Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure.

Cerebral organoids, three-dimensional cultures that model organogenesis, provide a new platform to investigate human brain development. High cost, variability, and tissue heterogeneity limit their broad applications. Here, we developed a miniaturized spinning bioreactor (SpinΩ) to generate forebrain-specific organoids from human iPSCs. These organoids recapitulate key features of human cortical development, including progenitor zone organization, neurogenesis, gene expression, and, notably, a distinct human-specific outer radial glia cell layer. We also developed protocols for midbrain and hypothalamic organoids. Finally, we employed the forebrain organoid platform to model Zika virus (ZIKV) exposure. Quantitative analyses revealed preferential, productive infection of neural progenitors with either African or Asian ZIKV strains. ZIKV infection leads to increased cell death and reduced proliferation, resulting in decreased neuronal cell-layer volume resembling microcephaly. Together, our brain-region-specific organoids and SpinΩ provide an accessible and versatile platform for modeling human brain development and disease and for compound testing, including potential ZIKV antiviral drugs.

N6-methyladenine DNA modification in Drosophila.

DNA N(6)-methyladenine (6mA) modification is commonly found in microbial genomes and plays important functions in regulating numerous biological processes in bacteria. However, whether 6mA occurs and what its potential roles are in higher-eukaryote cells remain unknown. Here, we show that 6mA is present in Drosophila genome and that the 6mA modification is dynamic and is regulated by the Drosophila Tet homolog, DNA 6mA demethylase (DMAD), during embryogenesis. Importantly, our biochemical assays demonstrate that DMAD directly catalyzes 6mA demethylation in vitro. Further genetic and sequencing analyses reveal that DMAD is essential for development and that DMAD removes 6mA primarily from transposon regions, which correlates with transposon suppression in Drosophila ovary. Collectively, we uncover a DNA modification in Drosophila and describe a potential role of the DMAD-6mA regulatory axis in controlling development in higher eukaryotes.

Lipid-mediated phase separation of AGO proteins on the ER controls nascent-peptide ubiquitination.

AGO/miRNA-mediated gene silencing and ubiquitin-mediated protein quality control represent two fundamental mechanisms that control proper gene expression. Here, we unexpectedly discover that fly and human AGO proteins, which are key components in the miRNA pathway, undergo lipid-mediated phase separation and condense into RNP granules on the endoplasmic reticulum (ER) membrane to control protein production. Phase separation on the ER is mediated by electrostatic interactions between a conserved lipid-binding motif within the AGOs and the lipid PI(4,5)P2. The ER-localized AGO condensates recruit the E3 ubiquitin ligase Ltn1 to catalyze nascent-peptide ubiquitination and coordinate with the VCP-Ufd1-Npl4 complex to process unwanted protein products for proteasomal degradation. Collectively, our study provides insight into the understanding of post-transcription-translation coupling controlled by AGOs via lipid-mediated phase separation.

The cellular and molecular basis of direction selectivity of Aδ-LTMRs.

The perception of touch, including the direction of stimulus movement across the skin, begins with activation of low-threshold mechanosensory neurons (LTMRs) that innervate the skin. Here, we show that murine Aδ-LTMRs are preferentially tuned to deflection of body hairs in the caudal-to-rostral direction. This tuning property is explained by the finding that Aδ-LTMR lanceolate endings around hair follicles are polarized; they are concentrated on the caudal (downward) side of each hair follicle. The neurotrophic factor BDNF is synthesized in epithelial cells on the caudal, but not rostral, side of hair follicles, in close proximity to Aδ-LTMR lanceolate endings, which express TrkB. Moreover, ablation of BDNF in hair follicle epithelial cells disrupts polarization of Aδ-LTMR lanceolate endings and results in randomization of Aδ-LTMR responses to hair deflection. Thus, BDNF-TrkB signaling directs polarization of Aδ-LTMR lanceolate endings, which underlies direction-selective responsiveness of Aδ-LTMRs to hair deflection.

Durvalumab after Chemoradiotherapy in Limited-Stage Small-Cell Lung Cancer.

