Mutations of the PML tumor suppressor gene in APL

From bloodjournal.hematologylibrary.org by guest on May 30, 2013. For personal use only.

Prepublished online November 20, 2003; doi:10.1182/blood-2003-07-2200

Mutations of the PML tumor suppressor gene in APL Carmela Gurrieri, Khedoudja Nafa, Taha Merghoub, Rosa Bernardi, Paola Capodieci, Andrea Biondi, Stephen Nimer, Dan Douer, Carlos Cordon-Cardo, Robert Gallagher and Pier Paolo Pandolfi

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From bloodjournal.hematologylibrary.org by guest on May 30, 2013. For DOI personal use only. Blood First Edition Paper, prepublished online November 20, 2003; 10.1182/blood-2003-07-2200

Mutations of the PML tumor suppressor gene in APL

Carmela Gurrieri, Khedoudja Nafa, Taha Merghoub, Rosa Bernardi, Paola Capodieci, Andrea Biondi, Stephen Nimer, Dan Douer, Carlos Cordon-Cardo, Robert Gallagher and Pier Paolo Pandolfi

From the Molecular Biology Program, Department of Pathology and Department of Medicine, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Division, Graduate School of Medical Sciences, Cornell University; Centro Ricerca M. Tettamanti, Clinica Pediatrica Universita' di Milano Bicocca, Monza, Italy; Division of Hematology, University of Southern California Medical School and Norris Cancer Center, CA; Montefiore Medical Center and Albert Einstein Cancer Center, NY.

Reprints and correspondence: Pier Paolo Pandolfi, Molecular Biology Program and Department of Pathology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, Box 110, 1275 York Avenue, New York, NY 10021 USA. Phone: 212-6396168; Fax: 212-7173102; e-mail: [email protected]

Copyright (c) 2003 American Society of Hematology


Abstract The PML tumor suppressor of APL is essential for a number of pro-apoptotic and growth suppressive pathways as well as for the activity of differentiating agents such as RA. In human APL, the dose of PML is reduced to heterozygosity given that one allele is involved in the chromosomal translocation while the status of the remaining PML allele is unknown. We have therefore screened by single strand conformational polymorphism (SSCP) and sequencing analysis, DNA from APL patients for mutations at the PML locus. We identified DNA sequence variations resulting in a truncated PML protein in APL cases that displayed RA resistance and a very poor prognosis. Mutation analysis also led to the identification of aberrant PML sequence variations in other hemopoietic malignancies. Complete functional loss of PML is therefore selected by the APL phenotype and associates with poor prognosis and RA unresponsiveness.

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Introduction The PML tumor suppressor gene has become the object of intense research due to its involvement in the pathogenesis of Acute Promyelocytic Leukemia (APL) where it is found fused to the retinoic acid (RA) receptor alpha (RAR ) gene as a consequence of the t(15;17) chromosomal translocation.1 PML is typically found concentrated in the nucleus in multiprotein speckled structures termed PML nuclear bodies (PML-NBs).2 These structures are disrupted in Pml-/- primary cells2 and in APL blasts.5 PML is therefore critical for the formation and stability of the NB in turn implying that the function of multiple NB components may be impaired in the APL blasts or in cells lacking PML. A number of findings support the role for PML as a tumor suppressor.3 First, its inactivation obtained by homologous recombination lends to cells of various histological origins a marked survival and proliferative advantage.4,5 Second, loss of PML function impairs cellular senescence in response to oncogenic stimuli.6,7 Lastly, PML inactivation impairs cellular differentiation upon differentiating agents such as RA and Vitamin D.4,8,9 PML modulates key tumor suppressive pathways such as the ones controlled by p53 and Rb, which are impaired in cells lacking its function.5-7,10-13 As a consequence PML-/- mice are tumor prone.4,14 Compelling evidence that PML opposes APL leukemogenesis has been obtained in mouse models of APL. The progressive reduction of the dose of PML obtained by crossing PML - RAR

transgenic mice with

PML-/- mice resulted, in fact, in a dramatic increase in the incidence of leukemia and in an acceleration of leukemia onset.9 In addition, in hemopoietic cells from PML - RAR transgenic mice, Pml inactivation resulted in impaired response to differentiating agents such as RA and vitamin D3 as well as in a marked survival advantage upon pro-apoptotic

