Raghavendra Gowda

Targeting V600EB-Raf and Akt3 Using Nanoliposomal-Small Interfering RNA Inhibits Cutaneous Melanocytic Lesion Development

Most events promoting early melanoma development are yet to be identified, but deregulation of the B-Raf and Akt3 signaling cascades is an important regulator of this process. Approximately 90% of normal moles and approximately 60% of early invasive cutaneous melanomas contain a T1799A B-Raf mutation ((V600E)B-Raf), leading to 10 times higher enzyme activity and constitutive activation of the mitogen-activated protein kinase pathway. Furthermore, approximately 70% of melanomas have elevated Akt3 signaling due to increased gene copy number and PTEN loss. Therefore, targeting (V600E)B-Raf and Akt3 signaling is necessary to prevent or treat cutaneous melanocytic lesions. Agents specifically targeting these proteins are needed, having fewer side effects than those inhibiting both normal and mutant B-Raf protein or targeting all three Akt isoforms. In this study, a unique nanoliposomal-ultrasound-mediated approach has been developed for delivering small interfering RNA (siRNA) specifically targeting (V600E)B-Raf and Akt3 into melanocytic tumors present in skin to retard melanoma development. Novel cationic nanoliposomes stably encapsulate siRNA targeting (V600E)B-Raf or Akt3, providing protection from degradation and facilitating entry into melanoma cells to decrease expression of these proteins. Low-frequency ultrasound using a lightweight four-cymbal transducer array enables penetration of nanoliposomal-siRNA complex throughout the epidermal and dermal layers of laboratory-generated or animal skin. Nanoliposomal-mediated siRNA targeting of (V600E)B-Raf and Akt3 led to a cooperatively acting approximately 65% decrease in early or invasive cutaneous melanoma compared with inhibition of each singly with negligible associated systemic toxicity. Thus, cationic nanoliposomes loaded with siRNA targeting (V600E)B-Raf and Akt3 provide an effective approach for targeted inhibition of early or invasive cutaneous melanomas.

Targeting casein kinase II restores Ikaros tumor suppressor activity and demonstrates therapeutic efficacy in high-risk leukemia

Ikaros (IKZF1) is a tumor suppressor that binds DNA and regulates expression of its target genes. The mechanism of Ikaros activity as a tumor suppressor and the regulation of Ikaros function in leukemia are unknown. Here, we demonstrate that Ikaros controls cellular proliferation by repressing expression of genes that promote cell cycle progression and the phosphatidylinositol-3 kinase (PI3K) pathway. We show that Ikaros function is impaired by the pro-oncogenic casein kinase II (CK2), and that CK2 is overexpressed in leukemia. CK2 inhibition restores Ikaros function as transcriptional repressor of cell cycle and PI3K pathway genes, resulting in an antileukemia effect. In high-risk leukemia where one IKZF1 allele has been deleted, CK2 inhibition restores the transcriptional repressor function of the remaining wild-type IKZF1 allele. CK2 inhibition demonstrated a potent therapeutic effect in a panel of patient-derived primary high-risk B-cell acute lymphoblastic leukemia xenografts as indicated by prolonged survival and a reduction of leukemia burden. We demonstrate the efficacy of a novel therapeutic approach for high-risk leukemia: restoration of Ikaros tumor suppressor activity via inhibition of CK2. These results provide a rationale for the use of CK2 inhibitors in clinical trials for high-risk leukemia, including cases with deletion of one IKZF1 allele.

