We have completed a screen of ∼10 6 small molecules to identify compounds that induce cell death in multipotent glioblastoma multiforme (GBM) cancer stem cells (CSCs). This resulted in the identification of a hit class (RIPGBM) that was found to induce apoptosis in GBM CSCs in a cell type-selective manner. Metabolite profiling experiments led to the identification of a proapoptotic derivative of RIPGBM (cRIPGBM), which was found to be selectively formed in GBM CSCs. Mechanistic studies revealed that cRIPGBM induces apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) in a mode that results in the formation of a proapoptotic RIPK2/caspase 1 complex. In a physiologically relevant orthotopic intracranial GBM CSC tumor xenograft mouse model, RIPGBM was found to significantly inhibit in vivo tumor formation.

Glioblastoma multiforme (GBM; grade IV astrocytoma) is the most prevalent and aggressive form of primary brain cancer. A subpopulation of multipotent cells termed GBM cancer stem cells (CSCs) play a critical role in tumor initiation, tumor maintenance, metastasis, drug resistance, and recurrence following surgery. Here we report the identification of a small molecule, termed RIPGBM, from a cell-based chemical screen that selectively induces apoptosis in multiple primary patient-derived GBM CSC cultures. The cell type-dependent selectivity of this compound appears to arise at least in part from redox-dependent formation of a proapoptotic derivative, termed cRIPGBM, in GBM CSCs. cRIPGBM induces caspase 1-dependent apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) and acting as a molecular switch, which reduces the formation of a prosurvival RIPK2/TAK1 complex and increases the formation of a proapoptotic RIPK2/caspase 1 complex. In an orthotopic intracranial GBM CSC tumor xenograft mouse model, RIPGBM was found to significantly suppress tumor formation in vivo. Our chemical genetics-based approach has identified a drug candidate and a potential drug target that provide an approach to the development of treatments for this devastating disease.

Stem cell mechanisms have been established to play critical roles in the development, progression, and recurrence of multiple cancer types (1⇓⇓⇓–5). One such case is the infiltrative brain cancer glioblastoma multiforme (GBM). For recurrent GBM, even with aggressive treatment, the median survival rate is presently 12–15 mo (6, 7). GBM cancer stem cells (CSCs) were among the first cancer stem cell populations to be isolated and characterized from solid tumors (8⇓⇓–11). These cells share features in common with neural stem cells (NPCs), namely the expression of NSC markers (e.g., Nestin and SOX2), the capacity for self-renewal, and the ability to differentiate and give rise to cell types of glial and neuronal lineages in response to inductive cues (8, 9, 11, 12). The highly infiltrative nature of GBM tumors is attributed to the ability of GBM CSCs to migrate within the brain, a feature also shared with NSCs (13). Further, GBM CSCs are thought to contribute to drug resistance (14, 15) and have even been demonstrated to have the potential to give rise to endothelial cells that enable tumor vascularization (16). Thus, efforts to develop new therapeutic strategies for the treatment of GBM have recently focused on targeting this stem cell population (17).

Here we have used an unbiased large-scale screening approach to identify drug-like small molecules that induce apoptosis in GBM CSCs in a cell type-selective manner. The use of expanded populations of proliferative nonstem/multipotent GBM cells for such screens is of limited utility, as such cell lines fail to recapitulate the in vitro and in vivo properties, including drug sensitivity, of the original tumor (9, 18⇓⇓⇓–22). In contrast, in vitro and in vivo preclinical models using cultured human tumor-derived GBM CSCs more accurately recapitulate the biology of the disease (9, 15, 23⇓⇓–26). Under defined serum-free adherent culture conditions, these patient-derived GBM CSCs can be expanded as stable cell lines that retain their in vitro differentiation potential, as well as their in vivo engraftment, tumor formation, and migration potential (17). In the present study, we have used patient-derived GBM CSC cultures to identify a potential drug candidate for the treatment of this devastating disease.

