Involvement In Chromosomal Translocations Research Articles

Finally, An Apoptosis-Targeting Therapeutic for Cancer.

Resistance to cell death represents one of the hallmarks of cancer. Various genetic and epigenetic changes in malignant cells afford cytoprotection in the face of genomic instability, oncogene activation, microenvironment stress, chemotherapy, targeted anticancer drugs, and even immunotherapy. Central among the regulators of cell life and death are Bcl-2 family proteins, with the founding member of the family (B-cell lymphoma/leukemia-2) discovered via its involvement in chromosomal translocations in lymphomas. The quest for therapeutics that target cell survival protein Bcl-2 represents a long road traveled, with many dead-ends, disappointments, and delays. Finally, a Bcl-2-targeting medicine has gained approval as a new class of anticancer agent. Cancer Res; 76(20); 5914-20. ©2016 AACR.

Abstract 4178: Tcl1 enhances keratinocytes’ survival/proliferation by promoting erk and jnk/sap phosphorylation at the expense of p38 and by controlling c-fos expression through miR-29b and miR-181a-1

Abstract The Tcl1 oncogene has been initially isolated for its involvement in chromosomal translocations of T-prolymphocytic leukemias and overexpression in B chronic lymphocytic leukemia by enhancing AKT nuclear translocation and allowing the Ser-473 transphosphorylation. Tcl1, also, acts as AP-1 transcriptional inhibitor, by interacting with c-fos and c-jun. More recent works have associated Tcl1 to proliferation and self-renewal ability of ES cells under the direct activation of OCT3/4, Zfx, KLF5 transcription factors, being Tcl1 expressed in preimplantation embryos where it allows the progression beyond the 4-cells stage. We have recently shown that the Tcl1 oncogene is expressed in epidermis and defines secondary hair germ (transient-amplifying, TA) cells differentiation at catagen-telogen (the degenerative-resting phase of the hair follicle (HF)) transition, allowing the proliferation of TA cells in anagen (regenerative phase of the HF), giving the slow-cycling stem cells, the ability to incorporate BrdU. In fact, Tcl1 mutant (Tcl1-/-) affects stem-cell marker CD34 expression and BrdU incorporation in the bulge and TA cells, resulting in skin defects in adults with the onset of alopecia followed by skin wounding. Phenomena that are almost completely rescued by K14-TCL1 transgenic expression, in vivo. Since Tcl1 has a role in maintenance of a normal skin homeostasis in mice, involving both hair growth and epidermis, we used the approach of the expression chip analysis to unravel the pathways that are affected by loss of function and overexpression of Tcl1 in epidermal keratinocytes, by using Tcl1-/- and K14-TCL1;Tcl1-/- mice models. Our findings show that Tcl1 function involves the MAPK pathway, since Tcl1-/- shows increasing in p38MAPK phosphorylation linked to terminal differentiation/senescence/apoptosis of keratinocytes, while K14-driven overexpression shows increasing of p-Erk and p-Sapk/p-Jnk phosphorylations, linked to proliferation/commitment of keratinocytes. These signals flow through the MAPK cascade lead to altered AP1 factor function. In particular, the phosphorylation of AP-1 subunit c-Jun and c-fos transcriptional regulation and cellular localization result also affected. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4178. doi:1538-7445.AM2012-4178

PAX5/TEL Has An Opposite Dominant Effect on Endogenous PAX5, Affecting Its Transcriptional Regulation, B Cell Adhesion and BCR Signaling In Murine Pre-BI Cells

