Ephrin receptor A10 monoclonal antibodies and the derived chimeric antigen receptor T cells exert an antitumor response in mouse models of triple-negative breast cancer.

Expression of the receptor tyrosine kinase ephrin receptor A10 (EphA10), which is undetectable in most normal tissues except for the male testis, has been shown to correlate with tumor progression and poor prognosis in several malignancies, including triple-negative breast cancer (TNBC). Therefore, EphA10 could be a potential therapeutic target, likely with minimal adverse effects. However, no effective clinical drugs against EphA10 are currently available. Here, we report high expression levels of EphA10 in tumor regions of breast, lung, and ovarian cancers as well as in immunosuppressive myeloid cells in the tumor microenvironment. Furthermore, we developed anti-EphA10 monoclonal antibodies (mAbs) that specifically recognize cell surface EphA10, but not other EphA family isoforms, and target tumor regions precisely in vivo with no apparent accumulation in other organs. In syngeneic TNBC mouse models, we found that anti-EphA10 mAb clone #4 enhanced tumor regression, therapeutic response rate, and T cell-mediated antitumor immunity. Notably, the chimeric antigen receptor T cells derived from clone #4 significantly inhibited TNBC cell viability in vitro and tumor growth in vivo. Together, our findings suggest that targeting EphA10 via EphA10 mAbs and EphA10-specific chimeric antigen receptor-T cell therapy may represent a promising strategy for patients with EphA10-positive tumors.

Ribonuclease 7-driven activation of ROS1 is a potential therapeutic target in hepatocellular carcinoma.

BACKGROUND & AIMS: There are currently limited therapeutic options for hepatocellular carcinoma (HCC), particularly when it is diagnosed at advanced stages. Herein, we examined the pathophysiological role of ROS1 and assessed the utility of ROS1-targeted therapy for the treatment of HCC. METHODS: Recombinant ribonucleases (RNases) were purified, and the ligand-receptor relationship between RNase7 and ROS1 was validated in HCC cell lines by Duolink, immunofluorescence, and immunoprecipitation assays. Potential interacting residues between ROS1 and RNase7 were predicted using a protein-protein docking approach. The oncogenic function of RNase7 was analyzed by cell proliferation, migration and invasion assays, and a xenograft mouse model. The efficacy of anti-ROS1 inhibitor treatment was evaluated in patient-derived xenograft (PDX) and orthotopic models. Two independent patient cohorts were analyzed to evaluate the pathological relevance of RNase7/ROS1. RESULTS: RNase7 associated with ROS1's N3-P2 domain and promoted ROS1-mediated oncogenic transformation. Patients with HCC exhibited elevated plasma RNase7 levels compared with healthy individuals. High ROS1 and RNase7 expression were strongly associated with poor prognosis in patients with HCC. In both HCC PDX and orthotopic mouse models, ROS1 inhibitor treatment markedly suppressed RNase7-induced tumorigenesis, leading to decreased plasma RNase7 levels and tumor shrinkage in mice. CONCLUSIONS: RNase7 serves as a high-affinity ligand for ROS1. Plasma RNase7 could be used as a biomarker to identify patients with HCC who may benefit from anti-ROS1 treatment. LAY SUMMARY: Receptor tyrosine kinases are known to be involved in tumorigenesis and have been targeted therapeutically for a number of cancers, including hepatocellular carcinoma. ROS1 is the only such receptor with kinase activity whose ligand has not been identified. Herein, we show that RNase7 acts as a ligand to activate ROS1 signaling. This has important pathophysiological and therapeutic implications. Anti-ROS1 inhibitors could be used to treatment patients with hepatocellular carcinoma and high RNase7 levels.

The impact of PD-L1 N-linked glycosylation on cancer therapy and clinical diagnosis.

