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Aug 8, 2010 - Juvenile myelomonocytic leukemia (JMML) is an aggres- sive childhood ... proliferative subtype of chronic myelomonocytic leukemia (CMML),.
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Germline CBL mutations cause developmental abnormalities and predispose to juvenile myelomonocytic leukemia Charlotte M Niemeyer1,18, Michelle W Kang2,18, Danielle H Shin2,18, Ingrid Furlan1, Miriam Erlacher1, Nancy J Bunin3, Severa Bunda4, Jerry Z Finklestein5, Kathleen M Sakamoto6, Thomas A Gorr1, Parinda Mehta7, Irene Schmid8, Gabriele Kropshofer9, Selim Corbacioglu10, Peter J Lang11, Christoph Klein12, Paul-Gerhard Schlegel13, Andrea Heinzmann1, Michaela Schneider1, Jan Starý14, Marry M van den Heuvel-Eibrink15, Henrik Hasle16, Franco Locatelli17, Debbie Sakai2, Sophie Archambeault2, Leslie Chen2, Ryan C Russell4, Stephanie S Sybingco4, Michael Ohh4, Benjamin S Braun2, Christian Flotho1 & Mignon L Loh2 CBL encodes a member of the Cbl family of proteins, which functions as an E3 ubiquitin ligase. We describe a dominant developmental disorder resulting from germline missense CBL mutations, which is characterized by impaired growth, developmental delay, cryptorchidism and a predisposition to juvenile myelomonocytic leukemia (JMML). Some individuals experienced spontaneous regression of their JMML but developed vasculitis later in life. Importantly, JMML specimens from affected children show loss of the normal CBL allele through acquired isodisomy. Consistent with these genetic data, the common p.371Y>H altered Cbl protein induces cytokine-independent growth and constitutive phosphorylation of ERK, AKT and S6 only in hematopoietic cells in which normal Cbl expression is reduced by RNA interference. We conclude that germline CBL mutations have developmental, tumorigenic and functional consequences that resemble disorders that are caused by hyperactive Ras/Raf/MEK/ERK signaling and include neurofibromatosis type 1, Noonan syndrome, Costello syndrome, cardiofaciocutaneous syndrome and Legius syndrome. Myeloproliferative neoplasms (MPNs) are clonal malignancies that are characterized by overproduction of immature and mature myeloid lineage cells. Juvenile myelomonocytic leukemia (JMML) is an aggressive childhood MPN that is characterized by malignant transformation

in the stem cell compartment with clonal proliferation of progeny that variably retain the capacity to differentiate1. Hematopoietic stem cell transplantation (HSCT) is the only cure for JMML; however, relapse rates approach 30% (ref. 2). Although spontaneous remissions occur in some infants3,4, the underlying mechanism for this is unknown. Germline and somatic mutations that deregulate Ras signaling are implicated as key initiating events in JMML; 60% of affected indivi­duals harbor an oncogenic mutation in the Ras signaling genes PTPN11, NRAS or KRAS and another 15% have mutations in the neurofibromatosis type 1 (NF1) gene (NF1) and show loss of the normal NF1 allele in leukemic cells5–9. Individuals with the myeloproliferative subtype of chronic myelomonocytic leukemia (CMML), a similar MPN in adults, frequently display NRAS, KRAS and JAK2 ­mutations10,11. Mouse models recapitulate these diseases, supporting the hypothesis that hyperactive Ras is necessary and sufficient to cause MPN12–15. A hallmark feature of JMML and CMML is the formation of abnormally high numbers of granulocyte-macrophage ­ colonyforming units (CFU-GM) in methylcellulose cultures containing low concentrations of granulocyte-macrophage colony-­stimulating factor (GM-CSF)16,17. Phosphorylation of the βc chain of the GM-CSF receptor creates docking sites for adapters and signal relay molecules, resulting in activation of the Ras pathway. Samples from individuals with MPNs can show copy-neutral loss of heterozygosity (acquired isodisomy) of a region on chromosome 11q and homozygous mutations in CBL18–21. Approximately 10–15%

1Department

of Pediatrics and Adolescent Medicine, University of Freiburg, Freiburg, Germany. 2Department of Pediatrics and the Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA. 3Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. 4Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. 5Miller Children’s Hospital/Harbor-UCLA, Jonathan Jaques Cancer Center, Long Beach, California, USA. 6Division of Hematology/Oncology, Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA. 7Division of Hematology-Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA. 8von Hauner Children’s Hospital, LMU Munich University, Munich, Germany. 9Department of Pediatrics and Adolescent Medicine, Medical University, Innsbruck, Austria. 10Department of Pediatrics, University of Ulm, Ulm, Germany. 11Department of Pediatrics, University of Tubingen, Tubingen, Germany. 12Department of Pediatrics, Medical School, Hannover, Hannover, Germany. 13Department of Pediatrics, University of Wuerzburg, Wuerzburg, Germany. 14Department of Pediatric Hematology and Oncology, Charles University Prague, Prague, Czech Republic. 15Department of Pediatric Oncology/Hematology, Erasmus Medical Center, Rotterdam, Netherlands. 16Department of Pediatrics, Aarhus University Hospital Skejby, Aarhus, Denmark. 17Department of Pediatric Hematology and Oncology, IRCCS Ospedale Bambino Gesu, University of Pavia, Rome, Italy. 18These authors contributed equally to this work. Correspondence should be addressed to M.L.L. ([email protected]) or C.M.N. ([email protected]). Received 4 December 2009; accepted 12 July 2010; published online 8 August 2010; doi:10.1038/ng.641

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VOLUME 42 | NUMBER 9 | SEPTEMBER 2010  Nature Genetics