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stimuli.9 In this report, we therefore investigated whether the remaining PML allele [the one not involved in the t(15;17)] would be found mutated in APL patients and whether complete PML loss would impact on disease onset, clinical outcome and/or response to therapy.

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Materials and methods Patients, Samples and Treatment Modalities and Response Criteria The study included 50 APL and 81 non-APL hematological malignancies patients (Table 2). All of the samples included in the present study were collected under Institutional Review Board approved protocols and with informed consent. For most of the APL patients, summaries of the treatments administered and clinical outcome have been presented elsewhere. Briefly, out of 50 APL patients twelve were first-relapse patients treated with all-trans retinoic acid (RA) and chemotherapy (daunorubicin and cytarabine)15, six were multiple-relapse patients relapsed at least twice (range 2-4) and treated with two or more courses of chemotherapy (range 2-4) containing RA16, seven were pretreatment samples from patients expressing the PML-RAR

V isoform17.

Among the APL patients from the Latino population in Los Angeles (n=15), eleven received liposomal or oral RA induction followed by consolidation with two or more (range 2-4) courses of chemotherapy on a variety of schedules, including idarubicin/cytarabine, etoposide/mitoxantrone and liposomal RA. Four patients were treated with liposomal or oral RA therapy only. Two patients at relapse were treated with arsenic trioxide. One received allogenic bone marrow transplantion (BMT). Twelve of the 15 patients, overall, achieved complete remission (CR). All juvenile APL patients (n=10; median age 6.4) received combined chemotherapy, followed by allogenic (two cases) or autologous (one case) BMT. Three out of ten patients achieved complete remission (CR). 17 out of 50 APL patients analyzed were resistant to RA. Criterion for RA-resistance was the reduced in vitro sensitivity of APL blasts to RA-induced differentiation as previously reported.15 All APL patients included in this study had the t(15:17) translocation demonstrated either by karyotyping or reverse transcriptionpolymerase chain reaction (RT-PCR) of the PML - RAR fusion gene. APL samples used to prepare DNA were obtained from bone marrow except for a few cases, which were obtained from peripheral blood. In all cases, except for one remission case (blasts 400-bp were digested with MboII (2nd fragment of exon 2 and fragment 2nd of exon 7b) or BamHI (1st fragment of exon 7b).The primers (M4, R7, M2, RJ) used to amplify and sequence the PML-RAR fusion gene are also reported.

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Table 2 PML mutations in hematological malignancies

Tumor type

APL

AML non APL

No. of samples analyzed

Exon/ Intron

DNA change

AA change (PML isoform 1)

Predicted alteration

50

IVS3

IVS3-1G A

Splice site

Frameshift

E5

1272delAG

Stop at 435

Frameshift

E9

1933C T

F645L

Missense

E9

1956C T

A652A

Silent

E9

2025C T

A675A

Silent

26

E1

45C

A

P15P

Silent

E1

46G A

A16T

Missense

None

Intronic/Splicing

IVS7

MDS Multiple Myeloma Lymphoma

IVS7-22C

T

15

5’UTR

-14C G

None

Posttranscriptional

12

IVS4

IVS4+24G C

None

Intronic/Splicing

28

IVS4

IVS4+24G C

None

Intronic/Splicing

The mutations were compared to the PML gene sequence, mRNA NM-033238, and confirmed by bi-directional analysis. Forty-four anonymized DNA samples from healthy volunteers were analyzed as normal controls. 5’UTR: 5’ untranslated region. E: exon. IVS: intron. Polymorphisms

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