Translational studies on antimetabolic therapies in paediatric oncology

Cure rates for paediatric and adult cancer patients have improved within the last decades. This can partly be explained by implementation of new technologies and methodologies such as the identification of new mutations after sequencing that can be directly targeted for treatment or the introduction of immunotherapy. However, there is an urgent need for improvement of survival particularly for patients with relapsed metastatic disease. More than 20 years ago, SAMHD1 was discovered and even though its key role in preventing viral HIV-1 infections was initially established, it was only later classified as the first deoxynucleoside triphosphate triphosphohydrolase that can remove the three phosphogroups of the dNTPs in a single reaction, which contributes to the dNTP pool homoeostasis by limiting potentially hazardous expansion of the intracellular dNTP pool. SAMHD1 is a homotetramer that is strictly regulated by the dNTP levels, with two allosteric sites (AS1 and AS2) and one catalytic site responsible for the dNTPase activity. Cancer cells are, among other hallmarks, characterized by loss of proliferation inhibition. It is therefore not surprising that in many cancer types, deregulation, or mutations of SAMHD1 have been reported that allow cells to circumvent dNTP shortage to permit further DNA replication. Many chemotherapeutic drugs target uncontrolled cancer proliferation. For example, a large group of these compounds are analogues of physiological nucleosides leading to inhibition of DNA replication. SAMHD1 has the capacity to use many of these analogues as substrates and through its dNTPase activity, it dephosphorylates them and prevents their incorporation into the nascent DNA chain. This can lead to treatment resistance effectively inactivating chemotherapy. One of these analogues frequently used in regimens against haematological malignancies is cytarabine. However, its active metabolite ara-CTP is a substrate for SAMHD1, hence SAMHD1- positive cancers might limit its cytotoxic efficacy. Therefore, SAMHD1 represents a promising therapeutic target, and its inhibition might enhance cytarabine efficacy. In the present thesis, we aimed to investigate whether there is an association between SAMHD1 expression and response to treatment with nucleoside analogues in two different haematological malignancies and whether SAMHD1 inhibition can improve current treatment protocols. In paper I, we performed a phenotypic screen of more than 33000 small molecules and discovered non-competitive inhibitors of ribonucleotide reductase to potentiate cytarabine in a SAMHD1-dependent manner. Inhibition of SAMHD1 activity towards ara- CTP occurred in an indirect manner as RNR inhibition led to dNTP ratio imbalances affecting SAMHD1 substrate specificity. As dCTP outcompeted dATP as dominant AS2 activator, SAMHD1 activity towards ara-CTP was gradually lost. Functionally, the RNR inhibitors hydroxyurea or gemcitabine acted synergistically with cytarabine, and sensitized cells to treatment in a SAMHD1-dependent manner, both in cell lines and in patient derived AML blasts. Furthermore, combination treatment prolonged survival in murine AML models. As a result, with this study we discovered already clinically available drugs that could act synergistically with cytarabine and improve treatment outcome. Hence, SAMHD1 can act as a biomarker for AML patients and combining cytarabine with RNR inhibitors might overcome SAMHD1-mediated resistance. In paper II, we showed that another nucleoside analogue, nelarabine, that is specifically cytotoxic against malignant T-cells was both an allosteric activator and a substrate for SAMHD1, thus limiting its cytotoxic efficacy. SAMHD1 depletion led to treatment sensitization and addition of hydroxyurea in SAMHD1 expressing cells, inhibited SAMHD1 catalytic activity and increased intracellular levels of the active metabolite ara-GTP. Finally, in T-ALL patient derived cells, addition of HU improved the efficacy of nelarabine treatment. All in all, we showed that SAMHD1 expression is a resistance factor in nelarabine treatment and inhibition with HU could have a potential clinical use. In paper III, based on our preclinical data we performed a small phase 1 clinical trial to validate the efficacy and safety of adding hydroxyurea to cytarabine-based treatment of AML patients. A total of nine patients were enrolled and they received a minimum of two cycles of treatment including daunorubicin, cytarabine and hydroxyurea. Analysis of blood mononuclear cells of patients showed that adding HU increased ara-CTP levels in vivo. All patients achieved complete remission (CR) without unexpected or unacceptable toxicities and MRD was negative in all eight patients that could be evaluated. Thus, CR of all patients combined with the pharmacokinetic studies, suggested that adding HU to alleviate the SAMHD1-based resistance barrier can be a rational strategy to improve treatment outcomes with cytarabine-based treatments, In paper IV, we investigated the correlation between SAMHD1 expression and its impact on induction and consolidation therapy of AML. In two independent patient cohorts (n=98 and n=124), SAMHD1 protein expression levels were assessed via immunohistochemistry. SAMHD1 was differentially expressed in AML blasts but was not expressed in several physiological hematopoietic cells. Based on their SAMHD1 expression, samples were allocated to three different groups and although no effect of SAMHD1 expression was evident during induction therapy, patients with low SAMHD1 levels at diagnosis had significantly prolonged event-free and overall survival rates. Therefore, evaluation of SAMHD1 levels can serve as a prognostic marker and might stratify personalized treatment strategies including SAMHD1 inhibitors. In summary, the results of this thesis show that SAMHD1 can be used as a prognostic biomarker for AML treated with cytarabine-based regimens and might stratify patients for enhanced treatment protocols adding the SAMHD1 inhibitor hydroxyurea to cytarabine. SAMHD1 might have a similar role for the nucleoside analogue nelarabine in Tlymphoblastic malignancies. Hence, SAMHD1 might constitute a universal resistance factor for a group of nucleoside analogues, irrespective of the specific oncological diagnosis. Targeting SAMHD1 thus promises to improve outcomes for a large group of cancers

