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A comprehensive constellation of somatic nonsilent mutations and copy-number (CN) variations in ocular adnexa marginal zone lymphoma (OAMZL) is unknown. By utilizing whole-exome sequencing in 69 tumors, we define the genetic landscape of OAMZL. Mutations and CN changes in CABIN1 (30%), RHOA (26%), TBL1XR1 (22%), and CREBBP (17%) and inactivation of TNFAIP3 (26%) were among the most common aberrations. Candidate cancer driver genes cluster in the B-cell receptor (BCR), NF-κB, NOTCH, and NFAT signaling pathways. One of the most commonly altered genes is CABIN1, a calcineurin inhibitor acting as a negative regulator of the NFAT and MEF2B transcriptional activity. CABIN1 deletions enhance BCR-stimulated NFAT and MEF2B transcriptional activity, while CABIN1 mutations enhance only MEF2B transcriptional activity by impairing binding of mSin3a to CABIN1. Our data provide an unbiased identification of genetically altered genes that may play a role in the molecular pathogenesis of OAMZL and serve as therapeutic targets. Significance: We report systematic application of whole-exome sequencing and CN variations in OAMZL, revealing common alterations in regulation of NFAT signaling pathway that may facilitate identification of new therapies.

In spite of newly emerging therapies and the improved survival of patients with non-Hodgkin lymphoma (NHL), relapses or primary refractory disease are commonly observed and associated with dismal prognosis. Although discovery of the anti-CD20 antibody rituximab has markedly improved outcomes in B-cell NHL, rituximab resistance remains an important obstacle to successful treatment of these tumors. To improve the efficacy of CD20-targeted therapy, we fused interleukin 21 (IL-21), which induces direct lymphoma cytotoxicity and activates immune effector cells, to the anti-CD20 antibody (αCD20-IL-21 fusokine). We observed substantially enhanced IL-21R-mediated signaling by the fusokine compared with native IL-21 at equimolar concentrations. Fusokine treatment led to direct apoptosis of lymphoma cell lines and primary tumors that otherwise were resistant to native IL-21 treatment. In addition to direct cytotoxicity, the fusokine enhanced NK cell activation, effector functions, and interferon γ production, resulting in greater antibody-dependent cell-mediated cytotoxicity compared with IL-21 and/or anti-CD20 antibody treatments. Further, the αCD20-IL-21 fusokine stabilizes IL-21 and prolongs its half-life. In vivo αCD20-IL-21 therapy resulted in a significant tumor control in the rituximab-resistant A20-huCD20 tumors. Collectively, the dual functional ability of the αCD20-IL-21 fusokine to induce direct apoptosis and activate immune effector cells may provide benefit over existing treatments for NHL.

Regulating B-cell receptor (BCR) signaling after antigenic stimulation is essential to properly control immune responses. Currently known mechanisms of inhibiting BCR signaling are via co-receptor stimulation and downstream immunoreceptor tyrosine-based inhibition motif (ITIM) phosphorylation. Herein we demonstrate that BCR stimulation induces rapid and reversible palmitoylation of the SCF-FBXO10 ubiquitin E3 ligase. This results in FBXO10 relocation to the cell membrane, where it targets the human germinal center-associated lymphoma (HGAL) protein for ubiquitylation and degradation, leading to decreases in both BCR-induced calcium influx and phosphorylation of proximal BCR effectors. Importantly, FBXO10 recognition and degradation of HGAL is phosphorylation independent and instead relies on a single evolutionarily conserved HGAL amino acid residue (H91) and FBXO10 relocalization to the cytoplasmic membrane. Together our findings demonstrate the first evidence of negative BCR signaling regulation from direct BCR stimulation and define the temporospatial functions of the FBXO10-HGAL axis. FBXO10 is infrequently mutated in DLBCL but some of these mutations deregulate BCR signaling. These observations may have important implications on lymphomagenesis and other immune processes.

Key Points CRISPR/Cas9 genomic editing of wild-type hematopoietic stem cells generates Npm1-Alk, leading to ALK+ large-cell lymphomas in recipients. CD30+ postthymic T-cell lymphomas are polyclonal but transplantable to secondary recipients with long latency.

GLI1 oncogene has been implicated in the pathobiology of several neoplasms including diffuse large B-cell lymphoma (DLBCL). However, mechanisms underlying GLI1-increased activity in DLBCL are poorly characterized. Herein, we demonstrate that IKKβ phosphorylates GLI1 in DLBCL. IKKβ activation increased GLI1 protein levels and transcriptional activity, whereas IKKβ silencing decreased GLI1 levels and transcriptional activity. Tumor necrosis factor-α (TNFα) mediated IKKβ activation-impaired GLI1 binding with the E3 ubiquitin ligase-ITCH, leading to decreased K48-linked ubiquitination/degradation of GLI1. We found 8 IKKβ-dependent phosphorylation sites that mediate GLI1 stability. Mutating or deleting these residues facilitated GLI1-ITCH interaction and decreased the protective effect of TNFα on GLI1 stability. IKKβ-GLI1 crosstalk is significant because combined inhibition of both molecules resulted in synergistic suppression of DLBCL viability in vivo and in vitro. By linking IKKβ-mediated nuclear factor-κB activity with GLI1, we identified a crosstalk between these 2 pathways that can inform the design of novel therapeutic strategies in DLBCL.

Tumor necrosis factor receptor-associated factor 6 (TRAF6), an (K63) E3-ligase, plays a role in many biological processes and its activity is relevant in diffuse large B cell lymphoma (DLBCL) biology. Although molecules that trigger TRAF6 activation have been defined, those that stabilize TRAF6 and/or enhance TRAF6 function remain largely unclear. We found that TRAF6 amplifies pAKT signaling in DLBCL. Moreover, TRAF6 activation and stabilization of its ubiquitination profile are facilitated by smoothened (SMO), signal transducer of canonical Hedgehog signaling. Here, we report that SMO is needed to facilitate and maintain TRAF6-dependent elevated pAKT levels, and that the SMO/TRAF6 axis contributes to doxorubicin resistance in DLBCL. Mechanistically, we found that SMO, through its C-terminal tail, stabilizes and protects TRAF6 from degradation, an effect mediated by ubiquitin-specific protease-8. Moreover, this functional link between SMO and TRAF6 is reflected in DLBCL patients where high expression of both molecules correlates with poor prognosis. In summary, our study reveals a novel cell survival mechanism in which SMO stabilizes and protects TRAF6 from degradation. The axis SMO/TRAF6/AKT is highly relevant in the biology of DLBCL and is involved in doxorubicin resistance.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease with a high proliferation rate. However, the molecular and genetic features that drive the aggressive clinical behavior of DLBCL are not fully defined. Here, we have demonstrated that activated Jun signaling is a frequent event in DLBCL that promotes dissemination of malignant cells. Downregulation of Jun dramatically reduces lymphoma cell adhesion to extracellular matrix proteins, subcutaneous tumor size in nude mice, and invasive behavior, including bone marrow infiltration and interaction with bone marrow stromal cells. Furthermore, using a combination of RNA interference and gene expression profiling, we identified Jun target genes that are associated with disseminated lymphoma. Among them, ITGAV, FoxC1, and CX3CR1 are significantly enriched in patients with 2 or more extranodal sites. Our results point to activated Jun signaling as a major driver of the aggressive phenotype of DLBCL.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Aberrant activation of Hedgehog (Hh) and nuclear factor (NF)-κB pathways is ubiquitously observed and known to mediate tumor growth, survival, and chemoresistance in DLBCL. Here, we find that activation of Hh signaling is positively correlated with NF-κB pathway in DLBCL tumors, and that smoothened (SMO), the signal transducer subunit of Hh pathway, contributes to NF-κB activation through recruiting G protein subunits Gαi and Gα12 to activate PKCβ/CARMA1/TRAF6/NEMO signaling axis followed by assembling of the CARMA1/BCL10/MALT1/TRAF6 complex to SMO. Moreover, functional inhibition of SMO enhances the cytotoxic effects of NF-κB inhibitor. Altogether, our study reveals a noncanonical Hh signaling pathway in which SMO activates trimeric G proteins and CARMA1-associated signaling complex, leading to NF-κB activation. This signaling cascade contributes to the survival of DLBCL and may serve as a potential target for combination therapies in DLBCL.

