Devalingam Mahalingam

TRAIL receptor signalling and modulation : Are we on the right TRAIL?

Tumour necrosis factor-related apoptosis-inducing ligand or Apo2 ligand (TRAIL/Apo2L) is a member of the tumour necrosis factor (TNF) superfamily of cytokines that induces apoptosis upon binding to its death domain-containing transmembrane receptors, death receptors 4 and 5 (DR4, DR5). Importantly, TRAIL preferentially induces apoptosis in cancer cells while exhibiting little or no toxicity in normal cells. To date, research has focused on the mechanism of apoptosis induced by TRAIL and the processes involved in the development of TRAIL resistance. TRAIL-resistant tumours can be re-sensitized to TRAIL by a combination of TRAIL with chemotherapeutics or irradiation. Studies suggest that in many cancer cells only one of the two death-inducing TRAIL receptors is functional. These findings as well as the aim to avoid decoy receptor-mediated neutralization of TRAIL led to the development of receptor-specific TRAIL variants and agonistic antibodies. These molecules are predicted to be more potent than native TRAIL in vivo and may be suitable for targeted treatment of particular tumours. This review focuses on the current status of TRAIL receptor-targeting for cancer therapy, the apoptotic signalling pathway induced by TRAIL receptors, the prognostic implications of TRAIL receptor expression and modulation of TRAIL sensitivity of tumour cells by combination therapies. The mechanisms of TRAIL resistance and the potential measures that can be taken to overcome them are also addressed. Finally, the status of clinical trials of recombinant TRAIL and DR4-/DR5-specific agonistic antibodies as well as the pre-clinical studies of receptor-selective TRAIL variants is discussed including the obstacles facing the use of these molecules as anti-cancer therapeutics.

Targeting HSP90 for cancer therapy

Heat-shock proteins (HSPs) are molecular chaperones that regulate protein folding to ensure correct conformation and translocation and to avoid protein aggregation. Heat-shock proteins are increased in many solid tumours and haematological malignancies. Many oncogenic proteins responsible for the transformation of cells to cancerous forms are client proteins of HSP90. Targeting HSP90 with chemical inhibitors would degrade these oncogenic proteins, and thus serve as useful anticancer agents. This review provides an overview of the HSP chaperone machinery and the structure and function of HSP90. We also highlight the key oncogenic proteins that are regulated by HSP90 and describe how inhibition of HSP90 could alter the activity of multiple signalling proteins, receptors and transcriptional factors implicated in carcinogenesis.

Inhibition of NEDD8-activating enzyme: a novel approach for the treatment of acute myeloid leukemia.

NEDD8 activating enzyme (NAE) has been identified as an essential regulator of the NEDD8 conjugation pathway, which controls the degradation of many proteins with important roles in cell-cycle progression, DNA damage, and stress responses. Here we report that MLN4924, a novel inhibitor of NAE, has potent activity in acute myeloid leukemia (AML) models. MLN4924 induced cell death in AML cell lines and primary patient specimens independent of Fms-like tyrosine kinase 3 expression and stromal-mediated survival signaling and led to the stabilization of key NAE targets, inhibition of nuclear factor-kappaB activity, DNA damage, and reactive oxygen species generation. Disruption of cellular redox status was shown to be a key event in MLN4924-induced apoptosis. Administration of MLN4924 to mice bearing AML xenografts led to stable disease regression and inhibition of NEDDylated cullins. Our findings indicate that MLN4924 is a highly promising novel agent that has advanced into clinical trials for the treatment of AML.

Autophagy inhibition enhances vorinostat-induced apoptosis via ubiquitinated protein accumulation

Autophagy is an evolutionarily conserved cell survival pathway that enables cells to recoup ATP and other critical biosynthetic molecules during nutrient deprivation or exposure to hypoxia, which are hallmarks of the tumour microenvironment. Autophagy has been implicated as a potential mechanism of resistance to anticancer agents as it can promote cell survival in the face of stress induced by chemotherapeutic agents by breaking down cellular components to generate alternative sources of energy. Disruption of autophagy with chloroquine (CQ) induces the accumulation of ubiquitin‐conjugated proteins in a manner similar to the proteasome inhibitor bortezomib (BZ). However, CQ‐induced protein accumulation occurs at a slower rate and is localized to lysosomes in contrast to BZ, which stimulates rapid buildup of ubiquitinated proteins and aggresome formation in the cytosol. The histone deacetylase (HDAC) inhibitor vorinostat (VOR) blocked BZ‐induced aggresome formation, but promoted CQ‐mediated ubiquitinated protein accumulation. Disruption of autophagy with CQ strongly enhanced VOR‐mediated apoptosis in colon cancer cells. Accordingly, knockdown of the essential autophagy gene Atg7 also sensitized cells to VOR‐induced apoptosis. Knockdown of HDAC6 greatly enhanced BZ‐induced apoptosis, but only marginally sensitized cells to CQ. Subsequent studies determined that the CQ/VOR combination promoted a large increase in superoxide generation that was required for ubiquitinated protein accumulation and cell death. Finally, treatment with the CQ/VOR combination significantly reduced tumour burden and induced apoptosis in a colon cancer xenograft model. Collectively, our results establish that inhibition of autophagy with CQ induces ubiquitinated protein accumulation and VOR potentiates CQ‐mediated aggregate formation, superoxide generation and apoptosis.

