of CSF-1-induced D2 expression. Analyses of CSF-1 receptor autophosphorylation mutants show that, although certain individual mutation has a partial inhibitory effect, only multiple combined mutations completely block induction of D2 in response to CSF-1. We report that at least three parallel pathways, the Src pathway, the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, and the c-myc pathway, are involved. Induction of D2 is partially inhibited in Src−/− bone marrow-derived macrophages and by Src inhibitor PP1 and is enhanced in v-Src-overexpressing cells. Activation of myc's transactivating activity selectively induces D2 but not D1. Blockade of c-myc expression partially blocks CSF-1-induced D2 expression. Complete inhibition of the MEK/ERK pathway causes 50% decrease of D2 expression. Finally, simultaneous inhibition of Src, MEK activation, and c-myc expression additively blocks CSF-1-induced D2 expression. This study indicates that multiple signaling pathways are involved in full induction of a single gene, and this finding may also apply broadly to other growth factor-inducible genes.

The importance of post-translational geranylgeranylation of the GTPase RhoA for its ability to induce cellular proliferation and malignant transformation is not well understood. In this manuscript we demonstrate that geranylgeranylation is required for the proper cellular localization of V14RhoA and for its ability to induce actin stress fiber and focal adhesion formation. Furthermore, V14RhoA geranylgeranylation was also required for suppressing p21(WAF) transcription, promoting cell cycle progression and cellular proliferation. The ability of V14RhoA to induce focus formation and enhance plating efficiency and oncogenic Ras anchorage-dependent growth was also dependent on its geranylgeranylation. The only biological activity of V14RhoA that was not dependent on its prenylation was its ability to induce serum response element transcriptional activity. Furthermore, we demonstrate that a farnesylated form of V14RhoA was also able to bind RhoGDI-1, was able to induce cytoskeleton organization, proliferation, and transformation, and was just as potent as geranylgeranylated V14RhoA at suppressing p21(WAF) transcriptional activity. These results demonstrate that RhoA geranylgeranylation is required for its biological activity and that the nature of the lipid modification is not critical.

The cytokine transforming growth factor-beta (TGF-beta) has multiple effects on a variety of cell types, modulating cell growth and differentiation as well as extracellular matrix deposition and degradation. In the present work, we demonstrate that TGF-beta1 produces a fourfold increase in transcription of the fibronectin gene in cultured human fetal lung fibroblasts with only a small increase in mRNA stability resulting in a significant increase in fibronectin mRNA steady state level. A corresponding increase in production of fibronectin protein accompanied the increase in mRNA. Through the use of specific inhibitors, we demonstrate that geranylgeranylated, but not farnesylated or acylated protein(s), protein kinase C-delta, phosphatidylcholine-specific phospholipse C, tyrosine kinase activity, and stress-activated protein kinase p38 are required for this TGF-beta1 effect. Trimeric G proteins and mitogen-activated protein kinases erk1 and erk2 do not appear to be involved. While these results emphasize the complexities involved in the control of extracellular matrix synthesis by TGF-beta, they also identify reaction sites that may be amenable to pharmacologic modulation. Such modulation could be of great advantage in the treatment of a wide variety of undesirable fibrotic reactions.

We recently showed that the farnesyltransferase inhibitor FTI-277 blocks interleukin 1β (IL-1β)-induced nitric oxide production in pulmonary vascular smooth muscle cells (SMC), whereas the geranylgeranyltransferase inhibitor GGTI-298 enhances this effect. Here we show that IL-1β and platelet-derived growth factor (PDGF) stimulate superoxide production by pulmonary vascular SMC and that this effect is blocked by both FTI-277 and GGTI-298, suggesting that farnesylated and geranylgeranylated proteins are required for superoxide production. We also show that FTI-277 and GGTI-298 block superoxide production stimulated by constitutively active mutant H-Ras. Furthermore, superoxide production by IL-1β, PDGF factor, and constitutively activated Ras is blocked by diphenyleneiodonium, implicating NAD(P)H oxidase as the generating enzyme. Given the role of oxidant radicals in vascular reactivity and injury, the action of both FTI-277 and GGTI-298 in suppressing superoxide generation by an inflammatory cytokine as well as by a potent smooth muscle mitogen may be therapeutically useful.

Farnesyltransferase inhibitors (FTIs) represent a novel class of anticancer drugs that exhibit a remarkable ability to inhibit malignant transformation without toxicity to normal cells. However, the mechanism by which FTIs inhibit tumor growth is not well understood. Here, we demonstrate that FTI-277 inhibits phosphatidylinositol 3-OH kinase (PI 3-kinase)/AKT2-mediated growth factor- and adhesion-dependent survival pathways and induces apoptosis in human cancer cells that overexpress AKT2. Furthermore, overexpression of AKT2, but not oncogenic H-Ras, sensitizes NIH 3T3 cells to FTI-277, and a high serum level prevents FTI-277-induced apoptosis in H-Ras- but not AKT2-transformed NIH 3T3 cells. A constitutively active form of AKT2 rescues human cancer cells from FTI-277-induced apoptosis. FTI-277 inhibits insulin-like growth factor 1-induced PI 3-kinase and AKT2 activation and subsequent phosphorylation of the proapoptotic protein BAD. Integrin-dependent activation of AKT2 is also blocked by FTI-277. Thus, a mechanism for FTI inhibition of human tumor growth is by inducing apoptosis through inhibition of PI 3-kinase/AKT2-mediated cell survival and adhesion pathway.

Publisher Summary This chapter discusses the inhibition of Rho guanosine 5'-triphosphates (GTPases) using protein geranylgeranyltransferase I inhibitors. Extracellular signals that induce cells to divide, differentiate, or die trigger a complex network of cellular signaling pathways. Most often this process relies on the transfer of biological information from cell surface receptors to the nucleus, and GTPases belonging to the Ras superfamily play a pivotal role in this transfer. One approach that has been taken to interfere with oncogenic signaling of the Ras superfamily of proteins is to design compounds that block their prenylation—a lipid posttranslational modification that is required for the transforming activity of the GTPases. The chapter also discusses the activity assays of protein geranylgeranyltransferase I (PGGTase I) and protein farnesyltransferase (PFTase) inhibitors.

The Ras protein undergoes a series of post-translational modifications at the C-terminal CAAX motif, which culminates with the anchoring of p21 Ras to the plasma membrane where it relays growth regulatory signals from receptor tyrosine kinases to various pathways of cell signal transduction. FTI-276 is a CAAX peptidomimetic of the carboxyl terminal of Ras proteins. Pharmacokinetic analysis of FTI-276 in A/J mice with a time-release pellet system showed a dose of 50 mg/kg body wt achieved an average serum level of 1.68 microg/ml for up to 30 days following implantation. In the present study, 4 week old A/J mice were initiated with a single dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (100 mg/kg), and monitored for 18 weeks. Mice were grouped for daily delivery (time-release pellet) of 50 mg/kg of FTI-276 for 30 days (n = 12) and the control group (n = 12). Analysis of tumors from time-release pellet treated animals showed a 60% reduction in tumor multiplicity and a 42% reduction in tumor incidence. Moreover, FTI-276 treatment resulted in a significant reduction in tumor volume (approximately 58%). Mutation analysis of the lung tumors from both treatment groups revealed that most of the tumors harbored mutations in the codon 12 of K-ras and there is no significant difference in the incidence and types of mutations between tumors from the treated and control animals. This is the first demonstration of chemotherapeutic efficacy of a synthetic CAAX peptidomimetic farnesyltransferase inhibitor in a primary lung tumor model.

Integrin-dependent leukocyte adhesion is modulated by alterations in receptor affinity or by post-receptor events. Pretreatment of Jurkat T-cells with the 3-hydroxymethylglutaryl-coenzyme A reductase inhibitor, lovastatin, markedly reduced (IC(50) approximately 1-2 microM) alpha(4)beta(1)-dependent adhesion to fibronectin (FN) stimulated by phorbol 12-myristate 13-acetate (PMA) which modulates post-receptor events. In contrast, lovastatin did not inhibit Jurkat cell adhesion to FN induced by the beta(1) integrin-activating monoclonal antibody (mAb) 8A2, which directly modulates beta(1) integrin affinity. Similarly, pretreatment of U937 cells with lovastatin inhibited PMA-stimulated, but not mAb 8A2-stimulated, alpha(6)beta(1)-dependent leukocyte adhesion to laminin. The inhibition of lovastatin on PMA-stimulated leukocyte adhesion was not mediated by mitogen-activated protein kinase or phosphatidylinositol 3-kinase pathway. The inhibitory effect of lovastatin on PMA-stimulated leukocyte adhesion was reversed by co-incubation with geranylgeraniol, but not with farnesol, with concurrent reversal of the inhibition of protein prenylation as shown by protein RhoA geranylgeranylation. The selective inhibition of protein geranylgeranylation by the specific protein geranylgeranyltransferase-I inhibitor, GGTI-298, blocked PMA-stimulated leukocyte adhesion but not mAb 8A2-induced leukocyte adhesion. The protein farnesyltransferase inhibitor, FTI-277, had no effect on leukocyte adhesion induced by either stimulus. These results demonstrate that protein geranylgeranylation, but not farnesylation, is required for integrin-dependent post-receptor events in leukocyte adhesion.

