Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation.

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered the biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.

FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy.

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of haematological malignancies such as acute lymphoblastic leukaemia, B cell lymphoma and multiple myeloma1-4, but the efficacy of CAR T cell therapy in solid tumours has been limited5. This is owing to a number of factors, including the immunosuppressive tumour microenvironment that gives rise to poorly persisting and metabolically dysfunctional T cells. Analysis of anti-CD19 CAR T cells used clinically has shown that positive treatment outcomes are associated with a more 'stem-like' phenotype and increased mitochondrial mass6-8. We therefore sought to identify transcription factors that could enhance CAR T cell fitness and efficacy against solid tumours. Here we show that overexpression of FOXO1 promotes a stem-like phenotype in CAR T cells derived from either healthy human donors or patients, which correlates with improved mitochondrial fitness, persistence and therapeutic efficacy in vivo. This work thus reveals an engineering approach to genetically enforce a favourable metabolic phenotype that has high translational potential to improve the efficacy of CAR T cells against solid tumours.

Grb7 Ablation in Mice Improved Glycemic Control, Enhanced Insulin Signaling, and Increased Abdominal fat Mass in Females.

OBJECTIVES: Growth factor receptor bound protein 7 (GRB7) is a multidomain signaling adaptor. Members of the Grb7/10/14 family, specifically Gbrb10/14, have important roles in metabolism. We ablated the Grb7 gene in mice to examine its metabolic function. METHODS: Global ablation of Grb7 in FVB/NJ mice was generated. Growth, organ weight, food intake, and glucose homeostasis were measured. Insulin signaling was examined by Western blotting. Fat and lean body mass was measured by nuclear magnetic resonance, and body composition after fasting or high-fat diet was assessed. Energy expenditure was measured by indirect calorimetry. Expression of adiposity and lipid metabolism genes was measured by quantitative PCR. RESULTS: Grb7-null mice were viable, fertile, and without obvious phenotype. Grb7 ablation improved glycemic control and displayed sensitization to insulin signaling in the liver. Grb7-null females but not males had increased gonadal white adipose tissue mass. Following a 12-week high-fat diet, Grb7-null female mice gained fat body mass and developed relative insulin resistance. With fasting, there was less decrease in fat body mass in Grb7-null female mice. Female mice with Grb7 ablation had increased baseline food intake, less energy expenditure, and displayed a decrease in the expression of lipolysis and adipose browning genes in gonadal white adipose tissue by transcript and protein analysis. CONCLUSION: Our study suggests that Grb7 is a negative regulator of glycemic control. Our results reveal a role for Grb7 in female mice in the regulation of the visceral adipose tissue mass, a powerful predictor of metabolic dysfunction in obesity.

Influence of the storage conditions of embryo culture media on mouse development.

The culture of preimplantation embryos in vitro is an important method for human and mouse reproductive technology. This study aims to investigate the influence of different conditions of culture media on the preimplantation stage of mouse embryos cultured in vitro, and monitor the post-implantation development of new mice after embryo transfer to surrogate females. We demonstrated here that mouse embryos cultured in vitro in fresh M16, KSOM, Global, and HTF embryo culture media from one cell to the blastocyst stage and the subsequent embryo transfer to surrogate females are able to proceed through post-implantation development and, after birth, develop into healthy mice. However, culture of embryos in differently aged media shows various (often unpredictable) results. To find the optimal storage conditions of culture media, we suggest that the freezing and long-term storage of these media at - 80°C will not influence the quality of the media. To test this hypothesis, we grew embryos from one cell to blastocysts in vitro in the selected media after thawing and subsequently transferring them to surrogate females. Embryo culture in these four media after thawing does not affect preimplantation and postnatal mouse development. Thus, we have shown that storage of embryo culture media at low temperature (- 80°C) does not impact the quality of the media, and subsequently, it can be used for the culture of embryos for the full preimplantation period, the same as in fresh media.

Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation.

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.

Rationally designed chimeric PI3K-BET bromodomain inhibitors elicit curative responses in MYC-driven lymphoma.

