Small molecules that induce protein interactions hold tremendous potential as new medicines, probes for molecular pathways and tools for agriculture. Explosive growth of targeted protein degradation drug development has spurred renewed interest in proximity-inducing molecules, especially molecular glue degraders (MGDs). These compounds catalyze the destruction of disease-causing proteins by reshaping protein surfaces and promoting cooperative binding between ubiquitylating enzymes and target proteins. MGD discovery for predefined targets is a major challenge in contemporary drug discovery. Here, we solve this important chemical challenge through 'chemocentric' MGD discovery of ZZ1, a BET-family protein degrader and a prodrug of a negatively charged glue. ZZ1 activation unmasks a sulfinic acid that binds the modular CTLH ubiquitin ligase complex through a basic pocket in its YPEL5 subunit. These findings demonstrate a previously unrecognized capacity of YPEL5 to recruit CTLH substrates and enable the discovery of MGDs for exceedingly common acidic and basic degrons.

Abstract Progression from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) is accompanied by profound remodeling of the bone marrow microenvironment (BME), yet the contribution of its non-immune compartment remains unclear. Using single-cell RNA sequencing in genetically engineered mouse models that recapitulate disease evolution, we transcriptionally profile endothelial cells (EC) and mesenchymal stem cells (MSC). EC adopt a stress-associated program at MGUS that precedes angiogenesis in MM, while MSC undergo early and sustained loss of differentiation capacity. We identify a coordinated interferon (IFN)-driven program across EC and MSC that defines MM in the BIcγ1 model but is absent in the more aggressive MI cγ1 model. Treatment with bortezomib, lenalidomide, and dexamethasone suppresses this IFN signature, promotes endothelial adaptation, and restores osteogenic potential in MSC. Validation in patient samples reveals enrichment of this IFN-signature across disease stages. These findings define dynamic and targetable alterations in the non-immune BME during myeloma progression.

Exposure to particles is a driver of several inflammatory diseases. Here, we investigated macrophage responses to monosodium urate crystals, calcium pyrophosphate crystals, aluminum salts, and silica nanoparticles. While each particle induced a distinct gene expression pattern, we identified a common inflammatory signature and acute activation of lysosomal acidification genes. Using monosodium urate crystals as a model, we demonstrated that this lysosomal gene program is regulated by a 5'-prime-AMP-activated protein kinase (AMPK)-dependent transcriptional network, including TFEB, TFE3, and the epigenetic regulators DNA methyl transferase 3a (DNMT3A) and DOT1L. This lysosomal acidification program operates in parallel with, but largely independently of, a JNK-AP-1-dependent network driving crystal-induced chemokine and cytokine expression. These findings reveal a bifurcation in pathways governing inflammatory and lysosomal responses, offering insights for treating particle-associated diseases.

Missense mutants of p53, such as the frequent hotspot variant R248Q, exert a dominant-negative effect (DNE) on wild-type (WT) p53 in cancer cells with monoallelic TP53 mutations. However, the precise functional and molecular mechanisms of the DNE have remained elusive due to a lack of appropriate model systems. In this study, we developed a variety of model systems, including CRISPR-edited human isogenic cell lines and transcriptional reporter cell lines, and targeted protein degradation assays that were combined with functional and molecular analyses to functionally characterize the DNE. Formation of heterotetramers between R248Q and WT p53 impaired proper WT p53 functionality by preventing DNA binding and subsequent target gene transactivation. Furthermore, the markedly increased protein half-life of R248Q led to supraphysiologic levels of R248Q, which was critically required for the DNE. Drug-induced targeted protein degradation of R248Q to lower the R248Q:WT ratio restored the transcriptional activity of WT p53, induced antiproliferative effects in cancer cells in vitro, and elicited strong therapeutic activity in vivo. Together, this study provides mechanistic insights into the DNE of p53 missense mutants and indicates that the DNE represents a promising therapeutic target.Significance: Heterotetramerization between R248Q mutant and wild-type p53 in conjunction with supraphysiologic p53R248Q accumulation underlies the dominant-negative effect, highlighting the need to develop pharmacologic strategies to decrease the elevated R248Q:WT ratio.See related commentary by Gencel-Augusto and Lozano, p. 1955