AbstractAbstract 2482 Introduction.PAX5 is a transcription factor with both activation and repression functions, essential for B-cell development. Recently, it has been found as frequent target of abnormalities in B-cell precursor ALL, showing point mutations, deletions or involvement in chromosomal translocations. The functional role of these lesions is still poorly understood. In previous experiments in mouse pre-BI cells, we showed that the PAX5/TEL protein acts as an aberrant transcription factor with repressor function, causing a block on B-cell differentiation, short-term IL-7 independence and resistance to the anti-proliferative and pro-apoptotic effects of TGFbeta1. Moreover, PAX5/TEL enhances cell migration towards CXCL12, with the over expression of CXCR4. Aim.The aim of the present study was to comprehensively understand how PAX5/TEL affects the transcription process and eventually interferes with PAX5 and TEL pathways and how these modulate cellular processes. Methods.We analyzed gene expression profiles in pre-BI cells transduced either by MIGR-PAX5/TEL-IRES-GFP or by MIGR-GFP (Affymetrix Gene Chip technology, Mouse array 430A 2.0). Validation of Differentially Expressed Genes has been performed by quantitative RQ-PCR and FACS analyses. In vitro adhesion assays have been performed on VCAM1-coated slides. Results.PAX5/TEL significantly modulated the transcription process: among 340 differentially expressed genes, 61% were down- and 39% up-regulated. Both up and down-regulated genes encompass numerous PAX5-target genes; in particular, PAX5/TEL represses genes which are normally activated by PAX5, and, vice versa, it activates genes physiologically repressed by PAX5. Moreover, gene function classification analyses suggested that PAX5/TEL modulates molecules which are related to fundamental cellular processes, such as phosphorylation, transcription, B cell receptor signaling, as well as adhesion. In particular, we demonstrated the modulation of surface antigens responsible of extra cellular binding as well as the modulation of intracellular molecules involved in the signaling of adhesion regulation, such as CD44, SDC4, EDG1, NEDD9, BCAR3, PLEKHA2, SPHK1. Moreover, in vitro adhesion assays on VCAM1-coated slides showed a significant reduction of adhesion capacities in PAX5/TEL positive cells. In agreement with our previous results, which showed down-regulation of CD19, BLNK/SLP-65 and MB-1/CD79a, both genes involved in BCR signaling, we demonstrated the additional repression of numerous key molecules fundamental for this pathway, such as SIGLECG/CD22, IRF4, LCP2/SLP-75, SLAMF6/LY108, PLEKHA2, PRKD2, IKZF2, IKZF3. Furthermore, we functionally validated the impairment of BCR-signaling and demonstrated that PAX/TEL transduced pre-BI cells are completely blocked in IgM protein expression, loosing the ability to complete the VDJ rearrangement and consequently express the m-chain on the cells surface, compared to the control cells. Conclusions.These analyses further sustain the role of PAX5/TEL as an aberrant transcription factor. Its effect on endogenous PAX5 does not represent a classical dominant negative role; indeed, we defined this effect as an ‘opposite dominance‘, since PAX5/TEL caused the up-regulation of PAX5-repressed genes and, vice versa, the down-regulation of PAX5-activated targets. The biological consequences of this aberrant transcriptional activity are the impairment of B cell receptor signaling and the reduced adhesion capacity, both fundamental processes in B-cells, potentially involved in tumor transformation and in leukemia. Disclosures:No relevant conflicts of interest to declare.

Transforming activity of AML1-ETO is independent of CBFβ and ETO interaction but requires formation of homo-oligomeric complexes

Although both heterodimeric subunits of core binding factors (AML1/RUNX1 and CBFbeta) essential for normal hematopoiesis are frequently mutated to form different chimeric fusion proteins in acute leukemia, the underlying molecular mechanisms and structural domains required for cellular transformation remain largely unknown. Despite the critical role of CBFbeta for wild-type AML1 function and its direct involvement in chromosomal translocation, we demonstrate that both the expression and interaction with CBFbeta are superfluous for AML1-ETO (AE)-mediated transformation of primary hematopoietic cells. Similarly, the hetero-oligomeric interaction with transcriptional repressor ETO family proteins and the highly conserved NHR1 domain in AE fusion are also dispensable for transforming activity. In contrast, AE-mediated transformation is critically dependent on the DNA binding and homo-oligomeric properties of the fusion. Abolishment of homo-oligomerization by a small-molecule inhibitor could specifically suppress AML1 fusion-mediated transformation of primary hematopoietic cells. Together, these results not only identify the essential molecular components but also potential avenues for therapeutic targeting of AE-mediated leukemogenesis.

