Abstract Mutations in the tumor suppressor p53 correlate with poor prognosis and high metastasis rates, which account for more than 90% of cancer-related mortality worldwide. Our recent work (Cancer Discovery PMC10841313), highlights that mutant p53 may exhibit neomorphic gain-of-function properties, driving squamous cell carcinoma (SCC) metastasis beyond its classic role via wild-type p53 loss. While our previous work and emerging studies have highlighted the neomorphic gain-of-function activities of mutant p53 in promoting metastasis, the tissue-specific transcriptional programs that underlie metastatic organotropism remain underexplored. To identify mutant p53-dependent mechanisms in facilitating organ-specific metastasis, we performed RNA-sequencing coupled with ChIP-sequencing on esophageal squamous cell carcinoma (ESCC)-derived lung and liver metastases harboring either mutant p53-R172H or p53 deletion. Analyses included differential gene expression (DEG), pathway enrichment, and comparisons between primary tumor and metastatic tumor transcriptomes. These studies were complemented by analyses of aero-digestive squamous cell carcinoma datasets from the AACR Project GENIE, TCGA, and tissue microarrays assessing mutant p53 in relation to patient survival and enriched molecular pathways. Mutant p53 contributed to distinct transcriptomic shifts in liver versus lung metastases. In lung metastases, 618 genes were upregulated and 667 downregulated dependent upon mutant p53-R172H, with enrichment in distinct immune response programs. By contrast, liver metastases showed 1,320 upregulated and 1,355 downregulated genes, with strong downregulation of interferon and inflammatory response pathways. Our analyses exhibited minimal overlap between up- or downregulated genes across tissues (103 shared downregulated and 77 shared upregulated genes), highlighting organ-specific p53-mediated transcriptional control. Furthermore, comparisons with primary tumors revealed subsets of DEGs uniquely dependent on p53-R172H across metastatic and primary contexts. Notably, only a small core set of genes (Acot1, Ldhb, Bcat1, Il18rap, Kif6, Paqr4, Il1rl1 and Cdc20) were upregulated in all three settings (primary, lung, liver), while most gene expressions were organ-specific. Overall, our results reveal novel insights into the mechanisms underlying mutant p53-mediated metastatic organotropism. These data highlight the necessity of incorporating the organ specific context metastatic cancers into therapeutic strategies driven by mutant p53. Citation Format: Gizem Efe, Raúl Navaridas, Katherine M. Cunningham, Dan Hasson, Chao Lu, James J. Manfredi, Carol L. Prives, Anil K. Rustgi. Mutant p53 neomorphic activities drive organ-specific metastatic programs through distinct transcriptional networks [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 3989.

Cancer immunotherapy with immune checkpoint inhibitors (ICIs) is often limited by an immunosuppressive tumor microenvironment (TME). Simultaneous targeting of the TME and immune checkpoints is a promising approach to address this limitation. Here we develop an inhalable exosome system that enables co-display of two inhibitory ligands and apply it to treat lung metastases of ICI-resistant melanoma. As immune exclusion in this context is often mediated by Wnt/β-catenin signaling, we harnessed the Alix sorting domain for tandem display of PD-1 and FZD8 to block PD-L1 and Wnt7b, which is overexpressed in ICI-resistant melanoma. This technology, called bispecific exosome activator of T cells (BEAT), enables uniform 1:1 co-display of two proteins on the exosome surface. We show that BEAT concurrently recruits and activates CD8⁺ T cells to reprogram the TME, yielding robust antitumor activity in ICI-resistant melanoma mouse models. Inhaled BEAT outperforms linked dual antibody targeting PD-L1 and Wnt7b in vivo. This approach to tandem protein display may be applicable to diverse ICI-resistant cancers. Engineered inhalable exosomes deliver a bispecific T cell activator to pembrolizumab-resistant metastatic melanoma.

