Hu, Guangzhen

Abstract Anaplastic large cell lymphomas (ALCLs) are CD30+ T-cell lymphomas that share pathologic features but differ in presentation, outcome, and genetics. Current classification incorporates clinical presentation and anaplastic lymphoma kinase (ALK) status but inadequately addresses molecular heterogeneity and therapeutic vulnerabilities. We studied 689 patients with ALCL in the LLMPP (Lymphoma/Leukemia Molecular Profiling Project) and performed expert consensus review, genetic subtyping (ALK, DUSP22, TP63, and triple negative), and immunohistochemistry for phosphorylated STAT3Tyr705. RNA sequencing with unsupervised gene expression profiling in 393 patients identified 2 main molecular types of ALCL that could be predicted with 91% accuracy based on the presence (type I) or absence (type II) of phosphorylated STAT3Y705 expression. Type I ALCLs included ALK+ ALCL and a subset of triple-negative ALCLs (TN-I); type II ALCLs included tumors with DUSP22 and/or TP63 rearrangements and the remaining triple-negative ALCLs (TN-II). Type I ALCLs were enriched for JAK-STAT3, whereas type II ALCLs were enriched for non–tyrosine kinase pathways, particularly epigenetic regulators such as EZH2. Immunohistochemistry showed overexpression of EZH2 and its trimethylated substrate H3K27. Prognosis in systemic ALCL was favorable for DUSP22-rearranged ALCL (5-year overall survival, 95%) and ALK+ ALCL (88%), intermediate for triple-negative ALCL (TN-I, 52% and TN-II, 37%), and poor for TP63-rearranged ALCL (0%). We introduce an integrated molecular classification that preserves currently diagnosed ALCL entities but identifies 4 molecularly distinct ALK− ALCL subtypes (DUSP22-rearranged, TP63-rearranged, TN-I, and TN-II). This classification can be easily implemented on paraffin tissue in routine practice or clinical trials, and stratifies ALCL into diagnostically, prognostically, biologically, and potentially therapeutically relevant subtypes.

Background: Anaplastic large cell lymphomas represent a group of T-cell lymphomas that have long been broadly classified by the presence or absence of ALK fusion proteins (ALK+ or ALK−, respectively). However, recent discovery of extensive additional molecular heterogeneity has both promoted refinement of disease classification and uncovered new opportunities for biomarker-guided targeted therapy. In addition to recognition of a new genetic subtype, ALK− ALCL with DUSP22 rearrangement (R), a new molecular classification has been proposed based on the recent discovery of 2 overarching molecular types of ALCL, defined by the presence (Type I) or absence (Type II) of a gene expression signature highly enriched for JAK-STAT3 activation. BCL2 is a mitochondrial membrane protein that functions to inhibit the actions of pro-apoptotic proteins such as BAX and BAK and can be targeted clinically by BCL2 small molecule inhibitors. BCL2 has been reported to be expressed more frequently in ALK− ALCL than in ALK+ ALCL, but its expression in the context of current molecular findings is unknown. Methods: ALCLs were centrally reviewed and classified by current WHO/ICC criteria. BCL2 was performed using IHC and scored as positive if ≥30% of tumor cells showed staining. DUSP22-R was assessed by fluorescence in situ hybridization (FISH) using a breakapart probe set and a dual-color, dual-fusion (D) probe set for the most common DUSP22 translocation, t(6;7)(p25.3;q32.3). pSTAT3Y705 IHC, which is >90% accurate for Type I/II assignment based on gene expression, was performed and scored based on a previously validated cut-off of ≥30%. Results: We evaluated 217 ALCLs (148M/69F; age: mean, 57 y; range, 5-93 y), including 55 systemic ALK+ ALCLs, 127 systemic ALK− ALCLs, and 35 primary cutaneous (pc) ALCLs. Overall, 86/217 cases were positive for BCL2 (40%). BCL2 was positive in 67/127 ALK− ALCLs (53%) and 15/35 pcALCLs (43%; P=NS), but in only 4/51 ALK+ (7%; P<0.0001). DUSP22-R was present in 90 cases (63 ALK− ALCL and 27 pcALCL). BCL2 was positive in 57/90 cases with DUSP22-R (63%) compared to 29/127 without DUSP22-R (23%; P<0.0001). Among 85 cases with DUSP22-R and informative D-FISH testing, BCL2 was positive in 24/36 cases with t(6;7)(p25.3;q32.3) (67%) and 31/49 cases without t(6;7)(p25.3;q32.3) (63%; P=NS). Among 213 cases with informative pSTAT3Y705 staining, BCL2 was positive in 13/88 pSTAT3+ cases (Type I, 15%) and 70/125 pSTAT3− cases (Type II, 56%; P<0.0001). Conclusions: We identified a complex pattern of BCL2 expression across ALCLs that strongly correlates with a recently introduced molecular classification based on gene expression profiling. While our findings are in keeping with the previously reported low expression in ALK+ ALCL, we found that the higher expression in both systemic ALK− ALCL and pcALCL is particularly associated with DUSP22-R and the absence of pSTAT3Y705 expression (Type II ALCLs). The discovery of 2 overarching types of ALCL associated with the presence or absence JAK-STAT3 pathway activation represents a new conceptual framework for classifying ALCL and understanding its therapeutic vulnerabilities. Of note, previous data demonstrated that Type II ALCLs are enriched for epigenetic alterations and particularly overexpress the histone methyltransferase EZH2. Correlation between BCL2 expression and EZH2 expression also is observed in other lymphomas such as diffuse large B-cell lymphoma, where dual targeting with EZH2 and BCL2 inhibitors has been proposed. Thus, our findings provide further rationale for stratifying Type I and Type II ALCLs and identify BCL2 as a differentially expressed oncoprotein that might represent a target for precision therapy approaches.

