CRISPR Dependency Screens in Primary Hematopoietic Stem Cells Identify KDM3B as a Genotype Specific Vulnerability in IDH2- and TET2-Mutant Cells.

Clonal hematopoiesis (CH) is a common premalignant state in the blood and confers an increased risk of blood cancers and all-cause mortality. Identification of therapeutic targets in CH has been hindered by the lack of an ex vivo platform amenable for studying primary hematopoietic stem and progenitor cells (HSPCs). Here, we utilize an ex vivo co-culture system of HSPCs with bone marrow endothelial cells to perform CRISPR/Cas9 screens in mutant HSPCs. Our data reveal that loss of the histone demethylase family members Kdm3b and Jmjd1c specifically reduces the fitness of Idh2- and Tet2-mutant HSPCs. Kdm3b loss in mutant cells leads to decreased expression of critical cytokine receptors including Mpl, rendering mutant HSPCs preferentially susceptible to inhibition of downstream JAK2 signaling. Our study nominates an epigenetic regulator and an epigenetically regulated receptor signaling pathway as genotype-specific therapeutic targets and provides a scalable platform to identify genetic dependencies in mutant HSPCs.

Mutation order in acute myeloid leukemia identifies uncommon patterns of evolution and illuminates phenotypic heterogeneity.

Acute myeloid leukemia (AML) has a poor prognosis and a heterogeneous mutation landscape. Although common mutations are well-studied, little research has characterized how the sequence of mutations relates to clinical features. Using published, single-cell DNA sequencing data from three institutions, we compared clonal evolution patterns in AML to patient characteristics, disease phenotype, and outcomes. Mutation trees, which represent the order of select mutations, were created for 207 patients from targeted panel sequencing data using 1 639 162 cells, 823 mutations, and 275 samples. In 224 distinct orderings of mutated genes, mutations related to DNA methylation typically preceded those related to cell signaling, but signaling-first cases did occur, and had higher peripheral cell counts, increased signaling mutation homozygosity, and younger patient age. Serial sample analysis suggested that NPM1 and DNA methylation mutations provide an advantage to signaling mutations in AML. Interestingly, WT1 mutation evolution shared features with signaling mutations, such as WT1-early being proliferative and occurring in younger individuals, trends that remained in multivariable regression. Some mutation orderings had a worse prognosis, but this was mediated by unfavorable mutations, not mutation order. These findings add a dimension to the mutation landscape of AML, identifying uncommon patterns of leukemogenesis and shedding light on heterogeneous phenotypes.

Mutation order in acute myeloid leukemia identifies uncommon patterns of evolution and illuminates phenotypic heterogeneity.

Acute myeloid leukemia (AML) has a poor prognosis and a heterogeneous mutation landscape. Although common mutations are well-studied, little research has characterized how the sequence of mutations relates to clinical features. Using published, single-cell DNA sequencing data from three institutions, we compared clonal evolution patterns in AML to patient characteristics, disease phenotype, and outcomes. Mutation trees, which represent the order of select mutations, were created for 207 patients from targeted panel sequencing data using 1 639 162 cells, 823 mutations, and 275 samples. In 224 distinct orderings of mutated genes, mutations related to DNA methylation typically preceded those related to cell signaling, but signaling-first cases did occur, and had higher peripheral cell counts, increased signaling mutation homozygosity, and younger patient age. Serial sample analysis suggested that NPM1 and DNA methylation mutations provide an advantage to signaling mutations in AML. Interestingly, WT1 mutation evolution shared features with signaling mutations, such as WT1-early being proliferative and occurring in younger individuals, trends that remained in multivariable regression. Some mutation orderings had a worse prognosis, but this was mediated by unfavorable mutations, not mutation order. These findings add a dimension to the mutation landscape of AML, identifying uncommon patterns of leukemogenesis and shedding light on heterogenous phenotypes.

Single-cell genotypic and phenotypic analysis of measurable residual disease in acute myeloid leukemia.

Measurable residual disease (MRD), defined as the population of cancer cells that persist following therapy, serves as the critical reservoir for disease relapse in acute myeloid leukemia and other malignancies. Understanding the biology enabling MRD clones to resist therapy is necessary to guide the development of more effective curative treatments. Discriminating between residual leukemic clones, preleukemic clones, and normal precursors remains a challenge with current MRD tools. Here, we developed a single-cell MRD (scMRD) assay by combining flow cytometric enrichment of the targeted precursor/blast population with integrated single-cell DNA sequencing and immunophenotyping. Our scMRD assay shows high sensitivity of approximately 0.01%, deconvolutes clonal architecture, and provides clone-specific immunophenotypic data. In summary, our scMRD assay enhances MRD detection and simultaneously illuminates the clonal architecture of clonal hematopoiesis/preleukemic and leukemic cells surviving acute myeloid leukemia therapy.