BACKGROUND: Adjuvant therapy with durvalumab, with or without tremelimumab, may have efficacy in patients with limited-stage small-cell lung cancer who do not have disease progression after standard concurrent platinum-based chemoradiotherapy. METHODS: In a phase 3, double-blind, randomized, placebo-controlled trial, we assigned patients to receive durvalumab at a dose of 1500 mg, durvalumab (1500 mg) plus tremelimumab at a dose of 75 mg (four doses only), or placebo every 4 weeks for up to 24 months. Randomization was stratified according to disease stage (I or II vs. III) and receipt of prophylactic cranial irradiation (yes vs. no). Results of the first planned interim analysis of the two primary end points of overall survival and progression-free survival (assessed on the basis of blinded independent central review according to the Response Evaluation Criteria in Solid Tumors, version 1.1) with durvalumab as compared with placebo (data cutoff date, January 15, 2024) are reported; results in the durvalumab-tremelimumab group remain blinded. RESULTS: A total of 264 patients were assigned to the durvalumab group, 200 to the durvalumab-tremelimumab group, and 266 to the placebo group. Durvalumab therapy led to significantly longer overall survival than placebo (median, 55.9 months [95% confidence interval {CI}, 37.3 to not reached] vs. 33.4 months [95% CI, 25.5 to 39.9]; hazard ratio for death, 0.73; 98.321% CI, 0.54 to 0.98; P = 0.01), as well as to significantly longer progression-free survival (median 16.6 months [95% CI, 10.2 to 28.2] vs. 9.2 months [95% CI, 7.4 to 12.9]; hazard ratio for progression or death, 0.76; 97.195% CI, 0.59 to 0.98; P = 0.02). The incidence of adverse events with a maximum grade of 3 or 4 was 24.4% among patients receiving durvalumab and 24.2% among patients receiving placebo; adverse events led to discontinuation in 16.4% and 10.6% of the patients, respectively, and led to death in 2.7% and 1.9%. Pneumonitis or radiation pneumonitis with a maximum grade of 3 or 4 occurred in 3.1% of the patients in the durvalumab group and in 2.6% of those in the placebo group. CONCLUSIONS: Adjuvant therapy with durvalumab led to significantly longer overall survival and progression-free survival than placebo among patients with limited-stage small-cell lung cancer. (Funded by AstraZeneca; ADRIATIC ClinicalTrials.gov number, NCT03703297.).

Amivantamab plus Lazertinib in Previously Untreated EGFR-Mutated Advanced NSCLC.

BACKGROUND: Amivantamab plus lazertinib (amivantamab-lazertinib) has shown clinically meaningful and durable antitumor activity in patients with previously untreated or osimertinib-pretreated EGFR (epidermal growth factor receptor)-mutated advanced non-small-cell lung cancer (NSCLC). METHODS: In a phase 3, international, randomized trial, we assigned, in a 2:2:1 ratio, patients with previously untreated EGFR-mutated (exon 19 deletion or L858R), locally advanced or metastatic NSCLC to receive amivantamab-lazertinib (in an open-label fashion), osimertinib (in a blinded fashion), or lazertinib (in a blinded fashion, to assess the contribution of treatment components). The primary end point was progression-free survival in the amivantamab-lazertinib group as compared with the osimertinib group, as assessed by blinded independent central review. RESULTS: Overall, 1074 patients underwent randomization (429 to amivantamab-lazertinib, 429 to osimertinib, and 216 to lazertinib). The median progression-free survival was significantly longer in the amivantamab-lazertinib group than in the osimertinib group (23.7 vs. 16.6 months; hazard ratio for disease progression or death, 0.70; 95% confidence interval [CI], 0.58 to 0.85; P<0.001). An objective response was observed in 86% of the patients (95% CI, 83 to 89) in the amivantamab-lazertinib group and in 85% of those (95% CI, 81 to 88) in the osimertinib group; among patients with a confirmed response (336 in the amivantamab-lazertinib group and 314 in the osimertinib group), the median response duration was 25.8 months (95% CI, 20.1 to could not be estimated) and 16.8 months (95% CI, 14.8 to 18.5), respectively. In a planned interim overall survival analysis of amivantamab-lazertinib as compared with osimertinib, the hazard ratio for death was 0.80 (95% CI, 0.61 to 1.05). Predominant adverse events were EGFR-related toxic effects. The incidence of discontinuation of all agents due to treatment-related adverse events was 10% with amivantamab-lazertinib and 3% with osimertinib. CONCLUSIONS: Amivantamab-lazertinib showed superior efficacy to osimertinib as first-line treatment in EGFR-mutated advanced NSCLC. (Funded by Janssen Research and Development; MARIPOSA ClinicalTrials.gov number, NCT04487080.).

Non-Hermitian topological whispering gallery.

