Abstract Introduction: Rhabdomyosarcoma (RMS) is a highly malignant soft tissue sarcoma. An aggressive subtype, fusion-positive RMS (FP-RMS), which is driven by PAX3/7::FOXO1 translocations, has a dismal 5-year overall survival rate of 13% for patients with metastatic disease. We hypothesized that targeting the oncogenic driver PAX3::FOXO1 with small molecules would be an effective treatment. To test this, we developed cell lines with endogenous PAX3::FOXO1 tagged with HiBiT epitope and performed a drug screen to identify drugs that downregulate PAX3::FOXO1 protein. Study Design: Using CRISPR-Cas9, we endogenously tagged PAX3::FOXO1 with HiBiT in two RMS cell lines (RH4 and SCMC), enabling the monitoring of PAX3::FOXO1 protein levels. We performed a drug screen using the Mechanism Interrogation Plate (MIPE) library of 2,480 compounds, of which 53% are FDA-approved or in clinical trials. NanoGlo Luciferase assays monitored levels of HiBiT-tagged PAX3::FOXO1, while CellTiterGlo measured cell viability at 24 hours. We selected hits that showed a difference in area under the curve (AUC) between the two readouts of ≥ 90 for drugs that preferentially reduce the fusion protein level over general cytotoxicity. We investigated whether inhibitors led to nuclear accumulation and reduced total protein levels using Western blot and immunofluorescent imaging. Candidates were validated in the parental cells and in vivo studies. Results and Conclusions: The screen identified 183 hits, including Eltanexor, an XPO1 inhibitor. XPO1 exports over 200 proteins from the nucleus by recognizing their nuclear export sequences (NESs). Since the fusion gene retains the NES of FOXO1, a known XPO1 target, we tested whether PAX3::FOXO1 is a substrate. We observed that Eltanexor enhanced PAX3::FOXO1 nuclear accumulation at 6 hours in RH4, and at 2 hours in SCMC, followed by protein downregulation at 24 hours by Western blotting. Furthermore, Eltanexor induced p53 nuclear accumulation, detectable at 6 hours in SCMC, suggesting that early accumulation of PAX3::FOXO1 may drive cytotoxicity. At 24 hours, RNA-seq in Eltanexor-treated cell lines demonstrated downregulation of PAX3::FOXO1 and MYCN signatures, components of the core regulatory network in FP-RMS. Preliminary in vivo studies also showed Eltanexor induces delays in tumor progression in an RMS xenograft model. Furthermore, Eltanexor in combination with Mivebresib, a validated BRD4 inhibitor, demonstrated significant synergy against FP-RMS cells. We will perform site-directed mutagenesis studies to disrupt PAX3::FOXO1’s NES and validate combination with Mivebresib in vivo. In conclusion, we identified Eltanexor, an XPO1 inhibitor, as a novel therapeutic agent that suppressed PAX3::FOXO1 activity and levels, induced nuclear accumulation and led to cytotoxicity in incurable FP-RMS. Citation Format: Soumili Dey, Yong Y. Kim, Katrina Jia, Mehal Churiwal, Michele Ceribelli, Teresa S. Hawley, Raj Chari, David Milewski, Young K. Song, Xinyu Wen, Hsien-Chao Chou, Vineela Gangalapudi, Jun S. Wei, Craig Thomas, Robert G. Hawley, Javed Khan. Small-molecule screening of HiBiT-tagged PAX3::FOXO1 rhabdomyosarcoma cell lines identifies eltanexor as a potent therapeutic agent against 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 6405.

Copper ions (Cu²⁺) trigger cuproptosis to suppress tumors, but glutathione (GSH) chelation and hypoxic suppression of lipoylated proteins in the tumor microenvironment (TME) limit efficacy. In this study, we design a pH-responsive copper-based metal-organic framework (Cu-MOF) loaded with a manganese complex (MnPC) to to synchronize reactive oxygen species (ROS) burst and proteotoxicity, triggering a Fenton-cuproptosis-DNA damage cascade for enhanced tumor therapy. In acidic TME, Cu-MOF releases Cu²⁺ ions, initiating a Fenton reaction that generates hydroxyl radicals for ROS-mediated damage. Concurrently, the released Cu²⁺ ions, after reduction by GSH to Cu⁺, accumulate in the mitochondrial matrix, inhibiting the tricarboxylic acid cycle. This leads to the aggregation of lipoylated proteins, a hallmark of cuproptosis, which induces strong proteotoxicity and amplifies cell death signals. Furthermore, the MnPC cause direct DNA double-strand breaks, creating a third layer of damage that reinforces tumor cell apoptosis. This multifaceted approach not only targets tumor cells directly but also modulates the tumor microenvironment, creating a synergistic effect that enhances overall treatment outcomes. This study provides a new perspective on the integration of ROS-based therapies with proteotoxicity induction, offering a promising avenue for the development of more effective and targeted cancer treatments.

