Lynn Kirkpatrick

In vivo molecular pharmacology and antitumor activity of the targeted Akt inhibitor PX-316.

Akt, a serine/threonine kinase that promotes cell survival, is activated by binding of its pleckstrin homology (PH) domain to membrane phosphatidylinositol (PtdIns)-3-phosphates formed by PtdIns-3-kinase. D-3-Deoxy-phosphatidyl-myo-inositols that cannot be phosphorylated on the 3-position of the myo-inositol group are inhibitors of the Akt PH domain. The most active compound is D-3-deoxy-phosphatidyl-myo-inositol 1-[(R)-2-methoxy-3-octadecyloxypropyl hydrogen phosphate] (PX-316). PX-316 administered intraperitoneally to mice at 150 mg/kg inhibits Akt activation in HT-29 human tumor xenografts up to 78% at 10 h with recovery to 34% at 48 h. Phosphorylation of GSK-3beta, a downstream target of Akt, is also inhibited. There is no decrease in PtdIns(3,4,5)-trisphosphate levels by PX-316, showing it is not an inhibitor of PtdIns-3-K in vivo. Gene expression profiling of HT-29 tumor xenografts shows many similarities between the effects of PX-316 and the PtdIns-3-K inhibitor wortmannin, with downregulation of several ribosomal-related genes, while PX-316 uniquely increases the expression of a group of mitochondrial-related genes. PX-316 has antitumor activity against early human MCF-7 breast cancer and HT-29 colon cancer xenografts in mice. PX-316 formulated in 20% hydroxypropyl-beta-cyclodextrin for intravenous administration is well tolerated in mice and rats with no hemolysis and no hematological toxicity. Thus, PX-316 is the lead compound of a new class of potential agents that inhibit Akt survival signaling.

Treatment with HIF-1α Antagonist PX-478 Inhibits Progression and Spread of Orthotopic Human Small Cell Lung Cancer and Lung Adenocarcinoma in Mice

Introduction: PX-478 is a potent small-molecule inhibitor of hypoxia-inducible factor 1&agr; (HIF-1&agr;). In prior preclinical studies, it had antitumor activity against various solid tumors in subcutaneous xenografts but had no measurable activity against a non-small cell lung cancer (NSCLC) xenograft. To determine the effectiveness of PX-478 against lung tumors, we investigated HIF-1&agr; expression in several lung cancer cell lines, both in vitro and in vivo, and treated orthotopic mouse models of human lung cancer with PX-478. Methods: Cells from two human lung adenocarcinoma cell models (PC14-PE6 and NCI-H441) or two human small cell lung cancer (SCLC) models (NCI-H187 and NCI-N417) were injected into the left lungs of nude mice and were randomized 16 to 18 days after injection with daily oral treatment with PX-478 or vehicle for 5 days. Results: In the PC14-PE6 NSCLC model, treatment with 20 mg/kg PX-478 significantly reduced the median primary lung tumor volume by 87% (p = 0.005) compared with the vehicle-treated group. PX-478 treatment also markedly reduced mediastinal metastasis and prolonged survival. Similar results were obtained in a second NSCLC model. In SCLC models, PX-478 was even more effective. In the NCI-H187 model, the median primary lung tumor volume was reduced by 99% (p = 0.0001). The median survival duration was increased by 132%. In the NCI-N417 model, the median primary lung tumor volume was reduced by 97% (p = 0.008). Conclusions: We demonstrated that the PX-478, HIF-1&agr; inhibitor, had significant antitumor activity against two orthotopic models of lung adenocarcinomas and two models of SCLC. These results suggest the inclusion of lung cancer patients in phase I clinical trials of PX-478.

The skin and hair as surrogate tissues for measuring the target effect of inhibitors of phosphoinositide-3-kinase signaling.

Background: The purpose of the study was to evaluate the use of phospho-Akt in mouse and human skin as a surrogate target for tumor phospho-Akt to measure the effect of antitumor inhibitors of phosphatidylinositol-3-kinase (PI-3-K)/Akt (protein kinase B) signaling. Method: The expression of phosphoSer473-Akt was quantitatively assessed by Western blotting in human HT-29 colon, MCF-7 breast, A-549 non small cell lung tumor xenografts in mice, and by immunohistochemistry in mouse skin and human hair. Results: The pattern of PI-3-K isoforms in human hair keratinocytes was similar to that in tumor but mouse hair keratinocytes showed a different pattern. A high level of phospho-Akt staining was present in keratinocytes of the external root sheath of the hair and was inhibited by the PI-3-K inhibitor PX-866 administered to mice, and in human hair exposed to PX-866 in culture. The inhibition of phospho-Akt by PX-866 in mouse hair keratinocytes was greater than inhibition of phospho-Akt in HT-29 and A-549 xenografts in the same mice. Phospho-Akt in mouse hair keratinocytes was inhibited by the Akt inhibitor PX-316 to a lesser degree than in MCF-7 tumor xenografts. Conclusions: Hair offers a way of measuring the effects of PI-3-K signaling inhibitors and, in cancer patients, may provide a readily obtainable surrogate tissue for assessing PI-3-K and phospho-Akt inhibition in tumor.

