The Vertebrate Codex Gene Breaking Protein Trap Library For Genomic Discovery and Disease Modeling Applications

The zebrafish is a powerful model to explore the molecular genetics and expression of the vertebrate genome. The gene break transposon (GBT) is a unique insertional mutagen that reports the expression of the tagged member of the proteome while generating Cre-revertible genetic alleles. This 1000+ locus collection represents novel codex expression data from the illuminated mRFP protein trap, with 36% and 87% of the cloned lines showcasing to our knowledge the first described expression of these genes at day 2 and day 4 of development, respectively. Analyses of 183 molecularly characterized loci indicate a rich mix of genes involved in diverse cellular processes from cell signaling to DNA repair. The mutagenicity of the GBT cassette is very high as assessed using both forward and reverse genetic approaches. Sampling over 150 lines for visible phenotypes after 5dpf shows a similar rate of discovery of embryonic phenotypes as ENU and retroviral mutagenesis. Furthermore, five cloned insertions were in loci with previously described phenotypes; embryos homozygous for each of the corresponding GBT alleles displayed strong loss of function phenotypes comparable to published mutants using other mutagenesis strategies (ryr1b, fras1, tnnt2a, edar and hmcn1). Using molecular assessment after positional cloning, to date nearly all alleles cause at least a 99+% knockdown of the tagged gene. Interestingly, over 35% of the cloned loci represent 68 mutants in zebrafish orthologs of human disease loci, including nervous, cardiovascular, endocrine, digestive, musculoskeletal, immune and integument systems. The GBT protein trapping system enabled the construction of a comprehensive protein codex including novel expression annotation, identifying new functional roles of the vertebrate genome and generating a diverse collection of potential models of human disease.

Use of chromosome engineering to model a segmental deletion of chromosome band 7q22 found in myeloid malignancies

Monosomy 7 and del(7q) are associated with adverse features in myeloid malignancies. A 2.5-Mb commonly deleted segment (CDS) of chromosome band 7q22 is implicated as harboring a myeloid tumor suppressor gene (TSG); however, molecular analysis of candidate TSGs has not uncovered loss of function. To determine whether haploinsufficiency for the 7q22 CDS contributes to myeloid leukemogenesis, we performed sequential gene targeting to flank a region of orthologous synteny on mouse chromosome band 5A3 with loxP sites. We then generated Mx1-Cre, 5A3fl mutant mice and deleted the targeted interval in vivo. Although excision was inefficient, we confirmed somatic deletion of the 5A3 CDS in the hematopoietic stem cell compartment. Mx1-Cre, 5A3fl mice show normal hematologic parameters and do not spontaneously develop myeloid malignancies. The 5A3fl deletion does not cooperate with oncogenic KrasG12D expression, Nf1 inactivation, or retroviral mutagenesis to accelerate leukemia development and did not modulate responsiveness to antileukemia drugs. These studies demonstrate that it is feasible to somatically delete a large chromosomal segment implicated in tumor suppression in hematopoietic cell populations in vivo; however, our data do not support the hypothesis that the 7q22/5A3 CDS interval contains a myeloid TSG.

A retroviral mutagenesis screen reveals strong cooperation between Bcl11a overexpression and loss of the Nf1 tumor suppressor gene

NF1 inactivation occurs in specific human cancers, including juvenile myelomonocytic leukemia, an aggressive myeloproliferative disorder of childhood. However, evidence suggests that Nf1 loss alone does not cause leukemia. We therefore hypothesized that inactivation of the Nf1 tumor suppressor gene requires cooperating mutations to cause acute leukemia. To search for candidate genes that cooperate with Nf1 deficiency in leukemogenesis, we performed a forward genetic screen using retroviral insertion mutagenesis in Nf1 mutant mice. We identified 43 common proviral insertion sites that contain candidate genes involved in leukemogenesis. One of these genes, Bcl11a, confers a growth advantage in cultured Nf1 mutant hematopoietic cells and causes early onset of leukemia of either myeloid or lymphoid lineage in mice when expressed in Nf1-deficient bone marrow. Bcl11a-expressing cells display compromised p21Cip1 induction, suggesting that Bcl11a's oncogenic effects are mediated, in part, through suppression of p21Cip1. Importantly, Bcl11a is expressed in human chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia samples. A subset of AML patients, who had poor outcomes, of 16 clusters, displayed high levels of BCL11A in leukemic cells. These findings suggest that deregulated Bcl11a cooperates with Nf1 in leukemogenesis, and a therapeutic strategy targeting the BCL11A pathway may prove beneficial in the treatment of leukemia.

Novel Mll-AF9 Cooperating Genes Identified in a Leukemogenesis Screen Using Retroviral Mutagenesis.

