WDR5 represents a therapeutically exploitable target for cancer stem cells in glioblastoma.

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy-resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1 family-mediated Lys4 methylation of histone H3 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown altered the stem cell state, disrupting CSC in vitro growth and self-renewal, as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture.

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular diversity, as well as complex interactions between tumor microenvironments, can make finding effective treatments a challenge. Traditional adherent or sphere culture methods can mask such complexities, whereas three-dimensional organoid culture can recapitulate regional microenvironmental gradients. Organoids are a method of three-dimensional GBM culture that better mimics patient tumor architecture, contains phenotypically diverse cell populations, and can be used for medium-throughput experiments. Although three-dimensional organoid culture is more laborious and time-consuming compared to traditional culture, it offers unique benefits and can serve to bridge the gap between current in vitro and in vivo systems. Organoids have established themselves as invaluable tools in the arsenal of cancer biologists to better understand tumor behavior and mechanisms of resistance, and their applications only continue to grow. Here, details are provided about methods for generating and maintaining GBM organoids. Instructions of how to perform organoid sample embedding and sectioning using both frozen and paraffin-embedding techniques, as well as recommendations for immunohistochemistry and immunofluorescence protocols on organoid sections, and measurement of total organoid cell viability, are all also described.

VRK1 Is a Synthetic-Lethal Target in VRK2-Deficient Glioblastoma.

Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic-lethal interactions by virtue of their redundant functions. Here, we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of VRK2, which is silenced by promoter methylation in approximately two thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2-M arrest and DNA damage. The VRK1-VRK2 synthetic-lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line-derived xenograft and patient-derived xenograft models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM. SIGNIFICANCE: A paralog synthetic-lethal interaction between VRK1 and VRK2 sensitizes VRK2-methylated glioblastoma to perturbation of VRK1 kinase activity, supporting VRK1 as a drug discovery target in this disease.

Three-dimensional organoid culture unveils resistance to clinical therapies in adult and pediatric glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor with a dismal prognosis. The inherent cellular diversity and interactions within tumor microenvironments represent significant challenges to effective treatment. Traditional culture methods such as adherent or sphere cultures may mask such complexities whereas three-dimensional (3D) organoid culture systems derived from patient cancer stem cells (CSCs) can preserve cellular complexity and microenvironments. The objective of this study was to determine if GBM organoids may offer a platform, complimentary to traditional sphere culture methods, to recapitulate patterns of clinical drug resistance arising from 3D growth. METHODS: Adult and pediatric surgical specimens were collected and established as organoids. We created organoid microarrays and visualized bulk and spatial differences in cell proliferation using immunohistochemistry (IHC) staining, and cell cycle analysis by flow cytometry paired with 3D regional labeling. We tested the response of CSCs grown in each culture method to temozolomide, ibrutinib, lomustine, ruxolitinib, and radiotherapy. RESULTS: GBM organoids showed diverse and spatially distinct proliferative cell niches and include heterogeneous populations of CSCs/non-CSCs (marked by SOX2) and cycling/senescent cells. Organoid cultures display a comparatively blunted response to current standard-of-care therapy (combination temozolomide and radiotherapy) that reflects what is seen in practice. Treatment of organoids with clinically relevant drugs showed general therapeutic resistance with drug- and patient-specific antiproliferative, apoptotic, and senescent effects, differing from those of matched sphere cultures. CONCLUSIONS: Therapeutic resistance in organoids appears to be driven by altered biological mechanisms rather than physical limitations of therapeutic access. GBM organoids may therefore offer a key technological approach to discover and understand resistance mechanisms of human cancer cells.

Altered lipid metabolism marks glioblastoma stem and non-stem cells in separate tumor niches.

