MARCH Proteins Mediate Responses to Antitumor Antibodies.

CD98, which is required for the rapid proliferation of both normal and cancer cells, and MET, the hepatocyte growth factor receptor, are potential targets for therapeutic antitumor Abs. In this study, we report that the antiproliferative activity of a prototype anti-CD98 Ab, UM7F8, is due to Ab-induced membrane-associated ring CH (MARCH) E3 ubiquitin ligase-mediated ubiquitination and downregulation of cell surface CD98. MARCH1-mediated ubiquitination of CD98 is required for UM7F8's capacity to reduce CD98 surface expression and its capacity to inhibit the proliferation of murine T cells. Similarly, CD98 ubiquitination is required for UM7F8's capacity to block the colony-forming ability of murine leukemia-initiating cells. To test the potential generality of the paradigm that MARCH E3 ligases can mediate the antiproliferative response to antitumor Abs, we examined the potential effects of MARCH proteins on responses to emibetuzumab, an anti-MET Ab currently in clinical trials for various cancers. We report that MET surface expression is reduced by MARCH1, 4, or 8-mediated ubiquitination and that emibetuzumab-induced MET ubiquitination contributes to its capacity to downregulate MET and inhibit human tumor cell proliferation. Thus, MARCH E3 ligases can act as cofactors for antitumor Abs that target cell surface proteins, suggesting that the MARCH protein repertoire of cells is a determinant of their response to such Abs.

CD98hc facilitates B cell proliferation and adaptive humoral immunity.

The proliferation of antigen-specific lymphocytes and resulting clonal expansion are essential for adaptive immunity. We report here that B cell-specific deletion of the heavy chain of CD98 (CD98hc) resulted in lower antibody responses due to total suppression of B cell proliferation and subsequent plasma cell formation. Deletion of CD98hc did not impair early B cell activation but did inhibit later activation of the mitogen-activated protein kinase Erk1/2 and downregulation of the cell cycle inhibitor p27. Reconstitution of CD98hc-deficient B cells with CD98hc mutants showed that the integrin-binding domain of CD98hc was required for B cell proliferation but that the amino acid-transport function of CD98hc was dispensable for this. Thus, CD98hc supports integrin-dependent rapid proliferation of B cells. We propose that the advantage of adaptive immunity favored the appearance of CD98hc in vertebrates.

Integrin Activation Controls Regulatory T Cell-Mediated Peripheral Tolerance.

Maintenance of the regulatory T (Treg) cell pool is essential for peripheral tolerance and prevention of autoimmunity. Integrins, heterodimeric transmembrane proteins consisting of α and ß subunits that mediate cell-to-cell and cell-to-extracellular matrix interactions, play an important role in facilitating Treg cell contact-mediated suppression. In this article, we show that integrin activation plays an essential, previously unappreciated role in maintaining murine Treg cell function. Treg cell-specific loss of talin, a ß integrin-binding protein, or expression of talin(L325R), a mutant that selectively abrogates integrin activation, resulted in lethal systemic autoimmunity. This dysfunction could be attributed, in part, to a global dysregulation of the Treg cell transcriptome. Activation of integrin α4ß1 led to increased suppressive capacity of the Treg cell pool, suggesting that modulating integrin activation on Treg cells may be a useful therapeutic strategy for autoimmune and inflammatory disorders. Taken together, these results reveal a critical role for integrin-mediated signals in controlling peripheral tolerance by virtue of maintaining Treg cell function.

Cutting Edge: Loss of T Cell RIAM Precludes Conjugate Formation with APC and Prevents Immune-Mediated Diabetes.

Rap1-interacting adaptor molecule (RIAM) is a Rap1 effector that mediates the recruitment of talin to integrins, thereby supporting their activation. In this study, we investigated the role of RIAM in an adoptive transfer model for type I diabetes and report that RIAM expression in T cells is necessary for diabetes development. Loss of RIAM did not prevent lymphocyte recruitment to draining lymph nodes 24 h after transfer, but it was required for Ag-driven proliferation and cytotoxic killing. RIAM is recruited to immune synapses along with talin and LFA-1, and loss of RIAM profoundly suppresses Ag-dependent conjugate formation in primary naive and effector T cells. These data identify the requirement of RIAM for formation of immunological synapses and in resulting T cell functions in autoimmunity. Moreover, because RIAM-null mice are healthy, fertile, and display no bleeding abnormalities, our results identify RIAM and its regulators as potential targets for therapies of T cell-mediated autoimmunity.

