Targeting Oral Squamous Cell Carcinoma with Combined Polo-Like-Kinase-1 Inhibitors and γ-Radiation Therapy.

Polo-like-kinase-1 (PLK-1) is a serine/threonine kinase that regulates the cell cycle and acts as an oncogene in multiple cancers, including oral squamous cell carcinoma (OSCC). The loss of PLK-1 can inhibit growth and induce apoptosis, making it an attractive therapeutic target in OSCC. We evaluated the efficacy of PLK-1 inhibitors as novel, targeted therapeutics in OSCC. PLK-1 inhibition using BI6727 (volasertib) was found to affect cell death at low nanomolar concentrations in most tested OSCC cell lines, but not in normal oral keratinocytes. In cell lines resistant to volasertib alone, pre-treatment with radiotherapy followed by volasertib reduced cell viability and induced apoptosis. The combinatorial efficacy of volasertib and radiotherapy was replicated in xenograft mouse models. These findings highlight the potential of adding PLK-1 inhibitors to adjuvant therapy regimens in OSCC.

Development and validation of a 21-gene prognostic signature in neuroblastoma.

Survival outcomes for patients with neuroblastoma vary markedly and reliable prognostic markers and risk stratification tools are lacking. We sought to identify and validate a transcriptomic signature capable of predicting risk of mortality in patients with neuroblastoma. The TARGET NBL dataset (n = 243) was used to develop the model and two independent cohorts, E-MTAB-179 (n = 478) and GSE85047 (n = 240) were used as validation sets. EFS was the primary outcome and OS was the secondary outcome of interest for all analysis. We identified a 21-gene signature capable of stratifying neuroblastoma patients into high and low risk groups in the E-MTAB-179 (HR 5.87 [3.83-9.01], p < 0.0001, 5 year AUC 0.827) and GSE85047 (HR 3.74 [2.36-5.92], p < 0.0001, 5 year AUC 0.815) validation cohorts. Moreover, the signature remained independent of known clinicopathological variables, and remained prognostic within clinically important subgroups. Further, the signature was effectively incorporated into a risk model with clinicopathological variables to improve prognostic performance across validation cohorts (Pooled Validation HR 6.93 [4.89-9.83], p < 0.0001, 5 year AUC 0.839). Similar prognostic utility was also demonstrated with OS. The identified signature is a robust independent predictor of EFS and OS outcomes in neuroblastoma patients and can be combined with clinically utilized clinicopathological variables to improve prognostic performance.

Spatial transcriptomics reveals distinct and conserved tumor core and edge architectures that predict survival and targeted therapy response.

The spatial organization of the tumor microenvironment has a profound impact on biology and therapy response. Here, we perform an integrative single-cell and spatial transcriptomic analysis on HPV-negative oral squamous cell carcinoma (OSCC) to comprehensively characterize malignant cells in tumor core (TC) and leading edge (LE) transcriptional architectures. We show that the TC and LE are characterized by unique transcriptional profiles, neighboring cellular compositions, and ligand-receptor interactions. We demonstrate that the gene expression profile associated with the LE is conserved across different cancers while the TC is tissue specific, highlighting common mechanisms underlying tumor progression and invasion. Additionally, we find our LE gene signature is associated with worse clinical outcomes while TC gene signature is associated with improved prognosis across multiple cancer types. Finally, using an in silico modeling approach, we describe spatially-regulated patterns of cell development in OSCC that are predictably associated with drug response. Our work provides pan-cancer insights into TC and LE biology and interactive spatial atlases ( http://www.pboselab.ca/spatial_OSCC/ ; http://www.pboselab.ca/dynamo_OSCC/ ) that can be foundational for developing novel targeted therapies.

Modulation of Reoviral Cytolysis (II): Cellular Stemness.

