Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

Single-cell spatial immune landscapes of primary and metastatic brain tumours.

Single-cell technologies have enabled the characterization of the tumour microenvironment at unprecedented depth and have revealed vast cellular diversity among tumour cells and their niche. Anti-tumour immunity relies on cell-cell relationships within the tumour microenvironment1,2, yet many single-cell studies lack spatial context and rely on dissociated tissues3. Here we applied imaging mass cytometry to characterize the immunological landscape of 139 high-grade glioma and 46 brain metastasis tumours from patients. Single-cell analysis of more than 1.1 million cells across 389 high-dimensional histopathology images enabled the spatial resolution of immune lineages and activation states, revealing differences in immune landscapes between primary tumours and brain metastases from diverse solid cancers. These analyses revealed cellular neighbourhoods associated with survival in patients with glioblastoma, which we leveraged to identify a unique population of myeloperoxidase (MPO)-positive macrophages associated with long-term survival. Our findings provide insight into the biology of primary and metastatic brain tumours, reinforcing the value of integrating spatial resolution to single-cell datasets to dissect the microenvironmental contexture of cancer.

Comprehensive genomic profiling of glioblastoma tumors, BTICs, and xenografts reveals stability and adaptation to growth environments.

Glioblastoma multiforme (GBM) is the most deadly brain tumor, and currently lacks effective treatment options. Brain tumor-initiating cells (BTICs) and orthotopic xenografts are widely used in investigating GBM biology and new therapies for this aggressive disease. However, the genomic characteristics and molecular resemblance of these models to GBM tumors remain undetermined. We used massively parallel sequencing technology to decode the genomes and transcriptomes of BTICs and xenografts and their matched tumors in order to delineate the potential impacts of the distinct growth environments. Using data generated from whole-genome sequencing of 201 samples and RNA sequencing of 118 samples, we show that BTICs and xenografts resemble their parental tumor at the genomic level but differ at the mRNA expression and epigenomic levels, likely due to the different growth environment for each sample type. These findings suggest that a comprehensive genomic understanding of in vitro and in vivo GBM model systems is crucial for interpreting data from drug screens, and can help control for biases introduced by cell-culture conditions and the microenvironment in mouse models. We also found that lack of MGMT expression in pretreated GBM is linked to hypermutation, which in turn contributes to increased genomic heterogeneity and requires new strategies for GBM treatment.

Eukaryotic initiation factor 5B (eIF5B) regulates temozolomide-mediated apoptosis in brain tumour stem cells (BTSCs).

Glioblastoma multiforme (GBM) is among the deadliest cancers, owing in part to complex inter- and intra-tumor heterogeneity and the presence of a population of stem-like cells called brain tumour stem cells (BTSCs/BTICs). These cancer stem cells survive treatment and confer resistance to the current therapies - namely, radiation and the chemotherapeutic, temozolomide (TMZ). TMZ induces cell death by alkylating DNA, and BTSCs resist this mechanism via a robust DNA damage response. Hence, recent studies aimed to sensitize BTSCs to TMZ using combination therapy, such as inhibition of DNA repair machinery. We have previously demonstrated in established GBM cell lines that eukaryotic initiation factor 5B (eIF5B) promotes the translation of pro-survival and anti-apoptotic proteins. Consequently, silencing eIF5B sensitizes these cells to TRAIL-induced apoptosis. However, established cell lines do not always recapitulate the features of human glioma. Therefore, we investigated this mechanism in patient-derived BTSCs. We show that silencing eIF5B leads to increased TMZ sensitivity in two BTSC lines: BT25 and BT48. Depletion of eIF5B decreases the levels of anti-apoptotic proteins in BT48 and sensitizes these cells to TMZ-induced activation of caspase-3, cleavage of PARP, and apoptosis. We suggest that eIF5B represents a rational target to sensitize GBM tumors to the current standard-of-care.

Multicentre, randomised, double-blind, placebo-controlled, proof of concept study of LSALT peptide as prevention of acute respiratory distress syndrome and acute kidney injury in patients infected with SARS-CoV-2 (COVID-19).

