Immune-monitoring of myelodysplastic neoplasms: Recommendations from the i4MDS consortium.

Advancements in comprehending myelodysplastic neoplasms (MDS) have unfolded significantly in recent years, elucidating a myriad of cellular and molecular underpinnings integral to disease progression. While molecular inclusions into prognostic models have substantively advanced risk stratification, recent revelations have emphasized the pivotal role of immune dysregulation within the bone marrow milieu during MDS evolution. Nonetheless, immunotherapy for MDS has not experienced breakthroughs seen in other malignancies, partly attributable to the absence of an immune classification that could stratify patients toward optimally targeted immunotherapeutic approaches. A pivotal obstacle to establishing "immune classes" among MDS patients is the absence of validated accepted immune panels suitable for routine application in clinical laboratories. In response, we formed International Integrative Innovative Immunology for MDS (i4MDS), a consortium of multidisciplinary experts, and created the following recommendations for standardized methodologies to monitor immune responses in MDS. A central goal of i4MDS is the development of an immune score that could be incorporated into current clinical risk stratification models. This position paper first consolidates current knowledge on MDS immunology. Subsequently, in collaboration with clinical and laboratory specialists, we introduce flow cytometry panels and cytokine assays, meticulously devised for clinical laboratories, aiming to monitor the immune status of MDS patients, evaluating both immune fitness and identifying potential immune "risk factors." By amalgamating this immunological characterization data and molecular data, we aim to enhance patient stratification, identify predictive markers for treatment responsiveness, and accelerate the development of systems immunology tools and innovative immunotherapies.

Sex and gender-based analysis and diversity metric reporting in acute care trials published in high-impact journals: a systematic review.

OBJECTIVE: To characterise sex and gender-based analysis (SGBA) and diversity metric reporting, representation of female/women participants in acute care trials and temporal changes in reporting before and after publication of the 2016 Sex and Gender Equity in Research guideline. DESIGN: Systematic review. DATA SOURCES: We searched MEDLINE for trials published in five leading medical journals in 2014, 2018 and 2020. STUDY SELECTION: Trials that enrolled acutely ill adults, compared two or more interventions and reported at least one clinical outcome. DATA ABSTRACTION AND SYNTHESIS: 4 reviewers screened citations and 22 reviewers abstracted data, in duplicate. We compared reporting differences between intensive care unit (ICU) and cardiology trials. RESULTS: We included 88 trials (75 (85.2%) ICU and 13 (14.8%) cardiology) (n=111 428; 38 140 (34.2%) females/women). Of 23 (26.1%) trials that reported an SGBA, most used a forest plot (22 (95.7%)), were prespecified (21 (91.3%)) and reported a sex-by-intervention interaction with a significance test (19 (82.6%)). Discordant sex and gender terminology were found between headings and subheadings within baseline characteristics tables (17/32 (53.1%)) and between baseline characteristics tables and SGBA (4/23 (17.4%)). Only 25 acute care trials (28.4%) reported race or ethnicity. Participants were predominantly white (78.8%) and male/men (65.8%). No trial reported gendered-social factors. SGBA reporting and female/women representation did not improve temporally. Compared with ICU trials, cardiology trials reported significantly more SGBA (15/75 (20%) vs 8/13 (61.5%) p=0.005). CONCLUSIONS: Acute care trials in leading medical journals infrequently included SGBA, female/women and non-white trial participants, reported race or ethnicity and never reported gender-related factors. Substantial opportunity exists to improve SGBA and diversity metric reporting and recruitment of female/women participants in acute care trials. PROSPERO REGISTRATION NUMBER: CRD42022282565.

The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts.

Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, and generally associate with more favorable prognosis. However, not all SF3B1 mutations are the same, and little is known about how distinct hotspots influence disease. Here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and a lack of the favorable survival seen with other SF3B1 mutations. Moreover, compared to other hotspot SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data have implications for our understanding of the functional diversity of spliceosome mutations, as well as the pathobiology, classification, prognosis, and management of SF3B1-mutant MDS.

Therapeutic targeting of EP300/CBP by bromodomain inhibition in hematologic malignancies.

