Vidya P. Ramamurthy

Galeterone and VNPT55 induce proteasomal degradation of AR/AR-V7, induce significant apoptosis via cytochrome c release and suppress growth of castration resistant prostate cancer xenografts in vivo

Galeterone (Gal) is a first-in-class multi-target oral small molecule that will soon enter pivotal phase III clinical trials in castration resistant prostate cancer (CRPC) patients. Gal disrupts androgen receptor (AR) signaling via inhibition of CYP17, AR antagonism and AR degradation. Resistance to current therapy is attributed to up-regulation of full-length AR (fAR), splice variants AR (AR-Vs) and AR mutations. The effects of gal and VNPT55 were analyzed on f-AR and AR-Vs (AR-V7/ARv567es) in LNCaP, CWR22Rv1 and DU145 (transfected with AR-Vs) human PC cells in vitro and CRPC tumor xenografts. Galeterone/VNPT55 decreased fAR/AR-V7 mRNA levels and implicates Mdm2/CHIP enhanced ubiquitination of posttranslational modified receptors, targeting them for proteasomal degradation. Gal and VNPT55 also induced significant apoptosis in PC cells via increased Bax/Bcl2 ratio, cytochrome-c release with concomitant cleavage of caspase 3 and PARP. More importantly, gal and VNPT55 exhibited strong in vivo anti-CRPC activities, with no apparent host toxicities. This study demonstrate that gal and VNPT55 utilize cell-based mechanisms to deplete both fAR and AR-Vs. Importantly, the preclinical activity profiles, including profound apoptotic induction and inhibition of CRPC xenografts suggest that these agents offer considerable promise as new therapeutics for patients with CRPC and those resistant to current therapy.

Novel C-4 Heteroaryl 13-cis-Retinamide Mnk/AR Degrading Agents Inhibit Cell Proliferation and Migration and Induce Apoptosis in Human Breast and Prostate Cancer Cells and Suppress Growth of MDA-MB-231 Human Breast and CWR22Rv1 Human Prostate Tumor Xenografts in Mice

The synthesis and in vitro and in vivo antibreast and antiprostate cancers activities of novel C-4 heteroaryl 13-cis-retinamides that modulate Mnk-eIF4E and AR signaling are discussed. Modifications of the C-4 heteroaryl substituents reveal that the 1H-imidazole is essential for high anticancer activity. The most potent compounds against a variety of human breast and prostate cancer (BC/PC) cell lines were compounds 16 (VNHM-1-66), 20 (VNHM-1-81), and 22 (VNHM-1-73). In these cell lines, the compounds induce Mnk1/2 degradation to substantially suppress eIF4E phosphorylation. In PC cells, the compounds induce degradation of both full-length androgen receptor (fAR) and splice variant AR (AR-V7) to inhibit AR transcriptional activity. More importantly, VNHM-1-81 has strong in vivo antibreast and antiprostate cancer activities, while VNHM-1-73 exhibited strong in vivo antibreast cancer activity, with no apparent host toxicity. Clearly, these lead compounds are strong candidates for development for the treatments of human breast and prostate cancers.

Dissecting major signaling pathways in prostate cancer development and progression: Mechanisms and novel therapeutic targets.

Prostate cancer (PCa) is the most frequently diagnosed non-cutaneous malignancy and leading cause of cancer mortality in men. At the initial stages, prostate cancer is dependent upon androgens for their growth and hence effectively combated by androgen deprivation therapy (ADT). However, most patients eventually recur with an androgen deprivation-resistant phenotype, referred to as castration-resistant prostate cancer (CRPC), a more aggressive form for which there is no effective therapy presently available. The current review is an attempt to cover and establish an understanding of some major signaling pathways implicated in prostate cancer development and castration-resistance, besides addressing therapeutic strategies that targets the key signaling mechanisms.

Targeting of protein translation as a new treatment paradigm for prostate cancer.

Purpose of review The current overview will summarize some of the developments in the area of protein translation, including their relation to the therapeutic targeting of prostate cancer. Recent findings Translational control, mediated by the rate-limiting eukaryotic translation initiation factor 4E (eIF4E), drives selective translation of several oncogenic proteins, thereby contributing to tumor growth, metastasis, and treatment resistance in various cancers, including prostate cancer. As an essential regulatory hub, several oncogenic hyperactive signaling pathways appear to converge on eIF4E to promote tumorigenesis. Several approaches that target the eIF4E-dependent protein translation network are being actively studied, and it is likely that some may ultimately emerge as promising anticancer therapeutics. Summary An array of inhibitors has shown promise in targeting specific components of the translational machinery in several preclinical models of prostate cancer. It is hoped that some of these approaches may ultimately have relevance in improving the clinical outcomes of patients with advanced prostate cancer.

