Methods for treating gastric cancer with specific diffuse driver pathway alterations

Abstract

This invention discloses a method for treating cancer in subjects using cyclin-dependent kinase 4 / 6 inhibitors such as abexilide. Specifically, the subjects carry specific diffuse driver pathway alterations, including ARHGAP fusions and CDH1 mutations. Specifically, the cancer is gastric cancer. A system for testing drug sensitivity using gastric cancer organoids is also provided, along with the use of this system for identifying biomarkers predicting sensitivity to CDK4 / 6 inhibitors in gastric cancer.

Description

Technical Field

[0001] This disclosure generally relates to methods for treating gastric cancer in subjects and to combination therapies for cancer. A system for testing drug sensitivity using gastric cancer organoids is also disclosed. Background Technology

[0002] Gastric cancer (GC) remains the fourth leading cause of cancer-related death and the fifth most common cancer worldwide (1). The high mortality rate underscores the ineffectiveness of current treatments and the urgent need for new therapies. Surgical resection combined with additional radiotherapy or standard chemotherapy is the primary treatment approach. A wide variety of approved cytotoxic chemotherapeutic agents are available, including 5-FU, cisplatin, epirubicin, paclitaxel, irinotecan or combinations thereof, and two targeted therapies, trastuzumab and ramucirumab, both in adjuvant and palliative settings. Unfortunately, despite these treatments, most patients are diagnosed at an advanced stage with a very poor prognosis. While immunotherapy targeting the PD-1 / PD-L1 pathway in GC has shown some success, only a fraction of patients with DNA mismatch repair deficiencies, EBV-related conditions, or ERBB2 amplification have demonstrated a response.

[0003] Gastric cancer is heterogeneous and exhibits diverse morphological growth patterns, characterized by adhesive glandular growth, known as the intestinal type, and a distinct, invasive, diffuse infiltrative growth pattern of solitary tumor cells, referred to as the diffuse type (Lauren classification). Some gastric cancers (GCs) can display a mixture of intestinal and diffuse growth patterns, hence the term mixed type. Subsequent molecular studies identified three key driver gene alterations highlighting diffuse GCs: mutations in CDH1 or RHOA; or chromosomal translocations that generate fusion proteins involving two ARHGAP genes (ARHGAP6 or ARHGAP26) known to inactivate RHO signaling, collectively referred to as ARHGAP fusions (2, 3). These three drivers collectively define the diffuse GC pathway molecularly, referred to herein as the diffuse molecular (m) group. While most diffuse molecular (m) GCs grow in a diffuse pattern, a minority may grow in an adhesive glandular or mixed pattern. Currently, this group of highly aggressive tumors shows the worst response to standard chemotherapy compared to other molecular subtypes of GC (4), and it remains unknown whether different driving factors can confer similar or different responses to specific therapeutic agents. Furthermore, patients with diffuse GC invariably develop peritoneal metastases (5, 6), which is generally considered an untreatable condition with low survival (7). Other known molecular alterations in GC include microsatellite instability (MSI) due to inactivation of DNA mismatch repair genes; EBV-related factors; and other microsatellite stable (MSS) cancers with intestinal or mixed growth patterns. Summary of the Invention

[0004] This article provides a method for treating gastric cancer and an organoid model-based drug testing system for identifying the sensitivity of gastric cancer with ARHGAP fusions or CDH1 mutations treated with cyclin-dependent kinase 4 / 6 inhibitors such as abecilibi, palbociclib, and / or ribociclib.

[0005] This article provides a method for treating gastric cancer in subjects with ARHGAP fusions or CDH1 mutations, which involves administering an effective amount of a cyclin-dependent kinase 4 / 6 inhibitor to the subject.

[0006] In some implementations, the method further includes additional therapies.

[0007] In some implementations, additional treatments include surgery, chemotherapy, targeted drug therapy, immunotherapy, and radiation therapy, or combinations thereof.

[0008] In some embodiments, the effective amount of the cyclin-dependent kinase 4 / 6 inhibitor is about 100 mg to 120 mg, 120 mg to 150 mg, 150 mg to 200 mg, 200 mg to 250 mg, 250 mg to 300 mg, 300 mg to 350 mg, 350 mg to 400 mg, 400 mg to 450 mg, 450 mg to 500 mg, 500 mg to 550 mg, 550 mg to 600 mg, 600 mg to 650 mg, or 650 mg to 700 mg.

[0009] In some implementations, cyclin-dependent kinase 4 / 6 inhibitors are abecilibi, palbociclib, and / or ribociclib.

[0010] In one implementation, the cyclin-dependent kinase 4 / 6 inhibitor is abexib.

[0011] In one embodiment, an effective dose of abexicillin is 200 mg twice daily. Optionally, an effective dose of abexicillin is 150 mg twice daily.

[0012] In one implementation, it is used as a monotherapy for gastric cancer patients with ARHGAP fusions at an effective dose of 150 mg or 200 mg twice daily.

[0013] In one implementation, an effective dose of 600 mg of ribociclib is administered once daily for 21 days.

[0014] In one implementation, an effective dose of 125 mg of abexilide is administered once daily for 21 days.

[0015] In one implementation scheme, the subjects' gastric cancer types included intestinal, diffuse, and mixed types.

[0016] In one implementation, the subject's gastric cancer type was diffuse.

[0017] In one implementation, the subject has ARHGAP fusion.

[0018] In one implementation, the subject has a CDH1 mutation.

[0019] In some implementations, the method includes testing the subject for ARHGAP fusions and / or CDH1 mutations prior to administration of an effective amount of abecilibi.

[0020] In one embodiment, the method includes administering an effective amount of a cyclin-dependent kinase 4 / 6 inhibitor in combination with one or more other therapies.

