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Phenol-soluble modulins (PSMs) are the primary proteinaceous component of Staphylococcus aureus biofilms. Residence in the protective environment of biofilms allows bacteria to rapidly evolve and acquire antimicrobial resistance, which can lead to persistent infections such as those caused by methicillin-resistant S. aureus (MRSA). In their soluble form, PSMs hinder the immune response of the host and can increase the virulence potential of MRSA. PSMs also self-assemble into insoluble functional amyloids that contribute to the structural scaffold of biofilms. The specific roles of PSM peptides in biofilms remain poorly understood. Here, we report the development of a genetically tractable yeast model system for studying the properties of PSMα peptides. Expression of PSMα peptides in yeast drives the formation of toxic insoluble aggregates that adopt vesicle-like structures. Using this system, we probed the molecular drivers of PSMα aggregation to delineate key similarities and differences among the PSMs and identified a crucial residue that drives PSM features. Biofilms are a major public health threat; thus, biofilm disruption is a key goal. To solubilize aggregates comprised of a diverse range of amyloid and amyloid-like species, we have developed engineered variants of Hsp104, a hexameric AAA+ protein disaggregase from yeast. Here, we demonstrate that potentiated Hsp104 variants counter the toxicity and aggregation of PSMα peptides. Further, we demonstrate that a potentiated Hsp104 variant can drive the disassembly of preformed S. aureus biofilms. We suggest that this new yeast model can be a powerful platform for screening for agents that disrupt PSM aggregation and that Hsp104 disaggregases could be a promising tool for the safe enzymatic disruption of biofilms.

BCR sequencing

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Based on gene expression profiles, diffuse large B-cell lymphoma (DLBCL) is subdivided into germinal center B-cell–like (GCB) and activated B-cell–like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88 as well as BCL2 copy-number gains. Here, we employ immune phenotyping, RNA sequencing, and whole-exome sequencing to characterize a Myd88- and BCL2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression compared with GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and PD-1 blockade significantly increased the overall survival of lymphoma-bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations.Significance:Oncogenic Myd88 and BCL2 cooperate in murine DLBCL lymphomagenesis. The resulting lymphomas display morphologic and transcriptomic features reminiscent of human ABC-DLBCL. Data derived from our Myd88/BCL2-driven autochthonous model demonstrate that combined BCL2 and PD-1 blockade displays substantial preclinical antilymphoma activity, providing preclinical proof-of-concept data, which pave the way for clinical translation.This article is highlighted in the In This Issue feature, p. 1

Supplementary Tables from An In Vivo CRISPR Screening Platform for Prioritizing Therapeutic Targets in AML

CRISPR–Cas9-based genetic screens have successfully identified cell type–dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro using established cell lines, evaluating the physiologic relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies in vivo, including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, further highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets.Significance:There is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, both of which represent novel therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 275

Supplementary Data from An In Vivo CRISPR Screening Platform for Prioritizing Therapeutic Targets in AML

Hsp104, a yeast protein disaggregase, can be potentiated via numerous missense mutations at disparate locations throughout the coiled-coil middle domain (MD). Potentiated Hsp104 variants can counter the toxicity and misfolding of TDP-43, FUS, and α-synuclein, proteins which are implicated in neurodegenerative disorders. However, potentiated MD variants typically exhibit off-target toxicity. Further, it has remained confounding how numerous seemingly degenerate mutations confer potentiation, hampering engineering of therapeutic Hsp104 variants. Here, we sought to comprehensively define the key drivers of Hsp104 potentiation. Using scanning mutagenesis, we iteratively studied the effects of modulation at each position in the Hsp104 MD. Screening this library to identify enhanced variants reveals that missense mutations at 26% of positions in the MD yield variants that counter FUS toxicity. Modulation of the helix 2-helix 3/4 MD interface potentiates Hsp104, whereas mutations in the analogous helix 1-2 interface do not. Surprisingly, we find that there is a higher likelihood of enhancing Hsp104 activity against human disease substrates than impairing Hsp104 native function. We find that single mutations can broadly destabilize the MD structure and lead to functional potentiation, suggesting this may be a common mechanism conferring Hsp104 potentiation. Using this approach, we have demonstrated that modulation of the MD can yield engineered variants with decreased off-target effects. This article is protected by copyright. All rights reserved.

FUS and EWSR1 are RNA-binding proteins with prion-like domains (PrLDs) that aggregate in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The FUS and EWSR1 genes are also prone to chromosomal translocation events, which result in aberrant fusions between portions of the PrLDs of FUS and EWSR1 and the transcription factors CHOP and FLI. The resulting fusion proteins, FUS-CHOP and EWS-FLI, drive aberrant transcriptional programs that underpin liposarcoma and Ewing's sarcoma, respectively. The translocated PrLDs alter the expression profiles of these proteins and promote their phase separation and aggregation. Here, we report the development of yeast models of FUS-CHOP and EWS-FLI toxicity and aggregation. These models recapitulated several salient features of sarcoma patient cells harboring the FUS-CHOP and EWS-FLI translocations. To reverse FUS and EWSR1 aggregation, we have explored Hsp104, a hexameric AAA+ protein disaggregase from yeast. Previously, we engineered potentiated Hsp104 variants to suppress the proteotoxicity, aggregation, and mislocalization of FUS and other proteins that aggregate in ALS/FTD and Parkinson's disease. Potentiated Hsp104 variants that robustly suppressed FUS toxicity and aggregation also suppressed the toxicity and aggregation of FUS-CHOP and EWS-FLI. We suggest that these new yeast models are powerful platforms for screening for modulators of FUS-CHOP and EWS-FLI phase separation. Moreover, Hsp104 variants might be employed to combat the toxicity and phase separation of aberrant fusion proteins involved in sarcoma.

SummaryHuman embryonic stem cells (hESCs) are highly sensitive to DNA damage and have low survival ability relative to differentiated cells. We investigated the source of this difference by comparing damage response pathways in hESCs and differentiated cells. We found that hESCs undergo more rapid p53-dependent apoptosis after DNA damage than differentiated cells do. However, p53 localization and function are similar between hESCs and differentiated cells, suggesting that p53 alone cannot explain the difference in sensitivity. Instead, we show that mitochondrial readiness for apoptosis, known as mitochondrial priming, differs between hESCs and differentiated cells. Specifically, the balance between proapoptotic and antiapoptotic proteins is shifted closer to the apoptotic threshold in hESCs than in differentiated cells. Altering this balance in differentiated cells increases their sensitivity and results in cell death, suggesting that manipulation of mitochondrial priming could potentially alter the sensitivity of other stem cells, including cancer stem cells.