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A fundamental requirement for growth of rapidly proliferating cells is metabolic adaptation to promote synthesis of biomass1. ATP citrate lyase (ACLY) is a critical enzyme responsible for synthesis of cytosolic acetyl-CoA, the key building component for de novo fatty acid synthesis and links vital pathways such as carbohydrate and lipid metabolism2. The mechanisms of ACLY regulation are not completely understood and the regulation of ACLY function by tyrosine phosphorylation is unknown. Here we show using mass-spectrometry-driven phosphoproteomics and metabolomics that ACLY is phosphorylated and functionally regulated at an evolutionary conserved residue, Y682. Physiologic signals promoting rapid cell growth such as epidermal growth factor stimulation in epithelial cells and T-cell receptor activation in primary human T-cells result in rapid phosphorylation of ACLY at Y682. In vitro kinase assays demonstrate that Y682 is directly phosphorylated by multiple tyrosine kinases, including ALK, ROS1, SRC, JAK2 and LTK. Oncogenically activating structural alterations such as gene-fusions, amplification or point mutations of ALK tyrosine kinase result in constitutive phosphorylation of ACLY in diverse forms of primary human cancer such as lung cancer, anaplastic large cell lymphoma (ALCL) and neuroblastoma. Expression of a phosphorylation-defective ACLY-Y682F mutant in NPM-ALK+ ALCL decreases ACLY activity and attenuates lipid synthesis. Metabolomic analyses reveal that ACLY-Y682F expression results in increased β-oxidation of 13C-oleic acid-labeled fatty acid with increased labeling of +2-citrate (pin vivo. Our results reveal a novel mechanism for direct ACLY regulation that is subverted by multiple oncogenically-activated tyrosine kinases in diverse human cancers. These findings have significant implications for novel therapies targeting ACLY in cancer and metabolism.

Introduction: B-cell immunophenotype could be swiftly assessed by flow cytometry on blood samples or bone marrow aspirate specimens. It provides crucial information later refined with histologic, genetic and molecular features to assert accurate diagnosis of chronic B-cell lymphoproliferative disorders (B-CLPD). Besides Matutes score we identified additional useful markers, i.e. CD148 and CD180 to classify mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL), respectively. Furthermore, CD200 is known to be highly expressed in chronic lymphoid leukemia (CLL) while absent in MCL. Hypothesis: The determination of CD148, CD180 and CD200 expression on B-cells by flow cytometry on blood samples and/or bone marrow aspirates could be a potent tool to accurately identify B-CLPD. We postulated the existence of the following specific expression patterns in B-CLPD: CD148 dim/CD180 dim/CD200 bright for CLL, CD148 dim/CD180 dim/CD200 dim for lymphoplasmocytic lymphoma (LPL), CD148 bright/CD180 dim/CD200 neg/dim for MCL and CD148 dim/CD180 bright/CD200 dim for MZL . Methods: In a prospective study we investigated the expression of CD148/CD180/CD200 on B-cells from 673 patients at the time of B-CLPD diagnosis in our hospital from 2014 to 2020. We analyzed 440 blood and 233 bone marrow aspirate specimens using a BD FACSCanto II flow cytometry instrument. Based solely on CD148/CD180/CD200 specific expression patterns we postulated a diagnosis of CLL, LPL, MCL or MZL. These postulated diagnoses were later confronted to the final diagnoses when all histologic, genetic and molecular features were finalized. Sensitivity, specificity, positive and negative predictive values of the expression profiles were determined. In addition, to investigate the relative importance of these three CD markers we then normalized their mean fluorescence intensities (MFI) and applied several supervised machine learning algorithms including Logistic Regression, Random Forest and Light Gradient Boosting Machine (LightGBM). Results: Out of the 673 clinical samples the CD148/CD180/CD200 expression patterns classified 212 specimens as CLL/SLL (30.8%), 160 as LPL (23.8%), 76 as MCL (11.28%) and 169 as MZL (25%). These diagnosis hypotheses were retrospectively compared to the final diagnoses based on all histologic, genetic and molecular features These diagnosis hypotheses of CLL, LPL, MCL and MZL were consistent with the final diagnosis in 583 out of the 617 corresponding cases (94%) with high positive and negative predictive values. The characteristics of the diagnosis accuracy are detailed in the table below. HCL and FL were not further investigated as their immunophenotype usually do not overlap with those of other B-CLPD. Seventeen out of 617 patients (17/617, 5.3%) did not displayed a clear CD148/CD180/CD200 pattern: 9 LPL, 4 CLL and 4 MZL. In sixteen patients (16/617, 5.0%) the diagnosis hypothesis based on this strategy was not confirmed after completion of the exploration including karyotype, MYD88 L265P mutational status, CCND1 overexpression and pathology explorations. We next investigated the relative importance of these 3 markers. We focused on MFI values of CD148, CD180 and CD200 and three categorical "positive or negative" markers (CD5, CD23, FMC7) that were assembled into a composite marker. After Cox-box normalization of CD148, CD180 and CD200 MFIs, a set of supervised machine learning algorithms including Logistic Regression, Random Forest and Light Gradient Boosting Machine (LightGBM) were applied to the cohort of CLL, LPL, MCL and MZL. We established that the highest diagnosis weights were obtained for CD200 in CLL, CD200 and CD148 in MCL (negatively and positively, respectively), CD180 in MZL. In LPL, CD148, CD180 and CD200 had the highest weights using LightGBM and Random Forest algorithms, while Logistic Regression determined that CD5 and CD23 had the highest (negative) weights. In conclusion, the determination of CD148/CD180/CD200 surface expression patterns by flow cytometry, along with morphology, allowed to assert an accurate diagnosis hypothesis in CLL, MCL, LPL and MZL with high positive and negative predictive values. Machine learning algorithms allowed to measure the relative importance of these markers, that could be of great help in case of discordant expression of the main diagnosis markers. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The spectrum of cutaneous CD30-positive lymphoproliferative disorders (LPDs) includes lymphomatoid papulosis and primary cutaneous anaplastic large cell lymphoma. Chromosomal translocations targeting tyrosine kinases in CD30-positive LPDs have not been described. Using whole-transcriptome sequencing, we identified a chimeric fusion involving NPM1 (5q35) and TYK2 (19p13) that encodes an NPM1-TYK2 protein containing the oligomerization domain of NPM1 and an intact catalytic domain in TYK2. Fluorescence in situ hybridization revealed NPM1-TYK2 fusions in 2 of 47 (4%) primary cases of CD30-positive LPDs and was absent in other mature T-cell neoplasms (n = 151). Functionally, NPM1-TYK2 induced constitutive TYK2, signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5 activation. Conversely, a kinase-defective NPM1-TYK2 mutant abrogated STAT1/3/5 signaling. Finally, short hairpin RNA-mediated silencing of TYK2 abrogated lymphoma cell growth. This is the first report of recurrent translocations involving TYK2, and it highlights the novel therapeutic opportunities in the treatment of CD30-positive LPDs with TYK2 translocations.