[0021] In one implementation, for patients with ARHGAP fusion, the IC50 value of abecilibi, palbociclib, or rebociclib is lower than the mean of the patient's steady-state plasma concentration.

[0022] This disclosure also provides the use of a cyclin-dependent kinase 4 / 6 inhibitor in the preparation of a medicament / pharmaceutical composition for treating gastric cancer in subjects with ARHGAP fusions or CDH1 mutations. In one particular embodiment, the cyclin-dependent kinase 4 / 6 inhibitor is abexicillin, palbociclib, and / or ribociclib. In a preferred embodiment, the cyclin-dependent kinase 4 / 6 inhibitor is abexicillin.

[0023] A system for testing drug sensitivity is provided, the system comprising: (i) obtaining a GC organoid culture; (ii) adding a test compound to the culture; and (iii) measuring the growth of the GC organoids in the culture, wherein reduced organoid growth compared to a control test compound indicates that the test compound is a candidate for treating GC, wherein the GC organoids contain an ARHGAP fusion or a CDH1 mutation. Preferably, the test compound is a cyclin-dependent kinase 4 / 6 inhibitor.

[0024] In one implementation, the cyclin-dependent kinase 4 / 6 inhibitor is abexib.

[0025] A system for screening regulated biomarkers in gastric cancer is provided, comprising: (i) obtaining GC organoid cultures from subjects with aberrant biomarkers; (ii) adding a CDK4 / 6 inhibitor to the cultures; and (iii) measuring the growth of GC organoids in the cultures, wherein reduced organoid growth compared to control organoid cultures indicates that the CDK4 / 6 inhibitor is a candidate for treating GC containing aberrant biomarkers. Attached Figure Description

[0026] Figure 1 A) A schematic diagram summarizing the current research. Sensitivity to abexicillin was investigated using a gastric cancer (GC) organoid biobank with in-depth molecular characterization. CRISPR / Cas9-engineered organoids and xenograft mouse models were used for validation. B) A summary of the GC organoid biobank used to test abexicillin sensitivity, its morphological classification via Lauren typing, and its molecular classification, including changes in key drivers of diffuse molecular (m) group, EBV, and MSI status.

[0027] Figure 2 AG. A) Survival force plot showing tumor organoids with different responses to abexicillin treatment, where tumor organoids with ARHGAP fusions are more sensitive than those with CDH1 mutations or wild-type (WT) tumor organoids (black). B) Scatter plot showing high reproducibility of drug response among biological replicates in terms of 1-AUC of abexicillin. C) Bar plot showing IC50 of abexicillin for each tumor organoid in ascending order. D) Scatter plot showing IC50 values ​​of abexicillin in gastric cancer organoids with ARHGAP fusions and CDH1 mutations, relative to wild-type organoids with these alterations. The ARHGAP fusion group showed significant sensitivity compared to other groups. E) Scatter plot showing that the mean IC50 of abexicillin in hormone receptor-positive HER-negative (HR+ / HER-) breast cancer cell lines was significantly lower than in other types of breast cancer cell lines, data adapted from O'Brien et al (22). F) Growth rate of organoids. G). A scatter plot showing the relationship between the growth rate of each tumor organoid and the abexicillin response. (DF) Solid lines for each group represent the mean IC50. (CE) Solid and dashed lines running through each plot represent the mean, 5th, and 95th percentiles of steady-state plasma concentrations after administration of 200 mg every 12 h in previous human pharmacokinetic studies of abexicillin. Statistical analysis between DF groups was performed using the Mann-Whitney U test.

[0028] Figure 3A) Abexicillin administration schedule (arrows) in mice carrying human Dif-ARHGAP fusion gastric cancer organoid xenografts and tumor size assessment by IVIS (arrow). B) Images show regression of the Dif-ARHGAP fusion xenografts after abexicillin treatment (12.5 mg / kg / day) at days 33 and 45, while tumors persisted in the mediator control. Color scales represent bioluminescence radiance (p / sec / cm). 2 C). Plot the bioluminescence signal for each experimental group against the number of treatment days. A significant reduction in tumor volume was observed on day 45 with abexicillin treatment; p-values ​​were obtained using the Mann-Whitney U test. D). Images show extensive peritoneal tumor masses in control NSG mice after intraperitoneal injection of Dif-ARHGAP-fused gastric cancer organoids on days 33 and 45, which regressed after abexicillin treatment. E). Plot the bioluminescence signal for each experimental group against the number of treatment days. A significant reduction in tumor burden was observed from day 33 onwards with abexicillin treatment and persisted until day 45; p-values ​​were obtained using the Mann-Whitney U test.

[0029] Figure 4 A) Representative bright-field and H&E images of CRISPR / Cas9-engineered human normal gastric organoids and their unique morphology, scale bar: 100 µm. B) Dose-response curves (left) and scatter plots (right) of abexicillin treatment from three technical replicates of normal CRISPR / Cas9-engineered organoids with and without CDH1 mutations. This experiment was repeated using organoids from different passages as biological replicates. Black lines represent mean; P, TP53. - / - ;C, CDH1 - / - A, APC - / - S, SMAD4 - / - Mutation. P-values ​​were obtained using Student's t-test. C) Abexicillin administration schedule in mice carrying Dif-CDH1 xenografts (shown by purple arrows) and tumor size assessment by IVIS (blue arrows). D) Images showing the status of xenografts after abexicillin treatment (25 mg / kg / day) at specified time intervals. Color scales represent bioluminescence radiance (p / sec / cm). 2 / sr). E). The bioluminescence signal of each experimental group was plotted against the number of days of treatment. Under abexicillin treatment, significant tumor regression was observed from day 19, and tumor growth inhibition continued until day 40. The p-value was obtained using the Mann-Whitney U test.