Tissue Factor Prothrombotic Activity Is Regulated by Integrin-arf6 Trafficking.

OBJECTIVE: Coagulation initiation by tissue factor (TF) is regulated by cellular inhibitors, cell surface availability of procoagulant phosphatidylserine, and thiol-disulfide exchange. How these mechanisms contribute to keeping TF in a noncoagulant state and to generating prothrombotic TF remain incompletely understood. APPROACH AND RESULTS: Here, we study the activation of TF in primary macrophages by a combination of pharmacological, genetic, and biochemical approaches. We demonstrate that primed macrophages effectively control TF cell surface activity by receptor internalization. After cell injury, ATP signals through the purinergic receptor P2rx7 induce release of TF+ microvesicles. TF cell surface availability for release onto microvesicles is regulated by the GTPase arf6 associated with integrin α4ß1. Furthermore, microvesicles proteome analysis identifies activation of Gαi2 as a participating factor in the release of microvesicles with prothrombotic activity in flowing blood. ATP not only prevents TF and phosphatidylserine internalization but also induces TF conversion to a conformation with high affinity for its ligand, coagulation factor VII. Although inhibition of dynamin-dependent internalization also exposes outer membrane procoagulant phosphatidylserine, the resulting TF+ microvesicles distinctly lack protein disulfide isomerase and high affinity TF and fail to produce fibrin strands typical for microvesicles generated by thrombo-inflammatory P2rx7 activation. CONCLUSIONS: These data show that procoagulant phospholipid exposure is not sufficient and that TF affinity maturation is required to generate prothrombotic microvesicles from a variety of cell types. These findings are significant for understanding TF-initiated thrombosis and should be considered in designing functional microvesicles-based diagnostic approaches.

Ubiquitylation of CD98 limits cell proliferation and clonal expansion.

CD98 heavy chain (SLC3A2) facilitates lymphocyte clonal expansion that enables adaptive immunity; however, increased expression of CD98 is also a feature of both lymphomas and leukemias and represents a potential therapeutic target in these diseases. CD98 is transcriptionally regulated and ectopic expression of the membrane-associated RING-CH (MARCH) E3 ubiquitin ligases MARCH1 or MARCH8 leads to ubiquitylation and lysosomal degradation of CD98. Here, we examined the potential role of ubiquitylation in regulating CD98 expression and cell proliferation. We report that blocking ubiquitylation by use of a catalytically inactive MARCH or by creating a ubiquitylation-resistant CD98 mutant, prevents MARCH-induced CD98 downregulation in HeLa cells. March1-null T cells display increased CD98 expression. Similarly, T cells expressing ubiquitylation-resistant CD98 manifest increased proliferation in vitro and clonal expansion in vivo. Thus, ubiquitylation and the resulting downregulation of CD98 can limit cell proliferation and clonal expansion.

Interaction of kindlin-2 with integrin ß3 promotes outside-in signaling responses by the αVß3 vitronectin receptor.

