Ras pathway mutations are prevalent in relapsed childhood acute lymphoblastic leukemia and confer sensitivity to MEK inhibition

Abstract

For most children who relapse with acute lymphoblastic leukemia (ALL), the prognosis is poor, and there is a need for novel therapies to improve outcome. We screened samples from children with B-lineage ALL entered into the ALL-REZ BFM 2002 clinical trial (www.clinicaltrials.gov, #NCT00114348) for somatic mutations activating the Ras pathway (KRAS, NRAS, FLT3, and PTPN11) and showed mutation to be highly prevalent (76 from 206). Clinically, they were associated with high-risk features including early relapse, central nervous system (CNS) involvement, and specifically for NRAS/KRAS mutations, chemoresistance. KRAS mutations were associated with a reduced overall survival. Mutation screening of the matched diagnostic samples found many to be wild type (WT); however, by using more sensitive allelic-specific assays, low-level mutated subpopulations were found in many cases, suggesting that they survived up-front therapy and subsequently emerged at relapse. Preclinical evaluation of the mitogen-activated protein kinase kinase 1/2 inhibitor selumetinib (AZD6244, ARRY-142886) showed significant differential sensitivity in Ras pathway-mutated ALL compared with WT cells both in vitro and in an orthotopic xenograft model engrafted with primary ALL; in the latter, reduced RAS-mutated CNS leukemia. Given these data, clinical evaluation of selumetinib may be warranted for Ras pathway-mutated relapsed ALL.

© 2014 by The American Society of Hematology.

Figures

Figure 1

Ras pathway mutations in relapsed ALL and their association with cytogenetics and event-free survival. Pie chart showing the proportion of patients with Ras pathway mutations (A). Histogram of Ras pathway mutations in relation to cytogenetic subgroups (B). Kaplan Meier overall and event-free survival curves of KRAS mutants (C and E) and NRAS mutants (D and F) compared with WT.

Figure 2

Backtracking and forward tracking in Ras pathway mutation–positive samples. Bar graph showing mutation status of matched diagnostic samples in those patients with Ras pathway mutations at relapse (A). Bar graph showing mutation status of Ras pathway–positive relapse samples at second relapse (B).

Figure 3

Selumetinib is active in Ras pathway–mutated ALL cells in vitro and is associated with reduced levels of p-ERK and induction of Bim and cleaved Parp. Histogram showing densitometry values of p-ERK levels relative to ERK as assessed by western analyses for both WT (black bars) and Ras pathway–mutated samples (gray bars) (A). Bar chart of GI50 values as assessed by 3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium assay after dosing with selumetinib for both Ras pathway–positive/p-ERK–positive samples (red bars, n = 5) and those negative for Ras pathway mutation and p-ERK (blue bars, n = 5) (B). Western analyses of ALL cell lysates from patients L897 (KRAS), L924 (KRAS), and L949 (WT) after treatment with a range of concentrations of selumetinib. Blots were probed for p-ERK, ERK2, Bim, cleaved Parp, and α-tubulin. In the case of L949, a positive control for p-ERK expression (CCRF-CEM cells) was included (C). Similar analyses of spleen cells from NOD SCID γ null mice engrafted with patient ALL cells as a source of primary-derived material (D). Primagrafts from duplicate mice implanted with blasts from patients L897 (KRAS) and L779 (NRAS) were treated with varying concentrations of selumetinib for 24 hours and processed for western analysis.

Figure 4

Selumetinib is active in Ras pathway–mutated ALL cells in an orthotopic primagraft model. Graphs showing log of the percent circulating leukemic cells in mice implanted with RAS WT (A,C) or Ras pathway mutant patient blasts (B,D). Mice were treated with either CV or selumetinib twice daily (100 mg/kg for B and 25 mg/kg for D) and the percent of circulating blasts quantified by flow cytometry during and at the end of treatment. Photographs of spleens (E,F) and graphs of spleen weights (G,H) after 30 drug doses of selumetinib in WT primagrafts (E,G) and RAS mutant primagrafts are shown (F,H).

Figure 5

Selumetinib eradicates CNS leukemia in an orthotopic primagraft model. Photomicrographs of brain sections stained with hematoxylin and eosin from mice engrafted with Ras pathway mutant ALL cells (L779) after treatment with selumetinib or CV (×20 objective) (A). Photomicrograph of 1 section from CV-treated mouse after increased magnification (×40) (B). Dot plot of the depth of leukemic infiltrate into the leptomeninges in CV- vs selumetinib-treated mice (C).

Figure 6

Pharmacodynamic analyses after selumetinib dosing in vivo shows prolonged inhibition of p-ERK and induction of apoptosis. Western analyses of spleen cells from mice engrafted with Ras pathway mutant ALL cells after 72 hours of 100 mg/kg (A, L897, KRAS) or 25 mg/kg BID (B, L779, NRAS). Flow cytometric analyses of the spleen harvests from L897 primagrafts stained with antibodies to mouse CD45, human CD19, CD10, and CD34, along with annexin V. Dot plot showing CD10 and CD19 expression of cells gated as lymphoid by light scatter and then for mouse CD45 negativity (C). CD10+CD19+ cells are then gated and displayed as histograms showing annexin-V fluorescence (D).