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Immunotherapy has emerged as a powerful strategy for halting cancer progression. However, primary malignancies affecting the brain have been exempt to this success. Indeed, brain tumors continue to portend severe morbidity and remain a globally lethal disease. Extensive efforts have been directed at understanding how tumor cells survive and propagate within the unique microenvironment of the central nervous system (CNS). Cancer genetic aberrations and metabolic abnormalities provoke a state of persistent endoplasmic reticulum (ER) stress that in turn promotes tumor growth, invasion, therapeutic resistance, and the dynamic reprogramming of the infiltrating immune cells. Consequently, targeting ER stress is a potential therapeutic approach. In this work, we provide an overview of how ER stress response is advantageous to brain tumor development, discuss the significance of ER stress in governing antitumor immunity, and put forth therapeutic strategies of regulating ER stress to augment the effect of immunotherapy for primary CNS tumors.

During therapy, adaptations driven by cellular plasticity are partly responsible for driving the inevitable recurrence of glioblastoma (GBM). To investigate plasticity-induced adaptation during standard-of-care chemotherapy temozolomide (TMZ), we performed in vivo single-cell RNA sequencing in patient-derived xenograft (PDX) tumors of GBM before, during, and after therapy. Comparing single-cell transcriptomic patterns identified distinct cellular populations present during TMZ therapy. Of interest was the increased expression of ribonucleotide reductase regulatory subunit M2 ( RRM2 ), which we found to regulate dGTP and dCTP production vital for DNA damage response during TMZ therapy. Furthermore, multidimensional modeling of spatially resolved transcriptomic and metabolomic analysis in patients’ tissues revealed strong correlations between RRM2 and dGTP. This supports our data that RRM2 regulates the demand for specific dNTPs during therapy. In addition, treatment with the RRM2 inhibitor 3-AP (Triapine) enhances the efficacy of TMZ therapy in PDX models. We present a previously unidentified understanding of chemoresistance through critical RRM2-mediated nucleotide production.

Stereotactic Radiosurgery (SRS) is one of the leading treatment modalities for oligo brain metastasis (BM), however no comprehensive genomic data assessing the effect of radiation on BM in humans exist. Leveraging a unique opportunity, as part of the clinical trial (NCT03398694), we collected post-SRS, delivered via Gamma-knife or LINAC, tumor samples from core and peripheral-edges of the resected tumor to characterize the genomic effects of overall SRS as well as the SRS delivery modality. Using these rare patient samples, we show that SRS results in significant genomic changes at DNA and RNA levels throughout the tumor. Mutations and expression profiles of peripheral tumor samples indicated interaction with surrounding brain tissue as well as elevated DNA damage repair. Central samples show GSEA enrichment for cellular apoptosis while peripheral samples carried an increase in tumor suppressor mutations. There are significant differences in the transcriptomic profile at the periphery between Gamma-knife vs LINAC.

The symbiotic interactions between cancer stem cells and the tumor microenvironment (TME) are critical for tumor progression. However, the molecular mechanism underlying this symbiosis in glioblastoma (GBM) remains enigmatic. Here, we show that circadian locomotor output cycles kaput (CLOCK) and its heterodimeric partner brain and muscle ARNT-like 1 (BMAL1) in glioma stem cells (GSC) drive immunosuppression in GBM. Integrated analyses of the data from transcriptome profiling, single-cell RNA sequencing, and TCGA datasets, coupled with functional studies, identified legumain (LGMN) as a direct transcriptional target of the CLOCK–BMAL1 complex in GSCs. Moreover, CLOCK-directed olfactomedin-like 3 (OLFML3) upregulates LGMN in GSCs via hypoxia-inducible factor 1-alpha (HIF1α) signaling. Consequently, LGMN promotes microglial infiltration into the GBM TME via upregulating CD162 and polarizes infiltrating microglia toward an immune-suppressive phenotype. In GBM mouse models, inhibition of the CLOCK–OLFML3–HIF1α–LGMN–CD162 axis reduces intratumoral immune-suppressive microglia, increases CD8+ T-cell infiltration, activation, and cytotoxicity, and synergizes with anti–programmed cell death protein 1 (anti–PD-1 therapy). In human GBM, the CLOCK-regulated LGMN signaling correlates positively with microglial abundance and poor prognosis. Together, these findings uncover the CLOCK–OLFML3–HIF1α–LGMN axis as a molecular switch that controls microglial biology and immunosuppression, thus revealing potential new therapeutic targets for patients with GBM.

