Abstract Fusion-positive rhabdomyosarcoma (FP-RMS) is a pediatric soft tissue carcinoma characterized by poor survival rate and limited therapeutic options. The fusion oncoprotein PAX3-FOXO1 (P3F) is the key driver for FP-RMS, and its transcriptional activity is dependent on histone acetyltransferase CBP/p300. Given the challenge of directly targeting the transcription factor P3F, CBP/p300 represents an attractive therapeutic target in treating FP-RMS. Through a focused screening of different epigenetic-modifying agents against FP-RMS, the inhibition of CBP/p300 emerged as the most effective strategy in selectively suppressing P3F-driven transcriptional activity. To overcome the limitations, such as toxicity and efficacy of the existing CBP/p300 inhibitors, we employed structure-based drug design to develop a potent CBP/p300 dual inhibitor, IHK-44. We used chemical genetic approaches to enable precise and temporal control of its function in investigating the underlying mechanism of CBP/p300 in the context of FP-RMS. In studying the basis of IHK-44’s selectivity and potency in FP-RMS compared to other CBP/p300 inhibitors and degraders, we conducted a comprehensive series of studies, including luciferase reporter assays, live-cell imaging, RNA-seq, ChIP-seq, and proteomics. These studies revealed that FP-RMS exhibits unique epigenetic dependencies, particularly at 3D clusters of histone acetylation lacking CpG islands. The predictor of gene responsiveness to CBP/p300 inhibition with IHK-44 is to be found by mapping the 3D enhancer/promoter loops into clusters and quantifying the extent of CpG island involvement in the elements that make up clusters. Genes driven CpG-poor clusters, often very rich in enhancers, are hypersensitive to IHK-44, while CpG-rich clusters, usually promoter-rich, are recalcitrant. This allows us to define clear categorization to distinct types of gene regulation hubs in the cancer epigenome. High-throughput screening across 900 cell lines revealed that FP-RMS is one of the cancer subtypes most sensitive to IHK-44, whereas normal cell lines and fusion-negative RMS were resistant. In summary, our findings suggest a novel therapeutic strategy for FP-RMS by targeting CBP/p300 and highlight the potential of IHK-44 as a promising candidate for further preclinical development. Citation Format: Md Imdadul H. Khan, Matthew Chang, Yaw Asante, Maya Al-Haddad, Jordyn Kelly, Bhavatharini Udhayakumar, Carrietta Farma-Hai, Berkley Gryder. Mechanistic understanding of a novel CBP/p300 inhibitor in fusion-positive rhabdomyosarcoma [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 7061.

