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Cytochrome P450 1B1 (CYP1B1) is found in tumor tissue and is suspected to play a role in oncogenesis and drug resistance. CYP1B1 gene polymorphisms have been associated with the risk of developing lung and other cancers. They may be associated with tumor response to anticancer drugs. We have determined 4 frequent nonsynonymous gene polymorphisms of CYP1B1 in the human tumor cell lines panels of the National Cancer Institute (NCI) and the Japanese Foundation for Cancer Research (JFCR): rs10012 (R48G), rs1056827 (A119S), rs1056836 (L432V), and rs1800440 (N453S). Numerous anticancer drugs have been tested against these panels that offer the opportunity to detect associations between gene polymorphisms and drug sensitivity. CYP1B1 single nucleotide polymorphisms were in marked linkage disequilibrium. The L432V allelic variants were significantly associated with reduced sensitivity to DNA-interacting anticancer agents, alkylators, camptothecins, topoisomerase II inhibitors, and some antimetabolites. For instance, in the NCI panel, cell lines homozygous for the V432 allele were globally 2-fold resistant to alkylating agents (P = 5 × 10−10) and 4.5-fold to camptothecins (P = 6.6 × 10−9) than cell lines homozygous for the L432 allele. Similar features were exhibited by the JFCR panel. Cell lines homozygous for the V432 allele were globally less sensitive to DNA-interfering drugs than cell lines having at least 1 common allele. There was no significant association between mRNA expression of CYP1B1 and CYP1B1 genotype, and no significant association between CYP1B1 mRNA expression and drug cytotoxicity. These observations open the way to clinical studies exploring the role of CYP1B1 gene polymorphisms for predicting tumor sensitivity to chemotherapy. Mol Cancer Ther; 9(12); 3315–21. ©2010 AACR.

Supplementary Data from Cytochrome P450 1B1 Gene Polymorphisms as Predictors of Anticancer Drug Activity: Studies with In vitro Models

Supplementary Material 1: Materials and Methods, and Supplementary data

Background: ATR plays a crucial role in maintaining genomic integrity by responding to a large spectrum of DNA damage, including double strand breaks (DSBs) that interfere with replication. We have previously demo started that ATR inhibition is associated with anti-tumor activity in pre-clinical models of soft-tissue sarcomas (STS) (Laroche et al., 2020). We decided to investigate the mechanisms of resistance to ATR inhibition in sarcomas. Methods: We used a CRISPRa was to identify genes that mediate resistance to AZD6738 in STS cells. The anti-tumor activity of AZD6738 (ATR inhibitor) and AZD0156 (ATM inhibitor) was investigated in a panel for 10 STS cell lines and two patient-derived xenografts (PDX) established at Institut Bergonié (Bordeaux, France). Results: We used a CRISPR approach in a STS cell line resistant to AZD6738 and we made a negative-selection screen, to identify genes whose silencing conferred sensitivity to low doses of ATR inhibition. Among these genes, we identified THRAP3 which encodes a protein phosphorylated in response to DNA damage and primarily by ATR. THRAP3 is involved in the maturation and further export of transcripts encoding the ATM and its depletion results in deficient processing of transcripts encoding the ATM kinase. We validate the result of our CRISPR analysis by demonstrating that selective THRAP3 inhibition reduce significantly ATRi IC50. Given the role of role of THRAP3 on the regulation of ATM activity, we decided to investigate the potential synergy between ATR and ATM inhibition in vivo and in vitro. AZD6738 and AZD0156 displayed a synergistic or additive effect in above all the STS cell lines tested. We noted that ATRi treatment reduced phosphorylation of the ATR kinase target CHK1 but increased ATM phosphorylation, suggesting that ATM pathway takes over for DNA Damage Response and cells survival. In contrast, both ATR and ATM pathway were inhibited when AZD6738 and AZD0156 were used in combination, as shown by reduction of CHK1 and ATM phosphorylation. We validated our in vitro results in an in vivo model of liposarcoma xenograft in which AZD6738 and AZD0156 had synergistic anti-tumor activity. Conclusion: Our study demonstrates synthetically lethal interactions between ATM and ATR in vitro and in vivo. This combination deserves further investigaitons in sarcomas. Citation Format: Mariella Spalato, Audrey Laroche-Clary, Vanessa Chaire, Antoine Italiano. Genome-wide CRISPR/Cas9 library screening identified THRAP3 as a critical driver for resistance to ATR inhibitor in sarcomas revealing synthetic lethality with ATM inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1509.

