
==== Front
Acta Pharm Sin B
Acta Pharm Sin B
Acta Pharmaceutica Sinica. B
2211-3835
2211-3843
Elsevier

S2211-3835(24)00232-6
10.1016/j.apsb.2024.06.001
Original Article
Molecular glue triggers degradation of PHGDH by enhancing the interaction between DDB1 and PHGDH
Huang Ziqi a†
Zhang Kun a†
Jiang Yurui a
Wang Mengmeng a
Li Mei a
Guo Yuda a
Gao Ruolin a
Li Ning a
Wang Chenyang a
Chen Jia a
Wang Jiefu jwang05@tmu.edu.cn
b⁎
Liu Ning liuning@nankai.edu.cn
a⁎
Liu Xiang liux@nankai.edu.cn
a⁎
Liu Shuangwei 1542366958@qq.com
a⁎
Wei Mingming mingmingshengwu@163.com
a⁎
Yang Cheng Cheng.yang@nankai.edu.cn
a⁎
Yang Guang Guang.yang@nankai.edu.cn
a⁎
a The State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, College of Pharmacy, Nankai University, Tianjin 300071, China
b Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, China
⁎ Corresponding authors. jwang05@tmu.edu.cnliuning@nankai.edu.cnliux@nankai.edu.cn1542366958@qq.commingmingshengwu@163.comCheng.yang@nankai.edu.cnGuang.yang@nankai.edu.cn
† These authors made equal contributions to this work.

06 6 2024
9 2024
06 6 2024
14 9 40014013
6 2 2024
6 5 2024
20 5 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Cancer stem cells (CSCs) play a pivotal role in tumor initiation, proliferation, metastasis, drug resistance, and recurrence. Consequently, targeting CSCs has emerged as a promising avenue for cancer therapy. Recently, 3-phosphoglycerate dehydrogenase (PHGDH) has been identified as being intricately associated with the regulation of numerous cancer stem cells. Yet, reports detailing the functional regulators of PHGDH that can mitigate the stemness across cancer types are limited. In this study, the novel “molecular glue” LXH-3-71 was identified, and it robustly induced degradation of PHGDH, thereby modulating the stemness of colorectal cancer cells (CRCs) both in vitro and in vivo. Remarkably, LXH-3-71 was observed to form a dynamic chimera, between PHGDH and the DDB1-CRL E3 ligase. These insights not only elucidate the anti-CSCs mechanism of the lead compound but also suggest that degradation of PHGDH may be a more viable therapeutic strategy than the development of PHGDH inhibitors. Additionally, compound LXH-3-71 was leveraged as a novel ligand for the DDB1-CRL E3 ligase, facilitating the development of new PROTAC molecules targeting EGFR and CDK4 degradation.

Graphical abstract

A novel "molecular glue", LXH-3-71, was discovered to degrade PHGDH by facilitating formation of the dynamic chimera between PHGDH and DDB1-CRL E3 ligase in CRC cells.Image 1

Key words

Molecular glue
Targeted protein degradation
PHGDH
Cancer stem cells
PROTACs
==== Body
pmc1 Introduction

Colorectal cancer (CRC) is the second leading cancer type, accompanied by a pronounced mortality rate1,2. While treatments like chemotherapy and radiotherapy can be beneficial for CRC patients’ post-surgery, the menace of recurrence remains significant1. A distinct subset of cells, termed as colorectal cancer stem cells (CRCSCs), is present within CRC3,4. These cells are characterized by their unique ability for self-renewal3, 4, 5. Several research initiatives6,7 suggest that CRCSCs play an integral role in tumor initiation, drug resistance8, facilitating metastasis9,10, and leading to relapse11. Alarmingly, certain therapeutic approaches like chemotherapy and radiotherapy might inadvertently enhance the CRCSCs population. However, a deep understanding of how to modulate the self-renewal, proliferation, differentiation and drug resistance of CRCSCs remains elusive12.

The enzyme 3-phosphoglycerate dehydrogenase (PHGDH) plays a crucial initial role in the serine biosynthetic pathway, diverging from glycolysis. Using NAD as a cofactor, PHGDH oxidizes the glycolytic compound 3-phosphoglycerate to produce phosphohydroxypyruvate13,14. Successive enzymatic reactions in this pathway, namely by PSAT1 through transamination and PSPH during phosphate ester hydrolysis, eventually lead to the formation of serine15. To date, a growing body of evidence has demonstrated the prevalent overexpression of PHGDH in various cancer types, underpinning its pivotal role in tumor initiation, invasion and metastasis, drug resistance, and recurrence16, 17, 18, 19. Consequently, PHGDH has emerged as a promising target, garnering substantial interest from medicinal chemists and biotechnology companies in the development of innovative anti-cancer molecules20, 21, 22, 23.

Previously, the research group identified a novel class of compounds derived from oleanolic acids, exhibiting potent anti-cancer stem cell activities across a broad spectrum of malignant tumors24. In the current study, the design and utilization of a pair of “differentiated active molecular probes” enabled the accurate identification of the direct target of the lead compound, LXH-3-71. Particularly, evidence substantiated that this compound could covalently bind to Cys281 of PHGDH, subsequently instigating a ubiquitin-proteasome system (UPS)-mediated degradation of this protein. Further results affirmed that LXH-3-71 acted as a novel “molecular glue”, facilitating dynamic proteolysis chimeras composed of PHGDH and DDB1-CRL E3 ligase. Notably, these findings also uncovered that selective degradation of PHGDH might represent a more promising strategy for cancer therapeutics compared to the development of PHGDH inhibitors.

2 Results and discussion

2.1 LXH-3-71 impairs the proliferation and stemness of colorectal cancer cells in vitro

Previously, a class of oleanolic acid analogues was identified as compounds with anti-cancer stem cells properties. Upon meticulous validation, the lead compound, LXH-3-71 (Fig. 1A), was selected, because of its potent anti-proliferative and anti-CSC activities against various colorectal cancer cells. Initially, the inhibitory effect of LXH-3-71 against several colorectal cancer cells was assessed through the CCK-8 assay, revealing that the lead compound could significantly inhibit the growth of all selected colorectal cancer cells at low micromolar concentrations (Fig. 1B). To contrast, LXH-3-71 incubationled to demonstrated relatively low cytotoxicity against some human cell lines (Supporting Information Fig. S1). Numerous reports have suggested that metastatic colorectal cancer (mCRC) cells often harbor high stemness, leading to drug resistance, recurrence, and poorer prognosis25, 26, 27. Subsequent studies determined whether LXH-3-71 could attenuate the stemness of mCRC cells. LXH-3-71 markedly reduced the colony numbers of HCT-116 and SW480 cells grown in soft agar (Fig. 1C‒E) and, for selected mCRC cells (HCT-116 and SW480), groups treated with LXH-3-71 not only diminished the sizes of tumorspheres but also reduced the number of tumorspheres in a dose-dependent manner compared to the control group (Fig. 1F‒K). Furthermore, LXH-3-71 was found to distinctly inhibit the proliferation of colorectal stem cells (CD133+ and CD44+) sorted by CSC makers in a dose-dependent manner (Fig. 1L and Supporting Information Fig. S2). In summary, these results indicated that LXH-3-71 could notably inhibit the growth of both colorectal cancer cells and colorectal cancer stem cells in vitro.Figure 1 The antiproliferative effects of LXH-3-71 on colorectal cancer cells and associated stem cells under in vitro conditions. (A) Chemical structure of lead compound LXH-3-71. (B) IC50 curve of lead compound LXH-3-71 against colorectal cancer cell lines. IC50 values were calculated using GraphPad Prism software, and were expressed as the mean ± SD of three independent experiments. (C) Inhibition of LXH-3-71 on the colony formation of colorectal cancer cells on soft agar. (D and E) Quantification of soft agar assay from (C). (F) Representative fields of tumorspheres treated by indicated concentrations of LXH-3-71 or DMSO for 72 h. (G and H) Quantitative analysis of the number of tumorspheres from (F). (I) Representative images of tumorspheres formed form HCT-116 and SW480 cells pre-treated with LXH-3‒71 (2.5 and 5 μmol/L) or DMSO for 3 days. (J, K) Quantitative analysis of the number of tumorspheres from (I). (L) Cell viability inhibition of LXH-3-71 on CD133+ CD44+ HCT-116 cells, and IC50 value (mean ± SD) was determined in triplicate. Statistical significance was determined by unpaired t-test. Error bars denoted SEM, n = 3, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 1

