Augustine T. Nkembo

Polyisoprenylated cysteinyl amide inhibitors disrupt actin cytoskeleton organization, induce cell rounding and block migration of non-small cell lung cancer

The malignant potential of Non-Small Cell Lung Cancer (NSCLC) is dependent on cellular processes that promote metastasis. F-actin organization is central to cell migration, invasion, adhesion and angiogenesis, processes involved in metastasis. F-actin remodeling is enhanced by the overexpression and/or hyper-activation of some members of the Rho family of small GTPases. Therefore, agents that mitigate hyperactive Rho proteins may be relevant for controlling metastasis. We previously reported the role of polyisoprenylated cysteinyl amide inhibitors (PCAIs) as potential inhibitors of cancers with hyperactive small GTPases. In this report, we investigate the potential role of PCAIs against NSCLC cells and show that as low as 0.5 μM PCAIs significantly inhibit 2D and 3D NCI-H1299 cell migration by 48% and 45%, respectively. PCAIs at 1 μM inhibited 2D and 3D NCI-H1299 cell invasion through Matrigel by 50% and 85%, respectively. Additionally, exposure to 5 μM of the PCAIs for 24 h caused at least a 66% drop in the levels of Rac1, Cdc42, and RhoA and a 38% drop in F-actin intensity at the cell membrane. This drop in F-actin was accompanied by a 73% reduction in the number of filopodia per cell. Interestingly, the polyisoprenyl group of the PCAIs is essential for these effects, as NSL-100, a non-farnesylated analog, does not elicit similar effects on F-actin assembly and organization. Our findings indicate that PCAIs disrupt F-actin assembly and organization to suppress cell motility and invasion. The PCAIs may be an effective therapy option for NSCLC metastasis and invasion control.

Suppression of focal adhesion formation may account for the suppression of cell migration, invasion and growth of non-small cell lung cancer cells following treatment with polyisoprenylated cysteinyl amide inhibitors

Migratory cells form extracellular matrix attachments called focal-adhesions. Focal adhesion assembly and disassembly are regulated by the Rho family of small GTPases. We previously reported that polyisoprenylated cysteinyl amide inhibitors (PCAIs) suppress Rho protein levels, disrupting F-actin cytoskeleton remodeling in the formation of lamellipodia and filopodia. In this study, we investigated whether these observations effect focal adhesion formation, which involves cell surface receptors known as integrins and several signaling/adaptor proteins such as vinculin, α-actinin, Rock kinases and phospho-Myosin Light Chain-2 (p-MLC-2), that foster the linkage of the actin cytoskeleton to the extracellular matrix. We observed that treatment of H1299 cells with 5 μM PCAIs for 24 h markedly diminished the level of full-length integrin α4 by at least 24% relative to controls. PCAIs at 5 μM, diminished the levels of vinculin by at least 50%. Immunofluorescent analysis showed at least a 76% decrease in the number of vinculin-focal adhesion punctates. In addition, PCAIs diminished Rock1 levels by 25% and its substrate, p-MLC-2 by 75%. PCAIs did not significantly alter the levels of integrin β5, α-actinin, and Rock2, suggesting that the effects of the PCAIs are target specific. Our data indicate that the PCAIs alter the levels of the Rho proteins and their effectors to abrogate their functions in cytoskeleton remodeling thereby suppressing focal adhesion formation. This in turn results in a PCAIs-induced decrease in cell invasion, thus making the PCAIs propitious agents for the inhibition of cancer growth and metastasis.

Abstract 2308: Sigma-2 receptor ligands induce apoptosis and inhibit proliferation in breast cancer cell line mda-mb-231

