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Despite improvement in the treatment of medulloblastoma over the last years, numerous patients with MYC- and MYCN-driven tumors still fail current therapies. Medulloblastomas have an intact retinoblastoma protein RB, suggesting that CDK4/6 inhibition might represent a therapeutic strategy for which drug combination remains understudied. We conducted high-throughput drug combination screens in a Group3 (G3) medulloblastoma line using the CDK4/6 inhibitor (CDK4/6i) ribociclib at IC20, referred to as an anchor, and 87 oncology drugs approved by FDA or in clinical trials. Bromodomain and extra terminal (BET) and PI3K/mTOR inhibitors potentiated ribociclib inhibition of proliferation in an established cell line and freshly dissociated tumor cells from intracranial xenografts of G3 and Sonic hedgehog (SHH) medulloblastomas in vitro. A reverse combination screen using the BET inhibitor JQ1 as anchor, revealed CDK4/6i as the most potentiating drugs. In vivo, ribociclib showed single-agent activity in medulloblastoma models whereas JQ1 failed to show efficacy due to high clearance and insufficient free brain concentration. Despite in vitro synergy, combination of ribociclib with the PI3K/mTOR inhibitor paxalisib did not significantly improve the survival of G3 and SHH medulloblastoma-bearing mice compared with ribociclib alone. Molecular analysis of ribociclib and paxalisib-treated tumors revealed that E2F targets and PI3K/AKT/MTORC1 signaling genes were depleted, as expected. Importantly, in one untreated G3MB model HD-MB03, the PI3K/AKT/MTORC1 gene set was enriched in vitro compared with in vivo suggesting that the pathway displayed increased activity in vitro. Our data illustrate the difficulty in translating in vitro findings in vivo. See related article in Mol Cancer Ther (2022) 21(8):1306–1317.

Supplementary Table S4

Supplementary Methods, Figures S1-S9 and Tables S3, S5-S7

Background Immunotherapy with chimeric antigen receptor (CAR) T cells is actively being explored for pediatric brain tumors in preclinical models and early phase clinical studies. At present, it is unclear which CAR target antigens are consistently expressed across different pediatric brain tumor types. In addition, the extent of HLA class I expression is unknown, which is critical for tumor recognition by conventional αβTCR T cells. Methods We profiled 49 low- and high-grade pediatric brain tumor patient-derived orthotopic xenografts (PDOX) by flow analysis for the expression of 5 CAR targets (B7-H3, GD2, IL-13Rα2, EphA2, and HER2), and HLA class I. In addition, we generated B7-H3-CAR T cells and evaluated their antitumor activity in vitro and in vivo. Results We established an expression hierarchy for the analyzed antigens (B7-H3 = GD2 >> IL-13Rα2 > HER2 = EphA2) and demonstrated that antigen expression is heterogenous. All high-grade gliomas expressed HLA class I, but only 57.1% of other tumor subtypes had detectable expression. We then selected B7-H3 as a target for CAR T-cell therapy. B7-H3-CAR T cells recognized tumor cells in an antigen-dependent fashion. Local or systemic administration of B7-H3-CAR T cells induced tumor regression in PDOX and immunocompetent murine glioma models resulting in a significant survival advantage. Conclusions Our study highlights the importance of studying target antigen and HLA class I expression in PDOX samples for the future design of immunotherapies. In addition, our results support active preclinical and clinical exploration of B7-H3-targeted CAR T-cell therapies for a broad spectrum of pediatric brain tumors.