CCS1477 (inobrodib) is a potent, selective EP300/CBP bromodomain inhibitor which induces cell-cycle arrest and differentiation in hematologic malignancy model systems. In myeloid leukemia cells, it promotes rapid eviction of EP300/CBP from an enhancer subset marked by strong MYB occupancy and high H3K27 acetylation, with downregulation of the subordinate oncogenic network and redistribution to sites close to differentiation genes. In myeloma cells, CCS1477 induces eviction of EP300/CBP from FGFR3, the target of the common (4; 14) translocation, with redistribution away from IRF4-occupied sites to TCF3/E2A-occupied sites. In a subset of patients with relapsed or refractory disease, CCS1477 monotherapy induces differentiation responses in AML and objective responses in heavily pre-treated multiple myeloma. In vivo preclinical combination studies reveal synergistic responses to treatment with standard-of-care agents. Thus, CCS1477 exhibits encouraging preclinical and early-phase clinical activity by disrupting recruitment of EP300/CBP to enhancer networks occupied by critical transcription factors.

Differentiation block in acute myeloid leukemia regulated by intronic sequences of FTO.

Iroquois transcription factor gene IRX3 is highly expressed in 20-30% of acute myeloid leukemia (AML) and contributes to the pathognomonic differentiation block. Intron 8 FTO sequences ∼220kB downstream of IRX3 exhibit histone acetylation, DNA methylation, and contacts with the IRX3 promoter, which correlate with IRX3 expression. Deletion of these intronic elements confirms a role in positively regulating IRX3. RNAseq revealed long non-coding (lnc) transcripts arising from this locus. FTO-lncAML knockdown (KD) induced differentiation of AML cells, loss of clonogenic activity, and reduced FTO intron 8:IRX3 promoter contacts. While both FTO-lncAML KD and IRX3 KD induced differentiation, FTO-lncAML but not IRX3 KD led to HOXA downregulation suggesting transcript activity in trans. FTO-lncAMLhigh AML samples expressed higher levels of HOXA and lower levels of differentiation genes. Thus, a regulatory module in FTO intron 8 consisting of clustered enhancer elements and a long non-coding RNA is active in human AML, impeding myeloid differentiation.

Single-cell multi-omics defines the cell-type-specific impact of splicing aberrations in human hematopoietic clonal outgrowths.

RNA splicing factors are recurrently mutated in clonal blood disorders, but the impact of dysregulated splicing in hematopoiesis remains unclear. To overcome technical limitations, we integrated genotyping of transcriptomes (GoT) with long-read single-cell transcriptomics and proteogenomics for single-cell profiling of transcriptomes, surface proteins, somatic mutations, and RNA splicing (GoT-Splice). We applied GoT-Splice to hematopoietic progenitors from myelodysplastic syndrome (MDS) patients with mutations in the core splicing factor SF3B1. SF3B1mut cells were enriched in the megakaryocytic-erythroid lineage, with expansion of SF3B1mut erythroid progenitor cells. We uncovered distinct cryptic 3' splice site usage in different progenitor populations and stage-specific aberrant splicing during erythroid differentiation. Profiling SF3B1-mutated clonal hematopoiesis samples revealed that erythroid bias and cell-type-specific cryptic 3' splice site usage in SF3B1mut cells precede overt MDS. Collectively, GoT-Splice defines the cell-type-specific impact of somatic mutations on RNA splicing, from early clonal outgrowths to overt neoplasia, directly in human samples.

Pathogen and human NDPK-proteins promote AML cell survival via monocyte NLRP3-inflammasome activation.

