Tumor suppressor function of FOXO1 in diffuse large B-cell lymphomas: mechanisms of regulation and personalized rational targeting strategies.

The project  entitled “Tumor suppressor function of FOXO1 in diffuse large B-cell lymphomas: mechanisms of regulation and personalized rational targeting strategies” is now completed.

 

FOXO proteins have emerged as important regulators of wide spectrum of cellular functions, including cell cycle, apoptosis, DNA damage response, glucose metabolism, and oxidative stress resistance. In humans, FOXO proteins are converging points integrating signaling pathways triggered by growth factors, cytokines, nutrients, oxidative stress and B-cell receptor activity. In normal B lymphocytes, nuclear exclusion of FOXO1 is a critical effector of pro-survival signaling mediated by B-cell receptor (BCR).  Diffuse large B-cell lymphomas (DLBCLs), the most common type of lymphoma in adults, are clinically and genetically heterogeneous tumours. In line with the clinical heterogeneity of DLBCLs, multiple signalling pathways have been identified that mediate survival benefit in different subsets of tumors. For example, a large subset of DLBCLs has surface expression of immunoglobulins and a constitutive B-cell receptor activity. Disruption of this signal with small molecule SYK or BTK inhibitors are capable of inducing apoptosis in DLBCL cells, the effect at least in part mediated by decreased AKT activity. Since decreased AKT activity leads to decrease in FOXO1 phosphorylation and its subsequent activation, we hypothesized that FOXO1 might be an important effector of BCR inhibitor-mediated toxicity in human BCR-dependent lymphomas. For this reason, we have first examined the role of AKT-FOXO1 pathway in BCR-dependent DLBCL cells treated with SYK inhibitor. In BCR-dependent DLBCL cell lines (DHL4,DHL6, Ly7, Ly1), AKT and FOXO1 phosphorylations were sensitive to SYK inhibition and decreased after incubation with a SYK inhibitor fostamatinib. Diminished FOXO1 phosphorylation resulted in its nuclear relocalization and induction of FOXO1-dependent gene expression. Complementation of AKT activity by its constitutively active form (myrAKT) disrupted the effect of SYK inhibitor on FOXO1 phosphorylations and rescued DLBCL cells from  fostamatinib toxicity. To assess whether the increased activity of FOXO1 is sufficient to induce apoptosis of DLBCL cells, we transduced DHL4 cells with a constitutively nuclear and transcriptionally active FOXO1 mutant. The mutant induced G1/S cell cycle arrest and triggered apoptosis, demonstrating that FOXO1 activation is sufficient to induce apoptosis of DLBCL cells. Next, we assessed the toxicity of SYK inhibitor in cells lacking FOXO1. Cells with silenced FOXO1 exhibited 70% lower sensitivity to SYK inhibitor, as measured with proliferation and apoptosis assays (p<0.0001). Since fostamatinib toxicity is dependent on the induction of a proapoptotic BCL2 family gene HRK, we assessed its expression in cells lacking FOXO1. Fostamatinib treatment induced 100-120-fold overexpression of HRK in control cells, whereas in cells with silenced FOXO1, HRK was not induced. Finally, we mechanistically linked FOXO1 with HRK expression via caspase dependent cleavage of HRK repressor, DREAM.  Consistent with its proapoptotic function, loss of FOXO1 in primary DLBCL cells was associated with significantly better prognosis. In addition, expression of FOXO1 was not observed in 10% of SYK positive tumors.

Since cancer cells can utilize multiple different mechanisms to inactivate the putative tumor suppressor, we explored other potential mechanism functionally inactivating proapototic function of FOXO1 in DLBCLs. In addition to the physiological, AKT-dependent mechanism of FOXO family members regulation, deacetylation of certain FOXO proteins is associated with altered transcriptional activity, reduced FOXO-dependent apoptosis and increased tolerance to oxidative stress. These observations are particularly interesting, since a subset of BCR-independent DLBCLs is characterized by increased expression of genes involved in mitochondrial function, electron transport and oxidative phosphorylation. We have found that FOXO1 in DLBCLs is acetylated by p300 acetyltransferase in response to oxidative stress. FOXO1 mutant with target lysines mutated to arginines induced significantly less expression of proapoptotic genes, demonstrating that acetylation augments its  proapoptotic potential.  The covalent association between FOXO1 and p300 is disrupted by thioredoxin, reducing the disulphide bond between C612  in FOXO1 and this acetyltransferase. Taken together, these results demonstrate the role AKT and FOXO1 as mediators of  proapoptotic activity of BCR/SYK blockade in DLBCLs. Since functional FOXO1 is required for proapoptotic activity of SYK inhibitor, these results highlight a potential role of FOXO1 in identifying patients unlikely to respond to this drug.  We also describe a mechanism of TXN-mediated attenuation of ROS-induced proapoptotic FOXO1 signaling that involves decreased FOXO1 acetylation. These studies also highlight a therapeutic potential of TXN inhibition in a specific DLBCL subset.

