Coordinated initiation of DNA replication is essential to ensure efficient and timely DNA synthesis. Yet, molecular mechanism describing how replication initiation is coordinated in eukar-yotic cells is not completely understood. Herein, we present data demonstrating a novel feature of RNAs transcribed in the proximity of actively replicating gene loci. We show that RNAs aN-Choring ORC1 (ANCORs) to the histone variant H2A.Z are licensors of the DNA replication process. This ANCOR-H2A.Z interaction is essential for cells to initiate duplication of their ge-netic material. Widespread and locus-specific perturbations of these transcripts correlate with anomalous replication patterns and a notable loss of the H2A.Z replicative marker at the origin site. Collectively, we present a previously undescribed RNA-mediated mechanism that is associ-ated with the generation of active replication origins in eukaryotic cells. Our findings delineate a strategy to modulate the origins of replication in human cells at a local and global level, with potentially broad biomedical implications.

Abstract Hypomethylating agents (HMAs), including azacitidine and decitabine, are widely used for high-risk myelodysplastic syndromes (MDS) and selected AML, yet most patients eventually relapse, and survival after HMA failure remains dismal. HMAs were long assumed to function primarily by reactivating tumor suppressor genes through DNA demethylation. However, our recent work demonstrated that HMAs paradoxically upregulate the oncofetal gene SALL4, and that this induction strongly correlates with inferior overall survival, challenging existing paradigms. Emerging data suggest that additional cancer-germline antigen genes (CGAGs) may be similarly reactivated, generating oncogenic programs that facilitate resistance and leukemic progression. To investigate this, we profiled paired pre- and post-HMA patient marrow samples using RNA-seq and methylation analysis. We observed consistent post-treatment upregulation and promoter hypomethylation of SALL4 and multiple CGAGs, including PIWIL2, HORMAD1, and DDX43. Induction was more pronounced in patients with progression or treatment failure. Functional studies in MDS cell models revealed that forced expression of SALL4 or selected CGAGs enhanced proliferation, impaired myeloid differentiation, and reduced sensitivity to HMAs. Conversely, shRNA-mediated depletion mitigated these phenotypes. CRISPR-DiR-driven locus-specific demethylation of SALL4 recapitulated HMA-associated activation and conferred a proliferative advantage, confirming a causal role for promoter demethylation in SALL4 reactivation. We next evaluated therapeutic vulnerabilities associated with this pathway. Treatment with a newly developed small-molecule SALL4 degrader (SH6) reduced viability in SALL4-high models and reversed HMA-induced resistance phenotypes. Combination treatment with HMA + SH6 demonstrated enhanced cytotoxicity compared to either agent alone. Ongoing studies are expanding this approach to additional CGAG targets and mapping resistant subpopulations using single-cell transcriptomic and epigenetic profiling. Together, these findings reveal that HMA therapy can trigger unintended activation of oncogenic CGAG programs that drive MDS progression. They support a new mechanistic framework in which treatment-induced epigenetic reawakening promotes malignant fitness, and they identify SALL4 and CGAGs as high-value biomarkers and therapeutic targets. This work motivates development of companion diagnostic strategies and combination regimens pairing HMAs with targeted degraders to prevent therapy-induced disease acceleration and improve patient outcomes. Citation Format: Junsu Kwon, Yanjing Liu, Mahmoud A. Bassal, Julie A.I. Thoms, Emiliano Fabiani, John Pimanda, Maria T. Voso, Daniel G. Tenen, Li Chai. HMA-induced oncogene reactivation as a driver of disease progression in MDS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5288.