A history of infection has been linked with increased risk of acute myeloid leukaemia (AML) and related myelodysplastic syndromes (MDS). Furthermore, AML and MDS patients suffer frequent infections because of disease-related impaired immunity. However, the role of infections in the development and progression of AML and MDS remains poorly understood. We and others previously demonstrated that the human nucleoside diphosphate kinase (NDPK) NM23-H1 protein promotes AML blast cell survival by inducing secretion of IL-1ß from accessory cells. NDPKs are an evolutionary highly conserved protein family and pathogenic bacteria secrete NDPKs that regulate virulence and host-pathogen interactions. Here, we demonstrate the presence of IgM antibodies against a broad range of pathogen NDPKs and more selective IgG antibody activity against pathogen NDPKs in the blood of AML patients and normal donors, demonstrating that in vivo exposure to NDPKs likely occurs. We also show that pathogen derived NDPK-proteins faithfully mimic the catalytically independent pro-survival activity of NM23-H1 against primary AML cells. Flow cytometry identified that pathogen and human NDPKs selectively bind to monocytes in peripheral blood. We therefore used vitamin D3 differentiated monocytes from wild type and genetically modified THP1 cells as a model to demonstrate that NDPK-mediated IL-1ß secretion by monocytes is NLRP3-inflammasome and caspase 1 dependent, but independent of TLR4 signaling. Monocyte stimulation by NDPKs also resulted in activation of NF-κB and IRF pathways but did not include the formation of pyroptosomes or result in pyroptotic cell death which are pivotal features of canonical NLRP3 inflammasome activation. In the context of the growing importance of the NLRP3 inflammasome and IL-1ß in AML and MDS, our findings now implicate pathogen NDPKs in the pathogenesis of these diseases.

Loss of TET2 in human hematopoietic stem cells alters the development and function of neutrophils.

Somatic mutations commonly occur in hematopoietic stem cells (HSCs). Some mutant clones outgrow through clonal hematopoiesis (CH) and produce mutated immune progenies shaping host immunity. Individuals with CH are asymptomatic but have an increased risk of developing leukemia, cardiovascular and pulmonary inflammatory diseases, and severe infections. Using genetic engineering of human HSCs (hHSCs) and transplantation in immunodeficient mice, we describe how a commonly mutated gene in CH, TET2, affects human neutrophil development and function. TET2 loss in hHSCs produce a distinct neutrophil heterogeneity in bone marrow and peripheral tissues by increasing the repopulating capacity of neutrophil progenitors and giving rise to low-granule neutrophils. Human neutrophils that inherited TET2 mutations mount exacerbated inflammatory responses and have more condensed chromatin, which correlates with compact neutrophil extracellular trap (NET) production. We expose here physiological abnormalities that may inform future strategies to detect TET2-CH and prevent NET-mediated pathologies associated with CH.

The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts.

Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, generally associate with more favorable prognosis, and serve as a predictive biomarker of response to luspatercept. However, not all SF3B1 mutations are the same, and here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and decreased survival. Moreover, in contrast to canonical SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data expand our knowledge of the functional diversity of spliceosome mutations, and they suggest that patients with E592K should be approached differently from low-risk, luspatercept-responsive MDS patients with ring sideroblasts and canonical SF3B1 mutations.

Vitamin B5 and succinyl-CoA improve ineffective erythropoiesis in SF3B1-mutated myelodysplasia.

Patients with myelodysplastic syndrome and ring sideroblasts (MDS-RS) present with symptomatic anemia due to ineffective erythropoiesis that impedes their quality of life and increases morbidity. More than 80% of patients with MDS-RS harbor splicing factor 3B subunit 1 (SF3B1) mutations, the founder aberration driving MDS-RS disease. Here, we report how mis-splicing of coenzyme A synthase (COASY), induced by mutations in SF3B1, affects heme biosynthesis and erythropoiesis. Our data revealed that COASY was up-regulated during normal erythroid differentiation, and its silencing prevented the formation of erythroid colonies, impeded erythroid differentiation, and precluded heme accumulation. In patients with MDS-RS, loss of protein due to COASY mis-splicing led to depletion of both CoA and succinyl-CoA. Supplementation with COASY substrate (vitamin B5) rescued CoA and succinyl-CoA concentrations in SF3B1mut cells and mended erythropoiesis differentiation defects in MDS-RS primary patient cells. Our findings reveal a key role of the COASY pathway in erythroid maturation and identify upstream and downstream metabolites of COASY as a potential treatment for anemia in patients with MDS-RS.

A three-gene leukaemic stem cell signature score is robustly prognostic in chronic myelomonocytic leukaemia.

Leukaemic stem cell (LSC) gene expression has recently been linked to prognosis in patients with acute myeloid leukaemia (17-gene LSC score, LSC-17) and myelodysplastic syndromes. Although chronic myelomonocytic leukaemia (CMML) is regarded as a stem cell disorder, the clinical and biological impact of LSCs on CMML patients remains elusive. Making use of multiple independent validation cohorts, we here describe a concise three-gene expression signature (LSC-3, derived from the LSC-17 score) as an independent and robust prognostic factor for leukaemia-free and overall survival in CMML. We propose that LSC-3 could be used to supplement existing risk stratification systems, to improve prognostic performance and guide management decisions.