Transforming Activity of AML1-ETO Oncoprotein Is Independent of CBFβ and ETO but Requires Homo-Oligomeric Interaction.

AML1/RUNX1 and CBFb encode two critical transcription factors essential for the generation of hematopoietic stem cells (HSC). In acute myeloid leukemia, where leukemic stem cells (LSCs) have been functionally identified, AML1 and CBFb also represent the most commonly mutated targets. While animal models indicate that AML1 fusions per se are not sufficient to induce full-blown leukemia, they enhance self-renewal and expand targeted HSC and early progenitors, a property also reported for other oncogenic transcription factors involved in acute leukemia. Although attempts had been made to identify the critical domains required for AML-ETO (AE) mediated transformation, conflicting results were presented from most of these studies using exploited wellestablished cell lines, which suffer from the pitfall of carrying irrelevant genetic aberrants that may not reflect the normal biology of the disease. The only available structure/ function data on primary cells was limited to NHR2 of the ETO portion of the fusion, but it does not distinguish the functional contribution between homo-oligomerization and hetero-oligomerization. The lack of comprehensive structure/function data has not only significantly impeded the progress of understanding the biology of the disease, but also hinders the development of specific therapeutics. To this end, we performed extensive functional analysis to identify the key components essential for AE-mediated transformation of primary hematopoietic cells. In spite of the critical role of CBFb for wild type AML1 functions and its direct involvement in chromosomal translocation, we demonstrate that multiple AE single point mutants defective in CBFb interaction were still capable of transforming primary hematopoietic cells. Consistently, shRNA mediated knockdown of the endogenous expression of CBFb in primary cells did not compromise the transforming activity of AE, strongly suggesting a dispensable function of CBFb in AE mediated transformation. On the other hand, we demonstrate that NHR2 as the only domain in the ETO portion of the fusion is essential for transformation, but its heterooligomeric function including interaction with transcriptional repressor ETO family proteins is dispensable for the transforming activity. In contrast, synthetic FKBP homooligomerization modules could functionally replace NHR2, indicating that AE mediated transformation is critically dependent on homo-oligomeric property of the resultant fusion. Moreover, the transformation can also be abolished by a small molecule inhibitor that specifically dissociates homo-oligomerization. Together, these results not only identify the essential components and refine potential avenues for therapeutic targeting of AE oncogenic complexes, but also strongly endorse a common homo-oligomerization dependent mechanism shared by the most prevalent leukemia associated transcription factors.

Chromatin control of gene expression: Mixed-lineage leukemia methyltransferase SETs the stage for transcription

DNA is tightly packaged inside the nucleus into chromatin, an intricate assembly of nucleic acid, histones, and accessory proteins. Under normal circumstances, the higher-order structure of chromatin is a formidable obstacle for almost all biochemical processes that involve DNA. To enable transcription, replication, and repair, all eukaryotic cells possess a whole array of sophisticated molecular machines with the sole purpose of modifying chromatin structure to allow unhindered access to DNA. Next to ATP-powered units that literally push nucleosomes out of the way, there are many enzymes that catalyze the covalent modification of histones. The various methylations, acetylations, phosphorylations, and ubiquitinations either directly change the packaging properties of the histone octamer or mark these proteins for recognition by other proteins that help to read out this so-called “histone code” (1). A flurry of recent publications describes an ever-growing list of histone modifications, the associated enzymes, and the respective correlation with an “activated” or “repressed” chromatin state. Still, important questions remain. For example, it is well established that histones in actively transcribed regions are acetylated and have a certain methylation pattern. However, we have no clue how modification activities are specifically recruited to coding regions that need to be transcribed. We comprehend neither what happens if a gene must be switched off nor how an enzyme “knows” where a gene starts and where it ends. In this issue of PNAS, Milne et al. (2) begin to address these questions in an elegant system involving the histone H3 lysine 4-specific methyltransferase mixed-lineage leukemia (MLL). The MLL gene has become notorious because of its frequent involvement in chromosomal translocations (11q23 translocations) that lead …