6517 Background: Clonal hematopoiesis (CH), an age-related condition involving somatic mutations in blood stem cells, increases the risk of myelodysplastic syndrome (MDS), blood cancers and cardiovascular disease through inflammatory pathways. Age-related macular degeneration (AMD), the leading cause of blindness in the developed world, is also characterized by chronic inflammation. An increased prevalence of AMD has been observed in older adults with MDS, but the association between CH and AMD remains unexplored. Understanding this relationship could reveal shared inflammatory mechanisms in age-related diseases and guide prevention strategies. Methods: This retrospective cohort study used exome sequencing and electronic medical records (EMRs) from 467,200 adults ≥40 years of age in the UK Biobank (UKB), recruited between 2006–2010 and followed until 2020. Participants with prevalent blood cancer, AMD, or with missing AMD diagnosis dates were excluded. CH was defined as pathogenic somatic mutations with a variant allele fraction (VAF) ≥0.02. Incident AMD was identified using ICD-10 codes (H35.3). Kaplan-Meier estimates and log-rank tests assessed cumulative incidence, while Cox regression models calculated hazard ratios (HRs), adjusted for age, sex, smoking and hypertension. A separate cohort of 4,079 patients from Dana-Farber Cancer Institute (DFCI) validated findings and enabled granular clinical data abstraction from EMRs. Results: CH was detected in 29,550 (6.8%) individuals of the UKB. The 12-year cumulative incidence (C.I.) of AMD was higher in individuals with CH (n=671, C.I. 2.45%) compared to those without (n=6,728, 1.61%; p<2x10-16). In unadjusted Cox models, individuals with CH had a 51% higher risk of AMD compared to those without (HR =1.51 (95% CI: 1.39–1.63; p < 2×10⁻¹⁶), remaining significant after adjusting for covariates (p=0.023). CH genotypes most associated with AMD risk included ASXL1 (HR: 1.32; p = 0.0146) and splicing factors (HR: 1.54; p = 0.0345). Individuals with CH and AMD had a 33% higher risk of progressing to blindness compared to those without CH, though this was not statistically significant (p = 0.242). In the DFCI cohort (n= 4,079), CH was present in 1,028 (25.2%) individuals. 86 (8.37%) individuals with CH had AMD diagnoses compared to those without CH (n= 86, 3.21%; p = 2.53×10⁻¹⁰), with exudative AMD, a more severe subtype, being more prevalent in CH patients (n=11; p = 1.1×10⁻⁵). Conclusions: There is a significant association between CH and AMD, suggesting that AMD prevalent in individuals with MDS is related to presence of CH in the pre-MDS state. Real world data support these findings, highlighting a trend towards severe AMD subtypes in individuals with CH. The identification of specific genes linked to AMD incidence suggests that certain CH genotypes may confer a higher risk for AMD, highlighting the role of AMD screening in individuals with myeloid malignancy precursor conditions.

TET2 is among the most commonly mutated genes in both clonal hematopoiesis and myeloid malignancies; thus, the ability to identify selective dependencies in TET2-deficient cells has broad translational significance. Here, we identify regulators of Tet2 knockout (KO) hematopoietic stem and progenitor cell (HSPC) expansion using an in vivo CRISPR-Cas9 KO screen, in which nucleotide barcoding enabled large-scale clonal tracing of Tet2-deficient HSPCs in a physiologic setting. Our screen identified candidate genes, including Ncoa4, that are selectively required for Tet2 KO clonal outgrowth compared with wild type. Ncoa4 targets ferritin for lysosomal degradation (ferritinophagy), maintaining intracellular iron homeostasis by releasing labile iron in response to cellular demands. In Tet2-deficient HSPCs, increased mitochondrial adenosine triphosphate production correlates with increased cellular iron requirements and, in turn, promotes Ncoa4dependent ferritinophagy. Restricting iron availability reduces Tet2 KO stem cell numbers, revealing a dependency in TET2-mutated myeloid neoplasms.

