Single-nucleotide polymorphism profiling by multimodal-targeted next-generation sequencing in methotrexate-resistant and -sensitive human osteosarcoma cell lines.

Introduction: Methotrexate (MTX) is one of the most important drugs included in the first-line protocols to treat high-grade osteosarcoma (HGOS). Although several polymorphisms have been reported to be associated with drug response or MTX-related toxicity in pharmacogenetic studies, their role in the development of MTX resistance in HGOS is still unclear. Methods: Therefore, in this study, 22 single nucleotide polymorphisms (SNPs) in 4 genes of the folate metabolism, 7 MTX transporter genes, and 2 SNPs of the tumor protein p53 (TP53) gene were investigated using a custom multimodal-targeted next-generation sequencing (mmNGS) approach in 8 MTX-resistant and 12 MTX-sensitive human HGOS cell lines. The panel was validated by TaqMan genotyping assays. Results: High instability of TP53 rs1642785 was observed in all U-2OS/MTX variants. Allele changes of the solute carrier family 19 member 1/replication factor C subunit 1 (SLC19A1, previously known as RFC1) and rs1051266 were identified in all Saos-2/MTX-resistant variants in both DNA- and RNA- derived libraries compared to the parental Saos-2 cell line. Allele changes of methylenetetrahydrofolate reductase (MTHFR) rs1801133 were identified only in the RNA-derived libraries of the two U2OS variants with the highest MTX resistance level. Significantly upregulated gene expression associated with the development of MTX resistance was revealed for dihydrofolate reductase (DHFR) whereas SLC19A1 was downregulated. In addition, a fusion transcript of DHFR (ex4) and MutS Homolog 3 (MSH3) (ex9) was identified in the RNA libraries derived from the two U-2OS variants with the highest MTX resistance level. Conclusion: This innovative mmNGS approach enabled the simultaneous exploration of SNPs at DNA and RNA levels in human HGOS cell lines, providing evidence of the functional involvement of allele changes associated with the development of MTX resistance.

Strategies to Overcome Resistance to Immune-Based Therapies in Osteosarcoma.

Improving the prognosis and cure rate of HGOSs (high-grade osteosarcomas) is an absolute need. Immune-based treatment approaches have been increasingly taken into consideration, in particular for metastatic, relapsed and refractory HGOS patients, to ameliorate the clinical results currently achieved. This review is intended to give an overview on the immunotherapeutic treatments targeting, counteracting or exploiting the different immune cell compartments that are present in the HGOS tumor microenvironment. The principle at the basis of these strategies and the possible mechanisms that HGOS cells may use to escape these treatments are presented and discussed. Finally, a list of the currently ongoing immune-based trials in HGOS is provided, together with the results that have been obtained in recently completed clinical studies. The different strategies that are presently under investigation, which are generally aimed at abrogating the immune evasion of HGOS cells, will hopefully help to indicate new treatment protocols, leading to an improvement in the prognosis of patients with this tumor.

Pharmacogenomic Profiling of Cisplatin-Resistant and -Sensitive Human Osteosarcoma Cell Lines by Multimodal Targeted Next Generation Sequencing.

Cisplatin (CDDP) is a drug for high-grade osteosarcoma (HGOS) treatment. Several germline pharmacogenetic studies have revealed associations between single nucleotide polymorphisms (SNPs) and CDDP-based therapy response or CDDP-related toxicity in patients with HGOS. Whether these variants could play a biological role in HGOS cells has not been studied so far. The aim of this study was to explore 28 SNPs of 14 genes in 6 CDDP-resistant and 12 drug-sensitive human HGOS cell lines. An innovative multimodal targeted next generation sequencing (mmNGS) approach with custom primers designed for the most commonly reported SNPs of genes belonging to DNA repair, CDDP transport or detoxification, or associated with CDPP-related toxicity was applied. The mmNGS approach was validated by TaqMan genotyping assays and emerged to be an innovative, reliable tool to detect genetic polymorphisms at both the DNA and RNA level. Allele changes in three SNPs (ERCC2 rs13181 and rs1799793, ERCC1 rs11615) were identified on both DNA and RNA derived libraries in association with CDDP resistance. A change of the GSTP1 rs1695 polymorphism from AA to AG genotype was observed in the RNA of all six CDDP-resistant variants. These SNPs emerged to be causally associated with CDDP resistance in HGOS cells.

