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LZTR1 is the substrate-specific adaptor of a CUL3-dependent ubiquitin ligase frequently mutated in sporadic and syndromic cancer. We combined biochemical and genetic studies to identify LZTR1 substrates and interrogated their tumor-driving function in the context of LZTR1 loss-of-function mutations. Unbiased screens converged on EGFR and AXL receptor tyrosine kinases as LZTR1 interactors targeted for ubiquitin-dependent degradation in the lysosome. Pathogenic cancer-associated mutations of LZTR1 failed to promote EGFR and AXL degradation, resulting in dysregulated growth factor signaling. Conditional inactivation of Lztr1 and Cdkn2a in the mouse nervous system caused tumors in the peripheral nervous system including schwannoma-like tumors, thus recapitulating aspects of schwannomatosis, the prototype tumor predisposition syndrome sustained by LZTR1 germline mutations. Lztr1– and Cdkn2a-deleted tumors aberrantly accumulated EGFR and AXL and exhibited specific vulnerability to EGFR and AXL coinhibition. These findings explain tumorigenesis by LZTR1 inactivation and offer therapeutic opportunities to patients with LZTR1-mutant cancer.Significance:EGFR and AXL are substrates of LZTR1-CUL3 ubiquitin ligase. The frequent somatic and germline mutations of LZTR1 in human cancer cause EGFR and AXL accumulation and deregulated signaling. LZTR1-mutant tumors show vulnerability to concurrent inhibition of EGFR and AXL, thus providing precision targeting to patients affected by LZTR1-mutant cancer.This article is highlighted in the In This Issue feature, p. 517

Phenotype alterations in the liver and brain of Lztr1-deleted mice

LZTR1 is the substrate-specific adaptor of a CUL3-dependent ubiquitin ligase frequently mutated in sporadic and syndromic cancer. We combined biochemical and genetic studies to identify LZTR1 substrates and interrogated their tumor-driving function in the context of LZTR1 loss-of-function mutations. Unbiased screens converged on EGFR and AXL receptor tyrosine kinases as LZTR1 interactors targeted for ubiquitin-dependent degradation in the lysosome. Pathogenic cancer-associated mutations of LZTR1 failed to promote EGFR and AXL degradation, resulting in dysregulated growth factor signaling. Conditional inactivation of Lztr1 and Cdkn2a in the mouse nervous system caused tumors in the peripheral nervous system including schwannoma-like tumors, thus recapitulating aspects of schwannomatosis, the prototype tumor predisposition syndrome sustained by LZTR1 germline mutations. Lztr1– and Cdkn2a-deleted tumors aberrantly accumulated EGFR and AXL and exhibited specific vulnerability to EGFR and AXL coinhibition. These findings explain tumorigenesis by LZTR1 inactivation and offer therapeutic opportunities to patients with LZTR1-mutant cancer.Significance:EGFR and AXL are substrates of LZTR1-CUL3 ubiquitin ligase. The frequent somatic and germline mutations of LZTR1 in human cancer cause EGFR and AXL accumulation and deregulated signaling. LZTR1-mutant tumors show vulnerability to concurrent inhibition of EGFR and AXL, thus providing precision targeting to patients affected by LZTR1-mutant cancer.This article is highlighted in the In This Issue feature, p. 517

Table S1. List of ubiquitylated peptides from DiGly proteomics assay. Table S2. List of proteins differentially abundant in LZTR1-/- compared toLZTR1+/+ cells quantified by TMT.Table S3. List of top scoring LZTR1 interacting proteins from immunoprecipitation/mass spectrometry.

The file contains supplementary Materials and Methods

Centrosome anomalies contribute to tumorigenesis, but it remains unclear how they are generated in lethal cancer phenotypes. Here, it is demonstrated that human microsatellite instable (MSI) and BRAFV600E-mutant colorectal cancers with a lethal rhabdoid phenotype are characterized by inactivation of centrosomal functions. A splice site mutation that causes an unbalanced dosage of rootletin (CROCC), a centrosome linker component required for centrosome cohesion and separation at the chromosome 1p36.13 locus, resulted in abnormally shaped centrosomes in rhabdoid cells from human colon tissues. Notably, deleterious deletions at 1p36.13 were recurrent in a subgroup of BRAFV600E-mutant and microsatellite stable (MSS) rhabdoid colorectal cancers, but not in classical colorectal cancer or pediatric rhabdoid tumors. Interfering with CROCC expression in near-diploid BRAFV600E-mutant/MSI colon cancer cells disrupts bipolar mitotic spindle architecture, promotes tetraploid segregation errors, resulting in a highly aggressive rhabdoid-like phenotype in vitro. Restoring near-wild-type levels of CROCC in a metastatic model harboring 1p36.13 deletion results in correction of centrosome segregation errors and cell death, revealing a mechanism of tolerance to mitotic errors and tetraploidization promoted by deleterious 1p36.13 loss. Accordingly, cancer cells lacking 1p36.13 display far greater sensitivity to centrosome spindle pole stabilizing agents in vitro. These data shed light on a previously unknown link between centrosome cohesion defects and lethal cancer phenotypes providing new insight into pathways underlying genome instability.Implications: Mis-segregation of chromosomes is a prominent feature of chromosome instability and intratumoral heterogeneity recurrent in metastatic tumors for which the molecular basis is unknown. This study provides insight into the mechanism by which defects in rootletin, a centrosome linker component causes tetraploid segregation errors and phenotypic transition to a clinically devastating form of malignant rhabdoid tumor. Mol Cancer Res; 16(9); 1385–95. ©2018 AACR.

Supplementary Table 1. Clinicopathological, immunohistochemical and molecular features of the 12 rhabdoid colorectal cancers7 Supplementary Table 2. Somatic mutations of candidate genes (15 out of 20 are reported) shared by the two rhabdoid cancers at exome sequencing Supplementary Table 3. Rhabdoid colorectal cancers (RC): molecular alterations, centrosome aberrations and ploidy Supplementary Table 4. Rhabdoid tumours of infants (RI): molecular alterations, centrosome aberrations, and ploidy Supplementary Table 5: Clinicopathologic and molecular features of classical colorectal cancers analyzed Supplementary Table 6. Sequencing and Reverse transcription PCR primers Supplementary Table 6B. SMARCB1 regions covered by the custom panel used for next-generation target sequencing Supplementary Table 7. CROCC regions covered by the custom panel used for next-generation targeted sequencing Supplementary Table 8. Antibodies employed and working conditions for immunohistochemistry, immunofluorescence or western blot analysis of tissues and cell lines Supplementary Table 9. Rhabdoid colorectal cancer reported in literature between 1993-2015

S1. Whole exome sequencing analysis of two rhabdoid colorectal cancers (RC). S2. Non-silent somatic mutations in rhabdoid tumours are enriched in centrosome-microtubule components. S3. Effect of CROCC mutations on centrosome phenotype. S4. CROCC expression profiling in classical colorectal cancers (CRCs). S5. Allelic loss at the 1p36.13 locus correlates with chromosomal instability in colorectal cancer cells. S6. Centrosome and chromosome instability in colorectal cancer cells. S7. Mitotic errors and impaired cell cycle caused by stable CROCC depletion in RKO cells. S8. Invasive behavior of colorectal cancer cells triggered by CROCC depletion. S9. Mitotic aberrations suppressed by CROCC or microtubule stabilizing agents in metastatic T84 and HT29 colorectal cancer cells.

