Loss of CBL E3-ligase activity in B-lineage childhood acute lymphoblastic leukaemia
Martinelli et al.
Supporting Information
Materials and Methods
Patients
Children and adolescents with B-ALL (N= 275) and T-ALL (N= 30), and adult patients (B-ALL, N= 37; T-ALL, N= 58) selected only on the basis of DNA availability were included in the study. Informed consent was obtained for each patient of the cohorts. Diagnosis was established according to standard morphologic, cytochemical and immunological criteria, as previously reported (Tartaglia et al., 2004; Flex et al., 2008).
Within the collaborative prospective Associazione Italiana di Ematologia Pediatrica and Berlin-Frankfurt-Munster (AIEOP-BFM ALL) 2000 study of childhood ALL, minimal residual disease (MRD) was assessed during follow up by monitoring the patient- and clone-specific rearrangements of the immunoglobulin (Ig) and T-cell receptor (TCR) genes as PCR targets. Patients were defined as MRD standard risk (MRD-SR) if MRD was found to be negative at both days 33 (time point 1 [TP1]) and 78 (TP2), using at least 2 molecular markers with sensitivity of 10-4 or more. If MRD levels differed between the 2 markers, the highest MRD level was chosen for the final MRD assessment. Patients were considered to be MRD intermediate risk (MRD-IR) when MRD was positive at 1 or both TPs but at a level of less than 10-3 at TP2 with at least 2 markers. Patients with MRD equal to 10-3 or more at TP2 were defined MRD high risk (MRD-HR), independent of the sensitivity and the number of markers. Patients with prednisone poor response (≥1000 leukemic blasts/ml in the peripheral blood on day 8) or failure to achieve remission (i.e., with 5% leukemic blasts in the bone marrow on day 33, or persistent extramedullary disease) after induction phase IA (induction failure) or positivity for MLL/AF4 fusion transcript were treated in the HR arm irrespective of their MRD results.
Molecular analysis
Mutational screening was carried out on the CBL (exons 8 and 9), HRAS, KRAS and NRAS (exons 1 and 2), and PTPN11 (exons 2-4, and 13) genes by denaturing high-performance liquid chromatography (DHPLC, Transgenomic) with the Wave 2100 System (Transgenomic) at column temperatures recommended by Navigator software, version 1.6.4.12 (Transgenomic). Our analysis was confined to the indicated exons as they represent the target for cancer-relevant mutations affecting these genes. PCR reactions were performed as previously described (Tartaglia et al., 2004; Martinelli et al., 2010). Amplimers with abnormal denaturing profiles were purified (Microcon PCR [Millipore]) and then sequenced bidirectionally with the ABI BigDye Terminator Sequencing Kit v.1.1 (Applied Biosystems) and ABI Prism 3500 (Applied Biosystems). Homozygous/hemizygous condition for CBL sequence variations was explored by DHPLC using pooled DNAs.
Loss of heterozygosity (LOH) analysis was achieved by amplification of microsatellite markers encompassing the CBL gene listed in Suppl. Table 2 and fluorescent detection (ABI PRISM Linkage Mapping Set v.2 and 3130XL, Applied Biosystems). Quantitative real-time PCR (qRT-PCR) was attained using SYBR green and ABI7900 Sequence Detection systems (Applied Biosystems). CBL copy number was determined relative to the 2N TERT copies, and normalized by using the average of 4 control DNAs as calibrator, according to the ∆∆Ct method (Livak and Schmittgen, 2001). Primer sequences and PCR conditions are reported in Suppl. Table 3.