To test the Kirsten-Ras (Ki-Ras) alternative prenylation hypothesis in malignant transformation, we used a novel farnesyltransferase inhibitor competitive to farnesyl-pyrophosphate, RPR130401, and a CaaX peptidomimetic geranylgeranyltransferase-1 inhibitor GGTI-298. In Ki-Ras-overexpressing transformed adrenocortical cells, RPR130401 at 1-10 microM inhibited very efficiently the [(3)H]farnesyl but not [(3)H]geranylgeranyl transfer to Ras. However, proliferation of these cells was only slightly sensitive to RPR130401 (IC(50)=30 microM). GGTI-298 inhibited the growth of these cells with an IC(50) of 11 microM but cell lysis was observed at 15 microM. The combination of 10 microM RPR130401 and 10 microM GGTI-298 inhibited efficiently (80%) cell proliferation. These combined inhibitors but not each inhibitor alone blocked the cell cycle in G(0)/G(1) and disrupted MAP kinase activation. Thus, combination of two inhibitors, at non-cytotoxic concentrations, acting on the farnesyl-pyrophosphate binding site of the farnesyltransferase and the CaaX binding site of the geranylgeranyltransferase-1 respectively is an efficient strategy for disrupting Ki-Ras tumorigenic cell proliferation.

In this paper, we describe the effect of the inhibitor of farnesyltransferase (FTI-277) on radioresistance induced by the 24-kDa isoform of FGF2 in human cells expressing wild-type RAS. Treatment with FTI-277 (20 μM) for 48 h prior to irradiation led to a significant decrease in survival of radioresistant cells expressing the 24-kDa isoform (HeLa 3A) but had no effect on the survival of control cells (HeLa PINA). The radiosensitizing effect of FTI-277 is accompanied by a stimulation of postmitotic cell death in HeLa 3A cells and by a reduction in ${\rm G}_{2}/{\rm M}\text{-phase}$ arrest in both cell types. These results clearly demonstrate that at least one farnesylated protein is involved in the regulation of the radioresistance induced by the 24-kDa isoform of FGF2. Furthermore, the radiation-induced ${\rm G}_{2}/{\rm M}\text{-phase}$ arrest is also under the control of farnesylated protein. This work also demonstrates that FTase inhibitors may be effective radiosensitizers of certain human tumors with...

We have recently reported that the geranylgeranyltransferase I inhibitor GGTI-298 arrests human tumor cells at the G1 phase of the cell cycle and increases the protein and RNA levels of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). Here, we show that GGTI-298 acts at the transcriptional level to induce p21(WAF1/CIP1) in a human pancreatic carcinoma cell line, Panc-1. This upregulation of p21(WAF1/CIP1) promoter was selective, since GGTI-298 inhibited serum responsive element- and E2F-mediated transcription. A functional analysis of the p21(WAF1/CIP1) promoter showed that a GC-rich region located between positions -83 and -74, which contains a transforming growth factor beta-responsive element and one Sp1-binding site, is sufficient for the upregulation of p21(WAF1/CIP1) promoter by GGTI-298. Electrophoretic mobility shift assays showed a small increase in the amount of DNA-bound Sp1-Sp3 complexes. Furthermore, the analysis of Sp1 transcriptional activity in GGTI-298-treated cells by using GAL4-Sp1 chimera or Sp1-chloramphenicol acetyltransferase reporter revealed a significant increase in Sp1-mediated transcription. Moreover, GGTI-298 treatment also resulted in increased Sp1 and Sp3 phosphorylation. These results suggest that GGTI-298-mediated upregulation of p21(WAF1/CIP1) involves both an increase in the amount of DNA-bound Sp1-Sp3 and enhancement of Sp1 transcriptional activity. To identify the geranylgeranylated protein(s) involved in p21(WAF1/CIP1) transcriptional activation, we analyzed the effects of the small GTPases Rac1 and RhoA on p21(WAF1/CIP1) promoter activity. The dominant negative mutant of RhoA, but not Rac1, was able to activate p21(WAF1/CIP1). In contrast, constitutively active RhoA repressed p21(WAF1/CIP1). Accordingly, the ADP-ribosyl transferase C3, which specifically inhibits Rho proteins, enhanced the activity of p21(WAF1/CIP1). Taken together, these results suggest that one mechanism by which GGTI-298 upregulates p21(WAF1/CIP1) transcription is by preventing the small GTPase RhoA from repressing p21(WAF1/CIP1) induction.

OBJECTIVE Preliminary studies have demonstrated that the Ras family and related guanosine 5'-triphosphate-dependent proteins (G-proteins) are overactivated in malignant gliomas and may function as indirect mediators of glial transformation initiated by deregulated upstream signaling elements. We postulated that inhibiting the activation of such proteins might represent a promising strategy for blocking the aberrant proliferation of these tumors. METHODS AND RESULTS Accordingly, we examined the therapeutic efficacy against malignant glioma cells in vitro of a series of selective peptidomimetic inhibitors of farnesylation (FTI-277) and geranylgeranylation (GGTI-286 and GGTI-298), which are critical steps in the post-translational processing (prenylation) of these proteins. We first defined concentration-response relationships for each of these agents, using MTS-based cell proliferation assays in the established malignant glioma cell lines U-87 and LN-Z308 and the low-passage malignant glioma cell line SG-388. FTI-277, GGTI-286, and GGTI-298 each produced a striking concentration-dependent antiproliferative effect on the glioma cell lines, with the median effective dose ranging from 2.5 to 15.5 micromol/L. We then assessed the effect of prenylation inhibition on cell viability using clonogenic growth assays. This demonstrated a steady drop in the number of colonies with increasing drug concentrations for all three inhibitors. Third, we examined whether the cytotoxic effects of one of these inhibitors (GGTI-298) were associated with the induction of apoptosis using a terminal transferase-catalyzed in situ end-labeling technique. This approach showed a time-dependent increase in apoptotic cell numbers, which correlated with a progressive decrease in the percentage of cells that were viable as assessed by trypan blue exclusion. CONCLUSION Our studies demonstrated that FTI-277, GGTI-286, and GGTI-298 each yielded significant antiproliferative effects in human malignant glioma cells in vitro at low micromolar concentrations, which have been achievable in vivo without major systemic toxicity. Extended periods of drug treatment produced cytotoxicity in the tumor cells, which correlated with the induction of apoptosis. We conclude that inhibition of Ras and related G-proteins offers a promising approach for blocking glioma proliferation that justifies further investigation in vivo.

The ability of Ras oncoproteins to cause malignant transformation requires their post-translational modifications by prenyl groups. Because K-Ras can be both farnesylated and geranylgeranylated it is not known whether both farnesyltransferase and geranylgeranyltransferase I inhibitors are required for suppressing human tumor growth in whole animals. In this paper we report that oncogenic Ras processing, MAP kinase activation and growth in nude mice are inhibited by the farnesyltransferase inhibitor FTI-276 in H- and N-Ras transformed NIH3T3 cells; whereas in KB-Ras transformed NIH3T3 cells both FTI-276 and the geranylgeranyltransferase I inhibitor GGTI-297 are required for inhibition. Furthermore, human lung A-549 and Calu-1 carcinoma cell lines were found to co-express H-, N- and K-Ras. In Calu-1 cells, the processing of H- and N-Ras is inhibited greatly by FTI-276 but only partially by GGTI-297 whereas K-Ras processing inhibition requires both FTI-276 and GGTI-297. In contrast, in A-549 cells the processing of H- and N-Ras is inhibited only by FTI-276 and K-Ras processing is resistant to co-treatment with FTI-276 and GGTI-297. Yet, the growth in nude mice of A-549 and Calu-1 xenografts, both of which express K-Ras mutations, is inhibited by FTI-276 (80% inhibition) and GGTI-297 (60%). Furthermore, FTI-276 inhibits tumor growth of NIH3T3 cells transformed by a form of oncogenic H-Ras that is exclusively geranylgeranylated and whose processing is resistant to this inhibitor. Taken together, the results demonstrate that both FTase and GGTase I inhibitors are required for inhibition of K-Ras processing but that each alone is sufficient to suppress human tumor growth in nude mice.