Single-cell analysis of circulating tumor cells identifies cumulative expression patterns of EMT-related genes in metastatic prostate cancer

Prostate tumors shed circulating tumor cells (CTCs) into the blood stream. Increased evidence shows that CTCs are often present in metastatic prostate cancer and can be alternative sources for disease profiling and prognostication. Here we postulate that CTCs expressing genes related to epithelial–mesenchymal transition (EMT) are strong predictors of metastatic prostate cancer.

A Phase I, Multicenter, Open-Label, First-in-Human, Dose-Escalation Study of the Oral Smoothened Inhibitor Sonidegib (LDE225) in Patients with Advanced Solid Tumors

Purpose: This phase I trial was undertaken to determine the maximum tolerated dose (MTD), dose-limiting toxicities (DLT), safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of the novel smoothened inhibitor sonidegib (LDE225), a potent inhibitor of hedgehog signaling, in patients with advanced solid tumors. Experimental Design: Oral sonidegib was administered to 103 patients with advanced solid tumors, including medulloblastoma and basal cell carcinoma (BCC), at doses ranging from 100 to 3,000 mg daily and 250 to 750 mg twice daily, continuously, with a single-dose pharmacokinetics run-in period. Dose escalations were guided by a Bayesian logistic regression model. Safety, tolerability, efficacy, pharmacokinetics, and biomarkers in skin and tumor biopsies were assessed. Results: The MTDs of sonidegib were 800 mg daily and 250 mg twice daily. The main DLT of reversible grade 3/4 elevated serum creatine kinase (18% of patients) was observed at doses ≥ the MTD in an exposure-dependent manner. Common grade 1/2 adverse events included muscle spasm, myalgia, gastrointestinal toxicities, increased liver enzymes, fatigue, dysgeusia, and alopecia. Sonidegib exposure increased dose proportionally up to 400 mg daily, and displayed nonlinear pharmacokinetics at higher doses. Sonidegib exhibited exposure-dependent reduction in GLI1 mRNA expression. Tumor responses observed in patients with medulloblastoma and BCC were associated with evidence of hedgehog pathway activation. Conclusions: Sonidegib has an acceptable safety profile in patients with advanced solid tumors and exhibits antitumor activity in advanced BCC and relapsed medulloblastoma, both of which are strongly associated with activated hedgehog pathway, as determined by gene expression. Clin Cancer Res; 20(7); 1900–9. ©2014 AACR.

Combined autophagy and HDAC inhibition: a phase I safety, tolerability, pharmacokinetic, and pharmacodynamic analysis of hydroxychloroquine in combination with the HDAC inhibitor vorinostat in patients with advanced solid tumors.

We previously reported that inhibition of autophagy significantly augmented the anticancer activity of the histone deacetylase (HDAC) inhibitor vorinostat (VOR) through a cathepsin D-mediated mechanism. We thus conducted a first-in-human study to investigate the safety, preliminary efficacy, pharmacokinetics (PK), and pharmacodynamics (PD) of the combination of the autophagy inhibitor hydroxychloroquine (HCQ) and VOR in patients with advanced solid tumors. Of 27 patients treated in the study, 24 were considered fully evaluable for study assessments and toxicity. Patients were treated orally with escalating doses of HCQ daily (QD) (d 2 to 21 of a 21-d cycle) in combination with 400 mg VOR QD (d one to 21). Treatment-related adverse events (AE) included grade 1 to 2 nausea, diarrhea, fatigue, weight loss, anemia, and elevated creatinine. Grade 3 fatigue and/or myelosuppression were observed in a minority of patients. Fatigue and gastrointestinal AE were dose-limiting toxicities. Six-hundred milligrams HCQ and 400 mg VOR was established as the maximum tolerated dose and recommended phase II regimen. One patient with renal cell carcinoma had a confirmed durable partial response and 2 patients with colorectal cancer had prolonged stable disease. The addition of HCQ did not significantly impact the PK profile of VOR. Treatment-related increases in the expression of CDKN1A and CTSD were more pronounced in tumor biopsies than peripheral blood mononuclear cells. Based on the safety and preliminary efficacy of this combination, additional clinical studies are currently being planned to further investigate autophagy inhibition as a new approach to increase the efficacy of HDAC inhibitors.

