R-Loop Accumulation in Spliceosome Mutant Leukemias Confers Sensitivity to PARP1 Inhibition by Triggering Transcription-Replication Conflicts.

RNA splicing factor (SF) gene mutations are commonly observed in patients with myeloid malignancies. Here we showed that SRSF2- and U2AF1-mutant leukemias are preferentially sensitive to PARP inhibitors (PARPi), despite being proficient in homologous recombination repair. Instead, SF-mutant leukemias exhibited R-loop accumulation that elicited an R-loop-associated PARP1 response, rendering cells dependent on PARP1 activity for survival. Consequently, PARPi induced DNA damage and cell death in SF-mutant leukemias in an R-loop-dependent manner. PARPi further increased aberrant R-loop levels, causing higher transcription-replication collisions and triggering ATR activation in SF-mutant leukemias. Ultimately, PARPi-induced DNA damage and cell death in SF-mutant leukemias could be enhanced by ATR inhibition. Finally, the level of PARP1 activity at R-loops correlated with PARPi sensitivity, suggesting that R-loop-associated PARP1 activity could be predictive of PARPi sensitivity in patients harboring SF gene mutations. This study highlights the potential of targeting different R-loop response pathways caused by spliceosome gene mutations as a therapeutic strategy for treating cancer. SIGNIFICANCE: Spliceosome-mutant leukemias accumulate R-loops and require PARP1 to resolve transcription-replication conflicts and genomic instability, providing rationale to repurpose FDA-approved PARP inhibitors for patients carrying spliceosome gene mutations.

BRD9 determines the cell fate of hematopoietic stem cells by regulating chromatin state.

ATP-dependent chromatin remodeling SWI/SNF complexes exist in three subcomplexes: canonical BAF (cBAF), polybromo BAF (PBAF), and a newly described non-canonical BAF (ncBAF). While cBAF and PBAF regulate fates of multiple cell types, roles for ncBAF in hematopoietic stem cells (HSCs) have not been investigated. Motivated by recent discovery of disrupted expression of BRD9, an essential component of ncBAF, in multiple cancers, including clonal hematopoietic disorders, we evaluate here the role of BRD9 in normal and malignant HSCs. BRD9 loss enhances chromatin accessibility, promoting myeloid lineage skewing while impairing B cell development. BRD9 significantly colocalizes with CTCF, whose chromatin recruitment is augmented by BRD9 loss, leading to altered chromatin state and expression of myeloid-related genes within intact topologically associating domains. These data uncover ncBAF as critical for cell fate specification in HSCs via three-dimensional regulation of gene expression and illuminate roles for ncBAF in normal and malignant hematopoiesis.

Therapeutic Targeting of RNA Splicing in Cancer.

RNA splicing is a key regulatory step in the proper control of gene expression. It is a highly dynamic process orchestrated by the spliceosome, a macro-molecular machinery that consists of protein and RNA components. The dysregulation of RNA splicing has been observed in many human pathologies ranging from neurodegenerative diseases to cancer. The recent identification of recurrent mutations in the core components of the spliceosome in hematologic malignancies has advanced our knowledge of how splicing alterations contribute to disease pathogenesis. This review article will discuss our current understanding of how aberrant RNA splicing regulation drives tumor initiation and progression. We will also review current therapeutic modalities and highlight emerging technologies designed to target RNA splicing for cancer treatment.

Verification of prognostic expression biomarkers is improved by examining enriched leukemic blasts rather than mononuclear cells from acute myeloid leukemia patients.

