Clinical insights into small cell lung cancer: Tumor heterogeneity, diagnosis, therapy, and future directions.

Small cell lung cancer (SCLC) is characterized by rapid growth and high metastatic capacity. It has strong epidemiologic and biologic links to tobacco carcinogens. Although the majority of SCLCs exhibit neuroendocrine features, an important subset of tumors lacks these properties. Genomic profiling of SCLC reveals genetic instability, almost universal inactivation of the tumor suppressor genes TP53 and RB1, and a high mutation burden. Because of early metastasis, only a small fraction of patients are amenable to curative-intent lung resection, and these individuals require adjuvant platinum-etoposide chemotherapy. Therefore, the vast majority of patients are currently being treated with chemoradiation with or without immunotherapy. In patients with disease confined to the chest, standard therapy includes thoracic radiotherapy and concurrent platinum-etoposide chemotherapy. Patients with metastatic (extensive-stage) disease are treated with a combination of platinum-etoposide chemotherapy plus immunotherapy with an anti-programmed death-ligand 1 monoclonal antibody. Although SCLC is initially very responsive to platinum-based chemotherapy, these responses are transient because of the development of drug resistance. In recent years, the authors have witnessed an accelerating pace of biologic insights into the disease, leading to the redefinition of the SCLC classification scheme. This emerging knowledge of SCLC molecular subtypes has the potential to define unique therapeutic vulnerabilities. Synthesizing these new discoveries with the current knowledge of SCLC biology and clinical management may lead to unprecedented advances in SCLC patient care. Here, the authors present an overview of multimodal clinical approaches in SCLC, with a special focus on illuminating how recent advancements in SCLC research could accelerate clinical development.

Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes.

Neuronal activity causes the rapid expression of immediate early genes that are crucial for experience-driven changes to synapses, learning, and memory. Here, using both molecular and genome-wide next-generation sequencing methods, we report that neuronal activity stimulation triggers the formation of DNA double strand breaks (DSBs) in the promoters of a subset of early-response genes, including Fos, Npas4, and Egr1. Generation of targeted DNA DSBs within Fos and Npas4 promoters is sufficient to induce their expression even in the absence of an external stimulus. Activity-dependent DSB formation is likely mediated by the type II topoisomerase, Topoisomerase IIß (Topo IIß), and knockdown of Topo IIß attenuates both DSB formation and early-response gene expression following neuronal stimulation. Our results suggest that DSB formation is a physiological event that rapidly resolves topological constraints to early-response gene expression in neurons.

Topoisomerase II-Induced Chromosome Breakage and Translocation Is Determined by Chromosome Architecture and Transcriptional Activity.

Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.

The RNase PARN Controls the Levels of Specific miRNAs that Contribute to p53 Regulation.

PARN loss-of-function mutations cause a severe form of the hereditary disease dyskeratosis congenita (DC). PARN deficiency affects the stability of non-coding RNAs such as human telomerase RNA (hTR), but these effects do not explain the severe disease in patients. We demonstrate that PARN deficiency affects the levels of numerous miRNAs in human cells. PARN regulates miRNA levels by stabilizing either mature or precursor miRNAs by removing oligo(A) tails added by the poly(A) polymerase PAPD5, which if remaining recruit the exonuclease DIS3L or DIS3L2 to degrade the miRNA. PARN knockdown destabilizes multiple miRNAs that repress p53 translation, which leads to an increase in p53 accumulation in a Dicer-dependent manner, thus explaining why PARN-defective patients show p53 accumulation. This work also reveals that DIS3L and DIS3L2 are critical 3' to 5' exonucleases that regulate miRNA stability, with the addition and removal of 3' end extensions controlling miRNA levels in the cell.

Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms.

Topoisomerase II (TOP2) unlinks chromosomes during vertebrate DNA replication. TOP2 "poisons" are widely used chemotherapeutics that stabilize TOP2 complexes on DNA, leading to cytotoxic DNA breaks. However, it is unclear how these drugs affect DNA replication, which is a major target of TOP2 poisons. Using Xenopus egg extracts, we show that the TOP2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces TOP2-dependent DNA breaks and TOP2-dependent fork stalling by trapping TOP2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to stall replication forks independently of TOP2. In human cells, etoposide stalls forks in a TOP2-dependent manner, while doxorubicin stalls forks independently of TOP2. However, both drugs exhibit TOP2-dependent cytotoxicity. Thus, etoposide and doxorubicin inhibit DNA replication through distinct mechanisms despite shared genetic requirements for cytotoxicity.