The comprehensive genetic alterations underlying the pathogenesis of T-cell prolymphocytic leukemia (T-PLL) are unknown. To address this, we performed whole-genome sequencing (WGS), whole-exome sequencing (WES), high-resolution copy-number analysis, and Sanger resequencing of a large cohort of T-PLL. WGS and WES identified novel mutations in recurrently altered genes not previously implicated in T-PLL including EZH2, FBXW10, and CHEK2. Strikingly, WGS and/or WES showed largely mutually exclusive mutations affecting IL2RG, JAK1, JAK3, or STAT5B in 38 of 50 T-PLL genomes (76.0%). Notably, gain-of-function IL2RG mutations are novel and have not been reported in any form of cancer. Further, high-frequency mutations in STAT5B have not been previously reported in T-PLL. Functionally, IL2RG-JAK1-JAK3-STAT5B mutations led to signal transducer and activator of transcription 5 (STAT5) hyperactivation, transformed Ba/F3 cells resulting in cytokine-independent growth, and/or enhanced colony formation in Jurkat T cells. Importantly, primary T-PLL cells exhibited constitutive activation of STAT5, and targeted pharmacologic inhibition of STAT5 with pimozide induced apoptosis in primary T-PLL cells. These results for the first time provide a portrait of the mutational landscape of T-PLL and implicate deregulation of DNA repair and epigenetic modulators as well as high-frequency mutational activation of the IL2RG-JAK1-JAK3-STAT5B axis in the pathogenesis of T-PLL. These findings offer opportunities for novel targeted therapies in this aggressive leukemia.

The mechanisms underlying the pathogenesis of the constitutively active tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressing anaplastic large cell lymphoma are not completely understood. Here we show using an integrated phosphoproteomic and metabolomic strategy that NPM-ALK induces a metabolic shift toward aerobic glycolysis, increased lactate production, and biomass production. The metabolic shift is mediated through the anaplastic lymphoma kinase (ALK) phosphorylation of the tumor-specific isoform of pyruvate kinase (PKM2) at Y105, resulting in decreased enzymatic activity. Small molecule activation of PKM2 or expression of Y105F PKM2 mutant leads to reversal of the metabolic switch with increased oxidative phosphorylation and reduced lactate production coincident with increased cell death, decreased colony formation, and reduced tumor growth in an in vivo xenograft model. This study provides comprehensive profiling of the phosphoproteomic and metabolomic consequences of NPM-ALK expression and reveals a novel role of ALK in the regulation of multiple components of cellular metabolism. Our studies show that PKM2 is a novel substrate of ALK and plays a critical role in mediating the metabolic shift toward biomass production and tumorigenesis.

Mechanisms by which NPM-ALK signaling regulates cell migration, invasion and contributes to the oncogenesis of anaplastic large cell lymphoma (ALCL) are not completely understood. In an attempt to identify novel actin signaling pathways regulated by NPM-ALK, a comprehensive phosphoproteome analysis of ALCL cell lines was performed in the presence or absence of NPM-ALK activity. Numerous phosphoproteins involved in actin dynamics including Wiskott-Aldrich syndrome protein (WASp) were regulated by NPM-ALK. Network analysis revealed that WASp is a central component of the NPM-ALK-dependent actin signaling pathway. Here we show that NPM-ALK phosphorylates WASp at its known activation site (Y290) as well as at a novel residue (Y102). Phosphorylation of WASp at Y102 negatively regulates its interaction with Wiskott-Aldrich interacting protein and decreases its protein stability. Phosphorylation of WASp at Y102 enhances anchorage-independent growth and tumor growth in an in vivo xenograft model and enhances invasive properties of ALCL. We show that knock-down of WASp or expression of Y102F mutant of WASp decreases colony formation and in vivo tumor growth. Our results show that WASp is a novel substrate of ALK and has a critical role in regulating invasiveness and oncogenesis of ALCL.

Background: Protein glycosylation is one of the most common posttranslational modifications and plays important roles in many biological processes. N-glycosylation facilitates protein trafficking to membranes and their secretion into the extracellular environment. N-glycoproteins are already used as routine biomarkers in clinical practice. These include cluster of differentiation (CD) proteins that define hematopoietic sub-populations and hematologic neoplasms. These proteins also represent important targets for therapeutic antibodies and immunotoxins. Although N-glycoproteins represent an appealing reservoir of potential biomarkers and therapeutic targets, their expression signatures in lymphoid neoplasia has not been established. Hypothesis: Unbiased characterization of N-glycoproteins of lymphoma cells from different subtypes of lymphoid neoplasia would constitute a valuable resource for identifying rational candidate biomarkers and therapeutic targets. Materials and Methods: We performed solid phase extraction of glycoproteins followed by identification of deglycosylated peptides using LC-MS/MS on 36 well-characterized cell lines representing 14 different human lymphoid neoplasm subtypes. Label-free spectral counting was used to quantify the relative abundance of N-glycoproteins. In addition, the N-glycoproteome profiles of 8 patient-derived lymphoma samples were compared in a blinded fashion to the profiles of the 36 cell lines. We employed orthogonal methods such as western blotting, flow cytometry and immunohistochemistry on cell lines and primary human lymphoma biopsies to validate several candidates. Pharmacological inhibition and RNAi-mediated silencing were used to functionally validate glycoproteins specific to ALK-positive anaplastic large cell lymphoma (ALCL, ALK+). Tumorigenicity was assessed in xenografts in SCID-BEIGE mice. Results: In all, 1,155 unique N-glycoproteins were identified from the analysis of the 36 cell lines. Of note, we identified 188 out of the 363 CD proteins listed by the Human Cell Differentiation Molecules consortium. Importantly, we detected virtually all CD proteins currently used for diagnostic evaluation of lymphoid neoplasia. Unsupervised hierarchical clustering of N-glycoproteomic data accurately designated the 36 cell lines according to their respective lineages, cells of origin and appropriate World Health Organization subtypes. Pearson's correlation between the 36 cell lines and the 8 primary biopsies demonstrated that N-glycoproteomic profiles accurately segregated all primary lymphoma samples within their original diagnostic categories. Using tissue microarrays of 644 primary biopsies of mantle cell lymphoma (n=34), follicular lymphoma (n=183), diffuse large B-cell lymphoma (n=245), classical Hodgkin lymphoma (n=150) and ALCL, ALK+ (n=32) we validated the differential expression of CD44, CD276 and paraoxonase 2 (PON2) in mantle cell lymphoma. In addition, our analyses revealed a distinctive cytokine network signature associated with ALCL, ALK+. The evaluation of CD25 expression in a well-characterized cohort of primary lymphoma biopsies (n=28) consisting of ALCL, ALK+ and ALCL, ALK- demonstrated statistically significant correlation between ALK and CD25 expression (p=0.002). In vitro, functional targeting of CD25 using a toxin-conjugated ligand abrogated ALCL, ALK+ cell growth and promoted cell death by apoptosis. This cytokine network signature also included the selective expression of oncostatin M receptor (OSMR) in ALCL, ALK+. We validated OSMR selective expression in ALCL, ALK+ cells by western blot and flow cytometry. Immunohistochemistry on 56 ALCL biopsies (ALK+ and ALK-) revealed a significant correlation between ALK and OSMR expression (p Conclusion: Our data reveal that several glycoproteins demonstrate characteristic and unique expression signatures in distinct forms of human malignant lymphoid neoplasia. Our results indicate that comprehensive profiling of N-glycoproteomes of cells offers opportunities for discovery of novel lymphoma biomarkers and therapeutic targets. Disclosures No relevant conflicts of interest to declare.