Introduction: Despite the reduction in mortality from breast cancer achieved in the last 30 years due to more effective targeted therapies, the survival rate for patients with triple negative breast cancer (TNBC) is poor and has virtually plateaued. Due to the absence of estrogen, progesterone, and human epidermal growth factor receptors 2, no targeted treatment options are currently available for TNBC. Moreover, the current standard systemic treatments are known to cause tremendous side effects. Thus, there is an urgent need to identify novel strategies for treating TNBC. Several reports have revealed that sigma-2 receptors are over-expressed in all solid tumors including TNBC, making it a plausible biomarker to explore for TNBC treatment. In this study, we investigated the effects of novel sigma-2 receptor ligands (XYZ-XI-14 and XYZ-VII-69) synthesized in our lab on the viability and survival of the TNBC, MDA-MB-231. Methods: MDA-MB-231 cells were treated for 48 h with XYZ-XI-14 and XYZ-VII-69, cell viability and proliferation were assessed using resazurin cell titre assay and cell count methods respectively. The effects of the ligands on spheroids formation, cell cycle, and mode of cell death were also investigated. Results: XYZ-XI-14 and XYZ-VII-69 decreased cell viability of MDA-MB-231 cells in a concentration-dependent manner. The EC50 for XYZ-XI-14 and XYZ-VII-69 were 12 and 13 µM respectively; and at 5 µM, the ligands inhibited cell proliferation, induced apoptosis, and arrested MDA-MB-231 cells at the G0/G1 phase of cell cycle. Additionally, concentrations of the ligand as low as 1 µM prevented the formation of spheroids as evidenced by the lack of compact spheroids, and caused the disintegration of preformed spheroids. Conclusion: This study indicates that targeting sigma-2 receptors with novel sigma-2 ligands (XYZ-XII-14 and XYZ-VII-69) effectively inhibits TNBC cancer cell growth by inducing apoptosis and cell cycle arrest, thus presenting a unique and effective pathway for treating TNBC. Additionally, the sigma-2 receptor ligands (XYZ-XI-14 and XYZ-VII-69) have the potential to halt tumor growth and prevent tumor relapse, as seen in our 3D culture assays. Thus, the sigma-2 receptor has the potential to be a valuable target for the development of novel agents for the treatment of TNBC. Citation Format: Gladys Asong, Felix Amissah, Olufisayo Salako, Rosemary Poku, Elizabeth Ntantie, Augustine Nkembo, Nazarius Lamango, Seth Ablordeppey. Sigma-2 receptor ligands induce apoptosis and inhibit proliferation in breast cancer cell line mda-mb-231 [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 2308. doi:10.1158/1538-7445.AM2017-2308

Abstract A03: Novel polyisoprenylated small molecules as potential new therapies for triple negative breast cancer

Breast cancer patients whose tumors do not overexpress estrogen, progesterone or HER2 receptors do not benefit from the effective therapies targeting these receptors. Knowing what drives the tumors in the latter group of patients, known as triple negative breast cancer (TNBC), is important for developing more effective and less toxic therapies directed at these drivers. We have developed a novel class of compounds, the polyisoprenylated cysteinyl amide inhibitors (PCAIs) that target the aberrant growth signaling by EGFR and/or constitutively active mutant K-Ras in a significant proportion of TNBC and other aggressive cancers such as of the pancreas, colon and lungs. The PCAIs induced apoptotic cell death with EC50 values ranging from 2.2 to 2.7 µM. The PCAIs induced apoptosis in MDA-MB-231 spheroids as well as inhibited the spheroid formation at 1 and 2 µM, respectively. The PCAIs, at 1 µM, inhibited over 80% colony formation in MDA-MB-231 cells. At 1 and 2 µM, MDA-MB-231 Transwell cell invasion was inhibited by 80 and 94%, respectively. The PCAIs blocked tube formation in cultured HUVEC cells at 0.2 to 0.5 µM and vessel formation in chick embryos at 0.12 µg/egg. The PCAIs can thus be used as targeted therapies to inhibit tumor growth, cell migration and angiogenesis in TNBC and other cancers with excessive growth factors receptor and mutant K-Ras signaling. Citation Format: Olufisayo O. Salako, Augustine T. Nkembo, Elizabeth Ntantie, Nazarius S. Lamango. Novel polyisoprenylated small molecules as potential new therapies for triple negative breast cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A03.