Intron retention (IR) is increasingly recognized as a feature of long noncoding RNAs (lncRNAs), yet the mechanisms that shape IR in lncRNAs and the functional consequences of this process remain largely unexplored. To investigate how IR contributes to lncRNA regulation, we performed a genome-wide screen to identify factors controlling IR in the lncRNA PURPL. This approach uncovered a prominent role for U2AF2, which promotes retention of a specific intron in PURPL through a weak polypyrimidine tract. IR of this intron drives nuclear enrichment of PURPL and enhances cell proliferation, revealing biological relevance. Transcriptome-wide analyses showed that although U2AF2 broadly supports canonical splicing consistent with its well-established function in promoting splicing, it also facilitates IR within a distinct subset of RNAs, including the nuclear speckle-associated lncRNA MALAT1. Loss of U2AF2 disrupts MALAT1 speckle localization and using MALAT1 knockout cells reconstituted with wild-type or intron deleted variants, we identified a single intron critical for MALAT1's speckle localization. Deletion of this intron from endogenous MALAT1 impaired speckle localization and reduced cell migration, phenocopying the loss of MALAT1. Together, these findings reveal IR as a key regulatory mechanism governing lncRNA localization and function and uncover an unexpected role for U2AF2 in promoting IR within specific lncRNA contexts.

Abstract Background: Pediatric tumors often co-opt normal developmental gene-regulatory programs, with errors in lineage-restricted progenitors that halt or reverse differentiation. Because these cancers arise within restricted developmental windows, display fetal-like programs, and carry relatively few driver mutations compared to adult tumors, we hypothesized that a pan-pediatric, transcriptome-inferred gene-regulatory network (GRN) analysis will discover lineage-specific regulons that anchor each tumor to a developmentally arrested state, which would identify actionable biomarkers and therapeutic targets. Methods: We analyzed 2541 bulk RNA-seq from 35 pediatric cranial and extracranial solid-tumor samples, after batch correction. We inferred a pan-pediatric GRN from gene expression data, integrating networks inferred by ARACNe-AP and GENIE3 into a consensus GRN across all tumor types. We used a one-vs-rest strategy to identify tumor-specific differentially expressed genes (DEGs) within the regulons. Using hypergeometric tests, we quantified transcription factor (TF) activity and their regulons across tumors by assessing the enrichment of tumor-specific DEGs within each regulon. To map genes to drugs, we queried drug libraries, including Mechanistic Interrogation PlatE, Profiling Relative Inhibition Simultaneously in Mixtures, ChEMBL, DrugBank, and DrugCentral. We filtered druggable genes among TFs, their regulon members, and their interactors using log fold change and adjusted p-values, and ranked candidates in 19 tumors with DepMap data by using effect size. Results: We identified 281 enriched TFs across tumors. The functional enrichment analyses showed that TF programs are usually restricted to specific tumor classes, mirroring their developmental cell-of-origin and highlighting candidate tumor-specific biomarkers. Examples include neurodevelopmental and neural-crest-related TFs (e.g., PHOX2B, ASCL1, and SOX10) in neuroblastoma (NB) and muscle-lineage TFs (e.g., MYOG, MYOD1, and PAX3/7) in fusion-positive rhabdomyosarcomas (FP-RMS). Our analysis suggests that TFs behave as robust, tumor-type-specific expression signatures and can distinguish tumors that may be histologically similar but arise from different developmental lineages. Furthermore, we used our TF-centric approach to identify known and new drug targets, such as SIX1, RRM2, AURKA, and BIRC5 in FP-RMS, and ACVR2B & BMPR1B in NB. Conclusions and Future Directions: A unified GRN framework analysis of pan pediatric solid tumors resolves lineage-specific regulons associated with tumorigenesis and yields a ranked set of druggable genetic dependencies. In vitro and in vivo validation studies are currently underway. Citation Format: Daniel Lee, Abid A. Reza, Syed A. Bukhari, Jun S. Wei, Hsein-Chao Chou, Xinyu Wen, Andrew S. Brohl, Javed Khan. A pan-pediatric gene-regulatory network analysis reveals druggable dependencies across pediatric solid tumors [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 4096.

