Search

Purpose: Anaplastic lymphoma kinase (ALK) aberrations have been identified in pediatric-type infant gliomas, but their occurrence across age groups, functional effects, and treatment response has not been broadly established. Experimental Design: We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric, and 10 congenital) with in vitro and in vivo validation of aberrations. Results: ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0–80 years). Recurrent as well as novel ALK fusions (LRRFIP1–ALK, DCTN1–ALK, PRKD3–ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to the treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages, and no gross effects on perinatal brain development were seen in pregnant mice treated with the ALK inhibitor ceritinib. Conclusions: These findings support use of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs.

Supplementary Figure 3: PPP1CB-ALK fusion is oncogenic and sensitive to targeted therapy. A. Immunoblot analysis of ALK downstream signaling proteins in cortical mNSC (CTX#6) and brainstem mNSC (BS#3) expressing PPP1CB-ALK. Cells were treated for 4 hours with ceritinib and lorlatinib at the indicated concentrations.B. Left panel: Soft agar colony-forming assay using stable mNSC PPP1CB-ALK demonstrated colony formation with dose-dependent inhibition by ceritinib. mNSC KRAS did not respond to ceritinib validating the drug specificity. Right panel: Quantification of colony formation under targeted drug inhibition using CellProfiler. Values represent colony counts relative to the untreated group ± s.d. The mean of three independent replicates is shown. Significance between treatments determined by the Mann-Whitney test. *P < 0.05, **P < 0.01.C. Cell viability of ceritinib-treated PPP1CB-ALK NIH-3T3 relative to eGFP-positive cells by CellTiterGlo.D. Relative viability of the ALK wild-type GBM line (BT164) and BT1857 lorlatinib-treated cells versus DMSO control.E. Relative viability of BT164 and BT1857 ceritinib-treated cells versus DMSO control.F. Relative viability of BT164 and BT1857 cells treated with an EGFR tyrosine kinase inhibitor neratinib.G. Pharmacodynamic analysis of NIH 3T3-PPP1CB-ALK s.c. tumors treated with vehicle or ceritinib at 30mg/kg for 5 days. Representative H&E stain, ALK, pSTAT3, pAKT S473 and Ki-67 IHC.H. Quantification of Ki-67-positive cells in vehicle and ceritinib-treated tumors using CellProfiler software. Error bars show standard error of the mean. *P < 0.05.I. Quantification of pSTAT3-positive cells in vehicle and ceritinib-treated tumors using CellProfiler software. Error bars show standard error of the mean. *P < 0.05.J. Tumor growth following subcutaneous implantation of PPP1CB-ALK and eGFP-expressing fibroblasts.K. Kaplan-Meier survival curves of SCID mice injected subcutaneously with mNSC CTX-PPP1CB-ALK (red, n = 5) or mNSC CTX-eGFP (black, n = 4) and treated with ceritinib at 30mg/kg/d for 15 days. Mice were euthanized at endpoint (tumor volume of 2000mm3). The grey area represents the treatment period.L. Vehicle or ceritinib-treated mouse weights at start and end of treatment.

Supplementary Figure 5: Single-cell RNA Sequencing and IHC examination of ALK in human neural development and immature neural cells. A. 2D representation of human brain cell subtypes (48 samples) during cortex development using a single cell RNA-seq atlas (cortex-dev.cells.ucsc.edu) shows that ALK transcript appears to be enriched in a certain cell type. Color-coded by ALK gene expression (beige to red) and age in weeks (rainbow).B. ALK-stained sagittal section of normal human cerebellum at 15 postconceptional weeks (pcw).C. ALK-stained sagittal section of human fetal neural stem cell pellets at 13 postconceptional weeks (pcw).D. ALK-stained sagittal section of human fetal neural stem cell pellets at 15 postconceptional weeks (pcw).

Purpose:Anaplastic lymphoma kinase (ALK) aberrations have been identified in pediatric-type infant gliomas, but their occurrence across age groups, functional effects, and treatment response has not been broadly established.Experimental Design:We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric, and 10 congenital) with in vitro and in vivo validation of aberrations.Results:ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0–80 years). Recurrent as well as novel ALK fusions (LRRFIP1–ALK, DCTN1–ALK, PRKD3–ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to the treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages, and no gross effects on perinatal brain development were seen in pregnant mice treated with the ALK inhibitor ceritinib.Conclusions:These findings support use of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs.

