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.

Overexpression of SHH enhances PCa cell lines resistance to paclitaxel. Higher level of SHH leads to increase in ABC transporters expression in a manner dependent on paclitaxel.

Abstract.HOXA genes encode transcription factors, which are crucial for embryogenesis and tissue differentiation and are involved in the early stages of hematopoiesis. Aberrations in HOXA genes and their cofactor MEIS1 are found in human neoplasms, including acute myeloid leukemia (AML). The present study investigated the role of HOXA4, HOXA5 and MEIS1 promoter DNA methylation and mRNA expression in AML. Samples from 78 AML patients and 12 normal bone marrow (BM) samples were included. The levels of promoter DNA methylation were determined using quantitative methylation-specific polymerase chain reaction (PCR; qMSP) and the relative expression levels were measured using reverse transcription quantitative PCR in Ficoll-separated BM mononuclear cells and in fluorescent activated cell sorting-sorted populations of normal hematopoietic progenitors. In total, 38.1 and 28.9% of the patients exhibited high methylation levels of HOXA4 and HOXA5, respectively, compared with the control samples, and MEIS1 methylation was almost absent. An inverse correlation between HOXA4 methylation and expression was identified in a group of patients with a normal karyotype (NK AML). An association between the genes was observed and correlation between the DNA methylation and expression levels of the HOXA gene promoter with the expression of MEIS1 was observed. Patients with favorable chromosomal aberrations revealed a low level of HOXA4 methylation and decreased expression levels of HOXA5 and MEIS1 compared with the NK AML and the adverse cytogenetic risk patients. The NK AML patients with NPM1 mutations exhibited elevated HOXA4 methylation and expression levels of HOXA5 and MEIS1 compared with the NPM1 wild-type patients. Comparison of the undifferentiated BM-derived hematopoietic CD34 + CD38 low , CD34 + CD38 + and CD15 + cells revealed a gradual decrease in the expression levels of these three genes and an increase in HOXA4 promoter methylation. This differentiation-associated variability was not observed in AML, which was classified according to the French-American-British system.