Abstract Originating from the neural crest, neuroblastoma is the most common extracranial solid tumor in children. Infiltrating immune cells contribute to the tumor’s growth and treatment response, as patients with high-risk disease, associated with MYCN amplification, benefit from anti-GD2 antibody immunotherapy. However, MYCN-amplified disease remains lethal in more than half of cases and has been associated with immunosuppression in bulk RNA studies. We thus hypothesize that MYCN activation, in conjunction with other molecular and clinical traits, influences the tumor microenvironment (TME), which can either support or suppress the disease. To comprehensively characterize the neuroblastoma TME, we performed CO-Detection by indEXing (CODEX), a multiplex immunohistochemistry technique, on 5 clinically annotated tissue microarrays containing 371 neuroblastic tumors from 179 patients representing all major disease subgroups and treatment protocols. In a subset of specimens, we also applied Visium HD spatial transcriptomics to identify regional malignant programs and their associated immune infiltrates. In parallel, we developed a novel natural language processing approach to detect generalizable spatial cell networks across these tissues. Interrogating more than 40 tumor-, immune-, and stroma-associated proteins revealed that neuroblastomas, despite downregulating MHC class I (MHC-I), harbor rich TMEs composed of about 20 phenotypically distinct cell populations, including multiple lymphoid- and myeloid-derived subsets. MYCN-amplified tumors are profoundly deficient in infiltrating helper, memory, and cytotoxic T cell lineages, whereas they form prominent tertiary structures in non-amplified disease. By contrast, antigen-presenting suppressor-like myeloid cells dominate the MYCN-amplified microenvironment, where they persist in chemotherapy-resistant tumors. These subtype-specific differences prompted functional studies of intrinsic immune and cytokine programs. RNA-seq of multiple human MYCN-amplified cell lines revealed that the differentiation therapy retinoic acid, while suppressing MYCN, drastically upregulated class I antigen presentation and pro-inflammatory cytokines. To further understand MYCN- and treatment-related changes in the inflammatory secretome, we are currently performing extracellular proteomics on these cell lines. Integrative spatial profiling by CODEX and Visium HD reveals that MYCN amplification fosters a T cell-poor, myeloid-rich microenvironment, in contrast to the organized lymphoid structures characteristic of non-amplified neuroblastoma. Together with the finding that retinoic acid restores MHC-I and pro-inflammatory programs in MYCN-amplified tumors, these results suggest that combining retinoic acid with cellular immunotherapies and myeloid-targeting approaches may significantly improve survival in patients with high-risk neuroblastoma. Citation Format: Joseph Seamus Toker, Katherine Elizabeth Masih, Noemi Kedei, Zahin Islam, Ben J. Somerville, Amir Jassim, Michail Mamalakis, Aysen Yuksel, Daniel R. Catchpoole, Li Zhou, Paul Aiyetan, Yong Yean Kim, David Milewski, Shaoli Das, Xinyu Wen, Yong Song, Jun Wei, Richard J. Gilbertson, Javed Khan. Distinct microenvironments define subtypes of neuroblastoma [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 639.

A high-throughput screening campaign designed to discover natural product inhibitors of rhabdomyosarcoma oncogene PAX3-FOXO1 gene expression identified partially purified fractions from an organic extract of the plant Gonystylus borneensis to have potent activity in the assay. Bioassay-guided isolation yielded five new 5,6-dihydro-α-pyrone natural products, namely, gonystylones A-E (1-5). Their structures were elucidated using 1D and 2D NMR experiments and their absolute configurations determined using semisynthetic and electronic circular dichroism methods. The gonystylones were found to be cytotoxic to rhabdomyosarcoma cells at low micromolar concentrations (3-19 μM).

