An instructive role for Interleukin-7 receptor α in the development of human B-cell precursor leukemia.

Kinase signaling fuels growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Yet its role in leukemia initiation is unclear and has not been shown in primary human hematopoietic cells. We previously described activating mutations in interleukin-7 receptor alpha (IL7RA) in poor-prognosis "ph-like" BCP-ALL. Here we show that expression of activated mutant IL7RA in human CD34+ hematopoietic stem and progenitor cells induces a preleukemic state in transplanted immunodeficient NOD/LtSz-scid IL2Rγnull mice, characterized by persistence of self-renewing Pro-B cells with non-productive V(D)J gene rearrangements. Preleukemic CD34+CD10highCD19+ cells evolve into BCP-ALL with spontaneously acquired Cyclin Dependent Kinase Inhibitor 2 A (CDKN2A) deletions, as commonly observed in primary human BCP-ALL. CRISPR mediated gene silencing of CDKN2A in primary human CD34+ cells transduced with activated IL7RA results in robust development of BCP-ALLs in-vivo. Thus, we demonstrate that constitutive activation of IL7RA can initiate preleukemia in primary human hematopoietic progenitors and cooperates with CDKN2A silencing in progression into BCP-ALL.

Perturbation of fetal hematopoiesis in a mouse model of Down syndrome's transient myeloproliferative disorder.

Children with Down syndrome develop a unique congenital clonal megakaryocytic proliferation disorder (transient myeloproliferative disorder [TMD]). It is caused by an expansion of fetal megakaryocyte-erythroid progenitors (MEPs) triggered by trisomy of chromosome 21 and is further enhanced by the somatic acquisition of a mutation in GATA1. These mutations result in the expression of a short-isoform GATA1s lacking the N-terminal domain. To examine the hypothesis that the Hsa21 ETS transcription factor ERG cooperates with GATA1s in this process, we generated double-transgenic mice expressing hERG and Gata1s. We show that increased expression of ERG by itself is sufficient to induce expansion of MEPs in fetal livers. Gata1s expression synergizes with ERG in enhancing the expansion of fetal MEPs and megakaryocytic precursors, resulting in hepatic fibrosis, transient postnatal thrombocytosis, anemia, a gene expression profile that is similar to that of human TMD and progression to progenitor myeloid leukemia by 3 months of age. This ERG/Gata1s transgenic mouse model also uncovers an essential role for the N terminus of Gata1 in erythropoiesis and the antagonistic role of ERG in fetal erythroid differentiation and survival. The human relevance of this finding is underscored by the recent discovery of similar mutations in GATA1 in patients with Diamond-Blackfan anemia.

Biased exonization of transposed elements in duplicated genes: A lesson from the TIF-IA gene.

BACKGROUND: Gene duplication and exonization of intronic transposed elements are two mechanisms that enhance genomic diversity. We examined whether there is less selection against exonization of transposed elements in duplicated genes than in single-copy genes. RESULTS: Genome-wide analysis of exonization of transposed elements revealed a higher rate of exonization within duplicated genes relative to single-copy genes. The gene for TIF-IA, an RNA polymerase I transcription initiation factor, underwent a humanoid-specific triplication, all three copies of the gene are active transcriptionally, although only one copy retains the ability to generate the TIF-IA protein. Prior to TIF-IA triplication, an Alu element was inserted into the first intron. In one of the non-protein coding copies, this Alu is exonized. We identified a single point mutation leading to exonization in one of the gene duplicates. When this mutation was introduced into the TIF-IA coding copy, exonization was activated and the level of the protein-coding mRNA was reduced substantially. A very low level of exonization was detected in normal human cells. However, this exonization was abundant in most leukemia cell lines evaluated, although the genomic sequence is unchanged in these cancerous cells compared to normal cells. CONCLUSION: The definition of the Alu element within the TIF-IA gene as an exon is restricted to certain types of cancers; the element is not exonized in normal human cells. These results further our understanding of the delicate interplay between gene duplication and alternative splicing and of the molecular evolutionary mechanisms leading to genetic innovations. This implies the existence of purifying selection against exonization in single copy genes, with duplicate genes free from such constrains.

