Preclinical Characterization of the Anti-Leukemia Activity of the CD33/CD16a/NKG2D Immune-Modulating TriNKET® CC-96191.

Increasing efforts are focusing on natural killer (NK) cell immunotherapies for AML. Here, we characterized CC-96191, a novel CD33/CD16a/NKG2D immune-modulating TriNKET®. CC-96191 simultaneously binds CD33, NKG2D, and CD16a, with NKG2D and CD16a co-engagement increasing the avidity for, and activation of, NK cells. CC-96191 was broadly active against human leukemia cells in a strictly CD33-dependent manner, with maximal efficacy requiring the co-engagement of CD16a and NKG2D. A frequent CD33 single nucleotide polymorphism, R69G, reduced CC-96191 potency but not maximal activity, likely because of reduced CD33 binding. Similarly, the potency, but not the maximal activity, of CC-96191 was reduced by high concentrations of soluble CD33; in contrast, the soluble form of the NKG2D ligand MICA did not impact activity. In the presence of CD33+ AML cells, CC-96191 activated NK cells but not T cells; while maximum anti-AML efficacy was similar, soluble cytokine levels were 10- to >100-fold lower than with a CD33/CD3 bispecific antibody. While CC-96191-mediated cytolysis was not affected by ABC transporter proteins, it was reduced by anti-apoptotic BCL-2 family proteins. Finally, in patient marrow specimens, CC-96191 eliminated AML cells but not normal monocytes, suggesting selectivity of TriNKET-induced cytotoxicity toward neoplastic cells. Together, these findings support the clinical exploration of CC-96191 as in NCT04789655.

An optimized IL-12-Fc expands its therapeutic window, achieving strong activity against mouse tumors at tolerable drug doses.

BACKGROUND: Interleukin-12 (IL-12) has emerged as one of the most potent cytokines for tumor immunotherapy due to its ability to induce interferon γ (IFNγ) and polarize Th1 responses. Clinical use of IL-12 has been limited by a short half-life and narrow therapeutic index. METHODS: We generated a monovalent, half-life-extended IL-12-Fc fusion protein, mDF6006, engineered to retain the high potency of native IL-12 while significantly expanding its therapeutic window. In vitro and in vivo activity of mDF6006 was tested against murine tumors. To translate our findings, we developed a fully human version of IL-12-Fc, designated DF6002, which we characterized in vitro on human cells and in vivo in cynomolgus monkeys in preparation for clinical trials. FINDINGS: The extended half-life of mDF6006 modified the pharmacodynamic profile of IL-12 to one that was better tolerated systemically while vastly amplifying its efficacy. Mechanistically, mDF6006 led to greater and more sustained IFNγ production than recombinant IL-12 without inducing high, toxic peak serum concentrations of IFNγ. We showed that mDF6006's expanded therapeutic window allowed for potent anti-tumor activity as single agent against large immune checkpoint blockade-resistant tumors. Furthermore, the favorable benefit-risk profile of mDF6006 enabled effective combination with PD-1 blockade. Fully human DF6002, similarly, demonstrated an extended half-life and a protracted IFNγ profile in non-human primates. CONCLUSION: An optimized IL-12-Fc fusion protein increased the therapeutic window of IL-12, enhancing anti-tumor activity without concomitantly increasing toxicity. FUNDING: This research was funded by Dragonfly Therapeutics.

Structures of activin ligand traps using natural sets of type I and type II TGFß receptors.

The 30+ unique ligands of the TGFß family signal by forming complexes using different combinations of type I and type II receptors. Therapeutically, the extracellular domain of a single receptor fused to an Fc molecule can effectively neutralize subsets of ligands. Increased ligand specificity can be accomplished by using the extracellular domains of both the type I and type II receptor to mimic the naturally occurring signaling complex. Here, we report the structure of one "type II-type I-Fc" fusion, ActRIIB-Alk4-Fc, in complex with two TGFß family ligands, ActA, and GDF11, providing a snapshot of this therapeutic platform. The study reveals that extensive contacts are formed by both receptors, replicating the ternary signaling complex, despite the inherent low affinity of Alk4. Our study shows that low-affinity type I interactions support altered ligand specificity and can be visualized at the molecular level using this platform.

