Claudia M. Espitia

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.

Disrupting Protein NEDDylation with MLN4924 Is a Novel Strategy to Target Cisplatin Resistance in Ovarian Cancer

Purpose: Ovarian cancer has the highest mortality rate of all female reproductive malignancies. Drug resistance is a major cause of treatment failure and novel therapeutic strategies are urgently needed. MLN4924 is a NEDDylation inhibitor currently under investigation in multiple phase I studies. We investigated its anticancer activity in cisplatin-sensitive and -resistant ovarian cancer models. Experimental Design: Cellular sensitivity to MLN4924/cisplatin was determined by measuring viability, clonogenic survival, and apoptosis. The effects of drug treatment on global protein expression, DNA damage, and reactive oxygen species generation were determined. RNA interference established natural born killer/bcl-2–interacting killer (NBK/BIK) as a regulator of therapeutic sensitivity. The in vivo effects of MLN4924/cisplatin on tumor burden and key pharmacodynamics were assessed in cisplatin-sensitive and -resistant xenograft models. Results: MLN4924 possessed significant activity against both cisplatin-sensitive and -resistant ovarian cancer cells and provoked the stabilization of key NEDD8 substrates and regulators of cellular redox status. Notably, MLN4924 significantly augmented the activity of cisplatin against cisplatin-resistant cells, suggesting that aberrant NEDDylation may contribute to drug resistance. MLN4924 and cisplatin cooperated to induce DNA damage, oxidative stress, and increased expression of the BH3-only protein NBK/BIK. Targeted NBK/BIK knockdown diminished the proapoptotic effects of the MLN4924/cisplatin combination. Administration of MLN4924 to mice bearing ovarian tumor xenografts significantly increased the efficacy of cisplatin against both cisplatin-sensitive and -resistant tumors. Conclusions: Our collective data provide a rationale for the clinical investigation of NEDD8-activating enzyme (NAE) inhibition as a novel strategy to augment cisplatin efficacy in patients with ovarian cancer and other malignancies. Clin Cancer Res; 19(13); 3577–90. ©2013 AACR.

Reovirus therapy stimulates endoplasmic reticular stress, NOXA induction, and augments bortezomib-mediated apoptosis in multiple myeloma

Oncolytic virotherapy with reovirus has demonstrated anti-cancer activity and minimal toxicity in clinical trials, but the mechanisms underlying these effects have not been fully elucidated. Reolysin, a proprietary formulation of reovirus for cancer therapy, stimulated selective viral replication and apoptosis in multiple myeloma (MM) cells. Reolysin-mediated apoptosis was associated with an induction of endoplasmic reticular (ER) stress-related gene expression, swelling of the endoplasmic reticulum, increases in intracellular calcium levels and a strong induction of the Bcl-2 homology 3 (BH3)-only pro-apoptotic protein NOXA. Knockdown of NOXA expression by short hairpin RNA significantly reduced the pro-apoptotic effects of Reolysin. We next showed that co-administration of Reolysin and bortezomib resulted in the dual accumulation of viral and ubiquitinated proteins, which led to enhanced ER stress, NOXA induction and apoptosis. Importantly, the combination of reovirus infection and proteasomal inhibition significantly decreased tumor burden in a xenograft and syngeneic bone disease model of MM without exhibiting adverse side effects. Our study establishes ER stress stimulation and NOXA induction as novel mediators of reovirus-induced apoptosis. Furthermore, reovirus infection can be used as a promising approach to augment the anti-myeloma activity of bortezomib by promoting additional stress to the endoplasmic reticulum of MM cells.

Progressive Visceral Leishmaniasis Is Driven by Dominant Parasite-induced STAT6 Activation and STAT6-dependent Host Arginase 1 Expression

The clinicopathological features of the hamster model of visceral leishmaniasis (VL) closely mimic active human disease. Studies in humans and hamsters indicate that the inability to control parasite replication in VL could be related to ineffective classical macrophage activation. Therefore, we hypothesized that the pathogenesis of VL might be driven by a program of alternative macrophage activation. Indeed, the infected hamster spleen showed low NOS2 but high arg1 enzyme activity and protein and mRNA expression (p<0.001) and increased polyamine synthesis (p<0.05). Increased arginase activity was also evident in macrophages isolated from the spleens of infected hamsters (p<0.05), and arg1 expression was induced by L. donovani in primary hamster peritoneal macrophages (p<0.001) and fibroblasts (p<0.01), and in a hamster fibroblast cell line (p<0.05), without synthesis of endogenous IL-4 or IL-13 or exposure to exogenous cytokines. miRNAi-mediated selective knockdown of hamster arginase 1 (arg1) in BHK cells led to increased generation of nitric oxide and reduced parasite burden (p<0.005). Since many of the genes involved in alternative macrophage activation are regulated by Signal Transducer and Activator of Transcription-6 (STAT6), and because the parasite-induced expression of arg1 occurred in the absence of exogenous IL-4, we considered the possibility that L. donovani was directly activating STAT6. Indeed, exposure of hamster fibroblasts or macrophages to L. donovani resulted in dose-dependent STAT6 activation, even without the addition of exogenous cytokines. Knockdown of hamster STAT6 in BHK cells with miRNAi resulted in reduced arg1 mRNA expression and enhanced control of parasite replication (p<0.0001). Collectively these data indicate that L. donovani infection induces macrophage STAT6 activation and STAT6-dependent arg1 expression, which do not require but are amplified by type 2 cytokines, and which contribute to impaired control of infection.

