Elimusertib has Antitumor Activity in Preclinical Patient-Derived Pediatric Solid Tumor Models.

The small-molecule inhibitor of ataxia telangiectasia and Rad3-related protein (ATR), elimusertib, is currently being tested clinically in various cancer entities in adults and children. Its preclinical antitumor activity in pediatric malignancies, however, is largely unknown. We here assessed the preclinical activity of elimusertib in 38 cell lines and 32 patient-derived xenograft (PDX) models derived from common pediatric solid tumor entities. Detailed in vitro and in vivo molecular characterization of the treated models enabled the evaluation of response biomarkers. Pronounced objective response rates were observed for elimusertib monotherapy in PDX, when treated with a regimen currently used in clinical trials. Strikingly, elimusertib showed stronger antitumor effects than some standard-of-care chemotherapies, particularly in alveolar rhabdomysarcoma PDX. Thus, elimusertib has strong preclinical antitumor activity in pediatric solid tumor models, which may translate to clinically meaningful responses in patients.

Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer.

DNA amplifications in cancer do not only harbor oncogenes. We sought to determine whether passenger coamplifications could create collateral therapeutic vulnerabilities. Through an analysis of >3,000 cancer genomes followed by the interrogation of CRISPR-Cas9 loss-of-function screens across >700 cancer cell lines, we determined that passenger coamplifications are accompanied by distinct dependency profiles. In a proof-of-principle study, we demonstrate that the coamplification of the bona fide passenger gene DEAD-Box Helicase 1 (DDX1) creates an increased dependency on the mTOR pathway. Interaction proteomics identified tricarboxylic acid (TCA) cycle components as previously unrecognized DDX1 interaction partners. Live-cell metabolomics highlighted that this interaction could impair TCA activity, which in turn resulted in enhanced mTORC1 activity. Consequently, genetic and pharmacologic disruption of mTORC1 resulted in pronounced cell death in vitro and in vivo. Thus, structurally linked coamplification of a passenger gene and an oncogene can result in collateral vulnerabilities. SIGNIFICANCE: We demonstrate that coamplification of passenger genes, which were largely neglected in cancer biology in the past, can create distinct cancer dependencies. Because passenger coamplifications are frequent in cancer, this principle has the potential to expand target discovery in oncology. This article is featured in Selected Articles from This Issue, p. 384.

Single-cell profiling of alveolar rhabdomyosarcoma reveals RAS pathway inhibitors as cell-fate hijackers with therapeutic relevance.

Rhabdomyosarcoma (RMS) is a group of pediatric cancers with features of developing skeletal muscle. The cellular hierarchy and mechanisms leading to developmental arrest remain elusive. Here, we combined single-cell RNA sequencing, mass cytometry, and high-content imaging to resolve intratumoral heterogeneity of patient-derived primary RMS cultures. We show that the aggressive alveolar RMS (aRMS) subtype contains plastic muscle stem-like cells and cycling progenitors that drive tumor growth, and a subpopulation of differentiated cells that lost its proliferative potential and correlates with better outcomes. While chemotherapy eliminates cycling progenitors, it enriches aRMS for muscle stem-like cells. We screened for drugs hijacking aRMS toward clinically favorable subpopulations and identified a combination of RAF and MEK inhibitors that potently induces myogenic differentiation and inhibits tumor growth. Overall, our work provides insights into the developmental states underlying aRMS aggressiveness, chemoresistance, and progression and identifies the RAS pathway as a promising therapeutic target.

Breaking the crosstalk of the Cellular Tumorigenic Network by low-dose combination therapy in lung cancer patient-derived xenografts.

Non-small cell lung cancer (NSCLC) is commonly diagnosed at advanced stages limiting treatment options. Although, targeted therapy has become integral part of NSCLC treatment therapies often fail to improve patient's prognosis. Based on previously published criteria for selecting drug combinations for overcoming resistances, NSCLC patient-derived xenograft (PDX) tumors were treated with a low dose combination of cabozantinib, afatinib, plerixafor and etoricoxib. All PDX tumors treated, including highly therapy-resistant adeno- and squamous cell carcinomas without targetable oncogenic mutations, were completely suppressed by this drug regimen, leading to an ORR of 81% and a CBR of 100%. The application and safety profile of this low dose therapy regimen was well manageable in the pre-clinical settings. Overall, this study provides evidence of a relationship between active paracrine signaling pathways of the Cellular Tumorigenic Network, which can be effectively targeted by a low-dose multimodal therapy to overcome therapy resistance and improve prognosis of NSCLC.

