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Cancers that appear pathologically similar often respond differently to the same drug regimens. Methods to better match patients to drugs are in high demand. We demonstrate a promising approach to identify robust molecular markers for targeted treatment of acute myeloid leukemia (AML) by introducing: data from 30 AML patients including genome-wide gene expression profiles and in vitro sensitivity to 160 chemotherapy drugs, a computational method to identify reliable gene expression markers for drug sensitivity by incorporating multi-omic prior information relevant to each gene’s potential to drive cancer. We show that our method outperforms several state-of-the-art approaches in identifying molecular markers replicated in validation data and predicting drug sensitivity accurately. Finally, we identify SMARCA4 as a marker and driver of sensitivity to topoisomerase II inhibitors, mitoxantrone, and etoposide, in AML by showing that cell lines transduced to have high  SMARCA4 expression reveal dramatically increased sensitivity to these agents. Identification of markers of drug response is essential for precision therapy. Here the authors introduce an algorithm that uses prior information about each gene’s importance in AML to identify the most predictive gene-drug associations from transcriptome and drug response data from 30 AML samples.

Type II topoisomerases (TOP2s) resolve the topological problems of DNA by transiently cleaving both strands of a DNA duplex to form a cleavage complex through which another DNA segment can be transported. Several widely prescribed anticancer drugs increase the population of TOP2 cleavage complex, which leads to TOP2-mediated chromosome DNA breakage and death of cancer cells. We present the crystal structure of a large fragment of human TOP2β complexed to DNA and to the anticancer drug etoposide to reveal structural details of drug-induced stabilization of a cleavage complex. The interplay between the protein, the DNA, and the drug explains the structure-activity relations of etoposide derivatives and the molecular basis of drug-resistant mutations. The analysis of protein-drug interactions provides information applicable for developing an isoform-specific TOP2-targeting strategy.

DNA topoisomerase II inhibitors are a major class of cancer chemotherapeutics, which are thought to eliminate cancer cells by inducing DNA double-strand breaks. Here we identify a novel activity for the anthracycline class of DNA topoisomerase II inhibitors: histone eviction from open chromosomal areas. We show that anthracyclines promote histone eviction irrespective of their ability to induce DNA double-strand breaks. The histone variant H2AX, which is a key component of the DNA damage response, is also evicted by anthracyclines, and H2AX eviction is associated with attenuated DNA repair. Histone eviction deregulates the transcriptome in cancer cells and organs such as the heart, and can drive apoptosis of topoisomerase-negative acute myeloid leukaemia blasts in patients. We define a novel mechanism of action of anthracycline anticancer drugs doxorubicin and daunorubicin on chromatin biology, with important consequences for DNA damage responses, epigenetics, transcription, side effects and cancer therapy. Anthracycline-based drugs can kill cancer cells by inhibiting topoisomerase II and promoting DNA double-strand breaks. Pang et al . show that anthracyclines also induce eviction of histones from open chromatin regions and, in doing so, modulate DNA repair and apoptosis in human cancer cells.

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. Last, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells.

Neuroblastoma (NB) is the most common cancer in infancy and most frequent cause of death from extracranial solid tumors in children. Ornithine decarboxylase (ODC) expression is an independent indicator of poor prognosis in NB patients. This study investigated safety, response, pharmacokinetics, genetic and metabolic factors associated with ODC in a clinical trial of the ODC inhibitor difluoromethylornithine (DFMO) ± etoposide for patients with relapsed or refractory NB. Twenty-one patients participated in a phase I study of daily oral DFMO alone for three weeks, followed by additional three-week cycles of DFMO plus daily oral etoposide. No dose limiting toxicities (DLTs) were identified in patients taking doses of DFMO between 500-1500 mg/m2 orally twice a day. DFMO pharmacokinetics, single nucleotide polymorphisms (SNPs) in the ODC gene and urinary levels of substrates for the tissue polyamine exporter were measured. Urinary polyamine levels varied among patients at baseline. Patients with the minor T-allele at rs2302616 of the ODC gene had higher baseline levels (p=0.02) of, and larger decreases in, total urinary polyamines during the first cycle of DFMO therapy (p=0.003) and had median progression free survival (PFS) that was over three times longer, compared to patients with the major G allele at this locus although this last result was not statistically significant (p=0.07). Six of 18 evaluable patients were progression free during the trial period with three patients continuing progression free at 663, 1559 and 1573 days after initiating treatment. Median progression-free survival was less among patients having increased urinary polyamines, especially diacetylspermine, although this result was not statistically significant (p=0.056). DFMO doses of 500-1500 mg/m2/day are safe and well tolerated in children with relapsed NB. Children with the minor T allele at rs2302616 of the ODC gene with relapsed or refractory NB had higher levels of urinary polyamine markers and responded better to therapy containing DFMO, compared to those with the major G allele at this locus. These findings suggest that this patient subset may display dependence on polyamines and be uniquely susceptible to therapies targeting this pathway. Clinicaltrials.gov NCT#01059071.

Liposomes have been well established as an effective drug delivery system, due to simplicity of their preparation and unique characteristics. However conventional liposomes are unsuitable for the on-demand content release, which limits their therapeutic utility. Here we report X-ray-triggerable liposomes incorporating gold nanoparticles and photosensitizer verteporfin. The 6 MeV X-ray radiation induces verteporfin to produce singlet oxygen, which destabilises the liposomal membrane and causes the release of cargos from the liposomal cavity. This triggering strategy is demonstrated by the efficiency of gene silencing in vitro and increased effectiveness of chemotherapy in vivo. Our work indicates the feasibility of a combinatorial treatment and possible synergistic effects in the course of standard radiotherapy combined with chemotherapy delivered via X-ray-triggered liposomes. Importantly, our X-ray-mediated liposome release strategy offers prospects for deep tissue photodynamic therapy, by removing its depth limitation. X-ray radiation has excellent tissue penetration depth, making it a useful trigger for deep tissue cancer therapy. Here, the authors design X-ray triggered drug/gene-loaded liposomes by embedding photosensitizers and gold nanoparticles in the liposome bilayer, and demonstrate their efficacy in cancer and gene therapy.