N-linked glycosylation is one of the most abundant posttranslational modifications of membrane-bound proteins in eukaryotes and affects a number of biological activities, including protein biosynthesis, protein stability, intracellular trafficking, subcellular localization, and ligand-receptor interaction. Accumulating evidence indicates that cell membrane immune checkpoint proteins, such as programmed death-ligand 1 (PD-L1), are glycosylated with heavy N-linked glycan moieties in human cancers. N-linked glycosylation of PD-L1 maintains its protein stability and interaction with its cognate receptor, programmed cell death protein 1 (PD-1), and this in turn promotes evasion of T-cell immunity. Studies have suggested targeting PD-L1 glycosylation as a therapeutic option by rational combination of cancer immunotherapies. Interestingly, structural hindrance by N-glycan on PD-L1 in fixed samples impedes its recognition by PD-L1 diagnostic antibodies. Notably, the removal of N-linked glycosylation enhances PD-L1 detection in a variety of bioassays and more accurately predicts the therapeutic efficacy of PD-1/PD-L1 inhibitors, suggesting an important clinical implication of PD-L1 N-linked glycosylation. A detailed understanding of the regulatory mechanisms, cellular functions, and diagnostic limits underlying PD-L1 N-linked glycosylation could shed new light on the clinical development of immune checkpoint inhibitors for cancer treatment and deepen our knowledge of biomarkers to identify patients who would benefit the most from immunotherapy. In this review, we highlight the effects of protein glycosylation on cancer immunotherapy using N-linked glycosylation of PD-L1 as an example. In addition, we consider the potential impacts of PD-L1 N-linked glycosylation on clinical diagnosis. The notion of utilizing the deglycosylated form of PD-L1 as a predictive biomarker to guide anti-PD-1/PD-L1 immunotherapy is also discussed.

A novel ligand-receptor relationship between families of ribonucleases and receptor tyrosine kinases.

Pancreatic ribonuclease is known to participate in host defense system against pathogens, such as parasites, bacteria, and virus, which results in innate immune response. Nevertheless, its potential impact to host cells remains unclear. Of interest, several ribonucleases do not act as catalytically competent enzymes, suggesting that ribonucleases may be associated with certain intrinsic functions other than their ribonucleolytic activities. Most recently, human pancreatic ribonuclease 5 (hRNase5; also named angiogenin; hereinafter referred to as hRNase5/ANG), which belongs to the human ribonuclease A superfamily, has been demonstrated to function as a ligand of epidermal growth factor receptor (EGFR), a member of the receptor tyrosine kinase family. As a newly identified EGFR ligand, hRNase5/ANG associates with EGFR and stimulates EGFR and the downstream signaling in a catalytic-independent manner. Notably, hRNase5/ANG, whose level in sera of pancreatic cancer patients, serves as a non-invasive serum biomarker to stratify patients for predicting the sensitivity to EGFR-targeted therapy. Here, we describe the hRNase5/ANG-EGFR pair as an example to highlight a ligand-receptor relationship between families of ribonucleases and receptor tyrosine kinases, which are thought as two unrelated protein families associated with distinct biological functions. The notion of serum biomarker-guided EGFR-targeted therapies will also be discussed. Furthering our understanding of this novel ligand-receptor interaction will shed new light on the search of ligands for their cognate receptors, especially those orphan receptors without known ligands, and deepen our knowledge of the fundamental research in membrane receptor biology and the translational application toward the development of precision medicine.

Targeting ALK averts ribonuclease 1-induced immunosuppression and enhances antitumor immunity in hepatocellular carcinoma.

Tumor-secreted factors contribute to the development of a microenvironment that facilitates the escape of cancer cells from immunotherapy. In this study, we conduct a retrospective comparison of the proteins secreted by hepatocellular carcinoma (HCC) cells in responders and non-responders among a cohort of ten patients who received Nivolumab (anti-PD-1 antibody). Our findings indicate that non-responders have a high abundance of secreted RNase1, which is associated with a poor prognosis in various cancer types. Furthermore, mice implanted with HCC cells that overexpress RNase1 exhibit immunosuppressive tumor microenvironments and diminished response to anti-PD-1 therapy. RNase1 induces the polarization of macrophages towards a tumor growth-promoting phenotype through activation of the anaplastic lymphoma kinase (ALK) signaling pathway. Targeting the RNase1/ALK axis reprograms the macrophage polarization, with increased CD8+ T- and Th1- cell recruitment. Moreover, simultaneous targeting of the checkpoint protein PD-1 unleashes cytotoxic CD8+ T-cell responses. Treatment utilizing both an ALK inhibitor and an anti-PD-1 antibody exhibits enhanced tumor regression and facilitates long-term immunity. Our study elucidates the role of RNase1 in mediating tumor resistance to immunotherapy and reveals an RNase1-mediated immunosuppressive tumor microenvironment, highlighting the potential of targeting RNase1 as a promising strategy for cancer immunotherapy in HCC.