Functional studies
The c.1256G>T nucleotide substitution was introduced in the hemagglutinin (HA)-tagged human CBL cDNA cloned in the pALTER-MAX vector (Promega) by site-directed mutagenesis (QuikChange Kit, Stratagene). HEK293T cells were transiently transfected with full-length EGFR (0.05 μg), and wild-type or mutant CBL (2 μg), by using Lipofectamine 2000 (Invitrogen). Twenty-four h post-transfection, cells were serum-starved (12 h) and then stimulated with EGF (GIBCO) (1 h, 150 ng/ml). Functional studies were performed as previously described (Martinelli et al., 2010; Checquolo et al., 2010), using the following antibodies: anti-ubiquitin, anti-HA, anti-ERK1/2, and anti-phospho-ERK1/2 (Santa-Cruz Biotechnology); anti-EGFR, anti-AKT, and anti-phospho-AKT (Cell Signaling); anti-phospho-Tyr (Millipore); anti-β-actin (Sigma). For FACS analysis, cells were stained with mouse monoclonal anti-EGFR antibody or its isotype control IgG2a (Santa-Cruz Biotechnology), incubated with a FITC-conjugated anti-mouse secondary antibody (BD Biosciences), and analyzed on a FACS-Calibur (BD Biosciences). The EGFR-specific mean fluorescence intensity (MFI) for each transfection was expressed relative to the MFI of the corresponding unstimulated cells (arbitrarily set at 100%).
Statistics
Exact confidence intervals (CI) of proportions were calculated based on binomial distribution.
References
Checquolo, S., Palermo, R., Cialfi, S., Ferrara, G., Oliviero, C., Talora, C. et al. (2010) Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia. Oncogene, 29, 1463-1474.
Flex, E., Petrangeli, V., Stella, L., Chiaretti, S., Hornakova, T., Knoops, L. et al. (2008) Somatically
acquired JAK1 mutations in adult acute lymphoblastic leukemia. Journal of Experimental Medicine, 205, 751-758.
Livak, K.J., Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25, 402-408.
Martinelli, S., De Luca, A., Stellacci, E., Rossi, C., Checquolo, S., Lepri, F. et al. (2010) Heterozygous germline mutations in the CBL tumor-suppressor gene cause a Noonan syndrome-like phenotype. American Journal of Human Genetics, 87, 250-257.
Tartaglia, M., Martinelli, S., Cazzaniga, G., Cordeddu, V., Iavarone, I., Spinelli, M. et al. (2004) Genetic evidence for lineage-related and differentiation stage-related contribution of somatic PTPN11 mutations to leukemogenesis in childhood acute leukemia. Blood, 104, 307-313.
Supplementary Table 1. Clinical and biological features of patients with mutation in CBL.a
Age at diagnosis (years) / 4.5 / 9.3
WBC/μl / 43900 / 68650
Gender / male / female
DNA index / 1.164 / 1.0
Prednisone response / Poor / Good
Risk group / Intermediate / Intermediate
Relapse, site and time (years after dx) / BM (3.6) / testis (0.9)
BMT, type and time (years after dx) / MUD (5.9) / MMUD (1.3)
Last follow up (after BMT) / alive (3.5) / alive (6.6)
aBoth patients were CD10+ (common ALL), with relatively high WBC counts at diagnosis, and were stratified as “risk group” according to minimal residual disease, and were negative for t(4;11), t(9;22) and t(12;21) chromosomal translocations. BM, bone marrow; BMT, bone marrow transplantation; MUD, matched unrelated donor; MMUD, mismatched unrelated donor.
Supplementary Table 2. List of microsatellite markers encompassing the CBL gene (11q 23.3) examined for LOH analysis.
D11S898 / Chr11:101,056,444 / Chr11:101,056,752
D11S4090 / Chr11:113,081,250 / Chr11:113,081,576
D11S908 / Chr11:115,287,301 / Chr11:115,287,506
CBL / Chr11:119,076,990 / Chr11:119,178,858
D11S925 / Chr11:120,828,211 / Chr11:120,828,546
D11S4094 / Chr11:123,290,234 / Chr11:123,290,234
D11S4151 / Chr11:126,291,971 / Chr11:126,292,341
D11S912 / Chr11:128,624,093 / Chr11:128,624,208
aMap positions are collected from UCSC (http://genome.ucsc.edu/; GRCh37/hg19).
Supplementary Table 3. Primer pairs used in qRT-PCR analysis.a
(bp)
CBL / Forward: TTCATTTGTTGCAGGTGGTG
Reverse: ATTCTTTAGCGCCAGCTTTG / 56
TERT / Forward: CTGTGACACTTCAGCCGCAA
Reverse: GAGGAACATGCGTCGCAAA / 51
aqRT-PCR conditions were as follows: two initial steps at 50 °C (2 min) and 95 °C (10 min) were followed by 40 cycles [95 °C (15 sec), 60 °C (1 min)].