Mouse models of medulloblastoma have proven to be instrumental in understanding disease mechanisms, particularly the role of epigenetic and molecular drivers, and establishing appropriate preclinical pipelines. To date, our research community has developed murine models for all four groups of medulloblastoma, each of which will be critical for the identification and development of new therapeutic approaches. Approaches to modeling medulloblastoma range from genetic engineering with CRISPR/Cas9 or in utero electroporation, to orthotopic and patient‐derived orthotopic xenograft systems. Each approach or model presents unique advantages that have ultimately contributed to an appreciation of medulloblastoma heterogeneity and the clinical obstacles that exist for this patient population.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor of the cerebellum, with four distinct molecular subgroups, each carrying different clinical prognoses. Group 3 (G3) is the most aggressive subgroup and is associated with amplifications of the MYC gene. MYC expression and function are controlled in part by epigenetic regulation, and interestingly, the few mutations that are found in G3 medulloblastoma are mostly located in genes encoding epigenetic regulators. Surprisingly, many tumors had no mutations raising the question as to whether non-mutated or non-amplified epigenetic regulators may be drivers of G3MB tumorigenesis. We previously found that dysregulation of the H3K27 methyltransferase EZH2 cooperates with MYC in G3MB, indicating that epigenetic regulators may provide a source of potential therapeutic targets. Therefore, to identify novel epigenetic drivers in G3MB, we performed an unbiased shRNA screen of 243 known chromatin regulators. The most intriguing candidate was SMYD3, a multidomain-containing protein with histone H4-lysine 5 (H4K5) methyltransferase activity. Knockdown of SMYD3 suppressed G3MB proliferation in vitro and tumor progression in vivo. SMYD3 protein is nuclear and associated with the MYC promoter. Deletion of SMYD3 results in loss of MYC, G2/M and epithelial-to-mesenchymal target gene expression. Using a multi-omics approach including CUT&RUN sequencing, RNA-seq, DNA methylation, cryo-electron microscopy (cryo-EM) and proteomics we aim to elucidate how SMYD3 controls G3MB tumorigenesis. These results highlight the role of a potentially targetable new epigenetic regulator of tumorigenesis in G3MB. Citation Format: Audrey L. Mercier, Jennifer L. Stripay, Silvija Bilokapic, Justin Williams, Stephanie Nance, Jingjing Liu, Steven Philips, Suresh Kandikonda, Marie Morfouace, Aude Bramoulle, Tamara Advedissian, Jiyang Yu, Junmin Peng, Aseem Ansari, Mario Halic, Adam Durbin, Christopher Vakoc, Martine F. Roussel. Interrogating the function of histone methyltransferases in MYC-driven medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2968.

Medulloblastoma (MB) consists of four core molecular subgroups with distinct clinical features and prognoses. Treatment consists of surgery, followed by radiotherapy and cytotoxic chemotherapy. Despite this intensive approach, outcome remains dismal for patients with certain subtypes of MB, namely MYC-amplified Group 3 and TP53-mutated SHH. Using high-throughput assays, six human MB cell lines were screened against a library of 3208 unique compounds. We identified 45 effective compounds from the screen and found that inhibition cell cycle checkpoint kinases (CHK1 and 2) synergistically enhanced the cytotoxic activity of clinically-used chemotherapeutics cyclophosphamide, cisplatin, and gemcitabine. To identify the best-in-class inhibitor, multiple CHK1/2 inhibitors were assessed in mice bearing intracranial MB. When combined with DNA-damaging chemotherapeutics, CHK1/2 inhibition reduced tumor burden and significantly increased survival of animals with high-risk MB, across multiple different models. In total, we tested 14 different models, representing distinct MB subgroups, and data were validated in three independent laboratories. Pharmacodynamics studies confirmed central nervous system penetration. In mice, combination treatment significantly increased DNA damage and apoptosis compared to chemotherapy alone, and studies with cultured cells showed that CHK inhibition disrupted chemotherapy-induced cell cycle arrest. Our findings indicated CHK1/2 inhibition, specifically with LY2606368 (prexasertib), has strong chemosensitizing activity in MB that warrants further clinical investigation. Moreover, these data demonstrated that we developed a robust and collaborative preclinical assessment platform that can be used to identify potentially effective new therapies for clinical evaluation for pediatric MB.

