The neo-open reading frame peptides that comprise the tumor framome are a rich source of neoantigens for cancer immunotherapy.

Identification of immunogenic cancer neoantigens as targets for therapy is challenging. Here, we integrate cancer whole genome and long-read transcript sequencing to identify the collection of novel open reading frame peptides (NOPs) expressed in tumors, termed the framome. NOPs represent tumor-specific peptides that are different from wild-type proteins and may be strongly immunogenic. We describe an uncharacterized class of hidden NOPs, which derive from structural genomic variants involving an upstream protein coding gene driving expression and translation of non-coding regions of the genome downstream of a rearrangement breakpoint. NOPs represent a vast amount of possible neoantigens particularly in tumors with many (complex) structural genomic variants and a low number of missense mutations. We show that NOPs are immunogenic and epitopes derived from NOPs can bind to MHC class I molecules. Finally, we provide evidence for the presence of memory T-cells specific for hidden NOPs in lung cancer patient peripheral blood.

Use, access, and initial outcomes of off-label ivosidenib in patients with IDH1 mutant glioma.

Background: Isocitrate dehydrogenase (IDH) is commonly mutated (mIDH) in gliomas, and this mutant enzyme produces the oncometabolite 2-hydroxyglutarate (2HG). 2HG promotes gliomagenesis and is implicated in epileptogenesis. Ivosidenib (IVO), a small molecule oral mIDH1 inhibitor, is FDA-approved for mIDH1 newly diagnosed and relapsed/refractory acute myeloid leukemia. Moreover, IVO has efficacy in clinical trials for recurrent mIDH1 gliomas. Given the lack of targeted treatments for gliomas, we initiated off-label IVO for mIDH glioma patients in October 2020. Methods: Retrospectively, we sought to assess early outcomes in our patients and describe their experience on IVO from October 2020 through February 2022. Our objective was to report on the following variables of off-label use of IVO: radiographic response, seizure control, tolerability, and access to the medication. All patients initially received single-agent IVO dosed at 500 mg orally once daily. Results: The cohort age range was 21-74 years. Tumor types included astrocytoma (n = 14) and oligodendroglioma (n = 16), with most being grade 2 (n = 21). The best radiographic response in nonenhancing disease (n = 22) was 12 stable diseases, 5 minor responses, 3 partial responses, and 2 progressive diseases. Seizure frequency was stable to improved for most patients (70%, n = 21). IVO was well-tolerated, with the most common toxicities being diarrhea, elevated creatine kinase, and QTc interval prolongation. Most patients (66.7%, n = 20) received drugs via the patient assistance program, with insurance initially covering a third of patients and with ongoing use, later covering 60%. Conclusions: Targeted therapies like IVO are options for mIDH glioma patients and can provide positive oncologic and neurological outcomes.

Gene expression analysis suggests immunosuppressive roles of endolysosomes in glioblastoma.

Targeting endolysosomes is a strategy extensively pursued for treating cancers, including glioblastomas (GBMs), on the basis that the intact function of these subcellular organelles is key to tumor cell autophagy and survival. Through gene expression analyses and cell type abundance estimation in GBMs, we showed that genes associated with the endolysosomal machinery are more prominently featured in non-tumor cells in GBMs than in tumor cells, and that tumor-associated macrophages represent the primary immune cell type that contributes to this trend. Further analyses found an enrichment of endolysosomal pathway genes in immunosuppressive (pro-tumorigenic) macrophages, such as M2-like macrophages or those associated with worse prognosis in glioma patients, but not in those linked to inflammation (anti-tumorigenic). Specifically, genes critical to the hydrolysis function of endolysosomes, including progranulin and cathepsins, were among the most positively correlated with immunosuppressive macrophages, and elevated expression of these genes is associated with worse patient survival in GBMs. Together, these results implicate the hydrolysis function of endolysosomes in shaping the immunosuppressive microenvironment of GBM. We propose that targeting endolysosomes, in addition to its detrimental effects on tumor cells, can be leveraged for modulating immunosuppression to render GBMs more amenable to immunotherapies.

PI3K/mTOR is a therapeutically targetable genetic dependency in diffuse intrinsic pontine glioma.