Stem cells reside in specialized microenvironments, termed niches, at several different locations in tissues1, 2-3. The differential functions of heterogeneous stem cells and niches are important given the increasing clinical applications of stem-cell transplantation and immunotherapy. Whether hierarchical structures among stem cells at distinct niches exist and further control aspects of immune tolerance is unknown. Here we describe previously unknown new hierarchical arrangements in haematopoietic stem cells (HSCs) and bone marrow niches that dictate both regenerative potential and immune privilege. High-level nitric oxide-generating (NOhi) HSCs are refractory to immune attack and exhibit delayed albeit robust long-term reconstitution. Such highly immune-privileged, primitive NOhi HSCs co-localize with distinctive capillaries characterized by primary ciliated endothelium and high levels of the immune-checkpoint molecule CD200. These capillaries regulate the regenerative functions of NOhi HSCs through the ciliary protein IFT20 together with CD200, endothelial nitric oxide synthase and autophagy signals, which further mediate immunoprotection. Notably, previously described niche constituents, sinusoidal cells and type-H vessels2, 3, 4, 5, 6, 7, 8, 9-10 co-localize with less immune-privileged and less potent NOlow HSCs. Together, we identify highly immune-privileged, late-rising primitive HSCs and characterize their immunoprotective niches comprising specialized vascular domains. Our results indicate that the niche orchestrates hierarchy in stem cells and immune tolerance, and highlight future immunotherapeutic targets.

Aberrant DNA methylation is a well-known feature of hematological malignancies, frequently leading to tumor suppressor genes silencing. The CCAAT enhancer binding protein alpha (CEBPA) gene, a critical regulator of myeloid differentiation and a known tumor suppressor, is often epigenetically repressed in leukemias by DNA methylation (Hackanson et al., 2007). Small activating RNAs (saRNAs) are a novel class of therapeutic agents capable of upregulating gene expression, although their mechanisms of action remain incompletely understood (Ghanbarian et al., 2021). Here, we studied the saRNA AW1-51, which targets the CEBPA locus and is currently in clinical trials for advanced liver cancer (ClinicalTrials.gov: NCT02716012; EudraCT 2021-005431-23) (Sarker et al., 2020) and advanced solid tumors (ClinicalTrials.gov: NCT04105335) (Plummer et al., 2025), to provide deeper insight into its molecular mechanism of action. Specifically, we assessed its ability to induce transcriptional activation of CEBPA through epigenetic modulation, since the CEBPA locus is methylation sensitive (Di Ruscio et al., 2013). Our study was developed using K562 chronic myeloid leukemia cells. Notably, K562 cells do not express detectable CEBPA protein (Perrotti et al., 2002), which provides a suitable system for investigating reactivation mechanisms specifically at the transcriptional level. To complement this model, since CEBPA is also a known tumor suppressor in lung cancer (Halmos et al., 2002), we also used A549 lung adenocarcinoma cells. A549 cells express basal CEBPA protein, as a surrogate system to study post-transcriptional events such as protein dynamics and mRNA stability in a context including CEBPA protein restoration. Upon AW1-51 transfection, we observed a specific demethylation of the CEBPA locus promoter region and a significant upregulation of CEBPA transcripts in both models. Remarkably, in A549 cells this was accompanied by an early CEBPA protein upregulation that preceded its mRNA upregulation, as detected by western blot and immunofluorescence. The described effects occurred in the absence of global changes in the genome DNA methylation profile. We are currently exploring the mechanisms underlying this early protein induction through AW1-51 pull-down mass spectrometry and mRNA stability assays. In conclusion, our findings reveal a novel locus-specific demethylation mechanism mediated by AW1-51, coupled with early protein upregulation, providing new insights into saRNA molecular mechanisms. These results may be also relevant for the development of saRNA-based therapies targeting silenced key tumor suppressor genes in hematologic malignancies, such as myeloid leukemias, where their restoration represents a promising therapeutic strategy.

