Next-generation humanized patient-derived xenograft mouse model for pre-clinical antibody studies in neuroblastoma.

Faithful tumor mouse models are fundamental research tools to advance the field of immuno-oncology (IO). This is particularly relevant in diseases with low incidence, as in the case of pediatric malignancies, that rely on pre-clinical therapeutic development. However, conventional syngeneic and genetically engineered mouse models fail to recapitulate the tumor heterogeneity and microenvironmental complexity of human pathology that are essential determinants of cancer-directed immunity. Here, we characterize a novel mouse model that supports human natural killer (NK) cell development and engraftment of neuroblastoma orthotopic patient-derived xenograft (O-PDX) for pre-clinical antibody and cytokine testing. Using cytotoxicity assays, single-cell RNA-sequencing, and multi-color flow cytometry, we demonstrate that NK cells that develop in the humanized mice are fully licensed to execute NK cell cytotoxicity, permit human tumor engraftment, but can be therapeutically redirected to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Although these cells share phenotypic and molecular features with healthy controls, we noted that they lacked an NK cell subset, termed activated NK cells, that is characterized by differentially expressed genes that are induced by cytokine activation. Because this subset of genes is also downregulated in patients with neuroblastoma compared to healthy controls, we hypothesize that this finding could be due to tumor-mediated suppressive effects. Thus, despite its technical complexity, this humanized patient-derived xenograft mouse model could serve as a faithful system for future testing of IO applications and studies of underlying immunologic processes.

ATRX In-Frame Fusion Neuroblastoma Is Sensitive to EZH2 Inhibition via Modulation of Neuronal Gene Signatures.

ATRX alterations occur at high frequency in neuroblastoma of adolescents and young adults. Particularly intriguing are the large N-terminal deletions of ATRX (Alpha Thalassemia/Mental Retardation, X-linked) that generate in-frame fusion (IFF) proteins devoid of key chromatin interaction domains, while retaining the SWI/SNF-like helicase region. We demonstrate that ATRX IFF proteins are redistributed from H3K9me3-enriched chromatin to promoters of active genes and identify REST as an ATRX IFF target whose activation promotes silencing of neuronal differentiation genes. We further show that ATRX IFF cells display sensitivity to EZH2 inhibitors, due to derepression of neurogenesis genes, including a subset of REST targets. Taken together, we demonstrate that ATRX structural alterations are not loss-of-function and put forward EZH2 inhibitors as a potential therapy for ATRX IFF neuroblastoma.

Reprogramming of mouse retinal neurons and standardized quantification of their differentiation in 3D retinal cultures.

Postmitotic differentiated neurons are among the most difficult cells to reprogram into induced pluripotent stem cells (iPSCs) because they have poor viability when cultured as dissociated cells. To overcome this, other protocols have required the inactivation of the p53 tumor suppressor to reprogram postmitotic neurons, which can result in tumorigenesis of the cells. We describe a method that does not require p53 inactivation but induces reprogramming in retinal cells from reprogrammable mice grown in aggregates with wild-type mouse retinal cells. After the first 10 d of reprogramming, the aggregates are then dispersed and plated on irradiated feeder cells to propagate and isolate individual iPSC clones. The reprogramming efficiency of different neuronal populations at any stage of development can be quantified using this protocol. Reprogramming retinal neurons using this protocol will take 56 d, and these retina-derived iPSCs can undergo retinal differentiation to produce retinae in 34 d. In addition, we describe a quantitative assessment of retinal differentiation from these neuron-derived iPSCs called STEM-RET. The procedure quantifies eye field specification, optic cup formation and retinal differentiation in 3D cultures using molecular, cellular and morphological criteria. An advanced level of cell culture experience is required to carry out this protocol.

Targeting the DNA repair pathway in Ewing sarcoma.

Ewing sarcoma (EWS) is a tumor of the bone and soft tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using three different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice.

Post-transcriptional gene expression control by NANOS is up-regulated and functionally important in pRb-deficient cells.

Inactivation of the retinoblastoma tumor suppressor (pRb) is a common oncogenic event that alters the expression of genes important for cell cycle progression, senescence, and apoptosis. However, in many contexts, the properties of pRb-deficient cells are similar to wild-type cells suggesting there may be processes that counterbalance the transcriptional changes associated with pRb inactivation. Therefore, we have looked for sets of evolutionary conserved, functionally related genes that are direct targets of pRb/E2F proteins. We show that the expression of NANOS, a key facilitator of the Pumilio (PUM) post-transcriptional repressor complex, is directly repressed by pRb/E2F in flies and humans. In both species, NANOS expression increases following inactivation of pRb/RBF1 and becomes important for tissue homeostasis. By analyzing datasets from normal retinal tissue and pRb-null retinoblastomas, we find a strong enrichment for putative PUM substrates among genes de-regulated in tumors. These include pro-apoptotic genes that are transcriptionally down-regulated upon pRb loss, and we characterize two such candidates, MAP2K3 and MAP3K1, as direct PUM substrates. Our data suggest that NANOS increases in importance in pRb-deficient cells and helps to maintain homeostasis by repressing the translation of transcripts containing PUM Regulatory Elements (PRE).