International audience; Background: Undifferentiated pleomorphic sarcoma (UPS) is the most frequent, aggressive and less-characterized sarcoma subtype. This study aims to assess UPS molecular characteristics and identify specific therapeutic targets. Methods: High-throughput technologies encompassing immunohistochemistry, RNA-sequencing, whole exomesequencing, mass spectrometry, as well as radiomics were used to characterize three independent cohorts of 110, 25 and 41 UPS selected after histological review performed by an expert pathologist. Correlations were made with clinical outcome. Cell lines and xenografts were derived from human samples for functional experiments. Findings: CD8 positive cell density was independently associated with metastatic behavior and prognosis. RNA-sequencing identified two main groups: the group A, enriched in genes involved in development and stemness, including FGFR2, and the group B, strongly enriched in genes involved in immunity. Immune infiltrate patterns on tumor samples were highly predictive of gene expression classification, leading to call the group B 'immune-high' and the group A 'immune-low'. This molecular classification and its prognostic impact were confirmed on an independent cohort of UPS from TCGA. Copy numbers alterations were significantly more frequent in immune-low UPS. Proteomic analysis identified two main proteomic groups that highly correlated with the two main transcriptomic groups. A set of nine radiomic features from conventional MRI sequences provided the basis for a radiomics signature that could select immune-high UPS on their pre-therapeutic imaging. Finally, in vitro and in vivo anti-tumor activity of FGFR inhibitor JNJ-42756493 was selectively shown in cell lines and patient-derived xenograft models derived from immune-low UPS. Interpretation: Two main disease entities of UPS, with distinct immune phenotypes, prognosis, molecular features and MRI textures, as well as differential sensitivity to specific anticancer agents were identified. Immune-high UPS may be the best candidates for immune checkpoint inhibitors, whereas this study provides rational for assessing FGFR inhibition in immune-low UPS. Funding: This work was partly founded by a grant from La Ligue.

We aimed to evaluate the therapeutic potential of the pan-FGFR inhibitor erdafitinib to treat dedifferentiated liposarcoma (DDLPS). FGFR expression and their prognostic value were assessed in a series of 694 samples of well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS). The effect of erdafitinib&mdash, alone or in combination with other antagonists&mdash, on tumorigenicity was evaluated in vitro and in vivo. We detected overexpression of FGFR1 and/or FGFR4 in a subset of WDLPS and DDLPS and demonstrated correlation of this expression with poor prognosis. Erdafitinib treatment reduced cell viability, inducing apoptosis and strong inhibition of the ERK1/2 pathway. Combining erdafitinib with the MDM2 antagonist RG7388 exerted a synergistic effect on viability, apoptosis, and clonogenicity in one WDLPS and two DDLPS cell lines. Efficacy of this combination was confirmed in vivo on a DDLPS xenograft. Importantly, we report the efficacy of erdafitinib in one patient with refractory DDLPS showing disease stabilization for 12 weeks. We provide evidence that the FGFR pathway has therapeutic potential for a subset of DDLPS and that an FGFR1/FGFR4 expression might constitute a powerful biomarker to select patients for FGFR inhibitor clinical trials. In addition, we show that combining erdafitinib with RG7388 is a promising strategy for patients with DDLPS that deserves further investigation in the clinical setting.

