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Recent advances in protein engineering have provided a wealth of methods that allow for the site-specific manipulation of proteinsin vitroand in cells. However, the efforts to expand these toolkits for use in live animals has been limited. Here, we report a new method for the semi-synthesis of site-specifically modified and chemically defined proteins in live animals. Importantly, we illustrate the usefulness of this methodology in the context of a challenging, chromatin bound N-terminal histone tail within rodent postmitotic neurons located in ventral striatum (Nucleus Accumbens/NAc). This approach provides the field with a precise and broadly applicable methodology for manipulating histonesin vivo, thereby serving as a unique template towards examining chromatin phenomena that may mediate transcriptomic and physiological plasticity within mammals.

Metastasis is the leading cause of cancer-related death among women with breast cancer. Chromosomal instability (CIN) has emerged as a hallmark of triple-negative breast cancer (TNBC) as it has recently been shown to promote metastasis. However, the underlying molecular mechanisms by which CIN drives metastasis are not completely understood. We have identified a discrete population of highly metastatic SOX2/OCT4+ cells expressing elevated levels of epigenetic regulator EZH2 associated with increased CIN in both human and mouse TNBC. EZH2 histone methyl transferase (HMT) is the catalytic subunit of the Polycomb repressive complex 2 (PRC2), represses target genes through trimethylation of Histone 3 at lysine 27 (H3K27me3). Importantly, genetic and pharmacologic inhibition of EZH2 lead to reduction of CIN and impaired metastasis. These findings led to the hypothesis that EZH2-mediated CIN constitutes a novel mechanism of metastasis regulation. To directly demonstrate epigenetic regulation of CIN, we used genome wide-Cleavage Under Targets and Release Using Nuclease (CUT&RUN) in parallel with RNA-seq. Gene set enrichment of EZH2-repressed target genes directly implicated the spindle formation pathway network. The central core of this network comprised of tankyrase (TNKS), a multifunctional poly (ADP-ribose) polymerase (PARP) previously implicated in DNA repair, telomere function and centrosome maturation. ChIP-PCR confirmed that EZH2 directly binds to the TNKS promoter, and CRISPR knockout of TNKS abrogated the ability of EZH2 inhibition in suppressing CIN, consistent with pharmacological inhibition of TNKS. More specifically, dysregulation of TNKS by EZH2 in OCT4/SOX2+ cells lead to increased numbers of centrosomes and multipolar mitosis. To directly demonstrate the role of aberrant H3K27me3 in regulating chromosomal segregation during mitosis, we used dCas9-EZH2 or dCas9-EZH2 catalytically dead mutant together with chromosome-specific CRISPR guides to ectopically enhance H3K27 trimethylation at the pericentromeric region of specific chromosomes. Ectopic deposition of EZH2 at pericentromeric regions lead to increased CIN. Conceptually, our work provides an unappreciated link between epigenetic regulation and CIN which have been hitherto studied in isolation. From a clinical perspective, demonstrating epigenetic regulation of CIN has opened the possibility for the development of first CIN suppressive therapeutic strategies targeting TNBC metastasis. Citation Format: Yang Bai, Albert Agustinus, Cem Meydan, Dylan R. McNally, Shira Yomtoubian, Liron Yoffe, Ari M. Melnick, Samuel Bakhoum, Vivek Mittal. Epigenetic regulation of chromosomal instability in triple-negative breast cancer [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 5811.

Amyloid-beta (Aβ) deposition occurs in the early stages of Alzheimer’s disease (AD), but the early detection of Aβ is a persistent challenge. Herein, we engineered a near-infrared optical nanosensor capable of detecting Aβ intracellularly in live cells and intracranially in vivo. The sensor is composed of single-walled carbon nanotubes functionalized with Aβ wherein Aβ-Aβ interactions drive the response. We found that the Aβ nanosensors selectively responded to Aβ via solvatochromic modulation of the near-infrared emission of the nanotube. The sensor tracked Aβ accumulation in live cells and, upon intracranial administration in a genetic model of AD, signaled distinct responses in aged mice. This technology enables the interrogation of molecular mechanisms underlying Aβ neurotoxicity in the development of AD in living systems.

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers [1-4], yet whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei [5, 6], and subsequent micronuclear envelope rupture [7] profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice as well as cancer and non-transformed cells. Some of the changes to histone PTMs occur due to micronuclear envelope rupture whereas others are inherited from mitotic abnormalities prior to micronucleus formation. Using orthogonal techniques, we show that micronuclei exhibit extensive differences in chromatin accessibility with a strong positional bias between promoters and distal or intergenic regions. Finally, we show that inducing CIN engenders widespread epigenetic dysregulation and that chromosomes which transit in micronuclei experience durable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, in addition to genomic copy number alterations, CIN can serve as a vehicle for epigenetic reprogramming and heterogeneity in cancer.

Chromosomal instability (CIN) and epigenetic alterations have been implicated in tumor progression and metastasis; yet how these two hallmarks of cancer are related remains poorly understood. By integrating genetic, epigenetic, and functional analyses at the single cell level, we show that progression of uveal melanoma (UM), the most common intraocular primary cancer in adults, is driven by loss of Polycomb Repressive Complex 1 (PRC1) in a subpopulation of tumor cells. This leads to transcriptional de-repression of PRC1-target genes and mitotic chromosome segregation errors. Ensuing CIN leads to the formation of rupture-prone micronuclei, exposing genomic double-stranded DNA (dsDNA) to the cytosol. This provokes tumor cell-intrinsic inflammatory signaling, mediated by aberrant activation of the cGAS-STING pathway. PRC1 inhibition promotes nuclear enlargement, induces a transcriptional response that is associated with significantly worse patient survival and clinical outcomes, and enhances migration that is rescued upon pharmacologic inhibition of CIN or STING. Thus, deregulation of PRC1 can promote tumor progression by inducing CIN and represents an opportunity for early therapeutic intervention., The molecular underpinnings driving uveal melanoma (UM) progression are unknown. Here the authors show that loss of Polycomb Repressive Complex 1 triggers chromosomal instability, which promotes inflammatory signaling and migration in UM.

