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Glioblastoma (GBM) is the most aggressive and common type of adult malignant brain tumor, with a median survival of only 21 months. To identify which genes drive its highly aggressive phenotype, we performed a genome-wide CRISPR-Cas9 knockout screen. Results showed substantial enrichment of ~160 novel essential oncogenic driver genes and pathways, including a previously unstudied gene THOC1—involved in RNA processing—that showed significant elevations in expression at RNA and protein levels (p< 0.05) in GBM, as well as a significant survival benefit in patient datasets when downregulated (p< 0.05). Knocking out THOC1 resulted in cell death in multiple GBM patient-derived xenograft (PDX) lines and extended survival compared to the controls (p< 0.01) in vivo. Overexpression of THOC1 in neural stem cells resulted in transformation to a cancerous phenotype, as evidenced by sphere formation in a soft agar assay (p< 0.01) and in vivo tumor engraftment assays. Further investigation of THOC1 through immunoprecipitation in neural stem cells and multiple GBM lines showed significant interaction in GBM with histone deacetylase complex SIN3A, involved in recruiting major histone deacetylases in order to close the DNA and prevent the accumulation of R-loops, RNA:DNA hybrids that pose a threat to genomic stability. Additional investigation revealed that THOC1-knockdowns in vitro induced R-loop formation and DNA damage, while THOC1-overexpression in vitro resulted in an untenable decrease in R-loops and DNA damage, suggesting that the THOC1-SIN3A axis is elevated in GBM in order to prevent the accumulation of genotoxic R-loops. Additionally, histone deacetylase activity was shown to be elevated in THOC1-overexpression conditions and reduced in THOC1-knockdown conditions, confirming that the THOC1-SIN3A axis functions to prevent R-loop accumulation through the epigenetic regulation. In summary, our whole-genome CRISPR-Cas9 knockout screen has identified a promising therapeutic target for GBM—a disease desperately in need of therapeutic innovations.

Glioblastoma (GBM) is the most common type of adult malignant brain tumor, with a median survival of only 21 months. This is partly due to the high rate of resistance to conventional therapy, including temozolomide (TMZ), leading to recurrence rates close to 100%. To identify the unknown genes driving the development of this resistance, we performed a genome-wide CRISPR knockout screen comparing a DMSO-treated population with a TMZ-treated population over 14 days. Results showed significant enrichment of ~200 novel genes and pathways. From this list, we identified 4 previously unstudied genes showing significant elevations in RNA expression (p< 0.05) when comparing recurrent and primary tumors in patient datasets, along with significant survival benefits corresponding to low gene expression (p< 0.05). Validation experiments in vitro showed significant elevations in RNA and protein expression in multiple patient-derived xenografts (PDX) lines during TMZ-treated conditions, while knocking out these genes also resulted in significantly heightened sensitivity to TMZ (p< 0.01). We investigated one particularly enriched gene, ARF4, known to be involved in retrograde trafficking. With previous studies showing that ARF4 is upregulated under ER stress, we first confirmed the increased expression of ER stress markers during TMZ treatment to explain the increased expression of ARF4 during treatment. Further investigation via live-cell imaging also showed a consequent increase in retrograde trafficking in TMZ-treated cells, as evidenced by significantly increased trafficking of transferrin receptors, a retrograde transport marker, as well as EGFR, known to play a role in promoting chemoresistance through strengthened DNA repair response. ARF4-overexpressed GBM cells similarly showed increased trafficking of transferrin receptors and EGFR to the nucleus, while ARF4-knockdowns showed decreased trafficking and nuclear EGFR expression. Ultimately, our CRISPR-Cas9 screen has identified a promising therapeutic target, ARF4, which may drive GBM’s robust resistance to chemotherapy through increased retrograde trafficking of chemoresistance-promoting nuclear EGFR.

