Protocol for mapping the metabolome and lipidome of medulloblastoma cells using liquid chromatography-mass spectrometry.

Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics and lipidomics have recently been used to show that MYC-amplified group 3 medulloblastoma tumors are driven by metabolic reprogramming. Here, we present a protocol to extract metabolites and lipids from human medulloblastoma brain tumor-initiating cells and normal neural stem cells. We describe untargeted LC-MS methods that can be used to achieve extensive coverage of the polar metabolome and lipidome. Finally, we detail strategies for metabolite identification and data analysis. For complete details on the use and execution of this protocol, please refer to Gwynne et al.1.

Dynamic profiling of medulloblastoma surfaceome.

Medulloblastoma (MB) is the most common type of malignant pediatric brain cancer. The current standard of care (SOC) involves maximal safe resection and chemoradiotherapy in individuals older than 3 years, often leading to devastating neurocognitive and developmental deficits. Out of the four distinct molecular subgroups, Group 3 and 4 have the poorest patient outcomes due to the aggressive nature of the tumor and propensity to metastasize and recur post therapy. The toxicity of the SOC and lack of response in specific subtypes to the SOC underscores the urgent need for developing and translating novel treatment options including immunotherapies. To identify differentially enriched surface proteins that could be evaluated for potential future immunotherapeutic interventions, we leveraged N-glycocapture surfaceome profiling on Group 3 MB cells from primary tumor, through therapy, to recurrence using our established therapy-adapted patient derived xenograft model. Integrin 𝛼5 (ITGA5) was one of the most differentially enriched targets found at recurrence when compared to engraftment and untreated timepoints. In addition to being enriched at recurrence, shRNA-mediated knockdown and small molecule inhibition of ITGA5 have resulted in marked decrease in proliferation and self-renewal in vitro and demonstrated a survival advantage in vivo. Together, our data highlights the value of dynamic profiling of cells as they evolve through therapy and the identification of ITGA5 as a promising therapeutic target for recurrent Group 3 MB.

Characterization of an RNA binding protein interactome reveals a context-specific post-transcriptional landscape of MYC-amplified medulloblastoma.

Pediatric medulloblastoma (MB) is the most common solid malignant brain neoplasm, with Group 3 (G3) MB representing the most aggressive subgroup. MYC amplification is an independent poor prognostic factor in G3 MB, however, therapeutic targeting of the MYC pathway remains limited and alternative therapies for G3 MB are urgently needed. Here we show that the RNA-binding protein, Musashi-1 (MSI1) is an essential mediator of G3 MB in both MYC-overexpressing mouse models and patient-derived xenografts. MSI1 inhibition abrogates tumor initiation and significantly prolongs survival in both models. We identify binding targets of MSI1 in normal neural and G3 MB stem cells and then cross referenced these data with unbiased large-scale screens at the transcriptomic, translatomic and proteomic levels to systematically dissect its functional role. Comparative integrative multi-omic analyses of these large datasets reveal cancer-selective MSI1-bound targets sharing multiple MYC associated pathways, providing a valuable resource for context-specific therapeutic targeting of G3 MB.

Cancer-selective metabolic vulnerabilities in MYC-amplified medulloblastoma.

MYC-driven medulloblastoma (MB) is an aggressive pediatric brain tumor characterized by therapy resistance and disease recurrence. Here, we integrated data from unbiased genetic screening and metabolomic profiling to identify multiple cancer-selective metabolic vulnerabilities in MYC-driven MB tumor cells, which are amenable to therapeutic targeting. Among these targets, dihydroorotate dehydrogenase (DHODH), an enzyme that catalyzes de novo pyrimidine biosynthesis, emerged as a favorable candidate for therapeutic targeting. Mechanistically, DHODH inhibition acts on target, leading to uridine metabolite scarcity and hyperlipidemia, accompanied by reduced protein O-GlcNAcylation and c-Myc degradation. Pyrimidine starvation evokes a metabolic stress response that leads to cell-cycle arrest and apoptosis. We further show that an orally available small-molecule DHODH inhibitor demonstrates potent mono-therapeutic efficacy against patient-derived MB xenografts in vivo. The reprogramming of pyrimidine metabolism in MYC-driven medulloblastoma represents an unappreciated therapeutic strategy and a potential new class of treatments with stronger cancer selectivity and fewer neurotoxic sequelae.

The proteomic landscape of glioblastoma recurrence reveals novel and targetable immunoregulatory drivers.

