Longitudinal molecular profiling elucidates immunometabolism dynamics in breast cancer.

Although metabolic reprogramming within tumor cells and tumor microenvironment (TME) is well described in breast cancer, little is known about how the interplay of immune state and cancer metabolism evolves during treatment. Here, we characterize the immunometabolic profiles of tumor tissue samples longitudinally collected from individuals with breast cancer before, during and after neoadjuvant chemotherapy (NAC) using proteomics, genomics and histopathology. We show that the pre-, on-treatment and dynamic changes of the immune state, tumor metabolic proteins and tumor cell gene expression profiling-based metabolic phenotype are associated with treatment response. Single-cell/nucleus RNA sequencing revealed distinct tumor and immune cell states in metabolism between cold and hot tumors. Potential drivers of NAC based on above analyses were validated in vitro. In summary, the study shows that the interaction of tumor-intrinsic metabolic states and TME is associated with treatment outcome, supporting the concept of targeting tumor metabolism for immunoregulation.

Chromatin damage generated by DNA intercalators leads to degradation of RNA Polymerase II.

In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much interest given their ability to inhibit RNA Polymerase I transcription (BMH-21), evict histones (Aclarubicin) or induce chromatin trapping of FACT (Curaxin CBL0137). Interestingly, these DNA intercalators lack the capacity to induce DNA damage while still retaining cytotoxic effects and stabilize p53. Herein, we report that these DNA intercalators impact chromatin biology by interfering with the chromatin stability of RNA polymerases I, II and III. These three compounds have the capacity to induce degradation of RNA polymerase II and they simultaneously enable the trapping of Topoisomerases TOP2A and TOP2B on the chromatin. In addition, BMH-21 also acts as a catalytic inhibitor of Topoisomerase II, resembling Aclarubicin. Moreover, BMH-21 induces chromatin trapping of the histone chaperone FACT and propels accumulation of Z-DNA and histone eviction, similarly to Aclarubicin and CBL0137. These DNA intercalators have a cumulative impact on general transcription machinery by inducing accumulation of topological defects and impacting nuclear chromatin. Therefore, their cytotoxic capabilities may be the result of compounding deleterious effects on chromatin homeostasis.

Actionable cancer vulnerability due to translational arrest, p53 aggregation and ribosome biogenesis stress evoked by the disulfiram metabolite CuET.

Drug repurposing is a versatile strategy to improve current therapies. Disulfiram has long been used in the treatment of alcohol dependency and multiple clinical trials to evaluate its clinical value in oncology are ongoing. We have recently reported that the disulfiram metabolite diethyldithiocarbamate, when combined with copper (CuET), targets the NPL4 adapter of the p97VCP segregase to suppress the growth of a spectrum of cancer cell lines and xenograft models in vivo. CuET induces proteotoxic stress and genotoxic effects, however important issues concerning the full range of the CuET-evoked tumor cell phenotypes, their temporal order, and mechanistic basis have remained largely unexplored. Here, we have addressed these outstanding questions and show that in diverse human cancer cell models, CuET causes a very early translational arrest through the integrated stress response (ISR), later followed by features of nucleolar stress. Furthermore, we report that CuET entraps p53 in NPL4-rich aggregates leading to elevated p53 protein and its functional inhibition, consistent with the possibility of CuET-triggered cell death being p53-independent. Our transcriptomics profiling revealed activation of pro-survival adaptive pathways of ribosomal biogenesis (RiBi) and autophagy upon prolonged exposure to CuET, indicating potential feedback responses to CuET treatment. The latter concept was validated here by simultaneous pharmacological inhibition of RiBi and/or autophagy that further enhanced CuET's tumor cytotoxicity, using both cell culture and zebrafish in vivo preclinical models. Overall, these findings expand the mechanistic repertoire of CuET's anti-cancer activity, inform about the temporal order of responses and identify an unorthodox new mechanism of targeting p53. Our results are discussed in light of cancer-associated endogenous stresses as exploitable tumor vulnerabilities and may inspire future clinical applications of CuET in oncology, including combinatorial treatments and focus on potential advantages of using certain validated drug metabolites, rather than old, approved drugs with their, often complex, metabolic profiles.

Small molecule-mediated disruption of ribosome biogenesis synergizes with FGFR inhibitors to suppress glioma cell growth.

