Targeting pregnane X receptor with a potent agonist-based PROTAC to delay colon cancer relapse

Bansard, Lucile; Laconde, Guillaume; Delfosse, Vanessa; Huet, Tiphaine; Ayeul, Margaux; Rigal, Emilie; Donati, Quentin; Gerbal-Chaloin, Sabine; Daujat-Chavanieu, Martine; Brunel, Luc; Legrand, Baptiste; Chavanieu, Alain; Martin, Anthony R.; Pannequin, Julie; Bourguet, William; Amblard, Muriel; Pascussi, Jean Marc

doi:10.1038/s41389-025-00573-2

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Published: 30 August 2025

Targeting pregnane X receptor with a potent agonist-based PROTAC to delay colon cancer relapse

Lucile Bansard¹^na1,
Guillaume Laconde²^na1,
Vanessa Delfosse ORCID: orcid.org/0000-0002-7436-7935³,
Tiphaine Huet³,
Margaux Ayeul¹,
Emilie Rigal⁴,
Quentin Donati²,
Sabine Gerbal-Chaloin⁴,
Martine Daujat-Chavanieu⁴,
Luc Brunel²,
Baptiste Legrand²,
Alain Chavanieu²,
Anthony R. Martin ORCID: orcid.org/0000-0001-6187-6979²,
Julie Pannequin¹^na1,
William Bourguet ORCID: orcid.org/0000-0002-0643-7719³^na1,
Muriel Amblard²^na1 &
…
Jean Marc Pascussi ORCID: orcid.org/0000-0002-5168-5383¹^na1

Oncogenesis volume 14, Article number: 34 (2025) Cite this article

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Abstract

Tumor recurrence is frequently attributed to drug-tolerant cancer cells. We previously demonstrated that downregulation of the Pregnane X Receptor (PXR, NR1I2) reduces chemoresistance and prevents colorectal cancer recurrence in xenograft mouse models. However, there is currently a lack of clinically-suitable PXR antagonists. In this study, we report the design and synthesis of a novel PXR agonist-based PROTAC (JMV7048) which promotes polyubiquitination and degradation of the human PXR protein via E3 CRBN ubiquitin ligase and 26S proteasome pathways. JMV7048 selectively degrades PXR in colon carcinoma, hepatoma, and pancreatic cancer cell lines, with no impact on primary human hepatocytes. Notably, JMV7048 reduces PXR protein expression in drug-tolerant colon cancer cells, sensitizing them to chemotherapy and significantly delaying cancer relapse in xenografted nude mice. These findings suggest that PXR-targeting PROTACs may serve as novel therapeutic agents to enhance the sensitivity of chemo-resistant cancer cells to chemotherapy.

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Introduction

Colorectal cancer is the third-most diagnosed cancer worldwide, accounting for 10% of cancer incidence and mortality; it remains a major therapeutic challenge despite mild advances in early detection and therapy [1]. Oxaliplatin, irinotecan, and 5-fluorouracil are the three chemotherapeutic drugs used to treat this disease. The addition of targeted drugs to these chemotherapy regimens has further slightly improved patient survival. Nevertheless, for patients, treatment is unsuccessful once the disease has metastasized to other organs. Tumors recur due to drug-tolerant cells: either Persister Cells [2] or Cancer Stem Cells [3, 4]. These cells share molecular and phenotypic markers such as enhanced ALDH1A1 and Aldefluor activity [5], cytotoxic chemicals metabolization activities and are thus highly enriched after therapy [4, 6]. Targeting these resistant self-renewing cells is therefore crucial to prevent colorectal cancer relapse after initial therapy.

We have previously shown that colon cancer recurrence following chemotherapy is partially due to chemo-resistant cancer cells that overexpress the Pregnane X Receptor protein (PXR, NR1I2) [7]. PXR is a ligand-activated transcription factor within the nuclear hormone receptor superfamily [8]. PXR regulates various metabolic pathways, particularly those involved in the metabolism and clearance of xenobiotics, including chemotherapeutic agents, in the liver and intestine [9, 10]. Indeed, PXR participates in the resistance of malignant tumor cells to numerous anti-tumor drugs [11] such as irinotecan, sorafenib, paclitaxel, etoposide, docetaxel, cisplatin, doxorubicin and adriamycin [12]. Accordingly, PXR expression is associated with poor survival in various types of cancer, including colorectal [13], hepatocellular [14] and pancreatic cancer [15]. In this context, we previously showed that the expression of key chemoresistance genes, including UGT1A1, ALDH1A1, CYP3A4, and ABCG2, depends on PXR expression levels in colorectal cancer and chemo-resistance cancer stem cells [7, 13]. In agreement, PXR knockdown, using either RNAi or drug-induced miRNA, delays post-chemotherapy tumor relapse by increasing the chemo-sensitivity of colorectal cancer cells [7, 16]. As a result, we [7] and others [17] found a negative correlation between PXR expression and recurrence-free survival in chemotherapy-treated colorectal cancer patients. More recently, CYP3A4 and PXR have been identified as markers of pancreatic ductal adenocarcinoma Persister cells enriched in residual disease and associated with poor survival after FOLFIRINOX treatment [18]. Together, these findings highlight PXR as a promising therapeutic target to increase the sensitivity of chemo-resistant cancer cells to antitumor drugs and prevent patient relapse. The race is now on to translate these recent findings into a clinical therapy.

However, although nuclear receptors generally respond to a specific set of high-affinity ligands, PXR is activated by a broad spectrum of low-affinity xenobiotics, and crystallographic studies have unveiled a ligand-binding domain (LBD) characterized by a large and malleable ligand binding pocket [19, 20]. This unique feature makes it challenging to identify PXR antagonists through in silico or conventional medicinal chemistry approaches [21]. Accordingly, only a few inhibitors, antagonists or inverse agonist, have been reported to date. They include SPA-70 and analogs [22, 23], l-sulforaphane [24], ketoconazole [25], and metformin [26]. However, despite significant interest in targeting PXR in cancers or in preventing drug toxicity, no compounds have yet demonstrated efficacy in humans or received approval for clinical use [27, 28].

Given the challenges associated with antagonizing PXR, we chose to target it for degradation using proteolysis targeting chimera (PROTAC) technology [29, 30]. PROTACs are bifunctional molecules that hijack the ubiquitin–proteasome system by simultaneously recruiting the target protein and an E3 ubiquitin ligase. This dual engagement triggers ubiquitination and subsequent degradation of the target protein through the proteasome pathway, even for proteins with low ligand-binding affinity. Unlike traditional small-molecule inhibitors, which typically exhibit reversible binding and short-lived effects upon drug removal, PROTAC-induced protein degradation can persist for an extended period [31]. This could be advantageous for achieving prolonged therapeutic responses and potentially lowering the risk of drug resistance [32]. As a result, PROTACs present an exciting avenue for the development of more effective and durable treatments.