Membrane-bound trafficking regulates nuclear transport of integral epidermal growth factor receptor (EGFR) and ErbB-2.

Nuclear localization of multiple receptor-tyrosine kinases (RTKs), such as EGF receptor (EGFR), ErbB-2, FGF receptor (FGFR), and many others, has been reported by several groups. We previously showed that cell surface EGFR is trafficked to the nucleus through a retrograde pathway from the Golgi to the endoplasmic reticulum (ER) and that EGFR is then translocated to the inner nuclear membrane (INM) through the INTERNET (integral trafficking from the ER to the nuclear envelope transport) pathway. However, the nuclear trafficking mechanisms of other membrane RTKs, apart from EGFR, remain unclear. The purpose of this study was to compare the nuclear transport of EGFR family proteins with that of FGFR-1. Interestingly, we found that digitonin permeabilization, which selectively releases soluble nuclear transporters from the cytoplasm and has been shown to inhibit nuclear transport of FGFR-1, had no effects on EGFR nuclear transport, raising the possibility that EGFR and FGFR-1 use different pathways to be translocated into the nucleus. Using the subnuclear fractionation assay, we further demonstrated that biotinylated cell surface ErbB-2, but not FGFR-1, is targeted to the INM, associating with Sec61ß in the INM, similar to the nuclear trafficking of EGFR. Thus, ErbB-2, but not FGFR-1, shows a similar trafficking pathway to EGFR for translocation to the nucleus, indicating that at least two different pathways of nuclear transport exist for cell surface receptors. This finding provides a new direction for investigating the trafficking mechanisms of various nuclear RTKs.

Nuclear EGFR suppresses ribonuclease activity of polynucleotide phosphorylase through DNAPK-mediated phosphorylation at serine 776.

Nuclear existence of epidermal growth factor receptor (EGFR) has been documented for more than two decades. Resistance of cancer to radiotherapy is frequently correlated with elevated EGFR expression, activity, and nuclear translocation. However, the role of nuclear EGFR (nEGFR) in radioresistance of cancers remains elusive. In the current study, we identified a novel nEGFR-associated protein, polynucleotide phosphorylase (PNPase), which possesses 3' to 5' exoribonuclease activity toward c-MYC mRNA. Knockdown of PNPase increased radioresistance. Inactivation or knock-down of EGFR enhanced PNPase-mediated c-MYC mRNA degradation in breast cancer cells, and also increased its radiosensitivity. Interestingly, the association of nEGFR with PNPase and DNA-dependent protein kinase (DNAPK) increased significantly in breast cancer cells after exposure to ionizing radiation (IR). We also demonstrated that DNAPK phosphorylates PNPase at Ser-776, which is critical for its ribonuclease activity. The phospho-mimetic S776D mutant of PNPase impaired its ribonuclease activity whereas the nonphosphorylatable S776A mutant effectively degraded c-MYC mRNA. Here, we uncovered a novel role of nEGFR in radioresistance, and that is, upon ionizing radiation, nEGFR inactivates the ribonuclease activity of PNPase toward c-MYC mRNA through DNAPK-mediated Ser-776 phosphorylation, leading to increase of c-MYC mRNA, which contributes to radioresistance of cancer cells.

RNA helicase A is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus.

