Combination Therapy In Glioblastoma Research Articles

Enhancing Glioblastoma Immunotherapy with Integrated Chimeric Antigen Receptor T Cells through the Re-Education of Tumor-Associated Microglia and Macrophages.

Glioblastoma (GBM) is an aggressive brain cancer that is highly resistant to treatment including chimeric antigen receptor (CAR)-T cells. Tumor-associated microglia and macrophages (TAMs) are major contributors to the immunosuppressive GBM microenvironment, which promotes tumor progression and treatment resistance. Hence, the modulation of TAMs is a promising strategy for improving the immunotherapeutic efficacy of CAR-T cells against GBM. Molecularly targeting drug pexidartinib (PLX) has been reported to re-educate TAMs toward the antitumorigenic M1-like phenotype. Here, we developed a cell-drug integrated technology to reversibly conjugate PLX-containing liposomes (PLX-Lip) to CAR-T cells and establish tumor-responsive integrated CAR-T cells (PLX-Lip/AZO-T cells) as a combination therapy for GBM. We used a mouse model of GBM to show that PLX-Lip was stably maintained on the surface of PLX-Lip/AZO-T cells in circulation and these cells could transmigrate across the blood-brain barrier and deposit PLX-Lip at the tumor site. The uptake of PLX-Lip by TAMs effectively re-educated them into the M1-like phenotype, which in turn boosted the antitumor function of CAR-T cells. GBM tumor growth was completely eradicated in 60% of the mice after receiving PLX-Lip/AZO-T cells and extended their overall survival time beyond 50 days; in comparison, the median survival time of mice in other treatment groups did not exceed 35 days. Overall, we demonstrated the successful fusion of CAR-T cells and small-molecule drugs with the cell-drug integrated technology. These integrated CAR-T cells provided a superior combination strategy for GBM treatment and presented a reference for the construction of integrated cell-based drugs.

EXTH-23. EFFECT OF EVOGLIPTIN AND TEMOZOLOMIDE COMBINATION THERAPY FOR THE TREATMENT OF GLIOBLASTOMA, IN VITRO AND IN VIVO

Abstract A recent study showed that high dipeptidyl-peptidase IV (DPP4) expression affects glioma progression. The purpose of this study is to investigate the synergistic effect of a DDP4 inhibitor, evogliptin and temzolomide (TMZ) combination therapy for glioblastoma (GBM) in vitro and in vivo. U87, U252, and A172 cells were treated with 50, 250, and 500 uM TMZ or with 5, 10, and 20 mM evogliptin for 24, 48 and 72 hrs. For in vivo experiment, GBM xenograft models were used. Three groups (n=5 mice) were treated with TMZ(15mg/kg/day), evogliptin (2mg/25g/day), and combination of TMZ and evogliptin via i.p. injection for 4 weeks. For evaluating the relevant mechanism, Affymetrix miRNA arrays are used. The MTT assay revealed that the combination treatment had higher cytotoxicity against U87, U251, and A172 cells than the monotherapy had after 48 and 72-hr treatment. mRNA DDP4 levels were much more decreased after combination administration. The median survival of each group was 43.6, 55.2, 53.2, 65.2, and 71.3 days in the control, evogliptin, TMZ, the first combination therapy with low-dose evogliptin (2mg/25g/day) and TMZ, and the second combination therapy with high-dose evogliptin (10mg/25g/day) and TMZ, respectively. In Affymetrix miRNA arrays, miR-1246, miR-149-3p, miR-3148, miR-4440, miR-4484, miR-4535, miR-6126, miR-6780b-5p, and miR-7110-5p were increased after evogliptin treatment. miR-149-5p, miR-23a-5p, miR-3195, miR-4330, miR-4497, miR-4669, miR-671-3p, and miR-744-5p were decreased. miR-4440 and miR-6780b-5p were also increased following combination therapy. In the TCGA dataset, the survival of glioma patients with low DPP4 expression is longer than that in patients with high DPP4. A DDP4 inhibitor, evoglipin regulates miRNA in GBM cell lines. The combination of evogliptin and TMZ had a moderate effect on the treatment of GBM in vitro and in vivo. miR-4440 and miR-6780b-5p could be associated with inhibiting the proliferation of GBM cells by targeting DPP4.

