A novel mechanism of regulation of the oncogenic transcription factor GLI3 by toll-like receptor signaling.

The transcription factor GLI3 is a member of the GLI family and has been shown to be regulated by canonical hedgehog (HH) signaling through smoothened (SMO). Little is known about SMO-independent regulation of GLI3. Here, we identify TLR signaling as a novel pathway regulating GLI3 expression. We show that GLI3 expression is induced by LPS/TLR4 in human monocyte cell lines and peripheral blood CD14+ cells. Further analysis identified TRIF, but not MyD88, signaling as the adapter used by TLR4 to regulate GLI3. Using pharmacological and genetic tools, we identified IRF3 as the transcription factor regulating GLI3 downstream of TRIF. Furthermore, using additional TLR ligands that signal through TRIF such as the TLR4 ligand, MPLA and the TLR3 ligand, Poly(I:C), we confirm the role of TRIF-IRF3 in the regulation of GLI3. We found that IRF3 directly binds to the GLI3 promoter region and this binding was increased upon stimulation of TRIF-IRF3 with Poly(I:C). Furthermore, using Irf3 -/- MEFs, we found that Poly(I:C) stimulation no longer induced GLI3 expression. Finally, using macrophages from mice lacking Gli3 expression in myeloid cells (M-Gli3-/- ), we found that in the absence of Gli3, LPS stimulated macrophages secrete less CCL2 and TNF-α compared with macrophages from wild-type (WT) mice. Taken together, these results identify a novel TLR-TRIF-IRF3 pathway that regulates the expression of GLI3 that regulates inflammatory cytokines and expands our understanding of the non-canonical signaling pathways involved in the regulation of GLI transcription factors.

Epigenetic targeting of Waldenström macroglobulinemia cells with BET inhibitors synergizes with BCL2 or histone deacetylase inhibition.

Aim: Waldenström macroglobulinemia (WM) is a low-grade B-cell lymphoma characterized by overproduction of monoclonal IgM. To date, there are no therapies that provide a cure for WM patients, and therefore, it is important to explore new therapies. Little is known about the efficiency of epigenetic targeting in WM. Materials & methods: WM cells were treated with BET inhibitors (JQ1 and I-BET-762) and venetoclax, panobinostat or ibrutinib. Results: BET inhibition reduces growth of WM cells, with little effect on survival. This finding was enhanced by combination therapy, with panobinostat (LBH589) showing the highest synergy. Conclusion: Our studies identify BET inhibitors as effective therapy for WM, and these inhibitors can be enhanced in combination with BCL2 or histone deacetylase inhibition.

GLI3: a mediator of genetic diseases, development and cancer.

The transcription factor GLI3 is a member of the Hedgehog (Hh/HH) signaling pathway that can exist as a full length (Gli3-FL/GLI3-FL) or repressor (Gli3-R/GLI3-R) form. In response to HH activation, GLI3-FL regulates HH genes by targeting the GLI1 promoter. In the absence of HH signaling, GLI3 is phosphorylated leading to its partial degradation and the generation of GLI3-R which represses HH functions. GLI3 is also involved in tissue development, immune cell development and cancer. The absence of Gli3 in mice impaired brain and lung development and GLI3 mutations in humans are the cause of Greig cephalopolysyndactyly (GCPS) and Pallister Hall syndromes (PHS). In the immune system GLI3 regulates B, T and NK-cells and may be involved in LPS-TLR4 signaling. In addition, GLI3 was found to be upregulated in multiple cancers and was found to positively regulate cancerous behavior such as anchorage-independent growth, angiogenesis, proliferation and migration with the exception in acute myeloid leukemia (AML) and medulloblastoma where GLI plays an anti-cancerous role. Finally, GLI3 is a target of microRNA. Here, we will review the biological significance of GLI3 and discuss gaps in our understanding of this molecule. Video Abstract.

Targeting IL-6 receptor reduces IgM levels and tumor growth in Waldenström macroglobulinemia.

The tumor microenvironment (TME) plays an important role in cancer cell biology and is implicated in resistance to therapy. In Waldenström macroglobulinemia (WM), a subtype of Non-Hodgkin lymphoma, the TME modulates WM biology by secreting cytokines that promote the malignant phenotype. In previous work, we have shown that TME-IL-6 promotes WM cell growth and IgM secretion in WM. Tocilizumab/Actemra is an anti-IL-6R antibody, which can competitively block IL-6 binding to the IL-6R. We investigated the efficacy of Tocilizumab in a preclinical mouse model of WM that considers the role of the TME in disease biology. Hairless SCID mice were subcutaneously implanted with BCWM.1 or RPCI-WM1 and bone marrow stromal cells. Groups of mice were treated with Tocilizumab or control antibody three times/week for 5 weeks and the effect on tumor burden and disease biology were evaluated. Although Tocilizumab had no effect on mice survival, there was a significant reduction in tumor growth rate in mice injected with RPCI-WM1 cells treated with Tocilizumab. In mice injected with BCWM.1 cells, there was a significant reduction in human IgM secretion in mice sera with Tocilizumab treatment. There was no significant change in mice weight suggesting Tocilizumab induced no toxicities to the mice. Taken together, our data found that administration of Tocilizumab to tumor bearing mice, results in a significant reduction in tumor volume and IgM secretion. Therefore, the evaluation of the role of Tocilizumab in WM patients may provide therapeutic efficacy by reducing IgM production and slowing the rate of tumor growth.