Identification of Novel Steroidal Androgen Receptor Degrading Agents Inspired by Galeterone 3β-Imidazole Carbamate.

Degradation of all forms of androgen receptors (ARs) is emerging as an advantageous therapeutic paradigm for the effective treatment of prostate cancer. In continuation of our program to identify and develop improved efficacious novel small-molecule agents designed to disrupt AR signaling through enhanced AR degradation, we have designed, synthesized, and evaluated novel C-3 modified analogues of our phase 3 clinical agent, galeterone (5). Concerns of potential in vivo stability of our recently discovered more efficacious galeterone 3β-imidazole carbamate (6) led to the design and synthesis of new steroidal compounds. Two of the 11 compounds, 3β-pyridyl ether (8) and 3β-imidazole (17) with antiproliferative GI50 values of 3.24 and 2.54 μM against CWR22Rv1 prostate cancer cell, are 2.75- and 3.5-fold superior to 5. In addition, compounds 8 and 17 possess improved (∼4-fold) AR-V7 degrading activities. Importantly, these two compounds are expected to be metabolically stable, making them suitable for further development as new therapeutics against all forms of prostate cancer.

VN/14-1 induces ER stress and autophagy in HP-LTLC human breast cancer cells and has excellent oral pharmacokinetic profile in female Sprague Dawley rats

Resistance to aromatase inhibitors is a major concern in the treatment of breast cancer. Long-term letrozole cultured (LTLC) cells represent a model of resistance to aromatase inhibitors. The LTLC cells were earlier generated by culturing MCF-7Ca, the MCF-7 human breast cancer cell line stably transfected with human placental aromatase gene for a prolonged period in the presence of letrozole. In the present study the effect of RAMBA, VN/14-1 on the sensitivity of LTLC cells upon multiple passaging and the mechanisms of action of VN/14-1 in such high passage LTLC (HP-LTLC) cells was investigated. We report that multiple passaging of LTLC cells (HP-LTLC cell clones) led to profound decrease in their sensitivity to VN/14-1. Additionally, microarray studies and protein analysis revealed that VN/14-1 induced marked endoplasmic reticulum (ER) stress and autophagy in HP-LTLC cells. We further report that VN/14-1 in combination with thapsigargin exhibited synergistic anti-cancer effect in HP-LTLC cells. Preliminary pharmacokinetics in rats revealed that VN/14-1 reached a peak plasma concentration (Cmax) within 0.17h after oral dosing. Its absolute oral bioavailability was >100%. Overall these results indicate potential of VN/14-1 for further clinical development as a potential oral agent for the treatment of breast cancer.

The retinamide VNLG‐152 inhibits f‐AR/AR‐V7 and MNK–eIF4E signaling pathways to suppress EMT and castration‐resistant prostate cancer xenograft growth

VNLG‐152 is a novel retinamide (NR) shown to suppress growth and progression of genetically diverse prostate cancer cells via inhibition of androgen receptor signaling and eukaryotic initiation factor 4E (eIF4E) translational machinery. Herein, we report therapeutic effects of VNLG‐152 on castration‐resistant prostate cancer (CRPC) growth and metastatic phenotype in a CRPC tumor xenograft model. Administration of VNLG‐152 significantly and dose‐dependently suppressed the growth of aggressive CWR22Rv1 tumors by 63.4% and 76.3% at 10 and 20 mg·kg−1 bw, respectively (P < 0.0001), vs. vehicle with no host toxicity. Strikingly, the expression of full‐length androgen receptor (f‐AR)/androgen receptor splice variant‐7 (AR‐V7), mitogen‐activated protein kinase‐interacting kinases 1 and 2 (MNK1/2), phosphorylated eIF4E and their associated target proteins, including prostate‐specific antigen, cyclin D1 and Bcl‐2, were strongly decreased in VNLG‐152‐treated tumors signifying inhibition of f‐AR/AR‐V7 and MNK–eIF4E signaling in VNLG‐152‐treated CWR22Rv1 tumors as observed in vitro. VNLG‐152 also suppressed the epithelial to mesenchymal transition in CWR22Rv1 tumors as evidenced by repression of N‐cadherin, β‐catenin, claudin, Slug, Snail, Twist, vimentin and matrix metalloproteinases (MMP‐2 and MMP‐9) with upsurge in E‐cadherin. These results highlight the promising use of VNLG‐152 in CRPC therapy and justify its further development towards clinical trials.

