ERBB and P-glycoprotein inhibitors break resistance in relapsed neuroblastoma through P-glycoprotein

Neuroblastoma is a pediatric tumor of the sympathetic nervous system that most commonly affects infants and young children. It is a disease with very variable outcomes, which range from spontaneous regression to aggressive disease. Particularly problematic are high-risk cases after relapse, with only 10% of patients surviving for longer than five years and chemotherapy resistance making treatment difficult. In this thesis, I examined differences between primary and relapsed patient gene expression data and between chemotherapy-sensitive and chemotherapy-resistant neuroblastoma cell lines to find a vulnerability that can be targeted to overcome chemotherapy resistance and lead to cell death. I further investigated which type of cell death was induced. My analysis of gene expression data from primary and relapsed neuroblastoma patients suggested that the ERBB family of receptor tyrosine kinases, particularly ERBB4, plays a role in relapsed high-risk neuroblastomas. The ERBB family is crucial in development and is well known for its link to cancer. Furthermore, I functionally examined the resistance breaking effect of 15 clinically relevant drugs on a neuroblastoma cell line that was treated to be resistant to the standard-of-care chemotherapeutic vincristine (VCR). Tariquidar, an inhibitor of P-glycoprotein (P-gp), and afatinib, a FDA approved inhibitor of the ERBB family, were the two most efficient drugs in breaking resistance in this screen. P-gp/ABCB1 is a transmembrane transporter that very efficiently eliminates drugs and other xenobiotics from the cell and has long been recognized for its contribution to chemotherapy resistance. Analysing gene expression datasets of more than 50 different neuroblastoma cell lines (primary and relapsed) and more than 160 neuroblastoma patient samples from the pediatric precision medicine platform INFORM (Individualized Therapy For Relapsed Malignancies in Childhood) confirmed a crucial role of P-gp in neuroblastoma resistance at relapse. The ERBB family appeared to play a minor part. I mechanistically investigated chemotherapy resistance through four pairs of vincristine- sensitive/resistant neuroblastoma sublines - each pair with the same genetic background - and two additional high-risk neuroblastoma cell lines. I effectively overcame resistance by the addition of the pan-ERBB family inhibitors afatinib and lapatinib, as well as the P-gp inhibitors tariquidar and verapamil, to VCR treatment, which synergistically reduced viability. Functional analyses of the ERBB downstream pathways, as well as ERBB4 knock-down, showed the resistance breaking effect of afatinib to be unrelated to ERBB signaling and suggested an off-target effect on P-gp. ABCB1 knock-down and analysis of transporter function confirmed that resistance was mediated through P-gp. My analysis of programmed cell death, in particular apoptosis and ferroptosis, in neuroblastoma cell lines showed that apoptosis was consistently induced, while ferroptosis contributed to cell death in the IMR-32 cell line. The combination treatments of VCR with afatinib or tariquidar in VCR-sensitive and VCR-resistant cell lines led to cell death by apoptosis. In summary, this study showed that P-gp is an important player in chemotherapy resistance in high-risk, relapsed neuroblastoma. ERBB4, although upregulated in individual cases or cell lines, did not contribute to resistance, and the sensitizing effect of the ERBB family inhibitor afatinib was due to an off-target effect on P-gp. I further showed that both apoptosis and ferroptosis are induced in neuroblastoma cell lines, with apoptosis being the dominant mechanism of cell death when chemotherapy resistance was broken

ERBB and P‐glycoprotein inhibitors break resistance in relapsed neuroblastoma models through P‐glycoprotein

Chemotherapy resistance is a persistent clinical problem in relapsed high-risk neuroblastomas. We tested a panel of 15 drugs for sensitization of neuroblastoma cells to the conventional chemotherapeutic vincristine, identifying tariquidar, an inhibitor of the transmembrane pump P-glycoprotein (P-gp/ABCB1), and the ERBB family inhibitor afatinib as the top resistance breakers. Both compounds were efficient in sensitizing neuroblastoma cells to vincristine in trypan blue exclusion assays and in inducing apoptotic cell death. The evaluation of ERBB signaling revealed no functional inhibition, i.e., dephosphorylation of the downstream pathways upon afatinib treatment but direct off-target interference with P-gp function. Depletion of ABCB1, but not ERRB4, sensitized cells to vincristine treatment. P-gp inhibition substantially broke vincristine resistance in vitro and in vivo (zebrafish embryo xenograft). The analysis of gene expression datasets of more than 50 different neuroblastoma cell lines (primary and relapsed) and more than 160 neuroblastoma patient samples from the pediatric precision medicine platform INFORM (Individualized Therapy For Relapsed Malignancies in Childhood) confirmed a pivotal role of P-gp specifically in neuroblastoma resistance at relapse, while the ERBB family appears to play a minor part

За кадры. 1974. № 50 (1806)

Кто он, абитуриент-74?На пороге вузаАвгуст - месяц надежд / А. БатуринЗачислены студентами / Р. Горская"Хочу быть геологом" / В. АлександровПусть мечты сбываются / В. ЛебедевОснованная Обручевым / В. Васильева, Н. Князюк, Л. РубанС оценкой "пять" / В. КарабатовКормить как следует / В. ГуськовАбитуриенты. Страницы истории / И. Т. Лозовский"Волшебная палочка" / Н. ЛевшуковНа прополке / В. Ивано