Small activating RNAs are short double-stranded RNAs designed to upregulate transcription of target genes. By this virtue, they can be used to restore expression of genes frequently silenced in cancer. AW1-51 (also referred to as CEBPA-51), the first small activating RNA therapeutic to enter clinical evaluation, has demonstrated biological activity and safety in Phase II trials for hepatocellular carcinoma, both as monotherapy and in combination with sorafenib, and in Phase 1a/1b in combination with pembrolizumab for patients with advanced solid tumors. It targets the master regulator CCAAT enhancer-binding protein alpha, abnormally silenced by DNA methylation in a wide range of hematological and non-hematological malignancies. However, the molecular events enabling this mechanism are only partially elucidated. In this study, we uncovered the molecular basis for AW1-51-induced transcriptional reactivation of CCAAT enhancer-binding protein alpha demonstrating that by directly promoting DNA demethylation of its promoter restores its expression, protein synthesis, and consequently cell differentiation. These findings unveil AW1-51 as a prototype for RNA-based precision medicine enabling conditional expression of CCAAT enhancer-binding protein alpha in diseases characterized by aberrant gene silencing and extending its potential therapeutic impact beyond cancer.

β-thalassemia and sickle cell disease are among the most prevalent genetic blood disorders globally. These conditions arise from mutations in the β-globin gene, leading to defective hemoglobin production and resulting in anemia. Current treatments include γ-globin inducers (eg, Hydroxyurea), blood transfusions, iron chelation therapy, and bone marrow transplantation. Recently approved disease-modifying agents and promising gene therapies offer hope, yet their broad application is constrained by scalability challenges. Traditionally, research and development for β-globinopathies have focused on γ-globin induction. However, the ε-globin variant, which is active during early embryonic development and subsequently silenced prenatally, was once considered noninducible by postnatal pharmacological means. Recent studies indicate that, akin to γ-globin, enhancing ε-globin expression could compensate for impaired β-globin synthesis, potentially ameliorating the clinical manifestations of β-globinopathies. This review critically examines the viability of ε-globin induction as a therapeutic strategy for β-thalassemia and sickle cell diseases. It also delves into the burgeoning research on the mechanisms governing ε-globin silencing and its pharmacological reactivation. We conclude with a discussion of prospective research directions and drug development initiatives aimed at exploiting ε-globin's therapeutic promise.

The transcription factor C/EBPα is a well characterized DNA binding protein with essential functions in controlling and regulating myeloid differentiation and lipid metabolism. Herein, we describe C/EBPα's separate and distinct RNA binding characteristics. Using Chromatin RNA Immunoprecipitation, we identified that C/EBPα interacts primarily with RNA introns in both HL-60 and THP-1 cells, with a preference for a palindromic GC-rich binding motif. Structural prediction in conjunction with RNA electrophoretic mobility shift assays show that C/EBPα interacts with RNA through two previously undescribed domains located towards the proteins N terminus. These domains are distinct from C/EBPα's DNA binding b-ZIP domain which is instead located on the proteins C terminus. Mouse bone marrow transplantation and in vitro cytokine assays reveal that C/EBPα RNA binding appears to be essential for macrophage maturation but not neutrophil differentiation. To better understand these phenotypic differences, we expanded our observations into the transcriptomic space by utilizing single-cell CITE-Seq. In line with biochemical data, notable mature cell populations were absent when the C/EBPα's RNA binding domains are excluded from the protein. Intriguingly, differential gene expression revealed strong, selective upregulation of Lipoprotein Lipase (Lpl) in monocyte, but not neutrophil populations (Panel 1). It has been previously reported that dysregulation of Lpl affects bone marrow monocyte progenitor differentiation and results in dysregulated cellular mobilization from the bone marrow, a phenotype that is commonly seen in cancers such as AML. The increased abundance of monocyte and neutrophil populations in the C/EBPα RNA deletion samples is therefore suggestive that C/EBPα RNA binding is critical in regulating terminal differentiation and mobilization circuits which may become dysregulated or perturbed in diseases such as AML. On-going analyses are investigating the RNA velocity trajectory of captured cell populations in an attempt to better understand at which stage C/EBPα RNA binding becomes essential for terminal maturation and differentiation of select cell populations. Taken together, we demonstrate that C/EBPα is also a bona fida RNA binding protein with unique functions distinct from its DNA binding activity that are essential for monocyte maturation, differentiation and mobilization. Panel 1 - Identified Cell Populations Following scCITE-Seq with C/EBPα Full-Length (WT) and C/EBPα RNA Binding Deletion (DD). Each circle represents a single cell presented in UMAP space. The left panel shows populations identified with the full-length C/EBPα construct. The right panel shows the cell populations identified with the RNA domain deletion construct. Notable differences in cell populations and numbers are evident. Overlayed onto the cells is the expression of Lipoprotein Lipase (Lpl). Expression is shown as a gradient from black up to green, representing a log2(4) expression. Lpl expression is shown to be expressed uniquely in the monocyte population of cells almost exclusively in the RNA deletion sample.