Only few drugs have shown activity in patients with advanced soft-tissue and the median overall survival is only 18 months. Alterations of genes involved in the DNA damage repair pathway have been associated with sarcoma risk and prognosis. ATR plays a crucial role in maintaining genomic integrity by responding to a large spectrum of DNA damage, including double strand breaks (DSBs) that interfere with replication. The objective of this study is to evaluate the pre-clinical activity of ATR inhibition in soft tissue sarcomas (STS). We explored the ability of the ATR inhibitor, VE-822, to prevent chemotherapy-induced intra-S-phase checkpoint activation and evaluated the antitumor potential of this combination in vitro and in vivo in STS cell lines and in a patient-derived xenograft model. The combination of VE-822 and gemcitabine in vitro was synergistic, inhibited cell proliferation, induced apoptosis, and accumulated in the S phase of the cell cycle with higher efficacy than either single agent alone. The combination also resulted in enhanced γH2AX intranuclear accumulation as a result of DNA damage induction. These effects were unrelated to the alternative lengthening of telomeres pathway. In vivo, the combination of VE-822 and gemcitabine significantly enhanced tumor growth inhibition and progression-free survival in an aggressive model of undifferentiated pleomorphic sarcoma. The combination of ATR inhibitor and chemotherapy is beneficial in pre-clinical models of soft-tissue sarcoma and deserves further exploration in the clinical setting.

Background: LRRC15 is a member of the LRR (leucine-rich repeat) superfamily present on tumor-associated fibroblasts (CAFs) and stromal cells. The expression of LRRC15 is upregulated by the pro-inflammatory cytokine TGFβ. ABBV-085 is a monomethyl auristatin E (MMAE)-containing antibody-drug conjugate (ADC) designed to target LRRC15, and which has shown significant anti-tumor activity in several tumor models. This is the first focused examination of LRRC15 expression and ABBV-085 activity in soft-tissue sarcomas (STS). Methods: We analyzed the LRRC15 expression profile by immunohistochemistry in 711 STS cases, covering a broad spectrum of STS histologies and sub-classifications. In vivo experiments were carried out by using LRRC15-positive and LRRC15-negative patient-derived xenograft (PDX) models of STS. Results: In contrast to patterns observed in epithelial tumors, LRRC15 was expressed not only by stromal cells but also by cancer cells in multiple subsets of STS with significant variations noted between histological subtypes. Overexpression of LRRC15 is positively correlated with grade and independently associated with adverse outcome. ABBV-085 has robust preclinical efficacy against LRRC15 positive STS patient-derived xenograft (PDX) models. Conclusion: We provide the first preclinical evidence that LRRC15 targeting with an antibody-drug conjugate is a promising strategy in LRRC15-positive STS. ABBV-085 is being evaluated in an ongoing clinical trial in STS and other malignancies.

Clinical trials have shown the benefits of FGFR inhibitors; notably, the oral pan-FGFR inhibitor JNJ-42756493 (erdafitinib) has recently been the first FGFR kinase inhibitor to receive US FDA approval for the treatment of patients with advanced or metastatic urothelial carcinoma. Our objectives were to show that the FGFR pathway is involved in liposarcoma (LPS) pathogenesis and to investigate the therapeutic potential of erdafitinib for LPS patients. LPS is the most common soft tissue sarcoma. Among LPS, well-differentiated/dedifferentiated LPS (WD/DDLPS) are the most frequent types. DDLPS have metastatic potential and a poor prognosis with a five-year mortality rate of 30%. Standard chemotherapies are inefficient and new therapeutic options are needed. FRS2, a crucial adaptor protein of the FGFR signaling pathway is overexpressed in most WD/DDLPS suggesting involvement of the FGFR pathway in LPS tumorigenesis. Immunohistochemistry was used to perform an exhaustive analysis of the expression pattern of FGFR1-4 in WD/DDLPS both in a large collection of 421 primary tumors and in our in-house panel of validated human WDLPS and DDLPS cell lines. Cells were exposed to erdafitinib - as a single agent or in combination - and apoptosis and signalling pathway perturbations were monitored by flow cytometry and western blotting. Xenograft mouse models were used to confirm our in vitro results in the in vivo setting. We have detected a high expression of FGFR1 and/or FGFR4 in a significant percentage of WDLPS and DDLPS and demonstrated that this expression is correlated with poor prognosis. Exposure of LPS cells to erdafitinib induced a decrease in cell viability, cell cycle arrest in G1 and apoptosis. Erdafitinib treatment had a strong inhibitory effect on the ERK1/2 pathway whereas the PI3K/AKT pathway was less affected. The PI3K/mTOR antagonist BEZ235 was not synergistic with erdafitinib in DDLPS cells. MDM2 overexpression being the hallmark of WD/DDLPS, we investigated the effects of the MDM2-TP53 interaction inhibitor RG7388 (idasanutlin) in combination with erdafitinib. Interestingly, we found that the combination of RG7388 and erdafitinib exerts a highly synergistic effect on both viability and apoptosis in all our WDLPS and DDLPS cell lines. We are currently investigating the mechanics of the synergy between the FGFR and the MDM2 antagonists. We confirmed the inhibitory effect of erdafitinib on tumor growth using a DDLPS xenograft model. Finally and importantly, we report the efficacy of FGFR inhibition in two metastatic DDLPS patients heavily pretreated and refractory to several lines of treatment with notably one patient on erdafitinib showing stable disease for 15 weeks. In conclusion, we have shown that the FGFR pathway has therapeutic potential for a subset of DDLPS patients and that FGFR expression might constitute a powerful biomarker to select patients for clinical trials testing FGFR inhibitors. Citation Format: Bérengère Dadone-Montaudié, Audrey Laroche-Clary, Aline Mongis, Vanessa Chaire, Ilaria Di Mauro, Renaud Schiappa, Emmanuel Chamorey, Bertucci François, Florence Pedeutour, Finetti Pascal, Jean-François Michiels, Antoine Italiano, Laurence Bianchini. Analysis of the role of FGFR signaling in liposarcomas: Dual targeting of FGFR and MDM2 is synergistic [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5185.