2.2 LXH-3-71 remarkably inhibits the growth and metathesis of mCRCs in vivo

Encouraged by promising in vitro experimental results, further evaluation of the in vivo anti-tumor activity of LXH-3-71 was conducted using mouse xenograft models. First of all, the results pharmacokinetic (PK) studies indicated that IP administration of LXH-3-71 exhibited excellent bioavailability in tested animals. (Please see Supporting Information Fig. S3 and Table S1). Next, in an HCT-116 subcutaneous xenograft model, LXH-3-71 administration effectively inhibited tumor growth in a dose-dependent manner, as illustrated in Fig. 2A–C, and D, and was compared to the reference drug, 5-FU. Negligible changes in body weight of the test animals indicated that both LXH-3-71 and 5-FU were well tolerated (Fig. 2B). Subsequent pathological analysis revealed a significant reduction in Ki67-positive cells in the LXH-3-71 groups, thereby confirming its suppressive impact on tumor proliferation (Fig. 2E). Moreover, the H&E staining of the spleen, liver, kidney and lung tissues showed that LXH-3-71 had none significant systemic toxicity (Supporting Information Fig. S4). Concurrently, an animal model simulating CRC lung metastasis was established to investigate the inhibitory effect of LXH-3-71 on metastatic colonization of CRCs. Mice treated with LXH-3-71 exhibited minimal fluorescence signals compared with the vehicle control and 5-FU treatment groups (Fig. 2F and G), indicating a significant reduction in lung metastatic burden as a result of LXH-3-71 administration. This observation was further substantiated by H&E staining, which demonstrated a notable reduction in lung metastasis following LXH-3-71 treatment (Fig. 2H).Figure 2 Effect of LXH-3-71 (1 and 3 mpk, i.p, QD) on CRC growth and metastatic potential in vivo. (A) Alterations in tumor volume throughout the administration in xenograft models. (B) Fluctuations in mice body weight during the treatment period in xenograft models. (C) Quantification of tumor weight across four groups upon sacrifice of mice. (D) Images of tumors excised from xenograft models on Day 17. (E) Tumor sections immunostained with anti-Ki67 antibody and imaged at 200× magnification. (F) Bioluminescence imaging of a lung metastasis animal model. (G) Quantification of bioluminescence signals for (F). (H) Hematoxylin and eosin (H&E) staining of lung sections. Statistical significance was determined by unpaired t-test. Error bars denoted SEM, n = 5, ∗P < 0.05, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 2

2.3 Design, synthesize and screen for differentiated chemical probes derived from LXH-3-71

The combined outcomes from previous investigations strongly imply that LXH-3-71 possesses significant efficacy against CRCSCs, both within laboratory settings and in vivo models. To further delve into the precise mechanism whereby LXH-3-71 counters the inherent stemness traits of mCRC cells, we designed and synthesized specific chemical probes, as depicted in Fig. 3A, to facilitate advanced affinity pull-down studies. It's increasingly recognized in scientific literature that moieties with α,β-unsaturated enone configurations can act as Michael acceptors. These configurations readily engage in forming a covalent C‒S linkage with distinct cysteine regions within the target protein28,29. Consequently, chemical probes, both with and without enone moieties, were developed through straightforward synthesis steps (Supporting Information Scheme S1). Additionally, biological evaluation results revealed that probe 10, which bears an enone moiety, maintained potent anti-CRCSCs activity at nearly 5 μmol/L, whereas its analogue, 13, exhibited no activity (Fig. 3B and C).Figure 3 Identification of targeted protein of LXH-3-71 based on chemical probes. (A) Chemical structure of probes originating from LXH-3-71. (B) Representative fields of tumorspheres treated by DMSO, positive probe 10, and negative probe 13. (C) Quantitative analysis of the number of tumorspheres form (B). Statistical significance was determined by unpaired t-test. Error bars denoted SEM, n = 3, ∗∗∗P < 0.001. (D) Streptavidin-agarose beads with chemical probes (probe 10, and probe 13) were incubated with HCT-116 cell lysates to pull down probes-bound proteins. Proteins were separated by 10% SDS-PAGE and stained with Comassie brilliant blue. (E) Information of targeted protein PHGDH from LC−MS/MS analysis. (F) Binding verification between LXH-3-71 and PHGDH through a combination of pull-down and western blotting approaches in HCT-116 cells. (G) Fusion MS/MS analysis of the recombinant human PHGDH with DMSO and LXH-3-71 incubation, respectively. (H) A hypothetical binding model for the interaction between LXH-3-71 and PHGDH. (I) Spearman analysis evaluated the correlations between PHGDH gene expression and stemness indices, utilizing corresponding clinical information for colon cancer (left) and rectal cancer (right) derived from the TCGA dataset.

