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Introduction and Objectives Myc-interacting zinc finger protein 1 (Miz-1; Zbtb17) is a BTB/POZ (POZ) domain transcription factor expressed in all mammalian tissues. The POZ domain is required for Miz-1 function and favors a stable association with chromatin. As a binding partner of the oncoprotein c-Myc, Miz-1 can modulate the expression of c-Myc target genes (e.g. p21Waf1, p15ink4b). c-Myc is frequently associated with a variety of leukemia and lymphoma. In particular, c-Myc takes part in the t(8;14) chromosomal translocation where it is placed under the control of transcriptional regulatory elements of the immunoglobulin heavy chain (IgH) locus. This chromosomal translocation is a hallmark of patients with Burkitt-type B-cell lymphoma (BL). Since epigenetic deregulation is often associated with tumor development, we wished to determine whether (i) Miz-1 is involved in the development of c-Myc-dependent B-cell lymphoma and whether (ii) Miz-1 influences c-Myc dependent chromatin and gene regulation. Methods We are using the Eµ-Myc mouse model to generate Burkitt type c-Myc dependent lymphomas to study the implication of Miz-1 in a c-Myc driven process of malignant transformation. Mice carrying the Eµ-Myc transgene (c-Myc gene placed under the control of Eµ enhancer of the IgH locus) overexpress c-Myc in lymphoid cells and develop a disease similar to Burkitt's lymphoma. Mice that express a conditional non-functional Miz-1 allele lacking the part coding for the POZ domain in B cells (Mb1-Cre-Miz-1DPOZ/flox mice, hereafter called DPOZ mice) were crossed with Eµ-Myc transgenic mice. Incidence and latency periods of the development of tumors in these animals were compared to Eµ-Myc animals that express a functional Miz-1 protein. Pre-cancerous mice of all genotypes were also investigated to evaluate the influence of Miz-1 during the development of the c-Myc dependant lymphomas. To address the role of Miz-1 in the deregulation of chromatin associated with c-Myc overexpression, chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) was performed on B-lymphoma and pre-tumoral B-cells and compared to normal B-cells. Binding to chromatin of Miz-1, c-Myc as well as histone marks will be assessed. RNA from each cell type will also be submitted to high throughput sequencing (RNA-seq) to correlate the binding of transcription factors with gene expression. Results Eµ-Myc mice that express the non-functional Miz-1 protein (DPOZ) in B-cells develop lymphoma later than Eµ-Myc mice that express a normal, functional Miz-1 (Figure 1). Tumors that grow in Eµ-Myc/DPOZ animals are phenotypically similar, but are smaller compared to those developing in Eµ-Myc mice. Additionally, blood analysis of sick animals reveal a lower amount of Large Unstained Cells (LUC) when Miz-1 is mutated. This suggests that Myc driven lymphoma and leukemia is impaired in the absence of a functional Miz-1 protein. Accordingly, in 40 days-old pre-tumoral Eµ-Myc/DPOZ animals compared to Eµ-Myc mice with wild type Miz-1, less pre-B and/or pro-B cells were observed in lymphoid organs and little or no LUC were present in the blood, suggesting that a c-Myc driven B-cell lymphoma develops with a longer latency period and progresses at a slower rate in the absence of a non-functional Miz-1 protein. Conclusion and ongoing work Our preliminary data indicate that Miz-1 is involved in the development of aggressive c-Myc driven B-cell lymphoma. To test the potency of Eµ-Myc/DPOZ tumor cells to initiate B-cell lymphoma, we will transplant these tumor cells into immune-deficient mice and follow the development of the transplanted cells. Also, to test whether Miz-1 would be a suitable therapeutic target for future leukemia and lymphoma treatment, we will use a conditional model (ROSA-CreER-Miz-1DPOZ/flox) where the POZ domain coding sequences of Miz-1 can be deleted after tamoxifen treatment after tumor initiation in Eµ-Myc animals. To better understand the mechanisms by which Miz-1 influences c-Myc in the process of lymphomagenesis, we are performing high throughput genomic analyses to identify genes that are differentially regulated in Eµ-Myc lymphomas when Miz-1 is functional or deficient. These analyzes should demonstrate whether a c-Myc/Miz-1 complex is required to malignantly transform B-cells or whether c-Myc and Miz-1 act independently in the development of B-cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