Clinical and Therapeutic Aspects of Sideroblastic Anaemia with B-Cell Immunodeficiency, Periodic Fever and Developmental Delay (SIFD) Syndrome: a Systematic Review.

BACKGROUND AND PURPOSE: Sideroblastic anaemia with B-cell immunodeficiency, periodic fever and developmental delay (SIFD) syndrome is a novel rare autoinflammatory multisystem disorder. We performed a systematic review of the available clinical and therapeutics aspects of the SIFD syndrome. METHODS: A systematic review according to PRISMA approach, including all articles published before the 30th of July 2021 in Pubmed and EMBASE database, was performed. RESULTS: The search identified 29 publications describing 58 unique patients. To date, 41 unique mutations have been reported. Onset of disease is very early with a median age of 4 months (range 0-252 months). The most frequent manifestations are haematologic such as microcytic anaemia or sideroblastic anaemia (55/58), recurrent fever (52/58), neurologic abnormalities (48/58), immunologic abnormalities in particular a humoral immunodeficiency (48/58), gastrointestinal signs and symptoms (38/58), eye diseases as cataract and retinitis pigmentosa (27/58), failure to thrive (26/58), mucocutaneous involvement (29/58), sensorineural deafness (19/58) and others. To date, 19 patients (35.85%) died because of disease course (16) and complications of hematopoietic cell stems transplantation (3). The use of anti-TNFα and hematopoietic cell stems transplantation (HCST) is dramatically changing the natural history of this disease. CONCLUSIONS: SIFD syndrome is a novel entity to consider in a child presenting with recurrent fever, anaemia, B-cell immunodeficiency and neurodevelopmental delay. To date, therapeutic guidelines are lacking but anti-TNFα treatment and/or HCST are attractive and might modify the clinical course of this syndrome.

Echocardiography findings in amniotic fluid embolism: a systematic review of the literature.

PURPOSE: Amniotic fluid embolism (AFE) is a leading cause of obstetrical cardiac arrest and maternal morbidity. The pathogenesis of hemodynamic collapse is thought to be from right ventricular (RV) failure; however, there is a paucity of data documenting echocardiography findings in this population. We undertook a systematic review of the literature to evaluate the echocardiography findings in patients with AFE. SOURCES: We retrieved all case reports and case series reporting AFE in Embase and MEDLINE from inception to 20 November 2021. Studies reporting AFE diagnosed by fulfilling at least one of three different proposed AFE criteria and echocardiography findings during hospitalization were included. Patient and echocardiographic data were retrieved, and univariate logistic regression analysis was performed for outcomes of interest. Bias was assessed using the Joanna Briggs Institute clinical appraisal tool for case series. PRINCIPAL FINDINGS: Eighty publications reporting on 84 patients were included in the final review. Fifty-five out of 82 patients with data (67%) showed RV dysfunction, including 11/82 (13%) with biventricular dysfunction; 14/82 (17%) had normal systolic function. No data on RV or left ventricular function were reported for two patients. The presence of RV dysfunction on echocardiography was associated with cardiac arrest (odds ratio [OR], 3.66; 95% confidence interval [CI], 1.39 to 9.67; P = 0.009), and a composite risk of cardiac arrest, maternal death or use of extracorporeal membrane oxygenation (OR, 3.86; 95% CI, 1.43 to 10.4; P = 0.007). A low risk of bias was observed in 15/84 (18%) cases. CONCLUSIONS: Right ventricular dysfunction on echocardiography is a common finding in AFE and is associated with a high risk of cardiac arrest. The finding of RV dysfunction on echocardiography may help diagnose AFE and help triage the highest risk patients with AFE. STUDY REGISTRATION: PROSPERO (CRD42021271323); registered 1 September 2021.