Epstein-Barr virus (EBV) -associated follicular lymphoma is only rarely reported. Herein, we report the largest series analyzing prevalence and clinicopathologic characteristics of EBV-associated follicular lymphoma occurring in unselected cases. Out of 382 analyzed cases, 10 EBV-positive follicular lymphomas were identified (prevalence=2.6%, 95% confidence interval 1.3-4.0%). All EBV-positive follicular lymphomas showed EBV-encoded small RNA-positive lymphoma cells present in a follicular distribution. Of these, eight also had tissue available for testing of expression of latent membrane protein 1 (LMP1), out of which six (75%) were positive. There was a significant association with grades 3A-3B follicular lymphoma (P

Hedgehog (Hh) signaling pathway is activated in diffuse large B-cell lymphoma (DLBCL). Genetic abnormalities that explain activation of Hh signaling in DLBCL are unknown. We investigate the presence of amplifications of Hh genes that might result in activation of this pathway in DLBCL. Our data showed few extra copies of GLI1 and SMO due to chromosomal aneuploidies in a subset of DLBCL cell lines. We also showed that pharmacologic inhibition of PI3K/AKT and NF-κB pathways resulted in decreased expression of GLI1 and Hh ligands. In conclusion, our data support the hypothesis that aberrant activation of Hh signaling in DLBCL mainly results from integration of deregulated oncogenic signaling inputs converging into Hh signaling.

Recently, reports using immunohistochemistry and a polyclonal antibody directed against the N-terminal region of PAX8 describe PAX8 expression in malignant lymphomas. As the N-terminal regions of PAX family members, including the B-cell transcription factor PAX5, have high sequence homology, we investigated PAX8 positivity in malignant lymphomas. Comparative sequence analysis between the N- and C-terminal regions of PAX8 and PAX5 proteins confirmed homologies of 70% and 39%, respectively. We then compared the results using N-terminal (high homology) and C-terminal (lower homology) anti-PAX8 antibodies to assess PAX8 expression in reactive tissues, diffuse large B-cell lymphoma and classical Hodgkin lymphoma, using routine immunohistochemical methods. Expression of PAX8 was also assessed in diffuse large B-cell lymphoma and classical Hodgkin lymphoma cell lines using real-time qRT-PCR methods. Our results show that reactive and neoplastic B-cells are positive for PAX8 using the N-terminal antibody, but negative for PAX8 when the C-terminal antibody was used. PAX8 mRNA levels were not detected in any of the B-cell lymphoma cell lines studied. These results indicate that benign and malignant B-cells do not express PAX8. We conclude that positivity for PAX8 reported by others in B-cell lymphomas is likely due to cross-reactivity between the N-terminal regions of PAX8 and PAX5, due to the high sequence homology of these two regions.

Successful treatment of diffuse large B-cell lymphoma (DLBCL) is frequently hindered by the development of resistance to conventional chemotherapy resulting in disease relapse and high mortality. High expression of antiapoptotic and/or drug transporter proteins induced by oncogenic signaling pathways has been implicated in the development of chemoresistance in cancer. Previously, our studies showed that high expression of adenosine triphosphate-binding cassette drug transporter ABCG2 in DLBCL correlated inversely with disease- and failure-free survival. In this study, we have implicated activated hedgehog (Hh) signaling pathway as a key factor behind high ABCG2 expression in DLBCL through direct upregulation of ABCG2 gene transcription. We have identified a single binding site for GLI transcription factors in the ABCG2 promoter and established its functionality using luciferase reporter, site-directed mutagenesis and chromatin-immunoprecipitation assays. Furthermore, in DLBCL tumor samples, significantly high ABCG2 and GLI1 levels were found in DLBCL tumors with lymph node involvement in comparison with DLBCL tumor cells collected from pleural and/or peritoneal effusions. This suggests a role for the stromal microenvironment in maintaining high levels of ABCG2 and GLI1. Accordingly, in vitro co-culture of DLBCL cells with HS-5 stromal cells increased ABCG2 mRNA and protein levels by paracrine activation of Hh signaling. In addition to ABCG2, co-culture of DLBCL cells with HS-5 cells also resulted in increase expression of the antiapoptotic proteins BCL2, BCL-xL and BCL2A1 and in induced chemotolerance to doxorubicin and methotrexate, drugs routinely used for the treatment of DLBCL. Similarly, activation of Hh signaling in DLBCL cell lines with recombinant Shh N-terminal peptide resulted in increased expression of BCL2 and ABCG2 associated with increased chemotolerance. Finally, functional inhibition of ABCG2 drug efflux activity with fumitremorgin C or inhibition of Hh signaling with cyclopamine-KAAD abrogated the stroma-induced chemotolerance suggesting that targeting ABCG2 and Hh signaling may have therapeutic value in overcoming chemoresistance in DLBCL.