Functionally diverse heteromeric traps for ligands of the transforming growth factor-ß superfamily.

Ligands of the transforming growth factor-ß (TGF-ß) superfamily are important targets for therapeutic intervention but present challenges because they signal combinatorially and exhibit overlapping activities in vivo. To obtain agents capable of sequestering multiple TGF-ß superfamily ligands with novel selectivity, we generated soluble, heterodimeric ligand traps by pairing the extracellular domain (ECD) of the native activin receptor type IIB (ActRIIB) alternately with the ECDs of native type I receptors activin receptor-like kinase 4 (ALK4), ALK7, or ALK3. Systematic analysis of these heterodimeric constructs by surface plasmon resonance, and comparison with their homodimeric counterparts, revealed that each type I receptor partner confers a distinct ligand-binding profile to the heterodimeric construct. Additional characterization in cell-based reporter gene assays confirmed that the heterodimeric constructs possessed different profiles of signaling inhibition in vitro, which translated into altered patterns of pharmacological activity when constructs were administered systemically to wild-type mice. Our results detail a versatile platform for the modular recombination of naturally occurring receptor domains, giving rise to inhibitory ligand traps that could aid in defining the physiological roles of TGF-ß ligand sets or be directed therapeutically to human diseases arising from dysregulated TGF-ß superfamily signaling.

ActRIIB:ALK4-Fc alleviates muscle dysfunction and comorbidities in murine models of neuromuscular disorders.

Patients with neuromuscular disorders suffer from a lack of treatment options for skeletal muscle weakness and disease comorbidities. Here, we introduce as a potential therapeutic agent a heterodimeric ligand-trapping fusion protein, ActRIIB:ALK4-Fc, which comprises extracellular domains of activin-like kinase 4 (ALK4) and activin receptor type IIB (ActRIIB), a naturally occurring pair of type I and II receptors belonging to the TGF-ß superfamily. By surface plasmon resonance (SPR), ActRIIB:ALK4-Fc exhibited a ligand binding profile distinctly different from that of its homodimeric variant ActRIIB-Fc, sequestering ActRIIB ligands known to inhibit muscle growth but not trapping the vascular regulatory ligand bone morphogenetic protein 9 (BMP9). ActRIIB:ALK4-Fc and ActRIIB-Fc administered to mice exerted differential effects - concordant with SPR results - on vessel outgrowth in a retinal explant assay. ActRIIB:ALK4-Fc induced a systemic increase in muscle mass and function in wild-type mice and in murine models of Duchenne muscular dystrophy (DMD), amyotrophic lateral sclerosis (ALS), and disuse atrophy. Importantly, ActRIIB:ALK4-Fc improved neuromuscular junction abnormalities in murine models of DMD and presymptomatic ALS and alleviated acute muscle fibrosis in a DMD model. Furthermore, in combination therapy ActRIIB:ALK4-Fc increased the efficacy of antisense oligonucleotide M12-PMO on dystrophin expression and skeletal muscle endurance in an aged DMD model. ActRIIB:ALK4-Fc shows promise as a therapeutic agent, alone or in combination with dystrophin rescue therapy, to alleviate muscle weakness and comorbidities of neuromuscular disorders.

Modified activin receptor IIB ligand trap mitigates ineffective erythropoiesis and disease complications in murine ß-thalassemia.

