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

Abstract

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 <0.1% after two cycles of chemotherapy. Treatment was well-tolerated, and median time to neutrophil recovery >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.

Keywords: SAMHD1; acute myeloid leukaemia; cytarabine; hydroxyurea; precision medicine; targeted therapy.

Fig. 1

Treatment outcome. The nine patients have been followed for 14.9–20.5 months. Six patients have undergone allogeneic haematopoietic stem cell transplantation (allo‐HSCT). Measurable residual disease (MRD) was performed in accordance with national guidelines after cycle 2 and cycle 4 or prior to allo‐HSCT. In non‐transplanted patients with NPM1, MRD by real‐time quantitative polymerase chain reaction (RT‐qPCR) was performed every 3 months. After allo‐HSCT, patients were routinely monitored every 3 months either by RT‐qPCR (NPM1) or flow cytometry (all others). Eight of nine patients remain in MRD negative remission (as defined by MRD <0.1% by flow cytometry or RT‐qPCR). *, Patient 1106 had bone marrow blasts 5.5% after cycle 1 and did not fulfil complete remission (CR) criteria until after cycle 2. However, blasts may have been elevated by granulocyte colony‐stimulating factor (G‐CSF) usage prior to sampling; †, patient 1106 relapsed after 11.9 months and was treated with a combination of azacitidine–venetoclax–gilteritinib (AZA‐VEN‐GILT) and is now in a second CR; ‡, in patient 1107, NPM1 RT‐qPCR MRD increased from negative to 0.00024%, was rechecked and was then negative again. Before receiving the result of the confirmatory MRD, the patient was put on azacitidine–venetoclax (AZA‐VEN), and three cycles were given as an additional consolidation due to the inconsistent results; §, patient 1108 received one cycle of AZA as bridging while waiting for allo‐HSCT.

Fig. 2

Leukemic clearance after cycle 2. Validated real‐time quantitative polymerase chain reaction (RT‐qPCR) markers for NPM1 and DEK::NUP214, respectively, were analysed in four patients, and all showed mutational clearance <0.1% (relative to the diagnostic level) after cycle 2. In the remaining five patients, one or two mutations were monitored for measurable residual disease (MRD) using deep sequencing (reporting the variant allele frequency). Mutation in RUNX1 was seen in four patients and was reduced to 0.74%–10.5% after cycle 2. In two of four patients with RUNX1, a FLT3‐TKD was also present, but was reduced below 0.05% after two cycles of treatment. After cycle 2, MRD levels below the detection limit for the method are indicated with open circles.

Fig. 3

SAMHD1 protein expression in acute myeloid leukaemia (AML) blasts and in remission bone marrow. (a) Upper images show haematoxylin and eosin routine staining in diagnostic bone marrow biopsies. The lower images show the corresponding SAMHD1‐CD68 double immunohistochemical staining. SAMHD1 (in brown) is mainly expressed in the nucleus of the AML blasts, while CD68⁺ macrophages (magenta) are strongly positive for SAMHD1 and serve as an internal positive control. Low, intermediate and high expression levels were defined as <25%, 25%–75% and >75% positive cells, respectively, as previously described [21]. (b) Upper images show haematoxylin–eosin routine staining in remission bone marrow biopsies from patients 1105 (left), 1101 (middle) and 1104 (right), respectively, with cellularity within normal range and morphology suggestive of reactive bone marrow. Lower images show SAMHD1/CD34 double immunostaining without double‐positive blasts. Occasionally, SAMHD1+/CD34− normal blasts are present. The SAMHD1+/CD34− cells are morphologically mostly histiocytes and reactive lymphocytes. All images are shown at 400× magnification.

Fig. 4

Effect of hydroxyurea on peak ara‐C triphosphate (ara‐CTP) levels in circulating mononuclear cells and ex vivo sensitivity to ara‐C. The left panel shows ara‐CTP levels measured in circulating mononuclear cells without hydroxyurea (w/o HU) as compared to ara‐CTP with hydroxyurea 1000 mg taken p.o. 1 h prior to start of ara‐C infusion, normalised to without hydroxyurea (n = 6). The right panel shows the fold reduction in IC₅₀ values of ara‐C in diagnostic bone marrow mononuclear cells treated ex vivo in the presence or absence of 100 μM hydroxyurea, normalised to the absence of hydroxyurea (n = 7). Individual dots correspond to individual patients; colours represent levels of SAMHD1 expression at diagnosis (green, <25%, orange 25%–75% and red >75%).