Varying Combination Strategies and their Influence on JH-RE-06 and Oxaliplatin Efficacy.
It is well-established that oxaliplatin, the primary platinum-based therapy for CRC18, creates DNA adducts or inter-strand crosslinks (ICLs). REV 1 promotes resistance to oxaliplatin in cancer cells by recruiting Pol ζ to bypass these DNA damage19. We hypothesized that combining JH-RE-06, a REV1 inhibitor, with oxaliplatin could improve therapeutic efficacy and alleviate resistance. JH-RE-06 was found to suppress the growth and proliferation of CRC cells in a concentration- and time-dependent manner (Figure S1A). SW620 cells were more sensitive to JH-RE-06 than HCT116 cells. We then investigated 3 drug combination protocols: simultaneous delivery, pre-treatment with one drug followed by the other, and vice versa (Fig. 1A). CCK8 assays revealed that the above drug combinations did not yield a synergistic effect on CRC cells (Fig. 1B). Interestingly, CRC cells remained resistant to oxaliplatin even after JH-RE-06 pre-treatment. However, the most effective treatment was sequential administration, with oxaliplatin followed by JH-RE-06 (OXA-JH), yielding an oxaliplatin IC50 of 2.1 µM. We then investigated whether JH-RE-06 enhanced oxaliplatin-induced DNA damage, as this synergy was the theoretical rationale. γH2AX foci levels were slightly elevated in the combination treatment group, but this increase was not statistically significant (Fig. 1C). This finding is supported by two other DNA damage markers, FANCD2 and 53BP1 (Fig. 1D). Furthermore, western blotting for caspase-3 and PARP1 cleavage showed reduced cleavage with simultaneous drug administration, indicating no synergistic effect on DNA damage-induced cell death (Fig. 1E, F). Finally, using an oxaliplatin-resistant HCT116 cell model (Figure S1B), we assessed the responsiveness of resistant cells to JH-RE-06. CCK8 assays and crystal violet staining indicate that JH-RE-06 effectively inhibits both the proliferation and clonogenic capacity of HCT116 cells(Figure S1C). Importantly, JH-RE-06 effectively suppressed tumor cell growth at concentrations non-toxic to normal cells. In conclusion, the cisplatin sensitizer JH-RE-06 induces elevated levels of cleaved-caspase3 and cleaved-PARP1 in CRC cells, yet does not enhance sensitivity to oxaliplatin. Notably, oxaliplatin-resistant HCT116 cells retain sensitivity to JH-RE-06, suggesting its potential as a second-line chemotherapy option for CRC.
The cellular signal triggered by JH-RE-06 and Oxaliplatin in HCT116 cells beyond the scope of DNA damage.
To understand the global proteomic changes induced by JH-RE-06 and oxaliplatin in CRC cells, we treated HCT116 cells with DMSO, 5 µM OXA, 3 µM JH-RE-06, or the combination (OXA-JH: 5 µM oxaliplatin + 3 µM JH-RE-06) for 24 hours and performed TMT proteomic sequencing (Figure S2A, B). Differentially expressed proteins were defined by a fold change of ≥1.2 or ≤1/1.2 (p-value < 0.05) compared to the DMSO control group. We identified 285, 650, and 570 differentially expressed proteins in the OXA, JH-RE-06, and OXA-JH groups, respectively (Fig. 2A, B, C). OXA treatment downregulated proteins related to ribosomes, ribosome biogenesis, and DNA replication. This aligns with previous research suggesting that oxaliplatin primarily induces ribosomal stress, rather than direct DNA damage, as its mechanism of action 20. Conversely, OXA treatment upregulated proteins enriched in the P53 signaling pathway (Fig. 2D, E). In the JH-RE-06 group, downregulated proteins were associated with DNA replication, oxidative phosphorylation, and cell cycle. Upregulated proteins were significantly enriched in the ferroptosis pathway, including SLC7A11, GCLC, SLC3A2, FTL, NCOA4, FTH1, GCLM, MAP1LC3B, HMOX1, and SLC39A14. These proteins are directly involved in iron metabolism and antioxidant transport, both core processes related to ferroptosis. Additionally, upregulated proteins showed significant enrichment in metabolic pathways such as steroid biosynthesis, cysteine and methionine metabolism, and the TCA cycle (Fig. 2F, G). The OXA-JH combination primarily downregulated proteins involved in DNA damage-related processes (DNA replication, mismatch repair, nucleotide excision repair, and base excision repair). Conversely, ferroptosis pathway proteins remained upregulated (Fig. 2H, I). To clarify, JH-RE-06, a tiny chemical designed to specifically inhibit the DNA damage repair pathway TLS, induces significant changes in the cell that transcend the DNA damage signal. Crucially, JH-RE-06 specifically elevated proteins associated with the ferroptosis pathway. Oxaliplatin, capable of inducing DNA adduct formation, was determined to elicit ribosome stress according to proteome sequencing results (Fig. 2J).