BACKGROUND: Studies have not systematically compared the ability to verify performance of prognostic transcripts in paired bulk mononuclear cells versus viable CD34-expressing leukemic blasts from patients with acute myeloid leukemia. We hypothesized that examining the homogenous leukemic blasts will yield different biological information and may improve prognostic performance of expression biomarkers. METHODS: To assess the impact of cellular heterogeneity on expression biomarkers in acute myeloid leukemia, we systematically examined paired mononuclear cells and viable CD34-expressing leukemic blasts from SWOG diagnostic specimens. After enrichment, patients were assigned into discovery and validation cohorts based on availability of extracted RNA. Analyses of RNA sequencing data examined how enrichment impacted differentially expressed genes associated with pre-analytic variables, patient characteristics, and clinical outcomes. RESULTS: Blast enrichment yielded significantly different expression profiles and biological pathways associated with clinical characteristics (e.g., cytogenetics). Although numerous differentially expressed genes were associated with clinical outcomes, most lost their prognostic significance in the mononuclear cells and blasts after adjusting for age and ELN risk, with only 11 genes remaining significant for overall survival in both cell populations (CEP70, COMMD7, DNMT3B, ECE1, LNX2, NEGR1, PIK3C2B, SEMA4D, SMAD2, TAF8, ZNF444). To examine the impact of enrichment on biomarker verification, these 11 candidate biomarkers were examined by quantitative RT/PCR in the validation cohort. After adjusting for ELN risk and age, expression of 4 genes (CEP70, DNMT3B, ECE1, and PIK3CB) remained significantly associated with overall survival in the blasts, while none met statistical significance in mononuclear cells. CONCLUSIONS: This study provides insights into biological information gained/lost by examining viable CD34-expressing leukemic blasts versus mononuclear cells from the same patient and shows an improved verification rate for expression biomarkers in blasts.

Protein biomarkers for response to XPO1 inhibition in haematologic malignancies.

XPO1 (Exportin-1) is the nuclear export protein responsible for the normal shuttling of several proteins and RNA species between the nucleocytoplasmic compartment of eukaryotic cells. XPO1 recognizes the nuclear export signal (NES) of its cargo proteins to facilitate its export. Alterations of nuclear export have been shown to play a role in oncogenesis in several types of solid tumour and haematologic cancers. Over more than a decade, there has been substantial progress in targeting nuclear export in cancer using selective XPO1 inhibitors. This has resulted in recent approval for the first-in-class drug selinexor for use in relapsed, refractory multiple myeloma and diffuse large B-cell lymphoma (DLBCL). Despite these successes, not all patients respond effectively to XPO1 inhibition and there has been lack of biomarkers for response to XPO1 inhibitors in the clinic. Using haematologic malignancy cell lines and samples from patients with myelodysplastic neoplasms treated with selinexor, we have identified XPO1, NF-κB(p65), MCL-1 and p53 protein levels as protein markers of response to XPO1 inhibitor therapy. These markers could lead to the identification of response upon XPO1 inhibition for more accurate decision-making in the personalized treatment of cancer patients undergoing treatment with selinexor.

A 13-Year-Old Male Patient With Severe Multifocal Pneumonia and Bronchiectasis That Required Extracorporeal Membrane Oxygenation.

CASE PRESENTATION: A 13-year-old male patient with intermittent asthma and joint hypermobility presented to the ED in acute hypoxemic respiratory distress. He reported experiencing cough, increased work of breathing, and worsening chest pain for 3 days before presentation. He also reported fatigue and decreased appetite for 2 weeks. He had no known fever, myalgias, or recent weight loss. His medical history included two hospitalizations during early childhood for viral respiratory illnesses, one of which required intubation at 8 months of age. He had a gastrostomy tube placed shortly after his hospitalization because of failure to thrive secondary to aspiration based on a swallow study. His weight gain and growth improved with adequate nutrition, and his gastrostomy tube was removed at 2 years of age. His newborn screen, which included immunoreactive trypsinogen, was normal. He was noted to have hypermobile joints on physical examination at a clinic visit in childhood, but his examination results were not concerning for a hypermobility syndrome, and further diagnosis was not pursued. His parents endorsed that he has been a "healthy child" overall other than the occasional cough, which was attributed to asthma. His lifestyle was described as sedentary; he did not play any sports or have any unusual hobbies. He did not take any daily medications and no environmental exposures were reported. There was no family history of pulmonary, autoimmune, or connective tissue disease.

Aberrant EVI1 splicing contributes to EVI1-rearranged leukemia.

Detailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drives inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer that upregulates transcription of EVI1. Here, we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 that is frequently present in inv(3)/t(3;3) acute myeloid leukemia (AML) and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly cooccurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of 6 amino acids at the 3' end of the second zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells, and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3' splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent cooccurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).

DEAD-Box RNA Helicases and Genome Stability.

DEAD-box RNA helicases are important regulators of RNA metabolism and have been implicated in the development of cancer. Interestingly, these helicases constitute a major recurring family of RNA-binding proteins important for protecting the genome. Current studies have provided insight into the connection between genomic stability and several DEAD-box RNA helicase family proteins including DDX1, DDX3X, DDX5, DDX19, DDX21, DDX39B, and DDX41. For each helicase, we have reviewed evidence supporting their role in protecting the genome and their suggested mechanisms. Such helicases regulate the expression of factors promoting genomic stability, prevent DNA damage, and can participate directly in the response and repair of DNA damage. Finally, we summarized the pathological and therapeutic relationship between DEAD-box RNA helicases and cancer with respect to their novel role in genome stability.

