Noncanonical functions of telomerase and telomeres in viruses-associated cancer.

The cause of cancer is attributed to the uncontrolled growth and proliferation of cells resulting from genetic changes and alterations in cell behavior, a phenomenon known as epigenetics. Telomeres, protective caps on the ends of chromosomes, regulate both cellular aging and cancer formation. In most cancers, telomerase is upregulated, with the telomerase reverse transcriptase (TERT) enzyme and telomerase RNA component (TERC) RNA element contributing to the maintenance of telomere length. Additionally, it is noteworthy that two viruses, human papillomavirus (HPV) and Epstein-Barr virus (EBV), utilize telomerase for their replication or persistence in infected cells. Also, TERT and TERC may play major roles in cancer not related to telomere biology. They are involved in the regulation of gene expression, signal transduction pathways, cellular metabolism, or even immune response modulation. Furthermore, the crosstalk between TERT, TERC, RNA-binding proteins, and microRNAs contributes to a greater extent to cancer biology. To understand the multifaceted roles played by TERT and TERC in cancer and viral life cycles, and then to develop effective therapeutic strategies against these diseases, are fundamental for this goal. By investigating deeply, the complicated mechanisms and relationships between TERT and TERC, scientists will open the doors to new therapies. In its analysis, the review emphasizes the significance of gaining insight into the multifaceted roles that TERT and TERC play in cancer pathogenesis, as well as their involvement in the viral life cycle for designing effective anticancer therapy approaches.

ALDH1A1 promotes PARP inhibitor resistance by enhancing retinoic acid receptor-mediated DNA polymerase θ expression.

Poly (ADP-ribose) Polymerase (PARP) inhibitors (PARPi) have been approved for both frontline and recurrent setting in ovarian cancer with homologous recombination (HR) repair deficiency. However, more than 40% of BRCA1/2-mutated ovarian cancer lack the initial response to PARPi treatment, and the majority of those that initially respond eventually develop resistance. Our previous study has demonstrated that increased expression of aldehyde dehydrogenase 1A1 (ALDH1A1) contributes to PARPi resistance in BRCA2-mutated ovarian cancer cells by enhancing microhomology-mediated end joining (MMEJ) but the mechanism remains unknown. Here, we find that ALDH1A1 enhances the expression of DNA polymerase θ (Polθ, encoded by the POLQ gene) in ovarian cancer cells. Furthermore, we demonstrate that the retinoic acid (RA) pathway is involved in the transcription activation of the POLQ gene. The RA receptor (RAR) can bind to the retinoic acid response element (RARE) located in the promoter of the POLQ gene, promoting transcription activation-related histone modification in the presence of RA. Given that ALDH1A1 catalyzes the biosynthesis of RA, we conclude that ALDH1A1 promotes POLQ expression via the activation of the RA signaling pathway. Finally, using a clinically-relevant patient-derived organoid (PDO) model, we find that ALDH1A1 inhibition by the pharmacological inhibitor NCT-505 in combination with the PARP inhibitor olaparib synergistically reduce the cell viability of PDOs carrying BRCA1/2 mutation and positive ALDH1A1 expression. In summary, our study elucidates a new mechanism contributing to PARPi resistance in HR-deficient ovarian cancer and shows the therapeutic potential of combining PARPi and ALDH1A1 inhibition in treating these patients.

The thioredoxin system determines CHK1 inhibitor sensitivity via redox-mediated regulation of ribonucleotide reductase activity.

Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i's) in combination with chemotherapy have shown promising results in preclinical studies but minimal efficacy with substantial toxicity in clinical trials. To explore novel combinational strategies that can overcome these limitations, we performed an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identified thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a novel determinant of CHK1i sensitivity. We established a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR1 inhibitor auronafin, an anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, these findings identify a new pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity.

Deep transfer learning of cancer drug responses by integrating bulk and single-cell RNA-seq data.