In 1878, Lord Rayleigh observed the highly celebrated phenomenon of sound waves that creep around the curved gallery of St Paul's Cathedral in London1,2. These whispering-gallery waves scatter efficiently with little diffraction around an enclosure and have since found applications in ultrasonic fatigue and crack testing, and in the optical sensing of nanoparticles or molecules using silica microscale toroids. Recently, intense research efforts have focused on exploring non-Hermitian systems with cleverly matched gain and loss, facilitating unidirectional invisibility and exotic characteristics of exceptional points3,4. Likewise, the surge in physics using topological insulators comprising non-trivial symmetry-protected phases has laid the groundwork in reshaping highly unconventional avenues for robust and reflection-free guiding and steering of both sound and light5,6. Here we construct a topological gallery insulator using sonic crystals made of thermoplastic rods that are decorated with carbon nanotube films, which act as a sonic gain medium by virtue of electro-thermoacoustic coupling. By engineering specific non-Hermiticity textures to the activated rods, we are able to break the chiral symmetry of the whispering-gallery modes, which enables the out-coupling of topological 'audio lasing' modes with the desired handedness. We foresee that these findings will stimulate progress in non-destructive testing and acoustic sensing.

Augmin complex activity finetunes dendrite morphology through non-centrosomal microtubule nucleation in vivo.

During development, neurons achieve a stereotyped neuron type-specific morphology, which relies on dynamic support by microtubules (MTs). An important player is the augmin complex (hereafter augmin), which binds to existing MT filaments and recruits the γ-tubulin ring complex (γ-TuRC), to form branched MTs. In cultured neurons, augmin is important for neurite formation. However, little is known about the role of augmin during neurite formation in vivo. Here, we have revisited the role of mammalian augmin in culture and then turned towards the class four Drosophila dendritic arborization (c4da) neurons. We show that MT density is maintained through augmin in cooperation with the γ-TuRC in vivo. Mutant c4da neurons show a reduction of newly emerging higher-order dendritic branches and in turn also a reduced number of their characteristic space-filling higher-order branchlets. Taken together, our data reveal a cooperative function for augmin with the γ-TuRC in forming enough MTs needed for the appropriate differentiation of morphologically complex dendrites in vivo.

Osimertinib with or without Chemotherapy in EGFR-Mutated Advanced NSCLC.

BACKGROUND: Osimertinib is a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that is selective for EGFR-TKI-sensitizing and EGFR T790M resistance mutations. Evidence suggests that the addition of chemotherapy may extend the benefits of EGFR-TKI therapy. METHODS: In this phase 3, international, open-label trial, we randomly assigned in a 1:1 ratio patients with EGFR-mutated (exon 19 deletion or L858R mutation) advanced non-small-cell lung cancer (NSCLC) who had not previously received treatment for advanced disease to receive osimertinib (80 mg once daily) with chemotherapy (pemetrexed [500 mg per square meter of body-surface area] plus either cisplatin [75 mg per square meter] or carboplatin [pharmacologically guided dose]) or to receive osimertinib monotherapy (80 mg once daily). The primary end point was investigator-assessed progression-free survival. Response and safety were also assessed. RESULTS: A total of 557 patients underwent randomization. Investigator-assessed progression-free survival was significantly longer in the osimertinib-chemotherapy group than in the osimertinib group (hazard ratio for disease progression or death, 0.62; 95% confidence interval [CI], 0.49 to 0.79; P<0.001). At 24 months, 57% (95% CI, 50 to 63) of the patients in the osimertinib-chemotherapy group and 41% (95% CI, 35 to 47) of those in the osimertinib group were alive and progression-free. Progression-free survival as assessed according to blinded independent central review was consistent with the primary analysis (hazard ratio, 0.62; 95% CI, 0.48 to 0.80). An objective (complete or partial) response was observed in 83% of the patients in the osimertinib-chemotherapy group and in 76% of those in the osimertinib group; the median response duration was 24.0 months (95% CI, 20.9 to 27.8) and 15.3 months (95% CI, 12.7 to 19.4), respectively. The incidence of grade 3 or higher adverse events from any cause was higher with the combination than with monotherapy - a finding driven by known chemotherapy-related adverse events. The safety profile of osimertinib plus pemetrexed and a platinum-based agent was consistent with the established profiles of the individual agents. CONCLUSIONS: First-line treatment with osimertinib-chemotherapy led to significantly longer progression-free survival than osimertinib monotherapy among patients with EGFR-mutated advanced NSCLC. (Funded by AstraZeneca; FLAURA2 ClinicalTrials.gov number, NCT04035486.).

Attractant and repellent induce opposing changes in the four-helix bundle ligand-binding domain of a bacterial chemoreceptor.