Background: Anaplastic large cell lymphomas (ALCLs) represent a heterogeneous group of T-cell lymphomas that currently are classified by the presence or absence of ALK tyrosine kinase (TK) fusion genes (ALK+ or ALK−) and clinical presentation (systemic, cutaneous, or breast implant-associated). Two overarching molecular types of ALCL recently were discovered, defined by the presence (Type I) or absence (Type II) of a gene expression signature highly enriched for JAK-STAT3 activation. Fusions involving non-ALK TK genes occur in some ALK− cases, but the fusion landscape of ALCL remains incompletely characterized. Methods: Expert consensus pathology review was conducted in the Lymphoma/Leukemia Molecular Profiling Project (LLMPP). RNAseq was performed and fusions were identified using FusionCatcher. Here, we focused on recurrent in-frame coding fusions. Previously unreported fusions were validated by RT-PCR. Type I/II was assigned using a previously validated gene expression-based model. Genes with adjusted (adj) P<0.05 were considered differentially expressed. Overall survival (OS) was assessed for systemic ALCL, when available. Results: We evaluated 379 ALCLs (229M/150F; mean age, 56 y). Of 199 candidate fusions (excluding reciprocal events), 28 were recurrent and passed quality metrics. At least 1 of these 28 fusions was present in 150 cases (40%). ALK fusions were present in 106 ALK+ ALCLs (all Type I; P<0.0001). Of these, NPM1::ALK was seen in 68/106 (64%). Alternate partners (X::ALK) included ATIC (N=24) and CTLC, COL1A2, MSN, MYH9, RNF213, SATB1, TFH, TPM3, and TRAF1 (1-3 cases each). X::ALK was associated with older age (mean, 52 y) than NPM1::ALK (32 y; P<0.0001) and showed relative overexpression of 49 genes, including multiple activators of small GTPases such as CGNL1 (FC, 5.6; Padj=2.4×10-6), SRGAP1, ALS2, DOCK1, and NCKAP1. ALK expression was similar in cases with NPM1::ALK and X::ALK and there was no significant difference in OS. Non-ALK TK fusions were seen in 17/273 ALK− cases (6%), including TYK2 (N=9); JAK2 (N=6); and ROS1 (N=2). All were Type I ALCLs (P<0.0001). ALK− cases with non-ALK TK fusions overexpressed 38 genes compared to ALK− cases without TK fusions, including cytokines involved in the IL17 signaling pathway such as CXCL6 (FC, 12.5; Padj=4.2×10-3), CXCL1, and CSF3. We then assessed the relationship between ALK− cases with non-ALK TK fusions and ALK+ cases by separately comparing each subset to ALK− cases without TK fusions. FC values for ALK− cases with non-ALK TK fusions were significantly correlated with FC values for ALK+ ALCLs over the set of expressed genes (R=0.54; P<0.0001). At a median follow-up of 32 months, 0/4 systemic ALCL patients with non-ALK TK fusions had died, while 30% of those with ALK− ALCL without TK fusions had died, but this was not statistically significant (P=0.26). The other 13 ALK− cases with non-ALK TK fusions were either localized ALCLs or did not have outcome data available. Of 20 TP63 fusions, 17 (85%) were TBL1XR1::TP63 and 3 were X::TP63. As previously reported, TP63 fusions were associated with poor OS (median OS, 10 months; not reached for other ALK− ALCLs; P=0.007). Four ALCLs had a novel NCL::UBTF fusion involving genes encoding the nucleolar proteins nucleolin and nucleolar (upstream binding) transcription factor-1. Four cases each had PARG::BMS1 and TNK1::GPS2 (the latter seen only in Type I ALCLs). Conclusions: This large consortium-based analysis of coding fusions further elucidates the molecular distinction between Type I and Type II ALCLs. Both ALK and non-ALK TK fusions were seen exclusively in Type I ALCLs, which we have shown previously are enriched for JAK-STAT3 pathway genes and correlate strongly with pSTAT3Y705 positivity by immunohistochemistry. These fusions were not identified in Type II ALCLs, which previous data suggest are associated with epigenetic alterations. ALK+ ALCLs with NPM1::ALK and X::ALK showed differences in gene expression, suggesting biological differences. Furthermore, ALCLs with non-ALK TK fusions shared gene expression features with ALK+ ALCL. Clinical fusion detection could enhance molecular classification of Type I and Type II ALCLs and may guide precision therapy.