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letters of children with de novo JMML are estimated to harbor homozygous CBL mutations20,22. CBL mutations are acquired somatically in adults with MPNs18,19,21. As children with NF1 and Noonan syndrome are predisposed to JMML8,9,23–25, we tested whether germline CBL mutations occur in affected children. Twenty-one children with JMML were found to have CBL mutations (16 of 21 were previously included in a screen of a larger international cohort20) and an unexpectedly high percentage of these children showed developmental delay, cryptorchidism and impaired growth (Tables 1 and 2). All children met diagnostic criteria for JMML26,27 but six individuals who were followed up for more than seven years did not undergo transplantation for various reasons. Of these, one died of progressive JMML (D088), but the MPN improved spontaneously in five others. All of these subjects continued to show variable degrees of splenomegaly in the presence of normal blood counts and had a persistent homozygous CBL mutation in CD4, CD8, CD14 and CD19 sorted cells from their peripheral blood at their last follow-up. In addition, four of these subjects developed clinical signs consistent with vascular pathology, including optic atrophy, hypertension and an acquired cardiomyopathy; one was diagnosed with Takayasu arteritis, type III by angiography (Fig. 1a). Another subject (D256) developed an intracranial germinoma harboring the same homozygous CBL mutation as in his bone marrow. Of note, among the patients treated with HSCT, there was a high rate of conversion to stable mixed chimerism (9 of 11 patients for whom data are available; Table 1). We analyzed normal tissues from 17 of these children and detected a heterozygous CBL mutation in each. Mutational analysis of parental DNA was informative in 13 families and confirmed autosomal inheritance of a CBL mutation in seven (Fig. 1b,c, Table 2 and Supplementary Fig. 1). Subjects UPN1333 and UPN1125 were from large pedigrees in which several individuals had died of JMML (Fig. 1b,c).

The proband in family 1 (UPN1333, V:1; Fig. 1b) was referred after transplantation for JMML. Initially diagnosed at 7 months of age, he received his first HSCT at 13 months and then developed mixed ­chimerism 6 months later. A blood sample showed a homozygous CBL mutation at c.1111T>C (p.371Y>H). Importantly, analysis of buccal swab DNA revealed a heterozygous lesion. Both maternal relatives died from progressive JMML. Peripheral blood or buccal swabs from extended family members revealed multiple heterozygous individuals (Fig. 1b). Detailed medical history from the affected maternal greatgrandmother revealed a history of infant leukemia characterized by a high white blood cell count and splenomegaly that resolved spontaneously. Sorted B (CD19) and T (CD3) cells and granulocytes from her peripheral blood showed a heterozygous c.1111T>C mutation. Leukemia cells from UPN1333 showed a classic pattern of GM-CSF hypersensitivity (Fig. 1d) and increased phosphorylation of STAT5 in response to low doses of GM-CSF28. Peripheral blood mono­nuclear cells from his heterozygous mother did not show either of these features, suggesting that homozygosity for the mutant CBL allele is essential for these characteristics (data not shown). The proband in family 2 (Fig. 1c; UPN1125, IV:3) was a girl diagnosed at 15 months of age with JMML. She also harbored a homozygous c.1111T>C CBL mutation in her bone marrow. Family history revealed that her mother had two male first cousins who were diagnosed with JMML and died before age 10. Frozen liver tissue from the first boy was available from autopsy and showed a heterozygous c.1111T>C mutation. Notably, he also developed clinical signs and laboratory test results consistent with small vessel vasculitis before his death. The mother of UPN1125 was found to carry a heterozygous CBL mutation (Fig. 1c). Three subjects showed homozygous splice site mutations (Table 2; I066, D647 and D347). RT-PCR showed several new splice products arising from these mutations (Fig. 2); most notable are the two splice

Table 1  Clinical features of 21 children with homozygous CBL mutations in JMML cells HSCTb

Age and hematological features at diagnosis ID A053 A054 D256 D048 D389 D088 NL075 CZ039 D451 D647 I066 SC084 D104 D347 D703 UPN1333 D251 UPN1778 UPN1125 D774 UPN1241

Sex

Age (y)

M F M F M M F M M F F M M M F M F M F F F

0.1 1.1 3.6 1.6 0.6 0.8 1.5 1.1 0.7 2.5 5.0 1.4 2.2 0.9 1.4 0.6 0.6 1.3 1.3 1.5 0.6

WBC

(×109/l)

112 36 13 23 14 46 55 29 27 67 21 33 27 72 27 36 31 22 196 22 43

% blasts PB/BM

Platelets

1/2 1/4 1/6 0/2 0/6 3/NA 5/2 3/4 2/3 1/0 2/10 0/0 0/2 4/6 2/1 3/4 0/20 0/1.5 4/5 0/4 5/3

(×109/l)

20 95 18 46 67 32 55 284 86 117 33 33 232 48 15 46 37 61 47 266 62

Spleen 9 7 10 9 8 12 12 6 6 6 5 13 5 10 4 4 4 5 4 3 4

sizea

Last follow-up

Karyotype

Age (y)

MC/CC

Alive/dead

Age (y)

Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal 45,XY,–16 46,XX+der (8) Normal Normal Normal Normal Normal Normal

– – – – – – 1.8 1.2 1.2 3.9 5.7 2.0 – 1.2 1.7 1.1 1.1 – 1.7 – 1.1

– – – – – – MC MC/CCc MC/CCd MC MC NA – MC MC MC CC – CC – MC

A A A D A D A A A A D A D D A D D A D A D

18.2 17.7 13.0 9.9 7.5 7.3 7.3 7.0 6.2 6.1 6.0 4.8 3.9 3.4 3.0 2.6 2.4 2.0 2.1 1.5 1.5

ID, subject identification; M, male; F, female; WBC, white blood count; PB, peripheral blood; BM, bone marrow; HSCT, hematopoietic stem cell transplantation; A, alive; D, dead; MC, mixed chimerism; CC, complete chimerism; NA, not analyzed. aSpleen

size given in cm below costal margin in left midclavicular line. bAll subjects had received a myeloablative preparative regimen before HSCT. One individual with MC died of progressive disease (UPN 1333) and the other deaths were due to transplant-related toxicities. cSubject converted back to CC after donor lymphocyte infusion. dSubject converted back to CC without therapy.

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letters Table 2  CBL mutation and non-hematological features in 21 children with JMML CBL mutation ID

Mutation

A053a A054 D256b D048c D389d D088 NL075 CZ039 D451 D647

p.396C>R p.371Y>H p.408W>R p.384C>R p.371Y>C p.371Y>D p.371Y>H p.404C>R p.371Y>H c.1228–2A>G splice site c.1228–2A>G splice site p.371Y>H p.384C>R c.1096–1G>C splice site

I066

© 2010 Nature America, Inc. All rights reserved.

SC084 D104e D347f D703 UPN1333 D251g UPN1778h UPN1125 D774i UPN1241

p.384C>R p.371Y>H p.380L>P p.371Y>N p.371Y>H p.398H>R p.371Y>H

Germ.

Mo.

Fa.