Oncogenic H-Ras requires farnesylation for its transforming activity. Lovastatin inhibits both protein farnesylation and geranylgeranylation by decreasing cellular pools of farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), respectively. Use of lovastatin as a chemotherapeutic agent has been precluded by its significant cytotoxic effects. In this report, we describe a novel approach utilizing a combination of lovastatin and geranylgeraniol (GGOH) to potentiate the ability of lovastatin to block oncogenic H-Ras signaling and concomitantly rescue lovastatin toxicity. GGOH co-treatment with lovastatin enhances inhibition of oncogenic H-Ras processing and constitutive activation of mitogen-activated protein kinase (MAPK), and preserves the processing of geranylgeranyltransferase (GGTase) I and GGTase II protein substrates. Moreover, co-treatment with GGOH significantly (15-fold) attenuates the cytotoxic effects of lovastatin as well as prevents lovastatin-induced cell rounding. These results demonstrate that GGOH potentiates the anti-oncogenic/anti-signaling activity of lovastatin while antagonizing its cytotoxicity. These opposing effects are due to a GGOH metabolite that serves simultaneously as a potent inhibitor for farneslyltransferase as well as a substrate for GGTases I and II.

The regulation of the tumor suppressor p53 by Ral GTPases and its contribution to malignant transformation are not known. Here we demonstrate that the expression of K-Ras, Ral A and Ral B, but not Akt1/2 and c-Raf, is required for maintaining low levels of p53 in human cancer cells that harbor mutant K-Ras and wild type p53. Depletion of Ral A and Ral B increases p53 protein levels by increasing its stability and this results in p53-dependent up regulation of p21waf and mdm2. Furthermore, depletion of Ral A and / or Ral B leads to a p53-dependent decrease in the proportion of cells in the S-phase of the cell cycle, inhibition of anchorage-independent growth and suppression of invasion. Thus, expression of Ral proteins is critical to maintaining low levels of p53, and this appears to be critical to the ability of Ral GTPase to induce malignant transformation. Citation Format: Awet Tecleab, Xiaolei Zhang, Said M. Sebti. RalGTPase regulation of p53 and its contribution to malignant transformation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 767. doi:10.1158/1538-7445.AM2013-767

Abstract: Lysophosphatidic acid acyltransferase-beta (LPAAT-β) catalyzes the production of phosphatidic acid (PA) from lysophosphatidic acid (LPA). PA is a lipid cofactor that contributes to the activation of c-Raf, BRAF, mTOR and PKC-ζ. LPAAT-β expression is a prognostic factor in gynecologic malignancies and is being investigated as a therapeutic target in a variety of tumor types. We previously reported that knockdown of LPAAT-β by siRNA led to a significant inhibition of both anchorage-dependent and -independent growth in pancreatic cancer cell lines. Having thus established LPAAT-β as a valid therapeutic target in pancreatic cancer, we screened small molecule libraries for compounds which block LPAAT-β enzymatic activity in vitro. Methods: We utilized a 96-well plate screening assay for LPAAT-β enzymatic activity modified from Aguado and Campbell, J Biol Chem 273(7):4096-4105, 1998. Briefly, compounds were introduced into a mixture containing full-length, recombinant, baculovirus-produced LPAAT-β (Blue Sky Biotechnology), oleoyl-LPA, and oleoyl coenzyme A. The ability of the compound to inhibit the enzyme activity was assayed by the measurement of the reaction of the thiol group of the released coenzyme A with the DTNB, resulting in an increase in the optical density of the sample when read at an absorbance of 413 nm. To rule out non-specific compounds identified from the DTNB plate assay, we confirmed activity using a secondary thin layer chromatography (TLC) assay that directly measures the ability of LPAAT-β to catalyze the synthesis of PA from oleoyl-LPA and a fluorochrome-labeled (NBD) palmitoyl coenzyme A. Results: We screened a total of ten libraries containing 11204 compounds, and achieved a 1.2% hit rate for inhibition of >50% at the tested concentration (100μM for nine libraries; 10μM for one library). Further testing identified several compounds with IC50s ranging from 0.1-15 μM. One hit identified with an IC50 of 100 nM was chosen for further investigation and for synthesis of analogs to optimize potency and undertake SAR. We verified the activity of this hit and its analogs against LPAAT-β by determining their inhibitory activity of the synthesis of NBD-labeled PA in a TLC assay. Finally, we demonstrated that the best analog from this group inhibited the proliferation of several pancreatic cancer cell lines. Conclusions: We have developed a library of compounds which show inhibitory activity against LPAAT-β in a screening assay, and have confirmed their ability to inhibit LPAAT-β directly in a TLC assay which monitors the production of PA. These compounds show low micromolar activity against LPAAT-β in vitro, and micromolar activity against the proliferation of pancreatic cancer cell lines in culture. Citation Format: Michelle A. Blaskovich, Harsukh Gevariya, Nicholas J. Lawrence, Saïd Sebti, Gregory Springett. Identification and evaluation of small molecule inhibitors of lysophosphatidic acid acyltransferase-beta. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2470. doi:10.1158/1538-7445.AM2013-2470

The ability of STAT3 to translocate to the nucleus, bind DNA and transcriptionally regulate STAT3 target genes depends on tyrosine phosphorylation STAT3-STAT3 dimerization. Our chemistry efforts led to the discovery of S3I-1757 that disrupts STAT3-STAT3 dimerization. Fluorescence polarization assays and molecular modeling show that S3I-1757 interacts with Y-705 binding site in the STAT3-SH2 domain and displaces fluorescein-labelled GpYLPQTV phosphotyrosine peptide from binding to STAT3. Furthermore, using co-immunoprecipitation and co-localization studies we show that S3I-1757 inhibits STAT3 dimerization and STAT3-EGF receptor binding in intact cells. Treatment of human cancer cells with S3I-1757 (but not a closely related analogue, S3I-1756, that does not inhibit STAT3 dimerization), inhibits selectively the phosphorylation of STAT3 over Akt and Erk1/2, nuclear accumulation of P-Y705-STAT3, STAT3-DNA binding and transcriptional activation and suppresses the expression levels of STAT3 target genes such as Bcl-xL, survivin, cyclin D1 and MMP9. Furthermore, S3I-1757 but not S3I-1756 inhibits anchorage-dependent and -independent growth, migration and invasion of human cancer cells which depend on STAT3 selectively over those that do not. Finally, STAT3-C, a genetically engineered mutant of STAT3 that forms a constitutively dimerized STAT3, rescues from S3I-1757 effects. Our results warrant further development of S3I-1757 as a STAT3-STAT3 dimerization inhibitor capable of blocking hyper activated STAT3 and suppressing malignant transformation of STAT3-dependent human tumors. Citation Format: Xiaolei Zhang, Ying Sun, Roberta Pireddu, Hua Yang, Murali K. Urlam, Harshani R. Lawrence, Wayne C. Guida, Nicholas J. Lawrence, Said M. Sebti. Discovery of S3I-1757, a STAT3-STAT3 dimerization disruptor that inhibits STAT3 activity, tumor survival, migration and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3254. doi:10.1158/1538-7445.AM2013-3254