Galeterone and its analogs inhibit Mnk-eIF4E axis, synergize with gemcitabine, impede pancreatic cancer cell migration, invasion and proliferation and inhibit tumor growth in mice

Survival rate for pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) is poor, with about 80% of patients presenting with the metastatic disease. Gemcitabine, the standard chemotherapeutic agent for locally advanced and metastatic PDAC has limited efficacy, attributed to innate/acquired resistance and activation of pro-survival pathways. The Mnk1/2-eIF4E and NF-κB signaling pathways are implicated in PDAC disease progression/metastasis and also associated with gemcitabine-induced resistance in PDAC. Galeterone (gal), a multi-target, agent in phase III clinical development for prostate cancer has also shown effects on the aforementioned pathways. We show for the first time, that gal/analogs (VNPT55, VNPP414 and VNPP433-3β) profoundly inhibited cell viability of gemcitabine-naive/resistance PDAC cell lines and strongly synergized with gemcitabine in gemcitabine-resistant PDAC cells. In addition, to inducing G1 cell cycle arrest, gal/analogs induced caspase 3-mediated cell-death of PDAC cells. Gal/analogs caused profound downregulation of Mnk1/2, peIF4E and NF-κB (p-p65), metastatic inducing factors (N-cadherin, MMP-1/-2/-9, Slug, Snail and CXCR4) and putative stem cell factors, (β-Catenin, Nanog, BMI-1 and Oct-4). Gal/analog also depleted EZH2 and upregulated E-Cadherin. These effects resulted in significant inhibition of PDAC cell migration, invasion and proliferation. Importantly, we also observed strong MiaPaca-2 tumor xenograft growth inhibition (61% to 92%). Collectively, these promising findings strongly support further development of gal/analogs as novel therapeutics for PDAC.Survival rate for pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) is poor, with about 80% of patients presenting with the metastatic disease. Gemcitabine, the standard chemotherapeutic agent for locally advanced and metastatic PDAC has limited efficacy, attributed to innate/acquired resistance and activation of pro-survival pathways. The Mnk1/2-eIF4E and NF-κB signaling pathways are implicated in PDAC disease progression/metastasis and also associated with gemcitabine-induced resistance in PDAC. Galeterone (gal), a multi-target, agent in phase III clinical development for prostate cancer has also shown effects on the aforementioned pathways. We show for the first time, that gal/analogs (VNPT55, VNPP414 and VNPP433-3β) profoundly inhibited cell viability of gemcitabine-naive/resistance PDAC cell lines and strongly synergized with gemcitabine in gemcitabine-resistant PDAC cells. In addition, to inducing G1 cell cycle arrest, gal/analogs induced caspase 3-mediated cell-death of PDAC cells. Gal/analogs caused profound downregulation of Mnk1/2, peIF4E and NF-κB (p-p65), metastatic inducing factors (N-cadherin, MMP-1/-2/-9, Slug, Snail and CXCR4) and putative stem cell factors, (β-Catenin, Nanog, BMI-1 and Oct-4). Gal/analog also depleted EZH2 and upregulated E-Cadherin. These effects resulted in significant inhibition of PDAC cell migration, invasion and proliferation. Importantly, we also observed strong MiaPaca-2 tumor xenograft growth inhibition (61% to 92%). Collectively, these promising findings strongly support further development of gal/analogs as novel therapeutics for PDAC.

Abstract B30: Inhibition of eIF4E/MNK axis by novel MNKDAs reduces breast cancer tumor growth, migration, invasion and metastasis