[0030] Figure 5 The scatter plot shows the similar mean IC50 (line) of palbociclib in gastric cancer organoids (A) and cell lines (B) with ARHGAP fusions and CDH1 mutations, as well as in these wild-type organoids and cell lines. The dashed line represents plasma concentration. Detailed Implementation

[0031] Gastric cancer (GC) is a deadly disease, and effective treatment remains an unmet clinical need. Diverse morphological and molecular subtypes exist, exhibiting varying drug responses and prognoses, necessitating tailored precision therapies. In this study, the potential utility of the cyclin-dependent kinase 4 / 6 inhibitor abexicillin (Verzenio, Eli Lilly, and Co) for the treatment of GC was explored using drug sensitivity testing with a previously established GC organoid biobank. Notably, abexicillin is FDA-approved as a monotherapy or in combination therapy for patients with hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer, but its efficacy in other cancer types remains unclear. Abexicillin responses from 66 tumor organoids from 56 GC patients were analyzed, including 9 ARHGAP fusion tumor organoid lines from 6 GC patients, which are poorly representative of 2D cancer cell lines. Interestingly, GC organoids with ARHGAP fusions were highly sensitive to abexicillin compared to other GC organoids without ARHGAP fusions (p<0.0001). Specifically, the IC50 value of abexicillin in the ARHGAP fusion group was consistently lower than the mean of the steady-state plasma concentration in patients and comparable to that in the HR+HER2- breast cancer cell line. Furthermore, two independent immunocompromised mouse models, mimicking primary and metastatic tumors respectively, implanted with ARHGAP fusion GC xenografts via orthotopic or intraperitoneal injection, showed significant tumor regression when treated with abexicillin at a therapeutic level well tolerated in humans (p<0.0079 and p<0.032, respectively).

[0032] The sensitivity of CDH1-mutated GC organoids to abexilide was supported by a lower mean IC50 than that of other non-diffuse GCs. However, further results showed that compared with parental normal controls or those using the intestinal GC driver TP53, the sensitivity was lower. - / - / APC - / - / SMAD4 - / -Compared to mutant-engineered organoids, normal organoids engineered with CDH1 mutations also exhibited higher sensitivity to abexicillin (p=0.04). More importantly, mice carrying diffuse CDH1-mutant GC xenografts at the peritoneum showed significant tumor regression from day 19 of abexicillin treatment (p=0.0023), with tumor growth inhibition persisting until day 40.

[0033] Overall, the results demonstrate high sensitivity to abexicillin in patients with GC who have both ARHGAP fusions and CDH1 mutations, thus representing a potential immediate clinical application. We also highlight the possibility of exploring combination therapies using abexicillin for the treatment of CDH1-mutant GC patients. Given the high sensitivity of ARHGAP fusion GC to abexicillin, this specific molecular subtype may be sensitive to other classes of CDK4 / 6 inhibitors already in clinical use or under development. There is also the potential to combine CDK4 / 6 inhibitors with other drugs to further enhance the efficacy of treating ARHGAP fusion GC. Finally, additional biomarkers that can predict responses to abexicillin or other CDK4 / 6 inhibitors in GC without ARHGAP fusions could be explored, potentially improving patient choice of therapies encompassing CDK4 / 6 inhibitors.

[0034] 5.1 In vitro model - gastric cancer organoids

[0035] The heterogeneity of GC and the lack of physiologically relevant in vitro models have long been key obstacles to advancements in the development of treatments for GC patients. This has also resulted in a scarcity of representative large-scale drug screening studies for GC until recently (8). We and others have previously established a large library of GC organoids that highly reproduce the histopathological, genetic, and transcriptomic features of primary tissues (9–11). To date, the number of accumulated GC organoids is three times the number of GC cell lines currently available in the field. Some of these GC organoids capture rare genetic alterations, such as ARHGAP fusions, or are derived from early stages that are either unavailable or underrepresented in cell line models. We and others have demonstrated the potential use of organoid culture to predict in vivo drug responses and the feasibility of scaling up high-throughput drug screening (9, 12–15). Recently, we further demonstrated that the in vitro cell-matrix adhesion-dependent phenotype of GC organoids is closely associated with in vivo infiltration patterns and predicts poor patient clinical outcomes (16), indicating the clinical relevance of organoid models.

[0036] 6. Examples

[0037] 6.1 Materials and Methods

[0038] 6.1.1 Patient-derived organoid cultures

[0039] As previously described, patient-derived organoid cultures were cultured in standard gastric medium (advanced DMEM / F12, 1xGlutaMax, 1x HEPES, 1x P / S, 50% Wnt3a, 10% RSPO-1, 10% Noggin, 1xB27, 50 ng / ml EGF, 200 ng / ml FGF10, 1 mM N-acetylcysteine, 1 nM gastrin, 2 μM A83-01) (9, 16). Normal organoids were passaged by mechanical shearing using glass Pasteur pipettes with narrow tip openings. Tumor organoids were passaged by TrypLE digestion for 5 minutes at 37°C. Mycoplasma contamination was periodically monitored in the organoids.