Impact of ABC Transporters in Osteosarcoma and Ewing's Sarcoma: Which Are Involved in Chemoresistance and Which Are Not?

The ATP-binding cassette (ABC) transporter superfamily consists of several proteins with a wide repertoire of functions. Under physiological conditions, ABC transporters are involved in cellular trafficking of hormones, lipids, ions, xenobiotics, and several other molecules, including a broad spectrum of chemical substrates and chemotherapeutic drugs. In cancers, ABC transporters have been intensely studied over the past decades, mostly for their involvement in the multidrug resistance (MDR) phenotype. This review provides an overview of ABC transporters, both related and unrelated to MDR, which have been studied in osteosarcoma and Ewing's sarcoma. Since different backbone drugs used in first-line or rescue chemotherapy for these two rare bone sarcomas are substrates of ABC transporters, this review particularly focused on studies that have provided findings that have been either translated to clinical practice or have indicated new candidate therapeutic targets; however, findings obtained from ABC transporters that were not directly involved in drug resistance were also discussed, in order to provide a more complete overview of the biological impacts of these molecules in osteosarcoma and Ewing's sarcoma. Finally, therapeutic strategies and agents aimed to circumvent ABC-mediated chemoresistance were discussed to provide future perspectives about possible treatment improvements of these neoplasms.

Drug Resistance in Osteosarcoma: Emerging Biomarkers, Therapeutic Targets and Treatment Strategies.

High-grade osteosarcoma (HGOS), the most common primary malignant tumor of bone, is a highly aggressive neoplasm with a cure rate of approximately 40-50% in unselected patient populations. The major clinical problems opposing the cure of HGOS are the presence of inherent or acquired drug resistance and the development of metastasis. Since the drugs used in first-line chemotherapy protocols for HGOS and clinical outcome have not significantly evolved in the past three decades, there is an urgent need for new therapeutic biomarkers and targeted treatment strategies, which may increase the currently available spectrum of cure modalities. Unresponsive or chemoresistant (refractory) HGOS patients usually encounter a dismal prognosis, mostly because therapeutic options and drugs effective for rescue treatments are scarce. Tailored treatments for different subgroups of HGOS patients stratified according to drug resistance-related biomarkers thus appear as an option that may improve this situation. This review explores drug resistance-related biomarkers, therapeutic targets and new candidate treatment strategies, which have emerged in HGOS. In addition to consolidated biomarkers, specific attention has been paid to the role of non-coding RNAs, tumor-derived extracellular vesicles, and cancer stem cells as contributors to drug resistance in HGOS, in order to highlight new candidate markers and therapeutic targets. The possible use of new non-conventional drugs to overcome the main mechanisms of drug resistance in HGOS are finally discussed.

Corrigendum: Next-Generation Sequencing Approaches for the Identification of Pathognomonic Fusion Transcripts in Sarcomas: The Experience of the Italian ACC Sarcoma Working Group.

[This corrects the article DOI: 10.3389/fonc.2020.00489.].

Pharmacogenomics and Pharmacogenetics in Osteosarcoma: Translational Studies and Clinical Impact.