Peroxisome proliferator-activated receptor γ agonist pioglitazone prevents the hyperglycemia caused by phosphatidylinositol 3-kinase pathway inhibition by PX-866 without affecting antitumor activity

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascade is an important component of the insulin signaling in normal tissues leading to glucose uptake and homeostasis and for cell survival signaling in cancer cells. Hyperglycemia is an on-target side effect of many inhibitors of PI3K/Akt signaling including the specific PI3K inhibitor PX-866. The peroxisome proliferator-activated receptor γ agonist pioglitazone, used to treat type 2 diabetes, prevents a decrease in glucose tolerance caused by acute administration of PX-866. Our studies have shown that pioglitazone does not inhibit the antitumor activity of PX-866 in A-549 non-small cell lung cancer and HT-29 colon cancer xenografts. In vitro studies also showed that pioglitazone increases 2-[1-14C]deoxy-d-glucose uptake in L-6 muscle cells and prevents inhibition of 2-deoxyglucose uptake by PX-866. Neither pioglitazone nor PX-866 had an effect on 2-deoxyglucose uptake in A-549 lung cancer cells. In vivo imaging studies using [18F]2-deoxyglucose (FDG) positron emission tomography showed that pioglitazone increases FDG accumulation by normal tissue but does not significantly alter FDG uptake by A-549 xenografts. Thus, peroxisome proliferator-activated receptor γ agonists may be useful in overcoming the increase in blood glucose caused by inhibitors of PI3K signaling by preventing the inhibition of normal tissue insulin-mediated glucose uptake without affecting antitumor activity. [Mol Cancer Ther 2009;8(1):94–100]

Abstract 3015: Selective inhibition of mutant KRAS cell and tumor growth by PHT-7.3, an inhibitor of the KRas signaling nanocluster protein Cnk1

Activating mutations of KRas is the most common proto-oncogenic event in human cancer but there remains no effective therapy for patients harboring mutated KRas (mut-KRas). Despite intense efforts, tight nucleotide binding, few defined pockets, and redundant localization signals have impeded the development of compounds that bind or inhibit KRas. We have identified connector enhancer of kinase suppressor of Ras 1 (Cnk1) as a critical mediator for growth driven by mut-KRas in human cancer cells. Cnk1 co-localizes with mutant KRas at the membrane and deletion of Cnk1 abrogates KRas activation and the activation of the Ras effectors Ral and Rho. Cnk1 deletion caused cells with mutant KRas to accumulate at the G1 checkpoint similar to selective deletion of mutant KRas itself. Following a screen and initial structural optimization a small molecule probe compound PHT-7.3 was identified and shown to bind selectively to the pleckstrin homology PH domain of Cnk1 preventing Cnk1 and mut-KRas co-localization. PHT-7.3 inhibited mut-KRas but not wt-KRas non small cell lung cancer (nsclc) cell growth, and selectively blocks mut-KRas downstream signaling in cells. PHT-7.3 exhibited cytostatic antitumor activity in the mut-KRas(G12S) A549 and mut-KRas(G12V) H441 nsclc xenografts, but not in the wt-KRas H1975 nsclc xenograft. Mut-KRas downstream signaling was inhibited by PHT-7.3 in the xenografts with downregulation of activated Rho and Ral signaling. PHT-7.3 showed further increased antitumor activity in A549 xenografts in combination with erlotinib or trametinib. Thus, the work identifies the PH domain of Cnk1 as a druggable target whose inhibition selectively blocks mutant-KRas activation, and PHT-7.3 as a lead agent in the development of therapies for KRas tumors. Citation Format: Roisin Delaney, Marco Maruggi, Martin Indarte, Robert Lemos, Geoff Grandjean, Lynn Kirkpatrick, Garth Powis. Selective inhibition of mutant KRAS cell and tumor growth by PHT-7.3, an inhibitor of the KRas signaling nanocluster protein Cnk1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3015. doi:10.1158/1538-7445.AM2017-3015