Oncolytic viruses (OVs) are an emerging cancer therapeutic that are intended to act by selectively targeting and lysing cancerous cells and by stimulating anti-tumour immune responses, while leaving normal cells mainly unaffected. Reovirus is a well-studied OV that is undergoing advanced clinical trials and has received FDA approval in selected circumstances. However, the mechanisms governing reoviral selectivity are not well characterised despite many years of effort, including those in our accompanying paper where we characterize pathways that do not consistently modulate reoviral cytolysis. We have earlier shown that reovirus is capable of infecting and lysing both certain types of cancer cells and also cancer stem cells, and here we demonstrate its ability to also infect and kill healthy pluripotent stem cells (PSCs). This led us to hypothesize that pathways responsible for stemness may constitute a novel route for the modulation of reoviral tropism. We find that reovirus is capable of killing both murine and human embryonic and induced pluripotent stem cells. Differentiation of PSCs alters the cells' reoviral-permissive state to a resistant one. In a breast cancer cell line that was resistant to reoviral oncolysis, induction of pluripotency programming rendered the cells permissive to cytolysis. Bioinformatic analysis indicates that expression of the Yamanaka pluripotency factors may be associated with regulating reoviral selectivity. Mechanistic insights from these studies will be useful for the advancement of reoviral oncolytic therapy.

Single-Cell Spatial Analysis Identifies Regulators of Brain Tumor-Initiating Cells.

Glioblastomas (GBM) are aggressive brain tumors with extensive intratumoral heterogeneity that contributes to treatment resistance. Spatial characterization of GBMs could provide insights into the role of the brain tumor microenvironment in regulating intratumoral heterogeneity. Here, we performed spatial transcriptomic and single-cell analyses of the mouse and human GBM microenvironment to dissect the impact of distinct anatomical regions of brains on GBM. In a syngeneic GBM mouse model, spatial transcriptomics revealed that numerous extracellular matrix (ECM) molecules, including biglycan, were elevated in areas infiltrated with brain tumor-initiating cells (BTIC). Single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing showed that ECM molecules were differentially expressed by GBM cells based on their differentiation and cellular programming phenotypes. Exogeneous biglycan or overexpression of biglycan resulted in a higher proliferation rate of BTICs, which was associated mechanistically with low-density lipoprotein receptor-related protein 6 (LRP6) binding and activation of the Wnt/ß-catenin pathway. Biglycan-overexpressing BTICs developed into larger tumors and displayed mesenchymal phenotypes when implanted intracranially in mice. This study points to the spatial heterogeneity of ECM molecules in GBM and suggests that the biglycan-LRP6 axis could be a therapeutic target to curb tumor growth. SIGNIFICANCE: Characterization of the spatial heterogeneity of glioblastoma identifies regulators of brain tumor-initiating cells and tumor growth that could serve as candidates for therapeutic interventions to improve the prognosis of patients.

PDGF gene expression and p53 alterations contribute to the biology of diffuse astrocytic gliomas.

Diffuse, histologically lower grade astrocytomas of adults (LGAs) are classified based on the mutational status of the isocitrate dehydrogenase (IDH) genes. While wild-type (WT) LGAs often evolve quickly to glioblastoma (GBM), mutant tumors typically follow an indolent course. To find possible effectors of these different behaviors, we compared their respective transcriptomes. Unlike mutant LGAs, platelet-derived growth factor (PDGF) signaling was significantly enriched in WT tumors, and PDGFA was the top overexpressed gene in the pathway. Moreover, methylation of the PDGFA and PDGFD promoters emerged as a possible mechanism for their low expression in mutant tumors. Copy number gain of chromosome 7 co-occurred with high expression of PDGFA in WT cases, and high expression of PDGFA was associated with aneuploidy, extracellular matrix (ECM)-related immunosuppressive features and poor prognosis. We also noted that high PDGFA expression in WT cases occurred irrespective of tumor grade and that multiple mechanisms of p53 pathway inactivation accompanied progression to GBM in PDGFA-overexpressing tumors. Conversely, TP53 point mutations were an early and constant feature of mutant LGAs. Our results suggest that members of the PDGF gene family, in concert with different p53 pathway alterations, underlie LGA behaviors.

NCBP2 and TFRC are novel prognostic biomarkers in oral squamous cell carcinoma.

