Tumorigenesis and metastasis are frequently attributed to the intricate interplay between cancer cells and the tumor microenvironment (TME). Comprehending the mechanisms and key regulators of cancer-immune crosstalk in the TME is imperative for developing efficacious immunotherapy. Through a series of in vivo CRISPR screens, we identified tumor-intrinsic ITGB1 as a critical regulator of triple-negative breast cancer (TNBC) development and deciphered its underlying mechanisms. Tumoral ITGB1 facilitated the establishment of pro-tumorigenic TME by orchestrating tumor-associated myeloid populations. Suppressing ITGB1 favored the enrichment of anti-tumorigenic myeloid cells and enhanced infiltration of CD4 and CD8 T cells, culminating in superior antitumor effects. CRISPR scanning pinpointed a previously unrecognized functional domain essential for ITGB1's pro-tumorigenic activity. This domain is distinct from all known ligand-binding sites in ITGB1. An antibody capable of sterically blocking this domain significantly impaired TNBC progression. These findings position tumoral ITGB1 as a promising therapeutic target for reprogramming the TME from a pro- to an anti-tumorigenic state, thereby effectively inhibiting TNBC development. Our study uncovers a novel mechanism of TNBC development and provides a unique therapeutic strategy for targeting ITGB1 in TNBC treatment.

Extrachromosomal DNA (ecDNA) is common in human cancers and is associated with poor clinical outcomes, yet how ecDNA-driven genetic heterogeneity is translated into functional heterogeneity remains unclear. Using single-cell multiomics sequencing and multiplexed IF-FISH, we show that asymmetric inheritance of ecDNA generates copy number heterogeneity that propagates to gene expression programs, including oncogenic signaling and cellular stress responses. Transgenerational live-cell lineage tracking directly shows that ecDNA heterogeneity arises within only a few cell divisions and modulates daughter cell division timing in a copy number-dependent manner, a property not observed for evenly inherited chromosomal amplicons. We identify an optimal middle ecDNA copy number range that maximizes proliferative fitness at baseline, while drug selection pressure induced by low-dose CHK1 inhibition selects for cells with a new optimal range at low ecDNA copy numbers. These low ecDNA copy number cells pre-exist in the population and can be generated de novo, driving copy number shifts promoting drug resistance. In vivo experiments further demonstrate that shifts toward ecDNA copy numbers that are optimal under the tumour microenvironment enhance tumourigenicity. Together, these findings establish ecDNA copy number plasticity as a central driver of tumour evolution.

