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Figure S1. Crizotinib resistant cells reveal a reprogrammed tumor metabolism. Figure S2. Crizotinib modulates fatty acid metabolism and mitochondrial parameters. Figure S3. Tracing analysis reveals distinct modulation of metabolism by MET inhibition. Figure S4. Acute and chronic MET inhibition drives oxygen consumption rate and ECAR. Figure S5. Specific silencing of MET enhances oxidative metabolism in patient-derived xenograft cells. Figure S6. Inhibition of complex I and complex V of the electron transport chain and MET synergistically reduce viability of glioblastoma cells. Figure S7. Gamitrinib and etomoxir synergize with crizotinib to reduce cellular viability in glioblastoma model systems. Figure S8. Combined Met inhibition and Gamitrinib treatment elicits cleavage of caspases and modulate the expression of Bcl2 family members. Figure S9. Crizotinib and gamitrinib elicit cell death with features of apoptosis. Figure S10. Gamitrinib and crizotinib is superior over kinase inhibitor combination treatments to elicit apoptosis. Figure S11. The combination treatment of etomoxir and crizotinib elicits a reduction in tumor proliferation without induction of organ toxicity. Supplementary Table 1. Primer sequences for real time PCR and chromatin immunoprecipitation qPCR

Purpose: Glioblastoma remains a challenge in oncology, in part due to tumor heterogeneity.Experimental Design: Patient-derived xenograft and stem-like glioblastoma cells were used as the primary model systems.Results: Based on a transcriptome and subsequent gene set enrichment analysis (GSEA), we show by using clinically validated compounds that the combination of histone deacetylase (HDAC) inhibition and bromodomain protein (BRD) inhibition results in pronounced synergistic reduction in cellular viability in patient-derived xenograft and stem-like glioblastoma cells. Transcriptome-based GSEA analysis suggests that metabolic reprogramming is involved with synergistic reduction of oxidative and glycolytic pathways in the combination treatment. Extracellular flux analysis confirms that combined HDAC inhibition and BRD inhibition blunts oxidative and glycolytic metabolism of cancer cells, leading to a depletion of intracellular ATP production and total ATP levels. In turn, energy deprivation drives an integrated stress response, originating from the endoplasmic reticulum. This results in an increase in proapoptotic Noxa. Aside from Noxa, we encounter a compensatory increase of antiapoptotic Mcl-1 protein. Pharmacologic, utilizing the FDA-approved drug sorafenib, and genetic inhibition of Mcl-1 enhanced the effects of the combination therapy. Finally, we show in orthotopic patient-derived xenografts of GBM, that the combination treatment reduces tumor growth, and that triple therapy involving the clinically validated compounds panobinostat, OTX015, and sorafenib further enhances these effects, culminating in a significant regression of tumors in vivo.Conclusions: Overall, these results warrant clinical testing of this novel, efficacious combination therapy. Clin Cancer Res; 24(16); 3941–54. ©2018 AACR.

Supplementary Data

Purpose: The goal of this study is to enhance the efficacy of imipridones, a novel class of AKT/ERK inhibitors that displayed limited therapeutic efficacy against glioblastoma (GBM).Experimental Design: Gene set enrichment, LC/MS, and extracellular flux analyses were used to determine the mechanism of action of novel imipridone compounds, ONC206 and ONC212. Orthotopic patient-derived xenografts were utilized to evaluate therapeutic potency.Results: Imipridones reduce the proliferation of patient-derived xenograft and stem-like glioblastoma cell cultures in vitro and in multiple xenograft models in vivo. ONC212 displayed the highest potency. High levels of c-myc predict susceptibility to growth inhibition and apoptosis induction by imipridones and increased host survival in orthotopic patient-derived xenografts. As early as 1 hour, imipridones elicit on-target inhibition, followed by dephosphorylation of GSK3β at serine 9. GSK3β promotes phosphorylation of c-myc at threonine 58 and enhances its proteasomal degradation. Moreover, inhibition of c-myc by BRD4 antagonists sensitizes for imipridone-induced apoptosis in stem-like GBM cells in vitro and in vivo. Imipridones affect energy metabolism by suppressing both glycolysis and oxidative phosphorylation, which is accompanied by a compensatory activation of the serine-one carbon-glycine (SOG) pathway, involving the transcription factor ATF4. Interference with the SOG pathway through novel inhibitors of PHGDH results in synergistic cell death induction in vitro and in vivo.Conclusions: These results suggest that c-myc expression predicts therapeutic responses to imipridones and that imipridones lead to suppression of tumor cell energy metabolism, eliciting unique metabolic vulnerabilities that can be exploited for clinical relevant drug combination therapies. Clin Cancer Res; 24(21); 5392–406. ©2018 AACR.