A Molecularly Characterized Preclinical Platform of Subcutaneous Renal Cell Carcinoma (RCC) Patient-Derived Xenograft Models to Evaluate Novel Treatment Strategies.

Renal cell carcinoma (RCC) is a kidney cancer with an onset mainly during the sixth or seventh decade of the patient's life. Patients with advanced, metastasized RCC have a poor prognosis. The majority of patients develop treatment resistance towards Standard of Care (SoC) drugs within months. Tyrosine kinase inhibitors (TKIs) are the backbone of first-line therapy and have been partnered with an immune checkpoint inhibitor (ICI) recently. Despite the most recent progress, the development of novel therapies targeting acquired TKI resistance mechanisms in advanced and metastatic RCC remains a high medical need. Preclinical models with high translational relevance can significantly support the development of novel personalized therapies. It has been demonstrated that patient-derived xenograft (PDX) models represent an essential tool for the preclinical evaluation of novel targeted therapies and their combinations. In the present project, we established and molecularly characterized a comprehensive panel of subcutaneous RCC PDX models with well-conserved molecular and pathological features over multiple passages. Drug screening towards four SoC drugs targeting the vascular endothelial growth factor (VEGF) and PI3K/mTOR pathway revealed individual and heterogeneous response profiles in those models, very similar to observations in patients. As unique features, our cohort includes PDX models from metastatic disease and multi-tumor regions from one patient, allowing extended studies on intra-tumor heterogeneity (ITH). The PDX models are further used as basis for developing corresponding in vitro cell culture models enabling advanced high-throughput drug screening in a personalized context. PDX models were subjected to next-generation sequencing (NGS). Characterization of cancer-relevant features including driver mutations or cellular processes was performed using mutational and gene expression data in order to identify potential biomarker or treatment targets in RCC. In summary, we report a newly established and molecularly characterized panel of RCC PDX models with high relevance for translational preclinical research.

Therapeutic targeting of ATR in alveolar rhabdomyosarcoma.

Despite advances in multi-modal treatment approaches, clinical outcomes of patients suffering from PAX3-FOXO1 fusion oncogene-expressing alveolar rhabdomyosarcoma (ARMS) remain dismal. Here we show that PAX3-FOXO1-expressing ARMS cells are sensitive to pharmacological ataxia telangiectasia and Rad3 related protein (ATR) inhibition. Expression of PAX3-FOXO1 in muscle progenitor cells is not only sufficient to increase sensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibit increased sensitivity to structurally diverse inhibitors of ATR. Mechanistically, ATR inhibition leads to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increases sensitivity of ARMS cells to PARP1 inhibition in vitro, and combined treatment with ATR and PARP1 inhibitors induces complete regression of primary patient-derived ARMS xenografts in vivo. Lastly, a genome-wide CRISPR activation screen (CRISPRa) in combination with transcriptional analyses of ATR inhibitor resistant ARMS cells identifies the RAS-MAPK pathway and its targets, the FOS gene family, as inducers of resistance to ATR inhibition. Our findings provide a rationale for upcoming biomarker-driven clinical trials of ATR inhibitors in patients suffering from ARMS.

Sex differences in physiological cardiac hypertrophy are associated with exercise-mediated changes in energy substrate availability.