Figure 3

2.4 LXH-3-71 could covalently bind to Cys281 of PHGDH

Employing these “differentiated active chemical probes”, subsequent “pull-down” experiments were conducted to identify the proteins which the active probe selectively bound to sustain its anti-CRCSC activity. To achieve that, HCT-116 cell lysates were incubated with the pair of probe molecules that were previously linked with streptavidin-containing beads. The bound proteins were precipitated, washed with buffers, resolved via SDS-PAGE, and subsequently stained. According to Fig. 3D, a distinct band, observed near 55 kDa, was specifically precipitated by positive probe 10 and was identified as PHGDH by LC‒MS/MS (Fig. 3E). Subsequent immunoblotting results further indicated that PHGDH was enriched in the precipitates of the positive probe 10 group and was scarcely detected in the negative probe 13 group (Fig. 3F). The provided data suggest a covalent reaction between LXH-3-71 and PHGDH. In our pursuit to pinpoint the exact Cys residue that binds, we subjected the full-length recombinant human PHGDH protein to purification and subsequent incubation with LXH-3-71. Post incubation, trypsin digestion was performed on the samples, followed by an LC‒MS/MS (Fushion) analysis. Referring to Fig. 3G, both computations and database searches highlighted a peptide exhibiting a mass of 2648.4499 Da. This weight was precisely 657.4326 Da greater than the mass of the peptide encompassing Cys281, identified as ALVDHENVISCPHLGASTK (with a calculated mass of 1991.0173 Da). Intriguingly, this mass variance (657.4326 Da) seamlessly aligned with the molecular weight of LXH-3-71. Tandem mass spectrometry analyses of the peptide pointed to this unique mass alteration spanning from the y1 to y18 fragment ions, suggesting a covalent interaction of LXH-3-71 with the thiol fraction of the Cys281 residue. However, capturing crystal data remained elusive, possibly due to the protein's pronounced flexibility. Notably, it was further confirmed that Cys281 of PHGDH was essential for maintaining the anti-CSCs activity of LXH-3-71 both in vitro and in vivo (Supporting Information Fig. S5). Additionally, analysis of the TCGA database revealed a positive correlation between PHGDH and the expression of tumor stem cell-related genes at the mRNA level in both colon and rectal cancers (Fig. 3I). Drawing conclusions from the results, PHGDH emerged as the immediate target of the anti-CSCs lead compound, demonstrating its ability to form a covalent bond at the Cys281 region of the protein.

2.5 LXH-3-71 triggers significant and selective degradation of PHGDH both in vitro and in vivo

Initial docking results unexpectedly revealed that the binding site for small molecules on PHGDH constitutes a shallow pocket situated on its surface, ostensibly unrelated to the previously reported catalytic domain of PHGDH30. The enzyme activity was initially probed to ascertain whether LXH-3-71 could inhibit PHGDH activity. NCT503, previously reported as a potent PHGDH inhibitor, served as a positive control31. Findings indicated that NCT503 presented an IC50 value of 7.17 ± 1.80 μmol/L32, while LXH-3-71 exhibited no inhibitory effect against PHGDH (IC50 > 50 μmol/L, Fig. 4A and B). Interestingly, NCT503 demonstrated a negligible impact on dispersing the spheroids of CRC cells (see Supporting Information Fig. S6), which suggested that PHGDH might modulate the stemness of CRC cells via an enzyme-independent mechanism. Subsequent cellular analyses revealed that LXH-3-71 treatment significantly downregulated PHGDH protein levels in CRC cells in both dose- and time-dependent manners (Fig. 4C and D). Furthermore, quantitative RT-PCR results suggested that LXH-3-71 likely does not impede the transcriptional process (Fig. 4E). Additionally, cycloheximide (CHX) treatment experiments revealed that LXH-3-71 administration could distinctly diminish PHGDH stability (Fig. 4F). Notably, this protein degradation could be counteracted by subsequent administration of MG-132, a potent proteasome inhibitor (Fig. 4G). LXH-3-71 treatment was also found to induce poly-ubiquitination of PHGDH, making it susceptible to proteasomal digestion (Fig. 4H). In vivo analyses of CRC tumor tissue samples using western blotting (Fig. 4I) and immunohistochemical staining (IHC) (Fig. 4J) exhibited a conspicuous downregulation of PHGDH levels across all samples following LXH-3-71 treatment. Moreover, selectivity for degradation was evaluated via quantitative proteomics analysis, identifying PHGDH as the most significantly downregulated protein (Fig. 4K). In summary, the accumulated data confirm that LXH-3-71 can notably induce the efficient and selective degradation of PHGDH, in both in vitro and in vivo contexts.Figure 4 Ubiquitin-dependent PHGDH degradation induced by LXH-3-71. (A and B) Inhibition of PHGDH enzyme activity by NCT-503 and LXH-3-71 conducted with the instruction from PHGDH activity assay kit. IC50 values were calculated with GraphPad Prism software. Data were represented as mean ± SD, n = 3. (C) Evaluation of PHGDH expression in HCT-116 and HCT-15 cells incubated with the indicated concentrations of LXH-3-71 for 24 h via Western blot. (D) Changes analysis of PHGDH protein level in HCT-116 cells treated with LXH-3-71 (4 μmol/L) for indicated time points by Western blot. (E) Detection of PHGDH mRNA levels in HCT-116 cells treated with different concentrations of LXH-3-71 (24 h) by qRT-PCR. Error bars denoted SEM, n = 3. (F) Identification of PHGDH protein level in CHX-treated cells with or without LXH-3-71 treatment (4 μmol/L) for indicated time points by Western blot. (G) Western blot analysis of PHGDH protein level in DMSO and LXH-3-71 (4 μmol/L) pre-treated cells (for 18 h) following treatments with MG-132 (10 μmol/L) for 6 h. (H) Western blot analysis of ubiquitin accumulation on PHGDH protein in DMSO and LXH-3-71 (4 μmol/L) pre-treated cells (for 18 h) following treatments with MG-132 (10 μmol/L) for 6 h. (I) Western blot scrutiny of PHGDH levels in tumor tissues from the groups after administration of LXH-3-71 (3 mpk) for indicated time points, compared to vehicle group. (J) Tumor sections from mice treated with vehicle, 5-FU, and LXH-3-71 were subjected to staining using anti-PHGDH antibodies, followed by photographic documentation. (K) A volcano plot depicts the differentially regulated proteins in cells treated with DMSO and LXH-3-71, with the x-axis showcasing the log2-transformed ratio in LXH-3-71 treated cells relative to control cells and the y-axis revealing the ‒log10-transformed P-value.