OBJECTIVE: Dipeptidase-1 (DPEP-1) is a recently discovered leucocyte adhesion receptor for neutrophils and monocytes in the lungs and kidneys and serves as a potential therapeutic target to attenuate inflammation in moderate-to-severe COVID-19. We aimed to evaluate the safety and efficacy of the DPEP-1 inhibitor, LSALT peptide, to prevent specific organ dysfunction in patients hospitalised with COVID-19. DESIGN: Phase 2a randomised, placebo-controlled, double-blinded, trial. SETTING: Hospitals in Canada, Turkey and the USA. PARTICIPANTS: A total of 61 subjects with moderate-to-severe COVID-19. INTERVENTIONS: Randomisation to LSALT peptide 5 mg intravenously daily or placebo for up to 14 days. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary endpoint was the proportion of subjects alive and free of respiratory failure and/or the need for renal replacement therapy (RRT). Numerous secondary and exploratory endpoints were assessed including ventilation-free days, and changes in kidney function or serum biomarkers. RESULTS: At 28 days, 27 (90.3%) and 28 (93.3%) of subjects in the placebo and LSALT groups were free of respiratory failure and the need for RRT (p=0.86). On days 14 and 28, the number of patients still requiring more intensive respiratory support (O2 ≥6 L/minute, non-invasive or invasive mechanical ventilation or extracorporeal membrane oxygenation) was 6 (19.4%) and 3 (9.7%) in the placebo group versus 2 (6.7%) and 2 (6.7%) in the LSALT group, respectively (p=0.14; p=0.67). Unadjusted analysis of ventilation-free days demonstrated 22.8 days for the LSALT group compared with 20.9 in the placebo group (p=0.4). LSALT-treated subjects had a significant reduction in the fold expression from baseline to end of treatment of serum CXCL10 compared with placebo (p=0.02). Treatment-emergent adverse events were similar between groups. CONCLUSION: In a Phase 2 study, LSALT peptide was demonstrated to be safe and tolerated in patients hospitalised with moderate-to-severe COVID-19. TRIAL REGISTRATION NUMBER: NCT04402957.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1::TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCCs), frustrating efforts to identify therapeutic targets for these rare cancers. Here, proteomic analysis identifies VCP/p97, an AAA+ ATPase with known segregase function, as strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1::TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1::TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributes with ASPSCR1::TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrate the oncogenic transcriptional signature of ASPSCR1::TFE3, by facilitating assembly of higher-order chromatin conformation structures demonstrated by HiChIP. Finally, ASPSCR1::TFE3 and VCP demonstrate co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

macroH2A2 antagonizes epigenetic programs of stemness in glioblastoma.

Self-renewal is a crucial property of glioblastoma cells that is enabled by the choreographed functions of chromatin regulators and transcription factors. Identifying targetable epigenetic mechanisms of self-renewal could therefore represent an important step toward developing effective treatments for this universally lethal cancer. Here we uncover an epigenetic axis of self-renewal mediated by the histone variant macroH2A2. With omics and functional assays deploying patient-derived in vitro and in vivo models, we show that macroH2A2 shapes chromatin accessibility at enhancer elements to antagonize transcriptional programs of self-renewal. macroH2A2 also sensitizes cells to small molecule-mediated cell death via activation of a viral mimicry response. Consistent with these results, our analyses of clinical cohorts indicate that high transcriptional levels of this histone variant are associated with better prognosis of high-grade glioma patients. Our results reveal a targetable epigenetic mechanism of self-renewal controlled by macroH2A2 and suggest additional treatment approaches for glioblastoma patients.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1-TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCC), frustrating efforts to identify therapeutic targets for these rare cancers. Proteomic analysis showed that VCP/p97, an AAA+ ATPase with known segregase function, was strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1-TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1-TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributed with ASPSCR1-TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrated the oncogenic transcriptional signature of ASPSCR1-TFE3, by facilitating assembly of higher-order chromatin conformation structures as demonstrated by HiChIP. Finally, ASPSCR1-TFE3 and VCP demonstrated co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

Dipeptidase-1 governs renal inflammation during ischemia reperfusion injury.