Supplementary Figure 6: Single cell RNA sequencing comparison of ALK expression and other infant/pediatric glioma kinase drivers in developing human and mouse brain. A. 2D representation of human brain cell subtypes (48 samples) during cortex development using a single cell RNA-seq atlas (http://cells.ucsc.edu/?ds=cortex-dev) shows that ALK positive cells make up less than 1% of all cells in the dataset and aren't localized within a specific cluster.B. Cluster-level summary of the human scRNA dataset shows that ALK expression (TPM) is decreased compared to EGFR, NTRK2, and NTRK3, and ALK positive cells make up a much lower percentage of each cluster compared to those 3 genes.C. 2D representation of developmental mouse forebrain (E12.5 – P6) in a single cell RNA-seq atlas (http://cc-shiny-01.functionalgenomics.ca/braindex/clusters) shows similar results as seen in the human scRNA Nowakowski dataset.D. Cluster-level summary of the mouse scRNA dataset corroborates results seen in the Nowkowski scRNA dataset; that Alk expression (log-normalized) is decreased, and less prevalent than EGFR, NTRK2, and NTRK3.E. 2D representation of RNA sequencing data for NTRK1-3 genes in the Nowakowski developing human cortex dataset. Compared to ALK expression, the NTRK genes have increased expression levels, increased prevalence in the dataset, and show increased localization to specific cluster.F. Similar to (E), the 2D representation of Ntrk genes in the developing mouse scRNA dataset shows increased expression and prevalence compared to Alk.

Supplementary Figure 2: T1 post-contrast axial and sagittal images of ALK-fused and non ALK-altered GBMs and ALK staining in ALK-fused congenital GBMs. A. Example images from patients with ALK aberrations (76, 89, 116, 197)B. In contrast to those without ALK aberrations (right columns). Contrast is gadolinium for those where applicable and show enhancement within both the tumor categories.C. Representative H&E and ALK IHCs of congenital GBM harboring an ALK fusion.

Supplementary Figure 4: Characterization of the lung cancer recurrent DCTN1-ALK fusion identified in cGBM (case ALK.232). A. Integrative Genomic Viewer (IGV) screenshot of the DCTN1-ALK variant.B. Predicted sequence of DCTN1-ALK fusion (DCTN1 exon 1-26, ALK exon 20-29 conserved).C. PDX DCTN1-ALK sub-renal capsule engraftment confimed by IHC.C. Relative viability of the ALK wild-type GBM line (BT164) and BT1857 ceritinib-treated cells versus DMSO control.D. Relative viability of BT164 and BT1857 lorlatinib-treated cells versus DMSO control.E. Relative viability of BT164 and BT1857 cells treated with an EGFR tyrosine kinase inhibitor neratinib.F. PDX DCTN1-ALK sub-renal capsule engraftment confirmed by IHC.

Supplementary Figure 1: Characterization of the novel LRRFIP1-ALK fusion (case ALK.223) A. Visualization of the read coverage at the fusion sites in ALK and LRRFIP1 genes using Integrative Genomic Viewer.B. Pile-up plot generated by SvABA using whole-genome sequencing data shows the breakpoint coordinates.C. Copy number ratio profile by WGS showing no somatic copy number alterations anywhere in the genome. The x-axis represents the chromosomes.D. Copy number ratio profile on chromosome 2 showing no copy number change of ALK and LRRFIP1 genes.E. Two RT-PCR products of 186bp (variant 1) and 189bp (variant 3) were detected by agarose gel electrophoresis with total RNA from tumor specimen ALK.223.F. Primer sequences of ALK-LRRFIP1 (variants 1 and 2) and LRRFIP1-ALK (variant 3) and the predicted DNA product size.G. Western Blot analysis of ALK signaling pathway in subcutaneous tumors treated with vehicle or lorlatinib. Normal brain was used as control.H. Representative 40x images and CellProfiler quantification of Cleaved Caspase 3- and Ki-67- stained subcutaneous tumors.

Additional information tables 1 to 8.

While cancer immunotherapy can produce dramatic responses, only a minority of patients respond to treatment. Reliable response biomarkers are needed to identify responders, and conventional imaging modalities have not proved adequate. Here, we provide a preclinical proof of concept for the use of granzyme B, a downstream effector of tumoral cytotoxic T cells, as an early biomarker for tumors responding to immunotherapy. We designed novel PET imaging probes for the murine and human granzyme B isoforms that specifically and quantitatively bind granzyme B. Immunotherapy-treated mice were imaged prior to therapy-induced tumor volume reduction. Imaging distinguished treated responders from nonresponders with excellent predictive ability. To assess the clinical value of a granzyme B imaging paradigm, biopsy specimens from melanoma patients on checkpoint inhibitor therapy were analyzed. A marked differential in granzyme B expression was observed between treated responders and nonresponders. Additionally, our human probe was able to specifically detect granzyme B expression in human samples, providing a clear candidate for clinical application. Overall, our results suggest granzyme B PET imaging can serve as a quantitatively useful predictive biomarker for efficacious responses to cancer immunotherapy. Cancer Res; 77(9); 2318–27. ©2017 AACR.

Rotating Maximum Intensity Projection Video of Mouse Treated with Anti-PD-1 and CTLA-4 Therapy

Rotating Maximum Intensity Projection Video of Vehicle Treated Mouse

This file contains supplementary figures pertaining to granzyme B heterogeneity, immunohistochemical and immunofluorescent analyses, peptide biodistribution and dynamic PET analysis.