Survival in primary hemophagocytic lymphohistiocytosis, 2016 to 2021: etoposide is better than its reputation.

ABSTRACT: Primary hemophagocytic lymphohistiocytosis (pHLH) is a life-threatening hyperinflammatory syndrome that develops mainly in patients with genetic disorders of lymphocyte cytotoxicity and X-linked lymphoproliferative syndromes. Previous studies with etoposide-based treatment followed by hematopoetic stem cell transplantation (HSCT) resulted in 5-year survival of 50% to 59%. Contemporary data are lacking. We evaluated 88 patients with pHLH documented in the international HLH registry from 2016-2021. In 12 of 88 patients, diagnosis was made without HLH activity, based on siblings or albinism. Major HLH-directed drugs (etoposide, antithymocyte globulin, alemtuzumab, emapalumab, ruxolitinib) were administered to 66 of 76 patients who were symptomatic (86% first-line etoposide); 16 of 57 patients treated with etoposide and 3 of 9 with other first-line treatment received salvage therapy. HSCT was performed in 75 patients; 7 patients died before HSCT. Three-year probability of survival (pSU) was 82% (confidence interval [CI], 72%-88%) for the entire cohort and 77% (CI, 64%-86%) for patients receiving first-line etoposide. Compared with the HLH-2004 study, both pre-HSCT and post-HSCT survival of patients receiving first-line etoposide improved, 83% to 91% and 70% to 88%. Differences to HLH-2004 included preferential use of reduced-toxicity conditioning and reduced time from diagnosis to HSCT (from 148 to 88 days). Three-year pSU was lower with haploidentical (4 of 9 patients [44%]) than with other donors (62 of 66 [94%]; P < .001). Importantly, early HSCT for patients who were asymptomatic resulted in 100% survival, emphasizing the potential benefit of newborn screening. This contemporary standard-of-care study of patients with pHLH reveals that first-line etoposide-based therapy is better than previously reported, providing a benchmark for novel treatment regimes.

ATM-dependent Phosphorylation of Nemo SQ Motifs Is Dispensable for Nemo-mediated Gene Expression Changes in Response to DNA Double-Strand Breaks.

In response to DNA double-strand breaks (DSBs), the ATM kinase activates NF-κB factors to stimulate gene expression changes that promote survival and allow time for cells to repair damage. In cell lines, ATM can activate NF-κB transcription factors via two independent, convergent mechanisms. One is ATM-mediated phosphorylation of nuclear NF-κB essential modulator (Nemo) protein, which leads to monoubiquitylation and export of Nemo to the cytoplasm where it engages the IκB kinase (IKK) complex to activate NF-κB. Another is DSB-triggered migration of ATM into the cytoplasm, where it promotes monoubiquitylation of Nemo and the resulting IKK-mediated activation of NF-κB. ATM has many other functions in the DSB response beyond activation of NF-κB, and Nemo activates NF-κB downstream of diverse stimuli, including developmental or proinflammatory stimuli such as LPSs. To elucidate the in vivo role of DSB-induced, ATM-dependent changes in expression of NF-κB-responsive genes, we generated mice expressing phosphomutant Nemo protein lacking consensus SQ sites for phosphorylation by ATM or related kinases. We demonstrate that these mice are viable/healthy and fertile and exhibit overall normal B and T lymphocyte development. Moreover, treatment of their B lineage cells with LPS induces normal NF-κB-regulated gene expression changes. Furthermore, in marked contrast to results from a pre-B cell line, primary B lineage cells expressing phosphomutant Nemo treated with the genotoxic drug etoposide induce normal ATM- and Nemo-dependent changes in expression of NF-κB-regulated genes. Our data demonstrate that ATM-dependent phosphorylation of Nemo SQ motifs in vivo is dispensable for DSB-signaled changes in expression of NF-κB-regulated genes.

Using energy to go downhill-a genoprotective role for ATPase activity in DNA topoisomerase II.