The mechanisms that drive leukocyte recruitment to the kidney are incompletely understood. Dipeptidase-1 (DPEP1) is a major neutrophil adhesion receptor highly expressed on proximal tubular cells and peritubular capillaries of the kidney. Renal ischemia reperfusion injury (IRI) induces robust neutrophil and monocyte recruitment and causes acute kidney injury (AKI). Renal inflammation and the AKI phenotype were attenuated in Dpep1-/- mice or mice pretreated with DPEP1 antagonists, including the LSALT peptide, a nonenzymatic DPEP1 inhibitor. DPEP1 deficiency or inhibition primarily blocked neutrophil adhesion to peritubular capillaries and reduced inflammatory monocyte recruitment to the kidney after IRI. CD44 but not ICAM-1 blockade also decreased neutrophil recruitment to the kidney during IRI and was additive to DPEP1 effects. DPEP1, CD44, and ICAM-1 all contributed to the recruitment of monocyte/macrophages to the kidney following IRI. These results identify DPEP1 as a major leukocyte adhesion receptor in the kidney and potential therapeutic target for AKI.

Gamma-secretase represents a therapeutic target for the treatment of invasive glioma mediated by the p75 neurotrophin receptor.

The multifunctional signaling protein p75 neurotrophin receptor (p75(NTR)) is a central regulator and major contributor to the highly invasive nature of malignant gliomas. Here, we show that neurotrophin-dependent regulated intramembrane proteolysis (RIP) of p75(NTR) is required for p75(NTR)-mediated glioma invasion, and identify a previously unnamed process for targeted glioma therapy. Expression of cleavage-resistant chimeras of p75(NTR) or treatment of animals bearing p75(NTR)-positive intracranial tumors with clinically applicable gamma-secretase inhibitors resulted in dramatically decreased glioma invasion and prolonged survival. Importantly, proteolytic processing of p75(NTR) was observed in p75(NTR)-positive patient tumor specimens and brain tumor initiating cells. This work highlights the importance of p75(NTR) as a therapeutic target, suggesting that gamma-secretase inhibitors may have direct clinical application for the treatment of malignant glioma.

Efficacy and safety/toxicity study of recombinant vaccinia virus JX-594 in two immunocompetent animal models of glioma.

The purpose of this study was to investigate the oncolytic potential of the recombinant, granulocyte macrophage colony-stimulating factor (GM-CSF)-expressing vaccinia virus (VV) JX-594 in experimental malignant glioma (MGs) in vitro and in immunocompetent rodent models. We have found that JX-594 killed all MG cell lines tested in vitro. Intratumoral (i.t.) administration of JX-594 significantly inhibited tumor growth and prolonged survival in rats-bearing RG2 intracranial (i.c.) tumors and mice-bearing GL261 brain tumors. Combination therapy with JX-594 and rapamycin significantly increased viral replication and further prolonged survival in both immunocompetent i.c. MG models with several animals considered "cured" (three out of seven rats >120 days, terminated experiment). JX-594 infected and killed brain tumor-initiating cells (BTICs) from patient samples grown ex vivo, and did so more efficiently than other oncolytic viruses MYXV, Reovirus type-3, and VSV(ΔM51). Additional safety/toxicity studies in nontumor-bearing rodents treated with a supratherapeutic dose of JX-594 demonstrated GM-CSF-dependent inflammation and necrosis. These results suggest that i.c. administered JX-594 triggers a predictable GM-CSF-mediated inflammation in murine models. Before proceeding to clinical trials, JX-594 should be evaluated in the brains of nonhuman primates and optimized for the viral doses, delivery routes as well as the combination agents (e.g., mTOR inhibitors).

Proliferation of human glioblastoma stem cells occurs independently of exogenous mitogens.