Intron retention (IR) is a form of alternative splicing in which an intron that is typically spliced out is retained in the mature RNA. Despite emerging evidence of widespread IR in protein-coding genes and lncRNAs, the underlying molecular mechanisms remain unclear. Here, we developed a genome-wide screen termed CRASP-seq, to investigate the mechanisms underlying IR in the lncRNA PURPL. Unexpectedly, the top hit was the essential splicing activator U2AF2 that promoted IR in PURPL by directly binding to a weak polypyrimidine tract. U2AF2 promoted IR in additional transcripts, including the nuclear speckle-localized MALAT1 whose localization to nuclear speckles was disrupted upon U2AF2 depletion. Importantly, retention of a specific endogenous MALAT1 intron was critical for its nuclear speckle localization and promoting cell migration. These findings uncover a non-canonical function of U2AF2 in promoting IR and reveal how IR contributes to the subcellular localization and functions of PURPL and MALAT1 .

The tumor suppressor p53 is a transcription factor that controls the expression of hundreds of genes. Emerging evidence indicates that the p53-induced RNA-binding protein ZMAT3 acts as a key splicing regulator that contributes to p53-dependent tumor suppression in vitro and in vivo. However, the mechanism by which ZMAT3 functions within the p53 pathway remains largely unclear. Here, we discovered a function of ZMAT3 in inhibiting transcription of HKDC1, a hexokinase that regulates glucose metabolism and mitochondrial respiration in human cancer cells. Quantitative proteomics revealed HKDC1 as the most significantly upregulated protein in ZMAT3-depleted colorectal cancer cells. ZMAT3 depletion resulted in increased mitochondrial respiration, which was rescued by simultaneous depletion of HKDC1, suggesting that HKDC1 is a critical downstream effector of ZMAT3. Unexpectedly, ZMAT3 did not bind to HKDC1 RNA or DNA; however, proteomic analysis of the ZMAT3 interactome identified its interaction with the oncogenic transcription factor JUN. ZMAT3 depletion enhanced JUN binding to the HKDC1 locus, leading to increased HKDC1 transcription that was rescued upon JUN depletion, suggesting that JUN activates HKDC1 transcription in ZMAT3-depleted cells. Collectively, these findings uncover a mechanism by which ZMAT3 regulates transcription through JUN and demonstrate that HKDC1 is a key component of the ZMAT3-regulated transcriptome in the context of mitochondrial respiration regulation.

RNASE1 is a ribonuclease secreted by cells and degrades extracellular RNAs. Here, we unexpectedly found that RNASE1, in addition to being secreted, is predominantly localized to the nucleus and functions to inhibit gene expression in human colorectal cancer (CRC) cells. RNASE1 expression is highly cell type-specific and is restricted to well-differentiated CRC cells where its transcription is activated by the pioneer transcription factor FOXA1. Using CRISPR interference utilizing three independent sgRNAs targeting the RNASE1 locus followed by RNA-seq, we found that upon depletion of RNASE1, most of the differentially expressed RNAs are modestly but significantly upregulated suggesting that RNASE1 predominantly functions to inhibit gene expression. In CRC patients, RNASE1 is significantly downregulated and high RNASE1 expression is associated with better patient survival, indicating a potential tumor suppressive function. Consistent with this, RNASE1 depletion results in increased proliferation and clonogenicity indicating that RNASE1 inhibits the growth of CRC cells. Finally, a promising RNASE1 target among the most significantly upregulated mRNAs upon RNASE1 depletion is DKK1 (Dickkopf inhibitor 1) which is upregulated in CRC and negatively regulated by RNASE1. Collectively, this initial characterization of endogenous RNASE1 uncovers a function of RNASE1 in inhibition of gene expression and CRC cell proliferation.