Abstract 1842: PHT-782 an inhibitor of the KRAS signaling nanocluster protein CNKSR1 blocks mutant KRAS signaling and cell growth but does not inhibit wild type KRAS cell growth

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL KRAS is the predominant form of mutated RAS (mut-KRAS) found in 25% of patient tumors across many cancer types. Mut-KRAS plays a critical role in driving tumor growth and resistance to therapy, and its effects are so powerful that it overrides the activity of many molecularly targeted signaling drugs being developed for cancer today such that they cannot be used in patients with mut-KRAS. Using a global siRNA screen and an isogenic MiaPaCa-2 pancreatic cell line with and without oncogenic KRAS, we searched for genes that when inhibited would block the growth of mut-KRAS cancer cells without affecting wild type-KRAS (wt-KRAS) cell growth. A top hit in the screen was CNKSR1 (connector enhancer of kinase suppressor of RAS 1), a protein associated with KRAS in the RAS membrane signaling nanocluster. siRNA knockdown of CNKSR1 inhibited the growth of mut-KRAS non small cell lung cancer (NSCLC) cell lines but not NSCLC cell lines with wt-KRAS. CNKSR1 is a multidomain protein with a pleckstrin homology (PH) domain, a 100 to 120 amino acid, highly conserved but low sequence identity 3D superfold. PH-domains are found in a number of signaling proteins where they are responsible for binding to membrane phosphatidylinositols-3-phosphates (PIP2/3). Over expression of the PH-domain of CNKSR1 in H1373 mut-KRAS NSCLC cells inhibits cell growth suggesting it has a dominant negative effect over native CNKSR1, and that the PH-domain of CNKSR1 is necessary for the effects on mut-KRAS activity. We used in-silico docking of large compound libraries and identified 7 small molecule compounds with predicted low micromolar binding affinity for the PH-domain of CNSKR1. Surface plasmon resonance spectroscopy confirmed the in-silico predictions with binding to the recombinant PH-domain of CNKSR1. The most active compound was PHT-782 (Kd=1.8 μM). PHT-782 inhibited mut-KRAS signaling and the growth of mut-KRAS isogenic and NSCLC cell lines (IC50 ∼ 30 μM) as effectively as siRNA knockdown of KRAS. Importantly, PHT-782 did not block the growth of wt-KRAS cell lines at concentrations up to 100 μM. In mice PHT-782 exhibited a plasma t12 > 4 hr, and had moderate antitumor activity against a mut-KRAS xenograft model without observable toxicity at doses up to 200 mg/kg. To improve potency and selectivity we have undertaken further iterative in-silico lead optimization using a proprietary computational modeling platform PHuDock®. We have identified several candidates with improved binding potency and optimal pharmacodynamic and pharmacokinetic properties. The results of our studies show that the PH domain of CNKSR1 can be targeted by small molecule inhibitors and that the agents selectively block mut-KRAS signaling and cell growth, thus creating a therapeutic potential for patients with oncogenic KRAS for which there is currently no effective therapy. 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 1842. doi:1538-7445.AM2012-1842

Abstract A197: Oncogenic KRAS is dependent on the scaffold protein CNKSR1 for cell growth and signaling.

KRAS is the predominant form of mutated RAS (mut-KRAS) and is found in 25% of patient tumors across many cancer types. Mut-KRAS is well known to play a critical role in driving tumor growth and resistance to therapy, and its effects are so powerful that it overrides the activity of many of the new molecularly targeted signaling drugs being developed for cancer today such that they cannot be used in patients with mut-KRAS. However, despite extensive effort, there is no effective treatment for mut-KRAS. The effects of mut-KRAS are mediated through multiple downstream signaling pathways which have been independently associated with tumorigenesis, including RAF1, RALGDS, and PI3K. Current efforts to treat mut-KRAS tumors employ concurrent treatment with inhibitors of the RAF and PI3K pathways but this does not address the potential contribution of other pathways for which there are currently no inhibitors. Using a global siRNA screen we searched for genes that when inhibited would block the growth of mut-KRAS cancer cells without affecting wild type-KRAS (wt-KRAS) cell growth using an isogenic MiaPaCa-2 pancreatic cell line with and without oncogenic KRAS, and validated these hits in a similar isogenic HCT-116 colon cell line. From the screen we identified CNKSR1 (connector enhancer of kinase suppressor of Ras 1) as a top hit. CNKSR1 is found associated with KRAS in the RAS membrane associated signaling nanocluster that KRAS has to be associated with to provide growth signals. Knockdown of CNKSR1 with siRNA inhibited the growth of a panel of 10 mut-KRAS non small cell lung cancer (NSCLC) cell lines but not of 4 NSCLC cell lines with wt-KRAS. CNKSR1 is a multidomain protein that has a potentially druggable plekstrin homology (PH) domain responsible for binding to membrane phosphatidylinositols-3-phosphates. In order to demonstrate whether the PH domain of CNKSR1 is necessary for mut-KRAS activity we over expressed the PH domain in H1373 mut-KRAS NSCLC cells and found that it acted as a dominant negative and inhibited cell growth. We suggest that the PH domain fragment competes with the full length CNKSR1 in the cell. We also showed that knockdown of CNKSR1 inhibited KRAS dependent phosphorylation of RAF1 in multiple non small cell lung cancer cell lines. Together our results suggest that the CNKSR1 protein, acting through it9s PH domain, is necessary for cell growth and down stream signaling by the KRAS oncogene. The PH domain can be targeted by inhibitors thus potentially providing agents that will selectively block mut-KRAS signaling and cell growth, creating a therapeutic potential for patients with oncogenic KRAS for which there is currently no effective therapy. 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 A197.