Acquired resistance to targeted therapies is the primary barrier to durable cancer remission. Therapy resistance is often associated with stemness and intermediate EMT programs, which are often viewed as proximal resistance mechanisms. On the other hand, a growing body of evidence suggests that these programs facilitate resistance through plasticity-mediated adaptations. Integrating computational modeling, functional experimental assays, and lineage tracing, we investigated the relationship between EMT and therapy resistance in experimental models of acquired resistance to ALK+ lung cancer. Our results support a model where phenotypic plasticity, associated with intermediate EMT, is a selectable trait, and selection for subpopulations with higher phenotypic plasticity is amplified under a multifactorial resistance scenario. Consequently, resistance to targeted therapy is associated with higher ability to adapt to orthogonal therapeutic and environmental stressors, as well as higher metastatic potential. These findings identify cellular plasticity as the fundamental substrate from which multifactorial resistance and metastatic competence evolve, indicating that targeting phenotypic plasticity can suppress the acquisition of resistance and prolong therapeutic responses. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Rhabdomyosarcoma (RMS) is a high-risk and lethal pediatric sarcoma that resembles developing skeletal muscle. RMS tumors have low mutation burdens, but these scant mutations often alter genes involved in transcriptional control. Transcriptional dysregulation is critical to RMS pathogenesis, supported by studies in both RMS tumors carrying mutationally derived chimeric transcription factors (“fusion positive (FP)”), or those without (“fusion negative” (FN)). However, mechanisms to selectively target dysregulated transcription in RMS remain outstanding. Here, we develop a novel approach targeting RMS transcription comprising simultaneous targeting of two distinctly acting transcriptional co-activators. We discover a common cell identity-controlling pan-RMS core regulatory circuit (CRC) composed of oncogenic and lineage-specific myogenic master transcription factors (mTFs). These mTFs are regulated by super-enhancers, and they co-bind genome-wide to control the malignant transcriptome of both FP- and FN-RMS. Using a super-enhancer-based reporter screen, we identify the EP300/CBP inhibitor A485 as a potent inhibitor of the pan-RMS CRC, though efficacy of this compound was limited by toxicity. To enhance on-target specificity, we identify the protein EYA2 as a co-factor that binds directly to SIX1, a member of the pan-RMS CRC and exploit a recently developed second-generation EYA1/2 tyrosine phosphatase inhibitor, LG1-34, to inactivate its function. While A485 and LG1-34 independently reduce mTF transcription and drive RMS cell death, in combination, these agents function synergistically to reduce RMS growth in vitro and in vivo. These results demonstrate that combined targeting of enhancer maintenance and CRC cofactors is a powerful strategy to suppress the RMS transcriptome and enforce RMS cell death. Citation Format: Annika Gustafson, Stephanie Nance, Berkley Gryder, Noha A. Shendy, Lars Wick, Grace McKay-Corkum, K. Elaine Ritter, Stephen Connor Purdy, Arthur R. Wolin, Sheera R. Rosenbaum, Sabateeshan Mathavarajah, Nickerson A. Demelfi, Yueyang Wang, Yang Zhang, Mark W. W. Zimmerman, Anoop M. Kavirayani, John Hardin, Alexander LaVeck, Xiang Wang, Neekesh V. Dharia, Andrew Hong, Guillaume Kugener, Jesse S. Boehm, Jennifer Roth, Javed Khan, Francisca Vasquez, Kristin B. Artinger, Rui Zhao, David M. Langenau, Jun Qi, Kimberly Stegmaier, Heide L. Ford, Adam D. Durbin, Brian J. Abraham. Synergistic targeting of EP300/CBP and EYA co-activators collapses the rhabdomyosarcoma core regulatory circuit [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 3500.

SUMMARY RNA Polymerase II (Pol2) transcribes genes through a complex life cycle (initiation, pausing, elongation, co-transcriptional splicing, termination, and recycling). Chromatin immunoprecipitation of Pol2 before and after chemical perturbation has identified promoter-proximal accumulation (pausing) as a critical step in the transcription genome-wide. However, the full landscape of Pol2 responses has not been well characterized. Here, we introduce a tool for comparing Pol2 Activity State Shifts (compPASS), a computational pipeline which uses data from paired ChIP-based approaches to assign genes to one of eight distinct modes by Pol2 response under different forms of perturbation. In multiple cancer types and drug contexts, we show that compPASS identifies previously undescribed Pol2 failure modes with important implications for gene regulation. By looking past pausing, compPASS exposes Pol2 failure modes (clogging, entry, gain, loss) that are rare but pinpoint the genes most relevant to cancer cell state changes in response to therapy, turning a single paired Pol2 ChIP-seq into a mechanistic map of shifting transcriptional states.

CoREST complexes (LSD1, HDAC1/2, and RCoR1/2/3) are pivotal in neurodevelopment and have long been recognized as transcriptional repressors across various cancers. However, distinct roles of RCoR factors remain underexplored. Here, we unveil non-canonical functions of RCoR2 in MYCN-amplified neuroblastoma (NB), underscoring its unique significance compared to its paralogs. This insight shifts the paradigm, highlighting RCoR2 as a key determinant of the NB chromatin landscape. Our findings demonstrate that RCoR2 is a super-enhancer-driven gene, which, unlike RCoR1, acts as a positive regulator of gene expression as a partner of the adrenergic NB core regulatory circuitry (CRC). We propose a model in which RCoR2 facilitates interactions between CRC-bound enhancers and their associated transcription start sites, thereby sustaining the expression of genes critical for NB cell survival. Thus, we identify RCoR2 as a critical vulnerability in high-risk NBs and a promising target for cancer therapeutics.

