ABSTRACT
-
Background/Aims
Biliary tract cancer (BTC) frequently harbors KRAS mutations, which are associated with resistance to traditional treatment and a poor prognosis. Synthetic lethality (SL) strategy may provide other targets of KRAS. Therefore, we aim to identify and validate potential therapeutic targets of KRAS for the treatment of BTC via SL.
-
Methods
The dependency (DepMap) projects were used to predict the synthetic lethal gene of KRAS. FDA-approved anticancer drug library was applied to screen potential drugs effective against KRAS-mutant BTC. Furthermore, the synthetic lethal effects or corresponding mechanisms of potential genes and drugs on BTC were investigated using KRAS-mutant and KRAS-wild type BTC cell lines, patient-derived xenografts (PDX), and KRAS oncogene-driven tumor models, as well as other KRAS-mutant cancer cell lines.
-
Results
Initially, we discovered that the loss of GATA2 reduced the viability of KRAS-mutant but not KRAS-wild-type BTC. Subsequently, the drug library screened out disulfiram, which primarily exerts a synthetic lethal effect by inhibiting interleukin-1β (IL-1β) in KRAS-mutant BTC. Mechanistically, GATA2 specifically enhanced the transcription of IL-1β to promote NF-κB signaling in KRAS-mutant BTC. IL-1β inhibition phenocopied GATA2 deficiency, leading to reduced KRAS-mutant BTC viability. These synthetically lethal effects were confirmed using PDX, a KRAS oncogene-driven tumor model, as well as in other KRAS-mutant cancer cell lines.
-
Conclusions
In summary, these results indicate that inhibiting GATA2/IL1β could be a therapeutic strategy in KRAS-mutant BTC and potentially other cancers.
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Keywords: KRAS mutation; Oncogene; Synthetic lethality; Biliary tract cancer; Drug screen
Study Highlights
• Transcription factor GATA2 has a synthetic lethal effect on KRAS-mutant BTC.
• Disulfiram, an FDA-approved drug, primarily exerts a synthetic lethal effect by inhibiting IL-1β in KRAS-mutant BTC.
• GATA2 specifically enhanced the transcription of IL-1β to promote NF-κB signaling for KRAS-mutant BTC survival.
• The synthetic lethal effect of inhibiting GATA2/IL1β could extend to other KRAS-mutant cancers.
Graphical Abstract
INTRODUCTION
Biliary tract cancer (BTC), which arises from intrahepatic or extrahepatic bile ducts or gallbladder, is an aggressive malignancy with a poor prognosis and rising incidence worldwide [
1,
2]. Despite surgery being the most effective treatment for BTC, nearly half of patients suffer from a recurrence within a year [
3,
4]. Moreover, due to the BTC’s asymptomatic behavior, most patients are diagnosed at an advanced or metastatic stage, limiting related treatment options, and resulting in less than 15% of patients surviving beyond five years [
3,
5,
6].
KRAS mutation is a common and significant gene alteration in BTC, and its mutation is associated with resistance to chemotherapy and systematic treatments [
7,
8]. Although
KRAS mutations have been studied in human cancers for 30 years, direct therapeutic targeting of
KRAS has only recently become a reality [
9-
11]. Sotorasib, a recently FDA-approved
KRAS mutation-selective inhibitor, has demonstrated efficacy against
KRASG12C-mutant non-small cell lung cancer. However, its benefits are limited by specificity to
KRASG12C-mutant tumors (which are rare) and by frequent acquired resistance [
12,
13]. Other KRAS inhibitors have shown promise but are in earlier stages of development. Almost 80% of mutations in BTC are
KRASG12D and
KRASG12V [
14-
16]. Therefore, developing new therapeutic strategies is crucial to overcoming this challenge and improving outcomes in
KRAS-mutant BTC.
Synthetic lethality (SL) is a phenomenon where a single genetic event is not enough to cause cell death, but the occurrence of multiple genetic events leads to cell death [
17,
18]. The concept of SL originated from a genetic phenomenon in fruit flies and has been exploited in anticancer therapeutics [
17-
19]. For instance, PARP inhibitors exploit SL by inducing cell death in BRCA-mutant cells while leaving normal cells unharmed [
20,
21]. With the development of various screening methods, including drug screens, RNA interference screens, clustered regularly interspaced short palindromic repeats (CRISPR) screens, and bioinformatics screens, more and more SL relationships were reported and the characterization of SL genetic interactions with tumor-specific mutations can be used to develop a conceptual framework for tackling those “undruggable” targets [
19,
22,
23]. Based on the above SL strategy, it seems to find new ways to target the pan-
KRAS-mutant BTCs.
In this study, we first performed genome-scale CRISPRCas9-based SL screens via the cancer dependency (Dep-Map) projects to uncover therapeutic targets for BTC with a KRAS mutation. We then evaluated the effect of inhibiting a potential target (GATA2) in KRAS-mutant BTC cells in vitro and in vivo. In parallel, we conducted a screening of an FDA-approved anticancer drug library and identified disulfiram as a compound that selectively induces synthetic lethal effect in KRAS-mutant BTC, primarily through the suppression of interleukin-1β (IL-1β) signaling. We further revealed the potential mechanism of interaction between GATA2 and IL-1β in KRAS-mutant BTC and examined its relevance in patient-derived xenograft (PDX) and an oncogenic KRAS-mutant BTC model, as well as in other types of KRAS-mutant cancer cell lines.
MATERIALS AND METHODS
Ethical regulations
All animal procedures were performed following the Guidelines for Care and Use of Laboratory Animals of Zhejiang University (Protocol No. 24622). In accordance with the requirements of the Laboratory Animal Welfare and Ethics Committee of Zhejiang University, the size of the subcutaneous tumor and body tumor of mice must not exceed 1,000 mm3, in which the diameter of any dimension must be less than 10 mm. Once this size is reached, euthanasia must be performed. In every animal experiment described in this article, the maximal tumor size/burden of the mouse was never exceeded.