EGF induces the translocation of EGF receptor (EGFR) from the cell surface to the nucleus where EGFR activates gene transcription through its binding to an AT-rich sequence (ATRS) of the target gene promoter. However, how EGFR, without a DNA-binding domain, can bind to the gene promoter is unclear. In the present study, we show that RNA helicase A (RHA) is an important mediator for EGFR-induced gene transactivation. EGF stimulates the interaction of EGFR with RHA in the nucleus of cancer cells. The EGFR/RHA complex then associates with the target gene promoter through binding of RHA to the ATRS of the target gene promoter to activate its transcription. Knockdown of RHA expression in cancer cells abrogates the binding of EGFR to the target gene promoter, thereby reducing EGF/EGFR-induced gene expression. In addition, interruption of EGFR-RHA interaction decreases the EGFR-induced promoter activity. Consistently, we observed a positive correlation of the nuclear expression of EGFR, RHA, and cyclin D1 in human breast cancer samples. These results indicate that RHA is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus.

Ribonuclease 1 Enhances Antitumor Immunity against Breast Cancer by Boosting T cell Activation.

The secretory enzyme human ribonuclease 1 (RNase1) is involved in innate immunity and anti-inflammation, achieving host defense and anti-cancer effects; however, whether RNase1 contributes to adaptive immune response in the tumor microenvironment (TME) remains unclear. Here, we established a syngeneic immunocompetent mouse model in breast cancer and demonstrated that ectopic RNase1 expression significantly inhibited tumor progression. Overall changes in immunological profiles in the mouse tumors were analyzed by mass cytometry and showed that the RNase1-expressing tumor cells significantly induced CD4+ Th1 and Th17 cells and natural killer cells and reduced granulocytic myeloid-derived suppressor cells, supporting that RNase1 favors an antitumor TME. Specifically, RNase1 increased expression of T cell activation marker CD69 in a CD4+ T cell subset. Notably, analysis of cancer-killing potential revealed that T cell-mediated antitumor immunity was enhanced by RNase1, which further collaborated with an EGFR-CD3 bispecific antibody to protect against breast cancer cells across molecular subtypes. Our results uncover a tumor-suppressive role of RNase1 through adaptive immune response in breast cancer in vivo and in vitro, providing a potential treatment strategy of combining RNase1 with cancer immunotherapies for immunocompetent patients.

Nuclear export signal mutation of epidermal growth factor receptor enhances malignant phenotypes of cancer cells.

Nuclear epidermal growth factor receptor (EGFR) has been shown to be correlated with drug resistance and a poor prognosis in patients with cancer. Previously, we have identified a tripartite nuclear localization signal (NLS) within EGFR. To comprehensively determine the functions and underlying mechanism of nuclear EGFR and its clinical implications, we aimed to explore the nuclear export signal (NES) sequence of EGFR that is responsible for interacting with the exportins. We combined in silico prediction with site-directed mutagenesis approaches and identified a putative NES motif of EGFR, which is located in amino acid residues 736-749. Mutation at leucine 747 (L747) in the EGFR NES led to increased nuclear accumulation of the protein via a less efficient release of the exportin CRM1. Interestingly, L747 with serine (L747S) and with proline (L747P) mutations were found in both tyrosine kinase inhibitor (TKI)-treated and -naïve patients with lung cancer who had acquired or de novo TKI resistance and a poor outcome. Reconstituted expression of the single NES mutant EGFRL747P or EGFRL747S, but not the dual mutant along with the internalization-defective or NLS mutation, in lung cancer cells promoted malignant phenotypes, including cell migration, invasiveness, TKI resistance, and tumor initiation, supporting an oncogenic role of nuclear EGFR. Intriguingly, cells with germline expression of the NES L747 mutant developed into B cell lymphoma. Mechanistically, nuclear EGFR signaling is required for sustaining nuclear activated STAT3, but not for Erk. These findings suggest that EGFR functions are compartmentalized and that nuclear EGFR signaling plays a crucial role in tumor malignant phenotypes, leading to tumorigenesis in human cancer.

Nuclear translocation of epidermal growth factor receptor by Akt-dependent phosphorylation enhances breast cancer-resistant protein expression in gefitinib-resistant cells.