Despite producing a panoply of potential cancer-specific targets, the proteogenomic characterization of human tumors has yet to demonstrate value for precision cancer medicine. Integrative multi-omics using a machine-learning network identified master kinases responsible for effecting phenotypic hallmarks of functional glioblastoma subtypes. In subtype-matched patient-derived models, we validated PKCδ and DNA-PK as master kinases of glycolytic/plurimetabolic and proliferative/progenitor subtypes, respectively, and qualified the kinases as potent and actionable glioblastoma subtype-specific therapeutic targets. Glioblastoma subtypes were associated with clinical and radiomics features, orthogonally validated by proteomics, phospho-proteomics, metabolomics, lipidomics and acetylomics analyses, and recapitulated in pediatric glioma, breast and lung squamous cell carcinoma, including subtype specificity of PKCδ and DNA-PK activity. We developed a probabilistic classification tool that performs optimally with RNA from frozen and paraffin-embedded tissues, which can be used to evaluate the association of therapeutic response with glioblastoma subtypes and to inform patient selection in prospective clinical trials.

Leucine-zipper transcription regulator 1 (LZTR1) is a highly mutated tumor suppressor gene, involved in the pathogenesis of several cancer types and developmental disorders. In proteasomal degradation, it acts as an adaptor protein responsible for the recognition and recruitment of substrates to be ubiquitinated in Cullin3-RING ligase E3 (CRL3) machinery. LZTR1 belongs to the BTB-Kelch family, a multi-domain protein where the Kelch propeller plays as the substrate recognition region and for which no experimental structure has been solved. Recently, large effort mutational analyses pointed to the role of disease-associated LZTR1 mutations in the RAS/MAPK signaling pathway and RIT1, a small Ras-related GTPase protein, has been identified by mass spectroscopy to interact with LZTR1. Hence, a better understanding of native structure, molecular mechanism, and substrate specificity would help clarifying the role of LZTR1 in pathological diseases, thus promoting advancement in the development of novel therapeutic strategies. Here, we address the interaction model between adaptor LZTR1 and substrate RIT1 by applying an integrated computational approach, including molecular modeling and docking techniques. We observe that the interaction model LZTR1-RIT1 is stabilized by an electrostatic bond network established between the two protein surfaces, which is reminiscent of homologous ubiquitin ligases complexes. Then, running MD simulations, we characterize differential conformational dynamics of the multi-domain LZTR1, offering interesting implications on the mechanistic role of specific point mutations. We identify G248R and R283Q as damaging mutations involved in the recognition process of the substrate RIT1 and R412C as a possible allosteric mutation from the Kelch to the C-term BTB-domain. Our findings provide important structural insights on targeting CRL3s for drug discovery.

Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF-1), the most common human genetic cancer predisposition syndrome. NF1 patients are prone to develop peripheral and central nervous system tumors, with glioma occurring in 15-20% of patients. The molecular profiles of glioma from patients with NF-1 have been previously explored by large scale DNA and RNA bulk analyses, providing the first comprehensive genetic landscape of NF-1 glioma. However, the intra-tumoral architecture and the microenvironment organization remained largely concealed within the global bulk profile. Here, to characterize the complexity of molecular states of both malignant and non-malignant cells, we profiled NF-1 glioma gene expression at individual cell resolution. We performed single-nuclei RNA sequencing (Nucseq) on frozen NF-1 glioma specimens, including high-grade and low-grade tumors. The functional classification of the transcriptional programs activated in the individual tumor cells identified three main molecular states that partially overlapped with the cellular subtypes of sporadic glioblastoma. Tumor cells of low-grade NF-1 glioma exhibited marked enrichment of neuronal functions, suggesting that the gliomagenesis process in the context of NF1 inactivation might be supported by specialized neuronal activities. Conversely, the proliferative state characterized high-grade NF-1 glioma, indicating that cell cycle-associated pathways are drivers of tumor progression. The single-cell deconvolution of immune microenvironment in NF-1 low grade glioma revealed infiltration of CD8+ T lymphocytes and the active crosstalk of these cells with pro-inflammatory macrophages and mature dendritic cells, supporting a possible anti-tumor cell-mediated response. The identification of divergent tumor cell states as well as the characterization of the immune infiltrates provide new insights for accurate stratification of NF-1 glioma patients and to inform personalized therapeutics.

The factors driving therapy resistance in diffuse glioma remain poorly understood. To identify treatment-associated cellular and genetic changes, we analyzed RNA and/or DNA sequencing data from the temporally separated tumor pairs of 304 adult patients with isocitrate dehydrogenase (IDH)-wild-type and IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions were associated with an increase in proliferating neoplastic cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their neoplastic cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a myeloid cell state defined by specific ligand-receptor interactions with neoplastic cells. Collectively, these recurrence-associated phenotypes represent potential targets to alter disease progression.

As the clinical failure of glioblastoma treatment is attributed by multiple components, including myelin-associated infiltration, assessment of the molecular mechanisms underlying such process and identification of the infiltrating cells have been the primary objectives in glioblastoma research. Here, we adopted radiogenomic analysis to screen for functionally relevant genes that orchestrate the process of glioma cell infiltration through myelin and promote glioblastoma aggressiveness. The receptor of the Nogo ligand (NgR1) was selected as the top candidate through Differentially Expressed Genes (DEG) and Gene Ontology (GO) enrichment analysis. Gain and loss of function studies on NgR1 elucidated its underlying molecular importance in suppressing myelin-associated infiltration in vitro and in vivo. The migratory ability of glioblastoma cells on myelin is reversibly modulated by NgR1 during differentiation and dedifferentiation process through deubiquitinating activity of USP1, which inhibits the degradation of ID1 to downregulate NgR1 expression. Furthermore, pimozide, a well-known antipsychotic drug, upregulates NgR1 by post-translational targeting of USP1, which sensitizes glioma stem cells to myelin inhibition and suppresses myelin-associated infiltration in vivo. In primary human glioblastoma, downregulation of NgR1 expression is associated with highly infiltrative characteristics and poor survival. Together, our findings reveal that loss of NgR1 drives myelin-associated infiltration of glioblastoma and suggest that novel therapeutic strategies aimed at reactivating expression of NgR1 will improve the clinical outcome of glioblastoma patients.