In this chapter, we describe several configurations of digital holographic microscopes operating with partially coherent illumination, including the implementation of color holography. The different ways of implementing partially coherent illumination are described and discussed, as well as the respective improvements they provide depending on the properties of the observed objects. Several significant applications in the biomedical and environmental fields are presented. In particular, we carry out researches on correlative quantitative phase-contrast-fluorescence imaging on blood with a special focus on the behavior of platelets in relation to relevant pathologies. In the environmental domain, we demonstrate the use of high-throughput digital holographic microscopy to monitor and analyze plankton and microalgal cultures.

SummaryPediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent oncogenic mechanism, which fosters genome instability and tumor cell growth in H3.3 mutant pHGG, thus opening new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme Polynucleotide Kinase 3’-Phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.

Purpose: Anaplastic Lymphoma Kinase (ALK) aberrations have been identified in pediatric type infant gliomas, but their occurrence across age groups, functional effects, and treatment response have not been broadly established. Experimental Design: We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly-generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric and 10 congenital) with in vitro and in vivo validation of aberrations. Results: ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0-80 years). Recurrent as well as novel ALK fusions (LRRFIP1-ALK, DCTN1-ALK, PRKD3-ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs, and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages and no gross effects on perinatal brain development was seen in pregnant mice treated with the ALK inhibitor ceritinib. Conclusions: These findings support expanded evaluation of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs. Citation Format: Anne-Florence Blandin, Ross Giglio, Maya Srikanth Graham, Guadalupe Garcia, Seth Malinowski, Jared K. Woods, Shakti Ramkissoon, Lori Ramkissoon, Frank Dubois, Kate Schoolcraft, Jessica W. Tsai, Dayle K. Wang, Robert Jones, Jayne Vogelzang, Kristine Pelton, Sarah Becker, Fiona Watkinson, Claire Sinai, Elizabeth Cohen, Matthew Booker, Michael Tolstorukov, Veerle Haemels, Liliana Goumnerova, Karen Wright, Mark Kieran, Katie Fehnel, David Reardon, Arnault Tauziede-Espariat, Rishi Lulla, Benjamin Carcamo, Stanley Chaleff, Alain Charest, Frederik De Smet, Azra H. Ligon, Adrian Dubuc, Melanie Pagès, Pascale Varlet, Patrick Wen, Brian Alexander, Susan Chi, Sanda Alexandrescu, Ralf Kittler, Robert Bachoo, Rameen Beroukhim, Pratiti Bandopadhayay, Keith L. Ligon. ALK amplification and rearrangements are recurrent targetable events in congenital and adult glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1201.

PDGFRA has been shown to be commonly altered in high-grade gliomas (HGGs), including histone 3 lysine 27-mutated diffuse midline gliomas (H3K27M DMG), a disease with almost no long-term survivors. Here, we performed comprehensive genomic and transcriptomic analysis of 260 high-grade glioma cases, which revealed PDGFRA genomic alterations (mutations and/or amplifications) in 13% of patients. H3K27M DMGs had significantly higher PDGFRA expression compared to H3 wild-type tumors, and PDGFRA gene amplification resulted in even higher expression levels in H3K27M DMGs as well as H3 wild-type HGGs. We tested a panel of patient- derived pHGG/H3K27M DMG models against a range of PDGFRA inhibitors, including avapritinib, a potent small molecule inhibitor with relatively selective activity against both wild-type and mutant PDGFRA. Avapritinib showed supra-micromolar blood-brain barrier penetration in our pre-clinical models and demonstrated significant survival impact in an aggressive patient-derived H3K27M DMG mouse xenograft model. Finally, building on this preclinical activity, we report here the first clinical experience using avapritinib in eight pediatric and young adult patients with high-grade glioma (H3K27M DMG and/or PDGFRA altered). Avapritinib has thus far been well tolerated with no significant acute toxicities. Most importantly, our preliminary data reveal radiographic response evaluated by RAPNO criteria in 50% of patients, a striking outcome rarely seen in this patient population. In summary, we report that avapritinib is a selective, CNS-penetrant small molecule inhibitor of PDGFRA that shows potent activity in preclinical models and produces promising clinical responses with good tolerability in patients with high-grade glioma. This suggests a promising role for avapritinib therapy in this population with previously dismal outcomes. Citation Format: Lisa Mayr, Maria Trissal, Kallen Schwark, Jenna Labelle, Andrew Groves, Julia Furtner-Srajer, Jeffrey Supko, Liesa Weiler-Wichtl, Olivia Hack, Jacob Rozowsky, Joana G. Marques, Eshini Pandatharatna, Ulrike Leiss, Verena Rosenmayr, Frank Dubois, Noah F. Greenwald, Sibylle Madlener, Armin S. Guntner, Hana Pálová, Natalia Stepien, Daniela Lötsch-Gojo, Christian Dorfer, Karin Dieckmann, Andreas Peyrl, Amedeo A. Azizi, Alicia Baumgartner, Ondřej Slabý, Petra Pokorná, Pratiti Bandopadhayay, Rameen Beroukhim, Keith Ligon, Christof Kramm, Annika Bronsema, Simon Bailey, Ana Guerreiro Stücklin, Sabine Mueller, David T. Jones, Natalie Jäger, Jaroslav Štěrba, Leonhard Müllauer, Christine Haberler, Chandan Kumar-Sinha, Arul Chinnaiyan, Rajen Mody, Mary Skrypek, Nina Martinez, Daniel C. Bowers, Carl Koschmann, Johannes Gojo, Mariella Filbin. Clinical response to the PDGFRα inhibitor avapritinib in high-grade glioma patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5719.