Targeting Aurora A kinase activity with the investigational agent alisertib increases the efficacy of cytarabine through a FOXO‐dependent mechanism

Novel therapies are urgently needed to improve clinical outcomes for patients with acute myeloid leukemia (AML). The investigational drug alisertib (MLN8237) is a novel Aurora A kinase inhibitor being studied in multiple Phase I and II studies. We investigated the preclinical efficacy and pharmacodynamics of alisertib in AML cell lines, primary AML cells and mouse models of AML. Here, we report that alisertib disrupted cell viability, diminished clonogenic survival, induced expression of the FOXO3a targets p27 and BIM and triggered apoptosis. A link between Aurora A expression and sensitivity to ara‐C was established, suggesting that Aurora A inhibition may be a promising strategy to increase the efficacy of ara‐C. Accordingly, alisertib significantly potentiated the antileukemic activity of ara‐C in both AML cell lines and primary blasts. Targeted FOXO3a knockdown significantly blunted the pro‐apoptotic effects of the alisertib/ara‐C combination, indicating that it is an important regulator of sensitivity to these agents. In vivo studies demonstrated that alisertib significantly augmented the efficacy of ara‐C without affecting its pharmacokinetic profile and led to the induction of p27 and BIM. Our collective data indicate that targeting Aurora A with alisertib represents a novel approach to increase the efficacy of ara‐C that warrants further investigation.

Targeting PIM kinase activity significantly augments the efficacy of cytarabine

Drug resistance is a major cause of treatment failure for patients with acute myeloid leukaemia (AML) and novel strategies that circumvent resistance mechanisms are urgently needed (Swords et al, 2010). The PIM kinases (PIM1, PIM2, PIM3) are a small family of proto-oncogenes within the CAMK superfamily that are frequently overexpressed in many forms of cancer including AML. PIM kinases have essential roles in the regulation of signal transduction cascades that promote cell survival, proliferation, and drug resistance (Amaravadi & Thompson, 2005; Giles, 2005; Nawijn et al, 2011). However, the specific roles of PIM kinases as regulators of AML pathogenesis and of the sensitivity to standard agents utilized in AML therapy remain to be fully elucidated. SGI-1776 is novel small molecule inhibitor of PIM kinase activity that has demonstrated preclinical activity in cancer models and has entered Phase I clinical trials (Chen et al, 2009; Mumenthaler et al, 2009). Considering the roles of the PIM kinases in the regulation of cell survival and proliferation and their high basal expression in AML cells, we hypothesized that SGI-1776 would possess significant anti-leukaemic activity in AML models. We first investigated the in vitro efficacy of SGI-1776 in a panel of nine human AML cell lines (Fig 1A). Treatment of AML cells with SGI-1776 led to a dose-dependent reduction in viability, impaired clonogenic survival (Fig 1B), and apoptotic cell death (Fig 1C, D). These effects were associated with a significant reduction in the phosphorylation of the PIM kinase substrate and apoptotic regulator Bad (Ser112), an event that increases its pro-apoptotic function. The drug-related reduction in Bad phosphorylation did not appear to be due to alterations in AKT activity as SGI-1776 treatment did not significantly affect the phosphorylation of AKT (Thr308) in MV4-11 cells, which have constitutive AKT activity (Fig 1E). Approximately 30% of patients with AML have constitutive fms-like tyrosine kinase-3 (FLT3) activity due to internal tandem duplication (ITD) or activating mutations. Considering that in vitro kinase activity screens with SGI-1776 and other PIM kinase inhibitors have demonstrated some offtarget inhibition of FLT3, we utilized MV4-11 cells with stable FLT3 knockdown to investigate whether these potential off-target effects were a critical factor underlying the antileukaemic activity of SGI-1776 (Swords et al, 2010). FLT3 knockdown caused a modest reduction in sensitivity to SGI1776 (Fig 1F), indicating that FLT3 inhibition contributes to the efficacy of SGI-1776, but is not its primary mechanism of action in AML. Several recent studies have suggested a mechanistic link between aberrant expression of PIM kinases and reduced sensitivity to certain anticancer agents (Xie et al, 2006, 2010). To address this issue in a manner relevant to AML therapy, we evaluated the expression levels of PIM1, PIM2, and PIM3 in paired HL-60 cells that are sensitive and resistant to cytarabine (ara-C). Our results showed that the levels of PIM1 and PIM3, but not PIM2, were significantly higher in ara-C-resistant HL60 cells (Fig 2A, B). Consistent with this observation, ara-C treatment led to increased PIM1 and PIM3 expression as assessed by immunoblotting (MOLM-13 cells, Fig 2C) and quantitative reverse transcription polymerase chain reaction (RT-PCR) (MOLM-13 cells and primary AML blasts, Fig 2D). We next investigated whether inhibiting PIM kinase signalling with SGI-1776 could augment the efficacy of ara-C. Treatment of AML cells with the combination of ara-C and SGI-1776 led to significantly greater diminished viability and inhibition of clonogenic survival over what was achieved by either single agent (Fig 2E, F). Propidium iodide/fluorescence-activated cell sorting (PI/FACS) analysis of the effects of SGI-1776, ara-C, and the combination of these agents on cell cycle distribution showed that SGI-1776 promoted the accumulation of cells with G1 DNA content (Fig 2G). HL-60 ara-C sensitive and resistant cells were utilized to investigate whether targeting PIM kinase activity with SGI1776 could be used as a strategy to overcome intrinsic ara-C resistance. Our results showed that SGI-1776 partially restored the sensitivity of ara-C resistant cells to ara-C (Fig 2H), indicating that ara-C resistance is a multifaceted problem with multiple underlying mechanisms including PIM overexpression (Fig 2A, B). Additionally, our findings show that abrogating PIM kinase activity could possibly be utilized as a novel approach to improve the therapeutic efficacy of ara-C including in circumstances of de novo ara-C resistance. In order to further investigate the therapeutic utility of this combination, we established AML xenografts in nude mice using the MOLM-13 AML cell line. Mice were randomized into groups of 10 and were administered vehicle, ara-C, SGI1776, or ara-C and SGI-1776 for 21 d. Treatment with the combination of these two agents was well tolerated and significantly increased the efficacy of single agent ara-C therapy (Fig 2I, J). Immunohistochemical analyzes of tumours from mice revealed that SGI-1776 significantly diminished Bad phosphorylation and cooperated with ara-C in vivo to promote correspondence

