An effective kinase inhibition strategy for metastatic recurrent childhood medulloblastoma.

PURPOSE: Medulloblastomas (MBs) constitute the most common malignant brain tumor in children and adolescents. MYC-amplified Group 3 MBs are characterized by disease recurrence, specifically in the leptomeninges, whereby patients with these metastatic tumors have a mortality rate nearing 100%. Despite limited research on such tumors, studies on MB metastases at diagnosis suggest targeting kinases to be beneficial. METHODS: To identify kinase inhibitors that eradicate cells driving therapy evasion and tumor dissemination, we utilized our established patient-derived xenograft (PDX) mouse-adapted therapy platform that models human MB metastatic recurrences following standard chemoradiotherapy. High-throughput screens of 640 kinase inhibitors were conducted against cells isolated from mouse spines in the PDX model and human fetal neural stem cells to reveal compounds that targeted these treatment-refractory, metastatic cells, whilst sparing healthy cells. Blood-brain barrier permeability assays and additional in vitro experimentation helped select top candidates for in vivo studies. RESULTS: Recurrent Group 3 MB PDX spine cells were therapeutically vulnerable to a selective checkpoint kinase 1 (CHK1) inhibitor and small molecular inhibitor of platelet-derived growth factor receptor beta (PDGFRß). Inhibitor-treated cells showed a significant reduction in MB stem cell properties associated with treatment failure. Mice also demonstrated survival advantage when treated with a CHK1 inhibitor ex vivo. CONCLUSION: We identified CHK1 and PDGFRß inhibitors that effectively target MB cells fueling treatment-refractory metastases. With limited research on effective therapies for Group 3 MB metastatic recurrences, this work highlights promising therapeutic options to treat these aggressive tumors. Additional studies are warranted to investigate these inhibitors' mechanisms and recommended in vivo administration.

An HLA-G/SPAG9/STAT3 axis promotes brain metastases.

Brain metastases (BM) are the most common brain neoplasm in adults. Current BM therapies still offer limited efficacy and reduced survival outcomes, emphasizing the need for a better understanding of the disease. Herein, we analyzed the transcriptional profile of brain metastasis initiating cells (BMICs) at two distinct stages of the brain metastatic cascade-the "premetastatic" or early stage when they first colonize the brain and the established macrometastatic stage. RNA sequencing was used to obtain the transcriptional profiles of premetastatic and macrometastatic (non-premetastatic) lung, breast, and melanoma BMICs. We identified that lung, breast, and melanoma premetastatic BMICs share a common transcriptomic signature that is distinct from their non-premetastatic counterparts. Importantly, we show that premetastatic BMICs exhibit increased expression of HLA-G, which we further demonstrate functions in an HLA-G/SPAG9/STAT3 axis to promote the establishment of brain metastatic lesions. Our findings suggest that unraveling the molecular landscape of premetastatic BMICs allows for the identification of clinically relevant targets that can possibly inform the development of preventive and/or more efficacious BM therapies.

The proteomic landscape of glioblastoma recurrence reveals novel and targetable immunoregulatory drivers.

Glioblastoma (GBM) is characterized by extensive cellular and genetic heterogeneity. Its initial presentation as primary disease (pGBM) has been subject to exhaustive molecular and cellular profiling. By contrast, our understanding of how GBM evolves to evade the selective pressure of therapy is starkly limited. The proteomic landscape of recurrent GBM (rGBM), which is refractory to most treatments used for pGBM, are poorly known. We, therefore, quantified the transcriptome and proteome of 134 patient-derived pGBM and rGBM samples, including 40 matched pGBM-rGBM pairs. GBM subtypes transition from pGBM to rGBM towards a preferentially mesenchymal state at recurrence, consistent with the increasingly invasive nature of rGBM. We identified immune regulatory/suppressive genes as important drivers of rGBM and in particular 2-5-oligoadenylate synthase 2 (OAS2) as an essential gene in recurrent disease. Our data identify a new class of therapeutic targets that emerge from the adaptive response of pGBM to therapy, emerging specifically in recurrent disease and may provide new therapeutic opportunities absent at pGBM diagnosis.

Dual Antigen T Cell Engagers Targeting CA9 as an Effective Immunotherapeutic Modality for Targeting CA9 in Solid Tumors.