Melanoma brain metastasis (MBM) frequently occurs in patients with advanced melanoma, yet our understanding of the underlying salient biology is rudimentary. Here, we performed single-cell/nucleus RNA-seq in 22 treatment-naïve MBM and 10 extracranial melanoma metastases (ECM), and matched spatial single-cell transcriptomics and T cell receptor (TCR)-seq. Cancer cells from MBM were more chromosomally unstable, adopted a neuronal-like cell state, and enriched for spatially variably expressed metabolic pathways. Key observations were validated in independent patient cohorts, patient-derived MBM/ECM xenograft models, RNA/ATAC-seq, proteomics, and multiplexed imaging. Integrated spatial analyses revealed distinct geography of putative cancer immune evasion, and evidence for more abundant intra-tumoral B to plasma cell differentiation in lymphoid aggregates in MBM. MBM harbored larger fractions of monocyte-derived macrophages and dysfunctional TOX(+)CD8(+) T cells with distinct expression of immune checkpoints. This work provides comprehensive insights into MBM biology and serves as a foundational resource for further discovery and therapeutic exploration.

Chromosomal instability is a hallmark of aggressive human cancers and it is often associated with metastasis, immune evasion, and therapeutic resistance. Chromosomally unstable tumors often contain rupture-prone micronuclei, which harbor mis-segregated chromosomes. Rupture of micronuclear envelopes represents a critical event in the evolution of chromosomally unstable tumors given its ability to catalyze the formation of genomic rearrangements known as chromothripsis, and to trigger the cytosolic DNA-sensing cGAS-STING inflammatory pathway, whose aberrant and chronic activation was found to promote metastatic progression [1]. Despite its central role in tumor progression, how micronuclear membranes collapse and why membrane repair mechanisms, which are functional at the primary nucleus, fail to restore micronuclear integrity, remains poorly understood. Here we show that the answer to these fundamental questions lays in mitochondria proximity to micronuclei: by means of advanced microscopy and cellular and molecular biology techniques, our results demonstrate that ruptured micronuclei are more heavily embedded in the mitochondrial matrix than the intact ones, and that ROS are the main cause of micronuclear rupture. We used a combination of ROS-inducing drugs on a variety of cell lines to show an increase in collapsed micronuclei, while using ROS-scavengers we reduced the physiological basal amount of micronuclear rupture. Nuclear membrane catastrophe has recently been identified as the main outcome of unrestrained activity of the ESCRT-III nuclear membrane repair complex, of which CHMP7 is the scaffolding protein [2]. We showed that cells under oxidative stress display an increase in CHMP7 content in micronuclei whose membrane is not yet collapsed, implicating a causative role of CHMP7 in micronuclear envelope rupture. Furthermore, by means of genetic manipulation we demonstrate a central role for CHMP7 in ROS-induced rupture, with evidences pointing towards a non-canonical role for CHMP7 in this process. We thus provide a mechanistic insight into a fundamental hub in advanced cancers metastatic onset: by understanding the players involved into micronuclear collapse, this work can bring to light new untapped drug targets that are specific for those cells that will undergo metastatic transformation. In conclusion, our study has the potential to develop new and more effective therapies for the treatment of aggressive chromosomally unstable cancers. 1. Bakhoum, S.F., et al., Chromosomal instability drives metastasis through a cytosolic DNA response. Nature, 2018. 553(7689): p. 467-472. 2. Vietri, M., et al., Unrestrained ESCRT-III drives micronuclear catastrophe and chromosome fragmentation. Nat Cell Biol, 2020. 22(7): p. 856-867 Citation Format: Melody Di Bona, Albert Agustinus, Yanyang Chen, Lorenzo Scipioni, Daniel Bronder, Samuel F. Bakhoum. Unraveling the mechanisms underlying micronuclear rupture, a seminal event in cancer 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 1589.

Alzheimer’s disease (AD) and other dementias are the 5th leading cause of death globally and pose an increasing global health challenge as the proportions of elderly population increase demographically. Although research aimed at providing treatments has been rigorously ongoing for the past few decades, no curative therapy has been established. Consequently, a focus on the identification of early, presymptomatic stages of the disease offer avenues for optimal timing of preventative treatments. Amyloid beta (Aß) accumulation and self-assembly is a central event in disease pathogenesis, and its use as a biomarker is established for disease prognosis and monitoring. Frontline diagnostic tools for detecting Aß, such as neuroimaging tools (Amyloid PET) and cerebral spinal fluid (CSF) analyses, are vital in classifying AD, yet detecting the early stages of dementia remains an ongoing challenge. High costs, limited availability, adverse effects, risk of complications, and exposure to radioactive isotopes limit the use of current Aß imaging tools to high risk individuals with measurements with limited measurements at short intervals. Therefore, there is a vital need to establish detection technologies that can specifically differentiate Aß burden from early stages and enhance identification of individuals that may progress to develop AD. We have developed a carbon nanotube (CNT) optical sensor that can monitor Aß biomarkers in live samples over time. We have designed and characterized a new class of bio-functionalized, intrinsically photoluminescent CNT sensor that can detect Aß via shifting of their intrinsic near-infrared emission wavelength. Our results show CNT sensors differentiate between AD models in-vitro and in in-vivo.