Abstract Metastatic esophageal cancer exhibits a strong predilection for dissemination to the lungs. Lung metastases are associated with poor survival outcomes, therapeutic resistance, and limited treatment options, underscoring the urgent need to identify targetable mechanisms driving lung colonization. Yet, these molecular mechanisms driving lung-specific tropism remain poorly understood, which serves as the basis for our novel approaches. Herein, we employed state-of-the-art in vivo CRISPR-Cas9 knockout screens using a sgRNA library targeting chromatin regulators to identify epigenetic modulators of lung metastasis in esophageal cancer. This screen targeted 600 genes with a pooled sgRNA library enriched for epigenetic regulators and chromatin remodelers. The library, containing 6 sgRNAs per gene and appropriate non-targeting controls, was transduced into isogenic cells with either mutant Trp53R172H/- (a frequently detected hotspot Trp53 mutation in the DNA binding domain) or its depletion at a low multiplicity of infection to ensure single sgRNA integration per cell. The transduced cells were injected orthotopically or via tail-vein into mice, and comparative abundance analysis was performed between the pre-implantation pool and lung metastatic lesions. Our screen uncovered key chromatin regulators that selectively promote lung colonization in the presence of mutant p53, including histone methyltransferases and demethylases (Kmt2d, Kdm1b, Kdm4d), histone deacetylases (Hdac4), and additional DNA and chromatin modifiers (Eya2, Prmt8, Parp14, Tox4, Eny2, and Gata2a). To elucidate the mechanisms by which these epigenetic regulators contribute to metastatic potential, we are performing comprehensive histone methylation and acetylation profiling in the corresponding KO clones, in combination with ATAC-seq and RNA-seq. This integrative approach will enable us to correlate histone modification landscapes with chromatin accessibility and gene expression programs, delineating how mutant p53 cooperates with specific chromatin regulators to reprogram enhancer networks and drive pro-metastatic transcriptional states. Collectively, these studies will define epigenetic mechanisms underlying lung tropism in esophageal cancer and identify actionable vulnerabilities for therapeutic targeting, with broader implications for TP53-mutant cancers. Citation Format: Raul Navaridas, Gizem Efe, Ali Iftikhar, Karen J. Dunbar, Katherine Cunningham, Emily Esquea, Noriyuki Noriyuki, Constanza Tapia Contreras, Alice E. Shin, Francisco J. Sánchez-Rivera, Chao Lu, Anil K. Rustgi. In vivo CRISPR screening of chromatin regulators reveals p53-dependent drivers of lung metastasis in esophageal cancer [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 6102.

Cardiac repair following cardiovascular diseases relies heavily on coordinated metabolism across different cells. Exosomes, or small extracellular vesicles secreted by cells, have emerged as pivotal regulators of the metabolic process. This review offers an introductory exploration of the intricate role played by exosomes in cardiac repair and regeneration. Specific metabolism regulation, including metabolism and immune response alterations, is emphasized and analyzed. Additionally, we explore some innovative engineering strategies for improving the therapeutic potential of exosomes in this field. This article aims to provide an integrated framework of comprehension toward exosomes and metabolism in cardiac repair and pave the way for novel therapy designs for cardiovascular diseases by outlining future directions for clinical translation.