Drug screening data from massive bulk gene expression databases can be analyzed to determine the optimal clinical application of cancer drugs. The growing amount of single-cell RNA sequencing (scRNA-seq) data also provides insights into improving therapeutic effectiveness by helping to study the heterogeneity of drug responses for cancer cell subpopulations. Developing computational approaches to predict and interpret cancer drug response in single-cell data collected from clinical samples can be very useful. We propose scDEAL, a deep transfer learning framework for cancer drug response prediction at the single-cell level by integrating large-scale bulk cell-line data. The highlight in scDEAL involves harmonizing drug-related bulk RNA-seq data with scRNA-seq data and transferring the model trained on bulk RNA-seq data to predict drug responses in scRNA-seq. Another feature of scDEAL is the integrated gradient feature interpretation to infer the signature genes of drug resistance mechanisms. We benchmark scDEAL on six scRNA-seq datasets and demonstrate its model interpretability via three case studies focusing on drug response label prediction, gene signature identification, and pseudotime analysis. We believe that scDEAL could help study cell reprogramming, drug selection, and repurposing for improving therapeutic efficacy.

ERK inactivation enhances stemness of NSCLC cells via promoting Slug-mediated epithelial-to-mesenchymal transition.

Rationale: The mitogen-activated protein kinase pathway (MAPK) is one of the major cancer-driving pathways found in non-small cell lung cancer (NSCLC) patients. ERK inhibitors (ERKi) have been shown to be effective in NSCLC patients with MAPK pathway mutations. However, like other MAPK inhibitors, ERKi rarely confers complete and durable responses. The mechanism of tumor relapse after ERKi treatment is yet defined. Methods: To best study the mechanism of tumor relapse after ERK inhibitor treatment in NSCLC patients, we treated various NSCLC cell lines and patient-derived xenograft (PDX) with ERK inhibitors and evaluated the enrichment of cancer stem cell (CSC) population. We then performed a Next-generation sequencing (NGS) to identify potential pathways that are responsible for the CSC enrichment. Further, the involvement of specific pathways was examined using molecular and cellular methods. Finally, we investigated the therapeutic benefits of ERKi treatment combined with JAK/STAT pathway inhibitor using cellular and xenograft NSCLC models. Results: We found that ERKi treatment expands the CSC population in NSCLC cells through enhanced epithelial-to-mesenchymal transition (EMT)-mediated cancer cell dedifferentiation. Mechanistically, ERK inactivation induces EMT via pSTAT3-mediated upregulation of Slug, in which, upregulation of miR-204 and downregulation of SPDEF, a transcription repressor of Slug, are involved. Finally, the JAK/STAT pathway inhibitor Ruxolitinib blocks the ERK inactivation-induced EMT and CSC expansion, as well as the tumor progression in xenograft models after ERKi treatment. Conclusions: This study revealed a potential tumor relapse mechanism of NSCLC after ERK inhibition through the unintended activation of the EMT program, ascertained the pSTAT-miR-204-SPDEF-Slug axis, and provided a promising combination inhibitor approach to prevent tumor relapse in patients.

A Novel Estrogen Receptor ß Agonist Diminishes Ovarian Cancer Stem Cells via Suppressing the Epithelial-to-Mesenchymal Transition.

Epithelial ovarian cancer is the most lethal malignancy of the female reproductive tract. A healthy ovary expresses both Estrogen Receptor α (ERα) and ß (ERß). Given that ERα is generally considered to promote cell survival and proliferation, thereby, enhancing tumor growth, while ERß shows a protective effect against the development and progression of tumors, the activation of ERß by its agonists could be therapeutically beneficial for ovarian cancer. Here, we demonstrate that the activation of ERß using a newly developed ERß agonist, OSU-ERb-12, can impede ovarian cancer cell expansion and tumor growth in an ERα-independent manner. More interestingly, we found that OSU-ERb-12 also reduces the cancer stem cell (CSC) population in ovarian cancer by compromising non-CSC-to-CSC conversion. Mechanistically, we revealed that OSU-ERb-12 decreased the expression of Snail, a master regulator of the epithelial-to-mesenchymal transition (EMT), which is associated with de novo CSC generation. Given that ERα can mediate EMT and facilitate maintenance of the CSC subpopulation and that OSU-ERb-12 can block the transactivity of ERα, we conclude that OSU-ERb-12 reduces the CSC subpopulation by inhibiting EMT in an ERα-dependent manner. Taken together, our data indicate that the ERß agonist OSU-ERb-12 could be used to hinder tumor progression and limit the CSC subpopulation with the potential to prevent tumor relapse and metastasis in patients with ovarian cancer.