BACKGROUND A Phase 2 multi-center study was conducted to evaluate the replication-competent oncolytic adenovirus, DNX-2401 (tasadenoturev), in combination with the anti-PD-1 antibody, pembrolizumab, in subjects with recurrent glioblastoma. METHODS Subjects ≥ 18 years with glioblastoma at first or second recurrence were treated with a single intratumoral injection of 5e8-5e10 vp DNX-2401 at the time of biopsy, followed by 200 mg pembrolizumab infusions every 3 weeks until progression or toxicity. RESULTS Forty-nine subjects were enrolled at first (79.6%) or second (20.4%) recurrence after prior surgery (89.8%), radiotherapy (100%), and temozolomide (100%). Forty-eight of 49 (98%) were treated with 5e8 vp (n=3), 5e9 vp (n=3), or 5e10 vp (n=42) DNX-2401 and pembrolizumab. Median treatment duration was 7 cycles (range 1–35) with one subject remaining on treatment (cycle 31). Adverse events were primarily mild to moderate, consistent with underlying disease, and manageable. Headache, brain edema, and fatigue are the most common events reported as related to the treatment regimen. Of subjects treated with full dose DNX-2401 and pembrolizumab (n=42), 5 subjects (11.9%) had confirmed responses, including 2 durable ongoing complete responses and 3 partial responses. One subject remains tumor free > 12 months after completing the planned 24 months of pembrolizumab. Median overall survival was 12.5 months; OS-12 and OS-18 were 54.5% and 20.8%, respectively. Four subjects, all of which have survived more than 21 months, continue to be followed for survival. CONCLUSIONS DNX-2401 plus pembrolizumab provides encouraging activity and is safe in patients with recurrent glioblastoma. A global, randomized, controlled Phase 3 study is planned.

Immunotherapy has revolutionized the treatment of many tumors. However, most glioblastoma (GBM) patients have not, so far, benefited from such successes. With the goal of exploring ways to boost anti-GBM immunity, we developed a B-cell-based vaccine (BVax) that consists of 4-1BBL+ B cells activated with CD40 agonism and IFNg stimulation. BVaxmigrate to key secondary lymphoid organs and are proficient at antigen cross-presentation, which promotes both the survival and functionality of CD8+ T cells. A combination of radiation, BVax, and PD-L1 blockade conferred tumor eradication in 80% of treated tumor-bearing animals. This treatment elicited immunologic memory that prevented the growth of new tumors upon subsequent re-injection in cured mice. GBM patient-derived BVax were successful in activating autologous CD8+ T cells; these T cells showed a strong ability to kill autologous glioma cells. In addition to the role in activating CD8+ T cells, BVax produce tumor-specific antibodies able to control tumor growth via antibody-mediated cell cytotoxicity. In conclusion, BVax tackles GBM immunosurveillance escape by using both cellular (CD8+ T-cell activation) and humoral (anti-tumor antibody production) immunity. Our study provides an efficient alternative to current immunotherapeutic approaches that can be readily translated to the clinic.

BACKGROUND This retrospective study evaluated the relationship between histologic subtype and treatment outcomes following SRS for the treatment of multiple brain metastases (MBM). METHODS We analyzed patients with MBM, defined here as >= 3 lesions, treated with SRS at our institution. Primary histologies examined were NSCLC, breast, and melanoma. Patients were categorized according to histology-specific subtypes (NSCLC-EGFR, ALK, KRAS, PD-L1%; breast-HER2, ER, PR; melanoma-BRAF). The primary outcome was local control (defined by RANO-BM) and secondary outcomes included intracranial progression-free survival (iPFS) and overall survival (OS). RESULTS 141 patients met inclusion criteria (66 NSCLC, 61 breast, and 14 melanoma). HER2+ and BRAF V600E+ lesions had increased rates of local control following SRS (P=0.0048 and P=0.0256, respectively) compared to other breast/melanoma subtypes. EGFR mutation was not associated with increased local control with SRS (71 vs 74%), but increased iPFS (P=0.0031). On multivariable analysis, EGFR+ was independently associated with a decreased time-dependent risk of death (P=0.011). The use of progressively newer generations of EGFR-directed therapies was associated with stepwise decreasing risk of intracranial progression and death. HER2+ disease had improved iPFS and OS (P=0.0058 and P< 0.0001, respectively; it was not an independent risk factor for progression or death; however, the use of HER2-directed antibodies was associated with decreased risk of death (p=0.036). The use of tyrosine kinase inhibitors (i.e. lapatanib) was not associated with improvements, although this was a small subset. CONCLUSIONS Some histologic subtypes appear to have better control with SRS, with HER2+ breast cancer and BRAF V600E+ melanoma associated with improved outcomes – this requires further validation; a volumetric analysis is pending. This the protective effect of EGFR mutation appears to be partly related to use of EGFR inhibitors, with the use of newer-generation therapies leading to improved outcomes; although local control with SRS remains excellent regardless of the mutation.