Glioblastoma (GBM) displays marked cellular and metabolic heterogeneity that varies among cellular microenvironments within a tumor. Metabolic targeting has long been advocated as a therapy against many tumors including GBM, but how lipid metabolism is altered to suit different microenvironmental conditions and whether cancer stem cells (CSCs) have altered lipid metabolism are outstanding questions in the field. We interrogated gene expression in separate microenvironments of GBM organoid models that mimic the transition between nutrient-rich and nutrient-poor pseudopalisading/perinecrotic tumor zones using spatial-capture RNA-sequencing. We revealed a striking difference in lipid processing gene expression and total lipid content between diverse cell populations from the same patient, with lipid enrichment in hypoxic organoid cores and also in perinecrotic and pseudopalisading regions of primary patient tumors. This was accompanied by regionally restricted upregulation of hypoxia-inducible lipid droplet-associated (HILPDA) gene expression in organoid cores and pseudopalisading regions of clinical GBM specimens, but not lower-grade brain tumors. CSCs have low lipid droplet accumulation compared to non-CSCs in organoid models and xenograft tumors, and prospectively sorted lipid-low GBM cells are functionally enriched for stem cell activity. Targeted lipidomic analysis of multiple patient-derived models revealed a significant shift in lipid metabolism between GBM CSCs and non-CSCs, suggesting that lipid levels may not be simply a product of the microenvironment but also may be a reflection of cellular state. CSCs had decreased levels of major classes of neutral lipids compared to non-CSCs, but had significantly increased polyunsaturated fatty acid production due to high fatty acid desaturase (FADS1/2) expression which was essential to maintain CSC viability and self-renewal. Our data demonstrate spatially and hierarchically distinct lipid metabolism phenotypes occur clinically in the majority of patients, can be recapitulated in laboratory models, and may represent therapeutic targets for GBM.

Painless Photodynamic Therapy Triggers Innate and Adaptive Immune Responses in a Murine Model of UV-induced Squamous Skin Pre-cancer.

Painless photodynamic therapy (p-PDT), which involves application of photosensitizer and immediate exposure to light to treat actinic keratosis (AK) in patients, causes negligible pain on the day of treatment but leads to delayed inflammation and effective lesion clearance (Kaw et al., J Am Acad Dermatol 2020). To better understand how p-PDT works, hairless mice with UV-induced AK were treated with p-PDT and monitored for 2 weeks. Lesion clearance after p-PDT was similar to clearance after conventional PDT (c-PDT). However, lesion biopsies showed minimal cell death and less production of reactive oxygen species (ROS) in p-PDT treated than in c-PDT-treated lesions. Interestingly, p-PDT triggered vigorous recruitment of immune cells associated with innate immunity. Neutrophils (Ly6G+) and macrophages (F4/80+) appeared at 4 h and peaked at 24 h after p-PDT. Damage-associated molecular patterns (DAMPs), including calreticulin, HMGB1, and HSP70, were expressed at maximum levels around 24 h post-p-PDT. Total T cells (CD3+) were increased at 24 h, whereas large increases in cytotoxic (CD8+) and regulatory (Foxp3+) T cells were observed at 1 and 2 weeks post-p-PDT. In summary, the ability of p-PDT to eliminate AK lesions, despite very little overt cellular damage, appears to involve stimulation of a local immune response.

Cutaneous Wounds in Mice Lacking TSG-6 Exhibit Delayed Closure and an Abnormal Inflammatory Response.

We investigated how loss of TSG-6 affects wound closure and skin inflammation. TSG-6 has several known biological functions, including the enzymatic transfer of heavy-chain proteins from inter-α-trypsin inhibitor to hyaluronan to form heavy-chain protein-hyaluronan complexes. TSG-6 and heavy-chain protein-hyaluronan are constitutively expressed in normal skin and increase post-wounding but are completely absent in TSG-6-null mice. Wound closure rates are significantly delayed in TSG-6-null mice relative to wildtype mice. Neutrophil recruitment is delayed in early wounds (12 hours and day 1), whereas late wounds (day 7) show elevated neutrophil accumulation. In addition, granulation phase resolution is delayed, with persistent blood vessels and reduced dermal collagen at 10 days. The proinflammatory cytokine TNFα is elevated >3-fold in unwounded TSG-6-null skin and increases further after wounding (from 12 hours to 7 days) before returning to baseline by day 10. Other cytokines examined, such as IL-6, IL-10, and monocyte chemotactic protein-1, showed no consistent differences. Reintroduction of TSG-6 into TSG-6-null wounds rescues both the delay in wound closure and the aberrant neutrophil phenotype. In summary, our study indicates that TSG-6 plays an important role in regulating wound closure and inflammation during cutaneous wound repair.

