SURG-05. Α5Β1 INTEGRIN TARGETING LIPOSOME DELIVERY OF MIR-603 SUPPRESSED GLIOBLASTOMA STEM CELL STATE AND ENHANCED RADIATION SENSITIVITY

INTRODUCTION Despite the potential for clinical efficacy, therapeutic delivery of microRNAs (miRNA) remains a major translational barrier. Here, we explore a surgery mediated polyethylenimine (PEI)/liposome-based strategy for the delivery of miR-603, a master regulatory miRNA that suppresses glioblastoma stem cell state by simultaneous down-regulation of insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R). METHODS miR-603 was complexed with PEI, a cationic polymer designed to facilitate miRNA from the endolysosomal compartment. The miR-603/PEI complex was encapsulated into liposomes decorated with polyethylene glycol (PEG) and PR_b, a fibronectin-mimetic peptide that specifically targets the α5β1 integrin that is overexpressed in glioblastoma. RESULTS Patient-derived glioblastoma cells internalized PR_b coated liposomes but not the non-coated liposomes. The internalization of PR_b liposomes encapsulating miR-603/PEI was associated with orders of magnitude increase in intra-cellular miR-603 levels and decreased IGF1 and IGF1R mRNA/protein levels. Moreover, treatment of glioblastoma cells with the PR_b liposomes encapsulating miR-603/PEI showed altered morphology and decreased expression of stem cell marker, suggesting the treated cells have exited the cancer stem cell state. Finally, treatment of the PR_b liposomes encapsulating miR-603/PEI sensitized glioblastoma cells to ionizing radiation (IR) in patient-derived glioblastoma cells. These results were not observed in liposomes missing the PR_b peptide, PEI, or miR-603. CONCLUSION These results suggest that intra-tumoral injection of PR_b functionalized PEGylated liposomes encapsulating miR-603/PEI complexes hold promise as a strategy for glioblastomas therapy. A first-in-human trial is currently underway to test this strategy.

C.5 Musashi-1 is a master regulator of aberrant translation in MYC-amplified Group 3 medulloblastoma

Background: Medulloblastoma (MB) is the most common solid malignant pediatric brain neoplasm. Group 3 (G3) MB, particularly MYC amplified G3 MB, is the most aggressive subgroup with the highest frequency of children presenting with metastatic disease, and is associated with a poor prognosis. To further our understanding of the role of MSI1 in MYC amplified G3 MB, we performed an unbiased integrative analysis of eCLIP binding sites, with changes observed at the transcriptome, the translatome, and the proteome after shMSI1 inhibition. Methods: Primary human pediatric MBs, SU_MB002 and HD-MB03 were kind gifts from Dr. Yoon-Jae Cho (Harvard, MS) and Dr. Till Milde (Heidelberg) and cultured for in vitro and in vivo experiments. eCLIP, RNA-seq, Polysome-seq, and TMT-MS were completed as previously described. Results:MSI1 is overexpressed in G3 MB. shRNA Msi1 interference resulted in a reduction in tumour burden conferring a survival advantage to mice injected with shMSI1 G3MB cells. Robust ranked multiomic analysis (RRA) identified an unconventional gene set directly perturbed by MSI1 in G3 MB. Conclusions: Our robust unbiased integrative analysis revealed a distinct role for MSI1 in the maintenance of the stem cell state in G3 MB through post-transcriptional modification of multiple pathways including identification of unconventional targets such as HIPK1.

Mechanical Compression Creates a Quiescent Muscle Stem Cell Niche

Skeletal muscles can regenerate throughout life time from resident Pax7-expressing (Pax7+) muscle stem cells (MuSCs). Pax7+ MuSCs are normally quiescent and localized at a niche in which they are attached to the extracellular matrix basally and compressed against the myofiber apically. Upon muscle injury, MuSCs lose apical contact with the myofiber and re-enter cell cycle to initiate regeneration. Prior studies on the physical niche of MuSCs focused on basal elasticity, and significance of the apical force exerted on MuSCs remains unaddressed. Here we simulate MuSCs' mechanical environment in vivo by applying physical compression to MuSCs' apical surface. We demonstrate that compression drives activated MuSCs back to a quiescent stem cell state, even when seeded on different basal elasticities. By mathematical modeling and manipulating cell tension, we conclude that low overall tension combined with high edge tension generated by compression lead to MuSC quiescence. We further show that apical compression results in up-regulation of Notch downstream genes, accompanied by increased levels of nuclear Notch. The compression-induced nuclear Notch is ligand-independent, as it does not require the canonical S2-cleavage of Notch by ADAM10/17. Our results fill the knowledge gap on the role of apical tension for MuSC fate. Implications to how stem cell fate and activity are interlocked with the mechanical integrity of its resident tissue are discussed.