Nucleophosmin mutations are among the most common aberrations in acute myeloid leukemia (AML). We previously uncovered a novel pivotal role for mutant NPM1 (NPM1c) in the nucleus, where it binds chromatin at key self-renewal gene loci, such as HOXA/B and MEIS1, directly regulating oncogenic transcription. How NPM1c regulates the transcriptional activity of its chromatin targets remains unclear. To identify potential nuclear cooperating partners of NPM1c, we employed TurboID proximity labeling of NPM1c and wild-type NPM1 (NPM1wt). Mass spectrometric analysis revealed an enrichment of nucleoporins in the NPM1c interactome, with Nucleoporin 98 (NUP98) emerging as a top candidate. NUP98, a key component of the nuclear pore complex (NPC), has been established as a critical oncogenic player in AML as part of oncogenic fusion proteins. In these oncofusions the N-terminal FG (phenylalanine-glycine) repeat domain of NUP98 is preserved and fused to a diverse set of partner genes. NUP98-fusion partners are believed to mediate the chromatin binding specificity of the oncoprotein while the NUP98 FG repeats have been shown to form biomolecular condensates that enhance transcriptional output. Interestingly, wild-type NUP98 (NUP98wt) itself has been linked to gene regulatory functions during embryonic development and normal hematopoiesis. However, the role of NUP98wt in transcriptional regulation in the context of leukemia remains unresolved. To explore a potential cooperation between NUP98wt and NPM1c we first validated their interaction by co-immunoprecipitation assays. We next wanted to determine whether NUP98 and NPM1c are recruited to the same chromatin sites. To this end, we performed ChIP-seq in an NPM1c endogenous degrader cell line. These experiments revealed co-occupancy of NUP98 at NPM1c target genes and a significant reduction of NUP98 binding upon degradation of NPM1c. These findings suggest that NPM1c is required for the recruitment of NUP98 to key oncogenic target loci. To dissect the functional role of NUP98wt in NPM1c AML, we performed loss of function studies. CRISPR mediated knock-out of NUP98 in NPM1 mutant (OCI-AML3) cells resulted in a significant repression of NPM1c target genes, including HOXA/B and MEIS1. Given the essential nature of NUP98, we also engineered an endogenous NUP98 degrader system in OCI-AML3 and NPM1wt (MV4-11) cells to allow for the study of the effects of rapid protein depletion. Upon degradation of NUP98, OCI-AML3 cells exhibited robust myeloid differentiation (~70% increase in CD11b) and increased apoptosis (~50% increase in Annexin V+). MV411 cells on the other hand did not show the same differentiation phenotype. To characterize the immediate transcriptional consequences of NUP98 depletion, we performed nascent RNAseq after 1 and 3 hours of degradation. In the first hours of degradation we observed a rapid and significant repression of NPM1c target genes such as HOXA/B cluster genes, MEIS1, SMC4, TNRC18, SATB1/2 and UNCX, thus demonstrating the strong dependency of the NPM1c-driven transcriptional program on the presence of NUP98. In contrast, in MV4-11 cells, NUP98 loss had no effect on HOXA/MEIS1 expression, suggesting a leukemia subtype-specific role of NUP98wt. To delineate the domains of NUP98 required for its chromatin function, we performed a CRISPR gene tiling screen of full-length NUP98 (635 sgRNAs) and based on this we generated several domain-deletion mutants for rescue experiments in the NUP98-degrader cell lines. Retroviral overexpression of the N-terminal FG-repeat containing domains of NUP98 was sufficient to rescue cell viability and prevent the differentiation phenotype typically observed upon NUP98 degradation. Strikingly, gene expression analysis showed that the NUP98 FG-repeats were sufficient to restore the expression of HOXA10, MEIS1, and HOXB9 expression. Overall, our study identifies NUP98wt as a critical cofactor for NPM1c-driven oncogenic transcription. NPM1c recruits NUP98 to its chromatin targets, functionally substituting for an oncogenic fusion partner to concentrate transcriptional condensates at oncogenic loci. This establishes a previously unrecognized, subtype-specific dependency on NUP98wt in leukemogenesis.

A systematic approach to identifying novel therapeutic strategies is essential for expanding curative options in hematopoietic malignancies such as acute myeloid leukemia (AML). We have recently established a high-resolution genetic platform that enables de novo therapeutic development through the CRISPR-Tiling Instructed Computer-Aided (CRISPR-TICA) workflow (Nat Struct Mol Biol 2024, PMID: 38316881). Using this approach, we further identified a new therapeutic pocket and its lead compound for AML treatment (Sci Adv 2024, PMID: 38394203). Here, we present an integrative pipeline, CRISPR-TICA.ai, which combines CRISPR-based functional genomics with generative artificial intelligence (GenAI)-driven ligand modeling to perform high-throughput evaluation of the top 100 leukemia-essential genes and their therapeutic potential in AML. We first used the CRISPR screen dataset published by Novartis (Cancer Discov 2016, PMID: 27260157), which tile-scanned 68 genes in three cancer cell lines, as the initial training dataset to establish the CRISPR-TICA.ai pipeline. Structurally, ligandable protein surface pockets were identified by employing the P2Rank algorithm (J Cheminform 2018, PMID: 30109435). For each pocket, we used the structure-guided diffusion model DiffSBDD (Nat Comput Sci 2024, PMID: 39653846) to generate previously unseen chemical structures that differ substantially from existing drugs, thereby enabling the exploration of novel chemical space. The model was further fine-tuned using direct preference optimization (NeurIPS 2023) against a curated dataset of known inhibitors to enrich for favorable binding profiles (low binding free energy, ΔG) and calibrated against drug-likeness (QED), synthetic accessibility (SA), lipophilicity (logP), and Lipinski's rule compliance. Additionally, our pipeline integrates ligand–protein contact maps with CRISPR-hypersensitive positions to assess spatial interactions, prioritizing ligands that effectively mask biologically essential residues with high affinity. The utility of CRISPR-TICA.ai in AML drug discovery was evaluated- we selected the top 100 leukemia-essential genes from the DepMap database (https://depmap.org/portal/) and generated custom CRISPR libraries for high-resolution gene tiling screens. These libraries comprised 20,233 sgRNAs to tile-scan all 100 genes (47,901 amino acid residues) in Molm13 AML cells at an average resolution of 2.4 amino acids per sgRNA. This dataset provided in-depth structural and functional input for CRISPR-TICA.ai to examine AI-enabled ligand design for over 300 high-confidence druggable pockets in AML. We further developed a user-friendly, web-based 3D visualization platform that interactively maps functional CRISPR hotspots alongside predicted pockets and ligands to enhance accessibility and reproducibility. This tool allows visualization of CRISPR tiling scores, predicted binding pockets, and ligand prioritization, enabling interactive exploration and application. To annotate therapeutically relevant sites, we incorporated data from multiple genomic and proteomic resources, including AlphaFold protein models, Pfam domain annotations, post-translational modification (PTM) sites, and COSMIC leukemia mutational data. Exportable files and GitHub integration are also provided for advanced users to deploy CRISPR-TICA.ai within their facilities.In summary, our study introduces a scalable, leukemia-focused framework for de novo therapeutic discovery. By integrating high-density CRISPR mutagenesis with diffusion-based GenAI modeling, CRISPR-TICA.ai provides a rational roadmap for compound design targeting residues essential for leukemia maintenance. Importantly, the CRISPR-TICA.ai pipeline is adaptable to CRISPR tiling datasets from solid tumors (e.g., the Novartis dataset), highlighting its versatility across diverse cancer types. This approach is especially promising for addressing historically undruggable and resistance-prone targets in AML and beyond.

