Nanoreporter Identifies Lysosomal Storage Disease Lipid Accumulation Intracranially.

Dysregulated lipid metabolism contributes to neurodegenerative pathologies and neurological decline in lysosomal storage disorders as well as more common neurodegenerative diseases. Niemann-Pick type A (NPA) is a fatal neurodegenerative lysosomal storage disease characterized by abnormal sphingomyelin accumulation in the endolysosomal lumen. The ability to monitor abnormalities in lipid homeostasis intracranially could improve basic investigations and the development of effective treatment strategies. We investigated the carbon nanotube-based detection of intracranial lipid content. We found that the near-infrared emission of a carbon nanotube-based lipid sensor responds to lipid accumulation in neuronal and in vivo models of NPA. The nanosensor detected lipid accumulation intracranially in an acid sphingomyelinase knockout mouse via noninvasive near-infrared spectroscopy. This work indicates a tool to improve drug development processes in NPA, other lysosomal storage diseases, and neurodegenerative diseases.

Activation of STING in Response to Partial-Tumor Radiation Exposure.

PURPOSE: To determine the mechanisms involved in partial volume radiation therapy (RT)-induced tumor response. METHODS AND MATERIALS: We investigated 67NR murine orthotopic breast tumors in Balb/c mice and Lewis lung carcinoma (LLC cells; WT, Crispr/Cas9 Sting KO, and Atm KO) injected in the flank of C57Bl/6, cGAS, or STING KO mice. RT was delivered to 50% or 100% of the tumor volume using a 2 × 2 cm collimator on a microirradiator allowing precise irradiation. Tumors and blood were collected at 6, 24, and 48 hours post-RT and assessed for cytokine measurements. RESULTS: There is a significant activation of the cGAS/STING pathway in the hemi-irradiated tumors compared with control and to 100% exposed 67NR tumors. In the LLC model, we determined that an ATM-mediated noncanonical activation of STING is involved. We demonstrated that the partial exposure RT-mediated immune response is dependent on ATM activation in the tumor cells and on the STING activation in the host, and cGAS is dispensable. Our results also indicate that partial volume RT stimulates a proinflammatory cytokine response compared with the anti-inflammatory profile induced by 100% tumor volume exposure. CONCLUSIONS: Partial volume RT induces an antitumor response by activating STING, which stimulates a specific cytokine signature as part of the immune response. However, the mechanism of this STING activation, via the canonical cGAS/STING pathway or a noncanonical ATM-driven pathway, depends on the tumor type. Identifying the upstream pathways responsible for STING activation in the partial RT-mediated immune response in different tumor types would improve this therapy and its potential combination with immune checkpoint blockade and other antitumor therapies.

Radiation-induced gastrointestinal (GI) syndrome as a function of age.

Previous studies show increased sensitivity of older mice (28-29 months) compared with young adult mice (3 months, possessing a mature immune system) to radiation-induced GI lethality. Age-dependent lethality was associated with higher levels of apoptotic stem cells in small intestinal crypts that correlated with sphingomyelinase activity, a source of pro-apoptotic ceramide. The objective of this study is to determine whether the cycling crypt base columnar cells (CBCs) in aging animals are specifically more sensitive to radiation effects than the CBCs in young adult mice, and to identify factors that contribute to increased radiosensitivity. Mortality induced by subtotal body radiation was assessed at different doses (13 Gy, 14 Gy, and 15 Gy) in young adult mice versus older mice. Each dose was evaluated for the occurrence of lethal GI syndrome. A higher death rate due to radiation-induced GI syndrome was observed in older mice as compared with young adult mice: 30 vs. 0% at 13 Gy, 90 vs. 40% at 14 Gy, and 100 vs. 60% at 15 Gy. Radiation-induced damage to crypts was determined by measuring crypt regeneration (H&E staining, Ki67 expression), CBC biomarkers (lgr5 and ascl2), premature senescence (SA-ß-gal activity), and apoptosis of CBCs. At all three doses, crypt microcolony survival assays showed that the older mice had fewer regenerating crypts at 3.5 days post-radiation treatment. Furthermore, in the older animals, baseline CBCs numbers per circumference were significantly decreased, correlating with an elevated apoptotic index. Analysis of tissue damage showed an increased number of senescent CBCs per crypt circumference in older mice relative to younger mice, where the latter was not significantly affected by radiation treatment. It is concluded that enhanced sensitivity to radiation-induced GI syndrome and higher mortality in older mice can be attributed to a decreased capacity to regenerate crypts, presumably due to increased apoptosis and senescence of CBCs.