Timing of umbilical cord occlusion, delayed vs early, in preterm babies: A randomized controlled trial (CODE-P Trial).

INTRODUCTION: Our hypothesis was that delayed cord clamping (DCC) (not earlier than 30 s; at 30-60 s) in premature neonates (born between 26.0 and 32.6 weeks of gestation), as compared with the usual early cord clamping (ECC), significantly reduces the need for blood transfusions and incidence of intraventricular haemorrhage (IVH) without an increased rate of maternal postpartum haemorrhage. MATERIAL AND METHODS: A prospective, open-label, randomized, controlled trial was conducted at Vall d'Hebron Hospital from July 2014 to December 2018. All pregnant women at risk of impending preterm birth (≥26.0-<33.0 weeks of gestation) who were admitted to the obstetrics emergency department were evaluated for eligibility. If they met the eligibility criteria, they were invited to participate in the study and, if they agreed, they signed an informed consent. Patients were randomly assigned to one of two groups: ECC group and DCC group. RESULTS: Our study included a total of 57 patients: 30 in the ECC group and 27 in the DCC group. Due to a lack of funding and low recruitment rates, the study was discontinued in 2018. Maternal characteristics and obstetric outcomes were similar between both groups. The intention-to-treat analysis did not reveal any differences between groups for neonatal red blood cell transfusions, neonatal IVH or maternal postpartum haemorrhage. There were no differences for secondary outcomes. Similarly, no differences were observed in the as-treated analysis. CONCLUSION: The primary and secondary outcomes of our study were not achieved. Therefore, more meta-analysis and trials are needed to evaluate the appropriate timing of cord clamping in preterm birth.

Chronic inflammation can transform the fate of normal and mutant hematopoietic stem cells.

Chronic inflammation, although subtle, puts the body in a constant state of alertness and is associated with many diseases, including cancer and cardiovascular diseases. It leads hematopoietic cells to produce and release proinflammatory cytokines, which trigger specific signaling pathways in hematopoietic stem cells (HSCs) that cause changes in proliferation, differentiation, and migration. This response is essential when HSCs are needed to produce specific blood cells to eliminate an intruder, such as a pathogenic virus, but mutant HSCs can use these proinflammatory signals to their advantage and accelerate the development of hematologic disease or malignancy. Understanding this complex process is vital for monitoring and controlling disease progression in patients. In the 2023 International Society for Experimental Hematology winter webinar, Dr. Eric Pietras (University of Colorado Anschutz Medical Campus, United States) and Dr. Katherine Y. King (Baylor College of Medicine, United States) gave a presentation on this topic, which is summarized in this review article.

Modification of Weak Localization in Metallic Thin Films Due to the Adsorption of Chiral Molecules.

We perform low-temperature magneto-conductance measurements on Cu and Au thin films with adsorbed chiral molecules and investigate their phase-coherent transport properties. Upon adsorption of chiral molecules, the spin-orbit coupling strength in Cu decreases and the Au films become ferromagnetic as evident from weak localization and antilocalization data. A theoretical model indicates that anisotropy in the molecular tilt angles, provided that the chiral molecules act as magnetic moments, induces a nonvanishing magnetic exchange interaction, causing changes in the spin-orbit coupling strength in Cu and Au. Our work adds a new viewpoint to the plethora of unique phenomena emerging from chiral molecule adsorption on materials.

Molecular predictors of immunophenotypic measurable residual disease clearance in acute myeloid leukemia.