BACKGROUND: High-grade gliomas are malignant brain tumors characterized by aggressiveness and resistance to chemotherapy. Prognosis remains dismal, highlighting the need to identify novel molecular dependencies and targets. Ribosome biogenesis (RiBi), taking place in the nucleolus, represents a promising target as several cancer types rely on high RiBi rates to sustain proliferation. Publicly available transcriptomics data of glioma patients revealed a positive correlation between RiBi rates and histological grades. We, therefore, hypothesized that glioma cells could be susceptible to RiBi inhibition. METHODS: Transcriptomics data from glioma patients were analyzed for RiBi-related processes. BMH-21, a small molecule inhibitor of RNA pol I transcription, was tested in adult and pediatric high-grade glioma cell lines and a zebrafish transplant model. Cellular phenotypes were evaluated by transcriptomics, cell cycle analysis, and viability assays. A chemical synergy screen was performed to identify drugs potentiating BMH-21-mediated effects. RESULTS: BMH-21 reduced glioma cell viability, induced apoptosis, and impaired the growth of transplanted glioma cells in zebrafish. Combining BMH-21 with TMZ potentiated cytotoxic effects. Moreover, BMH-21 synergized with Fibroblast Growth Factor Receptor (FGFR) inhibitor (FGFRi) Erdafitinib, a top hit in the chemical synergy screen. RiBi inhibition using BMH-21, POLR1A siRNA, or Actinomycin D revealed engagement of the FGFR-FGF2 pathway. BMH-21 downregulated FGFR1 and SOX2 levels, whereas FGF2 was induced and released from the nucleolus. CONCLUSIONS: This study conceptualizes the implementation of RiBi inhibition as a viable future therapeutic strategy for glioma and reveals an FGFR connection to the cellular response upon RiBi inhibition with potential translational value.

The exon-junction complex helicase eIF4A3 controls cell fate via coordinated regulation of ribosome biogenesis and translational output.

Eukaryotic initiation factor 4A-III (eIF4A3), a core helicase component of the exon junction complex, is essential for splicing, mRNA trafficking, and nonsense-mediated decay processes emerging as targets in cancer therapy. Here, we unravel eIF4A3's tumor-promoting function by demonstrating its role in ribosome biogenesis (RiBi) and p53 (de)regulation. Mechanistically, eIF4A3 resides in nucleoli within the small subunit processome and regulates rRNA processing via R-loop clearance. EIF4A3 depletion induces cell cycle arrest through impaired RiBi checkpoint-mediated p53 induction and reprogrammed translation of cell cycle regulators. Multilevel omics analysis following eIF4A3 depletion pinpoints pathways of cell death regulation and translation of alternative mouse double minute homolog 2 (MDM2) transcript isoforms that control p53. EIF4A3 expression and subnuclear localization among clinical cancer specimens correlate with the RiBi status rendering eIF4A3 an exploitable vulnerability in high-RiBi tumors. We propose a concept of eIF4A3's unexpected role in RiBi, with implications for cancer pathogenesis and treatment.

A chemical screen for modulators of mRNA translation identifies a distinct mechanism of toxicity for sphingosine kinase inhibitors.

We here conducted an image-based chemical screen to evaluate how medically approved drugs, as well as drugs that are currently under development, influence overall translation levels. None of the compounds up-regulated translation, which could be due to the screen being performed in cancer cells grown in full media where translation is already present at very high levels. Regarding translation down-regulators, and consistent with current knowledge, inhibitors of the mechanistic target of rapamycin (mTOR) signaling pathway were the most represented class. In addition, we identified that inhibitors of sphingosine kinases (SPHKs) also reduce mRNA translation levels independently of mTOR. Mechanistically, this is explained by an effect of the compounds on the membranes of the endoplasmic reticulum (ER), which activates the integrated stress response (ISR) and contributes to the toxicity of SPHK inhibitors. Surprisingly, the toxicity and activation of the ISR triggered by 2 independent SPHK inhibitors, SKI-II and ABC294640, the latter in clinical trials, are also observed in cells lacking SPHK1 and SPHK2. In summary, our study provides a useful resource on the effects of medically used drugs on translation, identified compounds capable of reducing translation independently of mTOR and has revealed that the cytotoxic properties of SPHK inhibitors being developed as anticancer agents are independent of SPHKs.

Human RTEL1 associates with Poldip3 to facilitate responses to replication stress and R-loop resolution.

