Alexey Efanov

Akt Phosphorylates and Regulates Pdcd4 Tumor Suppressor Protein

Programmed cell death 4 (Pdcd4) is a tumor suppressor protein that interacts with eukaryotic initiation factor 4A and inhibits protein synthesis. Pdcd4 also suppresses the transactivation of activator protein-1 (AP-1)-responsive promoters by c-Jun. The Akt (protein kinase B) serine/threonine kinase is a key mediator of phosphoinositide 3-kinase pathway involved in the regulation of cell proliferation, survival, and growth. Because Pdcd4 has two putative Akt phosphorylation sites at Ser(67) and Ser(457), we investigated whether Akt phosphorylates and regulates Pdcd4. Our results show that Akt specifically phosphorylates Ser(67) and Ser(457) residues of Pdcd4 in vitro and in vivo. We further show that phosphorylation of Pdcd4 by Akt causes nuclear translocation of Pdcd4. Using luciferase assay, we show that phosphorylation of Pdcd4 by Akt also causes a significant decrease of the ability of Pdcd4 to interfere with the transactivation of AP-1-responsive promoter by c-Jun.

Tcl1 functions as a transcriptional regulator and is directly involved in the pathogenesis of CLL

B cell chronic lymphocytic leukemia (B-CLL) is the most common human leukemia. Deregulation of the T cell leukemia/lymphoma 1 (TCL1) oncogene in mouse B cells causes a CD5-positive leukemia similar to aggressive human B-CLLs. To examine the mechanisms by which Tcl1 protein exerts oncogenic activity in B cells, we investigated the effect of Tcl1 expression on NF-κB and activator protein 1 (AP-1) activity. We found that Tcl1 physically interacts with c-Jun, JunB, and c-Fos and inhibits AP-1 transcriptional activity. Additionally, Tcl1 activates NF-κB by physically interacting with p300/CREB binding protein. We then sequenced the TCL1 gene in 600 B-CLL samples and found 2 heterozygous mutations: T38I and R52H. Importantly, both mutants showed gain of function as AP-1 inhibitors. The results indicate that Tcl1 overexpression causes B-CLL by directly enhancing NF-κB activity and inhibiting AP-1.

13q14 deletions in CLL involve cooperating tumor suppressors.

B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia. 13q14 deletions are most common chromosomal alterations in CLL. We previously reported that miR-15/16 is a target of 13q14 deletions and plays a tumor suppressor role by targeting BCL2. Because DLEU7 is located near miR-15/16 and is also positioned within a minimal deleted region, we investigated whether DLEU7 could also play a tumor suppressor role. Recent studies of transgenic mouse models demonstrated the importance of the nuclear factor-kappaB (NF-kappaB) pathway in CLL. To examine the possible role of DLEU7 in CLL, we investigated the effect of DLEU7 expression on NF-kappaB and nuclear factor of activated T cells (NFAT) activity. We found that DLEU7 functions as a potent NF-kappaB and NFAT inhibitor by physically interacting and inhibiting TACI and BCMA, members of the tumor necrosis factor (TNF) receptor family involved in B-CLL. In addition, DLEU7 expression in A549 lung cancer cells resulted in a decrease in S phase and increased apoptosis. The results suggest that loss of DLEU7 may cooperate with the loss of miR-15/16 in the pathogenesis of CLL.

Tcl1 interacts with Atm and enhances NF-κB activation in hematologic malignancies

The T-cell leukemia/lymphoma 1 (TCL1) oncogene is a target of chromosomal translocations and inversions at 14q31.2, and its rearrangement in T cells causes T-cell prolymphocytic leukemias. TCL1 dysregulation in B cells is responsible for the development of an aggressive form of chronic lymphocytic leukemia (CLL), the most common human leukemia. We have investigated the mechanisms underlying the oncogenic functions of Tcl1 protein using a mass spectrometry approach and have identified Atm (ataxia-telangiectasia mutated) as a candidate Tcl1-interacting protein. The Tcl1-Atm complex formation was validated by coimmunoprecipitation experiments. Importantly, we show that the association of Atm with Tcl1 leads to enhanced IκBα phosphorylation and ubiquitination and subsequent activation of the NF-κB pathway. Our findings reveal functional cross-talk between Atm and Tcl1 and provide evidence for a novel pathway that could be targeted in leukemias and lymphomas.