[0040] 6.1.2 Abeciliary Sensitivity Screening

[0041] As previously described (9, 17), abexicillin was used to treat 66 patient-derived tumor organoids from 56 patients, 10 of which were derived from a second region of the same tumor (Table 1). Briefly, organoids were trypsinized into single cells 2–3 days prior to plating to generate pools of organoids with homogeneous size. On plating day, organoids were mechanically dissociated from Matrigel and plated at 15,000–20,000 organoids / ml onto each well of a 384-well plate pre-coated with 50% Matrigel using a ThermoFisher Multidrop dispenser. The following day, the drug was dispensed onto the 384-well organoid plates using an Echo 555 acoustic dispenser (Labcyte), and the cells were cultured for another 4 days. On day 6, the effect of the drug on the cell viability of the tumor organoids was quantified using a Celltiter-Glo 2.0 assay (Promega). Abecitabine was screened at seven concentrations with a semi-logarithmic dilution, in triplicate, each using two different passages of organoids as biological replicates. Raw luminescent values ​​were obtained from a Thermofisher microplate reader. Data were compared with a positive control (10 mM MG132) and a negative control (DMSO). The half-maximal inhibitory concentration (IC50) and area under the dose-response curve (AUC) were generated using the gdscIC50 R package (18). For tumor organoids completely resistant to abecitabine, IC50 and AUC values ​​could not be generated, but for analytical purposes, the maximum IC50 or AUC of 1 for abecitabine was used in the organoid group.

[0042] 6.1.3 Generation of CRISPR / Cas9-engineered organoids for drug sensitivity screening

[0043] To investigate the response of abexicillin to GCs with CDH1 mutations, normal gastric organoids were genome-edited using either diffuse GC-driven factors (TP53 and CDH1 mutations) or intestinal GC-driven factors (TP53, APC, and SMAD4 mutations) to assess their response to abexicillin. Following the previously described protocol (19), the sgRNA oligos identified in Table 2 were cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 vector (Addgene plasmid #62988). Clones with correct insertions were confirmed by Sanger sequencing. As previously described (20), Amaxa was used… TM Mouse / Rat Hepatocytes - Nucleofector TM The kit allows for the simultaneous delivery of plasmids containing gRNAs targeting TP53 and CDH1, or targeting TP53, APC, and SMAD4, into normal gastric organoids via electroporation to induce diffuse and intestinal GC transformation, respectively. Forty-eight hours post-electroporation, cells are treated with 10 μM Nutlin3a to select for TP53. - / - / CDH1 - / - Mutant organoids (PCs) were obtained by simultaneously removing Wnt3a, Rspondin-1, and Noggin and adding 10 µM Nutlin3a, which yielded TP53⁻ / ⁻ / APC⁻ / ⁻ / SMAD4⁻ / ⁻ mutant organoids (PASs). Clonal organoids were selected and amplified for abexicillin treatment. Mutations in each gene were verified by PCR and Sanger sequencing using the primers listed in Table 3.

[0044] Table 2. List of sgRNAs targeting driver genes of interest

[0045]

[0046] Table 3 lists the primers used for PCR and Sanger sequencing of genomic DNA to detect mutations in organoids.

[0047]

[0048] 6.1.4 Mouse orthotopic xenograft model for drug therapy

[0049] To evaluate the efficacy of abexicillin in patients with diffuse GC having ARHGAP fusions or CDH1 mutations, a previously described orthotopic xenograft model was used (16). NOD.Cg-Prkdc cells were transplanted from 6-8 weeks of age before surgery. scid Il2rg tm1Wjl / SzJ(NSG) mice were fasted overnight. On the day of surgery, 2.5 x 10⁻⁶ mice were... 6Luciferase-labeled gastric tumor organoid cells (GX052-TO g1-Ctrl) carrying ARHGAP fusion were injected into the gastric submucosa of NSG mice to generate ARHGAP-fused GC tumor xenografts. To generate CDH1-mutant GC tumor xenografts, 2 x 10⁻⁶ cells were injected into the gastric submucosa of NSG mice. 5 Luciferase-labeled gastric tumor organoid cells carrying the CDH1 mutation (GX036-TO) with 2 x 10 5 Tumor-associated fibroblasts were co-injected into the gastric submucosa of NSG mice. Approximately 4 weeks post-implantation, mice were administered 100 mg kg⁻¹ D-luciferin via peritoneal injection to measure tumor volume using the IVIS® Spectrum in vivo imaging system. Mice were randomly assigned to two groups and administered either 1% hydroxyethylcellulose as a mediator (control) or abexicillin (12.5 mg / kg / day for ARHGAP fusion GC tumor xenograft GX052-TO g1-Ctrl, or 25 mg / kg / day for CDH1 mutant GC tumor xenograft GX036-TO) orally by gavage for 45 days. Tumor volume was monitored and recorded as bioluminescence intensity at specific time intervals between days 19 and 45 post-treatment using the IVIS® Spectrum in vivo imaging system. Mice were sacrificed and the stomachs were harvested for ex vivo imaging and histological analysis. Other internal organs were also examined for any abnormalities or potential metastases. All animal experiments were conducted in accordance with protocols approved by the Committee on the Use of Live Animals for Teaching and Research at the University of Hong Kong (CULATR No. 5084-19).

[0050] 6.1.5 Mouse peritoneal xenograft model for drug therapy

[0051] To evaluate the efficacy of abecilibaly in patients with diffuse GC having ARHGAP fusions or CDH1 mutations who have progressed to peritoneal metastasis, a peritoneal metastasis xenograft model was used. Briefly, 1 x 10 6Luciferase-labeled gastric tumor organoids carrying ARHGAP fusions (GX080-TO) or those carrying CDH1 mutations (GX036-TO) were injected into the peritoneal cavity of 6-8 week old NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice. Approximately 4 weeks post-implantation, 100 mg kg⁻¹ d-luciferin was administered intraperitoneally to the mice to measure tumor burden using the IVIS® Spectrum in vivo imaging system. Mice were randomly assigned to two groups and administered 1% hydroxyethylcellulose orally as a mediator control or abexicillin (12.5 mg / kg / day for GX080-TO, or 25 mg / kg / day for GX036-TO) for 40-45 days. Tumor burden was monitored and recorded as bioluminescence intensity at specific time intervals between day 12 and day 45 post-treatment using the IVIS® Spectrum in vivo imaging system. Mice were euthanized and their stomachs harvested for in vitro imaging and histological analysis. Other internal organs were also examined for any abnormalities or possible metastases. All experiments were conducted according to protocols approved by the Teaching and Research Live Animal Use Committee of the University of Hong Kong (CULATR Nos. 230-240).

