A small-molecule SUMOylation inhibitor activates antitumor immune responses and potentiates immune therapies in preclinical models.

SUMOylation, the covalent conjugation of small ubiquitin-like modifier (SUMO) proteins to protein substrates, has been reported to suppress type I interferon (IFN1) responses. TAK-981, a selective small-molecule inhibitor of SUMOylation, pharmacologically reactivates IFN1 signaling and immune responses against cancers. In vivo treatment of wild-type mice with TAK-981 up-regulated IFN1 gene expression in blood cells and splenocytes. Ex vivo treatment of mouse and human dendritic cells promoted their IFN1-dependent activation, and vaccination studies in mice demonstrated stimulation of antigen cross-presentation and T cell priming in vivo. TAK-981 also directly stimulated T cell activation, driving enhanced T cell sensitivity and response to antigen ex vivo. Consistent with these observations, TAK-981 inhibited growth of syngeneic A20 and MC38 tumors in mice, dependent upon IFN1 signaling and CD8+ T cells, and associated with increased intratumoral T and natural killer cell number and activation. Combination of TAK-981 with anti-PD1 or anti-CTLA4 antibodies improved the survival of mice bearing syngeneic CT26 and MC38 tumors. In conclusion, TAK-981 is a first-in-class SUMOylation inhibitor that promotes antitumor immune responses through activation of IFN1 signaling. TAK-981 is currently being studied in phase 1 clinical trials (NCT03648372, NCT04074330, NCT04776018, and NCT04381650) for the treatment of patients with solid tumors and lymphomas.

Preclinical evaluation of the selective small-molecule UBA1 inhibitor, TAK-243, in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy for which new therapeutic approaches are required. One such potential therapeutic strategy is to target the ubiquitin-like modifier-activating enzyme 1 (UBA1), the initiating enzyme in the ubiquitylation cascade in which proteins are tagged with ubiquitin moieties to regulate their degradation or function. Here, we evaluated TAK-243, a first-in-class UBA1 inhibitor, in preclinical models of AML. In AML cell lines and primary AML samples, TAK-243 induced cell death and inhibited clonogenic growth. In contrast, normal hematopoietic progenitor cells were more resistant. TAK-243 preferentially bound to UBA1 over the related E1 enzymes UBA2, UBA3, and UBA6 in intact AML cells. Inhibition of UBA1 with TAK-243 decreased levels of ubiquitylated proteins, increased markers of proteotoxic stress and DNA damage stress. In vivo, TAK-243 reduced leukemic burden and targeted leukemic stem cells without evidence of toxicity. Finally, we selected populations of AML cells resistant to TAK-243 and identified missense mutations in the adenylation domain of UBA1. Thus, our data demonstrate that TAK-243 targets AML cells and stem cells and support a clinical trial of TAK-243 in this patient population. Moreover, we provide insight into potential mechanisms of acquired resistance to UBA1 inhibitors.

A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment.

The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

Overcoming platinum resistance in preclinical models of ovarian cancer using the neddylation inhibitor MLN4924.

The nearly ubiquitous development of chemoresistant disease remains a major obstacle against improving outcomes for patients with ovarian cancer. In this investigation, we evaluated the preclinical activity of MLN4924, an investigational inhibitor of the NEDD8-activating enzyme, in ovarian cancer cells. Efficacy of MLN4924 both alone and in combination with platinum was assessed. Overall, single-agent MLN4924 exhibited moderate activity in ovarian cancer cell lines. However, the combination of MLN4924 with cisplatin or carboplatin produced synergistic effects in SKOV3 and ES2 cells, as well as in primary ovarian cancer cell lines established from high-grade serous, clear cell, and serous borderline ovarian tumors. The efficacy of cisplatin plus MLN4924 was also evident in several in vitro models of platinum-resistant ovarian cancer. Mechanistically, the combination of cisplatin and MLN4924 was not associated with DNA re-replication, altered platinum-DNA adduct formation, abrogation of FANCD2 monoubiquitination, or CHK1 phosphorylation. An siRNA screen was used to investigate the contribution of each member of the cullin RING ligase (CRL) family of E3 ubiquitin ligases, the best-characterized downstream mediators of MLN4924's biologic effects. Cisplatin-induced cytotoxicity was augmented by depletion of CUL3, and antagonized by siCUL1 in both ES2 and SKOV3 ovarian cancer cells. This investigation identifies inhibition of neddylation as a novel mechanism for overcoming platinum resistance in vitro, and provides a strong rationale for clinical investigations of platinum and MLN4924 combinations in ovarian cancer.