In oncology, patients treated with targeted therapies often have strong and durable responses early in treatment; however, the development of resistance hinders long-term therapeutic benefit. A subset of tumor cells can develop resistance, often from both genetic and non-genetic mechanisms through gradual, multi-step resistance. Nonetheless, predicting which cells will evolve to become resistant, and strategies to suppress adaptability contributing to resistance remain unknown. While resistance may emerge in cells harboring driver mutations, the adaptation process necessitates substantial modifications to the phenotype facilitated by cellular plasticity. Therefore, plasticity serves as a crucial facilitator of resistance by enhancing adaptability, rather than functioning as a direct cause of resistance. We hypothesize that the direct selection for therapy resistance leads to indirect selection for enhanced plasticity. We propose that as treatment selects for drug-resistant phenotypes, concurrent selection for cells exhibiting greater adaptability occurs. Thus, enhanced plasticity is indirectly favored during selection for drug resistance. We have found strong support for this hypothesis through in silico modeling, with subsequent validation through functional assays. In our functional assays, we utilize the H3122 cell line derived from a patient with ALK+ non-small cell lung cancer (NSCLC) to model targeted therapy resistance. This well-studied model effectively mimics the initial responsiveness and subsequent resistance observed in clinical settings. Utilizing single-cell sorting and the expansion of clonal isolates from the parental H3122 population, we identified heterogeneity in the ability to adapt where subgroups exhibited a range of high adaptation potential (HAP) to low adaptation potential (LAP) based on their response to numerous independent stressors, including targeted ALK inhibitors. We have found that therapeutic stressors lead to selection of subpopulations with higher adaptability potential, enriching for a more lethal subgroup than the original tumor. HAP cells display increased multi-drug resistance and metastatic potential compared to LAP cells. HAP cells are significantly enriched in plasticity-related pathways overlapping with those found in cells that have developed strong resistance to ALK inhibitors. In subcutaneous tumor models, LAP cells show a 1, 000-fold reduction in tumor burden, while HAP cells develop resistance. Our findings suggest that disrupting cellular adaptability can prevent resistance, maintaining cells in a therapy-sensitive state. This approach shifts the focus from killing tumor cells to blocking their ability to adapt, opening up a novel angle for overcoming resistance. Addressing adaptability reveals intervention points that conventional strategies might overlook, offering new, proactive strategies in cancer treatment. Alicia Bjornberg, Xierali Aobuli, Matthew Froid, Rowan Barker-Clarke, Jeff Maltas, Jacob Scott, Berkley Gryder, David Bassanta, Alexander Anderson, Virginia Turati, Andriy Marusyk. Cellular plasticity facilitates therapy resistance through enhancing adaptability. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2674.

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer. Most MCCs contain Merkel cell polyomavirus (virus-positive MCC; VP-MCC), and the remaining are virus-negative (VN-MCC). Immune checkpoint inhibitors are the first-line treatment for metastatic MCC, but durable responses are achieved in less than 50% of patients. To identify new treatments, we screen ~4,000 compounds for their ability to reduce MCC viability and demonstrate that VP-MCC and VN-MCC exhibit distinct response profiles. Aurora kinase inhibitors selectively reduce VP-MCC viability, with RNAi screening independently identifying AURKB as an essential gene for MCC survival, especially in VP-MCC. AZD2811, a selective AURKB inhibitor, induces mitotic dysregulation and apoptosis in MCC cells, with greater efficacy in VP-MCC. In mice, AZD2811 nanoparticles inhibit tumor growth and increase survival in both VP-MCC and VN-MCC xenograft models. Overall, our unbiased screens identify AURKB as a promising therapeutic target and AZD2811NP as a potential treatment for MCC.