[0052] 6.1.6 Statistical Analysis

[0053] Use the Student's t-test or the Mann-Whitney U test to compare differences between the two biological groups and indicate them in the corresponding legend.

[0054] 6.2 Results

[0055] 6.2.1 Testing the effect of abexicillin in gastric cancer

[0056] Previously, we described the GC organoid biobank, which covers a variety of tumor organoids of different subtypes and is capable of therapeutic screening. Figure 1 (9, 16). Currently, our tumor organoid biobank contains 58 GC patients, including 45 men and 13 women, aged between 29 and 83 years (Table 1). Our tumor organoids cover all common morphological and molecular signatures of gastric cancer, including the diffuse molecular (m) group carrying ARHGAP fusions, CDH1, or RHOA mutations (referred to as Dif-ARHGAP fusion, Dif-CDH1, and Dif-RHOA, respectively); EBV, MSI, and the remaining microsatellite stable group ( Figure 1(AB and Table 1). Specifically, this organoid library contains nine Dif-ARHGAP fusion organoids from six patients, which have not been previously described in 2D GC cell lines. In this study, we utilized this biobank to evaluate the clinical use of abexicillin in the treatment of GC. Abexicillin (trade name Verzenio, company: Eli Lilly and Co.) is a CDK4 / 6 inhibitor. It was the first CDK4 / 6 inhibitor approved by the FDA in 2017 as a monotherapy for the treatment of adult patients with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced or metastatic breast cancer who have experienced disease progression following endocrine therapy and, in metastatic cases, prior chemotherapy. Furthermore, it is approved as a combination therapy with aromatase inhibitors or fulvestrant for HR+ / HER- breast cancer. We sought to test the efficacy of abexicillin in GC patients using our patient-derived GC biobank. Abexib was screened at seven drug concentrations in triplicate, using two biological replicates from organoids from different passages. Figure 2 A). In summary, we have completed an analysis of abexicillin treatment on 66 tumor organoids from 56 patients using the previously described protocol (9). Two tumor organoids (GX024-TO and GX109-TO) in our biobank were excluded from drug treatment due to their slow growth rate (doubling time > 400 hours). Two independent abexicillin treatments on each organoid, as biological replicates, revealed high reproducibility of the drug treatment regimen, with a Pearson correlation coefficient of 0.84 at 1-AUC. Figure 2 B). When the IC50 of abexicillin for each organoid was calculated and plotted in ascending order, all Dif-ARHGAP fusion organoids tended to have low IC50 values ​​significantly lower than the mean steady-state plasma concentration in patients. Figure 2 C). Specifically, the mean abexilic acid IC50 of Dif-ARHGAP fusion organoids was significantly lower than that of other GC organoids, especially the non-diffuse molecular (m) group (p<0.0001), but also lower than that of the Dif-CDH1 group (p=0.004), while the Dif-RHO group was too small to be effectively compared. More importantly, the mean IC50 of Dif-ARHGAP fusion organoids was comparable to that of hormone receptor-positive (HR+) and HER2-negative breast cancer cell lines; cancer types with proven therapeutic efficacy ( Figure 1 E). This indicates that abecitabine is effective in treating GC patients with ARHGAP fusions within a well-tolerated treatment range. Although both Dif-ARHGAP and Dif-CDH1 organoids exhibit relatively higher in vitro growth rates than other organoids ( Figure 2F), but there was no correlation between abecili's IC50 and organoid growth rate. Figure 2 G), indicating that the results presented in this paper are not affected by in vitro growth conditions. We further adjusted two xenograft mouse models by orthotopic (16) or intraperitoneal injection to investigate the in vivo effects of abecicilline on tumor growth inhibition at the primary site and after metastasis. Figure 3 ). Luteinase-labeled Dif-AG tumor organoids (GX052-TO g1-Ctrl) were injected into the submucosa of the stomach of NSG mice. Xenografts were detected in approximately 50% of NSG mice approximately 4 weeks after tumor organoid injection. NSG mice with established xenografts were then randomly assigned to two groups and treated with either the mediator or 12.5 mg / kg abexilide daily for 45 days. The dose used in this study was within the Css (21) and less than the dose used to treat mice with HR+ / HER2- breast xenografts (50 mg / kg / day) (22). Tumor volume was measured based on luciferase signal transduction at days 19, 33, and 45. Figure 3 We found that abexicillin treatment for 45 days significantly reduced tumor volume (p=0.0079). Similarly, Dif-AG tumor organoids (GX080-TO) were injected intraperitoneally into NSG mice. NSG mice with established xenografts were then randomly assigned to two groups, each treated daily with either the mediator or 12.5 mg / kg abexicillin for 40 days. Figure 3 DE). Significant tumor volume reduction was observed from day 33 onwards after abecilibi treatment (p=0.032) and persisted until day 45 (p=0.047).