Combining APR-246 and HDAC-Inhibitors: A Novel Targeted Treatment Option for Neuroblastoma

APR-246 (Eprenetapopt/PRIMA-1Met) is a very potent anti-cancer drug in clinical trials and was initially developed as a p53 refolding agent. As an alternative mode of action, the elevation of reactive oxygen species (ROS) has been proposed. Through an in silico analysis, we investigated the responses of approximately 800 cancer cell lines (50 entities; Cancer Therapeutics Response Portal, CTRP) to APR-246 treatment. In particular, neuroblastoma, lymphoma and acute lymphocytic leukemia cells were highly responsive. With gene expression data from the Cancer Cell Line Encyclopedia (CCLE; n = 883) and patient samples (n = 1643) from the INFORM registry study, we confirmed that these entities express low levels of SLC7A11, a previously described predictive biomarker for APR-246 responsiveness. Combining the CTRP drug response data with the respective CCLE gene expression profiles, we defined a novel gene signature, predicting the effectiveness of APR-246 treatment with a sensitivity of 90% and a specificity of 94%. We confirmed the predicted APR-246 sensitivity in 8/10 cell lines and in ex vivo cultures of patient samples. Moreover, the combination of ROS detoxification-impeding APR-246 with approved HDAC-inhibitors, known to elevate ROS, substantially increased APR-246 sensitivity in cell cultures and in vivo in two zebrafish neuroblastoma xenograft models. These data provide evidence that APR-246, in combination with HDAC-inhibitors, displays a novel potent targeted treatment option for neuroblastoma patients

Adjuvant nivolumab plus ipilimumab or nivolumab monotherapy versus placebo in patients with resected stage IV melanoma with no evidence of disease (IMMUNED): a randomised, double-blind, placebo-controlled, phase 2 trial

Background Nivolumab and ipilimumab, alone or in combination, are widely used immunotherapeutic treatment options for patients with advanced-ie, unresectable or metastatic-melanoma. This criterion, however, excludes patients with stage IV melanoma with no evidence of disease. We therefore aimed to evaluate the safety and efficacy of adjuvant nivolumab plus ipilimumab or nivolumab monotherapy versus a placebo in this patient population. Methods We did a randomised, double-blind, placebo-controlled, phase 2 trial in 20 German academic medical centres. Eligible patients were aged 18-80 years with stage IV melanoma with no evidence of disease after surgery or radiotherapy. Key exclusion criteria included uveal or mucosal melanoma, previous therapy with checkpoint inhibitors, and any previous immunosuppressive therapy within the 30 days before study drug administration. Eligible patients were randomly assigned (1:1:1), using a central, interactive, online system, to the nivolumab plus ipilimumab group (1 mg/kg of intravenous nivolumab every 3 weeks plus 3 mg/kg of intravenous ipilimumab every 3 weeks for four doses, followed by 3 mg/kg of nivolumab every 2 weeks), nivolumab monotherapy group (3 mg/kg of intravenous nivolumab every 2 weeks plus ipilimumab-matching placebo during weeks 1-12), or double-matching placebo group. The primary endpoint was the recurrence-free survival in the intention-to-treat population. The results presented in this report reflect the prespecified interim analysis of recurrence-free survival after 90 events had been reported. This study is registered with ClinicalTrials.gov, NCT02523313, and is ongoing. Findings Between Sept 2, 2015, and Nov 20, 2018, 167 patients were randomly assigned to receive nivolumab plus ipilimumab (n=56), nivolumab (n=59), or placebo (n=52). As of July 2, 2019, at a median follow-up of 28.4 months (IQR 17.7-36.8), median recurrence-free survival was not reached in the nivolumab plus ipilimumab group, whereas median recurrence-free survival was 12.4 months (95% CI 5.3-33.3) in the nivolumab group and 6.4 months (3.3-9.6) in the placebo group. The hazard ratio for recurrence for the nivolumab plus ipilimumab group versus placebo group was 0.23 (97.5% CI 0.12-0.45; p<0.0001), and for the nivolumab group versus placebo group was 0.56 (0.33-0.94; p=0.011). In the nivolumab plus ipilimumab group, recurrence-free survival at 1 year was 75% (95% CI 61.0-84.9) and at 2 years was 70% (55.1-81.0); in the nivolumab group, 1-year recurrence-free survival was 52% (38.1-63.9) and at 2 years was 42% (28.6-54.5); and in the placebo group, this rate was 32% (19.8-45.3) at 1 year and 14% (5.9-25.7) at 2 years. Treatment-related grade 3-4 adverse events were reported in 71% (95% CI 57-82) of patients in the nivolumab plus ipilimumab group and in 27% (16-40) of those in the nivolumab group. Treatment-related adverse events of any grade led to treatment discontinuation in 34 (62%) of 55 patients in the nivolumab plus ipilimumab group and seven (13%) of 56 in the nivolumab group. Three deaths from adverse events were reported but were considered unrelated to the study treatment. Interpretation Adjuvant therapy with nivolumab alone or in combination with ipilimumab increased recurrencefree survival significantly compared with placebo in patients with stage IV melanoma with no evidence of disease. The rates of grade 3-4 treatment-related adverse events in both active treatment groups were higher than the rates reported in previous pivotal trials done in advanced melanoma with measurable disease. Copyright (c) 2020 Elsevier Ltd. All rights reserved