Oncogenic fusion genes are attractive therapeutic targets due to their tumor-specific expression and driver roles in cancer. PAX3::FOXO1 (P3F) is the dominant oncogenic driver of fusion-positive rhabdomyosarcoma (FP-RMS) with no current targeted therapy. HiBiT tag, an 11 amino acid peptide of NanoLuc luciferase, was inserted into the C-terminal end of the endogenous P3F using CRISPR. Western was used for HiBiT tag validation. RNA-seq and ChIP-seq were used to assess transcriptomics and DNA binding of HiBiT-tagged P3F (P3F-HiBiT). High-throughput drug screen was performed using the Mechanism Interrogation PlatE drug library with known mechanisms of action. Cell viability was measured using CellTiter-Glo. Mouse xenograft models were used to investigate in vivo efficacy. We validated the HiBiT tagging of P3F by Western. Both P3F-HiBiT and unmodified P3F activated the same gene sets in fibroblasts by RNA-seq Gene Set Enrichment Analysis (GSEA). ChIP-seq using HiBiT antibody verified that P3F-HiBiT binds to the same sites as P3F. A screen for compounds that downregulate P3F in both RH4 and SCMC identified 182 drugs. Filtering for drugs with ≥ 3 hits for the same target identified 14 drug classes, including HDAC inhibitors, BRD4 inhibitors, and CDK inhibitors. Focusing on CDK inhibitors, we found that FP-RMS was most sensitive to CDK7, CDK9 and multi-CDK inhibitors. TG02, a multi-CDK inhibitor with highest inhibition of CDK9 and currently in human trials, downregulated P3F protein. GSEA showed marked suppression of P3F targets after TG02 treatment. Western validated the inhibition of CDK9 with decreased RNA Pol2 Ser2 phosphorylation (Pol2S2). ChIP-seq for RNA Pol2 showed a decrease in transcription pause-release, indicating inhibition of transcription by TG02. Moreover, analysis of genes ranked by decreased Pol2S2 in the gene body showed significant enrichment for P3F targets (p<0.001). TG02 significantly delayed tumor progression without weight loss in a mouse xenograft model of FP-RMS. Also, we found that Vincristine (VCR) and Irinotecan (IRN) are synergistic with TG02 in vitro. Combinations of TG02 with VCR or IRN showed a significant delay in tumor progression compared to TG02 alone in mouse xenograft models. By HiBiT tagging the fusion oncogene P3F, we identified 182 drugs that suppress P3F levels. One of the top hits, TG02, showed in vivo efficacy, indicating that FP-RMS is susceptible to multi-CDK inhibition. Decreased occupancy of Pol2S2 in the gene body of P3F targets indicates that the mechanism of TG02 is primarily through transcriptional inhibition of P3F and its targets. This indicates that TG02 may be effective in transcriptionally addicted cancers such as FP-RMS. We also found synergy between TG02 with VCR and IRN showing promise for clinical translation in FP-RMS. Yong Yean Kim, Katrina Jia, Mehal Churiwal, Soumili Dey, Teresa S. Hawley, Silvia Pomella, Raj Chari, David Milewski, Ranuka Sinniah, Young K. Song, Hsien-Chao Chou, Xinyu Wen, Craig J. Thomas, Michele Ceribelli, Jun S. Wei, Robert G. Hawley, Javed Khan. Endogenous HiBiT-tagging of PAX3::FOXO1 reveals that CDK inhibitors downregulate the fusion oncogene, and demonstrate synergy effects when combined with vincristine and irinotecan [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 7037.