Targeting sphingosine kinase-1 to inhibit melanoma

Resistance to therapies develops rapidly for melanoma leading to more aggressive disease. Therefore, agents are needed that specifically inhibit proteins or pathways controlling the development of this disease, which can be combined, dependent on genes deregulated in a particular patient’s tumors. This study shows that elevated sphingosine‐1‐phosphate (S‐1‐P) levels resulting from increased activity of sphingosine kinase‐1 (SPHK1) occur in advanced melanomas. Targeting SPHK1 using siRNA decreased anchorage‐dependent and ‐independent growth as well as sensitized melanoma cells to apoptosis‐inducing agents. Pharmacological SPHK1 inhibitors SKI‐I but not SKI‐II decreased S‐1‐P content, elevated ceramide levels, caused a G2‐M block and induced apoptotic cell death in melanomas. Targeting SPHK1 using siRNA or the pharmacological agent called SKI‐I decreased the levels of pAKT. Furthermore, SKI‐I inhibited the expression of CYCLIN D1 protein and increased the activity of caspase‐3/7, which in turn led to the degradation of PARP. In animals, SKI‐I but not SKI‐II retarded melanoma growth by 25–40%. Thus, targeting SPHK1 using siRNAs or SKI‐I has therapeutic potential for melanoma treatment either alone or in combination with other targeted agents.

Selenium-containing histone deacetylase inhibitors for melanoma management

Melanoma incidence and mortality rates continue to increase each year. Lack of clinically viable agents, drug combinations, effective targeted delivery approaches and success inhibiting targets in tumor tissue have made this disease one of the most difficult to treat, which makes prevention an important option for decreasing disease incidence and mortality rates. Inhibiting histone deacetylases (HDAC) is an approach currently being explored to more effectively treat melanoma but use for prevention has not been explored. In this study, novel selenium containing derivatives of the FDA approved HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) called 5-phenylcarbamoylpentyl selenocyanide (PCP-SeCN) and Bis{5-phenylcarbamoylpentyl} diselenide (B(PCP)-2Se) were created and efficacy tested for preventing early melanocytic lesion development in skin. Topical application of PCP-SeCN and B(PCP)-2Se inhibited melanocytic lesion development in laboratory-generated skin by up to 87% with negligible toxicological effect. Mechanistically, PCP-SeCN and B(PCP)-2Se inhibited HDAC activity and had new inhibitory properties by moderating Akt activity to induce cellular apoptosis as demonstrated by an increase in the sub-G0-G1 cell population, and cleaved caspase-3 as well as PARP levels. Furthermore, PCP-SeCN and B(PCP)-2Se inhibited cell proliferation by inhibiting cyclin D1 expression and increasing p21 levels. Thus, PCP-SeCN and B(PCP)-2Se are potential melanoma chemopreventive agents with enhanced efficacy compared with SAHA due to new PI3 kinase pathway inhibitory properties.

Use of Nanotechnology to Develop Multi-Drug Inhibitors For Cancer Therapy.

Therapeutic agents that inhibit a single target often cannot combat a multifactorial disease such as cancer. Thus, multi-target inhibitors (MTIs) are needed to circumvent complications such as the development of resistance. There are two predominant types of MTIs, (a) single drug inhibitor (SDIs) that affect multiple pathways simultaneously, and (b) combinatorial agents or multi-drug inhibitors (MDIs) that inhibit multiple pathways. Single agent multi-target kinase inhibitors are amongst the most prominent class of compounds belonging to the former, whereas the latter includes many different classes of combinatorial agents that have been used to achieve synergistic efficacy against cancer. Safe delivery and accumulation at the tumor site is of paramount importance for MTIs because inhibition of multiple key signaling pathways has the potential to lead to systemic toxicity. For this reason, the development of drug delivery mechanisms using nanotechnology is preferable in order to ensure that the MDIs accumulate in the tumor vasculature, thereby increasing efficacy and minimizing off-target and systemic side effects. This review will discuss how nanotechnology can be used for the development of MTIs for cancer therapy and also it concludes with a discussion of the future of nanoparticle-based MTIs as well as the continuing obstacles being faced during the development of these unique agents.’