Disrupting protein NEDDylation with MLN4924 is a novel strategy to target cisplatin resistance in ovarian cancer.

PURPOSE: Ovarian cancer has the highest mortality rate of all female reproductive malignancies. Drug resistance is a major cause of treatment failure and novel therapeutic strategies are urgently needed. MLN4924 is a NEDDylation inhibitor currently under investigation in multiple phase I studies. We investigated its anticancer activity in cisplatin-sensitive and -resistant ovarian cancer models. EXPERIMENTAL DESIGN: Cellular sensitivity to MLN4924/cisplatin was determined by measuring viability, clonogenic survival, and apoptosis. The effects of drug treatment on global protein expression, DNA damage, and reactive oxygen species generation were determined. RNA interference established natural born killer/bcl-2-interacting killer (NBK/BIK) as a regulator of therapeutic sensitivity. The in vivo effects of MLN4924/cisplatin on tumor burden and key pharmacodynamics were assessed in cisplatin-sensitive and -resistant xenograft models. RESULTS: MLN4924 possessed significant activity against both cisplatin-sensitive and -resistant ovarian cancer cells and provoked the stabilization of key NEDD8 substrates and regulators of cellular redox status. Notably, MLN4924 significantly augmented the activity of cisplatin against cisplatin-resistant cells, suggesting that aberrant NEDDylation may contribute to drug resistance. MLN4924 and cisplatin cooperated to induce DNA damage, oxidative stress, and increased expression of the BH3-only protein NBK/BIK. Targeted NBK/BIK knockdown diminished the proapoptotic effects of the MLN4924/cisplatin combination. Administration of MLN4924 to mice bearing ovarian tumor xenografts significantly increased the efficacy of cisplatin against both cisplatin-sensitive and -resistant tumors. CONCLUSIONS: Our collective data provide a rationale for the clinical investigation of NEDD8-activating enzyme (NAE) inhibition as a novel strategy to augment cisplatin efficacy in patients with ovarian cancer and other malignancies.

Novel DNA damage checkpoints mediating cell death induced by the NEDD8-activating enzyme inhibitor MLN4924.

MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G(2)-M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G(2) phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924.

Treatment-emergent mutations in NAEß confer resistance to the NEDD8-activating enzyme inhibitor MLN4924.

MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE) in clinical trials for the treatment of cancer. MLN4924 is a mechanism-based inhibitor, with enzyme inhibition occurring through the formation of a tight-binding NEDD8-MLN4924 adduct. In cell and xenograft models of cancer, we identified treatment-emergent heterozygous mutations in the adenosine triphosphate binding pocket and NEDD8-binding cleft of NAEß as the primary mechanism of resistance to MLN4924. Biochemical analyses of NAEß mutants revealed slower rates of adduct formation and reduced adduct affinity for the mutant enzymes. A compound with tighter binding properties was able to potently inhibit mutant enzymes in cells. These data provide rationales for patient selection and the development of next-generation NAE inhibitors designed to overcome treatment-emergent NAEß mutations.

Identification and application of NEDD8 E1 inhibitors.

The NEDD8 conjugation pathway is initiated by the NEDD8 E1, also known as NEDD8 activating enzyme (NAE) or APPBP1/UBA3 (Gong, Yeh. J Biol Chem 274:12063-12042, 1999). The best described biological role for NEDD8 conjugation is to regulate the activity of the cullin RING ligase (CRL) family of ubiquitin E3 ligases (Gong, Yeh. J Biol Chem 274:12063-12042, 1999). In this way, the NEDD8 pathway regulates the turnover of a subset of ubiquitin proteasome system (UPS) substrates that are essential for cancer cell growth and survival (Soucy, Smith, Milhollen. Nature 458:732-737, 2009). We recently initiated clinical trials with a first-in-class small molecule inhibitor of NAE for the treatment of cancer (Soucy, Smith, Milhollen. Nature 458:732-737, 2009). Here we describe a biochemical and cell-based assay used to identify NAE inhibitors and monitor inhibition of the NEDD8 conjugation pathway.