High-grade osteosarcoma (HGOS) is a very aggressive bone tumor which primarily affects adolescents and young adults. Although not advanced as is the case for other cancers, pharmacogenetic and pharmacogenomic studies applied to HGOS have been providing hope for an improved understanding of the biology and the identification of genetic biomarkers, which may impact on clinical care management. Recent developments of pharmacogenetics and pharmacogenomics in HGOS are expected to: i) highlight genetic events that trigger oncogenesis or which may act as drivers of disease; ii) validate research models that best predict clinical behavior; and iii) indicate genetic biomarkers associated with clinical outcome (in terms of treatment response, survival probability and susceptibility to chemotherapy-related toxicities). The generated body of information may be translated to clinical settings, in order to improve both effectiveness and safety of conventional chemotherapy trials as well as to indicate new tailored treatment strategies. Here, we review and summarize the current scientific evidence for each of the aforementioned issues in view of possible clinical applications.

Next-Generation Sequencing Approaches for the Identification of Pathognomonic Fusion Transcripts in Sarcomas: The Experience of the Italian ACC Sarcoma Working Group.

This work describes the set-up of a shared platform among the laboratories of the Alleanza Contro il Cancro (ACC) Italian Research Network for the identification of fusion transcripts in sarcomas by using Next Generation Sequencing (NGS). Different NGS approaches, including anchored multiplex PCR and hybrid capture-based panels, were employed to profile a large set of sarcomas of different histotypes. The analysis confirmed the reliability of NGS RNA-based approaches in detecting sarcoma-specific rearrangements. Overall, the anchored multiplex PCR assay proved to be a fast and easy-to-analyze approach for routine diagnostics laboratories.

Genomics and Therapeutic Vulnerabilities of Primary Bone Tumors.

Osteosarcoma, Ewing sarcoma and chondrosarcoma are rare diseases but the most common primary tumors of bone. The genes directly involved in the sarcomagenesis, tumor progression and treatment responsiveness are not completely defined for these tumors, and the powerful discovery of genetic analysis is highly warranted in the view of improving the therapy and cure of patients. The review summarizes recent advances concerning the molecular and genetic background of these three neoplasms and, of their most common variants, highlights the putative therapeutic targets and the clinical trials that are presently active, and notes the fundamental issues that remain unanswered. In the era of personalized medicine, the rarity of sarcomas may not be the major obstacle, provided that each patient is studied extensively according to a road map that combines emerging genomic and functional approaches toward the selection of novel therapeutic strategies.

Cisplatin Resistance in Osteosarcoma: In vitro Validation of Candidate DNA Repair-Related Therapeutic Targets and Drugs for Tailored Treatments.

Treatment of high-grade osteosarcoma, the most common malignant tumor of bone, is largely based on administration of cisplatin and other DNA damaging drugs. Altered DNA repair mechanisms may thus significantly impact on either response or resistance to chemotherapy. In this study, by using a panel of human osteosarcoma cell lines, either sensitive or resistant to cisplatin, we assessed the value as candidate therapeutic targets of DNA repair-related factors belonging to the nucleotide excision repair (NER) or base excision repair (BER) pathways, as well as of a group of 18 kinases, which expression was higher in cisplatin-resistant variants compared to their parental cell lines and may be indirectly involved in DNA repair. The causal involvement of these factors in cisplatin resistance of human osteosarcoma cells was validated through gene silencing approaches and in vitro reversal of CDDP resistance. This approach highlighted a subgroup of genes, which value as promising candidate therapeutic targets was further confirmed by protein expression analyses. The in vitro activity of 15 inhibitor drugs against either these genes or their pathways was then analyzed, in order to identify the most active ones in terms of inherent activity and ability to overcome cisplatin resistance. NSC130813 (NERI02; F06) and triptolide, both targeting NER factors, proved to be the two most active agents, without evidence of cross-resistance with cisplatin. Combined in vitro treatments showed that NSC130813 and triptolide, when administered together with cisplatin, were able to improve its efficacy in both drug-sensitive and resistant osteosarcoma cells. This evidence may indicate an interesting therapeutic future option for treatment of osteosarcoma patients who present reduced responsiveness to cisplatin, even if possible effects of additive collateral toxicities must be carefully considered. Moreover, our study also showed that targeting protein kinases belonging to the mitogen-activated protein kinase (MAPK) or fibroblast growth factor receptor (FGFR) pathways might indicate new promising therapeutic perspectives in osteosarcoma, demanding for additional investigation.

