Blast crisis Ph+ chronic myeloid leukemia with NUP98/HOXA13 up-regulating MSI2
© Di Giacomo et al.; licensee BioMed Central Ltd. 2014
Received: 17 April 2014
Accepted: 13 June 2014
Published: 20 June 2014
Musashi2(Msi2)-Numb pathway de-regulation is a molecular mechanism underlying the transition of chronic phase Ph + CML to deadly blast crisis, particularly in cases with a NUP98/HOXA9 fusion from a t(7;11)(p15;p15). This study provides new insights on the mechanisms cooperating in driving MSI2 over-expression and progression of Ph-positive CML.
Herein we describe a t(7;11)(p15;p15) originating a NUP98 fusion with HOXA13, at 7p15, in a 39 year-old man in blast crisis of Ph-positive CML. Both MSI2 and HOXA9 were evaluated by quantitative RT-PCR in our patient and in a series of haematological malignancies. Up-regulation of both genes emerged only in the presence of NUP98/HOXA13 gene fusion. However, over-expression of MSI2, but not HOXA9, was found in 2 cases of Ph + blast crisis with additional chromosome aberrations other than t(7;11). To determine the mechanisms underlying MSI2 over-expression in our patient we performed Chromatin Immunoprecipitation and found that NUP98/HOXA13 fusion protein deregulates MSI2 gene by binding its promoter.
To the best of our knowledge, this is the first molecular characterization of NUP98/HOXA13 fusion in blast crisis of Ph + CML. Our findings suggest cooperative mechanisms of MSI2 over-expression driven by HOXA proteins and strongly supports MSI2 as a prognostic marker and a candidate in target treatment of CML.
KeywordsBlast crisis CML Fusion transcript MSI2 HOXA genes
MSI2 gene (17q22) is a member of the Musashi (Msi) family, which is well conserved during mammals evolution and distributed in the stem cell compartment of neural , hematopoietic , pancreatic  and epithelial tissues [4, 5]. Two translocations involving MSI2 gene at 17q22 have been described in myeloid malignancies [6, 7] and more than 50 point mutations have been reported in solid tumors and cell lines (COSMIC database , cBioPortal for Cancer Genomics ) . Msi2 expression is activated by cooperation between HoxA9 and Meis1 in Meis1-immortalized hematopoietic progenitors . In chronic myeloid leukemia (CML) Ito et al.  showed the Nup98/HoxA9 fusion contributed to blast crisis through HoxA9 homeodomain binding to the Msi2 promoter resulting in gene over-expression, down-regulation of the Notch1 inhibitor Numb, and loss of the proliferation/differentiation equilibrium in hematopoietic stem cells (HSCs) . De-regulation of the Musashi-Numb-Notch1 signaling axis is associated with poor prognosis in CML , acute myeloid leukemia (AML)  and B-cell acute lymphoblastic leukemia (B-ALL) . In addition to HOXA9, two genes of the HOXA cluster at 7p15, i.e., HOXA11 and HOXA13, rearrange with NUP98 in leukemia. All fusion transcripts contain the NUP98 N-terminal, with the FG/GLFG domains, that mediate both RNA and protein transport, and the HOX homeodomain, with its DNA binding ability . Thus all these chimeric proteins exhibit their oncogenic potential via transcriptional activation of downstream genes . Remarkably a single t(7;11)(p15;p15) can produce more than one NUP98/HOXA fusion . We investigated MSI2 regulation in the first case of Ph-positive CML in blast crisis with t(7;11) and NUP98/HOXA13 fusion gene.
A 39 year-old man was referred to our department because of leukocytosis, mild anemia, and thrombocytopenia (WBC 180000/mmc, Hb 11.8 g/dl, PLT 130000/mmc) associated with splenomegaly. Chronic Myeloid Leukemia in Blast Crisis was diagnosed on peripheral blood and bone marrow smears. Karyotype was 46,XY,t(9;22)(q34;q11)/46,XY,t(9;22)(q34;q11),t(7;11)(p15;p15) . Hydroxyurea therapy followed by Dasatinib 140 mg/day (40 days), were unsuccessful. The patient did not respond to high-dose ARA-C (3 g/m2/day, on days 1-3-5-7) and Daunorubicin (50 mg/m2/day on days 1-3-5). Rescue chemotherapy with Clofarabine (40 mg/m2/day on days 1–5) and Gemtuzumab-Ozogamicin (3 mg/m2/day on day 6) induced prolonged aplasia. Four months after diagnosis the patient underwent unsuccessful bone marrow transplantation from a HLA haploidentical brother.