Type II topoisomerases effect topological changes in DNA by cutting a single duplex, passing a second duplex through the break, and resealing the broken strand in an ATP-coupled reaction cycle. Curiously, most type II topoisomerases (topos II, IV and VI) catalyze DNA transformations that are energetically favorable, such as the removal of superhelical strain; why ATP is required for such reactions is unknown. Here, using human topoisomerase IIß (hTOP2ß) as a model, we show that the ATPase domains of the enzyme are not required for DNA strand passage, but that their loss elevates the enzyme's propensity for DNA damage. The unstructured C-terminal domains (CTDs) of hTOP2ß strongly potentiate strand passage activity in ATPase-less enzymes, as do cleavage-prone mutations that confer hypersensitivity to the chemotherapeutic agent etoposide. The presence of either the CTD or the mutations lead ATPase-less enzymes to promote even greater levels of DNA cleavage in vitro, as well as in vivo. By contrast, aberrant cleavage phenotypes of these topo II variants is significantly repressed when the ATPase domains are present. Our findings are consistent with the proposal that type II topoisomerases acquired ATPase function to maintain high levels of catalytic activity while minimizing inappropriate DNA damage.

Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts.

RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.

Naturally mutagenic sequence diversity in a human type II topoisomerase.

Type II topoisomerases transiently cleave duplex DNA as part of a strand passage mechanism that helps control chromosomal organization and superstructure. Aberrant DNA cleavage can result in genomic instability, and how topoisomerase activity is controlled to prevent unwanted breaks is poorly understood. Using a genetic screen, we identified mutations in the beta isoform of human topoisomerase II (hTOP2ß) that render the enzyme hypersensitive to the chemotherapeutic agent etoposide. Several of these variants were unexpectedly found to display hypercleavage behavior in vitro and to be capable of inducing cell lethality in a DNA repair-deficient background; surprisingly, a subset of these mutations were also observed in TOP2B sequences from cancer genome databases. Using molecular dynamics simulations and computational network analyses, we found that many of the mutations obtained from the screen map to interfacial points between structurally coupled elements, and that dynamical modeling could be used to identify other damage-inducing TOP2B alleles present in cancer genome databases. This work establishes that there is an innate link between DNA cleavage predisposition and sensitivity to topoisomerase II poisons, and that certain sequence variants of human type II topoisomerases found in cancer cells can act as DNA-damaging agents. Our findings underscore the potential for hTOP2ß to function as a clastogen capable of generating DNA damage that may promote or support cellular transformation.

Functional analysis of germline RAD51C missense variants highlight the role of RAD51C in replication fork protection.

Monoallelic or biallelic RAD51C germline mutations results in chromosome instability disorders such as Fanconi anemia and cancers. The bona fide function of RAD51C is to assist RAD51 nucleoprotein filament onto single-strand DNA to complete homologous recombination (HR) repair. In addition to HR repair, the role of RAD51C in DNA replication is emerging when replication forks are transiently or irreversibly stalled. We identified novel RAD51C variants of uncertain significance (VUS) from breast, ovarian, pancreatic and gastric cancer patients and functionally characterized the effect of these variants in replication fork protection and double-strand breaks (DSB's) repair. In RAD51C-deficient Chinese hamster CL-V4B cells, expression of RAD51C F164S, A87E, L134S and E49K variants heightened sensitivity to mitomycin C (MMC), etoposide and PARP inhibition. Differently, expression of subset of RAD51C variants R24L, R24W and R212H displayed mild sensitivity to MMC, etoposide and PARP inhibition. Further functional characterization of a subset of variants revealed that Rad51C F164S, A87E, L134S and E49K variants displayed reduced RAD51 foci formation and increased overall nuclear single strand DNA levels in the presence of replication stress. Additionally, DNA fiber assay revealed that RAD51C F164S, A87E, L134S and E49K variants displayed defective replication fork protection upon prolonged fork stalling. Investigations using patient-derived lymphoblastoid cell line carrying heterozygous RAD51C L134S variant showed an impairment in RAD51 chromatin association and replication fork protection, suggestive of deleteriousness of this VUS variant. Overall, our findings provide more insights into molecular roles of RAD51C in replication fork integrity maintenance and in DSB repair.

Assessing the efficacy and tolerability of PET-guided BrECADD versus eBEACOPP in advanced-stage, classical Hodgkin lymphoma (HD21): a randomised, multicentre, parallel, open-label, phase 3 trial.