The receptor kinase c-MET has emerged as a target for glioblastoma therapy. However, treatment resistance emerges inevitably. Here, we performed global metabolite screening with metabolite set enrichment coupled with transcriptome and gene set enrichment analysis and proteomic screening, and identified substantial reprogramming of tumor metabolism involving oxidative phosphorylation and fatty acid oxidation (FAO) with substantial accumulation of acyl-carnitines accompanied by an increase of PGC1α in response to genetic (shRNA and CRISPR/Cas9) and pharmacologic (crizotinib) inhibition of c-MET. Extracellular flux and carbon tracing analyses (U-13C-glucose, U-13C-glutamine, and U-13C-palmitic acid) demonstrated enhanced oxidative metabolism, which was driven by FAO and supported by increased anaplerosis of glucose carbons. These findings were observed in concert with increased number and fusion of mitochondria and production of reactive oxygen species. Genetic interference with PGC1α rescued this oxidative phenotype driven by c-MET inhibition. Silencing and chromatin immunoprecipitation experiments demonstrated that cAMP response elements binding protein regulates the expression of PGC1α in the context of c-MET inhibition. Interference with both oxidative phosphorylation (metformin, oligomycin) and β-oxidation of fatty acids (etomoxir) enhanced the antitumor efficacy of c-MET inhibition. Synergistic cell death was observed with c-MET inhibition and gamitrinib treatment. In patient-derived xenograft models, combination treatments of crizotinib and etomoxir, and crizotinib and gamitrinib were significantly more efficacious than single treatments and did not induce toxicity. Collectively, we have unraveled the mechanistic underpinnings of c-MET inhibition and identified novel combination therapies that may enhance its therapeutic efficacy.Significance:c-MET inhibition causes profound metabolic reprogramming that can be targeted by drug combination therapies.

Supplementary Methods

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BackgroundA fraction of COVID-19 patients develops severe disease requiring hospitalization, while the majority, including high-risk individuals, experience mild symptoms. Severe disease has been associated with higher levels of antibodies and inflammatory cytokines, but the association has often resulted from comparison of patients with diverse demographics and comorbidity status. This study examined patients with defined demographic risk factors for severe COVID-19 who developed mild vs. severe COVID-19.MethodsThis study evaluated hospitalized vs. ambulatory COVID-19 patients in the James J. Peters VA Medical Center, Bronx, NY. This cohort presented demographic risk factors for severe COVID-19: median age of 63, >80% male, >85% black and/or Hispanic. Sera were collected four to 243 days after symptom onset and evaluated for binding and functional antibodies as well as 48 cytokines/chemokines.FindingsAmbulatory and hospitalized patients showed no difference in SARS-CoV-2-specific antibody levels and functions. However, a strong correlation between anti-S2 antibody levels and the other antibody parameters was observed in hospitalized but not in ambulatory cases. Cytokine/chemokine levels also revealed differences, with notably higher IL-27 levels in hospitalized patients. Hence, among the older, mostly male patients studied here, SARS-CoV-2-specific antibody levels and functions did not distinguish hospitalized and ambulatory cases but a discordance in S2-specific antibody responses was noted in ambulatory patients, and elevated levels of specific cytokines were maintained in convalescent sera of hospitalized cases.InterpretationThe data indicate that antibodies against the relatively conserved S2 spike subunit and immunoregulatory cytokines such as IL-27 are potential immune determinants of COVID-19.Research in contextEvidence before this studyPrevious studies demonstrated that high levels of SARS-CoV-2 spike binding antibodies and neutralizing antibodies were associated with COVID-19 disease severity. However, the comparisons were often made without considering demographics and comorbidities. Correlation was similarly shown between severe disease and marked elevation of several plasma cytokines but again, most analyses of cytokine responses to COVID-19 were conducted by comparison of patient cohorts with diverse demographic characteristics and risk factors.Added value of this studyWe evaluated here a comprehensive profile of SARS-CoV-2-specific antibodies (total Ig, isotypes/subtypes, Fab- and Fc-mediated functions) and a panel of 48 cytokines and chemokines in serum samples from a cohort of SARS-CoV-2-infected patients with demographic risk factors for severe COVID-19: 81% were male, 79% were >50 years old (median of 63), and 85% belonged to US minority groups (black and/or Hispanic). Comparison of hospitalized vs. ambulatory patients within this cohort revealed two features that differed between severe vs. mild COVID-19 cases: a discordant Ab response to the S2 subunit of the viral spike protein in the mild cases and an elevated response of specific cytokines and chemokines, notably IL-27, in the severe cases.Implications of all the available evidenceData from the study identified key immunologic markers for severe vs. mild COVID-19 that provide a path forward for investigations of their roles in minimizing or augmenting disease severity.

