Oncogenesis

Internal-tandem-duplication of the receptor tyrosine kinase FLT3 (FLT3-ITD) generates ligand-independent signaling and is highly recurrent in acute myeloid leukemias (AMLs). One way signaling pathways can quickly influence cell fates is by phosphorylating key fate-determining proteins to trigger their proteolysis. We investigated the master transcription factor (MTF) driver of granulo-monocytic lineage-fates, CEBPA, for regulation by this mechanism because we found high CEBPA mRNA but little CEBPA protein in FLT3-ITD versus FLT3-wildtype AML cells, and inhibiting FLT3-ITD signaling with tyrosine kinase inhibitors (TKI) rapidly rescued CEBPA protein. Mass spectrometry analyses of CEBPA and its interactome demonstrated prominent interactions with major ubiquitin-proteosome pathway (UPP) components UHRF1 and USP7. TKI treatments decreased CEBPA and USP7 phosphorylations at serine 21 and serine 18 respectively alongside shifts in CEBPA interactions from degradative ubiquitin-ligase UHRF1 toward protective deubiquitinase USP7. The rescued CEBPA activated granulocytic-differentiation. Supporting that the serine-phosphorylations were phospho-degrons, UPP-inhibitors (bortezomib, MG132) increased phosphorylated and total CEBPA and USP7. The MTF regulator of apoptosis p53 is a known USP7 client, therefore, we also evaluated p53 status: TKIs and UPP-inhibitors stabilized USP7 and p53, triggering apoptosis in addition to granulocytic-differentiation specifically in FLT3-ITD but not FLT3-wildtype AML cells. UPP-inhibitors produced these consequences in TKI-resistant FLT3-ITD AML cells also. These data predicted genetic loss-of-function to CEBPA or TP53 is redundant in the FLT3-ITD context, borne out by mutual exclusivity of the mutations in clinical series. In summary, FLT3-ITD signals for CEBPA and p53 proteolysis to block lineage-maturation and apoptosis, positioning UPP-inhibitors as therapeutic candidates acting downstream of TKIs. KEY POINTSO_LIThe oncoprotein kinase FLT3-ITD signals for CEBPA and p53 proteolysis and hence suppresses lineage-differentiation and apoptosis C_LIO_LIProteosome-inhibitors are candidate remedies to restore CEBPA and p53, acting downstream of presently used FLT3-ITD kinase inhibitors C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/738455v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@6ae211org.highwire.dtl.DTLVardef@12003bforg.highwire.dtl.DTLVardef@d62eb9org.highwire.dtl.DTLVardef@1958693_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: High ambient temperature in early pregnancy has been linked to an increased risk of childhood acute lymphoblastic leukemia (ALL). To better understand biological mechanisms, the current study evaluated potential interaction between temperature and genetic characteristics. Methods: We used data from California birth records (1982-2008) and California Cancer Registry (1988-2011) to identify ALL cases (n=3,353) diagnosed <=14 years of age and non-cancer controls (n=3,530) matched 1:1 on sex, race, ethnicity, and birth year and month. Weekly ambient temperatures throughout pregnancy were assessed on a 1-km grid around the birth address, while genetic data were available from a genome-wide association study using neonatal blood spots. We evaluated the association between ambient temperature and ALL risk by quartiles of established genetic risk score for ALL. Next, we formally tested gene-temperature interactions in the association with ALL, correcting for multiple testing, for genes previously identified with epigenetic changes due to both temperature and ALL. All analyses were adjusted for potential confounders. Results: The elevated risk of ALL per 5 degrees C increase of weekly mean ambient temperature, confined to early pregnancy, was more pronounced among children with the lowest genetic susceptibility to ALL, especially among Latino children (first quartile: odds ratio [OR] = 1.50, 95% confidence interval [CI]: 1.14-1.97); fourth quartile: OR=1.03, 95% CI: 0.83-1.28). There were significant interactions (p<0.002) between ambient temperature and polymorphisms in BNC1 among non-Latino White children, and suggestive interactions (p<0.05) with TBPL2 and NRXN1 in the full population. Conclusions: Our findings suggest that there may be interactions between ambient temperature in early pregnancy and offspring genotype in the risk of childhood ALL. Impact: If replicated, these findings could help elucidate the biological mechanisms linking high ambient temperature in early pregnancy and the risk of childhood ALL.

