RAS Pathway Mutations Are Highly Prevalent In Relapsed Childhood Acute Lymphoblastic Leukaemia, Are Frequently Relapse-Drivers and Confer Sensitivity To MEK Inhibition

Abstract

For most children who relapse with acute lymphoblastic leukaemia (ALL), the prognosis is poor and there is a need for better prognostic biomarkers and novel therapies to improve outcome. Relapse samples from children with B lineage ALL entered into the ALL-REZ BFM 2002 clinical trial were screened for somatic mutations which activate the RAS pathway using DHPLC and Sanger sequencing. Mutations were found in 78 patients, giving an incidence of 37.9% and were made up of NRAS (n=30), KRAS (n=30), FLT3 (n=10) and PTPN11 (n=9); one patient had both a KRAS and FLT3 mutation. Clinically, mutated cases were associated with early relapse which was particularly significant for NRAS/KRAS mutated patients (p=0.001), with a 5 year probability of event free survival of 46.9% versus 60.1% for wild type (p=0.21). In addition, there was an over representation of NRAS/KRAS mutated patients with CNS involvement, 23.3% compared to 10.3% for the mutation negative group (p=0.014). Mutation screening of the matched diagnostic samples found many to be wild type (26 from 54) but using more sensitive allelic specific assays, low level mutated subpopulations were found in 57% of cases (8 from 14), suggesting these cells survived up front therapy and subsequently emerged at relapse. Assessment of p-ERK levels by western blotting in a large cohort of diagnostic ALL samples confirmed that RAS pathway mutated cases almost always had constitutive activation of the pathway (27 from 32; 84.3%), while relatively few wild type patients were p-ERK positive (9 from 48; 18.7%) (p<0.0001). Using MTS assay, differential sensitivity to the potent MEK1/2 inhibitor, selumetinib (AZD6244, ARRY-142886), was demonstrated in ALL blasts, with GI50 values being significantly lower for p–ERK/mutation positive samples (n=5, mean 250nM, range 18nM-918nM) compared to those that were negative (n=5, mean 68µM, range from 17.8µM-100µM; p=0.0079). This differential sensitivity was mirrored in an orthotopic mouse model using primagrafts derived from primary ALL cells. After intrafemoral injection and leukaemia engraftment, mice were randomised to receive either selumetinib or control vehicle (CV) (25 or 100mg/kg BID) and levels of circulating blasts monitored during treatment by flow cytometry. There was a dramatic reduction in circulating leukaemia cells in mice implanted with the RAS pathway mutant ALL cells (KRAS, G12D and NRAS Q61R) treated with selumetinib, with a mean fold decrease of >8 after 30 drug doses compared to a mean fold increase of >5 for CV. Mice treated with selumetinib and sacrificed at the end of the study showed a significant decrease in spleen size compared to CV treated mice. There was minimal effect of selumetinib in wild type ALLs. Histological analysis of post-mortem brains from mice engrafted with an NRAS mutant primagraft found extensive meningeal leukaemic infiltration in CV-treated, but not selumetinib-treated mice. Pharmacodynamic assessment in spleens of mice engrafted with mutant primagrafts showed absence of p-ERK and increased levels of the apoptotic biomarkers, Bim and cleaved PARP. In summary, we show that RAS pathway mutations are a common genetic abnormality in relapsed ALL and are associated with high risk features and are often relapse-drivers. Targeted MEK inhibition with selumetinib shows excellent activity in RAS mutated ALL both in vitro and in vivo and may offer clinical benefit for a substantial proportion of children with relapsed ALL. Given our findings, clinical trials of selumetinib in RAS pathway positive relapsed patients are warranted. Disclosures: No relevant conflicts of interest to declare.

Genomic characterization of pediatric T-cell acute lymphoblastic leukemia reveals novel recurrent driver mutations

“…Ras pathway mutations had significantly lower frequencies compared to these common drivers with a mean VAF = 0.33 (SD = 0.11) ( p = 0.006, Mann-Whitney- U test) ( Supplementary Figure S3 and Supplementary Information). Subclonality of these mutations corroborates previous reports [ 22 – 24 ] that describe a secondary role for Ras mutations in T-ALL occurring later in tumor progression. Fifteen patients (50%) harbored NOTCH1 mutations and two thirds (12/18) of identified NOTCH1 mutations were located in exons 26 and 27, coding for the extracellular heterodimerization domain and were mainly missense mutations (11/12).…”

RAS Pathway Mutations As Predictive Biomarker for Treatment Adaptation in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

“…These observations confirm that RAS pathway mutations are frequent at relapse in pediatric BCP-ALL. 28,29,63,64 The reduced mutational diversity at relapse suggests outgrowth of a single clone with stronger dependence on MAPK signaling, which may increase the chance of success for RAS-targeted therapy.…”

Upfront Treatment Influences the Composition of Genetic Alterations in Relapsed Pediatric B‐Cell Precursor Acute Lymphoblastic Leukemia

“…These studies show that recurrently affected genes and pathways at diagnosis, including those involved in B‐cell differentiation, cell cycle regulation, and Ras and JAK/STAT signaling, are also observed at relapse, although with different frequencies 4,6,12,20,21 . Alterations observed with a higher frequency at relapse, such as those affecting IKZF1 , TP53 , and genes involved in Ras signaling ( KRAS , NRAS , PTPN11, FLT3, and BRAF ), may play a role in therapy resistance and could be of prognostic value at the time of diagnosis 6,22–24 . By contrast, mutations in drug metabolism ( NT5C2 , PRPS1 ) or chromatin modification ( CREBBP ) regularly appear to arise during initial therapy, and become detectable only at relapse 13–16 …”

“…4,6,12,20,21 Alterations observed with a higher frequency at relapse, such as those affecting IKZF1, TP53, and genes involved in Ras signaling (KRAS, NRAS, PTPN11, FLT3, and BRAF), may play a role in therapy resistance and could be of prognostic value at the time of diagnosis. 6,[22][23][24] By contrast, mutations in drug metabolism (NT5C2, PRPS1) or chromatin modification (CREBBP) regularly appear to arise during initial therapy, and become detectable only at relapse. [13][14][15][16] Different chemotherapy regimens may have different effects on every leukemic subclone present in a patient, resulting in changes in clonal dynamics and, in some cases, survival of distinct subclones.…”