The context-dependent epigenetic and organogenesis programs determine 3D vs. 2D cellular fitness of MYC-driven cancer.

3D cellular-specific epigenetic and transcriptomic reprogramming is critical to organogenesis and tumorigenesis. Here we dissect the distinct cell fitness in 2D (normoxia vs. chronic hypoxia) vs 3D (normoxia) culture conditions. We identify over 600 shared essential genes and additional context-specific fitness genes and pathways. Knockout of the VHL-HIF1 pathway results in incompatible fitness defects under normoxia vs. 1% oxygen or 3D culture conditions. Moreover, deletion of each of the mitochondrial respiratory electron transport chain complex has distinct fitness outcomes. Notably, multicellular organogenesis signaling pathways including TGFß-SMAD specifically constrict the uncontrolled cell proliferation in 3D while inactivation of epigenetic modifiers (Bcor, Kmt2d, Mettl3 and Mettl14) has opposite outcomes in 2D vs. 3D. We further identify a 3D-dependent synthetic lethality with partial loss of Prmt5 due to a reduction of Mtap expression resulting from 3D-specific epigenetic reprogramming. Our study highlights unique epigenetic, metabolic and organogenesis signaling dependencies under different cellular settings.

Bone morphogenetic protein (BMP) signaling determines neuroblastoma cell fate and sensitivity to retinoic acid.

Retinoic acid (RA) is a standard-of-care neuroblastoma drug thought to be effective by inducing differentiation. Curiously, RA has little effect on primary human tumors during upfront treatment but can eliminate neuroblastoma cells from the bone marrow during post-chemo consolidation therapy-a discrepancy that has never been explained. To investigate this, we treated a large cohort of neuroblastoma cell lines with RA and observed that the most RA-sensitive cells predominantly undergo apoptosis or senescence, rather than differentiation. We conducted genome-wide CRISPR knockout screens under RA treatment, which identified BMP signaling as controlling the apoptosis/senescence vs differentiation cell fate decision and determining RA's overall potency. We then discovered that BMP signaling activity is markedly higher in neuroblastoma patient samples at bone marrow metastatic sites, providing a plausible explanation for RA's ability to clear neuroblastoma cells specifically from the bone marrow, seemingly mimicking interactions between BMP and RA during normal development.

RBM39 degrader invigorates natural killer cells to eradicate neuroblastoma despite cancer cell plasticity.

The cellular plasticity of neuroblastoma is defined by a mixture of two major cell states, adrenergic (ADRN) and mesenchymal (MES), which may contribute to therapy resistance. However, how neuroblastoma cells switch cellular states during therapy remains largely unknown and how to eradicate neuroblastoma regardless of their cell states is a clinical challenge. To better understand the lineage switch of neuroblastoma in chemoresistance, we comprehensively defined the transcriptomic and epigenetic map of ADRN and MES types of neuroblastomas using human and murine models treated with indisulam, a selective RBM39 degrader. We showed that cancer cells not only undergo a bidirectional switch between ADRN and MES states, but also acquire additional cellular states, reminiscent of the developmental pliancy of neural crest cells. The lineage alterations are coupled with epigenetic reprogramming and dependency switch of lineage-specific transcription factors, epigenetic modifiers and targetable kinases. Through targeting RNA splicing, indisulam induces an inflammatory tumor microenvironment and enhances anticancer activity of natural killer cells. The combination of indisulam with anti-GD2 immunotherapy results in a durable, complete response in high-risk transgenic neuroblastoma models, providing an innovative, rational therapeutic approach to eradicate tumor cells regardless of their potential to switch cell states.

Multi-Channel smFRET study reveals a Compact conformation of EF-G on the Ribosome.