Abstract Long noncoding RNAs (lncRNAs) are defined as RNA-like transcripts that are over 200 nucleotides and lack significant open reading frames. Some lncRNAs such as HOTAIR, MALAT1 and H19 have been found to be associated with clinical prognosis and are potential drivers of cancer progression in cancers of the breast, lung, and liver respectively. The role of lncRNAs in lymphoma is unknown. Dysregulation of eIF4E (a key component of the translation initiation complex eIF4F) influences global protein translation, especially the translation of “weak” mRNAs that can be malignancy-related. We and others have found that eIF4E is dysregulated in B-cell lymphoma. The aim of this study is to identify eIF4E-associated lncRNAs through next generation RNA-Sequencing (NGS RNA-Seq) and delineate their role in protein translation in lymphoma. RNA-immunoprecipitation (RNA-IP) was used to pull down eIF4E-bound lncRNA in lymphoma cells. eIF4E-bound lncRNAs were immunoprecipitated with eIF4E antibody or IgG control in Jeko, a mantle cell lymphoma (MCL) cell line and sent for microarray analysis and NGS-RNA-Seq for identification of lncRNAs. The microarray analysis showed that several lncRNAs were enriched with eIF4E antibody compared to IgG control. These included SNHG4 (13.6 fold), SNHG12 (4.8 fold), NCRNA00171 (4.8 fold) and IPW (4.6 fold), GNASAS (3.5 fold), SNHG7 (3.3 fold), NCRNA00182 (2.7 fold), NCRNA00094 (2.6 fold), NCRNA00188 (2.4 fold) and NCRNA00201 (2.1 fold). The binding of these lncRNAs to eIF4E was further confirmed by RT-PCR in Jeko, Mino and Granta MCL cell lines. Next, we looked the expression of these lncRNAs by qRT- PCR in the MCL cell lines and normal controls. We found SNHG4 and IPW to be overexpressed in all the MCL cell lines, while SNHG12 and NCRNA00201 were overexpressed in the selected cell lines. No significant difference was found for the expression of NCRNA00171 and NCRNA00182 in any of the MCL cell lines compared to controls. Overall, these data suggest that several lncRNA have altered expression in malignant B-cells. Considering that the microarray assay only covered a limited number of lncRNAs, we further confirmed eIF4E bound lncRNA by NGS RNA-Seq in Jeko MCL and normal control. The binding of 10/13 lncRNA mentioned above with eIF4E were found upregulated by NGS-RNA-Seq. In addition several novel lncRNAs such as SNHG1 (161.6), AC091814.2 (98.8) and RP11-304L19.5 (64.2) showed up in NGS-RNA-Seq data. These data suggest that lncRNAs, such as SNHG12, SNHG4, and SNHG1 bind to eIF4E with high affinity in malignant B-cells and might play a role in protein translation. We knocked down the expression of SNHG4 through siRNA and demonstrated that cell proliferation and global protein translation was inhibited in lymphoma cells. To further confirm the role of SNHG4 in translation regulation, a plasmid, which contains a renilla luciferase driven by SV40 promoter, was co-transfected with SNHG4 siRNA into Mino cells. The luciferase signal, decreased compared with the cells transfected with nontargeting siRNA. These data suggest that SNHG4 is involved in the regulation of protein translation. In order to clarify the mechanism of lncRNAs bound to eIF4E we searched for RNA binding sites or motifs in eIF4E protein using the web-based tools, BindN and PPRInt. Interestingly two RNA binding motifs, KNKRGGRWLITLNKQQRRS and SHADTATKSGSTTKNR, were found in eIF4E based on the prediction. To examine whether lncRNAs bind with eIF4E through these RNA binding motifs, an eIF4E mutant plasmid with both RNA binding motifs deleted (eIF4EDel), was constructed and transfected transiently into HEK-293T cells along with eIF4EWT plasmid. RNA-IP data showed that the lncRNAs SNHG12, SNHG4 and SNHG1 were not able to bind with eIF4E in eIF4EDel-transfected cells compared with that of eIF4EWT, suggesting that these lncRNAs complex with eIF4E through RNA-binding motifs within the eIF4E. Overall, our results show that the lncRNAs, SNHG1 and SNHG4 are able to bind with eIF4E and regulate protein translation. Since lncRNAs had