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma; DIPG), are uniformly fatal brain tumors that lack effective treatment. Analysis of CRISPR/Cas9 loss-of-function gene deletion screens identified PIK3CA and MTOR as targetable molecular dependencies across patient derived models of DIPG, highlighting the therapeutic potential of the blood-brain barrier-penetrant PI3K/Akt/mTOR inhibitor, paxalisib. At the human-equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic glucose feedback and increased insulin levels commensurate with patients using PI3K inhibitors. To exploit genetic dependence and overcome resistance while maintaining compliance and therapeutic benefit, we combined paxalisib with the antihyperglycemic drug metformin. Metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending survival of orthotopic models. DIPG models treated with paxalisib increased calcium-activated PKC signaling. The brain penetrant PKC inhibitor enzastaurin, in combination with paxalisib, synergistically extended the survival of multiple orthotopic patient-derived and immunocompetent syngeneic allograft models; benefits potentiated in combination with metformin and standard-of-care radiotherapy. Therapeutic adaptation was assessed using spatial transcriptomics and ATAC-Seq, identifying changes in myelination and tumor immune microenvironment crosstalk. Collectively, this study has identified what we believe to be a clinically relevant DIPG therapeutic combinational strategy.

Content analysis of IQOS direct mail and email marketing in the US.

Objective: Novel nicotine and tobacco products, including heated tobacco products (HTPs) like IQOS, are growing in global popularity. IQOS was the first HTP authorized for sale by the US Food and Drug Administration, entering the US market in 2019 and being removed in 2021 due to patent-related legal challenges, with the possibility of returning in 2024. Direct marketing is one method tobacco companies use to reach consumers of these products. The purpose of this study was to investigate the content of US IQOS direct mail and email marketing. Methods: Direct marketing items were collected between September 2019 and July 2021 by seven team members in the first US IQOS test market, Atlanta, Georgia. Results: Overall, 101 marketing items were collected, 59 of which were unique. Among the unique items that showed images of persons ("models"), 70 % showed models appearing to be from racial/ethnic minoritized groups, 86.8 % showed at least one female-presenting model, and 37.5 % showed models appearing to be young adults (18-29 years). Items often had an embedded link/URL (91.5 %) and mentioned topics such as online services (54.2 %; for example, online ordering and tutorials), user experience (49.2 %), social media (44.1 %), and purchasing locations (37.3 %). When examined for their main purpose, items focused on subjects such as store experience (37.7 %), product introduction (18.6 %), and product use (15.3 %). Conclusions: Our study highlights the importance of better understanding how novel tobacco products are marketed, which can inform policymakers' regulatory efforts and product authorization decisions.

Mechanisms of telomere maintenance and associated therapeutic vulnerabilities in malignant gliomas.

A majority of cancers (~85%) activate the enzyme telomerase to maintain telomere length over multiple rounds of cellular division. Telomerase-negative cancers activate a distinct, telomerase-independent mechanism of telomere maintenance termed Alternative lengthening of telomeres (ALT). ALT uses homologous recombination to maintain telomere length and exhibits features of break-induced DNA replication. In malignant gliomas, the activation of either telomerase or ALT is nearly ubiquitous in pediatric and adult tumors, and the frequency with which these distinct telomere maintenance mechanisms is activated varies according to genetically-defined glioma subtypes. In this review, we summarize the current state of the field of telomere maintenance mechanisms (TMMs) and their relevance to glioma biology and therapy. We review the genetic alterations and molecular mechanisms leading to telomerase activation or ALT induction in pediatric and adult gliomas. With this background, we review emerging evidence on strategies for targeting TMMs for glioma therapy. Finally, we comment on critical gaps and issues for moving the field forward to translate our improved understanding of glioma telomere maintenance into better therapeutic strategies for patients.

Elucidating cellular response to treatment with viral immunotherapies in pediatric high-grade glioma and medulloblastoma.

HSV G207, a double-stranded, DNA virus, and the polio:rhinovirus chimera, PVSRIPO, a single positive-strand RNA virus, are viral immunotherapies being used to treat pediatric malignant brain tumors in clinical trials. The purpose of this work is to elucidate general response patterns and putative biomarkers of response. Multiple pediatric high-grade glioma and medulloblastoma cell lines were treated with various multiplicities of infection of G207 or PVSRIPO. There was a significant inverse correlation between expression of one HSV cellular receptor, CD111, and the lethal dose of 50% of cells (LD50) of cells treated with G207 (r = -0.985, P<0.001) but no correlation between PVSRIPO cellular receptor expression (CD155) and LD50. RNA sequencing of control cells and cells treated for 8 and 24 h revealed that there were few shared differentially expressed (DE) genes between cells treated with PVSRIPO and G207: GCLM, LANCL2, and RBM3 were enriched whilst ADAMTS1 and VEGFA were depleted. Likewise, there were few shared DE genes enriched between medulloblastoma and high-grade glioma cell lines treated with G207: GPSM2, CHECK2, SEPTIN2, EIF4G2, GCLM, GDAP1, LANCL2, and PWP1.  Treatment with G207 and PVSRIPO appear to cause disparate gene enrichment and depletion suggesting disparate molecular mechanisms in malignant pediatric brain tumors.