Measurable residual disease (MRD) is a powerful prognostic factor in acute myeloid leukemia (AML). However, pre-treatment molecular predictors of immunophenotypic MRD clearance remain unclear. We analyzed a dataset of 211 patients with pre-treatment next-generation sequencing who received induction chemotherapy and had MRD assessed by serial immunophenotypic monitoring after induction, subsequent therapy, and allogeneic stem cell transplant (allo-SCT). Induction chemotherapy led to MRD- remission, MRD+ remission, and persistent disease in 35%, 27%, and 38% of patients, respectively. With subsequent therapy, 34% of patients with MRD+ and 26% of patients with persistent disease converted to MRD-. Mutations in CEBPA, NRAS, KRAS, and NPM1 predicted high rates of MRD- remission, while mutations in TP53, SF3B1, ASXL1, and RUNX1 and karyotypic abnormalities including inv (3), monosomy 5 or 7 predicted low rates of MRD- remission. Patients with fewer individual clones were more likely to achieve MRD- remission. Among 132 patients who underwent allo-SCT, outcomes were favorable whether patients achieved early MRD- after induction or later MRD- after subsequent therapy prior to allo-SCT. As MRD conversion with chemotherapy prior to allo-SCT is rarely achieved in patients with specific baseline mutational patterns and high clone numbers, upfront inclusion of these patients into clinical trials should be considered.

Crizotinib Has Preclinical Efficacy in Philadelphia-Negative Myeloproliferative Neoplasms.

PURPOSE: The Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) polycythemia vera, essential thrombocythemia, and primary myelofibrosis are characterized by JAK/STAT pathway activation. JAK inhibitors are approved for MPN treatment, but persistence has been observed, due to JAK/STAT reactivation. EXPERIMENTAL DESIGN: Using MPN patient samples, JAK2-mutated cell lines, and MPN mouse models, we examined both the efficacy and mechanism by which crizotinib, the ALK/MET/RON/ROS1 inhibitor approved for the treatment of non-small cell lung cancer, alters MPN cell proliferation and JAK/STAT activation. RESULTS: We found that crizotinib suppresses proliferation and activation of JAK/STAT signaling, and decreases the disease burden in the JAK2V617F mouse model of MPN. Furthermore, we found that crizotinib could overcome JAK inhibitor persistence to ruxolitinib. Interestingly, phosphorylation of the crizotinib target RON kinase was enhanced in ruxolitinib-persistent cells. We show that phospho-JAK2 and phospho-RON can physically interact to sustain JAK/STAT signaling, and that the combination of crizotinib and ruxolitinib disrupts this interaction. Furthermore, RON knockdown suppresses proliferation and activation of JAK/STAT signaling in JAK2-mutated cells, and RON deletion in a JAK2V617F mouse MPN model decreases the disease burden. We also observed RON hyperactivation in MPN patient cells, suggesting that RON may be an important target of crizotinib in MPN. CONCLUSIONS: In summary, we demonstrate that crizotinib has preclinical efficacy in MPN patient cells, JAK2-mutated cell lines, and a JAK2-mutated mouse model, and that the combination of crizotinib with JAK inhibitors suppresses JAK inhibitor persistence. Our work suggests that crizotinib should be investigated for the treatment of patients with MPN.

Respiratory technologists in the frontlines against COVID-19.

Background: The COVID-19 pandemic overwhelmed healthcare resources globally, but especially those of resource-limited countries. Strategies to supplement the number of healthcare workers attending COVID-19 patients had to be implemented. Several institutions used non-respiratory clinicians to work in COVID-19 wards. At Universitas Academic Hospital (UAH), Bloemfontein, South Africa, respiratory technologists were requested to assist with managing the oxygen supportive care of patients with severe COVID-19 and respiratory failure. Objectives: To highlight the contribution that respiratory technologists made in the management of severe COVID-19 pneumonia patients by describing the baseline characteristics and mortality of patients with COVID-19, whose oxygen supportive care was managed primarily by respiratory technologists at UAH. Methods: This was a retrospective study. The investigators extracted data from the hospital files of all adult patients admitted with severe COVID-19 to UAH and where respiratory technologists were involved in their care between 1 January and 31 December 2020. Results: A total of 781 patients were admitted to UAH, of whom 106 fulfilled the inclusion criteria. The majority of the patients were female (n=68; 64.1%), and the median age (interquartile range (IQR)) was 59.5 (51 - 68) years. Hypertension (n=69; 65.1%) and diabetes mellitus (n=39; 36.8%) were the most frequent comorbidities. At the time of admission, the median oxygen saturation was 92% and the median respiratory rate oxygenation (ROX) index was 3.2. The median length of stay was 7 days and the mortality was 41.5%. Conclusion: The clinical characteristics and mortality of patients whose oxygen support was managed by respiratory technologists were similar to those in previously reported studies from resource-limited settings. Respiratory technologists can form a valuable addition to the front-line team when clinicians and nurses are faced with overwhelming patient numbers in subsequent COVID-19 surges and where the mainstay of treatment is oxygen supportive care.