Epidermal growth factor receptor (EGFR), an aberrantly overexpressed or activated receptor-tyrosine kinase in many cancers, plays a pivotal role in cancer progression and has been an attractive target for cancer therapy. Gefitinib and erlotinib, two EGFR-tyrosine kinase inhibitors, have been approved for non-small cell lung cancer. However, durable clinical efficacy of these EGFR inhibitors is severely limited by the emergence of acquired resistance. For example, the expression of breast cancer-resistant protein (BCRP/ABCG2) has been shown to confer acquired resistance of wild-type EGFR (wtEGFR)-expressing cancer cells to gefitinib. However, the underlying molecular mechanisms still remain unclear. Here, we show that wtEGFR expression is elevated in the nucleus of acquired gefitinib-resistant cancer cells. Moreover, nuclear translocation of EGFR requires phosphorylation at Ser-229 by Akt. In the nucleus, EGFR then targets the proximal promoter of BCRP/ABCG2 and thereby enhances its gene transcription. The nuclear EGFR-mediated BCRP/ABCG2 expression may contribute at least in part to the acquired resistance of wtEGFR-expressing cancer cells to gefitinib. Our findings shed light on the role of nuclear EGFR in the sensitivity of wtEGFR-expressing cancer cells to EGFR tyrosine kinase inhibitors and also deciphered a putative molecular mechanism contributing to gefitinib resistance through BCRP/ABCG2 expression.

An optimized protocol for PD-L1 pathological assessment with patient sample deglycosylation to improve correlation with therapeutic response.

Immunotherapy via PD-1/PD-L1 blockade is a promising strategy to eradicate cancer cells. However, the PD-L1 pathological level is inconsistent with the therapeutic response and is not a reliable biomarker to stratify patients for anti-PD-1/PD-L1 therapy. Here, we describe patient sample deglycosylation in an immunohistochemistry (IHC) assay to resolve this challenge. This protocol facilitates antigen retrieval by removing N-glycans from surface antigens on formalin-fixed paraffin-embedded (FFPE) tissue slides and can be applied in medical pathology for multiple cancer types. For complete details on the use and execution of this profile, please refer to Lee et al. (2019).

De-glycosylated membrane PD-L1 in tumor tissues as a biomarker for responsiveness to atezolizumab (Tecentriq) in advanced breast cancer patients.

The atezolizumab (Tecentriq), a humanized antibody against human programmed death ligand 1 (PD-L1), combined with nab-paclitaxel was granted with accelerated approval to treat unresectable locally advanced or metastatic triple-negative breast cancer (TNBC) due to the encouraging positive results of the phase 3 IMpassion130 trial using PD-L1 biomarker from immune cells to stratify patients. However, the post-market study IMpassion131 did not support the original observation, resulting in the voluntary withdrawal of atezolizumab from the indication in breast cancer by Genentech in 2021. Emerging evidence has revealed a high frequency of false negative result using the standard immunohistochemical (IHC) staining due to heavy glycosylation of PD-L1. The removal of glycosylation prevents from the false negative staining, enabling more accurate assessment of PD-L1 levels and improving prediction for response to immune checkpoint therapy. In the present study, the natural and de-glycosylated PD-L1 expression in tumor and immune cells from nine TNBC patients were analyzed by using clone 28-8 monoclonal antibody to correlate with treatment outcome. Our results demonstrate that: (1) Removal of the glycosylation indeed enhances the detection of PD-L1 by IHC staining, (2) The PD-L1 levels on tumor cell surface after removal of the glycosylation correlates well with clinical responses for atezolizumab treatment; (3) The criteria used in the IMpassion130 and IMpassion131 trials which scored the natural PD-L1 in the immune cells failed to correlate with the clinical response. Taken together, tumor cell surface staining of PD-L1 with de-glycosylation has a significant correlation with the clinical response for atezolizumab treatment, suggesting that treatment of atezolizumab may be worthy of further consideration with de-glycosylation procedure as a patient stratification strategy. A larger cohort to validate this important issue is warranted to ensure right patient population who could benefit from the existing FDA-approved drugs.

DNA damage-induced paraspeckle formation enhances DNA repair and tumor radioresistance by recruiting ribosomal protein P0.