Abstract Background: Oncogenic fusion genes are attractive therapeutic targets due to their tumor-specific expression and driver roles in cancers. PAX3-FOXO1 (P3F) is the dominant oncogenic driver of fusion-positive rhabdomyosarcoma (FP-RMS) with no current targeted therapy. We developed methods to directly measure endogenous P3F protein levels amenable to high-throughput drug screens. Method: HiBiT tag, an 11 amino acid peptide of NanoLuc luciferase, was inserted into the endogenous P3F using CRISPR in FP-RMS cell lines RH4 and SCMC. Western blot was used for HiBiT tag validation. RNA-seq and ChIP-seq were used to assess transcriptomics and DNA binding of HiBiT-tagged P3F (P3F-HiBiT). High-throughput drug screen using Nano-Glo luciferase assay was performed using the Mechanism Interrogation PlatE (MIPE 5.0) drug library, a 2,480 drug library with known mechanisms of action. CellTiter-Glo was used to monitor cell viability. Mouse xenograft models of FP-RMS were used to investigate in vivo efficacy. Results: We validated HiBiT tagging of P3F by Western. Both P3F-HiBiT and unmodified P3F activated the same gene sets in fibroblasts by RNA-seq Gene Set Enrichment Analysis (GSEA). ChIP-seq using HiBiT antibody for P3F-HiBiT matched the genomic locations from ChIP-seq with P3F antibody in RH4 and SCMC. Using a cutoff value of > 90 (Area Under the Curve (AUC) of CellTiter-Glo minus AUC of Nano-Glo), in both RH4 and SCMC, we identified 182 compounds which downregulate P3F before cell death. Filtering for drugs with ≥ 3 hits for the same target identified 14 drug classes that suppressed P3F including HDAC inhibitors (3), BRD4 inhibitors (3), and CDK inhibitors (8). FP-RMS was sensitive to CDK1/2, CDK4/6, CDK9, and multi-CDK inhibitors. A multi-CDK inhibitor TG02, currently in human trials, downregulated P3F and RNA-seq GSEA showed marked suppression of P3F targets. TG02 also significantly delayed tumor progression in mouse xenograft model of FP-RMS without weight loss. Interestingly, the commonly used chemotherapeutic Vincristine (VCR) also downregulated P3F in vitro. TG02 with VCR showed synergy by Loewe analysis. In vivo, testing showed significant delay in tumor progression by the combination compared to TG02 or VCR alone. Of note, tumor RNA-seq GSEA following treatment with TG02, or VCR alone, and in combination significantly downregulated P3F targets. Conclusion And Future Directions: By HiBiT tagging the fusion oncogene P3F, we identified 182 drugs that suppress P3F levels of which TG02 was a top hit. TG02 showed in vivo efficacy indicating that FP-RMS is susceptible to CDK inhibition. We also found synergy between TG02 and VCR, resulting in a significant delay in FP-RMS tumor progression compared to single agents. Interestingly, we found that VCR alone can downregulate P3F and its targets. Combination therapy of TG02 with VCR shows promise for clinical translation in FP-RMS. Citation Format: Yong Yean Kim, Katrina Jia, Mehal Churiwal, Teresa Hawley, Silvia Pomella, Christine Evans, Raj Chari, David Milewski, Ranuka Sinniah, Young Song, Hsien-Chao Chou, Xinyu Wen, Craig Thomas, Michele Ceribelli, Jun Wei, Robert Hawley, Javed Khan. Endogenous HiBiT-tagging of PAX3-FOXO1 reveals downregulation of the fusion oncogene by CDK inhibitors and has synergy with vincristine [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 1089.

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.

Chimeric antigen receptor (CAR) T-cells targeting Fibroblast Growth Factor Receptor 4 (FGFR4), a highly expressed surface tyrosine receptor in rhabdomyosarcoma (RMS), are already in the clinical phase of development, but tumour heterogeneity and suboptimal activation might hamper their potency. Here we report an optimization strategy of the co-stimulatory and targeting properties of a FGFR4 CAR. We replace the CD8 hinge and transmembrane domain and the 4-1BB co-stimulatory domain with those of CD28. The resulting CARs display enhanced anti-tumor activity in several RMS xenograft models except for an aggressive tumour cell line, RMS559. By searching for a direct target of the RMS core-regulatory transcription factor MYOD1, we identify another surface protein, CD276, as a potential target. Bicistronic CARs (BiCisCAR) targeting both FGFR4 and CD276, containing two distinct co-stimulatory domains, have superior prolonged persistent and invigorated anti-tumor activities compared to the optimized FGFR4-specific CAR and the other BiCisCAR with the same 4-1BB co-stimulatory domain. Our study thus lays down the proof-of-principle for a CAR T-cell therapy targeting both FGFR4 and CD276 in RMS.