Detailed information refers to the supplementary materials.
RESULTS
GATA2 inhibition is synthetic lethal for KRAS-mutant BTC cells in vitro and in vivo
To discover novel genetic vulnerabilities in
KRAS-mutant BTC, we mined data from the cancer DepMap project [
24]. Seven genes that displayed the strongest DepMap in
KRAS mutant BTC cell lines were selected as synthetic lethal partner candidates: PYROXD1, MRE11, EIF4A1, INO80B, ARPC2, GATA2, and VGLL2 (
Fig. 1A). To verify this finding, we analyzed seven BTC cell lines and a human intrahepatic biliary epithelial cell line (HIBEC) to select two
KRAS-mutant cell lines (RBE, NOZ) and two
KRAS-wild type (WT) cell lines (CCLP1, GBC-SD) for subsequent experiments (
Supplementary Fig. 1A,
1B). Compared to the other six genes, short-term GATA2 suppression resulted in a distinct decrease in cell viability in
KRAS-mutant BTC cells (
Fig. 1B), indicating that GATA2 is a potential synthetic lethal partner of KRAS.
Then, the synthetic lethal effect of GATA2 on cell functions including apoptosis, proliferation, and migration was performed using a range of
in vitro assays using small interfering RNAs (siRNAs) that deplete KRAS or GATA2. As shown in
Figure 1C, GATA2 depletion induced caspase-3 activation and PARP cleavage in RBE and NOZ cells but did not in CCLP1, GBC-SD, and HIBEC cells, comparable to the effect seen with loss of KRAS itself. Similarly, flow cytometry analysis presented the same results (
Supplementary Fig. 1C). Cell counting kit-8 (CCK-8) assays demonstrated that knockdown of GATA2 substantially inhibited cell proliferation only in
KRAS-mutant BTC cells RBE and NOZ (
Fig. 1D). Colony formation assays further confirmed the inhibitory effect of GATA2 suppression on cell proliferation (
Supplementary Fig. 1D). Moreover, transwell assays demonstrated that cell migration was strongly impaired by the depletion of GATA2 in RBE and NOZ compared with WT cancer cells (
Fig. 1E). In wound healing migration assay, RBE and NOZ also exhibited delayed wound healing (
Supplementary Fig. 1E). These findings support the hypothesis that
KRAS-mutant BTC cells are vulnerable to GATA2 inhibition and may require GATA2 for tumor survival.
The BTC cells with stable knockdown of KRAS or GATA2 were implanted into nude mice to further examine the synthetic lethal effect
in vivo. Each mouse was monitored once every three days, and the mice were euthanized after three weeks. In the
KRAS-mutant tumor (NOZ) group, inhibition of KRAS or GATA2 significantly reduced the growth, size, and weight of tumors (
Fig. 1F,
Supplementary Fig. 1F). In contrast, these effects were not seen in
KRAS-WT tumors (GBC-SD). Hematoxylin and eosin (H&E) and Ki-67 immunostaining of xenograft tumor sections revealed similarly reduced cellularity and cell proliferation after KRAS or GATA2 inhibition in
KRAS-mutant BTC tumors (
Supplementary Fig. 1G). These findings suggest that
KRAS-mutant BTC cells depend upon GATA2 for survival.
Notably, previous studies have demonstrated that Rasmediated p38/ERK signaling promotes GATA-2 phosphorylation, which in turn enhances GATA-2 chromatin occupancy at its target gene loci, thereby up regulating the expression of IL-1β [
25]. Likewise, we observed similar regulatory patterns in BTC cell lines, as demonstrated by the elevated phosphorylation levels of ERK, p38 and GATA2 in
KRAS-mutant cell lines (RBE and NOZ) relative to non-
KRAS-mutant cell lines (CCLP1 and GBC-SD) (
Supplementary Fig. 1H).
Given the difficulty of directly targeting transcription factors such as GATA2 with small molecules, we next conducted a screen of 1,166 compounds using the FDA-approved anticancer drug library provided by MedChemExpress in RBE (
KRAS-mutant) and CCLP1 (
KRAS-WT) BTC cells. After cells were treated with the 1,166 inhibitors (1 μM) for 72 hours in 96-well plates using a “one well-one inhibitor” format, we determined the difference in the percent of viability [D=(% CCLP1 viability)−(% RBE viability)] for each compound based on the average of two screens.
Figure 2A shows that 870/1,166 compounds (75%) affected RBE and CCLP1 cell viability equally, with D values from −25% to +25%. Disulfiram (
Fig. 2A, inset) had the highest selectivity for inhibiting the viability of RBE versus CCLP1 cells (D=75%), inhibiting RBE cells by about 90% and 91% (two independent screens) but CCLP1 cells by only about 14 and 15% (
Fig. 2B). Notably, the top 20 compounds including disulfiram with the highest D value share a common feature: they are predominantly inhibitors of IL-1β or NF-κB. Because they showed similar anti-tumor effects to GATA2 against
KRAS-mutant BTC, the potential mechanism by which GATA2 regulates the survival of
KRAS-mutant BTC may be close to IL-1β/NF-κB and it needs to be further explored. Besides, we have presented the top 20 drugs with the most significant synthetic lethal effect by drug screening and cross-analyzed them in the published data in
Supplementary Table 1.
Next, we verified the synthetic lethal effect of disulfiram in
KRAS-mutant BTC cells
in vitro and
in vivo. First, we treated
KRAS-mutant,
KRAS-WT, and HIBEC cells with different concentrations of disulfiram, and cell proliferation was monitored by CCK-8. The 72 hours CCK-8 assays showed that RBE and NOZ cells were more sensitive to disulfiram at the same concentration compared to
KRAS-WT cells, suggesting disulfiram specifically inhibits the growth of
KRAS-BTC cells (
Fig. 2C). To solve the problem of distinct genetic backgrounds, we have constructed an isogenic cell model (G12D-SD) by introducing the KRAS G12D mutation into a KRAS wild-type cell line. Then, we have verified through CCK8 assays, flow cytometry-based apoptosis assay, and colony formation assays that both GATA2 and disulfiram exhibit significant SL in G12D-SD (
Supplementary Fig. 2A–
2C).
