Therapeutic Strategies for RB1-Deficient Cancers: Intersecting Gene Regulation and Targeted Therapy.

The retinoblastoma (RB) transcriptional corepressor 1 (RB1) is a critical tumor suppressor gene, governing diverse cellular processes implicated in cancer biology. Dysregulation or deletion in RB1 contributes to the development and progression of various cancers, making it a prime target for therapeutic intervention. RB1's canonical function in cell cycle control and DNA repair mechanisms underscores its significance in restraining aberrant cell growth and maintaining genomic stability. Understanding the complex interplay between RB1 and cellular pathways is beneficial to fully elucidate its tumor-suppressive role across different cancer types and for therapeutic development. As a result, investigating vulnerabilities arising from RB1 deletion-associated mechanisms offers promising avenues for targeted therapy. Recently, several findings highlighted multiple methods as a promising strategy for combating tumor growth driven by RB1 loss, offering potential clinical benefits in various cancer types. This review summarizes the multifaceted role of RB1 in cancer biology and its implications for targeted therapy.

Wearable Biosensor with Molecularly Imprinted Conductive Polymer Structure to Detect Lentivirus in Aerosol.

The coronavirus disease (COVID-19) pandemic has increased pressure to develop low-cost, compact, user-friendly, and ubiquitous virus sensors for monitoring infection outbreaks in communities and preventing economic damage resulting from city lockdowns. As proof of concept, we developed a wearable paper-based virus sensor based on a molecular imprinting technique, using a conductive polyaniline (PANI) polymer to detect the lentivirus as a test sample. This sensor detected the lentivirus with a 4181 TU/mL detection limit in liquid and 0.33% to 2.90% detection efficiency in aerosols at distances ranging from 30 cm to 60 cm. For fabrication, a mixture of a PANI monomer solution and virus were polymerized together to form a conductive PANI sensing element on a polyethylene terephthalate (PET) paper substrate. The sensing element exhibited formation of virus recognition sites after the removal of the virus via ultrasound sonication. A dry measurement technique was established that showed aerosol virus detection by the molecularly imprinted sensors within 1.5 h of virus spraying. This was based on the mechanism via which dispensing virus droplets on the PANI sensing element induced hybridization of the virus and molecularly imprinted virus recognition templates in PANI, influencing the conductivity of the PANI film upon drying. Interestingly, the paper-based virus sensor was easily integrated with a wearable face mask for the detection of viruses in aerosols. Since the paper sensor with molecular imprinting of virus recognition sites showed excellent stability in dry conditions for long periods of time, unlike biological reagents, this wearable biosensor will offer an alternative approach to monitoring virus infections in communities.

Unique Transcriptional Profiles Underlie Osteosarcomagenesis Driven by Different p53 Mutants.

Missense mutations in the DNA binding domain of p53 are characterized as structural or contact mutations based on their effect on the conformation of the protein. These mutations show gain-of-function (GOF) activities, such as promoting increased metastatic incidence compared with p53 loss, often mediated by the interaction of mutant p53 with a set of transcription factors. These interactions are largely context specific. To understand the mechanisms by which p53 DNA binding domain mutations drive osteosarcoma progression, we created mouse models, in which either the p53 structural mutant p53R172H or the contact mutant p53R245W are expressed specifically in osteoblasts, yielding osteosarcoma tumor development. Survival significantly decreased and metastatic incidence increased in mice expressing p53 mutants compared with p53-null mice, suggesting GOF. RNA sequencing of primary osteosarcomas revealed vastly different gene expression profiles between tumors expressing the missense mutants and p53-null tumors. Further, p53R172H and p53R245W each regulated unique transcriptomes and pathways through interactions with a distinct repertoire of transcription factors. Validation assays showed that p53R245W, but not p53R172H, interacts with KLF15 to drive migration and invasion in osteosarcoma cell lines and promotes metastasis in allogeneic transplantation models. In addition, analyses of p53R248W chromatin immunoprecipitation peaks showed enrichment of KLF15 motifs in human osteoblasts. Taken together, these data identify unique mechanisms of action of the structural and contact mutants of p53. SIGNIFICANCE: The p53 DNA binding domain contact mutant p53R245W, but not the structural mutant p53R172H, interacts with KLF15 to drive metastasis in somatic osteosarcoma, providing a potential vulnerability in tumors expressing p53R245W mutation.