Combining Radiotherapy with PI3K Isoform-Selectivity Inhibitor Increases Radiosensitivity And Suppresses Tumor Growth In Glioblastoma Cells

Glioblastoma (GBM) is the most malignant cancer that occurs within the brain and shows the dismal prognosis. The phosphoinositide 3-kinase (PI3K)-AKT signaling pathway plays a critical role in GBM. Also, the activation of Akt due to irradiation induces radioresistance. But, the pan-PI3K inhibitors have side-effects in clinical trial. Different PI3K isoforms play non-redundant roles in particular tumor. So, it is expected that selective inhibition of PI3K isoforms decrease side effects. Therefore, we investigated whether combining radiotherapy with the PI3K isoform inhibitor decreases radioresistance and tumor growth in GBM. Glioma 261 expressed luciferase (GL261-luc) cell lines were used to identify the effects of combination therapy. GL261-luc were irradiated 1Gy with or without PI3K isoform inhibitors. After irradiation, clonogenic assay was performed to confirm the GBM survival rate and observed inhibition of PI3K-AKT signaling pathway using western blot (WB) assay. In order to confirm to repair DNA double strand breaks (DSB), the expression of phospho-H2A.X was confirmed by immunofluorescence. Also, to observe the effects of combination therapy in GBM in vivo, an orthotopic mouse model was established by intracranial injection implanting GL261-luc. Irradiation was performed specifically to brain with 2Gy, 3Gy and 4Gy for 5 days with or without PI3K-α isoform inhibitor. We observed tumor growth using IVIS image and analyzed splenocyte RNA and protein level using RT-PCR and WB. According to inhibition of PI3K-AKT signaling, GL261-luc radiosensitivity was significantly increased. GL261-luc irradiated by 1Gy was suppressed cell growth about 70% compared to control (p<0.001). When irradiated with PI3K-isform inhibitor, each survival rate was about 18% (PI3K-pan, p<0.00001), 25% (PI3K-α, p<0.00001), 30% (PI3K-δ, p<0.00001), 45% (PI3K-γ/δ, p<0.00001). Also, the expression of phospho-H2A.X, DNA DSB marker, was increased by combination therapy. Also, GL261-luc irradiated with PI3K- isoform inhibitor was decreased the ability of migration. Especially, the effects of combining radiotherapy with PI3K-α inhibitor was similar as much as with PI3K-pan inhibitor (In vitro). So, we selected PI3K-α inhibitor and analyzed combination therapy effects in GBM in vivo. We demonstrated that combining radiotherapy with the PI3K-α isoform inhibitor was markedly delayed tumor growth than without inhibitor (p<0.0001). In addition, the expression of PD1, regulator of cancer immune system, in splenocyte was increased by combination therapy (p<0.001) (In vivo). Our findings demonstrate that combining radiotherapy with the PI3K-α isoform increased radiosensitivity. Also, GBM tumor growth was markedly suppressed and increased immunogenic marker such as PD1. Combining radiotherapy with the PI3K-α isoform inhibitor would be a potential strategy for overcoming radioresistance in GBM, but further study should be necessary.

Xenograft-based, platform-independent gene signatures to predict response to alkylating chemotherapy, radiation, and combination therapy for glioblastoma.

Predictive molecular biomarkers to select optimal treatment for patients with glioblastoma and other cancers are lacking. New strategies are needed when large randomized trials with correlative molecular data are not feasible. Gene signatures (GS) were developed from 31 orthotopic glioblastoma patient-derived xenografts (PDXs), treated with standard therapies, to predict benefit from radiotherapy (RT-GS), temozolomide (Chemo-GS), or the combination (ChemoRT-GS). Independent validation was performed in a heterogeneously treated clinical cohort of 502 glioblastoma patients with overall survival as the primary endpoint. Multivariate Cox analysis was used to adjust for confounding variables and evaluate interactions between signatures and treatment. PDX models recapitulated the clinical heterogeneity of glioblastoma patients. RT-GS, Chemo-GS, and ChemoRT-GS were correlated with benefit from treatment in the PDX models. In independent clinical validation, higher RT-GS scores were associated with increased survival only in patients receiving RT (P = 0.0031, hazard ratio [HR] = 0.78 [0.66-0.92]), higher Chemo-GS scores were associated with increased survival only in patients receiving chemotherapy (P < 0.0001, HR = 0.66 [0.55-0.8]), and higher ChemoRT-GS scores were associated with increased survival only in patients receiving ChemoRT (P = 0.0001, HR = 0.54 [0.4-0.74]). RT-GS and ChemoRT-GS had significant interactions with treatment on multivariate analysis (P = 0.0009 and 0.02, respectively), indicating that they are bona fide predictive biomarkers. Using a novel PDX-driven methodology, we developed and validated 3 platform-independent molecular signatures that predict benefit from standard of care therapies for glioblastoma. These signatures may be useful to personalize glioblastoma treatment in the clinic and this approach may be a generalizable method to identify predictive biomarkers without resource-intensive randomized trials.

