Casitas B lymphoma mutations in childhood acute lymphoblastic leukemia

GENES, CHROMOSOMES & CANCER 51:250–256 (2012)

Casitas B Lymphoma Mutations in Childhood AcuteLymphoblastic Leukemia

Lindsay Nicholson, Thomas Knight, Elizabeth Matheson, Lynne Minto, Marian Case, Maryna Sanichar,

Simon Bomken, Josef Vormoor, Andy Hall, and Julie Irving

Paediatric Oncologyand Haematology Group,Northern Institute for Cancer Research,Newcastle University,Newcastle uponTyne,UK

Casitas B-lineage lymphoma (CBL) proteins are RING finger ubiquitin E3 ligases that attenuate the signaling of receptor

tyrosine kinases and are mutated in a number of myeloid disorders. In this study, mutational screening of the linker-RING

domains of CBL and CBLB was performed by denaturing high performance liquid chromatography in a cohort of diagnostic

(n ¼ 180) or relapse (n ¼ 46) samples from children with acute lymphoblastic leukemia. Somatic mutations were identified

in three children, giving an overall incidence of 1.7% and involved small deletions affecting the intron/exon boundaries

of exon 8, leading to skipping of exon 8 and abolishing E3 ligase function. Mutated primary samples were associated

with constitutive activation of the RAS pathway and sensitivity to MEK inhibitors was shown. Thus, mutation of CBL is an

alternative route to activate the RAS pathway and may identify children who are candidates for MEK inhibitor clinical

trials. VVC 2011 Wiley Periodicals, Inc.

INTRODUCTION

Casitas B-lineage lymphoma (CBL) proteins

are a highly conserved family of RING finger

ubiquitin E3 ligases that serve to attenuate the

signaling of a variety of receptor tyrosine kinases

(RTK). Recently, somatic mutations of CBL have

been identified in a range of myeloid disorders

including acute myeloid leukemia (AML),

chronic myelo-monocytic leukemia (CMML), ju-

venile myelo-monocytic leukemia (JMML), and

myeloproliferative syndrome (Caligiuri et al.,

2007; Sargin et al., 2007; Dunbar et al., 2008;

Grand et al., 2009; Loh et al., 2009; Makishima

et al., 2009; Reindl et al., 2009; Kales et al., 2010;

Muramatsu et al., 2010; Ogawa et al., 2010).

Mutations principally affect the linker-RING do-

main of the CBL protein and are often associated

with acquired uniparental disomy (aUPD) at the

CBL gene locus at 11q23.3. Functional studies in

vitro and in vivo show that CBL mutations are

transforming and induce a myeloproliferative dis-

ease which can progress to leukemia and are asso-

ciated with enhanced RTK signaling, manifested

down numerous key signaling pathways including

the RAS, PKB and JAK-STAT pathways (Bandi

et al., 2009; Sanada et al., 2009; Rathinam et al.,

2010). In addition, somatic mutations of another

family member, CBLB, have also been reported

(Caligiuri et al., 2007; Makishima et al., 2009). In

JMML and CMML, CBL mutations were found

to be mutually exclusive with respect to RAS-

pathway activating mutations such as NRAS,

KRAS, and PTPN11, suggesting that CBL may

play an important a role in deregulating this key

survival pathway (Loh et al., 2009; Sanada et al.,

2009; Muramatsu et al., 2010).

Acute lymphoblastic leukemia (ALL) is the

most common cancer in children and is character-

ized by a clonal expansion of poorly differenti-

ated lymphoid precursors within the bone

marrow. While cure rates are high, novel thera-

pies are needed for those children that relapse.

ALL is a heterogeneous disease, with a vast array

of genomic aberrations and we recently showed

that mutations in genes impacting on the RAS

pathway, such as NRAS, KRAS, PTPN11, and

FLT3 represent one of the most common genetic

abnormalities, being found in a third of patients

(Case et al., 2008). Primary leukemic blasts with

mutations were associated with constitutive acti-

vation of the RAS pathway and differential sensi-

tivity to MEK inhibitors was shown in patients

with the highest levels of RAS pathway activa-

tion, suggesting that drugs such as MEK

Additional Supporting Information may be found in the onlineversion of this article.

Supported by: Leukaemia and Lymphoma Research.

*Correspondence to: Julie Irving, Northern Institute for CancerResearch, Paul O’Gorman Building, Framlington Place, Newcastleupon Tyne, Tyne and Wear, UK, NE2 4HH. E-mail: [email protected]

Received 3 June 2011; Accepted 6 October 2011

DOI 10.1002/gcc.20949

Published online 10 November 2011 inWiley Online Library (wileyonlinelibrary.com).

