Novel Role of Raft-Associated Smoothened (SMO) in AKT Signal Regulation in Diffuse Large B Cell Lymphoma

Constitutive PI3K/AKT activation is relevant to multiple aspects of tumor growth and survival in numerous cancers including diffuse large B cell lymphoma (DLBCL). For example, PTEN loss is one of the mechanisms leading to constitutive PI3K/AKT activation in a subset of DLBCL. Smoothened (SMO) is a seven transmembrane spanning and Frizzled-class G-protein coupled receptor that functions as a Hedgehog (Hh) signal transducer. SMO is overexpressed in DLBCL cell lines and tumors. While canonical Hh signaling culminates in the activation of GLI transcription factors and is best understood in the context of cilia, "noncanonical" Hh signaling does not involve GLI transcriptional activity and remains less well characterized. Here, we found that SMO is not only an integral component of lipid rafts but also plays an unexpected central role in the organization of raft microdomains (specialized glycolipid-enriched microdomains known to serve as a highly dynamic signaling platform for cell surface receptors and signaling proteins) and in the sorting of lipid raft-associated proteins. To address whether SMO co-localizes to lipid rafts in the context of DLBCL, HBL1 cells were engineered to stably overexpress a C-terminal SMO-mCherry-fusion protein and were incubated with FITC-Cholera toxin. Within 15 min, a large fraction of SMO-mCherry was co-localized with FITC-Cholera toxin in lipid raft clusters. We also performed immunostaining of endogenous SMO and CD59 in HBL1 cells. CD59 is a glycol-phosphatidyl inositol-anchored lipid raft protein. Immunostaining of live HBL1 cells revealed the colocalization of SMO and CD59 on the extracellular surface of HBL1 cells. No colocalization was found between SMO and the transferrin receptor (TF-R), a plasma membrane protein not associated with lipid rafts. Finally, immunoblotting analysis of detergent-free fractionation further corroborated SMO as a bona fide component of lipid rafts. We then tried to explore the functional relevance of SMO association to the lipid compartment. First, we examined the effects of SMO stable knockdown in DLBCL. We observed a marked decrease in the expression of raft-associated receptors and signaling proteins (e.g. IGFR1, EGFR, IRS1) while caveolin and flotillin, two functional components of lipid rafts, remained unaltered in their levels and distribution. Although only a portion of the overall pool of AKT and pAKT were localized to lipid rafts, SMO loss significantly reduced raft-localized total AKT and pAKT (T308/S473). Consistent with the well-established role of AKT in cell survival, SMO silencing also resulted in reduced DLBCL cell viability. To evaluate whether SMO regulated IGF1R expression at the transcriptional level, we analyzed the mRNA levels of GLI1, the immediate transcriptional target of canonical SMO signaling, and IGF1R in SMO-/- MEFs. Even if GLI1 transcript levels were reduced, consistent with the established mode of GLI1 regulation by SMO through the GLI2 transcription factor, IGF1R mRNA levels remained unchanged. Using GLI1-/- MEFs, we could confirm that the effect of SMO on surface receptors was independent of canonical Hh signaling. We also determined that the rate of IGFR degradation was comparable in the presence and absence of SMO. Total IGF1R receptor levels at steady state represent the balance of total protein synthesis and the fraction of existing receptors that is directed towards lysosomal degradation. To test whether the absence of SMO results in increased degradation, we inhibited lysosomal function with chloroquine (CHLQ). CHLQ had a far more pronounced impact on restoring IGF1R protein levels in SMO deficient cells than it did in control cells. These last results suggested that in the absence of SMO, a larger fraction of this receptor is directed towards lysosomal degradation, and thus resulting in lower steady state levels. In summary, our data confirm that SMO is localized to raft microdomains in lymphoma cells and play a novel role in the sorting of surface proteins for degradation or recycling. In particular, SMO increases the levels of raft resident receptors and facilitates the assembly of an AKT activating machinery to enhance lymphoma cell survival. This novel role of SMO in signal regulation at the level of lipid rafts has broad implications for cancer biology. Disclosures Vega: National Cancer Institute, national Institutes of Health: Other: Grant Funding-R01CA222918

