FKBP12 is a major regulator of ALK2 activity in multiple myeloma cells

Background The immunophilin FKBP12 binds to TGF-β family type I receptors, including the BMP type I receptor ALK2. FKBP12 keeps the type I receptor in an inactive state and controls signaling activity. Removal of FKBP12 with drugs such as the FKBP-ligand FK506 enhances BMP activity in various cell types. In multiple myeloma cells, activation of SMAD1/5/8 leads to apoptosis. We hypothesized that removing FKBP12 from ALK2 in myeloma cells would potentiate BMP-induced ALK2-SMAD1/5/8 activity and in consequence cell death. Methods Multiple myeloma cell lines were treated with FK506, or other FKBP-binding compounds, combined with different BMPs before analyzing SMAD1/5/8 activity and cell viability. SMAD1/5/8 activity was also investigated using a reporter cell line, INA-6 BRE-luc. To characterize the functional signaling receptor complex, we genetically manipulated receptor expression by siRNA, shRNA and CRISPR/Cas9 technology. Results FK506 potentiated BMP-induced SMAD1/5/8 activation and apoptosis in multiple myeloma cell lines. By using FKBP-binding compounds with different affinity profiles, and siRNA targeting FKBP12, we show that the FK506 effect is mediated by binding to FKBP12. Ligands that typically signal via ALK3 in myeloma cells, BMP2, BMP4, and BMP10, did not induce apoptosis in cells lacking ALK3. Notably, BMP10 competed with BMP6 and BMP9 and antagonized their activity via ALK2. However, upon addition of FK506, we saw a surprising shift in specificity, as the ALK3 ligands gained the ability to signal via ALK2 and induce apoptosis. This indicates that the receptor complex can switch from an inactive non-signaling complex (NSC) to an active one by adding FK506. This gain of activity was also seen in other cell types, indicating that the observed effects have broader relevance. BMP2, BMP4 and BMP10 depended on BMPR2 as type II receptor to signal, which contrasts with BMP6 and BMP9, that activate ALK2 more potently when BMPR2 is knocked down. Conclusions In summary, our data suggest that FKBP12 is a major regulator of ALK2 activity in multiple myeloma cells, partly by switching an NSC into an active signaling complex. FKBP12 targeting compounds devoid of immunosuppressing activity could have potential in novel treatment strategies aiming at reducing multiple myeloma tumor load

Chemical, Physical and Biological Triggers of Evolutionary Conserved Bcl-xL-Mediated Apoptosis

Background: The evidence that pan-Bcl-2 or Bcl-xL-specific inhibitors prematurely kill virus-infected or RNA/DNA-transfected cells provides rationale for investigating these apoptotic inducers further. We hypothesized that not only invasive RNA or DNA (biological factors) but also DNA/RNA-damaging chemical or physical factors could trigger apoptosis that have been sensitized with pan-Bcl-2 or Bcl-xL-specific agents; Methods: We tested chemical and physical factors plus Bcl-xL-specific inhibitor A-1155463 in cells of various origins and the small roundworms (C. elegans); Results: We show that combination of a A-1155463 along with a DNA-damaging agent, 4-nitroquinoline-1-oxide (4NQO), prematurely kills cells of various origins as well as C. elegans. The synergistic effect is p53-dependent and associated with the release of Bad and Bax from Bcl-xL, which trigger mitochondrial outer membrane permeabilization. Furthermore, we found that combining Bcl-xL-specific inhibitors with various chemical compounds or physical insults also induced cell death; Conclusions: Thus, we were able to identify several biological, chemical and physical triggers of the evolutionarily conserved Bcl-xL-mediated apoptotic pathway, shedding light on strategies and targets for novel drug development

The role of bone morphogenetic proteins in myeloma cell survival

Multiple myeloma is characterized by slowly growing clones of malignant plasma cells in the bone marrow. The malignant state is frequently accompanied by osteolytic bone disease due to a disturbed balance between osteoblasts and osteoclasts. Bone morphogenetic proteins (BMPs) are present in the bone marrow and are important for several aspects of myeloma pathogenesis including growth and survival of tumor cells, bone homeostasis, and anemia. Among cancer cells, myeloma cells are particularly sensitive to growth inhibition and apoptosis induced by BMPs and therefore represent good models to study BMP receptor usage and signaling. Our review highlights and discusses the current knowledge on BMP signaling in myeloma