been found to play roles in the regulation of gene expression, including transcription, splicing and mRNA stability, our results may broaden the view of the functional role of lncRNAs in translation in lymphoma cells and in other cancers. Furthermore, our results also suggested that SNHG4 lncRNAs might be served as potential biomarkers for MCL and other B cell lymphomas for translation therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2655 Diffuse large B cell lymphoma (DLBCL) has been classified into two distinct molecular subtypes: germinal center B cell–like (GCB), non-germinal centre-like (non-GCB). To improve outcomes for DLBCL patients, it is necessary to identify additional novel targets within GCB and non-GCB classifications to further stratify patients for prognosis and assist in choosing therapy for the individual patient. We have recently demonstrated that STAT3 activation is frequent in the DLBCL tumors, however the exact molecular mechanism(s) of STAT3 activation in DLBCL tumors are not known. Molecular mechanisms such as epigenetic silencing of negative regulators of the STAT3 pathway such as protein tyrosine phosphatase 6 (PTPN6) may contribute to STAT3 activation. We aimed to learn whether PTPN6 was expressed in GCB and non-GCB DLBCL, and if so, how that expression correlated with STAT3 activation and prognosis. We first performed epigenetic studies of PTPN6 in 38 DLBCL tumors and 6 DLBCL cell lines by methylation specific (MSP) PCR and high resolution melting array (HRM) methods. Surprisingly, PTPN6 promoter hypermethylation (0/38) was not detected in patient sample and cell lines by both the methods. Since the MSP PCR technique yields qualitative rather than quantitative data, we next performed pyrosequencing on the 38 DLBCL samples, and found results consistent with the MSP PCR analysis. Our data conclusively demonstrate that PTPN6 hypermethylation is absent in DLBCL tumors. We next sequenced the 600 bp upstream of the transcription initiation site of PTPN6 promoter 2 and 1 in 10 DLBCL tumors. We did not detect any point mutations in the promoter 2 and 1 core regions. Since PTPN6 promoter hypermethylation and mutations were absent in DLBCL tumors, we determined the expression level of the PTPN6 protein in 40 DLBCL tumors by molecular subtype. Formalin fixed paraffin-embedded DLBCL tumor samples were stained by immunohistochemistry (IHC) for the determination of molecular subtype using the Hans algorithm and the detection of PTPN6 expression. Using a threshold of ≥30%, 75% (30/40) of cases were PTPN6 positive with various levels of expression: 11 cases had 30–80% of tumor cells staining positive and 19 had >80% of cells PTPN6 positive. PTPN6 expression by IHC was only correlated with higher IPI scores and a trend towards a shorter event free survival (EFS) (p=0.07). Within the 29 GCB tumors 69% (20/29) were PTPN6 positive; 100% (10/10) of non-GCB cases were PTPN6 positive. These data clearly suggest that PTPN6 expression is found in both GCB and non-GCB with the latter being uniformly positive (p=0.03) and PTPN6 negative cases being uniformly GCB. PTPN6 mRNA and protein was detected in all three ABC lines (LY3, Ly10, DHL2). Within the GCB lines (DHL6, Ly1 and Ly19) DHL6 was weakly positive and Ly1 and Ly19 were negative for PTPN6 mRNA and protein. Furthermore, within the GCB group PTPN6 positive cases had inferior EFS as compared to PTPN6 negative cases. In the non-GCB group all cases were PTPN6 positive with an EFS similar to PTPN6 positive GCB cases. The role of PTPN6 in the persistent activation of the STAT3 pathway in DLBCL patients has not been investigated. We hypothesized that tumors with activated STAT3 would have loss of PTPN6. Interestingly, this hypothesis was disproven. Within the 15-pSTAT3 positive cases 12 (80%) were PTPN6 positive. Conversely, 26% (6/23) of the pSTAT3 negative cases were PTPN6 negative. The distribution of pSTAT3 and PTPN6 by IHC in samples was evaluated in both GCB and non-GCB groups. Within the PTPN6+/pSTAT3+ group out of the 12 cases most