Heparin inhibits proinflammatory and promotes anti-inflammatory macrophage polarization under hyperglycemic stress.

Monocytes are rapidly recruited to sites of diabetic complications and differentiate into macrophages. Previously, we showed that rat kidney mesangial cells dividing during hyperglycemic stress abnormally synthesize hyaluronan (HA) in intracellular compartments. This initiates a stress response, resulting in an extracellular HA matrix after division that recruits inflammatory cells. Cell-cell communication among macrophages that are recruited into the glomeruli and the damaged rat mesangial cells leads to diabetic nephropathy, fibrosis, and proteinurea, which are inhibited in heparin-treated diabetic rats. In this study, we found that murine bone marrow-derived macrophages (BMDMs) and a human leukemic cell line, U937 cells, dividing in hyperglycemia also accumulate intracellular HA and that heparin inhibits the HA accumulation. Both cell types expressed increased levels of proinflammatory markers: inducible nitric-oxide synthase and tumor necrosis factor-α, when cultured under hyperglycemic stress, which was inhibited by heparin. Furthermore, the abnormal intracellular HA was also observed in peripheral blood monocytes derived from three different hyperglycemic diabetic mouse models: streptozotocin-treated, high-fat fed, and Ins2Akita. Moreover, peripheral blood monocytes in humans with type 2 diabetes and poorly controlled blood glucose levels (hemoglobin A1c (HbA1c) levels of >7) also had intracellular HA, whereas those with HbA1c of <7, did not. Of note, heparin increased the anti-inflammatory markers arginase 1 and interleukin-10 in murine BMDMs. We conclude that heparin treatment of high glucose-exposed dividing BMDMs promotes an anti-inflammatory tissue-repair phenotype in these cells.

Painless versus conventional photodynamic therapy for treatment of actinic keratosis: Comparison of cell death and immune response in a murine model.

Aminolevulinic acid based photodynamic therapy (ALA-PDT) is a popular and efficacious treatment for actinic keratosis (AK). However, standard PDT can elicit stinging pain during illumination, and hence is not always favored by patients. In a new regimen called metronomic PDT (mPDT), similar to daylight PDT but using blue light, the illumination is delivered concurrently with ALA application rather than after a 1-hour pre-incubation (conventional regimen, cPDT). In the clinic, mPDT is not only painless but also nearly as effective as cPDT for AK lesion clearance. In this investigation, a murine AK model (generated by repeated UVB exposure) was treated with either mPDT or cPDT. Lesion clearance was followed by area measurement, and samples were harvested for mechanistic analyses. Compared to pretreatment (100%), the average lesion area was reduced to 47% and 32% in cPDT, and to 57% and 40% in mPDT at 1- and 2-weeks post PDT, respectively. Relative to untreated controls, enhanced cell death (histomorphology by H&E staining and apoptosis by TUNEL assay), and generation of Reactive Oxygen Species (ROS; CM-H2DCFDA staining) were observed in both cPDT and mPDT samples. Activation of cleaved Caspase-3 was specifically observed only in cPDT samples. Immunomodulation by inflammatory cells was observed by enhanced infiltration/retention of neutrophils and macrophages in metronomic PDT samples. Our results suggest that metronomic PDT can be just as effective as conventional PDT for treatment of AK, but the mechanisms may be quite different.