Background:This multi-institutional phase I trial aimed to determine the maximum tolerated dose (MTD), dose-limiting toxicities, pharmacokinetics, and preliminary antitumor activity of berubicin (WP744 or RTA744), designed to cross the blood-brain barrier, in patients with primary brain cancers. Methods:Thirty-five patients with recurrent or refractory primary brain cancers, including glioblastoma multiforme, received berubicin infusions over 2 h for 3 consecutive days (one course) every 21 days. Daily doses escalated from 1.2 to 9.6 mg/m2 using an accelerated titration design. Plasma levels of berubicin were measured via high-performance liquid chromatography-tandem mass spectrometry to estimate pharmacokinetic parameters. Results:The daily MTD was determined to be 7.5 mg/m2. Nonhematological toxicities were minimal; no cardiotoxicity was observed. In the intention-to-treat population (n = 35), 1 patient had a durable complete response, 1 had a partial response, and 9 had stable disease, corresponding to an objective response rate of 5.7% and a disease control rate of 31.4%. In the response-evaluable subset (n = 25), the corresponding rates were 8% and 44%, respectively; these secondary estimates should be interpreted with caution as patients who discontinued early were excluded. Pharmacokinetic analysis determined a mean half-life of 32.8 h. The area under the curve increased proportionally with dose. Conclusions:The tolerability and efficacy of berubicin, including one durable complete response, warrant continued development of the molecule. Berubicin shows activity over a range of doses, including a durable response at 2.4 mg/m2. The recommended phase II dose is 7.5 mg/m2 infusions over 2 h for 3 days every 3 weeks. Trial registration:NCT00526812.

Supplementary Figure 1 from A Novel Small Molecule Inhibitor of Signal Transducers and Activators of Transcription 3 Reverses Immune Tolerance in Malignant Glioma Patients

Supplementary Figure 3. Combination therapy with macitentan and TMZ produces durable responses in mice with established LN-229 glioblastomas.

Supplementary Figure 1. Combination therapy with macitentan and TMZ leads to regression of established LN-229 glioblastomas.

Supplementary Figure 4. Macitentan suppresses activation of AKT and MAPK signaling pathways on glioma cells and tumor-associated endothelial cells in TMZresistant D54Res glioblastomas.

Legends to Supplementary Figures 1-6

Demographic and Clinical Characteristics of Glioma Patients in the Parent Epidemiological Study (N=1247).

Supplementary Figure 5. MGMT promoter methylation in orthotopically implanted LN- 229, LN-229Res, and D54Res glioblastomas.

Supplementary Figure 6. Effects of treatment on glioblastoma-associated blood vessels.

Supplementary Figure 2. Combination therapy with macitentan plus TMZ eradicates experimental glioblastomas.

Background Repurposed memantine, mefloquine, and metformin have putative anticancer activity. The objective of this phase 1 study was to determine the maximum tolerated doses (MTDs) of combinations of these agents with temozolomide (TMZ). Methods Adults with newly diagnosed glioblastoma who completed chemoradiation were eligible. The patients were assigned to receive doublet, triplet, or quadruplet therapy with TMZ combined with mefloquine, memantine, and/or metformin. Dose-limiting toxicities (DLTs) were determined, using a 3 + 3 study design. Results Of 85 enrolled patients, 4 did not complete cycle 1 (the DLT observation period) for nontoxicity reasons, and 81 were evaluable for DLT. The MTDs for doublet therapy were memantine 20 mg twice daily, mefloquine 250 mg 3 times weekly, and metformin 850 mg twice daily. For triplet therapy, the MTDs were memantine 10 mg twice daily, mefloquine 250 mg 3 times weekly, and metformin 850 mg twice daily. For quadruplet therapy, the MTDs were memantine 10 mg twice daily, mefloquine 250 mg 3 times weekly, and metformin 500 mg twice daily. DLTs included dizziness (memantine) and gastrointestinal effects (metformin). Lymphopenia was the most common adverse event (66%). From study entry, the median survival was 21 months, and the 2-year survival rate was 43%. Conclusions Memantine, mefloquine, and metformin can be combined safely with TMZ in patients with newly diagnosed glioblastoma.

