Transposable elements (TEs) comprise a large proportion of long non-coding RNAs (lncRNAs). Here, we employed CRISPR to delete a short interspersed nuclear element (SINE) in Malat1, a cancer-associated lncRNA, to investigate its significance in cellular physiology. We show that Malat1 with a SINE deletion forms diffuse nuclear speckles and is frequently translocated to the cytoplasm. SINE-deleted cells exhibit an activated unfolded protein response and PKR and markedly increased DNA damage and apoptosis caused by dysregulation of TDP-43 localization and formation of cytotoxic inclusions. TDP-43 binds stronger to Malat1 without the SINE and is likely ‘hijacked’ by cytoplasmic Malat1 to the cytoplasm, resulting in the depletion of nuclear TDP-43 and redistribution of TDP-43 binding to repetitive element transcripts and mRNAs encoding mitotic and nuclear-cytoplasmic regulators. The SINE promotes Malat1 nuclear retention by facilitating Malat1 binding to HNRNPK, a protein that drives RNA nuclear retention, potentially through direct interactions of the SINE with KHDRBS1 and TRA2A, which bind to HNRNPK. Losing these RNA–protein interactions due to the SINE deletion likely creates more available TDP-43 binding sites on Malat1 and subsequent TDP-43 aggregation. These results highlight the significance of lncRNA TEs in TDP-43 proteostasis with potential implications in both cancer and neurodegenerative diseases.

The epithelial-mesenchymal transition (EMT) has been implicated in conferring stem cell properties and therapeutic resistance to cancer cells. Therefore, identification of drugs that can reprogram EMT may provide new therapeutic strategies. Here, we report that cells derived from claudin-low mammary tumors, a mesenchymal subtype of triple-negative breast cancer, exhibit a distinctive organoid structure with extended “spikes” in 3D matrices. Upon a miR-200 induced mesenchymal-epithelial transition (MET), the organoids switch to a smoother round morphology. Based on these observations, we developed a morphological screening method with accompanying analytical pipelines that leverage deep neural networks and nearest neighborhood classification to screen for EMT-reversing drugs. Through screening of a targeted epigenetic drug library, we identified multiple class I HDAC inhibitors and Bromodomain inhibitors that reverse EMT. These data support the use of morphological screening of mesenchymal mammary tumor organoids as a platform to identify drugs that reverse EMT.

Background: Cisplatin (CDDP) is commonly utilized in the treatment of advanced solid tumors including head and neck squamous cell carcinoma (HNSCC). Cisplatin response remains highly variable among individual tumors and development of cisplatin resistance is common. We hypothesized that development of cisplatin resistance is partially driven by metabolic reprogramming. Methods: Using a pre-clinical HNSCC model and an integrated approach to steady state metabolomics, metabolic flux and gene expression data we characterized the interaction between cisplatin resistance and metabolic reprogramming. Results: Cisplatin toxicity in HNSCC was driven by generation of intra-cellular oxidative stress. This was validated by demonstrating that acquisition of cisplatin resistance generates cross-resistance to ferroptosis agonists despite the fact that cisplatin itself does not trigger ferroptosis. Acquisition of cisplatin resistance dysregulated the expression of genes involved in amino acid, fatty acid metabolism and central carbon catabolic pathways, enhanced glucose catabolism and serine synthesis. Acute cisplatin exposure increased intra-tumoral levels of S-methyl-5-thiadenosine (MTA) precursors and metabotoxins indicative of generalized oxidative stress. Conclusions: Acquisition of cisplatin resistance is linked to metabolic recovery from oxidative stress. Although this portends poor effectiveness for directed metabolic targeting, it supports the potential for biomarker development of cisplatin effectiveness using an integrated approach.

Chordomas are rare spinal tumors addicted to expression of the developmental transcription factor brachyury. In chordomas, brachyury is super-enhancer associated and preferentially downregulated by pharmacologic transcriptional CDK inhibition, leading to cell death. To understand the underlying basis of this sensitivity, we dissect the brachyury transcription regulatory network and compare the consequences of brachyury degradation with transcriptional CDK inhibition. Brachyury defines the chordoma super-enhancer landscape and autoregulates through binding its super-enhancer, and its locus forms a transcriptional condensate. Transcriptional CDK inhibition and brachyury degradation disrupt brachyury autoregulation, leading to loss of its transcriptional condensate and transcriptional program. Compared with transcriptional CDK inhibition, which globally downregulates transcription, leading to cell death, brachyury degradation is much more selective, inducing senescence and sensitizing cells to anti-apoptotic inhibition. These data suggest that brachyury downregulation is a core tenet of transcriptional CDK inhibition and motivates developing strategies to target brachyury and its autoregulatory feedback loop.

A subset of environmental chemicals acts as “obesogens” as they increase adipose mass and lipid content in livers of treated rodents. One of the most studied class of obesogens are the tin-containing chemicals that have as a central moiety tributyltin (TBT), which bind and activate two nuclear hormone receptors, Peroxisome Proliferator Activated Receptor Gamma (PPARG) and Retinoid X Receptor Alpha (RXRA), at nanomolar concentrations. Here, we have tested whether TBT chloride at such concentrations may affect the neutral lipid level in two cell line models of human liver. Indeed, using high content image analysis (HCA), TBT significantly increased neutral lipid content in a time- and concentration-dependent manner. Consistent with the observed increased lipid accumulation, RNA fluorescence in situ hybridization (RNA FISH) and RT-qPCR experiments revealed that TBT enhanced the steady-state mRNA levels of two key genes for de novo lipogenesis, the transcription factor SREBF1 and its downstream enzymatic target, FASN. Importantly, pre-treatment of cells with 2-deoxy-D-glucose reduced TBT-mediated lipid accumulation, thereby suggesting a role for active glycolysis during the process of lipid accumulation. As other RXRA binding ligands can promote RXRA protein turnover via the 26S proteasome, TBT was tested for such an effect in the two liver cell lines. We found that TBT, in a time- and dose-dependent manner, significantly reduced steady-state RXRA levels in a proteasome-dependent manner. While TBT promotes both RXRA protein turnover and lipid accumulation, we found no correlation between these two events at the single cell level, thereby suggesting an additional mechanism may be involved in TBT promotion of lipid accumulation, such as glycolysis.