Background: Burkitt Lymphoma (BL) is the most common form of NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007, Cairo et al. JCO, 2012). The prognosis has improved with the addition of targeted immunotherapy with rituximab (Goldman/Cairo et al, Leukemia, 2013, Goldman/Cairo et al. BJH, 2014). However, a subset of patients with chemoimmunotherapy-resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012). Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of B-cell lymphomas, however, the extent to which they contribute to rituximab resistance is largely unknown (Barth et al. BJH, 2013). Obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab vs. RTX, a Type I CD20 Ab, mediates enhanced cell death & ADCC against diffuse B-cell lymphoma vs. RTX (Mössner et al. Blood, 2010), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate phosphorylation of signaling pathways are differentially altered following obinutuzumab vs RTX against RTX-sensitive/resistant BL. Methods: Raji (CD20+, ATCC, Manhass, VA) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. For in-vitro studies, tumor cells were incubated with 100 µg/ml obinutuzumab (supplied by Christian Klein, PhD, Roche Research & Early Development, Zurich), and/or RTX for 24 hrs. For Phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji/Raji4RH in the presence/absence of obinutuzumab or rituximab (100µg/ml for 24h) or isotype control. Six milligrams of protein from each condition were digested by trypsin and peptides and subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation. An LTQ Orbitrap XL, in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data that were searched with the Swissprot Human taxonomic protein database (McDonnell and Lim et al, Blood, 2013). Silencing of PLCG2 in Raji and Raji4RH cell lines was carried out according to the manufacturer's instructions (Dharmacon, PA, USA). Results: Four hundred and eighteen out of total 661 proteins in Raji and 377 out of total 534 proteins in Raji4RH were differentially phosphorylated (>1.5 fold) after obinutuzumab treatment. Of these proteins, 46 were expressed at significantly higher levels in obinutuzumab vs. RTX in Raji (Figure 1). However, Raji4RH, the RTX resistant cell line did not show significant increase in phosphorylation of protein following in obinutuzumab vs. RTX. Proteins differentially phosphorylated in response to obinutuzumab vs. RTX are involved in the BCR (PLCG2, BTK, GSK3B and RAF-1), FC gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF-1), and Natural Killer cell-mediated cytotoxicity (MAPK1, RAF-1, PLCG2 and MAPK3) signaling pathways in Raji vs. Raji4RH (Figure 2). Differential phosphorylation of BCR signaling pathways proteins (BTK, PLCG2 and GSK3B), validated by western blot studies after incubation with obinutuzumab vs. RTX in Raji/Raji4RH cell lines, revealed up-regulation of BTK and PLCY2 after obinutuzumab treatment vs. RTX treatment in Raji BL cell line. Silencing one of the BCR signaling pathway protein, PLCG2 significantly increased cell proliferation and decreased cell death after obinutuzumab vs. RTX treatment in Raji (P=0.0001 & 0.004) however, there was no change in Raji 4RH RTX resistant cell line. Conclusions: Obinutuzumab and RTX differentially phosphorylate BCR, phagocytosis and cytotoxicity signaling pathways in BL. Knockdown of PLCG2 significantly enhanced BL proliferation and reduced cell death after obinutuzumab vs. RTX treatment. These results offer insights into alternate therapeutic strategies in the treatment of RTX resistant BL. Disclosures Klein: Roche: Employment.

Introduction: Anaplastic large cell lymphoma (ALCL) is characterized by recurrent translocations involving anaplastic lymphoma kinase (ALK) and resulting in oncogenic chimeric fusion tyrosine kinases such as NPM-ALK. We integrated mass-spectrometry-driven phosphoproteomics and metabolomics of ALK+ ALCL and discovered that ATP citrate lyase (ACLY) is a candidate substrate of NPM-ALK at residue Y682. ACLY is a critical enzyme responsible for synthesis of acetyl-CoA and connects vital biosynthetic pathways such as carbohydrate and lipid metabolism. Functional regulation of ACLY by tyrosine phosphorylation is unknown and the significance of tyrosine phosphorylation of ACLY on cancer cell growth and metabolism have never been described. Methods: To identify novel substrates of NPM-ALK, we performed phospho-proteomic analyses of 3 ALK+ALCL derived cell lines. The MS-phosphoproteomic data were subjected to custom bioinformatic algorithms to identify highly statistically correlated phosphotyrosine proteins and phosphosites regulated by ALK kinase activity. We generated a phosphospecific antibody against a highly-ranked candidate (pACLY Y682) and utilized it in western blot analyses on ALK+ and ALK- ALCL lymphoma cell lines. Immunohistochemistry (IHC) on well-characterized primary human ALK+ (n=20) and ALK-ALCL biopsies (n=28) were performed. Co-immunoprecipitations (Co-IP) with HA-tagged ACLY-WT and ACLY-Y682F and ALK antibodies were performed and analyzed by western blot using the pACLY Y682 antibody. In vitro kinase assays were performed to investigate the direct role of NPM-ALK in phosphorylation of ACLY at Y682 using GST-tagged ACLY-WT or ACLY-Y682F peptides in the presence or absence of immunocomplexes containing active NPM-ALK or the kinase-defective K210R mutant. To study the impact of Y682F mutant on lipid metabolism, we generated ALCL-derived cell lines stably transduced to express GFP-tagged ACLY-WT or ACLY-Y682F and subjected them to metabolomic analysis for flux, β-oxidation or lipid synthesis using liquid chromatography/tandem mass spectrometry or gas chromatography platforms. The Seahorse XF24 Flux analyzer was used to measure oxygen consumption rates (OCR) in DEL stably expressing ACLY-WT and ACLY-Y682F. Cells stably expressing ACLY-WT and ACLY-Y682F were evaluated for cell proliferation, colony formation and tumor formation in SCID-BEIGE mouse xenograft models. Results: Phosphoproteomic analyses yielded a dataset containing a total of 626 tyrosine phosphoproteins. Bioinformatic analysis identified pACLY Y682 as a novel candidate substrate of NPM-ALK. Western blot analysis corroborated phosphoproteomic studies and demonstrated that phosphorylation of ALCY at Y682 is regulated by NPM-ALK activity. Expression of active NPM-ALK increased the phosphorylation of ACLY at Y682 in HEK293T cells compared to the K210R mutant. In vitro kinase assays demonstrated that NPM-ALK phosphorylates ACLY at Y682. Immunoprecipitation (IP) of ALK and co-IP (HA) demonstrated interaction of NPM-ALK and ACLY in co-transfected HEK293T cell lysates. Stable expression of ACLY-Y682F or pharmacologic antagonism of ALK with crizotinib resulted in decreased ACLY activity, decreased lipid synthesis and increased fatty acid β-oxidation when compared to ACLY-WT. β-oxidation of 13 C-oleic acid-labeled fatty acid demonstrated increased labeling of +2-citrate (p Conclusion: We identify for the first time a direct tyrosine kinase-mediated mechanism for ACLY regulation. In this regard, our studies demonstrate a novel oncogenic mechanism whereby NPM-ALK controls lipid synthesis and b-oxidation by phosphorylation of ACLY at Y682 and this mechanism contributes to ALCL pathogenesis. Our findings have significant implications for novel therapies targeting tumor metabolism in ALCLs. Disclosures No relevant conflicts of interest to declare.