HLF Expression Defines the Human Hematopoietic Stem Cell State.

Hematopoietic stem cells (HSCs) sustain blood cell homeostasis throughout life and can regenerate all blood lineages following transplantation. Despite this clear functional definition, highly enriched isolation of human HSCs can currently only be achieved through combinatorial assessment of multiple surface antigens. While several transgenic HSC reporter mouse strains have been described, no analogous approach to prospectively isolate human HSCs has been reported. To identify genes with the most selective expression in human HSCs, we profiled population- and single-cell transcriptomes of un-expanded and ex vivo cultured cord blood-derived HSPCs as well as peripheral blood, adult bone marrow, and fetal liver. Based on these analyses, we propose the master transcription factor HLF (Hepatic Leukemia Factor) as one of the most specific HSC marker genes. To directly track its expression in human hematopoietic cells, we developed a genomic HLF reporter strategy, capable of selectively labeling the most immature blood cells based on a single engineered parameter. Most importantly, HLF-expressing cells comprise all of the stem cell activity in culture and in vivo during serial transplantation. Taken together, these results experimentally establish HLF as a defining gene of the human hematopoietic stem cell state and outline a new approach to continuously mark these cells with high fidelity.

Inhibiting DNA-PK induces glioma stem cell differentiation and sensitizes glioblastoma to radiation in mice

Glioblastoma (GBM), a lethal primary brain tumor, contains glioma stem cells (GSCs) that promote malignant progression and therapeutic resistance. SOX2 is a core transcription factor that maintains the properties of stem cells, including GSCs, but mechanisms associated with posttranslational SOX2 regulation in GSCs remain elusive. Here, we report that DNA-dependent protein kinase (DNA-PK) governs SOX2 stability through phosphorylation, resulting in GSC maintenance. Mass spectrometric analyses of SOX2-binding proteins showed that DNA-PK interacted with SOX2 in GSCs. The DNA-PK catalytic subunit (DNA-PKcs) was preferentially expressed in GSCs compared to matched non–stem cell tumor cells (NSTCs) isolated from patient-derived GBM xenografts. DNA-PKcs phosphorylated human SOX2 at S251, which stabilized SOX2 by preventing WWP2-mediated ubiquitination, thus promoting GSC maintenance. We then demonstrated that when the nuclear DNA of GSCs either in vitro or in GBM xenografts in mice was damaged by irradiation or treatment with etoposide, the DNA-PK complex dissociated from SOX2, which then interacted with WWP2, leading to SOX2 degradation and GSC differentiation. These results suggest that DNA-PKcs–mediated phosphorylation of S251 was critical for SOX2 stabilization and GSC maintenance. Pharmacological inhibition of DNA-PKcs with the DNA-PKcs inhibitor NU7441 reduced GSC tumorsphere formation in vitro and impaired growth of intracranial human GBM xenografts in mice as well as sensitized the GBM xenografts to radiotherapy. Our findings suggest that DNA-PK maintains GSCs in a stem cell state and that DNA damage triggers GSC differentiation through precise regulation of SOX2 stability, highlighting that DNA-PKcs has potential as a therapeutic target in glioblastoma.

An NRF2 Perspective on Stem Cells and Ageing

Redox and metabolic mechanisms lie at the heart of stem cell survival and regenerative activity. NRF2 is a major transcriptional controller of cellular redox and metabolic homeostasis, which has also been implicated in ageing and lifespan regulation. However, NRF2’s role in stem cells and their functioning with age is only just emerging. Here, focusing mainly on neural stem cells, which are core to adult brain plasticity and function, we review recent findings that identify NRF2 as a fundamental player in stem cell biology and ageing. We also discuss NRF2-based molecular programs that may govern stem cell state and function with age, and implications of this for age-related pathologies.

