Tight regulation of a nuclear HAPSTR1-HUWE1 pathway essential for mammalian life.

The recently discovered HAPSTR1 protein broadly oversees cellular stress responses. This function requires HUWE1, a ubiquitin ligase that paradoxically marks HAPSTR1 for degradation, but much about this pathway remains unclear. Here, leveraging multiplexed proteomics, we find that HAPSTR1 enables nuclear localization of HUWE1 with implications for nuclear protein quality control. We show that HAPSTR1 is tightly regulated and identify ubiquitin ligase TRIP12 and deubiquitinase USP7 as upstream regulators titrating HAPSTR1 stability. Finally, we generate conditional Hapstr1 knockout mice, finding that Hapstr1-null mice are perinatal lethal, adult mice depleted of Hapstr1 have reduced fitness, and primary cells explanted from Hapstr1-null animals falter in culture coincident with HUWE1 mislocalization and broadly remodeled signaling. Notably, although HAPSTR1 potently suppresses p53, we find that Hapstr1 is essential for life even in mice lacking p53. Altogether, we identify novel components and functional insights into the conserved HAPSTR1-HUWE1 pathway and demonstrate its requirement for mammalian life.

The HAPSTR2 retrogene buffers stress signaling and resilience in mammals.

We recently identified HAPSTR1 (C16orf72) as a key component in a novel pathway which regulates the cellular response to molecular stressors, such as DNA damage, nutrient scarcity, and protein misfolding. Here, we identify a functional paralog to HAPSTR1: HAPSTR2. HAPSTR2 formed early in mammalian evolution, via genomic integration of a reverse transcribed HAPSTR1 transcript, and has since been preserved under purifying selection. HAPSTR2, expressed primarily in neural and germline tissues and a subset of cancers, retains established biochemical features of HAPSTR1 to achieve two functions. In normal physiology, HAPSTR2 directly interacts with HAPSTR1, markedly augmenting HAPSTR1 protein stability in a manner independent from HAPSTR1's canonical E3 ligase, HUWE1. Alternatively, in the context of HAPSTR1 loss, HAPSTR2 expression is sufficient to buffer stress signaling and resilience. Thus, we discover a mammalian retrogene which safeguards fitness.

C16orf72/HAPSTR1 is a molecular rheostat in an integrated network of stress response pathways.

All cells contain specialized signaling pathways that enable adaptation to specific molecular stressors. Yet, whether these pathways are centrally regulated in complex physiological stress states remains unclear. Using genome-scale fitness screening data, we quantified the stress phenotype of 739 cancer cell lines, each representing a unique combination of intrinsic tumor stresses. Integrating dependency and stress perturbation transcriptomic data, we illuminated a network of genes with vital functions spanning diverse stress contexts. Analyses for central regulators of this network nominated C16orf72/HAPSTR1, an evolutionarily ancient gene critical for the fitness of cells reliant on multiple stress response pathways. We found that HAPSTR1 plays a pleiotropic role in cellular stress signaling, functioning to titrate various specialized cell-autonomous and paracrine stress response programs. This function, while dispensable to unstressed cells and nematodes, is essential for resilience in the presence of stressors ranging from DNA damage to starvation and proteotoxicity. Mechanistically, diverse stresses induce HAPSTR1, which encodes a protein expressed as two equally abundant isoforms. Perfectly conserved residues in a domain shared between HAPSTR1 isoforms mediate oligomerization and binding to the ubiquitin ligase HUWE1. We show that HUWE1 is a required cofactor for HAPSTR1 to control stress signaling and that, in turn, HUWE1 feeds back to ubiquitinate and destabilize HAPSTR1. Altogether, we propose that HAPSTR1 is a central rheostat in a network of pathways responsible for cellular adaptability, the modulation of which may have broad utility in human disease.

The mTORC1-SLC4A7 axis stimulates bicarbonate import to enhance de novo nucleotide synthesis.

Bicarbonate (HCO3-) ions maintain pH homeostasis in eukaryotic cells and serve as a carbonyl donor to support cellular metabolism. However, whether the abundance of HCO3- is regulated or harnessed to promote cell growth is unknown. The mechanistic target of rapamycin complex 1 (mTORC1) adjusts cellular metabolism to support biomass production and cell growth. We find that mTORC1 stimulates the intracellular transport of HCO3- to promote nucleotide synthesis through the selective translational regulation of the sodium bicarbonate cotransporter SLC4A7. Downstream of mTORC1, SLC4A7 mRNA translation required the S6K-dependent phosphorylation of the translation factor eIF4B. In mTORC1-driven cells, loss of SLC4A7 resulted in reduced cell and tumor growth and decreased flux through de novo purine and pyrimidine synthesis in human cells and tumors without altering the intracellular pH. Thus, mTORC1 signaling, through the control of SLC4A7 expression, harnesses environmental bicarbonate to promote anabolic metabolism, cell biomass, and growth.