Designing PROTACs targeting PXR presents significant challenges due to the receptor’s complex ligand-binding pocket, which is deeply embedded within its ligand-binding domain [33]. Initial attempts to create a PXR-targeting PROTAC involved fusing the PXR inverse agonist SPA70 with thalidomide, an E3 ligase cereblon (CRBN) substrate receptor ligand [22]. However, this approach resulted in a molecular glue that degraded the GSPT1 translation termination factor protein instead of PXR [34]. Furthermore, recent studies have highlighted additional challenges in designing PXR-specific PROTACs, as several candidates unexpectedly acted as PXR agonists without inducing PXR degradation [35]. Here, we present JMV7048, the first in vivo effective PXR agonist-based PROTAC. This compound integrates a custom-engineered high-affinity human PXR ligand [36, 37] with thalidomide to recruit CRBN (patent WO2022243365). We evaluated the effects of JMV7048 on colon cancer phenotypes, including chemoresistance, self-renewal, tumor initiation, and relapse, in both in vitro and in vivo models, correlating its activity with PXR degradation. Alongside the recent report of a von Hippel-Lindau protein (VHL)-based PXR PROTAC that enhances chemotherapy efficacy in vitro [38], our findings emphasize the promise of this innovative approach for overcoming the challenges associated with targeting the PXR receptor. This work contributes to the development of new therapeutic strategies for chemo-resistant cancers.

Results

Rational development of a new PXR PROTAC

Given the challenging nature of inhibiting PXR activity, our strategy here was to convert a PXR agonist into a PROTAC to induce the degradation of PXR. Because PXR has the most extensive sequence diversity across vertebrate species in the ligand-binding domain of any nuclear receptor [39], with drastic pharmacologic differences between human and mouse PXRs [9], we selected the potent and human specific PXR agonist JMV6845 [36, 37] as our candidate molecule (Fig. 1A and Fig. S1A). After selecting the PXR ligand, it was crucial to initially identify the suitable position for introducing the linker between the ligand of the target protein and the ligase.

**Fig. 1: Transformation of a PXR agonist into PXR-PROTAC.**

Hence, to elucidate the binding mode of JMV6845, we solved the crystal structure of JMV6845-bound human PXR ligand-binding domain at a resolution of 2.45 Å (Supplementary Table 1). The PXR ligand-binding domain adopts the canonical active conformation of nuclear receptors, with its C-terminal activation helix H12 capping the ligand-binding pocket (Fig. S1B). JMV6845 could be positioned unambiguously in the electron density (Fig. S1C) that was well-defined for the entire compound, indicating that JMV6845 is well stabilized in the LBP, in line with its high affinity for PXR. JMV6845 mainly forms van der Waals interactions with residues in the ligand-binding pocket, and only one hydrogen bond is observed between the nitrogen atom of the sulfonamide moiety and S247 in helix H3 (Fig. 1B and Fig. S1C). Surprisingly, none of the remaining polar atoms of JMV6845 appear to be engaged in a direct or water-mediated hydrogen bond with any residues in the pocket. The mesityl sulfonamide moiety resides between helices H3 and H11, and it is adjacent to H12, whereas on the other side of the pocket, the benzyl group is nested into the so-called aromatic π-trap composed of F288, W299, and Y306. The benzyl and mesityl sulfonamide groups are linked by a central benzimidazole moiety between helix H5 and the loop linking helix H2’ and β-strand S1 (loop H2’-S1). Interestingly, despite being fully buried within the ligand-binding pocket, the C-H group of the imidazole ring of JMV6845 (indicated by a red asterisk in Fig. 1C) points toward several secondary structural elements in PXR that were previously shown to display high intrinsic dynamics [20]. Notably, this region of the PXR ligand-binding domain, comprising helix H2’, the following loop H2’-S1 and H6, is not visible in a number of previously published structures, including when the bulky PXR ligand rifampicin [8] is bound [40, 41] (Fig. S1D). In addition to the large volume of the LBP of this receptor, the structural flexibility of H2’ that can shift to accommodate a variety of distinct chemical structures clearly accounts for the promiscuity of PXR. The high malleability of this region has precluded for a long time the generation of antagonists that would destabilize the active conformation of PXR, and ligands using this expansion feature generally bind with lower affinity to the receptor [41, 42]. High dynamics of this LBD region could also be observed in the JMV6845-bound PXR structure as the thermal B-factors (representative of the disorder in the crystal) are the highest in the structure (Fig. S1E). We concluded from these observations that the imidazole C-H group could be the most suitable site for the derivatization of JMV6845 in the construction of PXR PROTACs.

Thus, guided by the binding mode of JMV6845 within the PXR ligand-binding domain, an aminoheptyl chain was introduced onto its imidazole C-H group, resulting in the analog JMV6944, (Fig. 1A). The crystal structure of the PXR ligand-binding domain bound to JMV6944 solved at 2.10 Å resolution (Supplementary Table 1 and Fig. S2A, B) unequivocally revealed that, owing to a large shift of helix H2’ (Fig. S2C), the aminoheptyl chain weaves its way between helices H2’, H6 and H7 to reach the outer surface of the domain (Fig.1C). The free amine of the linker forms a hydrogen bond with the surface residue C207, further stabilizing the interaction of JMV6944 with PXR. Next, we performed competitive binding assays using time-resolved fluorescence resonance energy transfer between a fluorescent PXR ligand and the purified human PXR ligand-binding domain (LanthaScreen TR-FRET PXR Competitive Binding Assay). This revealed that JMV6944 can bind to PXR (IC₅₀ = 719 nM ± 11) (Fig. 1D). Although it has lower affinity than the parental JMV6845 molecule (IC₅₀ = 26 nM ± 5), its binding affinity is comparable to that of compound SR12813 (IC₅₀ = 727 nM ± 6), which is known as one of the most potent human PXR agonists [43]. RT-qPCR analysis confirmed that JMV6944 is a potent inducer of CYP3A4 mRNA expression in freshly isolated human hepatocyte cultures (Fig. 1E), the gold standard for investigating PXR activity [44]. JMV6944 was also a potent inducer of CYP3A4 in LS174T cells (Fig. S2D).