In ß-thalassemia, unequal production of α- and ß-globin chains in erythroid precursors causes apoptosis and inhibition of late-stage erythroid differentiation, leading to anemia, ineffective erythropoiesis (IE), and dysregulated iron homeostasis. Here we used a murine model of ß-thalassemia intermedia (Hbb(th1/th1) mice) to investigate effects of a modified activin receptor type IIB (ActRIIB) ligand trap (RAP-536) that inhibits Smad2/3 signaling. In Hbb(th1/th1) mice, treatment with RAP-536 reduced overactivation of Smad2/3 in splenic erythroid precursors. In addition, treatment of Hbb(th1/th1) mice with RAP-536 reduced α-globin aggregates in peripheral red cells, decreased the elevated reactive oxygen species present in erythroid precursors and peripheral red cells, and alleviated anemia by promoting differentiation of late-stage erythroid precursors and reducing hemolysis. Notably, RAP-536 treatment mitigated disease complications of IE, including iron overload, splenomegaly, and bone pathology, while reducing erythropoietin levels, improving erythrocyte morphology, and extending erythrocyte life span. These results implicate signaling by the transforming growth factor-ß superfamily in late-stage erythropoiesis and reveal potential of a modified ActRIIB ligand trap as a novel therapeutic agent for thalassemia syndrome and other red cell disorders characterized by IE.

Transforming growth factor-ß superfamily ligand trap ACE-536 corrects anemia by promoting late-stage erythropoiesis.

Erythropoietin (EPO) stimulates proliferation of early-stage erythrocyte precursors and is widely used for the treatment of chronic anemia. However, several types of EPO-resistant anemia are characterized by defects in late-stage erythropoiesis, which is EPO independent. Here we investigated regulation of erythropoiesis using a ligand-trapping fusion protein (ACE-536) containing the extracellular domain of human activin receptor type IIB (ActRIIB) modified to reduce activin binding. ACE-536, or its mouse version RAP-536, produced rapid and robust increases in erythrocyte numbers in multiple species under basal conditions and reduced or prevented anemia in murine models. Unlike EPO, RAP-536 promoted maturation of late-stage erythroid precursors in vivo. Cotreatment with ACE-536 and EPO produced a synergistic erythropoietic response. ACE-536 bound growth differentiation factor-11 (GDF11) and potently inhibited GDF11-mediated Smad2/3 signaling. GDF11 inhibited erythroid maturation in mice in vivo and ex vivo. Expression of GDF11 and ActRIIB in erythroid precursors decreased progressively with maturation, suggesting an inhibitory role for GDF11 in late-stage erythroid differentiation. RAP-536 treatment also reduced Smad2/3 activation, anemia, erythroid hyperplasia and ineffective erythropoiesis in a mouse model of myelodysplastic syndromes (MDS). These findings implicate transforming growth factor-ß (TGF-ß) superfamily signaling in erythroid maturation and identify ACE-536 as a new potential treatment for anemia, including that caused by ineffective erythropoiesis.

A soluble bone morphogenetic protein type IA receptor increases bone mass and bone strength.

Diseases such as osteoporosis are associated with reduced bone mass. Therapies to prevent bone loss exist, but there are few that stimulate bone formation and restore bone mass. Bone morphogenetic proteins (BMPs) are members of the TGFß superfamily, which act as pleiotropic regulators of skeletal organogenesis and bone homeostasis. Ablation of the BMPR1A receptor in osteoblasts increases bone mass, suggesting that inhibition of BMPR1A signaling may have therapeutic benefit. The aim of this study was to determine the skeletal effects of systemic administration of a soluble BMPR1A fusion protein (mBMPR1A-mFc) in vivo. mBMPR1A-mFc was shown to bind BMP2/4 specifically and with high affinity and prevent downstream signaling. mBMPR1A-mFc treatment of immature and mature mice increased bone mineral density, cortical thickness, trabecular bone volume, thickness and number, and decreased trabecular separation. The increase in bone mass was due to an early increase in osteoblast number and bone formation rate, mediated by a suppression of Dickkopf-1 expression. This was followed by a decrease in osteoclast number and eroded surface, which was associated with a decrease in receptor activator of NF-κB ligand (RANKL) production, an increase in osteoprotegerin expression, and a decrease in serum tartrate-resistant acid phosphatase (TRAP5b) concentration. mBMPR1A treatment also increased bone mass and strength in mice with bone loss due to estrogen deficiency. In conclusion, mBMPR1A-mFc stimulates osteoblastic bone formation and decreases bone resorption, which leads to an increase in bone mass, and offers a promising unique alternative for the treatment of bone-related disorders.