Furthermore, we verified the synthetic lethal effect of disulfiram on the PDX animal models that originated from two advanced BTC patients (
KRAS-WT versus
KRASG12V mutation). In the
KRASG12V mutant model, the disulfiram treatment significantly inhibited the growth of tumors. This effect was not observed in the
KRAS-WT model (
Fig. 2D). To further validate these results
in vivo, we used a genetically engineered mouse model of
Kras-mutant BTC. Six-week-old B6-Kras-LSL-G12D homozygous mice were injected with pAV-ck19-cre packaged virus via the tail vein. About 8 weeks later, detailed histological analysis including H&E, CK7, CK19, CK20, CEA, HepPar-1, ARG1, GPC3, AFP and CD34 staining of the tumor tissue (
Supplementary Fig. 2D). The results of specific markers CK7, CK19 and CEA staining for BTC were positive, while the results of specific markers HepPar-1, ARG1, GPC3, AFP and CD34 staining for liver cancer were negative. In addition, H&E staining of tumors did not show the morphology of liver cancer. These results demonstrated the tumor tissue formed was indeed BTC.
Then, we randomized the mice with established BTC to treatment with disulfiram or PBS by intravenous injection three times a week for a total of 4 weeks. The body weights were recorded every 4 days. After treatment, some mice were sacrificed for follow-up experiments and others were used to measure overall survival. Notably, histopathological analysis of disulfiram treatment mice revealed a substantial delay in tumor growth (
Fig. 2E). The total tumor weights in mice treated with disulfiram for 4 weeks also presented major reduction (
Fig. 2F,
Supplementary Fig. 2E). Furthermore, normal body weight was maintained in mice treated with disulfiram (
Fig. 2G). In contrast, mice treated with PBS had a significant loss in body weight, which likely due to the severe tumor-induced liver dysfunction. H&E staining section analysis of vital organs (lung, liver, spleen, heart, and kidney) also showed no apparent differences between treated and untreated mice, indicating that the disulfiram had minimal adverse effects (
Supplementary Fig. 2F). Importantly, as shown in
Figure 2H, median overall survival of mice treated with disulfiram was 85 days, significantly longer than that of mice treated with PBS (40 days). Thus, disulfiram specifically inhibits the growth of
KRASG12V and
KRASG12d BTCs
in vitro and
in vivo.
Given that disulfiram is not just an inhibitor of IL-1β secretion, a series of phenotypes were seen upon the addition of 10 ng/mL exogenous mature IL-1β (mIL-1β) to disulfiram-treated (200 nM) RBE and NOZ. This helped to elucidate the precise mechanism by which disulfiram can recapitulate the synthetic lethal effect of GATA2 in
KRAS-mutant BTC. As shown in
Supplementary Figure 2G–
2K, mIL-1β can significantly reverse the phenotype such as apoptosis, proliferation, and migration, influenced by disulfiram. Furthermore, we transfected
KRAS-mutant BTC cells with two independent IL-1β shRNAs, then treated them with disulfiram and monitored cytotoxicity by CCK8 assays, flow cytometry-based apoptosis assay, and colony formation assays. As shown in
Supplementary Figure 2L–
2O, the cell viability was not continuously lost after disulfiram treatment. In conclusion, this result and IL-1β rescue experiments indicate that IL-1β is the sole target with the greatest potential.
Most agents that phenocopied the SL induced by GATA2 knockdown in
KRAS-mutant BTC cells were inhibitors of IL-1β and NF-κB signaling pathways according to the drug screen results. Importantly, disulfiram mainly induces the same synthetic lethal effect as GATA2 in
KRAS-mutant BTC by inhibiting IL-1β. In addition, IL-1β has been previously reported to activate the NF-κB signaling pathway by causing the p65/p50 complex formation and translocation into the nucleus [
26,
27]. Notably, KRAS mutation-driven phosphorylation of GATA2 has been shown to transcriptionally upregulate IL-1β expression [
25]. Thus, we hypothesized that phosphorylation of GATA2 enhances the transcription of IL-1β to regulate the NF-κB signaling pathway in
KRAS-mutant BTC cells. To test this hypothesis, we performed gene expression analysis on RBE and CCLP1 BTC cells expressing either control or GATA2 siRNA. As GATA2 was likely to operate via broad regulatory nodes, enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes pathway of differentially expressed genes was performed (
Supplementary Fig. 3A). Furthermore, the top 20 enriched pathways in
Supplementary Figure 3A were validated by gene set enrichment analysis, and the results revealed that the NF-κB signaling pathway was the most prominent candidate pathway negatively enriched in GATA2 knockdown cells (
Supplementary Fig. 3B). Meanwhile, JASPAR database (
https://jaspar.genereg.net/) predicts that there are multiple GATA2 potential binding sites in the IL-1β promoter region (
Supplementary Table 2). Finally, pharmacological inhibition of phosphorylation with SB203580 markedly downregulated GATA2 expression and its phosphorylation in
KRAS-mutant BTC cells (RBE, NOZ), accompanied by a reduction in IL-1β; in contrast, non-
KRAS-mutant cells (CCLP1, GBC-SD) showed no such response (
Supplementary Fig. 3C). These findings provided preliminary support for our hypothesis.