Li-Fraumeni Syndrome-Associated Dimer-Forming Mutant p53 Promotes Transactivation-Independent Mitochondrial Cell Death.

Cancer-relevant mutations in the oligomerization domain (OD) of the p53 tumor suppressor protein, unlike those in the DNA binding domain, have not been well elucidated. Here, we characterized the germline OD mutant p53(A347D), which occurs in cancer-prone Li-Fraumeni syndrome (LFS) patients. Unlike wild-type p53, mutant p53(A347D) cannot form tetramers and exists as a hyperstable dimeric protein. Further, p53(A347D) cannot bind or transactivate the majority of canonical p53 target genes. Isogenic cell lines harboring either p53(A347D) or no p53 yield comparable tumorigenic properties, yet p53(A347D) displays remarkable neomorphic activities. Cells bearing p53(A347D) possess a distinct transcriptional profile and undergo metabolic reprogramming. Further, p53(A347D) induces striking mitochondrial network aberration and associates with mitochondria to drive apoptotic cell death upon topoisomerase II inhibition in the absence of transcription. Thus, dimer-forming p53 demonstrates both loss-of-function (LOF) and gain-of-function (GOF) properties compared with the wild-type form of the protein. SIGNIFICANCE: A mutant p53 (A347D), which can only form dimers, is associated with increased cancer susceptibility in LFS individuals. We found that this mutant wields a double-edged sword, driving tumorigenesis through LOF while gaining enhanced apoptogenic activity as a new GOF, thereby yielding a potential vulnerability to select therapeutic approaches. See related commentary by Stieg et al., p. 1046. See related article by Gencel-Augusto et al., p. 1230. This article is highlighted in the In This Issue feature, p. 1027.

Rewired m6A epitranscriptomic networks link mutant p53 to neoplastic transformation.

N6-methyladenosine (m6A), one of the most prevalent mRNA modifications in eukaryotes, plays a critical role in modulating both biological and pathological processes. However, it is unknown whether mutant p53 neomorphic oncogenic functions exploit dysregulation of m6A epitranscriptomic networks. Here, we investigate Li-Fraumeni syndrome (LFS)-associated neoplastic transformation driven by mutant p53 in iPSC-derived astrocytes, the cell-of-origin of gliomas. We find that mutant p53 but not wild-type (WT) p53 physically interacts with SVIL to recruit the H3K4me3 methyltransferase MLL1 to activate the expression of m6A reader YTHDF2, culminating in an oncogenic phenotype. Aberrant YTHDF2 upregulation markedly hampers expression of multiple m6A-marked tumor-suppressing transcripts, including CDKN2B and SPOCK2, and induces oncogenic reprogramming. Mutant p53 neoplastic behaviors are significantly impaired by genetic depletion of YTHDF2 or by pharmacological inhibition using MLL1 complex inhibitors. Our study reveals how mutant p53 hijacks epigenetic and epitranscriptomic machinery to initiate gliomagenesis and suggests potential treatment strategies for LFS gliomas.

Bcl11b and Atoh8 Coordinate Cellular Plasticity for Reprogramming and Transformation.

By dissecting and comparing the transcriptional trajectories and epigenomic traits of reprogramming and transforming cells at the single-cell resolution, Huyghe et al discovered Bcl11b and Atoh8, two key transcription factors controlling cell plasticity during pluripotent reprogramming and oncogenic transformation.