Rationale: Despite substantial advancement in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), it remains a leading cause of cancer mortality in children due to the high relapse rate. Moreover, the long-term survival rates for adult B-ALL patients are still less than 40%. The B-ALL patients carrying MLL rearrangements or BCR-ABL fusion represent high-risk B-ALL subtypes that face particularly dismal prognoses. This study aims to identify innovative therapeutic vulnerability for high-risk B-ALL. Methods: The CRISPR-Cas9 screen was conducted to pinpoint genes essential for high-risk B-ALL cell survival/growth. Both in vitro and in vivo models were then employed to investigate the pathological role of ZNF217 in high-risk B-ALL. To characterize the downstream functionally essential targets of ZNF217, we performed RNA-seq and CUT&RUN-seq, followed by integrative bioinformatics analysis and experimental validation. Results: Through the focused CRISPR-Cas9 screening, ZNF217 emerged as the most essential gene for the cell survival/growth of B-ALL driven by MLL rearrangement or BCR-ABL. Through in vitro gain- and loss-of-function assays, we demonstrated that ZNF217 is indeed required for B-ALL cell survival/growth. Moreover, we established the B-ALL xenograft model and patient-derived xenograft (PDX) model and demonstrated that ZNF217 depletion significantly suppressed B-ALL progression and substantially extended the survival of recipient mice. Through integrative multiple-omics analysis, we elucidated that ZNF217 exerts its oncogenic role in B-ALL through both CoREST-dependent and CoREST-independent mechanisms. Furthermore, we characterized FOS as a functionally essential downstream target of ZNF217, and ZNF217 inhibited FOS expression in a CoREST-independent manner. Conclusions: Our findings highlight ZNF217 as a promising therapeutic target for the treatment of high-risk B-ALL, such as those carrying MLL-rearrangements or BCR-ABL fusion.

The catalytic subunit of Polycomb Repressive Complex 2 (PRC2), EZH2, is recurrently mutated in 25% of diffuse large B-cell lymphomas (DLBCL), causing increased H3K27me3 and decreased H3K27me2 levels. EZH2 inhibitors provide clinical benefit, but resistance frequently develops, highlighting the need for alternative therapeutic targets. Here, we identify the PRC2 accessory protein AEBP2 as a specific genetic dependency in EZH2-mutant DLBCL. While AEBP2 acts through PRC2, its essential role is surprisingly independent of canonical H3K27me3-mediated gene silencing. Instead, AEBP2 functions within a PRC2.2 complex lacking JARID2, using its zinc-finger domains to sample intergenic chromatin to sustain H3K27me2. Notably, loss of AEBP2 or NSD2 caused contrasting changes in intergenic H3K27me2 levels, driving sensitivity or resistance to PRC2 inhibitors, respectively. Our findings identify AEBP2-PRC2.2-maintained intergenic H3K27me2 as a therapeutic vulnerability in EZH2-mutant DLBCL and highlight dysregulated H3K27me2 as an underappreciated form of PRC2 dysfunction in cancer, with important therapeutic implications. ### Competing Interest Statement The authors have declared no competing interest.