Supplementary Figure from Circadian Regulator CLOCK Drives Immunosuppression in Glioblastoma

Full information pertaining to supplementary figure legends.

Morphology and proliferation of knockdown cells.

Primer sequence.

Brain metastases occur in about 10% to 30% of breast cancer patients, which culminates in a poor prognosis. It is, therefore, critical to understand the molecular mechanisms underlying brain metastatic processes to identify relevant targets. We hypothesized that breast cancer cells must express brain-associated markers that would enable their invasion and survival in the brain microenvironment. We assessed a panel of brain-predominant markers and found an elevation of several neuronal markers (βIII-tubulin, Nestin, and AchE) in brain metastatic breast cancer cells. Among these neuronal predominant markers, in silico analysis revealed overexpression of βIII-tubulin (TUBB3) in breast cancer brain metastases (BCBM) and its expression was significantly associated with distant metastases. TUBB3 knockdown studies were conducted in breast cancer models (MDA-Br, GLIM2, and MDA-MB-468), which revealed significant reduction in their invasive capabilities. MDA-Br cells with suppressed TUBB3 also demonstrated loss of key signaling molecules such as β3 integrin, pFAK, and pSrc in vitro. Furthermore, TUBB3 knockdown in a brain metastatic breast cancer cell line compromised its metastatic ability in vivo, and significantly improved survival in a brain metastasis model. These results implicate a critical role of TUBB3 in conferring brain metastatic potential to breast cancer cells. Mol Cancer Ther; 14(5); 1152–61. ©2015 AACR.

Supplementary Figure 1. Expression of GSC markers increases with therapy in vitro and in vivo; Supplementary Figure 2. In vitro evaluation of stemness and TMZ sensitivity in the TMZ rechallenged model; Supplementary Figure 3. In vitro Evaluation of GSC-specific reporter system; Supplementary Figure 4. Differentiation of PDX GBM line after culturing in the media with 10% FBS; Supplementary Figure 5. GSC-specific promoters functionally represent the GSC subpopulation; Supplementary Figure 6. Differentiation of PDX GBM line after culturing in the media Bone Morphogenic Protein 2 (BMP2); Supplementary Figure 7. HIFs can modulate the expression of GSC markers

Generation of TUBB3 knockdown cells.

PDF file, 477K, mRNA and protein expression of IDO.

Purpose:Paclitaxel (PTX) is one of the most potent and commonly used chemotherapies for breast and pancreatic cancer. Several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood–brain barrier for glioblastomas. Despite the widespread use of PTX for breast cancer, and the initiative to repurpose this drug for gliomas, there are no predictive biomarkers to inform which patients will likely benefit from this therapy.Experimental Design:To identify predictive biomarkers for susceptibility to PTX, we performed a genome-wide CRISPR knockout (KO) screen using human glioma cells. The genes whose KO was most enriched in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort using progression-free survival.Results:Combination of CRISPR screen results with outcomes from patients with taxane-treated breast cancer led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with susceptibility to PTX in breast cancer cells, glioma cells, and in multiple intracranial glioma xenografts models. KO of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. Mechanistically, SSR3 confers susceptibility to PTX through regulation of phosphorylation of ER stress sensor IRE1α.Conclusions:Our hypothesis generating study showed SSR3 as a putative biomarker for susceptibility to PTX, warranting its prospective clinical validation.

PDF file, 370K, Brain tumor derived IDO and peripheral Tregs.

(A) Sorted Tregs were placed the upper chamber of a 3.0 microm pore cell culture insert with or without 5 microM of the small molecule antagonist C 021 and chemokine-containing media was placed in the well. Migration index was determined after 4 hours through flow cytometric analysis. (B) A similar migration assay was performed as in A, except CCL21 and 10% FBS were used in the bottom chamber as a non-specific positive control. Data are representative of 3 independent experiments. Data are represented as mean {plus minus} SEM; *P < 0.05, by Student's t test.

Supplementary Table from Translocon-associated Protein Subunit SSR3 Determines and Predicts Susceptibility to Paclitaxel in Breast Cancer and Glioblastoma

(A-C) Vehicle control or C 021-treated mice were analyzed at 1 week post-GL261 injection for the accumulation of various T cell subsets and CD11b+Ly-6G+ MDSCs. (D-G) Quantification of flow cytometric analysis. (H-I) C 021 treatment improved the CD4/Foxp3 ratio and CD8/Foxp3 ratio. Data are representative of at least 2 independent experiments. Data are represented as mean {plus minus} SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student's t test.