Results

RIPGBM Is a Selective Inducer of Apoptosis in GBM CSCs. We have previously described an adapted system for the adherent in vitro expansion of patient-derived GBM CSCs that was successfully used to perform a kinomewide lentiviral RNAi screen in 384-well assay format (24). Importantly, these primary cell lines retain stem cell-like characteristics and differentiation properties, as well as the ability to engraft and form tumors that recapitulate the highly heterogeneous and infiltrative characteristics of high-grade gliomas in relevant rodent disease models (24). Here, we used one of these GBM CSC lines, termed GBM-1, to establish a robust 1,536-well format luciferase-based survival assay and completed a large-scale screen of ∼106 drug-like small molecules (1 μM) with the goal of identifying novel compounds that are selectively toxic to chemoresistant GBM CSCs (Fig. 1A). Fig. 1. A cell-based phenotypic screening approach identifies the small molecule RIPGBM, which induces apoptosis in GBM CSCs in a cell type-selective manner. (A) Schematic representation of the screening approach used to identify molecules that selectively induce apoptosis in GBM CSCs and structure of RIPGBM. (B) Bright-field image of GBM CSCs (GBM-1) or nondiseased human NPCs treated with RIPGBM (1 μM) for 96 h. (C) Immunofluorescent analysis of caspase 3 cleavage in GBM CSCs (GBM-1) following treatment with RIPGBM (1 μM) for 24 h. (D) Cell survival curves for GBM CSCs (GBM-1), human NPCs, primary human astrocyte cells, and primary HLFs treated with RIPGBM for 48 h. Values shown are mean ± SD. Confirmed primary hits (∼8,000, plate-based robust Z-score ≤−3) were evaluated by using a laser scanning cytometer-based cell death imaging assay (Acumen eX3; TTP Labtech; Fig. 1A). Selective cytotoxicity was determined by evaluating the primary hits at two concentrations (5 μM and 1 μM), using a panel of two patient-derived GBM CSC lines (GBM-1 and GBM-5, ref. 24) and 3 nondiseased cell types [primary human astrocytes, WA09 human ES cell-derived NPCs, and primary human lung fibroblasts (HLFs)]. Compounds found to kill GBM CSC lines with greater than fivefold toxicity index compared with control cell types were further characterized. A caspase 3/7 activation assay (Caspase-Glo 3/7; Promega) was used to evaluate the mechanism of induced cell death. The most potent and selective compound identified from these assays, termed RIPGBM (Fig. 1A), was found to selectively induce apoptosis in GBM CSC cell lines with an observed EC 50 of ≤500 nM and a selectivity index of at least fivefold compared with control cell types (Fig. 1 B–D and SI Appendix, Table S1). For comparison, the observed EC 50 for the standard-of-care drug temozolomide (TMZ) used to treat GBM is ≥20 μM for the same GBM CSC lines (SI Appendix, Table S1). Moreover, human ES-derived NSCs are at least twofold more sensitive to TMZ than GBM CSCs (SI Appendix, Fig. S1). The latter observation is in agreement with published data showing that GBM CSCs are resistant to chemotherapeutic agents (27, 28). We confirmed the ability of RIPGBM to induce apoptosis in GBM CSCs by immunofluorescent analysis using a cleaved caspase 3-recognizing antibody (Fig. 1C).

RIPGBM Is Converted to an Apoptosis-Inducing Derivative Selectively in GBM CSCs. Quinone-containing drugs represent a large and diverse class of antitumor agents approved for clinical use. For many of these drugs, cell type-selective reduction to reactive hydroquinone species has been shown to play a key role in their antitumor activity (29). As the naphthoquinone core of RIPGBM is found in various substrates for quinone oxidoreductase enzymes (e.g., NQO1), which generate hydroquinone or semihydroquinone species and are frequently up-regulated in various cancer cell types (30), we hypothesized that either of these events could lead to the selective formation of a proapoptotic species in GBM CSCs. We used an MS-based metabolite identification approach to evaluate whether RIPGBM undergoes selective conversion to a +1 (i.e., semihydroquinone) or +2 (i.e., hydroquinone) species in GBM CSCs. Cell pellets and growth media of GBM CSC and control cell types were extracted following drug treatment and subjected to quantitative high-resolution Orbitrap LC-MS analysis (Thermo Fisher Scientific). The formation of +1 or +2 species was not observed from samples derived from either cell type. However, RIPGBM was found to undergo significant conversion to a −18 species, potentially corresponding to a dehydration event, in GBM CSCs (∼50% in 24 h), and this conversion was found to occur selectively in diseased cells (Fig. 2A). An accurate mass measurement of 411.1506 m/z (SI Appendix, Fig. S2) and MS2 fragmentation data (SI Appendix, Fig. S3) were consistent with the cyclized imidazolium species, termed cRIPGBM, shown in Fig. 2B. In parallel experiments, it was found that, whereas RIPGBM is relatively stable in culture media (t 1/2 > 4 d), addition of the reducing agent NaBH 4 results in near-instantaneous formation of the −18 cRIPGBM species (SI Appendix, Fig. S4). Presumably, reduction of the quinone moiety makes the benzylic amine more nucleophilic, resulting in its addition to the acetamido group and subsequent loss of water. The putative cyclic derivative was synthesized and evaluated against a panel of GBM CSCs and nondisease cell lines. The cyclized species cRIPGBM was found to induce apoptosis in GBM CSCs with enhanced potency compared with the parent compound following 48 h of drug treatment (e.g., EC 50 = 68 nM vs. 220 nM in GBM-1), but with reduced selectivity compared with control cells (Fig. 2C and SI Appendix, Table S1). As such, the mechanism by which RIPGBM selectively induces cell death in GBM CSCs likely involves a redox-dependent prodrug-like process involving cell type-dependent formation of a proapoptotic species. Fig. 2. A metabolite of RIPGBM induces apoptosis in GBM CSCs by interacting with RIPK2. (A) Orbitrap MS-based metabolite identification studies in GBM-1 (GBM CSC) or primary HLF cells incubated with RIPGBM (1 μM) for 0, 12, 24, or 48 h. (B) Structure of the cyclized RIPGBM metabolite cRIPGBM generated in GBM CSCs. (C) Cell survival curves for GBM CSCs (GBM-1), human NPCs, primary human astrocyte cells, and HLFs treated with cRIPGBM for 48 h. (D) Structure of PAP reagent cRIPGBM-PAP. (E) In vitro binding of cRIPGBM-PAP to recombinant human full-length RIPK2 protein in the presence or absence of competition using underivatized cRIPGBM or RIPGBM. (F) Domain structure of RIPK2 and in vitro binding of cRIPGBM-PAP to recombinant full-length, truncated kinase domain, or truncated CARD domain human RIPK2 protein. (G) cRIPGBM-induced apoptosis in GBM-1 GBM CSCs following shRNA-mediated RIPK2 gene knockdown. Values shown are mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001).