HSF2 cooperates with HSF1 to drive a transcriptional program critical for the malignant state.

Heat shock factor 1 (HSF1) is well known for its role in the heat shock response (HSR), where it drives a transcriptional program comprising heat shock protein (HSP) genes, and in tumorigenesis, where it drives a program comprising HSPs and many noncanonical target genes that support malignancy. Here, we find that HSF2, an HSF1 paralog with no substantial role in the HSR, physically and functionally interacts with HSF1 across diverse types of cancer. HSF1 and HSF2 have notably similar chromatin occupancy and regulate a common set of genes that include both HSPs and noncanonical transcriptional targets with roles critical in supporting malignancy. Loss of either HSF1 or HSF2 results in a dysregulated response to nutrient stresses in vitro and reduced tumor progression in cancer cell line xenografts. Together, these findings establish HSF2 as a critical cofactor of HSF1 in driving a cancer cell transcriptional program to support the anabolic malignant state.

A network of core and subtype-specific gene expression programs in myositis.

Myositis comprises a heterogeneous group of skeletal muscle disorders which converge on chronic muscle inflammation and weakness. Our understanding of myositis pathogenesis is limited, and many myositis patients lack effective therapies. Using muscle biopsy transcriptome profiles from 119 myositis patients (spanning major clinical and serological disease subtypes) and 20 normal controls, we generated a co-expression network of 8101 dynamically regulated transcripts. This network organized the myositis transcriptome into a map of gene expression modules representing interrelated biological processes and disease signatures. Universally myositis-upregulated network modules included muscle regeneration, specific cytokine signatures, the acute phase response, and neutrophil degranulation. Universally myositis-suppressed pathways included a specific subset of myofilaments, the mitochondrial envelope, and nuclear isoforms of the anti-apoptotic humanin protein. Myositis subtype-specific modules included type 1 interferon signaling and titin (dermatomyositis), RNA processing (antisynthetase syndrome), and vasculogenesis (inclusion body myositis). Importantly, therapies exist to target influential proteins in many myositis-dysregulated modules, and nearly all modules contained understudied proteins and non-coding RNAs - many of which were extraordinarily dysregulated in myositis and may represent novel therapeutic targets. Finally, we apply our network to patient classification, finding that a deep learning algorithm trained on patient-level network "images" successfully assigned patients to clinical groups and further into molecular subclusters. Altogether, we provide a global resource to probe and contextualize differential gene expression in myositis.

FIREWORKS: a bottom-up approach to integrative coessentiality network analysis.

Genetic coessentiality analysis, a computational approach which identifies genes sharing a common effect on cell fitness across large-scale screening datasets, has emerged as a powerful tool to identify functional relationships between human genes. However, widespread implementation of coessentiality to study individual genes and pathways is limited by systematic biases in existing coessentiality approaches and accessibility barriers for investigators without computational expertise. We created FIREWORKS, a method and interactive tool for the construction and statistical analysis of coessentiality networks centered around gene(s) provided by the user. FIREWORKS incorporates a novel bias reduction approach to reduce false discoveries, enables restriction of coessentiality analyses to custom subsets of cell lines, and integrates multiomic and drug-gene interaction datasets to investigate and target contextual gene essentiality. We demonstrate the broad utility of FIREWORKS through case vignettes investigating gene function and specialization, indirect therapeutic targeting of "undruggable" proteins, and context-specific rewiring of genetic networks.

Quantitative and multiplexed chemical-genetic phenotyping in mammalian cells with QMAP-Seq.