In light of these results, which demonstrate the effective binding of JMV6944 to PXR and the accessibility of its amine function for derivatization, we designed and synthesized JMV6944-based PROTACs. Our primary approach focused on using the CRBN thalidomide ligand as a foundational element for developing PROTACs, as this E3 ligase ligand has been widely used to generate PROTACs currently in clinical trials [32, 45, 46]. To create the PROTAC JMV7048, we introduced a piperazine hexanamide linker to connect the two critical warheads: the CRBN-targeting thalidomide derivative and the PXR agonist JMV6845 (Fig. 1A). JMV7048 was synthesized following the procedure outlined in Supplementary Scheme S1 and described in the referenced patent WO2022243365. We then evaluated JMV7048 for its ability to degrade PXR in LS174T cells, using JMV6845 and JMV6944 as negative controls. We conducted siRNA transfection to precisely detected PXR expression by Western blot. As shown in Fig. 1F, we observed a significant reduction in PXR protein levels following the treatment of LS174T cells with 5 µM JMV7048 for 24 h. In contrast, PXR expression remained unchanged upon treatment with JMV6845 or the synthetic intermediate JMV6944 lacking a E3 ligase ligand. Building upon JMV7048 as a lead compound, we made slight modifications to the linker by extending its length by one or two methylene units, resulting in JMV7505 and JMV7506 respectively (Fig. 1A). Western blot analysis revealed that while the introduction of a single methylene unit within the JMV7048 linker (JMV7505) had no impact on PXR degradation efficiency, the addition of two units (JMV7506) strongly reduced PXR degradation (Fig. 1G). These findings underscore JMV7048 as our lead candidate for PXR degradation. Next, we investigated whether JMV7048 could inhibit downstream PXR signaling. Indeed, while rifampicin and JMV6944 stimulated CYP3A4 mRNA expression, JMV7048 significantly decreased CYP3A4 mRNA level in LS174T cells (Fig. 1H). In addition, JMV7048 inhibited both rifampicin-mediated CYP3A4 promoter activity (Fig. S2E) and protein level (Fig. S2F). These findings strongly suggest that JMV7048 likely functions as a PROTAC by binding to PXR thus promoting its degradation in colorectal cancer cells.

JMV7048 is a bona fide PXR PROTAC

We then performed a focused characterization of JMV7048-mediated PXR degradation. Firstly, dose-dependent assays were performed and demonstrated that JMV7048 potently and efficiently reduced endogenous PXR protein in the colorectal cancer cell line LS174T, with a half maximal degradation concentration (DC₅₀) of 379 ± 12 nM and a maximum degradation efficacy (D_Max) of 62 ± 10% (Fig. 2A). Because the first attempt to create a PXR PROTAC resulted in the generation of an indirect molecular glue (SJPYT-195) with low protein degradation kinetics instead of a bona fide PXR PROTAC [34], we performed comparative time-course analyses of PXR protein and mRNA expression upon JMV7048 treatment. PXR protein responded rapidly to JMV7048, being 50% degraded in one hour (Fig. 2B and Figure S3A). The effect was on the protein as there was no decrease of PXR mRNA level, as expected with a direct protein degrader. In addition, both real-time cell proliferation assays (Fig. 2B) and viability analysis 72 h after treatment exposure (Fig. 2C and Fig. S3B) demonstrate that JMV7048 shows no cytotoxic effects in all tested CRC and normal human colon cell lines. This is evident at a concentration as high as 20 μM of JMV7048 applied for 72 h, in comparison to the active metabolite from irinotecan (SN38).

Next, we performed washout experiments to assess the reversibility and durability of JMV7048 treatment. First, we exposed LS174T cells to JMV7048 for 6 h, then washed out and allowed to grow in PROTAC-free medium. The sustained effect of JMV7048 was revealed by a 50% recovery of PXR levels only after 160 h. The reappearance of PXR was observed only 184 h post-treatment (Fig. 2D). These results confirm a reversible and long lasting PXR degradation. To verify that PXR degradation is dependent on E3 CRBN, a JMV7048 analog incorporating a N-methyl group on the thalidomide moiety to prevent its binding to E3 CRBN [47] was used as negative control (JMV7159, Fig. 2E). At the concentration where the active JMV7048 PROTAC achieved maximal reduction in PXR expression (5 µM for 24 h), JMV7159 showed no impact (Fig. 2F). This highlights the essential role of the E3 ligand moiety in facilitating JMV7048-mediated PXR degradation. Importantly, in contrast to the previously reported PXR PROTAC [34], JMV7048 reduces PXR but not GSPT1 protein (Fig. 2F), which could be an of target. In addition, JMV7048 does not reduce the expression of other closely related nuclear receptors such as FXR (NR1H4), RXRα (NR2B1) or VDR (NR1I1). Both JMV7159 and the JMV7048 were detected in the cytosol of live cells (Fig. 2G). The lack of PXR degradation observed with JMV7159 is therefore not due to defective cell penetration. Additionally, flow cytometry analysis of JMV7048 and JMV7159 uptake in live cells showed similar profiles for both molecules (Fig. 2H and Fig. S3C).

To confirm the dual recruitment of PXR and CRBN by JMV7048, we conducted competitive experiments. TR-FRET PXR Competitive Binding Assay demonstrated that JMV7048 binds to PXR with an IC₅₀ of 1210 nM ± 19 (Fig. 3A). As expected, the CRBN ligand (JMV6945, corresponding to the commercially available pomalidomide analog, (Fig. 3B), was unable to displace the PXR ligand fluorophore. To confirm these data in cellulo, LS174T cells were pre-treated with an excess of the PXR ligand (JMV6944) or an excess of the E3 CRBN ligand JMV6945 (Fig. 3B) and PXR expression was assessed by Western blot. As shown in Fig. 3C both competitors prevented JMV7048-mediated PXR degradation. To confirm the involvement of the ubiquitin-proteasome system in JMV7048-induced PXR degradation, we used the E3 ligase inhibitor MLN4924 (Wu et al. [48]) and the 26S proteasome inhibitor Bortezomib (BZ) [49] (Fig. 3D). Cotreatment of LS174T cells with MLN4924 or BZ further demonstrated that the degradation of PXR protein was dependent on both the E3 CRBN ubiquitin ligase (Fig. 3E) and the 26S proteasome machinery (Fig. 3F), respectively. Finally, immunoprecipitation experiments revealed that JMV7048 significantly increased the accumulation of polyubiquitinated PXR when 26S proteasome activity was inhibited by bortezomib (Fig. 3F). Therefore, these results unequivocally demonstrate that JMV7048 induces PXR degradation through polyubiquitination via CRBN recruitment and the proteasome machinery, validating that our lead compound JMV7048 functions as a PXR PROTAC.

**Fig. 3: JMV7048 is a bona fide PXR PROTAC.**

JMV7048 degrades PXR in cancer cell lines but not in primary hepatocytes

Next, we tested whether JMV7048 degrades endogenously expressed PXR protein, in several cancer cell lines as well as in primary human hepatocyte cultures. We tested LS174T cells (human colon adenocarcinoma) as positive control, as well as HepG2 (human hepatoma) and AsPC-1 (human metastatic pancreatic adenocarcinoma), two cell lines known to express endogenous PXR [50, 51]. As shown in Fig. 4A, 24 h of treatment with 5 µM JMV7048 significantly reduced PXR expression. In contrast, JMV7048 failed to degrade PXR in primary cultures of human hepatocytes (Fig. 4B, C).

**Fig. 4: JMV7048 degrades PXR in pancreas and hepatoma cell lines but not in human hepatocytes.**

To decipher why JMV7048 cannot degrade PXR in human hepatocytes, we investigated JMV7048 metabolic stability. Accordingly, 400,000 cells (freshly isolated human hepatocytes, HepG2 or LS174T) were incubated with JMV7048 for five time-points (0, 5, 10, 60 minutes, and 24 h) and then analyzed by LC-MS/MS. As shown in Fig. 4D, and in agreement with the absence of JMV7048-triggered PXR degradation in human hepatocytes, there was significant acceleration of JMV7048 clearance (CL_int) within hepatocytes compared to colorectal cancer or hepatoma cell lines (8.7- and 5.1-fold, respectively). These results suggest a promising heightened vulnerability of JMV7048 to metabolic processes within human hepatocytes (T_1/2 = 10 h) in contrast to cancer cell lines (with T_1/2 = 3.76 days in LS174T cells and T_1/2 = 2.2 days in HepG2 cells). This is in line with the well-known notion that human hepatocytes harbor enhanced levels of drug transporters and drug-metabolizing enzymes compared to cancer cells as illustrated in Fig. 4E.