were non-GCB (8/12; 66%). However, within the PTPN6-/pSTAT3- group all the 5 cases were GCB (5/5; 100%). Survival analysis revealed that the groups with the best EFS were those with PTPN6-/pSTAT3- tumors (n=5); those with PTPN6+/pSTAT3+ group (n=12) had the shortest EFS; and those with PTPN6+/pSTAT3- tumors (n=17) being intermediate between the other groups. In summary, we have demonstrated for the first time that PTPN6 is highly expressed in DLBCL tumors, and a common abnormality in non-GCB subtypes, which is positively correlated with activated STAT3. PTPN6 expression in the DLBCLs is not regulated through SHP1 promoter hypermethylation or point mutations. The finding that SHP1 loss was found only in GCB cases and was especially favorable should be explored further and may provide an important new stratification factor for future DLBCL studies. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2690 Diffuse large B cell lymphoma (DLBCL) is the most common type of adult non-Hodgkin lymphoma. Aberrant gene expression and mutations are involved in the pathogenesis of DLBCL and may have prognostic and therapeutic relevance. We have shown that STAT3 (by immunohistochemistry) is constitutively activated in approximately 50% of new untreated DLBCL cases. To investigate whether genetic mutations play a role in the activation of JAK2/STAT3 signaling pathway in DLBCL, we sequenced the coding regions of the JAK2 gene and found no novel missense mutations. Since we did not find any missense mutations in the JAK2 gene, we proceeded to bi-directionally sequence all the 24 exons of the STAT3 gene in the same DLBCL patient tumors (n=40). We discovered mutations in 2 patients, one in exon 7 (T>A) and the other at the stop codon in exon 24 (G>A). While the G to A mutation in exon 24 was a silent mutation, the T to A mutation in exon 7 led to methionine substitution by lysine at codon 206 (M206K). Codon 206 is located in the coil-coiled domain of STAT3, which is a conserved amino acid in STAT3 from many different species. In order to delineate the functional significance of this STAT3 M206K mutation we generated STAT3 expression plasmids with the lentiviral expression vector pLEX-MCS. STAT3 constructs carrying the M206K mutation were generated by site directed mutagenesis of wild type STAT3. Since there are two STAT3 isoforms in humans, full-length STAT3α and the splicing variant STAT3β (which lacks the C-terminal transactivation domain), we constructed stable cell lines containing mutant and wild type gene of both STAT3α and STAT3β in HEK-293T cells. The mutation was found to increase the phosphorylation of Tyr705 in STAT3α mutant cell lines with no effect on STAT3β mutant cell line. Moreover the mutant STAT3α cell line was more sensitive to IL-10 in the activation of Tyr705 compared to its wild type while STAT3β mutant cell line was insensitive to IL-10. Both the STAT3 mutants (STAT3α and STAT3β) were insensitive to IL-6, which suggests that the mutant can sense the signal specifically from IL-10. Furthermore JAK2 inhibition through the pharmacological inhibitor TG 101348 dephosphorylated STAT3 in WT cells; however, had minimal effect in both the STAT3 mutants. Immunofluorescence staining showed that the M206K mutation did not affect the nuclear translocation of both STAT3α and STAT3β, which was confirmed by immuno-blotting on the cytosol and nucleus of the cells. Interestingly, the mutation activated the transcriptional activation activity of both STAT3α and STAT3β by luciferase reporter assay. Overall these results suggest that the M206K STAT3 gene mutation is rare (2.5%) in DLBCL tumors, leads to STAT3 activation, and enhances STAT3 transcriptional activation activity. Thus, STAT3 mutations in addition to cytokines can produce STAT3 pathway activation in DLBCL. Our data suggest that clinical trials of novel STAT3 inhibitors need not be restricted to tumors with STAT3 mutations but rather should focus on tumors with demonstrated aberrant STAT3 activation. Disclosures: No relevant conflicts of interest to declare.