While elongation factor G (EF-G) is crucial for ribosome translocation, the role of its GTP hydrolysis remains ambiguous. EF-G's indispensability is further exemplified by the phosphorylation of human eukaryotic elongation factor 2 (eEF2) at Thr56, which inhibits protein synthesis globally, but its exact mechanism is not clear. In this study, we developed a multi-channel single-molecule FRET (smFRET) microscopy methodology to examine the conformational changes of E. coli EF-G induced by mutations that closely aligned with eEF2's Thr56 residue. We utilized Alexa 488/594 double-labeled EF-G to catalyze the translocation of fMet-Phe-tRNAPhe-Cy3 inside Cy5-L27 labeled ribosomes, allowing us to probe both processes within the same complex. Our findings indicate that in the presence of either GTP or GDPCP, wild-type EF-G undergoes a conformational extension upon binding to the ribosome to promote normal translocation. On the other hand, T48E and T48V mutations did not affect GTP/GDP binding or GTP hydrolysis, but impeded Poly(Phe) synthesis and caused EF-G to adopt a unique compact conformation, which wasn't observed when the mutants interact solely with the sarcin/ricin loop. This study provides new insights into EF-G's adaptability and sheds light on the modification mechanism of human eEF2.

A CRISPR-drug perturbational map for identifying compounds to combine with commonly used chemotherapeutics.

Combination chemotherapy is crucial for successfully treating cancer. However, the enormous number of possible drug combinations means discovering safe and effective combinations remains a significant challenge. To improve this process, we conduct large-scale targeted CRISPR knockout screens in drug-treated cells, creating a genetic map of druggable genes that sensitize cells to commonly used chemotherapeutics. We prioritize neuroblastoma, the most common extracranial pediatric solid tumor, where ~50% of high-risk patients do not survive. Our screen examines all druggable gene knockouts in 18 cell lines (10 neuroblastoma, 8 others) treated with 8 widely used drugs, resulting in 94,320 unique combination-cell line perturbations, which is comparable to the largest existing drug combination screens. Using dense drug-drug rescreening, we find that the top CRISPR-nominated drug combinations are more synergistic than standard-of-care combinations, suggesting existing combinations could be improved. As proof of principle, we discover that inhibition of PRKDC, a component of the non-homologous end-joining pathway, sensitizes high-risk neuroblastoma cells to the standard-of-care drug doxorubicin in vitro and in vivo using patient-derived xenograft (PDX) models. Our findings provide a valuable resource and demonstrate the feasibility of using targeted CRISPR knockout to discover combinations with common chemotherapeutics, a methodology with application across all cancers.

Genome-wide mapping of cancer dependency genes and genetic modifiers of chemotherapy in high-risk hepatoblastoma.

A lack of relevant genetic models and cell lines hampers our understanding of hepatoblastoma pathogenesis and the development of new therapies for this neoplasm. Here, we report an improved MYC-driven hepatoblastoma-like murine model that recapitulates the pathological features of embryonal type of hepatoblastoma, with transcriptomics resembling the high-risk gene signatures of the human disease. Single-cell RNA-sequencing and spatial transcriptomics identify distinct subpopulations of hepatoblastoma cells. After deriving cell lines from the mouse model, we map cancer dependency genes using CRISPR-Cas9 screening and identify druggable targets shared with human hepatoblastoma (e.g., CDK7, CDK9, PRMT1, PRMT5). Our screen also reveals oncogenes and tumor suppressor genes in hepatoblastoma that engage multiple, druggable cancer signaling pathways. Chemotherapy is critical for human hepatoblastoma treatment. A genetic mapping of doxorubicin response by CRISPR-Cas9 screening identifies modifiers whose loss-of-function synergizes with (e.g., PRKDC) or antagonizes (e.g., apoptosis genes) the effect of chemotherapy. The combination of PRKDC inhibition and doxorubicin-based chemotherapy greatly enhances therapeutic efficacy. These studies provide a set of resources including disease models suitable for identifying and validating potential therapeutic targets in human high-risk hepatoblastoma.

Interaction among spring phytoplankton succession, water discharge patterns, and hydrogen peroxide dynamics in the Caloosahatchee River in southwest Florida.