Leelamine Mediates Cancer Cell Death through Inhibition of Intracellular Cholesterol Transport

Leelamine is a promising compound for the treatment of cancer; however, the molecular mechanisms leading to leelamine-mediated cell death have not been identified. This report shows that leelamine is a weakly basic amine with lysosomotropic properties, leading to its accumulation inside acidic organelles such as lysosomes. This accumulation leads to homeostatic imbalance in the lysosomal endosomal cell compartments that disrupts autophagic flux and intracellular cholesterol trafficking as well as receptor-mediated endocytosis. Electron micrographs of leelamine-treated cancer cells displayed accumulation of autophagosomes, membrane whorls, and lipofuscin-like structures, indicating disruption of lysosomal cell compartments. Early in the process, leelamine-mediated killing was a caspase-independent event triggered by cholesterol accumulation, as depletion of cholesterol using β-cyclodextrin treatment attenuated the cell death and restored the subcellular structures identified by electron microscopy. Protein microarray–based analyses of the intracellular signaling cascades showed alterations in RTK–AKT/STAT/MAPK signaling cascades, which was subsequently confirmed by Western blotting. Inhibition of Akt, Erk, and Stat signaling, together with abnormal deregulation of receptor tyrosine kinases, was caused by the inhibition of receptor-mediated endocytosis. This study is the first report demonstrating that leelamine is a lysosomotropic, intracellular cholesterol transport inhibitor with potential chemotherapeutic properties leading to inhibition of autophagic flux and induction of cholesterol accumulation in lysosomal/endosomal cell compartments. Importantly, the findings of this study show the potential of leelamine to disrupt cholesterol homeostasis for treatment of advanced-stage cancers. Mol Cancer Ther; 13(7); 1690–703. ©2014 AACR.

Simultaneous Targeting of COX-2 and AKT Using Selenocoxib-1-GSH to Inhibit Melanoma

Melanoma is a highly metastatic and deadly disease. An agent simultaneously targeting the COX-2, PI3K/Akt, and mitogen-activated protein kinase (MAPK) signaling pathways that are deregulated in up to 70% of sporadic melanomas might be an effective treatment, but no agent of this type exists. To develop a single drug inhibiting COX-2 and PI3K/Akt signaling (and increasing MAPK pathway activity to inhibitory levels as a result of Akt inhibition), a selenium-containing glutathione (GSH) analogue of celecoxib, called selenocoxib-1-GSH was synthesized. It killed melanoma cells with an average IC50 of 7.66 μmol/L compared with control celecoxib at 55.6 μmol/L. The IC50 range for normal cells was 36.3 to 41.2 μmol/L compared with 7.66 μmol/L for cancer cells. Selenocoxib-1-GSH reduced development of xenografted tumor by approximately 70% with negligible toxicity by targeting COX-2, like celecoxib, and having novel inhibitory properties by acting as a PI3K/Akt inhibitor (and MAPK pathway activator to inhibitory levels due to Akt inhibition). The consequence of this inhibitory activity was an approximately 80% decrease in cultured cell proliferation and an approximately 200% increase in apoptosis following 24-hour treatment with 15.5 μmol/L of drug. Thus, this study details the development of selenocoxib-1-GSH, which is a nontoxic agent that targets the COX-2 and PI3K/Akt signaling pathways in melanomas to inhibit tumor development. Mol Cancer Ther; 12(1); 3–15. ©2012 AACR.