The SNO+ experiment is located 2 km underground at SNOLAB in Sudbury, Canada. A low background search for neutrinoless double beta ($0������$) decay will be conducted using 780 tonnes of liquid scintillator loaded with 3.9 tonnes of natural tellurium, corresponding to 1.3 tonnes of $^{130}$Te. This paper provides a general overview of the SNO+ experiment, including detector design, construction of process plants, commissioning efforts, electronics upgrades, data acquisition systems, and calibration techniques. The SNO+ collaboration is reusing the acrylic vessel, PMT array, and electronics of the SNO detector, having made a number of experimental upgrades and essential adaptations for use with the liquid scintillator. With low backgrounds and a low energy threshold, the SNO+ collaboration will also pursue a rich physics program beyond the search for $0������$ decay, including studies of geo- and reactor antineutrinos, supernova and solar neutrinos, and exotic physics such as the search for invisible nucleon decay. The SNO+ approach to the search for $0������$ decay is scalable: a future phase with high $^{130}$Te-loading is envisioned to probe an effective Majorana mass in the inverted mass ordering region., 61 pages, 23 figures, 4 tables

Cell surface or intracellular antigens expressed in pediatric brain tumors are potential targets for chimeric antigen receptor (CAR) or ab (T-cell receptor) TCR T-cell immunotherapy. At present it remains unknown what cell surface antigens are suitable CAR targets for pediatric brain tumors; in addition, cell surface expression of HLA class I, a molecule critical for ab TCR T-cell recognition, has not been systemically studied in these tumors. Therefore, we set out to assess expression of five CAR targets (IL13Ra2, HER2, EphA2, B7-H3, GD2) and HLA class I. We established and validated a flow cytometry-based method to profile CAR targets and HLA class I expression from pediatric patient-derived xenograft (PDX) samples. To date, we profiled 53 PDX samples, including medulloblastoma, HGG, DIPG, ATRT, and ependymoma. We found that antigen expression has high intra- and inter-PDX sample variability with B7-H3 and IL13Ra2 being most consistently expressed. We confirmed these findings using conventional IHC for B7-H3 with PDX samples and patient tissue microarrays. HLA class I was present on the cell surface of HGGs and DIPGs, however significantly down-regulated in 26 out of 36 other brain tumor types. Finally, matched fresh tissue and PDX sample analysis revealed that cells derived from PDX models are indeed representative of fresh tissue. Our results indicate that more than one antigen needs to be targeted to achieve a more complete tumor clearance. In addition, variable expression of HLA class I suggests that pediatric brain tumors have developed immune evasion strategies to prevent recognition by conventional T cells.

Prexasertib (LY2606368) is a potent and selective small molecule inhibitor of cell-cycle checkpoint CHK1 and CHK2 protein kinases and is currently under clinical evaluation for treatment of pediatric malignancies. As a candidate therapy for pediatric Group 3 medulloblastoma (G3MB), prexasertib CNS penetration was evaluated in mice using cerebral microdialysis and pharmacokinetic modeling. A plasma pharmacokinetic study with a population-based design was performed in CD1 nude mice bearing G3MB orthotopically implanted in the brain and receiving a single dose of prexasertib (10 mg/kg, subcutaneously) to characterize prexasertib disposition and to establish a limited plasma sampling model for the microdialysis studies. The microdialysis studies were performed in both non-tumor bearing mice and in mice bearing G3MB receiving 10 mg/kg prexasertib subcutaneously, for up to 24 hours post-dose. Plasma and extracellular fluid (ECF) concentrations were quantified using validated LC MS/MS methods, and analyzed using a population pharmacokinetic model. Model-derived prexasertib tumor/ECF to plasma partition coefficient K(p,uu) (ratio of tumor/brain ECF to unbound plasma AUC(0–24h)) was significantly greater in G3MB tumor-bearing mice (0.17 ± 0.08) compared to non-tumor bearing mice (0.09 ± 0.04, p=0.04). A pharmacodynamic study was then performed in mice bearing G3MB (20 mg/kg, IV) to evaluate prexasertib-induced target engagement after a single dose. Phosphorylated CHK1 serine 345 (pCHK1 S345), phosphorylated Histone 2A variant (γ-H2AX), and cleaved caspase-3 were quantified in mouse G3MB tumor tissues by immunohistochemistry at different time points up to 24 hours post-dose. The induction of pCHK1 S345 and γ-H2AX peaked at 2 hours after the dose and was elevated above baseline for at least 6 hours, reflecting relevant CHK1 inhibition and DNA damage. Cleaved caspase-3 levels increased at 24 hours suggesting initiation of cell apoptosis. Adequate unbound prexasertib exposure reached the brain tumor site relative to target engagement in G3MB tumor bearing mice at a clinically relevant dosage. These results support further preclinical and clinical development of prexasertib to treat children with medulloblastoma.

