Faculty Bibliography

Identifying cellular programs that drive cancers to be stem-like and treatment resistant is critical to improving outcomes in patients. Here, we demonstrate that constitutive extracellular signal-regulated kinase 1/2 (ERK1/2) activation sustains a stem-like state in glioblastoma (GBM), the most common primary malignant brain tumor. Pharmacological inhibition of ERK1/2 activation restores neurogenesis during murine astrocytoma formation, inducing neuronal differentiation in tumorspheres. Constitutive ERK1/2 activation globally regulates miRNA expression in murine and human GBMs, while neuronal differentiation of GBM tumorspheres following the inhibition of ERK1/2 activation requires the functional expression of miR-124 and the depletion of its target gene SOX9. Overexpression of miR124 depletes SOX9 in vivo and promotes a stem-like-to-neuronal transition, with reduced tumorigenicity and increased radiation sensitivity. Providing a rationale for reports demonstrating miR-124-induced abrogation of GBM aggressiveness, we conclude that reversal of an ERK1/2-miR-124-SOX9 axis induces a neuronal phenotype and that enforcing neuronal differentiation represents a therapeutic strategy to improve outcomes in GBM.

As the field of neuro-oncology makes headway in uncovering the key oncogenic drivers in pediatric glioma, the role of precision diagnostics and therapies continues to rapidly evolve with important implications for the standard of care for clinical management of these patients. Four studies at major academic centers were published in the last year outlining the clinically integrated molecular profiling and targeting of pediatric brain tumors; all four demonstrated the feasibility and utility of incorporating sequencing into the care of children with brain tumors, in particular for children and young adults with glioma. Based on synthesis of the data from these studies and others, we provide consensus recommendations for the integration of precision diagnostics and therapeutics into the practice of pediatric neuro-oncology. Our primary consensus recommendation is that next-generation sequencing should be routinely included in the workup of most pediatric gliomas.

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is an aggressive type of brainstem cancer that targets young children. Complete resection is not possible, and chemotherapy and radiotherapy are currently only palliative. To identify potential therapeutic agents, we used a recently developed computational pipeline to perform an in silico screen for novel drugs. We then tested the identified drugs against a panel of patient-derived DIPG cell lines.
METHOD(S): We used a systematic computational approach with publicly available databases of gene expression signatures in DIPG, as well as gene expression data of cancer cell lines before and after treatment in conjunction with a massive library of clinically available drugs. We identified three drug hits with the ability to reverse a DIPG gene signature to one that matches normal tissue background. We used three DIPG cell lines (SF8628, DIPG4, DIPG-NYU) along with immortalized normal human astrocytes (NHA) as control to test these drugs. Flow cytometry, immunob-lot analysis, viability assays, and RNA-Seq before and after drug treatment were performed. In vivo experiments are currently underway.
RESULT(S): All three drugs showed potency against the primary DIPG4 cell line compared to NHA control. Triptolide showed the most potency against primary DIPG cell lines bearing H3K27M mutations (SF8628 and DIPG4, with IC50's in the nanomolar range after 72 hours treatment), while the effect on DIPG cell line without H3K27M mutation (DIPG-NYU) was less potent. Immunoblot data also suggested a correlation between drug potency and H3K27M levels in these cell lines.
CONCLUSION(S): Using a computational approach, we identified clinically available drugs with the ability to reverse DIPG gene expression signatures and signifcantly decreased the growth rate of primary DIPG cells in vitro. This novel approach can repurpose drugs and signif-cantly decrease the cost and time normally required in drug discovery