High grade gliomas (HGG) are aggressive primary brain malignancies typified by diffuse invasion, genetic heterogeneity, and a universally fatal outcome. MRI-defined contrast-enhancing (CE) tumor burden serves as the clinical standard that guides maximal surgical resection and post-therapy response assessment. However, HGGs also comprise an invasive non-enhancing (NE) tumor margin that extends beyond the CE core and harbors the cells that contribute to recurrence. Sampling restrictions have hindered the comprehensive study of these NE HGG cell populations driving tumor progression. Herein, we present an integrated multi-omic analysis of 313 spatially matched multi-regional CE and NE tumor biopsies from 68 HGG patients, performing whole exome and RNA sequencing of both IDH wild-type and IDH mutant HGGs. We report spatially restricted molecular profiles in IDH-mutant HGG, highlighting a concern for sampling bias given the importance of molecular diagnosis and prognostication in IDH-mutant HGG. Regardless of IDH status, we found that NE tumor regions harbored the highest proportion of private mutations, which reflects an increased development of regional genomic complexity in infiltrative tumor. The multiregional genomic profiling of our IDH wild-type HGG cohort reveals that EGFR and NF1 somatic alterations occur as mutually exclusive events in 98.7% of tumors. However, we resolved rare low allele frequency co-alterations of EGFR and NF1 within the NE region. We find this co-occurrence enriched in recurrent tumors, pointing to the early emergence of NF1 inactivation in the NE regions. We constructed genomic models predictive of recurrent disease from both NE and CE regions, which highlight the occurrence of clonal EGFR copy number alterations and NF1 loss as clonal or subclonal events, respectively, emphasizing the regional and temporal complexity of well-studied canonical driver alterations. We detailed the spatially unique acquisition of multiple distinct EGFR alterations giving rise to intra-tumoral EGFR mosaicism, a challenge in the implementation of EGFR directed therapies. Our study also identified two transcriptomic clusters delineated by the significant overrepresentation of neuronal (NEU) and glycolytic/plurimetabolic (GPM) pathway-based functional states in the NE region. NE regions of the NEU subtype harbor the greatest proportion of private mutations, suggesting these infiltrative tumor cells accumulate alterations without clonal expansion. GPM populations conversely displayed a less branched phylogeny and were transcriptionally enriched in immune cell signatures. This phenotypic dichotomy between GPM and NEU populations supports the growing body of evidence that invasive GBM cells either take on a neuronal phenotype for active invasion or a more metabolic phenotype involving interaction with astrocytes, other glial cells, and infiltrating immune cells. Citation Format: Mylan R. Blomquist, Leland S. Hu, Fulvio D'Angelo, Taylor M. Weiskittel, Francesca P. Caruso, Shannon P. Fortin Ensign, Christopher Sereduk, Gustavo De Leon, Lee Curtin, Javier Urcuyo, Ashlynn Gonzalez, Ashley Nespodzany, Teresa Noviello, Jennifer M. Eschbacher, Kris A. Smith, Peter Nakaji, Bernard R. Bendok, Richard S. Zimmerman, Chandan Krishna, Devi Patra, Naresh Patel, Mark Lyons, Kliment Donev, Maciej Mrugala, Alyx Porter, Anna Lasorella, Kristin R. Swanson, Michele Ceccarelli, Antonio Iavarone, Nhan L. Tran. Multiregional sampling of high grade glioma identifies regional biologic signatures [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 1507.

High grade glioma (HGG) represents a group of devastating diseases with dismal prognosis. Surgical resection of the contrast enhancing (CE) region of HGG remains the mainstay of treatment, but recurrence inevitably arises from the unresected non-contrast enhancing (NE) region, surgically inaccessible due to cancer cell invasion into healthy brain tissue. Due to its critical role in recurrence, understanding of the NE region is central to the improvement of clinical outcomes. We reveal the biological characteristics of this region through image localized multi-regional sampling. We linked microenvironmental characteristics measured by multi-parametric MRI to genomic mutations and transcriptional phenotypes using mixed effect modeling which allowed us to control for individualized patient effects. We first confirmed that T2 is a significant indicator of IDH mutation status in the NE region, being the first description of such a relationship in a HGG cohort. We found the combination of EGFR amplification and CDKN2A homozygous loss was associated with a significantly lower mean diffusivity (MD) compared to double wild type tumors in the NE region, indicating the presence of greater cellular packing and proliferation in EGFR amplification/CDKN2A loss regions. Finally, using single cell pathway based tumor classifications, we showed that nK2, a DSC-MRI metric representing cell size heterogeneity, correlated positively with neuronal signature and negatively with glycolytic/plurimetabolic signature within the NE tumor, indicating that glycolytic/plurimetobolic tumors possessed a high amount of cell size heterogeneity compared to neuronal samples. This hypothesis was supported using digital reference object (DRO) modeling which confirmed that cell size and heterogeneity drove the differential nK2 signal between neuronal and glycolytic/plurimetabolic samples. We identified immune cell infiltrate as one possible mechanism of increased cell size heterogeneity using transcriptomic signature analysis which found more immune cell signatures within glycolytic/plurimetobolic tumors compared to neuronal. Collectively this study demonstrates the central role of multi-parametric MRI as a non-invasive measure of tumor biology and a tool for understanding the clinically critical NE region which can then inform new therapies targeting this region of HGG recurrence. Citation Format: Matthew Flick, Taylor Weiskittel, Kevin Meng-Lin, Fulvio D'Angelo, Francesca Caruso, Shannon Ensign, Mylan Blomquist, Luija Wang, Christopher Sereduk, Gustavo De Leon, Ashley Nespodzany, Javier Urcuyo, Ashlynn Gonzalez, Lee Curtin, Kyle Singleton, Aliya Anil, Natenael Simmineh, Erika Lewis, Teresa Noviello, Reyna Patel, Panwen Wang, Junwen Wang, Jennifer Eschbacher, Andrea Hawkins-Daarud, Pamela Jackson, Kris Smith, Peter Nakaji, Bernard Bendok, Richard Zimmerman, Chandan Krishna, Devi Patra, Naresh Patel, Mark Lyons, Matthew Neal, Kliment Donev, Maciej Mrugala, Alyx Porter, Scott Beeman, Yuxiang Zhou, Leslie Baxter, Christopher Plaisier, Jing Li, Hu Li, Anna Lasorella, Chad Quarles, Kristin Swanson, Michele Ceccarelli, Antonio Iavarone, Nhan Tran, Leland Hu. Multi-parametric MRI maps regional heterogeneity of high grade glioma phenotypes. [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 5621.