[0057] 6.2.2 Testing the effect of abecitabine in gastric cancer with CDH1 mutation

[0058] Although not statistically significant, Dif-CDH1 organoids also showed a lower mean abexilic acid IC50 value than the non-diffuse molecular (m) group (p=0.41). Figure 2 D). Furthermore, the potential sensitivity of Dif-CDH1 to abecicilline was examined by reconstructing the CDH1 mutation in normal organoids using CRISPR / Cas9. A total of four independent normal organoids were engineered to have TP53. - / - / CDH1 - / - The mutation resulted in a diffuse tumor-like morphology, and an organoid (GX059-BO) was engineered to have TP53. - / - / APC - / - / SMAD4 - / - The mutation maintained the cystic nature and reproduced the intestinal type tumor. Figure 4A) As controls. These organoids were used together with two paired normal gastric organoids (GX052-AO and GX097-B1O) to test abexicillin response. Interestingly, the engineered organoids with the CDH1 mutation showed higher sensitivity to abexicillin (p=0.04) compared to either the engineered organoids with the intestinal-type driver mutation or the parental normal organoids. Figure 4 B) indicates that CDH1 mutations do indeed contribute to abexilide sensitivity in GC organoids.

[0059] Next, we used a CDH1-mutant xenograft mouse model to investigate the in vivo effects of abexicillin. Mice carrying CDH1-mutant xenografts at the peritoneum were treated with abexicillin for 40 days, and tumor size was monitored at specific time intervals. Figure 4 We found that abexicillin significantly reduced tumor volume on day 19 compared to the mediator control (p=0.0023). Tumor growth inhibition persisted until day 40, indicating that tumors with CDH1 mutations responded to abexicillin treatment.

[0060] Finally, we explored whether GC organoids with ARHGAP fusions or CDH1 mutations also responded to other CDK4 / 6 inhibitors such as palbociclib. Interestingly, no significant differences in sensitivity were observed in the GC subgroups. Figure 5 A). The same results were observed in gastric cancer cell lines from a public database (Genomics of Drug Sensitivity in Cancer). Figure 5 (B) Furthermore, palbociclib's IC50 for both GC organoids and cell lines was significantly higher than patient plasma concentrations, indicating potential toxic effects that can be observed in humans. Overall, the sensitivity response demonstrated in this study for GCs with ARHGAP fusions or CDH1 mutations is specific to abexicillin rather than applicable to general CDK4 / 6 inhibitors.

[0061] 6.3 Discussion

[0062] In this study, we tested the effects of abexicillin on our GC organoid biobank, which contains patient-derived organoids that are consistently underrepresented in 2D cell lines, such as ARHGAP fusion organoid cell models. By analyzing the abexicillin treatment response of our GC organoids, we found that Dif-ARHGAP fusion organoids were more sensitive to abexicillin than GC organoids without ARHGAP fusion. Figure 2 We further validated in vitro abexicillin sensitivity in two independent Dif-ARHGAP fusion xenografts, with tumors located at the primary site or peritoneum. Figure 3 ).

[0063] Abexicillin is the most potent of the three FDA-approved CDK4 / 6 inhibitors (including palbociclib and ribociclib) for the treatment of advanced HR+ breast cancer (23). Furthermore, based on multi-omics analyses of breast cancer cell lines, it exhibits broader inhibitory activity than other drugs (24). Current research highlights a new patient population that could benefit from abexicillin treatment, where clinical trials could be the next step in advancing this new finding. Indeed, numerous ongoing clinical trials focus on combining abexicillin treatment with other therapies to treat different types of solid cancer.

[0064] While the underlying mechanisms underlying abexicillin sensitivity in Dif-ARHGAP GCs remain to be elucidated, recent proteomic studies of GCs have observed elevated levels of CDK4 and Cdk6 proteins in morphologically defined diffuse GCs, suggesting CDK4 / 6 inhibitors as potential targets for the treatment of diffuse GCs (25). Although elevated levels do not always translate into a therapeutic response in biological systems due to the presence of reconnection and escape mechanisms, our study is the first to demonstrate that Dif-ARHGAP fusions and the Dif-CDH1 group constitute biomarkers predicting very high therapeutic responses, even in diffuse GCs. Further research is underway to identify additional biomarkers predicting abexicillin sensitivity in GCs without ARHGAP fusions or CDH1 mutations, as a small subset exhibits very good responses. Given the high sensitivity of ARHGAP fusion GCs to abexicillin, this particular molecular subtype has the potential to be sensitive to other classes of CDK4 / 6 inhibitors already in clinical use or under development. There is also the possibility of combining CDK4 / 6 inhibitors with other drugs to further increase the efficacy of treating ARHGAP fusion GC.

[0065] Table 1. Clinicopathological data, genomic data, and abeciliary data of tumor organoids derived from gastric cancer patients. IC50 value

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] ^Identification of ARHGAP fusions based on RNA sequencing data

[0075] Exemplary products, systems, and methods are described in the following:

[0076] 1. A method for treating gastric cancer in a subject with an ARHGAP fusion or CDH1 mutation, the method comprising administering to the subject an effective amount of a cyclin-dependent kinase 4 / 6 inhibitor.

[0077] 2. The method of item 1, which further includes additional therapies.

[0078] 3. The method of item 2, wherein the additional therapy includes surgery, chemotherapy, targeted drug therapy, immunotherapy and radiotherapy or a combination thereof.

[0079] 4. The method of any of the preceding items, wherein the effective amount of the cyclin-dependent kinase 4 / 6 inhibitor is about 100 mg to 120 mg, 120 mg to 150 mg, 150 mg to 200 mg, 200 mg to 250 mg, 250 mg to 300 mg, 300 mg to 350 mg, 350 mg to 400 mg, or 400 mg to 450 mg, 450 mg to 500 mg, 500 mg to 550 mg, 550 mg to 600 mg, 600 mg to 650 mg, or 650 mg to 700 mg.

[0080] 5. The method of any of the preceding items, wherein the cyclin-dependent kinase 4 / 6 inhibitor is abecilibi, palbociclib, or ribociclib.

[0081] 6. The method of any of the preceding items, wherein the cyclin-dependent kinase 4 / 6 inhibitor is abexilide.