JMV7048 reduces the number of chemo resistant-cancer cells

Both drug-tolerant persister cells (Persister cells) and cancer stem cells cancer stem cells are known to express more PXR and drug-metabolizing enzymes or transporters than bulk-cancer cells [7, 18]. We therefore tested the efficacy of JMV7048 in the ALDH-positive subpopulations, as this is a known marker of stem-like cancer cells [52] and Persister cells [5]. LS174T cells were first labeled with an Aldefluor kit (STEMCELL Technologies) and ALDH-negative and ALDH-positive cells were purified by flow cytometry and then treated for 24 h with 5 µM JMV7048 (Fig. 5A). Western blotting analysis confirmed the overexpression of PXR in ALDH-positive cells [7, 16] and revealed that JMV7048 reduces its expression in ALDH-positive cells. RT-qPCR analysis also confirmed that JMV7048 decreased PXR target genes level in LS174T ALDH-positive cells (Fig. 5B and Fig. S4A), including major drug-metabolizing enzymes (CYP3A4, ALDH1A1) and transporters (ABCG2). We then evaluated the effect of JMV7048 on the survival of ALDH-positive cells treated in vitro with a combination of CRC chemotherapy drugs (referred to as “Firi”), consisting of 5-FU and SN38, the active metabolite of irinotecan. As shown in Fig. 5C, treatment of ALDH-positive sorted cells with JMV7048 significantly decreased their survival after 72 h of treatment with Firi (EC50 = 0.69X) compared to DMSO-treated cells (EC50 = 1.33X, i.e., ~2-fold of reduction). Remarkably, the addition of JMV7048 as a PROTAC adjuvant rendered ALDH-positive cells as sensitive to chemotherapy as ALDH-negative cells. Consistent results were observed in ALDH-positive sorted HT29 cells (Fig. S4B, C).

**Fig. 5: JMV7048 decreases PXR expression in colon cancer stem cells and inhibits their population in vitro.**

Having demonstrated that JMV7048 promotes PXR degradation in ALDH-positive cells an sensitizes them to the chemotherapy, we then investigated its effect on cancer stem cell and Persister cell phenotypes. As shown in Fig. 5D, JMV7048 significantly decreased the proportion of cells exhibiting high ALDH activity in LS174T, HT29 and patient-derived colorectal cancer cells established in our lab, either from primary (CPP1) or metastatic (CPP19) tumors. In addition, JMV7048 significantly reduced both sphere formation efficiency and size (Fig. 5E). To continue assessing the impact of degrading PXR on cancer stem cells, we then tested JMV7048-treated cells for tumor initiation in subcutaneous xenografts in nude mice, recognized as the gold standard assay for challenging cancer stem cell potential [53]. CPP1 cells were first treated in vitro for 48 h with 5 µM of JMV7048 before being dissociated. Viable cells were purified by flow cytometry and then injected into nude mice (500 or 5000 cells/mouse, n = 5/group) for in vivo tumor initiation assays. JMV7048 significantly and strongly reduced the number of mice developing tumors (Fig. 5F). The calculated tumorigenicity (frequency of tumor-initiating cells [54]) of colon cancer cells was significantly reduced by JMV7048 (1/3557) compared to DMSO-treated cell (1/311, p = 0.0015).

Finally, we assessed the impact of JMV7048 on the survival of colorectal cancer cells treated with chemotherapy in vitro. HT29 cells were cultured as spheroids [55], while CRC-derived patient cells (CCP1 and CPP19) were maintained as organoids (tumoroids) onto Matrigel [56] to preserve their phenotypic heterogeneity. The cells were allowed to recover for 2 days, pre-treated for 48 h with 5 µM JMV7048 and then challenged with increasing concentrations of Firi. Spheres or tumoroids were then incubated for three days before cell viability measurement. As shown in Fig. 5G, pre-treatment with JMV7048 notably enhanced the sensitivity of HT29 cells to Firi. Specifically, after 72 h of exposure to Firi, spheroid survival was significantly reduced in the JMV7048 pre-treated group (EC₅₀JMV7048 = 0.003X). compared to the DMSO control (EC₅₀DMSO = 0.008X). In accordance, JMV7048 sensitized both CPP1 (EC₅₀DMSO = 0.1562X and EC₅₀JMV7048 = 0.0993X) and CPP19 (EC₅₀DMSO = 0.158X and EC₅₀JMV7048 = 0.0325X) patient-derived tumoroids to chemotherapy treatment, indicating increased drug efficacy. These results demonstrate that, JMV7048 can target chemo-resistant cells, decrease their expression of PXR, and reduce both self-renewal and chemoresistance.

JMV7048 inhibits PXR expression and delays tumor recurrence in xenografted tumors

We first conducted pharmacokinetic studies of JMV7048 in mice following 25 mg/kg intravenous (I.V), 50 mg/kg intraperitoneal (I.P) and 50 mg/kg oral administrations. A single injection of JMV7048 by either I.P. or I.V. route resulted in modest but significant drug exposure in plasma (5.19 μg/mL*h and 10.55 μg/mL*h, respectively). In contrast, oral administration showed very poor pharmacokinetic properties (Fig. 6A).

**Fig. 6: JMV7048 decreases PXR expression and tumor relapse.**

We therefore subsequently explored the efficacy of JMV7048 in preclinical mouse xenograft models. We subcutaneously xenografted 300,000 LS174T cells in SCID mice and allowed tumor volume to reach 100 mm³. Mice were then exposed to I.V. JMV7048 treatment (25 mg/kg/day) for 4 days. Western blotting analyses were then performed to determine PXR expression in excised xenograft tumors. JMV7048 induced a significant (~50%) PXR degradation (Fig. 6B). This demonstrates that this PXR PROTAC can be absorbed and distributed into tissues, at least in subcutaneous xenografts of human tumor cells, and retain its degradation activity in vivo.

We next evaluated the potential toxicity of prolonged I.V. injections of JMV7048 at 25 mg/kg/day, 5 days a week for 2 weeks, in immunodeficient nude and SCID mice (Fig. S5A). First, we did not notice any clinical signs of toxicity (diarrhea, skin ulcers, hyper/hypoactivity, or changes in motor activity), suggesting that JMV7048 is well tolerated by these mice. We then closely monitored changes in body weight, a crucial indicator of drug toxicity (Lewis et al. [57]). As shown in Fig. 6C and Fig. S5B, body weight fluctuations in the JMV7048-treated groups were generally negligible compared to the control groups in both mouse strains, whether administered alone or in combination with chemotherapy (FIRI: 50 mg/kg 5-FU and 25 mg/kg irinotecan, twice weekly).