Paraspeckles are mammal-specific membraneless nuclear bodies that participate in various biological processes. NONO, a central paraspeckle component, has been shown to play pivotal roles in DNA double-strand breaks (DSB) repair, whereas its underlying mechanism needs to be further disclosed. Here, using co-immunoprecipitation and mass spectrum, we identified ribosomal protein P0 (RPLP0) as a DSB-induced NONO-binding protein; RPLP0 binds to the RRM1 and RRM2 domains of NONO. Similar to NONO, RPLP0 enhances non-homologous end joining-mediated DSB repair, which was ascribed to a ribosome-independent manner. Interestingly, paraspeckles were induced as early as 15 min after irradiation; it further recruited nuclear RPLP0 to enhance its interaction with NONO. Radiation-induced NONO/RPLP0 complex subsequently anchored at the damaged DNA and increased the autophosphorylation of DNA-PK at Thr2609, thereby enhancing DSB repair. Consistently, in vivo and in vitro experiments showed that depletion of NONO sensitizes tumor cells to radiation. For patients with locally advanced rectal cancer, NONO expression was remarkably increased in tumor tissues and correlated with a poor response to radiochemotherapy. Our findings suggest a pivotal role of radiation-induced paraspeckles in DNA repair and tumor radioresistance, and provide a new insight into the ribosome-independent function of ribosomal proteins.

MRNIP condensates promote DNA double-strand break sensing and end resection.

The rapid recognition of DNA double-strand breaks (DSBs) by the MRE11/RAD50/NBS1 (MRN) complex is critical for the initiation of DNA damage response and DSB end resection. Here, we show that MRN complex interacting protein (MRNIP) forms liquid-like condensates to promote homologous recombination-mediated DSB repair. The intrinsically disordered region is essential for MRNIP condensate formation. Mechanically, the MRN complex is compartmentalized and concentrated into MRNIP condensates in the nucleus. After DSB formation, MRNIP condensates move to the damaged DNA rapidly to accelerate the binding of DSB by the concentrated MRN complex, therefore inducing the autophosphorylation of ATM and subsequent activation of DNA damage response signaling. Meanwhile, MRNIP condensates-enhanced MRN complex loading further promotes DSB end resection. In addition, data from xenograft models and clinical samples confirm a correlation between MRNIP and radioresistance. Together, these results reveal an important role of MRNIP phase separation in DSB response and the MRN complex-mediated DSB end resection.

Targeting the ALK-CDK9-Tyr19 kinase cascade sensitizes ovarian and breast tumors to PARP inhibition via destabilization of the P-TEFb complex.

Poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated promising clinical activity in multiple cancers. However, resistance to PARP inhibitors remains a substantial clinical challenge. In the present study, we report that anaplastic lymphoma kinase (ALK) directly phosphorylates CDK9 at tyrosine-19 to promote homologous recombination (HR) repair and PARP inhibitor resistance. Phospho-CDK9-Tyr19 increases its kinase activity and nuclear localization to stabilize positive transcriptional elongation factor b and activate polymerase II-dependent transcription of HR-repair genes. Conversely, ALK inhibition increases ubiquitination and degradation of CDK9 by Skp2, an E3 ligase. Notably, combination of US Food and Drug Administration-approved ALK and PARP inhibitors markedly reduce tumor growth and improve survival of mice in PARP inhibitor-/platinum-resistant tumor xenograft models. Using human tumor biospecimens, we further demonstrate that phosphorylated ALK (p-ALK) expression is associated with resistance to PARP inhibitors and positively correlated with p-Tyr19-CDK9 expression. Together, our findings support a biomarker-driven, combinatorial treatment strategy involving ALK and PARP inhibitors to induce synthetic lethality in PARP inhibitor-/platinum-resistant tumors with high p-ALK-p-Tyr19-CDK9 expression.

The translocon Sec61beta localized in the inner nuclear membrane transports membrane-embedded EGF receptor to the nucleus.