N-terminal PAX8 polyclonal antibody shows cross-reactivity with N-terminal region of PAX5 and is responsible for reports of PAX8 positivity in malignant lymphomas

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

The Inhibitor of NF-KB Kinase, IKKβ Regulates the Transcriptional Activity of GLI1 By Blocking Its Proteasomal Degradation

Abstract 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

ABCG2 is a Direct Transcriptional Target of Hedgehog Signaling and Involved in Stroma-Induced Drug Tolerance in Diffuse Large B-Cell Lymphoma

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. Previously, our studies showed high expression of ATP-binding cassette drug transporter ABCG2 in DLBCL correlated inversely with disease-free 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 to 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 (FTC) 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

CRISPR/Cas9 Generation of Npm1-Alk in Transplantable Murine Hematopoietic Stem Cells Accurately Models ALK-Positive Lymphoma in Recipients

Abstract 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

Jun-regulated genes promote interaction of diffuse large B-cell lymphoma with the microenvironment

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

CRISPR genome editing of murine hematopoietic stem cells to create Npm1-Alk causes ALK+ lymphoma after transplantation

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

Smoothened (SMO) regulates insulin-like growth factor 1 receptor (IGF1R) levels and protein kinase B (AKT) localization and signaling

The oncoprotein Smoothened (SMO), a Frizzled-class-G-protein-coupled receptor, is the central transducer of hedgehog (Hh) signaling. While canonical SMO signaling is best understood in the context of cilia, evidence suggests that SMO has other functions in cancer biology that are unrelated to canonical Hh signaling. Herein, we provided evidence that elevated levels of human SMO show a strong correlation with elevated levels of insulin-like growth factor 1 receptor (IGF1R) and reduced survival in diffuse large B-cell lymphoma (DLBCL). As an integral component of raft microdomains, SMO plays a fundamental role in maintaining the levels of IGF1R in lymphoma and breast cancer cells as well IGF1R-associated activation of protein kinase B (AKT). Silencing of SMO increases lysosomal degradation and favors a localization of IGF1R to late endosomal compartments instead of early endosomal compartments from which much of the receptor would normally recycle. In addition, loss of SMO interferes with the lipid raft localization and retention of the remaining IGF1R and AKT, thereby disrupting the primary signaling context for IGF1R/AKT. This activity of SMO is independent of its canonical signaling and represents a novel and clinically relevant contribution to signaling by the highly oncogenic IGF1R/AKT signaling axis

Detection of ABCC1 expression in classical Hodgkin lymphoma is associated with increased risk of treatment failure using standard chemotherapy protocols

BACKGROUND: The mechanisms responsible for chemoresistance in patients with refractory classical Hodgkin lymphoma (CHL) are unknown. ATP-binding cassette (ABC) transporters confer multidrug resistance in various cancers and ABCC1 overexpression has been shown to contribute to drug resistance in the CHL cell line, KMH2. FINDINGS: We analyzed for expression of five ABC transporters ABCB1, ABCC1, ABCC2, ABCC3 and ABCG2 using immunohistochemistry in 103 pre-treatment tumor specimens obtained from patients with CHL. All patients received first-line standard chemotherapy with doxorubicin (Adriamycin®), bleomycin, vinblastine, and dacarbazine (ABVD) or equivalent regimens. ABCC1 was expressed in Hodgkin and Reed-Sternberg (HRS) cells in 16 of 82 cases (19.5%) and ABCG2 was expressed by HRS cells in 25 of 77 cases (32.5%). All tumors were negative for ABCB1, ABCC2 and ABCC3. ABCC1 expression was associated with refractory disease (p = 0.01) and was marginally associated with poorer failure-free survival (p = 0.06). Multivariate analysis after adjusting for hemoglobin and albumin levels and age showed that patients with CHL with HRS cells positive for ABCC1 had a higher risk of not responding to treatment (HR = 2.84, 95%, CI: 1.12-7.19 p = 0.028). CONCLUSIONS: Expression of ABCC1 by HRS cells in CHL patients predicts a higher risk of treatment failure and is marginally associated with poorer failure-free survival using standard frontline chemotherapy regimens