Interplay between ATRX and IDH1 mutations governs innate immune responses in diffuse gliomas.

Stimulating the innate immune system has been explored as a therapeutic option for the treatment of gliomas. Inactivating mutations in ATRX, defining molecular alterations in IDH-mutant astrocytomas, have been implicated in dysfunctional immune signaling. However, little is known about the interplay between ATRX loss and IDH mutation on innate immunity. To explore this, we generated ATRX-deficient glioma models in the presence and absence of the IDH1R132H mutation. ATRX-deficient glioma cells are sensitive to dsRNA-based innate immune agonism and exhibit impaired lethality and increased T-cell infiltration in vivo. However, the presence of IDH1R132H dampens baseline expression of key innate immune genes and cytokines in a manner restored by genetic and pharmacological IDH1R132H inhibition. IDH1R132H co-expression does not interfere with the ATRX deficiency-mediated sensitivity to dsRNA. Thus, ATRX loss primes cells for recognition of dsRNA, while IDH1R132H reversibly masks this priming. This work reveals innate immunity as a therapeutic vulnerability of astrocytomas.

Understanding and therapeutically exploiting cGAS/STING signaling in glioblastoma.

Since the discovery that cGAS/STING recognizes endogenous DNA released from dying cancer cells and induces type I interferon and antitumor T cell responses, efforts to understand and therapeutically target the STING pathway in cancer have ensued. Relative to other cancer types, the glioma immune microenvironment harbors few infiltrating T cells, but abundant tumor-associated myeloid cells, possibly explaining disappointing responses to immune checkpoint blockade therapies in cohorts of patients with glioblastoma. Notably, unlike most extracranial tumors, STING expression is absent in the malignant compartment of gliomas, likely due to methylation of the STING promoter. Nonetheless, several preclinical studies suggest that inducing cGAS/STING signaling in the glioma immune microenvironment could be therapeutically beneficial, and cGAS/STING signaling has been shown to mediate inflammatory and antitumor effects of other modalities either in use or being developed for glioblastoma therapy, including radiation, tumor-treating fields, and oncolytic virotherapy. In this Review, we discuss cGAS/STING signaling in gliomas, its implications for glioma immunobiology, compartment-specific roles for STING signaling in influencing immune surveillance, and efforts to target STING signaling - either directly or indirectly - for antiglioma therapy.

The safety and accuracy of intratumoral catheter placement to infuse viral immunotherapies in children with malignant brain tumors: a multi-institutional study.

OBJECTIVE: Relatively little is known about the safety and accuracy of catheter placement for oncolytic viral therapy in children with malignant brain tumors. Accordingly, this study combines data from two phase I clinical trials that employed viral immunotherapy across two institutions to describe the adverse event profile, safety, and accuracy associated with the stereotactic placement and subsequent removal of intratumoral catheters. METHODS: Children with progressive/recurrent supratentorial malignant tumors were enrolled in two clinical trials (NCT03043391 and NCT02457845) and treated with either the recombinant polio:rhinovirus (lerapolturev) or the genetically modified oncolytic herpesvirus (G207). Age, sex, race, tumor diagnosis, and tumor location were analyzed. Events related to the catheter placement or removal were categorized. A catheter that was either pulled back or could not be used was defined as "misplaced." Neuronavigation software was used to analyze the accuracy of catheter placement for NCT03043391. Descriptive statistics were performed. RESULTS: Nineteen patients were treated across the two completed trials with a total of 49 catheters. The mean ± SD (range) age was 14.1 ± 3.6 (7-19) years. All tumors were grade 3 or 4 gliomas. Nonlobar catheter tip placement included the corpus callosum, thalamus, insula, and cingulate gyrus. Six of 19 patients (31.6%) had minor hemorrhage noted on CT; however, no patients were symptomatic and/or required intervention related to these findings. One of 19 patients had a delayed CSF leak after catheter removal that required oversewing of the surgical site. No patients developed infection or a neurological deficit. In 7 patients with accuracy data, the mean ± SD distance of the planned trajectory (PT) to the catheter tip was 1.57 ± 1.6 mm, the mean angle of the PT to the catheter was 2.43° ± 2.1°, and the greatest distance of PT to the catheter in the parallel plane was 1.54 ± 1.5 mm. Three of 49 (6.1%) catheters were considered misplaced. CONCLUSIONS: Although instances of minor hemorrhage were encountered, they were clinically asymptomatic. One of 49 catheters required intervention for a CSF leak. Congruent with previous studies in the literature, the stereotactic placement of catheters in these pediatric tumor patients was accurate with approximately 95% of catheters having been adequately placed.