Chromosomal rearrangements resulting in generation of novel fusion oncogenes are common in hematologic malignancies. These disease drivers are key therapeutic targets and form the basis of animal model development for preclinical studies. For example, retroviral introduction of the chronic myeloid leukemia (CML) fusion BCR-ABL to hematopoietic stem cells (HSCs) results in a myeloproliferative disease similar to accelerated-phase human CML when transplanted to recipient mice. This model and many others are established traditionally through retroviral or germline introduction of human fusion oncogenes to the murine genome. Recent advances in CRISPR/Cas9 technology now permit direct editing of the murine genome to create endogenous genotypes that more accurately reflect configurations found in human diseases. To date, these techniques have not been successfully applied to the modeling of fusion oncogene-driven hematologic malignancies. Anaplastic Large Cell lymphoma (ALCL) is a T-cell non-Hodgkin lymphoma common in adolescents and young adults and driven in ~70% of cases by chromosomal rearrangements involving Anaplastic Lymphoma Kinase (ALK). Most commonly, t(2:5; p23:q35) fuses the 3' ALK kinase domain to the 5' oligomerization domain of the constitutively expressed Nucleophosmin1 (NPM1) gene. An existing immunocompetent model of ALK+ lymphoma employs a CD4 promoter-driven NPM1-ALK transgene but results in immature T-lymphomas in about two-thirds and B-lineage plasma-cell lymphomas in the rest of animals. We employed CRISPR/Cas9 vectors containing guide strands designed to generate double-stranded DNA cleavage in mouse chromosomes 11 and 17 at breakpoints predicted to generate an in-frame Npm1-Alk fusion oncogene. Wild-type HSCs derived from fetal livers were divided and subjected to either transient CRISPR or mock transfection during a brief ex vivo passage followed by immediate transplantation to sub-lethally irradiated wild-type recipients. Mice initially transplanted with CRISPR-modified HSCs developed an ALK+ large cell lymphoma with a latency of ~9 months, while mock transfected controls sacrificed in parallel were phenotypically normal. qPCR analysis of lymphoid organs from mice that developed disease showed extremely high expression of Alk and Tnfrsf8, which encodes CD30. Pathologically, tumors contained large malignancy T cells with anaplastic morphology. Immunohistochemistry confirmed ALK protein expression, including nuclear localization classic for NPM1-ALK, and intense CD30 cell-surface staining. One recipient instead developed a CD30-negative ALK+ diffuse large B-cell lymphoma. Transplantation of primary T-lymphoma cells to secondary recipients resulted also in ALK+ T-cell lymphomas in recipients with more rapid onset infiltrating lymph nodes, spleen, liver, and other organs. T-cell receptor clonality analyses through β-chain deep sequencing show an oligoclonal T-cell disease in both primary and secondary recipients. We therefore demonstrate successful genomic editing of transplantable murine hematopoietic stem cells to generate a novel model of a fusion oncogene-driven hematologic malignancy. These methods are widely applicable to additional lymphomas and leukemias and could fuel development of improved in vivo preclinical model systems. Disclosures No relevant conflicts of interest to declare.

Background: Ocular adnexa lymphomas (OAL) consist of a heterogeneous group of tumors that can arise in the lacrimal gland, orbit, conjunctiva and eyelid. More than 95% of these lymphomas are of B-cell origin and extranodal marginal zone lymphoma (EMZL) of mucosa-associated lymphoid tissue is the most common subtype of OAL, accounting for 55-85% of the cases. Ocular adnexa extranodal marginal zone lymphoma (OA-EMZL) patients have a persistent risk of relapses and in 4% of the cases, the disease transforms into an aggressive lymphoma. A comprehensive list of all somatic nonsilent mutations remains currently unknown thus hampering the complete understanding of the disease and the development of new diagnostic and therapeutic approaches. Previous studies analyzed only a small number of OA-EMZL patients using gene targeted sequencing. In this study we utilized whole-exome sequencing to define the genetic landscape of 69 cases of OA-EMZL. Our data provide an unbiased identification of genetically altered genes and pathways that may play a role in the molecular pathogenesis of OA-EMZL. Methods: GATK HaplotypeCaller was used for joint variant calling on all samples, while MuTect was used to detect somatic variants in seven tumor samples with available paired normal germline DNA. We focused on variants that were exonic, not synonymous, and deleterious (CADD score > 10), while excluding any variants found with a frequency > 1% in control populations. For copy number (CN) analysis we used EXCAVATOR on each sequenced exome in pooled mode against the seven paired normal samples. The output was provided to GISTIC 2.0 to determine recurrent arm-level and gene-level CN changes. Results: We identified 125 candidate cancer driver genes with the following characteristics: 1) have genomic alteration in more than 5% of the cases, 2) are present in the list of COSMIC and DLBCL driver genes, 3) are considered essential genes or tumor suppressor genes as previously defined in a functional CRISPR screen (Reddy et al., Cell 2017). Among the most frequent alterations there were mutations and CN losses of CABIN1 (32%), inactivation of TNFAIP3 (26%) and KMT2D (22%), mutations and CN changes in CARD11 (25%), RHOA (25%), CREBBP (20%) and TBL1XR1 (23%). Gene set enrichment analysis of candidate driver genes showed statistically significant enrichment for genes involved in cell junction, adhesion, cytoskeleton regulation, chromatin organization and for genes harboring at least one highly conserved NFAT binding motif TGGAAA in their promoter. In addition, we identified recurrent lesions affecting several genes belonging to the NFkB, NOTCH and B cell receptor (BCR) signaling pathways. The most frequent alterations in the NFkB pathway included inactivation of TNFAIP3 (26%), mutations and CN alterations of CARD11 (25%), MYD88 (12%), BCL10 (10%) and CRD10 (9%). Mutations affecting NOTCH signaling were found in NOTCH3 (22%), DTX1 (12%), MAML1 (12%), MAML2 (12%), DLK1 (10%) and SPEN (9%). BCR signaling genes alterations were observed in PIK3R2 (12%), VAV1 (10%), PTPN6 (7%) and BTK (4%). CABIN1 was one of the most commonly altered genes. In T cells, CABIN1 is implicated in inhibiting calcineurin-mediated signal transduction, including NFAT activation. In reactive lymph node and tonsil tissue, CABIN1 was expressed in reactive B cells (CD20+ and CD3-) localized mainly in the mantel zone area, as demonstrated by IHC and IF. We hypothesized that it may act as a negative regulator of BCR signaling in MZL. To verify this possibility we used shRNAs to knock down CABIN1 in MZL cell lines and measured NFAT activity in response to IgM stimulation using a luciferase reporter system. IgM stimulation of CABIN1-depleted MZL cells induced a more pronounced activation of NFAT signaling compared to wild-type cells. Conclusions: The genetics of OA-EMZL is characterized by somatic alterations in NFkB, NOTCH, and BCR signaling pathways. Candidate cancer driver genes are implicated in chromatin organization, cytoskeleton regulation and in adhesion, and they are also enriched for NFAT target genes. One of the most altered candidate cancer driver genes was CABIN1, which is a calcineurin inhibitor that acts as a negative regulator of NFAT activity. CABIN1 depletion in MZL cells boosts BCR-stimulated NFAT activity. Our data suggest a widespread dysregulation of NFAT signaling cascade downstream of BCR stimulation as a possible mechanism of OA-EMZL pathogenesis. Disclosures Lossos: Affimed: Research Funding.

The pathogenesis of Chlamydophila psittaci-negative ocular adnexal extranodal marginal zone lymphomas (OAEMZLs) is poorly understood. OAEMZLs are monoclonal tumors expressing a biased repertoire of mutated surface immunoglobulins. Antigenic activation of the B-cell receptor (BCR) may have a role in the pathogenesis of these lymphomas. We have analyzed the reactivity of recombinant OAEMZL immunoglobulins. OAEMZL antibodies reacted with self-human antigens, as demonstrated by enzyme-linked immunosorbent assays, HEp-2 immunofluorescence and human protein microarrays. All the analyzed recombinant antibodies (rAbs) exhibited polyreactivity by comprehensive protein array antibody reactivity and some rAbs also demonstrated rheumatoid factor activity. The identity of several reactive antigens was confirmed by microcapillary reverse-phase high-performance liquid chromatography nano-electrospray tandem mass spectrometry. The tested rAbs frequently reacted with shared intracellular and extracellular self-antigens (for example, galectin-3). Furthermore, these self-antigens induced BCR signaling in B cells expressing cognate surface immunoglobulins derived from OAEMZLs. These findings indicate that interactions between self-antigens and cognate OAEMZL tumor-derived BCRs are functional, inducing intracellular signaling. Overall, our findings suggest that self-antigen-induced BCR stimulation may be implicated in the pathogenesis of C. psittaci-negative OAEMZLs.