We finally tested the impact of JMV7048 on post-treatment relapse using two preclinical mouse xenograft models and using two different administration routes (I.V. versus I.P.). For this purpose, we subcutaneously xenografted HT29, a mutant BRAF V600E cell line or CCP1, BRAF wt patient-derived cells, at 20,000 spheroid cells/mouse. Once tumors had reached 100 mm³, mice were randomized and received the following treatment regimens (Fig. S5C): FIRI alone (50 mg/kg 5-FU and 25 mg/kg irinotecan, twice a week), or FIRI plus JMV7048 (25 mg/kg/day I.V. for HT29 cells and 50 mg/kg/ day I.P. for CCP1 cells, 5 days/week). JMV7048 had no significant impact on tumor growth compared to control groups, either as a monotherapy (Fig. S5D) or in combination with FIRI in HT29 (Fig. 6D) and CRC1 (Fig. 6E) xenograft models. By the end of the co-treatment period, tumor volumes were nearly identical between both groups in these models, as JMV7048’s effect on the small but aggressive subpopulations was anticipated to manifest after chemotherapy cessation. Indeed, shortly following treatment, tumors in the control group progressed more rapidly, the gap widening over time with the difference becoming significant approximately two weeks post-treatment.

In assessing the dynamics of tumor growth, we calculated the tumor volume doubling time both during and following cotreatments for each group. During FIRI treatments, the doubling time for HT29 xenografts was 5.6 days regardless of the presence or absence of JMV7048. In CCP1 xenografts, the doubled slower in the presence of JMV7048 (15 days versus 13.3 days for FIRI treatment alone). Tumor relapse was observed in both xenograft models shortly after treatment ended, particularly in the control groups (treated with FIRI alone), as evidenced by a sharp acceleration in tumor growth. For example, after stopping FIRI treatment, the doubling time for HT29 tumors decreased more than threefold (from 5.6 days to 1.8 days), and for CCP1 tumors, it dropped 4.6-fold (from 13.4 days to 2.9 days). However, mice receiving FIRI combined with JMV7048 exhibited a delayed tumor relapse compared to those treated with FIRI alone: reduced by approximately 2.1-fold in HT29 tumors and 2.5-fold in CCP1 tumors, compared to the control FIRI groups. These results suggest that the most aggressive chemo-resistant tumor cells were effectively targeted. This delayed tumor relapse in preclinical models makes JMV7048 a promising adjuvant when combined with chemotherapy.

Discussion

Colorectal cancer research has seen considerable progress in diagnosis, but it remains mostly insensitive to therapy. While targeting cancer stem cells or persister cells appears promising, only a few molecules have successfully completed phase III trials. Indeed, most such strategies developed so far have aimed to block fundamental stemness signaling, with therapeutic limitations due to their impact on normal stem cells as well as cancer ones [58]. For this reason, we believe it is more prudent to target a specific factor associated with cancer stem cells or persister cells chemoresistance. In our previous work, we demonstrated that relapse-prone colon cancer cells exhibit high PXR expression and activity [7, 16]. Because developing PXR antagonists or inhibitors has faced challenges due to the nature of its ligand binding domain, we consequently shifted our focus to a more innovative strategy, the proteolysis targeting chimera (PROTAC).

PROTACs represent an innovative class of compounds that overcome traditional limitations, such as targeting low-affinity and low-specificity undruggable receptors, opening a new therapeutic modality. But at the same time, they challenge the rules used so far for drug discovery. For instance, their high molecular weight (>1000 Da) and the linker instability at the attachment points to ligands, mean that the delivery and bioavailability of PROTACs are significant hurdles to overcome on the way to the clinic [59]. Currently, approximately 20 protein degraders are in clinical trials [60]. The most advanced among these PROTACs is ARV-471 that simultaneously binds the intracellular E3 ligase CRBN and the ligand binding domain of Estrogen Receptor co-developed by Arvinas and Pfizer, which launched phase III clinical trials at the end of 2022 for the treatment of metastatic breast cancer [61]. However, the PROTAC technology is still maturing, and the design elements for successful PROTAC-based drugs are constantly evolving. Most PROTACs made to date have been constructed from antagonists of the target protein, with very few exceptions [62, 63]. However, the first attempt to create a PXR PROTAC from an antagonist led to the generation of a GSPT1 molecular glue instead of a bona fide PXR PROTAC [34]. In addition, several PROTACs designed for other targets act unusual PXR agonist and modulator, by preventing the formation of PXR-corepressor complex rather than promoting PXR-coactivator complex formation [35]. Thanks to crystallization and LBD PXR dynamics studies [20], we successfully developed the first agonist-based PXR PROTAC degrader, JMV7048, which can induce durable degradation of PXR in vitro and in vivo and exhibit therapeutic potential. Unlike previously identified PXR antagonists, which were mostly effective at the micromolar range and required a direct and sustained interaction with the PXR protein, JMV7048 remains active down to 400 nanomolar, and exhibits catalytically effects that endure for several days.

In our investigation, we noticed that while JMV7048 significantly decreased PXR protein expression in colon, hepatoma or pancreatic cancer cell lines, it was inefficient in non-cancer primary human hepatocyte cultures. Accordingly, metabolic stability analysis revealed JMV7048 is more vulnerable to metabolic processes in human hepatocytes than it is in cancer cell lines. These observations suggest that JMV7048 will not interfere with the crucial functions of PXR in the liver, thereby preserving the vital hepatic drug-metabolizing functions of PXR. Indeed, JMV7048 might be rapidly inactivated thought the first-pass effect. This selectivity of JMV7048 in its action on PXR between cancer cells and normal hepatocytes will likely contribute significantly to its development as a targeted therapeutic agent. This will potentially provide a more effective and safe treatment option for several types of cancer without compromising normal liver function and drug metabolism. On the other hand, the enhanced liver first-pass effect will also likely decrease the amount of active JMV7048 reaching the systemic circulation and thus it will require increased oral dosages or alternative administration routes. To circumvent this pitfall, we used parenteral administration in our preclinical tests.

Finally, we observed that JMV7048 is well tolerated, even at 50 mg/kg/day for 3–4 weeks, and does not induce any clinical signs of toxicity in mice. Despite its relatively poor pharmacokinetic properties -JMV7048 has a very short half-life—about 5 min (IV) and 30 min (IP)-, it significantly decreased PXR expression and delayed tumor relapse in xenograft tumor models. These observations support previous studies indicating that PROTACs establish a disconnection between pharmacokinetics and effectiveness [64]. PROTACs are known to function catalytically, meaning they do not rely on sustained drug-receptor binding to be effective. This allows them to work at lower receptor occupancy, often avoiding the need for high drug concentrations. This property is particularly advantageous for targeting low-affinity receptors like PXR or with compounds with low bioavailability, where traditional drugs may struggle to achieve sufficient binding. However, we agree there is now the need for extensive efforts to improve the in vivo efficiency and stability of this molecule. Indeed, minor structural changes, such as fluoration, phthaloyl ring opening, or the use of cognate ligands (e.g., lenalidomide, phenyldihydrouracil) to replace the thalidomide core could enhance bioavailability or address potential safety concerns. These strategies will be investigated in due course.