To further test this hypothesis, we carried out a series of experiments. Firstly, qRT-PCR was performed for IL-1β on
KRAS-mutant or -WT cells. Only
KRAS-mutant cells exhibited loss of IL-1β expression after GATA2 knockdown (
Fig. 3A). Second, chromatin immunoprecipitation (ChIP)-PCR and dual-luciferase assays were used in KRAS-mutated cells. ChIP was performed with a control antibody (IgG) or an antibody for GATA2 in RBE and NOZ cells, and PCR was performed for IL-1β. In RBE and NOZ cells, GATA2 only binds on one of the predicted sequences (in sixth segment P6: 5’-AGCTTATCTCC-3’) in the IL-1β promoter region (
Fig. 3B). Moreover, we observed a remarkable suppression of luciferase activity in
KRAS-mutant BTC cells when the sequence (5’-AGCTTATCTCC-3’) on the IL-1β promoter region to which GATA2 binds is artificially mutated (
Fig. 3C). Finally, western blotting analysis of NF-κB signaling pathway was performed for revealing the complete mechanism. As seen in Figure 3D, in RBE and NOZ cells, depletion of GATA2 resulted in decreased expression of IL-1β, nuclear p65, and nuclear p50, demonstrating the suppression of NF-κB signaling pathway compared with control groups. In contrast, GATA2 knockdown did not cause similar changes in
KRAS-WT BTC cells or HIBEC.
Finally, we sought to confirm that the changes in GATA2 that result in reduced viability are specific to
KRAS-mutant BTCs. The results of qRT-PCR showed that there was no difference in GATA2 gene expression between
KRAS-mutant and -WT BTC cells (
Supplementary Fig. 3D). On the contrary, protein expression of GATA2 was considerably greater in
KRAS-mutant cell lines compared to WT cells, as illustrated in
Supplementary Figure 3E. This suggests that GATA2 regulates the above mechanisms at the protein level. To determine the upstream-downstream relationship between IL-1β and NF-κB in this synthetic lethal process, we performed two corresponding overexpression and knockdown experiments [
28] in two
KRAS-mutant cell lines (RBE, NOZ) and observed the survival of them. As shown in
Supplementary Figure 3F and
3G, knockdown of IL-1β reduced the expression and nuclear translocation of NF-κB p65 and p50, and significantly decreased the survival of
KRAS-mutant tumor cells. On this basis, overexpression of NF-κB p65 significantly attenuated IL-1β-mediated death of
KRAS-mutant tumor cells. Similarly, p65 knockdown alone also reduced the viability of
KRAS-mutant tumor cells, and additional IL-1β overexpression did not significantly rescue them from death (
Supplementary Fig. 3H,
3I). Collectively, these findings indicate that GATA2 specifically enhances transcription of IL-1β, and then tumor cells secrete it in an autocrine fashion to up-regulate the NF-κB signaling pathway in
KRAS-mutant BTC cells.
The mechanism has also been verified in the oncogenic
KRAS-mutant BTC model mentioned above. Western blotting analysis of tumors in different groups showed that disulfiram inhibited the expression of IL-1β and nuclear translocation of the p65/p50 complex. There was a modest increase in GATA2 expression in the group that received disulfiram (
Fig. 3E). The secretion of IL-1β in tumor tissues was also analyzed by ELISA to demonstrate the inhibitory effect of disulfiram on the key target IL-1β
in vivo. The results showed obvious inhibition of IL-1β secretion by tumor cells after disulfiram administration (
Fig. 3F). Taken together, the mechanism of disulfiram against KRAS mutant tumor cells
in vivo corresponded to the
in vitro experiments described previously.
After the preliminary definition of the above mechanism and to determine the importance of IL-1β in the GATA2-mediated viability in
KRAS-mutant BTC cells, we carried out rescue experiments. Western blot results showed that overexpression of IL-1β could increase the entry of p65 and p50 into the nucleus to re-establish NF-κB signaling pathway function after GATA2 knockdown in RBE and NOZ cells (
Fig. 4A). However, overexpression of GATA2 could not reactivate NF-κB signaling pathway in the IL-1β inhibition group, indicating that IL-1β is a downstream mediator of the NF-κB pathway in this setting (
Supplementary Fig. 4A). Flow cytometry analysis showed that overexpression of IL-1β can restore the cell apoptosis after GATA2 knockdown, while GATA2 cannot rescue this phenotype induced by IL-1β knockdown (
Fig. 4B,
Supplementary Fig. 4B). CCK-8 assays presented the rescue of cell proliferation after IL-1β expression but not in shRNA (IL-1β)-infected cells overexpressing GATA2 (
Fig. 4C,
Supplementary Fig. 4C). Similarly, colony formation assays further confirmed this rescue effect on cell proliferation (
Fig. 4D,
Supplementary Fig. 4D). Furthermore, transwell and wound healing cell migration assays also presented the same results on cell migration function in
KRAS-mutant BTC cells (
Fig. 4E,
4F,
Supplementary Fig. 4E,
4F).
Due to the complex function of IL-1β involving multiple cellular signaling pathways [
29], it remains to be determined whether IL-1β maintains the survival of
KRAS-mutant BTC cells primarily by activating the NF-κB signaling pathway. IL-1β activates the NF-κB signaling pathway by binding to the IL-1 receptor (IL-1R) [
26,
27]. Thus, further validation of the mechanism in KRAS mutant BTC cells was performed by knocking down IL-1R and adding 10 ng/mL mIL-1β. We found that adding mIL-1β could not restore NF-κB pathway activity, cell apoptosis, decreased cell proliferation, or migration in IL-1R knockdown RBE and NOZ BTC cells (
Supplementary Fig. 5A–
5F).
Taken together, these data indicated that inhibition of IL-1β is sufficient to phenocopy the GATA2 deficiency that leads to reduced KRAS-mutant BTC cell viability.
DISCUSSION
In this study, we have shown that BTCs with
KRAS mutations depend upon activation of the transcription factor GATA2 for their survival
in vitro and
in vivo. This raises the possibility of inhibiting GATA2 for controlling
KRAS-mutant BTC growth. However, targeting transcription factors directly seems to be difficult [
30,
31]. This is because specific inhibition of the interaction of DNA-binding proteins with DNA is notoriously difficult, and the pharmaceutical industry has traditionally considered these targets undruggable [
9,
32]. Drug screening or repurposing may provide a novel solution to this problem by targeting the related upstream or downstream pathway. Besides, it has numerous advantages, such as reducing the risk of unsuccessful drug research and development, shortening the R&D cycle, and lowering R&D costs [
9,
33,
34]. Therefore, we used a large FDA-approved anticancer drug library for drug screening and discovered that disulfiram had the same effect as GATA2 on
KRAS-mutant BTCs.