The MDM2 gene is amplified in dedifferentiated liposarcoma (DDLPS). Treatment with MDM2 antagonists is a promising strategy to treat DDLPS, however, drug resistance is a major limitation when these drugs are used as a single agent. This study examined the impact of MDM2 antagonists on the mitogen-activated protein kinase (MAPK) pathway in DDLPS and investigated the potential synergistic activity of a MAPK kinase (MEK) inhibitor in combination with MDM2 antagonists. We identified a synergistic effect and identified the mechanism behind it. Combination effects of MDM2 antagonists and a MEK inhibitor were analyzed in a patient-derived xenograft mouse model and in DDLPS and leiomyosarcoma cell lines using different cell proliferation assays and immunoblot analysis. MDM2 antagonist (RG7388)-resistant IB115 [P4] cells and p53-silenced DDLPS cells were also established to understand the importance of functional p53. We found that MDM2 antagonists induced an upregulation of phosphorylated extracellular signal-regulated kinase (p-ERK) in DDLPS cells. The upregulation of p-ERK occurred due to mitochondrial translocation of p53, which resulted in increased production of reactive oxygen species, causing the activation of receptor tyrosine kinases (RTKs). Activated RTKs led to the activation of the downstream MEK/ERK signaling pathway. Treatment with a MEK inhibitor resulted in decreased expression of p-ERK, causing significant anti-tumor synergy when combined with MDM2 antagonists. Our results provide a framework for designing clinical studies of combination therapies in DDLPS patients.

Background Leiomyosarcoma (LMS) is one of the most frequent soft tissue sarcoma subtypes and is characterized by a consistent deregulation of the PI3K/mTOR pathway. Cancer stem cells (CSCs) have been poorly studied in soft tissue sarcomas. In this study, we aimed to evaluate the association between CSCs, the outcome of LMS patients, and the resistance to PI3K/mTOR pathway inhibition. Methods We investigated the relationships between aldehyde dehydrogenase 1 (ALDH1) expression, a cancer stem cell marker, and the outcome of LMS patients in two independent cohorts. We assessed the impact of CSCs in resistance to PI3K/mTOR pathway inhibition using LMS cell lines, a xenograft mouse model, and human tumor samples. Results We found that enhanced ALDH1 activity is a hallmark of LMS stem cells and is an independent prognostic factor. We also identified that secondary resistance to PI3K/mTOR pathway inhibition was associated with the expansion of LMS CSCs. Interestingly, we found that EZH2 inhibition, a catalytic component of polycomb repressive complex which plays a critical role in stem cell maintenance, restored sensitivity to PI3K/mTOR pathway inhibition. Importantly, we confirmed the clinical relevance of our findings by analyzing tumor samples from patients who showed secondary resistance after treatment with a PI3Kα inhibitor. Conclusions Altogether, our findings suggest that CSCs have a strong impact on the outcome of patients with LMS and that combining PI3K/mTOR and EZH2 inhibitors may represent a promising strategy in this setting. Electronic supplementary material The online version of this article (10.1186/s13045-018-0694-1) contains supplementary material, which is available to authorized users.