Osteosarcoma (OS) and Ewing sarcoma (EWS) are the most common malignant bone tumors in children and adolescents, with survival rates around 25% in metastatic disease and few advances in treatment in decades. High DDX3 expression has been reported across various sarcoma subtypes. Depending on the context, DDX3 appears to have opposing roles in regulating the tumor immune microenvironment. Within macrophages, DDX3 promotes inflammatory cytokine expression and supports immune cell function. In contrast, in tumor cells DDX3 suppresses a pro-inflammatory state by unwinding dsRNAs, preventing a Type I interferon response. We show that inhibiting DDX3 with RK-33 leads to dsRNA accumulation, inducing a Type I interferon response and broader inflammatory gene expression changes across multiple sarcoma models, shifting macrophage polarization toward a pro-inflammatory M1-like phenotype. To evaluate whether this innate immune microenvironmental remodeling could translate into clinical benefit, we assessed the therapeutic efficacy of RK-33 alone or in combination with mifamurtide, an immunostimulant, in immune competent mouse models of osteosarcoma, with metastatic burden as the primary outcome. We found that in the absence of MYC over-expression, the combination treatment significantly reduced metastatic spread. These findings support targeting DDX3 as a novel innate immune based therapeutic strategy and highlight that the tumors molecular landscape critically influences therapeutic responsiveness.

Clonal hematopoiesis (CH) is an age-associated condiion defined by over-representation of hematopoietic stem cells (HSCs) and their progeny carrying somatic mutations or variants that confer a selective advantage. CH is associated with increased risk of hematologic malignancies (1), cardiovascular disease and inflammatory bone loss (2, 3). Chronic inflammation is increasingly recognized as a central mediator of CH-mutant hematopoietic stem and progenitor cell (HSPC) expansion underlying CH (4). DNA methyltransferase 3a ( Dnmt3a )-mutant cells produce higher levels of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) (5), and blocking these pathways reduces the competitive advantage of Dnmt3a -mutant HSPCs (4, 6). The upstream mediators initiating inflammatory signaling in CH are unknown. Strong candidates are S100A8 and S100A9, members of the S100 calcium-binding protein family that regulate inflammatory signaling in the hematopoietic system. These proteins form a heterodimer complex and activate innate immune signaling through receptors including Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE) (7). S100A8/A9 signaling promotes production of pro-inflammatory cytokines and inflammasome activation leading to poor prognosis in myelodysplastic syndrome and myeloproliferative neoplasms (8, 9). Across multiple myeloid malignancies, neutrophils are the primary bone marrow (BM) source of this alarmin (8, 10, 11) and pharmacologic inhibition of S100A9 with tasquinimod reduces disease severity without disrupting normal hematopoiesis (10, 12). Given the role of S100A8/A9 in establishing an inflammatory milieu, here we investigated the role of S100A8/A9 in Dnmt3a -mutant hematopoiesis. We identify neutrophils as a major source of elevated S100A8/A9 in the BM of Dnmt3a -mutant mice and this increase correlates with production of the inflammatory cytokines TNFα and IL-6. We show that tasquinimod reduces TNFα and IL-6 levels and selectively reduces the Dnmt3a -mutant HSPC compartment.