The bidirectional signaling and hemostatic functions of platelet αIIbß3 are regulated by kindlin-3 through interactions with the ß3 cytoplasmic tail. Little is known about kindlin regulation of the related "vitronectin receptor," αVß3. These relationships were investigated in endothelial cells, which express αVß3 and kindlin-2 endogenously. "ß3ΔRGT" knock-in mice lack the 3 C-terminal ß3 tail residues, whereas in "ß3/ß1(EGK)" mice, RGT is replaced by the corresponding residues of ß1. The wild-type ß3 tail pulled down kindlin-2 and c-Src in vitro, whereas ß3ΔRGT bound neither protein and ß3/ß1(EGK) bound kindlin-2, but not c-Src. ß3ΔRGT endothelial cells, but not ß3/ß1(EGK) endothelial cells, exhibited migration and spreading defects on vitronectin and reduced sprouting in 3-dimensional fibrin. Short hairpin RNA silencing of kindlin-2, but not c-Src, blocked sprouting by ß3 wild-type endothelial cells. Moreover, defective sprouting by ß3ΔRGT endothelial cells could be rescued by conditional, forced interaction of αVß3ΔRGT with kindlin-2. Stimulation of ß3ΔRGT endothelial cells led to normal extracellular ligand binding to αVß3, pin-pointing their defect to one of outside-in αVß3 signaling. ß3ΔRGT mice, but not ß3/ß1(EGK) mice, exhibited defects in both developmental and tumor angiogenesis, responses that require endothelial cell function. Thus, the ß3/kindlin-2 interaction promotes outside-in αVß3 signaling selectively, with biological consequences in vivo.

Endothelial Cells Require CD98 for Efficient Angiogenesis-Brief Report.

OBJECTIVE: CD98 regulates integrin signaling and is critical for tumor cell proliferation. It is also expressed on endothelial cells (EC), but its role in angiogenesis is unclear. APPROACH AND RESULTS: We used specific genetic targeting and antibody blockade approaches to examine the function of CD98 in EC proliferation, blood vessel growth, and tumor angiogenesis. It is upregulated on angiogenic ECs, and EC-specific deletion of CD98 in mice inhibited tumor growth, retinal angiogenesis, and EC proliferation. Reconstitution with CD98 mutants showed that integrin and CD98 interaction is necessary for EC survival and growth. Moreover, anti-CD98 treatment inhibited vessel formation and reversed EC-assisted tumor growth. CONCLUSIONS: Our findings demonstrate a requirement for CD98 in EC growth and suggest that CD98-specific reagents could have a dual anticancer effect: directly by inhibiting tumor cell proliferation and indirectly by preventing tumor angiogenesis.

Antitumor activity of an anti-CD98 antibody.

CD98 is expressed on several tissue types and specifically upregulated on fast-cycling cells undergoing clonal expansion. Various solid (e.g., nonsmall cell lung carcinoma) as well as hematological malignancies (e.g., acute myeloid leukemia) overexpress CD98. We have identified a CD98-specific mouse monoclonal antibody that exhibits potent preclinical antitumor activity against established lymphoma tumor xenografts. Additionally, the humanized antibody designated IGN523 demonstrated robust tumor growth inhibition in leukemic cell-line derived xenograft models and was as efficacious as standard of care carboplatin in patient-derived nonsmall lung cancer xenografts. In vitro studies revealed that IGN523 elicited strong ADCC activity, induced lysosomal membrane permeabilization and inhibited essential amino acid transport function, ultimately resulting in caspase-3 and -7-mediated apoptosis of tumor cells. IGN523 is currently being evaluated in a Phase I clinical trial for acute myeloid leukemia (NCT02040506). Furthermore, preclinical data support the therapeutic potential of IGN523 in solid tumors.

CD98 at the crossroads of adaptive immunity and cancer.

Adaptive immunity, a vertebrate specialization, adds memory and exquisite specificity to the basic innate immune responses present in invertebrates while conserving metabolic resources. In adaptive immunity, antigenic challenge requires extremely rapid proliferation of rare antigen-specific lymphocytes to produce large, clonally expanded effector populations that neutralize pathogens. Rapid proliferation and resulting clonal expansion are dependent on CD98, a protein whose well-conserved orthologs appear restricted to vertebrates. Thus, CD98 supports lymphocyte clonal expansion to enable protective adaptive immunity, an advantage that could account for the presence of CD98 in vertebrates. CD98 supports lymphocyte clonal expansion by amplifying integrin signals that enable proliferation and prevent apoptosis. These integrin-dependent signals can also provoke cancer development and invasion, anchorage-independence and the rapid proliferation of tumor cells. CD98 is highly expressed in many cancers and contributes to formation of tumors in experimental models. Strikingly, vertebrates, which possess highly conserved CD98 proteins, CD98-binding integrins and adaptive immunity, also display propensity towards invasive and metastatic tumors. In this Commentary, we review the roles of CD98 in lymphocyte biology and cancer. We suggest that the CD98 amplification of integrin signaling in adaptive immunity provides survival benefits to vertebrates, which, in turn, bear the price of increased susceptibility to cancer.