Abstract A199: The plekstrin-homology-domain-containing protein PLEKHA7 is a novel target for selectively inhibiting mutant KRAS colon cancer cell proliferation.

KRAS harbors oncogenic mutations that yield a constitutively active protein in approximately 40% of colorectal cancer. Randomized clinical trials have indicated that the wild-type KRAS (wt-KRAS) is predictive for resistance to anti-EGFR therapy and there is currently no effective therapy for mutant KRAS (mut-KRAS) tumors that is associated with a poor prognosis. Therefore, mut-KRAS tumors are an unmet medical need in colorectal and other cancers. In a search for target genes that when down-regulated selectively inhibit the growth of cells with mut-KRAS but not the cells without mut-KRAS, we used a global synthetic lethal small interfering RNA screen of MiaPaCa-2 and HCT-116 cells (mut-KRAS). The screen identified the gene encoding PLEKHA7 (pleckstrin homology (PH) domain containing, family A member 7). Other PLEKHA family members did not show this effect. PLEKHA7 is a component of the epithelial adherens junction belt that links E-cadherin to the cytoskeleton, and through which KRAS may signal its effects on cell proliferation, invasion and metastasis. PLEKHA7 and KRAS levels were measured by RT-PCR in a panel of 23 colon cancer cell lines (10 wt-KRAS and 13 mut-KRAS) and showed a positive correlation with KRAS levels (R2 = 0.943), and a Wilcoxon Rank test showed that the median expression of PLEKHA7 was higher in cell lines with mut-KRAS than with wt-KRAS (P=0.02). Targeted knockdown of PLEKHA7 using siRNA resulted in growth inhibition for mut-KRAS cells (HCT116, DLD-1, SK-CO-1, SW480, HCT15, and LOVO), but not for wt-KRAS cells (SW48, CACO-2, SW1417, and HT29). Paradoxically, targeted knock-down of KRAS caused an increase in PLEKHA7 mRNA levels in mut-KRAS cells. PLEKHA7 contains a PH-domain in a three-dimensional protein fold that binds to plasma membrane phosphoinositide-3-phosphates (PIP3) formed by phosphatidyl-3-phosphate kinase. We have previously shown that PH-domains can be selectively targeted by small molecules, blocking the binding to PIP3, and inhibiting the function of PH-domain proteins such as Akt and PDPK1. The findings of the present study imply that PLEKHA7 offers a potentially druggable target for inhibiting mut-KRAS-related signaling and cancer-related functions, providing an approach for inhibiting cancer progression of colorectal cancer with KRAS mutation. 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 A199.

Abstract A213: Topical PHT-427, a dual AKT/PDPK1 PH domain inhibitor, for treatment of primary and metastatic skin cancer.