Disulfiram exhibits pleiotropic antitumor activity, including inhibition of ALDH, elevation of intracellular Cu
2+ levels, and, most critically, blockade of GSDMD pore formation [
35-
40]. Hu et al. [
39] first demonstrated that disulfiram covalently modifies GSDMD at Cys191/192, thereby preventing membrane pore formation and subsequent IL-1β release. Xu et al. [
41] extended the story to the tumor niche, showing that this same modification suppresses inflammasome activity in macrophages and slows tumor growth. To identify the specific mechanism of disulfiram action in BTC, we conducted specific experiments and found that exogenous mature IL-1β could significantly reverse phenotypes such as cell apoptosis, proliferation, and migration, when treated with disulfiram (
Supplementary Fig. 2G–
2K). This suggests that disulfiram can induce a synthetic lethal effect on
KRAS-mutant BTC mainly by inhibiting IL-1β.
The above mechanism of disulfiram was explored on
KRASG12V and
KRASG12D-mutant BTC animal models, while
in vitro experiments only contained
KRASG12V-mutant BTC cell lines. In addition to
KRAS mutations, these BTC cells harbor other (epi)-genetic alterations. Given the heterogeneity of BTC, it may not be conclusively determined that cell death induced by IL-1β inhibition is solely associated with the
KRAS mutations. To address these issues, we have developed a new cell model by introducing the KRAS G12D mutation into a KRAS wild-type cell line (GBC-SD) and validated (
Supplementary Fig. 6A,
6B) the mechanism of disulfiram in the two cell lines. As shown in
Supplementary Figure 6D–
6H, the similar effects of disulfiram were confirmed in
KRASG12D-mutant BTC cell lines.
We further found that GATA2 selectively enhances the transcription of IL-1β to regulate the NF-κB signaling pathway for survival in
KRAS-mutant BTC cells. IL-1β is necessary to mimic the phenotype of GATA2 inhibition, which reduces the viability of
KRAS-mutant BTC cells. Similarly, the same downstream mechanism of GATA2 regulation has also been verified in HuCCT1 and G12D-SD cell lines (
Supplementary Fig. 7). This GATA2/IL-1β/NF-κB axis echoes earlier lineage-specific studies: Kumar et al. [
9] demonstrated that GATA2 is an obligate effector of oncogenic KRAS in myeloid and lung epithelial compartments, while Ling et al. [
42] linked GATA2 dosage to IL-1β production and NF-κB activation in
KRAS-driven pancreatic and lung tumors. Furthermore, Hamarsheh et al. [
43] established that genetic or pharmacologic IL-1β blockade disrupts this circuitry and impairs tumor maintenance. Our results are consistent with findings in other solid cancers [
9,
44,
45]. Moreover, a recent phase II clinical study including 16 patients with
KRASG12D mutant lung adenocarcinoma revealed that NF-κB inhibitor treatment could achieve a disease control rate greater than 40% [
45]. Notably, another study revealed that overactivation of RAS can phosphorylate GATA2, thereby promoting the proliferation of acute myeloid leukemia cells [
25,
46]. This finding suggests that modifications such as phosphorylation may be responsible for the distinct protein-level variations in GATA between
KRAS-mutant and WT cells. Collectively, our findings proved the essential role of GATA2/IL-1β in
KRAS-mutant BTC.
Due to the promising SL activity of GATA2/IL-1β inhibition in
KRAS-mutant BTC, we examined whether this effect is seen in other
KRAS-mutant solid tumors. Analysis of data on digestive system tumors (including esophageal, stomach, and bowel cancer) from the DepMap project showed that GATA2 and IL-1β displayed a similar strong DepMap in
KRAS-mutant cells (
Supplementary Fig. 8A). Furthermore, the therapeutic effect of the same concentration of disulfiram was observed in
KRAS-mutant and WT digestive system tumor cells. It presented considerable synthetic lethal effects on
KRASG12C,
KRASG12D, and
KRASG12V cell lines (
Supplementary Fig. 8B), aligning with recent pan-cancer CRISPR screens that nominate IL-1β and inflammasome components as top SL partners of oncogenic KRAS [
47,
48]. Several agents targeting
KRAS-mutant cells have advanced to clinical trial testing [
49-
51]. To demonstrate that the synthetic lethal strategy is as effective as direct targeting strategies or superior, we have selected representative agents targeting
KRAS (RMC-7977 and MRTX1133, which is selective for the G12D type) to compare disulfiram’s antitumor effects on the G12D (HuCCT1) and G12V (NOZ) mutant CDX models. As shown in
Supplementary Figure 9, disulfiram had a similar antitumor effect on NOZ and HuCCT1 cells as RMC-7977. However, MRTX1133 was only effective against tumors with the G12D mutation. According to the above results, disulfiram treatment had significant efficacy
in vitro and
in vivo (
KRASG12V and
KRASG12D BTC cells), in PDX model (
KRASG12V BTC cells) or in an autochthonous
KRASG12D-driven murine BTC model. Thus, disulfiram may inhibit the growth across
KRAS-driven BTCs, irrespective of the mutation type, extending the potential for this approach to treat BTCs and potentially other
KRAS-driven cancers.
Despite the promising data, the problem of BTC resistance to therapy is likely inevitable irrespective of the targeted approach and is very complex [
52,
53]. Thus, it is likely that disulfiram will have to be combined with other treatments (for example, cytotoxics) to treat resistance and ensure durability of responses. Furthermore, IL-1β, an inflammatory factor, is closely associated with tumor immune microenvironment [
54,
55]. According to a recent study, IL-1β mediates immunotherapy resistance by inducing nicotinamide nucleotide transhydrogenase acetylation, and inhibition of IL-1β in combination with PD-1 antibody therapy can substantially enhance the therapeutic effect of tumor treatment [
56]. Thus, the SL strategy of combining disulfiram with standard immunotherapy is worthy of investigation in
KRAS-mutant BTC.