Human translocations involving the MLL gene are associated with a variety of myeloid and lymphoid leukemia. The MLL-AF9 translocation is common in acute myeloid leukemia (AML). C57BL/6 mice with a Mll-AF9 knock-in mimic the phenotype observed in human patients with this translocation: most develop AML but only after a preleukemic phase and prolonged latency. A minority of these mice develop acute lymphoblastic leukemia (ALL) (Dobson, et al. EMBO J. 1999, 18(13)). This model provides a system for understanding the evolution of AML initiated by a MLL fusion oncoprotein, including the identification of cooperating genetic events required for AML induction. The recombinant retrovirus MOL4070LTR (M4070) induces AML in some strains of mice (Wolff, et al. J Virol. 2003, 77(8)). We hypothesized that this virus could cooperate with the MLL-AF9 oncoprotein to accelerate AML by acting as an insertional mutagen, providing second hits in leukemia progression. We bred Mll-AF9 heterozygous C57BL/6 males to 129/SvJ wild type (WT) females, and injected the 280 offspring at 3 days of age with either M4070 virus (212) or a control supernatant (68). All mice were genotyped and observed for disease progression. Infected Mll-AF9/+ mice succumb to disease with a significantly reduced latency period when compared to virus treated WT mice (p < .0001) and uninfected Mll-AF9/+ mice (p < .0001), indicating that M4070 infection causes significant leukemia acceleration in these mice. Infected Mll - AF9 /+ Mice Succumb to Disease More Rapidly Than All Other Groups Infected Mll-AF9/+ Mice Succumb to Disease More Rapidly Than All Other Groups The gross pathology and analysis of the surface immunophentoype by flow cytometry and Southern blot indicate infected Mll-AF9/+ animals present with primarily myeloid leukemia while infected WT animals present with lymphoid leukemia. Retroviral insertion sites were cloned from 169 of our accelerated leukemia tissues using a shotgun-based, linker-mediated, cloning protocol to identify the genes most frequently mutated in these animals. From more than 20,000 sequence reads, over 4,700 independent proviral insertions were isolated and have been analyzed to identify 88 common insertion sites (CIS) present in at least 3 mice. We have found several CIS enriched in Mll-AF9/+ AMLs accelerated by M4070 insertion. We are examining the expression of CIS-associated genes in human AML with MLL gene translocations using human microarray data and in our murine leukemia samples using qRT-PCR to verify the viral insertions altered the expression of the candidate gene. We intend to test some of these genes for cooperation with MLL-AF9 in AML development using mouse transplantation assays. We are currently transducing bone marrow from Mll-AF9/+ mice with a retrovirus encoding a candidate gene to observe disease progression compared to controls.

Leukemogenesis by Truncated CBFβ-SMMHC Defective in Repressing RUNX1.

Chromosome 16 inversion, inv(16)(p13q22), is one of the most frequent chromosome abnormalities in human acute myeloid leukemia (AML), comprising almost 100% of subtype M4Eo and up to 15% of all AML. This inv(16) leads to an in-frame fusion of CBFB and MYH11 genes. CBFB-MYH11 encodes a fusion protein between CBFβ, which is an obligate partner of RUNX1 or AML1, and smooth muscle myosin heavy chain (SMMHC). Using knock-in mouse models we have previously demonstrated that Cbfb-MYH11 dominantly blocks Runx1/Cbfb function in hematopoiesis and predisposes mice to AML (requiring a second hit from ENU or retroviral mutagenesis). However, the molecular mechanism underlying these findings remains unclear. Current hypotheses, which are based on previous in vitro studies, focus on the ability of CBFβ-SMMHC to dominantly inhibit RUNX1/CBFβ and include CBFβ-SMMHC binding RUNX1 with higher affinity than CBFβ; CBFβ-SMMHC sequestration of RUNX1 in the cytoplasm; CBFβ-SMMHC stabilization of RUNX1 by decreasing RUNX1 ubiquitination; and CBFβ-SMMHC repression of RUNX1 transactivation, which is dependent on SMMHC multimerization and repressor recruitment. To test these hypotheses in vivo, we generated knock in chimeric and F1 heterozygous mice expressing CBFβ-SMMHC with C-terminal and internal deletions. One of the deletions removes only the domain responsible for high affinity binding of RUNX1 (HABD). The CBFβ-SMMHC protein with HABD deletion did not bind RUNX1 with higher affinity than CBFβ and was hypothesized to be unable to dominantly repress RUNX1. Consistent with this hypothesis, the HABD-deleted protein was less efficient in sequestering RUNX1 and caused less severe hematopoietic defects in F1 embryos than full length CBFβ-SMMHC. In contrast to mice expressing full length CBFβ-SMMHC, which develop AML only after ENU or retroviral mutagenesis, most HABD-deleted chimeric and all HABD-deleted F1 mice developed AML spontaneously shortly after birth. A larger deletion removed both the HABD and the RUNX1 stabilization domain (RSD). CBFβ-SMMHC with the HABD-RSD double deletion did not bind RUNX1 with high affinity and could not sequester RUNX1 in the cytoplasm. Mice expressing HABD-RSD double deleted CBFβ-SMMHC had normal hematopoiesis, did not develop leukemia spontaneously and developed T cell (not myeloid) malignancies after ENU treatment. These data suggest that HABD and RSD are important for CBFβ-SMMHC to block RUNX1 function and to dominantly impair hematopoiesis. The accelerated leukemogenesis associated with HABD deletion and induction of T cell malignancies in mice expressing HABD-RSD double deleted CBFβ-SMMHC strongly support the hypothesis that CBFβ-SMMHC can induce leukemia through RUNX1-inhibition-independent pathways.