Exercise-induced cardiac hypertrophy has been recently identified to be regulated in a sex-specific manner. In parallel, women exhibit enhanced exercise-mediated lipolysis compared with men, which might be linked to cardiac responses. The aim of the present study was to assess if previously reported sex-dependent differences in the cardiac hypertrophic response during exercise are associated with differences in cardiac energy substrate availability/utilization. Female and male C57BL/6J mice were challenged with active treadmill running for 1.5 h/day (0.25 m/s) over 4 wk. Mice underwent cardiac and metabolic phenotyping including echocardiography, small-animal PET, peri-exercise indirect calorimetry, and analysis of adipose tissue (AT) lipolysis and cardiac gene expression. Female mice exhibited increased cardiac hypertrophic responses to exercise compared with male mice, measured by echocardiography [percent increase in left ventricular mass (LVM): female: 22.2 ± 0.8%, male: 9.0 ± 0.2%; P < 0.05]. This was associated with increased plasma free fatty acid (FFA) levels and augmented AT lipolysis in female mice after training, whereas FFA levels from male mice decreased. The respiratory quotient during exercise was significantly lower in female mice indicative for preferential utilization of fatty acids. In parallel, myocardial glucose uptake was reduced in female mice after exercise, analyzed by PET {injection dose (ID)/LVM [%ID/g]: 36.8 ± 3.5 female sedentary vs. 28.3 ± 4.3 female training; P < 0.05}, whereas cardiac glucose uptake was unaltered after exercise in male counterparts. Cardiac genes involved in fatty acid uptake/oxidation in females were increased compared with male mice. Collectively, our data demonstrate that sex differences in exercise-induced cardiac hypertrophy are associated with changes in cardiac substrate availability and utilization.

Class I histone deacetylases (HDAC) critically contribute to Ewing sarcoma pathogenesis.

BACKGROUND: Histone acetylation and deacetylation seem processes involved in the pathogenesis of Ewing sarcoma (EwS). Here histone deacetylases (HDAC) class I were investigated. METHODS: Their role was determined using different inhibitors including TSA, Romidepsin, Entinostat and PCI-34051 as well as CRISPR/Cas9 class I HDAC knockouts and HDAC RNAi. To analyze resulting changes microarray analysis, qRT-PCR, western blotting, Co-IP, proliferation, apoptosis, differentiation, invasion assays and xenograft-mouse models were used. RESULTS: Class I HDACs are constitutively expressed in EwS. Patients with high levels of individual class I HDAC expression show decreased overall survival. CRISPR/Cas9 class I HDAC knockout of individual HDACs such as HDAC1 and HDAC2 inhibited invasiveness, and blocked local tumor growth in xenograft mice. Microarray analysis demonstrated that treatment with individual HDAC inhibitors (HDACi) blocked an EWS-FLI1 specific expression profile, while Entinostat in addition suppressed metastasis relevant genes. EwS cells demonstrated increased susceptibility to treatment with chemotherapeutics including Doxorubicin in the presence of HDACi. Furthermore, HDACi treatment mimicked RNAi of EZH2 in EwS. Treated cells showed diminished growth capacity, but an increased endothelial as well as neuronal differentiation ability. HDACi synergizes with EED inhibitor (EEDi) in vitro and together inhibited tumor growth in xenograft mice. Co-IP experiments identified HDAC class I family members as part of a regulatory complex together with PRC2. CONCLUSIONS: Class I HDAC proteins seem to be important mediators of the pathognomonic EWS-ETS-mediated transcription program in EwS and in combination therapy, co-treatment with HDACi is an interesting new treatment opportunity for this malignant disease.

Publisher Correction: mTORC1 and mTORC2 Differentially Regulate Cell Fate Programs to Coordinate Osteoblastic Differentiation in Mesenchymal Stromal Cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

mTORC1 and mTORC2 Differentially Regulate Cell Fate Programs to Coordinate Osteoblastic Differentiation in Mesenchymal Stromal Cells.