The presence of circulating villous lymphocytes (VLs) in lymphoma patients usually points to splenic marginal zone B-cell lymphoma (SMZL), even if the VLs can be found occasionally in other small B-cell lymphomas. However, those cells are variably described, and detailed cytologic characterization is often lacking. We identified lymphoma cases with numerous basophilic VLs among the large group of splenic lymphoma with VLs, and for further delineation, 37 cases with this particular cytology were analyzed. Patients, predominantly older men, presented with moderate lymphocytosis and splenomegaly without pancytopenia. The monoclonal B cells expressed IgM + D, IgM + G, IgM or IgG, as well as CD76 and CD11c, frequently CD103, and rarely CD123. Spleen sections were peculiar, with atrophic white pulp and a monomorphic diffuse lymphoma infiltration in a congested red pulp. Bone marrow infiltration was interstitial and intrasinusoidal without extensive fibrosis. Cytogenetic analysis showed a frequent absence of clonal aberrations (68%). Most cases (79%) were IgH mutated, with an overrepresentation of VH3 and VH4 gene families. These results, as well as the clinical evolution, show that those lymphoma cases represent a homogeneous group distinct from SMZL and reminiscent of hairy cell leukemia variant, perhaps corresponding to a separate lymphoma entity.

Multi-agent chemotherapy regimens have significantly improved long-term survival of pediatric Burkitt Lymphoma (BL) to over 80%. However, patients with refractory or relapsed disease have a dismal prognosis, stressing the need to identify mechanisms of therapeutic resistance and develop novel therapeutic approaches. Recent data demonstrated tonic B-cell receptor (BCR) activation of PI3K in BL (Srinivisan et al, Cell 2009); and implicated PI3K, in coordination with MYC, in Burkitt lymphomagenesis (Sander et al, Cancer Cell 2012). In order to investigate mechanisms of resistance, our laboratory generated several resistant BL cell lines. We exposed Raji cells to escalating doses of rituximab +/- human serum and generated several BL rituximab-resistant (Raji 7R and Raji 8RH) (RRCL) or therapy (rituximab-chemotherapy) resistant (Raji 2R and Raji 4RH) (TRCL) cell lines. We then screened for aberrant activation of signal transduction pathways using western blot, phospho-flow cytometry and phosphoproteomics to define pathways associated with the development of resistance. While total Akt expression was similar between all cell lines tested, Raji 2R and Raji 4RH cells had increased phosphorylation of Akt at Ser473 and Thr308, indicating Akt activation, when compared to Raji, Raji 7R and Raji 8RH by western blot and phospho-flow cytometry. Phosphoproteomic analysis comparing Raji and Raji 4RH identified an increase of at least 2 fold in phosphorylation of 315 proteins in Raji 4RH cells, including multiple direct targets of AKT (e.g. GSK3B, WEE1, FOXO1 and PRAS40). Altered phosphorylation of proteins downstream of Akt (e.g. BAD, 4EBP1, GSK3B and ERK) was also detected by western blot in Raji 2R and Raji 4RH compared to Raji, Raji 7R and Raji 8RH. These findings suggest that activation of PI3K/Akt may play a role in the development of chemoresistance noted in our cell line model. KEGG analysis of phosphoproteome and gene expression comparison of Raji to Raji 4RH cells identified the BCR pathway as one of the top pathways altered. Additionally, the microRNA (miR) 17-92 has been noted to increase Akt activation through its inhibitory effects on PTEN, a key regulator of Akt activation. A recent copy number analysis indicated that a recurrent gain in 13q, containing the MIR17HG locus, was also associated with higher mir17 RNA levels and early relapse in pediatric BL patients (Schiffman et al, Br J Haematol 2011). An analysis of miR expression in Raji 2R and Raji 4RH compared to Raji noted a 1.93 and 3.77 fold increase in miR17 respectively. These findings suggest possible mechanisms for increased PI3K/Akt activation in our chemoresistant cell lines. In vitro exposure to MK-2206, a pan-Akt inhibitor, resulted in dose- and time-dependent decreases in viability of TRCLs (IC50 at 72 hours: Raji 2R = 2.6µM; Raji 4RH = 3.2µM) and to a slightly lesser degree Raji and RRCL (Raji= 4.4µM; Raji 7R = 4.0µM; Raji 8RH = 5.2µM) (TRCL vs. Raji/RRCL, p Disclosures Cairo: Celgene: Research Funding.

