Activity In High-grade Gliomas Research Articles

EXTH-37. ALLOSTERIC MITOCHONDRIAL CLPP AGONIST ONC206 ALTERS STRESS RESPONSE, METABOLIC AND EPIGENETIC PROFILES TO ELICIT ANTI-CANCER EFFICACY IN HIGH-GRADE GLIOMAS

Abstract Dordaviprone (ONC201) is a dopamine receptor D2/3 (DRD2/3) antagonist and allosteric ClpP agonist that has demonstrated durable responses in recurrent H3 K27M-mutant glioma patients. ONC206, a derivative of ONC201 with nanomolar potency that also targets DRD2/3 and ClpP, is in Phase I trials for central nervous system tumors. We characterized target relevance, human ClpP binding, in vitro efficacy, downstream signaling and response determinants associated with ONC206 in high-grade gliomas (HGG). Co-crystallization with ClpP that assembles as a tetradecameric complex revealed an allosteric ligand interaction with distinctions in the ONC206-ClpP overall conformation relative to the ONC201-bound or apo complex, including an increase in the resolved pore size and decreased complex height. ONC206 was more potent than ONC201 in cell-free human ClpP casein/peptide degradation assays). Accordingly, HGG cell lines and patient-derived cells exhibited increased sensitivity to ONC206 relative to ONC201 and generally required a shorter duration of exposure. Unlike DRD2, CRISPR-mediated ClpP knockout in HGG cell lines blunted ONC206 sensitivity. Metabolomics revealed elevated α-ketoglutaric acid, α-hydroxyglutaric acid, and glutaric acid. Proteomics indicated inhibition of multiple mitochondrial pathways including OXPHOS and TCA cycle. TCA cycle enzyme α-ketoglutarate dehydrogenase (KGDH) was inhibited in a ClpP-dependent manner. Western blotting showed downregulated ClpX, and upregulated ATF4/CHOP, H3 K27me3 and H3 K36me3. Next-generation sequencing of acquired resistance cell lines, which exhibited cross-resistance to ONC201 and ONC206, revealed diverse ClpP missense mutations distributed across various monomer interfaces. Both intrinsic and well as ONC206-stimulated ClpP proteolysis were mitigated by these mutations, which was congruent with an absence of the tetradecameric assembly. These ClpP mutations recapitulated resistance and, conversely, wildtype ClpP overexpression restored sensitivity. Thus, allosteric ClpP agonism is a key determinant of ONC206 activity in HGG by altering stress response, metabolic, and epigenetic profiles.

P-137 - [18F]hGTS13 for imaging of xC− transporter activity in high-grade glioma

P-137 - [18F]hGTS13 for imaging of xC− transporter activity in high-grade glioma

Abstract 249: Combination of ONC201 with radiation exhibits synergistic efficacy in high grade gliomas and other advanced cancers

Abstract Background: Radiation is widely administered in many treatment settings for solid tumors. ONC201 is the first small molecule DRD2 antagonist for oncology that is being investigated in advanced cancer clinical trials as a single agent. ONC201 has exhibited preclinical and clinical anti-tumor activity in high grade gliomas and its immunostimulatory activity involving natural killer (NK) cells has been recently reported. Given that ONC201 exhibits broad synergy with anti-cancer drugs, excellent safety, and single activity in tumor types where radiation is used routinely, we evaluated the combination of ONC201 with radiation. Methods: Cell viability was evaluated in human and/or mouse breast, prostate and high grade glioma cell lines in response to ONC201 (1 - 10uM), radiation (2- 10 Gy), or the combination. Incubation times ranged from 24 to 96 hours and the sequence of the two agents in combination was varied. Results: Cell viability assays for ONC201 in combination with radiation in breast or prostate cancer cell lines revealed a cytotoxic response to the combination that was superior to either single agent. Western blot analysis of PC3 cells showed a synergistic induction of CHOP and ATF6 that are components of the integrated stress response. In glioblastoma or diffuse intrinsic pontine glioma (DIPG) cell lines, combination indices computed from cell viability experiments indicated modest synergy (~0.7 CI) for the combination ranging between 1-5 µM ONC201 and 2-10 Gy. In MDA-MB-468 breast cancer cells, Western blot analysis demonstrated a striking induction of PARP cleavage, a marker of caspase-mediated apoptosis, with 2 µM ONC201 in combination with 2 Gy radiation, whereas either single agent produced minimal PARP cleavage. In 4T1 murine triple-negative breast cancer subcutaneous tumors, the combination of oral ONC201 and radiation produced antitumor effects at subtherapeutic doses that required CD4+ T cells and NK cells. Conclusions: ONC201 combined with radiation produced additive or synergistic anti-cancer effects in high grade glioma, breast and prostate cancer in vitro and increased anti-tumor efficacy in an in vivo model of breast cancer that involved immune cells. Citation Format: Rohinton S. Tarapore, Sachin Jhawar, Mark Stein, Andrew Zloza, Sabine Mueller, Jie Zhang, Francesca Amoroso, Ian Mills, Wolfgang Oster, Joshua Allen. Combination of ONC201 with radiation exhibits synergistic efficacy in high grade gliomas and other advanced cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 249.