Targeted inhibitors of bromodomain and extraterminal (BET)-bromodomains and phosphatidylinositol-3-kinase (PI3K) signaling demonstrate potent but self-limited antilymphoma activity as single agents in the context of cellular Myelocytomatosis (cMYC) oncogene-dysregulation. However, combined PI3K and BET inhibition imparts synergistic anticancer activity with the potential for more sustained disease responses due to the mutual antagonism of compensatory epigenetic and signaling networks. Here, we describe the mechanistic and therapeutic validation of rationally designed dual PI3K/BET bromodomain inhibitors, built by linkage of established PI3K and BET inhibitor pharmacophores. The lead candidate demonstrates high selectivity, nanomolar range cellular potency, and compelling in vivo efficacy, including curative responses in the aggressive Eµ-Myc lymphoma model. These studies further support the therapeutic strategy of combined PI3K and BET inhibition and provide a potential step-change in approach to orthogonal MYC antagonism using optimized chimeric small-molecule technology.

The curious case of IDH mutant acute myeloid leukaemia: biochemistry and therapeutic approaches.

Of the many genetic alterations that occur in cancer, relatively few have proven to be suitable for the development of targeted therapies. Mutations in isocitrate dehydrogenase (IDH) 1 and -2 increase the capacity of cancer cells to produce a normally scarce metabolite, D-2-hydroxyglutarate (2-HG), by several orders of magnitude. The discovery of the unusual biochemistry of IDH mutations spurred a flurry of activity that revealed 2-HG as an 'oncometabolite' with pleiotropic effects in malignant cells and consequences for anti-tumour immunity. Over the next decade, we learned that 2-HG dysregulates a wide array of molecular pathways, among them a large family of dioxygenases that utilise the closely related metabolite α-ketoglutarate (α-KG) as an essential co-substrate. 2-HG not only contributes to malignant transformation, but some cancer cells become addicted to it and sensitive to inhibitors that block its synthesis. Moreover, high 2-HG levels and loss of wild-type IDH1 or IDH2 activity gives rise to synthetic lethal vulnerabilities. Herein, we review the biology of IDH mutations with a particular focus on acute myeloid leukaemia (AML), an aggressive disease where selective targeting of IDH-mutant cells is showing significant promise.

Hepatoprotective effect of the radiation countermeasure flagellin in the long term after irradiation of mice.

PURPOSE: Purpose is to study the hepatoprotective effect of a new promising radiation countermeasure flagellin, in the long term after irradiation. The results of the study can be useful for mitigating the consequences of man-made radiation accidents, protecting professional contingents, reducing the toxic effect of radiation therapy, and expanding the range of drug use. MATERIALS AND METHODS: Effect of flagellin was investigated 10 months after its administration of irradiated male of mice F1 (CBAхC57Bl/6). Flagellin (0.2 mg/kg) was administrated once intraperitoneally before exposure of mice to low-intensive (10 mGy/min) γ-radiation at a dose of 12.65 Gy. The effect was evaluated in three groups: control, irradiated mice without of flagellin and with the administration flagellin 30 minutes before irradiation. Cytogenetic and cytotoxic effect in bone marrow was studied with micronucleus assay (OECD 474), in liver - with the original technique for cytome analysis of hepatocytes after fixation of liver pieces with 10% formalin, dissociation of cells with 50% KOH, staining with aceto-orcein and light green. The proportion of cells 2n, 2n + 2n, 4n, 4n + 4n, ≥8n and ≥8n + 8n was determined. Cytogenetic disorders were counted as cells with micronuclei, nuclear buds, and internuclear bridges. The ploidy index and nuclearity index were defined. RESULTS: In all studied groups of mice, the frequency of polychromatic bone marrow erythrocytes with micronuclei and hepatocytes with cytogenetic disorders did not exceed the background level. A decrease in the ploidy index of hepatocytes by 4.3 times was established 10 months after exposure to low-power ionizing radiation. In mice treated with flagellin before irradiation, the ploidy index was normalized to control. CONCLUSIONS: A decrease in the ploidy of hepatocytes was revealed 10 months after exposure to a high dose of low-power ionizing radiation, which may indicate the initiation of carcinogenesis. For the first time, a new aspect of the anti-radiation effect of promising radiation countermeasure flagellin was established and its hepatoprotective properties were determined in the long term after exposure to ionizing radiation.