INTRODUCTION:Measuring the chromatin state of a tumor provides a powerful map of its epigenetic commitments; however, as these are generally bulk measurements, it has not yet been possible to connect changes in chromatin accessibility to the pathological signatures of complex tumors. In parallel, recent advances in computational pathology have enabled the identification of spatial features and immune cells within oral cavity tumors and their microenvironment. METHODS:Here, we present pathogenomic fingerprinting (PaGeFin), a novel method that integrates morphological tumor features with chromatin states using ATAC-seq. This framework links spatial morphologic and epigenetic features, offering insights into tumor progression and immune evasion within and across tumors. Morphologic features describing spatial relationships between tumor and lymphocyte cells that are prognostic of oral cavity squamous cell carcinoma (OSCC) were identified through AI-driven pathology analysis. These pathomic features were spatially colocalized within the epigenome of 4 distinct sections of 4 OSCC tumors. RESULTS:These key features pinpointed chromatin regions responsible for critical immune cell function through peak locations and enrichment analysis, highlighting loci of CD27+ memory B cells, helper CD4+ T cells, and cytotoxic CD8 naïve T cells that likely drive morphologic changes in the distribution of lymphocytes in the tumor microenvironment and promote aggressive tumor behavior. Gene Ontology analysis revealed that the CTLA4, CD79A, CD3D, and CCR7 genes were embedded in these regions. CONCLUSION:This computational approach is the first to assess the correlation between pathomic and epigenetic features in the context of cancer.

BACKGROUND:Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. Historically classified based on histology, advances in molecular profiling have allowed further sub-classification, which has improved risk stratification. Although molecular profiling has improved our understanding of disease progression and risk, the molecular evolution of therapy resistance in RMS remains poorly characterized. Transcriptomic profiling of patients with high-risk, relapsed RMS was undertaken with the goal of uncovering insights into the biology of RMS treatment failure. PROCEDURE:Formalin-fixed, paraffin-embedded (FFPE) tissue samples from patients with relapsed RMS who had samples archived at diagnosis and relapse were obtained. Histologic subtype and PAX3/7::FOXO1 fusion status were confirmed. Transcriptomic profiling of the FFPE tissue samples was performed using the high-throughput genomics (HTG) whole transcriptome panel. RESULTS:We identified 11 patients with relapsed RMS who had FFPE tissue samples archived at diagnosis and relapse following multimodality therapy. All patients were stratified as high risk, including five with PAX3/7::FOXO1 fusion-positive RMS (FP-RMS) and six with PAX3/7::FOXO1 fusion-negative RMS (FN-RMS). The transcriptomic analysis revealed that the myogenesis pathway and markers associated with myogenic differentiation were enriched pre-treatment in patients with FP-RMS and enriched post-treatment in patients with FN-RMS. Post-treatment enrichment of the inflammatory response pathway was observed in both FP-RMS and FN-RMS samples. CONCLUSIONS:Using a probe-based transcriptome panel to characterize matched pre- and post-treatment tissue samples from patients with RMS, we report that relapsed RMS follows a fusion status-dependent evolutionary trajectory, marked by differential expression of myogenesis-associated genes, myogenic differentiation markers, and inflammatory response pathways.

MOTIVATION:The genome interacts with itself within the volume of the cell nucleus to process information. These interactions mediate signal integration, gene regulation, and cell identity. The identification of new therapeutic targets from non-coding disease-associated variants relies critically on correctly assigning variants to genes through 3D interactions. Experimental techniques in 3D genomics, such as HiC and HiChIP, allow the mapping of interactions through sequencing. Bioinformatics for 3D genomics contends primarily with contact matrices that contain interaction frequencies for all possible element pairs, and BEDPE files that store element pairs that interact. Whereas the tools available for processing linear genomic data are mature, operating on contact matrices and BEDPE files remains cumbersome, opaque, and error-prone, as researchers have had to shoehorn tools originally designed for linear data. A genome arithmetic designed from the ground up for 3D genomics does not yet exist. RESULTS:We present AQuA Tools, a suite of shell- and R-based command-line tools that provide a set of core operations on contact matrices and BEDPE files motivated by key questions in population genetics, cancer research, and precision medicine. We have designed our core operations to be clear, reliable, intuitive and versatile. Core operations can be chained together along with standard UNIX commands. Our goal is to make AQuA Tools easy for the novice to learn and the go-to choice for power users. We hope our tools will motivate more researchers to use 3D genomic data in their projects. AVAILABILITY AND IMPLEMENTATION:We provide and maintain AQuA Tools at https://github.com/axiotl/aqua-tools.