Introduction: The CD30-positive cutaneous T-cell lymphoproliferative disorders (CD30-positive LPD) include lymphomatoid papulosis (LYP) and primary cutaneous anaplastic large cell lymphoma (ALCL). Recurrent chromosomal translocations frequently underlie the pathogenesis of several hematologic malignancies and often define molecular subtypes with distinct biological behavior. The genetic events that contribute to the pathogenesis of CD30-positive LPD are largely unknown. Goal: The goal of our study was to identify chromosomal translocations that result in the generation of oncogenic chimeric gene fusions that underlie the pathogenesis of CD30-positive LPD. Methods and Results: Paired end whole transcriptome sequencing (RNAseq) analysis was performed on the cutaneous T-cell lymphoma (CTCL) derived cell lines MyLa and HH. Bioinformatic analysis revealed an interchromosomal fusion between NPM1 (5q35) and TYK2 (19p13). The NPM1-TYK2 fusion was represented by 766 paired-end reads spanning the fusion junction of exon 9 of NPM1 and exon 16 of TYK2. Bi-directional Sanger sequencing confirmed predicted gene fusion sequence at the RNA level. SYBR Green I-based quantitative RT-PCR assays revealed specific expression of the NPM1-TYK2 fusion transcript only in MyLa and not in other CTCL (n = 2) or T-cell lymphoma-derived cell lines (n = 7). To establish that the NPM1-TYK2 gene fusion arose from a translocation event at the DNA level, we performed long range PCR which yielded a 1kb product containing the breakpoints and junctions in NPM1 (chr5:170,832,813) and TYK2 (chr19:10,469,817) introns that lead to juxtaposition of these genes by translocation. The fusion is predicted to encode an NPM1-TYK2 protein containing the oligomerization domain of NPM1 and an intact catalytic domain in TYK2. To determine if translocations targeting TYK2 were recurrent, we employed two independent fluorescence in situ hybridization (FISH) based assays, one using a TYK2 break-apart probe strategy to identify translocations targeting TYK2 and a second approach using an NPM1-TYK2 fusion probe strategy to specifically identify the NPM1-TYK2 translocations. These probes were used to screen patient-derived biopsies of CD30-positive LPD, CTCL and other T-cell neoplasms. FISH analyses revealed TYK2 translocations in 5 of 29 (17.2%) primary cases of CD30-positive LPDs (3/15 LYP; 2/14 cutaneous ALCLs) and was absent in other CTCL subtype (Mycosis fungoides, n = 44) and mature T-cell neoplasms (n = 107). Out of these 5 positive cases, one LYP case had an NPM1-TYK2translocation. Since TYK2 is a member of Jak family kinases that are integral components of the JAK-STAT signaling pathway, we investigated the downstream effects of NPM1-TYK2 fusion expression on TYK2 kinase activity and STAT protein pathway activation. Western blotting in MyLa, other cutaneous and mature T-cell lymphoma cell lines revealed hyperactivation of endogenous TYK2 kinase enzyme (pTYK2 levels) only in MyLa. Further, MyLa cells showed activation of downstream STAT signaling pathway proteins (pSTAT1, pSTAT3 and pSTAT5). Ectopic expression of NPM1-TYK2 wild type fusion gene in HEK293FT cells resulted in hyperactivation of TYK2 kinase enzyme and activation of STAT proteins, while a kinase-defective mutant NPM1-TYK2 K462R lacked TYK2 and STAT activity. Transcriptional activation assays for STAT proteins (STAT1, STAT3 and STAT5), showed more than two fold (P Conclusions: Our study demonstrates for the first time recurrent TYK2 gene translocations in CD30-positive LPD or in any form of primary cancer. We identify NPM1 as one of the gene-fusion partners of TYK2 and provide functional support for NPM1-TYK2 in mediating activation of STAT signaling to promote cell proliferation. Inactivation of TYK2 significantly diminishes proliferation, suggesting that TYK2 is an oncogenic driver kinase in a subset of CD30-positive LPD. Finally, our results raise the possibility that TYK2 may be a novel therapeutic target in a subset of CD30-positive LPDs. Disclosures No relevant conflicts of interest to declare.

Background Burkitt Lymphoma (BL) is the most common non-Hodgkin lymphoma (NHL) in children and adolescents and has an excellent prognosis (≥80% 5years, EFS) following short but intense multi-agent chemotherapy (Cairo et al JCO, 2012). The prognosis has improved following the addition of targeted immunotherapy with rituximab (Goldman/Cairo et al, Leukemia, 2013). However, a subset of patients with chemoimmunotherapy resistant disease have a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012 and Cairo et al Blood, 2007). Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of B-cell lymphomas, however the extent to which they contribute to rituximab resistance is largely unknown (Barth et al. BJH, 2013). Recently, Czuczman et al have generated a chemoimmunotherapy resistant BL cell line Raji4RH from the native Raji BL line after serial exposures to rituximab (Czuczman et al Clin Cancer Res, 2008). Objective We therefore sought to compare the in-vitro cytotoxicity of rituximab to Raji vs. Raji4RH and the resulting phosphoproteomics changes between untreated and rituximab treated Raji vs. Raji4RH by global phosphoproteomics. Methods In order to identify deregulated signaling proteins/pathways that play a role in BL rituximab resistance, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell line Raji, (CD20+,ATCC, Manhassas, VA) and its rituximab resistant derivative Raji 4RH in the presence or absence of Rituximab (100µg/ml for 24 hr) or isotype control. Six milligrams of protein from each condition were digested by trypsin and peptides were subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation using a cocktail of 3 anti-phosphotyrosine antibodies. An LTQ Orbitrap XL in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data which were searched with the Swissprot Human taxonomic protein database. Quantitation of identified phosphopeptides was based on spectral counts of phosphorylated peptides. The accuracy of the phospho-site localization was predicted using Luciphor program. Pathway analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Results Our in-vitro results shows that rituximab induced significantly more cell death against Raji vs. Raji4RH (32.7±6.8% vs.5.5±2.2%, p=0.001) compared to isotype control (9.35±0.75% vs.8.9±1.1%, p=NS) at 24 hrs. Furthermore, 2272 unique phosphopeptides were identified and quantified with a probability P> 0.7 and a false localization rate (FLR) < 0.01. These unique phosphopeptides corresponded to 850 unique phosphorylated proteins. Six hundred and nine proteins were differentially phosphorylated (>1.5-fold change) between Raji and Raji 4RH (Figure 1A). Out of these proteins, 374 were more phosphorylated in Raji4RH than in Raji and 235 were more phosphorylated in Raji than in Raji 4RH. Proteins in the spliceosome and the BCR signaling pathway were significantly represented. Changes in the phosphorylation of 430 proteins were induced by Rituximab in Raji cells and involved those in the B-cell receptor (BCR) signaling pathway (LYN, LCK, SLAMF1, PAG1, CD20), the spliceosome (SF3A1, SF3B1, SRSF6), the mTOR pathway (RICTOR, PDPK1, RPS6KA1, BRAF) and the chemokine signaling pathway (CXCR5, PTK2B, CCL1, PRKCD). After rituximab treatment, 559 proteins were differentially phosphorylated between Raji and Raji 4RH (Figure 1B). Rituximab treatment increased the phosphorylation of 262 proteins in Raji 4R while it increased the phosphorylation of 299 proteins in Raji. Conclusions The data indicate that Rituximab results in global changes in proteins in BL involved in not just BCR signaling but also in the spliceosome, the mTOR and the chemokine signaling pathway. Furthermore, aberrant phosphorylation-induced signaling events in the spliceosome and the BCR signaling pathways may contribute to resistance to rituximab. These data revealed the utility of unbiased phosphoproteome interrogation of rituximab mediated signaling events as well as characterizing signaling networks that may provide insights into pathogenetic mechanisms of rituximab resistance. AA and DR considered co-primary first authors, and ML and MC considered co-senior authors. Disclosures: Cairo: Roche/Genentech : advisory board Other.