Background Doxorubicin (DOX) is a small molecular cytotoxic agent that can be transferred efficiently to cancer cells by nanocarriers. This anthracycline antibiotic serves as an effective anti-neoplastic drug against both hematological and solid malignancies. Here, we set out to assess the capacity of a novel doxorubicin - transferrin conjugate (DOX-TRF) to provoke apoptosis in human normal and leukemia cells through free radicals produced via a redox cycle of doxorubicin (DOX) when released from its conjugate. Methods After DOX-TRF exposure, we determined the time-course of apoptotic and necrotic events, the generation of reactive oxygen species (ROS), changes in mitochondrial membrane potential, as well as alterations in cytochrome c levels and intracellular calcium concentrations in human leukemia-derived cell lines (CCRF-CEM, K562 and its doxorubicin-resistant derivative K562/DOX) and normal peripheral blood-derived mononuclear cells (PBMC). Results We found that DOX-TRF can induce apoptosis in all leukemia-derived cell lines tested, which was associated with morphological changes and decreases in mitochondrial membrane potential. In comparison to free DOX treated cells, we observed a time-dependency between a higher level of ROS generation and a higher drop in mitochondrial membrane potential, particularly in the doxorubicin-resistant cell line. In addition, we found that the apoptotic cell death induced by DOX-TRF was directly associated with a release of cytochrome c from the mitochondria and an increase in intracellular calcium level in all human leukemia-derived cell lines tested. Conclusions Our data indicate that DOX-TRF is considerably more cytotoxic to human leukemia cells than free DOX. In addition, we show that DOX-TRF can effectively produce free radicals, which are directly involved in apoptosis induction.

Background Inhibition of ChK1 appears as a promising strategy for selectively potentiate the efficacy of chemotherapeutic agents in G1 checkpoint-defective tumor cells such as those that lack functional p53 protein. The p53 pathway is commonly dysregulated in soft-tissue sarcomas (STS) through mutations affecting TP53 or MDM2 amplification. GDC-0575 is a selective ATP-competitive inhibitor of CHK1. Methods We have performed a systematic screening of a panel of 10 STS cell lines by combining the treatment of GDC-0575 with chemotherapy. Cell proliferation, cell death and cell cycle analysis were evaluated with high throughput assay. In vivo experiments were carried out by using TP53-mutated and TP53 wild-type patient-derived xenograft models of STS. Clinical activity of GDC-0575 combined with chemotherapy in patients with TP53-mutated and TP53 wild-type STS was also assessed. Results We found that GDC-0575 abrogated DNA damage-induced S and G2–M checkpoints, exacerbated DNA double-strand breaks and induced apoptosis in STS cells. Moreover, we observed a synergistic or additive effect of GDC-0575 together with gemcitabine in vitro and in vivo in TP53-proficient but not TP53-deficient sarcoma models. In a phase I study of GDC-0575 in combination with gemcitabine, two patients with metastatic TP53-mutated STS had an exceptional, long-lasting response despite administration of a very low dose of gemcitabine whereas one patient with wild-type TP53 STS had no clinical benefit. Genetic profiling of samples from a patient displaying secondary resistance after 1 year showed loss of one preexisting loss-of-function mutation in the helical domain of DNA2. Conclusion We provide the first preclinical and clinical evidence that potentiation of chemotherapy activity with a CHK1 inhibitor is a promising strategy in TP53-deficient STS and deserves further investigation in the phase II setting.