[0082] 7. The method of any of the preceding items, wherein the effective dose of abexicillin is 200 mg twice daily as a single therapy for patients with gastric cancer having ARHGAP fusion.

[0083] 8. The method of any of the preceding items, wherein the effective amount is 600 mg of ribociclib once daily.

[0084] 9. The method of any of the preceding items, wherein the effective amount is 125 mg of palbociclib once daily.

[0085] 10. The method of any of the preceding claims, wherein the method includes testing the subject for ARHGAP fusions and / or CDH1 mutations prior to administration of an effective amount of abecitabine.

[0086] 11. The method of any of the preceding items, wherein the subject has ARHGAP fusion.

[0087] 12. The method of any of the preceding items, wherein the subject has a CDH1 mutation.

[0088] 13. The method of any of the preceding claims, wherein the method comprises administering an effective amount of a cyclin-dependent kinase 4 / 6 inhibitor in combination with one or more other therapies.

[0089] 14. The method of any of the preceding items, wherein the subject has a CDH1 mutation.

[0090] 15. The method of any of the preceding items, wherein the subject has ARHGAP fusion.

[0091] 16. The method of any of the preceding items, wherein for patients with ARHGAP fusion, the IC50 value of abecilibi, palbociclib, or rebociclib is lower than the mean of the patient's steady-state plasma concentration.

[0092] 17. A system for testing drug sensitivity, the system comprising: (i) obtaining a GC organoid culture; (ii) adding a test compound to the culture; and (iii) measuring the growth of the GC organoid in the culture, wherein reduced organoid growth compared to a control test compound indicates that the test compound is a candidate for the treatment of GC, wherein the GC organoid contains an ARHGAP fusion or a CDH1 mutation and wherein the test compound is a cyclin-dependent kinase 4 / 6 inhibitor.

[0093] 18. The system in item 17, wherein the cyclin-dependent kinase 4 / 6 inhibitor is abexilide.

[0094] 19. A system for screening regulated biomarkers in gastric cancer, the system comprising: (i) obtaining GC organoid cultures from subjects with aberrant biomarkers; (ii) adding a CDK4 / 6 inhibitor to the cultures; and (iii) measuring the growth of GC organoids in the cultures, wherein reduced organoid growth compared to control organoid cultures indicates that the CDK4 / 6 inhibitor is a candidate for treating GC containing aberrant biomarkers.

[0095] The foregoing description of specific embodiments so fully reveals the general nature of this disclosure that those skilled in the art (including the content of cited and incorporated herein by reference) can readily modify and / or adapt such specific embodiments for various applications without departing from the general concepts of this disclosure, without excessive experimentation. Therefore, based on the teachings and guidance presented herein, such adaptations and modifications are intended to be within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology herein is for descriptive rather than limiting purposes, and that the terminology or terminology of this specification should be interpreted by those skilled in the art based on the teachings and guidance presented herein and in conjunction with the knowledge of those skilled in the art.

[0096] While various embodiments of this disclosure have been described above, it should be understood that they have been presented by way of example and not limitation. It will be apparent to those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of this disclosure. Therefore, this disclosure should not be limited to any of the exemplary embodiments described above, but should be defined solely by the appended claims and their equivalents.

[0097] All references cited in this article are incorporated herein by reference in their entirety, and for all purposes, to the same extent that each individual publication or patent or patent application is specifically and individually indicated to be incorporated herein by reference in its entirety for all purposes.

[0098] References

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[0100] 2. Network CGAR. Comprehensive molecular characterization of gastricadenocarcinoma. Nature. 2014;513(7517):202-9.

[0101] 3. Wang K, Yuen ST, Xu J, Lee SP, Yan HH, Shi ST, et al. Whole-genomesequencing and comprehensive molecular profiling identify new drivermutations in gastric cancer. Nat Genet. 2014;46(6):573-82.

[0102] 4. Sohn BH, Hwang JE, Jang HJ, Lee HS, Oh SC, Shim JJ, et al.Clinical Significance of Four Molecular Subtypes of Gastric Cancer Identifiedby The Cancer Genome Atlas Project. Clin Cancer Res. 2017;23(15):4441-9.

[0103] 5. Riihimäki M, Hemminki A, Sundquist K, Sundquist J, Hemminki K.Metastatic spread in patients with gastric cancer. Oncotarget. 2016;7(32):52307-16.

[0104] 6. Mori M, Sakaguchi H, Akazawa K, Tsuneyoshi M, Sueishi K, SugimachiK. Correlation between metastatic site, histological type, and serum tumormarkers of gastric carcinoma. Hum Pathol. 1995;26(5):504-8.

[0105] 7. Brigand C, Arvieux C, Gilly FN, Glehen O. Treatment of peritonealcarcinomatosis in gastric cancers. Dig Dis. 2004;22(4):366-73.

[0106] 8. Sa JK, Hong JY, Lee IK, Kim JS, Sim MH, Kim HJ, et al.Comprehensive pharmacogenomic characterization of gastric cancer. Genome Med.2020;12(1):17.

[0107] 9. Yan HHN, Siu HC, Law S, Ho SL, Yue SSK, Tsui WY, et al. AComprehensive Human Gastric Cancer Organoid Biobank Captures Tumor SubtypeHeterogeneity and Enables Therapeutic Screening. Cell Stem Cell. 2018;23(6):882-97 e11.

[0108] 10. Nanki K, Toshimitsu K, Takano A, Fujii M, Shimokawa M, Ohta Y, etal. Divergent Routes toward Wnt and R-spondin Niche Independency during HumanGastric Carcinogenesis. Cell. 2018;174(4):856-69.e17.

[0109] 11. Seidlitz T, Merker SR, Rothe A, Zakrzewski F, von Neubeck C, Grützmann K, et al. Human gastric cancer modelling using organoids. Gut. 2019;68(2):207-17.