Motile bacteria navigate toward favorable conditions and away from unfavorable environments using chemotaxis. Mechanisms of sensing attractants are well understood; however, molecular aspects of how bacteria sense repellents have not been established. Here, we identified malate as a repellent recognized by the MCP2201 chemoreceptor in a bacterium Comamonas testosteroni and showed that it binds to the same site as an attractant citrate. Binding determinants for a repellent and an attractant had only minor differences, and a single amino acid substitution in the binding site inverted the response to malate from a repellent to an attractant. We found that malate and citrate affect the oligomerization state of the ligand-binding domain in opposing way. We also observed opposing effects of repellent and attractant binding on the orientation of an alpha helix connecting the sensory domain to the transmembrane helix. We propose a model to illustrate how positive and negative signals might be generated.

Active N6-Methyladenine Demethylation by DMAD Regulates Gene Expression by Coordinating with Polycomb Protein in Neurons.

A ten-eleven translocation (TET) ortholog exists as a DNA N6-methyladenine (6mA) demethylase (DMAD) in Drosophila. However, the molecular roles of 6mA and DMAD remain unexplored. Through genome-wide 6mA and transcriptome profiling in Drosophila brains and neuronal cells, we found that 6mA may epigenetically regulate a group of genes involved in neurodevelopment and neuronal functions. Mechanistically, DMAD interacts with the Trithorax-related complex protein Wds to maintain active transcription by dynamically demethylating intragenic 6mA. Accumulation of 6mA by depleting DMAD coordinates with Polycomb proteins and contributes to transcriptional repression of these genes. Our findings suggest that active 6mA demethylation by DMAD plays essential roles in fly CNS by orchestrating through added epigenetic mechanisms.

Histone dynamics responding to internal and external cues underlying plant development.

Plants necessitate a refined coordination of growth and development to effectively respond to external triggers for survival and successful reproduction. This intricate harmonization of plant developmental processes and adaptability hinges on significant alterations within their epigenetic landscapes. In this review, we first delve into recent strides made in comprehending underpinning the dynamics of histones, driven by both internal and external cues. We encapsulate the prevailing working models through which cis/trans elements navigate the acquisition and removal of histone modifications, as well as the substitution of histone variants. As we look ahead, we anticipate that delving deeper into the dynamics of epigenetic regulation at the level of individual cells or specific cell types will significantly enrich our comprehension of how plant development unfolds under the influence of internal and external cues. Such exploration holds the potential to provide unprecedented resolution in understanding the orchestration of plant growth and development.

Skin keratinocyte-derived SIRT1 and BDNF modulate mechanical allodynia in mouse models of diabetic neuropathy.

Diabetic neuropathy is a debilitating disorder characterized by spontaneous and mechanical allodynia. The role of skin mechanoreceptors in the development of mechanical allodynia is unclear. We discovered that mice with diabetic neuropathy had decreased sirtuin 1 (SIRT1) deacetylase activity in foot skin, leading to reduced expression of brain-derived neurotrophic factor (BDNF) and subsequent loss of innervation in Meissner corpuscles, a mechanoreceptor expressing the BDNF receptor TrkB. When SIRT1 was depleted from skin, the mechanical allodynia worsened in diabetic neuropathy mice, likely due to retrograde degeneration of the Meissner-corpuscle innervating Aß axons and aberrant formation of Meissner corpuscles which may have increased the mechanosensitivity. The same phenomenon was also noted in skin-keratinocyte specific BDNF knockout mice. Furthermore, overexpression of SIRT1 in skin induced Meissner corpuscle reinnervation and regeneration, resulting in significant improvement of diabetic mechanical allodynia. Overall, the findings suggested that skin-derived SIRT1 and BDNF function in the same pathway in skin sensory apparatus regeneration and highlighted the potential of developing topical SIRT1-activating compounds as a novel treatment for diabetic mechanical allodynia.

Early Proteomic Characteristics and Changes in the Optic Nerve Head, Optic Nerve, and Retina in a Rat Model of Ocular Hypertension.