Development and Validation of Xenograft-Based Platform-Independent Gene Signatures That Predict Response to Alkylating Chemotherapy, Radiation, and Combination Therapy in Glioblastoma

Development and Validation of Xenograft-Based Platform-Independent Gene Signatures That Predict Response to Alkylating Chemotherapy, Radiation, and Combination Therapy in Glioblastoma

P01.071 Genomic profiling identifies tubulin mutations that may predict response to depatuxizumab mafodotin in patients with glioblastoma

Depatuxizumab mafodotin (depatux-m) is an antibody-drug conjugate (ADC) composed of an EGFR-targeted antibody conjugated to a microtubule inhibitor warhead, monomethyl auristatin F. In a Phase 1 study in glioblastoma (GBM), selection for EGFR amplification (amp) enriched for patients (pts) likely to respond. We sought to identify other determinants of sensitivity. We identified an index case with prolonged disease control but unexpectedly without EGFR amp. Whole exome sequencing (WES) of pre-treatment archival tissue identified a point mutation in TUBA3C (tubulin alpha 3c) which mechanistically affects sensitivity to the microtubule-directed warhead of depatux-m, as opposed to the EGFR-targeted antibody. WES analysis, with a focus on the tubulin gene family, was expanded to 48 pts with sufficient archival tissue. In vitro synthetic lethality experiments were performed to confirm the hypothesized importance of tubulin mutations on depatux-m sensitivity. WES revealed some tubulin genes were differentially mutated in depatux-m responders (n = 8) vs non-responders (n = 24), all with EGFR amp. TPPP2, TTLL11, TTLL6, TTLL7, TUBA3C, TUBA3D, TUBAL3, and TUBGCP4 mutations (1–2 per tumor) were observed preferentially in responders; gene knockdown enhanced response in vitro, consistent with mechanistic relevance to warhead sensitivity. TTLL2, TTLL4, TUBB2A, TUBB2B, TUBG1, TUBGCP2, TUBGCP5, TBCC, and TBCE mutations (1–3 per tumor) were observed exclusively in non-responders. Response to ADCs depends not only on the presence of the target antigen but also on sensitivity to the conjugated toxin. We identified tubulin gene family mutations correlating with response to the microtubule inhibitor warhead of the ADC depatux-m. While verification of this observation in an independent cohort is required, other experiments to elucidate the mechanism are ongoing. Further data will be presented. A proposed investigator-initiated trial encompassing tubulin mutations as an eligibility criterion is in development. These data related to warhead sensitivity of depatux-m may serve as a guide to support rational drug combination therapy in GBM.

Cytoplasmic p53 couples oncogene-driven glucose metabolism to apoptosis and is a therapeutic target in glioblastoma.

Cross-talk among oncogenic signaling and metabolic pathways may create opportunities for novel therapeutic strategies in cancer. Here we show that acute inhibition of EGFR-driven glucose metabolism induces minimal cell death, yet lowers the apoptotic threshold in a subset of patient-derived glioblastoma (GBM) cells. Mechanistic studies revealed that, following attenuated glucose consumption, Bcl-xL blocks cytoplasmic p53 from triggering intrinsic apoptosis. Consequently, pharmacological stabilization of p53 with the brain-penetrant small molecule, Idasanutlin, in combination with targeting EGFR-driven glucose metabolism promoted synthetic lethality in orthotopic xenograft models. Notably, neither inhibition of EGFR signaling, nor genetic analysis of EGFR, was sufficient to predict sensitivity to this new therapeutic combination. Conversely, rapid changes in 18F-fluorodeoxyglucose (18F-FDG) uptake using non-invasive positron emission tomography was an effective predictive biomarker of response in vivo. Together, these studies identify a critical link between oncogene signaling, glucose metabolism, and cytoplasmic p53, which could be exploited for combination therapy in GBM and potentially, other malignancies.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Single-Cell Phosphoproteomics Resolves Adaptive Signaling Dynamics and Informs Targeted Combination Therapy in Glioblastoma