Cell origin-dependent cooperativity of mutant Dnmt3a and Npm1 in clonal hematopoiesis and myeloid malignancy.

In adult acute myeloid leukemia (AML), the acquisition of driver somatic mutations may be preceded by a benign state termed clonal hematopoiesis (CH). To develop therapeutic strategies to prevent leukemia development from CH, it is important to understand the mechanisms by which CH-driving and AML-driving mutations cooperate. Here, we use mice with inducible mutant alleles common in human CH (DNMT3AR882; mouse Dnmt3aR878H) and AML (NPM1c; mouse Npm1cA). We find that Dnmt3aR878H/+ hematopoietic stem cells (HSCs), but not multipotent progenitor cell (MPP) subsets, have reduced cytokine expression and proinflammatory transcriptional signatures and a functional competitive advantage over their wild-type counterparts. Dnmt3aR878H/+ HSCs are the most potent cell type transformed by Npm1cA, generating myeloid malignancies in which few additional cooperating somatic mutation events were detected. At a molecular level, Npm1cA, in cooperation with Dnmt3aR878H, acutely increased the accessibility of a distinct set of promoters in HSCs compared with MPP cells. These promoters were enriched for cell cycling, PI3K/AKT/mTOR signaling, stem cell signatures, and targets of transcription factors, including NFAT and the chromatin binding factor HMGB1, which have been implicated in human AML. These results demonstrate cooperativity between preexisting Dnmt3aR878H and Npm1cA at the chromatin level, where specific loci altered in accessibility by Npm1cA are dependent on cell context as well as Dnmt3a mutation status. These findings have implications for biological understanding and therapeutic intervention in the transformation from CH to AML.

A CRISPR Activation Screen Identifies an Atypical Rho GTPase That Enhances Zika Viral Entry.

Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR-Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host-pathogen interactions.

Outcomes of Esophagogastric Cancer Surgery During Eight Years of Surgical Auditing by the Dutch Upper Gastrointestinal Cancer Audit (DUCA).

OBJECTIVE: To evaluate changes in treatment and outcomes of esophagogastric cancer surgery after introduction of the DUCA. In addition, the presence of risk-averse behavior was assessed. SUMMARY OF BACKGROUND DATA: Clinical auditing is seen as an important quality improvement tool; however, its long-term efficacy remains largely unknown. In addition, critics claim that enhancements result from risk-averse behavior rather than positive effects of auditing. METHODS: DUCA data were used from registration start (1-1-2011) until 31-12-2018. Trends in patient, tumor, hospital and treatment characteristics were univariably assessed. Trends in short-term outcomes were investigated using multilevel multivariable logistic regression. Presence of risk aversion was described by the corrected proportion of patients undergoing surgery, using data from the Netherlands Cancer Registry. To evaluate the impact of centralization on time trends identified, the association between hospital volume and outcomes was investigated. RESULTS: This study included 6172 patients with esophageal and 3,690 with gastric cancer who underwent surgery. Pathological outcomes (lymph node yield, radicality) improved and futile surgery decreased over the years. In-hospital/30-day mortality decreased for esophagectomy (4.2% to 2.5%) and for gastrectomy (7.1% to 4.3%). Reinterventions, (minor) complications and readmissions increased. Risk aversion appeared absent. Between 2011-2018, annual median hospital volumes increased from 38 to 53 for esophagectomy and from 14 to 29 for gastrectomy. Higher hospital volumes were associated with several improved outcomes measures. CONCLUSIONS: During 8 years of auditing, outcomes improved, with no signs of risk-averse behavior. These improvements occurred in parallel with centralization. Feedback on postoperative complications remains the focus of the DUCA.

N-Linked Glycosylation on Anthrax Toxin Receptor 1 Is Essential for Seneca Valley Virus Infection.