Epicutaneous (topical) therapy with molecularly targeted agents is an attractive modality for treatment of basal cell carcinoma (BCC), early UVB induced squamous cell carcinoma (SCC) and cutaneous metastatic disease (CMD) associated with metastatic breast cancer. There is evidence that Akt and PDKP1 play complimentary yet independent roles in PI3K pathway signaling associated with driving the growth of BCC, UVB induced SCC, and breast CMD. Both Akt and PDPK1 possess a pleckstrin homology (PH) domain, a highly conserved three-dimensional superfold with a high affinity for binding phosphatidylinositol-3-phosphates, causing Akt and PDPK1 to translocation to the plasma membrane where Akt is activated. Through reiterative molecular docking and structure refinement using a proprietary computational platform, we have identified PHT-427 as an agent that binds to the PH domains of Akt and PDPK1, inhibiting their activity. PHT-427 has antitumor activity when administered orally but importantly also following epicutaneous administration. We evaluated the antitumor potential of epicutaneous PHT-427 against CMD in an intradermal breast xenograft model and in an early UVB induced skin cancer model in mice. For the CMD investigation MCF-7 human breast cancer (mutant PIK3CA) was injected intradermally in the flank of female nu/nu mice. Groups of 5 mice were treated twice a day for 10 days with vehicle or 0.1ml PH-427 (50 mg/mL) applied to a 1 cm patch of skin over the tumor. The mice were euthanized 4 hr after the last application and blood, tumor and overlying skin were removed for analyzes. Topical PHT-427 produced 89% inhibition of tumor growth (3/5 no measurable tumor) with no change in body weight or skin toxicity and inhibited targets in skin and tumor. Levels of PHT-427 by HPL-MS were detected in the skin at the site of application, in tumor and in plasma. To evaluate the activity PHT-427 in early UVB induced skin cancer female SKH-1 hairless mice were exposed to UVB irradiation to induce Akt and the production of skin tumors. The mice were treated epicutaneously with PHT-427 or vehicle 3 times a week for 4 weeks at which point animals were sacrificed and skin, tumors and blood were obtained. In this study topical PHT-427 significantly reduced tumor incidence, tumor burden and tumor multiplicity. Following treatment with PHT-427 80% of mice were tumor free and those with tumors averaged fewer than 1 per mouse and those were 1mm in size. There was no change in body weight and no apparent skin toxicity. Thus, topical application of PHT-427 can deliver active drug to skin and tumor, inhibiting AKT and PDPK1, both of which drive the PI3K pathway important in UVB induced SCC and breast CMD, with significant inhibition of tumor growth without adverse effects on normal skin. 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 A213.

Abstract C137: Single-walled carbon nanotubes provide a safe and effective means for delivery of siRNA.

Carbon nanotubes have unique physical and chemical properties that are being widely explored for applications in biomedicine, especially as transporters of drugs, proteins, DNA and RNA. We have shown that siRNA/SWCNT complexes can be delivered safely to animals at high doses over an extended period with no weight loss or changes in blood hematology or chemistry, and produce excellent biological activity including target knockdown and antitumor activity. The current study aimed to prepare stable, optimized SWCNT solutions with consistent siRNA compositions and physical properties and explore cellular uptake. Atomic force microscopy revealed siRNA covers the SWCNT on a mass ratio basis and provided length distributions of the samples. Quantitation of siRNA payload on the SWCNT was achieved using electrophoretic separation of the siRNA from the SWCNT. The stability of siRNA/SWCNT was explored in ribonuclease at 37°C over time and found that siRNA in the complex was stable in 37°C with 80% remaining after 1 hr and 40% after 6 hr versus free siRNA where none remained in solution at 1 hr. Labeled Cy-3-siRNA whose fluorescence is quenched when complexed with SWCNT was used to determine whether the siRNA payload is released intracellularly from the SWCNT following delivery into cells. MiaPaCa pancreatic carcinoma and H2122 non-small cell lung cancer cells were exposed to Cy-3 siRNA/SWCNT for 30 min to 6 hrs. The cells were examined microscopically using near infrared fluorescence detection to observe intracellular SWCNT and visible fluorescence detection for intracellular Cy-3-siRNA. The siRNA/SWCNT complexes readily enter cells within 1 hr and the amount of intracellular SWCNT increase over time to 6 hr. Cy-3-siRNA was released within 1 hr and the siRNA is widely dispersed throughout the cytoplasm. Control cells showed no fluorescence under either condition and intact Cy-3-siRNA/SWCNT complex was not fluorescent due to quenching. We also used Cy-3 labeled siRNA to compare the transfection efficiency of siRNA/SWCNT to that of liposomal delivery. Using equivalent amounts of siRNA, SWCNT/siRNA complexes delivered and released siRNA intracellularly more quickly and distributed the siRNA more evenly within a 1 hr time frame as compared to liposomal delivery. Finally, in vivo delivery of Cy-3-siRNA/SWCNT appears to demonstrate a more prolonged and pronounced distribution of siRNA than that of free Cy-3 siRNA. The studies reported here provide data showing the stability of the SWCNT/siRNA complexes in vitro and in vivo and illustrate their cell penetrating ability with release of the payload intracellularly. We believe the low toxicity, the excellent membrane penetration ability, the protection afforded against blood breakdown of the siRNA payload and the good biological activity seen in vivo will allow SWCNTs to become universal transfection vehicles for siRNA and other RNAs for therapeutic applications. 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 C137.