cRIPGBM Targets RIPK2 in GBM CSCs. Structure–activity relationship studies revealed sites off the RIPGBM core tolerant of modification. This information was used to design and synthesize photoactivatable affinity probe (PAP) reagents. MS-based proteomic target identification studies involving incubation and photo-cross-linking with live GBM CSCs using cRIPGBM-PAP (Fig. 2D), which retained activity (EC 50 ) within fivefold of that of the parent molecule, resulted in the identification of receptor-interacting serine-threonine kinase 2 (RIPK2) as a candidate protein target for cRIPGBM (SI Appendix, Fig. S5). Based on its known role in regulating apoptosis and the degree of identified peptide coverage observed for this potential biomolecular target (SI Appendix, Fig. S5B), RIPK2 was explored in detail to examine its role in the mechanism of cRIPGBM-induced apoptosis. In in vitro binding assays involving purified recombinant proteins, cRIPGBM- PAP was observed to interact with full-length RIPK2 protein in a concentration-dependent manner at concentrations of 100 nM and greater (apparent K d of ∼2.3 µM; SI Appendix, Fig. S5 C and D). A 25-fold molar excess of cRIPGBM was found to inhibit this interaction, demonstrating the specificity of this interaction (Fig. 2E). Consistent with playing a relevant role in the induction of apoptosis, RIPK2 consists of an N-terminal autophosphorylation kinase domain and a C-terminal caspase 1 recruitment CARD domain separated by a domain of unknown function (Fig. 2F) (31). In vitro binding experiments suggest that cRIPGBM interacts with the kinase domain of this protein (Fig. 2F). However, the compound was not found to inhibit kinase activity at concentrations lower than 10 µM in an RIPK2 enzymatic assay (SI Appendix, Fig. S6). To further validate RIPK2 as the molecular target of cRIPGBM in GBM CSCs, we measured compound-induced cytotoxicity and caspase activation in cells in which RIPK2 levels were reduced by using shRNA. Consistent with the mechanistic relevance of this target, as demonstrated by the observed reduction of cRIPGBM-induced caspase 3/7 activation in GBM CSCs (Fig. 2G), RIPK2 suppression resulted in a significant decrease in drug sensitivity. The acyclic parent compound RIPGBM was not found to interact with RIPK2 (Fig. 2E). These results suggest that it is the cyclized derivative cRIPGBM that is responsible for RIPK2-dependent induction of apoptosis.