Medulloblastoma is an embryonal tumor that shows a predilection for distant metastatic spread and leptomeningeal seeding. For most patients, optimal management of medulloblastoma includes maximum safe resection followed by adjuvant craniospinal irradiation (CSI) and chemotherapy. Although CSI is crucial in treating medulloblastoma, the realization that medulloblastoma is a heterogeneous disease comprising four distinct molecular subgroups (wingless [WNT], sonic hedgehog [SHH], Group 3 [G3], and Group 4 [G4]) with distinct clinical characteristics and prognoses has refocused efforts to better define the optimal role of CSI within and across disease subgroups. The ability to deliver clinically relevant CSI to preclinical models of medulloblastoma offers the potential to study radiation dose and volume effects on tumor control and toxicity in these subgroups and to identify subgroup-specific combination adjuvant therapies. Recent efforts have employed commercial image-guided small animal irradiation systems as well as custom approaches to deliver accurate and reproducible fractionated CSI in various preclinical models of medulloblastoma. Here, we provide an overview of the current clinical indications for, and technical aspects of, irradiation of pediatric medulloblastoma. We then review the current literature on preclinical modeling of and treatment interventions for medulloblastoma and conclude with a summary of challenges in the field of preclinical modeling of CSI for the treatment of leptomeningeal seeding tumors.

Medulloblastoma (MB), the most common malignant pediatric brain tumor, is classified into four major molecularly and histopathologically distinct subgroups, among which MYC-driven Group 3 MBs confer a poor prognosis. The Cyclin D/CDK4/CDK6/RB pathway is frequently deregulated in MB leading to uncontrolled cell proliferation, but tumors express an intact RB protein (Northcott et al., Nature, 2017). Therefore, CDK4/6 inhibitor drugs offer a possible therapeutic approach to treat MBs. Because single agent therapy ultimately leads to drug resistance, we initiated in vitro combination drug screens to identify drug classes potentiating CDK4/6 inhibitors. We used Group 3 MB patient-derived orthotopic xenografts (PDOXs), a human cell line (HDMB03), and freshly dissociated tumor cells propagated only in the mouse brain. The drug screen included 90 compounds comprising targeted and cytotoxic drugs that are FDA approved or under active clinical investigation. Using a bioluminescence-based assay that measures ATP consumption (CellTiter-Glo) to evaluate the number of viable cells, these 90 compounds were screened in combination with a fixed concentration of ribociclib, one of the three FDA approved CDK4/6 inhibitors. The primary screen, carried out in HDMB03 cells, revealed several drugs with additive or synergistic potential when combined with ribociclib, including BET inhibitors, MEK inhibitors, PI3K/mTOR inhibitors and gemcitabine. We are currently evaluating the combination of brain penetrant compounds in Group 3 MB PDOXs. The identification of potent drug combinations should provide new therapeutic options for the treatment of Group 3 MB, one of the most difficult to treat.