Cancer cells utilize codon-biased translation to fuel tumorigenesis and drug resistance; however, underlying mechanisms remain poorly understood. In this study, we show that ALKBH1 is overexpressed in acute myeloid leukemia (AML) and essential for leukemia stem cell/leukemia-initiating cell self-renewal and AML development/maintenance but dispensable for normal hematopoiesis. ALKBH1 enhances mitochondrial assembly/function and oxidative phosphorylation, crucial for AML survival/proliferation and resistance to venetoclax, a potent BCL2 inhibitor and widely used first-line targeted therapy for AML in the clinic. Mechanistically, ALKBH1 catalyzes 5-formylcytosine at tRNA wobble positions, reprograming decoding and facilitating codon-biased translation, a mechanism we term "epitranslatomic Midas touch," which in turn drives leukemogenesis and drug resistance by promoting the synthesis of key oncogenic proteins like WDR43. Targeting ALKBH1, particularly together with venetoclax, exhibited potent antileukemia efficacy in preclinical models with favorable safety profiles. Collectively, our findings elucidate ALKBH1's pivotal role in codon-biased translation and tumorigenesis and propose a novel therapeutic strategy for cancer treatment. SIGNIFICANCE:This study uncovers that ALKBH1-driven tRNA-decoding reprogramming and codon-biased translation, termed "epitranslatomic Midas touch," is crucial for leukemogenesis, leukemia stem cell/leukemia-initiating cell self-renewal, mitochondria structure/function, and resistance to venetoclax. Targeting ALKBH1, especially in combination with venetoclax, offers a promising therapeutic strategy to combat drug resistance in AML and potentially other ALKBH1-overexpressing cancers.

Supplementary Figure S1. ALKBH1 is required for human AML cell survival/growth in vitro and human AML progression in vivo. Supplementary Figure S2. ALKBH1 is required for leukemic cell transformation, LSC/LIC self-renewal and AML maintenance. Supplementary Figure S3. ALKBH1 is dispensable for normal hematopoiesis. Supplementary Figure S4. ALKBH1 is required for mitochondrial functions in AML cells. Supplementary Figure S5. ALKBH1 specifically catalyzes the conversion of m5C/m5Cm to f5C in tRNAs and is crutial for protein translation in AML. Supplementary Figure S6. WDR43 is a direct target of ALKBH1 through tRNA f5C-mediaed codon biased translation and phenocopies the functions of ALKBH1 in AML. Supplementary Figure S7. Discovery of the ALKBH1/WDR43/BCL2 axis and the synergistic effect of targeting ALKBH1/WDR43/BCL2 axis together with venetoclax in treating AML.

