Dysregulation of autophagy in human follicular lymphoma is independent of overexpression of BCL-2.

Overexpression of the anti-apoptotic protein BCL-2 is characteristic of human follicular lymphoma (FL) and some cases of diffuse large B cell lymphoma (DLBCL). We aimed to determine autophagy status in primary FL and DLBCL samples and the BCL-2+/BCL-2- lymphoma cell lines using both autophagy PCR array and tissue microarray (TMA). A greater number of autophagy machinery genes were up-regulated in the BCL-2+ Su-DHL4 cell line compared with BCL-2- Su-DHL8 cells, at both the basal level and in response to autophagic stress. The autophagy-related gene expression profiles were determined in purified and unpurified malignant human lymph node biopsies. Seven autophagy machinery genes were up-regulated in purified FL B-cells compared with reactive B-cells. Only 2 autophagy machinery genes were up-regulated in DLBCL B-cells. In unpurified tissue biopsies, 20 of 46 genes in FL and 2 of 5 genes in DLBCL with increased expression were autophagy machinery genes. Expression of autophagy substrates p62 and LC3 were determined by TMAs. FL samples showed significantly decreased levels of both p62 and LC3 compared with reactive and DLBCL, indicative of an increased autophagy activity in FL. In summary, these results demonstrate that FL showed increased basal autophagy activity, regardless of overexpression of BCL-2 in this disease.

ranges from I to IV with a higher stage indicating more widespread disease. § The Follicular Lymphoma International Prognostic Index (FLIPI) score ranges from 1 to 4 where 1 = low risk (0-1 prognostic factor), 2 = intermediate risk (2 prognostic factors), 3 = high risk (3-5 prognostic factors) and 4 = not assessable; U = unknown. * CR= complete remission; CR(U)= undetermined complete remission; GPR= good partial remission; N/A= not applicable. † NHL= Non-Hodgkin lymphoma; N/A= not applicable. Progression ¶ Staging ranges from I to VI with a higher stage indicating more widespread disease. § The International Prognostic Index (IPI) score ranges from 0 to 5, with 0 indicating absence of all prognostic factors and 5 indicating all prognostic factors are present. * CR= complete remission; CR(U)= undetermined complete remission; GPR= good partial remission. † NHL= Non-Hodgkin lymphoma.

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3 Suppl Table 3: Summary of clinical characteristics of FL patients' samples used for the TMA ¶ Staging ranges from I to IV with a higher stage indicating more widespread disease. § The Follicular Lymphoma International Prognostic Index (FLIPI) score ranges from 1 to 4 where 1 = low risk (0-1 prognostic factor), 2 = intermediate risk (2 prognostic factors), 3 = high risk (3-5 prognostic factors) and 4 = not assessable; U = unknown.  Percentages of transformed.   v-akt murine thymoma viral oncogene homolog 1 Co-regulator of autophagy and apoptosis APP Amyloid beta (A4) precursor protein Co-regulator of autophagy and apoptosis ATG5 Autophagy related protein 5 Co-regulator of autophagy and apoptosis BAD BCL2-associated agonist of cell death (Bad) Co-regulator of autophagy and apoptosis BAK1 BCL2 antagonist/killer 1 Co-regulator of autophagy and apoptosis BAX Bcl-2 associated X protein Co-regulator of autophagy and apoptosis BCL2 B cell CLL/lymphoma 2 Co-regulator of autophagy and apoptosis BCL2L1 BCL2 like 1 Co-regulator of autophagy and apoptosis BID BH3 interacting domain death agonist Co-regulator of autophagy and apoptosis BNIP3 BCL2/adenovirus E1B 19kDa interacting protein 3 Co-regulator of autophagy and apoptosis CASP3, 8 Caspase-3, 8 Co-regulator of autophagy and apoptosis CDKN1B Cyclin-dependent kinase inhibitor 1B (p27) Co-regulator of autophagy and apoptosis and cell cycle CDKN2A Cyclin-dependent kinase inhibitor 2A (p16) Co-regulator of autophagy and apoptosis and cell cycle CLN3 Battenin Co-regulator of autophagy and apoptosis CXCR4 Chemokine (C-X-C motif) receptor 4 Co-regulator of autophagy and apoptosis DAPK1 Death-associated protein kinase 1 Co-regulator of autophagy and apoptosis EIF2AK3 Eukaryotic translation initiation factor 2 alpha kinase 3 Co-regulator of autophagy and apoptosis and autophagy induction FADD Fas (TNFRSF6)-associated via death domain Co-regulator of autophagy and apoptosis FAS Fas cell surface death receptor Co-regulator of autophagy and apoptosis HDAC1 Histone deacetylase 1 Co-regulator of autophagy and apoptosis HTT Huntingtin Co-regulator of autophagy and apoptosis IFNG Interferon  Co-regulator of autophagy and apoptosis and cell cycle and autophagy induction INS Insulin Co-regulator of autophagy and apoptosis MAPK8 Mitogen-activated protein kinase 8 Co-regulator of autophagy and apoptosis MTOR Mechanistic target of rapamycin Co-regulator of autophagy and apoptosis NFKB1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 Co-regulator of autophagy and apoptosis

PRKAA1
Protein Kinase, AMP-Activated, Alpha 1 Co-regulator of autophagy and apoptosis PTEN Phosphatase and tensin homolog Co-regulator of autophagy and apoptosis and cell cycle SNCA Synuclein, alpha Co-regulator of autophagy and apoptosis SQSTM1 Sequestosome 1 (p62) Co-regulator of autophagy and apoptosis TGFB1 Transforming growth factor beta 1 Co-regulator of autophagy and apoptosis and cell cycle TGM2 Transglutaminase 2 Co-regulator of autophagy and apoptosis TNF Tumour necrosis factor Co-regulator of autophagy and apoptosis TNFSF10 Tumour necrosis factor (ligand) superfamily, member 10 Co-regulator of autophagy and apoptosis

TP53
Tumour p53 Co-regulator of autophagy and apoptosis and cell cycle RB1 Retinoblastoma 1 Co-regulator of autophagy and cell cycle EIF4G1 Eukaryotic translation initiation factor 4 gamma, 1 Autophagy in response to other intracellular signals ESR1 Estrogen receptor 1 Autophagy in response to other intracellular signals GAA Glucosidase, α; acid Autophagy in response to other intracellular signals

HGS
Hepatocyte growth factor-regulated tyrosine kinase substrate Autophagy in response to other intracellular signals MAPK14 Mitogen-activated protein kinase 14 Autophagy in response to other intracellular signals PIK3C3 Phosphatidylinositol 3-kinase, catalytic subunit type 3 Autophagy in response to other intracellular signals PIK3R4 Phosphatidylinositol 3-kinase, regulatory subunit 4 Autophagy in response to other intracellular signals RPS6KB1 Ribosomal protein S6 kinase, 70kDa, polypeptide 1 Autophagy in response to other intracellular signals TMEM74 Transmembrane protein 74 Autophagy in response to other intracellular signals ULK2 Unc-51 like autophagy activating kinase 2 Autophagy in response to other intracellular signals UVRAG UV radiation resistance associated gene Autophagy in response to other intracellular signals HSP90AA1 Heat shock protein 90 alpha class A 1 Chaperone-mediated autophagy HSPA8 Heat shock protein 8 Chaperone-mediated autophagy Suppl