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the UKALLR3 trial, a poorer survival was seen in children with NRAS mutations.7 Thus, this genetic subtype of relapsed ALL clearly warrants exploratory therapies.
The Ras/Raf/Mek/Erk cascade regulates diverse cellular functions, including cell proliferation, survival, differentia- tion, angiogenesis and migration, and is deregulated in numerous cancers, including ALL.9-13 Classic activation is initiated by ligand binding to receptor tyrosine kinases at the cell surface and via Ras, then Raf activates MEK1/2 which has restricted substrate specify for extracellular sig- nal–regulated kinase 1 and 2 (Erk). ERK is a potent kinase with over 200 nuclear and cytoplasmic substrates includ- ing transcription factors such as the ETS family and pro- teins involved in the apoptotic machinery, such as the pro- apoptotic BIM. Phosphorylation of the predominant form of BIM (BIMEL) by ERK1/2, targets it for ubiquitination and proteasomal degradation and may also directly hinder its interactions with Bax14,15 and selumetinib-induced apopto- sis is associated with BIM induction.16
Relapsed ALL is generally more drug resistant than newly diagnosed disease and despite the use of more intensive chemotherapeutic regimens at ALL relapse, there are lower rates of complete remission and end-of- induction negativity for minimal residual disease.2,3 Assessment of in vitro drug sensitivity of primary ALL sam- ples has shown that blasts at relapse are significantly more resistant to many of the drugs used in upfront treatment protocols, with the highest level of drug resistance seen to glucocorticoids.17,18 Glucocorticoids, such as dexametha- sone, are pivotal agents in the treatment of all lymphoid malignancies because of their ability to specifically induce apoptosis in developing lymphocytes and induction of pro-apoptotic BIM is key to this effect.19 Thus, BIM is a common effector in both selumetinib- and dexametha- sone-induced apoptosis, suggesting the potential for syn- ergy. In addition, glucocorticoid resistance in ALL has been associated with enhanced activation of the pathway and its inhibition has led to glucocorticoid re-sensitiza- tion.20-22 These effects may be more pronounced in the context of RAS pathway-mutated ALL. We, therefore, preclinically evaluated the combination of dexamethasone and selumetinib in vitro and in an orthotopic mouse model engrafted with primary-derived ALL cells and showed pronounced drug synergism in RAS pathway-mutated ALL. These data suggest that this drug combination may
be highly effective in the significant subgroup of patients with this form of leukemia and has led to the Seludex trial, an international phase I/II expansion study on the treat- ment of relapsed/refractory RAS pathway-mutated ALL.
Methods
Compounds and formulation
Selumetinib was kindly provided by AstraZeneca (Cheshire, UK). For the in vitro studies, it was dissolved in dimethylsulfoxide to a concentration of 100 mM and stored in single-use aliquots at -20°C. Dexamethasone was purchased from Sigma-Aldrich (Dorset, UK), dissolved in ethanol at 20 mM and stored at -20°C. For in vivo studies, selumetinib was prepared as a suspension in 0.5% hydroxypropyl methylcellulose + 0.1% polysorbate 80.
Patients’ samples
In vitro drug sensitivity and synergy
Freshly harvested primagraft cells were suspended in RPMI1640
with 15% fetal bovine serum and plated out in triplicate at a den- sity of 5x105 cells/100 μL/well into 96-well plates and treated with a range of concentrations of dexamethasone (0.1 nM to 10 μM) or selumetinib (1 nM to 100 μM). After 96 h, cytotoxicity was assessed using the CellTiter 96 Aqueous One kit (Promega, Southampton, UK). The results were averaged and expressed as a percentage of the cytotoxicity of the control vehicle. Survival curves were plotted and half maximal growth inhibitory values (GI50) were calculated using GraphPad Prism software (GraphPad software Inc., San Diego, CA, USA). Drug combination experi- ments were analyzed for synergistic, additive, or antagonistic effects using the combination index method developed by Chou and Talalay.24 Briefly, primagraft cells were treated with fixed dose ratios based on the GI50 values of each drug (x0.25, x0.5, x1, x2 and x4) and evaluated by median effect analysis using CalcuSyn soft- ware (Cambridge, UK). The dose-effect curve for each drug alone
Table 1. Clinical features of patients and characterization of patient-derived xenografts.
Glucocorticoids and selumetinib in RAS pathway-mutated ALL
Primagrafts were generated in NOD SCID γ null (NSG) mice using ALL cells from bone marrow samples of children presenting or relapsing with ALL and accessed through the Newcastle Haematology Biobank, after appropriate consent (reference num- bers 2002/111 and 07/H0906). Clinical details of the patients are given in Table 1. Mutational screening for RAS pathway muta- tions and assessment of pathway activation by western blotting of p-ERK was performed as previously described.8,23
Patient ID Sex
L779 M
L897a M
Age at diagnosis (years)
5.5
Cytogenetics
High hyperdiploid
B other High hyperdiploid
High hyperdiploid t(17;19)
B other
B other
t(12;21)
End of induction MRD
Intermediate
High risk Low risk
High risk
High risk
High risk
Low risk
Low risk
Ras pathway mutation
NRAS (Q61R) KRAS (G12D)
Clonality
Clonal
Clonal Clonal
Clonal
Clonal
N/A
N/A
N/A
pERK
Positive
Positive Positive
Positive
Positive
Negative
Negative
Negative
L914
L829b relapse L707c
LX825
L920
16.8 F 7.3
CBL/FLT3
F 3.1 F 16.5 F 14.7 F 4.4
large del/D836 KRAS (G13D) KRAS (insertion) Wildtype Wildtype Wildtype
L848 M
2.5
aPatient suffered an on-treatment central nervous system relapse. bL829 at diagnosis was NRAS G12D. cPatient relapsed with the same KRAS mutation. B-other group: -; L897 is neg- ative by fluorescence in situ hybridization (FISH) for ETV6-RUNX1, BCR-ABL1, MLL and TCF3-PBX1/HLF. LX825 is negative by FISH for ETV6-RUNX1, BCR-ABL1, MLL, CRLF2, IKZF1,PAX5,IGHand PDGFRB.ID:identity;MRD:minimalresidualdisease;M:male;F:female;N/A:notavailable.
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