Immunomodulatory imide drugs (IMiDs) degrade specific C2H2 zinc finger degrons in transcription factors, making them effective against certain cancers. SALL4, a cancer driver, contains seven C2H2 zinc fingers in four clusters, including an IMiD degron in zinc finger cluster two (ZFC2). Surprisingly, IMiDs do not inhibit growth of SALL4 expressing cancer cells. To overcome this limit, we focused on a non-IMiD degron, SALL4 zinc finger cluster four (ZFC4). By combining AlphaFold and the ZFC4-DNA crystal structure, we identified a potential ZFC4 drug pocket. Utilizing an in silico docking algorithm and cell viability assays, we screened chemical libraries and discovered SH6, which selectively targets SALL4-expressing cancer cells. Mechanistic studies revealed that SH6 degrades SALL4 protein through the CUL4A/CRBN pathway, while deletion of ZFC4 abolished this activity. Moreover, SH6 led to significant 62% tumor growth inhibition of SALL4+ xenografts in vivo and demonstrated good bioavailability in pharmacokinetic studies. In summary, these studies represent a new approach for IMiD independent drug discovery targeting C2H2 transcription factors in cancer.

Transposable elements (TEs) are indispensable for human development, with critical functions in pluripotency and embryogenesis. TE sequences also contribute to human pathologies, especially cancer, with documented activities as cis/trans transcriptional regulators, as sources of non-coding RNAs, and as mutagens that disrupt tumor suppressors. Despite this knowledge, little is known regarding the involvement of TE-derived genes (TEGs) in tumor pathogenesis. Here, systematic analyses of TEG expression across human cancer reveal a prominent role for pogo TE derived with KRAB domain (POGK). We show that POGK acts as a tumor suppressor in triple-negative breast cancer (TNBC) cells and that it couples with the co-repressor TRIM28 to directly block the transcription of ribosomal genes RPS16 and RPS29, in turn causing widespread inhibition of ribosomal biogenesis. We report that POGK undergoes deactivation by isoform switching in clinical TNBC, altogether revealing its exapted activities in tumor growth control.

Oncofetal transcription factor SALL4 is essential for cancer cell survival. 1-5 Recently, several groups reported that immunomodulatory imide drugs (IMiDs) could degrade SALL4 in a proteasome-dependent manner. 6,7 Intriguingly, we observed that IMiDs had no effect on SALL4-positive cancer cells. Further studies demonstrated that IMiDs could only degrade SALL4A, one of the SALL4 isoforms. This finding raises the possibility that SALL4B, the isoform not affected by IMiDs, may be essential for SALL4-mediated cancer cell survival. SALL4B knockdown led to an increase in apoptosis and inhibition of cancer cell growth. SALL4B gain-of-function alone led to liver tumor formation in mice. Our observation that protein degraders can possess isoform-specific effects exemplifies the importance of delineating drug action and oncogenesis at the isoform level to develop more effective cancer therapeutics.