Diffuse glioma is characterized by a poor prognosis and a universal resistance to therapy, though the evolutionary processes behind this resistance remain unclear. The Glioma Longitudinal Analysis (GLASS) Consortium has previously demonstrated that therapy-induced selective pressures shape the genetic evolution of glioma in a stochastic manner. However, single-cell studies have revealed that malignant glioma cells are highly plastic and transition their cell state in response to diverse challenges, including changes in the microenvironment and the administration of standard-of-care therapy. To interrogate the factors driving therapy resistance in diffuse glioma, we collected and analyzed RNA- and/or DNA-sequencing data from temporally separated tumor pairs of over 300 adult patients with IDH-wild-type or IDH-mutant glioma. In a subset of these tumor pairs, we complemented these data with single-nucleus RNAseq and multiplex imaging mass cytometry at each time point. Recurrent tumors exhibited diverse changes that were attributable to changes in histological features, somatic alterations, and microenvironment interactions. IDH-wild-type tumors overall were more invasive at recurrence and exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. In contrast, recurrent IDH-mutant tumors exhibited a significant increase in proliferative expression programs that correlated with discrete genetic changes. Hypermutation and acquired CDKN2A homozygous deletions associated with an increase in proliferating stem-like malignant cells at recurrence in both glioma subtypes, reflecting active tumor expansion. A transition to the mesenchymal phenotype was associated with the presence of a specific myeloid cell state defined by unique ligand-receptor interactions with malignant cells, providing opportunities to target this transition through therapy. Collectively, our results uncover recurrence-associated changes in genetics and the microenvironment that can be targeted to shape disease progression following initial diagnosis.

LZTR1, the substrate-specific adaptor of a CUL3-dependent ubiquitin ligase is among the most frequently mutated ubiquitin ligase coding gene in syndromic and sporadic human cancers including glioblastoma multiforme, in which approximately 27% of cases harbor inactivating mutations and copy number loss. However, both the identity of the protein substrates targeted by LZTR1-mediated ubiquitylation and the biological contexts regulated by specific LZTR1-substrate(s) interactions remain uncertain. Here, we combined biochemical and genetic studies to identify LZTR1 substrates and interrogated their tumor-driving function in the context of LZTR1 loss-of-function mutations and in a new conditional Lztr1 knockout mouse. Multiple screens converged on the receptor tyrosine kinases EGFR and AXL as LZTR1 interactors targeted for ubiquitin-dependent degradation in the lysosome by LZTR1-CUL3 complexes. Pathogenic mutations affecting LZTR1 failed to promote degradation of EGFR and AXL in human tumors. Mice harboring conditional deletion of the LZTR1 gene combined with loss of CDKN2A in the neural progenitor compartment generated peripheral nervous system tumors including schwannoma like and malignant peripheral nervous system tumors (MPNST). Tumors from the LZTR1-mutant mouse model accumulated very high levels of EGFR and AXL and exhibited potent and specific vulnerability to the combinatorial inhibition of EGFR and AXL kinases. These findings explain the mechanism of tumorigenesis associated with LZTR1 inactivation and offer a therapeutic strategy to patients affected by tumors carrying mutations of LZTR1.

Glioblastoma multiforme (GBM) is the most common and malignant type of primary brain tumor. Despite intensive multimodal treatment, the prognosis of GBM patients is poor with the median overall survival under 15 months. Defining the genetic profiles of rare long-term GBM survivors may provide insights into the molecular basis of GBM progression. IDH mutations have been identified as a favorable prognostic factors, and define a clinically and molecularly distinct group of GBM. Additional molecular mechanisms underlying the prolonged survival in IDHwt GBM patients remained unclear. Therefore, we explored the molecular profiles of 71 IDHwt GBM homogeneously treated patients exhibiting disproportionate survival. The cohort included 36 short-term survivors that deceased within 1 year after diagnosis (median survival of 7 months) and 35 long-term survivors exhibiting an exceptional survival above 4 years (median survival of 88 months). The differential genetic landscape of short- and long-term IDHwt GBM survivors outlined similar mutation load and few differences in the frequency of recurrent alterations, including chromosomes 19 and 20 duplication significantly overrepresented in long-term survivors. To assess the potential impact of T lymphocyte neoantigen-mediated activation towards extended GBM survival, we inferred the restricted HLA binding affinity of the somatic mutations and applied the immune fitness model to accurately predict high-quality neoantigens. Although the general neoantigen burden remained unvaried between short- and long-term survivors, we found that the immune quality of the predicted neoantigens is significantly higher in the patients with exceptional long survival. Moreover, the immune deconvolution of the tumor microenvironment from the gene expression data indicated the presence of CD8+ T lymphocytes and pro-inflammatory macrophages in samples harboring high-quality neoantigens from long-term survivors. This study identified the neoantigen quality as a main distinctive feature of exceptionally long-term survivors supporting the key role of cellular-mediated immune response to restrict tumor progression in IDHwt GBM.

Current transcriptomic classification of Glioblastoma Multiforme (GBM) has been ineffective to predict survival and therapeutic vulnerabilities. Recently, we proposed a four-group functional classification of GBM that included proliferative/progenitor, neuronal, mitochondrial and glycolytic/plurimetabolic subtypes with prognostic and therapeutic implications as the mitochondrial subtype carries the best survival and exhibits distinct sensitivity to mitochondrial OXPHOS inhibitors. To uncover novel therapeutic targets for each functional GBM subtype, we focused on protein kinases for their attractive features as both drivers and drug targets, with current availability of 62 FDA-approved inhibitors available for cancer precision therapeutics. We designed an unbiased integrative, machine learning-based proteomics/phosphoproteomics network for the identification of Master Kinases (MKs) responsible for effecting key phenotypic hallmarks of each of the four GBM subtypes. Here we report the identification and validation of protein kinase C delta (PRKCd) and DNA-PKcs as MKs that sustain the glycolytic/plurimetabolic and proliferative/progenitor GBM subtypes, respectively. Genetic and pharmacologic inactivation of PKCd in GBM patient-derived organoids of the glycolytic/plurimetabolic subtype blocked glucose uptake and lipid accumulation, resulting in marked anti-tumor effects. We also validated the role of PKCd in oncometabolic processes at the intersection between insulin, IGF, and lipid metabolism. DNA-PKcs was qualified as MK for the proliferative/progenitor GBM subtype, which is characterized by high basal level of replication stress. Biochemical experiments showed activation of DNA-PK in GBM patient-derived organoids of the proliferative/progenitor subgroup. Targeting DNA-PK in proliferative/progenitor GBM organoids with the clinically tested DNA-PKcs inhibitor nedisertib in association with gamma irradiation, the essential component of the standard of care for patients with GBM, led to an unsustainable level of DNA damage and massive GBM cell death selectively in this GBM subtype. As DNA-PKcs inhibitors have been introduced into clinical trials, our findings suggest that pre-selection of patient with PPR tumors is likely to enhance therapeutic success.