Micropeptide PACMP inhibition elicits synthetic lethal effects by decreasing CtIP and poly(ADP-ribosyl)ation.

Synthetic lethality through combinatorial targeting DNA damage response (DDR) pathways provides exciting anticancer therapeutic benefit. Currently, the long noncoding RNAs (lncRNAs) have been implicated in tumor drug resistance; however, their potential significance in DDR is still largely unknown. Here, we report that a human lncRNA, CTD-2256P15.2, encodes a micropeptide, named PAR-amplifying and CtIP-maintaining micropeptide (PACMP), with a dual function to maintain CtIP abundance and promote poly(ADP-ribosyl)ation. PACMP not only prevents CtIP from ubiquitination through inhibiting the CtIP-KLHL15 association but also directly binds DNA damage-induced poly(ADP-ribose) chains to enhance PARP1-dependent poly(ADP-ribosyl)ation. Targeting PACMP alone inhibits tumor growth by causing a synthetic lethal interaction between CtIP and PARP inhibitions and confers sensitivity to PARP/ATR/CDK4/6 inhibitors, ionizing radiation, epirubicin, and camptothecin. Our findings reveal that a lncRNA-derived micropeptide regulates cancer progression and drug resistance by modulating DDR, whose inhibition could be employed to augment the existing anticancer therapeutic strategies.

Targeting Squalene Epoxidase Interrupts Homologous Recombination via the ER Stress Response and Promotes Radiotherapy Efficacy.

Over 50% of all patients with cancer are treated with radiotherapy. However, radiotherapy is often insufficient as a monotherapy and requires a nontoxic radiosensitizer. Squalene epoxidase (SQLE) controls cholesterol biosynthesis by converting squalene to 2,3-oxidosqualene. Given that SQLE is frequently overexpressed in human cancer, this study investigated the importance of SQLE in breast cancer and non-small cell lung cancer (NSCLC), two cancers often treated with radiotherapy. SQLE-positive IHC staining was observed in 68% of breast cancer and 56% of NSCLC specimens versus 15% and 25% in normal breast and lung tissue, respectively. Importantly, SQLE expression was an independent predictor of poor prognosis, and pharmacologic inhibition of SQLE enhanced breast and lung cancer cell radiosensitivity. In addition, SQLE inhibition enhanced sensitivity to PARP inhibition. Inhibition of SQLE interrupted homologous recombination by suppressing ataxia-telangiectasia mutated (ATM) activity via the translational upregulation of wild-type p53-induced phosphatase (WIP1), regardless of the p53 status. SQLE inhibition and subsequent squalene accumulation promoted this upregulation by triggering the endoplasmic reticulum (ER) stress response. Collectively, these results identify a novel tumor-specific radiosensitizer by revealing unrecognized cross-talk between squalene metabolites, ER stress, and the DNA damage response. Although SQLE inhibitors have been used as antifungal agents in the clinic, they have not yet been used as antitumor agents. Repurposing existing SQLE-inhibiting drugs may provide new cancer treatments. SIGNIFICANCE: Squalene epoxidase inhibitors are novel tumor-specific radiosensitizers that promote ER stress and suppress homologous recombination, providing a new potential therapeutic approach to enhance radiotherapy efficacy.

A Novel Role for RNF126 in the Promotion of G2 Arrest via Interaction With 14-3-3σ.

PURPOSE: Cell cycle checkpoints and DNA repair are important for cell survival after exogenous DNA damage. Both rapid blockage of G2 to M phase transition in the cell cycle and the maintenance of relatively slow G2 arrest are critical to protect cells from lethal ionizing radiation (IR). Checkpoint kinase 1 is pivotal in blocking the transition from G2 to M phases in response to IR. The 14-3-3σ protein is important for IR-induced G2 arrest maintenance in which p53-dependent 14-3-3σ transcription is involved. It has been demonstrated that Ring finger protein 126 (RNF126), an E3 ligase, is required to upregulate checkpoint kinase 1 expression. Thus, our goal was to study the role of RNF126 in the G2/M phase checkpoint. METHODS AND MATERIALS: The transition from G2 to M phases and G2 accumulation in response to IR were determined by flow cytometry through staining with phospho-histone H3 (pS10) antibody and propidium iodide, respectively. The interaction of RNF126 and 14-3-3σ was determined by GST-pulldown and coimmunoprecipitation assays. The stability of RNF126 and 14-3-3σ was determined by cycloheximide-based stability assay and ubiquitination detection by coimmunoprecipitation. The sequestering of cyclin-dependent kinase 1 and cyclin B1 from the nucleus was determined by immunofluorescence staining. RESULTS: RNF126 knockdown had no impact on the IR-induced transient blockage of G2 to M but impaired IR-induced G2 arrest maintenance in cells with or without wild-type p53. Mechanistically, RNF126 binds 14-3-3σ and prevents both proteins from ubiquitination-mediated degradation. Last, RNF126 is required for enforcing the cytoplasmic sequestration of cyclin B1 and cyclin-dependent kinase 1 proteins in response to IR. CONCLUSIONS: RNF126 promotes G2 arrest via interaction with 14-3-3σ in response to IR. Our study revealed a novel role for RNF126 in promoting G2 arrest, providing a new target for cancer treatment.