SARS-Cov-2 (severe acute respiratory disease coronavirus 2), which causes Coronavirus Disease 2019 (COVID19) was first detected in China in late 2019 and has since then caused a global pandemic. While molecular assays to directly detect the viral genetic material are available for the diagnosis of acute infection, we currently lack serological assays suitable to specifically detect SARS-CoV-2 antibodies. Here we describe serological enzyme-linked immunosorbent assays (ELISA) that we developed using recombinant antigens derived from the spike protein of SARS-CoV-2. Using negative control samples representing pre-COVID 19 background immunity in the general adult population as well as samples from COVID19 patients, we demonstrate that these assays are sensitive and specific, allowing for screening and identification of COVID19 seroconverters using human plasma/serum as early as two days post COVID19 symptoms onset. Importantly, these assays do not require handling of infectious virus, can be adjusted to detect different antibody types and are amendable to scaling. Such serological assays are of critical importance to determine seroprevalence in a given population, define previous exposure and identify highly reactive human donors for the generation of convalescent serum as therapeutic. Sensitive and specific identification of coronavirus SARS-Cov-2 antibody titers may, in the future, also support screening of health care workers to identify those who are already immune and can be deployed to care for infected patients minimizing the risk of viral spread to colleagues and other patients.

Fragile X syndrome (FXS) is the leading known inherited intellectual disability and the most common genetic cause of autism. The full mutation results in transcriptional silencing of the Fmr1 gene and loss of fragile X mental retardation protein (FMRP) expression. Defects in neuroenergetic capacity are known to cause a variety of neurodevelopmental disorders. Thus, we explored the integrity of forebrain mitochondria in Fmr1 knockout mice during the peak of synaptogenesis. We found inefficient thermogenic respiration due to futile proton leak in Fmr1 KO mitochondria caused by coenzyme Q (CoQ) deficiency and an open cyclosporine-sensitive channel. Repletion of mitochondrial CoQ within the Fmr1 KO forebrain closed the channel, blocked the pathological proton leak, restored rates of protein synthesis during synaptogenesis, and normalized the key phenotypic features later in life. The findings demonstrate that FMRP deficiency results in inefficient oxidative phosphorylation during the neurodevelopment and suggest that dysfunctional mitochondria may contribute to the FXS phenotype.

The B.1.1.529 (Omicron) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified in November of 2021 in South Africa and Botswana as well as in a sample of a traveler from South Africa in Hong Kong.1,2 Since then, B.1.1.529 has been detected in many countries globally. This variant seems to be more infectious than B.1.617.2 (Delta), has already caused super spreader events3 and has outcompeted Delta within weeks in several countries and metropolitan areas. B.1.1.529 hosts an unprecedented number of mutations in its spike gene and early reports have provided evidence for extensive immune escape and reduced vaccine effectiveness.2,4-6 Here, we investigated the neutralizing and binding activity of sera from convalescent, mRNA double vaccinated, mRNA boosted as well as convalescent double vaccinated and convalescent boosted individuals against wild type, B.1.351 and B.1.1.529 SARS-CoV-2 isolates. Neutralizing activity of sera from convalescent and double vaccinated participants was undetectable to very low against B.1.1.529 while neutralizing activity of sera from individuals who had been exposed to spike three or four times was maintained, albeit at strongly reduced levels. Binding to the B.1.1.529 receptor binding domain (RBD) and N-terminal domain (NTD) was reduced in convalescent not vaccinated but was mostly retained in vaccinated individuals.

Responses of Seropositive Persons to a Single SARS-CoV-2 Vaccine Some persons who have recovered from Covid-19 have antibodies to the spike protein of SARS-CoV-2. In 43 such persons who had receive...

