Frank J. Secreto

Wnt signaling as a therapeutic target for bone diseases

Background: There is a need to develop new bone anabolic agents because current bone regeneration regimens have limitations. The Wingless-type MMTV integration site (Wnt) pathway has emerged as a regulator of bone formation and regeneration. Objective: To review the molecular basis for Wnt pathway modulation and discuss strategies that target it and improve bone mass. Methods: Data in peer-reviewed reports and meeting abstracts are discussed. Results/conclusions: Neutralizing inhibitors of Wnt signaling have emerged as promising strategies. Small-molecule inhibitors of glycogen synthase kinase 3β increase bone mass, lower adiposity and reduce fracture risk. Neutralizing antibodies to Dickkopf 1, secreted Frizzled-related protein 1 and sclerostin produce similar outcomes in animal models. These drugs are exciting breakthroughs but are not without risks. The challenges include tissue-specific targeting and consequently, long-term safety.

Histone Deacetylase 3 Depletion in Osteo/Chondroprogenitor Cells Decreases Bone Density and Increases Marrow Fat

Histone deacetylase (Hdac)3 is a nuclear enzyme that contributes to epigenetic programming and is required for embryonic development. To determine the role of Hdac3 in bone formation, we crossed mice harboring loxP sites around exon 7 of Hdac3 with mice expressing Cre recombinase under the control of the osterix promoter. The resulting Hdac3 conditional knockout (CKO) mice were runted and had severe deficits in intramembranous and endochondral bone formation. Calvarial bones were significantly thinner and trabecular bone volume in the distal femur was decreased 75% in the Hdac3 CKO mice due to a substantial reduction in trabecular number. Hdac3-CKO mice had fewer osteoblasts and more bone marrow adipocytes as a proportion of tissue area than their wildtype or heterozygous littermates. Bone formation rates were depressed in both the cortical and trabecular regions of Hdac3 CKO femurs. Microarray analyses revealed that numerous developmental signaling pathways were affected by Hdac3-deficiency. Thus, Hdac3 depletion in osterix-expressing progenitor cells interferes with bone formation and promotes bone marrow adipocyte differentiation. These results demonstrate that Hdac3 inhibition is detrimental to skeletal health.

Suberoylanilide hydroxamic acid (SAHA; vorinostat) causes bone loss by inhibiting immature osteoblasts

Histone deacetylase (Hdac) inhibitors are used clinically to treat cancer and epilepsy. Although Hdac inhibition accelerates osteoblast maturation and suppresses osteoclast maturation in vitro, the effects of Hdac inhibitors on the skeleton are not understood. The purpose of this study was to determine how the pan-Hdac inhibitor, suberoylanilide hydroxamic acid (SAHA; a.k.a. vorinostat or Zolinza(TM)) affects bone mass and remodeling in vivo. Male C57BL/6J mice received daily SAHA (100mg/kg) or vehicle injections for 3 to 4weeks. SAHA decreased trabecular bone volume fraction and trabecular number in the distal femur. Cortical bone at the femoral midshaft was not affected. SAHA reduced serum levels of P1NP, a bone formation marker, and also suppressed tibial mRNA levels of type I collagen, osteocalcin and osteopontin, but did not alter Runx2 or osterix transcripts. SAHA decreased histological measures of osteoblast number but interestingly increased indices of osteoblast activity including mineral apposition rate and bone formation rate. Neither serum (TRAcP 5b) nor histological markers of bone resorption were affected by SAHA. P1NP levels returned to baseline in animals which were allowed to recover for 4weeks after 4weeks of daily SAHA injections, but bone density remained low. In vitro, SAHA suppressed osteogenic colony formation, decreased osteoblastic gene expression, induced cell cycle arrest, and caused DNA damage in bone marrow-derived adherent cells. Collectively, these data demonstrate that bone loss following treatment with SAHA is primarily due to a reduction in osteoblast number. Moreover, these decreases in osteoblast number can be attributed to the deleterious effects of SAHA on immature osteoblasts, even while mature osteoblasts are resistant to the harmful effects and demonstrate increased activity in vivo, indicating that the response of osteoblasts to SAHA is dependent upon their differentiation state. These studies suggest that clinical use of SAHA and other Hdac inhibitors to treat cancer, epilepsy or other conditions may potentially compromise skeletal structure and function.