Phytoplankton communities are major primary producers in the aquatic realm and are responsible for shaping aquatic ecosystems. The dynamics of algal blooms could be determined by a succession of variable taxonomic groups, which are altered based on complex environmental factors such as nutrient availability and hydraulic factors. In-river structures potentially increase the occurrence of harmful algal blooms (HABs) by increasing water residence time and deteriorating water quality. How flowing water stimulates cell growth and affects the population dynamics of phytoplankton communities is a prioritized question that needs to be addressed for water management tactics. The goal of this study was to determine if an interaction between water flow and water chemistry is present, furthermore, to determine the relationship among phytoplankton community successions in the Caloosahatchee River, a subtropical river strongly influenced by human-controlled water discharge patterns from Lake Okeechobee. Particularly we focused on how phytoplankton community shifts influence the natural abundance of hydrogen peroxide, the most stable reactive oxygen species and a byproduct of oxidative photosynthesis. High-throughput amplicon sequencing using universal primers amplify 23S rRNA gene in cyanobacteria and eukaryotic algal plastids revealed that Synechococcus and Cyanobium were the dominant cyanobacterial genera and their relative abundance ranged between 19.5 and 95.3% of the whole community throughout the monitoring period. Their relative abundance declined when the water discharge increased. On the contrary, the relative abundance of eukaryotic algae sharply increased after water discharge increased. As water temperature increased in May, initially dominant Dolichospermum decreased as Microcystis increased. When Microcystis declined other filamentous cyanobacteria such as Geitlerinema, Pseudanabaena, and Prochlorothreix increased in their relative abundances. Interestingly, a peak of extracellular hydrogen peroxide was observed when Dolichospermum dominance was ended, and M. aeruginosa numbers increased. Overall, phytoplankton communities were strongly impacted by human-induced water discharge patterns.

ZBP1-dependent inflammatory cell death, PANoptosis, and cytokine storm disrupt IFN therapeutic efficacy during coronavirus infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), continues to cause substantial morbidity and mortality in the ongoing global pandemic. Understanding the fundamental mechanisms that govern innate immune and inflammatory responses during SARS-CoV-2 infection is critical for developing effective therapeutic strategies. Whereas interferon (IFN)-based therapies are generally expected to be beneficial during viral infection, clinical trials in COVID-19 have shown limited efficacy and potential detrimental effects of IFN treatment during SARS-CoV-2 infection. However, the underlying mechanisms responsible for this failure remain unknown. In this study, we found that IFN induced Z-DNA-binding protein 1 (ZBP1)-mediated inflammatory cell death, PANoptosis, in human and murine macrophages and in the lungs of mice infected with ß-coronaviruses, including SARS-CoV-2 and mouse hepatitis virus (MHV). In patients with COVID-19, expression of the innate immune sensor ZBP1 was increased in immune cells from those who succumbed to the disease compared with those who recovered, further suggesting a link between ZBP1 and pathology. In mice, IFN-ß treatment after ß-coronavirus infection increased lethality, and genetic deletion of Zbp1 or its Zα domain suppressed cell death and protected the mice from IFN-mediated lethality during ß-coronavirus infection. Overall, our results identify that ZBP1 induced during coronavirus infection limits the efficacy of IFN therapy by driving inflammatory cell death and lethality. Therefore, inhibiting ZBP1 activity may improve the efficacy of IFN therapy, paving the way for the development of new and critically needed therapeutics for COVID-19 as well as other infections and inflammatory conditions where IFN-mediated cell death and pathology occur.

Complete Genome Sequence of Microcystis aeruginosa FD4, Isolated from a Subtropical River in Southwest Florida.

We report the first complete genome of Microcystis aeruginosa from North America. A harmful bloom that occurred in the Caloosahatchee River in 2018 led to a state of emergency declaration in Florida. Although strain FD4 was isolated from this toxic bloom, the genome did not have a microcystin biosynthetic gene cluster.