Myelodysplastic syndrome (MDS) is a group of heterogeneous diseases characterized by cytologic dysplasia and cytopenias resulting from ineffective hematopoiesis. Oncofetal protein SALL4 is a known oncogene in MDS and its baseline expression level serves as a prognostic biomarker for MDS at the time of diagnosis. In addition, a recent study showed that SALL4 upregulation following hypomethylating agent treatment in MDS patients correlates with poor outcomes. Despite its important mechanistic and diagnostic significance, the cellular identity of bone marrow cells with aberrant SALL4 expression in MDS patients remains unknown. In this study, we analyzed MDS bone marrow cells on single cell level by mass cytometry (CyTOF) and found that SALL4 was mainly aberrantly expressed in the hematopoietic stem and progenitor cells (HSPC) as well as myeloid lineages. Within the HSPC population from MDS patients, SALL4 and p53 were co-expressed, with the highest co-expressing clones harboring pathogenic TP53 mutations. Overall, our study characterizes for the first time the aberrant SALL4 expression in primary MDS patient samples at a single-cell level. Further studies on the SALL4/p53 network for in-depth mechanistic investigation are needed in the future. Key Points SALL4 expression in various MDS BM cells confirmed by mass cytometry (CyTOF). SALL4 and p53 double positive cells were predominantly found in the hematopoietic stem and progenitor cell (HSPC) population and associated with pathogenic TP53 mutation status.

DNA methylation is a fundamental epigenetic modification regulating gene expression. Aberrant DNA methylation is the most common molecular lesion in cancer cells. However, medical intervention has been limited to the use of broadly acting, small molecule-based demethylating drugs with significant side-effects and toxicities. To allow for targeted DNA demethylation, we integrated two nucleic acid-based approaches: DNMT1 interacting RNA (DiR) and RNA aptamer strategy. By combining the RNA inherent capabilities of inhibiting DNMT1 with an aptamer platform, we generated a first-in-class DNMT1-targeted approach – aptaDiR. Molecular modelling of RNA-DNMT1 complexes coupled with biochemical and cellular assays enabled the identification and characterization of aptaDiR. This RNA bio-drug is able to block DNA methylation, impair cancer cell viability and inhibit tumour growth in vivo. Collectively, we present an innovative RNA-based approach to modulate DNMT1 activity in cancer or diseases characterized by aberrant DNA methylation and suggest the first alternative strategy to overcome the limitations of currently approved non-specific hypomethylating protocols, which will greatly improve clinical intervention on DNA methylation.

Sal-like protein 4 (SALL4) is a C2H2 zinc finger transcription factor (TF) with two naturally occurring isoforms; SALL4A and SALL4B. It is typically detected in fetal tissues and silenced in most normal adult tissues. It is therefore intriguing that SALL4 is aberrantly re-expressed in about one-third of almost all primary human malignancies, presumably by demethylation dependent mechanisms. Direct evidence of the causative role of SALL4 in cancer has been demonstrated in SALL4 transgenic mice, which developed myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and/or liver tumors. Loss-of-function studies by SALL4 knock-down using shRNA showed cell growth inhibition and death in leukemias and solid tumors in culture and in in vivo xenotransplants. TF's, such as SALL4, which are critical for cancer development and survival, have historically been viewed as “undruggable”. However, rather than blocking activity, an alternative approach to target TFs is to induce protein degradation. Immunomodulatory imide drugs (IMiDs), including thalidomide, lenalidomide and pomalidomide, are used in treating patients with multiple myeloma (MM), MDS with 5q deletion, mantle cell lymphoma (MCL), and other hematological malignancies. Recently, several groups have reported that IMiDs can degrade SALL4 in a proteasome-dependent manner. To that end, we sought to investigate whether IMiDS could be utilized to treat SALL4-positive/expressing cancers. Initially, our studies observed that IMiDs had no effect on SALL4-positive cancer cells. Additional investigations thereafter demonstrated that IMiDs could only degrade SALL4A. These findings suggested that SALL4B may not be affected by IMiDs, and may be essential for SALL4-mediated cancer cell survival. Further investigation revealed that SALL4B knockdown led to an increase in apoptosis and inhibition of cancer cell growth. Moreover, through high-throughput screening, we identified a new non-IMiD SALL4 degrader that targets SALL4B via proteasomal degradation and which exhibited potent anti-cancer activity, inhibiting cancer cell proliferation in culture and in vivotumor growth by 70%. Our observation therefore suggest that protein degraders could possess isoform specific effects. Additionally, our results exemplify the importance of delineating drug action and oncogenesis at the isoform level to develop more effective cancer therapeutics.