Glioblastomas (GBM), the most common malignant primary adult brain tumors, are uniformly lethal and are in need of improved therapeutic modalities. GBM contain extensive regions of hypoxia and are enriched in therapy resistant brain tumor-initiating cells (BTICs). Carbonic anhydrase 9 (CA9) is a hypoxia-induced cell surface enzyme that plays an important role in maintenance of stem cell survival and therapeutic resistance. Here we demonstrate that CA9 is highly expressed in patient-derived BTICs. CA9+ GBM BTICs showed increased self-renewal and proliferative capacity. To target CA9, we developed dual antigen T cell engagers (DATEs) that were exquisitely specific for CA9-positive patient-derived clear cell Renal Cell Carcinoma (ccRCC) and GBM cells. Combined treatment of either ccRCC or GBM cells with the CA9 DATE and T cells resulted in T cell activation, increased release of pro-inflammatory cytokines and enhanced cytotoxicity in a CA9-dependent manner. Treatment of ccRCC and GBM patient-derived xenografts markedly reduced tumor burden and extended survival. These data suggest that the CA9 DATE could provide a novel therapeutic strategy for patients with solid tumors expressing CA9 to overcome treatment resistance. .

Real-time evaluation of a hydrogel delivery vehicle for cancer immunotherapeutics within embedded spheroid cultures.

Many protein immunotherapeutics are hindered by transport barriers that prevent the obtainment of minimum effective concentrations (MECs) in solid tumors. Local delivery vehicles with tunable release (infusion) rates for immunotherapeutics are being developed to achieve local and sustained release. To expedite their discovery and translation, in vitro models can identify promising delivery vehicles and immunotherapies that benefit from sustained release by evaluating cancer spheroid killing in real-time. Using displacement affinity release (DAR) within a hydrogel, we tuned the release of a CD133 targeting dual antigen T cell engager (DATE) without the need for further DATE or hydrogel modifications, yielding an injectable vehicle that acts as a tunable infusion pump. To quantify bioactivity benefits, a 3D embedded cancer spheroid model was developed for the evaluation of sustained protein release and combination therapies on T cell mediated spheroid killing. Using automated brightfield and fluorescent microscopy, the size of red fluorescent protein (iRFP670) expressing spheroids were tracked to quantify spheroid growth or killing over time as a function of controlled delivery. We demonstrate that sustained DATE release enhanced T cell mediated killing of embedded glioblastoma spheroids at longer timepoints, killing was further enhanced with the addition of anti-PD1 antibody (αPD1). The multi-cellular embedded spheroid model with automated microscopy demonstrated the benefit of extended bispecific release on T cell mediated killing, which will expedite the identification and translation of delivery vehicles such as DAR for immunotherapeutics.

CD70 as an actionable immunotherapeutic target in recurrent glioblastoma and its microenvironment.

PURPOSE: Glioblastoma (GBM) patients suffer from a dismal prognosis, with standard of care therapy inevitably leading to therapy-resistant recurrent tumors. The presence of cancer stem cells (CSCs) drives the extensive heterogeneity seen in GBM, prompting the need for novel therapies specifically targeting this subset of tumor-driving cells. Here, we identify CD70 as a potential therapeutic target for recurrent GBM CSCs. EXPERIMENTAL DESIGN: In the current study, we identified the relevance and functional influence of CD70 on primary and recurrent GBM cells, and further define its function using established stem cell assays. We use CD70 knockdown studies, subsequent RNAseq pathway analysis, and in vivo xenotransplantation to validate CD70's role in GBM. Next, we developed and tested an anti-CD70 chimeric antigen receptor (CAR)-T therapy, which we validated in vitro and in vivo using our established preclinical model of human GBM. Lastly, we explored the importance of CD70 in the tumor immune microenvironment (TIME) by assessing the presence of its receptor, CD27, in immune infiltrates derived from freshly resected GBM tumor samples. RESULTS: CD70 expression is elevated in recurrent GBM and CD70 knockdown reduces tumorigenicity in vitro and in vivo. CD70 CAR-T therapy significantly improves prognosis in vivo. We also found CD27 to be present on the cell surface of multiple relevant GBM TIME cell populations, notably putative M1 macrophages and CD4 T cells. CONCLUSION: CD70 plays a key role in recurrent GBM cell aggressiveness and maintenance. Immunotherapeutic targeting of CD70 significantly improves survival in animal models and the CD70/CD27 axis may be a viable polytherapeutic avenue to co-target both GBM and its TIME.

Preclinical Testing of CAR T Cells in a Patient-Derived Xenograft Model of Glioblastoma.