DDIS-16. ONC201 IN COMBINATION WITH RADIATION EXHIBITS SYNERGISTIC EFFICACY IN HIGH GRADE GLIOMAS AND OTHER ADVANCED CANCERS

INTRODUCTION ONC201 is the first small molecule DRD2 antagonist for oncology that is being investigated as a single agent in advanced cancer clinical trials. Downstream of DRD2 antagonism, ONC201 activates the integrated stress response pathway and apoptosis. ONC201 has exhibited preclinical and clinical anti-tumor activity in high grade gliomas. Given that ONC201 exhibits broad synergy with anti-cancer drugs, excellent safety, and single agent activity in tumor types where radiation is used routinely, we evaluated the combination of ONC201 with radiation in solid tumors.

FERMT3 contributes to glioblastoma cell proliferation and chemoresistance to temozolomide through integrin mediated Wnt signaling.

FERMT3, also known as kindlin-3, is one of three kindlin family members expressed in mammals. Kindlins are cytosolic, adaptor proteins that are important activators and regulators of integrin function. They have also been shown to play critical roles in the development and progression of various cancers. In the present study, we hypothesized that FERMT3 would enhance glioblastoma multiforme (GBM) cell survival. Indeed, expression level analyses showed significant FERMT3 upregulation in human glioma tissues as compared to normal brain tissues. The effect was particularly pronounced in high-grade gliomas. We then demonstrated that FERMT3 knockdown suppresses glioma cell proliferation and chemoresistance to temozolomide (TMZ). To determine the mechanism by which FERMT3 enhances glioma cell proliferation and chemoresistance, we examined the effects of FERMT3 on integrin activation and Wnt/β-catenin signaling. Through the use of western blot assays and TOPflash and FOPflash plasmid transfection into glioma cells lines, we demonstrated that FERMT3 regulates glioma cell activity through integrin-mediated Wnt/β-catenin signaling. These results suggest that FERMT3 activates integrin activity in high-grade gliomas to enhance glioma cell survival and chemoresistance. The present study thus indicates a potential role for FERMT3 as a genetic target in the treatment of GBM.

P08.21 Discovery of CT-179--a small molecule inhibitor of the transcription factor OLIG2 with potent anti-tumour activity in high-grade glioma

P08.21 Discovery of CT-179--a small molecule inhibitor of the transcription factor OLIG2 with potent anti-tumour activity in high-grade glioma

Altered splicing leads to reduced activation of CPEB3 in high-grade gliomas.