Intracellular iron homeostasis is increasingly recognized as a regulator of cancer cell behavior, but how iron distribution influences extracellular matrix (ECM) organization and invasion remains poorly understood. Here, we show that loss of divalent metal transporter 1 (DMT1/SLC11A2) disrupts intracellular iron homeostasis and promotes cancer cell invasion through an iron-ER-ECM axis. In MDA-MB-231 cells, DMT1 knockout (KO) reduced total iron content but increased the labile iron pool (LIP) in both 2D and 3D culture models, indicating altered intracellular iron distribution. Across transcriptomic and phenotypic readouts, DMT1-dependent effects were more evident in 3D than in 2D models, with DMT1 KO inducing endoplasmic reticulum (ER) stress and impaired collagen/ECM organization. Functionally, the DMT1-loss phenotype was marked by reduced 2D motility, whereas in 3D spheroid models DMT1 KO cells displayed enhanced invasive outgrowth in both Matrigel and collagen matrices. Iron chelation further modulated this phenotype in a DMT1-dependent manner. Pharmacologic induction of ER stress phenocopied the loose spheroid architecture and invasive behavior, supporting ER stress as a mechanistic link between altered iron handling and ECM destabilization. Together, these findings identify intracellular iron distribution, rather than total iron abundance alone, as a determinant of ECM integrity and context-dependent cancer cell invasion. Significance StatementOur study identifies an iron-ER-ECM axis through which intracellular iron homeostasis regulates cancer cell invasion. Total cellular iron content alone is insufficient to predict invasive behavior without considering how iron is distributed within the cell. By preserving intracellular iron homeostasis and ER function, DMT1 supports collagen synthesis and maintains ECM integrity. In contrast, DMT1 loss disrupts these processes, promoting formation of loosely aggregated spheroids and enhanced invasion in 3D tumor models despite reduced total iron levels. These findings challenge the assumption that lowering bulk iron uniformly suppresses invasive phenotypes and instead highlight intracellular iron trafficking as a potential therapeutic target for limiting cancer cell invasion.

Transcription factor CEBPA is mutated in 10-15% of acute myeloid leukaemia (AML), a haematopoietic malignancy with high mortality. CEBPA mutations show a distinct pattern, and most patients are biallelic, carrying both an in frame C-terminal mutation and a frameshift N-terminal mutation on opposing alleles. Rare N-terminal germline cases have 100% penetrance to AML, all with an acquired a C-terminal mutation. This suggests a selective pressure from one CEBPA mutation to develop another. Our zebrafish models faithfully recapitulate the human disease. All biallelic mutant combinations die by 4-6 weeks of age, with pre-leukaemic haematopoietic stem cell (HSC) expansion. C-terminal and N-terminal mutants show phenotypic differences in myeloid primed HSC and differences in the myeloid differentiation block. RNA-Seq identified differentially expressed genes in opposing vectors. We identified phosphatase receptor PTPRJ as a candidate driver of clonal selection, with knock-out of ptprja in our fish accelerating pre leukaemic expansion of HSC in C-terminal mutants, decelerating it in N-terminal mutants. Cebpa mutant murine cells exhibit changes in differentiation and a clonal advantage with loss of Ptprj, which perturbs key signalling pathways. Our data suggest that PTPRJ contributes to the mechanism of leukaemogenesis in CEBPA mutant AML by driving the selective pressure from each mutation to develop the other.

Long non-coding RNAs (lncRNAs) have attracted increasing interest because of their roles as modulators of tumor progression, acting either as oncogenic drivers or tumor suppressors, depending on the cellular context. LINC01133 has been implicated in regulation of multiple tumor-related mechanisms; however, its role in breast cancer, particularly in the triple-negative subtype, remains poorly characterized. In this study, we investigated the impact of LINC01133 depletion on malignant phenotypes and on the expression of migration- and invasion-associated genes using the Hs578T triple-negative breast cancer (TNBC) cell line, through comparative analyses of parental, control, and LINC01133-knockout cell lines, namely Hs578T_wt, Hs578T_ctr, and Hs578T_ko. Functional characterization included morphological analysis, growth assays, anchorage-independent colony formation, migration, invasion, and quantitative biomolecular experiments. Depletion of LINC01133 led to reduction of cell diameter, a significant increase in colony-forming capacity, and marked enhancement of migratory and invasive potential. At the molecular level, LINC01133 loss induced the expression of genes associated with extracellular matrix remodeling and cellular plasticity, including fibronectin, vimentin, integrins, FOXC1, and TWIST1, concomitant with reduced expression of ZEB1, TWIST2, and N-cadherin. Collectively, these data indicate that LINC01133 acts as a potential fine regulator of in vitro migration and invasion processes in TNBC, with its expression favoring a more asymptomatic mode of tumor progression, whereas its loss markedly enhances tumor malignancy.