BACKGROUND: Intensified systemic chemotherapy has the highest primary cure rate for advanced-stage, classical Hodgkin lymphoma but this comes with a cost of severe and potentially life long, persisting toxicities. With the new regimen of brentuximab vedotin, etoposide, cyclophosphamide, doxorubicin, dacarbazine, and dexamethasone (BrECADD), we aimed to improve the risk-to-benefit ratio of treatment of advanced-stage, classical Hodgkin lymphoma guided by PET after two cycles. METHODS: This randomised, multicentre, parallel, open-label, phase 3 trial was done in 233 trial sites across nine countries. Eligible patients were adults (aged ≤60 years) with newly diagnosed, advanced-stage, classical Hodgkin lymphoma (ie, Ann Arbor stage III/IV, stage II with B symptoms, and either one or both risk factors of large mediastinal mass and extranodal lesions). Patients were randomly assigned (1:1) to four or six cycles (21-day intervals) of escalated doses of etoposide (200 mg/m2 intravenously on days 1-3), doxorubicin (35 mg/m2 intravenously on day 1), and cyclophosphamide (1250 mg/m2 intravenously on day 1), and standard doses of bleomycin (10 mg/m2 intravenously on day 8), vincristine (1·4 mg/m2 intravenously on day 8), procarbazine (100 mg/m2 orally on days 1-7), and prednisone (40 mg/m2 orally on days 1-14; eBEACOPP) or BrECADD, guided by PET after two cycles. Patients and investigators were not masked to treatment assignment. Hierarchical coprimary objectives were to show (1) improved tolerability defined by treatment-related morbidity and (2) non-inferior efficacy defined by progression-free survival with an absolute non-inferiority margin of 6 percentage points of BrECADD compared with eBEACOPP. An additional test of superiority of progression-free survival was to be done if non-inferiority had been established. Analyses were done by intention to treat; the treatment-related morbidity assessment required documentation of at least one chemotherapy cycle. This trial was registered at ClinicalTrials.gov (NCT02661503). FINDINGS: Between July 22, 2016, and Aug 27, 2020, 1500 patients were enrolled, of whom 749 were randomly assigned to BrECADD and 751 to eBEACOPP. 1482 patients were included in the intention-to-treat analysis. The median age of patients was 31 years (IQR 24-42). 838 (56%) of 1482 patients were male and 644 (44%) were female. Most patients were White (1352 [91%] of 1482). Treatment-related morbidity was significantly lower with BrECADD (312 [42%] of 738 patients) than with eBEACOPP (430 [59%] of 732 patients; relative risk 0·72 [95% CI 0·65-0·80]; p<0·0001). At a median follow-up of 48 months, BrECADD improved progression-free survival with a hazard ratio of 0·66 (0·45-0·97; p=0·035); 4-year progression-free survival estimates were 94·3% (95% CI 92·6-96·1) for BrECADD and 90·9% (88·7-93·1) for eBEACOPP. 4-year overall survival rates were 98·6% (97·7-99·5) and 98·2% (97·2-99·3), respectively. INTERPRETATION: BrECADD guided by PET after two cycles is better tolerated and more effective than eBEACOPP in first-line treatment of adult patients with advanced-stage, classical Hodgkin lymphoma. FUNDING: Takeda Oncology.

Brentuximab Vedotin with Chemotherapy in Pediatric High-Risk Hodgkin's Lymphoma.