METTL14-mediated epitranscriptome modification of MN1 mRNA promote tumorigenicity and all-trans-retinoic acid resistance in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumor in adolescents. The molecular mechanism behind OS progression and metastasis remains poorly understood, which limits the effectiveness of current therapies. RNA N6-methyladenosine (m6A) modification plays a critical role in influencing RNA fate. However, the biological significance of m6A modification and its potential regulatory mechanisms in the development of OS remain unclear. METHODS: Liquid chromatography-tandem mass spectrometry (LC-MS/MS), dot blotting, and colorimetric ELISA were used to detect m6A levels. Western blotting, quantitative real-time PCR (RT-qPCR) and immunohistochemistry (IHC) were used to investigate METTL14 expression levels. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of METTL14. RNA pull-down and RNA immunoprecipitation (RIP) assays were conducted to explore the specific binding of target genes and relevant m6A "readers". RNA stability and polysome analysis assays were used to detect the half-lives and translation efficiencies of the downstream genes of METTL14. IHC and clinical data were applied to explore the clinical correlations of METTL14 and its downstream target genes with the prognosis of OS. FINDINGS: We observed the abundance of m6A modifications in OS and revealed that METTL14 plays an oncogenic role in facilitating OS progression. MeRIP-seq and RNA-seq revealed that MN1 is a downstream gene of METTL14. MN1 contributes to tumor progression and all-trans-retinoic acid (ATRA) chemotherapy resistance in OS. Mechanistically, MN1 is methylated by METTL14, specifically in the coding sequence (CDS) regions, and this modification is recognized by the specific m6A reader insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) to prevent MN1 mRNA degradation and promote it translation efficiency. IHC showed that MN1 expression was positively correlated with METTL14 and IGF2BP2 expression in OS tissues. The METTL14-IGF2BP2-MN1 panel demonstrated more promising prognostic value for OS patients than any of these molecules individually. INTERPRETATION: Our study revealed that METTL14 contributes to OS progression and ATRA resistance as an m6A RNA methylase by regulating the stability and translation efficiency of MN1 and thus provides both an underlying biomarker panel for prognosis prediction in OS patients. FUNDING: This work was supported by the National Natural Science Foundation of China (Grants 81972510 and 81772864).

Generation of a homozygous knock-in human embryonic stem cell line expressing mEos4b-tagged CTR1.

Copper transporter 1 (CTR1) is the major membrane protein responsible for cellular copper (Cu) uptake and mediates cellular copper homeostasis. To elucidate CTR1's behavior using imaging approaches, we generated a homozygous knock-in human embryonic stem cell (hESC) clone expressing photoconvertible fluorescence protein mEos4b-tagged endogenous CTR1 using CRISPR-Cas9 mediated homologous recombination. The engineered cells express functional CTR1-mEos4b fusion and have normal stem cell morphology. They remain pluripotent and can be differentiated into all three germ layers in vitro. This resource allows the study of CTR1 at an endogenous level in different cellular contexts using microscopy.

Hereditary retinoblastoma iPSC model reveals aberrant spliceosome function driving bone malignancies.

The RB1 gene is frequently mutated in human cancers but its role in tumorigenesis remains incompletely defined. Using an induced pluripotent stem cell (iPSC) model of hereditary retinoblastoma (RB), we report that the spliceosome is an up-regulated target responding to oncogenic stress in RB1-mutant cells. By investigating transcriptomes and genome occupancies in RB iPSC­derived osteoblasts (OBs), we discover that both E2F3a, which mediates spliceosomal gene expression, and pRB, which antagonizes E2F3a, coregulate more than one-third of spliceosomal genes by cobinding to their promoters or enhancers. Pharmacological inhibition of the spliceosome in RB1-mutant cells leads to global intron retention, decreased cell proliferation, and impaired tumorigenesis. Tumor specimen studies and genome-wide TCGA (The Cancer Genome Atlas) expression profile analyses support the clinical relevance of pRB and E2F3a in modulating spliceosomal gene expression in multiple cancer types including osteosarcoma (OS). High levels of pRB/E2F3a­regulated spliceosomal genes are associated with poor OS patient survival. Collectively, these findings reveal an undiscovered connection between pRB, E2F3a, the spliceosome, and tumorigenesis, pointing to the spliceosomal machinery as a potentially widespread therapeutic vulnerability of pRB-deficient cancers.

ZFP207 sustains pluripotency by coordinating OCT4 stability, alternative splicing and RNA export.

The pluripotent state is not solely governed by the action of the core transcription factors OCT4, SOX2, and NANOG, but also by a series of co-transcriptional and post-transcriptional events, including alternative splicing (AS) and the interaction of RNA-binding proteins (RBPs) with defined subpopulations of RNAs. Zinc Finger Protein 207 (ZFP207) is an essential transcription factor for mammalian embryonic development. Here, we employ multiple functional analyses to characterize its role in mouse embryonic stem cells (ESCs). We find that ZFP207 plays a pivotal role in ESC maintenance, and silencing of Zfp207 leads to severe neuroectodermal differentiation defects. In striking contrast to human ESCs, mouse ZFP207 does not transcriptionally regulate neuronal and stem cell-related genes but exerts its effects by controlling AS networks and by acting as an RBP. Our study expands the role of ZFP207 in maintaining ESC identity, and underscores the functional versatility of ZFP207 in regulating neural fate commitment.