Glioblastoma multiforme (GBM) is the most common aggressive tumor of the central nervous system (CNS) with 10,000 newly diagnosed cases annually, and an incidence rate of 3 in 100,000 individuals in the United States. Unfortunately, the current standard of care of radiotherapy with concomitant Temozolomide (TMZ) has not been effective in consistently controlling tumor growth, as the median age of survival for patients is 14.6 months after treatment, with inevitable recurrence beyond this time. One of the difficulties that emerge when treating GBM is tumor heterogeneity. For example, GBM cells upregulate different receptor tyrosine kinases (RTKs). Signaling through RTKs enables these cells to maintain their proliferative status to ensure their survival. The E3 ubiquitin ligase c-Cbl is a negative regulator of RTKs, and our lab has found it to be physically sequestered by ARGHEF7 (Cool-1) in GBM. We identified a combination of drugs, a c-Cbl restorative agent (CRA01) and an oxidizing agent (Tamoxifen [TMX]), that decreased c-Cbl/Cool-1 complex formation and activated c-Cbl through the Redox/Fyn/c-Cbl pathway (RFC) in GBM cells. Treatment with CRA01+TMX led to degradation of RTKs. Little is known about the RFC pathway in cancer regulation or its ability to prevent cell growth in TMZ resistant GBM cells. Here, we examine ways to regulate c-Cbl in GBM cells and seek to identify the mechanism(s) resulting in c-Cbl/Cool-1 complex formation. Furthermore, we aim to use a mouse model of TMZ resistance to determine the effects of CRA01+TMX in vivo. These results will provide important knowledge on the RFC pathway that can help properly identify patients with GBM and different cancers with c-Cbl dysregulation for whom this treatment could prove beneficial. Citation Format: Neal Shah, Jennifer Stripay. Analyzing the redox/fyn/c-Cbl pathway in glioblastoma multiforme: A mechanistic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4293.

Medulloblastoma (MB), the most common malignant pediatric brain tumor, is characterized by four molecularly and histopathologically distinct groups with different prognoses. With the advent of next generation sequencing, the field has gained new insights into somatically altered genes in MB, many of which are involved in epigenetic gene regulation and chromatin modification. A significant portion of Group 3 MB do not carry any mutations, suggesting that additional, non-mutated, epigenetic regulators may be implicated in shaping chromatin structure and driving tumor pathogenesis. Considering this possibility, we conducted two unbiased screens of epigenetic regulators in G3 MB to identify candidate drivers of medulloblastoma genesis. 1) In collaboration with Dr. Christopher Vakoc, we screened a library of shRNAs targeting 293 known chromatin modifiers in mouse G3 MB cells. Designed to assess reduction in viability, the dropout screen identified putative oncogenes, and of the top hits, SMYD3, a lysine methyltransferase, emerged as the most interesting candidate. SMYD3 is overexpressed and exhibits increased predicted activity in G3 MB compared to other subgroups. Perturbation of SMYD3 using shRNA or CRISPR/Cas9 results in significantly reduced tumor progression and significantly prolonged lifespan in tumor-bearing animals. 2) We also screened a pooled epigenetic CRISPR/Cas9 gRNA library in G3 MB cells where we assessed gRNA enrichment in vivo and thus, identified putative tumor suppressors. Based on biological relevance and computational prediction, PRDM2 was selected as the top candidate. Implicated in other cancers, PRDM2, a histone 3 lysine 9 methyltransferase, exhibits low expression and predicted activity in G3 MB compared to other subgroups. Current work is focused on dissecting the epigenetic, transcriptomic, and phenotypic consequences of PRDM2 perturbation in naïve neural progenitors and G3 MB cells. Taken together, these efforts will help us to understand how changes in chromatin modifiers affect disease pathogenesis and how these changes may represent therapeutic vulnerabilities.

In addition to dopaminergic neuron loss, it is clear that Parkinson disease includes other pathological changes, including loss of additional neuronal populations. As a means of addressing multiple pathological changes with a single therapeutically-relevant approach, we employed delayed transplantation of a unique class of astrocytes, GDAs(BMP), that are generated in vitro by directed differentiation of glial precursors. GDAs(BMP) produce multiple agents of interest as treatments for PD and other neurodegenerative disorders, including BDNF, GDNF, neurturin and IGF1. GDAs(BMP) also exhibit increased levels of antioxidant pathway components, including levels of NADPH and glutathione. Delayed GDA(BMP) transplantation into the 6-hydroxydopamine lesioned rat striatum restored tyrosine hydroxylase expression and promoted behavioral recovery. GDA(BMP) transplantation also rescued pathological changes not prevented in other studies, such as the rescue of parvalbumin(+) GABAergic interneurons. Consistent with expression of the synaptic modulatory proteins thrombospondin-1 and 2 by GDAs(BMP), increased expression of the synaptic protein synaptophysin was also observed. Thus, GDAs(BMP) offer a multimodal support cell therapy that provides multiple benefits without requiring prior genetic manipulation.