Receptor binding competition: A paradigm for regulating TGF-β family action

The transforming growth factor (TGF)-β family is a group of structurally related, multifunctional growth factors, or ligands that are crucially involved in the development, regulation, and maintenance of animal tissues. In humans, the family counts over 33 members. These secreted ligands typically form multimeric complexes with two type I and two type II receptors to activate one of two distinct signal transduction branches. A striking feature of the family is its promiscuity, i.e., many ligands bind the same receptors and compete with each other for binding to these receptors. Although several explanations for this feature have been considered, its functional significance has remained puzzling. However, several recent reports have promoted the idea that ligand-receptor binding promiscuity and competition are critical features of the TGF-β family that provide an essential regulating function. Namely, they allow a cell to read and process multi-ligand inputs. This capability may be necessary for producing subtle, distinctive, or adaptive responses and, possibly, for facilitating developmental plasticity. Here, we review the molecular basis for ligand competition, with emphasis on molecular structures and binding affinities. We give an overview of methods that were used to establish experimentally ligand competition. Finally, we discuss how the concept of ligand competition may be fundamentally tied to human physiology, disease, and therapy

Cytotoxic Effect on Human Myeloma Cells and Leukemic Cells by the Agaricus blazei Murill Based Mushroom Extract, Andosan™

Agaricus blazei Murill is an edible mushroom of the Basidiomycetes family, which has been found to contain a number of compounds with antitumor properties, such as proteoglycans and ergosterol. In the present investigation, we show that the commercial mushroom product Andosan, which contains 82.4% Agaricus blazei Murill, together with medicinal mushrooms Hericium erinaceus (14.7%) and Grifola frondosa (2.9%), has a cytotoxic effect on primary myeloma cells, other myeloma cell lines, and leukemia cell lines in vitro. Although the exact content and hence the mechanisms of action of the Andosan extract are unknown, we have found in this investigation indications of cell cycle arrest when myeloma cell lines are cultivated with Andosan. This may be one of the possible explanations for the cytotoxic effects of Andosan

Comparison Between 8-Methoxypsoralen and 5-Aminolevulinic Acid in Killing T Cells of Photopheresis Patients Ex Vivo

Background and Objective Extracorporeal photopheresis (ECP), an established modality for cutaneous T‐cell lymphoma (CTCL) and graft‐versus‐host disease, involves ex vivo treatment of isolated leukocytes of a patient with the photosensitizing drug 8‐methoxypsoralen (8‐MOP) and ultraviolet‐A (UV‐A) exposure before reinfusion back to the patient. However, 8‐MOP binds to both diseased and normal cells and thus kills both types of the cells after UV‐A illumination with little selectivity. Clinically, this modality gives only partial response in the majority of treated patients. 5‐Aminolevulinic acid (5‐ALA), a precursor of the potent photosensitizer protoporphyrin IX (PpIX), has been shown to selectively induce PpIX in activated T lymphocytes (T cells) and could be an alternative for 8‐MOP. The objectives of this study were to investigate ex vivo 5‐ALA dark toxicity, 5‐ALA‐induced PpIX production, and photodynamic effect on T cells obtained from clinical ECP patients after the treatment of 5‐ALA or 8‐MOP plus a built‐in certified UV‐A source in the commercial Therakos™ Photopheresis System. Materials and Methods Flow cytometry was used to study dark cytotoxic effects of 5‐ALA on human leukocytes, to measure the production of 5‐ALA‐induced PpIX in CD25+ activated T cells from both diluted mononuclear cells and undiluted buffy coat samples of ECP patients and to compare photodynamic effects on CD4+ and CD8+ T cells with 5‐ALA/UV‐A or 8‐MOP/UV‐A. Results No dark toxicity of 5‐ALA on the leukocytes of ECP patients was seen at concentrations up to 10 mM for an incubation of up to 20 hours. 5‐ALA‐induced PpIX was produced more in CD25+ activated T cells than resting T cells in both diluted mononuclear cells and undiluted buffy coat samples, although there was a huge variation of samples from different individual patients. The CD4+ and CD8+ T cells treated with 5‐ALA/UV‐A were killed more than those treated with 8‐MOP/UV‐A. Conclusion These results suggest that 5‐ALA/UV‐A may have the potential for improving the efficacy of ECP. Lasers Surg. Med. 50:469–475, 2018

TGF-β contamination of purified recombinant GDF15

Purified recombinant proteins for use in biomedical research are invaluable to investigate protein function. However, purity varies in protein batches made in mammalian expression systems, such as CHO-cells or HEK293-cells. This study points to caution while investigating effects of proteins related to the transforming growth factor (TGF)-β superfamily. TGF-β itself is a very potent cytokine and has effects on cells in the femtomolar range. Thus, even very small amounts of contaminating TGF-β in purified protein batches may influence the experimental results given that receptors for TGF-β are present. When we attempted to characterize possible receptors for the TGF-β superfamily ligand GDF15, striking similarities between GDF15-induced activities and known TGF-β activities were found. However, differences between batches of GDF15 were a concern and finally led us to the conclusion that the measured effects were caused by TGF-β and not by GDF15. Our results emphasize that purified recombinant proteins must be used with caution and warrant proper controls. Notably, some conclusions made about GDF15 in already published papers may not be supported by the results shown. Awareness about this issue in the scientific community may prevent spreading of false positive results