Figure 4

2.6 PHGDH modulates the stemness of CRCs

To elucidate the role of PHGDH in regulating the stemness of colorectal cancer cells, lentivirus-mediated shRNA knockdown and overexpression of PHGDH were performed in HCT-116 and HCT-15 cells respectively, and the PHGDH expression were validated by Western blot assay (Fig. 5A). Firstly, The proliferative effects of CRC cells were positively regulated by PHGDH alternation (Supporting Information Fig. S7). Tumorsphere formation experiments revealed that, in contrast to the control group, PHGDH knockdown significantly impeded cells' capacity to form pellets and diminished the dimensions of the tumorspheres. Conversely, PHGDH overexpression enhanced tumorsphere formation and CSCs’ self-renewal (Fig. 5B and C). Secondly, PHGDH knockdown substantially reduced the frequency of tumorsphere initiation, as evidenced by limited dilution assays (LDA) in vitro, while ectopic PHGDH expression notably ameliorated sphere-initiation efficacy (Fig. 5D). Additionally, colony formation assays in soft agar affirmed the attenuation of cell growth upon PHGDH depletion, while its overexpression markedly fostered malignant cloning formation (Fig. 5E and F). Moreover, given that certain colorectal cancer stem cell biomarkers (LGR5, CD44, CD133, and CD166) are pivotal for assessing the stemness of CRCs33, the influence of PHGDH on the regulation of these genes was examined. Both knockdown and overexpression of PHGDH in the HCT-116 cell line resulted in the respective reduction or increment of LGR5, CD44, CD133, and CD166 mRNA levels, as determined by qRT-PCR (Fig. 5G). Changes in the protein levels of LGR5 and CD44 were also corroborated by Western blotting (Fig. 5H). PHGDH knockdown also induced up-regulation of ROS level and down-regulation of GSH/GSSG ratio (Supporting Information Fig. S8). Furthermore, PHGDH deletion significantly inhibited tumor growth and diminished the tumor initiation frequency in vivo, as demonstrated by in vivo LDA experiments (Fig. 5I‒L). Then, the xenograft tumor tissue samples were analyzed using Western blotting (Fig. 5M) and IHC staining (Supporting Information Fig. S9). The expression levels of CD133, CD44, LGR5 and CD166 were downregulated in sh-PHGDH group compared with NC group (Fig. 5M). In summary, the results collectively substantiate that PHGDH expression is pivotal for maintaining the stemness of CRCs.Figure 5 The regulatory role of PHGDH in maintaining the stemness of CRC cells. (A) Western blot analysis of PHGDH protein level in HCT-116 cells with stable expression of scrambled or PHGDH shRNA, and in parallel or PHGDH overexpression HCT-15 cells. (B) Representative micrographs of tumorspheres from HCT-116 cells bearing scrambled or PHGDH shRNA (up) or PHGDH-overexpression HCT-15 cells (down). (C) Quantification of the numbers of tumorspheres in (B). (D) Limiting dilution analysis (LDA) to assess the sphere-initiation capacity of stable PHGDH knockdown or overexpression cells. Cells were planted into 96-well plates at densities of 8, 15, 31, or 62 cells per well and cultured for 10 days, followed by counting of wells containing tumorspheres and analysis utilizing ELDA software. (E) Representative images of colony formation in soft agar. (F) Quantification of colony numbers in (E). (G) Quantitative RT-PCR analysis of CRC stem cell markers (LGR5, CD133, CD44, and CD166). (H) Detection of PHGDH protein levels of CD44 and LGR5 by Western blot. (I) Limiting dilution tumorigenesis assays conducted by subcutaneously injecting scrambled or PHGDH shRNA cells (1 × 104, 1 × 105, and 1 × 106) into BALB/c nude mice. After 39 days, animals were sacrificed. The ratio of formed tumors to the total number in each group is presented in a table and illustrated in a pie chart. (J) Images of tumors excised from mice injected with scrambled or PHGDH shRNA cells. (K) Tumor tissue weight. (L) Tumor volume growth curve for each group. (M) Western blot analysis of CD133, CD44, LGR5 and CD166 level in sh-PHGDH group and NC group. Statistical significance was determined by unpaired t-test. Error bars denoted SEM, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 5

2.7 LXH-3-71 was identified as a “molecular glue” to furnish dynamic complex composed of PHGDH with DDB1-CUL4 ubiquitin ligase

To delve deeper into the molecular mechanism of degradation instigated by LXH-3-71, immunoprecipitation was performed a PHGDH specific antibody, with or without LXH-3-71 treatment. Upon resolution in SDS-PAGE, a conspicuous band emerged in the LXH-3-71 treatment group, later identified as DDB1 by LC‒MS/MS (Fig. 6B and C). DDB1 has been reported as a crucial component of the DDB1-CUL4 ubiquitin ligase complex, wherein, through interaction with adaptor proteins, it facilitates substrate recruitment to the complex for ubiquitination and eventual proteasomal degradation34, 35, 36. It was posited that LXH-3-71 may serve as a “molecular glue”, adhering PHGDH and the DDB1-CUL4 ubiquitin ligase, and conveying poly-ubiquitination to PHGDH. To substantiate this supposition, PHGDH/DDB1 complexes were precipitated and subjected to immunoblotting with anti-PHGDH or anti-DDB1 antibodies, respectively. The findings, illustrated in Fig. 6D and E, indicated that LXH-3-71 treatment markedly amplifies the interaction between PHGDH and DDB1. Subsequently, it was observed that the knockdown of DDB1 or introduction of MLN4924, a documented specific inhibitor of the DDB1-CUL4 complex, could notably disrupt the LXH-3-71-induced degradation of PHGDH (Fig. 6F and G). Additionally, an alternative biotin-containing probe 18 was designed and synthesized, connecting the biotin from the PHGDH binding site of LXH-3-71 (Fig. 6A). DDB1 was significantly enriched in pull-down experiments with 18 (Fig. 6H). To further verified the formation of dynamic E3 ligase complex in vitro, microscale thermophoresis method was used to measure the binding affinity of DDB1 and PHDGH in or out the presence of LXH-3-71. The results were delineated in Fig. 6I, the direct binding potency of DDB1 with PHGDH was weak, and the value of Kd was 26.4 μmol/L. To our delight, the covalent complex of PHGDH and LXH-3-71 (pre-incubated PHGDH with LXH-3-71) was exhibited enhanced binding capability to DDB1 (Kd = 2.7 μmol/L). Notably, the complex of PHGDHC281S and DDB1 could not be facilized by additional of LXH-3-71 (Supporting Information Fig. S10). Additionally, it was also determined that DDB1 knockdown attenuated anti-proliferation and anti-CSC activities of LXH-3-71 by markedly alleviating degradation of PHGDH (Supporting Information Fig. S11). In summary, these above-findings bolstered the hypothesis that LXH-3-71 substantially induces the formation of a dynamic complex between PHGDH and DDB1-CUL4 ubiquitin ligase, consequently facilitating the efficient cellular proteasomal degradation of PHGDH (Fig. 7).Figure 6 LXH-3-71 functions as a molecular glue facilitating PHGDH-DDB1 Interaction. (A) Chemical structure of compound 18. (B) Silver staining of immunoprecipitated proteins separated by SDS-PAGE. (C) Data pertaining to the DDB1-CUL4 ubiquitin ligase, derived from LC‒MS/MS analysis. (D, E) Co-IP assays were executed utilizing anti-PHGDH and anti-DDB1 antibodies, respectively, followed by Western blot analysis. (F) Western blot analysis of DDB1 protein level in HCT-116 cells transfected with siCON or siDDB1 oligos. (G) Western blot analysis of PHGDH protein level in MLN4924 (1 μmol/L) or DDB1 knockdown pre-treated HCT-116 cells followed by treatment with LXH-3-71 for 24 h. (H) A protein affinity pull-down assay, utilizing probe 18, was conducted with HCT-116 cell lysate and subsequently analyzed through a Western blotting assay. (I) Interaction studies between DDB1 and PHGDH (left) or PHGDH preincubated with LXH-3-71 (right) using microscale thermophoresis method. Data are represented as mean ± SD, n = 3.

Figure 6

Figure 7 Mechanism of LXH-3-71 as a molecular glue. LXH-3-71 induces the interaction between PHGDH and DDB1-CUL4 ubiquitin ligase, triggering degradation of PHGDH via the ubiquitination proteasome pathway and subsequently leading to inhibited cancer cell stemness.