PAX5 is genetically altered in over 30% of B-ALL cases, with point mutations representing the second most common type of alteration. These point mutations are highly enriched in the DNA binding (paired) domain and have been recognized as founder events in PAX5 P80R and PAX5alt B-ALL subtypes, which account for over 12% of B-ALL. However, the molecular features and underlying mechanisms of these mutations in B-ALL remain largely undefined, limiting the development of targeted therapies and resulting in poor outcomes, especially in adult patients. We systematically investigated the functional roles and oncogenic mechanisms of PAX5 paired domain mutations. Integrated CRISPR screening and genomic profiling revealed that the paired domain is critical for PAX5 function. Disruptions in this region significantly reduced cell viability in B-ALL cell lines, and analysis of 2,955 B-ALL samples showed that recurrent PAX5 missense mutations are highly enriched in this domain, further underscoring its functional importance. Functional analysis of knock-in mice with PAX5 mutations revealed that R38H and R140L severely impair DNA binding and block B-cell differentiation, while P34L and P80R retain binding ability but alter binding specificity and gene regulation. Mice carrying homozygous (P34L, R38H, P80R, R140L) or heterozygous (R38H, P80R) mutations developed spontaneous B-ALL within one year. To model co-occurring mutations observed in patients, Pax5 R38H/P34L and R38H/R140L mice were generated. These compound mutations exhibited greater leukemogenic potential than single mutants. Transcriptomic analysis of leukemia samples showed consistent up-regulation of metabolic pathways and Myc target genes. Notably, biallelic PAX5 alterations and additional genetic lesions in signaling pathways were detected in all PAX5R38H/+ and PAX5P80R/+ leukemias, faithfully recapitulating the genetic alteration features of patient B-ALL driven by PAX5 mutations. Among Pax5-mutant mouse strains, Pax5P80R/+ mice develop B-ALL with short latency and complete penetrance, making them an ideal model to study the mutational evolution and leukemogenesis of B-ALL. To characterize the stepwise leukemogenesis process, we performed serial bone marrow aspirations in the same Pax5P80R/+ mice. In all 17 mice analyzed, we identified early preleukemic clones with only Pax5 WT deletions, followed by the emergence of leukemic clones harboring Jak1/3 mutations. Single-cell RNA/BCR-seq revealed that Pax5 WT allele deletions relieve metabolic constraints, thereby promoting clonal expansion of preleukemic B cells blocked at the pre-B stage. These changes coincide with downregulation of metabolic checkpoint genes and upregulation of glycolytic enzymes, promoting genetic instability and facilitating malignant transformation. Clinically, PAX5 P80R patients have better outcomes than PAX5alt cases. Using our mouse models, we observed that leukemic cells driven by the PAX5 P80R mutation exhibit greater sensitivity to dexamethasone compared to the ones carrying other PAX5 mutations in PAX5alt subtype, potentially due to their heightened dependence on glucose uptake and glycolysis, suggesting that metabolic rewiring driven by PAX5 mutants may influence therapeutic response. Interestingly, we found that an aberrant isoform of MEGF10 (aMEGF10) is highly and specifically expressed in PAX5 P80R B-ALL. This N-terminal truncated isoform encodes a protein that retains the intracellular immunoreceptor tyrosine-based activation motifs (ITAMs). Using CUT&Tag, we showed that PAX5 P80R directly binds to the aMEGF10 promoter. By engineering human CD34⁺ cells, we established a humanized PAX5 P80R leukemia model that recapitulates the gene expression profile of patient samples, including robust activation of aMEGF10. Functionally, aMEGF10 binds to and activates SYK, thereby promoting leukemic cell proliferation. Importantly, this oncogenic effect depends on intact ITAM motifs and SYK signaling, revealing a novel targetable vulnerability in PAX5 P80R leukemias. Overall, the study establishes that PAX5 DNA binding domain mutations contribute to leukemogenesis through stepwise loss of PAX5 WT alleles and acquisition of signaling pathway mutations, involving impaired differentiation, metabolic deregulation, and novel oncogene activation. These findings not only deepen the understanding of PAX5-driven leukemias but also highlight aMEGF10–SYK signaling as a promising therapeutic target.

Metabolic reprogramming of amino acids represents a vulnerability in cancer cells, yet the mechanisms underlying serine metabolism in acute myeloid leukemia (AML) and leukemia stem/initiating cells (LSCs/LICs) remain unclear. Here, we identify RNA N6-methyladenosine (m6A) modification as a key regulator of serine biosynthesis in AML. Using a CRISPR/Cas9 screen, we find that depletion of m6A regulators IGF2BP3 or METTL14 sensitizes AML cells to serine and glycine (SG) deprivation. IGF2BP3 recognizies m6A on mRNAs of key serine synthesis pathway (SSP) genes (e.g., ATF4, PHGDH, PSAT1), stabilizing these transcripts and sustaining serine production to meet the high metabolic demand of AML cells and LSCs/LICs. IGF2BP3 silencing combined with dietary SG restriction potently inhibits AML in vitro and in vivo, while its deletion spares normal hematopoiesis. Our findings reveal the critical role of m6A modification in the serine metabolic vulnerability of AML and highlight the IGF2BP3/m6A/SSP axis as a promising therapeutic target.