Background: Pancreatic cancer, an aggressive and lethal cancer is the fourth leading cause of cancer-related deaths in the United States. Gemcitabine is currently the standard therapeutic agent for advanced pancreatic cancer, but has limited efficacy due to chemoresistance and dose escalation toxicity. We have shown that natural vitamin E δ-tocotrienol is the most bioactive tocotrienols against pancreatic cancer in vitro using pancreatic cancer cells lines as well as in vivo using xenograft models. In this study we evaluated the combination of δ-tocotrienol and gemcitabine on survival, tumor growth, apoptosis, angiogenesis and oncogenic signaling in a transgenic mouse model of pancreatic cancer Methods: Offspring of LSLKRASG12D x PDX-1-Cre x Trp53R172H intercrosses (triple positive by genotyping) were randomized to 4 groups: 1) Vehicle (olive oil, 1.0 ml/kg x 2/ day, PO and PBS 1.0 ml/kg x 2/week, IP), 2) Gemcitabine (100 mg/kg, x 2/week, IP), 3) δ-tocotrienol (200 mg/kg x 2/day, PO) and 4) Gemcitabine + δ-tocotrienol. The treatment was started at the age of 5 weeks and continued for 12 weeks. The survival of the mice in three groups was plotted by Kaplan-Meir graph. The weights of mice were recorded every week and the tumor weights were recorded at the end of the study. The apoptosis markers in plasma and pancreatic tumor, epithelial to mesenchymal transition (EMT), angiogenesis and kras/p53 signaling markers were determined by ELISA, Western blotting and immunostaining. Results: No significant difference in food intake and body weight gain between drug treatment and control groups was observed. δ-tocotrienol treatment alone significantly increased overall survival compared to vehicle (P The tumor weights were more significantly decreased in the combination group (p Conclusion: δ-tocotrienol synergizes with gemcitabine to inhibit pancreatic tumor growth through inhibition of angiogenesis, cell cycle, Kras signaling and induction of apoptosis in transgenic mouse model of pancreatic cancer. Citation Format: Kazim Husain, Barbara Centeno, Marta Perez, Chen Dung-Tsa, Said M. Sebti, Mokenge P. Malafa. Natural vitamin E delta-tocotrienol combined with gemcitabine prolongs the survival, induces apoptosis, inhibits tumor growth, angiogenesis and oncogenic signaling in a transgenic mouse model of pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2070. doi:10.1158/1538-7445.AM2013-2070

Signal transduction pathways that are mediated by ROCK1 and ROCK2 trigger cell migration, invasion and metastasis. This prompted us to design and synthesize novel Rho-kinase inhibitors (RKIs). Here, we show that one of our most potent RKIs, RKI-18 (IC50 397 nM (ROCK1) and 349 nM (ROCK2)) but not its inactive closely related analogue RKI-11 suppresses potently the phosphorylation of the ROCK substrate MLC2 in intact human cancer cells. Furthermore, RKI-18 is highly selective at inhibiting P-MLC2 over P-Akt, P-S6 and P-Erk ½ levels. RKI-18 suppresses ROCK-mediated actin fiber formation following stimulation with LPA as well as PAK-mediated lamelipodia and filopodia formation following bradykinin or PDGF stimulation. Furthermore, RKI-18 but not RKI-11 inhibits migration, invasion and anchorage-independent growth of human breast cancer cells. The fact that RKI-18, the active ROCK inhibitor, but not RKI-11, its inactive analogue, is effective at suppressing malignant transformation suggests that preventing migration, invasion and anchorage-independent growth with RKI-18 is likely due to inhibition of ROCK. Further advanced preclinical studies are required to determine the potential of RKI-18 as an anti-metastatic agent. Citation Format: Ronil A. Patel, Yan Lui, Binglin Wang, Ronshi Li, Said M. Sebti. Discovery of RKI-18, a small molecule that inhibits Rho kinases 1 and 2, migration and invasion of human tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5556. doi:10.1158/1538-7445.AM2013-5556

The O-tosylsalicylamide S3I-201 (10) was used as a starting point for design and synthesis of novel STAT-3 dimerization inhibitors with improved drug-like qualities. The phosphonic acid 12d and salicylic acids 13f, 13g with a shorter amide linker lacking the O-tosyl group had improved STAT-3 inhibitory activity. The equivalent potencies observed by the replacement of phosphonic acid moiety of 12d with 5-amino-2-hydroxybenzoic acid group as in 13f further validates 5-amino-2-hydroxybenzoic acid as a phosphotyrosine mimic. The salicylic acid 13f displayed improved whole cell activity. The focused library of salicylic acids 13 with benzamide linker indicated that hydrophobic heptyl and cyclohexyl are the best tolerated R groups and a biphenyl ether (as the Ar group) significantly contributes to STAT3 inhibitory activity. Our docking studies indicated that the acidic groups of 12d, 13f and 13g interact in the p-Tyr-705 binding site in a broadly similar manner, while the phenoxybenzoyl group and the cyclohexylbenzyl group occupying pY+1 and pY-X hydrophobic pockets respectively. The in vitro and cell based potency of 13f warrants further development of this scaffold as STAT3 inhibitors.

Background Multiple farnesylated proteins are involved in signal transduction in cancer. Farnesyltransferase inhibitors (FTIs) have been developed as a strategy to inhibit the function of these proteins. As FTIs inhibit proliferation of melanoma cell lines, we undertook a study to assess the impact of a FTI in advanced melanoma. As farnesylated proteins are also important for T cell activation, measurement of effects on T cell function was also pursued. Methods A 3-stage trial design was developed with a maximum of 40 patients and early stopping if there were no responders in the first 14, or fewer than 2 responders in the first 28 patients. Eligibility included performance status of 0–1, no prior chemotherapy, at most 1 prior immunotherapy, no brain metastases, and presence of at least 2 cutaneous lesions amenable to biopsy. R115777 was administered twice per day for 21 days of a 28-day cycle. Patients were evaluated every 2 cycles by RECIST. Blood and tumor were analyzed pre-treatment and during week 7. Results Fourteen patients were enrolled. Two patients had grade 3 toxicities, which included myelosuppression, nausea/vomiting, elevated BUN, and anorexia. There were no clinical responses. All patients analyzed showed potent inhibition of FT activity (85-98%) in tumor tissue; inhibition of phosphorylated ERK and Akt was also observed. T cells showed evidence of FT inhibition and diminished IFN-γ production. Conclusions Despite potent target inhibition, R115777 showed no evidence of clinical activity in this cohort of melanoma patients. Inhibition of T cell function by FTIs has potential clinical implications. Clinicaltrials.gov number NCT00060125

Background: We have shown that natural vitamin E γ-tocotrienol is the most bioactive tocotrienols in inhibiting the cell proliferation and induction of apoptosis in pancreatic cancer cells as well as in xenograft models. Therefore in this study we evaluated the chronic 12-month feeding of γ-tocotrienol on the prevention of the progression of PanIN lesions in a conditional KrasG12D mouse model. Methods: Offspring of LSLKRASG12D x PDX-1-Cre intercrosses (double positive by genotyping) were randomized to three groups: 1) No treatment (NT), 2) Vehicle (ethanol extracted olive oil, 1.0 ml/kg x 2/ day, PO) and 3) γ-tocotrienol (200 mg/kg x 2/day, PO). The treatment was started at the age of 10 weeks and continued for 12 months. The survival of the mice in three groups was plotted by Kaplan-Meir graph. The pancreas of KRASG12D; PDX-1-Cre mice was analyzed for the presence of murine PanINs. The apoptosis, cell cycle and signaling markers were determined by Western blotting and immunostaining. Results: No significant difference in food intake and body weight gain between γ-tocotrienol and control groups was observed during 12 months feeding. γ-tocotrienol treatment significantly increased survival of mice compared to control groups (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1613. doi:1538-7445.AM2012-1613

Abstract: Lysophosphatidic acid acyltransferase-beta (LPAAT-α) catalyzes the production of phosphatidic acid (PA) from lysophosphatidic acid (LPA). PA is a lipid cofactor that contributes to the activation of c-Raf, BRAF, mTOR and PKC-α. LPAAT-α expression is a prognostic factor in gynecologic malignancies and is being investigated as a therapeutic target in a variety of tumor types. We previously reported that LPAAT-α is expressed in pancreatic cancer cell lines and in human pancreatic adenocarcinoma tissues. We employed siRNA knockdown of LPAAT-α expression in pancreatic cancer cells in vitro. We find that reduction in the expression of LPAAT-α protein is associated with decreased proliferation and impairment in anchorage dependent growth of pancreatic cancer cells. Methods: MiaPaCa and Panc-1 cells were transfected with two different siRNAs targeted to LPAAT-α at 10 nM and 25 nM for 48 hrs and 72 hrs. A non-targeting siRNA was used as a control. The cells were then plated on tissue culture dishes or in soft agar and allowed to proliferate. Proliferation on plastic dishes was measured using Alamar Blue. Colony formation in soft agar was determined by directly counting macroscopic colonies. Results: Transfection of MiaPaCa and Panc-1 cells with siRNA at 25 nM for 72 hours achieved almost 100% inhibition of LPAAT-α protein expression by Western blotting. Under these conditions there was a 50% reduction in proliferation and colony formation. Studies to determine the effects of depleting LPAAT-α on the levels of p-PKC-α, p-MEK, p-4E-BP1 and p-S6Kinase are ongoing and will be presented. Conclusions: Suppression of expression of LPAAT-α protein in pancreatic cancer cell lines reduces proliferation and anchorage independent growth. This contributes to the validation of LPAAT-α as a therapeutic target in pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1854. doi:1538-7445.AM2012-1854