ABCA1/ABCB1 Ratio Determines Chemo- and Immune-Sensitivity in Human Osteosarcoma.

The ATP Binding Cassette transporter B1 (ABCB1) induces chemoresistance in osteosarcoma, because it effluxes doxorubicin, reducing the intracellular accumulation, toxicity, and immunogenic cell death induced by the drug. The ATP Binding Cassette transporter A1 (ABCA1) effluxes isopentenyl pyrophosphate (IPP), a strong activator of anti-tumor Vγ9Vδ2 T-cells. Recruiting this population may represent an alternative strategy to rescue doxorubicin efficacy in ABCB1-expressing osteosarcoma. In this work, we analyzed how ABCA1 and ABCB1 are regulated in osteosarcoma, and if increasing the ABCA1-dependent activation of Vγ9Vδ2 T-cells could be an effective strategy against ABCB1-expressing osteosarcoma. We used 2D-cultured doxorubicin-sensitive human U-2OS and Saos-2 cells, their doxorubicin-resistant sublines (U-2OS/DX580 and Saos-2/DX580), and 3D cultures of U-2OS and Saos-2 cells. DX580-sublines and 3D cultures had higher levels of ABCB1 and higher resistance to doxorubicin than parental cells. Surprisingly, they had reduced ABCA1 levels, IPP efflux, and Vγ9Vδ2 T-cell-induced killing. In these chemo-immune-resistant cells, the Ras/Akt/mTOR axis inhibits the ABCA1-transcription induced by Liver X Receptor α (LXRα); Ras/ERK1/2/HIF-1α axis up-regulates ABCB1. Targeting the farnesylation of Ras with self-assembling nanoparticles encapsulating zoledronic acid (NZ) simultaneously inhibited both axes. In humanized mice, NZ reduced the growth of chemo-immune-resistant osteosarcomas, increased intratumor necro-apoptosis, and ABCA1/ABCB1 ratio and Vγ9Vδ2 T-cell infiltration. We suggest that the ABCB1highABCA1low phenotype is indicative of chemo-immune-resistance. We propose aminobisphosphonates as new chemo-immune-sensitizing tools against drug-resistant osteosarcomas.

An update on emerging drugs in osteosarcoma: towards tailored therapies?

Introduction: Current treatment of conventional and non-conventional high-grade osteosarcoma (HGOS) is based on the surgical removal of primary tumor and, when possible, of metastases and local reccurrence, together with systemic pre- and post-operative chemotherapy with drugs that have been used since decades. Areas covered: This review is intended to summarize the new agents and therapeutic strategies that are under clinical evaluation in HGOS, with the aim to increase the cure probability of this highly malignant bone tumor, which has not significantly improved during the last 30-40 years. The list of drugs, compounds and treatment modalities presented and discussed here has been generated by considering only those that are included in presently ongoing and recruiting clinical trials, or which have been completed in the last 2 years with reported results, on the basis of the information obtained from different and continuously updated databases. Expert opinion: Despite HGOS is a rare tumor, several clinical trials are presently evaluating different treatment strategies, which may hopefully positively impact on the outcome of patients who experience unfavorable prognosis when treated with conventional therapies.

Hyaluronated liposomes containing H2S-releasing doxorubicin are effective against P-glycoprotein-positive/doxorubicin-resistant osteosarcoma cells and xenografts.