Tumor necrosis factor receptor-associated factor 6 (TRAF6), an (K63) E3-ligase, plays a crucial role in many biological processes and its activity is relevant in the biology of multiple cancers including diffuse large B cell lymphoma (DLBCL). Although molecules that trigger TRAF6 activation have been defined, those that stabilize TRAF6 levels and/or enhance TRAF6 function remain largely unclear. Previously, we found that activation of smoothened (SMO) with recombinant Hedgehog (Hh) ligand increased the binding between SMO with TRAF6, as well as TRAF6 protein levels (Blood 2013; 121:4718-28). In addition, transient overexpression of SMO resulted in increased K63-Ub of both TRAF6 and NEMO indicating stabilization of these proteins resulting in NF-kB activation. This is relevant, as more recently we found that TRAF6 amplifies pAKT signaling in DLBCL and that TRAF6 is the dominant E3 ligase for the K63-Ub of AKT in DLBCL. Moreover, TRAF6 recruitment to the cell membrane, and stabilization of its ubiquitination profile are facilitated by SMO. SMO is a member of the Frizzled-class G-protein-coupled receptor (GPCRs) and is traditionally known for its role as signal transducer in canonical Hedgehog (Hh) signaling. These observations prompted us to investigate whether the ability of SMO to increase TRAF6 levels is limited to ligand induced signaling, whether it contributes to chemoresistance in DLBCL cells, and whether SMO directly participates in controlling TRAF6 levels. To confirm the regulatory role of SMO in the TRAF6/AKT axis in DLBCL cells (HBL1 and HT) and further outline the nature of the underlying regulation, we measured the impact of activation of the Hh pathway with recombinant Shh ligand on TRAF6 levels, with and without SMO knockdown or recombinant SMO overexpression. Canonical Hh signaling results in the activation of the GLI1 transcription factor and the subsequent elevation of GLI1 mRNA levels is an established indicator of activation of the Hh pathway. However, neither SMO activation nor the knockdown of GLI1 had a significant impact on TRAF6 mRNA levels. These findings indicate that TRAF6 is not transcriptionally regulated by SMO signaling through GLI1 (canonical Hh signaling). In contrast, overexpression of SMO or siRNA knockdown of SMO resulted in an increase or decrease of TRAF6 protein levels, respectively. Consistent with the decrease of AKT activation (pAKT T308 and S473) after TRAF6 knockdown, the increase in TRAF6 levels that follows SMO overexpression resulted in an increase in the levels of AKT phosphorylation. Altogether, these observations suggest a post-translational regulation of TRAF6 by SMO. Indeed, stable knockdown of SMO dramatically reduces the half-life of TRAF6 in both HBL1 and HT cells in the presence of cyclohexamide. Furthermore, overexpression of SMO increases K63-Ub of both TRAF6 and AKT. In contrast, the SMO induced decrease in K48-Ub occurred only for TRAF6 but not for AKT. These data link the SMO-stimulated activation of TRAF6 to the enhancement of AKT signaling and protection of TRAF6 from proteasomal degradation. Mechanistically, we found that SMO, through its C-terminal tail, stabilizes TRAF6 and protects TRAF6 from proteosomal degradation, an effect mediated by ubiquitin-specific protease-8 (USP8). Importantly, this functional link between SMO and TRAF6 is reflected in DLBCL patient samples where high expression of both molecules correlates with poor prognosis. Resistance to DXR is a serious challenge in the treatment of DLBCL, and activated AKT is known to contribute to DXR resistance in multiple cancers including DLBCL. We evaluated whether SMO and TRAF6 support resistance to DXR in DLBCL cell lines. We exposed HT and HBL1 cells as well as their counterparts with stable knockdown of TRAF6 or SMO to DXR for 96hrs. Cell viability after exposure to DXR was determined by an Annexin V and PI staining assay. Silencing SMO or TRAF6 dramatically decreased cell survival after treatment with DXR. In summary, we report that SMO is needed to facilitate and maintain TRAF6-dependent elevated pAKT levels in DLBCL cell lines of germinal (GC) and non-GC subtypes, and that the SMO/TRAF6 axis contributes to DXR resistance in DLBCL. Our study reveals a novel and potential central cell survival signaling mechanism in which SMO stabilizes and protects TRAF6 from proteosomal degradation. Disclosures Lossos: Affimed: Research Funding.

Aberrant activation of Hedgehog signaling has been described in a growing number of cancers, including malignant lymphomas. Here, we report that canonical Hedgehog signaling modulates the transcriptional expression of AKT genes and that AKT1 is a direct transcriptional target of GLI1. We identified two putative binding sites for GLI1 in the AKT1 promoter region and confirmed their functionality using chromatin immunoprecipitation, luciferase reporter, and site-directed mutagenesis assays. Moreover, we provide evidence that GLI1 contributes to the survival of diffuse large B-cell lymphoma (DLBCL) cells and that this effect occurs in part through promotion of the transcription of AKT genes. This finding is of interest as constitutive activation of AKT has been described in DLBCL, but causative factors that explain AKT expression in this lymphoma type are not completely known. In summary, we demonstrated the existence of a novel cross-talk at the transcriptional level between Hedgehog signaling and AKT with biological significance in DLBCL.

Previously, we have demonstrated that inhibition of Hedgehog pathway induces predominantly apoptosis in diffuse large B-cell lymphoma (DLBCL) cell lines of activated B-cell (ABC) type but predominantly cell cycle arrest in those of germinal center (GC). Here, we explored the possibility of overcoming the resistance to apoptosis to SMO inhibitors in five DLBCL cells of GC type using the combination of the SMO inhibitor HhAntag (Genentech Inc) with the BH3 mimetic ABT-737 (Abbott Laboratories). As controls we have used two DLBCL of ABC type (OCI-LY10 and OCI-LY3). Combinatorial treatments were performed with increasing concentrations of the HhAntag with low doses (equal or less than the IC20) of ABT-737. MTS assays were used to detect changes in cell viability and Annexin-V and PARP1 cleavage assays were used to detect apoptosis. Combining low doses of ABT-737 with increasing concentrations of HhAntag in GC DLBCL cell lines resulted in significantly increase of apoptosis in comparison to treatments with the SMO inhibitor alone. We concluded that in GC DLBCL cell lines, in contrast to those of ABC type, functional inhibition of BCL2 family members is usually needed to overcome the resistance to apoptosis to SMO inhibitors. These findings provide a rationale to explore the use of SMO and BCL2 inhibitors as adjuvant therapy for treatment of DLBCL of GC type.