Abstract Background: Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor driven primarily by activating mutations in the RET proto-oncogene. It accounts for approximately 13% of thyroid cancer-related deaths. Total thyroidectomy is the main treatment option for MTC patients; however, its efficacy is limited due to the high prevalence of metastatic disease at diagnosis. Vandetanib, cabozantinib, and selpercatinib are RET signaling inhibitors (RETi), that have been approved for treating advanced MTC. Despite their effectiveness, many patients relapse or have refractory disease due to eventual resistance to these RETis. Methods: In our study, we employed genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) gene knockout (KO) in MTC cell lines treated with selpercatinib or vehicle to investigate the genetic mechanisms of resistance or sensitivity to RETi. Our overarching goal was to identify potential synergistic or additive drug combinations that may prevent or treat resistant disease. Results: Our research identified key genes and pathways confirming the sensitivity or resistance of MTC cells to RETi. Notably, apoptotic genes along with RAS and mTOR signaling pathways were found to play a significant role in resistance development. Knocking out negative regulators of these pathways (such as NF1 and TSC2) conferred growth advantages to cells treated with RETi. Alternatively, knocking out BCL2, an antiapoptotic protein, led to increased lethality. These findings suggested that targeting RAS, mTOR, or antiapoptotic pathways in conjunction with RETi may be synergistic or have additive activity. To validate the CRISPR screening, we selectively knocked down NF1 in MTC cells and found it led to a nearly two-fold increase in IC50 for selpercatinib. Furthermore, in-vitro and in-vivo testing confirmed significant synergistic activity when using the combination of trametinib, an MEK inhibitor (MEKi; downstream of the RAS pathway), with selpercatinib. Conclusions and Future Directions: We have identified a potent synergistic combination of MEKi with RETi as a novel synergistic drug combination to treat MTC, which may also prevent resistance to RETi. These results will be translated to patients through clinical trials conduced at the NCI. Citation Format: Abdelrahman Rahmy, Arwa Fallatah, David Milewski, Meijie Tian, Young Song, Yong Kim, Hsien-Chao Chou, Xinyu Wen, Chaoyu Wang, Jun Wei, Robert Hawley, John Glod, Javed Khan. Genome-Wide CRISPR gene knockout screens combined with selpercatinib identifies potent combination therapies for RET driven medullary thyroid carcinoma [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 5835.

Reduction of PAX3::FOXO1 protein expression does not alter proliferation rates of FP-RMS cell lines but reduces anchorage-independent growth.

Piperacetazine inhibits endogenous PAX3::FOXO1 activity in multiple cell lines. A, The effect of piperacetazine treatment on cell viability was measured using the CellTiter-Blue assay (black lines). The effect of piperacetazine treatment on PAX3::FOXO1 activity was measured via luciferase assay (blue lines), using the PAX3::FOXO1-responsive pGL3-PDGFRA luciferase reporter (RH30, RH41, RH28, U66788, RD), and PGK reporter (SN12C, negative control). B, Left: Luciferase activity was measured in HEK293T cells transfected with PAX3::FOXO1-responsive ASS1P reporter and an empty or PAX3::FOXO1 expression vector. Cells were treated with DMSO or piperacetazine for 48 hours. Matching Western blots are shown under each bar. Right: The same experimental setup was repeated with EWS::FLI1-responsive NR0B1 luciferase reporter and an EWS::FLI1 expression vector or empty vector. The cells were treated with the same concentration of piperacetazine or DMSO for 48 hours, and the normalized luciferase readouts and accompanying Western blots are shown. (*, P < 0.0001; Student t test; ns, P > 0.05)

Piperacetazine alters the expression of PAX3::FOXO1 target genes. A, Protein expression of PAX3::FOXO1 target genes was evaluated by Western blot analysis in RH30 cells treated with 10 µmol/L piperacetazine for 6 days. B, RNA-seq was performed in fusion-positive RH30 cells treated with 30 µmol/L piperacetazine for 24 hours. The ranked gene expression list was compared with existing lists of genes using GSEA. Gene set descriptions: Top left: genes upregulated during human skeletal muscle myoblast differentiation. Top right: Hallmark genes during myogenesis. Bottom left: Genes downregulated in SAOS-2 (osteosarcoma) cells upon expression of PAX3::FOXO1. Bottom right: Genes downregulated in fusion-positive versus fusion-negative RMS cell lines. NES = normalized enrichment score, FDR = false discovery rate). C, Piperacetazine does not cause PAX3::FOXO1 to shift its intracellular localization. RH30 cells were treated with 10 µmol/L piperacetazine for 24 hours prior to cellular fractionation, which were analyzed via Western blot analysis. Lamin A/C and alpha-tubulin were used as positive controls for nuclear and cytoplasmic fractions, respectively. D, Piperacetazine does not alter PAX3::FOXO1 protein levels or phosphorylation of Ser256, as evaluated by Western blot analysis. RH30 cells were treated with 10 µmol/L piperacetazine or vehicle for 3 days. E, PAX3::FOXO1 protein was immobilized on a CM5 chip, and double-stranded PAX3::FOXO1 oligonucleotide (100 nmol/L), piperacetazine (10 µmol/L), or a combination of the two were injected over the chip surface. Piperacetazine did not inhibit DNA binding to PAX3::FOXO1.