A high tumour mutational burden (hypermutation) is observed in some gliomas1–5; however, the mechanisms by which hypermutation develops and whether it predicts the response to immunotherapy are poorly understood. Here we comprehensively analyse the molecular determinants of mutational burden and signatures in 10,294 gliomas. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and mismatch repair (MMR) genes, and a more common post-treatment pathway, associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas that recur after treatment with the chemotherapy drug temozolomide. Experimentally, the mutational signature of post-treatment hypermutated gliomas was recapitulated by temozolomide-induced damage in cells with MMR deficiency. MMR-deficient gliomas were characterized by a lack of prominent T cell infiltrates, extensive intratumoral heterogeneity, poor patient survival and a low rate of response to PD-1 blockade. Moreover, although bulk analyses did not detect microsatellite instability in MMR-deficient gliomas, single-cell whole-genome sequencing analysis of post-treatment hypermutated glioma cells identified microsatellite mutations. These results show that chemotherapy can drive the acquisition of hypermutated populations without promoting a response to PD-1 blockade and supports the diagnostic use of mutational burden and signatures in cancer. Temozolomide therapy seems to lead to mismatch repair deficiency and hypermutation in gliomas, but not to an increase in response to immunotherapy.

In cardiovascular disorders, the study of thrombocytes, commonly known as platelets, is highly important since they are involved in blood clotting, essential in hemostasis, and they can in pathological situations affect the blood circulation. In this paper, single deposited platelets are measured using interferometric digital holographic microscopy. We have shown that the average optical height of platelets is significantly lower in healthy volunteers than in dialyzed patients, meaning a better spreading. It demonstrates the great interest for assessing this parameter in any patients, and therefore the high potential of analyzing single spread platelets using digital holographic microscopy in fundamental research as well as a diagnostic tool in routine laboratories, for usual blood tests.

Pediatric high-grade gliomas (pHGGs), encompassing hemispheric and diffuse midline gliomas (DMGs), remain a devastating disease. The last decade has revealed oncogenic drivers including single nucleotide variants (SNVs) in histones. However, the contribution of structural variants (SVs) to gliomagenesis has not been systematically explored due to limitations in early SV analysis approaches. Using SV algorithms, we recently created, we analyzed SVs in whole-genome sequences of 179 pHGGs including a novel cohort of treatment naïve samples–the largest WGS cohort assembled in adult or pediatric glioma. The most recurrent SVs targeted MYC isoforms and receptor tyrosine kinases, including a novel SV amplifying a MYC enhancer in the lncRNA CCDC26 in 12% of DMGs and revealing a more central role for MYC in these cancers than previously known. Applying de novo SV signature discovery, we identified five signatures including three (SVsig1-3) involving primarily simple SVs, and two (SVsig4-5) involving complex, clustered SVs. These SV signatures associated with genetic variants that differed from what was observed for SV signatures in other cancers, suggesting different links to underlying biology. Tumors with simple SV signatures were TP53 wild-type but were enriched with alterations in TP53 pathway members PPM1D and MDM4. Complex signatures were associated with direct aberrations in TP53, CDKN2A, and RB1 early in tumor evolution, and with extrachromosomal amplicons that likely occurred later. All pHGGs exhibited at least one simple SV signature but complex SV signatures were primarily restricted to subsets of H3.3K27M DMGs and hemispheric pHGGs. Importantly, DMGs with the complex SV signatures SVsig4-5 were associated with shorter overall survival independent of histone type and TP53 status. These data inform the role and impact of SVs in gliomagenesis and mechanisms that shape them.

Pediatric high-grade glioma (pHGG) is an incurable disease with a median survival of less than 6 months post-progression and no effective targeted therapy. PDGFRA is commonly altered in pHGG, but targeting PDGFRA in this disease has been unsuccessful, likely due to poor central nervous system (CNS) penetrance. Avapritinib is a novel and CNS-penetrant PDGFRA/KIT inhibitor that is FDA-approved for adults with unresectable or metastatic PDGFRA exon 18-mutant gastrointestinal stromal tumor (GIST) and is being studied in CNS tumors. We performed a pre-clinical and clinical assessment to determine the potential suitability of avapritinib therapy in PDGFRA-driven glioma. A multi-institutional cohort genetic analysis revealed PDGFRA amplification and mutation in 10.2% and 6.1% of pHGG, respectively. Additionally, PDGFRA expression in the absence of genetic events was significantly increased in H3K27-altered diffuse midline glioma (DMG) compared to H3-wildtype pHGG. Avapritinib performed well in: (i) mutant PDGFRA enzyme inhibition and wildtype inhibition at high dose, (ii) minimal off-target kinase inhibition, (iii) brain penetration (peak 10 µM), and (iv) proliferation/pPDGFRA reduction in PDGFRA-amplified and mutant pHGG cell lines. Avapritinib treatment in an aggressive PDX model of pHGG resulted in significant survival benefit. We pursued treatment of eight pediatric and young adult HGG patients with avapritinib across seven institutions. Patients were a mixture of local (N = 4) and metastatic disease (N = 4); all patients were post-initial radiation, with 7/8 having progressed on prior treatment. 7/8 patients had PDGFRA amplifications or mutations, and 7/8 had H3K27M mutations. Therapy was generally well-tolerated. 4/8 patients showed radiographic response to avapritinib, with one patient demonstrating complete response of target lesion and remains on therapy. Avapritinib levels in patients’ CSF and brain tumor tissue reached micromolar levels. These results demonstrate that avapritinib is a potent, selective, and CNS-penetrant PDGFRA/KIT inhibitor that is promising for further study in pHGG with relevant alterations.