Cytoplasmic polyadenylation element binding proteins (CPEBs) are auxiliary translational factors that associate with consensus sequences present in 3′UTRs of mRNAs, thereby activating or repressing their translation. Knowing that CPEBs are players in cell cycle regulation and cellular senescence prompted us to investigate their contribution to the molecular pathology of gliomas–most frequent of intracranial tumors found in humans. To this end, we performed methylation analyses in the promoter regions of CPEB1-4 and identified the CPEB1 gene to be hypermethylated in tumor samples. Decreased expression of CPEB1 protein in gliomas correlated with the rising grade of tumor malignancy. Abundant expression of CPEBs2-4 was observed in several glioma specimens. Interestingly, expression of CPEB3 positively correlated with tumor progression and malignancy but negatively correlated with protein phosphorylation in the alternatively spliced region. Our data suggest that loss of CPEB3 activity in high-grade gliomas is caused by expression of alternatively spliced variants lacking the B-region that overlaps with the kinase recognition site. We conclude that deregulation of CPEB proteins may be a frequent phenomenon in gliomas and occurs on the level of transcription involving epigenetic mechanism as well as on the level of mRNA splicing, which generates isoforms with compromised biological properties.

Is development of high-grade gliomas sulfur-dependent?

We characterized γ-cystathionase, rhodanese and 3-mercaptopyruvate sulfurtransferase activities in various regions of human brain (the cortex, thalamus, hypothalamus, hippocampus, cerebellum and subcortical nuclei) and human gliomas with II to IV grade of malignancy (according to the WHO classification). The human brain regions, as compared to human liver, showed low γ-cystathionase activity. The activity of rhodanese was also much lower and it did not vary significantly between the investigated brain regions. The activity of 3-mercaptopyruvate sulfurtransferase was the highest in the thalamus, hypothalamus and subcortical nuclei and essentially the same level of sulfane sulfur was found in all the investigated brain regions. The investigations demonstrated that the level of sulfane sulfur in gliomas with the highest grades was high in comparison to various human brain regions, and was correlated with a decreased activity of γ-cystathionase, 3-mercaptopyruvate sulfurtransferase and rhodanese. This can suggest sulfane sulfur accumulation and points to its importance for malignant cell proliferation and tumor growth. In gliomas with the highest grades of malignancy, despite decreased levels of total free cysteine and total free glutathione, a high ratio of GSH/GSSG was maintained, which is important for the process of malignant cells proliferation. A high level of sulfane sulfur and high GSH/GSSG ratio could result in the elevated hydrogen sulfide levels. Because of the disappearance of γ-cystathionase activity in high-grade gliomas, it seems to be possible that 3-mercaptopyruvate sulfurtransferase could participate in hydrogen sulfide production. The results confirm sulfur dependence of malignant brain tumors.

AT-04 * A RANDOMIZED PHASE II TRIAL OF VANDETANIB IN COMBINATION WITH CARBOPLATIN VERSUS CARBOPLATIN ALONE FOLLOWED BY VANDETANIB ALONE IN ADULTS WITH RECURRENT ANAPLASTIC ASTROCYTOMA

BACKGROUND: Vandetanib is a selective VEGFR and EGFR inhibitor that targets these key pathways implicated in gliomagenesis. While single-agent vandetanib has not shown significant clinical activity in high grade glioma but preclinical experience supports potential synergistic efficacy in combination with carboplatin. METHODS: This randomized non-comparative phase-II trial evaluates the efficacy and safety of vandetanib (300 mg/d NEIAED, 400 mg/d EIAED) in combination with carboplatin (AUC 6 q 4 weeks) versus carboplatin alone (AUC 6 q 4 weeks) in adults with recurrent anaplastic glioma. Upon progression with single-agent carboplatin, single-agent vandetanib was offered. Contrast-enhanced-MRI before subsequent cycles were compared to baseline and response assessed using RANO criteria. Primary end-point was progression free survival at 6 months (PFS-6). RESULTS: 23 patients were accrued to each group (n = 46). Gender, median age, performance status, number of recurrences, 1p/19q co-deletion status, and baseline steroid doses were comparable. 60% of patients in each group had failed prior VEGF-inhibitor therapy. Most frequent treatment related grade ≥3 toxicities were lymphopenia (n = 7), thrombocytopenia (n = 4), neutropenia (n = 3), hypertension (n = 3) and transaminitis (n = 3). All grade 4 toxicities were observed in the combination-group. There were 3 partial responses in the combination group, and 1PR in the sequential-group. PFS-6 was 8.7% (95% CI: 2.3%, 32.7%) and 17.4 % (95% CI: 7.1%, 42.4%), median-PFS (months) was 1.8 (95% CI: 1.15, 2.66) and 0.95 (95% CI:0.92,2.04)while median-OS (months) was 6.5 (95% CI: 5.42, 11.7) and 9.27 (95% CI: 5.52, 43.86) in combination-group and sequential-group respectively. CONCLUSION: Although this study was not powered to detect differences between groups and did not complete accrual as planned, no meaningful differences in PFS-6, PFS or OS were observed compared to historical controls. Majority of patients were VEGF therapy resistant, which may have limited efficacy. Combination therapy was more toxic.

