Metagenomic analysis during capecitabine therapy reveals microbial chemoprotective mechanisms and predicts drug toxicity in colorectal cancer patients.

Purpose: Unpredictable chemotherapy side effects are a major barrier to successful treatment. Cell culture and mouse experiments indicate that the gut microbiota is influenced by and influences anti-cancer drugs. However, metagenomic data from patients paired to careful side effect monitoring remains limited. Herein, we focus on the oral fluoropyrimidine capecitabine (CAP). We investigate CAP-microbiome interactions through metagenomic sequencing of longitudinal stool sampling from a cohort of advanced colorectal cancer (CRC) patients. Methods: We established a prospective cohort study including 56 patients with advanced CRC treated with CAP monotherapy across 4 centers in the Netherlands. Stool samples and clinical questionnaires were collected at baseline, during cycle 3, and post-treatment. Metagenomic sequencing to assess microbial community structure and gene abundance was paired with transposon mutagenesis, targeted gene deletion, and media supplementation experiments. An independent US cohort was used for model validation. Results: CAP treatment significantly altered gut microbial composition and pathway abundance, enriching for menaquinol (vitamin K2) biosynthesis genes. Transposon library screens, targeted gene deletions, and media supplementation confirmed that menaquinol biosynthesis protects Escherichia coli from drug toxicity. Microbial menaquinol biosynthesis genes were associated with decreased peripheral sensory neuropathy. Machine learning models trained in this cohort predicted hand-foot syndrome and dose reductions in an independent cohort. Conclusion: These results suggest treatment-associated increases in microbial vitamin biosynthesis serve a chemoprotective role for bacterial and host cells, with implications for toxicities outside the gastrointestinal tract. We provide a proof-of-concept for the use of microbiome profiling and machine learning to predict drug toxicities across independent cohorts. These observations provide a foundation for future human intervention studies, more in-depth mechanistic dissection in preclinical models, and extension to other cancer treatments.

Pharma[e]cology: How the Gut Microbiome Contributes to Variations in Drug Response.

Drugs represent our first, and sometimes last, line of defense for many diseases, yet despite decades of research we still do not fully understand why a given drug works in one patient and fails in the next. The human gut microbiome is one of the missing puzzle pieces, due to its ability to parallel and extend host pathways for drug metabolism, along with more complex host-microbiome interactions. Herein, we focus on the well-established links between the gut microbiome and drugs for heart disease and cancer, plus emerging data on neurological disease. We highlight the interdisciplinary methods that are available and how they can be used to address major remaining knowledge gaps, including the consequences of microbial drug metabolism for treatment outcomes. Continued progress in this area promises fundamental biological insights into humans and their associated microbial communities and strategies for leveraging the microbiome to improve the practice of medicine.

Expansion of a bacterial operon during cancer treatment ameliorates drug toxicity.

Dose-limiting toxicities remain a major barrier to drug development and therapy, revealing the limited predictive power of human genetics. Herein, we demonstrate the utility of a more comprehensive approach to studying drug toxicity through longitudinal study of the human gut microbiome during colorectal cancer (CRC) treatment (NCT04054908) coupled to cell culture and mouse experiments. 16S rRNA gene sequencing revealed significant shifts in gut microbial community structure during oral fluoropyrimidine treatment across multiple patient cohorts, in mouse small and large intestinal contents, and in patient-derived ex vivo communities. Metagenomic sequencing revealed marked shifts in pyrimidine-related gene abundance during oral fluoropyrimidine treatment, including enrichment of the preTA operon, which is sufficient for the inactivation of active metabolite 5-fluorouracil (5-FU). preTA + bacteria depleted 5-FU in gut microbiota grown ex vivo and the mouse distal gut. Germ-free and antibiotic-treated mice experienced increased fluoropyrimidine toxicity, which was rescued by colonization with the mouse gut microbiota, preTA + E. coli, or preTA-high CRC patient stool. Finally, preTA abundance was negatively associated with fluoropyrimidine toxicity in patients. Together, these data support a causal, clinically relevant interaction between a human gut bacterial operon and the dose-limiting side effects of cancer treatment. Our approach is generalizable to other drugs, including cancer immunotherapies, and provides valuable insights into host-microbiome interactions in the context of disease.

Associations between the Gut Microbiota, Race, and Ethnicity of Patients with Colorectal Cancer: A Pilot and Feasibility Study.