Café au lait spotsj

JXG

Cryptorchidism

Growth < 3rd percentile

Dev. delay

Hearing loss

Optic atrophy

+ + + NA + NA + + + +

− + − NA − NA − − − −

NA NA − NA + NA − − − −

− − + − + − − − − −

− + + − − − + − − −

− Female − Female + + Female − + Female

− − + + − + − − − +

+ − + + + − − − + +

+ − − − + − − − − −

+ − + + + − − − − −

+ − + + + − − − − −

+ − − + + − − − − −

NA

NA

NA





Female













+ NA +

+ NA −

− NA NA

− − +

− + +

− − +

− − +

+ − +

− − −

− − −

− − −

− − −

+ + + + + + +

− + NA − + + −

+ − NA NA − − −

− − − − − − +

− − + − − − −

Female − Female − Female Female Female

− − + − − − −

− + + − − − −

− + − − − − −

− − − − − − −

− − − − + − −

− − − − − − −

HyperCardiotensionk myopathy

ID, subject identification; Germ., germline; Mo., mother; Fa., father; NA, not analyzed; JXG, juvenile xanthogranuloma; Dev., developmental. aAutoimmune

thyroditis with anti-thyroglobulin antibodies; echocardiography with mitral insufficiency grade II, myocardial hypertrophy, hypoelastic and hypoplastic ascending aorta and arch of aorta. bDiagnosis of intracranial germinoma at age 9 y with homozygous CBL mutation in the brain tumor. cDiagnosed with Takayasu arteritis, type III, died 9 months after diagnosis of vasculitis and stenting of major arteries. dFather with history of Hodgkin lymphoma. eSudden death at home. fDied of cerebral hypoxia, also had a history of supraventricular tachycardia. gBefore HSCT, was diagnosed with erythroderma; post-HSCT with toxic epidermal necrolysis, acute liver failure, liver transplant, subtotal brain infarction, apallic syndrome. hPectus excavatum. iOlder sister with heterozygous mutation. jSubjects indicated by + have 1–2 café au lait spots. There was no subject with 6 or more café au lait spots. kMeasurements of blood pressure after HSCT were excluded from analysis.

site variants that delete either exon 8 (D347) or exon 9 (I066 and D647; Supplementary Fig. 2) or retain an interstitial intron (for example, intron 7 for D347). Each of the deletion splice variants encodes a protein that is predicted to lack essential regions of the linker and RING finger domains, whereas the retention of intron 7 introduces a premature stop codon to abort translation upstream of the RING finger domain. To investigate the functional properties of the mutant Cbl proteins encoded by homozygous point mutations, we first studied the effect of the common p.371Y>H substitution on the growth of primary hematopoietic cells from mouse fetal liver. This system reproduces the hypersensitivity to GM-CSF that is characteristic of JMML 16,29. Fetal liver cells transduced with retroviral vectors expressing wild-type or mutant Cbl proteins showed no increased sensitivity to GM-CSF (Fig. 3a). Similarly, expression of p.371Y>H Cbl in a BaF3-EpoR cell line did not confer cytokine independence (Fig. 3b). Based on the observation that JMML cells invariably lose the normal CBL allele and on recent findings21, we reasoned that reduction of Cbl expression might be mandatory to deregulate hematopoietic growth. Therefore, we introduced a short hairpin RNA, which reduced the expression of mouse Cbl in BaF3-EpoR cell lines (Fig. 3c). We next transduced these cells with a series of wild-type and mutant human constructs and found that exogenous Cbl was strongly expressed (Fig. 3c). In this context, we observed cytokine-independent proliferation (Fig. 3d) upon expression of p.371Y>H Cbl or a known mouse oncogenic Cbl protein (70Z) 30–32. The 70Z oncogenic protein deletes 17 amino acids from position 366–382 in the linker domain. Furthermore, cells transduced with p.371Y>H or 70Z showed hypersensitivity 796

to ­increasing ­concentrations of human EPO (Fig. 3e), mimicking the growth factor hypersensitivity seen in JMML (Fig. 1d). In cells depleted of endo­genous Cbl and expressing exogenous p.371Y>H or 70Z Cbl ­proteins, we observed constitutive phosphorylation of ERK, AKT and S6 in cells deprived of cytokine. We also found heightened responses to low doses of EPO (Fig. 3f). To determine whether mutant Cbl proteins retain E3 ligase acti­ vity, we assessed their ability to promote ubiquitylation of a known Cbl substrate. Soon after activation, the epidermal growth factor receptor (EGFR) undergoes Cbl-dependent polyubiquitylation and proteosomal degradation. HEK293 cells expressing hemagglutinintagged (HA)-Cbl(p.371Y>H) showed markedly elevated levels of phosphorylated EGFR upon EGF stimulation in comparison to cells expressing HA-Cbl(WT) (Fig. 4a), which indicates a possible defect in clearance of pEGFR by Cbl(p.371Y>H). The Cbl(p.384C>R) mutant showed a ­similar defect in pEGFR clearance (data not shown). Upon polyubiquitylation of targets, Cbl promotes its own polyubiquitylation and subsequent auto-degradation. Consistent with this model, levels of Cbl(WT), but not of Cbl(p.371Y>H or p.384C>R), rapidly diminished after EGF treatment in the absence of the proteasome inhibitor MG132 (Fig. 4a,b and data not shown,). However, Cbl(WT) levels were stabilized in the pre­sence of MG132 whereas Cbl(p.371Y>H) levels remained relatively high irrespective of MG132 (Fig. 4b), which suggests that the Cbl mutants p.371Y>H and p.384C>R have an inherent defect in E3 function. Consistent with this notion, Cbl(WT) promoted robust polyubiquitylation of pEGFR whereas p.371Y>H and p.384C>R showed diminished capacity to polyubiquitylate pEGFR in an in vitro ubiquitylation assay (data not shown). This diminished capacity is similar to that of 70Z, which is defective in E3 ligase VOLUME 42 | NUMBER 9 | SEPTEMBER 2010  Nature Genetics

letters a

2

II:

3 II: W4 T II: 5

6

I:

1 I:

II:

II T> : 2 C

1 1 kb 700 bp 500 bp

2

3

4

5

4

3

14

13

II: :7 III

II:

II T> I : 6 C

II:

12

I: 11

I: 10

CBL p.371Y>H WT

20

:8

0

0.01 0.10 1.00 10.00 GM-CSF dose (ng/ml)

III

:6

:7

III

III

0 :1 IV

:8

II:

40

V:

4

IV

II:

60

e IV W :9 T

:4 II T> I: 5 C

III

V:

3

IV :6 IV W :7 T

III

:3

II T> I: 2 C IV W :4 T IV T> : 5 C

:1 III

:3 IV

V W :2 T

IV T> : 2 C

IV W :1 T

V T :1 T> >C C

© 2010 Nature America, Inc. All rights reserved.