cRIPGBM Induces Caspase 1-Mediated Apoptosis. To elucidate the downstream mechanism of action by which cRIPGBM induces apoptosis, we assessed caspase activation in GBM CSCs. Compound treatment (250 nM) resulted in a time-dependent activation of caspase 1, caspase 9, and caspase 7, as well as PARP cleavage (Fig. 3A), providing further evidence that cRIPGBM induces cell death via an apoptotic mechanism. Correspondingly, the pan-caspase inhibitor Z-VAD-FMK significantly reduced compound-mediated GBM CSC death (Fig. 3B). PARP cleavage occurred in a time-dependent manner concomitant with the onset of DNA fragmentation as determined by using a TUNEL assay (SI Appendix, Fig. S7). In addition, we established that caspase 1 activation is upstream of caspase 9, caspase 7, and PARP cleavage by observing that pretreatment with the caspase 1 inhibitor Ac-YVAD-CHO effectively blocks caspase 9 cleavage in cRIPGBM-treated GBM CSCs (Fig. 3C). Fig. 3. cRIPGBM activates caspase 1-mediated apoptotic signaling in GBM CSCs by modulating the interaction of RIPK2 with TAK1 and caspase 1. (A) Time-dependent cRIPGBM (250 nM)-induced cleavage of caspase 1, caspase 9, caspase 7, and poly(ADP-ribose) polymerase (PARP) in GBM-1 GBM CSCs. (B) cRIPGBM-induced cell death in GBM-1 GBM CSCs following treatment with the pan-caspase inhibitor Z-VAD (20 μM). (C) cRIPGBM-induced (250 nM) caspase 9 cleavage in GBM-1 GBM CSCs following treatment with the caspase 1-selective inhibitor Ac-YVAD-CHO (20 μM). (D) Coimmunoprecipitation of cIAP1 and cIAP2 with anti-RIPK2 antibody in GBM-1 GBM CSCs following treatment with cRIPGBM. (E) Coimmunoprecipitation of TAK1 with anti-RIPK2 antibody in GBM-1 GBM CSCs following treatment with cRIPGBM (250 nM). (F) Quantification of TAK1 levels coimmunoprecipitated by using an anti-RIPK2 antibody in GBM-1 GBM CSCs following treatment with cRIPGBM (250 nM). (G) Coimmunoprecipitation of caspase 1 with anti-RIPK2 antibody in GBM-1 GBM CSCs following treatment with cRIPGBM (250 nM). (H) Quantification of caspase 1 levels coimmunoprecipitated by using an anti-RIPK2 antibody in GBM-1 GBM CSCs following treatment with cRIPGBM (250 nM). (I) Schematic representation of the proposed mechanism of action for cRIPGBM-induced apoptosis in GBMB CSCs. Values shown are mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001). Ubiquitination is a known key modification that regulates the ability of RIPK2 to act as a prosurvival or proapoptotic molecule (32, 33). Specifically, K63-ubiquitinated RIPK functions as a scaffold for the assembly of protein complexes that activate prosurvival signaling pathways (34⇓–36). The E3 ubiquitin ligases cIAP1 and cIAP2 have been previously shown to interact with and promote RIPK2 ubiquitination in various cell lines (33, 37, 38). Coimmunoprecipitation assays were used to determine if cRIPGBM treatment alters its interaction with cIAP1 and/or cIAP2 in GBM CSCs. Compound treatment reduced RIPK2 binding to cIAP1 (Fig. 3D) and significantly reduced binding to cIAP2 in a dose-dependent manner (Fig. 3D), which suggests that cIAP1 and cIAP2 are endogenous regulators of RIPK2 ubiquitination in GBM CSCs.

cRIPGBM Acts as a Molecular Switch That Modulates RIPK2 Binding Partners. On the basis of these observations, as well as previously established mechanisms that have been established for RIPK2 (34, 35), we hypothesized that cRIPGBM promotes cell death by modulating RIPK2 ubiquitination status, which could impact its interactions with a prosurvival molecule to favor its interaction with a proapoptotic adaptor protein. To test this notion, we determined if compound treatment alters RIPK2 binding partners by using coimmunoprecipitation experiments. Previous studies have established that K63-ubiquitinated RIPK2 associates with the prosurvival TAK1 complex (34, 35). Consistently, TAK1 was found to coimmunoprecipitate with RIPK2 under basal conditions in GBM CSCs (Fig. 3E). In cRIPGBM-treated cells, the interaction between RIPK2 and TAK1 was significantly decreased (Fig. 3 E and F). Conversely, because cRIPGBM treatment induces caspase 1-dependent cell death, we determined whether drug treatment correlated with an increased association between RIPK2 and caspase 1. Indeed, treatment with cRIPGBM for 6 h was found to result in a significant enhancement of RIPK2–caspase 1 interaction (Fig. 3 G and H). Taken together, these data suggest that cRIPGBM induces apoptosis in GBM CSCs by a mechanism that involves its interaction with RIPK2 in a mode that results in decreased association with TAK1 and increased association with and activation of caspase 1, which can lead to downstream activation of a caspase 1-mediated apoptotic signaling cascade (Fig. 3I). Additional future work will be required to determine a detailed understanding of how cRIPGBM/RIPK2 interaction impacts RIPK2 function in normal and disease cell types.