431P - Bevacizumab and Fotemustine for Recurrent High Grade Gliomas

ABSTRACT Aim: Primary tumours of the central nervous system account for approximately 3% of all cancers. High-grade gliomas (HGG) account for 70% of all gliomas and include anaplastic astrocytomas, anaplastic oligoastrocytomas and anaplastic oligodendrogliomas (WHO grade III), as well as glioblastomas (WHO grade IV). Median survival is 12-18 months for WHO grade IV and 2 to 5 years for WHO grade III. At recurrence there is no accepted standard of care. Recent phase II trials have demonstrated activity of nitrosoureas such as fotemustine (FTM) in recurrent HGG. Bevacizumab, a monoclonal antibody that binds to vascular endothelial growth factor, is the first antiangiogenic agent with documented activity in HGG. Methods: We have investigated the role of the combination of FTM and bevacizumab in glioblastoma (GBM) and WHO grade III gliomas (non-GBM) at first recurrence after prior therapy. From January 2011 to January 2014, data of 30 patients were collected. From recurrence, we estimated 6-month progression-free survival (PFS-6), overall survival (OS) and toxicities therapies-related. The treatment consisted of an induction phase with FTM at 75 mg/m2 intravenously on day 1, 8 and 15 and bevacizumab at 10 mg/kg intravenously on day 1 and 15, followed after an interval of 4-5 weeks by a maintenance phase with bevacizumab at 10 mg/kg every 2 weeks and FTM at 75 mg/m2 every 3 weeks until tumour progression or unacceptable toxicity. Patients who required progressive FTM dose reductions received bevacizumab alone. Results: The median time from original diagnosis to recurrence was 13 months for GBM and 35 months for non-GBM gliomas. All patients completed the induction phase and a median of 11 maintenance cycles were administered for GBM and 13 cycles for non-GBM patients. PFS-6 rate was 45% for GBM and 67% for non-GBM; median OS was 14 months for GBM and 21 months for non-GBM patients. Most patients experienced grade 1 toxicities (hypertension, fatigue, proteinuria); grade 2 toxicities were predominantly hematologic (thrombocitopenia). Conclusions: In our study the combination of FTM and bevacizumab was well tolerated and PFS-6 and OS confirmed published literature. Further studies are needed to demonstrate the benefit of this combination in the treatment of recurrent HGG. Disclosure: All authors have declared no conflicts of interest.

Plasma levels of IL-8 and g-CSF in high-grade gliomas treated with bevacizumab.

