Epigenetic control of immunoevasion in cancer stem cells.

Cancer stem cells (CSCs) are a poorly differentiated population of malignant cells that (at least in some neoplasms) is responsible for tumor progression, resistance to therapy, and disease relapse. According to a widely accepted model, all stages of cancer progression involve the ability of neoplastic cells to evade recognition or elimination by the host immune system. In line with this notion, CSCs are not only able to cope with environmental and therapy-elicited stress better than their more differentiated counterparts but also appear to better evade tumor-targeting immune responses. We summarize epigenetic modifications of DNA and histones through which CSCs evade immune recognition or elimination, and propose that such alterations constitute promising therapeutic targets to increase the sensitivity of some malignancies to immunotherapy.

Assessment of cell cycle progression and mitotic slippage by videomicroscopy.

Senescence is a state of irreversible cell cycle arrest accompanied by the acquisition of the senescence-associated secretory phenotype (SASP), which is activated in response to a variety of damaging stimuli, including genotoxic therapy. Accumulating evidence indicates that mitotic stress also promotes entry into senescence. This occurs via a mechanism involving defective mitoses and mitotic arrest, followed by abortion of cell division and slippage in the G1 phase. In this process, mitotic slippage leads to the generation of senescent cells characterized by a large cell body and a multinucleated and/or enlarged nuclear size. Here, we provide a detailed protocol for the assessment of cell proliferation and mitotic slippage in colorectal cancer cells upon pharmacological inhibition of the mitotic kinesin KIF11, best known as EG5. This approach can be used for preliminary characterization of senescence induction by therapeutics, but requires validation with standard senescence assays.

Apoptotic cell death in disease-Current understanding of the NCCD 2023.

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.

Type I IFNs promote cancer cell stemness by triggering the epigenetic regulator KDM1B.

Cancer stem cells (CSCs) are a subpopulation of cancer cells endowed with high tumorigenic, chemoresistant and metastatic potential. Nongenetic mechanisms of acquired resistance are increasingly being discovered, but molecular insights into the evolutionary process of CSCs are limited. Here, we show that type I interferons (IFNs-I) function as molecular hubs of resistance during immunogenic chemotherapy, triggering the epigenetic regulator demethylase 1B (KDM1B) to promote an adaptive, yet reversible, transcriptional rewiring of cancer cells towards stemness and immune escape. Accordingly, KDM1B inhibition prevents the appearance of IFN-I-induced CSCs, both in vitro and in vivo. Notably, IFN-I-induced CSCs are heterogeneous in terms of multidrug resistance, plasticity, invasiveness and immunogenicity. Moreover, in breast cancer (BC) patients receiving anthracycline-based chemotherapy, KDM1B positively correlated with CSC signatures. Our study identifies an IFN-I → KDM1B axis as a potent engine of cancer cell reprogramming, supporting KDM1B targeting as an attractive adjunctive to immunogenic drugs to prevent CSC expansion and increase the long-term benefit of therapy.

Catastrophic DNA replication in unscheduled tetraploid cells.

Unscheduled tetraploidy is a metastable state that rapidly evolves into aneuploidy. Recent findings reported by Gemble et al. demonstrate that freshly formed tetraploid cells fail to accumulate the required amounts of DNA replication factors during the first G1 phase after whole-genome duplication (WGD), culminating in genetic instability in the subsequent S phase and extensive karyotypic alterations.

Using epigenetic modifiers to target cancer stem cell immunoevasion.

Two recent reports in Nature highlight a novel mechanism of immunoevasion that relies on the SET domain bifurcated histone lysine methyltransferase 1 (SETDB1)-dependent epigenetic suppression of endogenous retroelements in melanoma cells. Because SETDB1 is highly expressed by the stem cell compartment, these findings delineate an innovative strategy for restoring cancer stem cell immunosurveillance.

BRIO: a web server for RNA sequence and structure motif scan.

The interaction between RNA and RNA-binding proteins (RBPs) has a key role in the regulation of gene expression, in RNA stability, and in many other biological processes. RBPs accomplish these functions by binding target RNA molecules through specific sequence and structure motifs. The identification of these binding motifs is therefore fundamental to improve our knowledge of the cellular processes and how they are regulated. Here, we present BRIO (BEAM RNA Interaction mOtifs), a new web server designed for the identification of sequence and structure RNA-binding motifs in one or more RNA molecules of interest. BRIO enables the user to scan over 2508 sequence motifs and 2296 secondary structure motifs identified in Homo sapiens and Mus musculus, in three different types of experiments (PAR-CLIP, eCLIP, HITS). The motifs are associated with the binding of 186 RBPs and 69 protein domains. The web server is freely available at http://brio.bio.uniroma2.it.