VVC 2011 Wiley Periodicals, Inc.

inhibitors which have already entered clinical trial

for adult solid tumors, may be a useful, novel

therapeutic approach in ALL (Case et al., 2008;

Bennouna et al., 2011). However, we also found

evidence for constitutive activation of the RAS

pathway in the absence of known activating

mutations and thus we hypothesized that CBLmutations were a likely alternative route to dysre-

gulate the RAS and possibly other key survival

pathways. Thus in this study, we mutationally

screened the linker-RING domains of CBL and

CBLB in a large cohort of ALL patients and

report the first identification of CBL mutations in

this disease.

MATERIALS AND METHODS

Patients

Bone marrow or peripheral blood samples were

obtained from children (less than 17 years) who

presented with ALL within the northern region

of England between 1994 and 2011. The study

had ethical approval (reference 2002/11 and 07/

H906). All diagnosis, remission, and relapse

events were pathologically confirmed. The major-

ity of this cohort were previously screened for

mutations in FLT3, PTPN11, NRAS, KRAS, andBRAF and were reported in Case et al. (2008).

Cell Lines and Cytotoxicity Assays

Cell lines used in the study were cultured in

RPMI 1640 medium containing L-glutamine, sup-

plemented with 10% fetal bovine serum, at 37�C,in an atmosphere of 5% CO2 and were routinely

tested for mycoplasma contamination using

MycoAlertVR

(Lonza, Basel, Switzerland). In vitro

cytotoxicity assays were performed using the

MTS assay (Promega, Southampton, UK) as pre-

viously described (Nicholson et al., 2010), which

assesses the capacity of metabolically active cells

to reduce formazan. Briefly, cell lines or patient

cells were plated out in triplicate at a seeding

density of 2 � 104 or 2 � 105 cells per well,

respectively, into 96-well plates and dosed with

either the FLT3 inhibitor PKC-412 (Sigma-

Aldrich, Dorset, UK), the MEK inhibitor U0126,

(Calbiochem, Nottingham, UK) or a control vehi-

cle (0.01% v/v DMSO). After 96-hr incubation,

the MTS reagent was added and the resulting ab-

sorbances were averaged and expressed as a per-

centage of the control vehicle. Survival curves

were plotted using GraphPad Prism software ver-

sion 4.0.

Mutational Screening and Sequencing/Cloning

Exons 8 and 9 (CBL) and exon 9 (CBLB), con-stituting the linker-RING domain, were screened

for mutations using denaturing high performance

liquid chromatography (dHPLC) on a Transge-

nomic WAVE machine using genomic DNA or in

some cases whole genome amplified DNA.

Amplicons were analyzed before and after spiking

with known wild type product to allow detection

of homozygous mutations. Primer sequences and

dHPLC conditions are shown in Supporting In-

formation Table 1. Direct sequencing and PCR

product cloning were performed using standard

techniques.

RT-PCR

Total RNA was extracted using a Qiagen

RNeasy kit and cDNA synthesized using a High

Capacity cDNA Reverse Transcription Kit

(Applied Biosystems). PCR was carried out using

primers described elsewhere (Reindl et al., 2009)

and the products visualized by electrophoresis

using a 2% agarose gel and GelRed Nucleic Acid

Stain (Biotium, Hayward, USA).

Western Blotting

Whole cell lysates were prepared using cell

lysis buffer supplied by Cell Signalling Technol-

ogy (NEB, UK) according to the manufacturers’

instructions. Protein was fractionated using 12%

SDS-polyacrylamide gels (BioRad, Hercules, CA)

and transferred onto PVDF membrane (BioRad).

Membranes were probed with antibodies directed

TABLE 1. Clinical Characteristics of CBL Mutant Positive Cases

Patient Age Sex WCC x 109/l FAB Immunophenotype Cytogenetics Day 28 MRD

L825 14 F >100 L1 CD10� B precursor ALL Normala 1.30%L914 7 F 9.6 L1 CD10þ B precursor ALL High hyperdiploid <0.01%L919 2 M 1.5 L1 CD10þ B precursor ALL Pseudodiploid Not done

aIn 16 metaphases.

F, female; M, male; WCC, white cell count; FAB, French American British morphology classification; MRD, minimal residual disease.

CBL MUTATIONS AND CHILDHOOD ALL 251

Genes, Chromosomes & Cancer DOI 10.1002/gcc

to p-STAT5, FLT-3, p-AKT, AKT (all purchased

from Cell Signaling Technology, Beverly, MA),

p-ERK and ERK (Santa Cruz Biotechnology,

Santa Cruz, CA) as previously described (Case

et al., 2008). All immunoblots were probed with

an antibody directed towards b-actin (Calbio-

chem, UK), which served as a loading control.