Hematopoiesis changes to adapt to the physiology of development and aging. Temporal changes in hematopoiesis parallel age-dependent incidences of blood diseases. Several heterochronic regulators of hematopoiesis have been identified, but how the master transcription factor (TF) circuitry of definitive hematopoietic stem cells (HSCs) adapts over the lifespan is unknown. Here, we show that expression of the ETS family TF Erg is adult-biased, and that programmed upregulation of Erg expression during juvenile to adult aging is evolutionarily conserved and required for complete implementation of adult patterns of HSC self-renewal and myeloid, erythroid, and lymphoid differentiation. Erg deficiency maintains fetal transcriptional and epigenetic programs, and persistent juvenile phenotypes in Erg haploinsufficient mice are dependent on deregulation of the fetal-biased TF Hmga2 . Finally, Erg haploinsufficiency in the adult results in fetal-like resistance to leukemogenesis. Overall, we identify a mechanism whereby HSC TF networks are rewired to specify stage-specific hematopoiesis, a finding directly relevant to age-biased blood diseases. SUMMARY:The hematopoietic system undergoes a process of coordinated aging from the juvenile to adult states. Here, we find that expression of ETS family transcription factor Erg is temporally regulated. Impaired upregulation of Erg during the hematopoietic maturation results in persistence of juvenile phenotypes.

The TBX2 subfamily of T-box transcription factors (e.g., Tbx2, Tbx3, Tbx4, Tbx5) plays an essential role in lung development. Down-regulation of these genes in human lung adenocarcinoma suggests that these genes may be tumor-suppressive; however, because down-regulation appears to occur primarily via epigenetic change, it remains unclear if these changes causally drive tumor progression or are merely the consequence of upstream events. Herein, we developed the first multiplexed mouse model to study the impact of TBX2 subfamily loss, alongside associated signaling genes (Egr1, Chd2, Tnfaip3a, and Atf3) in Ras-driven lung cancer. Using tumor-barcoding with high-throughput barcode sequencing (TuBa-seq), a high-throughput tumor-barcoding system, we quantified the growth effects of these knockouts during early and late tumorigenesis. Chd2 knockout suppressed both tumor initiation and progression, whereas Tnfaip3 knockout enhanced tumor initiation and overall tumor growth. Tbx2 loss showed stage-specific effects on tumor development. Notably, Egr1 emerged as a strong tumor suppressor and its knockout resulted in approximately a fivefold increase in tumor size at 20 weeks (two-sample t-test, P < 0.05), exceeding the impact observed with Rb1 loss. Transcriptomic analyses of Egr1-deficient tumors suggested immune dysregulation, including heightened inflammation and potential markers of T cell exhaustion in the tumor microenvironment. These findings indicate that Egr1 may play a role in suppressing tumor growth through modulating immune dynamics, offering new insights into the interplay between tumor progression and immune regulation in lung adenocarcinoma.

Abstract Nonbiased mutational screening to identify essential genes and pathways in cancer is a powerful approach for understanding underlying biology as well as the identification of potential therapeutic targets. Epigenetic (EPI) and transcriptional factors (TF) play an important role in the change of phenotype and lineage plasticity from adenocarcinoma to castration resistant prostate cancer (CRPC), double negative (DN) or treatment-induced neuroendocrine prostate cancer (NEPC). When RB1 loss occurs in combination with TP53 alterations, stemness and lineage plasticity increase with one consequence being differentiation to a neuroendocrine (NE) lineage. Here, we are identifying and testing the functional role of epigenetic and transcription factors required for the viability of CRPC, DN and NEPC relative to a matrix of genotypic and phenotypic characteristics. We applied CRISPR-CAS9 dropout screens in prostate organoids and cell lines to identify gene/s associated with CRPC, DN and NEPC viability. We successfully CRISPR-engineered LNCaP clones of single (LNCaP-TP53-SKO, LNCaP-RB1-SKO) and double (LNCaP-TP53-RB1-DKO) gene knockouts, which were subsequently characterized for immuno-phenotype and transcriptome. Our dropout screens, targeting all TFs and EPIs, identified 117 TF and 40 EPI genes responsible for the viability of DN, NEPC and LNCaP models. We found known genes such as FOXA1 and ASCL1, as well as novel genes including PROX1 and KLF3, which were validated through guide RNA competition assays. Our results showed PROX1 is essential for cell viability in a DN organoid model, and its expression is associated with increased aggressiveness, indicating PROX1 has role in stemness/lineage plasticity in treatment resistant adenocarcinoma. RNA-seq analysis following overexpression of PROX1 in the LNCaP-TP53-RB1 null adenocarcinoma model, with and without enzalutamide, identified pathways enriched for cell proliferation, differentiation, neuronal development, and prostate gland development. In summary, our study has suggested an important role for PROX1 in the growth and differentiation of poorly differentiated prostate cancer, which may lead to the identification of potential therapeutic targets. Citation Format: Sonam Raj, Brian Capaldo, Joel Bowman, Margaret White, Xavier Moore, Berkley Gryder, Aian N. Alilin, David Takeda, Kathleen Kelly. Determining the role of PROX1 in prostate cancer neuroendocrine trans-differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 554.