Chemical-genetic interaction profiling in model organisms has proven powerful in providing insights into compound mechanism of action and gene function. However, identifying chemical-genetic interactions in mammalian systems has been limited to low-throughput or computational methods. Here, we develop Quantitative and Multiplexed Analysis of Phenotype by Sequencing (QMAP-Seq), which leverages next-generation sequencing for pooled high-throughput chemical-genetic profiling. We apply QMAP-Seq to investigate how cellular stress response factors affect therapeutic response in cancer. Using minimal automation, we treat pools of 60 cell types-comprising 12 genetic perturbations in five cell lines-with 1440 compound-dose combinations, generating 86,400 chemical-genetic measurements. QMAP-Seq produces precise and accurate quantitative measures of acute drug response comparable to gold standard assays, but with increased throughput at lower cost. Moreover, QMAP-Seq reveals clinically actionable drug vulnerabilities and functional relationships involving these stress response factors, many of which are activated in cancer. Thus, QMAP-Seq provides a broadly accessible and scalable strategy for chemical-genetic profiling in mammalian cells.

Posttranslational Regulation of the Exon Skipping Machinery Controls Aberrant Splicing in Leukemia.

Splicing alterations are common in diseases such as cancer, where mutations in splicing factor genes are frequently responsible for aberrant splicing. Here we present an alternative mechanism for splicing regulation in T-cell acute lymphoblastic leukemia (T-ALL) that involves posttranslational stabilization of the splicing machinery via deubiquitination. We demonstrate there are extensive exon skipping changes in disease, affecting proteasomal subunits, cell-cycle regulators, and the RNA machinery. We present that the serine/arginine-rich splicing factors (SRSF), controlling exon skipping, are critical for leukemia cell survival. The ubiquitin-specific peptidase 7 (USP7) regulates SRSF6 protein levels via active deubiquitination, and USP7 inhibition alters the exon skipping pattern and blocks T-ALL growth. The splicing inhibitor H3B-8800 affects splicing of proteasomal transcripts and proteasome activity and acts synergistically with proteasome inhibitors in inhibiting T-ALL growth. Our study provides the proof-of-principle for regulation of splicing factors via deubiquitination and suggests new therapeutic modalities in T-ALL. SIGNIFICANCE: Our study provides a new proof-of-principle for posttranslational regulation of splicing factors independently of mutations in aggressive T-cell leukemia. It further suggests a new drug combination of splicing and proteasomal inhibitors, a concept that might apply to other diseases with or without mutations affecting the splicing machinery.This article is highlighted in the In This Issue feature, p. 1241.

Impact of Telemonitoring of Critically Ill Emergency Department Patients Awaiting ICU Transfer.

OBJECTIVES: Because of overcrowding and limited critical care resources, critically ill patients in the emergency department may spend hours to days awaiting transfer to the ICU. In these patients, often termed "ICU boarders," delayed ICU transfer is associated with poor outcomes. We implemented an emergency department-based, electronic ICU monitoring system for ICU boarders. Our aim was to investigate the effect of this initiative on morbidity, mortality, and ICU usage. DESIGN: Single-center, retrospective cohort study. SETTING: Nonprofit, tertiary care, teaching hospital with greater than 100,000 emergency department visits per year. PATIENTS: Emergency department patients with admission orders for the medical ICU, who spent more than 2 hours boarding in the emergency department after being accepted for admission to the medical ICU, were included in the study. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: During the study period, a total of 314 patients were admitted to the medical ICU from the emergency department, 214 of whom were considered ICU boarders with a delay in medical ICU transfer over 2 hours. Of ICU boarders, 115 (53.7%) were enrolled in electronic ICU telemonitoring (electronic ICU care), and the rest received usual emergency department care (emergency department care). Age, mean illness severity (Acute Physiology and Chronic Health Evaluation IVa scores), and admitting diagnoses did not differ significantly between ICU boarders receiving electronic ICU care and emergency department care. Forty-one electronic ICU care patients (36%) were ultimately transitioned to a less intensive level of care in lieu of ICU admission while still in the emergency department, compared with zero patients in the emergency department care group. Among all ICU boarders transferred to the ICU, in-hospital mortality was lower in the electronic ICU care cohort when compared with the emergency department care cohort (5.4% vs 20.0%; adjusted odds ratio, 0.08). CONCLUSIONS: In critically ill patients awaiting transfer from the emergency department to the medical ICU, electronic ICU care was associated with decreased mortality and lower ICU resource utilization.

Myositis Autoantigen Expression Correlates With Muscle Regeneration but Not Autoantibody Specificity.