Piperacetazine inhibits anchorage-independent growth of FP-RMS cells. A, Average 48 hour IC50 values for indicated cell lines. Sample size indicated above each bar. Red bars: FP-RMS, blue bars: FN-RMS, black bars: non-RMS. U48484, U66788, U37125, and U57810 are cell lines from transgenic RMS mouse models. B, RH30 and RD colonies in soft agar with DMSO or 15 µmol/L piperacetazine treatment. The top row images are whole well images taken with Gelcount and the bottom row images are microscope images of the colonies at 40X magnification. C, Gelcount quantification of colonies in B (*, P < 0.0001; Student t test; ns, P > 0.05).

Abstract Background: Oncogenic fusion genes are attractive therapeutic targets due to their tumor-specific expression and driver roles in cancers. PAX3-FOXO1 (P3F) is the dominant oncogenic driver of fusion-positive rhabdomyosarcoma (FP-RMS) with no targeted therapy. We developed methods to directly measure endogenous P3F protein levels amenable to high-throughput drug screens to identify suppressors of P3F. Methods: HiBiT tag, an 11 amino acid peptide of the small fragment of NanoLuc luciferase, was inserted into the endogenous P3F using CRISPR-Cas9 in FP-RMS cell lines RH4 and SCMC. Western analysis was used for HiBiT tag validation and confirmation of P3F suppression. RNA-seq and ChIP-seq were used to assess transcriptomics and DNA binding of HiBiT-tagged P3F (P3F-HiBiT) respectively. High-throughput drug screen using Nano-Glo luciferase assay was performed using the Mechanism Interrogation PlatE (MIPE 5.0) drug library, which included 2,480 drugs with known mechanisms of action. CellTiter-Glo was used to monitor cell viability. We identified drugs that suppressed P3F by Nano-Glo without acute cytotoxicity by CellTiter-Glo at an early 24-hour timepoint. Mouse xenograft model of FP-RMS was used to investigate in vivo efficacy of top hits. Results: We validated HiBiT tagging of P3F and not the wild-type FOXO1 by Western analysis. We showed that the HiBiT tag did not change the function of P3F by transducing human fibroblasts with P3F-HiBiT versus unmodified P3F. Gene Set Enrichment Analysis (GSEA) of RNA-seq showed that P3F-HiBiT activated the same downstream target genes as unmodified P3F. ChIP-seq using HiBiT antibody in HiBiT-tagged FP-RMS cell lines RH4 and SCMC matched the genomic locations from ChIP-seq with P3F antibody in parental RH4 and SCMC. Using a cutoff of Area Under the Curve (AUC) of CellTiter-Glo - AUC of Nano-Glo > 90, in both RH4 and SCMC, identified 182 compounds. Filtering for drugs with ≥ 3 hits for the same target identified 14 drug classes that suppressed P3F protein level including HDAC inhibitors (3), mTOR inhibitors (4), CDK inhibitors (8), and BRD4 inhibitors (3). One top hit was the CDK inhibitor TG02 (Zotiraciclib), currently in human trials. TG02 suppressed P3F protein levels by Nano-Glo and Western analysis. We confirmed induction of apoptosis by PARP cleavage in a panel of FP-RMS cell lines. GSEA analysis of RNA-seq after treatment with TG02 showed marked suppression of P3F target gene sets. TG02 also significantly delayed tumor progression of established tumors in a mouse xenograft model of FP-RMS without weight loss. Conclusion and Future Directions:By HiBiT tagging the fusion oncogene P3F, we identified 182 compounds that suppress P3F levels of which TG02 was a top hit that also showed in vivo efficacy. Drug combination studies are currently underway to identify synergistic suppressors of P3F protein levels that can be translated into clinical trials. Citation Format: Yong Yean Kim, Robert G. Hawley, Mehal Churiwal, Teresa S. Hawley, Christine N. Evans, Raj Chari, David Milewski, Ranuka Sinniah, Young K. Song, Hsien-Chao Chou, Xinyu Wen, Ying Pang, Jing Wu, Craig J. Thomas, Jun S. Wei, Michele Ceribelli, Javed Khan. Endogenous HiBiT-tagging of PAX3-FOXO1 identifies potent suppressors of PAX3-FOXO1 protein levels by high-throughput screening. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3538.

Supplementary Tables 1, 2, and 3