Activation of TAK1 by MYD88 L265P drives malignant B-cell Growth in non-Hodgkin lymphoma

Massively parallel sequencing analyses have revealed a common mutation within the MYD88 gene (MYD88L265P) occurring at high frequencies in many non-Hodgkin lymphomas (NHLs) including the rare lymphoplasmacytic lymphoma, Waldenströms macroglobulinemia (WM). Using whole-exome sequencing, Sanger sequencing and allele-specific PCR, we validate the initial studies and detect the MYD88L265P mutation in the tumor genome of 97% of WM patients analyzed (n=39). Due to the high frequency of MYD88 mutation in WM and other NHL, and its known effects on malignant B-cell survival, therapeutic targeting of MYD88 signaling pathways may be clinically useful. However, we are lacking a thorough characterization of the role of intermediary signaling proteins on the biology of MYD88L265P-expressing B cells. We report here that MYD88L265P signaling is constitutively active in both WM and diffuse large B-cell lymphoma cells leading to heightened MYD88L265P, IRAK and TRAF6 oligomerization and NF-κB activation. Furthermore, we have identified the signaling protein, TAK1, to be an essential mediator of MYD88L265P-driven signaling, cellular proliferation and cytokine secretion in malignant B cells. Our studies highlight the biological significance of MYD88L265P in NHL and reveal TAK1 inhibition to be a potential therapeutic strategy for the treatment of WM and other diseases characterized by MYD88L265P.

Estrogen receptor α/β isoforms, but not βcx, modulate unique patterns of gene expression and cell proliferation in Hs578T cells

The actions of 17β‐estradiol (E2) and selective estrogen receptor modulators (SERMs) have been extensively investigated regarding their ability to act through estrogen receptor‐α (ERα) to perturb estrogen receptor positive (ER+) breast cancer (BC) growth. However, many BCs also express ERβ, along with multiple estrogen receptor (ER) splice variants such as ERβcx, an ERβ splice variant incapable of binding ligand. To gain a more comprehensive understanding of ER action in BC cells, we stably expressed ERα, ERβ, or ERβcx under doxycycline (Dox) control in Hs578T cells. Microarrays performed on E2 or 4OH‐tamoxifen (4HT) treated Hs578T ERα and ERβ cells revealed distinct ligand and receptor‐dependent patterns of gene regulation, while the induction of ERβcx did not alter gene expression patterns. E2 stimulation of Hs578T ERβ cells resulted in a 27% decrease in cellular proliferation, however, no significant change in proliferation was observed following the exposure of Hs578T ERα or ERβ cells to 4HT. Expression of ERβcx in Hs578T cells did not effect cellular proliferation. Flow cytometry assays revealed a 50% decrease in E2‐stimulated Hs578T ERβ cells entering S‐phase, along with a 17% increase in G0/G1 cell‐cycle arrest. We demonstrate here that ERα and ERβ regulate unique gene expression patterns in Hs578T cells, and such regulation likely is responsible for the observed isoform‐specific changes in cell proliferation. Hs578T ER expressing cell‐lines provide a unique BC model system, permitting the comparison of ERα, ERβ, and ERβcx actions in the same cell‐line. J. Cell. Biochem. 101:1125–1147, 2007.

Runx2 and Bone Morphogenic Protein 2 Regulate the Expression of an Alternative Lef1 Transcript During Osteoblast Maturation

Lymphoid Enhancer Binding Factor (Lef) 1 is a transcriptional effector of the Wnt/Lrp5/β‐catenin signaling cascade, which regulates osteoblast differentiation, bone density, and skeletal strength. In this study, we describe the expression and function of an alternative Lef1 isoform in osseous cells. Lef1ΔN is a naturally occurring isoform driven by a promoter (p2) within the intron between exons 3 and 4 of Lef1. Lef1ΔN is induced during late osteoblast differentiation. This is converse to the expression pattern of the full‐length Lef1 protein, which as we previously showed, decreases during differentiation. Agonists of osteoblast maturation differentially affected Lef1ΔN expression. BMP2 stimulated Lef1ΔN expression, whereas Wnt3a blocked basal and BMP2‐induced expression of Lef1ΔN transcripts during osteoblast differentiation. We determined that the Lef1ΔN p2 promoter is active in osteoblasts and Runx2 regulates its activity. Stable overexpression of Lef1ΔN in differentiating osteoblasts induced the expression of osteoblast differentiation genes, osteocalcin and type 1 collagen. Taken together, our results suggest Lef1ΔN is a crucial regulator of terminal differentiation in osseous cells. J. Cell. Physiol. 221: 480–489, 2009.

t(X;14)(p11;q32) in MALT lymphoma involving GPR34 reveals a role for GPR34 in tumor cell growth

Genetic aberrations, including trisomies 3 and 18, and well-defined IGH translocations, have been described in marginal zone lymphomas (MZLs); however, these known genetic events are present in only a subset of cases. Here, we report the cloning of an IGH translocation partner on chromosome X, t(X;14)(p11.4;q32) that deregulates expression of an poorly characterized orphan G-protein-coupled receptor, GPR34. Elevated GPR34 gene expression was detected independent of the translocation in multiple subtypes of non-Hodgkin lymphoma and distinguished a unique molecular subtype of MZL. Increased expression of GPR34 was also detected in tissue from brain tumors and surface expression of GPR34 was detected on human MZL tumor cells and normal immune cells. Overexpression of GPR34 in lymphoma and HeLa cells resulted in phosphorylation of ERK, PKC, and CREB; induced CRE, AP1, and NF-κB-mediated gene transcription; and increased cell proliferation. In summary, these results are the first to identify a role for a GPR34 in lymphoma cell growth, provide insight into GPR34-mediated signaling, identify a genetically unique subset of MZLs that express high levels of GPR34, and suggest that MEK inhibitors may be useful for treatment of GPR34-expressing tumors.