The antiangiogenic effects of polyisoprenylated cysteinyl amide inhibitors in HUVEC, chick embryo and zebrafish is dependent on the polyisoprenyl moiety

Angiogenesis is essential for solid tumor growth, therapeutic resistance and metastasis, the latest accounting for 90% of cancer deaths. Although angiogenesis is essential for the malignant transformations in solid tumors and therefore is an attractive target, few drugs are available that block tumor angiogenesis. The focus has been to block signaling by receptor tyrosine kinases (RTKs), such as for vascular endothelial growth factor (VEGF), whose activation abrogate apoptosis and promote angiogenesis. The polyisoprenylated cysteinyl amide inhibitors (PCAIs) were designed to modulate aberrant polyisoprenylated small G-proteins such as mutant Ras whose constitutive activation promotes RTKs signaling. Since polyisoprenylation is essential for protein-protein interactions and functions of G-proteins, we hypothesized that the PCAIs would disrupt the monomeric G-protein signaling thereby effectively inhibiting angiogenesis. In this study we determined the effects of PCAIs on human umbilical vein endothelial cells (HUVEC) tube formation, cell viability, cell migration and invasion as well as in vivo using the chick chorioallantoic membrane (CAM) and zebrafish models. At sub- to low micromolar concentrations, the PCAIs inhibit the native and VEGF-stimulated cell migration and invasion as well as tube formation and angiogenesis in CAM and zebrafish embryos. The concentrations that block the angiogenic processes were lower than those that induce cell death. Since angiogenesis is essential for tumor growth but otherwise limited to wound healing, feeding fat cells and uterine wall repair in adults, it is conceivable that these compounds can be developed into safer therapeutics for cancers and retinal neovascularization that leads to loss of vision.

Abstract 1869: Dislodgement of K-Ras from plasma membranes, induction of apoptosis and tumor regression by PCAIs, a novel class of polyisoprenylated small molecules

Although mutation-induced dysregulation of Ras signaling constitutes the biochemical change that drives some of the most difficult-to-manage cancers, directly targeting the constitutively active mutant Ras GTPases has not resulted in clinically useful drugs. Therefore, modulating Ras activity for targeted treatment of cancer remains an urgent healthcare need. In the current study, we investigated a novel class of compounds, the polyisoprenylated cysteinyl amide inhibitors (PCAIs), for their anticancer molecular mechanisms using the NSCLC cell panel with K-Ras and/or other mutant genes. Treatment of the lung cancer cells with PCAIs, NSL-RD-035, NSL-BA-036, NSL-BA-040, NSL-BA-055 and NSL-BA-040 resulted in concentration-dependent cell death in both K-Ras mutant (A549 and NCI-H1573), N-Ras mutant (NCI-H1299) and other (NCI-H661, NCI-H460, NCI-H1975, NCI-H1563) NSCLC cells. The PCAIs at sub- to low micromolar 1.0 -10 μM concentrations induced the degeneration of 3D spheroid cultures, inhibited, clonogenic cell growth, and induced marked apoptosis and cell cycle arrest, together with a significant increase in active caspase 3 (p Citation Format: FELIX AMISSAH, ELIZABETH NTANTIE, ROSEMARY A. POKU, AUGUSTINE T. NKEMBO, OLUFISAYO O. SALAKO, HERNAN FLORES-ROZAS, NAZARIUS S. LAMANGO. Dislodgement of K-Ras from plasma membranes, induction of apoptosis and tumor regression by PCAIs, a novel class of polyisoprenylated small molecules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1869.

Abstract B89: Polyisoprenylated cysteinyl amide inhibitors of PMPMEase inhibit pancreatic cancer cell viability and migration: Implications for pancreatic cancer therapy

