Faculty Bibliography

BACKGROUND:Numerous studies suggest that radiation can boost antitumor immune response by stimulating release of tumor-specific antigens. However, the optimal timing between radiotherapy and immune checkpoint blockade to achieve potentially synergistic benefits is unclear. MATERIAL AND METHODS/METHODS:Multi-institutional retrospective analysis was conducted of ninety-nine metastatic melanoma patients from 2007 to 2014 treated with ipilimumab who later received stereotactic radiosurgery (SRS) for new brain metastases that developed after starting immunotherapy. All patients had complete blood count acquired before SRS. Primary outcomes were intracranial disease control and overall survival (OS). RESULTS:The median follow-up time was 15.5months. In the MD Anderson cohort, patients who received SRS after 5.5months (n=20) of their last dose of ipilimumab had significantly worse intracranial control than patients who received SRS within 5.5months (n=51) (median 3.63 vs. 8.09months; hazard ratio [HR] 2.07, 95% confidence interval [CI] 1.03-4.16, p=0.041). OS was not different between the two arms. The improvement in intracranial control was confirmed in an independent validation cohort of 28 patients treated at Yale-New Haven Hospital. Circulating absolute lymphocyte count before SRS predicted for treatment response as those with baseline counts >1000/µL had reduced risk of intracranial recurrence compared with those with ≤1000/µL (HR 0.46, 95% CI 0.0.23-0.94, p=0.03). CONCLUSIONS:In this multi-institutional study, patients who received SRS for new brain metastases within 5.5months after ipilimumab therapy had better intracranial disease control than those who received SRS later. Moreover, higher circulating lymphocyte count was associated with improved intracranial disease control.

ATG4B stimulates autophagy by promoting autophagosome formation through reversible modification of ATG8. We identify ATG4B as a substrate of mammalian sterile20-like kinase (STK) 26/MST4. MST4 phosphorylates ATG4B at serine residue 383, which stimulates ATG4B activity and increases autophagic flux. Inhibition of MST4 or ATG4B activities using genetic approaches or an inhibitor of ATG4B suppresses autophagy and the tumorigenicity of glioblastoma (GBM) cells. Furthermore, radiation induces MST4 expression, ATG4B phosphorylation, and autophagy. Inhibiting ATG4B in combination with radiotherapy in treating mice with intracranial GBM xenograft markedly slows tumor growth and provides a significant survival benefit. Our work describes an MST4-ATG4B signaling axis that influences GBM autophagy and malignancy, and whose therapeutic targeting enhances the anti-tumor effects of radiotherapy.

Glioblastoma (GBM) is the most common and lethal brain tumor. Gene expression profiling has classified GBM into distinct subtypes, including proneural, mesenchymal, and classical, and identifying therapeutic vulnerabilities of these subtypes is an extremely high priority. We leveraged The Cancer Genome Atlas (TCGA) data, in particular for microRNA expression, to seek druggable core pathways in GBM. The E2F1-regulated miR-17˜92 cluster and its analogs are shown to be highly expressed in proneural GBM and in GSC lines, suggesting the E2F cell cycle pathway might be a key driver in proneural GBM. Consistently, CDK4/6 inhibition with palbociclib preferentially inhibited cell proliferation in vitro in a majority of proneural GSCs versus those of other subtypes. Palbociclib treatment significantly prolonged survival of mice with established intracranial xenografts of a proneural GSC line. We show that most of these sensitive PN GSCs expressed higher levels of CDK6 and had intact Rb1, while two GSC lines with CDK4 overexpression and null Rb1 were highly resistant to palbociclib. Importantly, palbociclib treatment of proneural GSCs upregulated mesenchymal-associated markers and downregulated proneural-associated markers, suggesting that CDK4/6 inhibition induced proneural-mesenchymal transition and underscoring the enhanced role of the E2F cell cycle pathway in the proneural subtype. Lastly, the combination of palbociclib and N,N-diethylaminobenzaldehyde, an inhibitor of the mesenchymal driver ALDH1A3, showed strong synergistic inhibitory effects against proneural GSC proliferation. Taken together, our results reveal that proneural GBM has increased vulnerability to CDK4/6 inhibition, and the proneural subtype undergoes dynamic reprogramming upon palbociclib treatment-suggesting the need for a combination therapeutic strategy.

We leveraged IDH wild-type glioblastomas, derivative neurospheres, and single-cell gene expression profiles to define three tumor-intrinsic transcriptional subtypes designated as proneural, mesenchymal, and classical. Transcriptomic subtype multiplicity correlated with increased intratumoral heterogeneity and presence of tumor microenvironment. In silico cell sorting identified macrophages/microglia, CD4⁺ T lymphocytes, and neutrophils in the glioma microenvironment. NF1 deficiency resulted in increased tumor-associated macrophages/microglia infiltration. Longitudinal transcriptome analysis showed that expression subtype is retained in 55% of cases. Gene signature-based tumor microenvironment inference revealed a decrease in invading monocytes and a subtype-dependent increase in macrophages/microglia cells upon disease recurrence. Hypermutation at diagnosis or at recurrence associated with CD8⁺ T cell enrichment. Frequency of M2 macrophages detection associated with short-term relapse after radiation therapy.