Loss of T cell CD98 H chain specifically ablates T cell clonal expansion and protects from autoimmunity.

CD98 H chain (4F2 Ag, Slc3a2) was discovered as a lymphocyte-activation Ag. Deletion of CD98 H chain in B cells leads to complete failure of B cell proliferation, plasma cell formation, and Ab secretion. In this study, we examined the role of T cell CD98 in cell-mediated immunity and autoimmune disease pathogenesis by specifically deleting it in murine T cells. Deletion of T cell CD98 prevented experimental autoimmune diabetes associated with dramatically reduced T cell clonal expansion. Nevertheless, initial T cell homing to pancreatic islets was unimpaired. In sharp contrast to B cells, CD98-null T cells showed only modestly impaired Ag-driven proliferation and nearly normal homeostatic proliferation. Furthermore, these cells were activated by Ag, leading to cytokine production (CD4) and efficient cytolytic killing of targets (CD8). The integrin-binding domain of CD98 was necessary and sufficient for full clonal expansion, pointing to a role for adhesive signaling in T cell proliferation and autoimmune disease. When we expanded CD98-null T cells in vitro, they adoptively transferred diabetes, establishing that impaired clonal expansion was responsible for protection from disease. Thus, the integrin-binding domain of CD98 is required for Ag-driven T cell clonal expansion in the pathogenesis of an autoimmune disease and may represent a useful therapeutic target.

Fibronectin, DHPS and SLC3A2 Signaling Cooperate to Control Tumor Spheroid Growth, Subcellular eIF5A1/2 Distribution and CDK4/6 Inhibitor Resistance.

Extracellular matrix (ECM) protein expression/deposition within and stiffening of the breast cancer microenvironment facilitates disease progression and correlates with poor patient survival. However, the mechanisms by which ECM components control tumorigenic behaviors and responses to therapeutic intervention remain poorly understood. Fibronectin (FN) is a major ECM protein controlling multiple processes. In this regard, we previously reported that DHPS-dependent hypusination of eIF5A1/2 is necessary for fibronectin-mediated breast cancer metastasis and epithelial to mesenchymal transition (EMT). Here, we explored the clinical significance of an interactome generated using hypusination pathway components and markers of intratumoral heterogeneity. Solute carrier 3A2 (SLC3A2 or CD98hc) stood out as an indicator of poor overall survival among patients with basal-like breast cancers that express elevated levels of DHPS. We subsequently discovered that blockade of DHPS or SLC3A2 reduced triple negative breast cancer (TNBC) spheroid growth. Interestingly, spheroids stimulated with exogenous fibronectin were less sensitive to inhibition of either DHPS or SLC3A2 - an effect that could be abrogated by dual DHPS/SLC3A2 blockade. We further discovered that a subset of TNBC cells responded to fibronectin by increasing cytoplasmic localization of eIF5A1/2. Notably, these fibronectin-induced subcellular localization phenotypes correlated with a G0/G1 cell cycle arrest. Fibronectin-treated TNBC cells responded to dual DHPS/SLC3A2 blockade by shifting eIF5A1/2 localization back to a nucleus-dominant state, suppressing proliferation and further arresting cells in the G2/M phase of the cell cycle. Finally, we observed that dual DHPS/SLC3A2 inhibition increased the sensitivity of both Rb-negative and -positive TNBC cells to the CDK4/6 inhibitor palbociclib. Taken together, these data identify a previously unrecognized mechanism through which extracellular fibronectin controls cancer cell tumorigenicity by modulating subcellular eIF5A1/2 localization and provides prognostic/therapeutic utility for targeting the cooperative DHPS/SLC3A2 signaling axis to improve breast cancer treatment responses.

Integrin-associated proteins as potential therapeutic targets.

