Hala Elnakat Thomas

mTOR Inhibitors Synergize on Regression, Reversal of Gene Expression, and Autophagy in Hepatocellular Carcinoma

Combination therapy causes gene reprogramming, autophagy, and tumor regression in a mouse model approximating human HCC. Bridging the Generation Gap Kids of every generation disdain their elders—who clearly don’t understand them and are stuck in the past. Newer is better, after all. But sometimes, a blend of the old and new may be exactly what’s needed to solve a particular problem. Thomas et al. set out to see whether the new—the phosphatidylinositol 3-kinase/mammalian target of rapamycin (mTOR) adenosine triphosphate–site competitive inhibitor BEZ235—was better than the old—the U.S. Food and Drug Administration–approved mTOR-allosteric inhibitor RAD001. What they instead found was that these two drugs worked together to treat hepatocellular carcinoma (HCC). mTOR signaling is up-regulated in about 50% of HCCs. When the authors tested two mTOR-targeting drugs, BEZ235 and RAD001, on cultured HCC cells, they unexpectedly found that the drugs acted synergistically. In a mouse model that mimics human HCC, the two drugs induced a marked regression in tumor burden through a mechanism that involved down-regulation of genes involved in autophagy—where the cell degrades its own components. In patients with HCC, dysregulation of autophagy genes correlated with poor prognosis. The authors are now taking this observation into clinical trials to determine whether it holds true in people. By working together, old and new mTOR inhibitors may provide a new therapeutic option for HCC. Hepatocellular carcinoma (HCC) affects more than half a million people worldwide and is the third most common cause of cancer deaths. Because mammalian target of rapamycin (mTOR) signaling is up-regulated in 50% of HCCs, we compared the effects of the U.S. Food and Drug Administration–approved mTOR-allosteric inhibitor, RAD001, with a new-generation phosphatidylinositol 3-kinase/mTOR adenosine triphosphate–site competitive inhibitor, BEZ235. Unexpectedly, the two drugs acted synergistically in inhibiting the proliferation of cultured HCC cells. The synergistic effect closely paralleled eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) dephosphorylation, which is implicated in the suppression of tumor cell proliferation. In a mouse model approximating human HCC, the drugs in combination, but not singly, induced a marked regression in tumor burden. However, in the tumor, BEZ235 alone was as effective as the combination in inhibiting 4E-BP1 phosphorylation, which suggests that additional target(s) may also be involved. Microarray analyses revealed a large number of genes that reverted to normal liver tissue expression in mice treated with both drugs, but not either drug alone. These analyses also revealed the down-regulation of autophagy genes in tumors compared to normal liver. Moreover, in HCC patients, altered expression of autophagy genes was associated with poor prognosis. Consistent with these findings, the drug combination had a profound effect on UNC51-like kinase 1 (ULK1) dephosphorylation and autophagy in culture, independent of 4E-BP1, and in parallel induced tumor mitophagy, a tumor suppressor process in liver. These observations have led to an investigator-initiated phase 1B-2 dose escalation trial with RAD001 combined with BEZ235 in patients with HCC and other advanced solid tumors.

Phenformin-Induced Mitochondrial Dysfunction Sensitizes Hepatocellular Carcinoma for Dual Inhibition of mTOR

Purpose: Hepatocellular carcinoma (HCC) ranks second in cancer mortality and has limited therapeutic options. We recently described the synergistic effect of allosteric and ATP-site competitive inhibitors against the mTOR for the treatment of HCC. However, such inhibitors induce hyperglycemia and increase mitochondrial efficiency. Here we determined whether the mitochondrial complex I inhibitor phenformin could reverse both side effects, impose an energetic stress on cancer cells, and suppress the growth of HCC. Experimental Design: Human HCC cell lines were used in vitro to access the signaling and energetic impact of mTOR inhibitors and phenformin, either alone or in combination. Next, the therapeutic utility of these drugs alone or in combination was investigated preclinically in human orthotopic tumors implanted in mice, by analyzing their impact on the tumor burden and overall survival. Results: We found phenformin caused mitochondrial dysfunction and fragmentation, inducing a compensatory shift to glycolysis. In contrast, dual inhibition of mTOR impaired cell growth and glycolysis, while increasing mitochondrial fusion and efficiency. In a mouse model of human HCC, dual inhibition of mTOR, together with phenformin, was highly efficacious in controlling tumor burden. However, more strikingly, pretreatment with phenformin sensitized tumors to dual inhibition of mTOR, leading to a dramatic improvement in survival. Conclusions: Treatment of HCC cells in vitro with the biguanide phenformin causes a metabolic shift to glycolysis, mitochondrial dysfunction and fragmentation, and dramatically sensitizes orthotopic liver tumors to dual inhibition of mTOR. We therefore propose this therapeutic approach should be tested clinically in HCC. Clin Cancer Res; 24(15); 3767–80. ©2018 AACR.