BET-bromodomain inhibition (BETi) has shown pre-clinical promise for MYC-amplified medulloblastoma. However, the mechanisms for its action, and ultimately for resistance, have not been fully defined. Here, using a combination of expression profiling, genome-scale CRISPR/Cas9-mediated loss of function and ORF/cDNA driven rescue screens, and cell-based models of spontaneous resistance, we identify bHLH/homeobox transcription factors and cell-cycle regulators as key genes mediating BETi’s response and resistance. Cells that acquire drug tolerance exhibit a more neuronally differentiated cell-state and expression of lineage-specific bHLH/homeobox transcription factors. However, they do not terminally differentiate, maintain expression of CCND2, and continue to cycle through S-phase. Moreover, CDK4/CDK6 inhibition delays acquisition of resistance. Therefore, our data provide insights about the mechanisms underlying BETi effects and the appearance of resistance and support the therapeutic use of combined cell-cycle inhibitors with BETi in MYC-amplified medulloblastoma., BET-bromodomain inhibitors could be used to treat medulloblastoma tumors with Myc amplifications. Here, the authors show that both the response and resistance to BET inhibitors in mice is mediated by bHLH/homeobox transcription factors.

Structural variants (SVs) within the cancer genome reflect defects in DNA damage repair, impact gene regulation, and alter the genomic landscape. SVs impact a larger fraction of the cancer genome than any other genetic aberration, yet their effects on modulating therapeutic response have been largely uncharacterized. Patterns of rearrangements, otherwise known as signatures, vary across tumor and tissue type, and can indicate underlying biological mechanisms responsible for cancer etiology. We have extracted SV signatures from whole genome sequencing data from hundreds of cancer cell lines. Using datasets indicating the cell lines’ response to over a thousand small molecule inhibitors, we have identified associations between SV signatures and response to therapeutics. These results will enhance our understanding of mechanisms underlying rearrangement signatures and nominate new biomarkers of drug response. Citation Format: Simona Dalin, Frank Dubois, Shu Zhang, Alexander Crane, Jeremie Kalfon, Javad Noorbakhsh, Rameen Beroukhim. Associations between structural variant signatures and drug sensitivity in cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2710.

INTRODUCTION: Adamantinomatous craniopharyngiomas (ACPs) are rare brain tumors that primarily occur in children and impact long-term morbidity and mortality. The canonical driver mutation for ACP growth occurs in CTNNB1 and leads to constitutive activation of the Wnt/β-catenin signaling pathway. In this study, we outline the genomic, transcriptomic, and structural variant (SV) landscape in a cohort of 41 ACP samples. METHODS: We performed whole-genome sequencing (WGS) and RNA-sequencing of 41 ACP samples. Matched normal samples were also characterized by WGS. Mutect2 was used to detect single nucleotide variants (SNVs) and indels, and copy number data was generated using the GATK pipeline. SvABA was used to perform SV analysis and to identify significantly recurrent breakpoints and juxtapositions. DESeq2 was used to perform differential gene expression analysis based on clinical and molecular annotation data. RESULTS: 29/41 (70%) of the ACP samples harbored missense mutations in exon 3 of CTNNB1, all of which have previously been reported in ACP tumors. SV analysis identified a median of 11.5 events per tumor. Overall, 9.7% of events were interchromosomal. Of the remainder, the majority (78.6%) were deletions. No SVs occurred within CTNNB1. A positive correlation (r = 0.533) was observed between the frequency of SVs and SNVs within samples. Analysis of significantly recurring breakpoints (SRBs) did not identify recurrent breakpoint events. Differential gene expression analysis comparing samples with and without CTNNB1 variants identified 2,143 differentially expressed genes with q-value < 0.05. CONCLUSION: This study identifies activating mutations in exon 3 of CTNNB1 in a large cohort of ACP samples. We also integrate SV and transcriptomic data to comprehensively investigate ACP tumor genomes and identify putative novel tumorigenic mechanisms that advance our understanding of ACP biology.