Gait Abnormalities and Aberrant D2 Receptor Expression and Signaling in Mice Carrying the Human Pathogenic Mutation DRD2I212F.

A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder. That allelic variant D2-I212F is a constitutively active and G protein-biased receptor. We now describe mice engineered using CRISPR-Cas9-mediated gene editing technology to carry the D2-I212F variant. Drd2I212F mice exhibited gait abnormalities resembling those in other mouse models of chorea and/or dystonia and had striatal D2 receptor expression that was decreased approximately 30% per Drd2I212F allele. Electrically evoked inhibitory postsynaptic conductances in midbrain dopamine neurons and striatum from Drd2I212F mice, caused by G protein activation of potassium channels, exhibited slow kinetics (e.g., approximately four- to sixfold slower decay) compared with Drd2 +/+ mice. Current decay initiated by photolytic release of the D2 antagonist sulpiride from CyHQ-sulpiride was also ∼fourfold slower in midbrain slices from Drd2I212F mice than Drd2 +/+ mice. Furthermore, in contrast to Drd2 +/+ mice, in which dopamine is several-fold more potent at neurons in the nucleus accumbens than in the dorsal striatum, reflecting activation of Gα o versus Gα i, dopamine had similar potencies in those two brain regions of Drd2I212F mice. Repeated cocaine treatment, which decreases dopamine potency in the nucleus accumbens of Drd2 +/+ mice, had no effect on dopamine potency in Drd2 I212F mice. The results demonstrate the pathogenicity of the D2-I212F mutation and the utility of this mouse model for investigating the role of pathogenic DRD2 variants in early-onset hyperkinetic movement disorders. SIGNIFICANCE STATEMENT: The first dopamine receptor mutation to cause a movement disorder, D2-I212F, was recently identified. The mutation makes receptor activation of G protein-mediated signaling more efficient. To confirm the pathogenesis of D2-I212F, this study reports that mice carrying this mutation have gait abnormalities consistent with the clinical phenotype. The mutation also profoundly alters D2 receptor expression and function in vivo. This mouse model will be useful for further characterization of the mutant receptor and for evaluation of potential therapeutic drugs.

GPR39 Knockout Worsens Microcirculatory Response to Experimental Stroke in a Sex-Dependent Manner.

No current treatments target microvascular reperfusion after stroke, which can contribute to poor outcomes even after successful clot retrieval. The G protein-coupled receptor GPR39 is expressed in brain peri-capillary pericytes, and has been implicated in microvascular regulation, but its role in stroke is unknown. We tested the hypothesis that GPR39 plays a protective role after stroke, in part due to preservation of microvascular perfusion. We generated GPR39 knockout (KO) mice and tested whether GPR39 gene deletion worsens capillary blood flow and exacerbates brain injury and functional deficit after focal cerebral ischemia. Stroke was induced in male and female GPR39 KO and WT littermates by 60-min middle cerebral artery occlusion (MCAO). Microvascular perfusion was assessed via capillary red blood cell (RBC) flux in deep cortical layers in vivo using optical microangiography (OMAG). Brain injury was assessed by measuring infarct size by 2,3,5-triphenyltetrazolium chloride staining at 24 h or brain atrophy at 3 weeks after ischemia. Pole and cylinder behavior tests were conducted to assess neurological function deficit at 1 and 3 weeks post-stroke. Male but not female GPR39 KO mice exhibited larger infarcts and lower capillary RBC flux than WT controls after stroke. Male GPR39 KO mice also exhibited worse neurologic deficit at 1 week post-stroke, though functional deficit disappeared in both groups by 3 weeks. GPR39 deletion worsens brain injury, microvascular perfusion, and neurological function after experimental stroke. Results indicate that GPR39 plays a sex-dependent role in re-establishing microvascular flow and limiting ischemic brain damage after stroke.