Cell-intrinsic ceramides determine T cell function during melanoma progression.

Acid sphingomyelinase (Asm) and acid ceramidase (Ac) are parts of the sphingolipid metabolism. Asm hydrolyzes sphingomyelin to ceramide, which is further metabolized to sphingosine by Ac. Ceramide generates ceramide-enriched platforms that are involved in receptor clustering within cellular membranes. However, the impact of cell-intrinsic ceramide on T cell function is not well characterized. By using T cell-specific Asm- or Ac-deficient mice, with reduced or elevated ceramide levels in T cells, we identified ceramide to play a crucial role in T cell function in vitro and in vivo. T cell-specific ablation of Asm in Smpd1fl/fl/Cd4cre/+ (Asm/CD4cre) mice resulted in enhanced tumor progression associated with impaired T cell responses, whereas Asah1fl/fl/Cd4cre/+ (Ac/CD4cre) mice showed reduced tumor growth rates and elevated T cell activation compared to the respective controls upon tumor transplantation. Further in vitro analysis revealed that decreased ceramide content supports CD4+ regulatory T cell differentiation and interferes with cytotoxic activity of CD8+ T cells. In contrast, elevated ceramide concentration in CD8+ T cells from Ac/CD4cre mice was associated with enhanced cytotoxic activity. Strikingly, ceramide co-localized with the T cell receptor (TCR) and CD3 in the membrane of stimulated T cells and phosphorylation of TCR signaling molecules was elevated in Ac-deficient T cells. Hence, our results indicate that modulation of ceramide levels, by interfering with the Asm or Ac activity has an effect on T cell differentiation and function and might therefore represent a novel therapeutic strategy for the treatment of T cell-dependent diseases such as tumorigenesis.

Chemotherapy-induced acute vascular injury involves intracellular generation of ROS via activation of the acid sphingomyelinase pathway.

Several categories of chemotherapy confer substantial risk for late-term vascular morbidity and mortality. In the present study, we aimed to investigate the mechanism of acute chemotherapy-induced vascular injury in normal tissues. Specifically, we looked at activation of the acid sphingomyelinase (ASMase)/ceramide pathway, which leads to generation of reactive oxygen species (ROS) and induction of oxidative stress that may result in vascular injury. In particular, we focused on two distinct drugs, doxorubicin (DOX) and cisplatin (CIS) and their effects on normal endothelial cells. In vitro, DOX resulted in increased ASMase activity, intra-cellular ROS production and induction of apoptosis. CIS treatment generated significantly reduced effects in endothelial cells. In-vivo, murine femoral arterial blood flow was measured in real-time, during and after DOX or CIS administration, using fluorescence optical imaging system. While DOX caused constriction of small vessels and disintegration of large vessels' wall, CIS induced minor vascular changes in arterial blood flow, correlating with the in vitro findings. These results demonstrate that DOX induces acute vascular injury by increased ROS production, via activation of ASMase/ceramide pathway, while CIS increases ROS production and its immediate extracellular translocation, without causing detectable acute vascular injury. Our findings may potentially lead to the development of new strategies to prevent long-term cardiovascular morbidity in cancer survivors.

Chemotherapeutic Agents-Induced Ceramide-Rich Platforms (CRPs) in Endothelial Cells and Their Modulation.

The prevailing mechanism of action of chemotherapeutic drugs has been challenged by the role of ceramide, a second messenger, shown to induce apoptosis, differentiation, growth arrest, senescence, and autophagy in different cells (Chabner BA, Roberts TG Jr, Nat Rev Cancer 5:65-72, 2005; Jacobi J et al, Cell Signal 29:52-61, 2017; Rotolo J et al, J Clin Invest 122:1786-1790, 2012; Truman JP et al, PLoS One 5:e12310, 2010). Certain chemotherapeutic drugs activate the acid sphingomyelinase (ASMase)/ceramide pathway, generating ceramide in the tumor endothelium and this microvascular dysfunction is crucial for the tumor response. Ceramide has fusigenic properties and as such, when generated within the plasma membrane, initiates the oligomerization of ceramide-and cholesterol-rich domains in the outer leaflet of the plasma membrane, leading to the formation of ceramide-rich microdomains/platforms (CRP) (Jacobi J et al, Cell Signal 29:52-61, 2017; Truman JP et al, PLoS One 5:e12310, 2010; van Hell AJ et al, Cell Signal 34:86-91, 2017; Hajj C, Haimovitz-Friedman A, Handb Exp Pharmacol 216:115-130, 2013) known as "signaling platform." This chapter will discuss the generation, detection, and quantitation of CRP and their possible modulation in endothelial cells, in vitro and in vivo in response to certain chemotherapeutic drugs.