Primary glial tumors of the central nervous system, most commonly glioblastoma multiforme (GBM), are aggressive lesions with a dismal prognosis. Despite identification and isolation of human brain tumor stem cells (BTSCs), characteristics that distinguish BTSCs from neural stem cells remain to be elucidated. We cultured cells isolated from gliomas, using the neurosphere culture system, to understand their growth requirements. Both CD133(+) and CD133(-) adult GBM BTSCs proliferated in the absence of exogenous mitogenic stimulation and gave rise to multipotent GBM spheres that were capable of self-renewal. Epidermal growth factor (EGF) and fibroblast growth factor-2 enhanced GBM BTSC survival, proliferation, and subsequent sphere size. Blockade of EGF receptor (EGFR) signaling reduced exogenous mitogen-independent GBM sphere growth. Implantation of as few as 10 exogenous mitogen-independent GBM BTSCs led to the formation of highly invasive intracranial tumors, which closely resembled human GBMs, in immunocompromised mice. These results demonstrate that exogenous mitogen independence, mediated in part through EGFR signaling, is one characteristic that distinguishes CD133(+) and CD133(-) GBM BTSCs from neural stem cells. This novel experimental system will permit the elucidation of additional constitutively activated mechanisms that promote GBM BTSC survival, self-renewal, and proliferation.

The p75 neurotrophin receptor is a central regulator of glioma invasion.

The invasive nature of cancers in general, and malignant gliomas in particular, is a major clinical problem rendering tumors incurable by conventional therapies. Using a novel invasive glioma mouse model established by serial in vivo selection, we identified the p75 neurotrophin receptor (p75(NTR)) as a critical regulator of glioma invasion. Through a series of functional, biochemical, and clinical studies, we found that p75(NTR) dramatically enhanced migration and invasion of genetically distinct glioma and frequently exhibited robust expression in highly invasive glioblastoma patient specimens. Moreover, we found that p75(NTR)-mediated invasion was neurotrophin dependent, resulting in the activation of downstream pathways and producing striking cytoskeletal changes of the invading cells. These results provide the first evidence for p75(NTR) as a major contributor to the highly invasive nature of malignant gliomas and identify a novel therapeutic target.

Efficacy of systemically administered oncolytic vaccinia virotherapy for malignant gliomas is enhanced by combination therapy with rapamycin or cyclophosphamide.

PURPOSE: The oncolytic effects of a systemically delivered, replicating, double-deleted vaccinia virus has been previously shown for the treatment of many cancers, including colon, ovarian, and others. The purpose of this study was to investigate the oncolytic potential of double-deleted vaccinia virus alone or in combination with rapamycin or cyclophosphamide to treat malignant gliomas in vitro and in vivo. EXPERIMENTAL DESIGN: Rat (RG2, F98, C6) and human (A172, U87MG, U118) glioma cell lines were cultured in vitro and treated with live or UV-inactivated vaccinia virus. Viral gene [enhanced green fluorescent protein (EGFP)] expression by fluorescence-activated cell sorting, relative cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and assays for cytopathic effects were examined. S.c. murine tumor xenografts (U87MG, U118, C6) and i.c. (RG2, F98) tumor models in immunocompetent rats were treated with systemic administration of EGFP-expressing vaccinia virus (vvDD-EGFP), alone or in combination with rapamycin or cyclophosphamide, or controls. Tumor size, viral biodistribution, and animal survival were assessed. Lastly, the oncolytic effects of vvDD-EGFP on human malignant glioma explants were evaluated. RESULTS: vvDD-EGFP was able to infect and kill glioma cells in vitro. A single systemic dose of vvDD-EGFP significantly inhibited the growth of xenografts in athymic mice. Systemic delivery of vvDD-EGFP alone was able to target solitary and multifocal i.c. tumors and prolong survival of immunocompetent rats, whereas combination therapy with rapamycin or cyclophosphamide enhanced viral replication and further prolonged survival. Finally, vvDD-EGFP was able to infect and kill ex vivo primary human malignant gliomas. CONCLUSIONS: These results suggest that vvDD-EGFP is a promising novel agent for human malignant glioma therapy, and in combination with immunosuppressive agents, may lead to prolonged survival from this disease.