Mechanistic studies of substrate-assisted inhibition of ubiquitin-activating enzyme by adenosine sulfamate analogues.

Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin via an adenylate intermediate and catalyzes its transfer to a ubiquitin-conjugating enzyme (E2). MLN4924 is an adenosine sulfamate analogue that was identified as a selective, mechanism-based inhibitor of NEDD8-activating enzyme (NAE), another E1 enzyme, by forming a NEDD8-MLN4924 adduct that tightly binds at the active site of NAE, a novel mechanism termed substrate-assisted inhibition (Brownell, J. E., Sintchak, M. D., Gavin, J. M., Liao, H., Bruzzese, F. J., Bump, N. J., Soucy, T. A., Milhollen, M. A., Yang, X., Burkhardt, A. L., Ma, J., Loke, H. K., Lingaraj, T., Wu, D., Hamman, K. B., Spelman, J. J., Cullis, C. A., Langston, S. P., Vyskocil, S., Sells, T. B., Mallender, W. D., Visiers, I., Li, P., Claiborne, C. F., Rolfe, M., Bolen, J. B., and Dick, L. R. (2010) Mol. Cell 37, 102-111). In the present study, substrate-assisted inhibition of human UAE (Ube1) by another adenosine sulfamate analogue, 5'-O-sulfamoyl-N(6)-[(1S)-2,3-dihydro-1H-inden-1-yl]-adenosine (Compound I), a nonselective E1 inhibitor, was characterized. Compound I inhibited UAE-dependent ATP-PP(i) exchange activity, caused loss of UAE thioester, and inhibited E1-E2 transthiolation in a dose-dependent manner. Mechanistic studies on Compound I and its purified ubiquitin adduct demonstrate that the proposed substrate-assisted inhibition via covalent adduct formation is entirely consistent with the three-step ubiquitin activation process and that the adduct is formed via nucleophilic attack of UAE thioester by the sulfamate group of Compound I after completion of step 2. Kinetic and affinity analysis of Compound I, MLN4924, and their purified ubiquitin adducts suggest that both the rate of adduct formation and the affinity between the adduct and E1 contribute to the overall potency. Because all E1s are thought to use a similar mechanism to activate their cognate ubiquitin-like proteins, the substrate-assisted inhibition by adenosine sulfamate analogues represents a promising strategy to develop potent and selective E1 inhibitors that can modulate diverse biological pathways.

Inhibition of NEDD8-activating enzyme induces rereplication and apoptosis in human tumor cells consistent with deregulating CDT1 turnover.

Loss of NEDD8-activating enzyme (NAE) function by siRNA knockdown or inhibition by the small molecule NAE inhibitor MLN4924 leads to increased steady-state levels of direct Cullin-RING ligase (CRL) substrates by preventing their ubiquitination and proteasome-dependent degradation. Many of these CRL substrates are involved in cell cycle progression, including a critical DNA replication licensing factor CDT1. Cell cycle analysis of asynchronous and synchronous cultures after NAE inhibition revealed effects on cell cycle distribution and activation of DNA break repair signaling pathways similar to that reported for CDT1 overexpression. The siRNA knockdown of cullins critical for the turnover of CDT1 recapitulated the aberrant rereplication phenotype while CDT1 knockdown was suppressing. Although NAE inhibition leads to deregulation of many CRL substrates, these data demonstrate that CDT1 accumulation mediates the DNA rereplication phenotype resulting from loss of NAE function. DNA rereplication is an unrecoverable cellular insult and the small molecule inhibitor MLN4924, currently in phase I trials, represents an unprecedented opportunity to explore this mechanism of cytotoxicity for the treatment of cancer.

NEDD8-targeting drug MLN4924 elicits DNA rereplication by stabilizing Cdt1 in S phase, triggering checkpoint activation, apoptosis, and senescence in cancer cells.

