Waldenström Macroglobulinemia: Mechanisms of Disease Progression and Current Therapies.

Waldenström macroglobulinemia is an indolent, B-cell lymphoma without a known cure. The bone marrow microenvironment and cytokines both play key roles in Waldenström macroglobulinemia (WM) tumor progression. Only one FDA-approved drug exists for the treatment of WM, Ibrutinib, but treatment plans involve a variety of drugs and inhibitors. This review explores avenues of tumor progression and targeted drug therapy that have been investigated in WM and related B-cell lymphomas.

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.

MLL1 inhibition reduces IgM levels in Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM) is a B cell lymphoma characterized by the overproduction of a monoclonal IgM antibody, a leading pathogenic feature of the disease. Current therapies are based on our knowledge at the signaling and genetic scale, but recent research has identified epigenetic dysregulation as one of the important dynamics in the biology of this disease. In this study, we found that Mixed-lineage leukemia 1 (MLL1) histone methyltransferase and its chromatin tethering partner Menin are upregulated in WM patients. KMT2A knockdown using short hairpin RNA (shRNA) and inhibition of MLL1 function using the menin-MLL1 inhibitor (MI-2) in WM cells resulted in a significant reduction in IgM levels without significantly impacting WM cell growth and survival. Further analysis identified MLL1 binding at multiple sites in the 5' Eµ enhancer of the immunoglobulin heavy (IGH) chain. We found increased histone 3 lysine 4 trimethylation (H3K4me3) enrichment at multiple MLL1 binding sites upon LPS stimulation, a known inducer of IgM. Finally, we found that disruption of Menin-MLL1 complex using the MI-2 inhibitor in tumor-bearing mice significantly reduced human IgM levels in mice sera. Taken together, these results identify MLL1 as a regulator of IgM and define MLL1 as a new therapeutic target for WM.

Macrophage Polarization States in the Tumor Microenvironment.

The M1/M2 macrophage paradigm plays a key role in tumor progression. M1 macrophages are historically regarded as anti-tumor, while M2-polarized macrophages, commonly deemed tumor-associated macrophages (TAMs), are contributors to many pro-tumorigenic outcomes in cancer through angiogenic and lymphangiogenic regulation, immune suppression, hypoxia induction, tumor cell proliferation, and metastasis. The tumor microenvironment (TME) can influence macrophage recruitment and polarization, giving way to these pro-tumorigenic outcomes. Investigating TME-induced macrophage polarization is critical for further understanding of TAM-related pro-tumor outcomes and potential development of new therapeutic approaches. This review explores the current understanding of TME-induced macrophage polarization and the role of M2-polarized macrophages in promoting tumor progression.

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.

GLI1/GLI2 functional interplay is required to control Hedgehog/GLI targets gene expression.

The Hedgehog-regulated transcription factors GLI1 and GLI2 play overlapping roles in development and disease; however, the mechanisms underlying their interplay remain elusive. We report for the first time that GLI1 and GLI2 physically and functionally interact in cancer cells. GLI1 and GLI2 were shown to co-immunoprecipitate in PANC1 pancreatic cancer cells and RMS13 rhabdomyosarcoma cells. Mapping analysis demonstrated that the zinc finger domains of both proteins are required for their heteromerization. RNAi knockdown of either GLI1 or GLI2 inhibited expression of many well-characterized GLI target genes (BCL2, MYCN, PTCH2, IL7 and CCND1) in PANC1 cells, whereas PTCH1 expression was only inhibited by GLI1 depletion. qPCR screening of a large set of putative canonical and non-canonical Hedgehog/GLI targets identified further genes (e.g. E2F1, BMP1, CDK2) strongly down-regulated by GLI1 and/or GLI2 depletion in PANC1 cells, and demonstrated that ANO1, AQP1 and SOCS1 are up-regulated by knockdown of either GLI1 or GLI2. Chromatin immunoprecipitation showed that GLI1 and GLI2 occupied the same regions at the BCL2, MYCN and CCND1 promoters. Furthermore, depletion of GLI1 inhibited GLI2 occupancy at these promoters, suggesting that GLI1/GLI2 interaction is required for the recruitment of GLI2 to these sites. Together, these findings indicate that GLI1 and GLI2 co-ordinately regulate the transcription of some genes, and provide mechanistic insight into the roles of GLI proteins in carcinogenesis.