Numerous cell cycle, apoptosis and angiogenesis-regulatory proteins undergo ubiquitin proteasome-dependent proteolysis. A proteasome inhibitor Velcade was approved for the treatment of multiple myeloma. However, Velcade is a covalent and slowly reversible inhibitor and is associated with undesired side effects. Non-covalent inhibitors with potentially less toxicity are desirable. In this abstract we report on a family of non-covalent inhibitors that resulted from our HTS screen of a 50,000 compound library against the chymotrypsin-like (CT-L) activity of purified 20S proteasome. Our initial hit PI-1833 (IC50 value = 0.5 uM) was used as a starting point for structure activity relationship studies that led to highly potent (IC50 value = 20 nM) and cell permeable derivatives. In addition to inhibiting CT-L in human breast cancer cells, these derivatives also induced the accumulation of two proteasome target proteins Bax and p27KIP, indicating their ability to inhibit proteasome activity in intact cells. Furthermore, these proteasome inhibitors also induced apoptosis and inhibited tumor cell growth. Further mechanistic studies as well as in vivo anti-tumor efficacy studies are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2778. doi:1538-7445.AM2012-2778

The Aurora kinases are a family of serine-threonine kinases that play an important role in the regulation of cell division. The three members of the Aurora family Aurora A, B and C share a high degree of structural homology in their kinase domain but each kinase plays a different role in the control of mitosis. Aurora A and B have received the most attention to date as anticancer targets. Detailed studies have shown Aurora A and B are over expressed in a variety of cancers and are implicated in many aspects of tumor development. Aurora A is frequently over-expressed in tumors and cancer cell lines and has characteristics of an oncogene. Aurora B is also over expressed in many cancer types but does not have oncogenic properties. Aurora B plays important roles in M phase to ensure correct chromosome-microtubule alignment and attachment and chromosomal cytokinesis. Therefore, inhibition of Aurora kinase A and B is emerging as target-based therapy in cancer treatment. From our previous work HLM-8598 was identified from an in-house chemical library as a potent and highly selective inhibitor for Aurora A (IC50 value of 0.073 ± 0.002 μM) over Aurora B (IC50 = 5.4 ± 1.8 μM). X-ray crystallography studies of HLM-8598 bound to Aurora A confirmed HLM-8598 is a type-1 kinase inhibitor that interacts with the ATP binding site of the enzyme. The HLM-8598 bound to Aurora A structure-assisted synthesis of new analogs with IC50 < 2.5 nM affinity. We will present the synthesis of new analogs that contain an ortho-halogen substituted phenyl moiety, structure activity relationship studies as a part of our efforts to identify unusually potent Aurora A kinase inhibitors. We will discuss the use of kinase structural elements to rationally guide compound synthesis and improve design strategy. The in vitro and in vivo activities of ortho-halogen phenyl substituted pyrimidines will be presented as unusually potent Aurora A inhibitors and potential anti-cancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3902. doi:1538-7445.AM2012-3902

The development of proteosome inhibitors for CT-L activity has been the subject of considerable interest in the treatment of cancer due to its critical role in maintaining homeostasis and hence its key position in many cellular processes. Proteosome inhibitors are classified as reversible or irreversible inhibitors according to their chemical structure and their mechanism of inhibition. Irreversible inhibitors possess a chemically reactive group, such as aldehydes, boronic acids, epoxy ketones that react with catalytic Thr1-O covalently, where as reversible inhibitors inhibit proteosome non-covalently via H-bond interactions (electrostatic and/or van der Waals). Bortezomib, a peptide boronic acid analog, is the first clinically approved proteosome inhibitor and is found to be an irreversible or slow reversible inhibitor that forms covalent bond with the N-terminal nucleophilic Thr1 in the β5 subunit of the proteasome. Reversible proteosome inhibitors (peptidic molecules) reported to date inhibit the proteosome non-covalently. Similar to Bortezomib, MLN9708 is a modified dipeptidyl boronic acid, which hydroylses immediately in plasma to MLN2238, is a potent, reversible and specific inhibitor of the chymotrypsin-like subunit of the proteasome. Small molecules as reversible proteosome inhibitors are less extensively investigated. In this study, we present the discovery of PI-184, non-peptidic small molecule with a pyridine and oxadiazole pharmacore as a reversible, potent and a novel proteasome inhibitor. Structure Activity relationship (SAR) guided synthesis of analogs around PI-184 led to the discovery of highly potent compounds with in-vivo chymotrypsin-like inhibitory activity. Here we present the detailed SAR, in-vitro and in-vivo proteasome activity of PI-184 class of compounds as potential anti-cancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3894. doi:1538-7445.AM2012-3894

The Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) is a positive transducer of growth factor, cytokine, integrin, and hormone signaling pathways which regulates processes such as cell proliferation, differentiation, adhesion, migration, and apoptosis and plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with various kinds of leukemia, and solid tumors. This makes SHP2 an attractive target for anticancer therapy. We are developing SHP2 inhibitors as novel anticancer drugs. Previously, we have reported on SPI-112 and its methyl ester prodrug SPI-112Me. However, the poor solubility of SPI-112Me has become an obstacle for in vivo studies. In ongoing study, we generate SPI-112 analogs to further improve physicochemical properties of these SHP2 inhibitors, such as solubility and cell permeability. To this aim, we synthesized SPI-112 derivatives containing various phosphotyrosine mimics and their prodrugs. SPI-112 derivatives of SHP2 inhibitors having improved properties have been obtained in this effort. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2912. doi:1538-7445.AM2012-2912

Using high concentration biochemical assays and fragment-based screening, we designed and optimized a novel class of Rho-kinase inhibitors. Ligand Efficiency (LE) was employed to assess the binding potential of the fragments and to guide the optimization process. In addition, molecular modeling was used to aide the design of potent inhibitors. A series of fragments as hinge binders were designed and synthesized, and screened using high concentration biochemical assays. Both the spacer length and isomeric tail molecules played key roles in both activity and selectivity. Structure activity relationship studies led to optimization of fragments that yielded potent and selective ROCK 1 and ROCK 2 inhibitors. For instance, compound 24 was highly potent and selective for ROCK 2 (IC50 = 100 nM) over ROCK 1 (IC50 = 1690 nM) whereas compound 18 was potent and selective for ROCK 1 (IC50 = 460 nM) over ROCK 2 (IC50 = 700nM). Compound 24 but not its inactive stereoisomer 23, and compound 18 but not its inactive analog 11 inhibited the phosphorylation of Rho Kinase substrates in intact human cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A130.

Retinoblastoma tumor suppressor binding protein, Rb, is a major regulator of the mammalian cell cycle progression. Inactivation of Rb by a cascade of phosphorylation events leads to its inactivation, facilitating release of transcriptionally active E2F and S-phase entry. Rb phosphorylaiton is mediated mainly by CDK, but it was observed Raf-1 could bind and phosphorylate Rb early in the cell cycle, facilitating phosphorylation events. Although previous reports have suggested that K-Ras, which is mutated in over 80% of pancreatic cancers, activates MAPK through Raf/Mek signaling, inhibition of this pathway have proven clinically unsuccessful. We demonstrated that increased binding of Raf-1 to Rb, independent of the Mek/MAPK pathway, has contributed to tumor progression and inhibition of this interaction might be a suitable strategy for cancer therapies. Therefore, we examined whether targeting Rb-Raf-1 interaction with a small molecule inhibitor RRD-251 is a viable strategy against pancreatic cancer. To test our hypothesis, we initially probed a panel of pancreatic cancer cell lines (PANC-1, MiaPaCa-2, CD18/HPAF, L3.6pl) and demonstrated the disruption of Rb-Raf-1 interaction by RRD-251, using immunoprecipitation western blot experiments. To fully define the anti-tumor effects of RRD-251, cell cycle analyses, proliferation/cell viability (MTT), cell migration (wound healing assay), and invasion (Boyden chamber assay) experiments were performed. Animal studies were performed in nude mice with subcutaneous and orthotopic pancreas models with intraperitoneal injections of RRD-251 at 50 mpk/daily. RRD-251 significantly reduced proliferation by 3-fold and significantly reduced the ability of metastatic variant, L3.6pl, and other pancreatic cancer cell lines to migrate; there was also a a 5-fold decrease in invasion (p80%) of liver metastasis in the RRD-251 treated group. In this study, we demonstrate a disruption of the Rb-Raf-1 kinase interaction with RRD-251 significantly affects the malignant properties of pancreatic cancer cells. Treatment with combination RRD-251 and gemcitabine appears to be a viable strategy against pancreatic cancer. 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 2590. doi:10.1158/1538-7445.AM2011-2590

