Osteosarcoma is the most common primary malignant bone tumour in children and adolescents. Due to micrometastatic spread, radical surgery alone rarely results in cure. Introduction of combination chemotherapy in the 1970s, however, dramatically increased overall survival rates from 20% to approximately 70%. Unfortunately, large clinical trials aiming to intensify treatment in the past decades have failed to achieve higher cure rates. In this review, we revisit how the heterogenous nature of osteosarcoma as well as acquired and intrinsic resistance to chemotherapy can account for stagnation in therapy improvement. We summarise current osteosarcoma treatment strategies focusing on molecular determinants of treatment susceptibility and resistance. Understanding therapy susceptibility and resistance provides a basis for rational therapy betterment for both identifying patients that might be cured with less toxic interventions and targeting resistance mechanisms to sensitise resistant osteosarcoma to conventional therapies.

Antimetabolites, in particular nucleobase and nucleoside analogues, are cytotoxic drugs that, starting from the small field of paediatric oncology, in combination with other chemotherapeutics, have revolutionised clinical oncology and transformed cancer into a curable disease. However, even though combination chemotherapy, together with radiation, surgery and immunotherapy, can nowadays cure almost all types of cancer, we still fail to achieve this for a substantial proportion of patients. The understanding of differences in metabolism, pharmacokinetics, pharmacodynamics, and tumour biology between patients that can be cured and patients that cannot, builds the scientific basis for rational therapy improvements. Here, we summarise current knowledge of how tumour-specific and patient-specific factors can dictate resistance to nucleobase/nucleoside analogues, and which strategies of re-sensitisation exist. We revisit well-established hurdles to treatment efficacy, like the blood-brain barrier and reduced deoxycytidine kinase activity, but will also discuss the role of novel resistance factors, such as SAMHD1. A comprehensive appreciation of the complex mechanisms that underpin the failure of chemotherapy will hopefully inform future strategies of personalised medicine.

Cytoplasmic entry of HIV-1 requires binding of the viral glycoproteins to the cellular receptor and coreceptor, leading to fusion of viral and cellular membranes. Early studies suggested that productive HIV-1 infection occurs by direct fusion at the plasma membrane. Endocytotic uptake of HIV-1 was frequently observed but was considered to constitute an unspecific dead-end pathway. More recent evidence suggested that endocytosis contributes to productive HIV-1 entry and may even represent the predominant or exclusive route of infection. We have analyzed HIV-1 binding, endocytosis, cytoplasmic entry, and infection in T-cell lines and in primary CD4⁺T cells. Efficient cell binding and endocytosis required viral glycoproteins and CD4, but not the coreceptor. The contribution of endocytosis to cytoplasmic entry and infection was assessed by two strategies: (i) expression of dominant negative dynamin-2 was measured and was found to efficiently block HIV-1 endocytosis but to not affect fusion or productive infection. (ii) Making use of the fact that HIV-1 fusion is blocked at temperatures below 23°C, cells were incubated with HIV-1 at 22°C for various times, and endocytosis was quantified by parallel analysis of transferrin and fluorescent HIV-1 uptake. Subsequently, entry at the plasma membrane was blocked by high concentrations of the peptidic fusion inhibitor T-20, which does not reach previously endocytosed particles. HIV-1 infection was scored after cells were shifted to 37°C in the presence of T-20. These experiments revealed that productive HIV-1 entry occurs predominantly at the plasma membrane in SupT1-R5, CEM-ss, and primary CD4⁺T cells, with little, if any, contribution coming from endocytosed virions.IMPORTANCEHIV-1, like all enveloped viruses, reaches the cytoplasm by fusion of the viral and cellular membranes. Many viruses enter the cytoplasm by endosomal uptake and fusion from the endosome, while cell entry can also occur by direct fusion at the plasma membrane in some cases. Conflicting evidence regarding the site of HIV-1 fusion has been reported, with some studies claiming that fusion occurs predominantly at the plasma membrane, while others have suggested predominant or even exclusive fusion from the endosome. We have revisited HIV-1 entry using a T-cell line that exhibits HIV-1 endocytosis dependent on the viral glycoproteins and the cellular CD4 receptor; results with this cell line were confirmed for another T-cell line and primary CD4⁺T cells. Our studies show that fusion and productive entry occur predominantly at the plasma membrane, and we conclude that endocytosis is dispensable for HIV-1 infectivity in these T-cell lines and in primary CD4⁺T cells.

Recently, we demonstrated that sterile α motif and HD domain containing protein 1 (SAMHD1) is a major barrier in acute myelogenous leukemia (AML) cells to the cytotoxicity of cytarabine (ara-C), the most important drug in AML treatment. Ara-C is intracellularly converted by the canonical dNTP synthesis pathway to ara-CTP, which serves as a substrate but not an allosteric activator of SAMHD1. Using an AML mouse model, we show here that wild type but not catalytically inactive SAMHD1 reduces ara-C treatment efficacy in vivo. Expanding the clinically relevant substrates of SAMHD1, we demonstrate that THP-1 CRISPR/Cas9 cells lacking a functional SAMHD1 gene showed increased sensitivity to the antimetabolites nelarabine, fludarabine, decitabine, vidarabine, clofarabine, and trifluridine. Within this Extra View, we discuss and build upon both these and our previously reported findings, and propose SAMHD1 is likely active against a variety of nucleoside analog antimetabolites present in anti-cancer chemotherapies. Thus, SAMHD1 may constitute a promising target to improve a wide range of therapies for both hematological and non-haematological malignancies.

The deoxycytidine analogue cytarabine (ara‐C) remains the backbone treatment of acute myeloid leukaemia (AML) as well as other haematological and lymphoid malignancies, but must be combined with other chemotherapeutics to achieve cure. Yet, the underlying mechanism dictating synergistic efficacy of combination chemotherapy remains largely unknown. The dNTPase SAMHD1, which regulates dNTP homoeostasis antagonistically to ribonucleotide reductase (RNR), limits ara‐C efficacy by hydrolysing the active triphosphate metabolite ara‐CTP. Here, we report that clinically used inhibitors of RNR, such as gemcitabine and hydroxyurea, overcome the SAMHD1‐mediated barrier to ara‐C efficacy in primary blasts and mouse models of AML, displaying SAMHD1‐dependent synergy with ara‐C. We present evidence that this is mediated by dNTP pool imbalances leading to allosteric reduction of SAMHD1 ara‐CTPase activity. Thus, SAMHD1 constitutes a novel biomarker for combination therapies of ara‐C and RNR inhibitors with immediate consequences for clinical practice to improve treatment of AML.