Background: Despite recent advances in therapies, resistance to chemotherapy remains a critical problem in the clinical management of colorectal cancer (CRC). Cancer stem cells (CSCs) play a central role in therapy resistance. Thus, elimination of CSCs is crucial for effective CRC therapy; however, such strategies are limited. Autophagy promotes resistance to cancer therapy; however, whether autophagy protects CSCs to promote resistance to CRC-therapy is not well understood. Moreover, specific and potent autophagy inhibitors are warranted as clinical trials with hydroxychloroquine have not been successful. Methods: Colon cancer cells and tumoroids were used. Fluorescent reporter-based analysis of autophagy flux, spheroid and side population (SP) culture, and qPCR were done. We synthesized 36-077, a potent inhibitor of PIK3C3/VPS34 kinase, to inhibit autophagy. Combination treatments were done using 5-fluorouracil (5-FU) and 36-077. Results: The 5-FU treatment induced autophagy only in a subset of the treated colon cancer. These autophagy-enriched cells also showed increased expression of CSC markers. Co-treatment with 36-077 significantly improved efficacy of the 5-FU treatment. Mechanistic studies revealed that combination therapy inhibited GSK-3β/Wnt/β-catenin signaling to inhibit CSC population. Conclusion: Autophagy promotes resistance to CRC-therapy by specifically promoting GSK-3β/Wnt/β-catenin signaling to promote CSC survival, and 36-077, a PIK3C3/VPS34 inhibitor, helps promote efficacy of CRC therapy.

Identifying molecular characteristics that are associated with aggressive cancer phenotypes through gene expression profiling can help predict treatment responses and clinical outcomes. Claudins are deregulated in colorectal cancer (CRC). In CRC, increased claudin-1 expression results in epithelial-to-mesenchymal transition and metastasis, while claudin-7 functions as a tumor suppressor. In this study, we have developed a molecular signature based on claudin-1 and claudin-7 associated with poor patient survival and chemoresistance. This signature was validated using an integrated approach including publicly available datasets and CRC samples from patients who either responded or did not respond to standard-of-care treatment, CRC cell lines, and patient-derived rectal and colon tumoroids. Transcriptomic analysis from a patient dataset initially yielded 23 genes that were differentially expressed along with higher claudin-1 and decreased claudin-7. From this analysis, we selected a claudins-associated molecular signature including PIK3CA, SLC6A6, TMEM43, and ASAP-1 based on their importance in CRC. The upregulation of these genes and their protein products was validated using multiple CRC patient datasets, in vitro chemoresistant cell lines, and patient-derived tumoroid models. Additionally, blocking these genes improved 5-FU sensitivity in chemoresistant CRC cells. Our findings propose a new claudin-based molecular signature that associates with poor prognosis as well as characteristics of treatment-resistant CRC including chemoresistance, metastasis, and relapse.

The gut microbiome’s role in the pathogenesis of hematologic malignancies is actively being explored; yet studies on chronic lymphocytic leukemia (CLL) are limited. Using the Eμ-TCL1 murine model of CLL, we identify a unique and dysbiotic disease-associated gut microbiome that develops in mice over time. Leukemic mice show an increase in abundance of pathogenic bacteria, specifically members of the Proteobacteria phylum. We found that this dysbiotic microenvironment is associated with CLL involvement within the intestinal tract and high levels of markers indicative of altered tight junction permeability (e.g., claudin-2, soluble CD14, and zonulin). Moreover, utilizing the syngeneic model of CLL in tandem with an antibiotic-mediated microflora ablation approach, we found that leukemic mice receiving microflora-ablating antibiotics show marked changes to the gut microbiome and a delayed disease onset compared with mice receiving antibiotic-free water. Immunophenotyping of murine spleens showed that this delay in disease was accompanied by more tumor-reactive CD8+ T cells that coexpressed fewer inhibitory receptors (e.g., PD-1, LAG-3, and TIM-3). These findings confirm that a dysbiotic gut microbiome develops during CLL disease and demonstrate unique intestinal involvement and potential immune dysregulation occurring during CLL progression that may be influencing the overall microbial signature. Significance: There is a growing appreciation for the gut microbiome’s role in hematologic malignancies. Despite this, its role in CLL remains obscure. This study demonstrates a dysbiotic microbiome within CLL that may contribute to further intestinal and immune dysregulation present during CLL progression.

Figure S5. Antibiotic-mediated gut microflora ablation alters T-cell function in leukemic mice.

Figure S2. Relative abundance of microbiota in Eµ-TCL1 mice versus WT B6 mice.