Sustained androgen receptor (AR) signaling during relapse is a central driver of metastatic castration- resistant prostate cancer (mCRPC). Current AR antagonists, such as enzalutamide, fail to provide long- term benefit for the mCRPC patients who have dramatic increases in AR expression. Here, we report AR antagonists with efficacy in AR- overexpressing models. These molecules bind to the ligand- binding domain of the AR, promote AR localization to the nucleus, yet potently and selectively down- regulate AR- target genes. The molecules BG-15a and the pharmacokinetically optimized BG-15n elicit a decrease in cell and tumor growth in vitro and in vivo in models of mCRPC. BG-15a/n treatment causes the collapse of chromatin loops between enhancers and promoters at key genes in the AR- driven epigenome. AR binding in the promoter, as well as 3D chromatin clustering, is needed for genes to respond. BG-15a/n represent promising agents for treating patients with relapsed AR- driven mCRPC tumors.

Abstract BACKGROUND Diffuse midline glioma (DMG), H3K27-altered, is a lethal pediatric brain tumor driven by epigenetic dysregulation. Histone deacetylase (HDAC) inhibitors have been identified as a promising drug class; however, the precise mechanism of efficacy for these agents in DMG has not been fully explored. Furthermore, their translational potential has been limited by off-target effects and poor CNS penetration of clinically available drugs. METHODS We investigated the genetic and chemical dependencies of HDAC isoforms in DMG cell lines using CRISPR/Cas9 screening and an HDAC inhibitor toolbox. We identified an HDAC inhibitor termed “Compound 26” with class I isoform selectivity and excellent CNS penetration (brain/plasma AUC of >7). Effects of Compound 26 on DMG cells was evaluated in-vitro through assessment of cytotoxicity, proliferation and apoptosis. Mechanistic studies were undertaken using bulk RNAseq and HiChIP (H3K27ac, H3K27me3). Efficacy of Compound 26 in-vivo was determined using an aggressive orthotopic xenograft model. RESULTS We confirmed that HDAC class I isoforms are dependencies in DMG cell lines, with HDAC2 representing a selective dependency that is highly expressed in patient tumors. Compound 26 is cytotoxic in DMG cells, reducing proliferation through cell cycle arrest. Differential expression analysis confirmed profound effects on cycling programs with Compound 26 treatment. Furthermore, analysis of genome structure using HiChIP revealed global increases in H3K27ac contacts, with notable spreading along key cell cycle gene bodies such as p16 (CDKN2A). Interestingly, treatment with Compound 26 also significantly increased global H3K27me3 contacts, which is canonically decreased in H3K27-altered DMG. Most importantly, Compound 26 was well tolerated, effectively crossed the blood-brain barrier, and significantly extended survival in an aggressive DMG-orthotopic xenograft model. CONCLUSIONS This study comprehensively outlines the reliance of DMG cells on HDAC class I isoforms, elucidates novel mechanisms for this dependency, and highlights a compelling new agent to bypass prior translational pitfalls