Sil overexpression in lung cancer characterizes tumors with increased mitotic activity.

Sil (SCL interrupting locus) was cloned from the most common chromosomal rearrangement in T-cell acute lymphoblastic leukemia. It is an immediate early gene whose expression is associated with cell proliferation. Sil protein levels are tightly regulated during the cell cycle, reaching peak levels in mitosis and disappearing on transition to G1. A recent study found Sil to be one of 17 genes whose overexpression in primary adenocarcinomas predicts metastatic spread. We hypothesized that Sil might have a role in carcinogenesis. To address this question, we utilized several approaches. Using a multitumor tissue array, we found that Sil protein expression was increased mostly in lung cancer, but also at lower levels, in a subset of other tumors. Microarray gene expression analysis and immunohistochemistry of lung cancer samples verified these observations. Sil gene expression in lung cancer correlated with the expression of several kinetochore check-point genes and with the histopathologic mitotic index. These observations suggest that overexpression of the Sil gene characterizes tumors with increased mitotic activity.

Mutations in exon 2 of GATA1 are early events in megakaryocytic malignancies associated with trisomy 21.

Patients with Down syndrome (DS) frequently develop 2 kinds of clonal megakaryocytosis: a common, congenital, spontaneously resolving, transient myeloproliferative disorder (TMD) and, less commonly, childhood acute megakaryoblastic leukemia (AMKL). Recently, acquired mutations in exon 2 of GATA1, an X-linked gene encoding a transcription factor that promotes megakaryocytic differentiation, were described in 6 DS patients with AMKL. The mutations prevent the synthesis of the full-length GATA1, but allow the synthesis of a shorter GATA1 protein (GATA1s) that lacks the transactivation domain. To test whether mutated GATA1 is involved in the initiation of clonal megakaryoblastic proliferation or in the progression to AMKL, we screened 35 DS patients with either AMKL or TMD and 7 non-DS children with AMKL for mutations in exon 2 of GATA1. Mutations were identified in 16 of 18 DS patients with AMKL, in 16 of 17 DS patients with TMD, and in 2 identical twins with AMKL and acquired trisomy 21. Analysis revealed various types of mutations in GATA1, including deletion/insertions, splice mutations, and nonsense and missense point mutations, all of which prevent the generation of full-length GATA1, but preserve the translation of GATA1s. We also show that the likely mechanism of generation of GATA1 isoforms is alternative splicing of exon 2 rather than, or in addition to, alternative translation initiation, as was proposed before. These findings suggest that acquired intrauterine inactivating mutations in GATA1 and generation of GATA1s cooperate frequently with trisomy 21 in initiating megakaryoblastic proliferation, but are insufficient for progression to AMKL.

GLI3 is not mutated commonly in sporadic medulloblastomas.

BACKGROUND: Medulloblastoma is a malignant, invasive embryonic tumor of the cerebellum. Sonic hedgehog (SHH) is a secreted glycoprotein that has a major role in the developing cerebellum. Activation of the SHH pathway resulting from mutations in the PATCH gene, which is an inhibitor of the pathway, are associated with hereditary and sporadic medulloblastomas. The GLI3 protein is another negative regulator of SHH signaling. The authors hypothesized that mutations in GLI3 may be associated with meduloblastomas. METHODS: The authors describe a patient with hereditary Greig syndrome, which was caused by mutations in GLI3, and medulloblastoma. Another such patient was described in the literature. They also sequenced the GLI3 gene, including all exon-intron boundaries, in an additional 12 sporadic medulloblastomas. RESULTS: The authors detected a new nonsense germline mutation in a child with Greig syndrome and medulloblastoma. This mutation generates a stop codon in position 809 of GLI3 that has been predicted to result in massive truncation of the protein. Several new polymorphisms, but no tumor-associated mutations, were found in sporadic tumors. CONCLUSIONS: Gli3 is mutated rarely in medulloblastoma.