Organoids Reveal That Inherent Radiosensitivity of Small and Large Intestinal Stem Cells Determines Organ Sensitivity.

Tissue survival responses to ionizing radiation are nonlinear with dose, rather yielding tissue-specific descending curves that impede straightforward analysis of biologic effects. Apoptotic cell death often occurs at low doses, while at clinically relevant intermediate doses, double-strand break misrepair yields mitotic death that determines outcome. As researchers frequently use a single low dose for experimentation, such strategies may inaccurately depict inherent tissue responses. Cutting edge radiobiology has adopted full dose survival profiling and devised mathematical algorithms to fit curves to observed data to generate highly reproducible numerical data that accurately define clinically relevant inherent radiosensitivities. Here, we established a protocol for irradiating organoids that delivers radiation profiles simulating the organ of origin. This technique yielded highly similar dose-survival curves of small and large intestinal crypts in vivo and their cognate organoids analyzed by the single-hit multi-target (SHMT) algorithm, outcomes reflecting the inherent radiation profile of their respective Lgr5+ stem cell populations. As this technological advance is quantitative, it will be useful for accurate evaluation of intestinal (patho)physiology and drug screening. SIGNIFICANCE: These findings establish standards for irradiating organoids that deliver radiation profiles that phenocopy the organ of origin.See related commentary by Muschel et al., p. 927.

Photobiomodulation effects on head and neck squamous cell carcinoma (HNSCC) in an orthotopic animal model.

BACKGROUND: Photobiomodulation (PBM) has shown efficacy in preventing and treating cancer therapy-induced mucositis and dermatitis. However, there is contradictory information regarding the effect of PBM on (pre)malignant cells, which has led to questions regarding the safety of this technique. We address this issue using an orthotopic mouse model (Cal-33) with human squamous cell carcinoma of the oral cavity. METHODS: Mice with actively growing orthotopic Cal-33 head and neck carcinoma tumors were divided into 4 groups: control, PBM only, radiation therapy (RT) only, and PBM + RT. We performed three experiments: (1) PBM at 660 nm, 18.4 J/cm2, and 5 RT × 4 Gy doses delivered daily; (2) PBM at 660 nm, 18.4 J/cm2, and 1 × 15 Gy RT; and (3) PBM at 660 nm + 850 nm, 45 mW/cm2, 3.4 J/cm2, and 1 × 15 Gy RT. Mice were weighed daily and tumor volumes were evaluated by IVIS. Survival time was also evaluated. RESULTS: Animals treated with RT survived significantly longer and had significantly smaller tumor volume when compared with the control and PBM-only treatment groups. No significant differences were noted between the RT alone and PBM + RT groups in any of the experiments. CONCLUSION: Our results suggest that PBM at the utilized parameters does not provide protection to the tumor from the killing effects of RT.

An Antitumor Immune Response Is Evoked by Partial-Volume Single-Dose Radiation in 2 Murine Models.

PURPOSE: This study examined tumor growth delay resulting from partial irradiation in preclinical mouse models. METHODS AND MATERIALS: We investigated 67NR murine orthotopic breast tumors in both immunocompetent and nude mice. Treatment was delivered to 50% or 100% of the tumor using a 2 × 2 cm collimator on a microirradiator. Radiation response was modulated by treatment with anti-CD8 and anti-intercellular adhesion molecule (anti-ICAM) antibodies. Similar experiments were performed using the less immunogenic Lewis lung carcinoma mouse model. Tumor growth delay and γ-H2AX phosphorylation were measured, and immune response was assessed by immunofluorescence and flow cytometry at 1 and 7 days after radiation therapy. Tumor expression of cellular adhesion molecules was also measured at different times after radiation therapy. RESULTS: Partial irradiation led to tumor responses similar to those of fully exposed tumors in immunocompetent mice, but not in nude mice. After a single dose of 10 Gy, infiltration of CD8+ T cells was observed along with increased expression of ICAM. The response to 10 Gy in hemi-irradiated tumors was abrogated by treatment with either anti-CD8 or anti-ICAM antibodies. Similar responses were obtained in the less immunogenic Lewis lung carcinoma mouse model delivering 15 Gy to half the tumor volume. Treatment with FTY720, a compound that inhibits T-cell egress from lymph nodes, did not affect tumor response at the time of CD8+ T cells infiltration in the nonirradiated area of the tumor. This result indicated that the most likely source of these cells is the irradiated portion of the hemi-irradiated tumors. In addition, a significant abscopal effect was observed after partial irradiation with a single dose of 10 Gy in the 67NR model. CONCLUSIONS: In these models, radiation controls tumor growth both directly through cell killing and indirectly through immune activation. This outcome raises the possibility that this effect could be induced in the clinic.