Methods

General methods and synthesis of JMV7048

All chemicals and solvents were obtained directly from commercial sources. Non-commercial compounds (JMV- 7048, -6944) and intermediates were prepared according to the literature. NMR spectra were recorded on a Bruker 500 MHz spectrometer. Chemical shifts are reported in ppm (δ) and calibrated to the residual solvent signal at 2.50 ppm for 1H spectra in DMSO-d6 and 39.52 ppm for 13 C NMR spectra. 13 C NMR spectra are proton decoupled. Coupling constant are given in Hz. The purity of the final compound (JMV7048) was verified to be ≥95% purity by liquid chromatography analysis at 214 nm on an Agilent Infinity II LC/MSD iQ equipped with an ultraviolet (UV) detector, a SQ MS detector (ES+ mode) and a Poroshell 120 EC-C18 column (2.7 μm, 2.1 × 50 mm; gradient water–acetonitrile (1‰ FA): 100:0 to 0:100 in 5 min; flow = 0.8 ml.min−1). Synthesis procedure of compound JMV7048 is illustrated in Scheme S1.

PXR crystal structures

The human PXR ligand-binding domain was produced and purified as described [65]. Briefly, the relevant coding region (130–434) was expressed in E. coli as a fusion protein containing a fragment of the steroid receptor coactivator-1 (SRC-1, 678-700) in its C-terminal part to improve PXR stability. After an initial purification step by Ni-affinity chromatography, the SRC-1 fusion moiety was cleaved with thrombin prior to size exclusion chromatography. The purified protein was then concentrated at 4 mg.ml⁻¹. PXR complexes were co-crystalized with JMV6845 or JMV6944. 1 μl of protein with 3 molar equivalents of ligand was mixed with 1 μl of precipitant (100 mM imidazole pH 7.0-7.4, 10–13% (v/v) isopropanol), and equilibrated against a reservoir of 500 μl of precipitant. Crystals appeared in 24 h. Diffraction data were collected on the ID30A-3 (JMV6845) and ID23-2 (JMV6944) beamlines at the European Synchrotron Radiation Facilities (λ = 0.96769 Å and λ = 0.87313 Å, respectively, 100 K), Grenoble, France. Data were processed and scaled with XDS and XSCALE [66]. Crystals belong to space group P 43212. The structure was solved and refined using Phenix [67] and COOT [68]. The percentage of residues located in the favored Ramachandran plot region were 98.9% and 99.2% for the JMV6845 and the JMV6944 complexes, respectively (calculated with MolProbity [69]). Data collection and refinement statistics are summarized in Supplementary Table 1. Figures were prepared with PyMOL (http://pymol.org/).

TR-FRET PXR competitive binding assay

TR-FRET PXR (SXR) Competitive Binding Assay Kit (LanthaScreen™) was used to analyze the binding of PXR agonists to the PXR accordingly to the manufacturer’s instructions. Fluorescence reading was carried out using a PHERAstar (BMG Labtech).

Cell lines and patient-derived cells culture

The following cell lines were obtained from the ATCC: LS174T (human colon adenocarcinoma, CL-188), HT29 (human colon adenocarcinoma, HTB-38), AsPC-1 (human pancreas Adenocarcinoma, CRL-1682), HepG2 (human liver carcinoma, HB-8065) and CCP841-CON (normal human colon tissue).

The following cell lines were obtained from CCR patient biopsies: CPP1 (human colon adenocarcinoma), CPP14 (human colon adenocarcinoma), CPP19 (metastatic human colon adenocarcinoma). These biological samples were provided by CHU-Carémeau (Nîmes, France, ClinicalTrial.gov Identifier#NCT01577511) and patients’ clinical characteristics are presented in Supplementary Table 2.

Primary human hepatocytes were isolated as described previously (Pichard et al. [70]) from donor organs unsuitable for transplantation or from liver resections performed in adult patients for medical reasons unrelated to our research program. Liver samples were obtained from the Biological Resource Center of Montpellier University Hospital (CRB-CHUM; http://www.chu-montpellier.fr; Biobank ID: BB-0033-00031) and this study benefitted from the expertise of Dr Benjamin Rivière (hepatogastroenterology sample collection) and Dr Edouard Tuaillon (CRB-CHUM manager). The patients’ clinical characteristics are presented in Supplementary Table 3.

LS174T and AsPC-1 cell lines were cultured in RPMI medium (Thermo Fisher Scientific) containing 10% FBS (Eurobio). HT29, CRC1, CPP19, CCP841-CON cell lines were cultured in DMEM (Thermo Fisher Scientific) containing 10% FBS. The HepG2 cell line was cultured in MEM (Life) containing 10% FBS. All cell cultures were incubated in a humidified atmosphere with 5% CO₂ at 37 °C and divided at 80% confluency. Low passage numbers’ (<12 generations) cells were used. Cell lines were verified to be free of mycoplasma and human (hIMPACT Profile III) pathogens by PCR testing (Idexx Bioanalytics). Hepatocytes were seeded in collagen-coated dishes at 2.1 × 105 cells/cm² in ISOM medium containing 2% bovine serum (Isom et al., 1985). After 24 h of attachment, medium was changed to ISOM without serum.

For spheroid cultivation, the cells were seeded in flasks or wells coated with poly-HEMA (Sigma) at low density (1 cell/µl) in DMEM/F12 medium (Thermo Fisher Scientific) supplemented with Supplement N2 (Thermo Fisher Scientific), hEGF (Miltenyi), hFGF (Miltenyi), glucose 0.3% and insulin 0.002% (Sigma).

Cell viability assays

Cells were plated at 2000 cells per well in 96-well plates in DMEM or RPMI with 10% FBS. After 24 h, cells were treated with compounds for 72 h. Cell viability was assessed by Sulforhodamine B staining or CellTiter-Glo Luminescent cell viability assay (Promega) as previously described (Bansard et al., 2022).

Proliferation assays

The xCELLigence system makes it possible to measure the real-time toxicity, of a molecule, on cell proliferation. LS174T cells were plated at 2,000 cells per well in 8-well microplates in RPMI with 10% FBS and treated with JMV7048 or DMSO for 72 h. Cell proliferation was assessed by cell index calculation.

Transfection assay and oligonucleotides

PXR siRNA (siRNA NR1I2 silencer human, s16909, Life) transfection experiments were performed using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instructions. Final concentration of siRNA was 10 nM.

Luciferase activity

The LS174T cells stably expressing the PXR coding region expression cassette, and both CYP3A4 promoter-luciferase and CMV-driven eGFP constructs, have been described previously [7]. Cell lines were cultured in RPMI plus 5% FBS treated with activated carbon (Eurobio). Cells were lysed with passive lysis buffer (Promega) and GFP was read for viability data in a Tecan apparatus, then the luciferase substrate was added to read the luminescent signal (luc). Data are expressed as a ratio luc/eGFP.