To promote or inhibit glioma progression, that is the question for IL-33.

IL-33, a member of the IL-1 cytokine family has been shown to play a dual role within the body. First IL-33, similar to other IL-1 family members, is a secreted cytokine that binds to the cell surface receptor ST2 to induce a number of cell signaling pathways. Second, IL-33 enters the nucleus where it binds chromatin and directs transcriptional control of an array of growth factors and cytokines. Consistent with its complex cellular regulation, IL-33 mediates an array of biological functions by acting on a wide range of innate and adaptive immune cells. Recently, we found that IL-33 is expressed in a large number of human glioma patient specimens where its expression within the tumor correlates with the increased presence of Iba+ cells that include both resident microglia and recruited monocyte and macrophages. Strikingly, glioma derived expression of IL-33 correlates with a dramatic decrease in overall survival of tumor-bearing animals and thus supports its role as an influential factor in gliomagenesis. Notably however, when the nuclear localization function of IL-33 is crippled, the tumor microenvironment is programmed to be anti-tumorigenic and results in prolonged overall survival suggesting that when educated appropriately this could represent a novel therapeutic strategy for glioma (De Boeck et al. (2020), Nat Commun, doi: 10.1038/s41467-020-18569-4).

Development of a peptide-based delivery platform for targeting malignant brain tumors.

Despite extensive molecular characterization, human glioblastoma remains a fatal disease with survival rates measured in months. Little improvement is seen with standard surgery, radiotherapy and chemotherapy. Clinical progress is hampered by the inability to detect and target glioblastoma disease reservoirs based on a diffuse invasive pattern and the presence of molecular and phenotypic heterogeneity. The goal of this study was to target the invasive and stem-like glioblastoma cells that evade first-line treatments using agents capable of delivering imaging enhancers or biotherapeutic cargo. To accomplish this, a combinatorial phage display library was biopanned against glioblastoma cell model systems that accurately recapitulate the intra- and inter-tumor heterogeneity and infiltrative nature of the disease. Candidate peptides were screened for specificity and ability to target glioblastoma cells in vivo. Cargo-conjugated peptides delivered contrast-enhancing agents to highly infiltrative tumor populations in intracranial xenograft models without the obvious need for blood brain barrier disruption. Simultaneous use of five independent targeting peptides provided greater coverage of this complex tumor and selected peptides have the capacity to deliver a therapeutic cargo (oncolytic virus VSVΔM51) to the tumor cells in vivo. Herein, we have identified a series of peptides with utility as an innovative platform to assist in targeting glioblastoma for the purpose of diagnostic or prognostic imaging, image-guided surgery, and/or improved delivery of therapeutic agents to glioblastoma cells implicated in disease relapse.

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.

Oncolytic efficacy of recombinant vesicular stomatitis virus and myxoma virus in experimental models of rhabdoid tumors.

PURPOSE: Rhabdoid tumors are highly aggressive pediatric tumors that are usually refractory to available treatments. The purpose of this study was to evaluate the therapeutic potential of two oncolytic viruses, myxoma virus (MV) and an attenuated vesicular stomatitis virus (VSV(DeltaM51)), in experimental models of human rhabdoid tumor. EXPERIMENTAL DESIGN: Four human rhabdoid tumor cell lines were cultured in vitro and treated with live or inactivated control virus. Cytopathic effect, viral gene expression, infectious viral titers, and cell viability were examined at various time points after infection. To study viral oncolysis in vivo, human rhabdoid tumor cells were implanted s.c. in the hind flank or intracranially in CD-1 nude mice and treated with intratumoral (i.t.) or i.v. injections of live or UV-inactivated virus. Viral distribution and effects on tumor size and survival were assessed. RESULTS: All rhabdoid tumor cell lines tested in vitro were susceptible to productive lethal infections by MV and VSV(DeltaM51). I.t. injection of live MV or VSV(DeltaM51) dramatically reduced the size of s.c. rhabdoid tumor xenografts compared with control animals. I.v. administration of VSV(DeltaM51) or i.t. injection of MV prolonged the median survival of mice with brain xenografts compared with controls (VSV(DeltaM51): 25 days versus 21 days, log-rank test, P = 0.0036; MV: median survival not reached versus 21 days, log-rank test, P = 0.0007). Most of the MV-treated animals (4 of 6; 66.7%) were alive and apparently "cured" when the experiment was arbitrarily ended (>180 days). CONCLUSIONS: These results suggest that VSV(DeltaM51) and MV could be novel effective therapies against human rhabdoid tumor.