Importance:Clonal hematopoiesis of indeterminate potential (CHIP) is the age-related clonal expansion of hematopoietic stem cells with leukemia-associated mutations. Certain CHIP mutations promote atherosclerosis and heart failure through immune-related pathways. Objective:To test whether CHIP is associated with the development of myocarditis and pericarditis. Design, Setting, and Participants:This observational population-based cohort study used data from the UK Biobank. Enrollment occurred between 2006 and 2010. Participants with whole-exome sequencing, no prevalent cardiovascular disease or hematological malignancy, and complete covariate data were included. Follow-up occurred for a median of 13.6 (IQR, 12.8-14.2) years. Analyses were conducted from November 2024 to July 2025. Exposures:Any CHIP (variant allele frequency [VAF] ≥2%) and large CHIP (VAF ≥10%) constituted coprimary study exposures. Secondary analyses considered DNMT3A and TET2 CHIP as separate exposures. Main outcomes and measures:The primary outcome was a composite of incident myocarditis and pericarditis. Cox regression tested associations of CHIP with myocarditis and pericarditis, adjusting for age, sex, race and ancestry, and cardiovascular risk factors. Secondary analyses considered myocarditis and pericarditis as separate outcomes. Additional analyses compared associations of CHIP with myocarditis and pericarditis with those with other cardiovascular diseases, and tested the bidirectional associations between CHIP and noncardiac immune-mediated inflammatory diseases. Results:Among 335 426 participants (mean age, 56.1 years; 185 429 female [55.3%] and 149 997 male [44.7%]), 11 057 had any CHIP (3.3%), 7271 had large CHIP (2.2%), and 382 developed myocarditis or pericarditis (0.11%). Any and large CHIP were associated with multivariable-adjusted hazard ratios of 1.75 (95% CI, 1.14-2.68; P = .01) and 2.07 (95% CI, 1.28-3.33; P = .003), respectively, for the primary composite outcome of incident myocarditis and pericarditis. Increased risks were observed for DNMT3A and TET2 CHIP, with hazard ratios of 2.22 (95% CI, 1.17-4.21; P = .01) for DNMT3A with pericarditis and 3.65 (95% CI, 1.16-11.49; P = .03) for TET2 with myocarditis. CHIP associated with myocarditis and pericarditis more strongly than with other cardiovascular diseases (eg, coronary artery disease and heart failure). Any CHIP was also associated with 1.27-fold risk (95% CI, 1.16-1.39; P < .001) of developing noncardiac immune-mediated inflammatory diseases, without evidence for reverse causation. Conclusions and Relevance:In this study, CHIP was a strong risk factor for myocarditis and pericarditis among middle-aged adults. Targeting CHIP and its downstream pathways may represent a strategy for preventing or treating pericarditis and myocarditis.

ABSTRACT:Myelodysplastic syndrome (MDS) is driven by genetic mutations, but diagnosis relies on morphologic evaluation of bone marrow hematopoiesis. Only a small number of genetic abnormalities define specific bone marrow morphologic features in MDS, such as SF3B1 mutations and deletions of chromosome 5q. We hypothesized that additional genetic alterations are associated with specific dysplastic morphologic features in MDS. We assessed genetic-morphologic associations between commonly mutated genes and 10 morphologic features in a cohort of MDS bone marrows with a high degree of dysplasia. We replicated the association of SF3B1 mutations with ring sideroblasts and found that dysplastic megakaryocytes with separated nuclei were independently associated with STAG2 and/or ASXL1 mutations. In addition, STAG2 mutations were associated with abnormal myeloid nuclear segmentation and myeloid cell hypogranulation. These findings demonstrate that STAG2 and ASXL1 mutations are associated with specific morphologic abnormalities in MDS.

Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life. For example, such control can help avert any potential immunological and adverse events in clinical trials. Current genome editing technologies to control the half-life of Cas9 are slow, have lower activity, involve fusion of large response elements (> 230 amino acids), utilize expensive controllers with poor pharmacological attributes, and cannot be implemented in vivo on several CRISPR-based technologies. We report a general platform for half-life control using the molecular glue, pomalidomide, that binds to a ubiquitin ligase complex and a response-element bearing CRISPR-based technology, thereby causing the latter's rapid ubiquitination and degradation. Using pomalidomide, we were able to control the half-life of large CRISPR-based technologies (e.g., base editors, CRISPRi) and small anti-CRISPRs that inhibit such technologies, allowing us to build the first examples of on-switch for base editors. The ability to switch on, fine-tune and switch-off CRISPR-based technologies with pomalidomide allowed complete control over their activity, specificity, and genome editing outcome. Importantly, the miniature size of the response element and favorable pharmacological attributes of the drug pomalidomide allowed control of activity of base editor in vivo using AAV as the delivery vehicle. These studies provide methods and reagents to precisely control the dosage and half-life of CRISPR-based technologies, propelling their therapeutic development.

Organelles such as lysosomes and synaptic vesicles are acidified by V-ATPases, which consist of a cytosolically oriented V1 complex that hydrolyzes ATP and a membrane-embedded VO complex that pumps protons. In yeast, V1-VO association is facilitated by the RAVE (regulator of H+-ATPase of the vacuolar and endosomal membrane) complex, but how higher eukaryotes assemble V-ATPases remains unclear. Here we identify a metazoan RAVE complex (mRAVE) whose structure and composition are notably divergent from the ancestral counterpart. mRAVE consists of DMXL1 or DMXL2, WDR7 and the central linker ROGDI. DMXL1 and DMXL2 interact with subunits A and D of the inactive, isolated V1. On dissipation of proton gradients, mRAVE binds to V1 and VO, forming a supercomplex on the membrane. mRAVE then catalyzes V1-VO assembly, enabling lysosomal acidification, neurotransmitter loading into vesicles and ATG16L1 recruitment for LC3/ATG8 conjugation onto single membranes. Our findings provide a molecular basis for neurological disorders caused by mRAVE mutations.