Treatment with multi-agent chemotherapy regimens has significantly improved survival in pediatric Burkitt Lymphoma (BL) leading to long-term survival in over 80% of cases. The incorporation of rituximab in the treatment of pediatric B-cell non-Hodgkin lymphoma (B-NHL) has been slower than in adults, but may improve clinical outcomes in high risk pediatric BL patients when combined with chemotherapy regimens. On the other hand, BL patients with primary refractory or relapsed disease have a dismal prognosis, stressing the need to identify the mechanism(s) resulting in chemotherapy/rituximab resistance and to develop novel therapeutic approaches. To this end, we exposed a BL cell line (Raji) to escalating doses of rituximab with or without human serum and generated/characterized several BL rituximab-resistant (Raji 7R and Raji 8RH) (RRCL) or rituximab-chemotherapy resistant (Raji 2R and Raji 4RH) (RCRCL) cell lines. Subsequently, we screened for aberrant activation of signal transduction pathways between RSCL (Raji), RRCL (Raji 7R and Raji 8RH), or RCRCL (Raji 2R and Raji 4RH) in an attempt to define what pathways were associated with resistance to both rituximab and chemotherapy agents. This was accomplished by the analysis of phosphorylation patterns on key-regulatory members of pre-defined signal transduction pathways using Western blotting, phospho-flow cytometry studies and phosphoproteomics. Effects on cell viability or cell cycle distribution of RRCL or RCRCL following pharmacological inhibition of key-regulatory pathways identified was then performed using the alamar blue reduction assay or flow cytometry respectively. While total Akt (Protein kinase B) expression was similar between all the types of BL cell lines tested, we found that RCRCL (Raji 2R and Raji 4RH) had an increase in basal phosphorylation levels of Akt at the Ser473 and Thr308 phosphorylation sites when compared to RSCL (Raji cells) or RRCL (Raji 7R and Raji 8RH). These findings were confirmed by phospho-flow cytometry studies. Phosphoproteomic analysis comparing Raji (RSCL) against Raji 4RH (RCRCL) cells identified an increase of at least 2 fold in the phosphorylation of 315 proteins in RCRCL including several direct targets of AKT such as GSK3B, WEE1, FOXO1 and PRAS40. Altered phosphorylation of multiple Akt/mTOR downstream proteins (BAD, 4EBP1, GSK3B and ERK) was detected by western blot in RCRCLs (Raji 2R and Raji 4RH) compared to RSCL and RRCLs. In vitro exposure of RCRCL (Raji 2R and Raji 4RH) to escalating doses of MK-2206, a selective Akt inhibitor, or idelalisib, a selective PI3 kinase delta inhibitor, resulted in a dose- and time-dependent decrease in cell viability of RCRCL and to a lesser degree RRCL and RSCL (RCRCL vs. RRCL/RSCL, p Disclosures: Czuczman: Genetech, Onyx, Celgene, Astellas, Millennium, Mundipharma: Advisory Committees Other.

Abstract 896 Anaplastic Large Cell Lymphoma (ALCL) is a common form of pediatric non-Hodgkin lymphoma that is often characterized by the chromosomal translocation t(2;5)(p23;q35) and subsequent expression of the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein. NPM-ALK is a constitutively active tyrosine kinase that functions as a driving oncogenic mutation in ALCL. However, a complete understanding of the signaling mechanisms that contribute to its oncogenesis have not been elucidated. In an effort to identify novel mediators of NPM-ALK signaling, we pursued a phosphoproteomic strategy aimed at the unbiased identification of protein phosphorylation events regulated by NPM-ALK kinase. This analysis resulted in the identification of Pyruvate Kinase M2 (PKM2) as a candidate substrate of NPM-ALK in addition to other metabolic proteins. Phosphorylation of PKM2 is critical for inducing a metabolic shift which leads to accumulation of precursor biomass metabolites required for rapidly dividing cells. We hypothesized that NPM-ALK regulates an oncogenic metabolic shift through phosphorylation of PKM2. Western blot analysis and in vitro kinase assay confirmed that PKM2 is phosphorylated at the Y105 residue by NPM-ALK. The phosphorylation of PKM2 is inversely correlated with its enzymatic activity using an in vitro activity assay. Stable expression of Flag-PKM2 Y105F or treatment with PKM2 small molecule activators (NCGC00186528) results in increased PKM2 activity and induction of a metabolic switch as measured by decreased lactate (80% ± 6%, p Disclosures: No relevant conflicts of interest to declare.

Abstract 583 Background: Reversible phosphorylation is a fundamental post-translational modification which regulates cellular functions by modulation of the activity of critical proteins in cellular signal transduction pathways. Deregulation of protein phosphorylation underlies the pathogenesis of many human diseases, especially cancers. Large scale phosphoproteomic analysis offer an opportunity to catalogue and understand changes in protein phosphorylation associated with cancers and thus provide new possibilities for discovering novel diagnostic biomarkers and new therapeutic targets. Although B-cell non-Hodgkin lymphomas (NHLs) represent the seventh most common cancer in western countries, the extent to which phosphoprotein deregulation may be involved in their pathogenesis is largely unknown. Here we report results of a label-free quantitative phosphoproteomics study of 3 B-cell NHL entities and investigate the contribution of one of the identified protein in the germinal center-derived NHL proliferation. Material and methods: Six milligrams of protein from 13 human B-NHL-derived cell lines (4 mantle cell lymphomas (MCL), 3 Burkitt lymphomas (BL) and 6 follicular lymphomas (FL)) digested by trypsin were subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation using a cocktail of 3 anti-phosphotyrosine antibodies. Phosphopeptides were subjected to liquid chromatography (LC) and MS/MS. Spectra were searched against the UniProtKB database using X!Tandem with k-score. Search results were then post-processed with PeptideProphet and ProteinProphet. All proteins at false discovery rate of 1% were considered for further analysis. Quantitation of identified peptides was based on spectral counts of phosphorylated peptides. Immunoprecipitation and western blot studies were performed to validate the differential phosphorylation of a subset of proteins. The functional consequences of perturbation of an outlier tyrosine phosphorylated protein (PAG1) were explored using shRNA-mediated depletion followed by both colony formation and proliferation assays with or without BCR stimulation by anti-IgM or lipopolysaccharide (LPS). Results: Quantitative phosphoproteomics revealed a total of 7326 serine/threonine phosphorylatided peptides and 392 tyrosine phosphorylated peptides corresponding to 420, 290 and 356 phosphorylated proteins in BL, FL and MCL, respectively. While a subset of phosphoproteins was identified across all lymphoma entities, hierarchical clustering analysis revealed distinctive signatures for all 3 NHL subtypes. Regarding proteins identified with phosphorylated tyrosine residues, BL and FL cell lines were characterized by phosphorylation of proteins implicated in active B-cell receptor signaling such as BTK, LYN, LCK and SYK whereas MCL cell lines were characterized by phosphorylation of proteins implicated in cell cycle control such as CDK1 and CDK2. Interestingly, we identified PAG1, a negative regulator of B-cell receptor signaling, as the most highly tyrosine phosphorylated protein in the germinal center derived cell lines (BL and FL) exclusively. Differential phosphorylation of several proteins was validated by immunoprecipitation and western blot studies. PAG1 silencing in BJAB (BL cell line) by a specific PAG1 shRNA resulted in an increased propensity for colony formation when compared to a scramble shRNA. PAG1 depletion also enhanced significantly the proliferation of BJAB 48h after BCR stimulation by either anti-IgM (3.9-fold increase versus 3-fold increase, p = 0.016) or LPS (4.2-fold increase versus 3-fold increase, p = 0.015). Conclusion: Our study reveals the utility of unbiased phosphoproteome interrogation of B-NHLs to characterize signaling networks that may provide insights into the pathogenetic mechanisms. We identify more than 500 phosphorylated proteins in each NHL entity revealing distinct protein phosphorylation signatures between MCL and germinal center derived lymphomas. Activation of the BCR signaling pathway is characteristic of and pathologically relevant in germinal center derived NHLs. The results of our study reveal novel phosphorylation sites and signatures that may be exploited as diagnostic biomarkers or therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