Triple-negative breast cancer (TNBC) represents a molecularly heterogeneous subgrouping of high-risk breast tumors characterized by aggressive behavior, poor prognosis and lack of targeted therapeutic options. The translation initiation factor eIF4E, an oncogene essential for cap-dependent translation of specific mRNAs critical for cell division, cell survival, angiogenesis, and metastasis is commonly overexpressed in primary breast cancers including TNBC. eIF4E therapeutic targeting is considered novel and significant because tumor cells specifically overexpress eIF4E and develop an oncogene addiction to eIF4E, rendering the cells vulnerable to eIF4E inhibition, whereas normal cells are relatively insensitive. Phosphorylation of eIF4E at serine-209 is critical for the oncogenic activity of eIF4E, and it has been suggested that chemical compounds that prevent the phosphorylation of eIF4E could act as effective anticancer drugs. Herein, we report that a novel inhibitor of peIF4E function (Novel Retinamides, NRs), safely and potently suppresses breast tumor formation. Administration of novel retinamides (mnk degrading agents: MNKDAs) blocked the growth of primary breast tumors in MDA-MB-231 xenograft model, TNBC patient derived xenografts (PDX), and reduced the development of lung metastases in an invasive model. Mechanistically, NRs resulted in the degradation of MNKs (MNK1 and MNK2), a key kinase chiefly responsible for phosphorylating and activating eIF4E. This in turn hampered peIF4E involved translation initiation and activation of genes associated with cell proliferation and survival. Furthermore, MNK degradation by NRs also suppressed epithelial-to-mesenchymal transition, a process crucial for metastasis as evident by increased expression of E-cadherin and reduced expression of N-cadherin, fibronectin and vimentin; snail, slug, twist, fibronectin, claudin, lamin, TCF/ZEB-1, beta catenin, MMPs and vimentin. Taken together, the preclinical evidence of tumor regression and metastasis in TNBC models suggest that NRs offers considerable promise as new therapeutic candidate to target TNBC and metastatic breast cancer. Citation Format: Senthilmurugan Ramalingam, Lalji Gediya, Vidya Ramamurthy, Yong Choi, Rena Lapidus, Njar Vincent. Inhibition of eIF4E/MNK axis by novel MNKDAs reduces breast cancer tumor growth, migration, invasion and metastasis. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B30.

Abstract 2704: Simultaneous targeting of androgen receptor (AR) and Eukaryotic Initiation Factor 4E (eIF4E) dependent translation initiation by RAMBA Retinamides promotes apoptosis and impedes cell growth, cell proliferation and matrix invasion in androgen sensitive and castration-resistant prostate cancers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Prostate cancer (PCa) remains the second most leading cause of cancer-related death in Western male population. Although initially counteracted by androgen deprivation therapy (ADT), PCa cells eventually progress to a hormone-independent state thereby limiting treatment options and rendering the discovery of new therapeutic approaches a clinical priority. Direct targeting of translational machinery has gained enormous attention in recent years for treating castration resistant prostate cancers (CRPC). The present study aims to investigate the effect of our novel retinoic acid metabolism blocking agents (RAMBA) retinamides (RRs) on eukaryotic translation initiation factor 4E (eIF4E) translational machinery and androgen receptor (AR) signaling in prostate cancers. Methods: Androgen-sensitive (LNCaP), androgen-resistant (PC-3, C4-2B and CWR22Rv1), enzalutamide resistant (MR49F) prostate cancer cells and normal prostatic epithelial cells (PWR-1E) were used analyzing the effect of RRs on prostate cancers. Results: 24 h treatment of PCa cells to RRs resulted in substantial down-regulation of androgen receptor (AR), degradation of MAPK-interacting kinases (MNK), and attenuation of eukaryotic translation initiation factor 4E (eIF4E) cap dependent translation initiation. Down-regulation of AR and MNK mediated eIF4E translation initiation by RRs in PCa cells were in turn associated with inhibition of cell growth and proliferation, induction of cell cycle arrest, increased apoptosis, and inhibition of matrix invasion and metastasis. Most importantly, the effects of our RRs on AR degradation, eIF4E translation initiation and subsequent oncogenic program were far more potent than the clinically relevant retinoids, established MNK inhibitors (CGP 57380 and/or cercosporamide) and the FDA approved PCa drugs (Casodex, Enzalutamide and Abiraterone acetate). Conclusion: RAMBA retinamides which can exclusively target the degradation of both AR and MNK- the two divergent axes in PCa, at pharmacologically feasible concentrations are novel therapeutic compounds for treatment of both androgen-sensitive and androgen-resistant prostate cancers. Citation Format: Vidya priyadarsini Ramamurthy, Senthilmurugan Ramalingam, Lalji Gediya, Vincent C.O Njar. Simultaneous targeting of androgen receptor (AR) and Eukaryotic Initiation Factor 4E (eIF4E) dependent translation initiation by RAMBA Retinamides promotes apoptosis and impedes cell growth, cell proliferation and matrix invasion in androgen sensitive and castration-resistant prostate cancers. [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 2704. doi:10.1158/1538-7445.AM2014-2704