Patients with cancer experience a higher burden of SARS-CoV-2 infection, disease severity, complications, and mortality, than the general population. SARS-CoV-2 mRNA vaccines are highly effective in the general population; however, few data are available on their efficacy in patients with cancer. Using a prospective cohort, we assessed the seroconversion rates and anti-SARS-CoV-2 spike protein antibody titers following the first and second dose of BNT162b2 and mRNA-1273 SARS-CoV-2 vaccines in patients with cancer in US and Europe from January to April 2021. Among 131 patients, most (94%) achieved seroconversion after receipt of two vaccine doses. Seroconversion rates and antibody titers in patients with hematological malignancy were significantly lower than those with solid tumors. None of the patients with history of anti-CD-20 antibody in the 6 months before vaccination developed antibody response. Antibody titers were highest for clinical surveillance or endocrine therapy groups and lowest for cytotoxic chemotherapy or monoclonal antibody groups.

In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations., An analysis of mRNA vaccine-induced polyclonal antibodies and plasmablast derived monoclonal antibodies from individuals vaccinated against SARS-CoV-2 identifies a high proportion of non-neutralizing antibodies, the induction of cross-reactive antibodies to seasonal coronaviruses and also maps the regions in the spike protein that are targeted, even among viral variants.

Autosomal recessive thymidine kinase 2 (TK2) mutations causes TK2 deficiency, which typically manifests as a progressive and fatal mitochondrial myopathy in infants and children. Treatment with deoxycytidine and thymidine ameliorates mitochondrial defects and extends lifespan of Tk2 knock-in mouse (TK2−/−); however, efficacy is limited by age- and tissue-dependent expression of the cytosolic enzymes Tk1 and Dck. Thus, therapies aimed at systemic restoration of TK2 activity are needed. Here, we demonstrate that delivery of human TK2 cDNA to Tk2−/− mice using AAV9 efficiently rescued Tk2 activity in all the tissues tested except kidney, delayed disease onset, and increased lifespan. Sequential treatment of Tk2−/− mice with AAV9 first followed by AAV2 at different ages allowed us to reduce the viral dose while further prolonging the lifespan. Furthermore, addition of deoxycytidine and deoxythymidine supplementation to AAV9 + AAV2 treated Tk2−/− mice dramatically improved mtDNA copy numbers in liver and kidney, animal growth, and lifespan. These data indicate that combined pharmacological and gene therapies may be highly efficacious for human TK2 deficiency.

Mutations affecting mitochondrial coenzyme Q (CoQ) biosynthesis lead to kidney failure due to selective loss of podocytes, essential cells of the kidney filter. Curiously, neighboring tubular epithelial cells are spared early in disease despite higher mitochondrial content. We sought to illuminate noncanonical, cell-specific roles for CoQ, independently of the electron transport chain (ETC). Here, we demonstrate that CoQ depletion caused by Pdss2 enzyme deficiency in podocytes results in perturbations in polyunsaturated fatty acid (PUFA) metabolism and the Braf/Mapk pathway rather than ETC dysfunction. Single-nucleus RNA-Seq from kidneys of Pdss2(kd/kd) mice with nephrotic syndrome and global CoQ deficiency identified a podocyte-specific perturbation of the Braf/Mapk pathway. Treatment with GDC-0879, a Braf/Mapk-targeting compound, ameliorated kidney disease in Pdss2(kd/kd) mice. Mechanistic studies in Pdss2-depleted podocytes revealed a previously unknown perturbation in PUFA metabolism that was confirmed in vivo. Gpx4, an enzyme that protects against PUFA-mediated lipid peroxidation, was elevated in disease and restored after GDC-0879 treatment. We demonstrate broader human disease relevance by uncovering patterns of GPX4 and Braf/Mapk pathway gene expression in tissue from patients with kidney diseases. Our studies reveal ETC-independent roles for CoQ in podocytes and point to Braf/Mapk as a candidate pathway for the treatment of kidney diseases.