Specificity and structure of a high affinity activin receptor-like kinase 1 (ALK1) signaling complex.

Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-ß superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-ß family ligands BMP9 and BMP10 as well as the three type II TGF-ß family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies.

A novel therapeutic approach to treating obesity through modulation of TGFß signaling.

Obesity results from disproportionately high energy intake relative to energy expenditure. Many therapeutic strategies have focused on the intake side of the equation, including pharmaceutical targeting of appetite and digestion. An alternative approach is to increase energy expenditure through physical activity or adaptive thermogenesis. A pharmacological way to increase muscle mass and hence exercise capacity is through inhibition of the activin receptor type IIB (ActRIIB). Muscle mass and strength is regulated, at least in part, by growth factors that signal via ActRIIB. Administration of a soluble ActRIIB protein comprised of a form of the extracellular domain of ActRIIB fused to a human Fc (ActRIIB-Fc) results in a substantial muscle mass increase in normal mice. However, ActRIIB is also present on and mediates the action of growth factors in adipose tissue, although the function of this system is poorly understood. In the current study, we report the effect of ActRIIB-Fc to suppress diet-induced obesity and linked metabolic dysfunctions in mice fed a high-fat diet. ActRIIB-Fc induced a brown fat-like thermogenic gene program in epididymal white fat, as shown by robustly increased expression of the thermogenic genes uncoupling protein 1 and peroxisomal proliferator-activated receptor-γ coactivator 1α. Finally, we identified multiple ligands capable of reducing thermogenesis that represent likely target ligands for the ActRIIB-Fc effects on the white fat depots. These data demonstrate that novel therapeutic ActRIIB-Fc improves obesity and obesity-linked metabolic disease by both increasing skeletal muscle mass and by inducing a gene program of thermogenesis in the white adipose tissues.

Soluble endoglin specifically binds bone morphogenetic proteins 9 and 10 via its orphan domain, inhibits blood vessel formation, and suppresses tumor growth.

Endoglin (CD105), a transmembrane protein of the transforming growth factor ß superfamily, plays a crucial role in angiogenesis. Mutations in endoglin result in the vascular defect known as hereditary hemorrhagic telangiectasia (HHT1). The soluble form of endoglin was suggested to contribute to the pathogenesis of preeclampsia. To obtain further insight into its function, we cloned, expressed, purified, and characterized the extracellular domain (ECD) of mouse and human endoglin fused to an immunoglobulin Fc domain. We found that mouse and human endoglin ECD-Fc bound directly, specifically, and with high affinity to bone morphogenetic proteins 9 and 10 (BMP9 and BMP10) in surface plasmon resonance (Biacore) and cell-based assays. We performed a function mapping analysis of the different domains of endoglin by examining their contributions to the selectivity and biological activity of the protein. The BMP9/BMP10 binding site was localized to the orphan domain of human endoglin composed of the amino acid sequence 26-359. We established that endoglin and type II receptors bind to overlapping sites on BMP9. In the in vivo chick chorioallantoic membrane assay, the mouse and the truncated human endoglin ECD-Fc both significantly reduced VEGF-induced vessel formation. Finally, murine endoglin ECD-Fc acted as an anti-angiogenic factor that decreased blood vessel sprouting in VEGF/FGF-induced angiogenesis in in vivo angioreactors and reduced the tumor burden in the colon-26 mouse tumor model. Together our findings indicate an important role of soluble endoglin ECD in the regulation of angiogenesis and highlight efficacy of endoglin-Fc as a potential anti-angiogenesis therapeutic agent.