Fluorescence In Situ Hybridization (FISH)
NUP98_1083F 5’-ggtaataccagcaccataggacag-3’ and HOXA9_1036R 5’-tgtggcctgaggtttagagc-3’ or HOXA9_736R 5’-cagttccagggtctggtgtt-3’ for the first amplification round; NUP98_1252F 5’-cttactacatttggaagcac-3’ and HOXA9_736R or HOXA9_708R 5’-gggcaccgctttttccgagt-3’ for the second (NUP98: NM_00139131.3, HOXA9: NM_001164603.1).
NUP98_1083F and HOXA11_837R 5’-ctctcggatctggtacttggt-3’ for the first amplification round and NUP98_1252F or HOXA11_837R for the semi-nested PCR (HOXA11: NM_ 005523.5).
NUP98_1083F and HOXA13_992R 5’-cctcctataggagctggcat-3’ for the first amplification round and NUP98_1252F or NUP98_1400F 5’-acctgggactcttggaactg-3’ and HOXA13_992R for semi-nested PCRs (HOXA13: NM_000522.4).
PCR products were sub-cloned into the pGEM-Teasy vector (Promega, Madison, WI) and sequenced by Sanger’s method (ABI 3500 Genetic Analyzer, Applyed Biosystems, Foster City, CA). Molecular analysis revealed an in-frame fusion transcript. NUP98/HOXA13 had 3885 bp and a breakpoint between exon 16 (nt 2322) of NUP98 (NM_00139131.3) and exon 2 (nt 952) of HOXA13 gene (NM_000522.4). Three splicing variants had the same breakpoint but were lacking respectively NUP98 exon 11, 12 or 14 (which was out of frame) (Figure 1b and c).
Chromatin Immunoprecipitation (ChIP)
This study was important to enrich our knowledge on molecular events underlying the t(7;11)(p15;p15) in the blastic crisis of Ph + CML. For the first time NUP98/HOXA13 has been identified in CML, although it was reported in one case of AML  and one case of MDS . The pathogenetic role of NUP98/HOXA13 rearrangement in the evolution in this case is emphasized by the absence of transcript isoforms from alternative splicing, such as NUP98/HOXA11 and all NUP98/HOXA9, as previously reported . As we excluded the presence of the wild type HOXA13 transcript, MSI2 and HOXA9 were over-expressed in malignant cells (Figure 2a, b and c) as a consequence of NUP98/HOXA13 fusion. Notably, HOXA13, fused to NUP98, up-regulated MSI2 both directly by binding to its promoter and indirectly by binding to the HOXA9 promoter, thus inducing a synergistic effect between the two HOXA proteins (Figure 3c).
Over-expression of MSI2, but not HOXA9, emerged in two other cases of Ph + blast crisis (BC1 and BC2, Figure 2a and b) with additional cytogenetic rearrangements suggesting that, in addition to HOXA9 and HOXA13, alternative mechanisms may deregulate MSI2 in the presence of clonal evolution and acute phase progression of Ph + CML.
In conclusion this study provided new insights on the molecular heterogeneity of t(7;11)(p15;p15) in the blastic crisis of Ph + CML. Our report suggests that HOXA13 can bind to the promoter of MSI2 and may contribute to its activation in a patient that harbors NUP98/HOXA13 fusion.
Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
Ethical approval has been obtained for the protocol “In-depth genomic characterization of leukemia to provide new tools for personalized diagnosis and disease monitoring” ( AIRC 2011–2014) from the University Bioethics Committee of the University of Perugia (Prot. 1.X.2011).
Chronic myeloid leukemia
Acute myeloid leukemia
B-cell acute lymphoblastic leukemia
Hematopoietic stem cell
Fluorescence in situ hybridization
Reverse transcription polymerase chain reaction
We thank Dr. Cinzia Allegrucci, University of Nottingham, for a critical review of data and Dr. Enrico Gottardi, University of Turin, for kindly providing some samples. This work was funded by Associazione Italiana per la Ricerca sul Cancro (AIRC IG-11512), PRIN 2010–2011, Associazione Damiano per l’Ematologia (to DDG). Mutation data were obtained from the Sanger Institute Catalogue Of Somatic Mutations In Cancer web site, http://www.sanger.ac.uk/cosmic.
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