An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma.

Apicomplexan parasites possess secretory organelles called rhoptries that undergo regulated exocytosis upon contact with the host. This process is essential for the parasitic lifestyle of these pathogens and relies on an exocytic machinery sharing structural features and molecular components with free-living ciliates. However, how the parasites coordinate exocytosis with host interaction is unknown. Here, we performed a Tetrahymena-based transcriptomic screen to uncover novel exocytic factors in Ciliata and conserved in Apicomplexa. We identified membrane-bound proteins, named CRMPs, forming part of a large complex essential for rhoptry secretion and invasion in Toxoplasma. Using cutting-edge imaging tools, including expansion microscopy and cryo-electron tomography, we show that, unlike previously described rhoptry exocytic factors, TgCRMPs are not required for the assembly of the rhoptry secretion machinery and only transiently associate with the exocytic site-prior to the invasion. CRMPs and their partners contain putative host cell-binding domains, and CRMPa shares similarities with GPCR proteins. Collectively our data imply that the CRMP complex acts as a host-molecular sensor to ensure that rhoptry exocytosis occurs when the parasite contacts the host cell.

Inhibition of mutant IDH1 promotes cycling of acute myeloid leukemia stem cells.

Approximately 20% of acute myeloid leukemia (AML) patients carry mutations in IDH1 or IDH2 that result in over-production of the oncometabolite D-2-hydroxyglutarate (2-HG). Small molecule inhibitors that block 2-HG synthesis can induce complete morphological remission; however, almost all patients eventually acquire drug resistance and relapse. Using a multi-allelic mouse model of IDH1-mutant AML, we demonstrate that the clinical IDH1 inhibitor AG-120 (ivosidenib) exerts cell-type-dependent effects on leukemic cells, promoting delayed disease regression. Although single-agent AG-120 treatment does not fully eradicate the disease, it increases cycling of rare leukemia stem cells and triggers transcriptional upregulation of the pyrimidine salvage pathway. Accordingly, AG-120 sensitizes IDH1-mutant AML to azacitidine, with the combination of AG-120 and azacitidine showing vastly improved efficacy in vivo. Our data highlight the impact of non-genetic heterogeneity on treatment response and provide a mechanistic rationale for the observed combinatorial effect of AG-120 and azacitidine in patients.

Shared and Distinct Functional Effects of Patient-Specific Tbr1 Mutations on Cortical Development.

T-Box Brain Transcription Factor 1 (TBR1) plays essential roles in brain development, mediating neuronal migration, fate specification, and axon tract formation. While heterozygous loss-of-function and missense TBR1 mutations are associated with neurodevelopmental conditions, the effects of these heterogeneous mutations on brain development have yet to be fully explored. We characterized multiple mouse lines carrying Tbr1 mutations differing by type and exonic location, including the previously generated Tbr1 exon 2-3 knock-out (KO) line, and we analyzed male and female mice at neonatal and adult stages. The frameshift patient mutation A136PfsX80 (A136fs) caused reduced TBR1 protein in cortex similar to Tbr1 KO, while the missense patient mutation K228E caused significant TBR1 upregulation. Analysis of cortical layer formation found similar defects between KO and A136fs homozygotes in their CUX1+ and CTIP2+ layer positions, while K228E homozygosity produced layering defects distinct from these mutants. Meanwhile, the examination of cortical apoptosis found extensive cell death in KO homozygotes but limited cell death in A136fs or K228E homozygotes. Despite their discordant cortical phenotypes, these Tbr1 mutations produced several congruent phenotypes, including anterior commissure reduction in heterozygotes, which was previously observed in humans with TBR1 mutations. These results indicate that patient-specific Tbr1 mutant mice will be valuable translational models for pinpointing shared and distinct etiologies among patients with TBR1-related developmental conditions.SIGNIFICANCE STATEMENT Mutations of the TBR1 gene increase the likelihood of neurodevelopmental conditions such as intellectual disability and autism. Therefore, the study of TBR1 can offer insights into the biological mechanisms underlying these conditions, which affect millions worldwide. To improve the modeling of TBR1-related conditions over current Tbr1 knock-out mice, we created mouse lines carrying Tbr1 mutations identical to those found in human patients. Mice with one mutant Tbr1 copy show reduced amygdalar connections regardless of mutation type, suggesting a core biomarker for TBR1-related disorders. In mice with two mutant Tbr1 copies, brain phenotypes diverge by mutation type, suggesting differences in Tbr1 gene functionality in different patients. These mouse models will serve as valuable tools for understanding genotype-phenotype relationships among patients with neurodevelopmental conditions.

