Yadong Liu

Trimeric G protein-CARMA1 axis links smoothened, the hedgehog receptor transducer, to NF-κB activation in diffuse large B-cell lymphoma

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.

Transcriptional regulation of serine/threonine protein kinase (AKT) genes by glioma-associated oncogene homolog 1.

Background: Little is known regarding the transcriptional regulation of AKT. Results: GLI1 contributes to the survival of DLBC cells by promoting transcription of AKT genes. Conclusion: AKT1 is a novel direct downstream target of the Hedgehog transcriptional factor GLI1. Significance: Identifying target genes of GLI1 provides insights into the contribution of Hedgehog signaling in the pathobiology of malignant tumors. 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.

Functional inhibition of BCL2 is needed to increase the susceptibility to apoptosis to SMO inhibitors in diffuse large B-cell lymphoma of germinal center subtype

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.

Defining causative factors contributing in the activation of hedgehog signaling in diffuse large B-cell lymphoma

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.

Abstract 4114: Trimeric G protein-CARMA1 axis links smoothened to NF-κB activation in diffuse large B-cell lymphoma.

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