BACKGROUND: In adults with advanced-stage Hodgkin's lymphoma, the CD30-directed antibody-drug conjugate brentuximab vedotin combined with multiagent chemotherapy has been shown to have greater efficacy, but also more toxic effects, than chemotherapy alone. The efficacy of this targeted therapy approach in children and adolescents with Hodgkin's lymphoma is unclear. METHODS: We conducted an open-label, multicenter, randomized, phase 3 trial involving patients 2 to 21 years of age with previously untreated Hodgkin's lymphoma of stage IIB with bulk tumor or stage IIIB, IVA, or IVB. Patients were assigned to receive five 21-day cycles of brentuximab vedotin with doxorubicin, vincristine, etoposide, prednisone, and cyclophosphamide (brentuximab vedotin group) or the standard pediatric regimen of doxorubicin, bleomycin, vincristine, etoposide, prednisone, and cyclophosphamide (standard-care group). Slow-responding lesions, defined by a score of 4 or 5 (on a 5-point scale, with scores of 1 to 3 indicating rapid-responding lesions), were identified on centrally reviewed positron-emission tomography-computed tomography after two cycles. Involved-site radiation therapy was administered after the fifth cycle of therapy to slow-responding lesions and to large mediastinal adenopathy that was present at diagnosis. The primary end point was event-free survival, defined as the time until disease progression occurred, relapse occurred, a second malignant neoplasm developed, or the patient died. Safety and overall survival were assessed. RESULTS: Of 600 patients who were enrolled across 153 institutions, 587 were eligible. At a median follow-up of 42.1 months (range, 0.1 to 80.9), the 3-year event-free survival was 92.1% (95% confidence interval [CI], 88.4 to 94.7) in the brentuximab vedotin group, as compared with 82.5% (95% CI, 77.4 to 86.5) in the standard-care group (hazard ratio for event or death, 0.41; 95% CI, 0.25 to 0.67; P<0.001). The percentage of patients who received involved-site radiation therapy did not differ substantially between the brentuximab vedotin group and the standard-care group (53.4% and 56.8%, respectively). Toxic effects were similar in the two groups. Overall survival at 3 years was 99.3% (95% CI, 97.3 to 99.8) in the brentuximab vedotin group and 98.5% (95% CI, 96.0 to 99.4) in the standard-care group. CONCLUSIONS: The addition of brentuximab vedotin to standard chemotherapy resulted in superior efficacy, with a 59% lower risk of an event or death, and no increase in the incidence of toxic effects at 3 years. (Funded by the National Institutes of Health and others; AHOD1331 ClinicalTrials.gov number, NCT02166463.).

DNA Damage Response After Treatment of Cycling and Quiescent Cord Blood Hematopoietic Stem Cells With Distinct Genotoxic Noxae.

Hematopoietic stem cells (HSC) from cord blood can be applied as an alternative to bone marrow in transplantation to treat hematological diseases. Umbilical cord blood (UCB) consists of cycling and non-cycling CD34+/CD45low cells needed for long-term and short-term engraftment. After sorting and subsequent in vitro culture, quiescent HSCs enter the cell cycle. This enables the analysis of HSCs in 2 different cell cycle stages and the comparison of their responses to different genotoxic noxae. To analyze different mechanisms of DNA damage induction in cells, 2 different genotoxins were compared: etoposide, a topoisomerase II inhibitor that targets mitosis in the S/G2-phase of the cell cycle and the alkylating nitrosamine N-Nitroso-N-methylurea (MNU), which leads to the formation of methyl DNA adducts resulting in DNA double breaks during DNA replication and persistent mutations. Cycling cells recovered after treatment even with higher concentrations of etoposide (1.5µM/ 5µM/10µM), while sorted cells treated with MNU (0.1mM/0.3mM/0.5mM/1mM/3Mm/ 5mM) recovered after treatment with the lower MNU concentrations whereas high MNU concentrations resulted in apoptosis activation. Quiescent cells were not affected by etoposide treatment showing no damage upon entry into the cell cycle. Treatment with MNU, similarly to the cycling cells, resulted in a dose-dependent cell death. In conclusion, we found that depending on the genotoxic trigger and the cycling status, CD34+cells have distinct responses to DNA damage. Cycling cells employ both DDR and apoptosis mechanisms to prevent damage accumulation. Quiescent cells predominantly undergo apoptosis upon damage, but their cell cycle status protects them from certain genotoxic insults.

Etoposide promotes DNA loop trapping and barrier formation by topoisomerase II.

Etoposide is a broadly employed chemotherapeutic and eukaryotic topoisomerase II poison that stabilizes cleaved DNA intermediates to promote DNA breakage and cytotoxicity. How etoposide perturbs topoisomerase dynamics is not known. Here we investigated the action of etoposide on yeast topoisomerase II, human topoisomerase IIα and human topoisomerase IIß using several sensitive single-molecule detection methods. Unexpectedly, we found that etoposide induces topoisomerase to trap DNA loops, compacting DNA and restructuring DNA topology. Loop trapping occurs after ATP hydrolysis but before strand ejection from the enzyme. Although etoposide decreases the innate stability of topoisomerase dimers, it increases the ability of the enzyme to act as a stable roadblock. Interestingly, the three topoisomerases show similar etoposide-mediated resistance to dimer separation and sliding along DNA but different abilities to compact DNA and chirally relax DNA supercoils. These data provide unique mechanistic insights into the functional consequences of etoposide on topoisomerase II dynamics.