WNT11, a non-canonical WNT ligand, plays well-defined roles in development and tissue architecture; however, its function in cancer remains ambiguous. Here, we characterize WNT11 as a context-dependent suppressor of cancer stemness, invasion, and in vivo tumor formation in human epithelial cancer models. We show that WNT11 upregulation reduces the expression of stemness-promoting genes, suppresses epithelial-to-mesenchymal transition, and inhibits sphere formation and tumor growth. Conversely, downregulation of WNT11 enhances these aggressive malignant properties of cancer cells. Mechanistically, we found that the ability of WNT11 to inhibit RAC1 GTPase activation is essential for its regulation of invasion and self-renewal. In cells unresponsive to WNT11, the connectivity between WNT11 and RAC1 activity is disengaged. Direct manipulation of RAC1 activity in these cells recapitulates the phenotype and molecular signature of WNT11-responsive cells, establishing RAC1 as a critical effector of WNT11-mediated tumor suppression. Taken together, these findings identify the cellular context in which WNT11 suppresses RAC1 activation as a key determinant of its anti-tumor effects and provide a mechanistic framework for understanding the diverse, and sometimes opposing, roles of WNT11 reported in cancer.

In human acute myeloid leukemia (AML), a sub-population of leukemia stem cells (LSCs) drive disease initiation, therapeutic resistance, and relapse. However, the lack of reliable markers to distinguish LSCs from bulk leukemia cells has impeded progress in studying LSC pathogenesis and developing meaningful LSC-specific diagnostics and therapeutics. Existing LSC gene signatures, derived from bulk populations, cannot definitively identify LSCs at single-cell resolution. To address this, we analyzed large patient cohorts with bulk gene expression data and single-cell multi-omic assays to identify a prognostic gene signature that is specifically enriched in a clinically adverse AML sub-population. Using this signature, we defined and prospectively isolated CD34+CD90-CLL1-CD69+CD53- immunophenotypic LSCs that are significantly enriched for LSC content based on limiting dilution xenotransplantation assays. Our findings demonstrate the power of single-cell multi-omics to precisely identify a clinically relevant LSC population and establish a clear framework for future translational research in AML. Key PointsO_LISingle cell multi-omics identifies human AML LSCs at high resolution. C_LIO_LIHOPX and SOCS2 co-expression (hrLSC2) defines a prognostic gene signature in de novo acute myeloid leukemia. C_LIO_LIhrLSC2 marks an AML subpopulation (iLSCs) with a distinct immunophenotype. C_LIO_LIiLSCs can be purified using flow cytometry and are significantly enriched for LSCs. C_LI

Osteoblastic bone metastasis, in which disseminated tumor cells drive net bone formation, is a clinically distinct and mechanistically underexplored form of skeletal disease that is enriched in hormone receptor-positive breast cancers. Preclinical models of bone metastasis from breast cancer predominantly rely on immunodeficient hosts inoculated with osteolytic human breast cancer cell lines, limiting the study of immune-dependent mechanisms of bone remodeling. Here we describe the development and characterization of an immunocompetent, syngeneic osteoblastic bone metastasis model using intratibial injection of PyMT-CK(OB), a luciferase-expressing derivative of the MMTV-PyMT mammary carcinoma cell line, in FVB/N mice. PyMT-CK(OB) cells produced detectable bioluminescent signal after intratibial injection, enabling longitudinal monitoring of tumor progression. Micro-computed tomography (microCT) revealed significant increases in trabecular bone volume fraction and trabecular number at three and four weeks post-injection, consistent with osteoblastic remodeling. Histological analysis confirmed dense bone lesion formation in tumor-bearing bones. Critically, this osteoblastic phenotype was entirely absent in immunodeficient NOD SCID hosts, despite robust tumor growth, supporting a role for immune competence in tumor-induced bone formation. Loss of bioluminescent signal in immunocompetent mice reflected either immune pressure on reporter gene expression or limited space for cancer cell expansion in the bone, rather than tumor regression or hypoxia, as confirmed by hypoxia imaging and histological endpoint analysis. In contrast, a second PyMT cell subline, PyMT-CF, maintained sustained bioluminescent signal and produced predominantly osteolytic lesions, providing a complementary syngeneic model of osteolytic disease from the same parental background. In vitro hydrogel coculture experiments and protein array analysis of conditioned media revealed that the PyMT sublines have differing impact on MC3T3 osteoblast mineralization, identifying candidate mediators of divergent bone remodeling phenotypes. R7 mammary carcinoma cells derived from MMTV-RON transgenic mouse mammary tumors did not induce measurable bone remodeling under equivalent experimental conditions. Together, these models provide a validated, immunologically intact framework for studying the mechanistic basis of osteoblastic bone metastasis and evaluating therapeutic interventions targeting the tumor-bone microenvironment.