(A-G) Kaplan-Meier survival curves generated from TCGA data using GBM patients segregated by gene expression for a panel of putative Treg and MDSC recruiting chemokines. (H) GBM patient tissue array stained for CCL2. (I) Key showing CCL2 score corresponding to GBM patient cores. Scale bar = 7 mm. TCGA, The Cancer Genome Atlas. Survival curves were compared with the Log-rank test.

Supplementary Data from Translocon-associated Protein Subunit SSR3 Determines and Predicts Susceptibility to Paclitaxel in Breast Cancer and Glioblastoma

Supplementary Figure from Translocon-associated Protein Subunit SSR3 Determines and Predicts Susceptibility to Paclitaxel in Breast Cancer and Glioblastoma

Antibody Array Key (RayBiotech Cat# AAM-CYT-6, related to Figure 3.

In many aggressive cancers, such as glioblastoma multiforme, progression is enabled by local immunosuppression driven by the accumulation of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). However, the mechanistic details of how Tregs and MDSCs are recruited in various tumors are not yet well understood. Here we report that macrophages and microglia within the glioma microenvironment produce CCL2, a chemokine that is critical for recruiting both CCR4+ Treg and CCR2+Ly-6C+ monocytic MDSCs in this disease setting. In murine gliomas, we established novel roles for tumor-derived CCL20 and osteoprotegerin in inducing CCL2 production from macrophages and microglia. Tumors grown in CCL2-deficient mice failed to maximally accrue Tregs and monocytic MDSCs. In mixed-bone marrow chimera assays, we found that CCR4-deficient Treg and CCR2-deficient monocytic MDSCs were defective in glioma accumulation. Furthermore, administration of a small-molecule antagonist of CCR4 improved median survival in the model. In clinical specimens of glioblastoma multiforme, elevated levels of CCL2 expression correlated with reduced overall survival of patients. Finally, we found that CD163-positive infiltrating macrophages were a major source of CCL2 in glioblastoma multiforme patients. Collectively, our findings show how glioma cells influence the tumor microenvironment to recruit potent effectors of immunosuppression that drive progression. Cancer Res; 76(19); 5671–82. ©2016 AACR.

PDF file, 84K, Correlation of patient characteristics with IDO expression.

Word document file containing supplementary materials and methods, supplementary figure legends, and supplementary table

CD45.1+ recipient mice were irradiated and reconstituted with CD45.1+ WT marrow and CD45.2+ Ccr4-/- marrow. After at least 6 weeks of reconstitution, mice were implanted with GL261 tumors and brain tumor-infiltrating cells were quantified. Data are representative of at least 2 independent experiments. Data are represented as mean {plus minus} SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student's t test. n.s., not significant.

(A) GL261 cells were orthotopically implanted into the right hemisphere of syngeneic immunocompetent C57BL/6 mice. At 1 week post-intracranial (i.c.) injection whole brains were homogenized followed by Percoll gradient separation of leukocytes and non-leukocytes. qRT-PCR was performed for CCL2 in mice intracranially-injected with GL261 cells (n = 4) or PBS-injected controls (n = 3). (B) Detection of CCL2 protein in both brain hemispheres at 1 week-i.c. GL261 (n = 3). (C) Immunoreactivity for CCL2 was detected at 1 week post-GL261 injection. 20Ã- and 63Ã- magnification. Scale bar = 50 microm. (D) qRT-PCR for CCL1, CCL28, CCL2, CCL22, CCL17, and CCL20 from non-leukocytes (NL) and leukocytes (L) as in (A). Images in C are representative of 3 independent replicates. Data are representative of at least 2 independent experiments. Data are represented as mean {plus minus} SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA with Tukey's multiple comparisons test (A, D) or Student's t test (B).

(A) CCR4 and CCR2 expression on CD4+Foxp3- conventional CD4 T cells, CD4+Foxp3+ Tregs, and CD8+ T cells across various tissues at 1 week post-GL261 implantation. (B) Gating strategy for quantification of myeloid cells in brains of PBS-injected or GL261-implanted mice at 1-week post-intracranial implantation. Data are representative of at least 4 independent experiments.