MLN4924 is a first-in-class experimental cancer drug that inhibits the NEDD8-activating enzyme, thereby inhibiting cullin-RING E3 ubiquitin ligases and stabilizing many cullin substrates. The mechanism by which MLN4924 inhibits cancer cell proliferation has not been defined, although it is accompanied by DNA rereplication and attendant DNA damage. Here we show that stabilization of the DNA replication factor Cdt1, a substrate of cullins 1 and 4, is critical for MLN4924 to trigger DNA rereplication and inhibit cell proliferation. Even only 1 hour of exposure to MLN4924, which was sufficient to elevate Cdt1 for 4-5 hours, was found to be sufficient to induce DNA rereplication and to activate apoptosis and senescence pathways. Cells in S phase were most susceptible, suggesting that MLN4924 will be most toxic on highly proliferating cancers. Although MLN4924-induced cell senescence seems to be dependent on induction of p53 and its downstream effector p21(Waf1), we found that p53(-/-) and p21(-/-) cells were even more susceptible than wild-type cells to MLN4924. Our results suggested that apoptosis, not senescence, might be more important for the antiproliferative effect of MLN4924. Furthermore, our findings show that transient exposure to this new investigational drug should be useful for controlling p53-negative cancer cells, which often pose significant clinical challenge.

MLN4924, a NEDD8-activating enzyme inhibitor, is active in diffuse large B-cell lymphoma models: rationale for treatment of NF-{kappa}B-dependent lymphoma.

MLN4924 is a potent and selective small molecule NEDD8-activating enzyme (NAE) inhibitor. In most cancer cells tested, inhibition of NAE leads to induction of DNA rereplication, resulting in DNA damage and cell death. However, in preclinical models of activated B cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), we show that MLN4924 induces an alternative mechanism of action. Treatment of ABC DLBCL cells with MLN4924 resulted in rapid accumulation of pIkappaBalpha, decrease in nuclear p65 content, reduction of nuclear factor-kappaB (NF-kappaB) transcriptional activity, and G(1) arrest, ultimately resulting in apoptosis induction, events consistent with potent NF-kappaB pathway inhibition. Treatment of germinal-center B cell-like (GCB) DLBCL cells resulted in an increase in cellular Cdt-1 and accumulation of cells in S-phase, consistent with cells undergoing DNA rereplication. In vivo administration of MLN4924 to mice bearing human xenograft tumors of ABC- and GCB-DLBCL blocked NAE pathway biomarkers and resulted in complete tumor growth inhibition. In primary human tumor models of ABC-DLBCL, MLN4924 treatment resulted in NF-kappaB pathway inhibition accompanied by tumor regressions. This work describes a novel mechanism of targeted NF-kappaB pathway modulation in DLBCL and provides strong rationale for clinical development of MLN4924 against NF-kappaB-dependent lymphomas.

Substrate-assisted inhibition of ubiquitin-like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924 forms a NEDD8-AMP mimetic in situ.

The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.

The NEDD8 Conjugation Pathway and Its Relevance in Cancer Biology and Therapy.

Cancer cells depend on signals that promote cell cycle progression and prevent programmed cell death that would otherwise result from cumulative, aberrant stress. These activities require the temporally controlled destruction of specific intracellular proteins by the ubiquitin-proteasome system (UPS). To a large extent, the control points in this process include a family of E3 ubiquitin ligases called cullin-RING ligases (CRLs). The ligase activity of these multicomponent complexes requires modification of the cullin protein situated at their core with a ubiquitin-like protein called NEDD8. Neddylation results in conformational rearrangements within the CRL, which are necessary for ubiquitin transfer to a substrate. The NEDD8 pathway thus has a critical role in mediating the ubiquitination of numerous CRL substrate proteins involved in cell cycle progression and survival including the DNA replication licensing factor Cdt-1, the NF-κB transcription factor inhibitor pIκBα, and the cell cycle regulators cyclin E and p27. The initial step required for attachment of NEDD8 to a cullin is catalyzed by the E1, NEDD8-activating enzyme (NAE). The first-in-class inhibitor of NAE, MLN4924, has been shown to block the activity of NAE and prevent the subsequent neddylation of cullins. Preclinical studies have demonstrated antitumor activity in various solid tumors and hematological malignancies, and preliminary clinical data have shown the anticipated pharmacodynamic effects in humans. Here, we review the NEDD8 pathway, its importance in cancer, and the therapeutic potential of NAE inhibition.

