Inhibition of spleen tyrosine kinase (SYK) in tonic B-cell receptor (BCR) signal-dependent diffuse large B-cell lymphomas (DLBCLs) inhibits cellular proliferation, decreases cholesterol biosynthesis, and triggers apoptosis, at least in part via a mechanism involving decreased activity of phosphatidylinositol 3-kinase/AKT axis. Because forkhead box O1 (FOXO1) is a major effector of this pathway, we investigated the role of FOXO1 in toxicity of BCR pathway inhibition. Inhibition of SYK in DLBCL cells with tonic BCR signaling decreased phospho-AKT and phospho-FOXO1 levels and triggered FOXO1-driven gene expression. Introduction of constitutively active FOXO1 mutant triggered cell cycle arrest and apoptosis, indicating that increased FOXO1 activity is toxic to these DLBCL cells. Depletion of FOXO1 with short hairpin RNA led to almost complete resistance to chemical SYK inhibitor R406, demonstrating that FOXO1 is also required for R406-induced cell death. FOXO1 in these cells is also involved in regulation of expression of the critical master regulator of cholesterol biosynthesis, SREBP1. Because HRK is the key effector of SYK inhibition, we characterized a mechanism linking FOXO1 activation and HRK induction that involves caspase-dependent cleavage of HRK's transcriptional repressor DREAM. Because AKT in lymphoma cells can be regulated by other signals than BCR, we assessed the combined effects of the AKT inhibitor MK-2206 with R406 and found markedly synergistic FOXO1-dependent toxicity. In primary DLBCLs, FOXO1 expression was present in 80% of tumors, correlated with SYK activity, and was associated with longer overall survival. These results demonstrate that FOXO1 is required for SYK and AKT inhibitor-induced toxicity. (Blood. 2016;127(6):739-748)

Molecular profiling has led to identification of subtypes of diffuse large B-cell lymphomas (DLBCLs) differing in terms of oncogenic signaling and metabolic programs. The OxPhos-DLBCL subtype is characterized by enhanced mitochondrial oxidative phosphorylation. As increased oxidative metabolism leads to overproduction of potentially toxic reactive oxygen species (ROS), we sought to identify mechanisms responsible for adaptation of OxPhos cells to these conditions. Herein, we describe a mechanism involving the FOXO1-TXN-p300 redox-dependent circuit protecting OxPhos-DLBCL cells from ROS toxicity. We identify a BCL6-dependent transcriptional mechanism leading to relative TXN overexpression in OxPhos cells. We found that OxPhos cells lacking TXN were uniformly more sensitive to ROS and doxorubicin than control cells. Consistent with this, the overall survival of patients with high TXN mRNA expression, treated with doxorubicin-containing regimens, is significantly shorter than of those with low TXN mRNA expression. TXN overexpression curtails p300-mediated FOXO1 acetylation and its nuclear translocation in response to oxidative stress, thus attenuating FOXO1 transcriptional activity toward genes involved in apoptosis and cell cycle inhibition. We also demonstrate that FOXO1 knockdown in cells with silenced TXN expression markedly reduces ROS-induced apoptosis, indicating that FOXO1 is the major sensor and effector of oxidative stress in OxPhos-DLBCLs. These data highlight dynamic, context-dependent modulation of FOXO1 tumor-suppressor functions via acetylation and reveal potentially targetable vulnerabilities in these DLBCLs.

Resistance to glucocorticosteroids (GCs) is a major adverse prognostic factor in B-ALL, but the molecular mechanisms leading to GC resistance are not completely understood. Herein, we sought to elucidate the molecular background of GC resistance in B-ALL and characterize the therapeutic potential of targeted intervention in these mechanisms. Using exploratory bioinformatic approaches, we found that resistant cells exhibited significantly higher expression of MEK/ERK (MAPK) pathway components. We found that GC-resistant ALL cell lines had markedly higher baseline activity of MEK and small-molecule MEK1/2 inhibitor selumetinib increased GCs-induced cell death. MEK inhibitor similarly increased in vitro dexamethasone activity in primary ALL blasts from 19 of 22 tested patients. To further confirm these observations, we overexpressed a constitutively active MEK mutant in GC-sensitive cells and found that forced MEK activity induced resistance to dexamethasone. Since recent studies highlight the role GC-induced autophagy upstream of apoptotic cell death, we assessed LC3 processing, MDC staining and GFP-LC3 relocalization in cells incubated with either DEX, SEL or combination of drugs. Unlike either drug alone, only their combination markedly increased these markers of autophagy. These changes were associated with decreased mTOR activity and blocked 4E-BP1 phosphorylation. In cells with silenced beclin-1 (BCN1), required for autophagosome formation, the synergy of DEX and SEL was markedly reduced. Taken together, we show that MEK inhibitor selumetinib enhances dexamethasone toxicity in GC-resistant B-ALL cells. The underlying mechanism of this interaction involves inhibition of mTOR signaling pathway and modulation of autophagy markers, likely reflecting induction of this process and required for cell death. Thus, our data demonstrate that modulation of MEK/ERK pathway is an attractive therapeutic strategy overcoming GC resistance in B-ALL patients.