IL-21 in the bone marrow microenvironment contributes to IgM secretion and proliferation of malignant cells in Waldenstrom macroglobulinemia

Cytokines within the tumor microenvironment play an important role in supporting the growth and survival of B-cell malignancies. One such cytokine, IL-21, promotes the growth of myeloma and Hodgkin lymphoma cells while inducing apoptosis in chronic lymphocytic leukemia. However, the biologic significance of IL-21 has not been examined in Waldenstrom macroglobulinemia (WM), a B-cell lymphoma characterized by elevated serum IgM and a lymphoplasmacytic bone marrow infiltrate. We report here on the presence of IL-21 in the bone marrow of patients with WM and have identified activated T cells as the source of this cytokine. We readily detected the IL-21 receptor on malignant WM B cells and show that IL-21 significantly increases both IgM secretion and cellular proliferation of these cells with no effect on viability. IL-21 rapidly induces phosphorylation of STAT3 in WM cells, and treatment of the WM cell line MWCL-1 with a STAT3 inhibitor abolished the IL-21-mediated increases in cellular proliferation and IgM secretion. IL-21 also increased the expression of known STAT3 targets involved in B-cell differentiation, including BLIMP-1, XBP-1, IL-6, and IL-10. Overall, our data indicate that IL-21 in the bone marrow microenvironment significantly affects the biology of WM tumor cells through a STAT3-dependent mechanism.

Rbm20-deficient cardiogenesis reveals early disruption of RNA processing and sarcomere remodeling establishing a developmental etiology for dilated cardiomyopathy

Dilated cardiomyopathy (DCM) due to mutations in RBM20, a gene encoding an RNA-binding protein, is associated with high familial penetrance, risk of progressive heart failure and sudden death. Although genetic investigations and physiological models have established the linkage of RBM20 with early-onset DCM, the underlying basis of cellular and molecular dysfunction is undetermined. Modeling human genetics using a high-throughput pluripotent stem cell platform was herein designed to pinpoint the initial transcriptome dysfunction and mechanistic corruption in disease pathogenesis. Tnnt2-pGreenZeo pluripotent stem cells were engineered to knockdown Rbm20 (shRbm20) to determine the cardiac-pathogenic phenotype during cardiac differentiation. Intracellular Ca(2+) transients revealed Rbm20-dependent alteration in Ca(2+) handling, coinciding with known pathological splice variants of Titin and Camk2d genes by Day 24 of cardiogenesis. Ultrastructural analysis demonstrated elongated and thinner sarcomeres in the absence of Rbm20 that is consistent with human cardiac biopsy samples. Furthermore, Rbm20-depleted transcriptional profiling at Day 12 identified Rbm20-dependent dysregulation with 76% of differentially expressed genes linked to known cardiac pathology ranging from primordial Nkx2.5 to mature cardiac Tnnt2 as the initial molecular aberrations. Notably, downstream consequences of Rbm20-depletion at Day 24 of differentiation demonstrated significant dysregulation of extracellular matrix components such as the anomalous overexpression of the Vtn gene. By using the pluripotent stem cell platform to model human cardiac disease according to a stage-specific cardiogenic roadmap, we established a new paradigm of familial DCM pathogenesis as a developmental disorder that is patterned during early cardiogenesis and propagated with cellular mechanisms of pathological cardiac remodeling.

Soluble and Membrane-Bound TGF-β-Mediated Regulation of Intratumoral T Cell Differentiation and Function in B-Cell Non-Hodgkin Lymphoma

While the effect of TGF-β on malignant B cells in non-Hodgkin lymphoma (NHL) has been previously evaluated, studies to specifically define the role of TGF-β in tumor immunity in B-cell NHL are limited. We found that soluble TGF-β, secreted by both lymphoma cells and intratumoral T cells, is present in the serum of patients with B-cell NHL. Soluble TGF-β promoted regulatory T (Treg) cells by enhancing expression of Foxp3 in CD4+ T cells and suppressed effector helper T (TH) cells by inhibiting expression of IFN-γ and IL-17. Blockade of the IL-2 signaling pathway diminished the effect of soluble TGF-β on T cell differentiation. Furthermore, we found that membrane-bound TGF-β is expressed specifically on the surface of malignant B cells in B-cell NHL. TGF-β was able to bind to the surface of lymphoma B cells through an interaction with heparan sulfate (HS) but not through the TGF-β receptor. We showed that pretreatment of lymphoma B cells with TGF-β significantly inhibits the proliferation and cytokine production of intratumoral T cells. Taken together, these results suggest that tumor-associated soluble and membrane-bound TGF-β are involved in the regulation of intratumoral T cell differentiation and function in B-cell NHL.