Infertility with Defective Spermiogenesis in Mice Lacking AF5q31, the Target of Chromosomal Translocation in Human Infant Leukemia

AF5q31 (also called MCEF) was identified by its involvement in chromosomal translocation with the gene MLL (mixed lineage leukemia), which is associated with infant acute lymphoblastic leukemia. Several potential roles have been proposed for AF5q31 and other family genes, but the specific requirements of AF5q31 during development remain unclear. Here, we show that AF5q31 is essential for spermatogenesis. Although most AF5q31-deficient mice died in utero and neonatally with impaired embryonic development and shrunken alveoli, respectively, 13% of AF5q31-deficient mice thrived as wild-type mice did. However, the male mice were sterile with azoospermia. Histological examinations revealed the arrest of germ cell development at the stage of spermiogenesis, and virtually no spermatozoa were seen in the epididymis. AF5q31 was found to be preferentially expressed in Sertoli cells. Furthermore, mutant mice displayed severely impaired expression of protamine 1, protamine 2, and transition protein 2, which are indispensable to compact the haploid genome within the sperm head, and an increase of apoptotic cells in seminiferous tubules. Thus, AF5q31 seems to function as a transcriptional regulator in testicular somatic cells and is essential for male germ cell differentiation and survival. These results may have clinical implications in the understanding of human male infertility.

Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene.

The scl gene encodes a basic-helix-loop-helix transcription factor which was identified through its involvement in chromosomal translocations in T-cell leukemia. To elucidate its physiological role, scl was targeted in embryonic stem cells. Mice heterozygous for the scl null mutation were intercrossed and their offspring were genotyped. Homozygous mutant (scl-/-) pups were not detected in newborn litters, and analysis at earlier time points demonstrated that scl-/- embryos were dying around embryonic day 9.5. The scl-/- embryos were pale, edematous, and markedly growth retarded after embryonic day 8.75. Histological studies showed complete absence of recognizable hematopoiesis in the yolk sac of these embryos. Early organogenesis appeared to be otherwise normal. Culture of yolk sac cells of wild-type, heterozygous, and homozygous littermates confirmed the absence of hematopoietic cells in scl-/- yolk sacs. Reverse transcription PCR was used to examine the transcripts of several genes implicated in early hematopoiesis. Transcripts of GATA-1 and PU.1 transcription factors were absent from RNA from scl-/- yolk sacs and embryos. These results implicate scl as a crucial regulator of early hematopoiesis.

SCL and related hemopoietic helix-loop-helix transcription factors.

The helix-loop-helix (HLH) proteins are a family of transcription factors that include proteins critical to differentiation and development in species ranging from plants to mammals. Five members of this family (MYC, SCL, TAL-2, LYL-1 and E2A) are implicated in oncogenic events in human lymphoid tumors because of their consistent involvement in chromosomal translocations. Although activated in T cell leukemias, expression of SCL and LYL-1 is low or undetectable in normal T cell populations. SCL is expressed in erythroid, megakaryocyte and mast cell populations (the same cell lineages as GATA-1, a zinc-finger transcription factor). In addition, both SCL and GATA-1 undergo coordinate modulation during chemically induced erythroid differentiation of mouse erythroleukemia cells and are down-modulated during myeloid differentiation of human K562 cells, thus implying a role for SCL in erythroid differentiation events. However, in contrast to GATA-1, SCL is expressed in the developing brain. Studies of the function of SCL suggest it is also important in proliferation and self-renewal events in erythroid cells.

C-myc Involvement in chromosomal translocations in mice and men.

c-myc Involvement in chromosomal translocations in mice and men.