8

80

activity, and agrees with recent finidings that p.371Y>S and p.367Q>P mutants in human subjects also have diminished ubiquitylation acti­ vity21. The Y371 residue that is most commonly affected in germline CBL mutations has been the focus of extensive biochemical analysis21. This analysis supports the idea that Y371 in essential for maintaining the integrity of the alpha-helical structure of the linker region, which has a critical role in substrate specificity. Notably, Y371 has been found to be phosphorylated despite its predicted location away from the protein surface. Substitution of this residue with phenyl­ alanine results in a non-oncogenic form of the protein, which lacks E3 ligase activity. Conversely, substitution with a glutamate constitutively activates the E3 ligase activity33,34. CBL appears to function as a classic tumor suppressor gene in this cohort, with germline heterozygosity predisposing to neoplasia 1

9

100

0

a

II:

7

II:

II:

6

5

% maximum colony growth

d

II:

II:

IV T> : 3 C

:2 IV

b

II:

II T> I : 5 C

II W :4 T

3 II:

:3 III IV

:1

III W :4 T

:2 III

III

:1

II:

II:

1

2

I:

I:

1

2

c

6

7

8

b

TKB domain

upon reduction to homozygosity in target tissues. However, the predominance of specific missense mutations makes CBL distinct from most tumor suppressor genes such as RB and NF1, which have more severe loss of function mutations such as deletion or protein trun­ cation. This suggests that the mutant Cbl proteins retain an essential biochemical function20,21, which is supported by our data showing that exclusive expression of a mutant CBL allele has positive effects on cytokine signaling and proliferation. The specific disruption of E3 ligase acti­vity might leave intact adaptor functions, resulting in a relative ­imbalance of Cbl’s role in signal transduction. Sanada et al. hypothesized that this might result in inhibitory effects on Cbl-b, a related family member21. This is similar to what has been reported for p53, a classic tumor suppressor gene with specific gain-offunction mutations in the context of loss of heterozygosity35,36.

Linker

WT

exon 6

exon 7

D347-A

exon 6

exon 7

Figure 1  Autosomal dominant germline mutations in CBL are associated with a phenotype, GM-CSF hypersensitivity and vasculitis. (a) Angiograms from the aorta and left subclavian artery from subject D048 nine months after the diagnosis of Takayasu arteritis type III. (b) The family tree of UPN1333, whose diseased bone marrow showed a homozygous CBL c.1111T>C (red) mutation as well as a heterozygous lesion from his buccal swab (black). Only women appear to be heterozygote carriers, and only boys appear to be affected by JMML in this family. (c) The bone marrow of UPN1125 showed a homozygous CBL mutation. Her mother (III:5) is a known carrier, and two male cousins died from JMML (III:6, III:7). (d) GM-CSF hypersensitivity response on a colony assay for subjects with CBL mutations (n = 3) versus normal (n = 13). Error bars represent s.e.m. (e) A toddler (D703) diagnosed with JMML and a homozygous mutation at p.384C>R. She displays frontal bossing, downslanting palpebral fissures, hypertelorism, and a low nasal bridge. Her father harbors a heterozygous mutation at p.384C>R (Supplementary Fig. 1d). Both father and daughter also show bilateral ptosis. Informed consent to publish the photograph in e was obtained from the subject’s father.

RING finger exon 8

exon 8

Corresponding amplicon length

Pro-rich exon 9

exon 10

616 bp

exon 9

exon 10

544 bp

exon 9

exon 10

484 bp

∆72 bp

300 bp D347-B

exon 6

exon 7

100 bp

∆ exon 8 (132 bp) D347-C

exon 6

exon 7

*

intron 7

exon 8

exon 9

exon 10

937 bp

ins 321 bp Figure 2  Consequences of splice site mutations in cDNA from individuals * D647-A exon 9 exon 6 exon 10 exon 7 exon 8 570 bp D347, D647 and I066. (a) RT-PCR I066-A using an exon 6 forward primer and an ∆46 bp exon 10 reverse primer on cDNA generated * D647-B exon 10 exon 9 549 bp exon 6 exon 8 exon 7 from these subjects. The wild-type (WT) I066-B ∆67 bp amplicon is 616 bp long. Lane 1: MW ladder; lane 2: I066; lane 3: D347; D647-C exon 10 exon 6 exon 7 exon 8 412 bp I066-C lane 4: D647; lane 5: CBL point mutant; ∆ exon 9 (204 bp) lane 6: HM2833 CBL wild type; lane 7: genomic DNA control; lane 8: no template control. (b) Schematic representation of the splice site variants detected either recurrently (D347) or shared by I066 and D647 (see Supplementary Fig. 2 for sequences). Δ, deletions; ins, insertions; *premature stop codons.

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20 0

0

Number of cells (× 106)

200

50 kDa

Baf3-EpoR 2364 + pMIV 2364 + WT-CBL 2364 + 70Z 2364 + Y371H

100 50 0

1

2364 + 70Z

2364 + Y371H 0 0.01 0.10 1.00

1.00

2364 + WT-CBL

ERK pAKT AKT

e 150

2364 + pMIV

pERK

0

6

2364 + pMIV 2364 + WT-CBL 2364 + 70Z 2364 + Y371H 120 kDa

hEPO U/ml

0 1 2 3 4 5 6 7 8 9 10 Days

0

© 2010 Nature America, Inc. All rights reserved.