Glioblastoma (GBM) is characterized by extensive cellular and genetic heterogeneity. Its initial presentation as primary disease (pGBM) has been subject to exhaustive molecular and cellular profiling. By contrast, our understanding of how GBM evolves to evade the selective pressure of therapy is starkly limited. The proteomic landscape of recurrent GBM (rGBM), which is refractory to most treatments used for pGBM, are poorly known. We, therefore, quantified the transcriptome and proteome of 134 patient-derived pGBM and rGBM samples, including 40 matched pGBM-rGBM pairs. GBM subtypes transition from pGBM to rGBM towards a preferentially mesenchymal state at recurrence, consistent with the increasingly invasive nature of rGBM. We identified immune regulatory/suppressive genes as important drivers of rGBM and in particular 2-5-oligoadenylate synthase 2 (OAS2) as an essential gene in recurrent disease. Our data identify a new class of therapeutic targets that emerge from the adaptive response of pGBM to therapy, emerging specifically in recurrent disease and may provide new therapeutic opportunities absent at pGBM diagnosis.

Discovery of HDAC6-Selective Inhibitor NN-390 with in Vitro Efficacy in Group 3 Medulloblastoma.

Histone deacetylase 6 (HDAC6) has been targeted in clinical studies for anticancer effects due to its role in oncogenic transformation and metastasis. Through a second-generation structure-activity relationship (SAR) study, the design, and biological evaluation of the selective HDAC6 inhibitor NN-390 is reported. With nanomolar HDAC6 potency, >200-550-fold selectivity for HDAC6 in analogous HDAC isoform functional assays, potent intracellular target engagement, and robust cellular efficacy in cancer cell lines, NN-390 is the first HDAC6-selective inhibitor to show therapeutic potential in metastatic Group 3 medulloblastoma (MB), an aggressive pediatric brain tumor often associated with leptomeningeal metastases and therapy resistance. MB stem cells contribute to these patients' poor clinical outcomes. NN-390 selectively targets this cell population with a 44.3-fold therapeutic margin between patient-derived Group 3 MB cells in comparison to healthy neural stem cells. NN-390 demonstrated a 45-fold increased potency over HDAC6-selective clinical candidate citarinostat. In summary, HDAC6-selective molecules demonstrated in vitro therapeutic potential against Group 3 MB.

The Road to CAR T-Cell Therapies for Pediatric CNS Tumors: Obstacles and New Avenues.

Pediatric central nervous system (CNS) tumors are the most common solid tumors diagnosed in children and are the leading cause of pediatric cancer-related death. Those who do survive are faced with the long-term adverse effects of the current standard of care treatments of chemotherapy, radiation, and surgery. There is a pressing need for novel therapeutic strategies to treat pediatric CNS tumors more effectively while reducing toxicity - one of these novel modalities is chimeric antigen receptor (CAR) T-cell therapy. Currently approved for use in several hematological malignancies, there are promising pre-clinical and early clinical data that suggest CAR-T cells could transform the treatment of pediatric CNS tumors. There are, however, several challenges that must be overcome to develop safe and effective CAR T-cell therapies for CNS tumors. Herein, we detail these challenges, focusing on those unique to pediatric patients including antigen selection, tumor immunogenicity and toxicity. We also discuss our perspective on future avenues for CAR T-cell therapies and potential combinatorial treatment approaches.

CD70 as an actionable immunotherapeutic target in recurrent glioblastoma and its microenvironment.

PURPOSE: Glioblastoma (GBM) patients suffer from a dismal prognosis, with standard of care therapy inevitably leading to therapy-resistant recurrent tumors. The presence of cancer stem cells (CSCs) drives the extensive heterogeneity seen in GBM, prompting the need for novel therapies specifically targeting this subset of tumor-driving cells. Here, we identify CD70 as a potential therapeutic target for recurrent GBM CSCs. EXPERIMENTAL DESIGN: In the current study, we identified the relevance and functional influence of CD70 on primary and recurrent GBM cells, and further define its function using established stem cell assays. We use CD70 knockdown studies, subsequent RNAseq pathway analysis, and in vivo xenotransplantation to validate CD70's role in GBM. Next, we developed and tested an anti-CD70 chimeric antigen receptor (CAR)-T therapy, which we validated in vitro and in vivo using our established preclinical model of human GBM. Lastly, we explored the importance of CD70 in the tumor immune microenvironment (TIME) by assessing the presence of its receptor, CD27, in immune infiltrates derived from freshly resected GBM tumor samples. RESULTS: CD70 expression is elevated in recurrent GBM and CD70 knockdown reduces tumorigenicity in vitro and in vivo. CD70 CAR-T therapy significantly improves prognosis in vivo. We also found CD27 to be present on the cell surface of multiple relevant GBM TIME cell populations, notably putative M1 macrophages and CD4 T cells. CONCLUSION: CD70 plays a key role in recurrent GBM cell aggressiveness and maintenance. Immunotherapeutic targeting of CD70 significantly improves survival in animal models and the CD70/CD27 axis may be a viable polytherapeutic avenue to co-target both GBM and its TIME.