Glutarimide analogs, such as thalidomide, redirect the E3 ubiquitin ligase CRL4CRBN to induce degradation of certain zinc finger (ZF) proteins. Although the core structural motif recognized by CRBN has been characterized, it does not fully explain substrate specificity. To explore the role of residues adjacent to this core motif, we constructed a comprehensive ZF reporter library of 9,097 reporters derived from 1,655 human ZF proteins and conducted a library-on-library screen with 29 glutarimide analogs to identify compounds that collectively degrade 38 ZF reporters. Cryo-electron microscopy and crystal structures of ZFs in complex with CRBN revealed the importance of interactions beyond the core ZF degron. We used systematic mutagenesis of ZFs and CRBN to identify modes of neosubstrate recruitment requiring distinct amino acids. Finally, we found subtle chemical variations in glutarimide analogs that alter target scope and selectivity, thus providing a roadmap for their rational design.

Background: Clonal hematopoiesis of indeterminate potential (CHIP), the age-related clonal expansion of hematopoietic stem cells with preleukemic driver mutations, is a novel risk factor for cardiovascular diseases (CVD). While associations between CHIP and plasma proteins are emerging, the causal nature of these relationships remains undetermined. Hypothesis: We aimed to identify specific CHIP driver mutations that causally alter plasma protein levels using human genetics and validate key findings in experimental models. Approach: We analyzed 61,833 participants from the NHLBI TOPMed Program (SomaScan for proteomics measurement) and UK Biobank (Olink for proteomics measurement) with paired DNA sequencing and proteomics. Associations between CHIP variables (composite or drivers DNMT3A , TET2 , ASXL1 ) and proteins were analyzed separately by platform. Bi-directional Mendelian Randomization (MR) was performed to assess causal relations between CHIP, DNMT3A , or TET2 and protein levels. Proteins implicated by MR for TET2 (LCN2, MPO, FLT3LG) were selected for validation via ELISA in 8-9 week-old hematopoietic Tet2 -/- vs wild-type (WT) mice. Results: MR analyses identified multiple instances where CHIP likely causes proteomic changes, with less evidence for reverse causality. In TOPMed (SomaScan), we identified 9 causal pairs (FDR<0.05) among 35 (24 examined) significant CHIP-protein pairs, with TET2 causally increasing MPO levels being the strongest (beta [SE]:0.022[0.008]; P=3.6x10 -3 ). In UK Biobank (Olink), among 473 (318 examined) significant CHIP-protein pairs, 121 were causal, with TET2 showing the strongest effects on increased LCN2 (beta[SE]:0.056[0.008]; P=4.3x10 -11 ) and decreased FLT3LG (beta[SE]:-0.089[0.015]; P=1.1x10 -9 ). The causal effect of TET2 on LCN2 was consistent across platforms. Murine experiments corroborated these findings: hematopoietic Tet2 -/- mice exhibited significantly increased plasma MPO and LCN2 levels compared to WT controls. Plasma FLT3LG levels were not significantly different, though a decrease in Tet2 -/- mice was directionally consistent with human MR. Conclusions: This study provides robust human genetic and experimental evidence for causal effects of CHIP, particularly TET2 mutations, on the plasma proteome. This validation of MR-identified proteomic changes strengthens the causal link and offers potential mechanistic insights into how CHIP may influence downstream outcomes, including CVD.

Blood cancers are generally more common in males, and the prevalence of most mutations that drive clonal hematopoiesis and myeloid malignancies is higher in males. In contrast, hematopoietic DNMT3A mutations are more common in females. Among ∼450,000 participants in the UK Biobank, the prevalence of DNMT3A mutations and copy-number abnormalities is higher in females than males. In a murine model, Dnmt3a-mutant hematopoietic stem cells (HSCs) from unperturbed female mice had increased stemness gene expression compared to male and wild-type (WT) mice. Estrogen regulates HSCs, and we found that Dnmt3a mutations maintain stemness in the setting of estrogen-induced proliferative stress. Dnmt3a-mutant myeloid cells outcompeted WT cells under chronic estrogen treatment, an effect that was dependent on cell-intrinsic estrogen receptor alpha activity. Our studies indicate that estrogen might contribute to the female predominance of DNMT3A-mutant clonal hematopoiesis.