2083 Background: Bevacizumab, an anti-VEGF antibody, has shown significant activity in high grade gliomas (HGG). However, tumor recurrence inevitably occurs. Methods: We treated 63 recurrent HGG patients with poor prognostic factors with bevacizumab (10 mg/kg) and irinotecan (125 or 340 mg/m2) every 2 weeks, and investigated IL-12, IL-13, IL-17, FGF basic, G-CSF, MIP-1b, PDGF-beta, plasma levels before starting treatment and every 8 weeks by Bioplex. Ten age- and sex-matched healthy controls were used for comparison. Results: After a median follow-up of 27 weeks, median OS and PFS were 33 and 18 weeks, respectively. PFS at 6 and 12 months were 32% and 12%, respectively. OS at 6 months was 60%. Toxicity was mild. Baseline higher amounts of IL-13 (48±174 pg/ml vs 3.44±0.9 pg/ml, p=0.0001) and lower amounts of MIP-1b (35.3±20.9 pg/ml vs 67.2±18.8 pg/ml, p=0.0002), PDGF-beta (1585.5±1585 pg/ml vs 7098±1585 pg/ml, p=0.0001) and VEGF (27±39.8 pg/ml vs 54.5±32 pg/ml, p=0.001) were detected in patients than in healthy controls. In a cohort of 15 non-responders (patients who progressed 8 weeks after treatment onset), baseline IL-8 (15.7±10.8 pg/ml vs 10.9±9.4 pg/ml, p=0.03) and G-CSF (113.3±54 pg/ml vs 84.9±59.2 pg/ml, p=0.03) were significantly higher than in patients responding to treatment. In the same cohort no significant reduction of VEGF and other cytokines was observed after 8 weeks of treatment, while a decrease of plasma VEGF was observed in the remaining patients (26±32 pg/ml vs 13.3±28.5 pg/ml, p=0.001). Furthermore, in a cohort of 22 long-term responders (patients who progressed after more than 18 weeks of treatment), levels of VEGF decreased after 8 weeks of treatment when compared to baseline, whereas no difference was observed in baseline levels (23.9±22.6 pg/ml vs 9.8±9.4 pg/ml, p=0.001). Conclusions: Data suggest that high levels of IL-8 and G-CSF at baseline associated with a lack of VEGF decrease after 8 weeks of treatment identify patients who are resistant to bevacizumab. This hypothesis should be tested in a large number of patients.

Abstract 1383: High levels of CD109+ circulating endothelial cells and progenitors as marker of response to bevacizumab and irinotecan in high grade gliomas

Abstract Purpose: Bevacizumab, an anti-VEGF antibody, has shown significant activity in high grade gliomas (HGG). Previous studies emphasized the need for predictive markers of response. Experimental Design: We treated 63 recurrent HGG patients with poor prognostic factors with bevacizumab (10 mg/kg) and irinotecan (125 or 340 mg/m2) every 2 weeks, and investigated the predictive potential of circulating endothelial cells (CECs) and their progenitors (CEPs). Results: After a median follow-up of 27 weeks, median OS and PFS were 33 and 18 weeks, respectively. PFS at 6 and 12 months were 32% and 12%. OS at 6 months was 60%. No complete response but fourteen partial responses according to RANO criteria were observed. Toxicity and side effects were mild. Patients with distant intracerebral disease or leptomeningeal dissemination at baseline MRI had shorter PFS (p=0.002; p=0.01) and OS (p=0.005; p=0.03). Baseline CEP over 32.8 cells/ml (1st quartile) or CD45dimCD34+CD133+ hematopoietic committed progenitors over 27 cells/ml (1st quartile) were associated with an increased PFS (p=0.01; p=0.001, respectively). Baseline CD109+ CECs over 47.5 cells/ml (2nd quartile) were associated with longer PFS and OS (p= 0.001; p=0.02). Patients who progressed after 18 weeks of therapy or more (n=22) had baseline levels of CD109+ CECs and CD45dimCD34+VEGFR2+ cells significantly higher than others (p=0.008; p=0.04, respectively). At progression no significant change in CEC and CEP was detected. Conclusions: The data point to baseline CD109+ CECs, CEP and CD45dimCD34+CD133+ hematopoietic committed progenitors as promising markers for the selection of patients who could benefit from bevacizumab in the treatment of HGG. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1383. doi:1538-7445.AM2012-1383

Temozolomide and radiotherapy in malignant glioma: Final update of a phase II monoinstitutional study.