Secondary antibodies used were horseradish

peroxidise conjugates of either anti-rabbit or

anti-mouse immunoglobulins (Dako, Denmark).

Blots were visualized by ECL-Plus detection

(Amersham, UK) and exposed to Kodak Medical

Figure 1. Detection and characterization of CBL mutations in primary ALL. A: dHPLC chromato-grams of CBL run under non-denaturing conditions, showing aberrant peaks with reduced retention timein samples L825, L914 and L919, compared with the wild type control. B–D: Sequence chromatogramsfor CBL mutations in patients L825 (B), L914 (C) and L919 (D). E: Schematic of CBL mutations in ALL.

252 NICHOLSON ETAL.


X-ray film (GRI, UK). Cell lines which harbored

the relevant mutations or were wild-type served

as positive and negative controls, respectively,

and were ran alongside patient L825.

Flow Minimal Residual Disease

This was performed using the method

described in Irving et al. (2009). Briefly, samples

were collected into Acid Citrate Dextrose-A

tubes and red cell lysed using a standard ammo-

nium chloride procedure. At diagnosis, eight

antibody combinations were assessed with a

common CD34/CD19/CD10 spine and a mini-

mum of 50,000 events acquired on a FACSCali-

bur (Becton Dickinson, Oxford, UK).

Combinations, in which the leukemic blasts fell

into empty spaces, distinct from regions housing

normal B cell progenitors, were identified as leu-

kemia-associated immunophenotypes (LAIPs).

At least 2 LAIPS were then tracked in follow up

Day 28 samples and a minimum of 500,000

events acquired. Samples were considered posi-

tive if the number of leukemic cells identified

with one or more LAIP combinations was equal

to or greater than 0.01%.

RESULTS AND DISCUSSION

Mutational screening of CBL (exons 8 and 9)

and CBLB (exon 9) was performed by dHPLC in

a cohort of 180 diagnostic and 46 relapse ALL

samples. Heteroduplexes, indicative of a DNA

alteration, were identified in four samples, three

in CBL (patient numbers, L825, L914, and L919)

(Fig. 1A) and 1 in CBLB (L526). Direct sequenc-

ing of CBLB amplicons from patient L526

showed a T>C transition, P387P, which was also

present in the matched constitutive genomic

DNA and is a rare synonymous SNP (rs9657925).

For CBL, cloning and sequencing of amplicons

identified, from patient L825 a 4bp insertion/37

bp deletion at the exon 8/intron 8-9 boundary in

2 from 9 clones (22%; 95%CI, 5–56%); from

L914, a 6 bp insertion/21 bp deletion in 4 from

20 clones (20%; 95% CI, 8–42%) and from L919,

a 3 bp insertion/187 bp deletion in 5 from 9

clones (56%; 95% CI, 27–81%), the latter two

mutations affecting the intron 7-8/exon 8 bound-

ary (Figs. 1B–1E). DHPLC analyses of constitu-

tive DNA from these patients confirmed that

these deletions were acquired somatic mutations.

In addition, the mutational status of hot spot

exons of FLT3, NRAS, KRAS, and PTPN11 was

assessed and found to be wild type for patient

L825, while L914 also had a FLT3 I836 deletion.

Unfortunately, there was insufficient material to

perform additional mutational screening in L919.

All three samples had a high percentage of leuke-

mic cells (>90%) and unlike L914, CBL muta-

tions in L825 and L919 were present in the

majority of blasts, presumably heterozygously,

although without LOH analyses at the CBL

locus, we cannot rule out that the mutation exists

in a subpopulation of cells in a homozygous state,

concomitant with 11q aUPD.

RT-PCR of CBL with primers positioned in

exons 5 and 10 showed a wild type product of

752 bp in addition to a smaller 620 bp product in

bone marrow cells from patient L825 and L914

which was absent in a bone marrow aspirates

taken in clinical remission and also absent in the

PreB 697 cell line, which served as a wild type

control (Fig. 2A). Sequencing of the excised

smaller band in both patients identified CBLsequence, minus exon 8, which results in an in-

frame deletion (D8), disrupting the linker-RING

domain that is responsible for E3 ligase activity

(Fig. 2B). There was insufficient material to

Figure 2. Expression of mutant CBL in primary ALL. A: Agarosegel electrophoresis of CBL RT-PCR products generated from patientL825 and L914 diagnostic and remission samples, with the PreB 697cell line serving as a wild type control. B: Sequence chromatogramsof the mutant RT-PCR product of L825 showing splicing of CBL exon7 to exon 9.



perform this analysis for L919 but given the posi-

tion of the genetic lesion it is likely that this

patient too has exon 8 skipping. The CBL D8mutation has been previously reported in AML

and in a BAF3 cell line model it induced growth

factor-independence when cotransfected with

FLT3, causing autophosphorylation of FLT3 and

constitutive activation of the downstream targets

STAT5 and AKT (Caligiuri et al., 2007; Reindl

et al., 2009). FLT3 ligand-dependent hyperprolif-

eration of CBL mutant cells could be abrogated

by treatment with the FLT3 PTK inhibitor

PKC412, suggesting that CBL D8 AML may be

responsive to this class of targeted agent (Reindl

et al., 2009).