Anti-apoptotic members of the Bcl-2 protein family, such as Bcl-2, Bcl-xL and Mcl-1, are among the most effective inhibitors of apoptosis and are frequently upregulated in various types of human cancer. A number of pro-apoptotic proteins share only the BH3 domain with other members of the Bcl-2 family, so called BH3-only proteins, and appear to function essentially as transdominant inhibitors by binding to anti-apoptotic Bcl-2 family proteins and neutralizing their cell-survival activity. The development of small molecules to mimic the BH3 proteins has been an intense area of research. However, to date available Bcl-2 family proteins inhibitors are either pan-inhibitors such as (−)-gossypol (which simultaneously inhibits Bcl-xl/BH3, Bcl-2/BH3 and Mcl-1/BH3 interactions) or are selective for the Bcl-xl/BH3 and Bcl-2/BH3 interactions. Hence a selective Mcl-1/BH3 inhibitor would greatly aid investigating the role of Mcl-1 in cancer and in drug resistance. Our current study is centered on the identification and the synthesis of small molecule selective inhibitors of the Mcl-1/BH3 interaction. To this aim we interrogated the hit-list from inhibitors of the MLSCN screen of the Mcl-1-NOXA interaction [(Emory, AID 1417) TR-FRET screen for Mcl-1-NOXA inhibitors]. Selected compounds were purchased and assessed for activity in a fluorescence polarization and ELISA assay, to reveal the hit HLM050001 (FP IC50 0.8 ± 0.1 µM; ELISA IC50 19.3 ± 2.5 µM). HLM050001binding to Mcl-1 is currently being assessed via co-crystallization and other biophysical techniques. We will report the synthesis and biological evaluation of focused libraries based on the initial hit and designed by modeling and docking to Mcl-1. Structure activity relationship studies around the hit will be disclosed as well as the outcomes of further rounds of chemical design and biological assessment. 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 1354. doi:10.1158/1538-7445.AM2011-1354

The M-phase of cell division is controlled by phosphorylation events performed by several mitotic serine/threonine kinases. For example, the three human isoforms of Aurora kinase (A, B, and C) have been amply investigated as they are essential for the execution of numerous mitotic events and required for genome integrity and stability. Studies have shown that Aurora A and B are frequently over-expressed in various cancer cells. Aurora A also acts as an oncogene inducing malignant transformation when over-expressed. Since the discovery of the Aurora kinases and their association with cancer, there has been great interest in developing small molecule inhibitors of Aurora kinases as potential anticancer drugs.In-house screening campaign directed toward the discovery of novel Aurora A inhibitors, led to the identification of a new and potent class of oxindole derivatives as inhibitors of Aurora-A kinase. The initial hit HL1-058-1 was identified as an inhibitor of histone H3-Ser10 phosphorylation (IC50 1-5 μM). By using a Z’-lyte kinase assay kit, the inhibitory effect of HL1-058-1 was shown to be the direct consequence of the Aurora kinase inhibition (IC50 of 2.7 ± 0.3 µM). Based on the preliminary data, HL1-058-1 was selected for further study. Synthesis of a focused library of analogs based on this hit revealed RPM305 and RPM304 as potent inhibitors of Aurora A (IC50 0.020 ± 0.005 μM, and 0.038 ± 0.018 μM, respectively). Interestingly, compound RPM304 showed 26 fold selectivity over Aurora B (IC50 0.99 ± 0.08 μM). A coupled enzyme assay was also used to carry out further structure activity relationship studies of the most potent analogs. One of the lead compounds was co-crystallized with the kinase domain of Aurora A. The design and synthesis of further focused libraries of oxindoles, based on this X-ray structure will be described. Details of the cell activity of this class of compound to inhibit Aurora A activity and tumor cell growth will be described. 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 3253. doi:10.1158/1538-7445.AM2011-3253

Many low molecular weight GTPases such as Ras, Ral and Rho require posttranslational farnestylation or geranylgeranylation for mediating malignant transformation. This prompted the development of farnesyltransferase (FT) inhibitors (FTI) and geranylgeranyltransferase-I (GGT-1) inhibitors (GGTI) as potential anticancer agents. However, when cancer cells are treated with FTIs, K-Ras becomes geranylgeranylated suggesting that inhibition of both FT and GGT-1 may be required to inhibit tumors harboring K-Ras mutations. Recently our group has developed a series of dual FT and GGT-1 inhibitors based on an ethylenediamine scaffold. Among these dual inhibitors FGTI-2734 was most potent in vitro (FT IC50 = 250±190 nM and GGT-1 IC50 = 520±90 nM). In human cancer cells FGTI-2734 inhibits potently HDJ-2 farnesylation, Rap 1A geranylgeranylation and K-Ras prenylation. Furthermore, FGTI-2734 inhibits the phosphorylation of Erk1/2, Akt and S6K, induces p27 accumulation and inhibits potently tumor cell growth. Finally, FGTI-2734 significantly reduces survivin protein levels, and reduction of survivin levels is associated with induction of apoptosis. Thus, this single molecule with dual FT and GGT-1 inhibitory activities may have distinct advantage over selective FTIs and GGTIs and warrants further advanced pre-clinical studies. 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 620. doi:10.1158/1538-7445.AM2011-620

The retinoblastoma tumor suppressor protein Rb, is a vital regulator of the mammalian cell cycle which is inactivated by phosphorylation. We had shown that Raf-1 kinase binds and phosphorylates Rb early in the G1 phase facilitating its subsequent phosphorylation by cyclin dependent kinases. This phosphorlation released transcriptionally active E2Fs from Rb to facilitate S-phase entry. Disruption of Rb-Raf protein-protein interaction could prevent proliferation and angiogenesis, leading to inhibition of tumor growth in mouse models. We had earlier identified an orally active small molecule, Rb/Raf-1 disruptor 251 (RRD-251) that potently and selectively disrupts Rb/Raf-1 but not Rb/E2F, Rb/prohibitin, Rb/cyclin E or Rb/HDAC interactions. Here, we have made efforts to analyze additional analogues of RRD251; we focused on XW-19B and XW-35B. These compounds inhibited Rb-Raf interaction in vitro and in vivo as shown by ELISA and Immunoprecipitation-Western blot analysis. They also inhibited cell viability, proliferation, migration, invasion, angiogenesis and adherence-independent growth of human lung cancer cell lines A549. XW-19B inhibited tumor growth significantly in human A549 xenograft model in nude mice, compared to control while XW-35B showed certain amount of toxicity to animals at 50MPK. Overall, these results showed that Rb-Raf disruptors XW-19B and XW-35B have potential anticancer activity. Thus, selective targeting of Rb/Raf-1 interaction seems to be a promising approach for developing novel chemotherapeutic agents. 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 2108. doi:10.1158/1538-7445.AM2011-2108

Lysophosphatidic acid acyltransferase beta (LPAAT-β) is a new therapeutic target and prognostic marker in human cancer. The enzymatic function of LPAAT- β is to convert Lysophosphatidic Acid (LPA) to Phosphatidic Acid (PA), a lipid second messenger that functions to regulate the proliferation, adhesion, migration and survival of cancer cells. PA is an essential cofactor needed by the oncogenes c-Raf, BRAF, PKC-ζ and mTOR for their enzymatic and growth promoting activities in cancer. LPAAT-β protein over expression has been detected in tumors compared to normal tissue in ovarian, endometrial, breast and lung cancers. The over expression of LPAAT-β in ovarian tumors is associated with poor clinical outcome. Methods: In this study we determined LPAAT-β expression in paired specimens of pancreatic ductal adenocarcinoma and adjacent non-malignant pancreas obtained from 102 patients who underwent pancreatectomy with curative intent (stage IA-IIB). Formalin-fixed, paraffin-embedded core sections in a tissue microarray were immunostained using the avidin-biotin-peroxidase method with rabbit anti- LPAAT-β antibody. A semi-quantitative measure of LPAAT-β expression was determined as the product of immunostain intensity and percentage of cells stained, where intensity and percentage were each scored on a scale of 0 to 3, with 3 being maximal. Results: LPAAT-β expression (product score) in 57% (58/102) of pancreatic cancers was lower than in paired non-malignant pancreas, equal in 20% (20/102) of cancers, and greater in 23% (24/102) of cancers as compared to non-malignant pancreas. LPAAT-β expression in cancer (median product score = 3) was less than in non-malignant pancreas (median score = 4) (Wilcoxon signed-rank test, p < 0.0001). Conclusions: Low LPAAT-β expression in pancreatic cancer as compared to non-malignant pancreas stands in contrast to previous studies showing increased LPAAT-β expression in several other cancers. This finding indicates that there may be tissue-specific differences in the biologic activity of LPAAT-β. 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 1677. doi:10.1158/1538-7445.AM2011-1677