Pediatric high-grade gliomas (pHGGs), encompassing hemispheric and diffuse midline gliomas (DMGs), remain a devastating disease. The last decade has revealed oncogenic drivers including single nucleotide variants (SNVs) in histones. However, the contribution of structural variants (SVs) to gliomagenesis has not been systematically explored due to limitations in early SV analysis approaches. Using SV algorithms, we recently created, we analyzed SVs in whole-genome sequences of 179 pHGGs including a novel cohort of treatment naïve samples–the largest WGS cohort assembled in adult or pediatric glioma. The most recurrent SVs targeted MYC isoforms and receptor tyrosine kinases, including a novel SV amplifying a MYC enhancer in the lncRNA CCDC26 in 12% of DMGs and revealing a more central role for MYC in these cancers than previously known. Applying de novo SV signature discovery, we identified five signatures including three (SVsig1-3) involving primarily simple SVs, and two (SVsig4-5) involving complex, clustered SVs. These SV signatures associated with genetic variants that differed from what was observed for SV signatures in other cancers, suggesting different links to underlying biology. Tumors with simple SV signatures were TP53 wild-type but were enriched with alterations in TP53 pathway members PPM1D and MDM4. Complex signatures were associated with direct aberrations in TP53, CDKN2A, and RB1 early in tumor evolution, and with extrachromosomal amplicons that likely occurred later. All pHGGs exhibited at least one simple SV signature but complex SV signatures were primarily restricted to subsets of H3.3K27M DMGs and hemispheric pHGGs. Importantly, DMGs with the complex SV signatures SVsig4-5 were associated with shorter overall survival independent of histone type and TP53 status. These data inform the role and impact of SVs in gliomagenesis and mechanisms that shape them.

Red blood cells (RBCs) in pathological situations undergo biochemical and conformational changes, leading to alterations in rheology involved in cardiovascular events. The shape of RBCs in volunteers and stable and exacerbated chronic obstructive pulmonary disease (COPD) patients was analyzed. The effects of RBC spherization on platelet transport (displacement in the flow field caused by their interaction with RBCs) were studied in vitro and by numerical simulations. RBC spherization was observed in COPD patients compared with volunteers. In in vitro experiments at a shear rate of 100 s-1 , treatment of RBCs with neuraminidase induced greater sphericity, which mainly affected platelet aggregates without changing aggregate size. At 400 s-1 , neuraminidase treatment changes both the size of the aggregates and the number of platelet aggregates. Numerical simulations indicated that RBC spherization induces an increase of the platelet mean square displacement, which is traditionally linked to the platelet diffusion coefficient. RBCs of COPD patients are more spherical than healthy volunteers. Experimentally, RBC spherization induces increased platelet transport to the wall. Additional studies are needed to understand the link between the effect of RBCs on platelet transport and the increased cardiovascular events observed in COPD patients.

BACKGROUND AND PURPOSE: Recent advances in molecular techniques have characterized distinct subtypes of diffuse intrinsic pontine gliomas. Our aim was the identification of MR imaging correlates of these subtypes. MATERIALS AND METHODS: Initial MRIs from subjects with diffuse intrinsic pontine gliomas recruited for a prospective clinical trial before treatment were analyzed. Retrospective imaging analyses included FLAIR/T2 tumor volume, tumor volume enhancing, the presence of cyst and/or necrosis, median, mean, mode, skewness, kurtosis of ADC tumor volume based on FLAIR, and enhancement at baseline. Molecular subgroups based on EGFR and MGMT mutations were established. Histone mutations were also determined (H3F3A, HIST1H3B, HIST1H3C). Univariate Cox proportional hazards regression was used to test the association of imaging predictors with overall and progression-free survival. Wilcoxon rank sum, Kruskal-Wallis, and Fisher exact tests were used to compare imaging measures among groups. RESULTS: Fifty patients had biopsy and MR imaging. The median age at trial registration was 6 years (range, 3.3–17.5 years); 52% were female. On the basis of immunohistochemical results, 48 patients were assigned to 1 of 4 subgroups: 28 in MGMT–/epidermal growth factor receptor (EGFR)–, 14 in MGMT–/EGFR+, 3 in MGMT+/EGFR–, and 3 in MGMT+/EGFR+. Twenty-three patients had histone mutations in H3F3A, 8 in HIST1H3B, and 3 in HIST1H3C. Enhancing tumor volume was near-significantly different across molecular subgroups (P = .04), after accounting for the false discovery rate. Tumor volume enhancing, median, mode, skewness, and kurtosis ADC T2-FLAIR/T2 were significantly different (P ≤ .048) between patients with H3F3A and HIST1H3B/C mutations. CONCLUSIONS: MR imaging features including enhancement and ADC histogram parameters are correlated with molecular subgroups and mutations in children with diffuse intrinsic pontine gliomas.