50

d Baf3-EpoR

Tubulin

100

0.01 0.10 1.00 10.00 [GM-CSF] ng/ml

c

Total Cbl

150

Baf3-EpoR

0 0.01 0.10

6

40

200

0 0.01 0.10 1.00

60

Number of cells (× 10 )

80

f

BaF3-EpoR pMIG WT-CBL 70Z Y371H BaF3-EpoR + IL-3 pMIG + IL-3 WT-CBL + IL-3 70Z + IL-3 Y371H + IL-3

250

0 0.01 0.10 1.00

b

pMIG WT-CBL 70Z Y371H

100

Number of cells (× 10 )

% maximum colony growth

a

0 0.01 0.10 1.00

letters

3

5 Days

7

9

Baf3-EpoR 2364 + pMIV 2364 + WT-CBL 2364 + 70Z 2364 + Y371H

30

pSTAT5 STAT5 pS6

20

S6

10

Tubulin 0

11

0

0.01 0.10 [hEPO] U/ml

1.00

Figure 3  p.371Y>H does not confer cytokine sensitivity or cytokine independent growth until silencing of mouse Cbl. (a) Transduction of p.371Y>H or the known mouse oncogenic mutant 70Z in wild-type hematopoietic cells from fetal liver did not confer hypersensitivity to GM-CSF. (b) Expression of the same mutants in BaF3-EpoR cells did not result in cytokine-independent growth. (c) Protein blot showing that an shRNA to mouse Cbl (cbl.2364) caused near complete shutdown of expression in BaF3-EpoR cells with re-expression upon introduction of the human wild-type, 70Z or p.371Y>H Cbl proteins. (d) Both the p.371Y>H and 70Z Cbl mutants conferred cytokine-independent growth in the presence of cbl.2364. Controls included the Venus vector pMIV and BaF3-EpoR. Error bars for triplicate replicates (s.e.m.) are shown and when not visible, indicate tight clustering. Using a paired t-test: cbl.2364 + WT-Cbl compared with cbl.2364 + p.371Y>H at day 7, P = 0.017; at day 9, P < 0.001. (e) Serial transduction of the hairpin (cbl.2364) and p.371Y>H or 70Z constructs also conferred hypersensitive growth when we assessed cell proliferation on day 5 in increasing concentrations of Epo. Using a paired t-test at each concentration of Epo when comparing cbl.2364 + WT-Cbl with cbl.2364 + p.371Y>H: Epo 0 U/ml: P = 0.036; Epo 0.01 U/ml: P = 0.0015; Epo 0.1 U/ml: P = 0.029; Epo 1 U/ml (saturating dose), P = 0.697. (f) Cells containing either p.371Y>H or 70Z showed activation of pERK, pAKT and pS6 in the absence of Epo or in low dose 0.01 U/mL of Epo in comparison to negative controls. All cell proliferation work was done in triplicate.

Beyond missense mutations, truncated Cbl proteins also confer transforming effects by countering the negative action of full-length Cbl on RTK signaling37. Some individuals can experience spontaneous resolution of JMML but later develop clinical features consistent with vasculitis and other autoimmune phenomena. Mice that lack Cbl in T cells develop severe vascular lesions with massive infiltration of T cells and high concentrations of anti-double stranded DNA antibodies38. These T cells are hypersensitive to T cell receptor signaling and show prolonged ERK phosphorylation. Similarly, mice that lack Cbl in B cells develop a lupus-like syndrome associated with perivascular infiltration and hyperactivation of B cell receptor signaling39. In mice, loss of Cbl-b is required for these phenotypes. Oncogenic Cbl proteins might therefore inhibit Cbl-b in vivo, resulting in a functional loss of both Cbl and Cbl-b, which in turn contributes to dysregulated lymphocyte signaling and subsequent vasculitis. The redundant roles of Cbl and Cbl-b were explored by Sanada et al., who showed that c-Cbl p.371Y>S inhibits

wild-type Cbl-b21. Individuals with JMML and homozygous CBL mutations who undergo HSCT are not known to develop vasculitis later in life, implying that a normal immune system is essential for preventing this late manifestation. We describe a new syndrome in which affected children display several congenital anomalies that overlap with NF1, Noonan syndrome and Legius syndrome, suggesting that the affected proteins converge on the Ras/MAPK pathway. Indeed, several Ras/MAPK pathway proteins regulate developmental programs in multiple species: for instance, the Drosophila homologs of each of

a EGF(min)

HA-Cbl-WT + EGFR

HA-Cbl-Y371H + EGFR

0 15 30 60 120

0 15 30 60 120

Anti-pEGFR

Anti-EGFR

Figure 4  Cbl mutant proteins show prolonged protein turnover and are associated with increased phosphorylated EGFR upon EGF stimulation. (a) HEK293 cells transfected with plasmids encoding EGFR in combination with HA-Cbl(WT) or HA-Cbl(p.371Y>H) were serum starved for 18 h followed by 15 min EGF (50 ng/ml) stimulation. Cells were then washed and maintained in serum-free medium for the indicated periods of time. Equal amounts of whole-cell extracts were resolved on SDS-PAGE and immunoblotted with the indicated antibodies. (b) HEK293 cells transfected with plasmids encoding EGFR in combination with HA-Cbl(WT) or HA-Cbl(p.371Y>H) were serum starved for 18 h followed by 15 min EGF (50 ng/ml) stimulation. Cells were then washed and maintained in serum-free medium with (+) or without (−) MG132 for the indicated periods of time. Equal amounts of whole-cell extracts were resolved on SDS-PAGE and immunoblotted with the indicated antibodies.

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Anti-tubulin

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HA-Cbl-WT + EGFR

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VOLUME 42 | NUMBER 9 | SEPTEMBER 2010  Nature Genetics

© 2010 Nature America, Inc. All rights reserved.