RéSUMé: OBJECTIF: L'embolie amniotique (EA) est l'une des principales causes d'arrêt cardiaque obstétrical et de morbidité maternelle. Il est présumé que la pathogenèse du choc hémodynamique provient d'une défaillance ventriculaire droite (VD). Cependant, il y a peu de données documentant les constatations de l'examen échocardiographique dans cette population. Nous avons effectué une revue systématique des données probantes visant à évaluer l'utilité de l'échocardiographie chez les patientes atteintes d'embolie amniotique. SOURCES: Nous avons évalué tous les rapports de cas et séries de cas rapportant une EA dans les bases de données Embase et MEDLINE de leur création jusqu'au 20 novembre 2021. Les études rapportant une EA diagnostiquée en remplissant au moins l'un des trois critères d'EA proposés et les résultats échocardiographiques pendant l'hospitalisation ont été incluses. Les données sur les patientes et échocardiographiques ont été colligées, et une analyse de régression logistique univariée a été effectuée pour les issues cliniques d'intérêt. Le risque de biais a été évalué à l'aide de l'outil d'évaluation clinique de l'Institut Joanna Briggs pour les séries de cas. CONSTATATIONS PRINCIPALES: Quatre-vingts publications incluant 84 patientes ont été incluses dans la revue finale. Cinquante-cinq des 82 patientes présentant des données (67 %) avaient une dysfonction du VD incluant 11/82 (13 %) avec une dysfonction biventriculaire. Quatorze patientes sur 82 (17 %) avaient une fonction systolique normale. Aucune donnée sur la fonction du ventricule droit ou gauche n'a été rapportée pour deux patientes. La présence d'une dysfonction du VD à l'échocardiographie était associée à un arrêt cardiaque (rapport de cotes [RC], 3,66; intervalle de confiance à 95 % [IC], 1,39 à 9,67; P = 0,009), et à un risque composite d'arrêt cardiaque, de décès maternel ou d'utilisation de l'oxygénation par membrane extracorporelle (ECMO) (RC, 3,86; IC 95 %, 1,43 à 10,4; P = 0,007). Un faible risque de biais a été observé dans 15/84 (18 %) des cas. CONCLUSION: La dysfonction ventriculaire droite à l'échocardiographie est une constatation courante dans l'embolie amniotique et est associée à un risque élevé d'arrêt cardiaque. La découverte d'une dysfonction du VD à l'échocardiographie peut aider à diagnostiquer l'embolie amniotique et à identifier les patientes atteintes d'embolie amniotique les plus à risque. ENREGISTREMENT DE L'éTUD: PROSPERO (CRD42021271323); enregistrée le 1er septembre 2021.

Anticoagulation stewardship: Descriptive analysis of a novel approach to appropriate anticoagulant prescription.

Background: Anticoagulants are a leading cause of morbidity among hospitalized patients, with prescription errors commonly reported. Literature surrounding anticoagulation stewardship is scarce despite its documented effectiveness in the antimicrobial realm. Objective: To determine the proportion of accepted recommendations on inappropriate anticoagulant prescriptions suggested by a multidisciplinary anticoagulation stewardship program (ASP). Methods: We conducted a descriptive cohort study of hospitalized patients using therapeutic anticoagulation at a large Canadian tertiary care center between September 1, 2019, and February 28, 2020. A multidisciplinary ASP, composed of physicians and pharmacists, was implemented on June 1, 2019. Patient-, anticoagulant-, and admission-related characteristics were collected. The primary outcome was the proportion of accepted ASP team recommendations by the prescribing team. Results: A total of 381 patients were enrolled during the study period, resulting in 553 anticoagulant reviews (1.56 reviews/patient) by the ASP. The most common indications for anticoagulation were atrial fibrillation (n = 276, 72%) and venous thromboembolism (n = 84, 22%). Direct oral anticoagulants were most frequently prescribed (n = 253, 67%), followed by vitamin K antagonists (n = 88, 23%). Among the reviewed prescriptions, 355 of 553 (64%) generated a recommendation; 299 of 355 (84%) recommendations were accepted by the treating team. Dose adjustments were the leading category of recommendations (31%), followed by alerts regarding drug interactions (19%). Conclusion: Inpatient anticoagulant prescriptions were optimized following recommendations by the ASP team. The most frequent types of prescription changes concerned dose adjustments and drug interactions. Further research is required to assess the effect of an ASP on clinical outcomes.