Background/ObjectivesGlioblastoma is an aggressive primary brain tumor that develops within a chronically low-oxygen microenvironment, yet most preclinical studies are performed under atmospheric oxygen conditions that poorly reflect in vivo physiology. This study investigated how sustained culture under physiological oxygen tension (physioxia; 5% O{square}) influences glioblastoma cell behavior, signaling, and therapeutic response. MethodsMultiple patient-derived glioblastoma models were cultured under normoxia (21% O{square}) or sustained physioxia (5% O{square}) for at least seven days before experimentation. Cell migration, proliferation, cell cycle distribution, expression of the epithelial-to-mesenchymal transition-associated transcription factor Slug (SNAI2), PDGFR{beta}-associated signaling, and sensitivity to 5-fluorouracil were evaluated using transwell migration assays, cell counting, flow cytometry, RT-qPCR, immunoblotting, and BrdU incorporation assays. Additional patient-derived cultures established and maintained continuously under physioxia were used to examine the effects of oxygen history. ResultsSustained physioxia consistently increased migration across all glioblastoma models while reducing proliferation in normoxia-adapted cell lines through increased G0/G1 cell cycle arrest. Physioxia significantly increased Slug expression in all models and enhanced PDGFR{beta}, AKT, and ERK phosphorylation in a cell line-dependent manner. Therapeutic sensitivity to 5-fluorouracil was also altered, with physioxia conferring increased resistance in selected glioblastoma models but not universally. Patient-derived cultures maintained continuously under physioxia retained enhanced migratory capacity and exhibited increased proliferation compared with normoxia, indicating that prior oxygen exposure influences proliferative responses while the pro-migratory phenotype remains conserved. ConclusionsPhysiological oxygen tension is a major regulator of glioblastoma cell behavior, influencing migration, proliferation, signaling, and therapeutic response. These findings demonstrate that conventional normoxic culture conditions can obscure biologically relevant phenotypes and support incorporating physioxia into experimental design to improve the physiological and translational relevance of preclinical glioblastoma research.

Genetic uniqueness of the tumor microenvironment significantly influences cancer growth, survival, and response to therapy, independent of the cancer cells intrinsic properties or the adaptive immune system. Using genetically distinct Rag1-/- mouse models, this study shows that different strains exhibit varied tumor growth kinetics and survival outcomes when xenografted with identical leukemic and solid tumor cell lines. This study further highlights the critical role of the myeloid immune compartment and shows that disrupting both lymphoid and myeloid systems alters cancer progression. These results also reveal that the tumor microenvironment can permanently alter cancer cell phenotypes and significantly affect chemotherapy efficacy, as seen with Cisplatins varying effects across strains. These findings underscore the importance of considering genetic background in preclinical cancer models, suggesting that reliance upon a single mouse strain may lead to incomplete conclusions about cancer biology and treatment efficacy. SUMMARY STATEMENTPre-clinical xenograft mammalian models are used to study human diseases. Here we report that the genetic uniqueness of the tumor microenvironment, independent of the immune system, can determine the fate of cancer progression, survival, and therapy response.