The anti-apoptotic protein survivin is maintained at high levels in cancer cells relative to normal cells. The mechanism by which this occurs is ill-understood. Here we show in several human cancer cell lines including those derived from pancreatic, lung and ovarian cancers that knocking down K-Ras expression by siRNA results in decreased survivin levels in cancer cells harboring mutant but not wild type K-Ras. K-Ras knockdown decreases survivin mRNA and protein levels by 50% and 85%, respectively, suggesting that K-Ras regulates survivin transcription as well as protein stability. Consistent with this K-Ras knockdown decreases the levels of ectopically-expressed Myc-tagged survivin. Further support for the ability of K-Ras to regulate the protein stability of survivin is also supported by the fact that the proteasome inhibitor Velcade partially inhibits K-Ras siRNA from decreasing the levels of survivin. Taken together, the data suggest that K-Ras is a contributor to the maintenance of high levels of survivin in human cancer cells which harbor mutant but not wild type K-Ras. 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 3844. doi:10.1158/1538-7445.AM2011-3844

The ubiquitin-proteasome pathway plays a critical role in the regulated degradation of proteins involved in cell cycle control and tumor growth. Proteasome inhibitors cause selective apoptosis of malignant cells and represent a new class of antineoplastic agents. In 2003 Bortezomib (peptide boronate), a proteasome inhibitor was approved by FDA for treatment of multiple myeloma. Toxicity and tumor cell resistance against Bortezomib demand development of less toxic proteasome inhibitors with broader spectrum of tumor activity. Our aim is to develop non-peptidic small drug-like molecules as proteasome inhibitors. To this end, we have identified through in-house screening efforts HLM-030376 as a small molecule proteasome inhibitor with phenanthridine pharmacophore. HLM-030376 showed in-vitro chymotrypsin like, trypsin like and PGPH inhibitory activities with IC50 values 0.26, 0.19 and 2.07 µM respectively. Extensive synthetic modifications around the HLM-030376 pharmacophore were undertaken to aid the cell activity of this class of compounds. We will describe the synthesis and activity of analogs of HLM-030376 as potent proteasome inhibitors. The analogs of HLM-030376 showed in-vivo CT-L inhibitory activities (IC50 between 0.6-6.0 µM) against MDA-MB 468 breast cancer cell line as well as improved (IC50 between 70-200 nM) in-vitro CT-L inhibitory activities. The detailed in-vitro structure activity relationship (SAR) data of HLM-030376 and analogs will be discussed to understand the structural moieties responsible for proteasome activity. The whole cell activity data of HLM-030376 as well as in-silico docking studies will be presented as potential proteasome inhibitors for further development. 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 1362. doi:10.1158/1538-7445.AM2011-1362

The Shp2 protein tyrosine phosphatase (PTP) mediates signal transduction of growth factor receptors and regulates cellular activities critical to tumor growth and metastasis. In the basal state, the interactions between the N-SH2 and PTP domains keep the phosphatase in an auto inhibited closed conformation. Upon growth factor or cytokine stimulation, the SH2 domains of Shp2 binds to tyrosine phosphorylated docking proteins such as Gab1 and Gab2, which activates Src, Ras, and the Erk1/2 (Erk) mitogen-activated protein (MAP) kinase pathway. Shp2 gain-of-function mutations are found in leukemias and solid tumors and are linked to Noonan syndrome. Although Shp2 is involved in pathogenesis of human cancers, it is not clear how Shp2 mediates tumorigenesis. Shp2 is therefore believed to be an important enzyme for targeted cancer therapy. Since no potent and selective Shp2 PTP inhibitor is currently available for chemical biology studies and experimental therapy, we and others have been actively engaged in the search for such agents. In our continuing efforts to develop potent and selective Shp2 inhibitors to serve as both chemical probes to further our understanding of the role and signaling mechanisms of Shp2 in human diseases and as potential drugs for cancer therapy. We will report the screening of a focused library of a set of commercially available salicylic acid derivatives using the in vitro DIFMUP phosphatase assay for their ability to inhibit the Shp2 phosphatase domain. Activity of hits was confirmed using the Biomol Green assay to determine inhibition of dephosphorylation of a Shp2 peptide substrate derived from EphR. One such hit was a thiazole containing salicylic acid that inhibits Shp2 with an IC50 (DIFMUP) of 100 µM. We will report the design and synthesis of salicylic acid libraries along with their benzoic acid counterparts. The most potent compound to date is HM2-08-4 which has an IC50 (DIFMUP) 1.1 µM. Emerging structure-activity relationships as well as modeling and X-ray studies will be discussed. The preliminary use of selected compounds as tools to probe the pharmacological inhibition of Shp2 and elucidate its role in cancer will be described. 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 3248. doi:10.1158/1538-7445.AM2011-3248

Rho associated protein kinases (ROCKs) are Ser/Thr kinases which require activation by small GTPases of the Rho family. ROCK is often found to be over expressed in ovarian, testicular, bladder, and pancreatic cancers. ROCK has been implicated in cell motility, adhesion, smooth muscle contraction and stress fiber formation. Inhibition of Rho kinase is a novel approach for the treatment of cardiovascular disease, CNS disorders, and, more recently, cancer. ROCK has received increased attention for its affect on cell adhesion and invasion, two key steps in metastasis. We detail the development of ROCK inhibitors from initial small molecule library screening and in-house HTS screening data. We report on the identification and resynthesis of one screening hit (ROCK1 IC50 38.5 ± 3.4 µM). Focused chemical libraries were developed and structure activity relationships will be described. All compounds have been screened in a Z-lyte kinase assay to determine their activity against both ROCK1 and ROCK2. Hit-to-lead optimization and SAR studies resulted in compounds capable of inhibiting ROCK1 at low nanomolar concentrations, RPM1510 (ROCK1 IC50 0.15 ± 0.02 µM) and RPM1533 (ROCK1 IC50 0.12 ± 0.02 µM). Progress in obtaining structural information by X-ray crystallography will be reported along with ROCK inhibitory data. Trends in isoform and kinase selectivity and potency will also be discussed. Members of this class have been shown to inhibit potently, in human lung cancer cells, the phosphorylation of MYPT1, a surrogate for ROCK activity in vivo. 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 3243. doi:10.1158/1538-7445.AM2011-3243

Background: The human cellular apoptosis susceptibility protein (hCAS) is a Ran- binding protein playing a role as mitotic spindle checkpoint regulator and as nuclear export factor. hCAS expression is frequently altered in metastatic cancers. To date the role of hCAS in pancreatic cancer is unknown. Therefore this study aimed to investigate the role of hCAS in human pancreatic cancer in vitro and in vivo. Methods: The hCAS protein expression was determined using Western immunoblotting in human pancreatic cancer cell lines (AsPc-1, BxPc-3, Capan-2, MiaPaCa-2, Panc-1, and SW1990) and immortalized normal human pancreatic ductal epithelial cell (HPNE) and HPNE-Kras. siRNA induced hCAS knockdown pancreatic cancer cells were compared to scrambled siRNA cells with regard to proliferative (MTT assay), cell death, cell cycle (Trypan blue and Flow cytometry), malignant transformation (colony formation assay) and protein expression (Western and confocal microscopy). hCAS KD MiaPaCa-2 cells were xenografted in SCID nude mice. Tumor growth, expression of hCAS, proliferation index (Ki-67), and caspase-3 were immunostained in tumor tissues and compared with scr siRNA MiaPaCa-2 cells. Expression of hCAS was determined in human pancreatic tumor tissues using immunohistochemical stain. Results: hCAS protein expression was low in HPNE cells compared to HPNE-Kras. Lowest expression of hCAS noted in BxPc-3 cells compared to other pancreatic cancer cells. When compared to Scr-MiaPaCa-2, KD-MiaPaCa-2 cells demonstrated lower proliferation, G2/M phase cell cycle arrest, increased apoptosis, as well as inhibition of malignant transformation. Pancreatic ductal carcinomas (PCA) were hCAS strongly positive. Normal pancreatic ducts (ND) were hCAS weakly positive. hCAS KD-MiaPaCa-2 tumor growth was retarded in mice compared to Scr-MiaPaCa-2 tumors. hCAS KD-MiaPaCa-2 tumors showed hCAS depletion, lower proliferative index, increased caspase-3 immunostaining and PARP1 cleavage. Conclusion: This is the first report of hCAS expression in human pancreatic cancer. Our data suggest that hCAS is a regulator of cell proliferation, cell cycle regulation and apoptosis. hCAS may represent a potential chemopreventive and therapeutic target for human pancreatic cancer. 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 1667. doi:10.1158/1538-7445.AM2011-1667