A soluble activin receptor type IIb prevents the effects of androgen deprivation on body composition and bone health.

Androgen deprivation, a consequence of hypogonadism, certain cancer treatments, or normal aging in men, leads to loss of muscle mass, increased adiposity, and osteoporosis. In the present study, using a soluble chimeric form of activin receptor type IIB (ActRIIB) we sought to offset the adverse effects of androgen deprivation on muscle, adipose tissue, and bone. Castrated (ORX) or sham-operated (SHAM) mice received either TBS [vehicle-treated (VEH)] or systemic administration of ActRIIB-mFc, a soluble fusion protein comprised of a form of the extracellular domain of ActRIIB fused to a murine IgG2aFc subunit. In vivo body composition imaging demonstrated that ActRIIB-mFc treatment results in increased lean tissue mass of 23% in SHAM mice [19.02 +/- 0.42 g (VEH) versus 23.43 +/- 0.35 g (ActRIIB-mFc), P < 0.00001] and 26% in ORX mice [15.59 +/- 0.26 g (VEH) versus 19.78 +/- 0.26 g (ActRIIB-mFc), P < 0.00001]. Treatment also caused a decrease in adiposity of 30% in SHAM mice [5.03 +/- 0.48 g (VEH) versus 3.53 +/- 0.19 g (ActRIIB-mFc), NS] and 36% in ORX mice [7.12 +/- 0.53 g (VEH) versus 4.57 +/- 0.28 g (ActRIIB-mFc), P < 0.001]. These changes were also accompanied by altered serum levels of leptin, adiponectin, and insulin, as well as by prevention of steatosis (fatty liver) in ActRIIB-mFc-treated ORX mice. Finally, ActRIIB-mFc prevented loss of bone mass in ORX mice as assessed by whole body dual x-ray absorptiometry and micro-computed tomography of proximal tibias. The data demonstrate that treatment with ActRIIB-mFc restored muscle mass, adiposity, and bone quality to normal levels in a mouse model of androgen deprivation, thereby alleviating multiple adverse consequences of such therapy.

Characterization of the ligand binding functionality of the extracellular domain of activin receptor type IIb.

The single transmembrane domain serine/threonine kinase activin receptor type IIB (ActRIIB) has been proposed to bind key regulators of skeletal muscle mass development, including the ligands GDF-8 (myostatin) and GDF-11 (BMP-11). Here we provide a detailed kinetic characterization of ActRIIB binding to several low and high affinity ligands using a soluble activin receptor type IIB-Fc chimera (ActRIIB.Fc). We show that both GDF-8 and GDF-11 bind the extracellular domain of ActRIIB with affinities comparable with those of activin A, a known high affinity ActRIIB ligand, whereas BMP-2 and BMP-7 affinities for ActRIIB are at least 100-fold lower. Using site-directed mutagenesis, we demonstrate that ActRIIB binds GDF-11 and activin A in different ways such as, for example, substitutions in ActRIIB Leu(79) effectively abolish ActRIIB binding to activin A yet not to GDF-11. Native ActRIIB has four isoforms that differ in the length of the C-terminal portion of their extracellular domains. We demonstrate that the C terminus of the ActRIIB extracellular domain is crucial for maintaining biological activity of the ActRIIB.Fc receptor chimera. In addition, we show that glycosylation of ActRIIB is not required for binding to activin A or GDF-11. Together, our findings reveal binding specificity and activity determinants of the ActRIIB receptor that combine to effect specificity in the activation of distinct signaling pathways.

ALK1-Fc inhibits multiple mediators of angiogenesis and suppresses tumor growth.