BACKGROUND: Colorectal cancer (CRC) is more prevalent among some racial and ethnic minority and low socioeconomic status populations. Although the gut microbiota is a risk factor for CRC and varies with race and ethnicity, its role in CRC disparities remains poorly understood. METHODS: We examined the feasibility of recruiting sociodemographically diverse CRC patients for a microbiome study involving a home stool collection. We also explored whether race and ethnicity were associated with gut microbiome composition. We recruited Black/African American, Hispanic/Latino, and non-Hispanic White patients who were receiving care for active CRC to complete a comprehensive dietary and lifestyle survey, self-collect a stool sample, and complete an exit interview. Gut microbial diversity and composition were analyzed using 16S rRNA gene sequencing. RESULTS: 30 individuals consented (of 35 who were eligible and contacted) with 5 (17%) Black/African American, 11 (37%) Hispanic/Latino, and 14 (46%) non-Hispanic White. A total of 22 (73%) completed the dietary and lifestyle survey; 18 (63%) returned a stool sample. Even after controlling for socioeconomic, dietary, or treatment-related covariates, microbiome composition was associated with race and ethnicity. Fusobacteriota (a phylum associated with the development and progression of CRC) was significantly higher in the Black/African American group compared to others, and microbial diversity was higher in samples from non-Hispanic White individuals compared to Hispanic/Latino individuals. CONCLUSION: Our study shows that it is feasible to recruit and collect stool samples from diverse individuals with CRC and found significant associations in gut microbial structure with race and ethnicity.

Theranostic Targeting of CUB Domain-Containing Protein 1 (CDCP1) in Multiple Subtypes of Bladder Cancer.

PURPOSE: Despite recent approvals for checkpoint inhibitors and antibody-drug conjugates targeting NECTIN4 or TROP2, metastatic bladder cancer remains incurable and new treatment strategies are urgently needed. CUB domain-containing protein 1 (CDCP1) is a cell surface protein and promising drug target for many cancers. This study aimed to determine whether CDCP1 is expressed in bladder cancer and whether CDCP1 can be targeted for treatment with radiolabeled antibodies. EXPERIMENTAL DESIGN: CDCP1 expression was evaluated in four bladder cancer datasets (n = 1,047 biopsies). A tissue microarray of primary bladder cancer biopsies was probed for CDCP1 by IHC. CDCP1 expression was evaluated in patient-derived xenografts and cell lysates by immunoblot, flow cytometry, and saturation binding assays. Tumor detection in mouse bladder cancer models was tested using 89Zr-labeled 4A06, a monoclonal antibody targeting the ectodomain of CDCP1. 177Lu-4A06 was applied to mice bearing UMUC3 or HT-1376 xenografts to evaluate antitumor effects (CDCP1 expression in UMUC3 is 10-fold higher than HT-1376). RESULTS: CDCP1 was highest in the basal/squamous subtype, and CDCP1 was expressed in 53% of primary biopsies. CDCP1 was not correlated with pathologic or tumor stage, metastatic site, or NECTIN4 and TROP2 at the mRNA or protein level. CDCP1 ranged from 105 to 106 receptors per cell. Mechanism studies showed that RAS signaling induced CDCP1 expression. 89Zr-4A06 PET detected five human bladder cancer xenografts. 177Lu-4A06 inhibited the growth of UMUC3 and HT-1376 xenografts, models with high and moderate CDCP1 expression, respectively. CONCLUSIONS: These data establish that CDCP1 is expressed in bladder cancer, including TROP2 and NECTIN4-null disease, and suggest that bladder cancer can be treated with CDCP1-targeted radiotherapy.

Immunotherapeutic Targeting and PET Imaging of DLL3 in Small-Cell Neuroendocrine Prostate Cancer.

Effective treatments for de novo and treatment-emergent small-cell/neuroendocrine (t-SCNC) prostate cancer represent an unmet need for this disease. Using metastatic biopsies from patients with advanced cancer, we demonstrate that delta-like ligand 3 (DLL3) is expressed in de novo and t-SCNC and is associated with reduced survival. We develop a PET agent, [89Zr]-DFO-DLL3-scFv, that detects DLL3 levels in mouse SCNC models. In multiple patient-derived xenograft models, AMG 757 (tarlatamab), a half-life-extended bispecific T-cell engager (BiTE) immunotherapy that redirects CD3-positive T cells to kill DLL3-expressing cells, exhibited potent and durable antitumor activity. Late relapsing tumors after AMG 757 treatment exhibited lower DLL3 levels, suggesting antigen loss as a resistance mechanism, particularly in tumors with heterogeneous DLL3 expression. These findings have been translated into an ongoing clinical trial of AMG 757 in de novo and t-SCNC, with a confirmed objective partial response in a patient with histologically confirmed SCNC. Overall, these results identify DLL3 as a therapeutic target in SCNC and demonstrate that DLL3-targeted BiTE immunotherapy has significant antitumor activity in this aggressive prostate cancer subtype. SIGNIFICANCE: The preclinical and clinical evaluation of DLL3-directed immunotherapy, AMG 757, and development of a PET radiotracer for noninvasive DLL3 detection demonstrate the potential of targeting DLL3 in SCNC prostate cancer.