SUMMARY: Integrins are adhesion receptors important for hematopoiesis, leukocyte trafficking, and formation of immunological synapses; hence, they may provide targets for therapeutic intervention in leukocyte-driven pathologies. Blocking integrin-ligand binding is one strategy for inhibiting integrins; however, a complete loss of integrin function can lead to mechanism-based toxicities. Because integrin alpha and beta subunits interact with a variety of other proteins to receive and transmit cellular signals, targeting these integrin-associated proteins may utilize alternative sites for intervention that lead to therapies with fewer side effects. This review summarizes integrin-associated proteins in leukocytes and focuses on four of these proteins with perceived therapeutic potential. Specific mutations in the alpha4 integrin cytoplasmic tail block or enforce binding to paxillin and thus modulate integrin signaling required for efficient cell migration. Similarly, the association of RAPL(NORE1B) with beta2 integrins may participate in adhesive and migratory events in leukocytes. The beta integrin cytoplasmic tail-binding protein talin is critical for increasing the affinity of integrins (activation), and blockade of talin binding can prevent leukocyte arrest on the endothelium. Finally, the membrane protein CD98 mediates beta1 and beta3 integrin signaling and may be involved in leukocyte functions. Identification of biologically important interactions of integrins and signaling proteins can thus pave the way to new strategies for manipulating leukocyte functions.

Enhanced Integrin α4ß1-Mediated Adhesion Contributes to a Mobilization Defect of Endothelial Progenitor Cells in Diabetes.

Diabetes is associated with a deficit of circulating endothelial progenitor cells (EPCs), which has been attributed to their defective mobilization from the bone marrow. The basis for this mobilization defect is not completely understood, and we sought to determine if hyperglycemic conditions enhanced EPC adhesion. We found that culturing EPCs in high glucose media increased adhesion to bone marrow stromal cells. This enhanced adhesion was associated with decreased expression of protein kinase A regulatory subunit 1ß (PRKAR1ß), activation of protein kinase A (PKA), and phosphorylation of α4-integrin on serine 988. This potentiated adhesion was reversed by treatment with a PKA inhibitor, overexpression of PRKAR1ß, or expression of a phosphorylation-defective α4-integrin variant (α4[S988A]). Using a model of type 1 diabetes, we showed that α4(S988A)-expressing mice have more circulating EPCs than their wild-type counterparts. Moreover, diabetic α4(S988A) mice demonstrate enhanced revascularization after hind limb ischemia. Thus, we have identified a novel signaling mechanism activating PKA in diabetes (downregulation of an inhibitory regulatory subunit) that leads to deficits of circulating EPCs and impaired vascular repair, which could be reversed by α4-integrin mutation.

CD98-Mediated Adhesive Signaling Enables the Establishment and Propagation of Acute Myelogenous Leukemia.

Acute myelogenous leukemia (AML) is an aggressive disease associated with drug resistance and relapse. To improve therapeutic strategies, it is critical to better understand the mechanisms that underlie AML progression. Here we show that the integrin binding glycoprotein CD98 plays a central role in AML. CD98 promotes AML propagation and lethality by driving engagement of leukemia cells with their microenvironment and maintaining leukemic stem cells. Further, delivery of a humanized anti-CD98 antibody blocks growth of patient-derived AML, highlighting the importance of this pathway in human disease. These findings indicate that microenvironmental interactions are key regulators of AML and that disrupting these signals with targeted inhibitors such as CD98 antibodies may be a valuable therapeutic approach for adults and children with this disease.

Fine-tuning Tumor Immunity with Integrin Trans-regulation.

Inefficient T-cell homing to tissues limits adoptive T-cell immunotherapy of solid tumors. αLß2 and α4ß1 integrins mediate trafficking of T cells into tissues via engagement of ICAM-1 and VCAM-1, respectively. Inhibiting protein kinase A (PKA)-mediated phosphorylation of α4 integrin in cells results in an increase in αLß2-mediated migration on mixed ICAM-1-VCAM-1 substrates in vitro, a phenomenon termed "integrin trans-regulation." Here, we created an α4(S988A)-bearing mouse, which precludes PKA-mediated α4 phosphorylation, to examine the effect of integrin trans-regulation in vivo. The α4(S988A) mouse exhibited a dramatic and selective increase in migration of lymphocytes, but not myeloid cells, to sites of inflammation. Importantly, we found that the α4(S988A) mice exhibited a marked increase in T-cell entry into and reduced growth of B16 melanomas, consistent with antitumor roles of infiltrating T cells and progrowth functions of tumor-associated macrophages. Thus, increased α4 trans-regulation of αLß2 integrin function biases leukocyte emigration toward lymphocytes relative to myeloid cells and enhances tumor immunity.