letters these genes (CBL (D-cbl), PTPN11 (csw), NF1 and SPRED) perform crucial functions for growth and patterning40–43. Individuals with germline CBL mutations are at increased risk of developing JMML, which might follow an aggressive clinical course or resolve without treatment. Some affected individuals develop vasculitis later in life. The CBL mutations found in JMML can arise de novo or can be transmitted through the germline, and human leukemia samples invariably show loss of the normal CBL allele. Consistent with this tumor suppressor function, JMML-associated Cbl proteins confer cytokine hypersensitivity in transduced BaF3-EpoR cells in the absence of wild-type Cbl, have defective E3 ligase activity, and constitutively activate key Ras effector pathways. The role of ­aberrant Cbl signaling in vasculitis remains to be determined, and it will be interesting to investigate whether individuals with germline CBL mutations who have been cured of JMML after HSCT remain at risk of developing vasculitis. It is also of great interest that some of these individuals continue to show homozygous CBL mutations in their peripheral blood despite having improved blood counts. Finally, our data provide strong evidence that Cbl is a key negative regulator of Ras signaling networks in hematopoietic cells and it will be important to identify key targets of the Cbl ubiquitin ligase to uncover other biochemical mechanisms involved in growth control. Note added in proof: Heterozygous germline mutations in CBL have recently been reported to cause a Noonan Syndrome-like phenotype. Martinelli S. et al. Heterozygous germline mutations in the CBL tumor-suppressor gene cause a Noonan Syndrome-like phenotype. Am. J. Hum. Genet. published online, doi:10.1016/j.ajhg.2010.06.015 (7 July 2010). Methods Methods and any associated references are available in the online ersion of the paper at http://www.nature.com/naturegenetics/. Accession code. GenBank: nm_005188.2. Note: Supplementary information is available on the Nature Genetics website. Acknowledgments We gratefully acknowledge the generous participation of the families included in this report. Supported in part by the US National Institutes of Health (CA113557 to M.L.L.); the V Foundation for Cancer Research (M.L.L. and B.S.B.); NIH/NCI (K08 CA103868 to B.S.B., R01 CA104282 to M.L.L. and B.S.B.); The Leukemia Lymphoma Society (6059-09, 2157-08 to M.L.L.); the Frank A. Campini Foundation (M.L.L. and B.S.B.); The Concern Foundation (B.S.B.); Deutsche Forschungsgemeinschaft (KR3473/1-1 to C.F.); Deutsche Krebshilfe (108220 to C.M.N. and C.F.); Deutsche José Carreras Leukämie-Stiftung (R08/19 to C.F.); the Canadian Cancer Society (16056 to M.O.); and the National Institute of General Medical Sciences (T32GM007618 to D.H.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. AUTHOR CONTRIBUTIONS C.M.N. coordinated and collected clinical data from the subjects with EWOG-MDS and wrote the manuscript; M.W.K. collected clinical data, performed laboratory assays including sequencing, proliferation assays and prepared figures; D.H.S. performed laboratory assays including the shRNA experiments, the proliferation assays and protein blots; I.F. collected clinical data; M.E. collected subject samples and performed mutational analysis on highly purified populations of blood cells; N.J.B. contributed subject samples; S.B. performed ubiquitin assays; J.Z.F. and K.M.S. contributed subject samples; T.A.G. performed RNA isolation and cDNA sequencing; P.M. contributed subject samples; I.S., G.K., S.C., P.J.L., C.K. and P.G.S. contributed subject samples; A.H. provided age-matched control samples from children with asthma; M.S. performed mutational analysis; J.S., M.M.v.H., H.H. and F.L. contributed subject samples and collected clinical data; D.S. collected clinical data; S.A. performed colony assays and performed cDNA sequencing; L.C. collected clinical data; R.C.R. and S.S.S. performed ubiquitylation assays; M.O.

Nature Genetics  VOLUME 42 | NUMBER 9 | SEPTEMBER 2010

supervised the ubiquitylation assays and wrote the manuscript; B.S.B. oversaw the shRNA and cell proliferative experiments and wrote the manuscript; C.F. collected clinical data and performed sequencing; M.L.L. coordinated and collected clinical and laboratory data from the USA, oversaw all of the laboratory work, coordinated the data and wrote the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at http://www.nature.com/naturegenetics/. Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/.

1. Niemeyer, C.M. & Kratz, C.P. Paediatric myelodysplastic syndromes and juvenile myelomonocytic leukaemia: molecular classification and treatment options. Br. J. Haematol. 140, 610–624 (2008). 2. Locatelli, F. et al. Hematopoietic stem cell transplantation (HSCT) in children with juvenile myelomonocytic leukemia (JMML): results of the EWOG-MDS/EBMT trial. Blood 105, 410–419 (2005). 3. Matsuda, K. et al. Spontaneous improvement of hematologic abnormalities in patients having juvenile myelomonocytic leukemia with specific RAS mutations. Blood 109, 5477–5480 (2007). 4. Flotho, C. et al. Genotype-phenotype correlation in cases of juvenile myelomonocytic leukemia with clonal RAS mutations. Blood 111, 966–967 author reply 967–968 (2008). 5. Niemeyer, C.M. et al. Chronic myelomonocytic leukemia in childhood: a retrospective analysis of 110 cases. European Working Group on Myelodysplastic Syndromes in Childhood (EWOG-MDS). Blood 89, 3534–3543 (1997). 6. Kalra, R., Paderanga, D., Olson, K. & Shannon, K.M. Genetic analysis is consistent with the hypothesis that NF1 limits myeloid cell growth through p21ras. Blood 84, 3435–3439 (1994). 7. Loh, M.L. et al. Somatic mutations in PTPN11 implicate the protein tyrosine phosphatase SHP-2 in leukemogenesis. Blood 103, 2325–2331 (2004). 8. Shannon, K.M. et al. Loss of the normal NF1 allele from the bone marrow of children with type 1 neurofibromatosis and malignant myeloid disorders. N. Engl. J. Med. 330, 597–601 (1994). 9. Tartaglia, M. et al. Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat. Genet. 34, 148–150 (2003). 10. Levine, R.L. et al. The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia. Blood 106, 3377–3379 (2005). 11. Onida, F. et al. Prognostic factors and scoring systems in chronic myelomonocytic leukemia: a retrospective analysis of 213 patients. Blood 99, 840–849 (2002). 12. Braun, B.S. et al. Somatic activation of oncogenic Kras in hematopoietic cells initiates a rapidly fatal myeloproliferative disorder. Proc. Natl. Acad. Sci. USA 101, 597–602 (2004). 13. Chan, I.T. et al. Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease. J. Clin. Invest. 113, 528–538 (2004). 14. Le, D.T. et al. Somatic inactivation of Nf1 in hematopoietic cells results in a progressive myeloproliferative disorder. Blood 103, 4243–4250 (2004). 15. Mohi, M.G. et al. Prognostic, therapeutic, and mechanistic implications of a mouse model of leukemia evoked by Shp2 (PTPN11) mutations. Cancer Cell 7, 179–191 (2005). 16. Emanuel, P.D., Bates, L.J., Castleberry, R.P., Gualtieri, R.J. & Zuckerman, K.S. Seletive hypersensitivity to granulocyte-macrophage colony stimulating factor by juvenile chronic myeloid leukemia hematopoietic progenitors. Blood 77, 925–929 (1991). 17. Ramshaw, H.S., Bardy, P.G., Lee, M.A. & Lopez, A.F. Chronic myelomonocytic leukemia requires granulocyte-macrophage colony-stimulating factor for growth in vitro and in vivo. Exp. Hematol. 30, 1124–1131 (2002). 18. Dunbar, A.J. et al. 250K single nucleotide polymorphism array karyotyping identifies acquired uniparental disomy and homozygous mutations, including novel missense substitutions of c-Cbl, in myeloid malignancies. Cancer Res. 68, 10349–10357 (2008). 19. Grand, F.H. et al. Frequent CBL mutations associated with 11q acquired uniparental disomy in myeloproliferative neoplasms. Blood 113, 6181–6192 (2009). 20. Loh, M.L. et al. Mutations in CBL occur frequently in juvenile myelomonocytic leukemia. Blood 114, 1859–1863 (2009). 21. Sanada, M. et al. Gain-of-function of mutated C–CBL tumour suppressor in myeloid neoplasms. Nature 460, 904–908 (2009). 22. Makishima, H. et al. Mutations of e3 ubiquitin ligase cbl family members constitute a novel common pathogenic lesion in myeloid malignancies. J. Clin. Oncol. 27, 6109–6116 (2009). 23. Bader-Meunier, B. et al. Occurrence of myeloproliferative disorder in patients with the Noonan syndrome. J. Pediatr. 130, 885–889 (1997). 24. Tartaglia, M. et al. PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity. Am. J. Hum. Genet. 70, 1555–1563 (2002).