Childhood Medulloblastoma: An Overview.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor, representing 60% of childhood intracranial embryonal tumors. Despite multimodal advances in therapies over the last 20 years that have yielded a 5-year survival rate of 75%, high-risk patients (younger than 3 years, subtotal resection, metastatic lesions at diagnosis) still experience a 5-year overall survival of less than 70%. In this introductory chapter on pediatric MB, we describe the initial discrimination of MB based on histopathological examination and the more recent progress made in global gene expression profiling methods that have allowed scientists to more accurately subclassify and prognosticate on MB based on molecular characteristics. The identification of subtype-specific molecular drivers and pathways presents novel therapeutic targets that could lead to MB subtype-specific treatment modalities. Additionally, we detail how the cancer stem cell (CSC) hypothesis provides an explanation for tumor recurrence, and the potential for CSC-targeted therapies to address treatment-refractory MB. These personalized therapies can potentially increase MB survivorship and negate some of the long-term neurotoxicity associated with the current standard of care for MB patients.

Temporal profiling of therapy resistance in human medulloblastoma identifies novel targetable drivers of recurrence.

Medulloblastoma (MB) remains a leading cause of cancer-related mortality among children. The paucity of MB samples collected at relapse has hindered the functional understanding of molecular mechanisms driving therapy failure. New models capable of accurately recapitulating tumor progression in response to conventional therapeutic interventions are urgently needed. In this study, we developed a therapy-adapted PDX MB model that has a distinct advantage of generating human MB recurrence. The comparative gene expression analysis of MB cells collected throughout therapy led to identification of genes specifically up-regulated after therapy, including one previously undescribed in the setting of brain tumors, bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4). Subsequent functional validation resulted in a markedly diminished in vitro proliferation, self-renewal, and longevity of MB cells, translating into extended survival and reduced tumor burden in vivo. Targeting endothelial nitric oxide synthase, a downstream substrate of BPIFB4, impeded growth of several patient-derived MB lines at low nanomolar concentrations.

The Role of Serotonin in Breast Cancer Stem Cells.

Breast tumors were the first tumors of epithelial origin shown to follow the cancer stem cell model. The model proposes that cancer stem cells are uniquely endowed with tumorigenic capacity and that their aberrant differentiation yields non-tumorigenic progeny, which constitute the bulk of the tumor cell population. Breast cancer stem cells resist therapies and seed metastases; thus, they account for breast cancer recurrence. Hence, targeting these cells is essential to achieve durable breast cancer remissions. We identified compounds including selective antagonists of multiple serotonergic system pathway components required for serotonin biosynthesis, transport, activity via multiple 5-HT receptors (5-HTRs), and catabolism that reduce the viability of breast cancer stem cells of both mouse and human origin using multiple orthologous assays. The molecular targets of the selective antagonists are expressed in breast tumors and breast cancer cell lines, which also produce serotonin, implying that it plays a required functional role in these cells. The selective antagonists act synergistically with chemotherapy to shrink mouse mammary tumors and human breast tumor xenografts primarily by inducing programmed tumor cell death. We hypothesize those serotonergic proteins of diverse activity function by common signaling pathways to maintain cancer stem cell viability. Here, we summarize our recent findings and the relevant literature regarding the role of serotonin in breast cancer.

Antagonists of the serotonin receptor 5A target human breast tumor initiating cells.