BACKGROUND: Vemurafenib is an orally administered inhibitor of BRAF-V600E kinase approved for the treatment of BRAF-V600E melanoma. We have previously presented favorable radiographic response and safety data for vemurafenib in children with BRAF-V600E mutated brain tumors and report here an update on outcomes of this cohort, as well as pharmacokinetic (PK) data of patients taking whole (n=18) or crushed (n=6) tablets.
METHOD(S): Vemurafenib was given orally, either as crushed or whole tablets, beginning at the adult dose equivalent of 550 mg/m2, twice daily. Toxicity, PK, and response were assessed. Doses of 420 mg/m2 and 550 mg/m2 were assessed by a 3 + 3 design in a dose de-escalation study. PK plasma samples were analyzed using HPLC at Covance Inc and modeled by standard PK methods using Phoenix v8 (Certera, Princeton, New Jersey).
RESULT(S): PK analyses demonstrated a signifcant accumulation factor (approximately six-fold) over time with each vemurafenib dose. In the "crushed" tablet cohort, bioavailability was approximately 60% of that seen in patients receiving whole tablets. The steady-state area-under-the-curve (AUCss) median (range) was 666mg*h/L (321-920) in the whole tablet cohort and 459mg*h/L (147-1546) in the crushed tablet cohort. Updated best radiographic objective responses are: one complete response (CR), eleven partial responses (PR), and seven with stable disease (SD). Sustained responses were observed for over 50 months. Patients with SD had 30% lower AUCss (522mg*hr/L) compared PR+CR (730mg*hr/L). However, a logistic regression model using AUCss as a predictor of PR+CR was not signifcant given the observed variability.
CONCLUSION(S): Children with BRAF-V600E mutated brain tumors treated with vemurafenib have durable responses. PK modeling confrmed similar PK parameters in children as compared to adults treated for melanoma. Using crushed tablets to administer a liquid suspension of drug resulted in signifcantly decreased drug exposure and may require increases in dose to achieve comparable exposure

INTRODUCTION: Oncogenic, driver BRAF-V600E mutations are frequently found in pediatric gliomas. While small molecule inhibitors of BRAF and/or MEK kinases are showing early clinical effcacy against these tumors, no experimental strategies thus far have focused on the degradation of the mutant BRAF protein itself. Members of our team have recently discovered that in contrast to wildtype BRAF protein, BRAF-V600E protein binds to aurora kinase A. We therefore hypothesized that CD532, a novel small molecule allosteric aurora kinase A inhibitor, would preferentially degrade BRAF-V600E over wildtype BRAF, and lead to decreased cell growth in BRAF-V600E gliomas.
METHOD(S): We used an isogenic system of 3T3 fbroblasts transduced with empty vector, wildtype BRAF, KIAA1549:BRAF fusion, and BRAF-V600E, as well as established glioma cell lines AM38 (BRAF-V600E homozygous),

Pleomorphic xantoastrocytoma (PXA) is a typically well circumscribed astrocytic neoplasm that can progress to anaplastic PXA with an aggressive biologic behavior and worse prognosis. Histological features of anaplastic progression include increased proliferation, necrosis, microvascular proliferation, loss of pericellular reticulin, and increased infltrative growth. Genetically anaplastic PXA is characterized by the combination of CDKN2A biallelic inactivation and oncogenic RAF kinase signaling, as observed in PXA, as well as a relatively small number of additional genetic alterations, with the most common being TERT amplification or promoter mutation (Phillips et al 2018). Common to both PXA and anaplastic PXA is the perivascular accumulation of lymphocytes. Given the potential importance of the immune response in brain tumors we asked whether the immune response in anaplastic PXA was different than that in PXA and in IDH-mutant diffuse glioma. To do this we used quantitative multiplex immunofuorescence of scanned slides and quantified the populations and phenotypes of T cells and microglia/macrophages in tumors identified from the UCSF Brain Tumor Center Biorepository and from the archives of the UCSF Neuropathology Division. All tumors were molecularly characterized by immunostaining and/or comprehensive genomic profling. A total of 30 PXA, including 6 paired tumors before and after tumor progression, and 30 diffuse glioma were examined. Immunostaining for T cell subsets included CD3, CD8, and FOXP3 and for microglial/macrophages included Iba1, CD204, and CD163. Using quantitative multiplex immunostaining we assess immune cell subsets and their localization within the tissue highlighting the perivascular CD3+ lymphocytes. Our data suggest differences in the quantity and quality of immune cells in anaplastic PXA

The BRAF-V600E mutation is found in 15-20% of pediatric low grade gliomas (PLGG) and result in worse outcome and higher risk of transformation to high grade gliomas (PHGG). Although ongoing trials are assessing the role of BRAF inhibitors (BRAFi) in these children, data are still limited. We aimed to report overall response rates and predictors of outcome in childhood BRAF-V600E gliomas. We collected clinical, imaging and molecular information of patients treated with BRAFi outside trials from centers participating in the PLGG taskforce. Response was calculated by RANO criteria and follow up data were collected for all patients. Sixty-six patients were treated with BRAFi (55 PLGG and 11 PHGG); median follow-up time was 1.5 years (0.1-5y). In PLGG, objective response (tumor reduction of >25%) was observed in 77% compared to 15% in a cohort treated with conventional chemotherapy (pCDKN2A deletion was not associated with lack of response, while specifc enhancing patterns correlated strongly with response to BRAFi. Two-year PFS for the BRAF-V600E PLGG was 74% vs 47% for BRAFi vs chemotherapy, respectively (p=0.02). Our data reveal rapid, dramatic and sustained response of BRAF-V600E PLGG to BRAFi. These are in contrast to BRAF-V600E PHGG and non-enhancing PLGG. Additional molecular analyses are being performed to identify poor responders and emerging mechanisms of resistance in these tumors