Purpose: Glioblastoma multiforme (GBM) is an aggressive adult brain tumor with a poor prognosis. One hallmark of GBM is the accumulation of immunosuppressive and tumor-promoting CD4+FoxP3+GITR+ regulatory T cells (Tregs). Here, we investigated the role of indoleamine 2,3 dioxygenase (IDO) in brain tumors and the impact on Treg recruitment.Experimental Design: To determine the clinical relevance of IDO expression in brain tumors, we first correlated patient survival to the level of IDO expression from resected glioma specimens. We also used novel orthotopic and transgenic models of glioma to study how IDO affects Tregs. The impact of tumor-derived and peripheral IDO expression on Treg recruitment, GITR expression, and long-term survival was determined.Results: Downregulated IDO expression in glioma predicted a significantly better prognosis in patients. Coincidently, both IDO-competent and deficient mice showed a survival advantage bearing IDO-deficient brain tumors, when compared with IDO-competent brain tumors. Moreover, IDO deficiency was associated with a significant decrease in brain-resident Tregs, both in orthotopic and transgenic mouse glioma models. IDO deficiency was also associated with lower GITR expression levels on Tregs. Interestingly, the long-term survival advantage conferred by IDO deficiency was lost in T-cell–deficient mice.Conclusions: These clinical and preclinical data confirm that IDO expression increases the recruitment of immunosuppressive Tregs that lead to tumor outgrowth. In contrast, IDO deficiency decreases Treg recruitment and enhances T-cell–mediated tumor rejection. Thus, the data suggest a critical role for IDO-mediated immunosuppression in glioma and support the continued investigation of IDO–Treg interactions in the context of brain tumors. Clin Cancer Res; 18(22); 6110–21. ©2012 AACR.

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CCR Translation for This Article from IDO Expression in Brain Tumors Increases the Recruitment of Regulatory T Cells and Negatively Impacts Survival

PDF file, 79K.

Supplementary Materials and Methods from Inhibition of RAS-Mediated Transformation and Tumorigenesis by Targeting the Downstream E3 Ubiquitin Ligase Seven in Absentia Homologue

Supplementary Figure 1 from Inhibition of RAS-Mediated Transformation and Tumorigenesis by Targeting the Downstream E3 Ubiquitin Ligase Seven in Absentia Homologue

Supplementary Figure 3 from Inhibition of RAS-Mediated Transformation and Tumorigenesis by Targeting the Downstream E3 Ubiquitin Ligase Seven in Absentia Homologue

Supplementary Table 1 from Inhibition of RAS-Mediated Transformation and Tumorigenesis by Targeting the Downstream E3 Ubiquitin Ligase Seven in Absentia Homologue

Supplementary Figure 4 from Inhibition of RAS-Mediated Transformation and Tumorigenesis by Targeting the Downstream E3 Ubiquitin Ligase Seven in Absentia Homologue

Supplementary Figure 5 from Inhibition of RAS-Mediated Transformation and Tumorigenesis by Targeting the Downstream E3 Ubiquitin Ligase Seven in Absentia Homologue

Supplementary Figure 2 from Inhibition of RAS-Mediated Transformation and Tumorigenesis by Targeting the Downstream E3 Ubiquitin Ligase Seven in Absentia Homologue

Constitutively active RAS small GTPases promote the genesis of human cancers. An important goal in cancer biology is to identify means of countervailing activated RAS signaling to reverse malignant transformation. Oncogenic K-RAS mutations are found in virtually all pancreatic adenocarcinomas, making the RAS pathway an ideal target for therapeutic intervention. How to best contravene hyperactivated RAS signaling has remained elusive in human pancreatic cancers. Guided by the Drosophila studies, we reasoned that a downstream mediator of RAS signals might be a suitable anti-RAS target. The E3 ubiquitin ligase seven in absentia (SINA) is an essential downstream component of the Drosophila RAS signal transduction pathway. Thus, we determined the roles of the conserved human homologues of SINA, SIAHs, in mammalian RAS signaling and RAS-mediated tumorigenesis. We report that similar to its Drosophila counterpart, human SIAH is also required for oncogenic RAS signaling in pancreatic cancer. Inhibiting SIAH-dependent proteolysis blocked RAS-mediated focus formation in fibroblasts and abolished the tumor growth of human pancreatic cancer cells in soft agar as well as in athymic nude mice. Given the high level of conservation of RAS and SIAH function, our study provides useful insights into altered proteolysis in the RAS pathway in tumor initiation, progression, and oncogenesis. By targeting SIAH, we have found a novel means to contravene oncogenic RAS signaling and block RAS-mediated transformation/tumorigenesis. Thus, SIAH may offer a novel therapeutic target to halt tumor growth and ameliorate RAS-mediated pancreatic cancer. [Cancer Res 2007;67(24):11798–810]

