Balancing yield, purity and practicality: a modified differential ultracentrifugation protocol for efficient isolation of small extracellular vesicles from human serum.

Ultracentrifugation remains the gold standard for isolation of small extracellular vesicles (sEV), particularly for cancer applications. The objective of this study was to determine if a widely used ultracentrifugation protocol for isolation of serum sEV could be modified to reduce the number of ultracentrifugation cycles and increase efficiency, while maintaining equal or better sample purity and yield. Serum was obtained from two healthy subjects. sEVs were isolated from 1 mL aliquots using three different ultracentrifugation protocols. Co-isolation of RNA carrier protein was assessed by performing Western blots for ApoA-I, ApoB, and Ago2. Small RNA-sequencing was performed on the sEV isolates, and differential detection of small ncRNA was compared across isolation protocols. Reduction from three- to two-ultracentrifuge cycles with no sucrose cushion resulted in a much higher sEV yield but also had the highest levels of lipoprotein and Ago2 contamination. However, the two-ultracentrifugation cycle protocol that incorporated a 30% sucrose cushion into the first cycle resulted in slightly higher sEV yields with lower levels of protein contamination compared to the lengthier three-ultracentrifugation cycle approach, therefore presenting a more efficient alternative approach for isolation of serum sEVs. It was also notable that there were some differences in sEV ncRNA cargo according to protocol, although it was less than expected given the differences in co-isolated RNA carrier proteins. Our results suggest that use of the modified serum sEV isolation protocol with two ultracentrifugation cycles and incorporating a 30% sucrose cushion offers a more efficient approach in terms of efficiency and purity.

Mass spectrometry proteomics reveals a function for mammalian CALCOCO1 in MTOR-regulated selective autophagy.

Macroautophagy/autophagy is suppressed by MTOR (mechanistic target of rapamycin kinase) and is an anticancer target under active investigation. Yet, MTOR-regulated autophagy remains incompletely mapped. We used proteomic profiling to identify proteins in the MTOR-autophagy axis. Wild-type (WT) mouse cell lines and cell lines lacking individual autophagy genes (Atg5 or Ulk1/Ulk2) were treated with an MTOR inhibitor to induce autophagy and cultured in media with either glucose or galactose. Mass spectrometry proteome profiling revealed an elevation of known autophagy proteins and candidates for new autophagy components, including CALCOCO1 (calcium binding and coiled-coil domain protein 1). We show that CALCOCO1 physically interacts with MAP1LC3C, a key protein in the machinery of autophagy. Genetic deletion of CALCOCO1 disrupted autophagy of the endoplasmic reticulum (reticulophagy). Together, these results reveal a role for CALCOCO1 in MTOR-regulated selective autophagy. More generally, the resource generated by this work provides a foundation for establishing links between the MTOR-autophagy axis and proteins not previously linked to this pathway. Abbreviations: ATG: autophagy-related; CALCOCO1: calcium binding and coiled-coil domain protein 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain protein 2; CLIR: MAP1LC3C-interacting region; CQ: chloroquine; KO: knockout; LIR: MAP1LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MLN: MLN0128 ATP-competitive MTOR kinase inhibitor; MTOR: mechanistic target of rapamycin kinase; reticulophagy: selective autophagy of the endoplasmic reticulum; TAX1BP1/CALCOCO3: TAX1 binding protein 1; ULK: unc 51-like autophagy activating kinase; WT: wild-type.

Mitochondrial Complex I Activity Is Required for Maximal Autophagy.

Cells adapt to nutrient and energy deprivation by inducing autophagy, which is regulated by the mammalian target of rapamycin (mTOR) and AMP-activated 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.

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.

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.

Pharmacologic Targeting of S6K1 in PTEN-Deficient Neoplasia.

Genetic S6K1 inactivation can induce apoptosis in PTEN-deficient cells. We analyzed the therapeutic potential of S6K1 inhibitors in PTEN-deficient T cell leukemia and glioblastoma. Results revealed that the S6K1 inhibitor LY-2779964 was relatively ineffective as a single agent, while S6K1-targeting AD80 induced cytotoxicity selectively in PTEN-deficient cells. In vivo, AD80 rescued 50% of mice transplanted with PTEN-deficient leukemia cells. Cells surviving LY-2779964 treatment exhibited inhibitor-induced S6K1 phosphorylation due to increased mTOR-S6K1 co-association, which primed the rapid recovery of S6K1 signaling. In contrast, AD80 avoided S6K1 phosphorylation and mTOR co-association, resulting in durable suppression of S6K1-induced signaling and protein synthesis. Kinome analysis revealed that AD80 coordinately inhibits S6K1 together with the TAM family tyrosine kinase AXL. TAM suppression by BMS-777607 or genetic knockdown potentiated cytotoxic responses to LY-2779964 in PTEN-deficient glioblastoma cells. These results reveal that combination targeting of S6K1 and TAMs is a potential strategy for treatment of PTEN-deficient malignancy.

A Phase Ib Study of the Dual PI3K/mTOR Inhibitor Dactolisib (BEZ235) Combined with Everolimus in Patients with Advanced Solid Malignancies.

BACKGROUND: The combination of everolimus and the imidazoquinoline derivative, BEZ235 (dactolisib), a dual PI3K/mTOR inhibitor, demonstrated synergy in a preclinical model. OBJECTIVE: To establish clinical feasibility, a phase Ib dose-escalation trial investigating safety and pharmacokinetics of this combination in patients with advanced tumors was performed. PATIENTS AND METHODS: BEZ235 was orally administered daily in escalating doses of 200, 400, and 800 mg along with everolimus at 2.5 mg daily in 28-day cycles. Nineteen patients were enrolled. Adverse events and tumor responses were evaluated using CTCAE v4.0 and RECIST 1.1, respectively. Pharmacokinetic analyses were performed. RESULTS: Common toxicities observed included fatigue, diarrhea, nausea, mucositis, and elevated liver enzymes. No confirmed responses were observed. BEZ235 pharmacokinetics exhibited dose-proportional increases in Cmax and AUC0-24 over the three doses, with high inter-individual variability. Non-compartmental and population pharmacokinetic-based simulations indicated significant increases in everolimus Cmax and AUC0-24 on day 28 and decreased clearance to 13.41 L/hr. CONCLUSIONS: The combination of BEZ235 and everolimus demonstrated limited efficacy and tolerance. BEZ235 systemic exposure increased in a dose-proportional manner while oral bioavailability was quite low, which may be related to gastrointestinal-specific toxicity. The changes in steady-state pharmacokinetics of everolimus with BEZ235 highlight potential drug-drug interactions when these two drugs are administered together. Clinicaltrials.gov: NCT01508104.

mTOR inhibitors synergize on regression, reversal of gene expression, and autophagy in hepatocellular carcinoma.

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.