Therefore, pathway activation was investigated

in CBL mutated primary blasts, by western blot-

ting, using patient L825 cytosol for p-STAT5, p-

AKT expression, as well as for p-ERK, along

with appropriate positive and negative control

cell lines (Figs. 3A–3C). Patient L825 showed no

p-STAT5 or p-AKT but significant amounts of p-

ERK, indicative of activation of the RAS pathway

and at levels we have previously observed for

classic NRAS and KRAS mutations (Figs. 3A–3C)

(Case et al., 2008). Cell viability (MTS) assays

with patient L825 blasts were then performed

with both the MEK inhibitor U0126 and the

FLT3 inhibitor PKC412 (Figs. 3D and 3E,

respectively). For PKC412, sensitivity in patient

Figure 3. Pathway activation in CBL mutant primary ALL and cyto-toxicity to MEK and FLT3 inhibitors. Western blotting for: A: p-STAT5 expression in patient L825 with the HEL cell line (JAK2V617F) serving as a positive and PreB 697, a negative control andactin as loading control. B: p-ERK expression with CCRF CEM cells(KRAS G12D) serving as a positive and Nalm-6 cells as a negativecontrol, with ERK2 acting as a loading control. C: p-AKT expressionwith the PC3 cell line (PTEN deletion) serving as a positive and PreB697, serving as a negative control and AKT acting as a loading con-

trol. D: MTS viability assay with primary leukemic cells from patientL825 dosed with varying concentrations of the MEK inhibitor U0126and compared with the mean values (�SEM) calculated from resistantand sensitive primary ALL, previously characterized for RAS muta-tion, RAS pathway activation and sensitivity to U0126 and reportedin Case et al. (2008). E: MTS viability assays with L825 blasts, PreB697 cells (constitutively active FLT3) and Nalm-6 cells (FLT3 negative)dosed with varying concentrations of the FLT3 inhibitor PKC-412.

254 NICHOLSON ETAL.


L825 primary blasts was compared with cell line

controls that have been previously characterized

for FLT3 receptor activation and responsiveness

to FLT3 inhibition, i.e., PreB 697 serving as a

positive control (constitutively active FLT3; re-

sponsive) and Nalm-6 serving as a negative con-

trol (FLT3 receptor negative; nonresponsive)

(Brown et al., 2005). Patient L825 showed little

sensitivity to the drug, particularly when com-

pared with a cohort of MLL rearranged, FLT3

high, ALL primary samples reported by Stam

et al. (2005). However, L825 cells when dosed

with U0126 showed a significant cytotoxic

response and when compared with our published

primary ALL cohort characterized for RAS muta-

tion status, pathway activation and U0126 sensi-

tivity, were as sensitive as those with classic RAS

mutations and the highest levels of p-ERK

expression.

In terms of clinical details, CBL mutant posi-

tive patients were all B cell precursor ALL but

heterogeneous in terms of age, presenting white

cell count, CD10 expression, cytogenetics, and

clinical response (Table 1). For example, patient

L825, a 14-year-old girl, presented with a high

peripheral white cell count, apparently normal

cytogenetics and had a poor response to therapy

as gauged by an exceptionally high end of induc-

tion minimal residual disease (MRD) level of

1.3% and thus is at very high risk of relapse (Cou-

stan-Smith et al., 2000). The other two patients

were younger, presented with a low white cell

count, one pseudodiploid, one high hyperdiploid,

with the latter responding well to induction ther-

apy, with an end of induction MRD level of

<0.01%. In a large study of myeloid neoplasms,

the presence of CBL family mutations was associ-

ated with a poorer prognosis, particularly for indi-

viduals with homozygous mutations (Makishima

et al., 2009); however, other studies have found no

such relationship (Sanada et al., 2009).

In summary, this is the first report of somatic

mutation of CBL in ALL with a frequency of

1.7%, a similar incidence to AML. While uncom-

mon, it emphasizes the importance of RAS path-

way activation in the molecular pathology of

ALL and may identify children who, along with

other RAS pathway mutant positive cases, are

candidates for MEK inhibitor clinical trials.

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Casitas B lymphoma mutations in childhood acute lymphoblastic leukemia

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