RNA isolation and RT-PCR assays

Cells lines: Total RNA was extracted using the RNeasy mini kit (Qiagen) and treated with DNAse-1 as recommended by the manufacturer. The first-strand cDNA was synthesized using Superscript II (Invitrogen) and random hexamers, and gene expression was measured by real-time PCR.

Primary Human Hepatocytes: After extraction with Trizol reagent (Life Technologies), 500 ng of total RNA was reverse-transcribed using random hexamer and MMLV Reverse Transcriptase Kit (Life Technologies).

Quantitative polymerase chain reactions were performed using the Roche SYBER Green reagent and a LightCycler 480 apparatus (Roche Diagnostic, Meylan, France). Each cDNA sample was amplified in triplicate using SYBR Green (Roche) with a LC480 real-time PCR system (Roche). Primer sequences are presented in Supplementary Table 3. The following program was used: one step at 95 °C for 10 min and then 50 cycles of denaturation at 95 °C for 10 s, annealing at 62 °C for 15 s and elongation at 72 °C for 15 s.

Protein analysis

Cell lines: Cells were lysed with RIPA buffer and prepared for total protein extraction with protease inhibitors (Roche). After 20 min of centrifugation (>16,000 g), the supernatant was collected and protein concentrations were determined by the bicinchoninic acid method (Pierce Chemical, Rockford, IL). Bovine serum albumin (Pierce Chemical) was used as standard. 90 µg of protein were subjected to 10% SDS-PAGE and transferred to nitrocellulose membrane (Amersham). The following antibodies were used with catalog number and final dilutions shown: GADPH (sc-32233, 1/5000), FXR (sc-25309, 1/500), VDR (sc-1009, /1000), RXR-alpha (sc-515929, 1/500), CYP3A4 (sc-53850, 1/500), Ubiquitin (sc-166553, 1/500) and PXR (sc-48340, 1/500; or sc-48403, 1/500) from Santa Cruz, GSPT1 (ab126090, 1/500) and actin (ab253283, 1/1000) from Abcam and finally Actin (A4700, 1/5000) from Sigma. Band intensities were measured with Image Lab software (BIORAD).

Primary Human Hepatocytes: Cytosolic and nuclear fractions were prepared using the NE-PER cell fractionation kit (Thermo Fisher) according to the manufacturer’s instructions. The protein concentration was determined by the bicinchoninic acid method (Pierce Chemical, Rockford, IL). Bovine serum albumin (Pierce Chemical) was used as standard. Twenty µg of nuclear proteins were separated on precast 4–15% SDS-polyacrylamide gels, and then transferred onto polyvinylidene fluoride membranes (both Bio-Rad Laboratories). Membranes were incubated with mouse monoclonal anti-PXR (Santa-Cruz, clone H11) or rabbit anti-TATA-box binding protein (TBP, NB-22-7526, Neo Biotech, Clinisciences, France) antibodies. Immunocomplexes were detected with horseradish peroxidase-conjugated rabbit or mouse secondary antibodies (Sigma) followed by enhanced chemiluminescence reaction (Millipore, Molsheim, France). Chemiluminescence was monitored using a ChemiDoc-XRS⁺ apparatus (Bio-Rad Laboratories) and quantified with Image Lab software (version 6.1).

Xenografts tumors: snap-frozen tumor pieces (±300 mm³) were placed in a tube containing ceramic beads (Matrix D, MP Biomedicals) with 400μl of RIPA. The samples were subjected to 4 cycles of lysis in a FastPrep-24^TM apparatus (MP Biomedicals) for 20 s followed by incubation for 5 min on ice. After 20 min of centrifugation (>16,000 g), the supernatant was collected and protein concentration was determined by the bicinchoninic acid method (Pierce Chemical, Rockford, IL). Bovine serum albumin (Pierce Chemical) was used as standard. Ninety µg of protein were subjected to 10% SDS-PAGE and transferred to nitrocellulose membrane (Amersham).

Immunoprecipitation

LS174 PXR cells were plated at 4.10⁶ cells in 175 mm² flasks in RPMI with 10% FBS. After 6 hours, cells were treated with compounds for 16 h. Cells were lysed with 400 uL RIPA buffer and prepared for total protein extraction with protease inhibitors (Roche). One mg of protein was precleared with A/G agarose beads for 30 minutes before PXR antibody (1.6 μg) incubation (sc-48340, Santa Cruz) for 24 h. Immunoprecipitation was performed with A/G agarose beads for 4 h, beads were then extensively washed 3 times with TBS Tween 0.01% and 1 time with RIPA before resuspension in 50μL 2X Laemmli buffer.

Aldefluor assay and fluorescence-activated cell sorting (FACS)

The Aldefluor assay (Stem Cell Technologies) was performed according to the manufacturer’s instructions. ALDH-positive cells and ALDH-negative cells were identified by comparing the same sample with and without the ALDH inhibitor diethylamino benzaldehyde (DEAB). The gating strategy for flow cytometry analysis of Aldefluor-stained samples was as follows: cells were first stained using the ALDH assay, then stained with SYTOX Blue Dead Cell Stain (Invitrogen). All samples were analyzed by sequential gating including the main population (SSC vs FSC), single cells (SSC-A vs SSC-W), and viable (SYTOX Blue-negative) cells (SSC-A vs SYTOX Blue channel). Dead cells were excluded based on light scatter characteristics and SYTOX Blue Dead Cell Staining. Cells that had been incubated with the DEAB inhibitor were used to set the negative control gate, which was the basis for identification of the ALDH-positive subpopulation in the test samples (without DEAB inhibitor). Cells were sorted using a FACSAria II (BD) and analyzed using Flowing software (v 2.5.1; http://flowingsoftware.btk.fi/). ALDH activity was also analyzed using the MACSQUANT (Miltenyi) analyzer with the protocol defined above.

Microscopy

Cells were plated at 40,000 cells per well in 8-wells Lab-tek chamber slides (Nunc) in RPMI with 10% FBS. After 24 h, cells were treated for 24 further hours with 0.1% DMSO (control), JMV7048 or JMV7159 molecules (5 μM). Cells were then washed twice with 1X PBS, then maintained in 1X PBS 10% SVF and analyzed by Zeiss microscope in bright and EGFP (Ex: 490 nm, em: 510 nm) channels. Images were processed using OMERO software (dimensions XY: 2048 ×2048).

Fluorescence-activated cell sorting (FACS) to follow JMV molecules uptake in live cells

Cells were plated at 200,000 cells per well in 12-well plates in RPMI with 10% FBS. After 24 h, cells were treated with 0.1% DMSO (control), JMV7048 or JMV7159 molecules. After indicated duration of exposure, cells were then washed twice with 1X PBS and then dissociated using Accumax (Sigma-Aldrich) and resuspended in 300 μL of 1X PBS containing 5% FBS and SYTOX Green Dead Cell Stain (ThermoFisher, final dilution: 1/1000). All samples were analyzed by sequential gating including the main population (SSC vs FSC), single cells (SSC-A vs SSC-W), and viable (SYTOX Green-negative) cells (SSC-A vs SYTOX Green channel). Cells that had been incubated with DMSO were used to set the negative control gate, upon which identification of the JMV-positive subpopulation was based. Cells were analyzed using the MACSQUANT (Miltenyi) analyzer and Flowing software (v 2.5.1; http://flowingsoftware.btk.fi/).