SUMO-Targeted DNA Translocase Rrp2 Protects the Genome from Top2-Induced DNA Damage.

The action of DNA topoisomerase II (Top2) creates transient DNA breaks that are normally concealed inside Top2-DNA covalent complexes. Top2 poisons, including ubiquitously present natural compounds and clinically used anti-cancer drugs, trap Top2-DNA complexes. Here, we show that cells actively prevent Top2 degradation to avoid the exposure of concealed DNA breaks. A genome-wide screen revealed that fission yeast cells lacking Rrp2, an Snf2-family DNA translocase, are strongly sensitive to Top2 poisons. Loss of Rrp2 enhances SUMOylation-dependent ubiquitination and degradation of Top2, which in turn increases DNA damage at sites where Top2-DNA complexes are trapped. Rrp2 possesses SUMO-binding ability and prevents excessive Top2 degradation by competing against the SUMO-targeted ubiquitin ligase (STUbL) for SUMO chain binding and by displacing SUMOylated Top2 from DNA. The budding yeast homolog of Rrp2, Uls1, plays a similar role, indicating that this genome protection mechanism is widely employed, a finding with implications for cancer treatment.

Circumvention of Topoisomerase IIα Intron 19 Intronic Polyadenylation in Acquired Etoposide-Resistant Human Leukemia K562 Cells.

DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is an essential enzyme for proper chromosome dysjunction by producing transient DNA double-stranded breaks and is an important target for DNA damage-stabilizing anticancer agents, such as etoposide. Therapeutic effects of TOP2α poisons can be limited due to acquired drug resistance. We previously demonstrated decreased TOP2α/170 levels in an etoposide-resistant human leukemia K562 subline, designated K/VP.5, accompanied by increased expression of a C-terminal truncated TOP2α isoform (90 kDa; TOP2α/90), which heterodimerized with TOP2α/170 and was a determinant of resistance by exhibiting dominant-negative effects against etoposide activity. Based on 3'-rapid amplification of cDNA ends, we confirmed TOP2α/90 as the translation product of a TOP2α mRNA in which a cryptic polyadenylation site (PAS) harbored in intron 19 (I19) was used. In this report, we investigated whether the resultant intronic polyadenylation (IPA) would be attenuated by blocking or mutating the I19 PAS, thereby circumventing acquired drug resistance. An antisense morpholino oligonucleotide was used to hybridize/block the PAS in TOP2α pre-mRNA in K/VP.5 cells, resulting in decreased TOP2α/90 mRNA/protein levels in K/VP.5 cells and partially circumventing drug resistance. Subsequently, CRISPR/CRISPR-associated protein 9 with homology-directed repair was used to mutate the cryptic I19 PAS (AATAAA→ACCCAA) to prevent IPA. Gene-edited clones exhibited increased TOP2α/170 and decreased TOP2α/90 mRNA/protein and demonstrated restored sensitivity to etoposide and other TOP2α-targeted drugs. Together, results indicated that blocking/mutating a cryptic I19 PAS in K/VP.5 cells reduced IPA and restored sensitivity to TOP2α-targeting drugs. SIGNIFICANCE STATEMENT: The results presented in this study indicate that CRISPR/CRISPR-associated protein 9 gene editing of a cryptic polyadenylation site (PAS) within I19 of the TOP2α gene results in the reversal of acquired resistance to etoposide and other TOP2-targeted drugs. An antisense morpholino oligonucleotide targeting the PAS also partially circumvented resistance.

Identification of a druggable pocket of the calcium-activated chloride channel TMEM16A in its open state.

The calcium-activated chloride channel TMEM16A is a potential drug target to treat hypertension, secretory diarrhea, and several cancers. However, all reported TMEM16A structures are either closed or desensitized, and direct inhibition of the open state by drug molecules lacks a reliable structural basis. Therefore, revealing the druggable pocket of TMEM16A exposed in the open state is important for understanding protein-ligand interactions and facilitating rational drug design. Here, we reconstructed the calcium-activated open conformation of TMEM16A using an enhanced sampling algorithm and segmental modeling. Furthermore, we identified an open-state druggable pocket and screened a potent TMEM16A inhibitor, etoposide, which is a derivative of a traditional herbal monomer. Molecular simulations and site-directed mutagenesis showed that etoposide binds to the open state of TMEM16A, thereby blocking the ion conductance pore of the channel. Finally, we demonstrated that etoposide can target TMEM16A to inhibit the proliferation of prostate cancer PC-3 cells. Together, these findings provide a deep understanding of the TMEM16A open state at an atomic level and identify pockets for the design of novel inhibitors with broad applications in chloride channel biology, biophysics, and medicinal chemistry.