Single-dose radiotherapy disables tumor cell homologous recombination via ischemia/reperfusion injury.

Tumor cure with conventional fractionated radiotherapy is 65%, dependent on tumor cell-autonomous gradual buildup of DNA double-strand break (DSB) misrepair. Here we report that single-dose radiotherapy (SDRT), a disruptive technique that ablates more than 90% of human cancers, operates a distinct dual-target mechanism, linking acid sphingomyelinase-mediated (ASMase-mediated) microvascular perfusion defects to DNA unrepair in tumor cells to confer tumor cell lethality. ASMase-mediated microcirculatory vasoconstriction after SDRT conferred an ischemic stress response within parenchymal tumor cells, with ROS triggering the evolutionarily conserved SUMO stress response, specifically depleting chromatin-associated free SUMO3. Whereas SUMO3, but not SUMO2, was indispensable for homology-directed repair (HDR) of DSBs, HDR loss of function after SDRT yielded DSB unrepair, chromosomal aberrations, and tumor clonogen demise. Vasoconstriction blockade with the endothelin-1 inhibitor BQ-123, or ROS scavenging after SDRT using peroxiredoxin-6 overexpression or the SOD mimetic tempol, prevented chromatin SUMO3 depletion, HDR loss of function, and SDRT tumor ablation. We also provide evidence of mouse-to-human translation of this biology in a randomized clinical trial, showing that 24 Gy SDRT, but not 3×9 Gy fractionation, coupled early tumor ischemia/reperfusion to human cancer ablation. The SDRT biology provides opportunities for mechanism-based selective tumor radiosensitization via accessing of SDRT/ASMase signaling, as current studies indicate that this pathway is tractable to pharmacologic intervention.

An optical nanoreporter of endolysosomal lipid accumulation reveals enduring effects of diet on hepatic macrophages in vivo.

The abnormal accumulation of lipids within the endolysosomal lumen occurs in many conditions, including lysosomal storage disorders, atherosclerosis, nonalcoholic fatty liver disease (NAFLD), and drug-induced phospholipidosis. Current methods cannot monitor endolysosomal lipid content in vivo, hindering preclinical drug development and research into the mechanisms linking endolysosomal lipid accumulation to disease progression. We developed a single-walled carbon nanotube-based optical reporter that noninvasively measures endolysosomal lipid accumulation via bandgap modulation of its intrinsic near-infrared emission. The reporter detected lipid accumulation in Niemann-Pick disease, atherosclerosis, and NAFLD models in vivo. By applying the reporter to the study of NAFLD, we found that elevated lipid quantities in hepatic macrophages caused by a high-fat diet persist long after reverting to a normal diet. The reporter dynamically monitored endolysosomal lipid accumulation in vivo over time scales ranging from minutes to weeks, indicating its potential to accelerate preclinical research and drug development processes.

Phosphorylation state of Ser165 in α-tubulin is a toggle switch that controls proliferating human breast tumors.

Engineered overexpression of protein kinase Cα (PKCα) is known to phosphorylate Ser165 in α-tubulin resulting in stimulated microtubule dynamics and cell motility, and activation of an epithelial-mesenchymal transition (EMT) in non-transformed human breast cells. Here it is shown that endogenous phosphorylation of native α-tubulin in two metastatic breast cell lines, MDA-MB-231-LM2-4175 and MDA-MB-468 is detected at PKC phosphorylation sites. α-Tubulin mutants that simulated phosphorylated (S165D) or non-phosphorylated (S165 N) states were stably expressed in MDA-MB-231-LM2-4175 cells. The S165D-α-tubulin mutant engendered expression of the EMT biomarker N-cadherin, whereas S165 N-α-tubulin suppressed N-cadherin and induced E-cadherin expression, revealing a 'cadherin switch'. S165 N-α-tubulin engendered more rapid passage through the cell cycle, induced shorter spindle fibers and exhibited more rapid proliferation. In nude mice injected with MDA-MB-231-LM2-4175 cells, cells expressing S165 N-α-tubulin (but not the S165D mutant) produced hyper-proliferative lung tumors with increased tumor incidence and higher Ki67 expression. These results implicate the phosphorylation state of Ser165 in α-tubulin as a PKC-regulated molecular switch that causes breast cells to exhibit either EMT characteristics or hyper-proliferation. Evaluation of genomic databases of human tumors strengthens the clinical significance of these findings.