PURPOSE/OBJECTIVE:Isocitrate dehydrogenase (IDH) mutant gliomas are a distinct glioma molecular subtype for which no effective molecularly-directed therapy exists. Low-grade gliomas, which are 80-90% IDH mutant, have high RNA levels of the cell surface Notch ligand DLL3. We sought to determine DLL3 expression by immunohistochemistry in glioma molecular subtypes and the potential efficacy of an anti-DLL3 antibody drug conjugate (ADC), rovalpituzumab tesirine (Rova-T), in IDH mutant glioma. EXPERIMENTAL DESIGN/METHODS:We evaluated DLL3 expression by RNA using TCGA data and by immunohistochemistry in a discovery set of 63 gliomas and 20 non-tumor brain tissues and a validation set of 62 known IDH wildtype and mutant gliomas using a monoclonal anti-DLL3 antibody. Genotype was determined using a DNA methylation array classifier or by sequencing. The effect of Rova-T on patient-derived endogenous IDH mutant glioma tumorspheres was determined by cell viability assay. RESULTS:Compared to IDH wildtype glioblastoma, IDH mutant gliomas have significantly higher DLL3 RNA (P<1x10-15) and protein by immunohistochemistry (P=0.0014 and P<4.3x10-6 in the discovery and validation set, respectively). DLL3 immunostaining was intense and homogeneous in IDH mutant gliomas, retained in all recurrent tumors, and detected in only 1 of 20 non-tumor brains. Patient-derived IDH mutant glioma tumorspheres overexpressed DLL3 and were potently sensitive to Rova-T in an antigen-dependent manner. CONCLUSIONS:DLL3 is selectively and homogeneously expressed in IDH mutant gliomas and can be targeted with Rova-T in patient-derived IDH mutant glioma tumorspheres. Our findings are potentially immediately translatable and have implications for therapeutic strategies that exploit cell surface tumor-associated antigens.

Pleomorphic xanthoastrocytoma (PXA) is an astrocytic neoplasm that is typically well circumscribed and can have a relatively favorable prognosis. Tumor progression to anaplastic PXA (WHO grade III), however, is associated with a more aggressive biologic behavior and worse prognosis. The factors that drive anaplastic progression are largely unknown. We performed comprehensive genomic profiling on a set of twenty-three PXAs from 19 patients, including 15 with anaplastic PXA. Four patients had tumor tissue from multiple recurrences, including two with anaplastic progression. We find that PXAs are genetically defined by the combination of CDKN2A biallelic inactivation and RAF alterations that were present in all 19 cases, most commonly as CDKN2A homozygous deletion and BRAF p.V600E mutation but also occasionally BRAF or RAF1 fusions or other rearrangements. The third most commonly altered gene in anaplastic PXA was TERT, with 47% (7/15) harboring TERT alterations, either gene amplification (n=2) or promoter hotspot mutation (n=5). In tumor pairs analyzed before and after anaplastic progression, two had increased copy number alterations and one had TERT promoter mutation at recurrence. Less commonly altered genes included TP53, BCOR, BCORL1, ARID1A, ATRX, PTEN, and BCL6. All PXA in this cohort were IDH and histone H3 wildtype, and did not contain alterations in EGFR. Genetic profiling performed on six regions from the same tumor identified intratumoral genomic heterogeneity, likely reflecting clonal evolution during tumor progression. Overall, anaplastic PXA is characterized by the combination of CDKN2A biallelic inactivation and oncogenic RAF kinase signaling as well as a relatively small number of additional genetic alterations, with the most common being TERT amplification or promoter mutation. These data define a distinct molecular profile for PXA and suggest additional genetic alterations, including TERT, may be associated with anaplastic progression.