Improvement in Pulmonary Function Following Discontinuation of Vaping or E-Cigarette Use in Adolescents with EVALI.

Introduction: In 2019, an alarming number of cases coined as e-cigarette, or vaping, product use-associated lung injury (EVALI) were described in adolescents ranging from mild respiratory distress to fulminant respiratory failure. Limited data have been published on outcomes at short-term follow-up. We aimed to describe pulmonary manifestations, function, and radiologic findings after corticosteroid therapy in a cohort of adolescent patients. Methods: A retrospective chart analysis of all patients presenting to our institution between July 2019 and December 2019 with EVALI was conducted. Patients who had pulmonary follow-up were included. Spirometry was performed before discharge from the hospital and during outpatient follow-up. A paired t-test was used to compare serial spirometry data between visits. Results: Eight patients (6 males) were included. Two patients required intubation with mechanical ventilation, 2 required bilevel positive airway pressure, and 3 required oxygen supplementation. All patients received glucocorticoids (3 receiving pulse dosing). Initial spirometry revealed decreased forced expiratory volume in one second and forced vital capacity with clinically and statistically significant improvement at follow-up (mean follow-up was 46.5 days). Radiographic manifestations also improved after vaping was discontinued. Conclusion: In our cohort of patients with EVALI, at short-term follow-up, all normalized their spirometry parameters and showed clinical resolution of symptoms.

Manifestation and Pathogenesis of Sudden Infant Death Syndrome: A Review.

Historically known as "cot death," sudden infant death syndrome (SIDS) is one of the leading causes of postnatal death in infants. According to the Centers for Disease Control and Prevention in 2010, more than 2,000 U.S. infants died from SIDS. Sudden infant death syndrome is the unexplainable death of an infant, less than one year old, that is otherwise healthy. SIDS was first discovered in 1969, and it typically presents in infants with a peak incidence between 2 and 4 months of age. Death by SIDS is typically more prevalent in the winter months, making the infant increasingly vulnerable. Despite witnessing a significant decrease in deaths by SIDS due to awareness campaigns and medical advancements, SIDS remains the leading cause of infant mortality in Western countries, accounting for half of all postnatal deaths. Throughout this paper, we will focus on the environmental factors, physiological factors, and genetic factors that have been postulated to cause an infant to be susceptible to SIDS. The initially postulated pathogenesis of SIDS was explained as the triple risk hypothesis, which states that an increase in SIDS risk presents in situations where there is an overlap of three or more factors. The presence of three or more factors suggests that the trio of factors overrule the infant's threshold for survival; therefore, the infant's homeostasis is unable to protect against the dangers. Death by SIDS has declined considerably from 130.3 deaths per 100,000 live births in 1998 to 38.7 deaths per 100,000 live births in 2014.

E-cigarette or vaping product use-associated lung injury in the pediatric population: imaging features at presentation and short-term follow-up.

BACKGROUND: Cases of e-cigarette or vaping product use-associated lung injury (EVALI) have rapidly reached epidemic proportions, yet there remain limited reports within the literature on the associated imaging findings. OBJECTIVE: We describe the most common imaging findings observed on chest computed tomography (CT) and chest radiograph (CXR) at presentation and at short-term follow-up at our major pediatric hospital. MATERIALS AND METHODS: A retrospective review of the electronic medical records was performed on all patients with suspected EVALI who were treated at a major pediatric hospital and 11 patients were included for analysis. Two board-certified pediatric radiologists then categorized the CXRs as either normal or abnormal, and further performed a systematic review of the chest CTs for imaging findings in the lungs, pleura and mediastinum. Interrater discordance was reconciled by consensus review. RESULTS: The 11 patients (9 males:2 females) ranged in age from 14 to 18 years. Gastrointestinal and constitutional symptoms were present in all patients, whereas shortness of breath and cough were reported in 5/11 and 6/11 patients, respectively. The CXR was abnormal in 10/11 patients, whereas all chest CTs were abnormal. The most common CT findings included consolidation, ground-glass opacities, interlobular septal thickening, lymphadenopathy and crazy-paving pattern. Almost all patients demonstrated subpleural sparing, and less than half also demonstrated peribronchovascular sparing. There was complete or near-complete resolution of imaging abnormalities in 5/6 patients with a median follow-up duration of 114 days. CONCLUSION: Pulmonary opacities with subpleural and peribronchovascular sparing was a commonly observed pattern of EVALI in the pediatric population at this institution. A CXR may not be sufficiently sensitive in diagnosing EVALI, and radiologists and clinicians should exercise caution when excluding EVALI based on the lack of a pulmonary opacity. Caution should also be exercised when excluding EVALI solely based on the lack of respiratory symptoms. Despite extensive pulmonary involvement at presentation, findings may resolve on short-term follow-up imaging.

