Sensitisation of cancer cells to radiotherapy by serine and glycine starvation.

BACKGROUND: Cellular metabolism is an integral component of cellular adaptation to stress, playing a pivotal role in the resistance of cancer cells to various treatment modalities, including radiotherapy. In response to radiotherapy, cancer cells engage antioxidant and DNA repair mechanisms which mitigate and remove DNA damage, facilitating cancer cell survival. Given the reliance of these resistance mechanisms on amino acid metabolism, we hypothesised that controlling the exogenous availability of the non-essential amino acids serine and glycine would radiosensitise cancer cells. METHODS: We exposed colorectal, breast and pancreatic cancer cell lines/organoids to radiation in vitro and in vivo in the presence and absence of exogenous serine and glycine. We performed phenotypic assays for DNA damage, cell cycle, ROS levels and cell death, combined with a high-resolution untargeted LCMS metabolomics and RNA-Seq. RESULTS: Serine and glycine restriction sensitised a range of cancer cell lines, patient-derived organoids and syngeneic mouse tumour models to radiotherapy. Comprehensive metabolomic and transcriptomic analysis of central carbon metabolism revealed that amino acid restriction impacted not only antioxidant response and nucleotide synthesis but had a marked inhibitory effect on the TCA cycle. CONCLUSION: Dietary restriction of serine and glycine is a viable radio-sensitisation strategy in cancer.

Colorectal Cancer Develops Inherent Radiosensitivity That Can Be Predicted Using Patient-Derived Organoids.

Identifying colorectal cancer patient populations responsive to chemotherapy or chemoradiation therapy before surgery remains a challenge. Recently validated mouse protocols for organoid irradiation employ the single hit multi-target (SHMT) algorithm, which yields a single value, the D0, as a measure of inherent tissue radiosensitivity. Here, we translate these protocols to human tissue to evaluate radioresponsiveness of patient-derived organoids (PDO) generated from normal human intestines and rectal tumors of patients undergoing neoadjuvant therapy. While PDOs from adenomas with a logarithmically expanded Lgr5+ intestinal stem cell population retain the radioresistant phenotype of normal colorectal PDOs, malignant transformation yields PDOs from a large patient subpopulation displaying marked radiosensitivity due to reduced homologous recombination-mediated DNA repair. A proof-of-principle pilot clinical trial demonstrated that rectal cancer patient responses to neoadjuvant chemoradiation, including complete response, correlate closely with their PDO D0 values. Overall, upon transformation to colorectal adenocarcinoma, broad radiation sensitivity occurs in a large subset of patients that can be identified using SHMT analysis of PDO radiation responses. SIGNIFICANCE: Analysis of inherent tissue radiosensitivity of patient-derived organoids may provide a readout predictive of neoadjuvant therapy response to radiation in rectal cancer, potentially allowing pretreatment stratification of patients likely to benefit from this approach.

A Claudin-Based Molecular Signature Identifies High-Risk, Chemoresistant Colorectal Cancer Patients.

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.

Type of recurrence is associated with disease-free survival after salvage surgery for locally recurrent rectal cancer.

PURPOSE: To compare the characteristics and outcomes of rectal cancer patients with local recurrence at a perianastomotic site (PA), a surgical field (SF) site, or in lateral lymph nodes (LLN). METHODS: A total of 114 consecutive patients who underwent surgery for recurrent, non-metastatic rectal cancer at a single comprehensive cancer center between 1997 and 2012 were grouped on the basis of radiographic assessment of type of recurrence: PA, 76 (67%) patients; SF, 25 (22%) patients; LLN, 13 (11%) patients. Demographic, clinical, and pathological features were compared between the three groups, as were disease-free survival (DFS) and overall survival (OS). RESULTS: Recurrence type was associated with positive circumferential margin in the primary resection (PA, 4 [6%]; SF, 4 [19%]; LLN, 3 [25%]; P = 0.027), prior neoadjuvant therapy for the primary tumor (PA, 57 [75%]; SF, 18 [72%]; LLN, 4 [31%]; P = 0.007), and location of the primary tumor in the upper rectum (PA, 33 [45%]; SF, 5 [23%]; LLN, 1 [8%]; P < 0.001). Patients with PA had longer median DFS (PA, 5.1 years; SF, 1.5 years; LLN, 1.2 years; P = 0.036). There was a non-significant trend toward longer OS and higher rates of R0 resection for PA. CONCLUSION: Type of recurrence after salvage surgery for locally recurrent rectal cancer is associated with longer DFS in patients with PA recurrence.

Mismatch Repair-Deficient Rectal Cancer and Resistance to Neoadjuvant Chemotherapy.

