Malin Ageberg

Identification of a novel and myeloid specific role of the leukemia-associated fusion protein DEK-NUP214 leading to increased protein synthesis.

The t(6;9)(p22;q34) chromosomal translocation is found in a subset of patients with acute myeloid leukemia (AML). The translocation results in a fusion between the nuclear phosphoprotein DEK and the nucleoporin NUP214 (previously CAN). The mechanism by which the fusion protein DEK‐NUP214 contributes to leukemia development has not been identified, and disruptions of normal cellular functions by DEK‐NUP214 have previously not been described. In the present study, a novel effect of the DEK‐NUP214 fusion protein is demonstrated. Our findings reveal a substantial increase in global protein synthesis in DEK‐NUP214 expressing cells. Furthermore, we conclude that this effect is not the result of dysregulated transcription but merely due to increased translation. Consistent with the association with AML, the increased protein synthesis mediated by DEK‐NUP214 is restricted to cells of the myeloid lineage. Analysis of potential mechanisms for regulating protein synthesis shows that expression of DEK‐NUP214 correlates to the phosphorylation of the translation initiation protein, EIF4E. The present data provide evidence that increase of translational activity constitutes a mechanism by which the leukemogenic effect of DEK‐NUP124 may be mediated.

Forced expression of the DEK-NUP214 fusion protein promotes proliferation dependent on upregulation of mTOR

BackgroundThe t(6;9)(p23;q34) chromosomal translocation is found in 1% of acute myeloid leukemia and encodes the fusion protein DEK-NUP214 (formerly DEK-CAN) with largely uncharacterized functions.MethodsWe expressed DEK-NUP214 in the myeloid cell lines U937 and PL-21 and studied the effects on cellular functions.ResultsIn this study, we demonstrate that expression of DEK-NUP214 increases cellular proliferation. Western blot analysis revealed elevated levels of one of the key proteins regulating proliferation, the mechanistic target of rapamycin, mTOR. This conferred increased mTORC1 but not mTORC2 activity, as determined by the phosphorylation of their substrates, p70 S6 kinase and Akt. The functional importance of the mTOR upregulation was determined by assaying the downstream cellular processes; protein synthesis and glucose metabolism. A global translation assay revealed a substantial increase in the translation rate and a metabolic assay detected a shift from glycolysis to oxidative phosphorylation, as determined by a reduction in lactate production without a concomitant decrease in glucose consumption. Both these effects are in concordance with increased mTORC1 activity. Treatment with the mTORC1 inhibitor everolimus (RAD001) selectively reversed the DEK-NUP214-induced proliferation, demonstrating that the effect is mTOR-dependent.ConclusionsOur study shows that the DEK-NUP214 fusion gene increases proliferation by upregulation of mTOR, suggesting that patients with leukemias carrying DEK-NUP214 may benefit from treatment with mTOR inhibitors.

Inhibition of geranylgeranylation mediates sensitivity to CHOP-induced cell death of DLBCL cell lines.

Prenylation is a post-translational hydrophobic modification of proteins, important for their membrane localization and biological function. The use of inhibitors of prenylation has proven to be a useful tool in the activation of apoptotic pathways in tumor cell lines. Rab geranylgeranyl transferase (Rab GGT) is responsible for the prenylation of the Rab family. Overexpression of Rab GGTbeta has been identified in CHOP refractory diffuse large B cell lymphoma (DLBCL). Using a cell line-based model for CHOP resistant DLBCL, we show that treatment with simvastatin, which inhibits protein farnesylation and geranylgeranylation, sensitizes DLBCL cells to cytotoxic treatment. Treatment with the farnesyl transferase inhibitor FTI-277 or the geranylgeranyl transferase I inhibitor GGTI-298 indicates that the reduction in cell viability was restricted to inhibition of geranylgeranylation. In addition, treatment with BMS1, a combined inhibitor of farnesyl transferase and Rab GGT, resulted in a high cytostatic effect in WSU-NHL cells, demonstrated by reduced cell viability and decreased proliferation. Co-treatment of BMS1 or GGTI-298 with CHOP showed synergistic effects with regard to markers of apoptosis. We propose that inhibition of protein geranylgeranylation together with conventional cytostatic therapy is a potential novel strategy for treating patients with CHOP refractory DLBCL.