Aberrant expression of TMEM205 signaling promotes platinum resistance in ovarian cancer: An implication for the antitumor potential of DAP compound.

INTRODUCTION: TMEM205 is a novel transmembrane protein associated with platinum resistance (PR) in epithelial ovarian carcinoma (OC), however, the specific mechanisms associated with this resistance remain to be elucidated. METHODS: TMEM205 expression was evaluated in platinum-sensitive (PS) versus platinum resistant (PR) ovarian cancer cell lines and patient serum/tissues. Exosomal efflux of platinum was evaluated with inductively coupled plasma mass spectrometry (ICP-MS) after pre-treatment with small molecule inhibitors (L-2663/L-2797) of TMEM205 prior to treatment with platinum. Cytotoxicity of combination treatment was confirmed in vitro and in an in vivo model. RESULTS: TMEM205 expression was 10-20 fold higher in PR compared to PS ovarian cancer cell lines, serum samples, and tissues. Co-localization with CD1B was confirmed by in-situ proximity ligation assay suggesting that TMEM205 may mediate PR via the exosomal pathway. Exosomal secretion was significantly increased 5-10 fold in PR cell lines after treatment with carboplatin compared to PS cell lines. Pre-treatment with L-2663 prior to carboplatin resulted in significantly increased intracellular concentration of fluorescently-labeled cisplatin and decreased exosomal efflux of platinum. Decreased cell survival and tumor growth in vitro and in vivo was observed when PR cells were treated with a combination of L-2663 with carboplatin compared to carboplatin alone. CONCLUSION: TMEM205 appears to be involved in the development of PR in ovarian cancer through the exosomal efflux of platinum agents. This study provides pre-clinical evidence that TMEM205 could serve as a possible biomarker for PR as well as a therapeutic target in combination with platinum agents.

Determination of DNA lesion bypass using a ChIP-based assay.

DNA lesion bypass facilitates DNA synthesis across bulky DNA lesions, playing a critical role in DNA damage tolerance and cell survival after DNA damage. Assessing lesion bypass efficiency in the cell is important to better understanding of the mechanism of carcinogenesis and chemoresistance. Here we developed a chromatin immunoprecipitation (ChIP)-based method to measure lesion bypass activity across cisplatin-induced intrastrand crosslinks in cancer cells. DNA lesion bypass enables the replication to continue in the presence of replication blocks. Thus, the successful lesion bypass should result in the coexistence of DNA lesions and the newly synthesized DNA fragment opposite to this lesion. Using ChIP, we precipitated the cisplatin-induced intrastrand crosslinks, and quantitated the precipitated newly synthesized DNA that was labeled with BrdU. We validated this method on ovarian cancer cells with inhibited TLS activity. We then applied this method to show that ovarian cancer stem cells exhibit high lesion bypass activity relative to bulk cancer cells from the same cell line. In conclusion, this novel ChIP-based lesion bypass assay can detect the extent to which cisplatin-induced DNA lesions are bypassed in live cells. Our study may be applied more broadly to the study of other DNA lesions, as specific antibodies to these specific lesions are available.

Discovery of a Bicyclic Peptidyl Pan-Ras Inhibitor.