The hedgehog signaling pathway has been shown to play a pathogenic role in diffuse large B-cell lymphoma and anaplastic large cell lymphoma, but has not been assessed in classical Hodgkin lymphoma. Glioma-associated oncogene homologues 1, 2, and 3 are transcriptional effectors of the hedgehog pathway. In this study, we first used real-time quantitative polymerase chain reaction to investigate the expressions of GLI1, GLI2, and GLI3 in 3 classical Hodgkin lymphoma cell lines. GLI1 and GLI2 were variably expressed, but GLI3 was highly expressed in all cell lines. We then used immunohistochemistry to assess glioma-associated oncogene homologues 1, 2, and 3 in 39 classical Hodgkin lymphoma patient samples. Glioma-associated oncogene homologues 1 and 2 were weakly to variably expressed in a subset of classical Hodgkin lymphoma patient samples. In contrast, glioma-associated oncogene homologue 3 showed strong, uniform nuclear expression in virtually all Hodgkin/Reed-Stenberg cells. We then performed an immunohistochemical survey of glioma-associated oncogene homologue 3 expression in 13 cases of nodular lymphocyte predominant Hodgkin lymphoma and 218 non-Hodgkin lymphomas. Most other lymphoma types showed variable or no expression of glioma-associated oncogene homologue 3, with a minor subset of cases of nodular lymphocyte predominant Hodgkin lymphoma, ALK-positive and ALK-negative anaplastic large cell lymphoma, and B-cell lymphoma, unclassifiable with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma showing a glioma-associated oncogene homologue 3 staining pattern indistinguishable from classical Hodgkin lymphoma. Our data provide a rationale to further investigate the biologic significance of glioma-associated oncogene homologue 3 in classical Hodgkin lymphoma biology.

Background: Constitutive activation of NF-κB signaling is a hallmark of DLBCL. Activation of NF-κB is a multifactorial process resulting from oncogenic mutations (CARD11, MYD88…), chromosomal abnormalities, chronic activation of B-cell receptor signaling (BCR) as well as stimuli from the microenvironment. Chronic activation of BCR is not only the result of gene mutations (e.g.CD79B) but also is the result of stimuli generated from the lymphoma microenvironment. We previously found that smoothened (SMO), transducer of hedgehog (Hh) signaling, enhanced NF-κB activation in lymphoma cells, independently of the presence of oncogenic mutations (Changju et al., Blood 2013). Hh ligands are provided by the lymphoma microenvironment (e.g. stromal cells) to activate SMO in the lymphoma cells. Activated SMO, recruits and activates trimeric-G-coupled proteins to activate PKCβ/CARMA1/TRAF6/NEMO axis, followed by assembling of the CARMA1/BCL10/MALT1/TRAF6 complex to SMO resulting in NF-κB activation. Now, we reveal an additional mechanism by which SMO further contributes to the activation of NF-κB in DLBCL. Summary of results: We explored if recombinant Hh ligand can activate NF-κB signaling in the presence of the BTK inhibitor (PCI-32765). Hh stimulation resulted in phosphorylation of PLCg2 (downstream of BTK) and partially rescued the inhibitory effect of the BTK inhibitor on phosphorylation of PLCg2 suggesting a crosstalk between SMO and BCR receptor signaling. We identified that SMO forms a complex with TRAF-6 and PLCg2 using immunoprecipitation and Duolink in situ proximity ligation assays. We found that SMO stabilizes and protects TRAF-6 from proteosomal degradation. SMO-dependent TRAF6 stabilization is mediated by the ubiquitin-specific protease-8 (USP-8) by removing ubiquitin from K48-linked lysine of TRAF6. Conclusions: Collectively, our data reveals multilayer crosstalk between Hh and BCR/NF-κB pathways and provide significant insights into how SMO contributes to chemotolerance and progression of DLBCL. We expect that our results will delineate novel molecular mechanisms involved in the pathobiology of DLBCL that may serve as therapeutic targets. Disclosures Vega: Seatle Genetics: Honoraria; NIH: Research Funding.

GLI1 is a Hedgehog (Hh) related transcription factor originally discovered as an amplified product in gliomas. Inappropriate activation of the GLI1 has been shown in many cancers including diffuse large B cell lymphoma (DLBCL). We previously showed that GLI1 mediated canonical Hh signaling is constitutive active in DLBCL and contributes to cell survival, proliferation and enhances chemotolerance. Although the importance of GLI1 in tumor development is well recognized, the molecular mechanisms controlling the transcriptional activity of GLI1 are poorly characterized. To identify regulatory components that participate in the transcriptional activity of GLI1, we explored GLI1 putative interacting proteins by liquid chromatography tandem mass spectrometry following immunoprecipitation of endogenous GLI1. We detected that the inhibitor of NF-KB kinase, IKKβ, is one of the proteins associated with GLI1 transcription factor. Here we investigate the regulatory role of IKKβ in the transcriptional activity of GLI1. We show that IKKβ regulates the transcriptional activity of GLI1 by phosphorylating GLI1 in C-terminal region and modulating its protein stability. Short stimulation of SUDHL4 and DOHH2 cells with TNF-α (20ng/mL) resulted in increased GLI1 protein levels. Similar results were observed in 293T cells transiently transfected with GLI1 and IKKβ kinase constructs. Moreover, silencing of IKKβ using siRNA and shRNAs led to decreased GLI1 protein levels and its transcriptional activity in DLBCL cell lines with constitutive activation of the NF-KB. Next, we characterized nine probable IKKβ dependent GLI1 phosphorylation sites (S543-S548, S1070, S1071 and S1074 identified by nanospray ion trap mass spectrometry) using mutational and deletions studies. We show that IKKβ phosphorylates GLI1 at Thr1074 and decreases binding between GLI1 and HECT-type E3 ubiquitin ligase (ITCH) resulting in reduced GLI1 polyubiquitination and degradation. Point mutation of Threonine 1074 to Alanine prevents IKKβ-mediated GLI1 phosphorylation and facilitates GLI1-ITCH interaction, polyubiquitination and degradation of GLI1 in the proteasome. Collectively, our data links IKKβ-mediated NF-kB signaling to the transcriptional activity of GLI1 and illustrates a novel cross talk between these two pathways. This is of clinical interest because activation of the NF-kB pathway is frequent in DLBCL and the connection between Hh and NF-kB pathways may open novel therapeutic avenues for DLBCL. Disclosures No relevant conflicts of interest to declare.