Blighted Gotham Deaths caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in New York City (NYC) during the spring of 2020 have vastly exceeded those reported in China and many other countries. What were the early events that led to such a severe outbreak? Gonzalez-Reiche et al. sampled some of the early patients seeking assistance in February and March of 2020 at the Mount Sinai Health System. Phylogenetic analysis of virus sequences in these people, who were drawn from across NYC, showed that the virus had been independently introduced many times from Europe and elsewhere in the United States. Subsequent clusters of community transmission occurred. The focus of infection in NYC is a marker of the role this city plays as a two-way hub for human movement. Science this issue p. 297

Purpose: The goal of this study is to enhance the efficacy of imipridones, a novel class of AKT/ERK inhibitors that displayed limited therapeutic efficacy against glioblastoma (GBM). Experimental Design: Gene set enrichment, LC/MS, and extracellular flux analyses were used to determine the mechanism of action of novel imipridone compounds, ONC206 and ONC212. Orthotopic patient-derived xenografts were utilized to evaluate therapeutic potency. Results: Imipridones reduce the proliferation of patient-derived xenograft and stem-like glioblastoma cell cultures in vitro and in multiple xenograft models in vivo. ONC212 displayed the highest potency. High levels of c-myc predict susceptibility to growth inhibition and apoptosis induction by imipridones and increased host survival in orthotopic patient-derived xenografts. As early as 1 hour, imipridones elicit on-target inhibition, followed by dephosphorylation of GSK3β at serine 9. GSK3β promotes phosphorylation of c-myc at threonine 58 and enhances its proteasomal degradation. Moreover, inhibition of c-myc by BRD4 antagonists sensitizes for imipridone-induced apoptosis in stem-like GBM cells in vitro and in vivo. Imipridones affect energy metabolism by suppressing both glycolysis and oxidative phosphorylation, which is accompanied by a compensatory activation of the serine-one carbon-glycine (SOG) pathway, involving the transcription factor ATF4. Interference with the SOG pathway through novel inhibitors of PHGDH results in synergistic cell death induction in vitro and in vivo. Conclusions: These results suggest that c-myc expression predicts therapeutic responses to imipridones and that imipridones lead to suppression of tumor cell energy metabolism, eliciting unique metabolic vulnerabilities that can be exploited for clinical relevant drug combination therapies. Clin Cancer Res; 24(21); 5392–406. ©2018 AACR.

Nephrotic syndrome (NS), a frequent chronic kidney disease in children and young adults, is the most common phenotype associated with primary coenzyme Q(10) (CoQ(10)) deficiency and is very responsive to CoQ(10) supplementation, although the pathomechanism is not clear. Here, using a mouse model of CoQ deficiency-associated NS, we show that long-term oral CoQ(10) supplementation prevents kidney failure by rescuing defects of sulfides oxidation and ameliorating oxidative stress, despite only incomplete normalization of kidney CoQ levels and lack of rescue of CoQ-dependent respiratory enzymes activities. Liver and kidney lipidomics, and urine metabolomics analyses, did not show CoQ metabolites. To further demonstrate that sulfides metabolism defects cause oxidative stress in CoQ deficiency, we show that silencing of sulfide quinone oxido-reductase (SQOR) in wild-type HeLa cells leads to similar increases of reactive oxygen species (ROS) observed in HeLa cells depleted of the CoQ biosynthesis regulatory protein COQ8A. While CoQ(10) supplementation of COQ8A depleted cells decreases ROS and increases SQOR protein levels, knock-down of SQOR prevents CoQ(10) antioxidant effects. We conclude that kidney failure in CoQ deficiency-associated NS is caused by oxidative stress mediated by impaired sulfides oxidation and propose that CoQ supplementation does not significantly increase the kidney pool of CoQ bound to the respiratory supercomplexes, but rather enhances the free pool of CoQ, which stabilizes SQOR protein levels rescuing oxidative stress.

Purpose: Glioblastoma remains a challenge in oncology, in part due to tumor heterogeneity. Experimental Design: Patient-derived xenograft and stem-like glioblastoma cells were used as the primary model systems. Results: Based on a transcriptome and subsequent gene set enrichment analysis (GSEA), we show by using clinically validated compounds that the combination of histone deacetylase (HDAC) inhibition and bromodomain protein (BRD) inhibition results in pronounced synergistic reduction in cellular viability in patient-derived xenograft and stem-like glioblastoma cells. Transcriptome-based GSEA analysis suggests that metabolic reprogramming is involved with synergistic reduction of oxidative and glycolytic pathways in the combination treatment. Extracellular flux analysis confirms that combined HDAC inhibition and BRD inhibition blunts oxidative and glycolytic metabolism of cancer cells, leading to a depletion of intracellular ATP production and total ATP levels. In turn, energy deprivation drives an integrated stress response, originating from the endoplasmic reticulum. This results in an increase in proapoptotic Noxa. Aside from Noxa, we encounter a compensatory increase of antiapoptotic Mcl-1 protein. Pharmacologic, utilizing the FDA-approved drug sorafenib, and genetic inhibition of Mcl-1 enhanced the effects of the combination therapy. Finally, we show in orthotopic patient-derived xenografts of GBM, that the combination treatment reduces tumor growth, and that triple therapy involving the clinically validated compounds panobinostat, OTX015, and sorafenib further enhances these effects, culminating in a significant regression of tumors in vivo. Conclusions: Overall, these results warrant clinical testing of this novel, efficacious combination therapy. Clin Cancer Res; 24(16); 3941–54. ©2018 AACR.