2044 Background: Repurposing non-cancer drugs may represent a new source of novel therapies for glioblastoma (GBM). Memantine, mefloquine, and metformin have putative anticancer activity potentially relevant to gliomas. The aim of this phase I factorial study was to determine the maximum tolerated doses (MTD) of combinations of these agents with temozolomide (TMZ) for newly diagnosed GBM. Methods: Adults (≥18 years of age) with newly diagnosed GBM who received chemoradiation with TMZ, with no progressive disease on post-treatment imaging, were eligible. The patients were sequentially enrolled to one of seven treatment arms (doublet, triplet, or quadruplet therapy with TMZ combined with mefloquine, memantine, and/or metformin). Dose-limiting toxicities (DLTs) were determined over the first 28 days of treatment, using a 3+3 study design. Results: Of 85 enrolled patients, 81 patients completed the MTD period. The final MTDs for doublet therapy arms (TMZ plus 1 drug) were memantine 20 mg twice a day (BID), mefloquine 250 mg 3 times/week, and metformin 850 mg BID. For triplet therapy arms, the MTDs were memantine 10 mg BID, mefloquine 250 mg 3 times/week, and metformin 850 mg BID. For quadruplet therapy, the MTDs were memantine 10 mg BID, mefloquine 250 mg 3 times/week, and metformin 500 mg BID. DLTs included dizziness related to memantine and gastrointestinal effects related to metformin. Lymphopenia was the most common adverse event (66%); fatigue was the second most common (65%). From study entry, median survival was 21 months; the 2-year survival rate was 43%. Conclusions: Memantine, mefloquine, and metformin can be safely combined with TMZ as treatment for newly diagnosed GBM. The MTDs determined by this study can be used for subsequent clinical trials.

Purpose DNX-2401 (Delta-24-RGD; tasadenoturev) is a tumor-selective, replication-competent oncolytic adenovirus. Preclinical studies demonstrated antiglioma efficacy, but the effects and mechanisms of action have not been evaluated in patients. Methods A phase I, dose-escalation, biologic-end-point clinical trial of DNX-2401 was conducted in 37 patients with recurrent malignant glioma. Patients received a single intratumoral injection of DNX-2401 into biopsy-confirmed recurrent tumor to evaluate safety and response across eight dose levels (group A). To investigate the mechanism of action, a second group of patients (group B) underwent intratumoral injection through a permanently implanted catheter, followed 14 days later by en bloc resection to acquire post-treatment specimens. Results In group A (n = 25), 20% of patients survived > 3 years from treatment, and three patients had a ≥ 95% reduction in the enhancing tumor (12%), with all three of these dramatic responses resulting in > 3 years of progression-free survival from the time of treatment. Analyses of post-treatment surgical specimens (group B, n = 12) showed that DNX-2401 replicates and spreads within the tumor, documenting direct virus-induced oncolysis in patients. In addition to radiographic signs of inflammation, histopathologic examination of immune markers in post-treatment specimens showed tumor infiltration by CD8+ and T-bet+ cells, and transmembrane immunoglobulin mucin-3 downregulation after treatment. Analyses of patient-derived cell lines for damage-associated molecular patterns revealed induction of immunogenic cell death in tumor cells after DNX-2401 administration. Conclusion Treatment with DNX-2401 resulted in dramatic responses with long-term survival in recurrent high-grade gliomas that are probably due to direct oncolytic effects of the virus followed by elicitation of an immune-mediated antiglioma response.

2002 Background: DNX-2401 is a replication-competent, tumor-selective, oncolytic adenovirus with enhanced infectivity that causes durable tumor control by killing tumor cells and eliciting antitumor immunity. To increase immune activation, a phase 1b randomized study of intratumoral DNX-2401 alone versus DNX-2401 with interferon gamma (IFN) was conducted. Methods: A total of 27 patients with biopsy-confirmed glioblastoma at first or second recurrence received a single intratumoral injection of 3e10 vp DNX-2401. Patients were randomized in a 2:1 ratio to receive 50 mcg/m2 of subcutaneous IFN (Actimmune) Q3W initiated 14 days after DNX-2401 or to be followed without further treatment for safety and survival. Results: Twenty-seven (27) patients were enrolled following first (59%) or second (41%) recurrence having previously failed surgery, radiation, and temozolomide (100%). The median longest tumor diameter was 40 mm (range 20-77 mm). Patients were randomized to DNX-2401 followed by IFN (n = 18) or to DNX-2401 alone (n = 9). Due to the poor tolerability of IFN, the median duration of treatment was only 6 weeks (range 0-30 weeks), and two patients did not initiate treatment as scheduled due to early clinical deterioration. The most frequent grade 3-4 AEs across treatment groups were fatigue, headache, and seizures consistent with pre-existing symptoms, underlying disease and/or surgery. Based upon a preliminary intent-to-treat analysis, IFN did not appear to provide additional benefit. However, OS-12 and OS-18 for all patients enrolled was 33% and 22%, respectively regardless of treatment assignment. Three patients remain alive at 19, 21, and 22 months (DNX-2401, n = 1; DNX-2401 + IFN, n = 2). Interestingly, 50% of patients with a baseline tumor diameter of ≤ 42 mm survived beyond 12 months, potentially identifying a sub-population of patients that may live longer following intratumoral DNX-2401. Conclusions: DNX-2401 was well tolerated as monotherapy. Although the addition of IFN did not improve survival, clinical activity following a single injection of DNX-2401 is encouraging and supports an ongoing Phase II study of DNX-2401 for recurrent glioblastoma. Clinical trial information: NCT02197169.