Lymphoma microenvironment has a crucial role in lymphoma initiation, progression and drug resistance. Fibroblastic reticular cells (FRCs) participate dynamically in the cytokine microenvironment of the lymph node paracortex as well as the regulation of cell trafficking and access of T-cells into the paracortex. The FRC niche also participates in the cytokine microenvironment of lymphomas providing homing and survival signals including CCL19, CCL21, IL-7, CXCL12 and hedgehog (Hh) ligands. Hh signaling is evolutionary conserved signaling pathway that serves several physiological and development processes mediated by externally secreted Hh ligands. We previously demonstrated that the canonical Hh ligand-PTCH1-SMO-GLI axis is functional and contributes to cell survival, proliferation and enhances chemo tolerance in DLBCL. We further showed that GLI1, Hh signaling transcription factor is aberrantly activated in ∼87% of DLBCL and this activation is in part mediated by sonic Hh ligands secreted by stromal cells. Although the importance of GLI1 in tumor development is well recognized, the molecular mechanisms controlling the transcriptional activity of GLI1 is limited. To identify regulatory components that participate in the transcriptional activity of GLI1, we explored GLI1 putative interacting proteins by liquid chromatography tandem mass spectrometry following immunoprecipitation of endogenous GLI1. We identified the inhibitor of NF-ĸB kinase, IKKβ as a novel GLI1-binding protein. In addition, we found that the kinase activity of IKKβ is critical to recruit and phosphorylate GLI1 in the C-terminal fragment. We further showed that in DLBCL cells stimulated with TNFα, IKKβ phosphorylates GLI1 and restricts the binding between GLI1 with HECT-type E3 ubiquitin ligase (ITCH) resulting in GLI1 stability and increased oncogenic potential. Inhibition of IKKβ-mediated GLI1 phosphorylation restored the binding between GLI1 and ITCH and promoted ubiquitination and degradation of GLI1. Collectively, these results indicate that IKKβ regulates the transcriptional activity of GLI1 by phosphorylating GLI1 and modulating the binding between GLI1 and ITCH. In DLBCL, canonical NF-κB and GLI1 mediated Hh signaling pathways are strongly responsive with tumor microenvironment; our study will help to understand the cross talk between these two pathways which could represents a novel therapeutic approach for DLBCL. Citation Format: Nitin K. Agarwal, Kranthi Kunkalla, Francisco Vega. The inhibitor of NF-ĸB kinase, IKKβ, regulates the stability of GLI1 transcription factor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3605. doi:10.1158/1538-7445.AM2014-3605

Hedgehog (Hh) signaling plays an important role in the pathobiology of B-cell malignancies including diffuse large B cell lymphoma (DLBCL). The GLI family members that include GLI1, GLI2 and GLI3 are Hh signaling transcription factors. While most research has focused on smoothened (SMO), Hh transducer receptor subunit, many lines of evidence indicate that other signaling pathways can activate GLI transcription factors. GLI1 is a zinc finger transcription factor and established oncogene. GLI1 expression increases in response to Hh signaling activation and potentiates the transcriptional output of Hh signaling. We previously demonstrated that the canonical Hh ligand-PTCH1-SMO-GLI axis is functional and plays an important role in cell proliferation, survival and chemotolerance in DLBCL (Leukemia 2010 and Oncogene 2011). Moreover, using stable GLI1 knockdown DLBCL cell lines we found that GLI1 contributes to cell survival and proliferation of DLBCL (J Biol Chem 2013). However, our understanding of the mechanisms controlling the transcriptional activity of GLI1 is limited. To identify regulatory components that participate in the transcriptional regulation of GLI1, we explored GLI1 putative interacting proteins by liquid chromatography tandem mass spectrometry following immuno-precipitation (IP) of endogenous GLI1. We found that the NF-κB kinase, IKKβ, is one of the proteins associated with GLI1. This finding was of interest as we found a positive correlation between NF-kB activity and Hh signaling in DLBCL (Blood 2013). To validate the functional interaction between endogenous GLI1 with IKKβ in-vivo, lysates of 293T cells were IP with GLI1 or control antibody and subjected to immunoblot analysis with antibodies recognizing IKKβ subunit and SuFu (known partner of GLI1) respectively. IKKβ and SuFu were co-immunoprecipitated with GLI1. To confirm these findings, 293T cells were transiently co-transfected with plasmids carrying GLI1 and FLAG-tagged IKKα and IKKβ wild-type or kinase dead (K44A) forms. Both forms (IKKα and IKKβ) were detected in the immunoprecipitated GLI1 protein complex, however only transfection of IKKβ resulted in abundance of GLI1 protein suggesting a functional role of IKKβ kinase in the regulation of GLI1 protein levels. The association (and nuclear co-localization) between IKKβ and GLI1 was confirmed in DLBCL cell lines and 293T cells by immunofluorescence studies. To investigate the role of IKKβ in the transcriptional activity of GLI1, we knocked down IKKβ gene in HBL1 (cells with CD79B mutation) cells using two independent IKKβ shRNAs. Silencing IKKβ resulted in a decrease of IKKβ expression associated with decreased levels of GLI1 and GLI1 downstrean targets (PTCH1, CCND1 and BCL2). To better understand the functional role of IKKβ kinase activity in the transcriptional regulation of GLI1 target genes, we explore the IKKβ dependent GLI1 phosphorylation sites. We co-transfected full length GLI1 and IKKβ plasmids in 293T cells and IKKβ-GLI1 complex were purified using anti-GLI1 antibody. Peptides resulting from digestion of GLI1 with several different proteases were analyzed by nanospray ion trap mass spectrometry. Seven phosphorylation sites in GLI1 C-terminal residues were identified during analysis of trypsin-digested GLI1 by mass spectrometry. These data suggest that IKKβ phosphorylates GLI1 and plays an important role in the regulation of GLI1 transcriptional activity. Because activation of NF-κB is a frequent event in DLBCL, our study will help to understand the cross talk between NF-κB and Hh signaling pathways and will open potential new avenues for targeted therapy in DLBCL. Disclosures: Vega: NIH: Research Funding; Leukemia & Lymphoma Society: Research Funding.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Aberrant activation of Hedgehog (Hh) and NF-κB pathways is ubiquitously observed and known to mediate tumor growth, survival and chemo-resistance in DLBCL. Previously, we found that activation of Hh signaling is positively correlated with NF-κB pathway in DLBCL tumors and that modulating the activity of smoothened (SMO), the signal transducer subunit of Hh pathway, but not GLI1, transcriptional factor of Hh signaling, resulted in modulation of NF-kB pathway activation. Hereby, we investigate the molecular mechanisms that link SMO to NF-kB pathway. To elucidate whether there was interaction between G protein superfamily and SMO, we immunoprecipitated representative members of the Gα subfamilies (Gαi, Gα16, Gαq, Gαs and Gα12), and found that SMO was associated with Gαi, Gαq, and Gα12 in two DLBCL cell lines. Stimulation of SMO with Shh recombinant increased the recruitment of Gαi and Gα12 to SMO and also increased the GTPase activity of Gαi and Gα12. We also found that the activation of SMO with Shh in DLBCL cell lines was associated with increased total activity of PKC, phosphorylation of PKCβ1 and -2, and recruitment of CARMA1-MALT1-BCL10-TRAF6 to SMO receptor complex supporting that G proteins are involved in the transmission of the signal between SMO and NF-κB. Opposite results were seen when SMO was inhibited or silenced. As polyubiquitination of TRAF6 and NEMO (IKKγ) at lysine 63 (K63) are also important events in propagating NF-κB signaling, we examined the effect of SMO overexpression on K63-linked polyubiquitination of TRAF6 and NEMO. Transient overexpressing SMO resulted in increased polyubiquitination of TRAF6 and NEMO, supporting activation of both proteins by SMO. Moreover, functional inhibition of SMO (cyclopamine-KAAD) enhances the cytotoxic effect of NF-κB inhibitor (BAY-11-7082). Altogether, our study reveal a non-canonical Hh signaling pathway, in which SMO activates trimeric G proteins and CARMA1-associated signaling complex, leading to NF-κB activation. This signaling cascade contributes to the survival of DLBCL, and may serve as potential targets for combination therapies in DLBCL. Citation Format: Changju Qu, Yadong Liu, Kranthi Kunkalla, Rajesh Singh, Marzenna Blonska, Nitin Agarwal, Xin Lin, Francisco Vega. Trimeric G protein-CARMA1 axis links smoothened to NF-κB activation in diffuse large B-cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4114. doi:10.1158/1538-7445.AM2013-4114