OBJECTIVE: Although more than a dozen myositis-specific autoantibodies (MSAs) have been identified, most patients with myositis are positive for a single MSA. The specific overexpression of a given myositis autoantigen in myositis muscle has been proposed as initiating and/or propagating autoimmunity against that particular autoantigen. The present study was undertaken to test this hypothesis. METHODS: In order to quantify autoantigen RNA expression, RNA sequencing was performed on muscle biopsy samples from control subjects, MSA-positive patients with myositis, regenerating mouse muscles, and cultured human muscle cells. RESULTS: Muscle biopsy samples were available from 20 control subjects and 106 patients with autoantibodies recognizing hydroxymethylglutaryl-coenzyme A reductase (n = 40), signal recognition particles (n = 9), Jo-1 (n = 18), nuclear matrix protein 2 (n = 12), Mi-2 (n = 11), transcription intermediary factor 1γ (n = 11), or melanoma differentiation-associated protein 5 (n = 5). The increased expression of a given autoantigen in myositis muscle was not associated with autoantibodies recognizing that autoantigen (all q > 0.05). In biopsy specimens from both myositis muscle and regenerating mouse muscles, autoantigen expression correlated directly with the expression of muscle regeneration markers and correlated inversely with the expression of genes encoding mature muscle proteins. Myositis autoantigens were also expressed at high levels in cultured human muscle cells. CONCLUSION: Most myositis autoantigens are highly expressed during muscle regeneration, which may relate to the propagation of autoimmunity. However, factors other than overexpression of specific autoantigens are likely to govern the development of unique autoantibodies in individual patients with myositis.

Muscle endurance deficits in myositis patients despite normal manual muscle testing scores.

INTRODUCTION: It is unclear whether quantitating muscle endurance adds nonredundant information useful for the care of patients with muscular disease. METHODS: Records were retrospectively reviewed for all Johns Hopkins Myositis Center patients with a muscle endurance assessment (n = 128, 226 patient-visits). Muscle endurance and strength were quantitated with the Myositis Functional Index-2 (FI2) and manual muscle testing (MMT), respectively. RESULTS: Composite FI2 muscle endurance scores were comparable in inclusion body myositis (n = 58), dermatomyositis (n = 31), and polymyositis (n = 39). Overall, muscle endurance correlated with and evolved similarly to strength, inversely to serum creatine kinase. However, in patients with normal or near-normal strength (mean MMT > 9.75/10), muscle endurance was typically abnormal and highly variable (mean FI2, 5.6/10; interquartile range, 3.3-7.8/10). DISCUSSION: Muscle endurance testing may identify muscle impairment inadequately described by MMT, particularly in patients with high MMT scores. Muscle Nerve 59:70-75, 2019.

Calcium dysregulation, functional calpainopathy, and endoplasmic reticulum stress in sporadic inclusion body myositis.

Sporadic inclusion body myositis (IBM) is the most common primary myopathy in the elderly, but its pathoetiology is still unclear. Perturbed myocellular calcium (Ca2+) homeostasis can exacerbate many of the factors proposed to mediate muscle degeneration in IBM, such as mitochondrial dysfunction, protein aggregation, and endoplasmic reticulum stress. Ca2+ dysregulation may plausibly be initiated in IBM by immune-mediated membrane damage and/or abnormally accumulating proteins, but no studies to date have investigated Ca2+ regulation in IBM patients. We first investigated protein expression via immunoblot in muscle biopsies from IBM, dermatomyositis, and non-myositis control patients, identifying several differentially expressed Ca2+-regulatory proteins in IBM. Next, we investigated the Ca2+-signaling transcriptome by RNA-seq, finding 54 of 183 (29.5%) genes from an unbiased list differentially expressed in IBM vs. controls. Using an established statistical approach to relate genes with causal transcription networks, Ca2+ abundance was considered a significant upstream regulator of observed whole-transcriptome changes. Post-hoc analyses of Ca2+-regulatory mRNA and protein data indicated a lower protein to transcript ratio in IBM vs. controls, which we hypothesized may relate to increased Ca2+-dependent proteolysis and decreased protein translation. Supporting this hypothesis, we observed robust (4-fold) elevation in the autolytic activation of a Ca2+-activated protease, calpain-1, as well as increased signaling for translational attenuation (eIF2a phosphorylation) downstream of the unfolded protein response. Finally, in IBM samples we observed mRNA and protein under-expression of calpain-3, the skeletal muscle-specific calpain, which broadly supports proper Ca2+ homeostasis. Together, these data provide novel insight into mechanisms by which intracellular Ca2+ regulation is perturbed in IBM and offer evidence of pathological downstream effects.