MCL is a well-defined lymphoid neoplasm characterized by a proliferation of mature B lymphocytes expressing CD5 and with a genetic hallmark, the t(11;14) (q13;q32) translocation leading to the overexpression of cyclin D1, considered as the initial oncogenic event. However this entity may show a spectrum of morphological features broader than initially described. Clinically most of the patients have a poor prognosis with a rapid acquisition of chemoresistance. It has been recently described that some patients may follow an indolent clinical evolution, with the possibility of confusion in diagnosis because of overlapping morphological features with other small B cell lymphomas such as marginal zone lymphoma (MZL) and small lymphocytic lymphoma (SLL). The aim of our study was to analyze patients treated for a B cell lymphoma bearing the t(11;14)(q13;q32) who had a good outcome with a long survival. We retrospectively selected 21 patients (pts) with a t(11;14) B cell lymphoma and who followed an indolent outcome defined by an overall survival of more than 5 years after no more than 1 therapeutic regimen. Feature review included morphologic aspect, immunophenotype, cytogenetics, immunoglobulin (Ig) variable heavy chain (VH) gene usage and mutation patterns. Expression of CD5, CD10, CD23, and CD43, were evaluated on CD19+ cells by FCM. Cytogenetic analysis consisted in conventional cytogenetics on fresh tissue samples completed by a FISH analysis against 11q, 12p, 17p, and 18p realized on fresh frozen material. We further analysed the gene expression profiling of available samples using quantitative real-time PCR using microfluidic cards designed with selected genes of 3 independent signatures (Rosenwald A, et al. Cancer Cell 2003 - Ruiz-Ballesteros E, et al. Blood 2005 - Thieblemont C, et al. Blood 2004). Clinical characteristics showed that median age was 61 (range 45–81). All pts had a good PS and presented with a disseminated disease: bone marrow and blood involvement in 19 (90%) and 11/17 (65%) pts, respectively; peripheral and profound nodes involvement in 15 (71%); and splenomegaly in 13 (62%). LDH and b2-microglobulin levels were elevated in 4 pts. None of the pts had a monoclonal component. IPI score was low in 18 (86%) pts. Splenectomy alone or with chemotherapy (chlorambucil or fludarabine in 4; CHOP in 1) was proposed in 7 pts; monochemotherapy (chlorambucil or fludarabine) in 4 pts, CHOP +/− rituximab (R) in 2 pts, CHOP +/− R followed by autotransplant in 8. One pt did not receive any treatment. With a median follow-up at 6.44 years, median overall survival was 9.18 years. Morphologic review distinguished 2 groups of cases: true MCL cases (MCL, n = 8) and BorderLine Cases (BLC, n=13). Among the 8 MCL and 13 BCL, 7 and 12 were CD5+, 1 and 4 CD5/CD23+, 4 and 5 CD43+, respectively. All cases exhibited a t(11;14), associated with a complex caryotype in 5/8 (62%) MCL and in 9/13 (69%) BLC. Chromosome 7q deletion (n=1) and trisomy chr3/3q (n=3) were present only in BLC cases. IgVH gene was unmutated in 10 cases (3 MCL, 7 BLC) and mutated in 4 (2 MCL, 2 BLC). The most common VH families were VH1 (n=4), VH 3 (n=8) and VH4 (n=3). From the gene expression profiling studies we selected 70 genes specific of the diagnosis of MCL, MZL and SLL. A set of 5 MCL, 5 SLL, and 5 MZL control samples allowed us to validate 25 out of these 70 genes. These 25 genes were further used to analyse MCL and BLC samples. All these samples exhibited a more heterogeneous profile than control samples. Three MCL were more similar to MCL samples, whereas two were closer to MZL samples. All the BLC exhibited a profile close to that of MZL. We further analysed these samples with the 18 gene-signature predicting MCL survival. This signature could not discriminate survival of these MCL and BCL pts. In conclusion, pts with B cell lymphoma bearing the translocation t(11;14)(q13;q32) and with an indolent outcome can be distinguished in at least 2 groups with different characteristics in terms of morphology, immunophenotype, caryotype, and molecular profiles. Using a 25 gene-signature, we found profiles similar to either MCL or MZL for MCL samples, and similar to MZL for BCL samples. The signature discriminating the survival of MCL could not discriminate these indolent MCL. These findings will be validated on a larger series.

Background. Mantle cell lymphoma is one of the most refractory B-cell lymphomas. Despite the recent improvement of novel therapeutic strategies including high dose therapy and the introduction of immunotherapy, MCL patients are not cured by conventional therapeutic strategies. The increasing understanding of the MCL cell biology leads to the development of new drugs targeting to molecular mechanisms of the disease. Among the specific genes found to be overexpressed in MCL by gene expression profiling analysis, farnesyltransferase (FTase) encodes for an enzyme essential in the post- translational modifications of proteins that require prenylation for conversion to mature forms, allowing their participation in various signaling pathways regulating growth and survival. We recently demonstrated that inhibition of FTase by tipifarnib (Zarnestra) is associated in vitro with growth inhibition and apoptosis of MCL cell lines and in vivo with tumor xenograft stability. To determine the efficacy, the safety profile and the toxicity of tipifarnib in MCL, we conducted a phase II trial in patients with refractory MCL, and we evaluated the response considering published molecular predictors. Methods. Primary endpoint was the efficacy measured by the evaluation of the overall response rate (ORR) at 4 cycles, and, in case of response, at 6 cycles. Planned sample size of the study was 27 evaluable patients enrolled for an analysis based on an optimal two-stage design comprising 11 patients for the first stage and 16 for the second stage, under the hypothesis of an ORR of 35% to conclude for an effective drug and an ORR of 10% to conclude for an ineffective drug. Tipifarnib was administered at 300 mg orally twice daily for the first 21 days of each 28-days cycle for 4 to 6 cycles. Prediction of response was retrospectively evaluated in the initial tumor biopsy by the analysis of the 2-gene classifier, the RASGRP1/APTX gene expression ratio, and the AKAP13 expression level. Results. Eleven patients with refractory MCL were included in the analysis. Median number of lines of therapy before tipifarnib was 2.5 (range 1–7). Median age was 71 (range 66–79). All patients presented with stage IV disease and with good performance status. At 4 cycles of tipifarnib, 1 patient presented a complete response and 10 patients were in progressive disease. Two patients progressed during or after the first cycle, three patients after 3 cycles, and five patients after 4 cycles. No grade III-IV hematological toxicities were recorded. One patient presented an unrelated neurological symptom after the first dose administered. Evaluation of the molecular prediction of response to tipifarnib was realized for 3 patients: one responder patient and two non-responder patients. Results showed an increase in the RASGRP1/APTX gene expression ratio and a decrease in AKAP13 expression in the responder patient, and a decrease in the RASGRP1/APTX gene expression ratio and an increase in AKAP13 expression in the non-responder patients. This corresponds to the expected results of the response prediction to tipifarnib. Conclusion. Tipifarnib in refractory MCL was beneficial for only one patient. Response could be exactly predicted by specific molecular predictors of response evaluated in the initial tumor biopsy. These results demonstrate the necessity of categorizing molecularly the patients when targeted therapies are proposed to select those patients that might respond to.