mTOR Kinase Inhibition Effectively Decreases Progression of a Subset of Neuroendocrine Tumors that Progress on Rapalog Therapy and Delays Cardiac Impairment

Inhibition of mTOR signaling using the rapalog everolimus is an FDA-approved targeted therapy for patients with lung and gastroenteropancreatic neuroendocrine tumors (NET). However, patients eventually progress on treatment, highlighting the need for additional therapies. We focused on pancreatic NETs (pNET) and reasoned that treatment of these tumors upon progression on rapalog therapy, with an mTOR kinase inhibitor (mTORKi), such as CC-223, could overcome a number of resistance mechanisms in tumors and delay cardiac carcinoid disease. We performed preclinical studies using human pNET cells in vitro and injected them subcutaneously or orthotopically to determine tumor progression and cardiac function in mice treated with either rapamycin alone or switched to CC-223 upon progression. Detailed signaling and RNA sequencing analyses were performed on tumors that were sensitive or progressed on mTOR treatment. Approximately 57% of mice bearing pNET tumors that progressed on rapalog therapy showed a significant decrease in tumor volume upon a switch to CC-223. Moreover, mice treated with an mTORKi exhibited decreased cardiac dilation and thickening of heart valves than those treated with placebo or rapamycin alone. In conclusion, in the majority of pNETs that progress on rapalogs, it is possible to reduce disease progression using an mTORKi, such as CC-223. Moreover, CC-223 had an additional transient cardiac benefit on valvular fibrosis compared with placebo- or rapalog-treated mice. These results provide the preclinical rationale to further develop mTORKi clinically upon progression on rapalog therapy and to further test their long-term cardioprotective benefit in those NET patients prone to carcinoid syndrome. Mol Cancer Ther; 16(11); 2432–41. ©2017 AACR.

Immune checkpoint inhibitors in large cell neuroendocrine carcinoma: current status

Introduction Large cell neuroendocrine carcinomas (LCNEC) are a group of rare high grade neuroendocrine tumors that often behave clinically like small cell carcinoma (SCLC) and are treated as such. No major advancement in the management of these tumors has occurred in the last 30 years. Methods We present a case series of three cases from Markey Cancer center along with a review of 13 published cases in the literature wherein immune-checkpoint inhibitors were utilized in the management of LCNEC. Results Immune-checkpoint inhibitors might have clinical activity in LCNEC. Conclusion Role of immune-checkpoint inhibitors should be explored in prospective LCNEC clinical trials. We summarize current evidence regarding use of immune checkpoint inhibitors in the treatment of LCNEC.

Mitochondrial Complex I Activity Is Required for Maximal Autophagy

SUMMARY Cells adapt to nutrient and energy deprivation by inducing autophagy, which is regulated by the mammalian target of rapamycin (mTOR) and AMPactivated protein kinases (AMPKs). We found that cell metabolism significantly influences the ability to induce autophagy, with mitochondrial complex I function being an important factor in the initiation, amplitude, and duration of the response. We show that phenformin or genetic defects in complex I suppressed autophagy induced by mTOR inhibitors, whereas autophagy was enhanced by strategies that increased mitochondrial metabolism. We report that mTOR inhibitors significantly increased select phospholipids and mitochondrial-associated membranes (MAMs) in a complex I-dependent manner. We attribute the complex I autophagy defect to the inability to increase MAMs, limiting phosphatidylserine decarboxylase (PISD) activity and mitochondrial phosphatidylethanolamine (mtPE), which support autophagy. Our data reveal the dynamic and metabolic regulation of autophagy.