Tumor heterogeneity and broad therapeutic resistance are major challenges when treating patients with glioblastoma (GBM). Moreover, different from other cancer types, the absence of clinically useful classifiers for glioblastoma has hampered the translation of genomic and proteomic information to improve diagnosis and design precision therapeutics for patients. We analyzed a large dataset of human GBM to determine the multi-omics features characterizing the four GBM subtypes that we have recently identified through single-cell RNA sequencing and validated in bulk tumor analysis. Each subtype exhibits activation of unique functional traits that confer distinct therapeutic vulnerabilities. The examination of proteomics, phosphoproteomics, metabolomics, lipidomics and acetylomics data revealed that each GBM subtype has a coherent molecular structure driving the dominant function traceable in each analytical platform. Functional classes are not a specific attribute of GBM as we identified the same subtypes in breast and lung cancer. To test the translational impact of proteomic data in GBM, we developed an unbiased protein kinase signaling network approach for the selection of master kinases (MKs) aberrantly activated in each GBM subtype. We identified therapeutically actionable MKs and novel phosphorylation substrates that we experimentally validated. To provide rapid translation of the functional classifier for precision medicine in GBM, we developed a probabilistic classification tool which determines the probability that a patient’s GBM belongs to one of the four subtypes, exhibiting optimal performance when using RNA extracted from either frozen and paraffin-embedded tissues. The algorithm is publicly accessible and can be used to evaluate the association of therapeutic response with GBM subtypes and as tool for selection criteria in prospective clinical trials.

Pediatric low-grade gliomas (pLGGs) arise primarily at early stages of development. The molecular mechanisms of pLGG gliomagenesis are unclear, as is the progenitor cell of origin. In this issue of Developmental Cell, Jecrois et al. show that NF1-associated optic pathway gliomas originate from migrating glial progenitors that have distinct MEK/ERK dependency.

SummaryTumor adaptation or selection is thought to underlie therapy resistance of gliomas. To investigate the longitudinal epigenetic evolution of gliomas in response to therapeutic pressure, we performed an epigenomic analysis of 143 matched initial and recurrent patients with IDH-wildtype (IDHwt) and IDH-mutant (IDHmut) gliomas. IDHwt gliomas showed a longitudinally stable epigenome with relatively low levels of global methylation, whereas the epigenome of IDHmut gliomas showed initial high levels genome-wide of DNA methylation that was progressively reduced to levels similar to those of IDHwt tumors. By integrating DNA methylation and gene expression data, adaptive changes of putative master regulators of the cell cycle and of differentiation were seen in IDHmut recurrent tumors. Furthermore, relapses of IDHmut tumors were accompanied by histological progression which in turn influenced survival, as validated in an independent cohort. Finally, the initial cell composition of the tumor microenvironment differed between IDHwt and IDHmut tumors and changed differentially following treatment, suggesting increased neo-angiogenesis and T-cell infiltration upon treatment for IDHmut gliomas. Our study provides one of the largest cohorts of paired glioma samples profiled with epigenomics, transcriptomics and genomics; and our results demonstrate that the treatment of IDHmut gliomas reshapes the epigenome towards an IDHwt-like phenotype. Accordingly, the prevalent practice of early genotoxic treatment in this patient population may need to be revisited.

SummaryTo interrogate the factors driving therapy resistance in diffuse glioma, we collected and analyzed RNA and/or DNA sequencing data from temporally separated tumor pairs of 292 adult patients with IDH-wild-type or IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions associated with an increase in proliferating stem-like malignant cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their malignant cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a specific myeloid cell state defined by unique ligand-receptor interactions with malignant cells. Collectively, our results uncover recurrence-associated changes that could be targetable to shape disease progression following initial diagnosis.

The proteogenomic characterization of human tumors has begun to uncover molecular attributes enriched in different tumor subtypes and proposed a panoply of subtype-specific targets. As these studies lacked experimental testing of potential targets in high-quality cell models accurately reflecting individual tumor subtypes, it remains unclear whether any of the proposed targets retained value for precision medicine. The absence of clinically useful classifiers further hampered translation of proteogenomic information to better diagnose and treat cancer patients. In the case of the brain tumor glioblastoma (GBM), such challenges are exacerbated by tumor heterogeneity and broad therapeutic resistance. Here, we analyzed a large dataset of human GBM to determine the multi-omics features that characterize four GBM subtypes we recently identified through single cell RNAseq analysis. Each subtype exhibits activation of unique hallmark functional traits including distinct sensitivity to inhibitors of mitochondrial respiration for the mitochondrial subtype. The inspection of proteomics, phosphoproteomics, metabolomics, lipidomics and acetylomics data revealed that each GBM subtype has a coherent molecular structure that drives the dominant function and can be extracted by each analytical platform. Stratification of tumors in four functional classes is not a specific attribute of GBM as we were able to unbiasedly identify the same subtypes in breast and lung cancer. To identify actionable targets in GBM we developed an unbiased protein kinase signaling network approach for the selection of master kinases aberrantly activated in each GBM subtype. The experimental follow-up using a library of annotated GBM patient derived organoids (PDOs) established that inhibition of DNA-PK with the clinically tested compound nedisertib radiosensitizes proliferative/progenitor PDOs by exacerbating replication stress-induced DNA damage in this GBM subtype. Furthermore, genetic and pharmacological tools qualified PKCδ as vulnerable target for the broadly resistant glycolytic/plurimetabolic GBM subtype. Thus, together with the targeting of mitochondrial GBM with OXPHOS inhibitors, the functional classification delivers experimentally validated actionable targets for three GBM subtypes. To provide rapid translation of the classifier for precision medicine in GBM, we developed a probabilistic classification tool which determines the probability that a patient’s GBM belongs to one of the four subtypes based on transcriptomic features and exhibits optimal performance when using RNA extracted from either frozen and paraffin embedded tissues. The algorithm is publicly accessible. It can be used in retrospective studies to evaluate the association of therapeutic response with GBM subtypes and as tool for selection criteria in prospective clinical trials. Citation Format: Simona Migliozzi, Young Taek Oh, Luciano Garofano, Hasanain Mohammad, Fulvio D'Angelo, Ryan D. Najac, Franck Bielle, Karima Mokhtari, Jann N. Sarkaria, Michele Ceccarelli, Marc Sanson, Anna Lasorella, Antonio Iavarone. Pathway based analysis of glioblastoma by multi-omics with applications in targeted therapy, prognosis and probabilistic classification [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 4014.