Doxorubicin (dox) is one of the first-line drug in osteosarcoma treatment but its effectiveness is limited by the efflux pump P-glycoprotein (Pgp) and by the onset of cardiotoxicity. We previously demonstrated that synthetic doxs conjugated with a H2S-releasing moiety (Sdox) were less cardiotoxic and more effective than dox against Pgp-overexpressing osteosarcoma cells. In order to increase the active delivery to tumor cells, we produced hyaluronic acid (HA)-conjugated liposomes containing Sdox (HA-Lsdox), exploiting the abundance of the HA receptor CD44 in osteosarcoma. HA-Lsdox showed favorable drug-release profile and higher toxicity in vitro and in vivo than dox or the FDA-approved liposomal dox Caelyx® against Pgp-overexpressing osteosarcoma, displaying the same cardiotoxicity profile of Caelyx®. Differently from dox, HA-Lsdox delivered the drug within the endoplasmic reticulum (ER), inducing protein sulfhydration and ubiquitination, and activating a ER stress pro-apoptotic response mediated by CHOP. HA-Lsdox also sulfhydrated the nascent Pgp in the ER, reducing its activity. We propose HA-Lsdox as an innovative tool noteworthy to be tested in Pgp-overexpressing patients, who are frequently less responsive to standard treatments in which dox is one of the most important drugs.

Current understanding of pharmacogenetic implications of DNA damaging drugs used in osteosarcoma treatment.

INTRODUCTION: DNA damaging drugs are widely used for the chemotherapeutic treatment of high-grade osteosarcoma (HGOS). In HGOS patients, several germline polymorphisms have been reported to impact on the development of adverse toxic events related to DNA damaging drugs treatment. Some of these polymorphisms, when present in tumor cells, may also influence treatment response and prognosis of HGOS patients. Area covered: In this review, the authors have focused on pharmacogenetic markers (mainly germline polymorphisms) described in patients with HGOS, which have proved or indicated to be related to the susceptibility to adverse toxic reactions and/or to influence response to DNA damaging drugs. The concordant and discordant results reported in different studies have also been discussed. Expert opinion: Response and toxicity predisposition to DNA damaging drugs are influenced by genes encoding proteins involved in their uptake, efflux, activation, inactivation, and in DNA repair, activity of which may vary according to specific gene variations. In HGOS, there is a substantial medical need for biomarkers predictive for individual response and toxicity predisposition to DNA-targeting drugs, which may be used to tailor therapy in order to decrease the occurrence of adverse side effects and increase treatment efficacy and safety.

Genetic testing for high-grade osteosarcoma: a guide for future tailored treatments?

INTRODUCTION: Genetic characterization of osteosarcoma has evolved during the last decade, thanks to the integrated application of conventional and new candidate-driven and genome-wide technologies. Areas covered: This review provides an overview of the state of art in genetic testing applied to osteosarcoma, with particular regard to novel candidate genetic biomarkers that can be analyzed in tumor tissue and blood samples, which might be used to predict toxicity and prognosis, detect disease relapse, and improve patients' selection criteria for tailoring treatment. Expert commentary: Genetic testing based on modern technologies is expected to indicate new osteosarcoma-related prognostic markers and driver genes, which may highlight novel therapeutic targets and patients stratification biomarkers. The definition of tailored or targeted treatment approaches may improve outcome of patients with localized tumors and, even more, of those with metastatic disease, for whom progress in cure probability is highly warranted.

Pharmacogenomics of genes involved in antifolate drug response and toxicity in osteosarcoma.

INTRODUCTION: Antifolates are structural analogs of folates, which have been used as antitumor drugs for more than 60 years. The antifolate drug most commonly used for treating human tumors is methotrexate (MTX), which is utilized widely in first-line treatment protocols of high-grade osteosarcoma (HGOS). In addition to MTX, two other antifolates, trimetrexate and pemetrexed, have been tested in clinical settings for second-line treatment of recurrent HGOS with patients unfortunately showing modest activity. Areas covered: There is clinical evidence which suggsest that, like other chemotherapeutic agents, not all HGOS patients are equally responsive to antifolates and do not have the same susceptibility to experience adverse drug-related toxicities. Here, we summarize the pharmacogenomic information reported so far for genes involved in antifolate metabolism and transport and in MTX-related toxicity in HGOS patients. Expert opinion: Identification and validation of genetic biomarkers that significantly impact clinical antifolate treatment response and related toxicity may provide the basis for a future treatment modulation based on the pharmacogenetic and pharmacogenomic features of HGOS patients.