Endoplasmic reticulum stress-induced senescence in human lung fibroblasts.

Loss of proteostasis and cellular senescence have been previously established as characteristics of aging; however, their interaction in the context of lung aging and potential contributions to aging-associated lung remodeling remains understudied. In this study, we aimed to characterize endoplasmic reticulum (ER) stress response, cellular senescence, and their interaction in relation to extracellular matrix (ECM) production in lung fibroblasts from young (25-45 yr) and old (>60 yr) humans. Fibroblasts from young and old patients without significant preexisting lung disease were exposed to vehicle, MG132, etoposide, or salubrinal. Afterward, cells and cell lysates or supernatants were analyzed for ER stress, cellular senescence, and ECM changes using protein analysis, proliferation assay, and senescence-associated beta-galactosidase (SA-ß-Gal) staining. At baseline, fibroblasts from aging individuals showed increased levels of ER stress (ATF6 and PERK), senescence (p21 and McL-1), and ECM marker (COL1A1) compared to those from young individuals. Upon ER stress induction and etoposide exposure, fibroblasts showed an increase in senescence (SA-ß-Gal, p21, and Cav-1), ER stress (PERK), and ECM markers (COL1A1 and LUM) compared to vehicle. Additionally, IL-6 and IL-8 levels were increased in the supernatants of MG132- and etoposide-treated fibroblasts, respectively. Finally, the ER stress inhibitor salubrinal decreased the expression of p21 compared to vehicle and MG132 treatments; however, salubrinal inhibited COL1A1 but not p21 expression in MG132-treated fibroblasts. Our study suggests that ER stress response plays an important role in establishment and maintenance of a senescence phenotype in lung fibroblasts and therefore contributes to altered remodeling in the aging lung.NEW & NOTEWORTHY The current study establishes functional links between endoplasmic reticulum (ER) stress and cellular senescence per se in the specific context of aging human lung fibroblasts. Recognizing that the process of aging per se is complex, modulated by the myriad of lifelong and environmental exposures, it is striking to note that chronic ER stress may play a crucial role in the establishment and maintenance of cellular senescence in lung fibroblasts.

Interim PET-guided ABVD or ABVD/escalated BEACOPP for newly diagnosed advanced-stage classic Hodgkin lymphoma (JCOG1305).

This single-arm confirmatory study (JCOG1305) aimed to evaluate the utility of interim positron emission tomography (iPET)-guided therapy for newly diagnosed advanced-stage classic Hodgkin lymphoma (cHL). Patients aged 16-60 years with cHL received two cycles of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) and then underwent an iPET scan (PET2), which was centrally reviewed using a five-point Deauville scale. PET2-negative patients continued an additional four cycles of ABVD, whereas PET2-positive patients switched to six cycles of escalated bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone (eBEACOPP). The co-primary endpoints were 2-year progression-free survival (PFS) among all eligible and PET2-positive patients. Ninety-three patients were enrolled between January 2016 and December 2019. One patient was ineligible because of a diagnostic error. The median age of the 92 eligible patients was 35 (interquartile range, 28-48) years. Forty (43%) patients had stage III disease, and 43 (47%) had stage IV disease. The remaining nine (10%) patients had stage IIB disease with risk factors. Nineteen PET2-positive (21%) patients received eBEACOPP, 18 completed six cycles of eBEACOPP, 73 PET2-negative (79%) patients continued ABVD, and 70 completed an additional four cycles of ABVD. With a median follow-up period of 41.1 months, the 2-year PFS of 92 eligible patients and 19 PET2-positive patients were 84.8% (80% confidence interval [CI], 79.2-88.9) and 84.2% (80% CI, 69.7-92.1), respectively. Both primary endpoints were met at the prespecified threshold. This study demonstrates that iPET-guided therapy is a useful treatment option for younger patients with newly diagnosed advanced-stage cHL. Registration number: jRCTs031180218.