High-grade gliomas represent the most common type of primary adult malignant brain tumor historically diagnosed and graded from histologic criteria alone. Gliomas harboring isocitrate dehydrogenase (IDH) 1 or 2 mutations, which are present in more than 80% of World Health Organization (WHO) grade 2 or 3 tumors, portend a favorable prognosis as compared to IDH-wildtype tumors. However, recent molecular profiling has identified the presence of additional alterations in these lower grade tumors that transform the biology toward an aggressive high-grade phenotype clinically that are independent of histologic grading and confer a worse clinical prognosis. Of these, the loss of CDKN2A/B is the strongest implicated alteration and is sufficient to define a grade 4 tumor in IDH-mutant astrocytomas regardless of histologic appearance. However, there remain no effective therapies targeted at molecular subgroups in aggressive gliomas to date. Here we have profiled the intratumoral heterogeneity via whole exome and RNA sequencing and copy number alteration analysis within a cohort of de-identified IDH-mutant astrocytoma patient tumors collected through the Mayo Clinic and Barrow Neurological Institute (BNI) biorepositories to determine the spatial evolution of aggressive molecular features. Within this cohort we have collected 42 image-localized intratumoral biopsy specimens representing 13 IDH-mutant patient tumors. We identified 11 biopsies from 3 tumors that harbored loss of CDKN2A/B which included 1 tumor that displayed intratumoral heterogeneity of CDKN2A/B expression across subclones. We performed an unsupervised cluster analysis to profile the transcriptional signatures in IDH-mutant astrocytomas across both differential CDKN2A/B status and tumor geographic location comparing contrast enhancing versus non-enhancing regions, and we analyzed the MRI features in IDH-mutant tumors across CDKN2A/B status. We found that tumors with CDKN2A/B alteration demonstrated lower signal intensity on delayed post-contrast T2*W images, which could represent lower pre-contrast T2*, unique contrast agent kinetics, or dissimilar diffusion properties related to increased cellular density and/or greater heterogeneity of cell size. Interestingly, we uncovered spatial heterogeneity of additional molecular drivers of high-grade glioma including KRAS, MYCN, and PDGFRA. While EGFR alterations are rarely reported in IDH-mutant tumors, we identified one treatment naïve patient tumor that harbored EGFR alteration only in the contrast non-enhancing region. Based on this work we propose there is early spatial evolution of aggressive tumor subclones prior to primary tumor diagnosis, and multiregional molecular profiling may better inform tumor biology and predict therapeutic strategies in IDH-mutant astrocytomas. Citation Format: Shannon P. Fortin Ensign, Fulvio D'Angelo, Francesca P. Caruso, Christopher Sereduk, Kyle Singleton, Jennifer Eschbacher, Gina Mazza, Maciej Mrugala, Alyx Porter, Bernard Bendok, Richard Zimmerman, Kris Smith, Peter Nakaji, Kamala Swanson, Junwen Wang, Nathenael Semmineh, Christopher Quarles, Anna Lasorella, Kristin Swanson, Michele Ceccarelli, Antonio Iavarone, Leland Hu, Nhan L. Tran. Multiregional imaging and genomic analysis in high-grade IDH-mutant astrocytoma identifies intratumor subclones with aggressive features [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 770.

The transcriptomic classification of glioblastoma (GBM) has failed to predict survival and therapeutic vulnerabilities. A computational approach for unbiased identification of core biological traits of single cells and bulk tumors uncovered four tumor cell states and GBM subtypes distributed along neurodevelopmental and metabolic axes, classified as proliferative/progenitor, neuronal, mitochondrial and glycolytic/plurimetabolic. Each subtype was enriched with biologically coherent multiomic features. Mitochondrial GBM was associated with the most favorable clinical outcome. It relied exclusively on oxidative phosphorylation for energy production, whereas the glycolytic/plurimetabolic subtype was sustained by aerobic glycolysis and amino acid and lipid metabolism. Deletion of the glucose-proton symporter SLC45A1 was the truncal alteration most significantly associated with mitochondrial GBM, and the reintroduction of SLC45A1 in mitochondrial glioma cells induced acidification and loss of fitness. Mitochondrial, but not glycolytic/plurimetabolic, GBM exhibited marked vulnerability to inhibitors of oxidative phosphorylation. The pathway-based classification of GBM informs survival and enables precision targeting of cancer metabolism.

Glioblastoma (GBM) is the most aggressive nervous system cancer. Understanding its molecular pathogenesis is crucial to improving diagnosis and treatment. Integrated analysis of genomic, proteomic, post-translational modification and metabolomic data on 99 treatment-naive GBMs provides insights to GBM biology. We identify key phosphorylation events (e.g., phosphorylated PTPN11 and PLCG1) as potential switches mediating oncogenic pathway activation, as well as potential targets for EGFR-, TP53-, and RB1-altered tumors. Immune subtypes with distinct immune cell types are discovered using bulk omics methodologies, validated by snRNA-seq, and correlated with specific expression and histone acetylation patterns. Histone H2B acetylation in classical-like and immune-low GBM is driven largely by BRDs, CREBBP, and EP300. Integrated metabolomic and proteomic data identify specific lipid distributions across subtypes and distinct global metabolic changes in IDH-mutated tumors. This work highlights biological relationships that could contribute to stratification of GBM patients for more effective treatment. Wang et al. perform integrated proteogenomic analysis of adult glioblastoma (GBM), including metabolomics, lipidomics, and single nuclei RNA-Seq, revealing insights into the immune landscape of GBM, cell-specific nature of EMT signatures, histone acetylation in classical GBM, and the existence of signaling hubs which could provide therapeutic vulnerabilities.

Background Single-cell RNA sequencing is the reference technique for characterizing the heterogeneity of the tumor microenvironment. The composition of the various cell types making up the microenvironment can significantly affect the way in which the immune system activates cancer rejection mechanisms. Understanding the cross-talk signals between immune cells and cancer cells is of fundamental importance for the identification of immuno-oncology therapeutic targets. Results We present a novel method, single-cell Tumor–Host Interaction tool (scTHI), to identify significantly activated ligand–receptor interactions across clusters of cells from single-cell RNA sequencing data. We apply our approach to uncover the ligand–receptor interactions in glioma using 6 publicly available human glioma datasets encompassing 57,060 gene expression profiles from 71 patients. By leveraging this large-scale collection we show that unexpected cross-talk partners are highly conserved across different datasets in the majority of the tumor samples. This suggests that shared cross-talk mechanisms exist in glioma. Conclusions Our results provide a complete map of the active tumor–host interaction pairs in glioma that can be therapeutically exploited to reduce the immunosuppressive action of the microenvironment in brain tumor.