JH-RE-06 triggers ferroptosis in CRC cells.
Proteomic sequencing revealed enrichment in mitochondrial-related pathways, prompting us to investigate changes in the subcellular structures of JH-RE-06-treated CRC cells. Using transmission electron microscopy (TEM), we observed a significant decrease in mitochondrial abundance in both HCT116 and SW620 cells after 12 and 24 hours of JH-RE-06 treatment. Autophagic vacuoles containing engulfed mitochondria were evident (Fig. 3A, B). Given the proteomic data indicating changes in iron transport proteins and upregulation of ferroptosis markers, together with recent studies on the link between autophagy, the labile iron pool (LIP), and ferroptosis21, we hypothesized that JH-RE-06 induces ferroptosis in CRC cells. Using specialized assay kits, we found that intracellular free Fe2+ levels increased after 6 hours of treatment, peaking at 24 hours(Fig. 3C, D). Simultaneously, the lipid oxidation marker MDA rose (Fig. 3E, F), while GSH levels consistently decreased (Fig. 3G, H). Since cellular iron homeostasis is a key regulator of ferroptosis sensitivity, we co-treated CRC cells with the iron chelator deferoxamine (DFO) and JH-RE-06. At 12 hours, DFO decreased intracellular Fe2+ levels and by 24 hours, MDA levels were also reduced. This suggests that JH-RE-06 may promote Fe2+-dependent ferroptosis. We then used QPCR to analyze the transcription levels of ferritin (FTH and FTL) and glutathione-related genes (GCLC and GCLM) in HCT116. JH-RE-06 enhanced the transcription of ferroptosis-related proteins, and this effect was counteracted by DFO (Fig. 3I, J). These findings indicate that an iron-dependent mechanism was involved in the ferroptosis triggered by JH-RE-06 in CRC cells.
JH-RE-06 triggers cleaved-caspase3, cleaved-PARP1 and ferroptosis via NCOA4-mediated ferritinophagy
Proteomics sequencing revealed increased NCOA4 expression and decreased expression of MCM2-7 proteins, which are essential for DNA replication. REV1 inhibition can disrupt DNA gap filling, leading to the activation of DNA replication origins in cancer cells. Since NCOA4 regulates DNA replication origins by managing LIP levels and MCM2-7 proteins22,23, and promotes ferroptosis via ferritinophagy24,25, we hypothesized that JH-RE-06 elevates LIP in an NCOA4-dependent manner. To investigate, we used western blotting (WB) to assess changes in ferritinophagy-related proteins expression after JH-RE-06 treatment in CRC cells. We found that NCOA4, P62, and LC3 expression increased at 12h and 24h, while FTH1 increased at 12h but decreased at 24h. Consistent with prior findings, DFO alone increased NCOA4 and decreased FTH126. However, DFO did not alter these JH-RE-06-induced changes in our setting (Fig. 4A, B). We then silenced ATG7 (essential for autophagy) and treated cells with JH-RE-06 (Fig. 4C). WB analysis showed FTH1 upregulation in si-ATG7 cells compared to si-control cells, reversing the JH-RE-06-induced downregulation. The expression trends of NCOA4, LC3, and P62 were also partially reversed in si-ATG7 cells (Fig. 4D). Using FerroOrange to monitor LIP, we found that JH-RE-06 increased intracellular free iron levels in HCT116 cells. Importantly, this increase was not observed in both si-ATG7 and si-NCOA4 groups, indicating that JH-RE-06 influences free Fe2+ levels through NCOA4-mediated ferritinophagy (Fig. 4E). Similarly, MDA levels were reduced when ATG7 or NCOA4 was silenced prior to JH-RE-06 treatment (Fig. 4F). Interestingly, JH-RE-06 increased cleaved-caspase3 and cleaved-PARP1 levels after 24 hours, a response reversed by DFO, especially in si-ATG7 cells, suggesting JH-RE-06 triggers Caspase3 and PARP1 cleavage in a ferritinophagy-dependent manner (Fig. 4D). Based on the above findings, we propose that JH-RE-06 induces NCOA4-mediated ferritinophagy, ultimately leading to altered iron homeostasis and elevated levels of cleaved caspase-3, cleaved PARP1, and lipid peroxidation.