The ABNL-MARRO 001 study: a phase 1-2 study of randomly allocated active myeloid target compound combinations in MDS/MPN overlap syndromes.

BACKGROUND: Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) comprise several rare hematologic malignancies with shared concomitant dysplastic and proliferative clinicopathologic features of bone marrow failure and propensity of acute leukemic transformation, and have significant impact on patient quality of life. The only approved disease-modifying therapies for any of the MDS/MPN are DNA methyltransferase inhibitors (DNMTi) for patients with dysplastic CMML, and still, outcomes are generally poor, making this an important area of unmet clinical need. Due to both the rarity and the heterogeneous nature of MDS/MPN, they have been challenging to study in dedicated prospective studies. Thus, refining first-line treatment strategies has been difficult, and optimal salvage treatments following DNMTi failure have also not been rigorously studied. ABNL-MARRO (A Basket study of Novel therapy for untreated MDS/MPN and Relapsed/Refractory Overlap Syndromes) is an international cooperation that leverages the expertise of the MDS/MPN International Working Group (IWG) and provides the framework for collaborative studies to advance treatment of MDS/MPN and to explore clinical and pathologic markers of disease severity, prognosis, and treatment response. METHODS: ABNL MARRO 001 (AM-001) is an open label, randomly allocated phase 1/2 study that will test novel treatment combinations in MDS/MPNs, beginning with the novel targeted agent itacitinib, a selective JAK1 inhibitor, combined with ASTX727, a fixed dose oral combination of the DNMTi decitabine and the cytidine deaminase inhibitor cedazuridine to improve decitabine bioavailability. DISCUSSION: Beyond the primary objectives of the study to evaluate the safety and efficacy of novel treatment combinations in MDS/MPN, the study will (i) Establish the ABNL MARRO infrastructure for future prospective studies, (ii) Forge innovative scientific research that will improve our understanding of pathogenetic mechanisms of disease, and (iii) Inform the clinical application of diagnostic criteria, risk stratification and prognostication tools, as well as response assessments in this heterogeneous patient population. TRIAL REGISTRATION: This trial was registered with ClinicalTrials.gov on August 19, 2019 (Registration No. NCT04061421).

Serotonin Syndrome After Treatment of Nausea and Vomiting in Pregnancy.

BACKGROUND: Nausea and vomiting in pregnancy often require pharmacotherapy for symptom management. Serotonin syndrome is a rare clinical entity that can be precipitated by the medications used to treat nausea and vomiting in pregnancy. CASE: A 35-year-old pregnant individual with a history of hyperemesis gravidarum in an earlier pregnancy requiring prolonged hospitalization presented with nausea and vomiting at 7 weeks of gestation. She was incidentally found to have severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection when she was universally screened at the time of admission. She required pharmacotherapy, including prochlorperazine and ondansetron for treatment of nausea as well as sumatriptan for migraine. She developed acute spasticity, autonomic dysfunction, and temperature rise, precipitated by antiemetic therapy, consistent with serotonin syndrome. The syndrome resolved with supportive care and benzodiazepines. CONCLUSION: Serotonin syndrome is a serious clinical entity that can be provoked by the pharmacotherapy given to treat nausea and vomiting in pregnancy. This medical emergency requires early recognition and prompt management.

The Novel Application of Geometric Morphometrics with Principal Component Analysis to Existing G Protein-Coupled Receptor (GPCR) Structures.

The G protein-coupled receptor (GPCR) superfamily is a large group of membrane proteins which, because of their vast involvement in cell signalling pathways, are implicated in a plethora of disease states and are therefore considered to be key drug targets. Despite advances in techniques to study these receptors, current prophylaxis is often limited due to the challenging nature of their dynamic, complex structures. Greater knowledge and understanding of their intricate structural rearrangements will therefore undoubtedly aid structure-based drug design against GPCRs. Disciplines such as anthropology and palaeontology often use geometric morphometrics to measure variation between shapes and we have therefore applied this technique to analyse GPCR structures in a three-dimensional manner, using principal component analysis. Our aim was to create a novel system able to discriminate between GPCR structures and discover variation between them, correlated with a variety of receptor characteristics. This was conducted by assessing shape changes at the extra- and intracellular faces of the transmembrane helix bundle, analysing the XYZ coordinates of the amino acids at those positions. We have demonstrated that GPCR structures can be classified based on characteristics such as activation state, bound ligands and fusion proteins, with the most significant results focussed at the intracellular face. Conversely, our analyses provide evidence that thermostabilising mutations do not cause significant differences when compared to non-mutated GPCRs. We believe that this is the first time geometric morphometrics has been applied to membrane proteins on this scale, and believe it can be used as a future tool in sense-checking newly resolved structures and planning experimental design.