[0110] 12. van de Wetering M, Francies Hayley E, Francis Joshua M, BounovaG, Iorio F, Pronk A, et al. Prospective Derivation of a Living OrganoidBiobank of Colorectal Cancer Patients. Cell. 2015;161(4):933-45.

[0111] 13. Lo YH, Kolahi KS, Du Y, Chang CY, Krokhotin A, Nair A, et al. ACRISPR / Cas9-Engineered ARID1A-Deficient Human Gastric Cancer Organoid ModelReveals Essential and Nonessential Modes of Oncogenic Transformation. CancerDiscov. 2021;11(6):1562-81.

[0112] 14. Driehuis E, Kretzschmar K, Clevers H. Establishment of patient-derived cancer organoids for drug-screening applications. Nat Protoc. 2020;15(10):3380-409.

[0113] 15. Vlachogiannis G, Hedayat S, Vatsiou A, Jamin Y, Fernández-MateosJ, Khan K, et al. Patient-derived organoids model treatment response ofmetastatic gastrointestinal cancers. Science. 2018;359(6378):920-6.

[0114] 16. Tong Y, Cheng PSW, Or CS, Yue SSK, Siu HC, Ho SL, et al. Escapefrom cell-cell and cell-matrix adhesion dependence underscores diseaseprogression in gastric cancer organoid models. Gut. 2023;72 242-55.

[0115] 17. Francies HE, Barthorpe A, McLaren-Douglas A, Barendt WJ, GarnettMJ. Drug Sensitivity Assays of Human Cancer Organoid Cultures. In: Turksen K,editor. Organoids: Stem Cells, Structure, and Function. New York, NY:Springer New York; 2019. p. 339-51.

[0116] 18. Vis DJ, Bombardelli L, Lightfoot H, Iorio F, Garnett MJ, WesselsLF. Multilevel models improve precision and speed of IC50 estimates.Pharmacogenomics. 2016;17(7):691-700.

[0117] 19. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genomeengineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-308.

[0118] 20. Schwank G, Andersson-Rolf A, Koo BK, Sasaki N, Clevers H.Generation of BAC transgenic epithelial organoids. PLoS One. 2013;8(10):e76871.

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Claims

1. A method for treating gastric cancer in a subject with an ARHGAP fusion or CDH1 mutation, the method comprising administering to the subject an effective amount of a cyclin-dependent kinase 4 / 6 inhibitor.

2. The method of claim 1, further comprising one or more additional therapies.

3. The method of claim 2, wherein the additional therapy comprises surgery, chemotherapy, targeted drug therapy, immunotherapy, and radiotherapy, or a combination thereof.

4. The method according to any one of the preceding claims, wherein the effective amount of the cyclin-dependent kinase 4 / 6 inhibitor is about 100 mg to 120 mg, 120 mg to 150 mg, 150 mg to 200 mg, 200 mg to 250 mg, 250 mg to 300 mg, 300 mg to 350 mg, 350 mg to 400 mg, or 400 mg to 450 mg, 450 mg to 500 mg, 500 mg to 550 mg, 550 mg to 600 mg, 600 mg to 650 mg, or 650 mg to 700 mg.

5. The method according to any one of the preceding claims, wherein the cyclin-dependent kinase 4 / 6 inhibitor is abecilibi, palbociclib, or ribociclib.

6. The method according to any one of the preceding claims, wherein the cyclin-dependent kinase 4 / 6 inhibitor is abexilide.

7. The method according to claim 6, wherein the effective amount is 150 mg of abecitabine twice daily.

8. The method according to claim 6, wherein the effective amount is 200 mg of abecilibus twice daily.

9. The method according to any one of the preceding claims, wherein the effective amount is 600 mg of ribociclib once daily.

10. The method according to any one of the preceding claims, wherein the effective amount is 125 mg of palbociclib once daily.

11. The method according to any one of the preceding claims, wherein the gastric cancer type of the subject includes intestinal type, diffuse type, and mixed type.

12. The method according to any one of the preceding claims, wherein the subject has diffuse gastric cancer.

13. The method according to any one of the preceding claims, wherein the subject has a CDH1 mutation.

14. The method according to any one of the preceding claims, wherein the subject has ARHGAP fusion.

15. The method according to any one of the preceding claims, wherein the method comprises testing the subject for ARHGAP fusions and / or CDH1 mutations prior to administration of an effective amount of abecilibi.

16. The method according to any one of the preceding claims, wherein for patients with ARHGAP fusion, the IC50 value of abecilibi, palbociclib, or rebociclib is lower than the mean of the patient's steady-state plasma concentration.

17. A system for testing drug sensitivity, said system comprising: (i) Obtain GC organoid cultures; (ii) Add the test compound to the culture; (iii) Measure the growth of the GC organoids in the culture, wherein a decrease in the growth of the organoids compared with the control test compound indicates that the test compound is a candidate for the treatment of GC, wherein the GC organoids contain an ARHGAP fusion or a CDH1 mutation.

18. The system of claim 17, wherein the test compound is a cyclin-dependent kinase 4 / 6 inhibitor.

19. The system of claim 18, wherein the cyclin-dependent kinase 4 / 6 inhibitor is abexicillin.

20. A system for screening regulated biomarkers in gastric cancer, the system comprising: (i) Obtaining GC organoid cultures from subjects with abnormal biomarkers; (ii) Add CDK4 / 6 inhibitors to the culture; (iii) Measure the growth of the GC organoids in the culture, wherein a decrease in the growth of the organoids compared to a control organoid culture indicates that the CDK4 / 6 inhibitor is a candidate for treating GC containing the aberrant biomarker.

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