There are few prognostic biomarkers and targeted therapeutics currently in use for the clinical management of oral squamous cell carcinoma (OSCC) and patient outcomes remain poor in this disease. A majority of mutations in OSCC are loss-of-function events in tumour suppressor genes that are refractory to conventional modes of targeting. Interestingly, the chromosomal segment 3q22-3q29 is amplified in many epithelial cancers, including OSCC. We hypothesized that some of the 468 genes located on 3q22-3q29 might be drivers of oral carcinogenesis and could be exploited as potential prognostic biomarkers and therapeutic targets. Our integrative analysis of copy number variation (CNV), gene expression and clinical data from The Cancer Genome Atlas (TCGA), identified two candidate genes: NCBP2, TFRC, whose expression positively correlates with worse overall survival (OS) in HPV-negative OSCC patients. Expression of NCBP2 and TFRC is significantly higher in tumour cells compared to most normal human tissues. High NCBP2 and TFRC protein abundance is associated with worse overall, disease-specific survival, and progression-free interval in an in-house cohort of HPV-negative OSCC patients. Finally, due to a lack of evidence for the role of NCBP2 in carcinogenesis, we tested if modulating NCBP2 levels in human OSCC cell lines affected their carcinogenic behaviour. We found that NCBP2 depletion reduced OSCC cell proliferation, migration, and invasion. Differential expression analysis revealed the upregulation of several tumour-promoting genes in patients with high NCBP2 expression. We thus propose both NCBP2 and TFRC as novel prognostic and potentially therapeutic biomarkers for HPV-negative OSCC.

Identification of Altered Primary Immunodeficiency-Associated Genes and Their Implications in Pediatric Cancers.

BACKGROUND: Cancer is the leading cause of disease-related mortality in children and malignancies are more frequently observed in individuals with primary immunodeficiencies (PIDs). This study aimed to identify and highlight the molecular mechanisms, such as oncogenesis and immune evasion, by which PID-related genes may lead to the development of pediatric cancers. METHOD: We implemented a novel bioinformatics framework using patient data from the TARGET database and performed a comparative transcriptome analysis of PID-related genes in pediatric cancers between normal and cancer tissues, gene ontology enrichment, and protein-protein interaction analyses, and determined the prognostic impacts of commonly mutated and differentially expressed PID-related genes. RESULTS: From the Fulgent Genetics Comprehensive Primary Immunodeficiency panel of 472 PID-related genes, 89 genes were significantly differentially expressed between normal and cancer tissues, and 20 genes were mutated in two or more patients. Enrichment analysis highlighted many immune system processes as well as additional pathways in the mutated PID-related genes related to oncogenesis. Survival outcomes for patients with altered PID-related genes were significantly different for 75 of the 89 DEGs, often resulting in a poorer prognosis. CONCLUSIONS: Overall, multiple PID-related genes demonstrated the connection between PIDs and cancer development and should be studied further, with hopes of identifying new therapeutic targets.

Immune-based classification of HPV-associated oropharyngeal cancer with implications for biomarker-driven treatment de-intensification.

BACKGROUND: There is significant interest in treatment de-escalation for human papillomavirus-associated (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients given the generally favourable prognosis. However, 15-30% of patients recur after primary treatment, reflecting a need for improved risk-stratification tools. We sought to develop a molecular test to risk stratify HPV+ OPSCC patients. METHODS: We created an immune score (UWO3) associated with survival outcomes in six independent cohorts comprising 906 patients, including blinded retrospective and prospective external validations. Two aggressive radiation de-escalation cohorts were used to assess the ability of UWO3 to identify patients who recur. Multivariate Cox models were used to assess the associations between the UWO3 immune class and outcomes. FINDINGS: A three-gene immune score classified patients into three immune classes (immune rich, mixed, or immune desert) and was strongly associated with disease-free survival in six datasets, including large retrospective and prospective datasets. Pooled analysis demonstrated that the immune rich group had superior disease-free survival compared to the immune desert (HR = 9.0, 95% CI: 3.2-25.5, P = 3.6 × 10-5) and mixed (HR = 6.4, 95% CI: 2.2-18.7, P = 0.006) groups after adjusting for age, sex, smoking status, and AJCC8 clinical stage. Finally, UWO3 was able to identify patients from two small treatment de-escalation cohorts who remain disease-free after aggressive de-escalation to 30 Gy radiation. INTERPRETATION: With additional prospective validation, the UWO3 score could enable biomarker-driven clinical decision-making for patients with HPV+ OPSCC based on robust outcome prediction across six independent cohorts. Prospective de-escalation and intensification clinical trials are currently being planned. FUNDING: CIHR, European Union, and the NIH.