Chromosomal loops are CTCF–cohesin-mediated 3D genomic structures in the mammalian nucleus. In addition to these CTCF–cohesin loops, other epigenomic features can also form long-range interactions. Polycomb-targeted loci, regulated by the Polycomb repressive complex and trithorax group during development, form long-range chromatin interactions independent of CTCF–cohesin and are demarcated by regions of low DNA methylation—referred to as DNA methylation canyons. We previously identified extremely long Polycomb loops occurring between DNA methylation Canyon demarked Polycomb-targeted loci, spanning distances of up to 60 Mb (Zhang et al, 2020). These loops are found exclusively in self-renewing cells, such as human hematopoietic stem cells and mouse embryonic stem cells. In hematopoietic malignancies, both DNA methylation and Polycomb binding are significantly altered. To explore this further, we conducted a pan-cancer survey of long Polycomb loops across 223 tumor samples, with a focus on hematopoietic malignancies—given that their likely cell-of-origin, the hematopoietic stem cell, exhibits strong long Polycomb loops. Our cohort included 32 acute myeloid leukemias (AMLs), 24 T-cell lymphoblastic leukemias, 61 pediatric brain tumors, 80 prostate cancers, and 26 colon cancers. We found that most cancers—including all prostate and colon cancers—lack long Polycomb loops. However, long Polycomb loops are retained in pediatric brain tumors and a subset of AMLs. Interestingly, strong long Polycomb loop interactions are observed in normal developing brain tissue and hematopoietic stem cell. Our data suggest that the presence of long Polycomb loops is likely inherited from the epigenomic state of the cell of origin, as they are also observed in normal pediatric brain tissue and hematopoietic stem cells. Loss of long Polycomb loops in primary cancer samples is accompanied by DNA hypermethylation and reduced Polycomb binding at loop anchor loci, and these disruptions are further exacerbated in cultured cell lines. Interestingly, in AML, many of these previously silenced loci—often mesodermal transcription factors—become activated and form de novo 3D interaction anchors. For example, we observed the formation of a new domain around the leukemogenic ZEB2 gene, driven by leukemia-specific HOXA9 binding at an upstream enhancer within the TEX41 locus. Notably, AML samples displayed a wide range of long Polycomb loop strength. While most AMLs lose these loops, approximately 12% (4 out of 33) retain strong long Polycomb loops comparable to hematopoietic stem cells. These cases recurrently harbor somatic mutations in CEBPA (2 out of 4) and STAG2 (2 out of 4). These genes are not directly linked to Polycomb or DNA methylation machinery, suggesting alternative mechanisms of loop maintenance. The CEBPA mutant AMLs also exhibited H3K27me3 spreading to non-Polycomb target loci across the genome. We therefore tested whether EZH2 inhibition could disrupt long Polycomb loops in these AMLs and impact disease maintenance. Indeed, EZH2 inhibition attenuated long Polycomb loops, reduced colony-forming capacity, and promoted differentiation in long Polycomb loop–retaining AMLs. Treated AML cells activated a macrophage differentiation program and showed downregulation of cell cycle and DNA replication genes. These findings suggest that AMLs retaining long Polycomb loops are dependent on this 3D chromatin architecture. This rare but strong sensitivity to EZH2 inhibition in AML indicates that long Polycomb loops could serve as an epigenomic biomarker for EZH2 or other Polycomb-targeted therapies in cancer rather than the current genomic marker such as EZH2 gain-of-function mutations and SWI/SNF loss-of-function mutations. Overall, long Polycomb loops are commonly lost during leukemogenesis due to epigenomic disruption. However, a subset of AMLs maintains these loops from the cell of origin and appears to rely on the Polycomb network to sustain self-renewal.

Abstract Treatment with highly potent anti-androgens, such as enzalutamide and abiraterone promotes lineage plasticity in metastatic castration-resistant prostate cancer (mCRPC), which results in intra-tumor heterogeneity and emergence of mCRPC subtypes. The histological transformation from adenocarcinoma to aggressive neuroendocrine prostate cancer (CRPC-NE) has been associated with a loss of dependency on lineage-survival signals, leading to targeted drug resistance. Epigenomic reprogramming might be a fundamental driver of lineage plasticity. To determine CRPC-NE vulnerabilities, we have performed image-based screening using a small library of antibodies targeting different histone marks on CRPC-NE organoids derived from genetically engineered mice. We find that the histone mark H3K36me2 and the histone methyltransferase NSD2 play important roles in maintain the CRPC-NE epigenetic state. Knockout of NSD2 or ablation of H3K36me2 using an oncohistone mutant H3.3K36M reverted CRPC-NE to an adenocarcinoma phenotype. Simultaneous profiling of the transcriptome and epigenome from single cells verified this lineage conversion and the generation of cell states with canonical AR signaling. Moreover, H3K36me2 or NSD2 depleted mouse and human CRPC-NE organoids responded to enzalutamide treatment in vitro and in vivo, suggesting a reversal of castration-resistance. Most importantly, a small molecule inhibitor of NSD2 in combination with enzalutamide leads to growth suppression or apoptosis of mouse and human CRPC-NE organoids, as well as organoids of other CRPC subtypes. In conclusion, inhibition of NSD2 reverts the epigenomic state of CRPC-NE, reversing lineage plasticity and restoring anti-androgen sensitivity. Thus, we suggest that the combination of NSD2 inhibition with AR inhibition may represent a novel therapeutic approach for patients with CRPC-NE or even other CRPC subtypes. Citation Format: Jia J. Li, Alessandro Vasciaveo, Dimitrios Karagiannis, Xiao Chen, Chen Yu, Andrea Califano, Chao Lu, Michael M. Shen. Targeting NSD2 to reverse lineage plasticity and drug resistance in neuroendocrine prostate cancer and subtypes of mCRPC [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 7546.