Vascular regeneration depends on intact function of progenitors of vascular smooth muscle cells such as pericytes and their circulating counterparts, mesenchymal stromal cells (MSC). Deregulated MSC differentiation and maladaptive cell fate programs associated with age and metabolic diseases may exacerbate arteriosclerosis due to excessive transformation to osteoblast-like calcifying cells. Targeting mTOR, a central controller of differentiation and cell fates, could offer novel therapeutic perspectives. In a cell culture model for osteoblastic differentiation of pluripotent human MSC we found distinct roles for mTORC1 and mTORC2 in the regulation of differentiation towards calcifying osteoblasts via cell fate programs in a temporally-controlled sequence. Activation of mTORC1 with induction of cellular senescence and apoptosis were hallmarks of transition to a calcifying phenotype. Inhibition of mTORC1 with Rapamycin elicited reciprocal activation of mTORC2, enhanced autophagy and recruited anti-apoptotic signals, conferring protection from calcification. Pharmacologic and genetic negative interference with mTORC2 function or autophagy both abolished regenerative programs but induced cellular senescence, apoptosis, and calcification. Overexpression of the mTORC2 constituent rictor revealed that enhanced mTORC2 signaling without altered mTORC1 function was sufficient to inhibit calcification. Studies in mice reproduced the in vitro effects of mTOR modulation with Rapamycin on cell fates in vascular cells in vivo. Amplification of mTORC2 signaling promotes protective cell fates including autophagy to counteract osteoblast differentiation and calcification of MSC, representing a novel mTORC2 function. Regenerative approaches aimed at modulating mTOR network activation patterns hold promise for delaying age-related vascular diseases and treatment of accelerated arteriosclerosis in chronic metabolic conditions.

A synthetic epoxyeicosatrienoic acid analogue prevents the initiation of ischemic acute kidney injury.

AIM: Imbalances in cytochrome P450 (CYP)-dependent eicosanoid formation may play a central role in ischemic acute kidney injury (AKI). We reported previously that inhibition of 20-hydroxyeicosatetraenoic acid (20-HETE) action ameliorated ischemia/reperfusion (I/R)-induced AKI in rats. Now we tested the hypothesis that enhancement of epoxyeicosatrienoic acid (EET) actions may counteract the detrimental effects of 20-HETE and prevent the initiation of AKI. METHODS: Male Lewis rats underwent right nephrectomy and ischemia was induced by 45 min clamping of the left renal pedicle followed by up to 48 h of reperfusion. Circulating CYP-eicosanoid profiles were compared in patients who underwent cardiac surgery with (n = 21) and without (n = 38) developing postoperative AKI. RESULTS: Ischemia induced an about eightfold increase of renal 20-HETE levels, whereas free EETs were not accumulated. To compensate for this imbalance, a synthetic 14,15-EET analogue was administered by intrarenal infusion before ischemia. The EET analogue improved renal reoxygenation as monitored by in vivo parametric MRI during the initial 2 h reperfusion phase. The EET analogue improved PI3K- as well as mTORC2-dependent rephosphorylation of Akt, induced inactivation of GSK-3ß, reduced the development of tubular apoptosis and attenuated inflammatory cell infiltration. The EET analogue also significantly alleviated the I/R-induced drop in creatinine clearance. Patients developing postoperative AKI featured increased preoperative 20-HETE and 8,9-EET levels. CONCLUSIONS: Pharmacological interventions targeting the CYP-eicosanoid pathway could offer promising new options for AKI prevention. Individual differences in CYP-eicosanoid formation may contribute to the risk of developing AKI in clinical settings.

Renal Ischemia/Reperfusion Injury in Soluble Epoxide Hydrolase-Deficient Mice.

AIM: 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP)-dependent eicosanoids that play opposite roles in the regulation of vascular tone, inflammation, and apoptosis. 20-HETE aggravates, whereas EETs ameliorate ischemia/reperfusion (I/R)-induced organ damage. EETs are rapidly metabolized to dihydroxyeicosatrienoic acids (DHETs) by the soluble epoxide hydrolase (sEH). We hypothesized that sEH gene (EPHX2) deletion would increase endogenous EET levels and thereby protect against I/R-induced acute kidney injury (AKI). METHODS: Kidney damage was evaluated in male wildtype (WT) and sEH-knockout (KO)-mice that underwent 22-min renal ischemia followed by two days of reperfusion. CYP-eicosanoids were analyzed by liquid chromatography tandem mass spectrometry. RESULTS: Contrary to our initial hypothesis, renal function declined more severely in sEH-KO mice as indicated by higher serum creatinine and urea levels. The sEH-KO-mice also featured stronger tubular lesion scores, tubular apoptosis, and inflammatory cell infiltration. Plasma and renal EET/DHET-ratios were higher in sEH-KO than WT mice, thus confirming the expected metabolic consequences of sEH deficiency. However, CYP-eicosanoid profiling also revealed that renal, but not plasma and hepatic, 20-HETE levels were significantly increased in sEH-KO compared to WT mice. In line with this finding, renal expression of Cyp4a12a, the murine 20-HETE-generating CYP-enzyme, was up-regulated both at the mRNA and protein level, and Cyp4a12a immunostaining was more intense in the renal arterioles of sEH-KO compared with WT mice. CONCLUSION: These results indicate that the potential beneficial effects of reducing EET degradation were obliterated by a thus far unknown mechanism leading to kidney-specific up-regulation of 20-HETE formation in sEH-KO-mice.