Activin receptor-like kinase-1 (ALK1) is a type I, endothelial cell-specific member of the transforming growth factor-beta superfamily of receptors known to play an essential role in modulating angiogenesis and vessel maintenance. In the present study, we sought to examine the angiogenic and tumorigenic effects mediated upon the inhibition of ALK1 signaling using a soluble chimeric protein (ALK1-Fc). Of 29 transforming growth factor-beta-related ligands screened by surface plasmon resonance, only bone morphogenetic protein (BMP9) and BMP10 displayed high-affinity binding to ALK1-Fc. In cell-based assays, ALK1-Fc inhibited BMP9-mediated Id-1 expression in human umbilical vein endothelial cells and inhibited cord formation by these cells on a Matrigel substrate. In a chick chorioallantoic membrane assay, ALK1-Fc reduced vascular endothelial growth factor-, fibroblast growth factor-, and BMP10-mediated vessel formation. The growth of B16 melanoma explants was also inhibited significantly by ALK1-Fc in this assay. Finally, ALK1-Fc treatment reduced tumor burden in mice receiving orthotopic grafts of MCF7 mammary adenocarcinoma cells. These data show the efficacy of chimeric ALK1-Fc proteins in mitigating vessel formation and support the view that ALK1-Fc is a powerful antiangiogenic agent capable of blocking vascularization.

A soluble activin type IIB receptor improves function in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurologic disease characterized by progressive weakness that results in death within a few years of onset by respiratory failure. Myostatin is a member of the TGF-beta superfamily that is predominantly expressed in muscle and acts as a negative regulator of muscle growth. Attenuating myostatin has previously been shown to produce increased muscle mass and strength in normal and disease animal models. In this study, a mouse model of ALS (SOD1(G93A) transgenic mice) was treated with a soluble activin receptor, type IIB (ActRIIB.mFc) which is a putative endogenous signaling receptor for myostatin in addition to other ligands of the TGF-beta superfamily. ActRIIB.mFc treatment produces a delay in the onset of weakness, an increase in body weight and grip strength, and an enlargement of muscle size whether initiated pre-symptomatically or after symptom onset. Treatment with ActRIIB.mFc did not increase survival or neuromuscular junction innervation in SOD1(G93A) transgenic mice. Pharmacologic treatment with ActRIIB.mFc was superior in all measurements to genetic deletion of myostatin in SOD1(G93A) transgenic mice. The improved function of SOD1(G93A) transgenic mice following treatment with ActRIIB.mFc is encouraging for the development of TGF-beta pathway inhibitors to increase muscle strength in patients with ALS.

A soluble activin type IIA receptor induces bone formation and improves skeletal integrity.

Diseases that affect the regulation of bone turnover can lead to skeletal fragility and increased fracture risk. Members of the TGF-beta superfamily have been shown to be involved in the regulation of bone mass. Activin A, a TGF-beta signaling ligand, is present at high levels in bone and may play a role in the regulation of bone metabolism. Here we demonstrate that pharmacological blockade of ligand signaling through the high affinity receptor for activin, type II activin receptor (ActRIIA), by administration of the soluble extracellular domain of ActRIIA fused to a murine IgG2a-Fc, increases bone formation, bone mass, and bone strength in normal mice and in ovariectomized mice with established bone loss. These observations support the development of this pharmacological strategy for the treatment of diseases with skeletal fragility.

Visualization of protein interactions in living cells using bimolecular fluorescence complementation (BiFC) analysis.

Protein interactions integrate stimuli from different signaling pathways and developmental programs. Bimolecular fluorescence complementation (BiFC) analysis has been developed for visualization of protein interactions in living cells. This approach is based on complementation between two fragments of a fluorescent protein when they are brought together by an interaction between proteins fused to the fragments, and it enables visualization of the subcellular locations of protein interactions in the normal cellular environment. It can be used for the analysis of many protein interactions and does not require information about the structures of the interaction partners. A multicolor BiFC approach has been developed for simultaneous visualization of interactions with multiple alternative partners in the same cell, based on complementation between fragments of engineered fluorescent proteins that produce bimolecular fluorescent complexes with distinct spectral characteristics. This enables comparison of subcellular distributions of different protein complexes in the same cell and allows analysis of competition between mutually exclusive interaction partners.