In summary, we provide evidence for a new approach to selectively target the KRAS-mutant subset of BTCs. This strategy may have great potential in the treatment and clinical application of pan-cancer. It also should be further validated in clinical studies, alone or in combination with standard chemo- and immunotherapy.
FOOTNOTES
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Authors’ contributions
S.L., Y.S., and T.C. designed the experiments, analyzed the data, and wrote the manuscript. M.C. supervised the project and revised the manuscript. S.L., Y.S., T.C., W.T., S.J., Z.L., Y.S., T.X., R.S., L.H., J.C., G.C., J.L., X.M., P.G., D.G.D., and X.C. performed the experiments and revised the manuscript. All authors discussed the results throughout the project and approved the final version of the manuscript.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (No. 82473007, 82202873 and 32200566), the Key Research and Development Program, Science and Technology Department of Zhejiang Province, China (No. 2021C03061), the Fundamental Research Funds for the Central Universities (226-2025-00172) and State Key Laboratory of Advanced Drug Delivery and Release Systems (No. DSQZ-QN-202303). The figures in this article were created using Adobe Illustrator, Adobe Photoshop, BioRender, and Microsoft PowerPoint.
Supplementary information is available for this paper. Correspondence and requests for materials should be addressed to M.C.
All data generated or analyzed during this study are included in this published article (and its Supplementary Information files). All other data are available from the corresponding authors upon request.
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Conflicts of Interest
The authors have no conflicts to disclose.
SUPPLEMENTARY MATERIAL
Supplementary material is available at Clinical and Molecular Hepatology website (
http://www.e-cmh.org).
Supplementary Figure 1.
GATA2 is synthetic lethal in combination with KRAS mutation BTC in vitro and vivo. (A) The partial results of exon sequencing for BTC cell lines (RBE, HuCCT1, and NOZ). (B) HIBEC and BTC cell lines were transfected with control siRNA and KRAS siRNA. Cell numbers were counted 72 hours later. (C) Flow cytometry measurements were performed to determine the apoptosis of cells transfected with control siRNA, KRAS siRNA, or GATA2. Error bars represent. (D) Representative images of colonies formed of cells transfected with control siRNA, KRAS siRNA, or GATA2. (E) The effect of KRAS or GATA2 suppression on cell migration was determined by wound healing assays. (F) Tumor images and tumor weights in the different groups of NOZ and GBC-SD xenograft models. (G) Representative images of H&E and Ki67 staining of collected tumor sections from different groups of NOZ and GBC-SD xenograft models. Scale bar=100 μm. (H) Western blotting for RBE, NOZ, CCLP1, and GBC-SD cells expressing KRAS, p38, p-p38 (Thr180/Tyr182), ERK, p-ERK (Thr202/Tyr204), GATA2, p-GATA2 (Ser192) and β-actin. Error bars represent SD (n=3 in vitro, n=6 in vivo). All **P<0.01.
Supplementary Figure 2.
Disulfiram recapitulates the synthetic lethal effect of GATA2 in vitro and in vivo. (A) CCK-8 assays were performed to assess the proliferation of GBC-SD and G12D-SD cells after treatment with 316 nM disulfiram or transfection with control siRNA or GATA2 siRNA. (B) Apoptosis measurement by flow cytometry of GBC-SD and G12D-SD cells after treatment with 316 nM disulfiram or transfection with control siRNA or GATA2 siRNA. (C) Representative images of colonies formed in GBC-SD and G12D-SD cells after treatment with 316 nM disulfiram or transfection with control siRNA or GATA2 siRNA. (D) Representative images of H&E, CK7, CK19, CK20, CEA, HepPar-1, ARG1, GPC3, AFP and CD34 staining of collected tumor sections from tumor-bearing B6-Kras-LSL-G12D mice. Scale bar=100 μm. (E) Representative images of livers and tumors removed from the different groups of tumor-bearing B6-Kras-LSL-G12D mice after treatment. Red arrows indicate BTC tumors. (F) H&E staining analysis for disulfiram in vivo toxicology. Scale bar=250 μm. (G) Apoptosis measurement by flow cytometry of disulfiram-treated RBE and NOZ cells with or without added exogenous mature IL-1β. (H) CCK-8 assays were performed to determine the proliferation of disulfiram-treated RBE and NOZ cells with or without added exogenous mature IL-1β. (I) Representative images of colonies formed in disulfiram-treated RBE and NOZ cells with or without added exogenous mature IL-1β. (J, K) Cell migration of disulfiram-treated RBE and NOZ cells with or without added exogenous mature IL-1β was determined by transwell and wound healing assays. (L) RBE and NOZ were transfected with control shRNA or dual IL-1β shRNA for Western blotting for IL-1β and β-actin. (M) CCK-8 assays were performed to assess the proliferation of RBE and NOZ cells after transfection with control shRNA or dual IL-1β shRNA and added 316 nM disulfiram or not. (N) Apoptosis measurement by flow cytometry of RBE and NOZ cells after transfection with control shRNA or dual IL-1β shRNA and added 316 nM disulfiram added or not. (O) Representative images of colonies formed in RBE and NOZ cells after transfection with control shRNA or dual IL-1β shRNA and 316 nM disulfiram added or not. Error bars represent SD (n=3 in vitro and vivo). All **P<0.01.
Supplementary Figure 3.