Accumulating evidence indicates that endocytosis plays an essential role in the nuclear transport of the ErbB family members, such as epidermal growth factor receptor (EGFR) and ErbB-2. Nevertheless, how full-length receptors embedded in the endosomal membrane pass through the nuclear pore complexes and function as non-membrane-bound receptors in the nucleus remains unclear. Here we show that upon EGF treatment, the biotinylated cell surface EGFR is trafficked to the inner nuclear membrane (INM) through the nuclear pore complexes, remaining in a membrane-bound environment. We further find that importin ß regulates EGFR nuclear transport to the INM in addition to the nucleus/nucleoplasm. Unexpectedly, the well known endoplasmic reticulum associated translocon Sec61ß is found to reside in the INM and associate with EGFR. Knocking down Sec61ß expression reduces EGFR level in the nucleoplasm portion and accumulates it in the INM portion. Thus, the Sec61ß translocon plays an unrecognized role in the release of the membrane-anchored EGFR from the lipid bilayer of the INM to the nucleus. The newly identified Sec61ß function provides an alternative pathway for nuclear transport that can be utilized by membrane-embedded proteins such as full-length EGFR.

NONO phase separation enhances DNA damage repair by accelerating nuclear EGFR-induced DNA-PK activation.

Radioresistance is one of the main causes of cancer treatment failure, which leads to relapse and inferior survival outcome of cancer patients. Liquid-liquid phase separation (LLPS) of proteins is known to be involved in various biological processes, whereas its role in the regulation of radiosensitivity remains largely unknown. In this study, we characterized NONO, an RNA/DNA binding protein with LLPS capacity, as an essential regulator of tumor radioresistance. In vitro assay showed that NONO involved in DNA repair via non-homologous end joining (NHEJ) manner. NONO knockout significantly reduced DNA damage repair and sensitized tumor cells to irradiation in vitro and in vivo. NONO overexpression was correlated with an inferior survival outcome in cancer patients. Mechanically, NONO was associated with nuclear EGFR (nEGFR). Both irradiation and EGF treatment induced nEGFR accumulation, thereby increased the association between NONO and nEGFR. However, NONO was not a substrate of EGFR kinase. Furthermore, NONO promoted DNA damage-induced DNA-PK phosphorylation at T2609 by enhancing the interaction between EGFR and DNA-PK. Importantly, NONO protein formed high concentration LLPS droplets in vitro, and recruited EGFR and DNA-PK. Disruption of NONO droplets with LLPS inhibitor significantly reduced the interaction between EGFR and DNA-PK, and suppressed DNA damage-induced phosphorylation of T2609-DNA-PK. Taken together, LLPS of NONO recruits nuclear EGFR and DNA-PK and enhances their interaction, further increases DNA damage-activated pT2609-DNA-PK and promotes NHEJ-mediated DNA repair, finally leads to tumor radioresistance. NONO phase separation-mediated radioresistance may serve as a novel molecular target to sensitize tumor cell to radiotherapy.

PRMT1 enhances oncogenic arginine methylation of NONO in colorectal cancer.

Arginine methylation is an important posttranslational modification catalyzed by protein arginine methyltransferases (PRMTs). However, the role of PRMTs in colorectal cancer (CRC) progression is not well understood. Here we report that non-POU domain-containing octamer-binding protein (NONO) is overexpressed in CRC tissue and is a potential marker for poor prognosis in CRC patients. NONO silencing resulted in decreased proliferation, migration, and invasion of CRC cells, whereas overexpression had the opposite effect. In a xenograft model, tumors derived from NONO-deficient CRC cells were smaller than those derived from wild-type (WT) cells, and PRMT1 inhibition blocked CRC xenograft progression. A mass spectrometry analysis indicated that NONO is a substrate of PRMT1. R251 of NONO was asymmetrically dimethylated by PRMT1 in vitro and in vivo. Compared to NONO WT cells, NONO R251K mutant-expressing CRC cells showed reduced proliferation, migration, and invasion, and PRMT1 knockdown or pharmacological inhibition abrogated the malignant phenotype associated with NONO asymmetric dimethylation in both KRAS WT and mutant CRC cells. Compared to adjacent normal tissue, PRMT1 was highly expressed in the CRC zone in clinical specimens, which was correlated with poor overall survival in patients with locally advanced CRC. These results demonstrate that PRMT1-mediated methylation of NONO at R251 promotes CRC growth and metastasis, and suggest that PRMT1 inhibition may be an effective therapeutic strategy for CRC treatment regardless of KRAS mutation status.