Targeting human medulloblastoma: oncolytic virotherapy with myxoma virus is enhanced by rapamycin.

We have shown previously the oncolytic potential of myxoma virus in a murine xenograft model of human glioma. Here, we show that myxoma virus used alone or in combination with rapamycin is effective and safe when used in experimental models of medulloblastoma in vitro and in vivo. Nine of 10 medulloblastoma cell lines tested were susceptible to lethal myxoma virus infection, and pretreatment of cells with rapamycin increased the extent of in vitro oncolysis. Intratumoral injection of live myxoma virus when compared with control inactivated virus prolonged survival in D341 and Daoy orthotopic human medulloblastoma xenograft mouse models [D341 median survival: 21 versus 12.5 days; P = 0.0008; Daoy median survival: not reached (three of five mice apparently "cured" after 223 days) versus 75 days; P = 0.0021]. Rapamycin increased the extent of viral oncolysis, "curing" most Daoy tumor-bearing mice and reducing or eliminating spinal cord and ventricle metastases. Rapamycin enhanced tumor-specific myxoma virus replication in vivo and prolonged survival of D341 tumor-bearing mice (median survival of mice treated with live virus (LV) and rapamycin, versus LV alone, versus rapamycin alone, versus inactivated virus: 25 days versus 19, 13, and 11 days, respectively; P < 0.0001). Rapamycin increased the levels of constitutively activated Akt in Daoy and D341 cells, which may explain its ability to enhance myxoma virus oncolysis. These observations suggest that myxoma virus may be an effective oncolytic agent against medulloblastoma and that combination therapy with signaling inhibitors that modulate activity of the phosphatidylinositol 3-kinase/Akt pathway will further enhance the oncolytic potential of myxoma virus.

Intratumoral Genetic and Functional Heterogeneity in Pediatric Glioblastoma.

Pediatric glioblastoma (pGBM) is a lethal cancer with no effective therapies. To understand the mechanisms of tumor evolution in this cancer, we performed whole-genome sequencing with linked reads on longitudinally resected pGBM samples. Our analyses showed that all diagnostic and recurrent samples were collections of genetically diverse subclones. Clonal composition rapidly evolved at recurrence, with less than 8% of nonsynonymous single-nucleotide variants being shared in diagnostic-recurrent pairs. To track the origins of the mutational events observed in pGBM, we generated whole-genome datasets for two patients and their parents. These trios showed that genetic variants could be (i) somatic, (ii) inherited from a healthy parent, or (iii) de novo in the germlines of pGBM patients. Analysis of variant allele frequencies supported a model of tumor growth involving slow-cycling cancer stem cells that give rise to fast-proliferating progenitor-like cells and to nondividing cells. Interestingly, radiation and antimitotic chemotherapeutics did not increase overall tumor burden upon recurrence. These findings support an important role for slow-cycling stem cell populations in contributing to recurrences, because slow-cycling cell populations are expected to be less prone to genotoxic stress induced by these treatments and therefore would accumulate few mutations. Our results highlight the need for new targeted treatments that account for the complex functional hierarchies and genomic heterogeneity of pGBM. SIGNIFICANCE: This work challenges several assumptions regarding the genetic organization of pediatric GBM and highlights mutagenic programs that start during early prenatal development.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/9/2111/F1.large.jpg.