Influence of Half-life and Smoking/Nonsmoking Ratio on Biomarker Consistency between Waves 1 and 2 of the Population Assessment of Tobacco and Health Study.

BACKGROUND: Biomarkers of exposure are tools for understanding the impact of tobacco use on health outcomes if confounders like demographics, use behavior, biological half-life, and other sources of exposure are accounted for in the analysis. METHODS: We performed multiple regression analysis of longitudinal measures of urinary biomarkers of alkaloids, tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons, volatile organic compounds (VOC), and metals to examine the sample-to-sample consistency in Waves 1 and 2 of the Population Assessment of Tobacco and Health (PATH) Study including demographic characteristics and use behavior variables of persons who smoked exclusively. Regression coefficients, within- and between-person variance, and intra-class correlation coefficients (ICC) were compared with biomarker smoking/nonsmoking population mean ratios and biological half-lives. RESULTS: Most biomarkers were similarly associated with sex, age, race/ethnicity, and product use behavior. The biomarkers with larger smoking/nonsmoking population mean ratios had greater regression coefficients related to recency of exposure. For VOC and alkaloid metabolites, longer biological half-life was associated with lower within-person variance. For each chemical class studied, there were biomarkers that demonstrated good ICCs. CONCLUSIONS: For most of the biomarkers of exposure reported in the PATH Study, for people who smoke cigarettes exclusively, associations are similar between urinary biomarkers of exposure and demographic and use behavior covariates. Biomarkers of exposure within-subject consistency is likely associated with nontobacco sources of exposure and biological half-life. IMPACT: Biomarkers measured in the PATH Study provide consistent sample-to-sample measures from which to investigate the association of adverse health outcomes with the characteristics of cigarettes and their use.

Tobacco Constituents, Flavorants, and Paper Permeability of Factory-Made and Roll-Your-Own Cigarettes on the Australian Market.

INTRODUCTION: Roll-your-own (RYO) tobacco is a popular choice in Australia, with some people who smoke finding these products more attractive than factory-made cigarettes (FMC). Differences in visual and tactile properties and in the feel and taste of the smoke may contribute to this attractiveness. These differences may be driven by variation in tobacco constituents and wrapping paper permeability. However, to date, there has been no comparison of RYO and FMC products on the Australian market. AIMS AND METHODS: Chemical constituents, pH, flavorants, and paper permeability were compared in unburned RYO tobacco and tobacco from FMC. RYO and FMC products from matched brands were compared, as were products from the most popular FMC and RYO brands on the Australian market in 2018. RESULTS: RYO tobacco had higher moisture and humectant content (glycerol and propylene glycol) than FMC tobacco. RYO tobacco also had higher amounts of total and reducing sugars and lower nicotine when comparing the most popular brands. RYO papers were less permeable than FMC papers. Both RYO and FMC tobacco contained many chemicals identified as flavorants, including fourteen with known potential health risks. For most measured constituents and flavorants, RYO tobaccos had more in common with other RYO than FMC, with the commonalities remaining even when matched brands were compared. CONCLUSIONS: Higher levels of moisture, humectants, and sugars in Australian RYO tobacco compared to FMC may be increasing attractiveness of RYO by reducing the harsh taste of the smoke and increasing the moist feel of the tobacco. IMPLICATIONS: While price is the main factor driving the use of RYO tobacco, some people who smoke find these products more attractive. This study has shown that Australian RYO tobacco contains higher amounts of glycerol, propylene glycol, and sugars than FMC. These chemicals may be improving the taste of the tobacco, as well as creating a moist feel that is falsely perceived as indicating that the tobacco is "fresh" and "less chemically." Ironically, it may be that higher amounts of some added chemicals in RYO contribute to false perceptions of a more natural and less harmful product.

Reactions to Messages About a Nicotine Reduction Policy: A Focus Group Study Among People Who Use Little Cigars and Cigarillos.