Blast cells surviving acute myeloid leukemia induction therapy are in cycle with a signature of FOXM1 activity.

BACKGROUND: Disease relapse remains common following treatment of acute myeloid leukemia (AML) and is due to chemoresistance of leukemia cells with disease repopulating potential. To date, attempts to define the characteristics of in vivo resistant blasts have focused on comparisons between leukemic cells at presentation and relapse. However, further treatment responses are often seen following relapse, suggesting that most blasts remain chemosensitive. We sought to characterise in vivo chemoresistant blasts by studying the transcriptional and genetic features of blasts from before and shortly after induction chemotherapy using paired samples from six patients with primary refractory AML. METHODS: Leukemic blasts were isolated by fluorescence-activated cell sorting. Fluorescence in situ hybridization (FISH), targeted genetic sequencing and detailed immunophenotypic analysis were used to confirm that sorted cells were leukemic. Sorted blasts were subjected to RNA sequencing. Lentiviral vectors expressing short hairpin RNAs were used to assess the effect of FOXM1 knockdown on colony forming capacity, proliferative capacity and apoptosis in cell lines, primary AML cells and CD34+ cells from healthy donors. RESULTS: Molecular genetic analysis revealed early clonal selection occurring after induction chemotherapy. Immunophenotypic characterisation found leukemia-associated immunophenotypes in all cases that persisted following treatment. Despite the genetic heterogeneity of the leukemias studied, transcriptional analysis found concerted changes in gene expression in resistant blasts. Remarkably, the gene expression signature suggested that post-chemotherapy blasts were more proliferative than those at presentation. Resistant blasts also appeared less differentiated and expressed leukemia stem cell (LSC) maintenance genes. However, the proportion of immunophenotypically defined LSCs appeared to decrease following treatment, with implications for the targeting of these cells on the basis of cell surface antigen expression. The refractory gene signature was highly enriched with targets of the transcription factor FOXM1. shRNA knockdown experiments demonstrated that the viability of primary AML cells, but not normal CD34+ cells, depended on FOXM1 expression. CONCLUSIONS: We found that chemorefractory blasts from leukemias with varied genetic backgrounds expressed a common transcriptional program. In contrast to the notion that LSC quiescence confers resistance to chemotherapy we find that refractory blasts are both actively proliferating and enriched with LSC maintenance genes. Using primary patient material from a relevant clinical context we also provide further support for the role of FOXM1 in chemotherapy resistance, proliferation and stem cell function in AML.

Enhancer recruitment of transcription repressors RUNX1 and TLE3 by mis-expressed FOXC1 blocks differentiation in acute myeloid leukemia.

Despite absent expression in normal hematopoiesis, the Forkhead factor FOXC1, a critical mesenchymal differentiation regulator, is highly expressed in ∼30% of HOXAhigh acute myeloid leukemia (AML) cases to confer blocked monocyte/macrophage differentiation. Through integrated proteomics and bioinformatics, we find that FOXC1 and RUNX1 interact through Forkhead and Runt domains, respectively, and co-occupy primed and active enhancers distributed close to differentiation genes. FOXC1 stabilizes association of RUNX1, HDAC1, and Groucho repressor TLE3 to limit enhancer activity: FOXC1 knockdown induces loss of repressor proteins, gain of CEBPA binding, enhancer acetylation, and upregulation of nearby genes, including KLF2. Furthermore, it triggers genome-wide redistribution of RUNX1, TLE3, and HDAC1 from enhancers to promoters, leading to repression of self-renewal genes, including MYC and MYB. Our studies highlight RUNX1 and CEBPA transcription factor swapping as a feature of leukemia cell differentiation and reveal that FOXC1 prevents this by stabilizing enhancer binding of a RUNX1/HDAC1/TLE3 transcription repressor complex to oncogenic effect.