The pathogenesis of glaucoma is still unknown. There are few studies on the dynamic change of tissue-specific and time-specific molecular pathophysiology caused by ocular hypertension (OHT). This study aimed to identify the early proteomic alterations in the retina, optic nerve head (ONH), and optic nerve (ON). After establishing a rat model of OHT, we harvested the tissues from control and glaucomatous eyes and analyzed the changes in protein expression using a multiplexed quantitative proteomics approach (TMT-MS3). Our study identified 6403 proteins after 1-day OHT and 4399 proteins after 7-days OHT in the retina, 5493 proteins after 1-day OHT and 4544 proteins after 7-days OHT in ONH, and 5455 proteins after 1-day OHT and 3835 proteins after 7-days OHT in the ON. Of these, 560 and 489 differential proteins were identified on day 1 and 7 after OHT in the retina, 428 and 761 differential proteins were identified on day 1 and 7 after OHT in the ONH, and 257 and 205 differential proteins on days 1 and 7 after OHT in the ON. Computational analysis on day 1 and 7 of OHT revealed that alpha-2 macroglobulin was upregulated across two time points and three tissues stably. The differentially expressed proteins between day 1 and 7 after OHT in the retina, ONH, and ON were associated with glutathione metabolism, mitochondrial dysfunction/oxidative phosphorylation, oxidative stress, microtubule, and crystallin. And the most significant change in retina are crystallins. We validated this proteomic result with the Western blot of crystallin proteins and found that upregulated on day 1 but recovered on day 7 after OHT, which are promising as therapeutic targets. These findings provide insights into the time- and region-order mechanisms that are specifically affected in the retina, ONH, and ON in response to elevated IOP during the early stages.

CBL family E3 ubiquitin ligases control JAK2 ubiquitination and stability in hematopoietic stem cells and myeloid malignancies.

Janus kinase 2 (JAK2) is a central kinase in hematopoietic stem/progenitor cells (HSPCs), and its uncontrolled activation is a prominent oncogenic driver of hematopoietic neoplasms. However, molecular mechanisms underlying the regulation of JAK2 have remained elusive. Here we report that the Casitas B-cell lymphoma (CBL) family E3 ubiquitin ligases down-regulate JAK2 stability and signaling via the adaptor protein LNK/SH2B3. We demonstrated that depletion of CBL/CBL-B or LNK abrogated JAK2 ubiquitination, extended JAK2 half-life, and enhanced JAK2 signaling and cell growth in human cell lines as well as primary murine HSPCs. Built on these findings, we showed that JAK inhibitor (JAKi) significantly reduced aberrant HSPCs and mitigated leukemia development in a mouse model of aggressive myeloid leukemia driven by loss of Cbl and Cbl-b Importantly, primary human CBL mutated (CBLmut ) leukemias exhibited increased JAK2 protein levels and signaling and were hypersensitive to JAKi. Loss-of-function mutations in CBL E3 ubiquitin ligases are found in a wide range of myeloid malignancies, which are diseases without effective treatment options. Hence, our studies reveal a novel signaling axis that regulates JAK2 in normal and malignant HSPCs and suggest new therapeutic strategies for treating CBLmut myeloid malignancies.

Informing the Need for a SARS-CoV-2 Booster Based on the Immune Responses Among Young Healthy Adults to Variants Circulating in Late 2023.

BACKGROUND: COVID-19 remains a global public health challenge due to new immune-evasive SARS-CoV-2 variants and heterogeneous immunity. METHODS: In this cross-sectional study, we evaluated the adaptive immune responses in US active duty personnel who completed a COVID-19 primary vaccine series and had heterogenous SARS-CoV-2 vaccination and infection histories to 3 previously dominant variants (ancestral, Delta, BA.5) and 3 circulating variants (XBB.1.5, EG.5, and BA.2.86) in late 2023. Analyses were based on the most recent exposure in terms of timing (within or beyond 12 months) and type (vaccine or infection). RESULTS: Significant reduction was observed in binding antibodies, neutralization antibodies, memory B cells, and CD8+ T cells against circulating variants when compared with previous variants. The reduction in antibody response was more pronounced in those whose most recent exposure was >12 months from enrollment. In contrast, the CD4+ T-cell response was largely consistent across all tested variants. The type of most recent exposure was not a significant factor in determining the magnitude of current immune responses. CONCLUSIONS: Administration of the XBB.1.5-based booster is likely to enhance cross-reactive humoral responses against SARS-CoV-2 circulating lineages. Ongoing surveillance of immune responses to emerging variants is needed for informing vaccine composition and timing.

Dengue NS1 antibodies are associated with clearance of viral NS1.