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letters activation by tyrosine to glutamate point mutations. J. Biol. Chem. 279, 28017–28027 (2004). 34. Thien, C.B. & Langdon, W.Y. Cbl: many adaptations to regulate protein tyrosine kinases. Nat. Rev. Mol. Cell Biol. 2, 294–307 (2001). 35. Dittmer, D. et al. Gain of function mutations in p53. Nat. Genet. 4, 42–46 (1993). 36. Lang, G.A. et al. Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome. Cell 119, 861–872 (2004). 37. Robertson, H., Hime, G.R., Lada, H. & Bowtell, D.D. A Drosophila analogue of v-Cbl is a dominant-negative oncoprotein in vivo. Oncogene 19, 3299–3308 (2000). 38. Naramura, M. et al. c-Cbl and Cbl-b regulate T cell responsiveness by promoting ligand-induced TCR down-modulation. Nat. Immunol. 3, 1192–1199 (2002). 39. Kitaura, Y. et al. Control of the B cell-intrinsic tolerance programs by ubiquitin ligases Cbl and Cbl-b. Immunity 26, 567–578 (2007). 40. Hashimoto, S., Nakano, H., Singh, G. & Katyal, S. Expression of Spred and Sprouty in developing rat lung. Mech. Dev. 119Suppl 1, S303–S309 (2002). 41. Oishi, K. et al. Transgenic Drosophila models of Noonan syndrome causing PTPN11 gain-of-function mutations. Hum. Mol. Genet. 15, 543–553 (2006). 42. Pai, L.M., Barcelo, G. & Schupbach, T. D-cbl, a negative regulator of the Egfr pathway, is required for dorsoventral patterning in Drosophila oogenesis. Cell 103, 51–61 (2000). 43. The, I. et al. Rescue of a Drosophila NF1 mutant phenotype by protein kinase A. Science 276, 791–794 (1997).

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25. Rauen, K.A. et al. Proceedings from the 2009 genetic syndromes of the Ras/MAPK pathway: from bedside to bench and back. Am. J. Med. Genet. A 152A, 4–24 (2009). 26. Chan, R.J., Cooper, T., Kratz, C.P., Weiss, B. & Loh, M.L. Juvenile myelomonocytic leukemia: A report from the 2nd International JMML Symposium. Leuk. Res. 33, 355–362 (2008). 27. Hasle, H. et al. A pediatric approach to the WHO classification of myelodysplastic and myeloproliferative diseases. Leukemia 17, 277–282 (2003). 28. Kotecha, N. et al. Single-cell profiling identifies aberrant stat5 activation in myeloid malignancies with specific clinical and biologic correlates. Cancer Cell 14, 335–343 (2008). 29. Schubbert, S. et al. Functional analysis of leukemia-associated PTPN11 mutations in primary hematopoietic cells. Blood 106, 311–317 (2005). 30. Langdon, W.Y., Hyland, C.D., Grumont, R.J. & Morse, H.C. III. The c-cbl protooncogene is preferentially expressed in thymus and testis tissue and encodes a nuclear protein. J. Virol. 63, 5420–5424 (1989). 31. Andoniou, C.E., Thien, C.B. & Langdon, W.Y. Tumour induction by activated abl involves tyrosine phosphorylation of the product of the cbl oncogene. EMBO J. 13, 4515–4523 (1994). 32. Blake, T.J., Shapiro, M., Morse, H.C. III & Langdon, W.Y. The sequences of the human and mouse c–cbl proto-oncogenes show v–cbl was generated by a large truncation encompassing a proline-rich domain and a leucine zipper-like motif. Oncogene 6, 653–657 (1991). 33. Kassenbrock, C.K. & Anderson, S.M. Regulation of ubiquitin protein ligase activity in c-Cbl by phosphorylation-induced conformational change and constitutive