The Targeting of MRE11 or RAD51 Sensitizes Colorectal Cancer Stem Cells to CHK1 Inhibition.

Cancer stem cells (CSCs) drive not only tumor initiation and expansion, but also therapeutic resistance and tumor relapse. Therefore, CSC eradication is required for effective cancer therapy. In preclinical models, CSCs demonstrated high capability to tolerate even extensive genotoxic stress, including replication stress, because they are endowed with a very robust DNA damage response (DDR). This favors the survival of DNA-damaged CSCs instead of their inhibition via apoptosis or senescence. The DDR represents a unique CSC vulnerability, but the abrogation of the DDR through the inhibition of the ATR-CHK1 axis is effective only against some subtypes of CSCs, and resistance often emerges. Here, we analyzed the impact of druggable DDR players in the response of patient-derived colorectal CSCs (CRC-SCs) to CHK1/2 inhibitor prexasertib, identifying RAD51 and MRE11 as sensitizing targets enhancing prexasertib efficacy. We showed that combined inhibition of RAD51 and CHK1 (via B02+prexasertib) or MRE11 and CHK1 (via mirin+prexasertib) kills CSCs by affecting multiple genoprotective processes. In more detail, these two prexasertib-based regimens promote CSC eradication through a sequential mechanism involving the induction of elevated replication stress in a context in which cell cycle checkpoints usually activated during the replication stress response are abrogated. This leads to uncontrolled proliferation and premature entry into mitosis of replication-stressed cells, followed by the induction of mitotic catastrophe. CRC-SCs subjected to RAD51+CHK1 inhibitors or MRE11+CHK1 inhibitors are eventually eliminated, and CRC-SC tumorspheres inhibited or disaggregated, via a caspase-dependent apoptosis. These results support further clinical development of these prexasertib-based regimens in colorectal cancer patients.

Control of replication stress and mitosis in colorectal cancer stem cells through the interplay of PARP1, MRE11 and RAD51.

Cancer stem cells (CSCs) are tumor subpopulations driving disease development, progression, relapse and therapy resistance, and their targeting ensures tumor eradication. CSCs display heterogeneous replication stress (RS), but the functionality/relevance of the RS response (RSR) centered on the ATR-CHK1 axis is debated. Here, we show that the RSR is efficient in primary CSCs from colorectal cancer (CRC-SCs), and describe unique roles for PARP1 and MRE11/RAD51. First, we demonstrated that PARP1 is upregulated in CRC-SCs resistant to several replication poisons and RSR inhibitors (RSRi). In these cells, PARP1 modulates replication fork speed resulting in low constitutive RS. Second, we showed that MRE11 and RAD51 cooperate in the genoprotection and mitosis execution of PARP1-upregulated CRC-SCs. These roles represent therapeutic vulnerabilities for CSCs. Indeed, PARP1i sensitized CRC-SCs to ATRi/CHK1i, inducing replication catastrophe, and prevented the development of resistance to CHK1i. Also, MRE11i + RAD51i selectively killed PARP1-upregulated CRC-SCs via mitotic catastrophe. These results provide the rationale for biomarker-driven clinical trials in CRC using distinct RSRi combinations.

Intratumoral heterogeneity in cancer progression and response to immunotherapy.

Most (if not all) tumors emerge and progress under a strong evolutionary pressure imposed by trophic, metabolic, immunological, and therapeutic factors. The relative impact of these factors on tumor evolution changes over space and time, ultimately favoring the establishment of a neoplastic microenvironment that exhibits considerable genetic, phenotypic, and behavioral heterogeneity in all its components. Here, we discuss the main sources of intratumoral heterogeneity and its impact on the natural history of the disease, including sensitivity to treatment, as we delineate potential strategies to target such a detrimental feature of aggressive malignancies.

Relative Information Gain: Shannon entropy-based measure of the relative structural conservation in RNA alignments.

