Lineage-specific canonical and non-canonical activity of EZH2 in advanced prostate cancer subtypes.

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and emerging therapeutic target that is overexpressed in most castration-resistant prostate cancers and implicated as a driver of disease progression and resistance to hormonal therapies. Here we define the lineage-specific action and differential activity of EZH2 in both prostate adenocarcinoma (PRAD) and neuroendocrine prostate cancer (NEPC) subtypes of advanced prostate cancer to better understand the role of EZH2 in modulating differentiation, lineage plasticity, and to identify mediators of response and resistance to EZH2 inhibitor therapy. Mechanistically, EZH2 modulates bivalent genes that results in upregulation of NEPC-associated transcriptional drivers (e.g., ASCL1) and neuronal gene programs, and leads to forward differentiation after targeting EZH2 in NEPC. Subtype-specific downstream effects of EZH2 inhibition on cell cycle genes support the potential rationale for co-targeting cyclin/CDK to overcome resistance to EZH2 inhibition.

Partial gene suppression improves identification of cancer vulnerabilities when CRISPR-Cas9 knockout is pan-lethal.

BACKGROUND: Hundreds of functional genomic screens have been performed across a diverse set of cancer contexts, as part of efforts such as the Cancer Dependency Map, to identify gene dependencies-genes whose loss of function reduces cell viability or fitness. Recently, large-scale screening efforts have shifted from RNAi to CRISPR-Cas9, due to superior efficacy and specificity. However, many effective oncology drugs only partially inhibit their protein targets, leading us to question whether partial suppression of genes using RNAi could reveal cancer vulnerabilities that are missed by complete knockout using CRISPR-Cas9. Here, we compare CRISPR-Cas9 and RNAi dependency profiles of genes across approximately 400 matched cancer cell lines. RESULTS: We find that CRISPR screens accurately identify more gene dependencies per cell line, but the majority of each cell line's dependencies are part of a set of 1867 genes that are shared dependencies across the entire collection (pan-lethals). While RNAi knockdown of about 30% of these genes is also pan-lethal, approximately 50% have selective dependency patterns across cell lines, suggesting they could still be cancer vulnerabilities. The accuracy of the unique RNAi selectivity is supported by associations to multi-omics profiles, drug sensitivity, and other expected co-dependencies. CONCLUSIONS: Incorporating RNAi data for genes that are pan-lethal knockouts facilitates the discovery of a wider range of gene targets than could be detected using the CRISPR dataset alone. This can aid in the interpretation of contrasting results obtained from CRISPR and RNAi screens and reinforce the importance of partial gene suppression methods in building a cancer dependency map.

A Ubiquitination Cascade Regulating the Integrated Stress Response and Survival in Carcinomas.

Systematic identification of signaling pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 1,086 cancer cell lines to identify selective coessentiality modules and found that a ubiquitin ligase complex composed of UBA6, BIRC6, KCMF1, and UBR4 is required for the survival of a subset of epithelial tumors that exhibit a high degree of aneuploidy. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization of the heme-regulated inhibitor, a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy. SIGNIFICANCE: We describe the identification of a heretofore unrecognized ubiquitin ligase complex that prevents the aberrant activation of the ISR in a subset of cancer cells. This provides a novel insight on the regulation of ISR and exposes a therapeutic opportunity to selectively eliminate these cancer cells. See related commentary Leli and Koumenis, p. 535. This article is highlighted in the In This Issue feature, p. 517.

Sparse dictionary learning recovers pleiotropy from human cell fitness screens.

In high-throughput functional genomic screens, each gene product is commonly assumed to exhibit a singular biological function within a defined protein complex or pathway. In practice, a single gene perturbation may induce multiple cascading functional outcomes, a genetic principle known as pleiotropy. Here, we model pleiotropy in fitness screen collections by representing each gene perturbation as the sum of multiple perturbations of biological functions, each harboring independent fitness effects inferred empirically from the data. Our approach (Webster) recovered pleiotropic functions for DNA damage proteins from genotoxic fitness screens, untangled distinct signaling pathways upstream of shared effector proteins from cancer cell fitness screens, and predicted the stoichiometry of an unknown protein complex subunit from fitness data alone. Modeling compound sensitivity profiles in terms of genetic functions recovered compound mechanisms of action. Our approach establishes a sparse approximation mechanism for unraveling complex genetic architectures underlying high-dimensional gene perturbation readouts.