The KEAP1-NRF2-ARE pathway is involved in regulating JH-RE-06 induced programmed cell death in CRC cells.
To investigate whether JH-RE-06-induced cell death could be reversed, we pre-treated CRC cells with a series of programmed cell death inhibitors, including apoptosis inhibitor Z-VAD-FMK, necroptosis inhibitor Necrostatin-1, autophagy inhibitor Chloroquine, and Brefeldin A, ferroptosis inhibitor DFO and ferrostatin-1, and cysteine regenerator L-Penicillamine (L-Pen), 2-Mercaptoethanol (2-ME) and N-acetylcysteine (NAC). CCK8 assays showed that while JH-RE-06 increased MDA levels, neither ferroptosis inhibitor prevented cell death. However, co-treatment with a disulfidptosis inhibitor completely rescued JH-RE-06-induced cell death (Fig. 5A, B). Given that disulfidptosis involves cytoskeletal collapse, we used phalloidin staining after drug treatment. In SW620 cells, cytoskeletal collapse began 12h post-JH-RE-06 treatment and worsened by 24h. Co-treatment with NAC, 2-ME, and L-Pen significantly reversed this collapsed morphology (Fig. 5C). Considering that L-Pen, 2-ME, and NAC all play roles in cysteine regeneration and antioxidant activity, we speculate that the cells experience oxidative stress following drug induction.Based on the results of proteomic sequencing, we focused on NRF2 and its downstream gene SLC7A11, GCLC, SLC3A2 (4F2hc), GCLM, HMOX1, and SLC39A1427. We isolated proteins from HCT116 and SW620 cells treated with JH-RE-06 for 12 and 24 hours. WB revealed time-dependent KEAP1 degradation and increased NRF2 levels at 12 hours following drug treatment. Simultaneously, the ARE proteins SLC7A11 and SLC3A2 (4F2hc, 75KD band) were upregulated. Pre-treatment with DFO reversed the increase in SLC7A11 expression but did not affect SLC3A2. We also observed a time-dependent decrease in glutathione peroxidase 4 (GPX4) levels (Fig. 5D, E). Immunofluorescence confirmed that JH-RE-06 induced NRF2 nuclear translocation from 6 to 24 hours, correlating with KEAP1 degradation (Fig. 5F, G). Furthermore, MDA levels increased significantly in NRF2 knockdown cells treated with JH-RE-06 compared to the si-control group (Figure S3A, B). The qPCR results suggest that the upregulation of SLC7A11 and SLC3A2 occurs at the transcriptional level (Figure S3C). This, suggests that KEAP1-NRF2-ARE pathway is involved in regulating JH-RE-06 induced programmed cell death in CRC cells.
JH-RE-06 inhibited the growth of oxaliplatin-resistant tumors in vivo.
While the combination of oxaliplatin and JH-RE-06 yielded inconsistent therapeutic effects across various drug combination methods, the ability of JH-RE-06 to inhibit oxaliplatin-resistant cancer cells suggests its potential application in second-line chemotherapy for colorectal cancer. To investigate this potential, we evaluated the therapeutic efficacy of JH-RE-06 in vivo using a xenograft mouse model established with the oxaliplatin-resistant HCT116 cells. Mice were divided into four groups and treated with oxaliplatin and JH-RE-06 for 40 days following a 12-day tumor growth period (Fig. 6A). No significant differences in body weight were observed among the treated groups. As expected, the tumors in the DMSO group were significantly larger, and oxaliplatin treatment failed to control tumor growth, confirming the resistance of the cell line. Conversely, the JH-RE-06 treatment group exhibited the most significant control of tumor volume, demonstrating its efficacy against oxaliplatin-resistant tumors (Fig. 6B, C). While pre-treatment with oxaliplatin did not abolish the effectiveness of JH-RE-06, it did not lead to a higher therapeutic effect. We further evaluated the safety of JH-RE-06 by examining the hearts, livers, spleens, lungs, and kidneys of treated mice through H&E staining. No significant pathological damage was observed in these major organs(Fig. 6D). Additionally, immunohistochemistry for proliferation markers Ki67 and PCNA revealed that JH-RE-06 significantly suppressed tumor growth and blood vessel formation. Consistent with previous findings suggesting JH-RE-06-induced caspase3 cleavage in resistant cells, immunohistochemistry confirmed an increase in cleaved-caspase3 levels in the tumor tissue (Fig. 6E). These findings suggest that JH-RE-06 effectively suppresses the proliferation of oxaliplatin-resistant CRC cells in vivo with minimal adverse effects. This data supports the potential of JH-RE-06 as a viable second-line chemotherapeutic option for CRC treatment.