Activins as dual specificity TGF-β family molecules: SMAD-activation via activin- and BMP-type I receptors

Activins belong to the transforming growth factor (TGF)-β family of multifunctional cytokines and signal via the activin receptors ALK4 or ALK7 to activate the SMAD2/3 pathway. In some cases, activins also signal via the bone morphogenetic protein (BMP) receptor ALK2 causing activation of the SMAD1/5/8 pathway. We here aimed to describe more carefully activation of the two main SMAD branches by activin A and activin B homodimers, and activin AB and activin AC heterodimers. We compared the activin-induced signaling kinetics of ALK4/7-SMAD2/3 and ALK2-SMAD1/5 in a multiple myeloma cell line. Signaling via ALK2 to SMAD1/5 varied more between ligands than signaling via ALK4/ALK7 to SMAD2/3. Interestingly, activin B and activin AB very potently activated SMAD1/5, resembling the activation commonly seen with BMPs. SMAD1/5 was also activated by activins in other cell types, indicating a general mechanism. The antagonist follistatin inhibited signaling by all the tested activins, whereas cerberus specifically antagonized activin B. Interestingly, ALK2-mediated activation of SMAD1/5 was blunted by mutations in the follistatin-binding region of activin A, whereas ALK4-mediated SMAD2/3 activation remained unchanged. Taken together, we propose that activins may be considered dual-specificity TGF-β family members and that this may affect the way activins are targeted clinically

Activins as Dual Specificity TGF-β Family Molecules: SMAD-Activation via Activin- and BMP-Type 1 Receptors

Activins belong to the transforming growth factor (TGF)-β family of multifunctional cytokines and signal via the activin receptors ALK4 or ALK7 to activate the SMAD2/3 pathway. In some cases, activins also signal via the bone morphogenetic protein (BMP) receptor ALK2, causing activation of the SMAD1/5/8 pathway. In this study, we aimed to dissect how activin A and activin B homodimers, and activin AB and AC heterodimers activate the two main SMAD branches. We compared the activin-induced signaling dynamics of ALK4/7-SMAD2/3 and ALK2-SMAD1/5 in a multiple myeloma cell line. Signaling via the ALK2-SMAD1/5 pathway exhibited greater differences between ligands than signaling via ALK4/ALK7-SMAD2/3. Interestingly, activin B and activin AB very potently activated SMAD1/5, resembling the activation commonly seen with BMPs. As SMAD1/5 was also activated by activins in other cell types, we propose that dual specificity is a general mechanism for activin ligands. In addition, we found that the antagonist follistatin inhibited signaling by all the tested activins, whereas the antagonist cerberus specifically inhibited activin B. Taken together, we propose that activins may be considered dual specificity TGF-β family members, critically affecting how activins may be considered and targeted clinically

SMAD-activation by recombinant GDF15 in myeloma cell lines.

<p>A. Phosphorylation of SMAD1/5 or SMAD2 was determined using immunoblotting in IH-1 cells treated with BMP-9 (0.5 ng/mL), activin A (25 ng/mL) or indicated concentrations of GDF15 (100–400 ng/mL) for 1 hour. B. INA-6 cells were treated with GDF15 (200 ng/mL) and the inhibitor SB431542 (0–2.5 μM) for 1 hour before immunoblotting with anti-phospho-SMAD2. C. INA-6 cells were transiently transfected with siRNAs targeting <i>ACVR1B/ALK4</i>, <i>ACVR1C/ALK7</i>, <i>TGFBR1/ALK5</i> and a non-targeting control siRNA. Two days after transfection the cells were treated with GDF15 (200 ng/mL) for 1 hour before immunoblotting with anti-phospho-SMAD2. D. Knock-down of receptors by siRNA in cells used in (C) as shown by QRT-PCR. Gene expression was calculated with the comparative ΔCt-method with <i>GAPDH</i> as housekeeping gene. The error bars indicate SEM of three independent experiments. Asterisks above bars indicate the degree of significance for downregulation of each gene compared to control (*, P≤0.05; **, P≤0.01; and ***, P≤0.001). E. INA-6 cells were treated with GDF15 (100 ng/mL) and a neutralizing TGFBR2 antibody (10–15 μM) for 1 hour before immunoblotting with anti-phospho-SMAD2. F. INA-6 cells were treated with GDF15 (100 ng/mL) and the indicated soluble receptors (5 μg/mL for all except endoglin, which was 1 μg/mL) for 1 hour before immunoblotting with anti-phospho-SMAD2. Antibody staining towards GAPDH was used as loading control for all Western blots. The experiments were performed 2–3 times each. GDF15 used in this figure was from R&D Systems, Lot# EHF1713081.</p