Integrative mapping of the dog epigenome: Reference annotation for comparative intertissue and cross-species studies.

Dogs have become a valuable model in exploring multifaceted diseases and biology relevant to human health. Despite large-scale dog genome projects producing high-quality draft references, a comprehensive annotation of functional elements is still lacking. We addressed this through integrative next-generation sequencing of transcriptomes paired with five histone marks and DNA methylome profiling across 11 tissue types, deciphering the dog's epigenetic code by defining distinct chromatin states, super-enhancer, and methylome landscapes, and thus showed that these regions are associated with a wide range of biological functions and cell/tissue identity. In addition, we confirmed that the phenotype-associated variants are enriched in tissue-specific regulatory regions and, therefore, the tissue of origin of the variants can be traced. Ultimately, we delineated conserved and dynamic epigenomic changes at the tissue- and species-specific resolutions. Our study provides an epigenomic blueprint of the dog that can be used for comparative biology and medical research.

Multi-targeted therapy resistance via drug-induced secretome fucosylation.

Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post- translational cancer hallmark and the consequences thereof remain elusive. Here, we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In cancer cell cultures, xenograft mouse models, and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycoproteomes identified fucosylation of the antioxidant PON1 as a critical component of the therapy-induced secretome (TIS). N-glycosylation of TIS and target core fucosylation of PON1 are mediated by the fucose salvage-FUT8-SLC35C1 axis with PON3 directly modulating GDP-Fuc transfer on PON1 scaffolds. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Global and PON1-specific secretome de-N-glycosylation both limited the expansion of resistant clones in a tumor regression model. We defined the resistance-associated transcription factors (TFs) and genes modulated by the N-glycosylated TIS via a focused and transcriptome-wide analyses. These genes characterize the oxidative stress, inflammatory niche, and unfolded protein response as important factors for this modulation. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance.

Dual Targeting of EGFR with PLK1 Exerts Therapeutic Synergism in Taxane-Resistant Lung Adenocarcinoma by Suppressing ABC Transporters.

To overcome the limitations of chemoresistance, combination therapies using druggable targets have been investigated. Our previous studies led us to hypothesize that the downregulation of PLK1 expression or activity can be one strategy to overcome the hurdles of taxane resistance by the downregulation of ABC transporters. To explore this, various versions of PLK1 including a constitutively active version, kinase-dead form, and polo-box domain mutant were expressed in paclitaxel-resistant lung adenocarcinoma (LUADTXR). Targeting PLK1 using shRNA or non-functional mutants downregulated ABCB1, ABCC9, and ABCG2 in LUADTXR cells, which was similar to the downregulation effects from treatment with PLK1 inhibitors. The high expression of EGFR in LUAD led us to administer gefitinib, showing a markedly reduced EGFR level in LUADTXR cells. When gefitinib and PLK1 inhibitors were combined, LUADTXR cells tended to undergo apoptosis more effectively than parental cells, showing a synergistic effect on the downregulation of ABC transporters through c-Myc and AP-1. Clinical data provide evidence for the relevance between survival rates and expressions of PLK1 and EGFR in LUAD patients. Based on these results, we suggest that a combination of gefitinib and PLK1 inhibitors exerts strong synergism in LUADTXR, which helps to overcome the limitations associated with taxanes.

Chemotherapy confers a conserved secondary tolerance to EGFR inhibition via AXL-mediated signaling bypass.

Drug resistance remains the major culprit of therapy failure in disseminated cancers. Simultaneous resistance to multiple, chemically different drugs feeds this failure resulting in cancer relapse. Here, we investigate co-resistance signatures shared between antimitotic drugs (AMDs) and inhibitors of receptor tyrosine kinases (RTKs) to probe mechanisms of secondary resistance. We map co-resistance ranks in multiple drug pairs and identified a more widespread occurrence of co-resistance to the EGFR-tyrosine kinase inhibitor (TKI) gefitinib in hundreds of cancer cell lines resistant to at least 11 AMDs. By surveying different parameters of genomic alterations, we find that the two RTKs EGFR and AXL displayed similar alteration and expression signatures. Using acquired paclitaxel and epothilone B resistance as first-line AMD failure models, we show that a stable collateral resistance to gefitinib can be relayed by entering a dynamic, drug-tolerant persister state where AXL acts as bypass signal. Delayed AXL degradation rendered this persistence to become stably resistant. We probed this degradation process using a new EGFR-TKI candidate YD and demonstrated that AXL bypass-driven collateral resistance can be suppressed pharmacologically. The findings emphasize that AXL bypass track is employed by chemoresistant cancer cells upon EGFR inhibition to enter a persister state and evolve resistance to EGFR-TKIs.

Prior acquired resistance to paclitaxel relays diverse EGFR-targeted therapy persistence mechanisms.

Secondary drug resistance stems from dynamic clonal evolution during the development of a prior primary resistance. This collateral type of resistance is often a characteristic of cancer recurrence. Yet, mechanisms that drive this collateral resistance and their drug-specific trajectories are still poorly understood. Using resistance selection and small-scale pharmacological screens, we find that cancer cells with primary acquired resistance to the microtubule-stabilizing drug paclitaxel often develop tolerance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), leading to formation of more stable resistant cell populations. We show that paclitaxel-resistant cancer cells follow distinct selection paths under EGFR-TKIs by enriching the stemness program, developing a highly glycolytic adaptive stress response, and rewiring an apoptosis control pathway. Collectively, our work demonstrates the alterations in cellular state stemming from paclitaxel failure that result in collateral resistance to EGFR-TKIs and points to new exploitable vulnerabilities during resistance evolution in the second-line treatment setting.