Structural characterization of RNAs is a dynamic field, offering many modelling possibilities. RNA secondary structure models are usually characterized by an encoding that depicts structural information of the molecule through string representations or graphs. In this work, we provide a generalization of the BEAR encoding (a context-aware structural encoding we previously developed) by expanding the set of alignments used for the construction of substitution matrices and then applying it to secondary structure encodings ranging from fine-grained to more coarse-grained representations. We also introduce a re-interpretation of the Shannon Information applied on RNA alignments, proposing a new scoring metric, the Relative Information Gain (RIG). The RIG score is available for any position in an alignment, showing how different levels of detail encoded in the RNA representation can contribute differently to convey structural information. The approaches presented in this study can be used alongside state-of-the-art tools to synergistically gain insights into the structural elements that RNAs and RNA families are composed of. This additional information could potentially contribute to their improvement or increase the degree of confidence in the secondary structure of families and any set of aligned RNAs.

Targeting Cancer Heterogeneity with Immune Responses Driven by Oncolytic Peptides.

Accumulating preclinical and clinical evidence indicates that high degrees of heterogeneity among malignant cells constitute a considerable obstacle to the success of cancer therapy. This calls for the development of approaches that operate - or enable established treatments to operate - despite such intratumoral heterogeneity (ITH). In this context, oncolytic peptides stand out as promising therapeutic tools based on their ability to drive immunogenic cell death associated with robust anticancer immune responses independently of ITH. We review the main molecular and immunological pathways engaged by oncolytic peptides, and discuss potential approaches to combine these agents with modern immunotherapeutics in support of superior tumor-targeting immunity and efficacy in patients with cancer.

Tuning Cancer Fate: Tumor Microenvironment's Role in Cancer Stem Cell Quiescence and Reawakening.

Cancer cell dormancy is a common feature of human tumors and represents a major clinical barrier to the long-term efficacy of anticancer therapies. Dormant cancer cells, either in primary tumors or disseminated in secondary organs, may reawaken and relapse into a more aggressive disease. The mechanisms underpinning dormancy entry and exit strongly resemble those governing cancer cell stemness and include intrinsic and contextual cues. Cellular and molecular components of the tumor microenvironment persistently interact with cancer cells. This dialog is highly dynamic, as it evolves over time and space, strongly cooperates with intrinsic cell nets, and governs cancer cell features (like quiescence and stemness) and fate (survival and outgrowth). Therefore, there is a need for deeper insight into the biology of dormant cancer (stem) cells and the mechanisms regulating the equilibrium quiescence-versus-proliferation are vital in our pursuit of new therapeutic opportunities to prevent cancer from recurring. Here, we review and discuss microenvironmental regulations of cancer dormancy and its parallels with cancer stemness, and offer insights into the therapeutic strategies adopted to prevent a lethal recurrence, by either eradicating resident dormant cancer (stem) cells or maintaining them in a dormant state.

Caspase 2 and p53 Reunited in Tumor Control.

Recent findings (Tsabar et al.) demonstrate that human cancer cells that evade the cell cycle blockage normally imposed by DNA damage experience sustained p53 signaling upon MDM2 degradation by caspase 2. Such a response may be connected to the delivery of immunostimulatory signals to ensure the elimination of genetically instable cancer cells.

Consensus guidelines for the definition, detection and interpretation of immunogenic cell death.

Cells succumbing to stress via regulated cell death (RCD) can initiate an adaptive immune response associated with immunological memory, provided they display sufficient antigenicity and adjuvanticity. Moreover, multiple intracellular and microenvironmental features determine the propensity of RCD to drive adaptive immunity. Here, we provide an updated operational definition of immunogenic cell death (ICD), discuss the key factors that dictate the ability of dying cells to drive an adaptive immune response, summarize experimental assays that are currently available for the assessment of ICD in vitro and in vivo, and formulate guidelines for their interpretation.

Cytofluorometric assessment of dendritic cell-mediated uptake of cancer cell apoptotic bodies.

Dendritic cells (DCs) are specialized antigen presenting cells (APCs) able to intake and crosspresent antigens (Ags) on major histocompatibility complex (MHC) class I and II molecules to T cells thus initiating primary and memory immune responses. DC-mediated Ag uptake and crosspresentation represent crucial steps toward cancer recognition and eventually elimination. Cytofluorometry is a standardized procedure to study phagocytosis. By fast and reproducible single cell measurements, flow cytometry allows for simultaneous biochemical and functional analyses of Ag intake. In this chapter, we discuss a two-color flow cytometric analysis of DC-mediated uptake of apoptotic bodies. We also show data on the adjuvanticity of Type-I-interferons (Type-I-IFNs) during Ag retention as we offer a guideline and a range of advice on sample preparation and acquisition.