BACKGROUND: Breast tumor initiating cells (BTIC) are stem-like cells that initiate and sustain tumor growth, and drive disease recurrence. Identifying therapies targeting BTIC has been hindered due primarily to their scarcity in tumors. We previously reported that BTIC frequency ranges between 15% and 50% in multiple mammary tumors of 3 different transgenic mouse models of breast cancer and that this frequency is maintained in tumor cell populations cultured in serum-free, chemically defined media as non-adherent tumorspheres. The latter enabled high-throughput screening of small molecules for their capacity to affect BTIC survival. Antagonists of several serotonin receptors (5-HTRs) were among the hit compounds. The most potent compound we identified, SB-699551, selectively binds to 5-HT5A, a Gαi/o protein coupled receptor (GPCR). METHODS: We evaluated the activity of structurally unrelated selective 5-HT5A antagonists using multiple orthogonal assays of BTIC frequency. Thereafter we used a phosphoproteomic approach to uncover the mechanism of action of SB-699551. To validate the molecular target of the antagonists, we used the CRISPR-Cas9 gene editing technology to conditionally knockout HTR5A in a breast tumor cell line. RESULTS: We found that selective antagonists of 5-HT5A reduced the frequency of tumorsphere initiating cells residing in breast tumor cell lines and those of patient-derived xenografts (PDXs) that we established. The most potent compound among those tested, SB-699551, reduced the frequency of BTIC in ex vivo assays and acted in concert with chemotherapy to shrink human breast tumor xenografts in vivo. Our phosphoproteomic experiments established that exposure of breast tumor cells to SB-699551 elicited signaling changes in the canonical Gαi/o-coupled pathway and the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) axis. Moreover, conditional mutation of the HTR5A gene resulted in the loss of tumorsphere initiating cells and BTIC thus mimicking the effect of SB-699551. CONCLUSIONS: Our data provide genetic, pharmacological and phosphoproteomic evidence consistent with the on-target activity of SB-699551. The use of such agents in combination with cytotoxic chemotherapy provides a novel therapeutic approach to treat breast cancer.

Monoamine oxidase-A activity is required for clonal tumorsphere formation by human breast tumor cells.

BACKGROUND: Breast tumor growth and recurrence are driven by an infrequent population of breast tumor-initiating cells (BTIC). We and others have reported that the frequency of BTIC is orders of magnitude higher when breast tumor cells are propagated in vitro as clonal spheres, termed tumorspheres, by comparison to adherent cells. We exploited the latter to screen > 35,000 small molecules to identify agents capable of targeting BTIC. We unexpectedly discovered that selective antagonists of serotonin signaling were among the hit compounds. To better understand the relationship between serotonin and BTIC we expanded our analysis to include monoamine oxidase-A (MAO-A), an enzyme that metabolizes serotonin. METHODS: We used the Nanostring technology and Western blotting to determine whether MAO-A is expressed in human breast tumor cell lines cultured as tumorspheres by comparison to those grown as adherent cells. We then determined whether MAO-A activity is required for tumorsphere formation, a surrogate in vitro assay for BTIC, by assessing whether selective MAO-A inhibitors affect the frequency of tumorsphere-forming cells. To learn whether MAO-A expression in breast tumor cells is associated with other reported properties of BTIC such as anticancer drug resistance or breast tumor recurrence, we performed differential gene expression analyses using publicly available transcriptomic datasets. RESULTS: Tumorspheres derived from human breast tumor cell lines representative of every breast cancer clinical subtype displayed increased expression of MAO-A transcripts and protein by comparison to adherent cells. Surprisingly, inhibition of MAO-A activity with selective inhibitors reduced the frequency of tumorsphere-forming cells. We also found that increased MAO-A expression is a common feature of human breast tumor cell lines that have acquired anticancer drug resistance and is associated with poor recurrence-free survival (RFS) in patients that experienced high-grade, ER-negative (ER-) breast tumors. CONCLUSIONS: Our data suggests that MAO-A activity is required for tumorsphere formation and that its expression in breast tumor cells is associated with BTIC-related properties. The discovery that a selective MAO-A inhibitor targets tumorsphere-forming cells with potencies in the nanomolar range provides the first evidence of this agent's anticancer property. These data warrant further investigation of the link between MAO-A and BTIC.

The Antiarrhythmic Drug, Dronedarone, Demonstrates Cytotoxic Effects in Breast Cancer Independent of Thyroid Hormone Receptor Alpha 1 (THRα1) Antagonism.

Previous research has suggested that thyroid hormone receptor alpha 1 (THRα1), a hormone responsive splice variant, may play a role in breast cancer progression. Whether THRα1 can be exploited for anti-cancer therapy is unknown. The antiproliferative and antitumor effects of dronedarone, an FDA-approved anti-arrhythmic drug which has been shown to antagonize THRα1, was evaluated in breast cancer cell lines in vitro and in vivo. The THRα1 splice variant and the entire receptor, THRα, were also independently targeted using siRNA to determine the effect of target knockdown in vitro. In our study, dronedarone demonstrates cytotoxic effects in vitro and in vivo in breast cancer cell lines at doses and concentrations that may be clinically relevant. However, knockdown of either THRα1 or THRα did not cause substantial anti-proliferative or cytotoxic effects in vitro, nor did it alter the sensitivity to dronedarone. Thus, we conclude that dronedarone's cytotoxic effect in breast cancer cell lines are independent of THRα or THRα1 antagonism. Further, the depletion of THRα or THRα1 does not affect cell viability or proliferation. Characterizing the mechanism of dronedarone's anti-tumor action may facilitate drug repurposing or the development of new anti-cancer agents.