The ATP-dependent ubiquitin-proteosome pathway is responsible for the controlled degradation of proteins in eukaryotic cells. The 26S proteosome is a multifunctional complex, consisting of a 19S regulatory particle (RP) and a 20S core particle (CP). The three main catalytic activities of the proteasome; peptidylglutamyl peptide hydrolysing (PGPH), trypsin-like (T-L), and chymotrypsin-like (CT-L), are mediated by three distinct catalytic β-1, β-2, and β-5 subunits, respectively. Owing to the central role of proteosome in maintaining homeostasis and hence its key position in many cellular processes, the development of inhibitors for CT-L activity has been the subject of considerable interest in the treatment of cancer. Selective inhibition of the CT-L activity of 20S proteasome represents a successful strategy in cancer therapy as demonstrated by Bortezomib (Velcade), a clinically approved drug for multiple myeloma and mantle lymphoma. Toxicity and tumor cell resistance against Bortezomib demand the development of improved and selective proteasome inhibitors. The potent proteasome inhibitors reported to date have been developed as aldehydes, boronates, vinylsulfones and expoxyketones and these compounds function through covalent modification of the N-terminal threonine residue of β-5 subunit. Small, drug-like synthetic proteasome inhibitors that are selective for cancer over normal cells are rare, but clearly would have a potential advantage over the existing inhibitors. PI-1833 was identified in our program as a ‘hit’ with in-vitro CT-L proteasome activity (IC50 = 0.58 μM). PI-1833 is a small molecule with oxadiazole pharmacophore and presents 3 points of diversity amenable for focused library synthesis. Lack of a reactive chemical moiety (or electrophilic moiety) to form proteasome adducts is an important feature of PI-1833. The first generation analogs of PI-1833 have shown promising SAR (in-vitro and in-vivo) toward proteasome inhibition with the IC50 values ranging from 0.3-0.7 μM. In this work we will present in-vitro and in-vivo structure activity relationship (SAR) data, synthesis and selectivity of a PI-1833 library and discuss the mode of proteasome inhibition (co-valent vs non co-valent) by this class of compounds. 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 1359. doi:10.1158/1538-7445.AM2011-1359

Shp2 is a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. Shp2 mediates proliferative signaling induced by growth factors. Gain-of-function PTPN11 mutations that encode constitutively active Shp2 are leukemic oncogenes. In a continuing effort to identify new Shp2 PTP inhibitors, we screened a small molecule library comprising the National Cancer Institute (NCI) Approved Oncology Drug set and the NIH Clinical Collection. After evaluation of initial hits, estramustine phosphate was verified as a Shp2 PTP inhibitor. A focused structure-activity relationship study indicated that the 17-phosphate group is required for the Shp2 PTP inhibitor activity of estramustine phosphate. A search for estramustine phosphate analogs led to identification of two triperpenoids, enoxolone and celastrol, having Shp2 PTP inhibitor activity. With the previously reported PTP1B inhibitor trodusquemine, our study reveals steroids and triterpenoids with negatively charged phosphate, carboxylate, or sulfonate groups as novel pharmacophores of selective PTP inhibitors. These findings point to a rich natural source for discovery of lead compounds of novel PTP inhibitors. 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 3239. doi:10.1158/1538-7445.AM2011-3239

MDM2 and MDMX function as key regulators of p53 by binding to its N terminus, inhibiting its transcriptional activity, and promoting its degradation. In particular, MDM2 is overexpressed in some of human tumors, and with MDMX contributes directly to loss of p53 function during the development of nearly 50% of human cancers. Due to p53 inactivation, MDM2 in many tumors confers tumor survival; therefore it is an important molecular target for anticancer therapy. Several studies showed that reactivation of wild type p53 in tumor cells can be obtained by disrupting the MDM2/p53 interaction with peptidic, peptidomimetic, and small molecule p53-mimetics. Specific successful examples include the Nutlins and spirooxindole analogs (MI-219 and MI-63). Amongst the peptidic and peptidomimetic inhibitors examined to date, none is nearly as effective as Nutlins and MI-219 in tumor killing in vitro. Hence, new inhibitors against MDM2 and/or MDMX are needed: as cell permeable chemical probes of the p53 pathway in cancer biology, and as templates for structure-based rational design of p53 activators for future therapeutic use. As part of our drug discovery program to identify antagonists of the p53/MDM2 and p53/MDMx protein-protein interactions, a high-throughput in-silico screen of a 3.2 millions virtual library of compounds (from Schrödinger, Inc.). A physical restraint was applied during the screen, in order to mimic binding to the hydrophobic cleft of MDM2 normally occupied by three p53 side chains (F19, W23, and L26) that are critical for MDM2/p53 binding. The top highest ranked 160 compounds were then assessed for their ability to block p53 interaction with MDM2 and MDMx in an ELISA assay. This resulted in the identification of E12/DP3-117, a small molecule disruptor of the p53/MDM2 protein-protein interaction with an IC50 value of 47 ± 14 μM. We will report the synthesis and biological evaluation of focused libraries based on the initial hit and on compounds showing improved activity. Structure activity relationship studies around the hits will be disclosed as well as the outcomes of further rounds of chemical design and biological assessment. Binding of E12/DP3-117 to MDM2 is currently being assessed via co-crystallization and other biophysical techniques. We will describe the use of the crystal structure of p53-like mutant peptides in complex with the N-terminal domains of Mdm2, as the basis for rational design of more potent MDM2 small-molecule/peptide hybrid inhibitors. 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 3242. doi:10.1158/1538-7445.AM2011-3242

Abstract 3986 Background: P-glycoprotein (P-gp or ABCB1) is a common cause of multidrug resistance (MDR) in cancer and leukemia that serves to extrude amphipathic drugs from the plasma membrane. P-gp is an ATP-binding cassette (ABC) transporter that exports a wide range of anti-neoplastics that are structurally or functionally unrelated. The vast majority of P-gp inhibitors tested clinically act as competitive export channel inhibitors to promote antineoplastic retention. We investigated the in vitro and in vivo effects of a novel substituted quinoline P-gp modulator, HG-829, in MDR cell lines and xenograft models. Methods: In vitro activity of HG-829 was evaluated in the K562-daunomycin-selected (K562-R) cell line and the ABCB1-transfected human embryonic kidney-293 cell line (HEK-293-B1) using a variety of P-gp substrates (daunomycin, doxorubicin, taxol, etoposide, vincristine) in a 72h MTT proliferation assay, and results compared to the effects of cyclosporine-A (CsA). Rhodamine 123 export and retention was assessed by flow cytometry. Flank injections of K562-R and parental K562-S cells in female SCID beige mice were performed for xenograft models. ANOVA and Turkey's multiple comparison tests were used to determine significant differences between groups in the proliferation assay and xenograft studies. Differences in rhodamine intracellular retention and efflux were assessed by Student's t-test. Results: Treatment with HG-829 at 0.5uM and 1uM completely reversed resistance to each of the antineoplastics studied in both the K562-R and HEK-293-B1 cell lines, but did not sensitize parental cells. HG-829 sensitized K562-R cells to daunomycin 57-fold and 97-fold at concentrations of 0.5uM and 1uM, respectively (p Conclusions: HG-829 is a potent non-substrate inhibitor of P-glycoprotein with a prolonged duration of action. Clinical testing of HG-829 is warranted. Disclosures: No relevant conflicts of interest to declare.