While therapy resistance in acute myeloid leukemia (AML) is often attributed to leukemic stem cells (LSCs), their functional properties are not fully captured by their well-established genetic landscape and cell lineage transcriptional programs. Here, we explore AML cell states and their associations to drug response and systemic immune context. We performed integrated single-cell transcriptomics and immunophenotyping on diagnostic AML samples (n=6) to define transcriptional cell state gene signatures. These were projected onto bulk RNA-seq data from 448 AML patients to assess associations with drug sensitivity, plasma proteomics, clinical features, and established prognostic scores. Longitudinal single-cell data from external cohorts and cell-cell communication analyses were used to examine treatment dynamics and microenvironmental signaling. We defined nine AML cell states, including progenitor-like, stromal-like, antigen-presenting, and monocytic programs. Stemness features were distributed across multiple states, with lymphoid-primed and stress-adapted progenitors showing the strongest alignment with established stemness scores. Distinct drug sensitivities emerged, including cell cycle checkpoint inhibitor sensitivity in stress-adapted progenitors and kinase inhibitor sensitivity in cycling progenitors, alongside shared resistance to BH3 mimetics in monocytic states. Stress-adapted progenitors were associated with adverse clinical features and expanded following venetoclax-based therapy. Monocytic states acted as immunosuppressive hubs via TIGIT signaling, while stromal-associated states received niche-derived survival signals. Overall, we define a framework that associates AML cell states with stemness, drug response, and microenvironmental interactions. These findings highlight distributed stemness, state-specific vulnerabilities, and niche-driven resistance mechanisms, informing more precise therapeutic strategies in AML.

Background: Human induced pluripotent stem cells (hiPSCs) hold promise for vascular regeneration, but preliminary research often relies on neonatal donors, whereas clinical applications will use cells derived from aged individuals. Although the impact of donor age on reprogramming efficiency has been studied, its effect on the functionality of hiPSC-derived endothelial progenitors (hiPSC-EPs) remains unclear. This question is the focus of the current study. Methods and Results: We derived EPs from iPSCs sourced from three neonatal donors (ND) and three mature donors (MD) matched 1:1 for sex and somatic cell origin. We assessed their functional, epigenetic, and transcriptomic characteristics. Despite higher CD34? yields from MD-iPSCs, MD-hiPSC-EPs formed poorly interconnected and non-lumenized vascular structures in 3D hydrogels, compared to neonatal donor (ND) lines. In 2D culture, MD-hiPSC-EPs exhibited reduced cell density and aberrant VE-Cadherin localization. DNA methylation analysis revealed that somatic cell origin was the dominant driver of variance, but consistent differences in methylation of mesoderm commitment, angiogenesis, ECM remodeling, and cytoskeleton-related genes were observed between age groups. Epigenetic age prediction showed MD-hiPSC-EPs had more developmentally advanced signatures, potentially explaining their shift away from vasculogenic competence. Our RNA-sequencing findings confirm trends seen in the DNA methylation data and show differential expression of pathways linked to mitochondrial regulation and nitric oxide signaling. Conclusions: Donor age significantly alters the vasculogenic function of hiPSC-EPs. These findings underscore the necessity of donor-specific considerations in hiPSC-based vascular engineering and highlight potential barriers to translating hiPSC-derived therapeutics into aged patient populations.

Despite continuous progress in diagnosis and therapy, breast carcinoma (BC) remains a major health problem. Triple-negative (Estrogen Receptor-/Progesterone Receptor-/HER2-) breast cancer (TNBC) is the most aggressive subtype due to its high metastatic potential and resistance to chemotherapy. The Y-box binding protein 1 (YB-1) transcription factor, a protein present in both cytoplasm and nucleus, is a driver of TNBC malignancy as it stimulates its cancer stem cell phenotype and disrupts cell cycle progression. Here, we hypothesized that YB-1-containing sEVs deliver YB-1 to the nuclear compartment of recipient cancer cells and play a major role in the activation of the metastatic process. We found a selective enrichment of YB-1 in sEVs from MDA and 4T1 cells, with [~]65% and 50% of all sEVs positive for YB-1 by d-STORM. Administration of sEVs from wild-type MDA and 4T1 to their YB-1 knockout counterparts resulted in nuclear translocation of sEV-associated YB-1 and increased tumorsphere formation. Pharmacological blockade of the nuclear transport machinery based on the inhibition of the formation of the "VOR" complex (VAP-A-ORP3-Rab7) by PRR851 impaired both nuclear translocation and the YB-1-induced increase in tumorsphere formation. YB-1 phosphorylation at S102 was required for nuclear localization. In fact, loss of YB-1 phosphorylation inhibited tumorsphere growth and stemness of cancer cells and YB-1-positive sEVs restored the oncogenic behavior of cancer cells expressing phospho-mutant YB-1. Moreover, PRR851 inhibited the nuclear translocation of the phosphorylated form of YB-1 and the oncogenic behavior of the TNBC cells. These data support the conclusion that the nuclear translocation of sEV-associated phosphorylated YB-1 is an important factor in the malignant behavior of TNBC and a potential therapeutic target.