An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.

The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin-proteasome system might prove useful for the treatment of human malignancies. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer.

The angiogenesis inhibitor NM-3 is active against human NSCLC xenografts alone and in combination with docetaxel.

The novel isocoumarin 2-(8-hydroxy-6-methoxy-1-oxo-1 H-2-benzopyran-3-yl) propionic acid (NM-3) has completed phase I clinical evaluation as an orally bioavailable angiogenesis inhibitor. NM-3 directly kills both endothelial and tumor cells in vitro at low mM concentrations and is effective in the treatment of diverse human tumor xenografts in mice. The present work has assessed the activity of NM-3 against human non-small-cell lung cancer (NSCLC) cells when used alone and in combination with docetaxel. The results demonstrate that NM-3 decreases clonogenic survival of NSCLC cells at clinically achievable concentrations. The results also demonstrate that NM-3 is effective in the treatment of NSCLC (A549, NCI-H460) tumor xenografts in mice. Moreover, NM-3 potentiated the antitumor activity of docetaxel against NSCLC xenografts without increasing toxicity. Our findings indicate that NM-3 may be effective alone or in combination with docetaxel in the treatment of patients with NSCLC.

2-(8-Hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl)propionic acid, a small molecule isocoumarin, potentiates dexamethasone-induced apoptosis of human multiple myeloma cells.

2-(8-Hydroxy-6-methoxy-1-oxo-1Eta-2-benzopyran-3-yl)propionic acid (NM-3) is a small molecule isocoumarin derivative that has recently entered clinical trials as an orally bioavailable anticancer agent. NM-3 induces lethality of human carcinoma cells by both apoptotic and nonapoptotic mechanisms and potentiates the effects of cytotoxic chemotherapeutic agents. The present studies have evaluated the effects of NM-3 on human multiple myeloma (MM) cells. The results demonstrate that NM-3 potentiates dexamethasone-induced killing of both dexamethasone-sensitive MM1.S and dexamethasone-resistant RPMI8226 and U266 MM cells. We show that NM-3 enhances dexamethasone-induced release of the mitochondrial apoptogenic factors cytochrome c and Smac/DIABLO. The results also demonstrate that NM-3 enhances dexamethasone-induced activation of the intrinsic caspase-9->caspase-3 apoptotic pathway. In concert with these results, NM-3 potentiates dexamethasone-induced apoptosis of MM1.S cells. Moreover, NM-3 acts synergistically with dexamethasone in inducing apoptosis of the dexamethasone-resistant RPMI8226 and U266 MM cells. These findings indicate that NM-3 may be effective in combination with dexamethasone in the treatment of MM.

Antineoplastic effects of chemotherapeutic agents are potentiated by NM-3, an inhibitor of angiogenesis.

Antiangiogenic therapy, although effective in shrinking tumors, has not yet been established as a standalone treatment for cancer. This therapeutic limitation can be overcome by combining angiogenesis inhibitors with chemotherapeutic agents. NM-3, a small molecule isocoumarin, is a recently discovered angiogenesis inhibitor. Here we demonstrate that NM-3 inhibits the proliferation of human umbilical vein endothelial cells in vitro, at concentrations 10-fold less than those required to inhibit normal fibroblasts or tumor cells (HT29, MKN28, and MCF-7). NM-3 alone inhibits endothelial sprouting and tube formation in vitro. The results also show that synergistic antiproliferative activity is observed when human umbilical vein endothelial cells are treated with NM-3 in combination with 5-fluorouracil. The effects of treatment with NM-3 and various chemotherapeutic agents were also evaluated in tumor xenografts. The results demonstrate that combined treatment with NM-3 and chemotherapeutic agents significantly reduced mean tumor volume compared with either treatment alone, with no effects on body weight changes. Taken together, these findings demonstrate that NM-3 is a well-tolerated angiogenesis inhibitor that significantly increases the efficacy of existing antineoplastic agents.