Abstract Although major mechanisms of resistance to targeted therapies include mutation of the drug target, recent evidence suggests cell-adaptive mechanisms as alternative avenues for escaping treatment. In several cancers, tumor cells become plastic in response to target inhibition, changing their identity and molecular programs without acquiring further mutations. In the case of castration-resistant prostate cancer (CRPC) treated with standard-of-care, next-generation anti-androgens enzalutamide, tumor cells often switch lineage to survive drug pressure, acquiring neuroendocrine (NE) features and thereby transforming into neuroendocrine prostate cancer (CRPC-NE). Existing targeted therapies for CRPC and CRPC-NE provide limited benefit, representing an unmet clinical need. Hence, the elucidation of tumor plasticity and consequent heterogeneity that contribute to drug resistance is of paramount importance to identify novel therapeutic opportunities. We have recently developed a network-based precision cancer medicine framework to characterize molecular and drug sensitivity profiles of ~100 Genetically-Engineered Mouse Models (GEMM)-derived tumors of prostate cancer (Vasciaveo, et al. 2023). This study revealed that tumors derived from GEMMs with the loss of function of both Pten and Trp53 (NPp53) align with CRPC patients and exhibit high heterogeneity in regulatory programs and histopathologic features, including NE differentiation. We then adopted the NPp53 model to generate several 3D organoid lines in order to study tumor plasticity and heterogeneity. To elucidate their tumors’ regulatory programs, we reverse-engineered genome-wide regulatory networks using multimodal single-cell RNASeq and ATACSeq data to identify Master Regulator (MR) proteins implementing tumor cell identities and characterize their epigenetic landscape. Our network-based regulatory analysis identified several distinct cell populations, including cells activating epithelial-to-mesenchymal (EMT) programs and NE differentiation, all resistant to enzalutamide treatment. Cross-species analysis aligned these cells to human CRPC and CRPC-NE tumors. In particular, our analyses have shed light into the roles of NSD2, a known MR of lethal prostate cancer, whose ablation restored sensitivity to enzalutamide. In summary, we leveraged single-cell, multimodal data from CRPC and CRPC-NE 3D mouse organoids to elucidate regulatory programs of drug resistant, lethal prostate cancer and characterize its heterogeneity. Importantly, we also identified a mechanism that can be exploited for therapeutic intervention to restore tumor cells’ sensitivity Citation Format: Alessandro Vasciaveo, Jia J. Li, Dimitrios Karagiannis, Xiao Chen, Andrea Califano, Chao Lu, Michael M. Shen. Reversing lineage plasticity and drug resistance in lethal prostate cancer [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 6545.