Targeting Multiple Key Signaling Pathways in Melanoma Using Leelamine

Melanoma is a highly drug-resistant cancer with resistance developing to agents targeting single proteins. To circumvent this problem, a new class of agent inhibiting multiple key pathways important in this disease is being developed to reduce the likelihood of developing resistant disease. The phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), and STAT3 pathways are constitutively activated in 50% to 70% of melanomas, promoting disease development. To identify a drug simultaneously targeting the PI3K, MAPK, and STAT3 cascades, a natural product library was screened to identify leelamine as a potential inhibitor. Leelamine was 4.5-fold more effective at inhibiting cultured melanoma cell survival than normal cells, with average IC50 values of 2 and 9.3 μmol/L, respectively. It inhibited cellular proliferation at a concentration of 2.5 μmol/L by 40% to 80% and longer exposure increased apoptosis 600%. Leelamine inhibited the growth of preexisting xenografted melanoma tumors by an average of 60% by targeting the PI3K, MAPK, and STAT3 pathways without affecting animal body weight or blood markers of major organ function. The mechanism of action of leelamine is mediated by disruption of cholesterol transport, causing decreased cellular proliferation and consequently leading to increased tumor cell apoptosis as well as decreased tumor vascularization. Thus, a unique agent and novel mechanism of action has been identified for the treatment of melanoma that acts by inhibiting the activity of three major signaling pathways regulating the development of this disease. Mol Cancer Ther; 13(7); 1679–89. ©2014 AACR.

Identification of Aurora Kinase B and Wee1-Like Protein Kinase as Downstream Targets of V600EB-RAF in Melanoma

BRAF is the most mutated gene in melanoma, with approximately 50% of patients containing V600E mutant protein. (V600E)B-RAF can be targeted using pharmacological agents, but resistance develops in patients by activating other proteins in the signaling pathway. Identifying downstream members in this signaling cascade is important to design strategies to avoid the development of resistance. Unfortunately, downstream proteins remain to be identified and therapeutic potential requires validation. A kinase screen was undertaken to identify downstream targets in the (V600E)B-RAF signaling cascade. Involvement of aurora kinase B (AURKB) and Wee1-like protein kinase (WEE1) as downstream proteins in the (V600E)B-RAF pathway was validated in xenografted tumors, and mechanisms of action were characterized in size- and time-matched tumors. Levels of only AURKB and WEE1 decreased in melanoma cells, when (V600E)B-RAF, mitogen-activated protein kinase 1/2, or extracellular signal-regulated kinase 1/2 protein levels were reduced using siRNA compared with other identified kinases. AURKB and WEE1 were expressed in tumors of patients with melanoma at higher levels than observed in normal human melanocytes. Targeting these proteins reduced tumor development by approximately 70%, similar to that observed when inhibiting (V600E)B-RAF. Furthermore, protein or activity levels of AURKB and WEE1 decreased in melanoma cells when pharmacological agents targeting upstream (V600E)B-RAF or mitogen-activated protein kinase were used to inhibit the (V600E)B-RAF pathway. Thus, AURKB and WEE1 are targets and biomarkers of therapeutic efficacy, lying downstream of (V600E)B-RAF in melanomas.

Identification of glycogen synthase kinase 3α as a therapeutic target in melanoma.

Deregulated expression or activity of kinases can lead to melanomas, but often the particular kinase isoform causing the effect is not well established, making identification and validation of different isoforms regulating disease development especially important. To accomplish this objective, an siRNA screen was undertaken that which identified glycogen synthase kinase 3α (GSK3α) as an important melanoma growth regulator. Melanocytes and melanoma cell lines representing various stages of melanoma tumor progression expressed both GSK3α and GSK3β, but analysis of tumors in patients with melanoma showed elevated expression of GSK3α in 72% of samples, which was not observed for GSK3β. Furthermore, 80% of tumors in patients with melanoma expressed elevated levels of catalytically active phosphorylated GSK3α (pGSK3αY279), but not phosphorylated GSK3β (pGSK3βY216). siRNA‐mediated reduction in GSK3α protein levels reduced melanoma cell survival and proliferation, sensitized cells to apoptosis‐inducing agents and decreased xenografted tumor development by up to 56%. Mechanistically, inhibiting GSK3α expression using siRNA or the pharmacological agent AR‐A014418 arrested melanoma cells in the G0/G1 phase of the cell cycle and induced apoptotic death to retard tumorigenesis. Therefore, GSK3α is a key therapeutic target in melanoma.