// Benjamin Fourneaux 1, 2 , Vanessa Chaire 1, 2 , Carlo Lucchesi 2 , Marie Karanian 3 , Raphael Pineau 1 , Audrey Laroche-Clary 1, 2 , Antoine Italiano 2, 3 1 Universite de Bordeaux, Bordeaux, France 2 Institut National de la Sante et de la Recherche Medicale (INSERM), Institut Bergonie, Bordeaux, France 3 Department of Medical Oncology, Institut Bergonie, Bordeaux, France Correspondence to: Antoine Italiano, email: A.Italiano@bordeaux.unicancer.fr Keywords: leiomyosarcomas, PI3K/AKT/mTOR pathway, BEZ235, BKM120, everolimus Received: September 14, 2016 Accepted: December 05, 2016 Published: December 16, 2016 ABSTRACT The PI3K/AKT/mTOR pathway plays a crucial role in the development of leiomyosarcomas (LMSs). In this study, we tested the efficacy of dual PI3K/mTOR (BEZ235), PI3K (BKM120) and mTOR (everolimus) inhibitors in three human LMS cell lines. In vitro and in vivo studies using LMS cell lines showed that BEZ235 has a significantly higher anti-tumor effect than either BKM120 or everolimus, resulting in a greater reduction in tumor growth and more pronounced inhibitory effects on mitotic activity and PI3K/AKT/mTOR signaling. Strikingly, BEZ235 but neither BKM120 nor everolimus markedly enhanced the ERK pathway. This effect was reproduced by the combination of BKM120 and everolimus, suggesting the involvement of mTORC2 via a PI3K-independent mechanism. Silencing of RICTOR in LMS cells confirmed the role of mTORC2 in the regulation of ERK activity. Combined treatment with BEZ235 and GSK1120212, a potent MEK inhibitor, resulted in synergistic growth inhibition and apoptosis induction in vitro and in vivo . These findings document for the first time that dual PI3K/mTOR inhibition in leiomyosarcomas suppress a negative feedback loop mediated by mTORC2, leading to enhanced ERK pathway activity. Thus, combining a dual PI3K/mTOR inhibitor with MEK inhibitors may be a relevant approach to increase anti-tumor activity and prevent drug resistance in patients with LMS.

Aim: Tumor gene-expression profiling may define signatures capable of discriminating between responders and nonresponders to chemotherapy. Patients & methods: Fifty seven metastatic colorectal cancer patients were prospectively included and 40 tumors were analyzed. Patients were treated in first line with 5-fluorouracil associated with irinotecan or oxaliplatin. Response was evaluated using WHO criteria every 2 months after chemotherapy. Gene-expression profiling was performed using Applied Biosystems microarrays (Human Genome Survey Microarray v2.0; Paris, France). Data were analyzed using Bioconductor packages. Differential-expression analysis was performed by fitting a linear model. Moderated t-statistics were computed and p-values were adjusted for false-discovery rate. Pearson correlations tests were evaluated between gene expression and progression-free and overall survival. Results: Nonsupervised analysis did not show any clustering of expression levels according to treatment response. Supervised analysis compared expression levels between responders and nonresponders, within each treatment group and independently from treatment. No genes were identified as differentially expressed at a p-value of 10-3 and false-discovery rate of 30%. No correlation between expression levels and survival data was found. Conclusion: These negative results show that the determinants of response to chemotherapy should be sought not only in the tumor characteristics, but also among the processes leading to drug availability to the tumor. Original submitted 7 July 2011; Revision submitted 17 October 2011

The fact that conventional and newly emerging treatment procedures like chemotherapy, catalytic therapy, photodynamic therapy, and radiotherapy have not succeeded in reversing the outcome of many cancers alternative treatment options has been explored. This study documents the identification of component(s) from the Mediterranean sponge, Spongia officinalis that have anti-inflammatory and antiproliferative activities. In the present study we investigated the efficacy of a crude extract and its semi-purified fractions (F1–F3) of the defensive secretion from Spongia officinalis for in vivo anti-inflammatory activity using the carrageenan-induced paw edema assay in rats and their in vitro antiproliferative effects against three human cancer cell lines (A549, lung cell carcinoma; HCT15, colon cell carcinoma; and MCF7, breast adenocarcinoma). Among the series, the crude extract exhibited interesting anti-inflammatory activity associated with significant growth and concentration-related colony inhibitory effects against the three cell lines. The fractions F2 and F3 showed, respectively, interesting anti-inflammatory and antiproliferative activities in a dose-dependent manner. The purification and the determination of chemical structures of compounds of these active fractions are under investigation. Drug Dev Res 71: 412–418, 2010. © 2010 Wiley-Liss, Inc.