Abstract Patient-derived organoid models provide a highly amenable experimental platform to study human tumor biology. Over the past seven years, our group has generated a living biobank of 82 organoid lines established from urothelial carcinomas. These organoids have been generated from all stages, are capable of unlimited expansion in vitro, and recapitulate histopathological and molecular features of their corresponding primary tumors. In addition, we have characterized the genomic, transcriptomic, and chromatin accessibility profiles of these organoid lines. Using these lines, we have identified a novel form of lineage plasticity in which a subset of organoids derived from luminal NMIBC tumors can acquire basal features in culture, but reverse back to a luminal phenotype following orthotopic xenografting in immunodeficient mice. We have focused our studies on this luminal-to-basal (L-B) plasticity since the acquisition of basal phenotypes is linked to secondary MIBC. In ongoing studies, we have examined the genomic correlates of L-B plasticity and found that loss-of-function (LOF) mutations in the epigenetic regulator KMT2D are enriched in plastic lines derived from NMIBC and in stable basal lines from basal/squamous MIBC. We also found that tumors with squamous histology in the TCGA cohort are enriched for KMT2D LOF mutations. KMT2D encodes a histone methyltransferase that adds H3K4me1 active histone marks to enhancer regions and is frequently mutated in urothelial cancers. To investigate its functional role, we performed CRISPR knock-out of KMT2D in a stable luminal organoid line and found that isogenic knock-out organoids acquired basal phenotypes. Using a barcoded lineage-tracing approach known as CellTag, we have found that acquisition of basal phenotypes occurs in a clonal fashion at the single-cell level. In parallel, we have performed a small molecule screen to identify compounds that can reverse L-B plasticity, using 17 compounds that target a range of epigenetic regulators. We found that GSK-LSD1, an inhibitor of KDM1A, can restore luminal phenotypes to plastic organoid lines in culture and increase the genomic distribution of the H3K4me1 mark, as detected by CUT&Tag. Interestingly, KDM1A is a histone demethylase that removes H3K4me1/2 marks, and thus can antagonize KMT2D. Using CellTag, we found that reversal of the basal phenotype occurs in a non-clonal fashion and represents a cell state transition at the level of single cells. In further studies, we have shown that KDM1A inhibitor treatment of fresh patient samples can prevent the L-B transition. We have also demonstrated in an organoid outgrowth assay that KDM1A inhibition can prevent invasive behavior in vitro. Moreover, an orally available KDM1A inhibitor, ORY-1001, can also reverse phenotypic plasticity, and we are currently testing its activity in xenografts to determine whether it can reverse invasive phenotypes. Overall, these results demonstrate the utility of patient-derived organoid models to investigate novel features of bladder cancer and develop therapeutic approaches. Citation Format: John R. Christin, Alana Nguyen, Talal Syed, Kwanghee Kim, Clementine Le Coz, Caroline J. Laplaca, Luis A. Pina, Reuben Akabas, Hanina Hibshoosh, Andrew T. Lenis, Guarionex J. Decastro, Christopher B. Anderson, James M. McKiernan, Chao Lu, Hikmat A. Al-Ahmadie, David B. Solit, Michael M. Shen. A living biobank of patient-derived organoids reveals a role for KMT2D in maintaining luminal identity in urothelial carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A028.

Supplementary Table S2. List of antibodies and dilutions used in the study.

Supplementary Figure S5. CSF-1/CSF-1R signaling associates positively with clinical outcomes, and leukocyte infiltration ESCC.

Supplementary Figure S2. Trp53R172H-dependent Csf-1/Csf-1r signaling promotes metastasis only in the presence of p53-R172H, but not p53 null or wild-type p53.

Supplementary Table S3. Statistics of aligned reads from the CUT&RUN-seq analysis of H3K27ac in ESCC cells with Trp53R172H/- (n=3) and Trp53+/+ (n=2).

Supplementary Figure S4. Depletion of Brd4 reduces Csf1 secretion and ESCC tumor cell invasion.

Supplementary Figure S6. Graphical abstract depicting the pro-metastatic role and mechanism of Trp53R172H-dependent Csf-1/Csf-1r signaling.

Esophageal squamous cell carcinoma (ESCC) is a devastating and common disease worldwide. Despite recent advances annotating the genetic and epigenetic changes underlying the initiation and development of ESCC and its precursor lesion, esophageal squamous dysplasia, more work is needed to translate these findings into novel therapeutic strategies. This gap is due in part to the lack of tractable models of ESCC initiation and development that can then be manipulated ex vivo for preclinical testing. Currently, the large-scale studies characterizing the aforementioned molecular changes have used primary tissue for their analyses and have subsequently been unable to test the specific hypotheses that arise from their data using the original tissue that they profiled. To address this gap, we have generated a large (n = 54) three-dimensional organoid library from the esophagi of mice treated with the carcinogen 4-Nitroquinoline N-oxide (4-NQO) representing each stage of esophageal squamous cell initiation and progression from early intraepithelial dysplasia through lung metastasis. We have determined that these organoids faithfully recapitulate the molecular biology, histology, and oncogenic phenotype of their tissue of origin. Through integrative analysis of both RNA sequencing and whole exome sequencing data, we have identified several novel therapeutic targets that contribute to ESCC initiation and development including the DNA damage response and NOTCH1 signaling. Together, our work capitalizes on the intersection of large-scale multiomics and sophisticated 3D organoid models to identify novel therapeutic targets for the treatment of a devastating disease. Citation Format: Samuel Flashner, Masataka Shimonosono, Satoshi Takada, Norihiro Matsuura, Yasuto Tomita, Xiao Chen, Alison Taylor, Andres Klein-Szanto, Fatameh Momen-Heravi, J. Alan Diehl, Chao Lu, Hiroshi Nakagawa. Leveraging a 3D organoid library to identify novel therapeutic targets during ESCC initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3077.