There are multiple published studies correlating DNA methylation with gene expression, but to date, most are associative in nature without precise localization of causal elements. Multiple CpG destinations have been described with inconsistent evidence supporting their causal association with transcription. This has hampered adoption and use of methylation signatures as biomarkers or for targeted therapies. This has had consequent effects in hindering mechanistic understanding in how global demethylation agents lead to responses in some patients, but not others. With this state of the literature in mind, we set out to better understand and locate causal methylation regulatory loci genome-wide. We hypothesized that causal methylation regulatory elements possess heightened sensitivity to regulatory signals such as “demethylate”; signals that can be introduced using hypo-methylating agents (HMAs) typically used in treatment of Myelodysplastic syndromes and Acute Myeloid Leukemia. We thus analyzed 19 whole-genomic bisulfite sequenced sample pairs (pre/post-demethylation in patients, cell lines, and in murine in vivo samples) and identified previously undescribed DNA methylation sensitive elements throughout the genome, which we call Methylation Mesas (MMs) based on the profile shape observed in naïve and post-HMA treated sample. These narrow-width MMs range typically between 45-300bp wide and are present throughout the genome with greater abundance in noncoding and intragenic regions than in promoter-CpG islands. We also demonstrate that MM show an up to 90% concordant overlap with primed and active histone marks by means of a compiled histone mark footprint map using 60 human and 59 murine ENCODE datasets for each respective species, suggesting a functional role for MMs as regulatory elements with transcriptional potential. While HMA's enable identification of MM's genome-wide, their mode of action renders them unsuitable for ascertaining the direct transcriptional potential of any singular locus. Therefore, in order to ascertain the causal role of MM for transcriptional regulation, we developed a fine-resolution targeted demethylation technology(CRISPR-DiR), leveraging our previously published findings that DNA methyltransferase I (DNMT1) can be blocked by DNMT1-interacting RNAs (DiR). Compared with other targeted demethylation technologies with wider effect windows (eg. CRISPR-TET1), CRISPR-DiR shows superior locus specificity thus serving as an ideal tool to identify causal regulatory elements. Using tumor suppressor p16 as an example, we elucidated that demethylation of a single MM in the first exon is sufficient to trigger locus and distal chromatin rewiring events that natively initiate expression to a significantly greater extent than promoter CpG island targeting alone. Similar CRISPR-DiR activation is also observed in four additional, natively silenced tumor suppressor genes, one of which has no annotated CpG island. In summary, we have identified a previously undescribed narrow-width DNA methylation sensitive regulatory element, consistent across all epigenomes investigated, located largely independent of the promoter CpG island dogma, yet initiating gene transcription more potently. Identification of said elements paves the way for future studies to better understand the mechanisms of how mutations in DNA methyltransferases lead to disease, how global hypomethylating agents can lead to a response in diseases such as MDS and AML but can also activate oncogenes, and finally development of targeted therapies.

Cellular metabolism (or energetics) and epigenetics are tightly coupled cellular processes. It is arguable that of all the described cancer hallmarks, dysregulated cellular energetics and epigenetics are the most tightly coregulated. Cellular metabolic states regulate and drive epigenetic changes while also being capable of influencing, if not driving, epigenetic reprogramming. Conversely, epigenetic changes can drive altered and compensatory metabolic states. Cancer cells meticulously modify and control each of these two linked cellular processes in order to maintain their tumorigenic potential and capacity. This review aims to explore the interplay between these two processes and discuss how each affects the other, driving and enhancing tumorigenic states in certain contexts.