Figure 7

2.8 LXH-3-71 was utilized for development of new PROTAC molecules to degrade EGFR and CDK4

Based on the elucidation of the crucial mechanism of the “molecular glue” LXH-3-71 for the degradation of PHGDH in CRC cells, the endeavor aimed to chemically convert small molecular drugs into novel PROTAC molecules for targeted protein degradation (TPD), utilizing LXH-3-71 analogues as a novel ligand of E3 ligase. Initially, Gefitinib was selected as the ligand for the target protein in a preliminary attempt to develop a new EGFR degrader. As illustrated in Supporting Information Scheme S2, chemical linkers of varying types and lengths were employed to conjugate the core of Gefitinib with LXH-3-71, resulting in the preparation of eight potential TPD molecules. The TPD efficacies of these molecules were biologically evaluated through in vitro western blotting within a 24-h frame (Supporting Information Fig. S12). Compound 37 was identified as the most efficient molecule for EGFR degradation, exhibiting efficacy in a dose-dependent manner (Fig. 8A and B). Subsequent to this success in optimizing the chemical linker, a potent CDK4 TPD molecule, 45, was synthesized and identified, employing the structure of Ribociclib (Supporting Information Scheme S3, Supporting Information Fig. S13, Fig. 8C and D). To verify whether the down-regulation of EGFR and CDK4 were PROTAC-mediated degradation, the HCT-116 cells were firstly pre-incubated with MG-132, compound 47, Gefitinib and Ribociclib, respectively (Supporting Information Scheme S4, Fig. 8F and G). As revealed from the comparison with DMSO group, the degradations of EGFR and CDK4 triggered by both compounds were obviously attenuated. Without the active proteasome, inhibited by MG-132, the degradation of EGFR and CDK4 were completely inhibited. Since compound 47 was the potential ligand of PHGDH and DDB1, and Gefitinib and Ribociclib were ligands of EGFR and CDK4, respectively, the pre-treatment with high concentrations of these compounds will occupy the pockets of proteins and result in disturbing the formation of ternary complex between DDB1, target proteins and PROTACs, which will significantly inhibit the UPS-mediated degradation of the target proteins. Pre-treated with MLN4924 (CRL inhibitor) or DDB1 knockdown could also terminate the degradation process of both EGFR and CDK4. Collectively, these findings may offer novel and alternative strategies for the design and discovery of new TPD molecules, which could induce CRL4-mediated protein degradation.Figure 8 Targeted protein degradation induced by PROTAC derived from LXH-3-71. (A, C) Chemical structures of compounds 37 and 45, respectively. (B, D) Cells were incubated with either compound 37 or compound 45 at specified concentrations for 24 h, followed by an assessment of targeted protein levels via Western blot analysis. (E) Chemical structure of compound 47. (F) Western blot analysis of EGFR protein level in MG-132 (10 μmol/L), compound 47 (10 μmol/L), Gefitinib (10 μmol/L), MLN4924 (1 μmol/L) or DDB1 knockdown pre-treated HCT-116 cells followed by treatment with compound 37 for 24 h. (G) Western blot analysis of CDK4 protein level in MG-132 (10 μmol/L), compound 47 (10 μmol/L), Ribociclib (10 μmol/L), MLN4924 (1 μmol/L) or DDB1 knockdown pre-treated HCT-116 cells followed by treatment with compound 45 for 24 h.

Figure 8

3 Conclusions

In conclusion, the current study unveiled a novel “molecular glue”, LXH-3-71, which instigates potent and selective degradation of PHGDH, subsequently modulating the stemness of CRCs both in vitro and in vivo. It has been observed that LXH-3-71 can facilitate the formation of dynamic complexes, amalgamating PHGDH with DDB1-CUL4 ubiquitin ligase, which consequently induces rapid poly-ubiquitination and proteasomal degradation of PHGDH. The elimination of PHGDH appears to attenuate the stemness of CRCs in an enzyme-independent manner. These findings not only elucidate the anti-CSCs mechanism of the lead compound but also demonstrate that selective degradation of PHGDH could emerge as a more promising strategy for cancer therapeutics compared to PHGDH enzyme inhibitors. Moreover, this work furnishes a novel ligand of DDB1-CRL E3 ligase, paving the way for designing new PROTAC molecules.

4 Experimental

4.1 Cell culture

The cell lines used in this study were purchased from Procell Life Science&Technology Co., Ltd. Colorectal cancer cell lines (HCT-116, SW480, HCT-15, LoVo, RKO and CT26) and normal cell lines (MCF 10A, BEAS-2B, LO2 and 293T) were incubated at 37 °C in a humidified atmosphere containing 5% CO2. HCT-116 cells were cultured in McCoy's 5A medium; HCT-15, LoVo and CT26 cells in RPMI1640; SW480, BEAS-2B, LO2 and 293T in Dulbecco's modified Eagle's medium (DMEM) and RKO in MEM. All media were supplemented with 10% fetal bovine serum (FBS, Procell) and 1% penicillin-streptomycin (YEASEN). MCF 10A cells were cultured in DMEM/F12 with 5% HS, 20 ng/mL EGF, 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 1% NEAA and 1% penicillin-streptomycin.

4.2 Virus production and cell infection

HCT-116 and HCT-15 cells were infected with lentivirus to construct a stable knockdown or overexpression cell line, respectively. Briefly, shRNA fragments were cloned into pLKO.1-puro plasmid using AgeI/EcoRI sites. Full length sequence of human PHGDH was synthesized and cloned into pCDH-CMV-MCS-EF1-copGFP vector, which was contracted by HANBIO. For lentivirus production, HEK293FT cells were transfected with recombinant plasmid and the packaging vectors PSPAX2 and pMD2G using polyethylenimine (YEASEN). Medium containing the virus was collected at 24 and 48 h after transfection. HCT-116 or HCT-15 cells were infected with the collected virus supernatant in the presence of 8 μg/mL polybrene (YEASEN). The shRNA sequences were listed as follows:

sh-PHGDH-1: 5′-CCCTGTAGTACAGCAATAA-3′;

sh-PHGDH-2: 5′-CGCAGAACTCACTTGTGGAAT-3′;

sh-DDB1-1: 5′-CGACCGTAAGAAGGTGACTTT-3′;

sh-DDB1-2: 5′-CCTTGATTGGTGTTGCCAGTT-3′.

4.3 Fluorescence activated cell sorting

Flow cytometry was performed to sort CD133+ CD44+ subpopulation form HCT-116 cells. The cells were harvested, washed twice with PBS, and suspended in 500 μL PBS. Cells were then treated with 25 μL FcR Blocking Reagent for 15 min, and incubated with antibodies (human anti-CD44-FITC and human anti-CD133-APC, BD Pharmingen, USA) for 30 min. Subsequently, cells were washed twice with PBS and centrifuged. The cell pellet was resuspended in 100 μL McCoy's 5A medium. CD133+CD44+ cells were sorted by a cell sorter (BD FACSAria III).

4.4 Cell cytotoxicity assay

CCK-8 assays were used to assess the sensitivity of different cancer cell lines to the drug. Cells were seeded at a density of 5 × 103‒8 × 103 cells/well into 96 well-plates. Then, LXH-3-71 was added at different concentrations after 24 h. After drug treatment for 72 h, 10 μL of CCK-8 (Vazyme, A311-01) solution was added to each well and incubated for another 2 h. The absorbance at 450 nm was measured with a microplate reader. The concentration for 50% growth inhibition (IC50) were calculated using GraphPad Prism software.

