Chen, Renee

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

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

Gene fusions involving tumor protein p63 gene (TP63) occur in multiple T and B cell lymphomas and portend a dismal prognosis for patients. The function and mechanisms of TP63 fusions remain unclear, and there is no target therapy for patients with lymphoma harboring TP63 fusions. Here, we show that TP63 fusions act as bona fide oncogenes and are essential for fusion-positive lymphomas. Transgenic mice expressing TBL1XR1::TP63, the most common TP63 fusion, develop diverse lymphomas that recapitulate multiple human T and B cell lymphomas. Here, we identify that TP63 fusions coordinate the recruitment of two epigenetic modifying complexes, the nuclear receptor corepressor (NCoR)—histone deacetylase 3 (HDAC3) by the N-terminal TP63 fusion partner and the lysine methyltransferase 2D (KMT2D) by the C-terminal TP63 component, which are both required for fusion-dependent survival. TBL1XR1::TP63 localization at enhancers drives a unique cell state that involves up-regulation of MYC and the polycomb repressor complex 2 (PRC2) components EED and EZH2. Inhibiting EZH2 with the therapeutic agent valemetostat is highly effective at treating transgenic lymphoma murine models, xenografts, and patient-derived xenografts harboring TP63 fusions. One patient with TP63 -rearranged lymphoma showed a rapid response to valemetostat treatment. In summary, TP63 fusions link partner components that, together, coordinate multiple epigenetic complexes, resulting in therapeutic vulnerability to EZH2 inhibition.

Abstract Recurrent chromosomal rearrangements are a hallmark of hematologic malignancies and play critical roles in pathogenesis. The TP53 analog TP63 is rearranged in 5-10% of diverse subtypes of both aggressive T- and B-cell lymphomas. Patients with TP63-rearranged lymphomas have dismal outcomes, with 5-year overall survival rates between 0-17%, depending on cohorts. The function and mechanisms of TP63 rearrangements and TP63 fusion proteins in tumorigenesis are poorly understood. As a result, attempts to treat these patients to date have been largely empiric. Thus, there is an urgent need to understand how TP63 fusions contribute to tumorigenesis and to translate the findings into novel therapeutic options for these patients. Here, we demonstrated that TP63 fusions are essential for the propagation of T-cell lymphomas (TCLs). Knockdown of TP63 fusions with specific shRNAs in TCL cell lines harboring TP63 fusions suppressed both cell growth in vitro and tumor growth in vivo. Retroviral expression of TBL1XR1-TP63, the most common TP63 fusion, conferred cytokine independence in Ba/F3 cells, consistent with its role as an oncogene. To investigate the role of TP63 fusions in T- and B-cell lymphomagenesis, we engineered a CAG-Loxp-Stop-Loxp-TBL1XR1-TP63 conditional knock-in mouse model and crossed with hCD2-Cre mice. This results in expression beginning during early lymphoid development. As observed in patients, transgenic mice developed multiple subtypes of both T- and B-cell lymphoma. To define the effects and mechanisms of TP63 fusions within T cells, we performed CRISPR scanning, transcriptomic, epigenomic, and proteomic analyses. Our data showed that domains within both the N-terminal TBL1XR1 and C-terminal TP63 portions contribute to the function of this fusion. We found that the N-terminal component of TP63 fusions interacts with components of the NCOR/SMRT complex. At the same time, the C-terminal portion of TP63 (which recapitulates the deltaN-p63 isoform expressed in some carcinomas) interacts with the enhancer modifier KMT2D and its complex members. TBL1XR1-TP63 binds to a novel distal enhancer to drive MYC expression, and thus upregulates the expression of the histone H3K27 methylase EZH2. Finally, we assessed whether EZH2 is a vulnerability of TP63-rearranged lymphomas. We found that knockdown of EZH2 in TP63-rearranged lines significantly impaired cell growth, as did treatment with the EZH2 and 1 dual inhibitor valemetostat. Valemetostat, which is now being tested in patients with lymphoma, counteracted the oncogenic effects of TP63 fusions in multiple preclinical models in vivo. Together, our results identify the TP63 fusion as a highly unique oncogenic driver in lymphomagenesis capable of recruiting multiple epigenetic modifier complexes and inducing a targetable dependence on EZH2. Citation Format: Gongwei Wu, Noriaki Yoshida, Jihe Liu, Xiaoyang Zhang, Yuan Xiong, Tayla Heavican-Foral, Huiyun Liu, Geoffrey Nelson, Lu Yang, Renee Chen, Katherine Donovan, Marcus Jones, Mikhail Roshal, Yanming Zhang, Ran Xu, Ajit Nirmal, Salvia Jain, Catharine Leahy, Kristen Jones, Kristen Stevenson, Natasha Galasso, Nivetha Ganesan, Tiffany Chang, Wen-Chao Wu, Abner Louissaint, Lydie Debaize, Hojong Yoon, Paola Dal Cin, Wing Chan Chan, Shannan Ho Sui Ho Sui, Samuel Ng, Andrew Feldman, Steven M. Horwitz, Mathew Meyerson, Karen Adelman, Eric Fischer, Chun-Wei Chen, David Weinstock, Myles Brown. TP63 fusions drive enhancer rewiring, lymphomagenesis, and dependence on EZH2. [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 5755.