Vorinostat Enhances the Activity of Temsirolimus in Renal Cell Carcinoma Through Suppression of Survivin Levels

Purpose: The mammalian target of rapamycin (mTOR) inhibitor temsirolimus has exhibited promising anticancer activity for the treatment of renal cell cancers (RCC). Survivin expression has been implicated in drug resistance and reducing its levels with the histone deacetylase (HDAC) inhibitor vorinostat may enhance the anticancer activity of temsirolimus. Experimental Design: The sensitivity of RCC cell lines to the combination of temsirolimus and vorinostat was determined by measuring cell viability, clonogenic survival, and apoptosis. The effects of this combination on survivin levels were determined in vitro and in vivo. Survivin expression was silenced using small interfering RNA to evaluate its role in determining sensitivity to temsirolimus and vorinostat. The effect of the combination on angiogenesis was also determined in RCC xenograft models. Results: Vorinostat synergistically improved the anticancer activity of temsirolimus in a panel of RCC cell lines in vitro and in two xenograft models in vivo. While each single agent led to a modest decrease in survivin levels, the combination dramatically reduced its expression, which correlated with an induction of apoptosis. Silencing survivin levels induced apoptosis and significantly improved the efficacy of temsirolimus and vorinostat. In addition, the temsirolimus/vorinostat combination led to a strong reduction in angiogenesis. Conclusions: Vorinostat augmented the anticancer activity of temsirolimus in both in vitro and in vivo models of RCC. The effectiveness of the combination was due to a decrease in survivin levels and corresponding induction of apoptosis, and enhanced inhibition of angiogenesis. Targeting survivin may be a promising therapeutic strategy to improve RCC therapy. Clin Cancer Res; 16(1); 141–53

Lucanthone is a novel inhibitor of autophagy that induces cathepsin D-mediated apoptosis

Cellular stress induced by nutrient deprivation, hypoxia, and exposure to many chemotherapeutic agents activates an evolutionarily conserved cell survival pathway termed autophagy. This pathway enables cancer cells to undergo self-digestion to generate ATP and other essential biosynthetic molecules to temporarily avoid cell death. Therefore, disruption of autophagy may sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis. Chloroquine and its analog hydroxychloroquine are the only clinically relevant autophagy inhibitors. Because both of these agents induce ocular toxicity, novel inhibitors of autophagy with a better therapeutic index are needed. Here we demonstrate that the small molecule lucanthone inhibits autophagy, induces lysosomal membrane permeabilization, and possesses significantly more potent activity in breast cancer models compared with chloroquine. Exposure to lucanthone resulted in processing and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, but impaired autophagic degradation as revealed by transmission electron microscopy and the accumulation of p62/SQSTM1. Microarray analysis, qRT-PCR, and immunoblotting determined that lucanthone stimulated a large induction in cathepsin D, which correlated with cell death. Accordingly, knockdown of cathepsin D reduced lucanthone-mediated apoptosis. Subsequent studies using p53+/+ and p53−/− HCT116 cells established that lucanthone induced cathepsin D expression and reduced cancer cell viability independently of p53 status. In addition, lucanthone enhanced the anticancer activity of the histone deacetylase inhibitor vorinostat. Collectively, our results demonstrate that lucanthone is a novel autophagic inhibitor that induces apoptosis via cathepsin D accumulation and enhances vorinostat-mediated cell death in breast cancer models.

The emerging safety profile of mTOR inhibitors, a novel class of anticancer agents

Mammalian target of rapamycin (mTOR) has emerged as an important target for cancer therapy. Rapamycin has a distinct, well-documented toxicity profile and most of the toxicity data has been reported in patients with organ transplantation. Newer mTOR inhibitors have slightly different pharmacokinetic properties, yet they present toxicity profiles similar to rapamycin. Most of these toxicities are mild to moderate in severity and can be managed clinically by dose modification and supportive measures. Mucositis and pneumonitis are the most commonly reported toxicities, but they rarely lead to treatment discontinuation. Pathogenesis of pneumonitis is uncertain, but various hypotheses have been suggested, including cell-mediated immune response to the drug.