The p53 target gene TRIM22 directly or indirectly interacts with the translation initiation factor eIF4E and inhibits the binding of eIF4E to eIF4G

The interferon (IFN)‐inducible protein TRIM22 (Staf50) is a member of the tripartite motif protein family and has been suggested a role in the regulation of viral replication as well as of protein ubiquitylation. In addition, we have previously shown that TRIM22 is a direct target gene for the tumour suppressor p53. Consistently, over‐expression of TRIM22 inhibits the clonogenic growth of monoblastic U937 cells, suggesting anti‐proliferative or cell death‐inducing effects.

Abstract 3085: The IFN inducible p53 target gene TRIM22 binds to the translation initation factor eIF4E and represses translation through disturbing the assembly of the eIF4F complex

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL TRIM22 (Staf50) is an interferon (IFN)-inducible protein as well as a p53 target gene. It belongs to the TRIM (TRIpartite Motif) family proteins, characterized of a conserved tripartite motif consisting of a RING finger, B-box and a coiled-coil domain. The TRIM family includes more than 70 proteins, involved in various biological processes; e.g. apoptosis and cell proliferation. Consistently, several TRIM family members are involved in cancer. TRIM22 has been suggested to be an antiviral protein, capable of restricting HIV-, hepatitis B- and encephalomyocarditis replication. The molecular mechanisms for TRIM22 are unclear, but the antiviral activity may be dependent on the ubiquitylation capacity of TRIM22. We have previously shown it to suppress the clonogenic growth of leukemic U-937 cells, suggesting anti-proliferative or cell death-inducing effects. Now, we demonstrate that TRIM22 binds to the translation initiation factor eIF4E, and inhibits the binding of eIF4E to eIF4G, thus disturbing the assembly of the eIF4F complex, vital for cap dependent translation of mRNA. As a consequence, TRIM22 represses translation, as shown by decreased reporter protein levels, as well as global protein synthesis. Pulse-chase and cycloheximide experiments reveal that the repressive effects on the reporter protein is not due to increased protein degradation. Taken together, our data show TRIM22 to repress the binding of eIF4E to eIF4G, suggesting a novel translation regulating mechanism, possibly mediated in response to p53 and/or IFN signaling. 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 3085. doi:10.1158/1538-7445.AM2011-3085

Abstract 4005: The leukemia-associated fusion protein DEK-NUP214 induces proliferation through an mTOR-dependent mechanism

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Acute myeloid leukemia (AML) is characterized by the dysregulated proliferation and impaired differentiation of myeloid precursor cells. The majority of these leukemias harbor genetic translocations, which determine both the molecular mechanistics and the prognosis of the disease. The t(6;9)(p22;q34) chromosomal translocation is found in 1% of AML, where it is associated with young age and poor prognosis. The translocation occurs between specific introns in the gene DEK on chromosome 6 and the gene NUP214 on chromosome 9, creating the fusion gene DEK-NUP214. The role of DEK-NUP214 in leukemogenesis is still largely uncharacterized. To obtain an experimental model of the disease, we expressed the fusion gene in the myeloid cell line U937 and studied the phenotype of the stable clones. We show that cells expressing DEK-NUP214 proliferate faster and also sustain their proliferative capacity longer in culture than their normal counterparts. Cellular proliferation is regulated by a wide range of signaling pathways, but many converge on the activating phosphorylation of the mechanistic target of rapamycin (mTOR) at Ser2448. Western blot analysis of the clones revealed that cells expressing DEK-NUP214 have higher levels of both phosphorylated and total mTOR protein. To determine the effect on downstream cellular functions, we proceeded to study mTOR-dependent translation and metabolism. We performed a global translation assay where the incorporation of radioactively labeled amino acids into newly synthesized proteins reflects the rate of translation. The results show that cells expressing DEK-NUP214 have a markedly increased translation rate. Cellular metabolism was studied by measuring the consumption of glucose and the production of lactate in cell supernatant. We demonstrate that cells expressing DEK-NUP214 produce less lactate, despite equal glucose consumption and increased proliferation. In concordance with increased mTOR activity, our results suggest that cells expressing DEK-NUP214 shift their metabolism from glycolysis to oxidative phosphorylation. The increased activity of mTOR thus leads to translational and metabolic changes that could play a role in the leukemogenic effect of DEK-NUP214. Interestingly, proliferation induced by DEK-NUP214 is highly dependent on mTOR. Treatment with the mTOR inhibitor everolimus (RAD001) reduces the proliferation of the DEK-NUP214 cells to the level of the control cells, without affecting the control cells. This pivotal role of mTOR suggests that leukemias harboring the t(6;9)(p22;q34) translocation may be susceptible to treatment with either rapamycin or the novel mTOR inhibitors that are approaching the clinic. 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 4005. doi:10.1158/1538-7445.AM2011-4005