Hepatocellular carcinomas (HCC) are genetically heterogeneous cancers frequently characterized by MYC gene amplification or hyperactivating {beta}-catenin (CTNNB1) mutations. Analysis of TCGA transcriptomics revealed that MYC-driven HCC tumors have decreased expression of mtDNA-encoded genes, but increased expression of nuclear-encoded mitochondrial genes. To investigate this apparent discrepancy, we generated MYC- and CTNNB1-driven murine HCCs, all of which displayed aberrant mitochondrial metabolism. Notably, MYC-driven tumors exhibited significant reductions in OXPHOS and TCA cycle activity that correlated with increased ROS levels, as well as elevated mitochondrial turnover through mitochondrial fission and mitophagy. MYC induces the expression of nuclear respiratory factor 1 (NRF1), which regulates DRP1 and other genes to promote receptor-mediated mitophagy. Knocking out DRP1 reduced mitophagy and ROS levels and promoted survival of HCC-bearing mice. These results identify elevated mitochondrial turnover as a potential therapeutic target in MYC-driven HCC. SignificanceHepatocellular carcinoma can arise from multiple oncogenes, making targeted therapy more difficult. Here we show that tumors with MYC amplification lose mitochondrial function via fission and mitophagy upregulation. Targeting mitochondrial quality control results in increased survival suggesting a therapeutic window in MYC-driven HCC.

Glioblastoma (GBM) is a highly lethal brain cancer with limited therapeutic durability, where the majority of patients develop recurrent or persistent disease after standard chemoradiotherapy. Meanwhile, tRNA-derived fragments (tRFs) have become increasingly relevant to cancer biology; however, their clinical relevance in GBM remains undefined. Here, we report that a specific family of tRFs, 5-tRNA halves (tiR5s) dominates the small RNA landscape of GBM patient tumors and associates with worse overall survival, post-therapeutic disease persistence, and pro-invasive proteogenomic pathways across two independent GBM patient cohorts. This association between elevated tiR5 levels and therapeutic resistance re-emerges in radiation-resistant GBM xenograft models. Our findings reveal that tiR5s are an underappreciated molecular feature of highly aggressive GBM tumors, supporting further investigation into their biological roles and prognostic utility in GBM. HighlightsO_LItiR5s are the predominant tRF family in primary GBM patient tumors C_LIO_LIElevated tiR5 expression distinguishes primary GBM tumors that develop persistent disease after first-line therapy C_LIO_LIRadiation-resistant GBM PDX models show elevated tiR5 expression C_LIO_LIElevated tiR5 expression associates with poor overall patient survival and pro-invasive molecular programs in GBM patient tumors C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/738483v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@184ddc1org.highwire.dtl.DTLVardef@1faadc2org.highwire.dtl.DTLVardef@a5ae02org.highwire.dtl.DTLVardef@1431506_HPS_FORMAT_FIGEXP M_FIG C_FIG

Myelodysplastic syndromes (MDS) are driven by somatic mutations in hematopoietic stem and progenitor cells (HSPCs), leading to clonal expansion and ineffective hematopoiesis. Hypomethylating agents (HMAs; azacitidine or decitabine) are the standard of care for higher-risk MDS. However, their effects on the bone marrow (BM) microenvironment, and the extent to which these changes correlate with clinical response, remain poorly understood. We performed longitudinal analyses of BM aspirates, trephine biopsies, and peripheral blood samples from MDS patients treated with azacitidine in a clinical trial (NCT03493646), integrating CyTOF, 5' single-cell RNA and TCR sequencing, plasma proteomics, and multiplex immunofluorescence microscopy to characterize changes associated with azacitidine response. Clinical responders showed expansion of GzmBCD56CD8 T cells together with increased type I and type II interferon signaling within the T-cell compartment. Responders also exhibited marked alterations in circulating platelet- and myeloid-derived factors with the potential to remodel the BM niche. Spatial analyses revealed expansion of neighborhoods enriched for CXCL12-abundant reticular cells and CD8 T cells in responders, whereas HSPC-enriched neighborhoods were largely unchanged. In contrast, several HSPC-enriched neighborhoods expanded in non-responders. These microenvironmental changes were accompanied by evidence of enhanced myelopoiesis in clinical responders. Our findings support a model in which azacitidine response extends beyond direct effects on malignant hematopoietic cells to involve coordinated remodeling of the BM microenvironment which may be reinforced by platelet- and myeloid-derived signals that establish a feed-forward circuit promoting productive hematopoiesis.