Made available in DSpace on 2018-12-11T17:22:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-01 Chronic myeloid leukemia (CML) is a stem cell derived malignant disorder result of translocation t(9;22)(q34;q11) called Philadelphia chromosome (Ph+). microRNAS (miRNAs) are involved in several biological processes, altering the progression of various pathologies, including CML. This study evaluated whether circulating miRNAs display differential expression profiles in peripheral blood of CML-Chronic Phase (CML-CP) patients newly diagnosed in comparison with CML-CP treated with imatinib. We obtained peripheral blood samples from CML-CP Ph+ patients divided among group 1 (untreated newly diagnosed) and group 2 (treated with imatinib). A pool of total leukocytes from healthy donors was considered as control group. Expression analyses were performed for 768 miRNAs by RT-qPCR array. Bioinformatic tools were used to identify significant pathways and interaction networks. We found 80 deregulated miRNAs between the groups and, according to bioinformatic analysis, they are involved in different pathways, including molecular mechanisms of cancer. The study allows better understanding of disease molecular behavior, and it is useful for possible monitoring CML treatment and prognostic biomarkers identification. Department of Internal Medical São Paulo State University (UNESP-FMB) Laboratory of Molecular Biology Hospital Dr. Amaral Carvalho Department of Science and Technology Sannio University Department of Internal Medical São Paulo State University (UNESP-FMB)

Diffuse glioma is an aggressive brain cancer that is characterized by a poor prognosis and a universal resistance to therapy. The evolutionary processes behind this resistance remain unclear. Previous studies by the Glioma Longitudinal Analysis (GLASS) Consortium have indicated that therapy-induced selective pressures shape the genetic evolution of glioma in a stochastic manner. However, single-cell studies have revealed that malignant glioma cells are highly plastic and transition their cell state in response to diverse challenges, including changes in the microenvironment and the administration of standard-of-care therapy. Interactions between these factors remain poorly understood, making it difficult to predict how a patient’s tumor will evolve from diagnosis to recurrence. To interrogate the factors driving therapy resistance in diffuse glioma, we collected and analyzed RNA- and/or DNA-sequencing data from temporally separated tumor pairs of 292 adult patients with IDH-wild-type or IDH-mutant glioma. Recurrent tumors exhibited diverse changes that were attributable to changes in anatomic composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A homozygous deletions associated with an increase in proliferating stem-like malignant cells at recurrence in both glioma subtypes, reflecting active tumor expansion. IDH-wild-type tumors were more invasive at recurrence, and their malignant cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a specific myeloid cell state defined by unique ligand-receptor interactions with malignant cells, providing opportunities to target this transition through therapy. Collectively, our results uncover recurrence-associated changes in genetics and the microenvironment that can be targeted to shape disease progression following initial diagnosis.

Diffuse glioma is an aggressive brain cancer that is characterized by a poor prognosis and a universal resistance to therapy. The evolutionary processes behind this resistance remain unclear. Previous studies by the Glioma Longitudinal Analysis (GLASS) Consortium have indicated that therapy-induced selective pressures shape the genetic evolution of glioma in a stochastic manner. However, single cell studies have revealed that malignant glioma cells are highly plastic, and capable of changing their cell state in response to diverse challenges in their microenvironment. The tumor immune response has been implicated as a major driver of these malignant cell state transitions, and is known to be affected by the administration of therapy, but the extent to which tumor genetics, therapy, and the different components of the immune response interact to influence a glioma’s evolutionary trajectory are poorly understood. To further investigate these factors, we collected DNA and RNA sequencing data on pre- and post-treatment tumor pairs from over 150 glioma patients that have received chemotherapy, radiotherapy, and/or immune checkpoint blockade agents. By integrating mutation, copy number, and in silico deconvolution analyses of bulk transcriptome data across the three molecular subtypes of diffuse glioma, we show that longitudinal increases in chromosomal instability and gene fusions associate with decreased immune infiltrate and altered cell states at recurrence. We additionally find that specific molecular alterations and malignant cell states associate with unique inflammatory and immunosuppressive programs in tumor-associated macrophages and microglia. Lastly, we show that the abundance of T cells in the tumor microenvironment does not associate with changes in neoantigen depletion and the acquisition of antigen presentation machinery defects, suggesting minimal immunoediting activity over time. Collectively, our results indicate that the administration of therapy can alter the dynamics of tumor-immune interactions in glioma, resulting in new steady-states at recurrence that can be subsequently targeted.

Water reabsorption along the collecting duct is dependent on the function of aquaporin 2 (AQP2). Currently, information on microRNA (miRNA)-mediated, post-transcriptional regulation of AQP2, which may influence water reabsorption, is limited. In mice, ablation of the Dicer enzyme (crucial for miRNA maturation) in AQP2-expressing cells induces nephrogenic diabetes insipidus (NDI) with dysregulation of the miRNA profile. A major finding is the identification of miRNAs associated with NDI through mediating epigenetic control of AQP2. This study offers novel targets for AQP2 regulation and potential treatment for governing renal water reabsorption.Background MicroRNAs (miRNAs), formed by cleavage of pre-microRNA by the endoribonuclease Dicer, are critical modulators of cell function by post-transcriptionally regulating gene expression.Methods Selective ablation of Dicer in AQP2-expressing cells (DicerAQP2Cre+ mice) was used to investigate the role of miRNAs in the kidney collecting duct of mice.Results The mice had severe polyuria and nephrogenic diabetes insipidus, potentially due to greatly reduced AQP2 and AQP4 levels. Although epithelial sodium channel levels were decreased in cortex and increased in inner medulla, amiloride-sensitive sodium reabsorption was equivalent in DicerAQP2Cre+ mice and controls. Small-RNA sequencing and proteomic analysis revealed 31 and 178 significantly regulated miRNAs and proteins, respectively. Integrated bioinformatic analysis of the miRNAome and proteome suggested alterations in the epigenetic machinery and various transcription factors regulating AQP2 expression in DicerAQP2Cre+ mice. The expression profile and function of three miRNAs (miR-7688-5p, miR-8114, and miR-409-3p) whose predicted targets were involved in epigenetic control (Phf2, Kdm5c, and Kdm4a) or transcriptional regulation (GATA3, GATA2, and ELF3) of AQP2 were validated. Luciferase assays could not demonstrate direct interaction of AQP2 or the three potential transcription factors with miR-7688-5p, miR-8114, and miR-409textendash3p. However, transfection of respective miRNA mimics reduced AQP2 expression. Chromatin immunoprecipitation assays demonstrated decreased Phf2 and significantly increased Kdm5c interactions at the Aqp2 gene promoter in DicerAQP2Cre+ mice, resulting in decreased RNA Pol II association.Conclusions Novel evidence indicates miRNA-mediated epigenetic regulation of AQP2 expression.