Reolysin is a novel reovirus-based agent that induces endoplasmic reticular stress-mediated apoptosis in pancreatic cancer

Activating mutation of KRas is a genetic alteration that occurs in the majority of pancreatic tumors and is therefore an ideal therapeutic target. The ability of reoviruses to preferentially replicate and induce cell death in transformed cells that express activated Ras prompted the development of a reovirus-based formulation for cancer therapy called Reolysin. We hypothesized that Reolysin exposure would trigger heavy production of viral products leading to endoplasmic reticular (ER) stress-mediated apoptosis. Here, we report that Reolysin treatment stimulated selective reovirus replication and decreased cell viability in KRas-transformed immortalized human pancreatic duct epithelial cells and pancreatic cancer cell lines. These effects were associated with increased expression of ER stress-related genes, ER swelling, cleavage of caspase-4, and splicing of XBP-1. Treatment with ER stress stimuli including tunicamycin, brefeldin A, and bortezomib (BZ) augmented the anticancer activity of Reolysin. Cotreatment with BZ and Reolysin induced the simultaneous accumulation of ubiquitinated and viral proteins, resulting in enhanced levels of ER stress and apoptosis in both in vitro and in vivo models of pancreatic cancer. Our collective results demonstrate that the abnormal protein accumulation induced by the combination of Reolysin and BZ promotes heightened ER stress and apoptosis in pancreatic cancer cells and provides the rationale for a phase I clinical trial further investigating the safety and efficacy of this novel strategy.

Targeting PIM kinase enhances the activity of sunitinib in renal cell carcinoma

Background:Upregulation of PIM kinase expression has been reported in many malignancies, suggesting that inhibition of PIM kinase activity may be an attractive therapeutic strategy. We hypothesised that inhibition of PIM kinase activity with SGI-1776, a novel small molecule inhibitor of PIM kinase activity, would reduce the viability of renal cell carcinoma (RCC) cells and enhance the activity of sunitinib.Methods:Immunoblotting, qRT–PCR, and gene expression arrays were carried out to identify genes modulated by SGI-1776 treatment. The anticancer activity of SGI-1776 and sunitinib was determined by viability and apoptosis assays and in tumour xenografts in vivo.Results:Treatment with SGI-1776 led to a decrease in phosphorylated and total c-Myc levels, which resulted in the modulation of c-Myc target genes. SGI-1776 in combination with sunitinib induced a further reduction in c-Myc levels, which was associated with enhanced anticancer activity. siRNA-mediated knockdown of c-Myc demonstrated that its expression has a key role in regulating the sensitivity to the combination of SGI-1776 and sunitinib. Importantly, the combination significantly reduced tumour burden in two RCC xenograft models compared with single-agent therapy and was very well tolerated.Conclusion:These data indicate that targeting PIM kinase signalling is a promising treatment strategy for RCC.