birinapant and Colonic-Neoplasms

Colorectal cancer is a molecularly heterogeneous disease. Responses to genotoxic chemotherapy in the adjuvant or palliative setting vary greatly between patients, and colorectal cancer cells often resist chemotherapy by evading apoptosis. Antagonists of an inhibitor of apoptosis proteins (IAPs) can restore defective apoptosis signaling by degrading cIAP1 and cIAP2 proteins and by inhibition of XIAP. Due to the multiple molecular mechanisms-of-action of these targets, responses to IAP antagonist may differ between molecularly distinct colon cancer cells. In this study, responses to the IAP antagonist Birinapant and oxaliplatin/5-fluorouracil (5-FU) were investigated in 14 colon cancer cell lines, representing the consensus molecular subtypes (CMS). Treatment with Birinapant alone did not result in a substantial increase in apoptotic cells in this cell line panel. Annexin-V/PI assays quantified by flow cytometry and high-content screening showed that Birinapant increased responses of CMS1 and partially CMS3 cell lines to oxaliplatin/5-FU, whereas CMS2 cells were not effectively sensitized. FRET-based imaging of caspase-8 and -3 activation validated these differences at the single-cell level, with CMS1 cells displaying sustained activation of caspase-8-like activity during Birinapant and oxaliplatin/5-FU co-treatment, ultimately activating the intrinsic mitochondrial apoptosis pathway. In CMS2 cell lines, Birinapant exhibited synergistic effects in combination with TNFα, suggesting that Birinapant can restore extrinsic apoptosis signaling in the context of inflammatory signals in this subtype. To explore this further, we co-cultured CMS2 and CMS1 colon cancer cells with peripheral blood mononuclear cells. We observed increased cell death during Birinapant single treatment in these co-cultures, which was abrogated by anti-TNFα-neutralizing antibodies. Collectively, our study demonstrates that IAP inhibition is a promising modulator of response to oxaliplatin/5-FU in colorectal cancers of the CMS1 subtype, and may show promise as in the CMS2 subtype, suggesting that molecular subtyping may aid as a patient stratification tool for IAP antagonists in this disease.

Topics: Apoptosis; Colonic Neoplasms; Dipeptides; Humans; Indoles

Apoptosis resistance contributes to treatment failure in colorectal cancer (CRC). New treatments that reinstate apoptosis competency have potential to improve patient outcome but require predictive biomarkers to target them to responsive patient populations. Inhibitor of apoptosis proteins (IAPs) suppress apoptosis, contributing to drug resistance; IAP antagonists such as TL32711 have therefore been developed. We developed a systems biology approach for predicting response of CRC cells to chemotherapy and TL32711 combinations in vitro and in vivo. CRC cells responded poorly to TL32711 monotherapy in vitro; however, co-treatment with 5-fluorouracil (5-FU) and oxaliplatin enhanced TL32711-induced apoptosis. Notably, cells from genetically identical populations responded highly heterogeneously, with caspases being activated both upstream and downstream of mitochondrial outer membrane permeabilisation (MOMP). These data, combined with quantities of key apoptosis regulators were sufficient to replicate in vitro cell death profiles by mathematical modelling. In vivo, apoptosis protein expression was significantly altered, and mathematical modelling for these conditions predicted higher apoptosis resistance that could nevertheless be overcome by combination of chemotherapy and TL32711. Subsequent experimental observations agreed with these predictions, and the observed effects on tumour growth inhibition correlated robustly with apoptosis competency. We therefore obtained insights into intracellular signal transduction kinetics and their population-based heterogeneities for chemotherapy/TL32711 combinations and provide proof-of-concept that mathematical modelling of apoptosis competency can simulate and predict responsiveness in vivo. Being able to predict response to IAP antagonist-based treatments on the background of cell-to-cell heterogeneities in the future might assist in improving treatment stratification approaches for these emerging apoptosis-targeting agents.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Colonic Neoplasms; Dipeptides; Drug Therapy, Combination; Female; Fluorouracil; Humans; Indoles; Inhibitor of Apoptosis Proteins; Mice; Mice, Inbred BALB C; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Models, Theoretical; Oxaliplatin; Receptor-Interacting Protein Serine-Threonine Kinases; RNA Interference; RNA, Small Interfering; X-Linked Inhibitor of Apoptosis Protein

Induction of apoptosis in tumors is considered a desired goal of anticancer therapy. We investigated whether the dynamic temporal and spatial evolution of apoptosis in response to cytotoxic and mechanism-based therapeutics could be detected noninvasively by the caspase-3 radiotracer [(18)F]ICMT-11 and positron emission tomography (PET).. The effects of a single dose of the alkylating agent cyclophosphamide (CPA or 4-hydroperoxycyclophosphamide), or the mechanism-based small molecule SMAC mimetic birinapant on caspase-3 activation was assessed in vitro and by [(18)F]ICMT-11-PET in mice bearing 38C13 B-cell lymphoma, HCT116 colon carcinoma, or MDA-MB-231 breast adenocarcinoma tumors. Ex vivo analysis of caspase-3 was compared to the in vivo PET imaging data.. Drug treatment increased the mean [(18)F]ICMT-11 tumor uptake with a peak at 24 hours for CPA (40 mg/kg; AUC40-60: 8.04 ± 1.33 and 16.05 ± 3.35 %ID/mL × min at baseline and 24 hours, respectively) and 6 hours for birinapant (15 mg/kg; AUC40-60: 20.29 ± 0.82 and 31.07 ± 5.66 %ID/mL × min, at baseline and 6 hours, respectively). Voxel-based spatiotemporal analysis of tumor-intrinsic heterogeneity suggested that discrete pockets of caspase-3 activation could be detected by [(18)F]ICMT-11. Increased tumor [(18)F]ICMT-11 uptake was associated with caspase-3 activation measured ex vivo, and early radiotracer uptake predicted apoptosis, distinct from the glucose metabolism with [(18)F]fluorodeoxyglucose-PET, which depicted continuous loss of cell viability.. The proapoptotic effects of CPA and birinapant resulted in a time-dependent increase in [(18)F]ICMT-11 uptake detected by PET. [(18)F]ICMT-11-PET holds promise as a noninvasive pharmacodynamic biomarker of caspase-3-associated apoptosis in tumors.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Azides; Breast Neoplasms; Caspase 3; Colonic Neoplasms; Cyclophosphamide; Dipeptides; Enzyme Activation; Female; HCT116 Cells; Humans; Indoles; Lymphoma, B-Cell; Mice; Mice, Inbred C3H; Positron-Emission Tomography; Radiopharmaceuticals; Xenograft Model Antitumor Assays