The interaction of germline variation and somatic cancer driver mutations is under-investigated. Here we describe the genomic mitochondrial landscape in adult acute myeloid leukaemia (AML) and show that rare variants affecting the nuclear- and mitochondrially-encoded complex I genes show near-mutual exclusivity with somatic driver mutations affecting isocitrate dehydrogenase 1 ( IDH1 ), but not IDH2 suggesting a unique epistatic relationship. Whereas AML cells with rare complex I variants or mutations in IDH1 or IDH2 all display attenuated mitochondrial respiration, heightened sensitivity to complex I inhibitors including the clinical-grade inhibitor, IACS-010759, is observed only for IDH1 -mutant AML. Furthermore, IDH1 mutant blasts that are resistant to the IDH1-mutant inhibitor, ivosidenib, retain sensitivity to complex I inhibition. We propose that the IDH1 mutation limits the flexibility for citrate utilization in the presence of impaired complex I activity to a degree that is not apparent in IDH2 mutant cells, exposing a mutation-specific metabolic vulnerability. This reduced metabolic plasticity explains the epistatic relationship between the germline complex I variants and oncogenic IDH1 mutation underscoring the utility of genomic data in revealing metabolic vulnerabilities with implications for therapy.

Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) is a technique used to identify protein-DNA interaction sites through antibody pull-down, sequencing and analysis; with enrichment 'peak' calling being the most critical analytical step. Benchmarking studies have consistently shown that peak callers have distinct selectivity and specificity characteristics that are not additive and seldom completely overlap in many scenarios, even after parameter optimization. We therefore developed ChIP-AP, an integrated ChIP-seq analysis pipeline utilizing four independent peak callers, which seamlessly processes raw sequencing files to final result. This approach enables (1) better gauging of peak confidence through detection by multiple algorithms, and (2) more thoroughly surveys the binding landscape by capturing peaks not detected by individual callers. Final analysis results are then integrated into a single output table, enabling users to explore their data by applying selectivity and sensitivity thresholds that best address their biological questions, without needing any additional reprocessing. ChIP-AP therefore presents investigators with a more comprehensive coverage of the binding landscape without requiring additional wet-lab observations.

The phosphoinositide 3-kinase (PI3K) pathway represents the most hyperactivated oncogenic pathway in triple-negative breast cancer (TNBC), a highly aggressive tumor subtype encompassing ∼15% of breast cancers and which possesses no targeted therapeutics. Despite critical contributions of its signaling arms to disease pathogenesis, PI3K pathway inhibitors have not achieved expected clinical responses in TNBC, owing largely to a still-incomplete understanding of the compensatory cascades that operate downstream of PI3K. Here, we investigated the contributions of long noncoding RNAs (lncRNAs) to PI3K activities in clinical and experimental TNBC and discovered a prominent role for LINC01133 as a PI3K-AKT signaling effector. We found that LINC01133 exerted protumorigenic roles in TNBC and that it governed a previously undescribed mTOR Complex 2 (mTORC2)-dependent pathway that activated AKT in a PI3K-independent manner. Mechanistically, LINC01133 induced the expression of the mTORC2 component PROTOR1/PRR5 by competitively coupling away its negative messenger RNA (mRNA) regulator, the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1). PROTOR1/PRR5 in turn was sufficient and necessary for LINC01133-triggered functions, casting previously unappreciated roles for this Rictor-binding protein in cellular signaling and growth. Notably, LINC01133 antagonism undermined cellular growth, and we show that the LINC01133-PROTOR1/PRR5 pathway was tightly associated with TNBC poor patient survival. Altogether, our findings uncovered a lncRNA-driven signaling shunt that acts as a critical determinant of malignancy downstream of the PI3K pathway and as a potential RNA therapeutic target in clinical TNBC management.