CUB Domain-Containing Protein 1 (CDCP1) Is a Target for Radioligand Therapy in Castration-Resistant Prostate Cancer, including PSMA Null Disease.

PURPOSE: With the improvement in overall survival with 177Lu-PSMA 617, radioligand therapy (RLT) is now a viable option for patients with metastatic castration-resistant prostate cancer (mCRPC). However, responses are variable, in part due to low PSMA expression in 30% of patients. Herein, we evaluated whether the cell surface protein CUB domain-containing protein 1 (CDCP1) can be exploited to treat mCRPC with RLT, including in PSMA-low subsets. EXPERIMENTAL DESIGN: CDCP1 levels were evaluated using RNA sequencing from 119 mCRPC biopsies. CDCP1 levels were assessed in 17 post-enzalutamide- or abiraterone-treated mCRPC biopsies, 12 patient-derived xenografts (PDX), and prostate cancer cell lines. 4A06, a recombinant human antibody that targets the CDCP1 ectodomain, was labeled with Zr-89 or Lu-177 and tested in tumor-bearing mice. RESULTS: CDCP1 expression was observed in 90% of mCRPC biopsies, including small-cell neuroendocrine (SCNC) and adenocarcinomas with low FOLH1 (PSMA) levels. Fifteen of 17 evaluable mCRPC biopsies (85%) demonstrated membranous CDCP1 expression, and 4 of 17 (23%) had higher CDCP1 H-scores compared with PSMA. CDCP1 was expressed in 10 of 12 PDX samples. Bmax values of approximately 22,000, 6,200, and 2,800 fmol/mg were calculated for PC3, DU145, and C4-2B human prostate cancer cells, respectively. 89Zr-4A06 PET detected six human prostate cancer xenografts, including PSMA-low tumors. 177Lu-4A06 significantly suppressed growth of DU145 and C4-2B xenografts. CONCLUSIONS: The data provide the first evidence supporting CDCP1-directed RLT to treat mCRPC. Expanded studies are warranted to determine whether CDCP1 is a viable drug target for patients with mCPRC.

TROP2 Expression Across Molecular Subtypes of Urothelial Carcinoma and Enfortumab Vedotin-resistant Cells.

Sacituzumab govitecan (SG) is an antibody-drug conjugate (ADC) targeting TROP2, which has recently been approved for treatment-refractory metastatic urothelial cancer (UC). However, the variability of TROP2 expression across different bladder cancer (BC) subtypes, as well as after enfortumab vedotin (EV) exposure, remains unknown. Using gene expression data from four clinical cohorts with >1400 patient samples of muscle-invasive BC and a BC tissue microarray, we found that TROP2 mRNA and protein are highly expressed across basal, luminal, and stroma-rich subtypes, but depleted in the neuroendocrine subtype. In addition, TROP2 mRNA levels are correlated with NECTIN4 mRNA but are more highly expressed than NECTIN4 mRNA in patient cohorts and BC cell lines. Moreover, CRISPR/Cas9-mediated knockdown of TROP2 demonstrates that its expression is one factor governing SG sensitivity. After prolonged EV exposure, cells can downregulate NECTIN4, leading to EV resistance, but retain TROP2 expression and remain sensitive to SG, suggesting nonoverlapping resistance mechanisms to these ADCs. While our findings warrant further validation, they have significant implications for biomarker development, patient selection, and treatment sequencing in the clinic as well as clinical trial design and stratification for metastatic BC patients. PATIENT SUMMARY: In this report, we investigated the expression levels of the drug target TROP2 across different molecular subtypes of bladder cancer in multiple patient cohorts and cell lines. We found high levels of TROP2 in most subtypes except in the neuroendocrine subtype. Overall, TROP2 gene expression is higher than NECTIN4 gene expression, and cells resistant to enfortumab vedotin (EV), a NECTIN4-targeting antibody-drug conjugate, remain sensitive to sacituzumab govitecan (SG). Our findings suggest that SG may be effective across most bladder cancer subtypes, including the bladder cancers previously treated with EV.