GLI2-Mediated Inflammation in the Tumor Microenvironment.

The tumor microenvironment (TME) plays an important role in the development and progression of cancer and has been shown to contribute to resistance to therapy. Inflammation is one of the hallmarks of cancer implicated in disease phenotype. Therefore, understanding the mechanisms that regulate inflammation in cancer and consequently how inflammatory mediators promote cancer progression is important for our understanding of cancer cell biology. The transcription factor GLI2 was initially identified as a member of the Hedgehog (HH) signaling pathway. During the last decade, studies have shown a novel mechanism of GLI2 regulation independent of HH signaling, where GLI2 consequently modulated several cytokine genes in the TME. These studies highlight a novel role for GLI2 as an inflammatory mediatory independent of HH stimulation. This chapter will discuss canonical and noncanonical pathways of GLI2 regulation and some of the downstream cytokine target genes regulated by GLI2.

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 MYC activity in double-hit lymphoma with MYC and BCL2 and/or BCL6 rearrangements with epigenetic bromodomain inhibitors.

Double/triple-hit lymphomas (DHL/THL) account for 5-10% of diffuse large B cell lymphoma (DLBCL) with rearrangement of MYC and BCL2 and/or BCL6 resulting in MYC overexpression. Despite the poor prognosis of DHL, R-CHOP chemotherapy remains the treatment backbone and new targeted therapy is needed. We performed comprehensive cytogenetic studies/fluorescence in situ hybridization on DLBCL and Burkitt lymphoma cell lines (n = 11) to identify the DHL/THL DLBCL in vitro model. We identified MYC/IG in Raji and Ramos (single hit); MYC/IG-BCL2 (DHL) in DOHH2, OCI-LY1, SUDHL2, and OCI-LY10; MYC/IG-BCL2/BCL6 (THL) in VAL; and no MYC rearrangement in U2932 and HBL1 (WT-MYC). Targeting MYC in the DHL/THL DLBCLs through bromodomain extra-terminal inhibitors (BETi) (JQ1, I-BET, and OTX015) significantly (p < 0.05) reduced proliferation, similar to WT-MYC cells, accompanied by decreased MYC but not BCL2 protein. Moreover, BETi suppressed MYC transcription and decreased BRD4 binding to MYC promoter in DHL cells. CD47 and PD-L1 are immunoregulatory molecules often expressed on tumors and regulated by MYC. High levels of surface CD47 but not surface PD-L1 was observed in DHL/THL, which was reduced by JQ1 treatment. BETi in combination with Pan-HDAC inhibitor had a limited effect on survival of DHL/THL, while combination of BETi and BCL2 inhibitor (ABT-199) had a significant (p < 0.005) inhibitory effect on survival followed by BCL-XL inhibition. Overall, the data suggests that MYC-expressing DLBCLs are probably addicted to the MYC-oncogenic effect regardless of MYC rearrangements. In summary, we identified an in vitro model for DHL/THL DLBCLs and provide evidence for the therapeutic potential of BET inhibitor alone or in combination with BCL2 inhibitor.

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.

Bone marrow stromal cells interaction with titanium; Effects of composition and surface modification.