The role of cancer-associated fibroblasts (CAFs) in glioblastoma remains unclear, as their existence in the brain tumor microenvironment is still debated, given that the normal brain parenchyma is devoid of fibroblasts. It is unclear whether cells described as CAFs represent a distinct stromal population or a transcriptional state of perivascular cells such as pericytes. The aim of this study was to determine the identity, origin, and functional relevance of CAFs in glioblastoma. We analyzed 54 single-cell RNA sequencing datasets together with 88 bulk RNA sequencing samples. We identified a continuous transcriptional spectrum linking endothelial cells, pericytes, and CAFs, supporting pericytes as the most likely source of CAFs in glioblastoma. We further derived and validated robust CAF- and pericyte-specific gene signatures, enabling clear separation of these populations across cohorts. Reproducible CAF-associated ligand-receptor interactions were enriched in angiogenesis and immune modulation pathways. In bulk RNA-seq data, both CAF signature scoring and deconvolution consistently demonstrated increased CAF abundance in IDH-wildtype gliomas and further enrichment after chemoradiotherapy, while selective CYP1B1 expression in CAFs suggested a potential association with therapy-induced tumor adaptation. Overall, CAFs represent a distinct, pericyte-related stromal population in glioblastoma with conserved transcriptional and signaling programs. High CAF signature scores were associated with poorer overall and progression-free survival and were enriched in IDH-wildtype and post-chemoradiotherapy gliomas, suggesting a role for CAFs in therapy-associated remodeling of the tumor microenvironment in aggressive disease.

Transcriptional condensates anchored by chromatin readers are increasingly recognized as organizing hubs for gene expression, but how their assembly and stability are regulated remains poorly understood. Here, we identify an acetylation-dependent feed-forward circuit that controls the integrity of the Super Elongation Complex (SEC), a key driver of transcriptional elongation. We show that the SAGA histone acetyltransferase catalytic subunits KAT2A/KAT2B license acetylation of both histone H3 lysine 9 (H3K9ac) and SEC components themselves, including ENL, AFF1, and AFF3. Loss of this dual acetylation activity, achieved via a cereblon-recruiting PROTAC (GSK983/GSK699), displaces the chromatin reader ENL from target loci, dissolves ENL-anchored transcriptional condensates, and disrupts SEC-dependent transcriptional output - linking histone and non-histone acetylation to the physical integrity of a core transcriptional machine. Using genome-scale dependency data, we show that the SAGA complex is a selective chromatin dependency in acute myeloid leukemia (AML) AML and hematological malignancies and disrupting this feed-forward transcriptional circuit in AML demonstrates subtype independent antileukemia effects. KAT2A/B degradation drives potent, broad-spectrum antileukemic activity across genetically diverse AML cell lines, primary patient samples, and an isogenic KMT2A-rearranged model bearing cooperating oncogenic mutations, with H3K9ac loss concentrated asymmetrically at core AML oncogene loci such as MYC, MYB, and the HOXA cluster. Together, these findings define an acetylation-dependent circuit governing SEC integrity and establish KAT2A/B degradation as a mechanism-based, pan-AML therapeutic strategy, with implications for transcriptional condensate regulation beyond leukemia. HIGHLIGHTSO_LIThe SAGA complex is a selectively essential chromatin dependency across hematological malignancies and particularly in AML C_LIO_LIKAT2A/B degradation drives broad anti-leukemic activity across genetically diverse AML subtypes including chemo-refractory disease C_LIO_LIKAT2A/B degradation depletes H3K9ac at AML oncogene loci and dismantles ENL-anchored condensates C_LIO_LIKAT2A/B licenses regulation of super elongation complex acetylation and ENL interaction with SEC complex components C_LI