Purpose: HSP90, a highly conserved molecular chaperone that regulates the function of several oncogenic client proteins, is altered in glioblastoma. However, HSP90 inhibitors currently in clinical trials are short-acting, have unacceptable toxicities, or are unable to cross the blood-brain barrier (BBB). We examined the efficacy of onalespib, a potent, long-acting novel HSP90 inhibitor as a single agent and in combination with temozolomide (TMZ) against gliomas in vitro and in vivoExperimental Design: The effect of onalespib on HSP90, its client proteins, and on the biology of glioma cell lines and patient-derived glioma-initiating cells (GSC) was determined. Brain and plasma pharmacokinetics of onalespib and its ability to inhibit HSP90 in vivo were assessed in non-tumor-bearing mice. Its efficacy as a single agent or in combination with TMZ was assessed in vitro and in vivo using zebrafish and patient-derived GSC xenograft mouse glioma models.Results: Onalespib-mediated HSP90 inhibition depleted several survival-promoting client proteins such as EGFR, EGFRvIII, and AKT, disrupted their downstream signaling, and decreased the proliferation, migration, angiogenesis, and survival of glioma cell lines and GSCs. Onalespib effectively crossed the BBB to inhibit HSP90 in vivo and extended survival as a single agent in zebrafish xenografts and in combination with TMZ in both zebrafish and GSC mouse xenografts.Conclusions: Our results demonstrate the long-acting effects of onalespib against gliomas in vitro and in vivo, which combined with its ability to cross the BBB support its development as a potential therapeutic agent in combination with TMZ against gliomas. Clin Cancer Res; 23(20); 6215-26. ©2017 AACR.

Background:Glioblastoma (GBM) is the most common primary malignant brain tumor. Nomograms are often used for individualized estimation of prognosis. This study aimed to build and independently validate a nomogram to estimate individualized survival probabilities for patients with newly diagnosed GBM, using data from 2 independent NRG Oncology Radiation Therapy Oncology Group (RTOG) clinical trials. Methods:This analysis included information on 799 (RTOG 0525) and 555 (RTOG 0825) eligible and randomized patients with newly diagnosed GBM and contained the following variables: age at diagnosis, race, gender, Karnofsky performance status (KPS), extent of resection, O6-methylguanine-DNA methyltransferase (MGMT) methylation status, and survival (in months). Survival was assessed using Cox proportional hazards regression, random survival forests, and recursive partitioning analysis, with adjustment for known prognostic factors. The models were developed using the 0525 data and were independently validated using the 0825 data. Models were internally validated using 10-fold cross-validation, and individually predicted 6-, 12-, and 24-month survival probabilities were generated to measure the predictive accuracy and calibration against the actual survival status. Results:A final nomogram was built using the Cox proportional hazards model. Factors that increased the probability of shorter survival included greater age at diagnosis, male gender, lower KPS, not having total resection, and unmethylated MGMT status. Conclusions:A nomogram that assesses individualized survival probabilities (6-, 12-, and 24-mo) for patients with newly diagnosed GBM could be useful to health care providers for counseling patients regarding treatment decisions and optimizing therapeutic approaches. Free software for implementing this nomogram is provided: http://cancer4.case.edu/rCalculator/rCalculator.html.

Background:Human mesenchymal stem cells (hMSCs) have been shown to reside as stromal cells in human gliomas as glioma-associated hMSCs (GA-hMSCs), but their biological role remains unclear. Because recent evidence indicates that GA-hMSCs drive tumor cell proliferation and stemness, we hypothesized that a higher percentage of GA-hMSCs in tumors predicts poor patient prognosis. Method:We determined the percentage of cells coexpressing GA-hMSC markers CD105+/CD73+/CD90+ from patients with newly diagnosed high-grade glioma and analyzed the association between this percentage and overall survival (OS) in 3 independent cohorts: fresh surgical glioblastoma specimens (cohort 1, N = 9), cultured tumor specimens at passage 3 (cohort 2, N = 28), and The Cancer Genome Atlas (TCGA) database. Results:In all cohorts, patient OS correlated with the percentages of GA-hMSCs in tumors. For cohort 1, the median OS of patients with tumors with a low percentage of triple-positive cells was 46 months, and for tumors with a high percentage of triple-positive cells, it was 12 months (hazard ratio [HR] = 0.24; 95% CI: 0.02-0.5, P = .02). For cohort 2, the median OS of patients with tumors with a low percentage of GA-hMSCs was 66 months, and for tumors with a high percentage, it was 11 months (HR = 0.38; 95% CI: 0.13-0.9, P = .04). In the database of TCGA, the median OS times in patients with high and low coexpression levels of CD105/CD73/CD90 were 8.4 months and 13.1 months (HR = 0.4; 95% CI: 0.1-0.88; P = .04), respectively. Conclusions:The percentage of GA-MSCs inversely correlates with OS, suggesting a role for GA-MSCs in promoting aggressive behavior of gliomas.