An Open-Source, Vender Agnostic Hardware and Software Pipeline for Integration of Artificial Intelligence in Radiology Workflow.

Although machine learning (ML) has made significant improvements in radiology, few algorithms have been integrated into clinical radiology workflow. Complex radiology IT environments and Picture Archiving and Communication System (PACS) pose unique challenges in creating a practical ML schema. However, clinical integration and testing are critical to ensuring the safety and accuracy of ML algorithms. This study aims to propose, develop, and demonstrate a simple, efficient, and understandable hardware and software system for integrating ML models into the standard radiology workflow and PACS that can serve as a framework for testing ML algorithms. A Digital Imaging and Communications in Medicine/Graphics Processing Unit (DICOM/GPU) server and software pipeline was established at a metropolitan county hospital intranet to demonstrate clinical integration of ML algorithms in radiology. A clinical ML integration schema, agnostic to the hospital IT system and specific ML models/frameworks, was implemented and tested with a breast density classification algorithm and prospectively evaluated for time delays using 100 digital 2D mammograms. An open-source clinical ML integration schema was successfully implemented and demonstrated. This schema allows for simple uploading of custom ML models. With the proposed setup, the ML pipeline took an average of 26.52 s per second to process a batch of 100 studies. The most significant processing time delays were noted in model load and study stability times. The code is made available at " http://bit.ly/2Z121hX ". We demonstrated the feasibility to deploy and utilize ML models in radiology without disrupting existing radiology workflow.

DNA methylation disruption reshapes the hematopoietic differentiation landscape.

Mutations in genes involved in DNA methylation (DNAme; for example, TET2 and DNMT3A) are frequently observed in hematological malignancies1-3 and clonal hematopoiesis4,5. Applying single-cell sequencing to murine hematopoietic stem and progenitor cells, we observed that these mutations disrupt hematopoietic differentiation, causing opposite shifts in the frequencies of erythroid versus myelomonocytic progenitors following Tet2 or Dnmt3a loss. Notably, these shifts trace back to transcriptional priming skews in uncommitted hematopoietic stem cells. To reconcile genome-wide DNAme changes with specific erythroid versus myelomonocytic skews, we provide evidence in support of differential sensitivity of transcription factors due to biases in CpG enrichment in their binding motif. Single-cell transcriptomes with targeted genotyping showed similar skews in transcriptional priming of DNMT3A-mutated human clonal hematopoiesis bone marrow progenitors. These data show that DNAme shapes the topography of hematopoietic differentiation, and support a model in which genome-wide methylation changes are transduced to differentiation skews through biases in CpG enrichment of the transcription factor binding motif.

Mutations in the RNA Splicing Factor SF3B1 Promote Tumorigenesis through MYC Stabilization.

Although mutations in the gene encoding the RNA splicing factor SF3B1 are frequent in multiple cancers, their functional effects and therapeutic dependencies are poorly understood. Here, we characterize 98 tumors and 12 isogenic cell lines harboring SF3B1 hotspot mutations, identifying hundreds of cryptic 3' splice sites common and specific to different cancer types. Regulatory network analysis revealed that the most common SF3B1 mutation activates MYC via effects conserved across human and mouse cells. SF3B1 mutations promote decay of transcripts encoding the protein phosphatase 2A (PP2A) subunit PPP2R5A, increasing MYC S62 and BCL2 S70 phosphorylation which, in turn, promotes MYC protein stability and impair apoptosis, respectively. Genetic PPP2R5A restoration or pharmacologic PP2A activation impaired SF3B1-mutant tumorigenesis, elucidating a therapeutic approach to aberrant splicing by mutant SF3B1. SIGNIFICANCE: Here, we identify that mutations in SF3B1, the most commonly mutated splicing factor gene across cancers, alter splicing of a specific subunit of the PP2A serine/threonine phosphatase complex to confer post-translational MYC and BCL2 activation, which is therapeutically intervenable using an FDA-approved drug.See related commentary by O'Connor and Narla, p. 765.This article is highlighted in the In This Issue feature, p. 747.