Inflammation and implant loosening are major concerns when using titanium implants for hard tissue engineering applications. Surface modification is one of the promising tools to enhance tissue-material integration in metallic implants. Here, we used anodization technique to modify the surface of commercially pure titanium (CP-Ti) and titanium alloy (Ti-6Al-4V) samples. Our results show that electrolyte composition, anodization time and voltage dictated the formation of well-organized nanotubes. Although electrolyte containing HF in water resulted in nanotube formation on Ti, the presence of NH4F and ethylene glycol was necessary for successful nanotube formation on Ti-6Al-4V. Upon examination of the interaction of bone marrow stromal cells (BMSCs) with the modified samples, we found that Ti-6Al-4V without nanotubes induced cell proliferation and cluster of differentiation 40 ligand (CD40L) expression which facilitates B-cell activation to promote early bone healing. However, the expression of glioma associated protein 2 (GLI2), which regulates CD40L, was reduced in Ti-6Al-4V and the presence of nanotubes further reduced its expression. The inflammatory cytokine interleukin-6 (IL-6) expression was reduced by nanotube presence on Ti. These results suggest that Ti-6Al-4V with nanotubes may be suitable implants because they have no effect on BMSC growth and inflammation.

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.

Calf melanin immunomodulates RPE cell attachment to extracellular matrix protein.

PURPOSE: It is widely accepted that RPE melanin has a protective effect against oxidative damage in RPE cells. It is possible that an additional protective characteristic of melanin is the ability to modulate RPE cell immune response. In this study, in vitro modeling was used to probe the relationship between RPE pigmentation and immune response by monitoring IL-6 expression and secretion in calf melanin pigmented ARPE-19 cells seeded onto glycated extracellular matrix as a stressor. METHODS: ARPE-19 cells were left unpigmented or were pigmented with either calf melanin or latex beads, and were then seeded onto RPE-derived extracellular matrix (ECM) or tissue culture-treated plates (no ECM). ECMs were modified by glycation. IL-6 expression was measured using qPCR and IL-6 secretion was determined using an ELISA, both at 30 min and 24 h after seeding. MTT assay was used to quantify cell attachment to glycated matrices 30 min after seeding. In unpigmented ARPE-19 cells, rate of cell attachment to substrate was monitored for 60 min after seeding using a hemacytometer to count unattached cells. Additionally, cell viability was evaluated using the Neutral Red assay 24 h after seeding. RESULTS: A significant increase in IL-6 expression was observed in calf melanin pigmented cells versus latex bead and unpigmented controls (p < 0.0001) 30 min after seeding onto ECM. Twenty-four hours after seeding, a significant decrease in IL-6 expression was observed in calf melanin pigmented cells (p < 0.0001) versus controls, implicating down-regulation of the cytokine. Additionally, calf melanin pigmented cell populations showed significant increase in attachment compared to unpigmented controls on either no ECM or unmodified ECM. CONCLUSIONS: Pigmentation of RPE cells with calf melanin resulted in significant changes in IL-6 expression regardless of ECM modification, in vitro. These findings suggest that melanin in the RPE may participate in immune response modulation in the retina with particular regard to cell attachment to protein substrates. The results of this study further implicate the role of chemical changes to melanin in regulating inflammation in retinal disease.

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.

rtfA controls development, secondary metabolism, and virulence in Aspergillus fumigatus.

Invasive aspergillosis by Aspergillus fumigatus is a leading cause of infection-related mortality in immune-compromised patients. In order to discover potential genetic targets to control A. fumigatus infections we characterized rtfA, a gene encoding a putative RNA polymerase II transcription elongation factor-like protein. Our recent work has shown that the rtfA ortholog in the model fungus Aspergillus nidulans regulates morphogenesis and secondary metabolism. The present study on the opportunistic pathogen A. fumigatus rtfA gene revealed that this gene influences fungal growth and conidiation, as well as production of the secondary metabolites tryptoquivaline F, pseurotin A, fumiquinazoline C, festuclavine, and fumigaclavines A, B and C. Additionally, rtfA influences protease activity levels, the sensitivity to oxidative stress and adhesion capacity, all factors important in pathogenicity. Furthermore, rtfA was shown to be indispensable for normal virulence using Galleria mellonella as well as murine infection model systems.

Modulation of the IL-6 Receptor α Underlies GLI2-Mediated Regulation of Ig Secretion in Waldenström Macroglobulinemia Cells.