PD-1 independent of PD-L1 ligation promotes glioblastoma growth through the NFκB pathway.

Brain tumor­initiating cells (BTICs) drive glioblastoma growth through not fully understood mechanisms. Here, we found that about 8% of cells within the human glioblastoma microenvironment coexpress programmed cell death 1 (PD-1) and BTIC marker. Gain- or loss-of-function studies revealed that tumor-intrinsic PD-1 promoted proliferation and self-renewal of BTICs. Phosphorylation of tyrosines within the cytoplasmic tail of PD-1 recruited Src homology 2­containing phosphatase 2 and activated the nuclear factor kB in BTICs. Notably, the tumor-intrinsic promoting effects of PD-1 did not require programmed cell death ligand 1(PD-L1) ligation; thus, the therapeutic antibodies inhibiting PD-1/PD-L1 interaction could not overcome the growth advantage of PD-1 in BTICs. Last, BTIC-intrinsic PD-1 accelerated intracranial tumor growth, and this occurred in mice lacking T and B cells. These findings point to a critical role for PD-1 in BTICs and uncover a nonimmune resistance mechanism of patients with glioblastoma to PD-1­ or PD-L1­blocking therapies.

The KrasG12D;Trp53fl/fl murine model of undifferentiated pleomorphic sarcoma is macrophage dense, lymphocyte poor, and resistant to immune checkpoint blockade.

Sarcomas are rare, difficult to treat, mesenchymal lineage tumours that affect children and adults. Immunologically-based therapies have improved outcomes for numerous adult cancers, however, these therapeutic strategies have been minimally effective in sarcoma so far. Clinically relevant, immunologically-competent, and transplantable pre-clinical sarcoma models are essential to advance sarcoma immunology research. Herein we show that Cre-mediated activation of KrasG12D, and deletion of Trp53, in the hindlimb muscles of C57Bl/6 mice results in the highly penetrant, rapid onset undifferentiated pleomorphic sarcomas (UPS), one of the most common human sarcoma subtypes. Cell lines derived from spontaneous UPS tumours can be reproducibly transplanted into the hindlimbs or lungs of naïve, immune competent syngeneic mice. Immunological characterization of both spontaneous and transplanted UPS tumours demonstrates an immunologically-'quiescent' microenvironment, characterized by a paucity of lymphocytes, limited spontaneous adaptive immune pathways, and dense macrophage infiltrates. Macrophages are the dominant immune population in both spontaneous and transplanted UPS tumours, although compared to spontaneous tumours, transplanted tumours demonstrate increased spontaneous lymphocytic infiltrates. The growth of transplanted UPS tumours is unaffected by host lymphocyte deficiency, and despite strong expression of PD-1 on tumour infiltrating lymphocytes, tumours are resistant to immunological checkpoint blockade. This spontaneous and transplantable immune competent UPS model will be an important experimental tool in the pre-clinical development and evaluation of novel immunotherapeutic approaches for immunologically cold soft tissue sarcomas.

Fibrinogen in the glioblastoma microenvironment contributes to the invasiveness of brain tumor-initiating cells.