17ß-Estradiol regulates mTORC2 sensitivity to rapamycin in adaptive cardiac remodeling.

Adaptive cardiac remodeling is characterized by enhanced signaling of mTORC2 downstream kinase Akt. In females, 17ß-estradiol (E2), as well as Akt contribute essentially to sex-related premenopausal cardioprotection. Pharmacologic mTOR targeting with rapamycin is increasingly used for various clinical indications, yet burdened with clinical heterogeneity in therapy responses. The drug inhibits mTORC1 and less-so mTORC2. In male rodents, rapamycin decreases maladaptive cardiac hypertrophy whereas it leads to detrimental dilative cardiomyopathy in females. We hypothesized that mTOR inhibition could interfere with 17ß-estradiol (E2)-mediated sexual dimorphism and adaptive cell growth and tested responses in murine female hearts and cultured female cardiomyocytes. Under physiological in vivo conditions, rapamycin compromised mTORC2 function only in female, but not in male murine hearts. In cultured female cardiomyocytes, rapamycin impaired simultaneously IGF-1 induced activation of both mTOR signaling branches, mTORC1 and mTORC2 only in presence of E2. Use of specific estrogen receptor (ER)α- and ERß-agonists indicated involvement of both estrogen receptors (ER) in rapamycin effects on mTORC1 and mTORC2. Classical feedback mechanisms common in tumour cells with upregulation of PI3K signaling were not involved. E2 effect on Akt-pS473 downregulation by rapamycin was independent of ERK as shown by sequential mTOR and MEK-inhibition. Furthermore, regulatory mTORC2 complex defining component rictor phosphorylation at Ser1235, known to interfere with Akt-substrate binding to mTORC2, was not altered. Functionally, rapamycin significantly reduced trophic effect of E2 on cell size. In addition, cardiomyocytes with reduced Akt-pS473 under rapamycin treatment displayed decreased SERCA2A mRNA and protein expression suggesting negative functional consequences on cardiomyocyte contractility. Rictor silencing confirmed regulation of SERCA2A expression by mTORC2 in E2-cultured female cardiomyocytes. These data highlight a novel modulatory function of E2 on rapamycin effect on mTORC2 in female cardiomyocytes and regulation of SERCA2A expression by mTORC2. Conceivably, rapamycin abrogates the premenopausal "female advantage".

Sex-specific mTOR signaling determines sexual dimorphism in myocardial adaptation in normotensive DOCA-salt model.

The deoxycorticosterone acetate (DOCA)-salt mouse model exhibits adverse cardiac remodeling in male mice and cardiac protection in female mice, even when blood pressure is normalized. We hypothesized that intact mammalian target of rapamycin (mTOR) signaling is necessary for cardiac protection in females. We first tested sex differences and intracellular signaling after mTOR targeting with rapamycin in wild-type mice. Radio-telemetric blood pressure was maintained at normal for 6 weeks. Rapamycin significantly reduced left ventricular hypertrophy, preserved ejection fraction, inhibited fibrosis, and maintained capillary structure in male mice. Decreased mTORC1 and increased mTORC2 activity were detected in rapamycin-treated male mice compared with vehicle controls. In contrast, female mice developed dilative left ventricular hypertrophy, cardiac fibrosis, and capillary loss similar to DOCA-salt females lacking the estrogen receptor ß (ERß(-/-)) that we described earlier. Because rapamycin downregulated ERß in female mice, we next studied ERß(-/-) normotensive DOCA-salt females. Vehicle-treated wild-type females maintained their high constitutive mTORC1 and mTORC2 in response to DOCA-salt. In contrast to males, both mTORCs were decreased by rapamycin, in particular mTORC2 by 60%. ERß(-/-) DOCA-salt females showed similar mTORC1 and mTORC2 response patterns. We suggest that ERß-dependent regulation involves sex-specific use of mTOR signaling branches. Maintenance of both mTORC1 and mTORC2 signaling seems to be essential for adaptive cardiac remodeling in females and supports a rationale for sex-specific therapeutic strategies in left ventricular hypertrophy.