Serotonergic system antagonists target breast tumor initiating cells and synergize with chemotherapy to shrink human breast tumor xenografts.

Breast tumors comprise an infrequent tumor cell population, termed breast tumor initiating cells (BTIC), which sustain tumor growth, seed metastases and resist cytotoxic therapies. Hence therapies are needed to target BTIC to provide more durable breast cancer remissions than are currently achieved. We previously reported that serotonergic system antagonists abrogated the activity of mouse BTIC resident in the mammary tumors of a HER2-overexpressing model of breast cancer. Here we report that antagonists of serotonin (5-hydroxytryptamine; 5-HT) biosynthesis and activity, including US Federal Food and Drug Administration (FDA)-approved antidepressants, targeted BTIC resident in numerous breast tumor cell lines regardless of their clinical or molecular subtype. Notably, inhibitors of tryptophan hydroxylase 1 (TPH1), required for 5-HT biosynthesis in select non-neuronal cells, the serotonin reuptake transporter (SERT) and several 5-HT receptors compromised BTIC activity as assessed by functional sphere-forming assays. Consistent with these findings, human breast tumor cells express TPH1, 5-HT and SERT independent of their molecular or clinical subtype. Exposure of breast tumor cells ex vivo to sertraline (Zoloft), a selective serotonin reuptake inhibitor (SSRI), reduced BTIC frequency as determined by transplanting drug-treated tumor cells into immune-compromised mice. Moreover, another SSRI (vilazodone; Viibryd) synergized with chemotherapy to shrink breast tumor xenografts in immune-compromised mice by inhibiting tumor cell proliferation and inducing their apoptosis. Collectively our data suggest that antidepressants in combination with cytotoxic anticancer therapies may be an appropriate treatment regimen for testing in clinical trials.

Serotonin transporter antagonists target tumor-initiating cells in a transgenic mouse model of breast cancer.

Accumulating data suggests that the initiation and progression of human breast tumors is fueled by a rare subpopulation of tumor cells, termed breast tumor-initiating cells (BTIC), which resist radiotherapy and chemotherapy. Consequently, therapies that abrogate BTIC activity are needed to achieve durable cures for breast cancer patients. To identify such therapies we used a sensitive assay to complete a high-throughput screen of small molecules, including approved drugs, with BTIC-rich mouse mammary tumor cell populations. We found that inhibitors of the serotonin reuptake transporter (SERT) and serotonin receptors, which include approved drugs used to treat mood disorders, were potent inhibitors of mouse BTIC activity as determined by functional sphere-forming assays and the initiation of tumor formation by transplant of drug-exposed tumor cells into syngeneic mice. Moreover, sertraline (Zoloft), a selective serotonin reuptake inhibitor (SSRI), synergized with docetaxel (Taxotere) to shrink mouse breast tumors in vivo. Hence drugs targeting the serotonergic system might be repurposed to treat breast cancer patients to afford more durable breast cancer remissions.

The C-terminus of Bienertia sinuspersici Toc159 contains essential elements for its targeting and anchorage to the chloroplast outer membrane.

Most nucleus-encoded chloroplast proteins rely on an N-terminal transit peptide (TP) as a post-translational sorting signal for directing them to the organelle. Although Toc159 is known to be a receptor for specific preprotein TPs at the chloroplast surface, the mechanism for its own targeting and integration into the chloroplast outer membrane is not completely understood. In a previous study, we identified a novel TP-like sorting signal at the C-terminus (CT) of a Toc159 homolog from the single-cell C4 species, Bienertia sinuspersici. In the current study, we have extended our understanding of the sorting signal using transient expression of fluorescently-tagged fusion proteins of variable-length, and with truncated and swapped versions of the CT. As was shown in the earlier study, the 56 residues of the CT contain crucial sorting information for reversible interaction of the receptor with the chloroplast envelope. Extension of this region to 100 residues in the current study stabilized the interaction via membrane integration, as demonstrated by more prominent plastid-associated signals and resistance of the fusion protein to alkaline extraction. Despite a high degree of sequence similarity, the plastid localization signals of the equivalent CT regions of Arabidopsis thaliana Toc159 homologs were not as strong as that of the B. sinuspersici counterparts. Together with computational and circular dichroism analyses of the CT domain structures, our data provide insights into the critical elements of the CT for the efficient targeting and anchorage of Toc159 receptors to the dimorphic chloroplasts in the single-cell C4 species.