Dysregulated Ca2+ handling is prevalent during sepsis and postulated to perpetuate the aberrant inflammation underlying subsequent organ dysfunction and death. The signal transduction cascades mediating these processes are unknown. Here, we identify that CaMKIα mediates the Mφ response to LPS in vitro and the inflammation and organ dysfunction of sepsis in vivo. We show that LPS induced active pThr177-CaMKIα in RAW 264.7 cells and murine peritoneal Mφ, which if inhibited biochemically with STO609 (CaMKK inhibitor) or by RNAi, reduces LPS-induced production of IL-10. Transfection of constitutively active CaMKIα (CaMKI293), but not a kinase-deficient mutant (CaMKI293K49A), induces IL-10 release. This production of IL-10 is mediated by CaMKIα-dependent regulation of p38 MAPK activation. CaMKIα activity also mediates the cellular release of HMGB1 by colocalizing with and regulating the packaging of HMGB1 into secretory lysosomes. During endotoxemia, mice receiving in vivo CaMKIαRNAi display reduced systemic concentrations of IL-10 and HMGB1 in comparison with mice receiving NTRNAi. These data support the biological relevance of CaMKIα-dependent IL-10 production and HMGB1 secretion. In a CLP model of sepsis, CaMKIαRNAi mice display reduced systemic concentrations of IL-10, IL-6, TNF-α, and HMGB1 in comparison with NTRNAi mice, which correlate with reductions in the development of renal dysfunction. These data support that CaMKIα signaling is integral to the Mφ responding to LPS and may also be operant in vivo in regulating the inflammation and organ dysfunction consequent to sepsis.

Background and aims: Treatment of luminal breast cancer (LBC) with tamoxifen (TMX) is associated with the emergence of resistance in ~40% of patients. Such resistance is associated with emergence of tumor initiating cells (TICs, also known as cancer stem cells), resistance to other therapeutic agents and multiple other obstacles to successful treatment. This study aims to investigate the role of tumor suppressor gene c-Cbl in TMX resistance and identify novel therapeutic approaches in treatment of acquired TMX resistant LBC that address unmet clinical needs. Methods: TMX resistant MCF7 and T47D cell lines were generated by chronic exposure to TMX in vitro. Unbiased drug screening using the NINDS-II library revealed previously approved drugs with unexpected activities to restore c-Cbl activity. Assays, including dose-response studies, western blot, co-immunoprecipitation analyses and tumorsphere assays, were used to determine the mechanisms of action and utility of our lead agent (CRA-1). Finally, response to treatment was verified in xenograft mouse models. Findings: In TMX-resistant LBC cells, c-Cbl was inhibited due to complex formation with the Cool-1/ßpix (C1ßp) protein. Restoration of c-Cbl function by C1ßp knockdown or by treatment with our lead c-Cbl restorative agent (CRA-1, which is approved for other purposes and is suitable for prolonged use in patients) restored sensitivity to TMX and decreased TIC function. Treatment with CRA-1+TMX increased c-Cbl activation via the non-canonical redox/Fyn/c-Cbl (RFC) pathway by harnessing TMX's estrogen receptor-independent pro-oxidative activity. This treatment also decreased levels of the c-Cbl/C1ßp inhibitory complex, and of levels and/or activity of multiple direct and indirect c-Cbl targets important in TMX resistance (e.g. EGFR, HER2, Notch-1, sox2, ß-catenin). Treatment with CRA-1+TMX also increased sensitivity to cyclophosphamide (CPP), one of the standard chemotherapeutic agents to treat TMX resistant LBCs. In vivo, treatment with MPT+TMX + ultra-low-dose of CPP completely suppressed tumor growth in a xenograft animal model for 20 weeks without any obvious toxicity. Tumors thus far have not recurred (15 weeks) after treatment was stopped. Conclusions: Our study offers a new approach to overcoming acquired TMX resistance that also: (i) has the potential for rapid clinical translation due to the use of drugs already approved for other purposes; (ii) eliminates TICs; (iii) suppresses tumor growth in vivo with only a small fraction of clinically relevant CPP dosages; and (iv) exhibits greatly reduced toxicity effects and applicability for extended treatment periods. Citation Format: Xiaowen Wang, Hsing-Yu Chen, Jennifer Stripay, Mark Noble. Targeting the c-Cbl tumor suppressor protein as a novel therapeutic approach in overcoming acquired tamoxifen resistance in luminal breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1930.