Survival for glioblastoma (GBM) patients with an unmethyated MGMT promoter in their tumor is generally worse than methylated MGMT tumors, as temozolomide (TMZ) response is limited. How to better treat patients with unmethylated MGMT is unknown. We performed a trial combining erlotinib and bevacizumab in unmethylated GBM patients after completion of radiation (RT) and TMZ. GBM patients with an unmethylated MGMT promoter were trial eligible. Patient received standard RT (60 Gy) and TMZ (75 mg/m2 × 6 weeks) after surgical resection of their tumor. After completion of RT they started erlotinib 150 mg daily and bevacizumab 10 mg/kg every 2 weeks until progression. Imaging evaluations occurred every 8 weeks. The primary endpoint was overall survival. Of the 48 unmethylated patients enrolled, 46 were evaluable (29 men and 17 women); median age was 55.5 years (29-75) and median KPS was 90 (70-100). All patients completed RT with TMZ. The median number of cycles (1 cycle was 4 weeks) was 8 (2-47). Forty-one patients either progressed or died with a median progression free survival of 9.2 months. At a follow up of 33 months the median overall survival was 13.2 months. There were no unexpected toxicities and most observed toxicities were categorized as CTC grade 1 or 2. The combination of erlotinib and bevacizumab is tolerable but did not meet our primary endpoint of increasing survival. Importantly, more trials are needed to find better therapies for GBM patients with an unmethylated MGMT promoter.

Brain metastasis is a major cause of morbidity and mortality in patients with breast cancer. Our previous studies indicated that Stat3 plays an important role in brain metastasis. Here, we present evidence that Stat3 functions at the level of the microenvironment of brain metastases. Stat3 controlled constitutive and inducible VEGFR2 expression in tumor-associated brain endothelial cells. Furthermore, inhibition of Stat3 by WP1066 decreased the incidence of brain metastases and increased survival in a preclinical model of breast cancer brain metastasis. WP1066 inhibited Stat3 activation in tumor-associated endothelial cells, reducing their infiltration and angiogenesis. WP1066 also inhibited breast cancer cell invasion. Our results indicate that WP1066 can inhibit tumor angiogenesis and brain metastasis mediated by Stat3 in endothelial and tumor cells.