Hematopoiesis is sustained by a pool of multipotent hematopoietic stem cells (HSCs) that have the capacity to differentiate into cells of all blood cell lineages. The pool of long-lived HSCs is maintained throughout life by the self-renewal ability of HSCs. New evidence suggests the process of alternative splicing is an important regulator of the maturation and activation of blood and immune effector cells. It is presently estimated that almost all multi-exon genes in human genome undergo alternative pre-mRNA splicing, and aberrant splicing has been linked to a variety of human pathologies. However, the role that pre-mRNA splicing may have for HSCs behaviour remains largely unexplored. Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is an RNA-binding protein that regulates alternative splicing by binding exonic splicing silencers elements (ESS) resulting in exon exclusion from the mature mRNA. RT-PCR analyses showed that hnRNPL is expressed in early stages of hematopoiesis including HSCs and lineage restricted hematopoietic progenitors. To test the role of hnRNPL in hematopoietic differentiation, we have generated conditional deficient mice, since a constitutive deletion of hnRNPL results in early embryonic lethality. Animals carrying two hnRNPL-floxed alleles (hnRNPLfl/fl) can be deleted at adult stage by the pIpC inducible MxCre transgene or by the VavCre transgene, which is expressed in all hematopoietic cells starting at embryonic stage E14. VavCre+hnRNPLfl/fl mice were not viable and did not progress further in their development than embryonic stage E17.5 and ablation of hnRNPL by pIpC injection caused a high rate of mortality in adult MxCre+hnRNPLfl/fl mice compared to control animals. Both the fetal liver (FL) of VavCre+hnRNPLfl/fl mice and the bone marrow (BM) of adult MxCre+hnRNPLfl/fl mice had a significantly reduced cellularity. Furthermore, flow cytometric analysis revealed in both FL and BM a significant reduction in frequency and absolute numbers of all mature blood cells, the lymphoid and myeloid precursors, CLPS, CMPs and GMPs and to a lesser extent the erythroid/megakaryocytic precursors (MEPs). Methylcellulose and both competitive and non-competitive transplantation assays demonstrated that HSCs lacking hnRNPL cannot generate lineage-committed progenitors and have lost their self-renewal capacity and reconstitution potential. A genome-wide analysis of mRNA expression and splicing through next-generation RNA sequencing of wild-type (WT) or VavCre+hnRNPLfl/fl E14.5 Lin- c-kit+ fetal liver cells (FLCs) revealed that hnRNPL deficiency affects not only alternative splicing but also gene expression levels in hematopoietic progenitors. In the absence of hnRNPL, genes implicated in regulating apoptosis, DNA damage response and cell division where found up-regulated in Lin- c-kit+ FLCs. Among those genes, many were p53 effector genes such as Cdkn1a, Ccng1, Trp53inp1, TrailR2, Bax and Zmat3. In addition genes that are known to be required for normal hematopoiesis and HSCs functions such as Gfi1, CD34, Csfr1, Egr1 and Runx1 were found down-regulated in those cells. Further analyses by qPCR and Western blots confirmed those findings and also showed that the level of p53 protein expression was upregulated in VavCre+hnRNPLfl/fl FLCs although the mRNA level is the same as in the WT cells suggesting that hnRNPL affects p53 mRNA translation efficiency. Similarly, several genes found differentially spliced are implicated in cell cycle progression or required for normal hematopoiesis in FL such as Bcl11a, Cdk4, Ccnd2 and TRP53bp1. These results together with an increased level of Reactive Oxygen Species (ROS) and elevated levels of phosphorylated histone H2AX (γ-H2AX, a sensor for double strand DNA breaks) suggest that hnRNPL regulates the activation of a p53 dependent DNA damage response pathway in hematopoietic stem cells. As a consequence loss of hnRNPL results in a loss of hematopoietic stem and progenitor cells. Our data also suggest that hnRNPL does not only regulate alternative splicing but also expression levels of a set of specific effector genes involved in HSC survival, proliferation, ultimately affecting self-renewal. Disclosures: No relevant conflicts of interest to declare.