Akt Phosphorylates Tal1 Oncoprotein and Inhibits Its Repressor Activity

The helix-loop-helix transcription factor Tal1 is required for blood cell development and its activation is a frequent event in T-cell acute lymphoblastic leukemia. The Akt (protein kinase B) kinase is a key player in transduction of antiapoptotic and proliferative signals in T cells. Because Tal1 has a putative Akt phosphorylation site at Thr90, we investigated whether Akt regulates Tal1. Our results show that Akt specifically phosphorylates Thr90 of the Tal1 protein within its transactivation domain in vitro and in vivo. Coimmunoprecipitation experiments showed the presence of Tal1 in Akt immune complexes, suggesting that Tal1 and Akt physically interact. We further showed that phosphorylation of Tal1 by Akt causes redistribution of Tal1 within the nucleus. Using luciferase assay, we showed that phosphorylation of Tal1 by Akt decreased repressor activity of Tal1 on EpB42 (P4.2) promoter. Thus, these data indicate that Akt interacts with Tal1 and regulates Tal1 by phosphorylation at Thr90 in a phosphatidylinositol 3-kinase-dependent manner.

Heat shock protein 70 regulates Tcl1 expression in leukemia and lymphomas

T-cell leukemia/lymphoma 1 (TCL1) is an oncogene overexpressed in T-cell prolymphocytic leukemia and in B-cell malignancies including B-cell chronic lymphocytic leukemia and lymphomas. To date, only a limited number of Tcl1-interacting proteins that regulate its oncogenic function have been identified. Prior studies used a proteomic approach to identify a novel interaction between Tcl1 with Ataxia Telangiectasia Mutated. The association of Tcl1 and Ataxia Telangiectasia Mutated leads to activation of the NF-κB pathway. Here, we demonstrate that Tcl1 also interacts with heat shock protein (Hsp) 70. The Tcl1-Hsp70 complex was validated by coimmunoprecipitation experiments. In addition, we report that Hsp70, a protein that plays a critical role in the folding and maturation of several oncogenic proteins, associates with Tcl1 protein and stabilizes its expression. The inhibition of the ATPase activity of Hsp70 results in ubiquitination and proteasome-dependent degradation of Tcl1. The inhibition of Hsp70 significantly reduced the growth of lymphoma xenografts in vivo and down-regulated the expression of Tcl1 protein. Our findings reveal a functional interaction between Tcl1 and Hsp70 and identify Tcl1 as a novel Hsp70 client protein. These findings suggest that inhibition of Hsp70 may represent an alternative effective therapy for chronic lymphocytic leukemia and lymphomas via its ability to inhibit the oncogenic functions of Tcl1.

Tal1 transgenic expression reveals absence of B lymphocytes.

TAL1 oncogene encodes a helix-loop-helix transcription factor, Tal1, which is required for blood cell development, and its activation is a frequent event in T-cell acute lymphoblastic leukemia. Tal1 interacts and inhibits other helix-loop-helix factors such as E47 and HEB. To investigate the function of Tal1 in B cells, we generated Emu-TAL1 transgenic mouse line, expressing Tal1 in mouse B-cell lineage. Fluorescence-activated cell sorting (FACS) analysis of lymphocytes isolated from spleens of five out of five founders reveals complete absence of IgM- or CD19-expressing cells. Only 2% to 3% of these cells were B220+ and 100% of B220+ cells were CD43+, indicating that these mice were able to make pro-B cells. Similarly, FACS analysis of bone marrow cells in Emu-TAL1 mice revealed complete absence of B220+IgM+ and B220+CD19+ cells. Analysis of the recombination status of IgH genes revealed the presence of D-J but absence or drastic reduction of V-D-J rearrangements. Our results suggest that Tal1 overexpression in B cells results in a phenotype similar to that of B cells of E47/E2A knockout animals. This represents first in vivo evidence that Tal1 can completely inhibit E47/E2A function.