BACKGROUND DNX-2401 (tasadenoturev) is a replication-competent, tumor-selective, oncolytic adenovirus. Phase I studies in adults with recurrent glioblastoma (rGBM) have demonstrated safety and encouraging clinical activity. A Phase II open-label, dose-escalating multi-center study in rGBM was initiated to evaluate DNX-2401 with pembrolizumab. Planned enrollment of 48 subjects is complete. METHODS Subjects ≥ 18 years, with a single tumor, KPS ≥ 70% and adequate organ function were enrolled sequentially into 3 cohorts of DNX-2401 (5e8vp, 5e9vp, 5e10vp). A single intratumoral injection of DNX-2401 was administered followed 7 days later by pembrolizumab (200 mg IV). Thereafter, pembrolizumab was infused Q3wks up to 24 months until progression or toxicity. 5e10vp was determined as the optimal dose and this cohort was expanded. Safety monitoring, assessments of response and survival follow-up are ongoing. RESULTS Nine subjects were treated in the escalation phase; 42 subjects received 5e10vp. No dose-limiting toxicity or unexpected safety issues were identified by an independent review committee, and there were no treatment-related deaths. Adverse events were primarily consistent with underlying disease, effects of the neuro-procedure, expected effects of pembrolizumab, and concomitant use of steroids/anticonvulsants per the standard of care. The majority of events were mild to moderate, and unrelated to DNX-2401. Headache and manageable vasogenic edema were the most common events related to DNX-2401 with pembrolizumab. For the 48 subjects who received pembrolizumab (median 6 cycles), median OS from DNX-2401 administration was 12 months (95% CI, 10.6–14.7), OS6 was 91%, and 47% experienced clinical benefit (stable disease or better). Four subjects (5e10) had a partial response (two with > 94% regression of tumor), and three (5e8; 5e10[2]) are alive > 20 months. Updated safety and efficacy results will be presented. CONCLUSIONS The data continue to demonstrate that DNX-2401 administered with pembrolizumab has an acceptable safety profile. Long-term survival and clinical benefit remain compelling.

We have conducted experiments with intracranial patient-derived xenograft (PDX) models of glioblastoma (GBM) in which PDX recurrence, following the administration of radiotherapy (RT) and temozolomide (TMZ) to animal subjects, is monitored by bioluminescence imaging. Recurrent tumors were resected from brain then sequentially propagated as subcutaneous xenografts through three successive mouse hosts that received no treatment during tumor propagation. Subcutaneous tumor from the third mouse host was used for establishing intracranial tumors in a new series of mice that received the same treatments as in the initial experiment, in order to assess whether recurrent, treated tumors respond differently to the same treatments used in treating corresponding therapy naïve tumors. We observed no indication of tumor recurring from one cycle of RT + TMZ treatment as displaying resistance to the same treatments used in treating corresponding therapy naïve tumor. However, recurrent tumor that was subjected to two additional cycles of TMZ treatment, prior to resection from brain and subcutaneous propagation through 3 successive mice (107 days total subcutaneous propagation while receiving no treatment), displayed TMZ resistance when tested for response to the same treatments as corresponding naïve tumor. Median TMZ treatment survivals for mice intracranially engrafted with naïve, RT + 1 cycle TMZ treated, and RT + 3 cycles TMZ treated tumor cells were 80, 74, and 42 days, respectively, and survival results yielded log-rank p-values

Topoisomerase IIB (TOP2B) is known to decoil and decatenate DNA, releasing chromatin torsional forces. Whereas TOP2B has been implicated in transcript splicing, direct evidence supporting this function, through localization of TOP2B to specific chromatin sites and in association with specific types of cancer, is modest at best. Here, we report our preliminary activities that are intended to address this knowledge gap. GSEA differential expression analysis using TCGA glioblastoma data showed that high TOP2B expression is associated with elevated expression of cell cycle-related genes (p-value= 1.67E(-05)). To investigate the possible association of TOP2B with specific gene sequences we used TOP2B ChIP-seq in analyzing glioma cell lines TS543 and BT142, and found TOP2B associations with the introns of especially long genes (gen length ≥ 100Kb, p-value= 0.001). Functional annotation analysis for genes with TOP2B intron binding showed a significant enrichment for genes with multiple known splice variants (p-value= 1.9E(-11)). To determine effects of TOP2B inhibition on splice variant expression, we treated BT142 cells with the TOP2 inhibitor ICRF-193 and examined treatment effects on splice variant expression using paired-end RNA-seq. We observed altered splicing of 319 genes following TOP2 inhibition, with increased expression of splice variant CDK4-009 being the most significant of all splice variant expression changes (p-value= 1E(-54)). This result corresponded well with TCGA data whose analysis showed a strong positive correlation between TOP2B and CDK4-009 expression (rho= -0.3 and p-value= 7.28E(-05)). CONCLUSIONS: Our results support TOP2B expression and activity as being influential in regulating the levels of CDK4 splice variant 009 in GBM.