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Introduction The B-cell receptor (BCR)- activated PI3K pathway plays a critical pro-survival function in normal B-lymphocytes and certain B-cell malignancies. Multiple mechanisms are involved in triggering and modulation of the BCR signal amplitude. MiR-155 has emerged as a positive regulator of PI3K signaling in multiple malignancies, including DLBCL, through targeting negative modulator of this pathway, SHIP-1. In the present study we have searched for new targets of miR-155, which might play a role in the deregulation of AKT and NFkB signaling in DLBCL. Methods MiR-155 target prediction was performed with PicTar, Miranda and TagetScan algorithms. Predicted miR-155 targets were validated with 3'UTR luciferase reporter assays in HEK293 cells. MiR-155 expression was modulated through transfection with miR-155 mimic or miR-155 inhibitor. The consequences of the miR-155 perturbations were assessed in DLBCL cell lines by proliferation assays (MTS) and immunoblotting with antibodies against predicted miR-155 targets and p-AKT. The DEPTOR silencing in DLBCL cells was achieved with retroviral shRNA vector and its consequences were assessed using proliferation assays and immunoblotting. DEPTOR mRNA expression and survival of DLBCL patients was determined using publicly available microarray data (Lenz et al, 2008, GEO accession GSE10846). Results Using miRNA target finding algorithms, we identified miR-155-matching sequences in 3'UTRs of two genes involved in SYK/PI3K/AKT pathway regulation: c-CBL (SYK ubiquitin E3 ligase) and DEPTOR (an mTOR phosphatase). MiR-155 suppressed the luciferase activities of vectors containing 3'UTR fragments from SHIP-1, c-CBL and DEPTOR genes with wild-type, but not mutant miR-155 seed sequence. To establish a link between miR-155 and c-CBL or DEPTOR, DLBCL cell lines were transfected with a control non-targeting miR or miR-155 mimic. The control miR did not affect the protein level of SHIP-1, c-CBL or DEPTOR, whereas introduction of miR-155 resulted in a decrease in expression of these proteins. The repression of miR-155 target genes was accompanied by increased phosphorylation of AKT. As expected, neutralization of endogenous miR-155 in U2932 cell line by anti-miR-155 exhibited opposite effects. In addition to modulation of SYK/PI3K/AKT pathway, miR-155 has been reported to modulate NFĸB activity. To determine whether miR-155 affects NFĸB in DLBCL, we assessed transcript abundance of the genes regulated by NFĸB (CD40, BFL-1, RelB, IĸBα, A20, MIR155HG) in U2932 cell line with reduced miR-155 level. Repression of endogenous miR-155 in these cells led to marked downregulation of NFĸB-controlled genes, indicating that miR-155 amplifies NFĸB signaling in this DLBCL cell line. Consistent with this, anti-miR-155 sensitized U2932 cells to ibrutinib. Since the function of a newly identified miR-155 target, the mTOR phosphatase DEPTOR, in DLBCL has not been elucidated, we silenced the expression of this protein with shRNA. Attenuated protein level of DEPTOR enhanced activity of AKT and promoted proliferation of SU-DHL4 cell line. To determine whether expression level of DEPTOR correlates with patient survival, we analyzed data from gene expression studies. Higher DEPTOR mRNA level in primary DLBCL biopsies was associated with longer overall survival (log rank test, p=0.018). Collectively, these data suggest that DEPTOR plays a tumor suppressor function in DLBCL. Conclusions Our data underscore the role of miR-155 in the regulation of AKT and NFkB prosurvival signaling in DLBCL. MiR-155 regulates AKT signaling not only by decreasing expression of SHIP-1, but also by modulating the abundance of c-CBL and DEPTOR. DEPTOR modulates AKT activity and its silencing promotes proliferation of DLBCL cells, suggesting that DEPTOR functions as a tumor suppressor in DLBCL. Disclosures No relevant conflicts of interest to declare.