Microenvironment drives cell state, plasticity, and drug response in pancreatic cancer.

Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo, we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo-relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities.

Macromolecular fraction (MMF) from 3D ultrashort echo time cones magnetization transfer (3D UTE-Cones-MT) imaging predicts meniscal degeneration and knee osteoarthritis.

OBJECTIVE: Meniscal degeneration is strongly associated with osteoarthritis (OA). We aimed to evaluate a 3D ultrashort-echo-time Cones magnetization transfer (UTE-Cones-MT) sequence for quantification of macromolecular fraction (MMF) and MT ratio (MTR) in menisci of healthy volunteers and patients with different degrees of OA. METHODS: Patients with mild OA (n = 19; 37-86 years; 10 males) or advanced OA (n = 12; 52-88 years; 4 males) and healthy volunteers (n = 17; 20-49 years; 7 males) were scanned with T2-FSE and UTE-Cones-MT sequences at 3T. Morphological assessment was performed using meniscal whole-organ magnetic resonance imaging score (WORMS). MMF and MTR were calculated for menisci, and correlated with age and meniscal WORMS scores. The diagnostic efficiency was performed by using receiver operating characteristic (ROC) curve and the area under the curve (AUC) analyses. RESULTS: Decreased MMF and MTR were observed in menisci of patients with mild or advanced OA compared with healthy subjects, and in menisci with tears (Grade 2-4) compared with normal menisci (Grade 0). Significant negative correlations were observed between MMF (r = -0.769, P < 0.01), MTR (r = -0.320, P < 0.01), and meniscal WORMS score. There was a mild negative correlation between MMF (r = -0.438, P < 0.01), MTR (r = -0.289, P < 0.01), and age. The AUC values of MMF and MTR in the four horns of meniscus and the posterior horn medial meniscus for differentiating OA patients from healthy volunteers were 0.762 and 0.699, and 0.835 and 0.883, respectively. CONCLUSION: The 3D UTE-Cones-MT biomarkers of MTR and especially MMF can detect compositional changes in meniscus and differentiate healthy subjects from patients with mild or advanced knee OA.

Repeat expansions confer WRN dependence in microsatellite-unstable cancers.

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair1-4. Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides5. Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.

A randomized trial comparing colloid preload to coload during spinal anesthesia for elective cesarean delivery.

BACKGROUND: Hypotension after spinal anesthesia for cesarean delivery is common. Previous studies have demonstrated that a crystalloid fluid "coload" (rapid administration of a fluid bolus starting at the time of intrathecal injection) is superior to the conventional crystalloid preload (fluid administered before the intrathecal injection) for preventing hypotension. Colloid preload provides a sustained increase in central blood volume. We hypothesized that, in contrast to crystalloid, a colloid preload may be more effective than colloid coload for reducing the incidence of spinal anesthesia-induced hypotension. METHODS: In this double-blind study, 178 patients were randomly assigned to receive a preload of 500 mL of hydroxyethyl starch over a period of 15-20 min before initiation of spinal anesthesia (n = 90) or an identical fluid bolus of hydroxyethyl starch starting at the time of identification of cerebrospinal fluid (n = 88). Vasopressors (ephedrine or phenylephrine) were administered if systolic arterial blood pressure decreased less than 80% of the baseline pressure and <100 mm Hg, or with smaller decreases in blood pressure if accompanied by nausea, vomiting, or dizziness. The primary outcome was the incidence of hypotension (defined as the administration of at least one dose of vasopressor). RESULTS: There was no significant difference between the groups in the incidence of hypotension (68% in preload group and 75% in coload group, 95% confidence interval of difference -6%-20%; P = 0.28), doses of ephedrine and phenylephrine, and number of vasopressor unit doses. The incidence of severe hypotension (systolic blood pressure <80 mm Hg) was 16% in the preload group and 22% in the coload group (P = 0.30). There were no differences in the incidence of nausea and/or vomiting, or neonatal outcome between the groups. CONCLUSION: There was no difference in the incidence of hypotension in women who received colloid administration before the initiation of spinal anesthesia compared with at the time of initiation of anesthesia. Both modalities are inefficient as single interventions to prevent hypotension.