Abstract 2399 Activation of the Hedgehog (Hh)/glioma-associated oncogene (GLI) pathway has been found in a growing number of malignancies. We have provided evidence that canonical Hh signaling is required for cell survival and proliferation of DLBCL cell lines. To confirm the pathogenic role of GLI1 in DLBCL, we established GLI1 knock down DLBCL cell lines (OCI-Ly19, HBL-1 and BJAB) using a lentiviral shRNA system and performed cell viability and apoptosis assays. Cell viability assays demonstrated that GLI1 knockdown DLBCL cells experienced a statistically significantly decrease in the number of viable cells in comparison with control cells harboring scramble shRNA. To examine whether decreases number of cell viability in GLI1 knock down cells were due to apoptosis, we performed annexin V and PI assays. We observed marked increase of apoptosis in GLI1 knock down DLBCL cells versus controls (2.5 fold increase for OCI-Ly10, and 5 fold for HBL1 and BJAB). To investigate the mechanism by which GLI1 regulates tumorigenesis and cell survival, we searched for whole genome GLI1-target genes in DLBCL cells using CHIP sequencing technique and identified AKT genes as potential targets of GLI1. Using pharmacological and silencing approaches, we observed that Hh signaling modulates the expression of AKT genes in DLBCL cells. We further identified two putative binding sites for GLI1 in the AKT1 promoter region and confirmed their functionality using chromatin immunoprecipitation, luciferase reporter and site-directed mutagenesis assays. To investigate whether there is any correlation between AKT1 and GLI1 mRNA expression in human DLBCL tumors, we performed quantitative real-time PCR analyses in 17 frozen DLBCL specimens including apharesis samples from pleural effusions. The real time PCR analysis revealed a strong Spearmen correlation coefficient (R2=0.9) between GLI1 and AKT1 mRNA expression. In summary, we provide evidence of the role of GLI1 in the pathobiology of DLBCL and demonstrated a cross talk, at the transcriptional level, between Hh signaling and AKT in DLBCL. A link between these 2 pathways at the trasncriptional level was not previoulsy documented. This finding is of clinical interest as AKT has a key role in lymphoma cell survival and constitutive activation of AKT has been described in DLBCL. Disclosures: No relevant conflicts of interest to declare.

Abstract 1298 Aberrant activation of hedgehog (Hh) and NF-kB pathways contribute to tumor cell growth, survival and chemotolerance in diffuse large B-cell lymphoma (DLBCL). Previously, we documented a functional crosstalk between hedgehog (Hh) and NF-kB pathways that contribute to tumor cell growth and survival in diffuse large B-cell lymphoma (DLBCL). However, the molecular mechanisms that link Hh with NF-kB pathway have not been defined. Based on that smoothened (SMO) has been associated with heterotrimeric G protein members of the Gα i family and established as a GPCRs-like protein, we hypothesize that GPCRs-related mechanisms such as the protein kinase C (PKC)-CARMA1-BCL10/MALT1/TRAF6 axis may contribute to SMO-dependent activation of NF-kB. First, we confirmed that SMO contributes to the activity of total PKC in 293T and DLBCL cells. Transiently or stably silencing of SMO resulted in decreased total PKC activity in comparison with the controls, while overexpression of SMO resulted in increased activity of PKC. Cyclopamine-KAAD or recombinant Shh N-terminal peptide resulted in decrease or increase of the total activity of PKC, respectively. As PKC isoforms β-1 and β-2 are the major isoforms expressed in B-lymphocytes that mediates NF-kB activation induced by activation of the B-cell receptor, we assessed the activation status of these two isoforms in response to changes of SMO activity. Inhibiting SMO with cyclopamine-KAAD or silencing SMO by siRNA decreased the phosphorylation status of PKCβ-1 and −2. In contrast, activating SMO with Shh N-terminal peptide increased the phosphorylation of PKCβ-1 and −2. Next, we assessed if SMO can modulated the activity of CARMA1. CARMA1 is a scaffold protein that serves to integrate the upstream signal of PKCs with downstream effectors in hematopoietic cells. We found that cyclopamine-KAAD or silencing SMO by siRNA decreased the phosphorylation status (inactivation) of CARMA1 and that activation of SMO with Shh N-terminal peptide increased the phosphorylation (activation) of CARMA 1. Because the polyubiquitination of TRAF6 and NEMO (IKKg) at lysine 63 (K63) are important events in propagating NF-kB signaling we examined the effect of overexpressing SMO on K63 polyubiquitination of TRAF6 and NEMO. Overexpression of SMO resulted in increased polyubiquitination of TRAF6 and NEMO at K63 supporting NF-kB pathway activation. TRAF6 is involved in activation of TAK1 and the IKK complex, resulting in translocation of NF-kB to the nucleus and activation of NF-kB. Silencing SMO by shRNA decreased total levels of TRAF6 that was associated with faster proteosomal degradation of TRAF6 in 293T cells. Inhibition of SMO with cyclopamine-KAAD also decreased the total levels of TRAF6 in DLBCL cells. In addition, overexpression of SMO (including a constitutively active mutated SMO) in 293T cells caused increase of TRAF6 expression by decreased polyubiquitination of TRAF6 at lysine 48 (K48) that targets TRAF6 for proteosomal degradation. In summary, altogether, these findings support that at least one mechanism by which SMO contributes to modulate activation of NF-kB is through the activation of the axis PKCβ/CARMA1/TRAF6/NEMO and stabilization and blocking degradation of TRAF6. Disclosures: No relevant conflicts of interest to declare.