Patient-derived iPSCs link elevated mitochondrial respiratory complex I function to osteosarcoma in Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive genetic disorder characterized by poikiloderma, small stature, skeletal anomalies, sparse brows/lashes, cataracts, and predisposition to cancer. Type 2 RTS patients with biallelic RECQL4 pathogenic variants have multiple skeletal anomalies and a significantly increased incidence of osteosarcoma. Here, we generated RTS patient-derived induced pluripotent stem cells (iPSCs) to dissect the pathological signaling leading to RTS patient-associated osteosarcoma. RTS iPSC-derived osteoblasts showed defective osteogenic differentiation and gain of in vitro tumorigenic ability. Transcriptome analysis of RTS osteoblasts validated decreased bone morphogenesis while revealing aberrantly upregulated mitochondrial respiratory complex I gene expression. RTS osteoblast metabolic assays demonstrated elevated mitochondrial respiratory complex I function, increased oxidative phosphorylation (OXPHOS), and increased ATP production. Inhibition of mitochondrial respiratory complex I activity by IACS-010759 selectively suppressed cellular respiration and cell proliferation of RTS osteoblasts. Furthermore, systems analysis of IACS-010759-induced changes in RTS osteoblasts revealed that chemical inhibition of mitochondrial respiratory complex I impaired cell proliferation, induced senescence, and decreased MAPK signaling and cell cycle associated genes, but increased H19 and ribosomal protein genes. In summary, our study suggests that mitochondrial respiratory complex I is a potential therapeutic target for RTS-associated osteosarcoma and provides future insights for clinical treatment strategies.

Cardiotoxicity of Antineoplastic Therapies and Applications of Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

The therapeutic landscape for the treatment of cancer has evolved significantly in recent decades, aided by the development of effective oncology drugs. However, many cancer drugs are often poorly tolerated by the body and in particular the cardiovascular system, causing adverse and sometimes fatal side effects that negate the chemotherapeutic benefits. The prevalence and severity of chemotherapy-induced cardiotoxicity warrants a deeper investigation of the mechanisms and implicating factors in this phenomenon, and a consolidation of scientific efforts to develop mitigating strategies. Aiding these efforts is the emergence of induced pluripotent stem cells (iPSCs) in recent years, which has allowed for the generation of iPSC-derived cardiomyocytes (iPSC-CMs): a human-based, patient-derived, and genetically variable platform that can be applied to the study of chemotherapy-induced cardiotoxicity and beyond. After surveying chemotherapy-induced cardiotoxicity and the associated chemotherapeutic agents, we discuss the use of iPSC-CMs in cardiotoxicity modeling, drug screening, and other potential applications. Improvements to the iPSC-CM platform, such as the development of more adult-like cardiomyocytes and ongoing advances in biotechnology, will only enhance the utility of iPSC-CMs in both basic science and clinical applications.

The Function of the Mutant p53-R175H in Cancer.

Wild-type p53 is known as "the guardian of the genome" because of its function of inducing DNA repair, cell-cycle arrest, and apoptosis, preventing the accumulation of gene mutations. TP53 is highly mutated in cancer cells and most TP53 hotspot mutations are missense mutations. Mutant p53 proteins, encoded by these hotspot mutations, lose canonical wild-type p53 functions and gain functions that promote cancer development, including promoting cancer cell proliferation, migration, invasion, initiation, metabolic reprogramming, angiogenesis, and conferring drug resistance to cancer cells. Among these hotspot mutations, p53-R175H has the highest occurrence. Although losing the transactivating function of the wild-type p53 and prone to aggregation, p53-R175H gains oncogenic functions by interacting with many proteins. In this review, we summarize the gain of functions of p53-R175H in different cancer types, the interacting proteins of p53-R175H, and the downstream signaling pathways affected by p53-R175H to depict a comprehensive role of p53-R175H in cancer development. We also summarize treatments that target p53-R175H, including reactivating p53-R175H with small molecules that can bind to p53-R175H and alter it into a wild-type-like structure, promoting the degradation of p53-R175H by targeting heat-shock proteins that maintain the stability of p53-R175H, and developing immunotherapies that target the p53-R175H-HLA complex presented by tumor cells.