This review discusses a unique discovery path starting with novel findings on redox regulation of precursor cell and signaling pathway function and identification of a new mechanism by which relatively small changes in redox status can control entire signaling networks that regulate self-renewal, differentiation, and survival. The pathway central to this work, the redox/Fyn/c-Cbl (RFC) pathway, converts small increases in oxidative status to pan-activation of the c-Cbl ubiquitin ligase, which controls multiple receptors and other proteins of central importance in precursor cell and cancer cell function. Integration of work on the RFC pathway with attempts to understand how treatment with systemic chemotherapy causes neurological problems led to the discovery that glioblastomas (GBMs) and basal-like breast cancers (BLBCs) inhibit c-Cbl function through altered utilization of the cytoskeletal regulators Cool-1/βpix and Cdc42, respectively. Inhibition of these proteins to restore normal c-Cbl function suppresses cancer cell division, increases sensitivity to chemotherapy, disrupts tumor-initiating cell (TIC) activity in GBMs and BLBCs, controls multiple critical TIC regulators, and also allows targeting of non-TICs. Moreover, these manipulations do not increase chemosensitivity or suppress division of nontransformed cells. Restoration of normal c-Cbl function also allows more effective harnessing of estrogen receptor-α (ERα)-independent activities of tamoxifen to activate the RFC pathway and target ERα-negative cancer cells. Our work thus provides a discovery strategy that reveals mechanisms and therapeutic targets that cannot be deduced by standard genetics analyses, which fail to reveal the metabolic information, isoform shifts, protein activation, protein complexes, and protein degradation critical to our discoveries.

Calcium plays an essential role in nearly all cellular processes. As such, cellular and systemic calcium concentrations are tightly regulated. During sepsis, derangements in such tight regulation frequently occur, and treating hypocalcemia with parenteral calcium administration remains the current practice guideline.We investigated whether calcium administration worsens mortality and organ dysfunction using an experimental murine model of sepsis and explored the mechanistic role of the family of calcium/calmodulin-dependent protein kinases in mediating these physiological effects. To highlight the biological relevance of these observations, we conducted a translational study of the association between calcium administration, organ dysfunction, and mortality among a cohort of critically ill septic ICU patients.Prospective, randomized controlled experimental murine study and observational clinical cohort analysis.University research laboratory and eight ICUs at a tertiary care center.A cohort of 870 septic ICU patients.C57Bl/6 and CaMKK mice.Mice underwent cecal ligation and puncture polymicrobial sepsis and were administered with calcium chloride (0.25 or 0.25 mg/kg) or normal saline.Administering calcium chloride to septic C57Bl/6 mice heightened systemic inflammation and vascular leak, exacerbated hepatic and renal dysfunction, and increased mortality. These events were significantly attenuated in CaMKK mice. In a risk-adjusted analysis of septic patients, calcium administration was associated with an increased risk of death, odds ratio 1.92 (95% CI, 1.00-3.68; p = 0.049), a significant increase in the risk of renal dysfunction, odds ratio 4.74 (95% CI, 2.48-9.08; p0.001), and a significant reduction in ventilator-free days, mean decrease 3.29 days (0.50-6.08 days; p = 0.02).Derangements in calcium homeostasis occur during sepsis that is sensitive to calcium administration. This altered calcium signaling, transduced by the calmodulin-dependent protein kinase kinase cascade, mediates heightened inflammation and vascular leak that culminates in elevated organ dysfunction and mortality. In the clinical management of septic patients, calcium supplementation provides no benefit and may impose harm.