Vaccination With Oral Polio Vaccine Reduces COVID-19 Incidence.

Background: Effective response to emerging pandemic threats is complicated by the need to develop specific vaccines and other medical products. The availability of broadly specific countermeasures that could be deployed early in the pandemic could significantly alter its course and save countless lives. Live attenuated vaccines (LAVs) were shown to induce non-specific protection against a broad spectrum of off-target pathogens by stimulating innate immune responses. The purpose of this study was to evaluate the effect of immunization with bivalent Oral Poliovirus Vaccine (bOPV) on the incidence of COVID-19 and other acute respiratory infections (ARIs). Methods and Findings: A randomized parallel-group comparative study was conducted in Kirov Medical University. 1115 healthy volunteers aged 18 to 65 were randomized into two equal groups, one of which was immunized orally with a single dose of bOPV "BiVac Polio" and another with placebo. The study participants were monitored for three months for respiratory illnesses including COVID-19. The endpoint was the incidence of acute respiratory infections and laboratory confirmed COVID-19 in both groups during 3 months after immunization. The number of laboratory-confirmed cases of COVID-19 was significantly lower in the vaccinated group than in placebo (25 cases vs. 44, p=0.036). The difference between the overall number of clinically diagnosed respiratory illnesses in the two groups was not statistically significant. Conclusions: Immunization with bOPV reduced the number of laboratory-confirmed COVID-19 cases, consistent with the original hypothesis that LAVs induce non-specific protection against off-target infections. The findings are in line with previous observations of the protective effects of OPV against seasonal influenza and other viral and bacterial pathogens. The absence of a statistically significant effect on the total number of ARIs may be due to the insufficient number of participants and heterogeneous etiology of ARIs. OPV could be used to complement specific coronavirus vaccines, especially in regions of the world where the vaccines are unavailable, and as a stopgap measure for urgent response to future emerging infections. Clinical trial registration number NCT05083039 at clinicaltrals.gov https://clinicaltrials.gov/ct2/show/NCT05083039?term=NCT05083039&draw=2&rank=1.

Inhibition of pyrimidine biosynthesis targets protein translation in acute myeloid leukemia.

The mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) catalyzes one of the rate-limiting steps in de novo pyrimidine biosynthesis, a pathway that provides essential metabolic precursors for nucleic acids, glycoproteins, and phospholipids. DHODH inhibitors (DHODHi) are clinically used for autoimmune diseases and are emerging as a novel class of anticancer agents, especially in acute myeloid leukemia (AML) where pyrimidine starvation was recently shown to reverse the characteristic differentiation block in AML cells. Herein, we show that DHODH blockade rapidly shuts down protein translation in leukemic stem cells (LSCs) and has potent and selective activity against multiple AML subtypes. Moreover, we find that ablation of CDK5, a gene that is recurrently deleted in AML and related disorders, increases the sensitivity of AML cells to DHODHi. Our studies provide important molecular insights and identify a potential biomarker for an emerging strategy to target AML.