Abstract 2185: DLEU7 is a putative tumor suppressor in glioblastoma

DLEU7 was first identified as a tumor suppressor in B-CLL. We previously showed that Dleu7 functions as a potent NF-kappaB and NFAT inhibitor. To investigate possible function of Dleu7 in brain tumorigenesis we carried out the real-time PCR of RNAs isolated from different parts of human brain, brain tumors and brain tumor cell lines and found that expression of DLEU7 significantly decreased in brain tumor cell lines and brain tumors (glioblastoma) in comparison with normal brain tissues. We further investigated the effect of DLEU7 expression in HTB-15 glioblastoma cell line. Our results showed that Dleu7 overexpression resulted in apoptosis and cell cycle halt. In addition, adenovirus-mediated DLEU7 expression resulted in 100 percent increase of caspase 3/7 activation compared to AdenoGFP control in HTB-15 cells. Our data indicate that DLEU7 may play a tumor suppressor role in glioblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2185. doi:10.1158/1538-7445.AM2011-2185

Abstract LB-352: B-CLL phenotype in Eµ-miR-29 transgenic mice

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the western world. Human B-CLL occurs in two forms: aggressive (showing high ZAP-70 expression and unmutated IgH VH) and indolent (showing low ZAP-70 expression and mutated IgH VH). We found that miR-29a is upregulated in indolent human B-CLL compared to aggressive B-CLL and normal CD19+ B-cells. To study the role of miR-29 in B-CLL, we generated Eµ-miR-29 transgenic mice overexpressing miR-29 in mouse B-cells. Flow cytometric analysis revealed a markedly expanded CD5+ population in the spleen of these mice starting at 2 months of age. Over 80% of miR-29 transgenic mice exhibited an expanded population of CD5+ B-cells, a characteristic of the B-CLL phenotype. An average of 56% of the B-cell population in these transgenics were CD5 positive. At the age of 2 years these mice showed significantly enlarged spleens and an increase in CD5+ B-cell population of up to 100% of B-cells. Over 8% (3/36) of Eµ-miR-29 transgenic mice developed frank leukemia and prematurely died from the disease at the age of 24-26 months. The expanded CD5+ B-cell population was found to be proliferative, with an increased number of cells in the S-phase of the cell cycle, compared to wild type CD19+ B-cells. These results suggest that deregulation of miR-29 can cause, or at least significantly contribute to the pathogenesis of indolent B-CLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-352.

Abstract 401: CD5+CD23+ leukemic cell populations in Tcl1 transgenic mice show significantly increased proliferation and Akt phosphorylation

B-cell chronic lymphocytic leukemia (B-CLL) is the most common human leukemia. Deregulation of the T-cell leukemia/lymphoma 1 (TCL1) oncogene in mouse B-cells causes a CD5 positive leukemia similar to aggressive human B-CLLs. We recently reported that TCL1 expression in B-CLL is regulated by miR29 and miR181. To determine whether treatment with microRNAs can inhibit B-CLL in mice by targeting TCL1 we generated TCL1 transgenic mice using a construct containing 3’ and 5’ UTRs of TCL1 under B-cell specific Eμ promoter (Eμ-TCL1fl). At the age of 12-18 months these mice showed B-CLL like disease. Immunophenotyping revealed accumulation of CD5+CD23+B220+ population in spleens and lymph nodes. Our results show that CD5+CD23+ B-cell populations from Eμ-TCL1fl mice actively proliferate and display significantly increased levels of phospho-Akt. Eμ-TCL1fl mice showed immunological abnormalities similar to human B-CLL, including hypoimmunoglobulinemia, abnormal levels of cytokines, and impaired immune response. These findings revealed biochemical and immunological similarities between Tcl1 driven B-CLL in mice and human B-CLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 401.