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most deadly neoplasms due to lack of effective targeted therapies. This vacuum stems from the fact that over 90% of pancreatic cancer cases harbor the K-Ras mutation which has been very challenging to drug. The numerous efforts to drug oncogenic Ras have involved inhibiting the post-translational modifications that are essential to its proliferative activity. These drug discovery efforts have largely ignored a critical step in the metabolism of Ras and related proteins involving polyisoprenylated methylated protein methyl esterase (PMPMEase). The objectives of these studies were to test the hypothesis that PMPMEase hyperactivity promotes pancreatic cancer progression. The potential for polyisoprenylated cysteinyl amide inhibitors (PCAIs) of PMPMEase as potential anticancer agents was evaluated against PDAC cell lines MIAPaCa-2 with K-Ras mutation and BxPC-3 with wild type K-Ras for their effects on cell viability, migration, and cytoskeletal organization. The PCAIs inhibited the viability of MIAPaCa-2 and BxPC-3 cells with 48 h EC50 values as low as 1.9 and 3.2 µM, respectively. Erlotinib and Salirasib (FTS) were ineffective at concentrations in excess of 20 µM. The PCAIs also inhibited MIAPaCa-2 cell migration by up to 50% at 0.5 µM and disrupted F-actin organization at 5 µM. Further investigations showed that at the EC50 concentrations the PCAIs caused the pancreatic cancer cells to die by apoptosis. The cell cycle analysis for MIAPaCa-2 cells showed that the PCAIs block the cells growth at G0/G1 phase. The results showed that the PCAIs are over 10-fold more effective than Salirasib and Erlotinib against PDAC cell viability. Furthermore, their effects against cell migration and actin filament organization suggest their potential for treating tumor growth and metastasis in not only PDAC but other cancers with oncogenic Ras. Citation Format: Augustine T. Nkembo, Byron J. aguilar, Randolph Duverna, Felix Amissah, Nazarius S. Lamango. Polyisoprenylated cysteinyl amide inhibitors of PMPMEase inhibit pancreatic cancer cell viability and migration: Implications for pancreatic cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B89.

Abstract 2035: Targeting the hyperactive polyisoprenylated monomeric G-proteins functions in pancreatic cancer

Pancreatic cancer (PC) incidence is a major healthcare problem as the five-year survival rate remains below 5%. Over 90% of PC cases harbor the hyperactive K-Ras mutant protein. Polyisoprenylation processes facilitate the proper localization and signaling of K-Ras whose gain-in-function mutations drive uncontrolled cell survival, differentiation and proliferation. Efforts to target and disrupt this pathway and signaling for cancer therapy have been unsuccessful. The purpose of this study was to determine the effects of polyisoprenylated cysteinyl amide inhibitors (PCAIs) as a potential novel class of small molecules for PC therapy. The effects of the PCAIs on cell viability, proliferation, survival, apoptosis, migration, colony formation and cytoskeletal F-actin organization in four human PC cell lines representing wild type K-Ras (BxPC-3), Mutant K-Ras (MIAPaCa-2 and Panc10.05) and mucins (HPAF-II) were examined. PCAIs induced apoptosis with EC50 values ranging from 2.4 to 5.1, 2.0 to 7.3, 1.8 to 3.3 and 7.0 to 33 μM in MIAPaCa-2, BxPC-3, Panc10.05 and HPAF-II cells, respectively. At 0.2 μM, PCAIs arrested MIAPaCa-2 cells at the G0/G1 phase and significantly inhibited cell migration in the wound-healing assay. MIAPaCa-2 cells transfected with a plasmid expressing a protein that fluoresces red when interacting with F-actin were used to determine the PCAIs effects on F-actin organization. Treatment with PCAIs disrupted the F-actin structures causing the cells to shrink and become rounded upon treatment with PCAIs concentrations as low as 0.2 μM. The ability of PCAIs to disrupt these biological phenomena in pancreatic cancer cells shows that PCAIs may halt progression in tumors with the hyperactive K-Ras mutations. Citation Format: Augustine T. Nkembo, Olufisayo Salako, Rosemary Poku, Tryphon Mazu, Byron Aguilar, Hernan Flores-Rozas, Nazarius S. Lamango. Targeting the hyperactive polyisoprenylated monomeric G-proteins functions in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2035. doi:10.1158/1538-7445.AM2015-2035

Abstract 660: Polyisoprenylated cysteinyl amide inhibitors exhibit anti-tumor effects in human breast cancer cell lines