4.5 Colony formation assays in soft agar

Compounds were diluted with 2 × McCoy's 5A or DMEM containing 20% FBS to specific concentrations. 1.2% agar and the 2 × medium were 1:1 mixed, then 1 mL of the mixture was layered onto a 35 mm petri dish. Subsequently, 0.6% agar mixed with 2 × McCoy's 5A or DMEM supplemented with 20% FBS, then 800 HCT-116 and 1200 SW480 cells were plated on top of the solidified bottom layer. For PHGDH knockdown or PHGDH-overexpressed cells, culture dishes were coated with 1 mL culture medium mixed by 1.2% agar and 2 × McCoy's 5A. Then 800 indicated cells were re-suspended in 1 mL of growth medium mixed by 0.6% agar and 2 × McCoy's 5A, then plated on top of the solidified bottom layer. The embedded cells were incubated at 37 °C and 5% CO2 until the arising colonies were stained with 0.001% crystal violet.

4.6 Sphere formation assay and limited dilution analysis (LDA)

For sphere formation assay, 1000 HCT-116 and SW480 cells were harvested, and plated into a 96-well ultralow attachment plate (Corning) with serum-free DMEM/F12 medium containing B27 supplement, 20 ng/mL epidermal growth factor and 10 ng/ml basic fibroblast growth factor. Tumorspheres were treated with or without LXH-3-71 at indicated concentrations after 3 days of cell plating. On Day 6, the sphere numbers in each well were quantified. PHGDH knockdown and PHGDH-overexpressed cells were plated as 500 cells per well, tumorspheres numbers were counted after 3 days of incubation.

For in vitro LDA assay, PHGDH knockdown and PHGDH-overexpressed cells were sorted into 96-well ultralow attachment plate at densities of 62, 31, 15 or 8 cell per well. About 10 days later, wells containing spheres were counted and the results were analyzed using a web-based tool (ELDA, http://bioinf.wehi.edu.au/software/elda/index.html).

4.7 Western blotting

HCT-116 and HCT-15 cells were seeded at 3 × 105 cells/well in 6 well-plates. The cells were treated with drugs at the indicated concentrations for the appropriate times, and lysed in RIPA lysis buffer supplemented with a protease inhibitor cocktail/PMSF and dephosphorylase inhibitor NaF. The protein concentration was measured with the BCA Protein Assay Kit (Beyotime Biotechnology, P0009). Equal amounts of proteins were electrophoresed by sodium dodecyl sulfate−polyacrylamide gel electrophoresis under denaturing conditions and transferred onto the PDVF membranes (Millipore Corp., IPVH00010). Membranes were blocked in 5% nonfat milk (BD Difco, 232100) and then incubated with primary antibodies. After being washed, the membranes were incubated with secondary antibodies and detected by a Chemiluminescence Imaging System (MiniChemi610). The following antibodies were used in this study: anti-PHGDH (14719-1-AP, Proteintech), anti-ubiquitin (10201-2-AP), anti-CD44 (15675-1-AP, Proteintech), anti-LGR5 (DF2816, Affinity), anti-CD166 antibody (21972-1-AP, Proteintech), anti-CD133 antibody (18470-1-AP), anti-CDK4 (11026-1-AP, Proteintech), anti-EGFR (sc-373746, Santa Cruz Biotechnology), anti-DDB1 (11380-1-AP, Proteintech), anti-β-actin (66009-1-Ig, Proteintech).

4.8 Biotin−streptavidin affinity pull-down assay

The pull-down assay was performed using a streptavidin-coated agarose bead (Thermo Fisher Scientific). First, the agarose beads were aliquoted and washed with PBS three times. The beads were resuspended in 1 mL of lysis buffer (20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100), and incubated with shaking at 4 °C for 6 h with biotin probes (100 μmol/L). Next, HCT-116 cells were extracted in lysis buffer. The lysates were centrifuged at 12,000 rpm for 10 min and the suspension was kept at 4 °C. The beads were collected, washed three times with PBS, and incubated with shaking with 800 μL of the suspended cell lysate solution at 4 °C overnight. The lysate solution was removed and the beads were washed five times with wash buffer (20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 10% glycerol, 0.1% Triton X-100), resuspended in 100 μL of 2 × sodium dodecyl sulfate (SDS) loading buffer, boiled at 100 °C for 15 min, and centrifuged at 12,000 rpm for 10 min. The supernatant was then analyzed by SDS PAGE. Specific bound proteins were excised for subsequent in-gel digestion and analysis by LC−MS/MS (Fusion).

4.9 Protein expression and purification

The full-length PHGDH open reading frame (Seajet Scientific, Beijing, China) was amplified by polymerase chain reaction (PCR). The PCR fragments were ligated into the pET21a (+) vector, confirmed by DNA sequencing (Genewiz, Beijing, China), and transformed to the BL21 (DE3) strain of Escherchia coli. Recombinant cell was cultivated at 37 °C until the OD600 reached 0.6–0.8. Then, PHGDH expression was induced and the cells were grown for another 18 h at 18 °C. Cells were harvested by centrifugation (5000 rpm, 10 min) and broken by sonication. Insoluble material was separated by centrifugation (16,000 rpm, 45 min) and the supernatant was purified using a nickel-nitrilotriacetic column (HisTrap HP; GE Healthcare). The protein purity was assessed by SDS-PAGE. Protein concentration was measured via Nanodrop 2000 (Thermo Scientific, USA)37.

4.10 Kinase inhibition assay

The kinase activities of PHGDH were assessed using PHGDH activity assay kit (K569, BioVision).

4.11 Immunoprecipitation assay

Cells were lysed in lysis buffer (20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.5% NP-40, protease inhibitor cocktail/PMSF, dephosphorylase inhibitor NaF). Lysate was incubated with 5 μg anti-PHGDH (14719-1-AP, Proteintech) or anti-DDB1 (11380-1-AP, Proteintech) antibodies and immunocomplexes were precipitated using protein A/G PLUS-Agarose (sc-2003, Santa Cruz). Immunoprecipitates were washed six times with wash buffer (20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.05% NP-40, protease inhibitor cocktail/PMSF, dephosphorylase inhibitor NaF) and proteins were eluted with 2 × SDS loading buffer. The supernatant was then analyzed by SDS PAGE.

4.12 siRNA transfection

Cells were transfected with siRNAs using Lipofectamine® RNAiMAX transfection reagent (Thermo Fisher Scientific, Invitrogen, 13778075) following the manufacturer's protocol and analyzed 72 h later. siRNA knockdown efficiencies were validated by western blotting. The sequences of the siRNAs used were as follows:

siDDB1-1: 5′-UAACAUGAGAACUCUUGUC-3′;

siDDB1-2: 5′-GCAAGGACCUGCUGUUUAU-3′;

siCON: 5′-UUCUCCGAACGUGUCACGU-3′.