Glioblastoma (GBM) is the most common malignant adult brain tumor that is resistant to the standard care therapy. Advances in chimeric antigen receptor (CAR) T cell therapies have spurred renewed interest in developing CAR T cell therapies to target chemoradiotherapy-resistant brain tumor-initiating cells. This protocol shows how to isolate peripheral blood mononuclear cells from healthy donors and generate CAR T cells for the antigens of interest, and how to intracranially inject the CAR T cells into a patient-derived xenograft mouse model of GBM. For complete details on the use and execution of this protocol, please refer to Vora et al. (2020).

Wnt activation as a therapeutic strategy in medulloblastoma.

Medulloblastoma (MB) is defined by four molecular subgroups (Wnt, Shh, Group 3, Group 4) with Wnt MB having the most favorable prognosis. Since prior reports have illustrated the antitumorigenic role of Wnt activation in Shh MB, we aimed to assess the effects of activated canonical Wnt signaling in Group 3 and 4 MBs. By using primary patient-derived MB brain tumor-initiating cell (BTIC) lines, we characterize differences in the tumor-initiating capacity of Wnt, Group 3, and Group 4 MB. With single cell RNA-seq technology, we demonstrate the presence of rare Wnt-active cells in non-Wnt MBs, which functionally retain the impaired tumorigenic potential of Wnt MB. In treating MB xenografts with a Wnt agonist, we provide a rational therapeutic option in which the protective effects of Wnt-driven MBs may be augmented in Group 3 and 4 MB and thereby support emerging data for a context-dependent tumor suppressive role for Wnt/ß-catenin signaling.

The Rational Development of CD133-Targeting Immunotherapies for Glioblastoma.

CD133 marks self-renewing cancer stem cells (CSCs) in a variety of solid tumors, and CD133+ tumor-initiating cells are known markers of chemo- and radio-resistance in multiple aggressive cancers, including glioblastoma (GBM), that may drive intra-tumoral heterogeneity. Here, we report three immunotherapeutic modalities based on a human anti-CD133 antibody fragment that targets a unique epitope present in glycosylated and non-glycosylated CD133 and studied their effects on targeting CD133+ cells in patient-derived models of GBM. We generated an immunoglobulin G (IgG) (RW03-IgG), a dual-antigen T cell engager (DATE), and a CD133-specific chimeric antigen receptor T cell (CAR-T): CART133. All three showed activity against patient-derived CD133+ GBM cells, and CART133 cells demonstrated superior efficacy in patient-derived GBM xenograft models without causing adverse effects on normal CD133+ hematopoietic stem cells in humanized CD34+ mice. Thus, CART133 cells may be a therapeutically tractable strategy to target CD133+ CSCs in human GBM or other treatment-resistant primary cancers.

Degeneration of rapid eye movement sleep circuitry underlies rapid eye movement sleep behavior disorder.

During healthy rapid eye movement sleep, skeletal muscles are actively forced into a state of motor paralysis. However, in rapid eye movement sleep behavior disorder-a relatively common neurological disorder-this natural process is lost. A lack of motor paralysis (atonia) in rapid eye movement sleep behavior disorder allows individuals to actively move, which at times can be excessive and violent. At first glance this may sound harmless, but it is not because rapid eye movement sleep behavior disorder patients frequently injure themselves or the person they sleep with. It is hypothesized that the degeneration or dysfunction of the brain stem circuits that control rapid eye movement sleep paralysis is an underlying cause of rapid eye movement sleep behavior disorder. The link between brain stem degeneration and rapid eye movement sleep behavior disorder stems from the fact that rapid eye movement sleep behavior disorder precedes, in the majority (∼80%) of cases, the development of synucleinopathies such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, which are known to initially cause degeneration in the caudal brain stem structures where rapid eye movement sleep circuits are located. Furthermore, basic science and clinical evidence demonstrate that lesions within the rapid eye movement sleep circuits can induce rapid eye movement sleep-specific motor deficits that are virtually identical to those observed in rapid eye movement sleep behavior disorder. This review examines the evidence that rapid eye movement sleep behavior disorder is caused by synucleinopathic neurodegeneration of the core brain stem circuits that control healthy rapid eye movement sleep and concludes that rapid eye movement sleep behavior disorder is not a separate clinical entity from synucleinopathies but, rather, it is the earliest symptom of these disorders. © 2017 International Parkinson and Movement Disorder Society.