Stress granules (SGs) and processing bodies (PBs), assembled via liquid-liquid phase separation (LLPS), are critical for spatial regulation of gene expression in the cytoplasm. However, their roles in tumorigenesis remain poorly understood. Here, we show DEAD-box helicase 6 (DDX6) as the most promising vulnerability in acute myeloid leukemia (AML) through in vitro and in vivo CRISPR screenings using a specialized library targeting RNA-binding proteins enriched in SGs and PBs. Knockout (KO) of DDX6 significantly delays leukemogenesis with minimal impact on normal hematopoiesis. Importantly, the functions of DDX6 in AML depend largely on its ability to trigger LLPS and PB assembly. Mechanistically, PBs serve as "reservoirs" for the mRNAs interacting directly with DDX6 and having low GC content. DDX6 KO leads to rapid PB dissolution and release of PB-enriched mRNAs, such as BCAT1, into the cytosol, where these transcripts undergo degradation. By reducing BCAT1 levels, DDX6 KO reprograms amino acid metabolism and sensitizes AML cells to cytarabine chemotherapy.

Accumulating myelin damage and impaired remyelination are central pathological features of leukoencephalopathies, including Multiple Sclerosis and Alexander Disease, where astrocytes play essential roles in maintaining central nervous system homeostasis and mediating astrocyte-oligodendrocyte interactions. Chloride Ion Channel 2 (ClC-2), encoded by CLCN2, is functionally expressed in astrocytes and is likely critical to white matter integrity; however, its precise roles and interactions remain unclear. Clarifying the mechanisms by which astrocytic ClC-2 influences white matter is essential for developing treatments for CLCN2-related leukoencephalopathy (CC2L) and potentially other white matter disorders. In this study, we demonstrate that dysfunctional ClC-2 in astrocytes—derived from both mouse models and human-induced pluripotent stem cells (hiPSCs)—impairs oligodendrocyte lineage cell development in vitro and delays remyelination in vivo. Transcriptomic analyses identified SPP1 as a key inhibitory factor on remyelination, secreted from ClC-2-deficient astrocytes. This inhibition was validated through astrocyte-specific modulation of SPP1 expression, where overexpression exacerbated, and downregulation alleviated, demyelination effects. Furthermore, we discovered that SPP1 upregulation in astrocytes with abnormal ClC-2 negatively impacts remyelination by interacting with CD44 on oligodendrocyte progenitor cells. Finally, we confirmed that increased SPP1 expression and the resulting suppression of oligodendrocyte lineage cell development were also present in hiPSC-derived astrocytes harboring a CLCN2 mutation from a CC2L patient. Collectively, these findings reveal that astrocytic ClC-2 is intricately linked to white matter integrity through SPP1 regulation, positioning it as a potential therapeutic target for CC2L and other leukoencephalopathies.

B-cell receptor (BCR) signaling plays an important role in the pathogenesis of mantle cell lymphoma (MCL), but the detailed mechanisms are not fully understood. In this study, through a genome-wide loss-of-function screen, we identify carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) as an essential factor in a subset of MCL tumors. Our signal transduction studies reveal that CEACAM1 plays a critical role in BCR activation through involvement in two dynamic processes. First, following BCR engagement, CEACAM1 co-localizes to the membrane microdomains (lipid rafts) by anchoring to the F-actin cytoskeleton through the adaptor protein filamin A. Second, CEACAM1 recruits and increases the abundance of SYK in the BCR complex leading to BCR activation. These activities of CEACAM1 require its cytoplasmic tail and the N-terminal ectodomain. Considering that previous studies have extensively characterized CEACAM1 as an ITIM-bearing inhibitory receptor, our findings regarding its activating role are both surprising and context-dependent, which may have implications for BCR-targeting therapies.

As one amino acid can often be encoded by multiple co-dons,the genetic code is redundant,which accounts for synonymous mutations in protein-coding regions.1 Since synonymous mutations do not cause any alterations in amino acid sequence,it was previously believed that they do not change the structure and function of the proteins and thus are functionally silent.1