Abstract 3483 Activated Hedgehog (HH) signaling was identified in our laboratory to contribute to cell survival, proliferation and chemotolerance of diffuse large B-cell lymphoma (DLBCL) (Leukemia 2010;24:1025 & Oncogene 2011, in press). The HH receptor complex is integrated by two main proteins, smoothened (SMO) and patched-1 (PTCH1). SMO is a seven-transmembrane G protein-coupled receptor that transduces HH signal to the cytoplasm and has glioma-associated oncogene homologue (GLI) proteins as major signaling transcription effectors. PTCH1 is a 12 transmembrane protein that inhibits SMO in the absence of HH ligands. Here, we investigated potential cross talk between SMO with the activation status of PI3K/AKT and NF-kB, two relevant oncogenic pathways in DLBCL. Using a small interfering (si)RNA approach and DLBCL cell lines of germinal center (GC) and activated B-cell (ABC) type we found that the expression levels of SMO modulate the activation status of AKT and canonical NF-KB pathways in DLBCL cells of GC type and mainly AKT in those of ABC type. In DLBCL cells of GC cell type, silencing SMO resulted in decrease of the phosphorylation levels of ser473p-AKT and ser536p-P65 and silencing PTCH1 resulted in increase of the phosphorylation levels of both proteins. The same silencing experimental approach in DLBCL cells of ABC type resulted in similar modulation in the activation status of the AKT but not, or to less extent, in the activation of NF-kB pathway. The modulation of the activation status of the NF-KB pathway was also confirmed using protein nuclear extracts and DNA binding ELISA assays. In cells of both DLBCL subtypes, silencing of the SMO transcriptional effector GLI1 showed no changes in the activation status of both pathways. The modulation in the activation status of AKT and NF-KB was also detected using SMO inhibitors, cyclopamine-KAAD and HhAntag (Genentech Inc) or activators, purmorphamine and recombinant HH protein. Combinatorial treatments with increasing concentrations of SMO inhibitors (cyclopamine and HhAntag [1.6, 3.2 and 4.8 μM]) with minimal lethal doses of Ly294002 (PI3K inhibitor) or BAY-11 (NF-KB inhibitor) were also performed. Using cell viability, and apoptosis (Annexin-V) assays, we found that combined treatments of PI3K or NF-KB inhibitors with a SMO inhibitor resulted in an additive/synergistic decrease of cell viability and increase of apoptosis in comparison to the treatments with SMO inhibitors alone. Taken together, our data shed novel light on the contribution of SMO on the activation of PI3K/AKT and NF-kB pathways in DLBCL. Our data also provide a rationale to use SMO inhibitors in combination with inhibitors of other oncogenic pathways such as PI3K/AKT and/or NF-KB for the treatment of patients with DLBCL. Disclosures: No relevant conflicts of interest to declare.

Abstract 2479 Generally in cancers, high expression of anti-apoptotic proteins induced by oncogenic signaling pathways is a major cause for treatment failure. Previously, we have demonstrated that Hedgehog (Hh) signaling is activated and functional in diffuse large B-cell lymphoma (DLBCL) and that its inhibition, using the Smoothened (SMO) inhibitor cyclopamine-KAAD, induces apoptosis in cell lines of DLBCL of activated B-cell (ABC) subtype and cell cycle arrest (G1-phase) in those of germinal center (GC) subtype. Induction of apoptosis in ABC-DLBCL was due to decrease of BCL2 expression (protein and mRNA levels), a direct target of Hh signaling (Leukemia 2010;24:1025–36). We also found that BCL2 is upregulated in the tumor cells in the presence of stroma and that Hh inhibition abrogates this upregulation (Oncogene 2011, in press). Here, we explored the possibility of overcoming resistance to apoptosis-induced by Hh inhibition in GC-DLBCL using a combination of the SMO inhibitor “Hh antagonist” (Genentech Inc) with the BH3 mimetic ABT-737 (Abbot laboratories) in a panel of 5 GC type DLBCL cell lines, 4 of them with the t(14:18)(q32;q21) (DOHH2, SUDHL4, Toledo and OCI-LY19) and one without the translocation (HT). ABT-737 is a functional inhibitor of three major anti-apoptotic proteins of the BCL2 family (BCL2, BCL-xL and BCL-W). Combinatorial treatments were performed with increasing concentrations of the Hh antagonist (2.5, 5.0 and 7.5 μM) and with variable but minimal lethal doses of ABT-737 (inducing cell death less than 20%). Using cell viability, apoptosis (annexin-V) and cell cycle based assays, we found that the combined treatments resulted in a additive/synergistic decrease of cell viability and increase of apoptosis in comparison to the treatment with the Hh antagonist alone. In contrast to ABC type DLBCL cell lines, in DLBCL cells with t(14;18), Hh antagonist did not result in decrease expression of BCL2, BCL-xL and BCL-W indicating that the additive effect of ABT-737 was due to their functional inhibition and not by suppressing expression levels of these proteins. On the other hand, treatments with the Hh antagonist resulted in a concentration dependent decrease of BCL2 protein levels in HT cells. In summary, our data confirm that in the presence the t(14;18), inhibition of SMO does not result in decrease of BCL2 expression. In these cases, concomitant inhibition of BCL2 function and SMO is needed to induce apoptosis. Combined inhibition of Hh signaling and BCL2 function may have a therapeutic value for lymphomas with the t(14;18)(q32;q21). Disclosures: No relevant conflicts of interest to declare.

Abstract 972 Successful treatment of diffuse large B-cell lymphoma (DLBCL) is frequently hindered by development of resistance to conventional chemotherapy resulting in disease relapse and high mortality. High expression of anti-apoptotic and/or drug transporter proteins induced by oncogenic signaling pathways has been implicated in the development of chemoresistance in cancer. However, the cellular mechanisms responsible for drug resistance and treatment failure in DLBCL are poorly understood and their elucidation could lead to development of new therapeutic approaches. Activated Hedgehog (HH) signaling was identified in our laboratory to contribute to DLBCL cell survival and proliferation. We also found that inhibiting HH signaling resulted in decrease of BCL-2 protein and mRNA levels in ABC DLBCL cells but not in GC DLBCL cells, and that the resistance to apoptosis to HH inhibition observed in GC DLBCL cells can be overcome using the BCL2 inhibitor YC137. (Leukemia 2010;24:1025). We also found that the drug transporter protein ABCG2 is a potential prognostic factor in DLBCL given that its expression (as detected by immunohistochemistry) inversely correlated with overall survival and failure-free survival (Mod Pathol 2009;22:1312). Here, we investigate potential mechanistic connections between HH signaling and chemoresistance in DLBCL. Using chromatin immunoprecipitation (ChIP), site directed mutagenesis and luciferase reporter based assays, we confirmed the presence of a GLI-1 transcription factor-binding site in the ABCG2 gene promoter and we established ABCG2 gene as a direct downstream target of HH signaling. We also characterized the baseline expression and functionality of ABCG2 in DLBCL cell lines and explored the role of stroma in modulating its expression levels as well as the expression of other drug transporters (MDR1 and ABCC1) and the levels of the anti-apoptotic proteins, BCL-2, BCL2A1 and BCL-xL. Co-culturing DLBCL cell lines (BJAB and DOHH2) with human bone marrow stromal cells (HS5) resulted in decreased chemosensitivity to doxorubicin and methotrexate that was associated to marked increased expression levels of the drug transporters ABCG2, MDR1 and ABCC1 as well as of the expression levels of BCL2, BCL2A1 and BCL-xL. Our results also showed that pharmacologic inhibition of the activity of ABCG2, using fumitremorgin C and glefanine, significantly increased the chemosensitivity of BJAB and DOHH2 cells in the presence of stromal cells. However, this effect was not seen in the absence of stromal cells. Collectively, our data confirm that the stromal cells plays a role in inducing chemoresistance in DLBCL and that this effect is in part mediated by inducing high expression of drug protein transporters and antiapoptotic proteins. Inhibition of HH signaling as well as inhibition of drug transporters abrogates the stroma-induced chemoresistance suggesting that targeting these molecules may have a therapeutic value for the treatment of DLBCL. Disclosures: No relevant conflicts of interest to declare.