Targeted protein degradation has emerged as a promising approach in drug discovery, utilizing small molecules like molecular glue degraders to harness the ubiquitin-proteasome pathway for selective degradation of disease-driving proteins. Based on results from proteomics screens we investigated the potential of niclosamide, an FDA-approved anthelmintic drug with a 50 year history in treating tapeworm infections, as a molecular glue degrader targeting the proto-oncogene cyclin D1. Proteomics screens in HCT116 colon carcinoma and KELLY neuroblastoma cells, found that niclosamide induces rapid cyclin D1 degradation through a mechanism involving the ubiquitin-proteasome pathway. A genetic CRISPR screen identified the E3 ligase CRL4AMBRA1 as a key player in this process. Structure-activity relationship studies highlighted critical features of niclosamide necessary for cyclin D1 degradation, demonstrating a correlation between mitochondrial membrane potential (MMP) disruption and cyclin D1 downregulation. Notably, various mitochondrial uncouplers and other compounds with similar drug sensitivity profiles share this correlation suggesting that MMP disruption can trigger cyclin D1 degradation, and that the cellular signal driving the degradation differs from previously described mechanism involving CRL4AMBRA1. Our findings underscore the complexities of proteostatic mechanisms and the multitude of mechanisms that contribute to degrader drug action.

Plasma proteomic profiles associated with subclinical somatic mutations in blood cells may offer insights into downstream clinical consequences. Here we explore these patterns in clonal hematopoiesis of indeterminate potential (CHIP), which is linked to several cancer and non-cancer outcomes, including coronary artery disease (CAD). Among 61,833 participants (3881 with CHIP) from TOPMed and UK Biobank (UKB) with blood-based DNA sequencing and proteomic measurements (1,148 proteins by SomaScan in TOPMed and 2917 proteins by Olink in UKB), we identify 32 and 345 proteins from TOPMed and UKB, respectively, associated with CHIP and most prevalent driver genes (DNMT3A, TET2, and ASXL1). These associations show substantial heterogeneity by driver genes, sex, and race, and were enriched for immune response and inflammation pathways. Mendelian randomization in humans, coupled with ELISA in hematopoietic Tet2-/- vs wild-type mice validation, disentangle causal proteomic perturbations from TET2 CHIP. Lastly, we identify plasma proteins shared between CHIP and CAD.

Superficial plaque erosion causes many acute coronary syndromes. However, mechanisms of plaque erosion remain poorly understood, and we lack directed therapeutics for thrombotic complication. Human eroded plaques can harbor neutrophil extracellular traps (NETs) that propagate endothelial damage at experimental arterial lesions that recapitulate superficial erosion. Clonal Hematopoiesis of Indeterminate Potential (CHIP) denotes age-related clonal expansion of bone marrow-derived cells harboring somatic mutations in the absence of overt hematological disease. CHIP heightens the risk of cardiovascular disease, with the greatest increase seen in individuals with JAK2

Our understanding of the progression from clonal hematopoiesis (CH) to aggressive leukemias such as acute myelogenous leukemia enables a potential opportunity for intervention. Many mutations in CH are in genes encoding epigenetic regulators (e.g. DNMT3A, TET2 and ASXL1) presenting an opportunity for epigenetic targeted therapies to be used to prevent leukemia. However, the lack of representative human models of CH has hampered the development of effective therapeutic strategies. We have developed an in vitro competition model of TET2 loss-of-function CH that allows for chemical and genetic screening to identify key vulnerabilities.We have optimized culturing conditions to expand total CD34+ hematopoietic stem and progenitor cells (HSPCs) for up to 8-weeks. Using these culturing conditions, we knocked-out TET2 in CD34+ cells from cord blood and adult bone marrow using CRISPR-Cas9 and observed an increase in proliferation, immature phenotype (e.g. CD34+) and colony formation compared to controls. By employing distinct fluorescent markers for TET2-null (mNeonGreen) and AAVS1 control (mScarlet), we observed the competitive advantage of TET2-null cells over time by flow cytometry. We performed a chemical screen of 31 epigenetic targeting compounds using this competitive culture. This screen identified expected hits such as the known hypomethylating agents azacitidine and decitabine, as well as unique candidates that preferentially target TET2-null HSPCs. We validated two of the candidate molecules in differentiation, colony formation and in vivo murine competitive transplant assays. Furthermore, we performed a targeted CRISPR screen and identified the known target one of our candidate compounds as a top hit. This data demonstrates the feasibility of this in vitro model for scalable chemical and genetic screening to identify vulnerabilities in CH.