INTRODUCTION: The U.S. Food and Drug Administration (FDA) has proposed rulemaking to reduce the nicotine content in cigarettes and other combusted tobacco products to non-addictive levels. This qualitative study documents reactions to messages communicating this policy among people who use little cigars and cigarillos (LCCs). AIMS AND METHODS: We conducted eight focus groups with participants from four populations with the highest prevalence of cigar use (African American males and females, white males and females). Participants described their reactions to eight messages about the policy: Three messages about the equal risk of LCCs with regular and low nicotine levels; three quit efficacy messages about low nicotine LCCs being easier to quit; one "compensation" message to correct misperceptions about the policy causing people to smoke more to get desired nicotine; and one message about using alternative nicotine sources (eg, e-cigarettes). RESULTS: Participants perceived risk messages as the most motivating to quit, whereas efficacy messages made some participants feel that the policy would cause former users of LCCs to relapse. Many participants expressed favorable responses to the compensation message. The message about using alternative nicotine sources sparked intense responses, with many participants expressing outrage and mistrust of the message. Participants' beliefs that they were not addicted to LCCs dampened their perceptions of the effectiveness of the policy. CONCLUSIONS: Perceptions of the addictiveness and relative harms of LCCS influenced responses to policy messages. The FDA should consider using different messages to communicate with people who use LCCs because they perceive LCCs as different from cigarettes. IMPLICATIONS: This is the first study to document affective and cognitive responses to the FDA's reduced nicotine policy among people who use LCCs. The false belief that cigar products are less harmful than cigarettes may be influencing people's lack of support for the reduced nicotine policy and difficulty in understanding its potential positive impact. To maximize the public health benefit of the reduced nicotine policy, the FDA should include LCC products in the policy; however, it is crucial that they use educational messaging to clarify misperceptions regarding nicotine and harm as it applies to LCCs.

Physician, patient, and caregiver support for a formal certification in pediatric neuro-oncology: A survey-based report from the SNO pediatrics working group.

Background: Although CNS tumors are the most common pediatric cancer in the United States, most physicians caring for these patients are not formally certified in the subspecialty. To determine support for developing a formal certification process in pediatric neuro-oncology, the Society for Neuro-Oncology's Pediatrics Special Interest Track Training and Credentialing working group performed a cross-sectional survey-based study of physicians and patients/caregivers of children with a CNS tumor history. Methods: Surveys were built in Survey Monkey and were available for 3 months. The physician survey had 34 questions and was open to doctors currently caring for pediatric neuro-oncology patients. The patient/caregiver survey had 13 questions. Both surveys were completed anonymously. Results: The physician survey was completed by 193 participants, the majority of whom self-identified as oncologists. Only 5.6% of survey participants had ever been board-certified in neuro-oncology; the majority of participating physicians were either unaware that this certification existed or thought they were not eligible due to training in pediatrics rather than neurology or internal medicine. Almost half of the self-identified pediatric neuro-oncologists had not completed any specific clinical neuro-oncology training. Over 75% of physicians were supportive of the implementation of a formal certification process in pediatric neuro-oncology. A total of 30 participants completed the patient/caregiver survey. Although the majority of survey participants were highly satisfied with their oncologist, 70% would have been more comfortable if their oncologist had been specifically certified in pediatric neuro-oncology. Conclusions: There is support from physicians, patients, and caregivers to establish a formal certification process in pediatric neuro-oncology.

Ataxia-telangiectasia mutated ( Atm ) disruption sensitizes spatially-directed H3.3K27M/TP53 diffuse midline gliomas to radiation therapy.

Diffuse midline gliomas (DMGs) are lethal brain tumors characterized by p53-inactivating mutations and oncohistone H3.3K27M mutations that rewire the cellular response to genotoxic stress, which presents therapeutic opportunities. We used RCAS/tv-a retroviruses and Cre recombinase to inactivate p53 and induce K27M in the native H3f3a allele in a lineage- and spatially-directed manner, yielding primary mouse DMGs. Genetic or pharmacologic disruption of the DNA damage response kinase Ataxia-telangiectasia mutated (ATM) enhanced the efficacy of focal brain irradiation, extending mouse survival. This finding suggests that targeting ATM will enhance the efficacy of radiation therapy for p53-mutant DMG but not p53-wildtype DMG. We used spatial in situ transcriptomics and an allelic series of primary murine DMG models with different p53 mutations to identify transactivation-independent p53 activity as a key mediator of such radiosensitivity. These studies deeply profile a genetically faithful and versatile model of a lethal brain tumor to identify resistance mechanisms for a therapeutic strategy currently in clinical trials.