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin Lymphoma and is characterized by deregulation of several signal transduction pathways. Approximately 50% patients with DLBCL are shown to have aberrant activation of the signal transducer and activator of transcription (STAT) pathway. However, mechanism of aberrant STAT3 signaling in DLBCL is not well understood. Protein tyrosine phosphatases (PTPs) are important enzymes that control the activity of multiple signaling pathways downstream of tyrosine kinases; mutations in PTPN11 have been found associated with the development of myeloproliferative disorder. In the current study, we sequenced a set of 38 DLBCL tumor samples for genetic mutations of PTPN6. Bi-directional sequencing reactions were performed on genomic DNA, and all exons of PTPN6 were amplified and analyzed by Sanger sequencing. We identified 2 novel heterozygous missense mutations in PTPN6 gene in 2 separate patients (2/38; 5.2%). The first missense mutation occurred in the exon 7, resulted in an Asp226 to lysine substitution (N226K), while other missense mutation found on exon 15 resulted in an Ala552 to valine substitution (A552V). In order to elucidate the functional significance of these mutations, we performed site directed mutagenesis to mutate wild type (WT) PTPN6 at codon 226 and 552. Both, wild-type PTPN6 (PTPN6WT) or those containing the N226K (PTPN6N226K) and A552V (PTPN6A552V) mutations were cloned and stably expressed in the HEK-293T cells with a lentiviral expression vector and PTPN6 phosphatase activity was measured. Our results showed marked reduction (70%) in the PTPN6 phosphatase activity in both the PTPN6 mutants as compared to the WT control. Interestingly both the PTPN6 mutants (PTPN6N226K and PTPN6A552V) promoted cell proliferation of HEK-293 cells as compared to WT PTPN6. In order to evaluate that whether or not PTPN6 mutations modulate STATs signaling, we analyzed STAT1, STAT3, STAT5 and STAT6 phosphorylation at tyrosine residue in cells stably transfected with WT or mutant PTPN6. While overexpression of WT PTPN6 reduced STAT3 phosphorylation, however no effect was observed on STAT1, STAT5 and STAT6 phosphorylation. Unlike WT-PTPN6, none of the PTPN6 mutants was able to dephosphorylate STAT3. In addition, PTPN6 A552V mutation was found to decrease the ability of PTPN6 to dephosphorylate STAT3 after induction with interleukin-10. Furthermore JAK2/JAK1 inhibition through the pharmacological inhibitor ruxolitinib (5μM) dephosphorylated STAT3 in WT PTPN6 as well as in PTPN6 mutants within 4 hours. Inhibition of JAK3 through a pharmacological inhibitor WHI-P154 (5μM) was able to completely dephosphorylated STAT3, however both the PTPN6 mutants were found resistant to JAK3 inhibition. Moreover, in a luciferase reporter assay compared to WT PTPN6, expressing either of the PTPN6 mutants (N226K and A552V) enhanced transcriptional activity of STAT3. To determine the effect of the PTPN6 mutations on the downstream targets of the JAK/STAT pathway, we demonstrated that Mcl-1, survivin and Bcl-2 protein level was significantly elevated in both mutant cells than that seen in the WT PTPN6 cells. Overall these results demonstrate that the N226K and A552V PTPN6 mutations occur in 5% of DLBCL tumors. Mutations we have found are both able to cause loss-of-function of PTPN6 that leads to increased STAT3 phosphorylation and increased transcriptional activity resulting in accumulation of Mcl-1, Bcl-2 and survivin proteins. Our data suggest that clinical trials of JAK1/JAK2 inhibitors need not be restricted to tumors with PTPN6 mutations but rather should focus on tumors with demonstrated aberrant STAT3 activation. Taken together, our data suggest that PTPN6 mutations may guide the clinical use of inhibitors of JAK3 kinase in DLBCL. Disclosures No relevant conflicts of interest to declare.