4.13 Microscale thermophoresis analysis

The purified PHGDH protein and PHGDH protein preincubated with LXH-3-71 (100 μmol/L) were labeled with the NT-647-NHS dye using a protein-labeling kit (NanoTemper Technologies), then diluted into 400 nmol/L with assay buffer including 0.05% Tween-20. Purified DDB1 protein (ab114333, Abcam) were diluted by two-fold and then mixed with the labeled protein 1:1 (10 μL final volume). The samples were loaded into standard capillaries (NanoTemper Technologies) and analyzed on a Monolith NT.115 MST Instrument (NanoTemper Technologies, 20% excitation power, and high MST power). The interactions were tested in three replicates.

4.14 Quantitative RT-PCR

To measure relative gene expression by qRT-PCR, total RNA was extracted using EZ-press RNA Purification Kit (EZBioscience). The cDNA was synthesized from 1 μg of total RNA by HiScript III 1st Strand cDNA Synthesis Kit (Vazyme). PCR amplification performed with the Hieff UNICON®qPCR SYBR Green Master Mix (YEASEN), and the PCR-amplified gene products were analyzed. Levels of mRNA expression were quantified after normalization to GAPDH endogenous control using the ΔCT (difference between cycle thresholds) method.

The mRNA expression of CD133, CD44, LGR5 and CD166 was determined by qRT-PCR. Primers used for qRT-PCR:

CD133, forward 5′-CACTACCAAGGACAAGGCGTTC-3′, reverse 5′-CAACGCCTCTTTGGTCTCCTTG-3′

CD44, forward 5′-CCAGAAGGAACAGTGGTTTGGC-3′, reverse 5′-ACTGTCCTCTGGGCTTGGTGTT-3′

CD166, forward 5′-TCCAGAACACGATGAGGCAGAC-3′, reverse 5′-GTAGACGACACCAGCAACAAGG-3′

LGR5, forward 5′-CCTGCTTGACTTTGAGGAAGACC-3′, reverse 5′-CCAGCCATCAAGCAGGTGTTCA-3′

GAPDH, forward 5′-GGAGCGAGATCCCTCCAAAAT-3′, reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′

4.15 Animal study

The animal studies were approved by the Institutional Review Board of Nankai University. All animal studies were conducted according to protocols approved by the Animal Ethics Committee of Nankai University. A total volume of 0.1 mL of PBS containing 5.0 × 106 HCT-116 cells was injected subcutaneously into six-week-old female BALB/c nude mice. When the tumor volume reached about 50–100 mm3, the mice were randomly divided to vehicle control, 5-FU (10 mg/kg/day, i.p., n = 5) and LXH-3-71 (1 and 3 mg/kg/day, i.p, n = 5) and were treated as above dose. Mice were executed and tumors were harvested on Day 17. Ki67-positive cells and PHGDH-positive cells were staining with anti-Ki67 antibody (27309-1-AP, Proteintech) and anti- PHGDH antibody (14719-1-AP, Proteintech).

For HCT-116 intravenous xenograft, a total volume of 0.1 mL of PBS containing 1.0 × 106 HCT-116 cells with luciferase gene was injected into the tail vein of six-week-old female BALB/c nude mice. Subsequently, the mice were randomly divided into vehicle control (n = 5), 5-FU (10 mg/kg/day, i.p., n = 5), and LXH-3-71 (1 and 3 mg/kg/day, i.p., n = 5) and were treated as above. Bioluminescence signaling was acquired with Imaging System at Day 28 after treatment. Finally, mice were killed at Day 28, and lung tissues were harvested and fixed in 4% paraformaldehyde. Lung sections were stained with hematoxylin and eosin (H&E).

For the tumor initiation assay, a gradient of scramble or PHGDH shRNA cells (1 × 104, 1 × 105 and 1 × 106) were subcutaneously injected into Balb/c nude mice (n = 10). The number of tumors formed was determined 39 days post injection. On Day 39, mice were euthanized and the tumors were excised as well as weighed. CD44-positive cells and CD166-positive cells were staining with anti-CD44 antibody (15675-1-AP, Proteintech) and anti-CD166 antibody (21972-1-AP, Proteintech).

4.16 Statistical analysis

All statistical analyses were conducted using Graph Prism software. Statistically significant differences were determined by Student's t-test, and P values less than 0.05 were considered statistically significant in all cases.

Acknowledgments

This work was supported by the 10.13039/501100001809 National Natural Science Foundation of China (NSFC, No. 82003186 , 82073691 and 82373134 ), the International Science and Technology Cooperation Project of China (No. 2022YFE0133300 ), 10.13039/501100007928 Ningbo Science and Technology Bureau under CM2025 Programme (2020Z092 , China), 10.13039/501100004791 Shenzhen Science and Technology Foundation (JCYJ20210324122006017 , China), Tianjin Natural Science Fund (21JCQNJC01910 , China), China Postdoctoral Science Foundation – Tianjin Joint Support Program (No. 2023T029TJ).

Author contributions

Ziqi Huang: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. Kun Zhang: Data curation, Investigation, Methodology, Writing – review & editing. Yurui Jiang: Data curation, Investigation. Mengmeng Wang: Data curation, Investigation. Mei Li: Data curation, Investigation. Yuda Guo: Data curation, Investigation. Ruolin Gao: Data curation, Formal analysis, Methodology. Ning Li: Data curation. Chenyang Wang: Data curation. Jia Chen: Data curation. Jiefu Wang: Project administration, Supervision, Writing – review & editing. Ning Liu: Data curation, Methodology, Writing – review & editing. Xiang Liu: Methodology, Supervision, Writing – review & editing. Shuangwei Liu: Methodology, Supervision, Writing – review & editing. Mingming Wei: Data curation, Project administration, Supervision, Validation, Writing – review & editing. Cheng Yang: Funding acquisition, Writing – review & editing. Guang Yang: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – original draft, Writing – review & editing.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary information

The following is the Supporting Information to this article:Multimedia component 1

Multimedia component 1

Peer review under the responsibility of Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences.

Appendix A Supporting information to this article can be found online at https://doi.org/10.1016/j.apsb.2024.06.001.
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References