Intratumoral heterogeneity of signaling networks may contribute to targeted cancer therapy resistance, including in the highly lethal brain cancer glioblastoma (GBM). We performed single-cell phosphoproteomics on a patient-derived in vivo GBM model of mTOR kinase inhibitor resistance and coupled it to an analytical approach for detecting changes in signaling coordination. Alterations in the protein signaling coordination were resolved as early as 2.5 days after treatment, anticipating drug resistance long before it was clinically manifest. Combination therapies were identified that resulted in complete and sustained tumor suppression in vivo. This approach may identify actionable alterations in signal coordination that underlie adaptive resistance, which can be suppressed through combination drug therapy, including non-obvious drug combinations.

Abstract PR09: Single cell phosphoproteomics identifies adaptive network dynamics of mTOR inhibitor resistance and defines effective combination therapy in glioblastoma

Abstract Background: Resistance to single-agent targeted cancer therapy is almost universal. Resistance can occur when drug-resistant tumor cell subpopulations expand to drive recurrence in a process akin to Darwinian-type evolution under the selection pressure of the drug. An alternative resistance mechanism is the one in which cancer cells targeted by the inhibitor adapt to that drug, so as to maintain the signal flux through those networks that are required for tumor maintenance and growth. The main goal of this study is to identify the mechanisms of resistance in a targeted therapy by analyzing single cells and to provide a strategy to design a more effective therapy that suppresses resistance. Methods: We investigated a clinically relevant model of acquired cancer drug resistance in glioblastoma (GBM). GBM39 is a human-derived primary GBM line that is maintained by serial transplantation in xenografts. GBM39 expresses high levels of the epidermal growth factor receptor (EGFR) variant(v)III oncogene, which sensitizes tumor cells to inhibitors of the mammalian target of rapamycin (mTOR). Therefore, we used this model to study acquired resistance to the mTOR kinase inhibitor CC214-2, which suppresses the proliferation of EGFRvIII expressing GBMs. We utilized a microfluidic device as a main tool that enables multiplex single cell proteomics. Relative to bulk assays, it can provide deeper insights into the signaling activity within a phosphoprotein signaling network, and how that activity is influenced by a drug. In addition to providing average protein levels, such assays also yield information on network signaling coordination that can be extracted from the variance for any given protein, as well as correlations between proteins. We determined the structure of the hyperactivated phosphoprotein signaling networks from statistical numbers of single cancer cells separated from the tumor for the untreated vehicle, during response to CC214-2, and after resistance to CC214-2. Results: We find evidence that the tumors can rapidly adapt (within 2-3 days) to specific targeted inhibitors. The details of how the cells adapt, which are uniquely resolved by the single cell assays, can suggest effective therapy combinations. For a GBM murine model, tumor cells rapidly adapted to mTOR inhibition by activating ERK/Src signaling, suggesting that mTOR and ERK/Src signaling provided two independent druggable pathways for that tumor. Consonant with this hypothesis, we devised three therapeutic combinations and showed them to be highly effective in causing sustained clinical remission in vivo. A retrospective analysis revealed that mTOR signaling was a major driver of tumor growth in the untreated tumor, while ERK/Src signaling promoted resistance to mTOR inhibition. An additional study of a low passage, patient derived GBM cell line was also shown to exhibit rapid adaptation to single-agent targeted therapy in a manner that suggested effective combination therapies for cell killing. Finally, the analysis was shown to be feasible on a freshly resected patient GBM tumor. Conclusions: The data and analysis presented here provide evidence that tumor cells can respond rapidly to a therapy and restores the growth characteristics temporarily disrupted by the therapy. This resistance mechanism is intrinsic; the same cells that respond to the therapy also adapt and develop resistance to it. For the GBM models explored here, a single cell proteomics analysis of the phosphoprotein signaling networks can resolve the independent signaling modes that drive tumor growth in both the untreated and drug resistant states. Such an analysis can be rapidly carried out using untreated tumor biopsies, and so may represent a new approach for guiding the selection of targeted combination therapies with low anticipated resistance. Citation Format: Young Shik Shin, Wei Wei, Beatrice Gini, Paul Mischel, James Heath. Single cell phosphoproteomics identifies adaptive network dynamics of mTOR inhibitor resistance and defines effective combination therapy in glioblastoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR09.