Gadolinium borate and iron oxide bioconjugates: Nanocomposites of next generation with multifunctional applications.

The systematic investigations concerning the bioconjugation of GdBO3-Fe3O4 nanocomposite and their in vitro biocompatibility with cancer cell lines are reported. The nanocomposites were prepared hydrothermally from magnetite (Fe3O4), borax or boric acid and a Gd3+ salt. Bioconjugation processes were performed with citric acid and fluorescein isothiocyanate-doped silica, followed by the treatment with folic acid. Overall, the procedure involved "bare or PEGylated Fe3O4 as the magnetic core" and "vaterite- or triclinic-type of GdBO3 as the surface borate layer" for comparative evaluation of the results. The successful vectorization of the nanocomposite particles was demonstrated by quantitative and qualitative analytical data. All bioconjugates displayed soft ferromagnetic properties and negative zeta potential values that are appropriate for biological applications. The 10B and 157Gd contents were ca. 1014 atom/µg making them promising agents for BNCT, GdNCT and the combined GdBNCT. The Gd/Fe molar ratios (0.27-0.63) provided the capability for T1- or dual (T1 + T2) magnetic resonance imaging (MRI). In vitro studies were conducted to investigate the efficiency of targeted FA-conjugated versus non-FA conjugated nanoformulations on Mia-Pa-Ca-2, HeLa and A549 cells. Fluorescence microscopy and flow cytometry data unveiled the essential role of the zeta potential competing with folate targeting in the uptake mechanism. The bioconjugated nanoplatforms of GdBO3-Fe3O4 composite, introduced herein, proved to have potential features of next generation agents for magnetically targeted therapy, fluorescence imaging, magnetic resonance imaging/diagnosis and Neutron Capture Therapy.

Novel Molecular Mechanism of Regulation of CD40 Ligand by the Transcription Factor GLI2.

The interaction between tumor cells and their surrounding microenvironment is essential for the growth and persistence of cancer cells. This interaction is mediated, in part, by cytokines. Although the role of cytokines in normal and malignant cell biology is well established, many of the molecular mechanisms regulating their expression remain elusive. In this article, we provide evidence of a novel pathway controlling the transcriptional activation of CD40L in bone marrow-derived stromal cells. Using a PCR-based screening of cytokines known to play a role in the biology of bone marrow malignancies, we identified CD40L as a novel GLI2 target gene in stromal cells. CD40L plays an important role in malignant B cell biology, and we found increased Erk phosphorylation and cell growth in malignant B cells cocultured with CD40L-expressing stromal cells. Further analysis indicated that GLI2 overexpression induced increased CD40L expression, and, conversely, GLI2 knockdown reduced CD40L expression. Using luciferase and chromatin immunoprecipitation assays, we demonstrate that GLI2 directly binds and regulates the activity of the CD40L promoter. We found that the CCR3-PI3K-AKT signaling modulates the GLI2-CD40L axis, and GLI2 is required for CCR3-PI3K-AKT-mediated regulation of the CD40L promoter. Finally, coculture of malignant B cells with cells stably expressing human CD40L results in increased Erk phosphorylation and increased malignant B cell growth, indicating that CD40L in the tumor microenvironment promotes malignant B cell activation. Therefore, our studies identify a novel molecular mechanism of regulation of CD40L by the transcription factor GLI2 in the tumor microenvironment downstream of CCR3 signaling.

Re-engineered p53 chimera with enhanced homo-oligomerization that maintains tumor suppressor activity.

The use of the tumor suppressor p53 for gene therapy of cancer is limited by the dominant negative inactivating effect of mutant endogenous p53 in cancer cells. We have shown previously that swapping the tetramerization domain (TD) of p53 with the coiled-coil (CC) from Bcr allows for our chimeric p53 (p53-CC) to evade hetero-oligomerization with endogenous mutant p53. This enhances the utility of this construct, p53-CC, for cancer gene therapy. Because domain swapping to create p53-CC could result in p53-CC interacting with endogenous Bcr, which is ubiquitous in cells, modifications on the CC domain are necessary to minimize potential interactions with Bcr. Hence, we investigated the possible design of mutations that will improve homodimerization of CC mutants and disfavor hetero-oligomerization with wild-type CC (CCwt), with the goal of minimizing potential interactions with endogenous Bcr in cells. This involved integrated computational and experimental approaches to rationally design an enhanced version of our chimeric p53-CC tumor suppressor. Indeed, the resulting lead candidate p53-CCmutE34K-R55E avoids binding to endogenous Bcr and retains p53 tumor suppressor activity. Specifically, p53-CCmutE34K-R55E exhibits potent apoptotic activity in a variety of cancer cell lines, regardless of p53 status (in cells with mutant p53, wild-type p53, or p53-null cells). This construct overcomes the dominant negative effect limitation of wt p53 and has high significance for future gene therapy for treatment of cancers characterized by p53 dysfunction, which represent over half of all human cancers.