Glioblastomas (GBMs) are highly aggressive, recurrent, and lethal brain tumors that are maintained via brain tumor-initiating cells (BTICs). The aggressiveness of BTICs may be dependent on the extracellular matrix (ECM) molecules that are highly enriched within the GBM microenvironment. Here, we investigated the expression of ECM molecules in GBM patients by mining the transcriptomic databases and also staining human GBM specimens. RNA levels for fibronectin, brevican, versican, heparan sulfate proteoglycan 2 (HSPG2), and several laminins were high in GBMs compared to normal brain, and this was corroborated by immunohistochemistry. While fibrinogen transcript was at normal level in GBM, its protein immunoreactivity was prominent within GBM tissues. These ECM molecules in tumor specimens were in proximity to, and surrounding BTICs. In culture, fibronectin and pan-laminin induced the adhesion of BTICs onto the plastic substratum. However, fibrinogen increased the size of the BTIC spheres by facilitating the adhesive property, motility, and invasiveness of BTICs. These features of elevated invasiveness were corroborated in resected GBM specimens by the close proximity of fibrinogen with matrix metalloproteinase (MMP)-2 and-9, which are proteases implicated in metastasis. Moreover, the effect of fibrinogen-induced invasiveness was attenuated in BTICs where MMP-2 and -9 have been inhibited with siRNAs or pharmacological inhibitors. Our results implicate fibrinogen in GBM as a mediator of the invasive properties of BTICs, and as a target for therapy to reduce BTIC tumorigenecity.

TGF-ß Mediated Immune Evasion in Cancer-Spotlight on Cancer-Associated Fibroblasts.

Various components of the tumor microenvironment (TME) play a critical role in promoting tumorigenesis, progression, and metastasis. One of the primary functions of the TME is to stimulate an immunosuppressive environment around the tumor through multiple mechanisms including the activation of the transforming growth factor-beta (TGF-ß) signaling pathway. Cancer-associated fibroblasts (CAFs) are key cells in the TME that regulate the secretion of extracellular matrix (ECM) components under the influence of TGF-ß. Recent reports from our group and others have described an ECM-related and CAF-associated novel gene signature that can predict resistance to immune checkpoint blockade (ICB). Importantly, studies have begun to test whether targeting some of these CAF-associated components can be used as a combinatorial approach with ICB. This perspective summarizes recent advances in our understanding of CAF and TGF-ß-regulated immunosuppressive mechanisms and ways to target such signaling in cancer.

Glioma-derived IL-33 orchestrates an inflammatory brain tumor microenvironment that accelerates glioma progression.

Despite a deeper molecular understanding, human glioblastoma remains one of the most treatment refractory and fatal cancers. It is known that the presence of macrophages and microglia impact glioblastoma tumorigenesis and prevent durable response. Herein we identify the dual function cytokine IL-33 as an orchestrator of the glioblastoma microenvironment that contributes to tumorigenesis. We find that IL-33 expression in a large subset of human glioma specimens and murine models correlates with increased tumor-associated macrophages/monocytes/microglia. In addition, nuclear and secreted functions of IL-33 regulate chemokines that collectively recruit and activate circulating and resident innate immune cells creating a pro-tumorigenic environment. Conversely, loss of nuclear IL-33 cripples recruitment, dramatically suppresses glioma growth, and increases survival. Our data supports the paradigm that recruitment and activation of immune cells, when instructed appropriately, offer a therapeutic strategy that switches the focus from the cancer cell alone to one that includes the normal host environment.

Correction: Combined poly-ADP ribose polymerase and ataxia-telangiectasia mutated/Rad3-related inhibition targets ataxia-telangiectasia mutated-deficient lung cancer cells.

This Article was originally published under Nature Research's License to Publish, but has now been made available under a CC BY 4.0 license. This has now been corrected in both the PDF and HTML versions of the Article.

Control of brain tumor growth by reactivating myeloid cells with niacin.

Glioblastomas are generally incurable partly because monocytes, macrophages, and microglia in afflicted patients do not function in an antitumor capacity. Medications that reactivate these macrophages/microglia, as well as circulating monocytes that become macrophages, could thus be useful to treat glioblastoma. We have discovered that niacin (vitamin B3) is a potential stimulator of these inefficient myeloid cells. Niacin-exposed monocytes attenuated the growth of brain tumor-initiating cells (BTICs) derived from glioblastoma patients by producing anti-proliferative interferon-α14. Niacin treatment of mice bearing intracranial BTICs increased macrophage/microglia representation within the tumor, reduced tumor size, and prolonged survival. These therapeutic outcomes were negated in mice depleted of circulating monocytes or harboring interferon-α receptor-deleted BTICs. Combination treatment with temozolomide enhanced niacin-promoted survival. Monocytes from glioblastoma patients had increased interferon-α14 upon niacin exposure and were reactivated to reduce BTIC growth in culture. We highlight niacin, a common vitamin that can be quickly translated into clinical application, as an immune stimulator against glioblastomas.