Thymidine kinase gene delivery using curcumin loaded peptide micelles as a combination therapy for glioblastoma.

The effect of the combination therapy of curcumin and the herpes simplex virus thymidine kinase (HSVtk) gene using R7L10 as a carrier was evaluated in a glioblastoma animal model. Curcumin was loaded into the cores of R7L10 peptide micelles using an oil-in-water emulsion/solvent evaporation method to generate curcumin loaded R7L10 micelles (R7L10-Cur), which were used as a carrier to deliver the HSVtk gene. The plasmid DNA (pDNA)/R7L10-Cur complex was confirmed by gel retardation, heparin competition, and dynamic light scattering analyses. Transfection efficiency and cytotoxicity were measured using luciferase, MTT, and TUNEL assays. Intracellular delivery of curcumin was determined by fluorescence and absorbance. In the glioblastoma animal model, the effects of the intratumoral delivery of curcumin and the HSVtk gene were evaluated according to tumor size, immunohistochemistry, and TUNEL assays. R7L10-Cur delivered pDNA into the cells more efficiently than PLL and R7L10. In addition, R7L10-Cur delivered curcumin into the cells more efficiently than curcumin alone. The pHSVtk/R7L10-Cur complex induced cell death efficiently both in vitro and in vivo. Likewise, the combination of curcumin and the HSVtk gene using the pHSVtk/R7L10-Cur complex reduced tumor size more efficiently than the pHSVtk/PEI and pHSVtk/R7L10 complexes in a glioblastoma animal model. R7L10 is an efficient carrier for delivery of curcumin and the HSVtk gene, which may be a useful combination therapy for glioblastoma.

Using proton MRSI to predict response to vorinostat treatment in recurrent GBM.

3055 Background: A major impediment to the development of new therapies for glioblastoma (GBM) is a lack of biomarkers indicating response. Epigenetic modifications are now recognized as a frequent occurrence in the early phases of tumorigenesis, playing a central role in tumor development. Epigenetic alterations differ significantly from genetic modifications in that they may be reversed by ‘‘epigenetic drugs’’ such as histone deacetylase inhibitors (HDACis). As a promising new modality for cancer therapy, the first generation of HDACi is currently being tested in phase I/II clinical trials. Methods: GBM alterations from therapy with HDACis, such as vorinostat (SAHA), include tumor redifferentiation/cytostasis rather than tumor size reduction limits the utility of traditional imaging methods such as MRI. Magnetic resonance spectroscopic imaging (MRSI) quantitates various metabolite levels in tumor and normal brain, allowing characterization of metabolic processes in live tissue. Results: In our preclinical model, MRS detected metabolic response to SAHA after only 3 days of treatment: reduced alanine and lactate and elevated myo-inositol, N-acetyl aspartate and creatine; each returning toward normal brain levels. This led to our clinical study of MRSI to evaluate the metabolic response of recurrent GBMs to SAHA + temozolomide. After only 7 days of SAHA treatment, MRSI can distinguish metabolic responders (normalization/restoration of tumor metabolites towards normal brain-like metabolism) from non-responders (no significant change in tumor metabolites). Our initial cohort (n=6) consists of 3 responders and 3 non-responders with highly significant differences in their change in metabolite levels (p &lt; 0.001). Conclusions: Our results provide exciting insights into the mechanisms by which HDACi exerts its effect on GBMs. Tumor cells have increased biosynthetic needs requiring reprogramming of cellular metabolism. This creates increased energy demands, making tumor cells even more vulnerable to interventions targeting their metabolism. HDACi may induce redifferentiation in tumors by targeting tumor metabolism. Thus, MRSI provides a novel modality to predict response to HDACi-containing combination therapy in GBM.