PURPOSE: Evaluate response of mismatch repair-deficient (dMMR) rectal cancer to neoadjuvant chemotherapy. EXPERIMENTAL DESIGN: dMMR rectal tumors at Memorial Sloan Kettering Cancer Center (New York, NY) were retrospectively reviewed for characteristics, treatment, and outcomes. Fifty patients with dMMR rectal cancer were identified by IHC and/or microsatellite instability analysis, with initial treatment response compared with a matched MMR-proficient (pMMR) rectal cancer cohort. Germline and somatic mutation analyses were evaluated. Patient-derived dMMR rectal tumoroids were assessed for chemotherapy sensitivity. RESULTS: Of 21 patients receiving neoadjuvant chemotherapy (fluorouracil/oxaliplatin), six (29%) had progression of disease. In comparison, no progression was noted in 63 pMMR rectal tumors (P = 0.0001). Rectal cancer dMMR tumoroids reflected this resistance to chemotherapy. No genomic predictors of chemotherapy response were identified. Of 16 patients receiving chemoradiation, 13 (93%) experienced tumor downstaging; one patient had stable disease, comparable with 48 pMMR rectal cancers. Of 13 patients undergoing surgery, 12 (92%) had early-stage disease. Forty-two (84%) of the 50 patients tested positive for Lynch syndrome with enrichment of germline MSH2 and MSH6 mutations when compared with 193 patients with Lynch syndrome-associated colon cancer (MSH2, 57% vs 36%; MSH6, 17% vs 9%; P < 0.003). CONCLUSIONS: Over one-fourth of dMMR rectal tumors treated with neoadjuvant chemotherapy exhibited disease progression. Conversely, dMMR rectal tumors were sensitive to chemoradiation. MMR status should be performed upfront in all locally advanced rectal tumors with careful monitoring for response on neoadjuvant chemotherapy and genetic testing for Lynch syndrome in patients with dMMR rectal cancer.

A rectal cancer organoid platform to study individual responses to chemoradiation.

Rectal cancer (RC) is a challenging disease to treat that requires chemotherapy, radiation and surgery to optimize outcomes for individual patients. No accurate model of RC exists to answer fundamental research questions relevant to patients. We established a biorepository of 65 patient-derived RC organoid cultures (tumoroids) from patients with primary, metastatic or recurrent disease. RC tumoroids retained molecular features of the tumors from which they were derived, and their ex vivo responses to clinically relevant chemotherapy and radiation treatment correlated with the clinical responses noted in individual patients' tumors. Upon engraftment into murine rectal mucosa, human RC tumoroids gave rise to invasive RC followed by metastasis to lung and liver. Importantly, engrafted tumors displayed the heterogenous sensitivity to chemotherapy observed clinically. Thus, the biology and drug sensitivity of RC clinical isolates can be efficiently interrogated using an organoid-based, ex vivo platform coupled with in vivo endoluminal propagation in animals.

PAF promotes stemness and radioresistance of glioma stem cells.

An integrated genomic and functional analysis to elucidate DNA damage signaling factors promoting self-renewal of glioma stem cells (GSCs) identified proliferating cell nuclear antigen (PCNA)-associated factor (PAF) up-regulation in glioblastoma. PAF is preferentially overexpressed in GSCs. Its depletion impairs maintenance of self-renewal without promoting differentiation and reduces tumor-initiating cell frequency. Combined transcriptomic and metabolomic analyses revealed that PAF supports GSC maintenance, in part, by influencing DNA replication and pyrimidine metabolism pathways. PAF interacts with PCNA and regulates PCNA-associated DNA translesion synthesis (TLS); consequently, PAF depletion in combination with radiation generated fewer tumorspheres compared with radiation alone. Correspondingly, pharmacological impairment of DNA replication and TLS phenocopied the effect of PAF depletion in compromising GSC self-renewal and radioresistance, providing preclinical proof of principle that combined TLS inhibition and radiation therapy may be a viable therapeutic option in the treatment of glioblastoma multiforme (GBM).

Tumor Evolution of Glioma-Intrinsic Gene Expression Subtypes Associates with Immunological Changes in the Microenvironment.

We leveraged IDH wild-type glioblastomas, derivative neurospheres, and single-cell gene expression profiles to define three tumor-intrinsic transcriptional subtypes designated as proneural, mesenchymal, and classical. Transcriptomic subtype multiplicity correlated with increased intratumoral heterogeneity and presence of tumor microenvironment. In silico cell sorting identified macrophages/microglia, CD4+ T lymphocytes, and neutrophils in the glioma microenvironment. NF1 deficiency resulted in increased tumor-associated macrophages/microglia infiltration. Longitudinal transcriptome analysis showed that expression subtype is retained in 55% of cases. Gene signature-based tumor microenvironment inference revealed a decrease in invading monocytes and a subtype-dependent increase in macrophages/microglia cells upon disease recurrence. Hypermutation at diagnosis or at recurrence associated with CD8+ T cell enrichment. Frequency of M2 macrophages detection associated with short-term relapse after radiation therapy.

Epigenetic Activation of WNT5A Drives Glioblastoma Stem Cell Differentiation and Invasive Growth.

Glioblastoma stem cells (GSCs) are implicated in tumor neovascularization, invasiveness, and therapeutic resistance. To illuminate mechanisms governing these hallmark features, we developed a de novo glioblastoma multiforme (GBM) model derived from immortalized human neural stem/progenitor cells (hNSCs) to enable precise system-level comparisons of pre-malignant and oncogene-induced malignant states of NSCs. Integrated transcriptomic and epigenomic analyses uncovered a PAX6/DLX5 transcriptional program driving WNT5A-mediated GSC differentiation into endothelial-like cells (GdECs). GdECs recruit existing endothelial cells to promote peritumoral satellite lesions, which serve as a niche supporting the growth of invasive glioma cells away from the primary tumor. Clinical data reveal higher WNT5A and GdECs expression in peritumoral and recurrent GBMs relative to matched intratumoral and primary GBMs, respectively, supporting WNT5A-mediated GSC differentiation and invasive growth in disease recurrence. Thus, the PAX6/DLX5-WNT5A axis governs the diffuse spread of glioma cells throughout the brain parenchyma, contributing to the lethality of GBM.