Abstract Background Effective and tolerable treatment for patients with platinum-resistant recurrent ovarian cancer remains a great challenge in clinical practice. This study aimed to evaluate the efficacy and safety of envafolimab, the first subcutaneously administered programmed death ligand 1 (PD-L1) inhibitor, combined with lenvatinib and etoposide in patients with platinum-resistant recurrent ovarian cancer. Methods This was an open-label, single-arm, phase 2 trial (ENLEN-OC-001). Patients with platinum-resistant recurrent ovarian cancer were eligible for inclusion who were administered envafolimab subcutaneously on day 1 and lenvatinib and etoposide orally on days 1-14, with 21 days as a cycle. After 6-10 cycles, envafolimab and lenvatinib were taken as maintenance therapy until intolerable toxicity, disease progression, withdrawal of consent, or finishing 24 months of treatment. The primary endpoint was objective response rate (ORR), and the secondary endpoints included disease control rate (DCR), progression-free survival (PFS), overall survival (OS), and safety. Results Of the screened 28 patients, 21 were included, with 18 being assessed for the efficacy and safety. The ORR was 44.4% (95% CI 21.5%-69.2%), and the DCR was 83.3% (95% CI 58.6%-96.4%). The median PFS was 10.2 months (95% CI 5.6-not applicable [NA]), and the median OS was 21.3 months (95% CI 6.8-NA). The most common grade 3/4 adverse events were leukopenia (27.8%) and thrombocytopenia (16.7%). No serious adverse events or treatment-related deaths were reported. No changes were observed in the depression, anxiety, and quality of life following treatment. Conclusion Envafolimab combined with lenvatinib and etoposide showed promising efficacy and tolerable safety for patients with platinum-resistant recurrent ovarian cancer. ClinicalTrials.gov identifier NCT05422183

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 IIβ 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. Using single-molecule biophysics methods, Le et al. discovered that etoposide, a chemotherapeutic poison of topoisomerase II (topo II), promotes topo II to compact DNA, trap DNA loops and pause DNA supercoiling relaxation, thus converting topo II into a strong roadblock to DNA processing.

Nanoparticle technology in cancer chemotherapy is a promising approach to enhance active ingredient pharmacology and pharmacodynamics. Indeed, drug nanoparticles display various assets such as extended blood lifespan, high drug loading and reduced cytotoxicity leading to better drug compliance. In this context, organic nanocrystal suspensions for pharmaceutical use have been developed in the past ten years. Nanocrystals offer new possibilities by combining the nanoformulation features with the properties of solid dispersed therapeutic ingredients including (i) high loading of the active ingredient, (ii) its bioavailability improvement, and (iii) reduced drug systemic cytotoxicity. However, surprisingly, no antitumoral drug has been marketed as a nanocrystal suspension until now. Etoposide, which is largely used as an anti-cancerous agent against testicular, ovarian, small cell lung, colon and breast cancer in its liquid dosage form, has been selected to develop injectable nanocrystal suspensions designed to be transferred to the clinic. The aim of the present work is to provide optimized formulations for nanostructured etoposide solutions and validate by means of in vitro and in vivo evaluations the efficiency of this multiphase system. Indeed, the etoposide formulated as a nanosuspension by a bottom-up approach showed higher blood life span, reduced tumor growth and higher tolerance in a murine carcinoma cancer model. The results obtained are promising for future clinical evaluation of these etoposide nanosuspensions.

Background Tumor associated macrophages (TAMs) are present in high density in solid tumors. TAMs share many characteristics with alternatively activated macrophages, also called M2. They have been shown to favor tumor development and a role in chemoresistance has also been suggested. Here, we investigated the effects of M2 in comparison to M1 macrophages on cancer cell sensitivity to etoposide. Methods We set up a model of macrophage polarization, starting from THP-1 monocytes differentiated into macrophages using PMA (Phorbol 12-myristate 13-acetate). Once differentiated (M0 macrophages), they were incubated with IL-4 and IL-13 in order to obtain M2 polarized macrophages or with IFN-gamma and LPS for classical macrophage activation (M1). To mimic the communication between cancer cells and TAMs, M0, M1 or M2 macrophages and HepG2 or A549 cancer cells were co-cultured during respectively 16 (HepG2) or 24 (A549) hours, before etoposide exposure for 24 (HepG2) or 16 (A549) hours. After the incubation, the impact of etoposide on macrophage polarization was studied and cancer cell apoptosis was assessed by western-blot for cleaved caspase-3 and cleaved PARP-1 protein, caspase activity assay and FACS analysis of Annexin V and PI staining. Results mRNA and protein expression of M1 and M2 markers confirmed the polarization of THP-1-derived macrophages, which provide a new, easy and well-characterized model of polarized human macrophages. Etoposide-induced cancer cell apoptosis was markedly reduced in the presence of THP-1 M2 macrophages, while apoptosis was increased in cells co-cultured with M1 macrophages. On the other hand, etoposide did not influence M1 or M2 polarization. Conclusions These results evidence for the first time a clear protective effect of M2 on the contrary to M1 macrophages on etoposide-induced cancer cell apoptosis.