BACKGROUND: Cytoreductive surgery is considered controversial for primary central nervous system lymphoma (PCNSL). OBJECTIVE: To investigate survival following craniotomy or biopsy for PCNSL METHODS: The National Cancer Database-Participant User File (NCDB, n = 8936), Surveillance, Epidemiology, and End Results Program (SEER, n = 4636), and an institutional series (IS, n = 132) were used. We retrospectively investigated the relationship between craniotomy, prognostic factors, and survival for PCNSL using case–control design. RESULTS: In NCDB, craniotomy was associated with increased median survival over biopsy (19.5 vs 11.0 mo), independent of subsequent radiation and chemotherapy (hazard ratio [HR] 0.80, P

Topoisomerase II (TOP2B) A/B regulate transcription by inducing double-strand DNA breaks and enhancing chromatin accessibility in other contexts, but their role in gliomas has not been elucidated. We investigated the effect of TOP2A/B on epigenetic regulation in glioma cell lines and explored the targeting of this process as a therapeutic strategy.

Topoisomerase II (TOP2) is highly expressed on proneural gliomas, but its specific function on these tumors is unknown. We found that TOP2 is expressed in gliomas from early stages, increases during progression, and is elevated in radiographically localized non-enhancing regions of human glioblastoma, an area that is challenging to target systemically, and a common source of recurrence. TOP2A is highly expressed by infiltrating tumor cells, in the tumor center and at the edges, and low on surrounding brain. TOP2 enzymatic activity was detected on proneural glioma cell lines. TOP2 poison etoposide and 3 TOP2 inhibitors showed significant cytotoxicity on a proneural glioma cells (IC50 0.3-4 mM). Moreover, intracranial etoposide convection-enhanced delivery (CED) (80 mM) was tolerated and led to a significant survival benefit in mouse proneural gliomas. In contrast, etoposide CED (4 mM) showed transient decrease in tumor growth but failed to prolong survival, consistent with ineffective tumoral concentrations in trials of systemic etoposide for recurrent gliomas. TOP2 transcriptional modulation on proneural gliomas was suggested as the activity of many key transcription factors correlates with TOP2 levels across glioblastoma patients. TOP2 pharmacological targeting is a promising treatment target for some gliomas. Refining patient selection and drug delivery is key for optimizing this therapy.

BACKGROUND: The primary treatment of GBM is surgical resection of the contrast enhancing mass, while regions of non-enhancing tumor are left behind, and inevitably gives rise to recurrence. Thus, there is therapeutic significance to understanding the cellular and molecular features of the non-enhancing margins of GBM. Advances in comparative genetic and molecular analyses have uncovered patterns of gene expression and genetic alterations in GBM, and led to the recognition of clinically significant tumor subtypes. However, most previous studies have been based on analysis of tissue that was removed during surgery without documenting the radiographic location. This has precluded the systematic study of intra-tumoral heterogeneity, and its correlation with radiographic regions. We propose a method of sequential image guided biopsies, allowing for specimen localization, intra-tumoral comparative analysis, and the systematic study of infiltrated tissue beyond the borders of contrast enhancement. METHODS: In 77 patients with GBM, multiple stereotactic-guided biopsies were obtained from radiographically distinct regions prior to resection. The radiographic localization of each biopsy was mapped using the 3D intraoperative neuro-navigation system (BrainLab). Sampled regions included areas of contrast enhancement (CE) and adjacent non-enhancing FLAIR+ (NEF+). Samples from each region were divided into pieces processed for RNA-seq and histological analyses. RESULTS: There were significant differences in the cellular density and cellular composition between the CE and NEF+ samples. The CE samples had higher cellularity than NEF+ samples, and were more likely to contain the histological hallmarks of GBM, including vascular proliferation and pseudopalisading necrosis. In contrast, the NEF+ regions showed the histological features of diffusely infiltrating glioma with neoplastic glial cells intermingled with non-neoplastic and reactive cells. RNA-seq showed that the CE and NEF+ regions also had markedly different expression patterns. Comparisons between our image guided biopsies and the TCGA data set showed that the CE regions were enriched for genes associated with the mesenchymal, classical and proneural subtypes while the NEF+ region were enriched for genes of the neural subtype. Further analysis revealed that region-specific and subtype-specific differences in expression patterns were predominantly due to differences in the cellular composition. CONCLUSIONS: Cellular composition is a major determinant of the expression patterns seen at the non-enhancing margins of GBM. The different GBM subtypes show distinct expression patterns that relate the contrast enhancing centers to the non-enhancing margins of the tumors. Understanding these patterns provides a means to infer the molecular and cellular features of glioma tissue left behind after surgery. SECONDARY CATEGORY: Tumor Biology.