GATA2 specifically regulates NF-κB signaling pathway via enhancing transcription of IL-1β in KRAS-mutated BTC cells. (A) Top 20 enrichment pathways based on the analysis of KEGG. (B) GSEA enrichment plot for the “NFKAPPAB PATHWAY” pathway. (C) RBE, NOZ, CCLP1, and GBC-SD were treated with PBS or kinase inhibitor (SB203580) and processed 48 h later for western blotting for GATA2, p-GATA2 (Ser192), IL-1β, and β-actin. (D) qRT-PCR was performed for RBE, NOZ, CCLP1, GBC-SD and HIBEC cells expressing GATA2. (E) Western blotting was performed for RBE, NOZ, CCLP1, GBC-SD and HIBEC cells expressing GATA2 and β-actin. (F) Western blotting was performed on shRNA-infected RBE and NOZ cells expressing empty vectors or NF-κB p65 for IL-1β, NF-κB p50, NF-κB p65, β-actin and Histone-3 in the cytoplasm or/and nucleus. (G) CCK-8 assays were performed to assess the proliferation of shRNA-infected RBE and NOZ cells expressing empty vectors or NF-κB p65. (H) Western blotting was performed on shRNA-infected RBE and NOZ cells expressing empty vectors or IL-1β for NF-κB p50, NF-κB p65, IL-1β and β-actin. (I) CCK-8 assays were performed to assess the proliferation of shRNA-infected RBE and NOZ cells expressing empty vectors or IL-1β. Error bars represent SD (n=3). All **P<0.01.
Supplementary Figure 4.
IL-1β is necessary to phenocopy GATA2 loss for synthetic lethality in KRAS-mutated BTC cells. (A) Western blotting was performed on shRNA-infected RBE and NOZ cells expressing empty vectors or GATA2 for NF-κB p50, NF-κB p65, β-actin and Lamin B1 in the cytoplasm or/and nucleus. (B) Apoptosis measurement by flow cytometry of shRNA-infected RBE and NOZ cells expressing empty vectors or GATA2. (C) CCK-8 assays were performed to determine the proliferation of shRNA-infected RBE and NOZ cells expressing empty vectors or GATA2. (D) Representative images of colonies formed in shRNA-infected RBE and NOZ cells expressing empty vectors or GATA2. (E, F) Cell migration of shRNA-infected RBE and NOZ cells expressing empty vectors or GATA2 was determined by transwell and wound healing assays. Error bars represent SD (n=3). ns, not significant. All **P<0.01.
Supplementary Figure 5.
IL-1β is necessary to phenocopy GATA2 loss for synthetic lethality in KRAS-mutated BTC cells. (A) Western blotting was performed on shRNA-infected RBE and NOZ cells with or without added exogenous mature IL-1β for NF-κB p50, NF-κB p65, β-actin and Lamin B1 in the cytoplasm or/and nucleus. (B) Apoptosis measurement by flow cytometry of shRNA-infected RBE and NOZ cells with or without added exogenous mature IL-1β. (C) CCK-8 assays were performed to determine the proliferation of shRNA-infected RBE and NOZ cells added with or without exogenous mature IL-1β. (D) Representative images of colonies formed in shRNA-infected RBE and NOZ cells with or without added exogenous mature IL-1β. (E, F) Cell migration of shRNA-infected RBE and NOZ cells, with or without added exogenous mature IL-1β, was determined by transwell and wound healing assays. Error bars represent SD (n=3). ns, not significant. All **P<0.01.
Supplementary Figure 6.
Disulfiram induces a synthetic lethal effect on KRASG12D -mutant BTC mainly by inhibiting IL-1β. (A) qRT-PCR was performed in GBC-SD and G12D-SD cells for KRAS G12D. (B) Western blotting was performed on GBC-SD and G12D-SD cells for KRAS G12D and β-actin. (C) 72 hours viability of HuCCT1 and G12D-SD cells exposed to different concentrations of disulfiram. (D) CCK-8 assays were performed to determine the proliferation of disulfiram (316 nM)-treated HuCCT1 and G12D-SD cells with or without the addition of exogenous mature IL-1β (10 ng/mL). (E) Western blotting was performed on disulfiram-treated HuCCT1 and G12D-SD cells with or without added exogenous mature IL-1β for NF-κB p50, NF-κB p65, β-actin and Lamin B1 in the cytoplasm or/and nucleus. (F) Apoptosis measurement by flow cytometry of disulfiram-treated HuCCT1 and G12D-SD cells with or without added exogenous mature IL-1β. (G) Representative images of colonies formed in disulfiram-treated HuCCT1 and G12D-SD cells with or without added exogenous mature IL-1β. (H) Cell migration of disulfiram-treated HuCCT1 and G12D-SD cells with or without added exogenous mature IL-1β was determined by transwell. Error bars represent SD (n=3). All **P<0.01.
Supplementary Figure 7.
GATA2 regulates IL-1β/NF-κB signaling for KRASG12D -mutant BTC cells’ survival. (A) CCK-8 assays were performed to determine the proliferation of shRNA-infected HuCCT1 and G12D-SD cells expressing empty vectors or IL-1β. (B) Western blotting was performed on shRNA-infected HuCCT1 and G12D-SD cells expressing empty vectors or IL-1β for NF-kB p50, NF-kB p65, β-actin, and Lamin B1 in the cytoplasm or/and nucleus. (C) Apoptosis measurement by flow cytometry of shRNA-infected HuCCT1 and G12D-SD cells expressing empty vectors or IL-1β. (D) Representative images of colonies formed in shRNA-infected HuCCT1 and G12D-SD cells expressing empty vectors or IL-1β. (E) Cell migration of shRNA-infected HuCCT1 and G12D-SD cells expressing empty vectors or IL-1β was determined by transwell. Error bars represent SD (n=3). All **P<0.01.
Supplementary Figure 8.
GATA2/IL-1β is synthetic lethal for KRAS-mutated digestive system cancer cells. (A) Volcano plot of gene dependencies in digestive system cancer cell lines from the DepMap project grouped according to their KRAS mutation status. (B) CCK-8 assays of various digestive system cancer cell lines (including esophageal, stomach, and bowel cancer) exposed to the same concentration of disulfiram (1 μM). Error bars represent SD (n=3). **P<0.01.
Supplementary Figure 9.
inal tumor images and tumor volume changes in the different groups of HuCCT1 and NOZ xenograft models. Error bars represent SD (n=3). All **P<0.01.