BACKGROUND: Dengue vascular permeability syndrome is the primary cause of death in severe dengue infections. The protective versus potentially pathogenic role of dengue NS1 antibodies are not well understood. The main goal of this analysis was to characterize the relationship between free NS1 concentration and NS1 antibody titers in primary and secondary dengue infection in order to better understand the presence and duration of NS1 antibody complexes in clinical dengue infections. METHODS: Hospitalized participants with acute dengue infection were recruited from Northern Colombia between 2018 to 2020. Symptom assessment including dengue signs and symptoms, chart review and blood collection was performed. Primary versus secondary Dengue was assessed serologically. NS1 titers and anti-NS1 antibodies were measured daily. RESULTS: Patients with secondary infection have higher antibody titers than those in primary infection, and we find a negative correlation between anti-NS1 antibody titer and NS1 protein. We demonstrate that in a subset of secondary infection, there are indeed NS1 antibody-antigen complexes at the admission day during the febrile phase that are not detectable by the recovery phase. Furthermore, dengue infection status is associated with higher circulating sialidases. DISCUSSION: The negative correlation between antibody and protein suggests that antibodies may play a role in clearing this viral protein.

Baicalin alleviates angiotensin II-induced cardiomyocyte apoptosis and autophagy and modulates the AMPK/mTOR pathway.

As a main extraction compound from Scutellaria baicalensis Georgi, Baicalin exhibits various biological activities. However, the underlying mechanism of Baicalin on hypertension-induced heart injury remains unclear. In vivo, mice were infused with angiotensin II (Ang II; 500 ng/kg/min) or saline using osmotic pumps, followed by intragastrically administrated with Baicalin (5 mg/kg/day) for 4 weeks. In vitro, H9C2 cells were stimulated with Ang II (1 µM) and treated with Baicalin (12.5, 25 and 50 µM). Baicalin treatment significantly attenuated the decrease in left ventricular ejection fraction and left ventricular fractional shortening, increase in left ventricular mass, left ventricular systolic volume and left ventricular diastolic volume of Ang II infused mice. Moreover, Baicalin treatment reversed 314 differentially expressed transcripts in the cardiac tissues of Ang II infused mice, and enriched multiple enriched signalling pathways (including apoptosis, autophagy, AMPK/mTOR signalling pathway). Consistently, Baicalin treatment significantly alleviated Ang II-induced cell apoptosis in vivo and in vitro. Baicalin treatment reversed the up-regulation of Bax, cleaved-caspase 3, cleaved-caspase 9, and the down-regulation of Bcl-2. Meanwhile, Baicalin treatment alleviated Ang II-induced increase of autophagosomes, restored autophagic flux, and down-regulated LC3II, Beclin 1, as well as up-regulated SQSTM1/p62 expression. Furthermore, autophagy inhibitor 3-methyladenine treatment alleviated the increase of autophagosomes and the up-regulation of Beclin 1, LC3II, Bax, cleaved-caspase 3, cleaved-caspase 9, down-regulation of SQSTM1/p62 and Bcl-2 expression after Ang II treated, which similar to co-treatment with Baicalin. Baicalin treatment reduced the ratio of p-AMPK/AMPK, while increased the ratio of p-mTOR/mTOR. Baicalin alleviated Ang II-induced cardiomyocyte apoptosis and autophagy, which might be related to the inhibition of the AMPK/mTOR pathway.

Deubiquitinase USP9X stabilizes RNA m6A demethylase ALKBH5 and promotes acute myeloid leukemia cell survival.

Post-translational modifications including protein ubiquitination regulate a plethora of cellular processes in distinct manners. RNA N6-methyladenosine is the most abundant post-transcriptional modification on mammalian mRNAs and plays important roles in various physiological and pathological conditions including hematologic malignancies. We previously determined that the RNA N6-methyladenosine eraser ALKBH5 is necessary for the maintenance of acute myeloid leukemia (AML) stem cell function, but the post-translational modifications involved in ALKBH5 regulation remain elusive. Here, we show that deubiquitinase ubiquitin-specific peptidase 9X (USP9X) stabilizes ALKBH5 and promotes AML cell survival. Through the use of mass spectrometry as an unbiased approach, we identify USP9X and confirm that it directly binds to ALKBH5. USP9X stabilizes ALKBH5 by removing the K48-linked polyubiquitin chain at K57. Using human myeloid leukemia cells and a murine AML model, we find that genetic knockdown or pharmaceutical inhibition of USP9X inhibits leukemia cell proliferation, induces apoptosis, and delays AML development. Ectopic expression of ALKBH5 partially mediates the function of USP9X in AML. Overall, this study uncovers deubiquitinase USP9X as a key for stabilizing ALKBH5 expression and reveals the important role of USP9X in AML, which provides a promising therapeutic strategy for AML treatment in the clinic.