The receptor kinase c-MET has emerged as a target for glioblastoma therapy. However, treatment resistance emerges inevitably. Here, we performed global metabolite screening with metabolite set enrichment coupled with transcriptome and gene set enrichment analysis and proteomic screening, and identified substantial reprogramming of tumor metabolism involving oxidative phosphorylation and fatty acid oxidation (FAO) with substantial accumulation of acyl-carnitines accompanied by an increase of PGC1α in response to genetic (shRNA and CRISPR/Cas9) and pharmacologic (crizotinib) inhibition of c-MET. Extracellular flux and carbon tracing analyses (U-13C-glucose, U-13C-glutamine, and U-13C-palmitic acid) demonstrated enhanced oxidative metabolism, which was driven by FAO and supported by increased anaplerosis of glucose carbons. These findings were observed in concert with increased number and fusion of mitochondria and production of reactive oxygen species. Genetic interference with PGC1α rescued this oxidative phenotype driven by c-MET inhibition. Silencing and chromatin immunoprecipitation experiments demonstrated that cAMP response elements binding protein regulates the expression of PGC1α in the context of c-MET inhibition. Interference with both oxidative phosphorylation (metformin, oligomycin) and β-oxidation of fatty acids (etomoxir) enhanced the antitumor efficacy of c-MET inhibition. Synergistic cell death was observed with c-MET inhibition and gamitrinib treatment. In patient-derived xenograft models, combination treatments of crizotinib and etomoxir, and crizotinib and gamitrinib were significantly more efficacious than single treatments and did not induce toxicity. Collectively, we have unraveled the mechanistic underpinnings of c-MET inhibition and identified novel combination therapies that may enhance its therapeutic efficacy. Significance: c-MET inhibition causes profound metabolic reprogramming that can be targeted by drug combination therapies.

Liver‐X‐receptor (LXR) agonists are known to bear anti‐tumor activity. However, their efficacy is limited and additional insights regarding the underlying mechanism are necessary. By performing transcriptome analysis coupled with global polar metabolite screening, we show that LXR agonists, LXR623 and GW3965, enhance synergistically the anti‐proliferative effect of BH3 mimetics in solid tumor malignancies, which is predominantly mediated by cell death with features of apoptosis and is rescued by exogenous cholesterol. Extracellular flux analysis and carbon tracing experiments (U‐13C‐glucose and U‐13C‐glutamine) reveal that within 5 h, activation of LXRβ results in reprogramming of tumor cell metabolism, leading to suppression of mitochondrial respiration, a phenomenon not observed in normal human astrocytes. LXR activation elicits a suppression of respiratory complexes at the protein level by reducing their stability. In turn, energy starvation drives an integrated stress response (ISR) that up‐regulates pro‐apoptotic Noxa in an ATF4‐dependent manner. Cholesterol and nucleotides rescue from the ISR elicited by LXR agonists and from cell death induced by LXR agonists and BH3 mimetics. In conventional and patient‐derived xenograft models of colon carcinoma, melanoma, and glioblastoma, the combination treatment of ABT263 and LXR agonists reduces tumor sizes significantly stronger than single treatments. Therefore, the combination treatment of LXR agonists and BH3 mimetics might be a viable efficacious treatment approach for solid malignancies.

Deregulation of the mevalonate pathway is known to be involved in a number of diseases that exhibit a systemic inflammatory phenotype and often neurological involvements, as seen in patients suffering from a rare disease called mevalonate kinase deficiency (MKD). One of the molecular mechanisms underlying this pathology could depend on the shortage of isoprenoid compounds and the subsequent mitochondrial damage, leading to oxidative stress and pro-inflammatory cytokines’ release. Moreover, it has been demonstrated that cellular death results from the balance between apoptosis and pyroptosis, both driven by mitochondrial damage and the molecular platform inflammasome. In order to rescue the deregulated pathway and decrease inflammatory markers, exogenous isoprenoid compounds were administered to a biochemical model of MKD obtained treating a murine monocytic cell line with a compound able to block the mevalonate pathway, plus an inflammatory stimulus. Our results show that isoprenoids acted in different ways, mainly increasing the expression of the evaluated markers [apoptosis, mitochondrial dysfunction, nucleotide-binding oligomerization-domain protein-like receptors 3 (NALP3), cytokines and nitric oxide (NO)]. Our findings confirm the hypothesis that inflammation is triggered, at least partially, by the shortage of isoprenoids. Moreover, although further studies are necessary, the achieved results suggest a possible role for exogenous isoprenoids in the treatment of MKD.