e12521 Background: Temozolomide (TMZ), an oral imidazotetrazine second-generation alkylating agent that enters the cerebrospinal fluid, has demonstrated antitumor activity in high-grade glioma when given as adjuvant therapy concomitant with RT, and then alone (Stupp R., NEJM,2005). Methods: The aim was to evaluate disease free (DFS) overall survival (OS) and safety TMZ in combination with RT in high-grade cerebral glioma, after surgery. RT to limited fields was administered once a day at 2 Gy/fraction, 5 days a week for 6 weeks, for a total of 60 Gy, with concomitant TMZ 75 mg/sqm daily, followed by 6 cycles of TMZ 150-200 mg/sqm daily on days 1->5 q 28 days. Results: From 11/2004 to 9/2007, we accrued 23 consecutive pts, median age 63 years (range 34-72), 18 M, 5 F; ECOG PS was 0 in 21 pts and 3 in remaining 2. 10 pts. (43,5%) had R0 resection; 7.(30,4%) underwent not-radical resection, and 6 (26,1%) were considered inoperable. Pathology was as follows: glioblastoma 14 (60,9%), anaplastic astrocytoma 4 (17,4%), astrocytoma GII 4 (17,4%), anaplastic oligodendroglyoma 1 (4,3%). 19 pts (82,6%) completed concomitant RT/CT treatment and 8 (34,7%) also completed the subsequent 6 cycles of TMZ as planned. 2 pts. (8,7%), R2 resected and inoperable respectively, suspended TMZ/RT program due to early progression and the other 2 (8,7%) PS 3 pts., received no treatment. 10/19 (52,6%) pts., who completed the first part of the planned treatment, delayed TMZ cause of PD after a median of 3 cycles (range 1-5); 1/19 (5,3%) did not receive TMZ due to PD after TMZ/RT. No G3/4 toxicity occurred DFS was 5,5 + months (range 1-47+). Second line CT with PCV was given to 5/23 pts (21,7%), TMZ dose dense in 2/23 (8,7%) and 1/23 (4,3%) underwent third line CT with fotemustine.1/23 (4,3%)was treated with rescue surgeryand 2/23 (8,7%) with stereotassic radiotherapy. Median OS was 10,1+ months (range 1,1-72+). Conclusions: Even if debulking surgery occurred in only 74% pts, this schedule is safe, feasible and active in malignant glioma. During following years, due to improvement of OS, more pts underwent further lines of CT or stereotassic radiotherapy or novel surgery, unthinkable since few years ago.

Treatment of recurrent high-grade gliomas

Although therapies for patients with recurrent high-grade gliomas are limited, there has been important progress recently. This review summarizes current treatments for recurrent high-grade gliomas with an emphasis on more novel approaches. There is increasing evidence that antiangiogenic therapies have activity in high-grade gliomas. Recently, the United States Food and Drug Administration granted accelerated approval of bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor, for the treatment of recurrent glioblastomas. Bevacizumab decreases corticosteroid requirements and improves radiologic response rates and progression-free survival. The extent to which these agents prolong overall survival remains to be determined. There is also emerging evidence that other therapies such as dose-dense and metronomic temozolomide regimens, targeted molecular agents, and other antiangiogenic therapies may have activity in recurrent high-grade gliomas. Although there has been progress in treating recurrent high-grade gliomas, the prognosis remains poor and much work still needs to be done to improve on the current results of antiangiogenic and targeted molecular therapies, as well as other novel therapies.

Paraoxonase 192 gene polymorphism and serum paraoxonase activity in high grade gliomas and meningiomas

The purpose of this study was to demonstrate the relationship between serum PON1 activity and PON 192 polymorphism in brain tumours. The distribution of PON 192 polymorphism in 42 high grade gliomas and 42 meningiomas were determined by polymerase chain reaction--based restriction fragment length polymorphism analysis and compared with 50 healthy control subjects. Serum paraoxonase1 activities were also measured and compared in the same population. We found that in both tumour groups serum PON1 activity was significantly lower than the control group (p < 0.001), but did not differ between meningiomas and high grade gliomas. There was no significant difference either in distribution of the AA, AB and BB genotypes or in the allelic frequencies, between the patient group and control subjects (p > 0.05). Our results suggest that serum PON1 as a part of the lipid peroxidation scavenging systems might be involved in the tumourigenesis of brain tumours.