Abstract Epigenetic silencing of tumor-suppressor genes has potential therapeutic relevance if the diagnostic and prognostic utility of particular DNA methylation patterns could be established. SHP1 is a tumor-suppressor gene that can be downregulated in various neoplasms. The aims of this study are (i) whether SHP1 was lost/ suppressed in diffuse large B cell lymphoma (DLBCL) (ii) Understanding the role of various epigenetic mechanisms including CpG hypermethylation and histone modifications in SHP1 loss, and (iii) whether SHP1 in DLBCL can be re-expressed with epigenetic therapy. We initially analyzed SHP1 mRNA expression in DLBCL (n=9) patient specimens and normal B-cells by QRT-PCR. Decreased expression of SHP1 mRNA was observed in all the DLBCL patient samples as compared to normal B cells. DLBCL tumor samples (n=37) along with normal tonsils (n=10) were stained for SHP1 protein by immunohistochemistry (IHC). SHP1 expression was lost or suppressed in 53% of DLBCL tumors compared to normal controls. These data, when taken together, confirm that SHP1 is strongly expressed in normal B-cells and can be lost or suppressed in DLBCL tumors. We performed epigenetic studies of SHP1 CpG promoter 2 (named CPG1 P2) in 37 DLBCL tumors and 6 DLBCL cell lines by methylation specific (MSP1) PCR and pyrosequencing methods. Surprisingly, SHP1 CpG1 P2 hypermethylation (0/37) was not detected in patient samples or cell lines by both methods. Our data conclusively demonstrate that SHP1 CpG1 P2 hypermethylation is absent in DLBCL tumors. The finding that SHP1 was lost or silenced in many of the clinical samples without finding any CpG1 P2 hypermethylation led us to extend our methylation studies to a novel, more proximal CpG island within P2 (named CpG2) that has not been previously studied in any hematological malignancies. We designed the primers specific for these CpG2 sites and ran the pyrosequencing on the same 37 DLBCL tumors as used above for CpG1 methylation. Using a cut off of >25% (intermediate to high methylation) 57% (21/37) of patient samples were methylated. The Ly3, Ly10, and DHL2 DLBCL cell lines were also hypermethylated at CpG2. To confirm the role of CpG2 methylation in SHP1 expression, we treated the Ly3 and DHL2 cells with the DNA methyltransferase inhibitor, 5-Azacytidine (Aza). We observed increased SHP1 expression compared to untreated control cells in Ly3 and DHL2 cells followed by a progressive demethylation of SHP1 CpG2, as shown by positive U-MSP with increasing amplification intensity. This resulted in a corresponding down-regulation of activated STAT3 in a similar time dependent manner. Treatment of Ly3 and DHL2 cells with 5-Aza for 0-6 days significantly (p=0.0008 for Ly3 and p=0.03 for DHL2) reduced the cell survival (40% decrease at day 6). To further investigate epigenetic control of SHP1 in DLBCL, we proceeded to study whether histone modifications in the SHP1 promoter were involved in the regulation of SHP1 expression. Chromatin immunoprecipitation assays were performed in the Ly3, Raji cell line and normal B cells, by using antibodies to repressing marks on histones. We observed that the SHP1 promoter in the Ly3 and Raji cells was enriched for both H3K9me3 and H3K27me3 repressing marks as compared to normal B cells. To confirm the role of histone modifications in SHP1 expression, we treated Ly3 cells with the histone methyltransferase inhibitor (HMT) DZNEP and the HDAC inhibitor LBH589 (Novartis Pharmaceuticals). DZNEP has been shown to be very specific for histone suppressive marks H3K27me3 that are mediated through EZH2. Treatment with DZNEP partially reverted the H3K27me3 histone marks in the SHP1 promoter followed by increased the SHP1 expression in Ly3 cells. LBH589 treatment was not able to inhibit H3K27me3 marks, but was able to inhibit the CpG2 methylation within the SHP1 promoter 2 alone or in combination with 5- Aza. In conclusion, these data provide a comprehensive characterization of the epigenetic mechanisms leading to SHP1 deregulation in the DLBCL tumors. These findings support the notion that control of SHP1 P2 in DLBCL tumors is associated with both DNA promoter methylation and histone methylation, which explains our results that treatment with 5-Aza, DZNEP and LBH589 could reactivate SHP1 expression and inhibit DLBCL cell survival. Therefore, SHP1 may prove useful diagnostic and prognostic marker and provide a rationale to use epigenetic therapy for patients with DLBCL. Disclosures: No relevant conflicts of interest to declare.