NUTMEG: A randomized phase II study of nivolumab and temozolomide versus temozolomide alone in newly diagnosed older patients with glioblastoma.

Background: There is an immunologic rationale to evaluate immunotherapy in the older glioblastoma population, who have been underrepresented in prior trials. The NUTMEG study evaluated the combination of nivolumab and temozolomide in patients with glioblastoma aged 65 years and older. Methods: NUTMEG was a multicenter 2:1 randomized phase II trial for patients with newly diagnosed glioblastoma aged 65 years and older. The experimental arm consisted of hypofractionated chemoradiation with temozolomide, then adjuvant nivolumab and temozolomide. The standard arm consisted of hypofractionated chemoradiation with temozolomide, then adjuvant temozolomide. The primary objective was to improve overall survival (OS) in the experimental arm. Results: A total of 103 participants were randomized, with 69 in the experimental arm and 34 in the standard arm. The median (range) age was 73 (65-88) years. After 37 months of follow-up, the median OS was 11.6 months (95% CI, 9.7-13.4) in the experimental arm and 11.8 months (95% CI, 8.3-14.8) in the standard arm. For the experimental arm relative to the standard arm, the OS hazard ratio was 0.85 (95% CI, 0.54-1.33). In the experimental arm, there were three grade 3 immune-related adverse events which resolved, with no unexpected serious adverse events. Conclusions: Due to insufficient evidence of benefit with nivolumab, the decision was made not to transition to a phase III trial. No new safety signals were identified with nivolumab. This complements the existing series of immunotherapy trials. Research is needed to identify biomarkers and new strategies including combinations.

Reply to Foxon, F.; Shiffman, S. Comment on "Wang et al. Consumption of JUUL vs. Other E-Cigarette Brands among U.S. E-Cigarette Users: Evidence from Wave 5 of the PATH Study. Int. J. Environ. Res. Public Health 2022, 19, 10837".

In this reply, we respond to the comments by Foxon and Shiffman [...].

Pooled genetic screens to identify vulnerabilities in TERT-promoter-mutant glioblastoma.

Pooled genetic screens represent a powerful approach to identify vulnerabilities in cancer. Here we used pooled CRISPR/Cas9-based approaches to identify vulnerabilities associated with telomerase reverse transcriptase (TERT) promoter mutations (TPMs) found in >80% of glioblastomas. We first developed a platform to detect perturbations that cause long-term growth defects in a TPM-mutated glioblastoma cell line. However, we could not detect dependencies on either TERT itself or on an E-twenty six transcription (ETS) factor known to activate TPMs. To explore this finding, we cataloged TPM status for 441 cell lines and correlated this with genome-wide screening data. We found that TPM status was not associated with differential dependency on TERT, but that E-twenty six (ETS) transcription factors represent key dependencies in both TPM+ and TPM- lines. Further, we found that TPMs are associated with expression of gene programs regulated by a wide array of ETS-factors in both cell lines and primary glioblastoma tissues. This work contributes a unique TPM cell line reagent, establishes TPM status for many deeply-profiled cell lines, and catalogs TPM-associated vulnerabilities. The results highlight challenges in executing genetic screens to detect TPM-specific vulnerabilities, and suggest redundancy in the genetic network that regulates TPM function with therapeutic implications.

Repurposing Clemastine to Target Glioblastoma Cell Stemness.

Brain tumor-initiating cells (BTICs) and tumor cell plasticity promote glioblastoma (GBM) progression. Here, we demonstrate that clemastine, an over-the-counter drug for treating hay fever and allergy symptoms, effectively attenuated the stemness and suppressed the propagation of primary BTIC cultures bearing PDGFRA amplification. These effects on BTICs were accompanied by altered gene expression profiling indicative of their more differentiated states, resonating with the activity of clemastine in promoting the differentiation of normal oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes. Functional assays for pharmacological targets of clemastine revealed that the Emopamil Binding Protein (EBP), an enzyme in the cholesterol biosynthesis pathway, is essential for BTIC propagation and a target that mediates the suppressive effects of clemastine. Finally, we showed that a neural stem cell-derived mouse glioma model displaying predominantly proneural features was similarly susceptible to clemastine treatment. Collectively, these results identify pathways essential for maintaining the stemness and progenitor features of GBMs, uncover BTIC dependency on EBP, and suggest that non-oncology, low-toxicity drugs with OPC differentiation-promoting activity can be repurposed to target GBM stemness and aid in their treatment.