Glioblastoma (GBM) is an ideal candidate disease for signal transduction targeted therapy because the majority of these tumors harbor genetic alterations that result in aberrant activation of growth factor signaling pathways. Loss of heterozygosity of chromosome 10, mutations in the tumor suppressor gene PTEN, and PI3K mutations are molecular hallmarks of GBM and indicate poor prognostic outcomes in many cancers. Consequently, inhibiting the PI3K pathway may provide therapeutic benefit in these cancers. PI3K inhibitors generally block proliferation rather than induce apoptosis. To restore the sensitivity of GBM to apoptosis induction, targeted agents have been combined with conventional therapy. However, the molecular heterogeneity and infiltrative nature of GBM make it resistant to traditional single agent therapy. Our objectives were to test a dual PI3K/mTOR inhibitor that may cross the blood-brain barrier (BBB) and provide the rationale for using this inhibitor in combination regimens to chemotherapy-induced synergism in GBM. Here we report the preclinical potential of a novel, orally bioavailable PI3K/mTOR dual inhibitor, DS7423 (hereafter DS), in in-vitro and in-vivo studies. DS was tested in mice, and DS plasma and brain concentrations were determined. DS crossed the BBB and led to potent suppression of PI3K pathway biomarkers in the brain. The physiologically relevant concentration of DS was tested in 9 glioma cell lines and 22 glioma-initiating cell (GIC) lines. DS inhibited the growth of glioma tumor cell lines and GICs at mean 50% inhibitory concentration values of less than 250 nmol/L. We found that PI3K mutations and PTEN alterations were associated with cellular response to DS treatment; with preferential inhibition of cell growth in PI3KCA-mutant and PTEN altered cell lines. DS showed efficacy and survival benefit in the U87 and GSC11 orthotopic models of GBM. Furthermore, administration of DS enhanced the antitumor efficacy of temozolomide against GBM in U87 glioma models, which shows that PI3K/mTOR inhibitors may enhance alkylating agent-mediated cytotoxicity, providing a novel regimen for the treatment of GBM. Our present findings establish that DS can specifically be used in patients who have PI3K pathway activation and/or loss of PTEN function. Further studies are warranted to determine the potential of DS for glioma treatment.

The classification of brain tumors has traditionally depended on microscopic examination of hematoxylin and eosin-stained tissue sections. The increased understanding of clinically relevant genetic alterations has led to the incorporation of molecular signatures as part of the diagnosis of brain malignancies. Advances in sequencing technologies have facilitated the use of next-generation sequencing (NGS) assays in clinical laboratories. We performed a retrospective analysis of sequencing results for 381 brain tumors tested by NGS at our institution using a validated, commercially available panel. The results of the NGS assay were analyzed in conjunction with the results of immunohistochemical stains. A genetic alteration was detected in approximately two thirds of the cases. The most commonly mutated genes were TP53 (37.2%), IDH1 (29.4%), PIK3CA (8%), PTEN (8%), and EGFR (7.5%). BRAF mutations were detected in ∼3% of the cases, including 50% of gangliogliomas and ∼20% of gliosarcomas. No mutations were detected in 6 medulloblastomas. PIK3CA and CTNNB1 mutations were detected in 1 rosette-forming glioneuronal tumor and 1 adamantinomatous craniopharyngioma, respectively. Approximately 23% of cases showed amplification of 1 or more of the genes included in the NGS panel. This analysis demonstrates the utility of NGS for detecting genetic alterations in brain tumors in the clinical setting.

Background/UNASSIGNED:Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Prognosis remains poor despite multimodal therapy. Developing alternative treatments is essential. Drugs targeting kinases within the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) effectors of receptor tyrosine kinase (RTK) signaling represent promising candidates. Methods/UNASSIGNED:We previously developed a non-germline genetically engineered mouse model of GBM in which PI3K and MAPK are activated via Pten deletion and KrasG12D in immortalized astrocytes. Using this model, we examined the influence of drug potency on target inhibition, alternate pathway activation, efficacy, and synergism of single agent and combination therapy with inhibitors of these 2 pathways. Efficacy was then examined in GBM patient-derived xenografts (PDX) in vitro and in vivo. Results/UNASSIGNED:PI3K and mitogen-activated protein kinase kinase (MEK) inhibitor potency was directly associated with target inhibition, alternate RTK effector activation, and efficacy in mutant murine astrocytes in vitro. The kinomes of GBM PDX and tumor samples were heterogeneous, with a subset of the latter harboring MAPK hyperactivation. Dual PI3K/MEK inhibitor treatment overcame alternate effector activation, was synergistic in vitro, and was more effective than single agent therapy in subcutaneous murine allografts. However, efficacy in orthotopic allografts was minimal. This was likely due to dose-limiting toxicity and incomplete target inhibition. Conclusion/UNASSIGNED:Drug potency influences PI3K/MEK inhibitor-induced target inhibition, adaptive kinome reprogramming, efficacy, and synergy. Our findings suggest that combination therapies with highly potent, brain-penetrant kinase inhibitors will be required to improve patient outcomes.