The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure.

The SWI/SNF-like chromatin-remodeling protein ATRX has emerged as a key factor in the regulation of α-globin gene expression, incorporation of histone variants into the chromatin template and, more recently, as a frequently mutated gene across a wide spectrum of cancers. Therefore, the availability of a functional ATRX cDNA for expression studies is a valuable tool for the scientific community. We have identified two independent transposon insertions of a bacterial IS10 element into exon 8 of ATRX isoform 2 coding sequence in two different plasmids derived from a single source. We demonstrate that these insertion events are common and there is an insertion hotspot within the ATRX cDNA. Such IS10 insertions produce a truncated form of ATRX, which significantly compromises its nuclear localization. In turn, we describe ways to prevent IS10 insertion during propagation and cloning of ATRX-containing vectors, including optimal growth conditions, bacterial strains, and suggested sequencing strategies. Finally, we have generated an insertion-free plasmid that is available to the community for expression studies of ATRX.

Regulation of base excision repair: Ntg1 nuclear and mitochondrial dynamic localization in response to genotoxic stress.

Numerous human pathologies result from unrepaired oxidative DNA damage. Base excision repair (BER) is responsible for the repair of oxidative DNA damage that occurs in both nuclei and mitochondria. Despite the importance of BER in maintaining genomic stability, knowledge concerning the regulation of this evolutionarily conserved repair pathway is almost nonexistent. The Saccharomyces cerevisiae BER protein, Ntg1, relocalizes to organelles containing elevated oxidative DNA damage, indicating a novel mechanism of regulation for BER. We propose that dynamic localization of BER proteins is modulated by constituents of stress response pathways. In an effort to mechanistically define these regulatory components, the elements necessary for nuclear and mitochondrial localization of Ntg1 were identified, including a bipartite classical nuclear localization signal, a mitochondrial matrix targeting sequence and the classical nuclear protein import machinery. Our results define a major regulatory system for BER which when compromised, confers a mutator phenotype and sensitizes cells to the cytotoxic effects of DNA damage.

Mitochondrial DNA oxidative damage and mutagenesis in Saccharomyces cerevisiae.

Mutation of human mitochondrial DNA (mtDNA) has been linked to maternally inherited neuromuscular disorders and is implicated in more common diseases such as cancer, diabetes, and Parkinson's disease. Mutations in mtDNA also accumulate with age and are therefore believed to contribute to aging and age-related pathology. Housed within the mitochondrial matrix, mtDNA encodes several of the proteins involved in the production of ATP via the process of oxidative phosphorylation, which involves the flow of high-energy electrons through the electron transport chain (ETC). Because of its proximity to the ETC, mtDNA is highly vulnerable to oxidative damage mediated by reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, and hydroxyl radicals that are constantly produced by this system. Therefore, it is important to be able to measure oxidative mtDNA damage under normal physiologic conditions and during environmental or disease-associated stress. The budding yeast, Saccharomyces cerevisiae, is a facile and informative model system in which to study such mtDNA oxidative damage because it is a unicellular eukaryotic facultative anaerobe that is conditionally dependent on mitochondrial oxidative phosphorylation for viability. Here, we describe methods for quantifying oxidative mtDNA damage and mutagenesis in S. cerevisiae, several of which could be applied to the development of similar assays in mammalian cells and tissues. These methods include measuring the number of point mutations that occur in mtDNA with the erythromycin resistance assay, quantifying the amount of oxidative DNA damage utilizing a modified Southern blot assay, and measuring mtDNA integrity with the "petite induction" assay.

Dynamic compartmentalization of base excision repair proteins in response to nuclear and mitochondrial oxidative stress.

DNAs harbored in both nuclei and mitochondria of eukaryotic cells are subject to continuous oxidative damage resulting from normal metabolic activities or environmental insults. Oxidative DNA damage is primarily reversed by the base excision repair (BER) pathway, initiated by N-glycosylase apurinic/apyrimidinic (AP) lyase proteins. To execute an appropriate repair response, BER components must be distributed to accommodate levels of genotoxic stress that may vary considerably between nuclei and mitochondria, depending on the growth state and stress environment of the cell. Numerous examples exist where cells respond to signals, resulting in relocalization of proteins involved in key biological transactions. To address whether such dynamic localization contributes to efficient organelle-specific DNA repair, we determined the intracellular localization of the Saccharomyces cerevisiae N-glycosylase/AP lyases, Ntg1 and Ntg2, in response to nuclear and mitochondrial oxidative stress. Fluorescence microscopy revealed that Ntg1 is differentially localized to nuclei and mitochondria, likely in response to the oxidative DNA damage status of the organelle. Sumoylation is associated with targeting of Ntg1 to nuclei containing oxidative DNA damage. These studies demonstrate that trafficking of DNA repair proteins to organelles containing high levels of oxidative DNA damage may be a central point for regulating BER in response to oxidative stress.