The results of our research is published in the following papers and meeting abstracts:

1.       Szydlowski M, Kiliszek P, Sewastianik T , Jablonska E , BialopiotrowiczE , Gorniak P, Polak A , Markowicz S , Nowak E , Grygorowicz MA , Prochorec-Sobieszek M , Szumera-Cieckiewicz A , Malenda A , Lech-Maranda E, Warzocha K , Juszczynski P. FOXO1 activation is an effector of SYK and AKT inhibition in tonic BCR  signal dependent diffuse large B-cell lymphomas. Blood 2015, Epub 19 Nov 2015, doi:10.1182/blood-2015-06-654111

Sewastianik T, Prochorec-Sobieszek M, Chapuy B, Juszczyński P. MYC deregulation in lymphoid tumors: Molecular mechanisms, clinical consequences and therapeutic implications. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 2014; 1846: 457-67
Szydłowski M, Jabłońska E, Juszczyński P. FOXO1 Transcription Factor: A Critical Effector of the PI3K-AKT Axis in B-Cell Development. Int Rev Immunol. 2014 ;33:146-57
4.       Sewastianik T, Szydlowski M, Bialopiotrowicz E, Jablonska E, Kiliszek P, Polak A, Prochorec-Sobieszek M, Szumera-Cieckiewicz A, Kaminski TS, Gorniak P, Markowicz S, Nowak E, Grygorowicz M, Warzocha K, Juszczynski P. FOXO1-p300-Txn Circuit Regulates Oxidative Stress Responses in Diffuse Large B-Cell Lymphomas Characterized By Enhanced Oxidative Phosphorylation. Amerian Society of Hematology Annual Meeting, Orlando, FL December 4-8, 2015. Oral presentation; Blood 2015 126:466; 2015

5.       Jablonska E, Gorniak P, Prusisz W, Kiliszek P, Szydlowski M, Sewastianik T, Bialopiotrowicz E, Polak A, Prochorec-Sobieszek M, Szumera-Cieckiewicz A, Warzocha K, Juszczynski P. MiR-155 Amplifies AKT and NFkB Signaling By Targeting Multiple Regulators of BCR Signal in DLBCL. Amerian Society of Hematology Annual Meeting, Orlando, FL December 4-8, 2015. Poster presentation. Blood 2015 126:2455

6.       Kiliszek P, Szydlowski M, Sewastianik T, Jablonska E, Bialopiotrowicz E, Gorniak P, Polak A, Markowicz S, Nowak E, Grygorowicz MA, Prochorec-Sobieszek M, Szumera-Cieckiewicz A, Malenda A, Lech-Maranda E, Warzocha K, Juszczynski P.  FOXO1 Activation Is an Effector of SYK and AKT Inhibition in Tonic BCR Signal-Dependent Diffuse Large B-Cell Lymphomas. Amerian Society of Hematology Annual Meeting, Orlando, FL December 4-8, 2015. Oral presentation. Blood 2015 126:314

7.       Sewastianik T, Szydlowski M, Bialopiotrowicz E, Jablonska E, Kiliszek P, Gorniak P, Polak A, Prochorec-Sobieszek M, Szumera-Cieckiewicz A, Kaminski TS, Markowicz S, Nowak E, Grygorowicz E, Warzocha K, Juszczynski P. TXN overexpression in diffuse large B-cell lymphoma cells attenuates oxidative stress-induced proapoptoticactivity of  FOXO1 transcription factor. 13 International Conference on Malignant Lymphoma, Lugano, Switzerland, 17-20 June 2015, Hematol Oncol 2015; 33, Suppl.1: 182

8.       Jablonska E, Gorniak P, Prusisz W, Kiliszek P, Szydlowski M, Sewastianik T, Bialopiotrowicz E, Polak A, Prochorec-Sobieszek M, Szumera-Cieckiewicz A, Warzocha K, Juszczynski P. MiR-155 amplifies BCR signalling by targeting its multiple negative regulators in DLBCL.  13 International Conference on Malignant Lymphoma, Lugano, Switzerland, 17-20 June 2015, Hematol Oncol 2015; 33, Suppl.1: 183

9.       Kiliszek P, Szydlowski M, Sewastianik T, Bialopiotrowicz E, Jablonska E, Polak A, Gorniak P, Markowicz S, Nowak E, Grygorowicz MA, Bojarczuk K, Winiarska M, Gołąb J, Lech-Marańda E, Warzocha K, Juszczyński P. SYK inhibition triggers DLBCL cell death via a mechanism linking FOXO1 activation, DREAM cleavage and expression of a proapoptotic BCL2 family member, HRK. 13 International Conference on Malignant Lymphoma, Lugano, Switzerland, 17-20 June 2015, Hematol Oncol 2015; 33, Suppl.1: 234

10.   Sewastianik T, Szydłowski M, Białopiotrowicz E, Jabłońska E, Kiliszek P, Polak A, Markowicz S, Nowak E, Grygorowicz M, Juszczyński P. Thioredoxin Reduces Proapoptotic Activity of FOXO1 by Altering Its Acetylation Status in DLBCLs. 2014 ASH Meeting on Lymphoma Biology, Aug 10-13, 2014, Colorado Springs, CO, USA

11.   Kiliszek P,  Szydłowski M, Sewastianik T, Białopiotrowicz E, Jabłońska E, Polak A, Prusisz W, Markowicz S, Nowak E, Grygorowicz MA, Bojarczuk K, Winiarska M, Gołąb J, Juszczyński P. FOXO1 transcription factor mediates toxicity of SYK inhibition in DLBCL.  2014 ASH Meeting on Lymphoma Biology, Aug 10-13, 2014, Colorado Springs, CO, USA

12.   Kiliszek P, Szydłowski M, Sewastianik T, Białopiotrowicz E, Jabłońska E, Polak A, Prusisz W, Markowicz S, Nowak E, Grygorowicz MA, Bojarczuk K, Winiarska M, GołąbJ, Juszczyński P. Foxo1 transcription factor mediates toxicity of SYK inhibition in DLBCL. 19th Congress of the European Hematology Association, 12-15 June, 2014, Milan, Italy . Haematologica, 104, Suppl. 1; 99:  137

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DOTACJE NA INNOWACJE

Projekt realizowany w ramach programu TEAM Fundacji na rzecz Nauki Polskiej,
współfinansowany przez Unię Europejską z Europejskiego Funduszu Rozwoju Regionalnego