Purpose: Paclitaxel (PTX) is one of the most potent and commonly used chemotherapies for breast and pancreatic cancer. Several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood–brain barrier for glioblastomas. Despite the widespread use of PTX for breast cancer, and the initiative to repurpose this drug for gliomas, there are no predictive biomarkers to inform which patients will likely benefit from this therapy. Experimental Design: To identify predictive biomarkers for susceptibility to PTX, we performed a genome-wide CRISPR knockout (KO) screen using human glioma cells. The genes whose KO was most enriched in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort using progression-free survival. Results: Combination of CRISPR screen results with outcomes from patients with taxane-treated breast cancer led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with susceptibility to PTX in breast cancer cells, glioma cells, and in multiple intracranial glioma xenografts models. KO of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. Mechanistically, SSR3 confers susceptibility to PTX through regulation of phosphorylation of ER stress sensor IRE1α. Conclusions: Our hypothesis generating study showed SSR3 as a putative biomarker for susceptibility to PTX, warranting its prospective clinical validation.

The symbiotic interactions between cancer stem cells and the tumor microenvironment (TME) are critical for tumor progression. However, the molecular mechanism underlying this symbiosis in glioblastoma (GBM) remains enigmatic. Here, we show that circadian locomotor output cycles kaput (CLOCK) and its heterodimeric partner brain and muscle ARNT-like 1 (BMAL1) in glioma stem cells (GSC) drive immunosuppression in GBM. Integrated analyses of the data from transcriptome profiling, single-cell RNA sequencing, and TCGA datasets, coupled with functional studies, identified legumain (LGMN) as a direct transcriptional target of the CLOCK–BMAL1 complex in GSCs. Moreover, CLOCK-directed olfactomedin-like 3 (OLFML3) upregulates LGMN in GSCs via hypoxia-inducible factor 1-alpha (HIF1α) signaling. Consequently, LGMN promotes microglial infiltration into the GBM TME via upregulating CD162 and polarizes infiltrating microglia toward an immune-suppressive phenotype. In GBM mouse models, inhibition of the CLOCK–OLFML3–HIF1α–LGMN–CD162 axis reduces intratumoral immune-suppressive microglia, increases CD8+ T-cell infiltration, activation, and cytotoxicity, and synergizes with anti–programmed cell death protein 1 (anti–PD-1 therapy). In human GBM, the CLOCK-regulated LGMN signaling correlates positively with microglial abundance and poor prognosis. Together, these findings uncover the CLOCK–OLFML3–HIF1α–LGMN axis as a molecular switch that controls microglial biology and immunosuppression, thus revealing potential new therapeutic targets for patients with GBM.

Objective With the advent of high-throughput genome analysis, we are increasingly able to sequence and hence understand the pathogenic processes underlying individual cancers. Recently, consortiums such as The Cancer Genome Atlas (TCGA) have performed large-scale projects to this end, providing significant amounts of information regarding the genetic landscapes of several cancers. Patients and methods We performed a narrative review of studies from the TCGA and other major studies. We aimed to summarise data exploring the clinical implications of specific genetic alterations, both prognostically and therapeutically, in four major urological cancers. These were renal cell carcinoma, muscle-invasive bladder cancer/carcinoma, prostate cancer, and testicular germ cell tumours. Results With these four urological cancers, great strides have been made in the molecular characterisation of tumours. In particular, recent studies have focussed on identifying molecular subtypes of tumours with characteristic genetic alterations and differing prognoses. Other prognostic alterations have also recently been identified, including those pertaining to epigenetics and microRNAs. In regard to treatment, numerous options are emerging for patients with these cancers such as including immune checkpoint inhibition, epigenetic-based treatments, and agents targeting MAPK, PI3K, and DNA repair pathways. There are a multitude of trials underway investigating the effects of these novel agents, the results of which are eagerly awaited. Conclusions As medicine chases the era of personalised care, it is becoming increasingly important to provide individualised prognoses for patients. Understanding how specific genetic alterations affects prognosis is key for this. It will also be crucial to provide highly targeted treatments against the specific genetics of a patient's tumour. With work performed by the TCGA and other large consortiums, these aims are gradually being achieved. Our review provides a succinct overview of this exciting field that may underpin personalised medicine in urological oncology.