Autophagy is an evolutionarily conserved cytoplasmic process regulated by the energy rheostats mammalian target of rapamycin and AMP kinase (AMPK) that recycles damaged or unused proteins and organelles. It has been described as an important effector arm of immune cells. We have shown that the cytoplasmically oriented calcium/calmodulin-dependent protein kinase (CaMK)Iα regulates the inflammatory phenotype of the macrophage (Mϕ). In this study, we hypothesize that CaMKIα mediates Mϕ autophagy. LPS induced autophagy in RAW 264.7 cells and murine peritoneal Mϕ that was attenuated with biochemical CaMK inhibition or CaMKIα small interfering RNA (siRNA). Inhibition of CaMKIα reduced LPS-induced p-Thr172AMPK and target of rapamycin complex-1 activity, and expression of a constitutively active CaMKIα but not a kinase-deficient mutant induced p-Thr172AMPK and autophagy that was attenuated by the AMPK inhibitor compound C. Coimmunoprecipitation and in vitro kinase assays demonstrated that CaMKIα activates AMPK, thereby inducing ATG7, which also localizes to this CaMKIα/AMPK complex. During LPS-induced lung inflammation, C57BL/6 mice receiving CaMKIαsiRNA displayed reduced lung and bronchoalveolar immune cell autophagy that correlated with reduced neutrophil recruitment, myeloperoxidase activity, and air space cytokine concentration. Independently inhibiting autophagy, using siRNA targeting the PI3K VPS34, yielded similar reductions in lung autophagy and neutrophil recruitment. Thus, a novel CaMKIα/AMPK pathway is rapidly activated in Mϕ exposed to LPS and regulates an early autophagic response, independent of target of rapamycin complex-1 inhibition. These mechanisms appear to be operant in vivo in orchestrating LPS-induced lung neutrophil recruitment and inflammation.

Glioblastoma multiforme is the most common primary brain tumor in adults, the most malignant of all intracranial tumors, and is associated with inevitable recurrence and 15 month mean survival despite multimodal therapy. The majority of GBM tumors maintain heterogeneous amplifications (many in the absence of genetic mutation) in receptor tyrosine kinases (RTKs), which are mediators of growth factor signaling. Our lab has discovered that control over RTK levels in normal CNS progenitors is maintained by the Redox/Fyn/c-Cbl (RFC) pathway whereby increased oxidative state results in activation of the E3 ubiquitin ligase c-Cbl and the subsequent internalization and degradation of target RTKs. RFC signaling is disrupted in GBM due to a physical sequestration of c-Cbl, preventing pro-oxidative chemotherapeutics from reducing pro-survival signaling through Epidermal Growth Factor Receptor, Platelet-Derived Growth Factor Receptor, etc. Using a triangulating drug screen, we identified candidate FDA-approved small molecules capable of reducing signaling downstream of c-Cbl-targeted RTKs. We found that candidate URMC-06, an antidepressant with a good clinical safety profile, could rescue RFC signaling and significantly enhance the sensitivity of GBM cells to a pro-oxidative agent (Tamoxifen) in a c-Cbl-dependent manner. There is significant evidence to suggest that a tumor initiating cell population mediates the malignancy of GBM tumors. These cells express stem-cell associated markers and exhibit self-renewal capacity. We observed a c-Cbl-dependent inhibition of neurosphere formation (an in vitro measure of self-renewal) and a reduction in the expression of stem cell markers Sox2, beta-catenin, FoxM1 and Nestin in GBM cells treated with URMC-06. Our candidate also facilitated reduction in heat shock signaling and an inhibition of autophagy (both putative survival mechanisms) in GBM cells. URMC-06 was effective, in combination with clinically relevant agents (Temozolomide and Tamoxifen), at reducing tumor progression in an in vivo xenograft model of GBM. Our work supports the hypothesis that c-Cbl is a critical player in GBM that integrates multiple nodes of cancer control and that pharmacological rescue of c-Cbl represents a potentially powerful therapeutic approach. Citation Format: Jennifer L. Stripay, Brett M. Stevens, Addie L. Bardin, Mark D. Noble. Targeting a network of cancer control nodes through rescue of c-Cbl: A novel therapeutic approach in GBM. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B14.