PURPOSE: Triple negative breast cancer (TNBC) is the most aggressive and fatal form of breast cancer. TNBC has no effective therapies since it is not driven by the estrogen (ER), progesterone (PR), and human epidermal growth factor type 2 (Her2/Neu) receptors for which there are targeted drugs. Human epidermal growth factor receptors (EGFR) are overexpressed in 92% of TNBC cases, however, anti-EGFR therapies are ineffective. Monomeric G-proteins that regulate cell proliferation, angiogenesis, migration and apoptosis operate downstream of EGFR and other signal transduction pathways. Polyisoprenylated methylated protein methyl esterase (PMPMEase) metabolizes monomeric G-proteins, allowing them to function properly. PMPMEase is overexpressed in many cancers and may serve as a biomarker and target for cancer therapy. We have synthesized polyisoprenylated cysteinyl amide inhibitors that may inhibit PMPMEase and/or disrupt polyisoprenylated protein function. We hypothesize that the PCAIs will promote anti-tumorigenic effects, inducing apoptosis, while inhibiting cell survival, proliferation, migration, and invasion. The purpose of this study was to test the effectiveness of PCAIs against the biological hallmarks of TNBC. METHODS: Four TNBC cell lines were treated with the PCAIs and analyzed for their effects on cell survival, viability, F-actin organization, cell migration and apoptosis. RESULTS: Treatment of TNBC cells with the PCAIs resulted in a concentration-dependent death of the TNBC cells with EC50 values ranging from 2.13 to 2.78 μM for NSL-BA-040 and NSL-BA-055 in media containing 5% FBS. AO/EB staining revealed early apoptosis at 1 μM. NSL-RD-036 inhibited MDA-MB-231 cell migration by 71% (163±10.7 cells in control versus 47±6.9 in treated (±SEM, N = 3). PCAIs induced complete disruption of F-actin filament organization at 2 μM in MDA-MB-468 cells. CONCLUSION: The PCAIs’ ability to induce apoptosis, and inhibit cell migration by disrupting F-actin filaments suggest that their potential therapeutic value could involve the inhibition of TNBC tumor growth, metastasis, and angiogenesis, which are all essential hallmarks for breast cancer progression. The PCAIs may ultimately fill the targeted therapy void in TNBC therapy. Citation Format: Olufisayo O. Salako, Rosemary A. Poku, Augustine T. Nkembo, Typhon Mazu, Hernan Flores-Rozas, Nazarius S. Lamango. Polyisoprenylated cysteinyl amide inhibitors exhibit anti-tumor effects in human breast cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 660. doi:10.1158/1538-7445.AM2015-660

Abstract LB-14: Polyisoprenylated cysteinyl amides as targeted small molecule anti-Ras therapies

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Pancreatic cancer harbors the highest reported rates of K-Ras mutations, lacks effective therapies and is the most challenging to treat. It is thus the most deadly neoplasm with a 5-year survival rate of only 6%. We reveal here that polyisoprenylated methylated protein methyl esterase (PMPMEase) is overexpressed in 93% of pancreatic duct adenocarcinoma. We further present novel compounds with the potential to serve as effective targeted therapies for pancreatic and other cancers with hyperactive growth signaling pathways mediated by Ras and related G-proteins. The design and synthesis of the polyisoprenylated cysteinyl amide inhibitors (PCAIs) of PMPMEase incorporate three key elements to obtain compounds to selectively disrupt polyisoprenylation-mediated protein-protein interactions; the farnesyl group for high affinity interactions, a substituted amide bioisostere of the scissile ester bond of the endogenous substrates and an ionizable appendage group designed to mitigate the excessive hydrophobicity of the farnesyl cysteinyl amide that constitutes the pharmacophore. The PCAIs inhibited PMPMEase with Ki values ranging from 3.7 to 20 μM. The 48 h EC50 values for pancreatic cancer Mia PaCa-2 and BxPC-3 cell lines were as low as 1.9 μM while Salirasib (farnesylthiosalicylic acid, FTS) and farnesylthiosalicylamide were not effective even at 20 μM. The PCAIs thus have the potential to serve as a novel class of targeted therapies for cancers with hyperactive G-proteins and the growth factor receptors whose signals they transmit. Citation Format: Nazarius S. Lamango, Byron J. Aguilar, Augustine T. Nkembo, Randolph Duverna, Felix Amissah, Rosemary A. Poku, Seth Y. Ablordeppey. Polyisoprenylated cysteinyl amides as targeted small molecule anti-Ras therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-14. doi:10.1158/1538-7445.AM2014-LB-14