Background Tumor heterogeneity underlies resistance and disease progression in glioblastoma (GBM), and tumors most commonly recur adjacent to the surgical resection margins in contrast non-enhancing (NE) regions. To date, no targeted therapies have meaningfully altered overall patient survival in the up-front setting. The aim of this study was to characterize intratumoral heterogeneity in recurrent GBM using bulk samples from primary resection and recurrent samples taken from contrast-enhancing (EN) and contrast NE regions. Methods Whole exome and RNA sequencing were performed on matched bulk primary and multiple recurrent EN and NE tumor samples from 16 GBM patients who received standard of care treatment alone or in combination with investigational clinical trial regimens. Results Private mutations emerge across multi-region sampling in recurrent tumors. Genomic clonal analysis revealed increased enrichment in gene alterations regulating the G2M checkpoint, Kras signaling, Wnt signaling, and DNA repair in recurrent disease. Subsequent functional studies identified augmented PI3K/AKT transcriptional and protein activity throughout progression, validated by phospho-protein levels. Moreover, a mesenchymal transcriptional signature was observed in recurrent EN regions, which differed from the proneural signature in recurrent NE regions. Conclusions Subclonal populations observed within bulk resected primary GBMs transcriptionally evolve across tumor recurrence (EN and NE regions) and exhibit aberrant gene expression of common signaling pathways that persist despite standard or targeted therapy. Our findings provide evidence that there are both adaptive and clonally mediated dependencies of GBM on key pathways, such as the PI3K/AKT axis, for survival across recurrences.

Single-cell RNA sequencing is the reference technique to characterize the heterogeneity of tumor microenvironment and can be efficiently used to discover cross-talk mechanisms between immune cells and cancer cells. We present a novel method, single cell Tumor-Host Interaction tool (scTHI), to identify significantly activated ligand-receptor interactions across clusters of cells from single-cell RNA sequencing data. We apply our approach to uncover the ligand-receptor interactions in glioma using six publicly available human glioma datasets encompassing 71 patients. We provide a comprehensive map of the signalling mechanisms between malignant cells and non-malignant cells in glioma uncovering potential novel therapeutic targets.

Pancreatic organogenesis is a multistep process that requires the cooperation of several signaling pathways. In this context, the role of pancreatic mesenchyme is important to define the epithelium development, nevertheless, the precise space&ndash, temporal signaling activation still needs to be clarified. This study reports a dissection of the pancreatic embryogenesis, highlighting the molecular network surrounding the epithelium&ndash, mesenchyme interaction. To investigate this crosstalk, pancreatic epithelium and surrounding mesenchyme, at embryonic day 10.5, were collected through laser capture microdissection (LCM) and characterized based on their global gene expression. We performed a bioinformatic analysis to hypothesize crosstalk interactions, validating the most promising genes and verifying the precise localization of their expression in the compartments, by RNA in situ hybridization (ISH). Our analyses pointed out also the c-Met gene, a very well-known factor involved in stimulating motility, morphogenesis, and organ regeneration. We also highlighted the potential crosstalk between Versican (Vcan) and Syndecan4 (Sdc4) since these genes are involved in pancreatic tissue repair, strengthening the concept that the same signaling pathways required during pancreatic embryogenesis are also involved in tissue repair. This finding leads to novel strategies for obtaining functional pancreatic stem cells for cell replacement therapies.

Diffuse glioma is an aggressive brain cancer that is characterized by a poor prognosis and a universal resistance to therapy. The evolutionary processes behind this resistance remain unclear. Previous studies by the Glioma Longitudinal Analysis (GLASS) Consortium have indicated that therapy-induced selective pressures shape the genetic evolution of glioma in a stochastic manner. However, single cell studies have revealed that malignant glioma cells are highly plastic and transition their cell state in response to diverse challenges, including changes in the immune response and the administration of standard-of-care therapy. Interactions between these factors remain poorly understood, making it difficult to predict how a patient's tumor will evolve from diagnosis to recurrence. To investigate these factors, we collected RNAseq data from 151 pre- and post-treatment tumor pairs, 101 of which also had matched whole exome or whole genome sequencing. Together, this dataset represents the largest collection of longitudinal multiomic glioma data yet assembled. We integrated this dataset with representative glioma single cell RNAseq data to implement digital cytometry approaches that quantified the microenvironmental composition of each tumor and reconstructed their tumor and immune cell state-specific gene expression profiles. In both IDHwt and IDHmut glioma, the tumor microenvironment was dominated by myeloid cells. The myeloid compartment of IDHwt tumors more closely resembled blood-derived macrophages, while myeloid cells in IDHmut tumors were more similar to microglia. While therapy did not alter the balance between macrophages and microglia in IDHwt tumors, myeloid cells in IDHmut tumors that increased grade following therapy had fewer microglia characteristics. Myeloid cells in mesenchymal glioma exhibited a distinct gene expression signature compared to those in non-mesenchymal tumors, and this signature was upregulated in the myeloid cells of tumors that acquired this phenotype following therapy. Receptor-ligand analyses revealed that this mesenchymal phenotype was associated with interactions between differentiated-like tumor cells and myeloid cells, suggesting a potential mechanism of mesenchymal transformation. Radiation therapy did not alter the composition or transcriptional activity of the glioma microenvironment but did associate with increased synaptic plasticity in stem cell-like and differentiated-like tumor cells. Collectively, our results suggest that tumor-myeloid cell interactions in glioma are dynamic and capable of shaping tumor evolution following the administration of therapy. Clinically targeting these interactions may allow for better control of malignant cell state shifts over time, creating new opportunities for disease management. Citation Format: Frederick S. Varn, Kevin C. Johnson, Floris P. Barthel, Hoon Kim, Taylor Wade, Disha Lodha, Shoaib Ajaib, Nazia Ahmed, Luciano Garofano, Fulvio D'Angelo, Lucy Stead, Houtan Noushmehr, Antonio Iavarone, Roel Verhaak, The GLASS Consortium. Tumor-myeloid cell interactions are dynamic and influence the evolutionary trajectory of adult diffuse glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2169.