Preliminary data suggest drug resistance plays a major role in mantle cell lymphoma (MCL) and is linked to the aberrant expression of molecules such as Glutathion S-Transferase pi (GST pi) that catalyzes the nuclear conjugation of a broad variety of reactive electrophiles to the glutathion. We investigated in vitro the effect of the inhibition of GSTpi activity on the chemosensitivity of MCL cell lines by inhibiting the nuclear transport of GSTpi with Agaricus bisporus leptine (ABL). Methods. Four MCL cell lines (Granta, NCEB, REC and UPN1) were analyzed for GSTP1 transcript expression by RT-PCR analysis and for GSTP1 genetic Ile105Val polymorphism. The effect of ABL on the 4 MCL cell lines viability was examined using cells pre-treated with ABL (40μg/ml) for 10 h followed by treatment with various concentrations of doxorubicin (DOX), cisplatin (CDDP), cytarabin and bortezomib for 48 h. The cell viability was estimated by MTT assay repeated three times. Results. All the 4 cell lines expressed GSTPI transcript. Two of them (UPN1 and REC) were heterozygous for Ile105Val polymorphism, whereas NCEB and GRANTA were homozygous GSTP1-105Ile which has been correlated with an enzyme of high activity. DOX, CDDP, cytarabin and bortezomib have cytotoxic effects compared to non-treated cells, without any difference between the 4 MCL cell lines when considering their genetic polymorphism status. Co-administration of ABL caused a significant increasis of the cytotoxicity of CDDP and bortezomib in all cell lines (Student test: p between 0.0004 to 0.02 for CDDP; and p between 0.013 and 0.05 for velcade, depending on the cell lines). Co-administration of ABL caused an increasis of the cytotoxicity of cytarabin in 3 cell lines, the 4th being actually tested. No influence of ABL was detected on the cytotoxic effect of DOX. The analysis of the nuclear and the cytoplasmic localizations of GSTpi is currently realized by immunohistochemistry. Conclusion. These results suggest that inhibition of the nuclear transfer of GSTpi increases in vitro the MCL sensitivity to CDDP, cytarabin and velcade but not DOX.

Non-germinal centre small B-cell lymphomas represent a heterogeneous group of non-hodgkin lymphomas which most frequent histologic subtypes are small lymphocytic lymphoma (SLL), marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL). These three lymphoma entities have very different clinical outcomes but may be difficult to distinguish either histologically or clinically. We previously identified transcriptomic signatures specific of these 3 lymphoma subtypes. We further analyzed these lymphomas using Surface-Enhanced Laser Desorption/Ionisation Time of Flight (SELDI-TOF). A total of 58 tumors, including 20 SLL (all lymph nodes), 20 MZL (1 lymph node and 19 spleens) and 18 MCL (19 lymph nodes and 1 spleen) were analyzed. In addition, we included 7 controls obtained from traumatic normal spleens. The spectra were generated on weak cation exchange (CM10), strong anion exchange (Q10) and reversed-phase (H50) ProteinChip arrays. Protein patterns of all samples were comparatively analysed using two distinct strategies. We first used a binary recursive partitioning method with the Biomarker Pattern software (Ciphergen®), and second a hierarchical clustering method to visualized patterns of protein peaks completed with a supervised method (discriminating score) to point out individual peaks distinguishing the three histological subtypes (SLL, MZL and MCL). Spectra analyses revealed a very homogeneous protein patterns among all lymphoma samples. However specific SLL, MZL and MCL signatures based on 34 protein peaks with differential expression could be identified and allowed to classify 95% of the samples in their respective entity. SLL signature included 9 peaks, MZL signature 16 peaks and MCL signature 9 peaks. The binary recursive partitioning analysis was concordant but identified only the five most discriminant peaks. Further identification of the discriminating peaks is currently realized using SELDI-assisted purification. We are focusing on peaks at 9942 Da for SLL and at 11324 Da for MCL. Functional genomic studies can distinguish non-germinal small B-cell lymphomas at the transcriptomic level (our previous study) and at the proteomic level. This will provide new markers for diagnosis and potentially new therapeutic targets.

Glutathione-S-transferase π (GSTπ), whose gene is located in 11q13, is belonging to a complex multigene family of enzymes that may detoxify electrophilic xenobiotics. Recent gene expression profiling analysis showed that GSTπ transcript is belonging to the mantle cell lymphoma (MCL) signature and constitutes one of the most expressed mRNA in MCL[*][1]. Furthermore it has been reported by immunohistochemistry that overexpression of cyclin D1 in MCL was associated with a high level of GSTπ protein expression. We then looked for the specificity of a high level of GSTπ protein expression in MCL, and particularly when comparing to marginal zone lymphoma (MZL) with t(11;14). Material and Methods: Immunochemistry analysis of GSTπ and cyclinD1 expression was performed 111 non Hodgkin’s lymphoma including MCL, MZL, small lymphocytic lymphoma (SLL), follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL) cases with or without t(11;14). Results: A strong GSTπ expression in more than 50% of the cells was observed in 98% of the MCL. None of the MZL cases with t(11;14) expressed GSTπ, except one case that expressed a weak signal in less than 20% of the cells. None of the MZL without t(11;14) expressed GSTpi. Only two (13%) FL graded 3 according to OMS classification expressed GSTπ in less than 50% of the cells. A high GSTπ expression (>50% cells stained) was detected in 29% of the DLBCL and 43% of the SLL. | | n | GST π expression >50% | GST π expression 5–50% | π GST expression