Visualization of protein interactions in living cells using bimolecular fluorescence complementation (BiFC) analysis.

Protein interactions integrate stimuli from different signaling pathways and developmental programs. Bimolecular fluorescence complementation (BiFC) analysis has been developed for visualization of protein interactions in living cells. This approach is based on complementation between two fragments of a fluorescent protein when they are brought together by an interaction between proteins fused to the fragments, and it enables visualization of the subcellular locations of protein interactions in the normal cellular environment. It can be used for the analysis of many protein interactions and does not require information about the structures of the interaction partners. A multicolor BiFC approach has been developed for simultaneous visualization of interactions with multiple alternative partners in the same cell, based on complementation between fragments of engineered fluorescent proteins that produce bimolecular fluorescent complexes with distinct spectral characteristics. This enables comparison of subcellular distributions of different protein complexes in the same cell and allows analysis of competition between mutually exclusive interaction partners.

Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells.

Myc and Mad family proteins play opposing roles in the control of cell growth and proliferation. We have visualized the subcellular locations of complexes formed by Myc/Max/Mad family proteins using bimolecular fluorescence complementation (BiFC) analysis. Max was recruited to different subnuclear locations by interactions with Myc versus Mad family members. Complexes formed by Max with Mxi1, Mad3, or Mad4 were enriched in nuclear foci, whereas complexes formed with Myc were more uniformly distributed in the nucleoplasm. Mad4 was localized to the cytoplasm when it was expressed separately, and Mad4 was recruited to the nucleus through dimerization with Max. The cytoplasmic localization of Mad4 was determined by a CRM1-dependent nuclear export signal located near the amino terminus. We compared the relative efficiencies of complex formation among Myc, Max, and Mad family proteins in living cells using multicolor BiFC analysis. Max formed heterodimers with the basic helix-loop-helix leucine zipper (bHLHZIP) domain of Myc (bMyc) more efficiently than it formed homodimers. Replacement of two amino acid residues in the leucine zipper of Max reversed the relative efficiencies of homo- and heterodimerization in cells. Surprisingly, Mad3 formed complexes with Max less efficiently than bMyc, whereas Mad4 formed complexes with Max more efficiently than bMyc. The distinct subcellular locations and the differences between the efficiencies of dimerization with Max indicate that Mad3 and Mad4 are likely to modulate transcription activation by Myc at least in part through distinct mechanisms.

Both Max and TFE3 cooperate with Smad proteins to bind the plasminogen activator inhibitor-1 promoter, but they have opposite effects on transcriptional activity.

Transforming growth factor (TGF)-beta regulates gene expression in large part through combinatorial interactions between members of the Smad family and other transcription factors. The basic helix-loop-helix leucine zipper (bHLHZIP) protein TFE3 and Smad3 synergistically activate transcription of the plasminogen activator inhibitor-1 (PAI-1) as well as other genes. We investigated interactions among different bHLHZIP and Smad family proteins. TFE3, TFEB, and Max associated with Smad3 and Smad4 in the absence of DNA and at the PE2.1 element of the PAI-1 promoter. These interactions were mediated by the leucine zipper and MH1 regions of the respective proteins. No interactions were observed with the E47 bHLH family protein. Chimeric proteins, in which leucine zippers from bHLHZIP or bZIP proteins were fused to heterologous bHLH domains, associated with Smad proteins both in the absence of DNA and at the PE2.1 element. The kinetics of bHLHZIP and Smad protein binding at the PE2.1 element were examined using surface plasmon resonance analysis. TFE3 exhibited cooperative DNA binding with Smad proteins, whereas no cooperativity was observed between E47 and Smads. Max inhibited transcription activation by Smad3 and TGF-beta at the PAI-1 promoter, whereas TFE3 and TFEB stimulated transcription activation. These results suggest that Smad family proteins can interact with several bHLHZIP proteins, resulting in different transcriptional outcomes.