Supplementary Table 1.
Top 20 drugs with the most significant synthetic lethal effect on KRAS-mutant BTC and their latest research (PubMed database) in cancer treatment
Figure 1.GATA2 is synthetic lethal in combination with KRAS mutation BTC in vitro and in vivo. (A) Volcano plot of gene dependencies in BTC cell lines from the DepMap project grouped according to their KRAS mutation status. (B) Normal biliary epithelial cells (HIBEC), KRAS-mutant (RBE, NOZ) and WT (CCLP1, GBC-SD) BTC cells were transfected with control siRNA, PYROXD1 siRNA, MRE11 siRNA, EIF4A1 siRNA, INO80B siRNA, ARPC2 siRNA, GATA2 siRNA, or VGLL2 siRNA. CCK-8 assays were used to measure viability 72 hours later. (C) RBE, NOZ, CCLP1, GBC-SD, and HIBEC were transfected with control siRNA, KRAS siRNA, or GATA2 siRNA and processed 72 hours later for western blotting for KRAS, GATA2, c-caspase3, c-PARP, and β-actin. (D) CCK-8 assays were performed to determine the proliferation of cells transfected with control siRNA, KRAS siRNA, or GATA2. (E) The effect of KRAS or GATA2 suppression on cell migration was determined by transwell assays. (F) Tumor volume changes in the different groups of NOZ and GBC-SD xenograft models. Error bars represent SD (n=3 in vitro, n=6 in vivo). BTC, biliary tract cancer; CCK-8, cell counting kit-8; DepMap, dependency; HIBEC, human intrahepatic biliary epithelial cell line. All **P<0.01.
Figure 2.Disulfiram recapitulates the synthetic lethal effect of GATA2 in vitro and in vivo. (A) RBE (KRAS mutation) and CCLP1 (WT) cells were treated for 72 hours in 96-well plates with 1,166 kinase inhibitors (1 μM) using a one-well-one inhibitor format. D [(% viability of CCLP1)−(% viability of RBE)] was determined for each compound based on the average of two screens. (B) Effects of disulfiram on percent cell viability from both screens. (C) 72 hours viability of RBE, NOZ, CCLP1, GBC-SD, and HIBEC cells exposed to different concentrations of disulfiram. (D) Tumor images and tumor volume changes in the different treatment groups of KRAS-G12V mutation or WT PDX animal models. (E) Representative histological images of livers from tumor-bearing B6-Kras-LSL-G12D mice treated with disulfiram or PBS for 4 weeks. Black arrows indicate BTC tumors. Scale bar=1 mm. (F) Total tumor weights of mice treated with disulfiram or PBS for 4 weeks. (G) Body weight changes of tumor-bearing B6-Kras-LSL-G12D mice in different groups during treatment. (H) Kaplan–Meier analysis of tumor-bearing B6-Kras-LSL-G12D mice in different groups. Error bars represent SD (n=3 for the PDX model, n=5 for B6-Kras-LSL-G12D mice). BTC, biliary tract cancer; PBS, phosphate buffer saline; PDX, patient-derived xenograft; WT, wild type. All **P<0.01.
Figure 3.GATA2 specifically regulates the NF-kB signaling pathway by enhancing transcription of IL-1β in KRAS-mutated BTC cells. (A) qRT-PCR was performed in control or GATA2 in siRNA-transfected RBE, NOZ, CCLP1, GBC-SD and HIBEC cells for IL-1β. (B) ChIP was performed for IgG and GATA2 in RBE and NOZ cells and PCR was performed using primers targeting IL-1β promoter region. (C) Dual-Luciferase assays were performed in RBE and NOZ cells with different treatments. (D) RBE, NOZ, CCLP1, GBC-SD and HIBEC were transfected with control siRNA, or GATA2 siRNA and processed 72 hours later for western blotting for GATA2, IL-1β, NF-kB p50, NF-kB p65, IkB, β-actin and Lamin B1 in the cytoplasm and/or nucleus. (E) B6-Kras-LSL-G12D mice that have extensive tumor burden were intravenously injected with disulfiram or PBS. After 4 weeks of treatments, animals were sacrificed, and western blotting was performed on tumor tissues for GATA2, IL-1β, NF-kB p50, NF-kB p65, IkB, β-actin, and Lamin B1 in the cytoplasm or/and nucleus. (F) IL-1β content in tumor tissues of mice treated with disulfiram or PBS for 4 weeks was analyzed by ELISA. Error bars represent SD (n=3 in vitro, n=5 in vivo). BTC, biliary tract cancer; HIBEC, human intrahepatic biliary epithelial cell line; IL-1β, interleukin-1β; PBS, phosphate buffer saline. All **P<0.01.
Figure 4.IL-1β is necessary to phenocopy GATA2 loss for synthetic lethality in KRAS-mutated BTC cells. (A) Western blotting was performed on shRNA-infected RBE and NOZ cells expressing empty vectors or IL-1β for NF-kB p50, NF-kB p65, β-actin, and Lamin B1 in the cytoplasm or/and nucleus. (B) Apoptosis measurement by flow cytometry of shRNA-infected RBE and NOZ cells expressing empty vectors or IL-1β. (C) CCK-8 assays were performed to determine the proliferation of shRNA-infected RBE and NOZ cells expressing empty vectors or IL-1β. (D) Representative images of colonies formed in shRNA-infected RBE and NOZ cells expressing empty vectors or IL-1β. (E) and (F) Cell migration of shRNA-infected RBE and NOZ cells expressing empty vectors or IL-1β was determined by transwell and wound healing assays. Error bars represent SD (n=3). BTC, biliary tract cancer; CCK-8, cell counting kit-8; IL-1β, interleukin-1β. All **P<0.01.
Abbreviations
chromatin immunoprecipitation
clustered regularly interspaced short palindromic repeat
human intrahepatic biliary epithelial cell line
patient-derived xenograft
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