The PI3K-mTOR Pathway

Phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling is required for normal development, growth, and physiology. Mutations in multiple key regulators of this pathway have been reported to occur leading to aberrant signaling and have been implicated in a number of pathologies, including metabolic syndrome. This chapter will review the major proteins involved in PI3K/mTOR signaling and discuss the negative feedback loops which maintain homeostasis. The therapeutic advantages and limitations of PI3K and/or catalytic mTOR inhibitors, which are currently in clinical development, will be discussed. We also report studies using these inhibitors along with genetic models to delete or overexpress key players in PI3K/mTOR signaling pathways in yeast, worms, drosophila, and mice, which have been instrumental in elucidating the functions of these proteins in normal and disease states. Particular attention has been focused on the role of PI3K/mTOR signaling in proliferation, translation, metabolism (including energy balance regulation and metabolic syndrome), autophagy, and differentiation.

Abstract A53: Dual inhibition of mTOR in hepatocellular carcinoma: Autophagy friend or foe?

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and ranks second in cancer deaths. HCC is associated with chronic liver damage due to viral infection, excess alcohol, and most recently, non-alcoholic hepatitis (NASH), linked to obesity. Despite the recent success of the multikinase inhibitor sorafenib in HCC, for those patients with advanced stage disease the prognosis remains poor. A promising target in the treatment of HCC is mTOR, which is hyperactivated in 50% of HCCs. However, the FDA-approved mTOR-allosteric inhibitor, RAD001 has failed as a single agent in HCC, most likely due to its incomplete suppression of mTOR Complex 1 (mTORC1). Consistent with this finding we recently showed that RAD001 had minimal effects on the proliferation of HCC cells in culture, but when combined with BEZ235, a PI3K/mTOR-ATP site competitive inhibitor, synergistically blocked the growth of HCC tumor cells in culture and caused tumor regression in a mouse model approximating human HCC with bad prognosis (Elnakat et al., 2012). Further studies revealed the down regulation of a number of genes implicated in autophagy, which acts as a tumor suppressor in liver. This led to the finding that RAD001/BEZ235 causes the induction of autophagy in culture and induced mitophagy in tumors. Despite the finding that RAD001/BEZ235 caused tumor regression in vivo, it is argued that a small population of human CD133+ HCC stem-like cells (HSCs) is protected by mTOR inhibitors. In contrast, the biguanides, phenformin and metformin, also potent inhibitors of mTORC1, are reported to selectively suppress the proliferation of human cancer stem cells (CSC). These effects are argued to be through inhibition of mitochondrial oxidative phosphorylation, blocking ATP production, activating AMPK, and inducing autophagy. Given the ability of biguanides to inhibit mTORC1 and to selectively inhibit the proliferation of CSCs, we set out to determine its effects on tumor progression, using human HCC cell lines in a mouse orthotopic model, as compared to the RAD001/BEZ235. In parallel, we analyzed the effect of either drug treatment on whole cell energy consumption and autophagy. In our preliminary studies, similar to our results in the syngeneic mouse model, we found that orthotopic human HCC tumor progression is strongly impaired by RAD001/BEZ235, and also by phenformin as a single agent. RAD001/BEZ235 treated tumors show on-target effects as decreased phosphorylation of RPS6 and 4E-BP1, and lower proliferation indices. Similar on-target effects are observed after phenformin treatment. In vitro, we found that RAD001/BEZ235 inhibits mitochondrial respiration (25-40%), with mass decreasing (10-20%), but that mitochondria conserve their coupled vs uncoupled ATP oxygen consumption ratio, with no apparent compensatory shift towards glycolysis. In contrast, phenformin induces a greater decrease in mitochondrial respiration (80%) and a more rapid loss of mitochondrial mass (30-40%), with only 50% of mitochondrial oxygen consumption being coupled to ATP production, and a stronger induction of ROS. This strong mitochondrial impairment leads to higher glycolitic capacity, but is not sufficient to prevent cell death. Combining mTOR inhibition and mitochondrial complex 1 targeting results in even a greater decrease in mitochondrial function (>80%), with the same uncoupling and mass loss seen in phenformin treatment alone. Finally, using either a cargo-based or flux autophagic assay, implied, unexpectedly, that the benefits of phenformin in suppressing tumor progression are independent of autophagy, but consistent with mitochondrial dysfunction. Note: This abstract was not presented at the conference. Citation Format: Sonia Pereira Da Veiga, Carol Mercer, Ge Xuemei, Hala Elnakat Thomas, Sara Kozma, George Thomas. Dual inhibition of mTOR in hepatocellular carcinoma: Autophagy friend or foe? [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A53.