ATM-Deficient Cancers Provide New Opportunities for Precision Oncology.

Poly-ADP ribose polymerase (PARP) inhibitors are currently used in the treatment of several cancers carrying mutations in the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2, with many more potential applications under study and in clinical trials. Here, we discuss the potential for extending PARP inhibitor therapies to tumours with deficiencies in the DNA damage-activated protein kinase, Ataxia-Telangiectasia Mutated (ATM). We highlight our recent findings that PARP inhibition alone is cytostatic but not cytotoxic in ATM-deficient cancer cells and that the combination of a PARP inhibitor with an ATR (ATM, Rad3-related) inhibitor is required to induce cell death.

Demeclocycline Reduces the Growth of Human Brain Tumor-Initiating Cells: Direct Activity and Through Monocytes.

Myeloid cells that infiltrate into brain tumors are deactivated or exploited by the tumor cells. We previously demonstrated that compromised microglia, monocytes, and macrophages in malignant gliomas could be reactivated by amphotericin-B to contain the growth of brain tumorinitiating cells (BTICs). We identified meclocycline as another activator of microglia, so we sought to test whether its better-tolerated derivative, demeclocycline, also stimulates monocytes to restrict BTIC growth. Monocytes were selected for study as they would be exposed to demeclocycline in the circulation prior to entry into brain tumors to become macrophages. We found that demeclocycline increased the activity of monocytes in culture, as determined by tumor necrosis factor-α production and chemotactic capacity. The conditioned medium of demeclocycline-stimulated monocytes attenuated the growth of BTICs generated from human glioblastoma resections, as evaluated using neurosphere and alamarBlue assays, and cell counts. Demeclocycline also had direct effects in reducing BTIC growth. A global gene expression screen identified several genes, such as DNA damage inducible transcript 4, frizzled class receptor 5 and reactive oxygen species modulator 1, as potential regulators of demeclocycline-mediated BTIC growth reduction. Amongst several tetracycline derivatives, only demeclocycline directly reduced BTIC growth. In summary, we have identified demeclocycline as a novel inhibitor of the growth of BTICs, through direct effect and through indirect stimulation of monocytes. Demeclocycline is a candidate to reactivate compromised immune cells to improve the prognosis of patients with gliomas.

In Vitro Investigation Demonstrates IGFR/VEGFR Receptor Cross Talk and Potential of Combined Inhibition in Pediatric Central Nervous System Atypical Teratoid Rhabdoid Tumors.

BACKGROUND: Atypical teratoid rhabdoid tumor of the central nervous system (CNS ATRT) is a malignancy that commonly affects young children. The biological mechanisms contributing to tumor aggressiveness and resistance to conventional therapies in ATRT are unknown. Previous studies have shown the activity of insulin like growth factor-I receptor (IGF-1R) in ATRT tumor specimens and cell lines. IGF-1R has been shown to cross-talk with other receptor tyrosine kinases (RTKs) in a number of cancer types, leading to enhanced cell proliferation. OBJECTIVE: This study aims to evaluate the role of IGF-1 receptor cross-talk in ATRT biology and the potential for therapeutic targeting. METHODS: Cell lines derived from CNS ATRT specimens were analyzed for IGF-1 mediated cell proliferation. A comprehensive receptor tyrosine kinase (RTK) screen was conducted following IGF-1 stimulation. Bioinformatic analysis of publicly available cancer growth inhibition data to identify correlation between IC50 of a VEGFR inhibitor and IGF-1R expression. RESULTS: Comprehensive RTK screen identified VEGFR-2 cross-activation following IGF-1 stimulation. Bioinformatics analysis demonstrated a positive correlation between IC50 values of VEGFR inhibitor Axitinib and IGF-1R expression, supporting the critical influence of IGF-1R in modulating response to anti-angiogenic therapies. CONCLUSION: Overall, our data present a novel experimental framework to evaluate and utilize receptor cross-talk mechanisms to select effective drugs and combinations for future therapeutic trials in ATRT.