The Ras subfamily of small GTPases is mutated in ∼30% of human cancers and represents compelling yet challenging anticancer drug targets owing to their flat protein surface. We previously reported a bicyclic peptidyl inhibitor, cyclorasin B3, which binds selectively to Ras-GTP with modest affinity and blocks its interaction with downstream effector proteins in vitro but lacks cell permeability or biological activity. In this study, optimization of B3 yielded a potent pan-Ras inhibitor, cyclorasin B4-27, which binds selectively to the GTP-bound forms of wild-type and mutant Ras isoforms (KD = 21 nM for KRasG12V-GppNHp) and is highly cell-permeable and metabolically stable (serum t1/2 > 24 h). B4-27 inhibits Ras signaling in vitro and in vivo by blocking Ras from interacting with downstream effector proteins and induces apoptosis of Ras-mutant cancer cells. When administered systemically (i.v.), B4-27 suppressed tumor growth in two different mouse xenograft models at 1-5 mg/kg of daily doses.

MiRNA-200C expression in Fanconi anemia pathway functionally deficient lung cancers.

The Fanconi Anemia (FA) pathway is essential for human cells to maintain genomic integrity following DNA damage. This pathway is involved in repairing damaged DNA through homologous recombination. Cancers with a defective FA pathway are expected to be more sensitive to cross-link based therapy or PARP inhibitors. To evaluate downstream effectors of the FA pathway, we studied the expression of 734 different micro RNAs (miRNA) using NanoString nCounter miRNA array in two FA defective lung cancer cells and matched control cells, along with two lung tumors and matched non-tumor tissue samples that were deficient in the FA pathway. Selected miRNA expression was validated with real-time PCR analysis. Among 734 different miRNAs, a cluster of microRNAs were found to be up-regulated including an important cancer related micro RNA, miR-200C. MiRNA-200C has been reported as a negative regulator of epithelial-mesenchymal transition (EMT) and inhibits cell migration and invasion by promoting the upregulation of E-cadherin through targeting ZEB1 and ZEB2 transcription factors. miRNA-200C was increased in the FA defective lung cancers as compared to controls. AmpliSeq analysis showed significant reduction in ZEB1 and ZEB2 mRNA expression. Our findings indicate the miRNA-200C potentially play a very important role in FA pathway downstream regulation.

A Novel miR-146a-POU3F2/SMARCA5 Pathway Regulates Stemness and Therapeutic Response in Glioblastoma.

Rapid tumor growth, widespread brain-invasion, and therapeutic resistance critically contribute to glioblastoma (GBM) recurrence and dismal patient outcomes. Although GBM stem cells (GSC) are shown to play key roles in these processes, the molecular pathways governing the GSC phenotype (GBM-stemness) remain poorly defined. Here, we show that epigenetic silencing of miR-146a significantly correlated with worse patient outcome and importantly, miR-146a level was significantly lower in recurrent tumors compared with primary ones. Further, miR-146a overexpression significantly inhibited the proliferation and invasion of GBM patient-derived primary cells and increased their response to temozolomide (TMZ), both in vitro and in vivo. Mechanistically, miR-146a directly silenced POU3F2 and SMARCA5, two transcription factors that mutually regulated each other, significantly compromising GBM-stemness and increasing TMZ response. Collectively, our data show that miR-146a-POU3F2/SMARCA5 pathway plays a critical role in suppressing GBM-stemness and increasing TMZ-response, suggesting that POU3F2 and SMARCA5 may serve as novel therapeutic targets in GBM. IMPLICATIONS: miR-146a predicts favorable prognosis and the miR-146a-POU3F2/SMARCA5 pathway is important for the suppression of stemness in GBM.

DNA polymerase eta: A potential pharmacological target for cancer therapy.