Nucleobase and Nucleoside Analogues: Resistance and Re-Sensitisation at the Level of Pharmacokinetics, Pharmacodynamics and Metabolism

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

ALK+ Anaplastic Large Cell Lymphoma (ALCL)-Derived Exosomes Carry ALK Signaling Proteins and Interact with Tumor Microenvironment

The oncogenic pathways activated by the NPM-ALK chimeric kinase of ALK+ anaplastic large cell lymphoma (ALCL) are well characterized; however, the potential interactions of ALK signaling with the microenvironment are not yet known. Here we report that ALK+ ALCL-derived exosomes contain critical components of ALK signaling as well as CD30, and that exosome uptake by lymphoid cells led to increased proliferation and expression of critical antiapoptotic proteins by the recipient cells. The bone marrow fibroblasts highly uptake ALK+ ALCL-derived exosomes and acquire a cancer-associated fibroblast (CAF) phenotype. Moreover, exosome-mediated activation of stromal cells altered the cytokine profile of the microenvironment. These interactions may contribute to tumor aggressiveness and possibly resistance to treatment

Low-level expression of SAMHD1 in acute myeloid leukemia (AML) blasts correlates with improved outcome upon consolidation chemotherapy with high-dose cytarabine-based regimens

Abstract Sterile alpha motif and histidine/aspartic acid domain containing protein 1 (SAMHD1) limits the efficacy of cytarabine (ara-C) used in AML by hydrolyzing its active metabolite ara-CTP and thus represents a promising therapeutic target. SAMHD1 has also been implicated in DNA damage repair that may impact DNA damage-inducing therapies such as anthracyclines, during induction therapy. To determine whether SAMHD1 limits ara-C efficacy during induction or consolidation therapy, SAMHD1 protein levels were assessed in two patient cohorts of de novo AML from The University of Texas MD Anderson Cancer Center (USA) and the National University Hospital (Singapore), respectively, using immunohistochemistry and tissue microarrays. SAMHD1 was expressed at a variable level by AML blasts but not in a broad range of normal hematopoietic cells in reactive bone marrows. A sizeable patient subset with low SAMHD1 expression (<25% of positive blasts) was identified, which was significantly associated with longer event-free (EFS) and overall (OS) survival in patients receiving high-dose cytarabine (HDAC) during consolidation. Therefore, evaluation of SAMHD1 expression level in AML blasts at diagnosis, may stratify patient groups for future clinical trials combining HDAC with novel SAMHD1 inhibitors as consolidation therapy

Targeting SAMHD1 with hydroxyurea in first-line cytarabine-based therapy of newly diagnosed acute myeloid leukaemia : Results from the HEAT-AML trial

Background: Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine (ara-C) and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that the addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-C triphosphate (ara-CTP) levels through targeted inhibition of SAMHD1. Objectives: In this phase 1 trial, we evaluated the feasibility, safety and efficacy of the addition of hydroxyurea to standard chemotherapy with cytarabine/daunorubicin in newly diagnosed AML patients. Methods: Nine patients were enrolled and received at least two courses of ara-C (1 g/m2/2 h b.i.d. d1-5, i.e., a total of 10 g/m2 per course), hydroxyurea (1–2 g d1-5) and daunorubicin (60 mg/m2 d1-3). The primary endpoint was safety; secondary endpoints were complete remission rate and measurable residual disease (MRD). Additionally, pharmacokinetic studies of ara-CTP and ex vivo drug sensitivity assays were performed. Results: The most common grade 3-4 toxicity was febrile neutropenia (100%). No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in median ara-CTP levels (1.5-fold; p = 0.04) in patients receiving doses of 1 g hydroxyurea. Ex vivo, diagnostic leukaemic bone marrow blasts from study patients were significantly sensitised to ara-C by a median factor of 2.1 (p = 0.0047). All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow cytometry or real-time quantitative polymerase chain reaction [RT-qPCR]) had an MRD level 1.0 × 109/L and to platelet recovery >50 × 109/L after the start of cycle 1 was 19 days and 22 days, respectively. Six of nine patients underwent allogeneic haematopoietic stem-cell transplantation (allo-HSCT). With a median follow-up of 18.0 (range 14.9–20.5) months, one patient with adverse risk not fit for HSCT experienced a relapse after 11.9 months but is now in second complete remission. Conclusion: Targeted inhibition of SAMHD1 by the addition of hydroxyurea to conventional AML therapy is safe and appears efficacious within the limitations of the small phase 1 patient cohort. These results need to be corroborated in a larger study