Integrated clinical and genomic evaluation of guadecitabine (SGI-110) in peripheral T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) is a rare, heterogenous malignancy with dismal outcomes at relapse. Hypomethylating agents (HMA) have an emerging role in PTCL, supported by shared mutations with myelodysplasia (MDS). Response rates to azacitidine in PTCL of follicular helper cell origin are promising. Guadecitabine is a decitabine analogue with efficacy in MDS. In this phase II, single-arm trial, PTCL patients received guadecitabine on days 1-5 of 28-day cycles. Primary end points were overall response rate (ORR) and safety. Translational sub-studies included cell free plasma DNA sequencing and functional genomic screening using an epigenetically-targeted CRISPR/Cas9 library to identify response predictors. Among 20 predominantly relapsed/refractory patients, the ORR was 40% (10% complete responses). Most frequent grade 3-4 adverse events were neutropenia and thrombocytopenia. At 10 months median follow-up, median progression free survival (PFS) and overall survival (OS) were 2.9 and 10.4 months respectively. RHOAG17V mutations associated with improved PFS (median 5.47 vs. 1.35 months; Wilcoxon p = 0.02, Log-Rank p = 0.06). 4/7 patients with TP53 variants responded. Deletion of the histone methyltransferase SETD2 sensitised to HMA but TET2 deletion did not. Guadecitabine conveyed an acceptable ORR and toxicity profile; decitabine analogues may provide a backbone for future combinatorial regimens co-targeting histone methyltransferases.

Epigenetic Activation of Plasmacytoid DCs Drives IFNAR-Dependent Therapeutic Differentiation of AML.

Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies. SIGNIFICANCE: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397.

ANKRD24 organizes TRIOBP to reinforce stereocilia insertion points.

The stereocilia rootlet is a key structure in vertebrate hair cells, anchoring stereocilia firmly into the cell's cuticular plate and protecting them from overstimulation. Using superresolution microscopy, we show that the ankyrin-repeat protein ANKRD24 concentrates at the stereocilia insertion point, forming a ring at the junction between the lower and upper rootlets. Annular ANKRD24 continues into the lower rootlet, where it surrounds and binds TRIOBP-5, which itself bundles rootlet F-actin. TRIOBP-5 is mislocalized in Ankrd24KO/KO hair cells, and ANKRD24 no longer localizes with rootlets in mice lacking TRIOBP-5; exogenous DsRed-TRIOBP-5 restores endogenous ANKRD24 to rootlets in these mice. Ankrd24KO/KO mice show progressive hearing loss and diminished recovery of auditory function after noise damage, as well as increased susceptibility to overstimulation of the hair bundle. We propose that ANKRD24 bridges the apical plasma membrane with the lower rootlet, maintaining a normal distribution of TRIOBP-5. Together with TRIOBP-5, ANKRD24 organizes rootlets to enable hearing with long-term resilience.

Non-genetic determinants of malignant clonal fitness at single-cell resolution.

All cancers emerge after a period of clonal selection and subsequent clonal expansion. Although the evolutionary principles imparted by genetic intratumour heterogeneity are becoming increasingly clear1, little is known about the non-genetic mechanisms that contribute to intratumour heterogeneity and malignant clonal fitness2. Here, using single-cell profiling and lineage tracing (SPLINTR)-an expressed barcoding strategy-we trace isogenic clones in three clinically relevant mouse models of acute myeloid leukaemia. We find that malignant clonal dominance is a cell-intrinsic and heritable property that is facilitated by the repression of antigen presentation and increased expression of the secretory leukocyte peptidase inhibitor gene (Slpi), which we genetically validate as a regulator of acute myeloid leukaemia. Increased transcriptional heterogeneity is a feature that enables clonal fitness in diverse tissues and immune microenvironments and in the context of clonal competition between genetically distinct clones. Similar to haematopoietic stem cells3, leukaemia stem cells (LSCs) display heritable clone-intrinsic properties of high, and low clonal output that contribute to the overall tumour mass. We demonstrate that LSC clonal output dictates sensitivity to chemotherapy and, although high- and low-output clones adapt differently to therapeutic pressure, they coordinately emerge from minimal residual disease with increased expression of the LSC program. Together, these data provide fundamental insights into the non-genetic transcriptional processes that underpin malignant clonal fitness and may inform future therapeutic strategies.