Menin inhibitor MI-3454 induces remission in MLL1-rearranged and NPM1-mutated models of leukemia.

The protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations. When applied as a single agent, MI-3454 induced complete remission or regression of leukemia in mouse models of MLL1-rearranged or NPM1-mutated leukemia, including patient-derived xenograft models, through downregulation of key genes involved in leukemogenesis. We also identified MEIS1 as a potential pharmacodynamic biomarker of treatment response with MI-3454 in leukemia, and demonstrated that this compound is well tolerated and did not impair normal hematopoiesis in mice. Overall, this study demonstrates, for the first time to our knowledge, profound activity of the menin-MLL1 inhibitor as a single agent in clinically relevant PDX models of leukemia. These data provide a strong rationale for clinical translation of MI-3454 or its analogs for leukemia patients with MLL1 rearrangements or NPM1 mutations.

Coordinated alterations in RNA splicing and epigenetic regulation drive leukaemogenesis.

Transcription and pre-mRNA splicing are key steps in the control of gene expression and mutations in genes regulating each of these processes are common in leukaemia1,2. Despite the frequent overlap of mutations affecting epigenetic regulation and splicing in leukaemia, how these processes influence one another to promote leukaemogenesis is not understood and, to our knowledge, there is no functional evidence that mutations in RNA splicing factors initiate leukaemia. Here, through analyses of transcriptomes from 982 patients with acute myeloid leukaemia, we identified frequent overlap of mutations in IDH2 and SRSF2 that together promote leukaemogenesis through coordinated effects on the epigenome and RNA splicing. Whereas mutations in either IDH2 or SRSF2 imparted distinct splicing changes, co-expression of mutant IDH2 altered the splicing effects of mutant SRSF2 and resulted in more profound splicing changes than either mutation alone. Consistent with this, co-expression of mutant IDH2 and SRSF2 resulted in lethal myelodysplasia with proliferative features in vivo and enhanced self-renewal in a manner not observed with either mutation alone. IDH2 and SRSF2 double-mutant cells exhibited aberrant splicing and reduced expression of INTS3, a member of the integrator complex3, concordant with increased stalling of RNA polymerase II (RNAPII). Aberrant INTS3 splicing contributed to leukaemogenesis in concert with mutant IDH2 and was dependent on mutant SRSF2 binding to cis elements in INTS3 mRNA and increased DNA methylation of INTS3. These data identify a pathogenic crosstalk between altered epigenetic state and splicing in a subset of leukaemias, provide functional evidence that mutations in splicing factors drive myeloid malignancy development, and identify spliceosomal changes as a mediator of IDH2-mutant leukaemogenesis.

Spliceosomal disruption of the non-canonical BAF complex in cancer.

SF3B1 is the most commonly mutated RNA splicing factor in cancer1-4, but the mechanisms by which SF3B1 mutations promote malignancy are poorly understood. Here we integrated pan-cancer splicing analyses with a positive-enrichment CRISPR screen to prioritize splicing alterations that promote tumorigenesis. We report that diverse SF3B1 mutations converge on repression of BRD9, which is a core component of the recently described non-canonical BAF chromatin-remodelling complex that also contains GLTSCR1 and GLTSCR1L5-7. Mutant SF3B1 recognizes an aberrant, deep intronic branchpoint within BRD9 and thereby induces the inclusion of a poison exon that is derived from an endogenous retroviral element and subsequent degradation of BRD9 mRNA. Depletion of BRD9 causes the loss of non-canonical BAF at CTCF-associated loci and promotes melanomagenesis. BRD9 is a potent tumour suppressor in uveal melanoma, such that correcting mis-splicing of BRD9 in SF3B1-mutant cells using antisense oligonucleotides or CRISPR-directed mutagenesis suppresses tumour growth. Our results implicate the disruption of non-canonical BAF in the diverse cancer types that carry SF3B1 mutations and suggest a mechanism-based therapeutic approach for treating these malignancies.