PI3K inhibitors, such as idelalisib and duvelisib, have demonstrated efficacy in relapsed/refractory chronic lymphocytic leukemia (CLL) but severe autoimmune toxicities limit their clinical use. We have previously shown that idelalisib toxicity is driven by immune dysregulation, with a decline in CD4+ regulatory T cells (Tregs) and a shift toward effector T cells and Th17 pathway activation. However, these informative CyTOF studies did not address three critical questions: what underlies Treg dysfunction, how does idelalisib affect effector cells, and what drives Th17 pathway hyperactivation. We therefore performed single-cell RNA sequencing (scRNAseq) with TCR sequencing (scTCRseq) on T-cell-enriched PBMCs from 8 CLL patients with and without idelalisib-induced toxicity (n=4 for each group, matched pre- and on treatment samples). Differential abundance analysis revealed significantly higher baseline mucosal-associated invariant T (MAIT) and CD8+ naive cells in toxicity patients (tox pts), with lower MAIT, CD8+ effector memory (TEM) and CD4+ TEM cells on treatment. Supporting a pathogenic role for MAIT cells, differential expression (DE) analysis showed idelalisib increased IL7R, FOS, and STAT1 expression, suggesting increased Th17 differentiation in tox pts. GSEA demonstrated that Tregs in toxicity patients displayed upregulated ZEB1 and BACH2 (transcriptional repressors of Treg function) alongside downregulated EGR1 (a FOXP3 activator) on treatment, leading to impaired critical TNFA signaling via NFKB. Additionally, underexpression of key T cell migration markers (CFL1, RAC2, ACTB, CORO1A, and TMSB4X) in Tregs from tox pts suggested reduced migratory ability to inflammation sites thus impairing their immunity suppression capacity. Integration of scRNAseq and scTCRseq data revealed that T cells in toxicity-associated samples exhibited a greater proportion of unique clones at both pre- (tox vs nontox: 87 vs 72%) and on-treatment (tox vs nontox: 88 vs 68%) timepoints and higher clonal diversity compared to non-toxicity controls, with minimal changes following idelalisib exposure. Patient-specific clonal analysis revealed that idelalisib treatment induced expansion of new T cell clones primarily from CD8+ naive, CD8+ TEM, CD4+ central memory (TCM), and MAIT cells in both groups. In tox pts, major clones on treatment were new clones, whereas non-tox patients showed expansion of pre-existing clones. DE analysis revealed these expanded clones express distinct transcriptional programs between the two cohorts. In toxicity patients, emergent clones of CD8+ naive cells showed TH17/TH1 polarization (TBX21 and IFNG) with higher levels of and coexpression of cytotoxic (GZMB) and migratory (CXCR3) markers compared to new clones of nontox pts, suggesting these emergent clones may contribute to toxicity. CD4+ TCM new clones in the tox pts exhibited metabolic reprogramming activating the JAK-STAT pathway, as evidenced by enrichment of IFN-γ/α response (STAT1, JAK2, IRF1, TBX21 and IFNG) and allograft rejection pathways (CXCR3, PRF1 and IFNGR1). Key metabolic drivers of this expansion including MTHFD2, LDHA, and ACLY were co-expressed with cytotoxic and migratory phenotype markers in toxicity samples on treatment but not at baseline. These toxicity-associated CD4+ TCM clones also showed enrichment in TH17/TH1 differentiation and glycolysis pathway genes compared to pre- and other on-treatment clones. Lineage trajectory analysis revealed that a small subset of these CD4+ TCM new clones exhibited TH17/TH1 differentiation in tox pts while co-expressing cytotoxic and migratory markers, coinciding with relatively activated PI3K-AKT-mTOR signaling. Notably, this pattern was absent in non-toxicity controls, highlighting toxicity-specific metabolic reprogramming that leads to aberrant effector functions linked with autoimmune-like early immune toxicity. This aligns with prior findings that CD4+ T cells reprogram metabolism and promote Th17 responses via HIF-1α and GLUT1 in rheumatoid arthritis, an autoimmune disease. Overall, our findings suggest idelalisib-induced toxicity develops in patients with a predisposed immune microenvironment with sustained higher clonal diversity and metabolic-immune dysregulation, leading to the expansion of CD8+ and CD4+ T cell clones with TH17/TH1 polarization, cytotoxicity, and migratory phenotypes. These insights into PI3Kδ inhibitor-induced toxicity may suggest potential targets to mitigate adverse effects.