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ONLINE METHODS

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Subjects. Subjects were diagnosed and treated either in Europe under the auspices of the European Working Group of Myelodysplastic Syndromes in Childhood (EWOG-MDS) or enrolled as research subjects at the University of California, San Francisco. The Committees on Human Research at each of the institutions in EWOG-MDS and at UCSF approved these studies. Informed consent was obtained from parents or guardians, and in the case of pedigree analysis, all screened relatives. Family and clinical histories were reviewed, as were physical exams at diagnosis. Additional details are provided in a Supplementary Note. Mutation screening. Bone marrow or peripheral blood samples at diagnosis were obtained. Mononuclear cells were isolated using standard Histopaque 1111. Buccal swabs, fibroblasts or tissues unaffected by tumor were also obtained when available. Genomic DNA was extracted using PureGene ­reagents (Qiagen). Subjects were screened for mutations in CBL, NRAS, KRAS and PTPN11 as described6,7,20. RNA was prepared according to standard methods. cDNA was generated using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). Splice variants were identified through PCR using an annealing temperature of 58 °C and the following primers: 5′-TTGAGGGAACACATACTCGCT-3′ and 5′-TATGTTACTGCTGATGGGAACA-3′. Splice variants were gel extracted using the QIAquick Gel Extraction Kit (Qiagen). The resulting fragments were subcloned using the TOPO TA Cloning Kit (Invitrogen) according to the manufacturer’s instructions. Positive colonies were picked, mini prepped using the QIAprep Spin Miniprep Kit (Qiagen), and sequenced with standard M13 Forward (−20) and M13 Reverse primers. Cbl and shRNA expression constructs. For the CFU-GM experiments, Gateway technology (Invitrogen) was used to clone wild-type and mutant Cbl cDNAs into the murine stem cell virus (MSCV) backbone containing a green fluorescent protein (GFP) cassette driven by an internal ribosome entry site (IRES) downstream of the Cbl sequence (pMIG). The human wildtype and 70Z Cbl plasmids were a gift from H. Band (Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA)44. For the subsequent experiments using Ba/F3 cells, the same Cbl cDNAs were cloned into an MSCV-IRES-Venus (pMIV) backbone to allow co-transduction with the GFP-tagged shRNA. MiR30 based shRNA sequences targeting mouse Cbl were designed by J. Zuber and S. Lowe (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA). We selected one of these putative sequences (cbl.2364) and custom ordered a single 110-bp oligonucleotide (Bioneer) to serve as a template for PCR amplification. PCR products were digested with XhoI and EcoRI and ligated into the LTR-driven MiR30 SV40-GFP (LMS) MSCVbased vector (also provided by the Lowe laboratory) to produce the LMS-2364 retrovirus encoding the Cbl shRNA. The sequence for cbl.2364 is TCGAGA AGGTATATTGCTGTTGACAGTGAGCGATACCTATGAAGCGATGTATA ATAGTGAAGCCACAGATGTATTATACATCGCTTCATAGGTACTGCCT ACTGCCTCGG. Hematopoietic progenitor assays. All experimental procedures involving mice were reviewed and approved by the UCSF Committee on Animal Research. These assays were performed as described previously using mouse fetal liver cells transduced with MSCV-Cbl-IRES-GFP retroviruses engineered to express wild-type or mutant Cbl proteins. For the human CFU-GM assays, mono­ nuclear cells from peripheral blood or bone marrow were plated in MethoCult H4230 (StemCell Technologies), supplemented with recombinant human GM-CSF (Peprotech) and counted 14 days later, as described28. For the CFU-GM assays, GFP-positive cells were sorted using a FACS Aria (BD Biosciences) and then seeded in methylcellulose medium (M3231; StemCell Technologies)45, supplemented with recombinant mouse GM-CSF (Peprotech). Colonies were counted by indirect microscopy after 8 d. Cell viability and proliferation assays and protein blots. Mouse pro-B Ba/F3 cells were transduced with MSCV-EpoR-IRES-puro as described 46. These cells were maintained in RPMI-1640 with 10% FCS (HyClone),

doi:10.1038/ng.641

­ enicillin, ­streptomycin, l-glutamine, 10 ng/ml puromycin (Calbiochem) and p 10 μg/ml mouse interleukin-3 (IL-3) (Peprotech). The Ba/F3-EpoR cells were transduced with the LMS-2364 construct or the LMS vector alone. GFP-positive cells were sorted on a FACS Aria (BD Biosciences) and then transduced with the MSCV-Cbl-IRES-Venus retroviruses expressing wild-type or mutant Cbl. Cells positive for both GFP and YFP expression were sorted on the FACS Aria and studied in proliferation and protein blot assays. For the proliferation assays, cells were washed 3 times and then cultured for 6 h in cytokine-free medium before being plated in 6-well plates at a density of 500,000 cells per ml at increasing doses of hEPO (R&D Systems). Growth was monitored every other day using a ViCell cell counter (Beckman Coulter). For protein blot analysis, cells were washed 3 times and cultured for 6 h in cytokine-free medium before being stimulated for 15 min with increasing doses of hEPO (R&D Systems). Whole-cell lysates were blotted and probed with the following antibodies: anti-phospho-p44/42 mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (ERK1/2) (Thr202/Tyr204, cat. 9101), anti-phospho-AKT (S473, cat. 4060), anti-phospho-S6 (Ser235/236, cat. 2211) (all from Cell Signaling Technology); anti-phospho-STAT5 (cat. 44-390G) (Invitrogen) and anti-α-tubulin (Abnova). ERK1/2 (cat. 9102), AKT (cat. 9272), S6 (2217), and STAT5 (9363) antibodies were from Cell Signaling Technology. HEK cells. HEK293 cells were obtained from the American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated FBS (Wisent) at 37 °C in a humidified 5% CO2 atmosphere. Antibodies. Mouse monoclonal antibodies against HA (12CA5) and α-tubulin were obtained from Boehringer Ingelheim and Sigma, respectively. Rabbit polyclonal antibodies against pEGFR, EGFR and ubiquitin were obtained from Upstate, Santa Cruz Biotechnology and DAKO Canada, respectively. EGF ligand was obtained from Sigma. MG132 proteasome inhibitor was obtained from Boston Biochem. Plasmids. Plasmids encoding HA-CBL(WT, 70Z, Y371H, C384R) were subcloned into the pcDNA-DEST4.0 vector using Gateway Cloning technology (Invitrogen) and confirmed by DNA sequencing. The plasmid encoding EGFR was generated as described47. Immunoblotting and immunoprecipitation. Immunoprecipitation and immunoblotting were performed as described48. Cells were lysed in EBC buffer (50 mM Tris pH 8.0, 120 mM NaCl and 0.5% NP-40) supplemented with protease and phosphatase inhibitors (Roche). Cell lysates were immuno­ precipitated with indicated antibodies in the presence of Protein-A agarose beads (Repligen). Bound proteins were washed 5 times with NETN buffer (20 mM Tris pH 8.0, 120 mM NaCl, 1 mM EDTA and 0.5% NP-40), eluted by boiling in sodium dodecyl sulfate (SDS)-containing sample buffer, and resolved by SDS-PAGE (PAGE). In vitro ubiquitylation assay. In vitro ubiquitylation assay was performed as described49. 44. Levkowitz, G. et al. c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev. 12, 3663–3674 (1998). 45. Schubbert, S. et al. Biochemical and functional characterization of germ line KRAS mutations. Mol. Cell. Biol. 27, 7765–7770 (2007). 46. Mullighan, C.G. et al. JAK mutations in high-risk childhood acute lymphoblastic leukemia. Proc. Natl. Acad. Sci. USA 106, 9414–9418 (2009). 47. Wang, X., Huang, D.Y., Huong, S.M. & Huang, E.S. Integrin alphavbeta3 is a coreceptor for human cytomegalovirus. Nat. Med. 11, 515–521 (2005). 48. Ohh, M. et al. The von Hippel-Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix. Mol. Cell 1, 959–968 (1998). 49. Ohh, M. et al. Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein. Nat. Cell Biol. 2, 423–427 (2000).

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