1 Brenner H. Kloor M. Pox C.P. Colorectal cancer Lancet 383 2014 1490 1502 24225001
2 Li K. Wu J.L. Qin B. Fan Z. Tang Q. Lu W. ILF3 is a substrate of SPOP for regulating serine biosynthesis in colorectal cancer Cell Res 30 2019 163 178 31772275
3 Henderson K. Kirkland S.C. Multilineage differentiation of cloned HRA-19 cells in serum-free medium: a model of human colorectal epithelial differentiation Differentiation 60 1996 259 268 8765056
4 Vermeulen L. Todaro M. de Sousa Mello F. Sprick M.R. Kemper K. Alea M.P. Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity Proc Natl Acad Sci U S A 105 2008 13427 13432 18765800
5 Vermeulen L. Snippert H.J. Stem cell dynamics in homeostasis and cancer of the intestine Nat Rev Cancer 14 2014 468 480 24920463
6 Ricci-Vitiani L. Lombardi D.G. Pilozzi E. Biffoni M. Todaro M. Peschle C. Identification and expansion of human colon-cancer-initiating cells Nature 445 2007 111 115 17122771
7 Dalerba P. Dylla S.J. Park I.K. Liu R. Wang X. Cho R.W. Phenotypic characterization of human colorectal cancer stem cells Proc Natl Acad Sci U S A 104 2007 10158 10163 17548814
8 Lopez-Bertoni H. Kozielski K.L. Rui Y. Lal B. Vaughan H. Wilson D.R. Bioreducible polymeric nanoparticles containing multiplexed cancer stem cell regulating miRNAs inhibit glioblastoma growth and prolong survival Nano Lett 18 2018 4086 4094 29927251
9 Rasanen K. Herlyn M. Paracrine signaling between carcinoma cells and mesenchymal stem cells generates cancer stem cell niche via epithelialmesenchymal transition Cancer Discov 2 2012 775 777 22969117
10 Lenos K.J. Miedema D.M. Lodestijn S.C. Nijman L.E. van den Bosch T. Romero Ros X. Stem cell functionality is microenvironmentally defined during tumour expansion and therapy response in colon cancer Nat Cell Biol 20 2018 1193 1202 30177776
11 Govaere O. Wouters J. Petz M. Vandewynckel Y.P. Van den Eynde K. Van den Broeck A. Laminin-332 sustains chemoresistance and quiescence as part of the human hepatic cancer stem cell niche J Hepatol 64 2016 609 617 26592953
12 Wang D. Fu L. Sun H. Guo L. DuBois R.N. Prostaglandin E2 promotes colorectal cancer stem cell expansion and metastasis in mice Gastroenterology 149 2015 1884–95.e4
13 Achouri Y. Rider M.H. Schaftingen E.V. Robbi M. Cloning, sequencing and expression of rat liver 3-phosphoglycerate dehydrogenase Biochem J 323 1997 365 370 9163325
14 Walsh D.A. Sallach H.J. Purification and properties of chicken liver d-3-phosphoglycerate dehydrogenase Biochemistry 4 1965 1076 1085 4378782
15 Snell K. Enzymes of serine metabolism in normal, developing and neoplastic rat tissues Adv Enzyme Regul 22 1984 325 400 6089514
16 Sharif T. Martell E. Dai C. Ghassemi-Rad M.S. Lee K. Singh S.K. Phosphoglycerate dehydrogenase inhibition induces p-mTOR-independent autophagy and promotes multilineage differentiation in embryonal carcinoma stem-like cells Cell Death Dis 9 2018 990 30250195
17 Samanta D. Semenza G.L. Serine synthesis helps hypoxic cancer stem cells regulate redox Cancer Res 76 2016 6458 6462 27811150
18 Li Q. Qiu J. Yang H. Sun G. Hu Y. Zhu D. Kinesin family member 15 promotes cancer stem cell phenotype and malignancy via reactive oxygen species imbalance in hepatocellular carcinoma Cancer Lett 482 2020 112 125 31733289
19 Jeon M.J. You M.H. Han J.M. Sim S. Yoo H.J. Lee W.K. High phosphoglycerate dehydrogenase expression induces stemness and aggressiveness in thyroid cancer Thyroid 30 2020 1625 1638 32438862
20 Mullarky E. Lucki N.C. Beheshti Zavareh R. Anglin J.L. Gomes A.P. Nicolay B.N. Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers Proc Natl Acad Sci U S A 113 2016 1778 1783 26831078
21 Guo J. Gu X. Zheng M. Zhang Y. Chen L. Li H. Azacoccone E inhibits cancer cell growth by targeting 3-phosphoglycerate dehydrogenase Bioorg Chem 87 2019 16 22 30852233
22 Rohde J.M. Brimacombe K.R. Liu L. Pacold M.E. Yasgar A. Cheff D.M. Discovery and optimization of piperazine-1-thiourea-based human phosphoglycerate dehydrogenase inhibitors Bioorg Med Chem 26 2018 1727 1739 29555419
23 Pacold M.E. Brimacombe K.R. Chan S.H. Rohde J.M. Lewis C.A. Swier L.J. A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate Nat Chem Biol 12 2016 452 458 27110680
24 Liu X.H. Li B.L. Zhang Z. Wei Y.J. Xu Z.X. Qin S.L. Synthesis and discovery novel anti-cancer stem cells compounds derived from the natural triterpenoic acids J Med Chem 61 2018 10814 10833 30433783
25 Todaro M. Francipane M.G. Medema J.P. Stassi G. Colon cancer stem cells: promise of targeted therapy Gastroenterology 138 2010 2151 2162 20420952
26 Botchkina G. Colon cancer stem cells–from basic to clinical application Cancer Lett 338 2013 127 140 22537805
27 Hervieu C. Christou N. Battu S. Mathonnet M. The role of cancer stem cells in colorectal cancer: from the basics to novel clinical trials Cancers 13 2021 1092 33806312
28 Jackson P.A. Widen J.C. Harki D.A. Brummond K.M. Covalent modifiers: a chemical perspective on the reactivity of α,β-unsaturated carbonyls with thiols via hetero-Michael addition reactions J Med Chem 60 2017 839 885 27996267
29 Zhao Z. Liu Q. Bliven S. Xie L. Bourne P.E. Determining cysteines available for covalent inhibition across the human kinome J Med Chem 60 2017 2879 2889 28326775
30 Tan Y. Zhou X. Gong Y. Gou K. Luo Y. Jia D. Biophysical and biochemical properties of PHGDH revealed by studies on PHGDH inhibitors Cell Mol Life Sci 79 2021 27 34971423
31 Oh S. Yeom J. Cho H.J. Kim J.H. Yoon S.J. Kim H. Integrated pharmaco-proteogenomics defines two subgroups in isocitrate dehydrogenase wild-type glioblastoma with prognostic and therapeutic opportunities Nat Commun 11 2020 3288 32620753
32 Zhao J.Y. Feng K.R. Wang F. Zhang J.W. Cheng J.F. Lin G.Q. A retrospective overview of PHGDH and its inhibitors for regulating cancer metabolism Eur J Med Chem 61 2018 10814 10833
33 Cho Y.H. Ro E.J. Yoon J.S. Mizutani T. Kang D.W. Park J.C. 5-FU promotes stemness of colorectal cancer via p53-mediated WNT/β-catenin pathway activation Nat Commun 11 2020 5321 33087710
34 Hu J. McCall C.M. Ohta T. Xiong Y. Targeted ubiquitination of CDT1 by the DDB1–CUL4A–ROC1 ligase in response to DNA damage Nat Cell Biol 6 2004 1003 1009 15448697
35 Angers S. Li T. Yi X. MacCoss M.J. Moon R.T. Zheng N. Molecular architecture and assembly of the DDB1–CUL4A ubiquitin ligase machinery Nature 443 2006 590 593 16964240
36 Tao S. Pan S. Gu C. Wei L. Kang N. Xie Y. Characterization and engineering of broadly reactive monoclonal antibody against hepatitis B virus X protein that blocks its interaction with DDB1 Sci Rep 9 2019 20323
37 Wang Q. Liberti M.V. Liu P. Deng X. Liu Y. Locasale J.W. Rational design of selective allosteric inhibitors of PHGDH and serine synthesis with anti-tumor activity Cell Chem Biol 24 2017 55 65 28042046