In familial and sporadic multiple system atrophy (MSA) patients, deficiency of coenzyme Q10 (CoQ10) has been associated with mutations in COQ2, which encodes the second enzyme in the CoQ10 biosynthetic pathway. Cerebellar ataxia is the most common presentation of CoQ10 deficiency, suggesting that the cerebellum might be selectively vulnerable to low levels of CoQ10. To investigate whether CoQ10 deficiency represents a common feature in the brains of MSA patients independent of the presence of COQ2 mutations, we studied CoQ10 levels in postmortem brains of 12 MSA, 9 Parkinson disease (PD), 9 essential tremor (ET) patients, and 12 controls. We also assessed mitochondrial respiratory chain enzyme activities, oxidative stress, mitochondrial mass, and levels of enzymes involved in CoQ biosynthesis. Our studies revealed CoQ10 deficiency in MSA cerebellum, which was associated with impaired CoQ biosynthesis and increased oxidative stress in the absence of COQ2 mutations. The levels of CoQ10 in the cerebella of ET and PD patients were comparable or higher than in controls. These findings suggest that CoQ10 deficiency may contribute to the pathogenesis of MSA. Because no disease modifying therapies are currently available, increasing CoQ10 levels by supplementation or upregulation of its biosynthesis may represent a novel treatment strategy for MSA patients.

Recent data suggest that glioblastomas (GBM) activate the c-MET signaling pathway and display increased levels in anti-apoptotic Bcl-2 family members. Therefore, targeting these two deregulated pathways for therapy might yield synergistic treatment responses. We applied extracellular flux analysis to assess tumor metabolism. We found that combined treatment with ABT263 and Crizotinib synergistically reduces the proliferation of glioblastoma cells, which was dependent on dual inhibition of Bcl-2 and Bcl-xL. The combination treatment led to enhanced apoptosis with loss of mitochondrial membrane potential and activation of caspases. On the molecular level, c-MET-inhibition results in significant energy deprivation with a reduction in oxidative phosphorylation, respiratory capacity and a suppression of intracellular energy production (ATP). In turn, loss of energy levels suppresses protein synthesis, causing a decline in anti-apoptotic Mcl-1 levels. Silencing of Mcl-1 enhanced ABT263 and MET-inhibitor mediated apoptosis, but marginally the combination treatment, indicating that Mcl-1 is the central factor for the induction of cell death induced by the combination treatment. Finally, combined treatment with BH3-mimetics and c-MET inhibitors results in significantly smaller tumors than each treatment alone in a PDX model system of glioblastoma. These results suggest that c-MET inhibition causes a selective vulnerability of GBM cells to Bcl-2/Bcl-xL inhibition.

Multiple System Atrophy is a severe neurodegenerative disorder which is characterized by a variable clinical presentation and a broad neuropathological spectrum. The pathogenic mechanisms are almost completely unknown. In the present study, we established a cellular model of MSA by using fibroblasts' primary cultures and performed several experiments to investigate the causative mechanisms of the disease, with a particular focus on mitochondrial functioning. Fibroblasts' analyses (7 MSA-P, 7 MSA-C and 6 healthy controls) displayed several anomalies in patients: an impairment of respiratory chain activity, in particular for succinate Coenzyme Q reductase (p

Summary Multiple system atrophy (MSA) is a progressive neurodegenerative disease that affects several areas of the CNS, whose pathogenesis is still widely unclear and for which an effective treatment is lacking. We have generated induced pluripotent stem cell-derived dopaminergic neurons from four MSA patients and four healthy controls and from two monozygotic twins discordant for the disease. In this model, we have demonstrated an aberrant autophagic flow and a mitochondrial dysregulation involving respiratory chain activity, mitochondrial content, and CoQ10 biosynthesis. These defective mechanisms may contribute to the onset of the disease, representing potential therapeutic targets., Highlights • An iPSC-based neuronal model of MSA is described • Mitochondria are dysfunctional in MSA neurons • Autophagic machinery is impaired in MSA neurons, Monzio Compagnoni et al. present an iPSC-based neuronal in vitro model of multiple system atrophy. Patients' dopaminergic neurons display a dysregulation of mitochondrial functioning and autophagy, suggesting new hints for the comprehension of the pathogenesis of the disease.