Ig secretion by terminally differentiated B cells is an important component of the immune response to foreign pathogens. Its overproduction is a defining characteristic of several B cell malignancies, including Waldenström macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Therefore, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for this disease. In this study, we define a novel pathway involving the oncogenic transcription factor GLI2 modulating IgM secretion by WM malignant cells. Pharmacological and genetic inhibition of GLI2 in WM malignant cells resulted in a reduction in IgM secretion. Screening for a mechanism identified the IL-6Rα (gp80) subunit as a downstream target of GLI2 mediating the regulation of IgM secretion. Using a combination of expression, luciferase, and chromatin immunoprecipitation assays we demonstrate that GLI2 binds to the IL-6Rα promoter and regulates its activity as well as the expression of this receptor. Additionally, we were able to rescue the reduction in IgM secretion in the GLI2 knockdown group by overexpressing IL-6Rα, thus defining the functional significance of this receptor in GLI2-mediated regulation of IgM secretion. Interestingly, this occurred independent of Hedgehog signaling, a known regulator of GLI2, as manipulation of Hedgehog had no effect on IgM secretion. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets.

The oncogenic transcription factor IRF4 is regulated by a novel CD30/NF-κB positive feedback loop in peripheral T-cell lymphoma.

Peripheral T-cell lymphomas (PTCLs) are generally aggressive non-Hodgkin lymphomas with poor overall survival rates following standard therapy. One-third of PTCLs express interferon regulatory factor-4 (IRF4), a tightly regulated transcription factor involved in lymphocyte growth and differentiation. IRF4 drives tumor growth in several lymphoid malignancies and has been proposed as a candidate therapeutic target. Because direct IRF4 inhibitors are not clinically available, we sought to characterize the mechanism by which IRF4 expression is regulated in PTCLs. We demonstrated that IRF4 is constitutively expressed in PTCL cells and drives Myc expression and proliferation. Using an inhibitor screen, we identified nuclear factor κB (NF-κB) as a candidate regulator of IRF4 expression and cell proliferation. We then demonstrated that the NF-κB subunits p52 and RelB were transcriptional activators of IRF4. Further analysis showed that activation of CD30 promotes p52 and RelB activity and subsequent IRF4 expression. Finally, we showed that IRF4 transcriptionally regulates CD30 expression. Taken together, these data demonstrate a novel positive feedback loop involving CD30, NF-κB, and IRF4; further evidence for this mechanism was demonstrated in human PTCL tissue samples. Accordingly, NF-κB inhibitors may represent a clinical means to disrupt this feedback loop in IRF4-positive PTCLs.

Factors regulating immunoglobulin production by normal and disease-associated plasma cells.

Immunoglobulins are molecules produced by activated B cells and plasma cells in response to exposure to antigens. Upon antigen exposure, these molecules are secreted allowing the immune system to recognize and effectively respond to a myriad of pathogens. Immunoglobulin or antibody secreting cells are the mature form of B lymphocytes, which during their development undergo gene rearrangements and selection in the bone marrow ultimately leading to the generation of B cells, each expressing a single antigen-specific receptor/immunoglobulin molecule. Each individual immunoglobulin molecule has an affinity for a unique motif, or epitope, found on a given antigen. When presented with an antigen, activated B cells differentiate into either plasma cells (which secrete large amounts of antibody that is specific for the inducing antigen), or memory B cells (which are long-lived and elicit a stronger and faster response if the host is re-exposed to the same antigen). The secreted form of immunoglobulin, when bound to an antigen, serves as an effector molecule that directs other cells of the immune system to facilitate the neutralization of soluble antigen or the eradication of the antigen-expressing pathogen. This review will focus on the regulation of secreted immunoglobulin by long-lived normal or disease-associated plasma. Specifically, the focus will be on signaling and transcriptional events that regulate the development and homeostasis of long-lived immunoglobulin secreting plasma cells.