Estrogen receptor-beta signals left ventricular hypertrophy sex differences in normotensive deoxycorticosterone acetate-salt mice.

We found earlier that deoxycorticosterone acetate-salt treatment causes blood pressure-independent left ventricular hypertrophy, but only in male mice. To test the hypothesis that the estrogen receptor-ß (ERß) protects the females from left ventricular hypertrophy, we treated male and female ERß-deficient (ERß(-/-)) mice and their male and female littermates (wild-type [WT]) with deoxycorticosterone acetate-salt and made them telemetrically normotensive with hydralazine. WT males had increased (+16%) heart weight/tibia length ratios compared with WT females (+7%) at 6 weeks. In ERß(-/-) mice, this situation was reversed. Female WT mice had the greatest heart weight/tibia length ratio increases of all of the groups (+23%), even greater than ERß(-/-) males (+10%). Echocardiography revealed concentric left ventricular hypertrophy in male WT mice, whereas ERß(-/-) females developed dilative left ventricular hypertrophy. The hypertrophic response in female ERß(-/-) mice was accompanied by the highest degree of collagen deposition, indicating maladaptive remodeling. ERß(+/+) females showed robust protective p38 and extracellular signal-regulated kinase 1/2 signaling relationships compared with other groups. Calcineurin Aß expression and its positive regulator myocyte-enriched calcineurin-interacting protein 1 were increased in deoxycorticosterone acetate-salt female ERß(-/-) mice, yet lower than in WT males. Endothelin increased murine cardiomyocyte hypertrophy in vitro, which could be blocked by estradiol and an ERß agonist. We conclude that a functional ERß is essential for inducing adaptive p38 and extracellular signal-regulated kinase signaling, while reducing maladaptive calcineurin signaling in normotensive deoxycorticosterone acetate female mice. Our findings address the possibility of sex-specific cardiovascular therapies.

Deoxycorticosterone acetate-salt mice exhibit blood pressure-independent sexual dimorphism.

We tested the hypothesis that female and male mice differ in terms of cardiac hypertrophy after deoxycorticosterone acetate (DOCA)+salt hypertension (uninephrectomy and 1% saline in drinking water) and focused on calcineurin signaling. We excluded confounding effects of blood pressure elevation or sex-related blood pressure differences by treating DOCA-salt mice with hydralazine (250 mg/L in drinking water). We found that directly measured mean arterial blood pressure was lowered to control values with hydralazine and corroborated this finding in separate mouse groups with radiotelemetry. Male mice were more responsive to DOCA-salt-related effects. They developed more left ventricular hypertrophy and more renal hypertrophy after 6 weeks of DOCA-salt+hydralazine compared with female mice. In hearts, transcripts for calcineurin Abeta and for myocyte-enriched calcineurin interacting protein 1 were upregulated in male but not in female mice. Enhanced activity of calcineurin Abeta, as indicated by diminished phosphorylation of NFATc2 in male mice, accounted for this sex-specific difference. Stretch-related, inflammatory, and profibrotic responses were also accentuated in male mice, as shown by higher transcript levels of atrial natriuretic peptide, monocyte chemoattractant protein-1, and transforming growth factor-beta. Our results support sex-specific regulation of the calcineurin pathway in response to largely blood pressure-independent mineralocorticoid action. We suggest that sex-specific calcineurin activation determines the maladaptive cardiac and renal hypertrophic responses and accompanying organ injury in male mice.