Temporal regulation of super-enhancer (SE) driven transcription factors (TFs) underlies normal developmental programs. Neuroblastoma (NB) arises from an inability of sympathoadrenal progenitors to exit a self-renewal program and terminally differentiate. To identify SEs driving TF regulators, we use all-trans retinoic acid (ATRA) to induce NB growth arrest and differentiation. Time-course H3K27ac ChIP-seq and RNA-seq reveal ATRA coordinated SE waves. SEs that decrease with ATRA link to stem cell development (MYCN, GATA3, SOX11). CRISPR-Cas9 and siRNA verify SOX11 dependency, in vitro and in vivo. Silencing the SOX11 SE using dCAS9-KRAB decreases SOX11 mRNA and inhibits cell growth. Other TFs activate in sequential waves at 2, 4 and 8 days of ATRA treatment that regulate neural development (GATA2 and SOX4). Silencing the gained SOX4 SE using dCAS9-KRAB decreases SOX4 expression and attenuates ATRA-induced differentiation genes. Our study identifies oncogenic lineage drivers of NB self-renewal and TFs critical for implementing a differentiation program. This study identifies temporal and coordinately regulated cell-state-specific super-enhancers driving the expression of transcription factors that control circuits needed to switch neuroblastoma tumor cells from self-renewal to differentiation.

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.

Abstract Rhabdomyosarcoma (RMS) is the most frequent pediatric soft tissue sarcoma. Fusion-positive (FP-)RMS expressing the oncogenic chimeric transcription factor (TF) PAX3-FOXO1 (P3F) is at high risk of recurrence. FP-RMS cells show survival dependency on P3F. Despite P3F, as a TF, is considered undruggable, we and others demonstrated that P3F levels/functions can be epigenetically modulated. The BET protein and epigenetic reader BRD4 binds super-enhancers (SEs) to foster oncogenic transcription in cancer. We showed that P3F rewires the RMS enhancer landscape by recruiting BRD4 on oncogenic SEs. Accordingly, JQ1, a BET inhibitor (BETi) shuts down P3F functions halting tumor growth in vitro and in vivo. Therefore, BRD4 inhibition in FP-RMS results in a tumor subtype-specific vulnerability. Nonetheless, as for other targeted therapies, cancer cells can acquire resistance to BETi, thus, we investigated whether FP-RMS cells could develop BETi resistance. Therefore, we established resistant cells from two sensitive FP-RMS cell lines chronically exposed to escalating doses of JQ1, as a model of naturally acquired resistance. Our data show that the two established FP-RMS cell lines, acquired resistance by increasing JQ1 IC50 over the sensitive cells both in 2D and 3D settings. Compared to the sensitive ones, both the resistant cell lines show cross-resistance to other monovalent and bivalent BETi and degraders. Resistant cells also maintain the ability to form colonies in a clonogenic assay and to invade in a 3D Matrigel assay in the presence of JQ1. Moreover, conversely to sensitive cells, JQ1 treatment is unable to down-regulate P3F, MYCN and BCL2 (P3F target) levels in resistant cells. Furthermore, integration of transcriptomic and proteomic data reveals differentially modulated subset of genes/proteins among which MYCN and P3F targets, suggesting a restored activity of the TFs circuitry in resistant cells. Accordingly, MYCN silencing completely halts growth of resistant cells. In line, MYCN protein levels are reduced by JQ1 in a dose-dependent manner in sensitive but not in resistant cells and its half-life is 3 folds longer in resistant than in sensitive cells. Being MYCN protein stability affected by specific post-translational modifications among which the phosphorylation at Ser62 by ERKs, we evaluated the expression of phosphorylated MYCN and the activated form of ERK (pERK) and found both of them increased in resistant cells. In agreement, resistant cells are more sensitive to MEK-ERKs pathway inhibition, showing a lower IC50 of Trametinib (MEKi) compared to sensitive cells. Moreover, Trametinib and JQ1 co-treatment downregulates MYCN protein levels in resistant but not in sensitive cells, suggesting that ERKs overactivation could participate to BETi acquired resistance by stabilizing MYCN, thus representing an acquired vulnerability. The study has received funding from Ministero della Salute to SP. Citation Format: Matteo Cassandri, Erika Ferraro, Lucrezia D'Archivio, Francesca Aiello, Hsien-Chao Chou, Young K. Song, Berkley E. Gryder, Jun S. Wei, Simone Sidoli, Robert G. Hawley, Rossella Rota, Franco Locatelli, Javed Khan, Silvia Pomella. Targeting resistance to BET inhibitors in Fusion-Positive Rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4739.

Gene Signatures Prognosticating Survival after IC Treatment