Targeting CDKs with Roscovitine Increases Sensitivity to DNA Damaging Drugs of Human Osteosarcoma Cells.

Cyclin-dependent kinase 2 (CDK2) has been reported to be essential for cell proliferation in several human tumours and it has been suggested as an appropriate target to be considered in order to enhance the efficacy of treatment regimens based on the use of DNA damaging drugs. We evaluated the clinical impact of CDK2 overexpression on a series of 21 high-grade osteosarcoma (OS) samples profiled by using cDNA microarrays. We also assessed the in vitro efficacy of the CDKs inhibitor roscovitine in a panel of drug-sensitive and drug-resistant human OS cell lines. OS tumour samples showed an inherent overexpression of CDK2, and high expression levels at diagnosis of this kinase appeared to negatively impact on clinical outcome. CDK2 expression also proved to be relevant for in vitro OS cells growth. These findings indicated CDK2 as a promising candidate therapeutic marker for OS and therefore we assessed the efficacy of the CDKs-inhibitor roscovitine in both drug-sensitive and -resistant OS cell lines. All cell lines resulted to be responsive to roscovitine, which was also able to increase the activity of cisplatin and doxorubicin, the two most active DNA damaging drugs used in OS chemotherapy. Our results indicated that combined treatment with conventional OS chemotherapeutic drugs and roscovitine may represent a new candidate intervention approach, which may be considered to enhance tumour cell sensitivity to DNA damaging drugs.

Targeting ABCB1 and ABCC1 with their Specific Inhibitor CBT-1® can Overcome Drug Resistance in Osteosarcoma.

Clinical treatment response achievable with conventional chemotherapy in high-grade osteosarcoma (OS) is severely limited by the presence of intrinsic or acquired drug resistance, which in previous studies has been mainly addressed for overexpression of ABCB1 (MDR1/P-glycoprotein). This study was aimed to estimate the impact on OS drug resistance of a group of ATP binding cassette (ABC) transporters, which in other human tumors have been associated with unresponsiveness to the drugs that represent the backbone of multidrug treatment regimens for OS (doxorubicin, methotrexate, cisplatin). By using a group of 6 drug-sensitive and 20 drug-resistant human OS cell lines, the most relevant transporter which proved to be associated with the degree of drug resistance in OS cells, in addition to ABCB1, was ABCC1. We therefore evaluated the in vitro activity of the orally administrable ABCB1/ABCC1 inhibitor CBT-1(®) (Tetrandrine, NSC-77037). We found that in our OS cell lines this agent was able to revert the ABCB1/ABCC1-mediated resistance against doxorubicin, as well as against the drugs used in second-line OS treatments that are substrates of these transporters (taxotere, etoposide, vinorelbine). Our findings indicated that inhibiting ABCB1 and ABCC1 with CBT-1(®), used in association with conventional chemotherapeutic drugs, may become an interesting new therapeutic option for unresponsive or relapsed OS patients.

Role of pharmacogenetics of drug-metabolizing enzymes in treating osteosarcoma.

INTRODUCTION: Drug-metabolizing enzymes (DMEs) biotransform several toxins and xenobiotics in both tumor and normal cells, resulting in either their detoxification or their activation. Since DMEs also metabolize several chemotherapeutic drugs, they can significantly influence tumor response to chemotherapy and susceptibility of normal tissues to collateral toxicity of anticancer treatments. AREAS COVERED: This review discusses the pharmacogenetics of DMEs involved in the metabolism of drugs which constitute the backbone of osteosarcoma (OS) chemotherapy, highlighting what is presently known for this tumor and their possible impact on the modulation of future treatment approaches. EXPERT OPINION: Achieving further insight into pharmacogenetic markers and biological determinants related to treatment response in OS may ultimately lead to individualized treatment regimens, based on a combination of genotype and tumor characteristics of each patient.