Phase contrast microscopy is highly valuable in medicine, biology, fluid dynamics, etc., as it allows in focus observation of transparent or semi-transparent objects, which are difficult to analyze with conventional bright field microscopy. Indeed, such samples mainly affect the phase of the optical field, i.e., the shape of the wavefront, but not the light intensity. Consequently, techniques providing quantitative phase contrast in microscopy, e.g., digital holography, are suitable for transparent object characterization: assessing the thickness, or more precisely the optical thickness, of an object directly from its phase profile is a very common approximation. However, the phase profile in the object median plane is generally different from its thickness profile, as actual three-dimensional objects cause wavefront distortion. This paper discusses the validity and limitations of this approximation. The presented study considers simulated homogeneous, transparent, spherical particles. The optical field behind the particle, computed using Mie theory, is backpropagated to the object plane by means of Rayleigh–Sommerfeld propagation equation. We have shown that the approximation is better for larger spheres and, to a certain extent, for lesser refractive index difference between the object and the surrounding medium. Moreover, the error in assessing the object thickness directly from the central value of the phase profile, has been studied. Considering, for example, a siliceous sphere in oil or in air, the error increases rapidly above 5% for diameters smaller than the illumination wavelength. The impact of a slight defocus has also been studied.

RationaleThere are important interactions between Red Blood Cells (RBCs) and platelets in the bloodstream. These interactions lead to a phenomenon called margination. RBCs in pathological situations undergo biochemical and conformational changes leading to alterations in blood rheology.AimRBCs shape in volunteers (21), stable (42) and exacerbated (31) COPD patients was analyzed. We studied the effect of the RBCs spherization on the platelets transport experimentally, in vitro, and by using numerical simulations.MethodsRBC shape was estimated by the second moment of Pearson obtained through flow cytometry on fsc histogram. In vitro experiments were performed to analyze the effect of RBC shape on platelets adhesion/aggregation in dynamic conditions. Neuraminidase treatment was used to induce RBCs spherization. Numerical simulation were performed to determine the effect of RBCs spherization on platelets mean square displacement (MSD) to provide a physical explanation.ResultsSignificant increase of RBC sphericity was observed in COPD patients compared to volunteers (Kruskal-Wallis: pIn vitro experiments, at shear rate of 100 s-1, we observed that RBCs treated with neuraminidase mainly affect the number of platelet aggregates (p = 0.004). There was no change in the aggregates size. At a shear rate of 400 sec-1 neuraminidase treatment changes both the size of the aggregates (p = 0.009) and the number of platelet aggregates (p = 0.008).Numerical simulations indicated that RBCs spherization induces an increase of MSD and the effect was more pronounced when the shear rate increased.ConclusionOur results show that the RBCs of COPD patients are more spherical than those of healthy volunteers. Experimentally we observe that the RBCs spherization induces an increase platelet transport to the wall. Additional studies are needed to better understand the possible association between the RBCs effect on the platelets transport and the increased cardiovascular events observed in COPD patients.

The longwave oscillatory Marangoni convection in a horizontal two-layer film heated from above, in the presence of an interfacial heat consumption, is considered. The effect of gravity is taken into account. The action of a time-periodic parameter modulation on the nonlinear Marangoni waves is investigated. The problem is studied numerically in the framework of longwave amplitude equations. The alternating rolls patterns and the oscillating squares have been found. In some intervals of the modulation frequency the phenomenon of synchronization has been observed.

Carbon nanotubes and silica nanoparticles are allowed to self-assemble into a nanocomposite by first forming an aqueous suspension, then depositing one drop after the other and finally letting them evaporate. Two types of composites are prepared. One by forming alternate layers and the other by forming several layers of a pre-mixed suspension. The thickness, thermal and electrical conductivity of the composites are measured versus the number of depositions. The pre-mixed composites showed an increase in the values in both the parallel and perpendicular directions of both the electrical and thermal conductivities, making them suitable for electrodes or battery-like applications. The values of the electrical and thermal conductivities in the perpendicular direction for the first composite decrease and increase, respectively, while for the parallel direction the values are significantly constant. As such, they would be useful as electrical insulators for optimal cooling. Thickness measurements showed that the pre-mixed composite is the denser one, due to a better alignment of the carbon nanotubes.

An influence of a spatial temperature modulation of the interfacial heat release/consumption on nonlinear convective flows in the 47v2 silicone oil - water system, is studied. Rigid heat-insulated lateral walls, corresponding to the case of closed cavities, have been considered. Transitions between the flows with different spatial structures, have been investigated. It is shown that the spatial modulation can change the sequence of bifurcations and lead to the appearance of specific steady and oscillatory flows in the system.