Background: Mevalonate Kinase Deficiency (MKD, OMIM #610377) is a rare autosomal recessive metabolic and inflammatory disease. In MKD, defective function of the enzyme mevalonate kinase, due to a mutation in the MVK gene, leads to the shortage of mevalonate- derived intermediates, which results in unbalanced prenylation of proteins and altered metabolism of sterols. These defects lead to a complex multisystem inflammatory and metabolic syndrome. Objective: Although biologic therapies aimed at blocking the inflammatory cytokine interleukin- 1 can significantly reduce inflammation, they cannot completely control the clinical symptoms that affect the nervous system. For this reason, MKD can still be considered an orphan drug disease. The availability of MKD models reproducing the MKD-systematic inflammation, is crucial to improve the knowledge on its pathogenesis, which is still unknown. New therapies are also required in order to improve pateints’ conditions and their quality of life. Methods: MKD-cellular models can be obtained by biochemical inhibition of mevalonatederived isoprenoids. Of note, these cells present an exaggerated response to inflammatory stimuli that can be reduced by treatment with zaragozic acid, an inhibitor of squalene synthase, thus increasing the availability of isoprenoids intermediates upstream the enzymatic block. Results: A similar action might be obtained by lapaquistat acetate (TAK-475, Takeda), a drug that underwent extensive clinical trials as a cholesterol lowering agent 10 years ago, with a good safety profile. Conclusions: Here we describe the preclinical evidence supporting the possible repositioning of TAK-475 from its originally intended use to the treatment of MKD and discuss its potential to modulate the mevalonate pathway in inflammatory diseases.

Mevalonic aciduria, a rare autosomal recessive disease, represents the most severe form of the periodic fever, known as Mevalonate Kinase Deficiency. This disease is caused by the mutation of the MVK gene, which codes for the enzyme mevalonate kinase, along the cholesterol pathway. Mevalonic aciduria patients show recurrent fever episodes with associated inflammatory symptoms, severe neurologic impairments, or death, in early childhood. The typical neurodegeneration occurring in mevalonic aciduria is linked both to the intrinsic apoptosis pathway (caspase-3 and -9), which is triggered by mitochondrial damage, and to pyroptosis (caspase-1). These cell death mechanisms seem to be also related to the assembly of the inflammasome, which may, in turn, activate pro-inflammatory cytokines and chemokines. Thus, this particular molecular platform may play a crucial role in neuroinflammation mechanisms. Nowadays, a specific therapy is still lacking and the pathogenic mechanisms involving neuroinflammation and neuronal dysfunction have not yet been completely understood, making mevalonic aciduria an orphan drug disease. This review aims to analyze the relationship among neuroinflammation, mitochondrial damage, programmed cell death, and neurodegeneration. Targeting inflammation and degeneration in the central nervous system might help identify promising treatment approaches for mevalonic aciduria or other diseases in which these mechanisms are involved.

Mevalonate Kinase Deficiency (MKD) is a rare autosomal recessive inborn disorder of cholesterol biosynthesis caused by mutations in the mevalonate kinase (MK) gene, leading to MK enzyme decreased activity. The consequent shortage of mevalonate-derived isoprenoid compounds results in an inflammatory phenotype, caused by the activation of the NALP3 inflammasome that determines an increased caspase-1 activation and IL-1βrelease. In MKD, febrile temperature can further decrease the residual MK activity, leading to mevalonate pathway modulation and to possible disease worsening. We previously demonstrated that the administration of exogenous isoprenoids such as geraniol or the modulation of the enzymatic pathway with drugs, such as Tipifarnib, partially rescues the inflammatory phenotype associated with the defective mevalonic pathway. However, it has not been investigated yet how temperature can affect the success of these treatments. Thus, we investigated the effect of temperature on primary human monocytes from MKD patients. Furthermore the ability of geraniol and Tipifarnib to reduce the abnormal inflammatory response, already described at physiological temperature in MKD, was studied in a febrile condition. We evidenced the role of temperature in the modulation of the inflammatory events and suggested strongly considering this variable in future researches aimed at finding a treatment for MKD.