RNA m6A modification orchestrates a LINE-1-host interaction that facilitates retrotransposition and contributes to long gene vulnerability.

The molecular basis underlying the interaction between retrotransposable elements (RTEs) and the human genome remains poorly understood. Here, we profiled N6-methyladenosine (m6A) deposition on nascent RNAs in human cells by developing a new method MINT-Seq, which revealed that many classes of RTE RNAs, particularly intronic LINE-1s (L1s), are strongly methylated. These m6A-marked intronic L1s (MILs) are evolutionarily young, sense-oriented to hosting genes, and are bound by a dozen RNA binding proteins (RBPs) that are putative novel readers of m6A-modified RNAs, including a nuclear matrix protein SAFB. Notably, m6A positively controls the expression of both autonomous L1s and co-transcribed L1 relics, promoting L1 retrotransposition. We showed that MILs preferentially reside in long genes with critical roles in DNA damage repair and sometimes in L1 suppression per se, where they act as transcriptional "roadblocks" to impede the hosting gene expression, revealing a novel host-weakening strategy by the L1s. In counteraction, the host uses the SAFB reader complex to bind m6A-L1s to reduce their levels, and to safeguard hosting gene transcription. Remarkably, our analysis identified thousands of MILs in multiple human fetal tissues, enlisting them as a novel category of cell-type-specific regulatory elements that often compromise transcription of long genes and confer their vulnerability in neurodevelopmental disorders. We propose that this m6A-orchestrated L1-host interaction plays widespread roles in gene regulation, genome integrity, human development and diseases.

Generation of a homozygous knock-in human embryonic stem cell line expressing SNAP-tagged SOD1.

Superoxide Dismutase 1 (SOD1) is an antioxidant enzyme that protects the cells from radical oxygen species. To study the behavior of endogenous SOD1 under a microscope, we genetically modified H1 human embryonic stem cells (hESCs) to express SOD1 fused with a SNAP-tag, a protein tag that can be covalently labeled with a variety of synthetic probes. The engineered homozygous clone expressing SOD1-SNAP fusion proteins has normal stem cell morphology and karyotype, expresses pluripotency markers, and can be differentiated into all three germ layers in vitro, providing a versatile platform for imaging-based studies of SOD1.

An Esrrb and Nanog Cell Fate Regulatory Module Controlled by Feed Forward Loop Interactions.

Cell fate decisions during development are governed by multi-factorial regulatory mechanisms including chromatin remodeling, DNA methylation, binding of transcription factors to specific loci, RNA transcription and protein synthesis. However, the mechanisms by which such regulatory "dimensions" coordinate cell fate decisions are currently poorly understood. Here we quantified the multi-dimensional molecular changes that occur in mouse embryonic stem cells (mESCs) upon depletion of Estrogen related receptor beta (Esrrb), a key pluripotency regulator. Comparative analyses of expression changes subsequent to depletion of Esrrb or Nanog, indicated that a system of interlocked feed-forward loops involving both factors, plays a central part in regulating the timing of mESC fate decisions. Taken together, our meta-analyses support a hierarchical model in which pluripotency is maintained by an Oct4-Sox2 regulatory module, while the timing of differentiation is regulated by a Nanog-Esrrb module.

Modeling of osteosarcoma with induced pluripotent stem cells.

Osteosarcoma is the most common type of bone cancer. Osteosarcoma is commonly associated with TP53 inactivation (around 95% of cases) and RB1 inactivation (around 28% of cases). With the discovery of reprogramming factors to induce pluripotency even in terminally differentiated cells, induced pluripotent stem cells (iPSCs) have emerged as a promising disease model. iPSC-based disease modeling uniquely recapitulates disease phenotypes and can support discoveries into disease etiology and is used extensively today to study a variety of diseases, including cancers. This paper focuses on iPSC-based modeling of Li-Fraumeni syndrome (LFS), an autosomal dominant disorder commonly associated with TP53 mutation and high osteosarcoma incidence. As iPSCs are increasingly utilized as a platform for cancer modeling, the experimental approaches that we discuss here may serve as a guide for future studies.