Supplementary Table S1: Effects of ALKBH1 KD on f5C34 in mitochondrial tRNA-Met Supplementary Table S2: Genetic background of the AML patient samples. Supplementary Table S3: List of oligonucleotide sequences

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor characterized by chemoresistance and poor prognosis. This study investigates the role of GPRC5A in GBM, focusing on its association with chemoresistance to temozolomide (TMZ) and its impact on patient outcomes. Through integrative analyses of the Cancer Therapeutics Response Portal (CTRP) and The Cancer Genome Atlas (TCGA) databases, we identified GPRC5A as significantly upregulated in GBM, correlating with poorer survival and TMZ resistance. Functional assays demonstrated that GPRC5A overexpression enhances tumor sphere formation and stem cell marker expression, such as CD133, suggesting its role in promoting stem-cell-like properties and TMZ resistance. Bioinformatic analyses revealed GPRC5A's involvement in regulating hypoxia and glucose metabolism via the HIF-1 signaling and glycolytic pathways. Mechanistically, GPRC5A interacts with the glycolytic transporter GLUT1, influencing glucose uptake and glycolysis. Silencing GPRC5A increased TMZ sensitivity by reducing GLUT1 stability and glucose uptake, an effect reversible by GLUT1 overexpression. In vivo studies confirmed that GPRC5A silencing reduced tumor growth and improved survival, highlighting its potential as a therapeutic target for overcoming chemoresistance in GBM. These findings underscore the critical role of GPRC5A in GBM and suggest that targeting the GPRC5A-GLUT1 interaction could improve patient outcomes.

Quantitative proteomics after ALKBH1 knockdown in MOLM13 cells.

Diffuse midline glioma (DMG) is a fatal childhood brain tumor characterized primarily by mutant histone H3 (H3K27M). H3K27M causes a global reduction in Polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation (H3K27me3). Paradoxically, PRC2 is essential in DMG cells, although the downstream molecular mechanisms are poorly understood. Here, we have discovered a specific form of canonical PRC1 (cPRC1) containing CBX4 and PCGF4 that drives oncogenic gene repression downstream of H3K27me3 in DMG cells. Via a novel functional region, CBX4 preferentially associates with PCGF4-containing cPRC1. The characteristic H3K27me3 landscape in DMG rewires the distribution of cPRC1 complexes, with CBX4/PCGF4-cPRC1 accumulating at H3K27me3-enriched CpG islands. Despite comprising <5% of cPRC1 in DMG cells, the unique repressive functions of CBX4/PCGF4-cPRC1 are essential for DMG growth. Our findings link the altered distribution of H3K27me3 to imbalanced cPRC1 function, which drives oncogenic gene repression in DMG, highlighting potential therapeutic opportunities for this incurable childhood brain cancer.

Background While liver cancer stem cells (CSCs) play a crucial role in hepatocellular carcinoma (HCC) initiation, progression, recurrence, and treatment resistance, the mechanism underlying liver CSC self-renewal remains elusive. We aim to characterize the role of Methyltransferase 16 (METTL16), a recently identified RNA N 6 -methyladenosine (m 6 A) methyltransferase, in HCC development/maintenance, CSC stemness, as well as normal hepatogenesis. Methods Liver-specific Mettl16 conditional KO (cKO) mice were generated to assess its role in HCC pathogenesis and normal hepatogenesis. Hydrodynamic tail-vein injection (HDTVi)-induced de novo hepatocarcinogenesis and xenograft models were utilized to determine the role of METTL16 in HCC initiation and progression. A limiting dilution assay was utilized to evaluate CSC frequency. Functionally essential targets were revealed via integrative analysis of multi-omics data, including RNA-seq, RNA immunoprecipitation (RIP)-seq, and ribosome profiling. Results METTL16 is highly expressed in liver CSCs and its depletion dramatically decreased CSC frequency in vitro and in vivo. Mettl16 KO significantly attenuated HCC initiation and progression, yet only slightly influenced normal hepatogenesis. Mechanistic studies, including high-throughput sequencing, unveiled METTL16 as a key regulator of ribosomal RNA (rRNA) maturation and mRNA translation and identified eukaryotic translation initiation factor 3 subunit a ( eIF3a ) transcript as a bona-fide target of METTL16 in HCC. In addition, the functionally essential regions of METTL16 were revealed by CRISPR gene tiling scan, which will pave the way for the development of potential inhibitor(s). Conclusions Our findings highlight the crucial oncogenic role of METTL16 in promoting HCC pathogenesis and enhancing liver CSC self-renewal through augmenting mRNA translation efficiency.