In the last two decades, intensive research has been carried out to improve the survival rates of cancer patients. However, the development of chemoresistance that ultimately leads to tumor relapse poses a critical challenge for the successful treatment of cancer patients. Many cancer patients experience tumor relapse and ultimately die because of treatment failure associated with acquired drug resistance. Cancer cells utilize multiple lines of self-defense mechanisms to bypass chemotherapy and radiotherapy. One such mechanism employed by cancer cells is translesion DNA synthesis (TLS), in which specialized TLS polymerases bypass the DNA lesion with the help of monoubiquitinated proliferating cell nuclear antigen. Among all TLS polymerases (Pol η, Pol ι, Pol κ, REV1, Pol ζ, Pol µ, Pol λ, Pol ν, and Pol θ), DNA polymerase eta (Pol η) is well studied and majorly responsible for the bypass of cisplatin and UV-induced DNA damage. TLS polymerases contribute to chemotherapeutic drug-induced mutations as well as therapy resistance. Therefore, targeting these polymerases presents a novel therapeutic strategy to combat chemoresistance. Mounting evidence suggests that inhibition of Pol η may have multiple impacts on cancer therapy such as sensitizing cancer cells to chemotherapeutics, suppressing drug-induced mutagenesis, and inhibiting the development of secondary tumors. Herein, we provide a general introduction of Pol η and its clinical implications in blocking acquired drug resistance. In addition; this review addresses the existing gaps and challenges of Pol η mediated TLS mechanisms in human cells. A better understanding of the Pol η mediated TLS mechanism will not merely establish it as a potential pharmacological target but also open possibilities to identify novel drug targets for future therapy.

A Genome-Wide Pooled shRNA Screen Identifies PPP2R2A as a Predictive Biomarker for the Response to ATR and CHK1 Inhibitors.

There is currently a lack of precise predictive biomarkers for patient selection in clinical trials of inhibitors targeting replication stress (RS) response proteins ATR and CHK1. The objective of this study was to identify novel predictive biomarkers for the response to these agents in treating non-small cell lung cancer (NSCLC). A genome-wide loss-of-function screen revealed that tumor suppressor PPP2R2A, a B regulatory subunit of protein phosphatase 2 (PP2A), determines sensitivity to CHK1 inhibition. A synthetic lethal interaction between PPP2R2A deficiency and ATR or CHK1 inhibition was observed in NSCLC in vitro and in vivo and was independent of p53 status. ATR and CHK1 inhibition resulted in significantly increased levels of RS and altered replication dynamics, particularly in PPP2R2A-deficient NSCLC cells. Mechanistically, PPP2R2A negatively regulated translation of oncogene c-Myc protein. c-Myc activity was required for PPP2R2A deficiency-induced alterations of replication initiation/RS and sensitivity to ATR/CHK1 inhibitors. We conclude that PPP2R2A deficiency elevates RS by upregulating c-Myc activity, rendering cells reliant on the ATR/CHK1 axis for survival. Our studies show a novel synthetic lethal interaction and identify PPP2R2A as a potential new predictive biomarker for patient stratification in the clinical use of ATR and CHK1 inhibitors. SIGNIFICANCE: This study reveals new approaches to specifically target PPP2R2A-deficient lung cancer cells and provides a novel biomarker that will significantly improve treatment outcome with ATR and CHK1 inhibitors.

Prognostic Value of DNA Damage Response Genomic Alterations in Relapsed/Advanced Urothelial Cancer.

BACKGROUND: DNA damage response (DDR) genomic alterations may play an important role in clinical outcomes of patients with urothelial cancer (UC). However, data on the prognostic role of DDR gene alterations in patients with advanced UC remain unclear. MATERIALS AND METHODS: We retrospectively collected data of three independent patient cohorts with relapsed or advanced UC including 81 and 91 patients from four institutions who underwent FoundationOne genomic sequencing as well as 129 patients selected from The Cancer Genome Atlas bladder cohort. Fisher's exact test was used to determine differences of mutation frequency among the three cohorts. Logistic regression analysis was performed to calculate odds ratio (OR) and 95% confidence interval (CI). Overall survival (OS) was measured from time of initial diagnosis and Cox proportional hazard regression analysis was performed to calculate the hazard ratio (HR) and 95% CI. RESULTS: DDR genomic alterations were present in 76.5% (62/81), 40.7% (37/91), and 51.2% (66/129) of the three cohorts. ATM alterations consistently correlated with significantly shorter OS, whereas other DDR alterations (excluding ATM) were associated with better prognosis. In 152 patients treated with platinum pooled from the three cohorts, the prognostic value of alterations in ATM as compared with other predefined DDR genes was substantially different (ATM: adjusted HR [HR], 2.03; 95% CI, 1.03-4; p = .04; other DDR: adjusted HR, 0.49; 95% CI, 0.31-0.8; p = .003). CONCLUSIONS: Genomic alterations in ATM and other DDR genes may have opposite prognostic value in relapsed and/or advanced UC. ATM may have a complex role in UC progression. IMPLICATIONS FOR PRACTICE: Somatic mutations of DNA damage response (DDR) genes are frequently found in urothelial cancer and appear to play an important role in tumorigenesis, progression, treatment response, and outcomes. In a set of DDR genes, ATM alterations were associated with worse survival, while other alterations were associated with better survival in advanced urothelial cancer. The results of this study suggest a complex role of ATM in tumor progression and call for further studies to determine the underlying mechanisms and biomarker clinical utility.