The RNA helicases DDX5 and DDX17 facilitate neural differentiation of human pluripotent stem cells NTERA2

Understanding human neurogenesis is critical toward regenerative medicine for neurodegeneration. However, little is known how neural differentiation is regulated by RNA helicases, which comprise a diverse class of RNA remodeling enzymes. We show here that expression of the DEAD box-containing RNA helicases DDX5 and DDX17 is abundant throughout retinoic acid-induced neural differentiation of the human pluripotent stem cell (hPSC) line NTERA2, and is mostly localized within the nucleus. Using ChIP-seq, we identify that the two RNA helicases occupy chromatin genome-wide at regions associated with neurogenesis- and differentiation-related genes in both hPSCs and their neural derivatives. Further, RNA-seq analyses indicate both DDX5 and DDX17 are mutually required for controlling transcriptional expression of these genes. We show that the two RNA helicases are not important for maintenance of stem cell state of hPSCs. In contrast, they facilitate early neural differentiation of hPSCs, generation of neurospheres from the stem cells, and expression of key neurogenic transcription factors during neural differentiation. Importantly, DDX5 and DDX17 are important for differentiation of hPSCs toward NESTIN- and TUBB3-positive cells, which represent neural progenitors and mature neurons. Collectively, our findings suggest the role of DDX5 and DDX17 in transcriptional regulation of genes involved in neurogenesis, and hence in neural differentiation of hPSCs.

Endoplasmic reticulum stress regulates the intestinal stem cell state through CtBP2

Enforcing differentiation of cancer stem cells is considered as a potential strategy to sensitize colorectal cancer cells to irradiation and chemotherapy. Activation of the unfolded protein response, due to endoplasmic reticulum (ER) stress, causes rapid stem cell differentiation in normal intestinal and colon cancer cells. We previously found that stem cell differentiation was mediated by a Protein kinase R-like ER kinase (PERK) dependent arrest of mRNA translation, resulting in rapid protein depletion of WNT-dependent transcription factor c-MYC. We hypothesize that ER stress dependent stem cell differentiation may rely on the depletion of additional transcriptional regulators with a short protein half-life that are rapidly depleted due to a PERK-dependent translational pause. Using a novel screening method, we identify novel transcription factors that regulate the intestinal stem cell fate upon ER stress. ER stress was induced in LS174T cells with thapsigargin or subtilase cytotoxin (SubAB) and immediate alterations in nuclear transcription factor activity were assessed by the CatTFRE assay in which transcription factors present in nuclear lysate are bound to plasmid DNA, co-extracted and quantified using mass-spectrometry. The role of altered activity of transcription factor CtBP2 was further examined by modification of its expression levels using CAG-rtTA3-CtBP2 overexpression in small intestinal organoids, shCtBP2 knockdown in LS174T cells, and familial adenomatous polyposis patient-derived organoids. CtBP2 overexpression organoids were challenged by ER stress and ionizing irradiation. We identified a unique set of transcription factors with altered activation upon ER stress. Gene ontology analysis showed that transcription factors with diminished binding were involved in cellular differentiation processes. ER stress decreased CtBP2 protein expression in mouse small intestine. ER stress induced loss of CtBP2 expression which was rescued by inhibition of PERK signaling. CtBP2 was overexpressed in mouse and human colorectal adenomas. Inducible CtBP2 overexpression in organoids conferred higher clonogenic potential, resilience to irradiation-induced damage and a partial rescue of ER stress-induced loss of stemness. Using an unbiased proteomics approach, we identified a unique set of transcription factors for which DNA-binding activity is lost directly upon ER stress. We continued investigating the function of co-regulator CtBP2, and show that CtBP2 mediates ER stress-induced loss of stemness which supports the intestinal stem cell state in homeostatic stem cells and colorectal cancer cells.

Mitochondrial Control of Stem Cell State and Fate: Lessons From Drosophila

Over the years, Drosophila has served as a wonderful genetically tractable model system to unravel various facets of tissue-resident stem cells in their microenvironment. Studies in different stem and progenitor cell types of Drosophila have led to the discovery of cell-intrinsic and extrinsic factors crucial for stem cell state and fate. Though initially touted as the ATP generating machines for carrying various cellular processes, it is now increasingly becoming clear that mitochondrial processes alone can override the cellular program of stem cells. The last few years have witnessed a surge in our understanding of mitochondria’s contribution to governing different stem cell properties in their subtissular niches in Drosophila. Through this review, we intend to sum up and highlight the outcome of these in vivo studies that implicate mitochondria as a central regulator of stem cell fate decisions; to find the commonalities and uniqueness associated with these regulatory mechanisms.