Tremendous efforts are currently dedicated to the development of novel therapies targeting the androgen receptor (AR), the major driver of prostate cancer disease and its progression to castration resistance. The ability to noninvasively interrogate AR expression over time in murine models of prostate cancer would permit longitudinal preclinical analysis of novel compounds that could not otherwise be accomplished ex vivo. Although PET imaging with 16β-18F-fluoro-5α-dihydrotestosterone (18F-FDHT) has successfully quantified AR levels clinically, no rodent model of 18F-FDHT imaging has been reported so far. One difference between humans and rodents is the absence in the latter of the sex hormone-binding globulin (SHBG), a glycoprotein that binds to testosterone in the bloodstream, Here, we explore the role of SHBG in developing a working model of rodent AR imaging. Methods: Three human prostate cancer cell lines and xenografts (LNCaP, 22Rv1, and PC3) were used to examine the uptake of free 18F-FDHT and SHBG-bound 18F-FDHT. Both ligands were examined for stability and competitive binding to AR over time in vitro before in vivo studies. PET/CT imaging was used to dynamically measure the uptake of both tracers over 4 h, whereas specificity was determined by competitive binding with the AR antagonist enzalutamide. Results: AR levels correlated with the uptake of both 18F-FDHT and SHBG-18F-FDHT in prostate cancer cell lines. Interestingly, whereas both free and SHBG-bound 18F-FDHT had a similar cellular accumulation at 1 and 2.5 h, SHBG-18F-FDHT accumulated at significantly higher levels after 4 h-evidence that receptor-mediated uptake of SHBG accounted for later time-point differences. This observation was also seen in 22Rv1 tumor-bearing mice, in which SHBG-18F-FDHT exhibited a significantly increased uptake (average tumor-to-background ratio [TBR], 1.62 ± 0.62) in comparison to unbound 18F-FDHT (TBR, 0.81 ± 0.08) at 4 h. Furthermore, the specificity of the SHBG-18F-FDHT accumulation at 4 h was demonstrated by a reduced tumor uptake after AR blockade with enzalutamide (TBR, 1.07 ± 0.13). Conclusion: Prebinding of 18F-FDHT to SHBG allows accurate and quantitative PET imaging of AR levels in murine models of prostate cancer. This procedure may permit the use of PET imaging to study the longitudinal effects of AR-targeting therapies, accelerating novel-drug development.

Nonlinear convective flows developed under the joint action of buoyant and thermocapillary effects in a two-layer system with rigid heat-insulated lateral walls, have been investigated. The influence of a temperature-dependent interfacial heat release/consumption on oscillatory regimes, has been studied. It is shown that sufficiently strong temperature dependence of interfacial heat sources and heat sinks can lead to the change of the sequence of bifurcations and the development of new nonlinear regimes. Specifically, the period doubling bifurcations have been found. (C) 2017 Elsevier Masson SAS. All rights reserved.

Progressive kidney diseases are often associated with scarring of the kidney’s filtration unit, a condition called focal segmental glomerulosclerosis (FSGS). This scarring is due to loss of podocytes, cells critical for glomerular filtration, and leads to proteinuria and kidney failure. Inherited forms of FSGS are caused by Rac1-activating mutations, and Rac1 induces TRPC5 ion channel activity and cytoskeletal remodeling in podocytes. Whether TRPC5 activity mediates FSGS onset and progression is unknown. We identified a small molecule, AC1903, that specifically blocks TRPC5 channel activity in glomeruli of proteinuric rats. Chronic administration of AC1903 suppressed severe proteinuria and prevented podocyte loss in a transgenic rat model of FSGS. AC1903 also provided therapeutic benefit in a rat model of hypertensive proteinuric kidney disease. These data indicate that TRPC5 activity drives disease and that TRPC5 inhibitors may be valuable for the treatment of progressive kidney diseases., National Institutes of Health (U.S.) (Grant DK095045), National Institutes of Health (U.S.) (Grant DK099465), National Institutes of Health (U.S.) (Grant DK103658), National Institutes of Health (U.S.) (Grant DK083511), National Institutes of Health (U.S.) (Grant DK093746)

BACKGROUND Pediatric High-Grade Gliomas (pHGGs) show recurrent high-level amplifications around the oncogenes MET, MYCN and EGFR. However what drives expression of the oncogenes from these amplicons remains unclear. We aim to discover enhancers on these amplicons that are responsible for oncogene expressions and the core regulatory transcription factors (TFs) they bind. METHOD Using RNA-seq from 12 pHGG cell lines, we identified groups of high and low-expressing pHGG lines for MET, MYCN and EGFR. We then compared the H3K27Ac ChIP-seq between the two groups using diffbind. This allowed us to identify statistically significant peaks that are differentially activated in the oncogene-high v.s. oncogene-low expressing groups. Additionally, we overlapped the positions of these candidate oncogene enhancers with the regions that are recurrently incorporated into high-level amplicons based on published whole genome sequencing data. Using a previously defined set of core regulatory TFs we determined which TF binds the amplified oncogene enhancers and could be driving oncogenic expressions of MET, MYCN and EGFR in pHGGs. RESULTS We identify 3 cell lines for both the high- and low-expressing groups for each oncogene. Cell lines with high expression of the oncogene showed distinct enhancers with significant enrichment in H3K27Ac compared to the cell lines with low expression for each oncogene. Of all enhancers with enrichment high oncogene expression groups those with binding sites for known pHGG core regulatory circuit TF were preferentially incorporated into the high-level amplicons of the oncogene. We also identified core TFs that bind enhancers for MYCN, EGFR and MET as well as core TFs that are unique to a single oncogene. CONCLUSION We identified candidate core transcription factor that drives expression of multiple oncogenes in pHGG. These could serve as a potential novel therapeutic target for pHGGs with addiction to MYCN or RTK signaling.