Sphere formation assays

Percentage of Cell Forming Spheres (SFE) was determined after plating 200 cells/well in M11 medium in 96 well plates in ultra-low attachment plates (PolyHeme coating). Number and size of spheres (diameter exceeded 50 μM) were measured using ImageJ software between 7 and 10 days.

Tumororoids viability assays

To cultivate tumoroids, 1000 cells were seeded on 96-well plates at a density of 1000 cells in 40 µL of a DMEM/Matrigel mixture (v:v) (Corning). The plates were incubated at 37 °C for 20 min to allow dome formation, after which the domes were overlaid with 100 µL of DMEM/F12 medium (Thermo Fisher Scientific) supplemented with Supplement N2 (Thermo Fisher Scientific), hEGF (Miltenyi), hFGF (Miltenyi), 0.3% glucose, and 0.002% insulin (Sigma). Two days post-seeding, cells were treated with or without PROTACs (5 µM final concentration) for 16 hours and then exposed to increasing concentrations of chemotherapies for 72 h. Drug toxicity in the tumoroids was assessed using the CellTiter-Glow 3D viability assay (Promega), following the manufacturer’s instructions.

In vivo experiments

All experiments were performed according to the European Union (Council directive 86/609EEC) and institutional/local guidelines on laboratory animal usage. Animal protocols were approved by the French ethical committee for animal testing (authorization referral #38102). All efforts were directed at minimizing animal discomfort and to reduce the number of animals used (3 R rule). Rodent housing conditions used in this study were: temperature set point: 22 °C; high limit: 23 °C; low limit: 21 °C Humidity set point: 45%; high limit: 55%; low limit: 40%. Light cycle: 12 h light/dark. No mouse exhibited severe loss of body weight (>15%) or evidence of infections or wounds. Female 4- to 6-week-old athymic nude mice, Crt/NU (NCr)Foxn1nu, BALB/c SCID BCySmn.cb17-prkcdscid/J*) mice (LS174T) were purchased from the Charles River. Cancer cell lines (CRC1 and HT-29) were suspended and counted in cold DMEM or RPMI medium with 50% of Matrigel, and injected into mice subcutaneously. Tumor volume ([length x width²/2) was measured with a caliper. Randomization in order to homogenize tumor sizes across experimental groups and treatment started once tumor volume reached 100 mm3, and mice were sacrificed when tumors reached 1500 mm³.

Tumor initiation assays: CPP1 cells were pre-treated in vitro for 48 h at 5uM then 7AAD negative cells (i.e., viable cells) were resuspended in 100μL cold DMEM medium with 50% of Matrigel, and injected into Crt/NU (NCr)Foxn1nu mice subcutaneously (5000 or 500 cells/mouse). Tumor initiating cell frequencies were determined as previously described (Bansard et al., 2022).

Mice treatment: JMV7048 was dissolved in a vehicle composed by 20% kolliphor hs 15, 5% EtOH qsp water with 5% dextrose and injected by codal (IV) or intraperitoneal injections (IP). FIRI cocktail was dissolved in PBS and IP injected into mice at a dose of 50 mg/kg for 5-FU and 25 mg/kg for irinotecan two times a week.

Statistical analysis

For each experiment, data are shown as mean ± S.D. Statistical analyses were performed using GraphPad Prism 7 software. Comparisons were made between two groups at a time (control versus a single treatment group) using the Mann–Whitney test. The significance level was set at α = 0.05.

Data availability

All data presented in the article and its associated Supplementary information, are available from the corresponding author upon reasonable request.

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Acknowledgements

We acknowledge the contribution of iExplore animal facility (IGF, Montpellier). We thank C. Duperray (IRBM, Montpellier) from the Montpellier RIO Imaging platform for flow cytometry experiments. We acknowledge experimental assistance from the staff of the European Synchrotron Radiation Facility (Grenoble, France) during crystallographic data collection.

Funding

This work was supported by Grants from the ANR (Agence Nationale de la recherche, France), AxLR (INSERM) and CNRS INSB maturation programs, MITI Prime-80 (CNRS), INCA- Cancéropôle GSO, Association pour la Recherche contre le Cancer (France), Ligue contre le cancer (France), Key Initiative Muse “Biomarkers and Therapy”, GEFLUC and SIRIC of Montpellier (France).

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These authors contributed equally: Lucile Bansard, Guillaume Laconde, Julie Pannequin, William Bourguet, Muriel Amblard, Jean Marc Pascussi.

Authors and Affiliations

Institute of Functional Genomics (IGF), Univ. Montpellier, Inserm, CNRS, Montpellier, France
Lucile Bansard, Margaux Ayeul, Julie Pannequin & Jean Marc Pascussi
Institute of Biomolecules Max Mousseron (IBMM), Univ. Montpellier, CNRS, ENSCM, Montpellier, France
Guillaume Laconde, Quentin Donati, Luc Brunel, Baptiste Legrand, Alain Chavanieu, Anthony R. Martin & Muriel Amblard
Center for Structural Biology (CBS), Univ. Montpellier, Inserm, CNRS, Montpellier, France
Vanessa Delfosse, Tiphaine Huet & William Bourguet
Institute for Regenerative Medicine and Biotherapy (IRBM), Univ. Montpellier, INSERM, CHU Montpellier, Montpellier, France
Emilie Rigal, Sabine Gerbal-Chaloin & Martine Daujat-Chavanieu

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Lucile Bansard
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Guillaume Laconde
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Vanessa Delfosse
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Tiphaine Huet
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Luc Brunel
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Anthony R. Martin
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Muriel Amblard
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Jean Marc Pascussi
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Contributions

GL, AC, JP, MA and J-MP conceived the work. LB, GL, JP, MA and JM-P, designed parts of the experiments. LB, GL, VD, LB, TH, MA, ER, QD, SG-C, M-C, LB, BL, and ARM performed the experiments. LB, GL, LB, VD, TH, MA, ER, QD, SG-C, M-C, BL, and ARM analyzed the data. JP, WB, MA, and J-MP wrote the manuscript. All authors have given approval to the final version of the manuscript.

Corresponding authors

Correspondence to Muriel Amblard or Jean Marc Pascussi.

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Supplementary information

original data file Western blot

original data file RT-qPCR

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Bansard, L., Laconde, G., Delfosse, V. et al. Targeting pregnane X receptor with a potent agonist-based PROTAC to delay colon cancer relapse. Oncogenesis 14, 34 (2025). https://doi.org/10.1038/s41389-025-00573-2

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Received: 20 May 2025
Revised: 04 July 2025
Accepted: 24 July 2025
Published: 30 August 2025
DOI: https://doi.org/10.1038/s41389-025-00573-2