ALDH1A1 Contributes to PARP Inhibitor Resistance via Enhancing DNA Repair in BRCA2-/- Ovarian Cancer Cells.

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are approved to treat recurrent ovarian cancer with BRCA1 or BRCA2 mutations, and as maintenance therapy for recurrent platinum-sensitive ovarian cancer (BRCA wild-type or mutated) after treatment with platinum. However, the acquired resistance against PARPi remains a clinical hurdle. Here, we demonstrated that PARP inhibitor (olaparib)-resistant epithelial ovarian cancer (EOC) cells exhibited an elevated aldehyde dehydrogenase (ALDH) activity, mainly contributed by increased expression of ALDH1A1 due to olaparib-induced expression of BRD4, a member of bromodomain and extraterminal (BET) family protein. We also revealed that ALDH1A1 enhanced microhomology-mediated end joining (MMEJ) activity in EOC cells with inactivated BRCA2, a key protein that promotes homologous recombination (HR) by using an intrachromosomal MMEJ reporter. Moreover, NCT-501, an ALDH1A1-selective inhibitor, can synergize with olaparib in killing EOC cells carrying BRCA2 mutation in both in vitro cell culture and the in vivo xenograft animal model. Given that MMEJ activity has been reported to be responsible for PARPi resistance in HR-deficient cells, we conclude that ALDH1A1 contributes to the resistance to PARP inhibitors via enhancing MMEJ in BRCA2-/- ovarian cancer cells. Our findings provide a novel mechanism underlying PARPi resistance in BRCA2-mutated EOC cells and suggest that inhibition of ALDH1A1 could be exploited for preventing and overcoming PARPi resistance in EOC patients carrying BRCA2 mutation.

Loss of BRUCE reduces cellular energy level and induces autophagy by driving activation of the AMPK-ULK1 autophagic initiating axis.

Autophagy is an intracellular catabolic system. It delivers cellular components to lysosomes for degradation and supplies nutrients that promote cell survival under stress conditions. Although much is known regarding starvation-induced autophagy, the regulation of autophagy by cellular energy level is less clear. BRUCE is an ubiquitin conjugase and ligase with multi-functionality. It has been reported that depletion of BRUCE inhibits starvation-induced autophagy by blockage of the fusion step. Herein we report a new function for BRUCE in the dual regulation of autophagy and cellular energy. Depletion of BRUCE alone (without starvation) in human osteosarcoma U2OS cells elevated autophagic activity as indicted by the increased LC3B-II protein and its autophagic puncta as well as further increase of both by chloroquine treatment. Such elevation results from enhanced induction of autophagy since the numbers of both autophagosomes and autolysosomes were increased, and recruitment of ATG16L onto the initiating membrane structure phagophores was increased. This concept is further supported by elevated lysosomal enzyme activities. In contrast to starvation-induced autophagy, BRUCE depletion did not block fusion of autophagosomes with lysosomes as indicated by increased lysosomal cleavage of the GFP-LC3 fusion protein. Mechanistically, BRUCE depletion lowered the cellular energy level as indicated by both a higher ratio of AMP/ATP and the subsequent activation of the cellular energy sensor AMPK (pThr-172). The lower energy status co-occurred with AMPK-specific phosphorylation and activation of the autophagy initiating kinase ULK1 (pSer-555). Interestingly, the higher autophagic activity by BRUCE depletion is coupled with enhanced cisplatin resistance in human ovarian cancer PEO4 cells. Taken together, BRUCE depletion promotes induction of autophagy by lowering cellular energy and activating the AMPK-ULK1-autophagy axis, which could contribute to ovarian cancer chemo-resistance. This study establishes a BRUCE-AMPK-ULK1 axis in the regulation of energy metabolism and autophagy, as well as provides insights into cancer chemo-resistance.