Abstract Long non-coding RNAs (lncRNA) are key regulatory RNAs that do not code for protein. Recently lncRNAs have been recognized as key regulators of gene expression and chromatin organization. Although, deregulation of lncRNAs such as HOTAIR, MALAT1 and H19 has been reported in solid cancers, role of lncRNAs in lymphoma or other hematologic malignancies is not known. The aim of this study is to identify lymphoma-associated lncRNAs and elucidate their role in disease pathogenesis. Next generation RNA-sequencing and validation studies were carried out on mantle cell lymphoma (MCL) patient samples along with normal controls. As a result, hundreds of lncRNAs were found overexpressed in the MCL samples as compared with normal B cells, including RP11-24J23.2 (50 fold), RP11-12A2.3 (36 fold) and DLEU7-AS1 (19 fold). Quantitative RT-PCR (QRT-PCR) confirmed higher expression of RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 in MCL patient samples as compared to normal B cells. To investigate the biological significance of lncRNAs in MCL cells, we overexpressed RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 lncRNAs and analyzed effects on cell proliferation and apoptosis. To achieve overexpression lncRNAs, RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 were cloned individually in expression pcDNA3.1 generating constructs: pcDNA3RP11-24J23.2, pcDNA3RP11-12A2.3 and pcDNA3DLEU7-AS1and then transfected into MCL cell lines along with pcDNA3.1. QRT-PCR revealed effective overexpression (~100 fold increase over baseline) for each transfected lncRNAs construct in Granta and Mino cells. Analysis of proliferation and survival showed that overexpressing RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1 promotes cell proliferation and survival in these cells as compared to the vector controls. Most of the lncRNAs found so far have been linked to the transcriptional regulation of the target genes through their association with chromatin remodeling factors such as Polycomb Repressive Complex 2 (PRC2). To identify the functional relevance of RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 lncRNAs, we examined the binding of the lymphoma specific lncRNAs with PRC2 complex, by performing RNA immunoprecipitation (RNA-IP) using specific antibodies to EZH2, EED and SUZ12. The RNA-IP results demonstrate high affinity binding of all three lncRNAs with SUZ12 and EED but a weak binding with EZH2. Furthermore, RNA-IP using antibody to histone 3-lysine 27 trimethylation (H3K27me3), a suppressive histone mark associated with PRC2 complex, also showed efficient binding with RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1. These results suggest that RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 lncRNAs can directly associated with PRC2 complex at chromatin. To understand the mechanism of increased cell growth after overexpression of lncRNAs, RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1, we analyzed expression level of important genes signature of MCL pathogenesis that include cyclin D1, cyclin D3, c-Myc, P14, and SOX11, a member of the group C SOX (SRY-related HMG-box) transcription factor family. Our results demonstrate that overexpression of lncRNAs RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1 enhanced the levels of cyclin D1 (>2 fold) mRNA without any impact on cyclin D3 or c-Myc transcript levels. In contrast overexpression of RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1 lncRNAs decreased SOX11 (0.08, 0.1 and 0.06 respectively) and P14 (0.06, 0.7 and 0.40 respectively) mRNA expression in Mino cells. To gain understanding of the mechanism involved in the suppression of SOX11 by the lncRNAs, we performed Chromatin immunoprecipitation assays with EZH2, EED and SUZ12 antibodies on Mino cells transfected with pcDNA3RP11-24J23.2, pcDNA3RP11-12A2.3 and pcDNA3DLEU7-AS1 or vector control. The data showed that overexpression of RP11-12A2.3 and DLEU7-AS1 significantly increased the binding of EED and to a lesser extent of EZH2 to SOX11 promoter while the binding of SUZ12 to SOX11 promoter was not changed as compared to vector alone. In conclusion, our results imply that lncRNAs are overexpressed in the MCL. Moreover, lymphoma associated lncRNAs (RP11-24J23.2, RP11-12A2.3 and DLEU7-AS1) regulate SOX11 expression via PRC2 complex contributing towards growth of MCL cells. Specific nature of lncRNAs found in lymphoma samples suggests that lncRNAs can be used as potential biomarkers for mantle cell lymphoma. Disclosures No relevant conflicts of interest to declare.

AbstractAnaplastic large cell lymphomas (ALCLs) represent a relatively common group of T-cell non-Hodgkin lymphomas (T-NHLs) that are unified by similar pathologic features but demonstrate marked genetic heterogeneity. ALCLs are broadly classified as being anaplastic lymphoma kinase (ALK)+ or ALK−, based on the presence or absence of ALK rearrangements. Exome sequencing of 62 T-NHLs identified a previously unreported recurrent mutation in the musculin gene, MSCE116K, exclusively in ALK− ALCLs. Additional sequencing for a total of 238 T-NHLs confirmed the specificity of MSCE116K for ALK− ALCL and further demonstrated that 14 of 15 mutated cases (93%) had coexisting DUSP22 rearrangements. Musculin is a basic helix-loop-helix (bHLH) transcription factor that heterodimerizes with other bHLH proteins to regulate lymphocyte development. The E116K mutation localized to the DNA binding domain of musculin and permitted formation of musculin–bHLH heterodimers but prevented their binding to authentic target sequence. Functional analysis showed MSCE116K acted in a dominant-negative fashion, reversing wild-type musculin-induced repression of MYC and cell cycle inhibition. Chromatin immunoprecipitation–sequencing and transcriptome analysis identified the cell cycle regulatory gene E2F2 as a direct transcriptional target of musculin. MSCE116K reversed E2F2-induced cell cycle arrest and promoted expression of the CD30–IRF4–MYC axis, whereas its expression was reciprocally induced by binding of IRF4 to the MSC promoter. Finally, ALCL cells expressing MSCE116K were preferentially targeted by the BET inhibitor JQ1. These findings identify a novel recurrent MSC mutation as a key driver of the CD30–IRF4–MYC axis and cell cycle progression in a unique subset of ALCLs.