RTEL1 helicase is a component of DNA repair and telomere maintenance machineries. While RTEL1's role in DNA replication is emerging, how RTEL1 preserves genomic stability during replication remains elusive. Here we used a range of proteomic, biochemical, cell, and molecular biology and gene editing approaches to provide further insights into potential role(s) of RTEL1 in DNA replication and genome integrity maintenance. Our results from complementary human cell culture models established that RTEL1 and the Polδ subunit Poldip3 form a complex and are/function mutually dependent in chromatin binding after replication stress. Loss of RTEL1 and Poldip3 leads to marked R-loop accumulation that is confined to sites of active replication, enhances endogenous replication stress, and fuels ensuing genomic instability. The impact of depleting RTEL1 and Poldip3 is epistatic, consistent with our proposed concept of these two proteins operating in a shared pathway involved in DNA replication control under stress conditions. Overall, our data highlight a previously unsuspected role of RTEL1 and Poldip3 in R-loop suppression at genomic regions where transcription and replication intersect, with implications for human diseases including cancer.

The antimalarial drug amodiaquine stabilizes p53 through ribosome biogenesis stress, independently of its autophagy-inhibitory activity.

Pharmacological inhibition of ribosome biogenesis is a promising avenue for cancer therapy. Herein, we report a novel activity of the FDA-approved antimalarial drug amodiaquine which inhibits rRNA transcription, a rate-limiting step for ribosome biogenesis, in a dose-dependent manner. Amodiaquine triggers degradation of the catalytic subunit of RNA polymerase I (Pol I), with ensuing RPL5/RPL11-dependent stabilization of p53. Pol I shutdown occurs in the absence of DNA damage and without the subsequent ATM-dependent inhibition of rRNA transcription. RNAseq analysis revealed mechanistic similarities of amodiaquine with BMH-21, the first-in-class Pol I inhibitor, and with chloroquine, the antimalarial analog of amodiaquine, with well-established autophagy-inhibitory activity. Interestingly, autophagy inhibition caused by amodiaquine is not involved in the inhibition of rRNA transcription, suggesting two independent anticancer mechanisms. In vitro, amodiaquine is more efficient than chloroquine in restraining the proliferation of human cell lines derived from colorectal carcinomas, a cancer type with predicted susceptibility to ribosome biogenesis stress. Taken together, our data reveal an unsuspected activity of a drug approved and used in the clinics for over 30 years, and provide rationale for repurposing amodiaquine in cancer therapy.

TPL2 kinase is a suppressor of lung carcinogenesis.

Lung cancer is a heterogeneous disease at both clinical and molecular levels, posing conceptual and practical bottlenecks in defining key pathways affecting its initiation and progression. Molecules with a central role in lung carcinogenesis are likely to be targeted by multiple deregulated pathways and may have prognostic, predictive, and/or therapeutic value. Here, we report that Tumor Progression Locus 2 (TPL2), a kinase implicated in the regulation of innate and adaptive immune responses, fulfils a role as a suppressor of lung carcinogenesis and is subject to diverse genetic and epigenetic aberrations in lung cancer patients. We show that allelic imbalance at the TPL2 locus, up-regulation of microRNA-370, which targets TPL2 transcripts, and activated RAS (rat sarcoma) signaling may result in down-regulation of TPL2 expression. Low TPL2 levels correlate with reduced lung cancer patient survival and accelerated onset and multiplicity of urethane-induced lung tumors in mice. Mechanistically, TPL2 was found to antagonize oncogene-induced cell transformation and survival through a pathway involving p53 downstream of cJun N-terminal kinase (JNK) and be required for optimal p53 response to genotoxic stress. These results identify multiple oncogenic pathways leading to TPL2 deregulation and highlight its major tumor-suppressing function in the lung.

Tpl2 kinase signal transduction in inflammation and cancer.

The activation of mitogen-activated protein kinases (MAPKs) is critically involved in inflammatory and oncogenic events. Tumor progression locus 2 (Tpl2), also known as COT and MAP3 kinase 8 (MAP3K8), is a serine-threonine kinase with an important physiological role in tumor necrosis factor, interleukin-1, CD40, Toll-like receptor and G protein-coupled receptor-mediated ERK MAPK signaling. Whilst the full characterization of the biochemical events that lead to the activation of Tpl2 still represent a major challenge, genetic and molecular evidence has highlighted interesting interactions with the NF-κB network. Here, we provide an overview of the multifaceted functions of Tpl2 and the molecular mechanisms that govern its regulation.