Seneca Valley virus (SVV) is a picornavirus with potency in selectively infecting and lysing cancerous cells. The cellular receptor for SVV mediating the selective tropism for tumors is anthrax toxin receptor 1 (ANTXR1), a type I transmembrane protein expressed in tumors. Similar to other mammalian receptors, ANTXR1 has been shown to harbor N-linked glycosylation sites in its extracellular vWA domain. However, the exact role of ANTXR1 glycosylation on SVV attachment and cellular entry was unknown. Here we show that N-linked glycosylation in the ANTXR1 vWA domain is necessary for SVV attachment and entry. In our study, tandem mass spectrometry analysis of recombinant ANTXR1-Fc revealed the presence of complex glycans at N166, N184 in the vWA domain, and N81 in the Fc domain. Symmetry-expanded cryo-EM reconstruction of SVV-ANTXR1-Fc further validated the presence of N166 and N184 in the vWA domain. Cell blocking, co-immunoprecipitation, and plaque formation assays confirmed that deglycosylation of ANTXR1 prevents SVV attachment and subsequent entry. Overall, our results identified N-glycosylation in ANTXR1 as a necessary post-translational modification for establishing stable interactions with SVV. We anticipate our findings will aid in selecting patients for future cancer therapeutics, where screening for both ANTXR1 and its glycosylation could lead to an improved outcome from SVV therapy.

Spin-orbit coupling suppression and singlet-state blocking of spin-triplet Cooper pairs.

An inhomogeneous magnetic exchange field at a superconductor/ferromagnet interface converts spin-singlet Cooper pairs to a spin-polarized triplet state. Although the decay envelope of triplet pairs within ferromagnetic materials is well studied, little is known about their decay in nonmagnetic metals and superconductors and, in particular, in the presence of spin-orbit coupling (SOC). Here, we investigate devices in which singlet and triplet supercurrents propagate into the s-wave superconductor Nb. In the normal state of Nb, triplet supercurrents decay over a distance of 5 nm, which is an order of magnitude smaller than the decay of spin-singlet pairs due to the SOC. In the superconducting state of Nb, triplet supercurrents are not able to couple with the singlet wave function and are thus blocked by the absence of available equilibrium states in the singlet gap. The results offer insight into the dynamics between s-wave singlet and s-wave triplet states.

Failure to Cure in Patients Undergoing Surgery for Gastric Cancer: A Nationwide Cohort Study.

BACKGROUND: This study aimed to describe the incidence of failure to cure (a composite outcome measure defined as surgery not meeting its initial aim), and the impact of hospital variation in the administration of neoadjuvant therapy on this outcome measure. METHODS: All patients in the Dutch Upper Gastrointestinal Cancer Audit undergoing curatively intended gastric cancer surgery in 2011-2019 were included. Failure to cure was defined as (1) 'open-close' surgery; (2) irradical surgery (R1/R2); or (3) 30-day/in-hospital mortality. Case-mix-corrected funnel plots, based on multivariable logistic regression analyses, investigated hospital variation. The impact of a hospital's tendency to administer neoadjuvant chemotherapy on the heterogeneity in failure to cure between hospitals was assessed based on median odds ratios and multilevel logistic regression analyses. RESULTS: Some 3862 patients from 28 hospitals were included. Failure to cure was noted in 22.3% (hospital variation: 14.5-34.8%). After case-mix correction, two hospitals had significantly higher-than-expected failure to cure rates, and one hospital had a lower-than-expected rate. The failure to cure rate was significantly higher in hospitals with a low tendency to administer neoadjuvant chemotherapy. Approximately 29% of hospital variation in failure to cure could be attributed to different hospital policies regarding neoadjuvant therapy. CONCLUSIONS: Failure to cure has an incidence of 22% in patients undergoing gastric cancer surgery. Higher failure to cure rates were seen in centers administering less neoadjuvant chemotherapy, which confirms the Dutch guideline recommendation on the administration of neoadjuvant chemotherapy. Failure to cure provides short loop feedback and can be used as a quality indicator in surgical audits.

Controllable Enhancement of p-Wave Superconductivity via Magnetic Coupling to a Conventional Superconductor.

Unconventional superconductors are of high interest due to their rich physics, a topical example being topological edge states associated with p-wave superconductivity. A practical obstacle in studying such systems is the very low critical temperature T_{c} that is required to realize a p-wave superconducting phase in a material. We predict that the T_{c} of an intrinsic p-wave superconductor can be significantly enhanced by coupling to a conventional s-wave or d-wave superconductor with a higher critical temperature via an atomically thin ferromagnetic (F) layer. We show that this T_{c} boost is tunable via the direction of the magnetization in F. Moreover, we show that the enhancement in T_{c} can also be achieved using the Zeeman effect of an external magnetic field. Our findings provide a way to increase T_{c} in p-wave superconductors in a controllable way and make the exotic physics associated with such materials more easily accessible experimentally.

TMEM41B Is a Pan-flavivirus Host Factor.

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.