Paraoxonase-1 (PON1) induces metastatic potential and apoptosis escape via its antioxidative function in lung cancer cells.

Paraoxonase-1 (PON1) gene polymorphisms have been closely associated with the development of advanced cancers while PON1 secretion to the serum is linked with inhibition of oxidized high-density lipoprotein by its antioxidative function. Our group previously demonstrated that post-translational modification of serum PON1 in form of fucosylated PON1 is a potential biomarker of small cell lung cancer. Here, we interrogated the role of PON1 in the pathobiology of lung cancer (LC) by addressing cell-autonomous mechanisms using gain-of-function and loss-of-function approaches and protein expression profiling of tissue samples in our clinical biobank. PON1 expression in LC patient tissues varied between overexpression in squamous cell carcinoma and minimal loss in adenocarcinoma sub-types. Simultaneous overexpression of PON1 both at the gene and protein stability levels induced pro-oncogenic characteristics in LC cells and xenografts. PON1 overexpression supported metastatic progression of LC by decreasing G1/S ratio and LC cell senescence involving p21Waf1/Cip1. PON1 suppressed drug- and ligand-induced cell death and protected LC cells from genotoxic damages with maintained ATP levels, requiring p53-directed signals. PON1 promoted ROS deregulation protecting the mitochondria from dysregulation. PON1 knockdown resulted in the blockage of its antioxidant function in LC cells through Akt signaling with reduced invasive signature as a consequence of scant expression. Targeted glycolysis stimulated PON1 antioxidant activity regulating phosphorylation of AMPK-α. The functional data imply that exploitation of the antioxidative function of PON1 is consequential in driving LC pathogenesis at the cell-autonomous mechanistic level with consequences on tumor growth.

Paclitaxel-resistant cancer cell-derived secretomes elicit ABCB1-associated docetaxel cross-resistance and escape from apoptosis through FOXO3a-driven glycolytic regulation.

Chemotherapy-induced cancer cell secretomes promote resistance due, in part, to a predominant glycolytic energy metabolism, which drives aggressive cancer cell proliferation. However, the characterization of these secretomes and the molecular events that associate them with acquired drug resistance remain poorly understood. In this study, we show that secretomes of cancer cells with high-level paclitaxel resistance stimulated cell proliferation and suppressed drug-induced apoptosis of drug-sensitive cells. We also found that drug (docetaxel)-stimulated induction of interferon-α (IFN-α), IFN-λ and tumor necrosis factor-α (TNF-α) release in drug-sensitive cells was lowered by these secretomes. The promotion of cell proliferation by paclitaxel-resistant (PacR) cancer cell secretomes was associated, in part, with an increase in S phase of the cell cycle and downregulation of the cell death pathway that supports escape from apoptosis. In addition, we also found that the regulation of targeted glycolysis in PacR cancer cells alters the effects of the secretomes on cell growth, apoptosis, ATP generation and acquired drug resistance. Further study revealed that the deletion of FOXO3a transcription exacerbates glycolytic shift-induced apoptosis by rescuing TRAIL expression. By generating a docetaxel-cross-resistant PacR cancer cell line (PacR/DCT), we further clarified the role of FOXO3a in glycolysis-associated mediation of P-glycoprotein/ABCB1 hyperactivity that induces docetaxel cross-resistance. These findings suggest that suppression of the cellular energy supply by targeting glycolysis may inhibit the multiplicity of acquired chemotherapy resistance. Therefore, the therapeutic inhibition of FOXO3a might direct glycolysis to induce apoptosis and overcome multidrug resistance in cancer cells.

Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation.

Acquired drug resistance is a primary obstacle for effective cancer therapy. The correlation of point mutations in class III ß-tubulin (TUBB3) and the prominent overexpression of ATP-binding cassette P-glycoprotein (ABCB1), a multidrug resistance gene, have been protruding mechanisms of resistance to microtubule disruptors such as paclitaxel (PTX) for many cancers. However, the precise underlying mechanism of the rapid onset of cross-resistance to an array of structurally and functionally unrelated drugs in PTX-resistant cancers has been poorly understood. We determined that our established PTX-resistant cancer cells display ABCB1/ABCC1-associated cross-resistance to chemically different drugs such as 5-fluorouracil, docetaxel, and cisplatin. We found that feedback activation of TUBB3 can be triggered through the FOXO3a-dependent regulation of ABCB1, which resulted in the accentuation of induced PTX resistance and encouraged multiplicity in acquired cross-resistance. FOXO3a-directed regulation of P-glycoprotein (P-gp) function suggests that control of ABCB1 involves methylation-dependent activation. Consistently, transcriptional overexpression or downregulation of FOXO3a directs inhibitor-controlled protease-degradation of TUBB3. The functional PI3K/Akt signaling is tightly responsive to FOXO3a activation alongside doxorubicin treatment, which directs FOXO3a arginine hypermethylation. In addition, we found that secretome factors from PTX-resistant cancer cells with acquired cross-resistance support a P-gp-dependent association in multidrug resistance (MDR) development, which assisted the FOXO3a-mediated control of TUBB3 feedback. The direct silencing of TUBB3 reverses induced multiple cross-resistance, reduces drug-resistant tumor mass, and suppresses the impaired microtubule stability status of PTX-resistant cells with transient cross-resistance. These findings highlight the control of the TUBB3 response to ABCB1 genetic suppressors as a mechanism to reverse the profuse development of multidrug resistance in cancer.