2039 Background: Vorinostat (Vor), a histone deacetylase inhibitor, has shown preliminary activity in recurrent GBM. Preclinical studies demonstrated that Vor can overcome resistance to isotretinoin (cRA) and temozolomide (TMZ). We hypothesized that Vor could overcome resistance to cRA and cytotoxic chemotherapy in the treatment of recurrent gliomas. Carboplatin (CBP) was originally selected as the cytotoxic drug since eligible patients would have failed prior standard TMZ therapy. Methods: We conducted a Phase I study of combinations of these agents preceding a proposed adaptive randomized 3-arm Phase II study. Adults with recurrent malignant glioma were enrolled into one of 3 arms. Arm 1: Vor + cRA, Arm 2: CBP + cRA, or Arm 3: Vor + cRA + CBP. Dose escalation was by a 3 + 3 design to define the maximum tolerated dose (MTD). Due to excessive toxicity in arms containing CBP, this drug was replaced by dose-dense TMZ due to preliminary evidence of activity in recurrent gliomas. Results: A total of 55 patients (8 anaplastic gliomas, 47 GBM) were enrolled from 11/2007 to 03/2012. Among 52 evaluable patients, a total of 11 DLTs were seen, but none after introduction of TMZ to Arm 3. MTDs are summarized in Table. Toxicities included: Arm 1 – neutropenia, thrombocytopenia, pulmonary embolism, elevated AST (DLT), and hypertriglyceridemia (DLT); Arm 2/CBP – neutropenia, thrombocytopenia (DLT), and hypertriglyceridemia; Arm 3A/CBP - thrombocytopenia (DLT) and hypokalemia (DLT); Arm 3B/TMZ – thrombocytopenia, hypertriglyceridemia, fatigue. Best response was stable disease in 26 patients, for ≥ 4 months in 15 patients; 10 patients achieved 6-month progression-free survival; 7 of whom had GBM. Conclusions: Two and 3 drug combinations of Vor, cRA, and dose-dense TMZ were well tolerated with MTD that will be used in a multicenter adaptive randomized Phase II study in the near future. Clinical trial information: NCT00555399. MTD Arm 1 (n=14) Vor 400 mg/day, D 1-14 cRA 100 mg/m2/day, D 1-21 Arm 2/CBP (n=12)* CBP AUC 5, D 1 cRA 100 mg/m2/day, D 1-21 Arm 3A/CBP (n=19)* De-escalation to dose level -3 (Vor 300 mg/day x 14 days; cRA 100 mg/m2/day x 21 days; CBP AUC 4) Arm 3B/TMZ (n =10) Vor 500 mg/day, D 1-7 & 15-21 cRA 100 mg/m2/day, D 1-21 TMZ 150 mg/m2/day, D 1-7 &15-21 *Not going to Phase II

Antiangiogenic therapy can rapidly reduce vascular permeability and cerebral edema but high doses of bevacizumab may induce selective pressure to promote resistance. This trial evaluated the efficacy of low dose bevacizumab in combination with lomustine (CCNU) compared to standard dose bevacizumab in patients with recurrent glioblastoma. Patients (N = 71) with recurrent glioblastoma who previously received radiation and temozolomide were randomly assigned 1:1 to receive bevacizumab monotherapy (10 mg/kg) or low dose bevacizumab (5 mg/kg) in combination with lomustine (90 mg/m 2 ). The primary end point was progression-free survival (PFS) based on a blinded, independent radiographic assessment of post-contrast T1-weighted and non-contrast T2/FLAIR weighted magnetic resonance imaging (MRI) using RANO criteria. For 69 evaluable patients, median PFS was not significantly longer in the low dose bevacizumab + lomustine arm (4.34 months, CI 2.96–8.34) compared to the bevacizumab alone arm (4.11 months, CI 2.69–5.55, p = 0.19). In patients with first recurrence, there was a trend towards longer median PFS time in the low dose bevacizumab + lomustine arm (4.96 months, CI 4.17–13.44) compared to the bevacizumab alone arm (3.22 months CI 2.5–6.01, p = 0.08). The combination of low dose bevacizumab plus lomustine was not superior to standard dose bevacizumab in patients with recurrent glioblastoma. Although the study was not designed to exclusively evaluate patients at first recurrence, a strong trend towards improved PFS was seen in that subgroup for the combination of low dose bevacizumab plus lomustine. Further studies are needed to better identify such subgroups that may most benefit from the combination treatment.

Abstract Hyperpolarized [1-13C]-pyruvate has shown tremendous promise as an agent for imaging tumor metabolism with unprecedented sensitivity and specificity. Imaging hyperpolarized substrates by magnetic resonance is unlike traditional MRI because signals are highly transient and their spatial distribution varies continuously over their observable lifetime. Therefore, new imaging approaches are needed to ensure optimal measurement under these circumstances. Constrained reconstruction algorithms can integrate prior information, including biophysical models of the substrate/target interaction, to reduce the amount of data that is required for image analysis and reconstruction. In this study, we show that metabolic MRI with hyperpolarized pyruvate is biased by tumor perfusion and present a new pharmacokinetic model for hyperpolarized substrates that accounts for these effects. The suitability of this model is confirmed by statistical comparison with alternates using data from 55 dynamic spectroscopic measurements in normal animals and murine models of anaplastic thyroid cancer, glioblastoma, and triple-negative breast cancer. The kinetic model was then integrated into a constrained reconstruction algorithm and feasibility was tested using significantly undersampled imaging data from tumor-bearing animals. Compared with naïve image reconstruction, this approach requires far fewer signal-depleting excitations and focuses analysis and reconstruction on new information that is uniquely available from hyperpolarized pyruvate and its metabolites, thus improving the reproducibility and accuracy of metabolic imaging measurements. Cancer Res; 75(22); 4708–17. ©2015 AACR.