CD98 is a potential target for ablating B cell clonal expansion and autoantibody in multiple sclerosis.

Current B cell-directed therapies for multiple sclerosis impact multiple B cell functions. CD98hc enables B cell clonal expansion and antibody production. I probed the relative importance of autoantibody secretion vs. other B cell functions in MS and targeted CD98hc as a possible therapeutic strategy. I report that the loss of CD98hc function in B cells largely prevents autoantibody production while preserving antigen-presenting and T cell-directing capacities. Mice lacking CD98hc in B cells are protected from EAE; importantly this is overcome with autoantibody-containing plasma. Thus CD98hc blockade is a possible avenue to treat MS by inhibiting clonal expansion and autoantibody.

Recruitment and activation of macrophages by pathogenic CD4 T cells in type 1 diabetes: evidence for involvement of CCR8 and CCL1.

Adoptive transfer of diabetogenic CD4 Th1 T cell clones into young NOD or NOD.scid recipients rapidly induces onset of diabetes and also provides a system for analysis of the pancreatic infiltrate. Although many reports have suggested a role for macrophages in the inflammatory response, there has been little direct characterization of macrophage activity in the pancreas. We showed previously that after migration to the pancreas, diabetogenic CD4 T cell clones produce a variety of inflammatory cytokines and chemokines, resulting in the recruitment of macrophages. In this study, we investigated mechanisms by which macrophages are recruited and activated by T cells. Analysis of infiltrating cells after adoptive transfer by the diabetogenic T cell clone BDC-2.5 indicates that large numbers of cells staining for both F4/80 and CD11b are recruited into the pancreas where they are activated to make IL-1beta, TNF-alpha, and NO, and express the chemokine receptors CCR5, CXCR3, and CCR8. Diabetogenic CD4 T cell clones produce several inflammatory chemokines in vitro, but after adoptive transfer we found that the only chemokine that could be detected ex vivo was CCL1. These results provide the first evidence that CCR8/CCL1 interaction may play a role in type 1 diabetes through macrophage recruitment and activation.

Effector function of diabetogenic CD4 Th1 T cell clones: a central role for TNF-alpha.

Effector function of T cells in autoimmune diabetes has been widely studied with mixed populations of lymphoid T cells stimulated ex vivo, but this approach does not permit evaluation of the contribution by a single T cell clone in the inflammatory site during pathogenesis. We have investigated cytokine production both in vitro and in vivo in a panel of diabetogenic CD4 Th1 T cell clones derived from the NOD mouse. SuperArray analysis showed a common pattern of mRNA expression for inflammatory cytokines and receptors in vitro after TCR stimulation. Ex vivo intracellular cytokine staining demonstrated that two important inflammatory cytokines, IFN-gamma and TNF-alpha, were being made by these T cells recovered from the pancreas 6 days following adoptive transfer. TNF-alpha produced in the pancreas by pathogenic T cell clones and recruited macrophages was not the membrane-bound form. Secreted TNF-alpha can lead to production of multiple inflammatory chemokines, as were observed in the pathogenic clones by intracellular cytokine staining. Our results not only define the nature of an inflammatory cytokine response critical to development of diabetes, but also suggest its role in the regulation of other events during pathogenesis induced by CD4 T cells. Similar analyses in other models demonstrated that disease induced by CD4 T cell clones closely resembles spontaneous autoimmune diabetes in which both CD4 and CD8 T cells are required. Thus, cloned T cells in effect amplify effector function of T cells which otherwise may be difficult to detect without ex vivo stimulation.