Pazopanib radio-sensitization of human sarcoma tumors.

Recent data in our laboratory indicate that engagement of host-derived microenvironmental elements impact tumor response to single high dose radiation therapy (SDRT). In these studies we showed that microvascular endothelial damage plays a critical role in tumor response as regulator of direct lethal damage of SDRT. Using a genetic model of Acid Sphingomyelinase (ASMase)-deficient mice we showed that activation of this enzyme by SDRT-induced damage in the endothelium is mandatory for tumor cure. ASMase activation triggers ceramide-mediated apoptosis, and therein microvascular dysfunction, which increased the vulnerability of tumor cells to lethal damage by radiation. Angiogenic factors repressed this activity while a monoclonal antibody targeting VEGF, de-repressed ASMase activity and radiosensitized tumor endothelium when delivered immediately prior to SDRT. In this study, we tested the effect of SDRT in combination with the short-acting anti-angiogenic agent, Pazopanib (anti-VEGFR-1/2/3, PDGF-α/ß and c-kit), in two xenograft models of human sarcoma. Pre-treatment with a single dose of Pazopanib increased SDRT-induced ASMase activity and endothelial dysfunction in vitro and in vivo, enhancing SDRT tumor cure, and exhibiting critical dependence on timing relative to SDRT exposure, suggesting a mechanism of action identical to that demonstrated for anti-VEGF/VEGFR2 antibodies. These results demonstrate the ability of Pazopanib to shift the response towards tumor cure and could therefore have a significant impact on clinical trial development in combination with SDRT for sarcoma cancer patients.

Logarithmic expansion of LGR5+ cells in human colorectal cancer.

Stem cells of the small and large intestine are marked by expression of the Wnt target gene LGR5, a leucine-rich-repeat-containing G protein-coupled receptor. Previous studies reported increased expression of LGR5 in human colorectal cancer (CRC) compared to normal tissue either by immunohistochemistry or in situ hybridization (ISH). However, as these studies were semi-quantitative they did not provide a numerical estimate of the magnitude of this effect. While we confirm that LGR5+ cells are exclusively located at the base of normal human small and large intestinal crypts, representing approximately 6% of total crypt cells, we show this cell population is 10-fold expanded in all grades of CRC, representing as much as 70% of the cells of tumor crypt-like structures. This expansion of the LGR5 compartment coincides with maintenance of crypt-like glandular structure (adenomas, and well and moderately differentiated adenocarcinomas), and is reduced in poorly differentiated CRC, where crypt-like glandular architecture is lost, accompanied by reduced epithelial terminal differentiation. Altogether these results indicate that LGR5+ cell expansion is a hallmark of CRC tumorigenesis occurring during progression to adenoma, supporting CRC as a stem cell disease with implications for CRC therapy.

A Combination of Radiation and the Hypoxia-Activated Prodrug Evofosfamide (TH-302) is Efficacious against a Human Orthotopic Pancreatic Tumor Model.

This study was designed to investigate the effect of single-dose radiation therapy (RT) in combination with evofosfamide (TH-302), a hypoxia-activated prodrug, in a pre-clinical model of pancreatic cancer. AsPC1 tumors were implanted orthotopically in the pancreas of nude mice. Tumors were treated with 15 Gy of RT, using a 1 cm diameter field, and delivered as a continuous arc. Image-guidance to center the field on the tumor was based on CT imaging with intraperitoneal contrast. Evofosfamide (100 mg/kg, i.p.) was administered 3 hours before RT. Tumor volumes were measured using ultrasound, and regrowth curves were plotted. Tumor hypoxia and cell proliferation were measured using pimonidazole and the thymidine analog EdU, respectively. In vitro clonogenic assays were performed. Tumors were shown to contain substantial areas of hypoxia, as calculated by percent pimonidazole staining. Evofosfamide was active in these tumors, as demonstrated by a significant reduction in uptake of the thymidine analog EdU. This effect was visible in oxygenated tissue, consistent with the previously reported bystander effects of evofosfamide. RT produced significant regrowth delay, as did evofosfamide. The combination of both agents produced a growth delay that was at least equal to the sum of the two treatments given separately. The improvement in tumor response when evofosfamide is combined with RT supports the hypothesis that hypoxia is a cause of radioresistance in high dose RT for pancreatic cancer. Assessing the efficacy and safety of stereotactic radiation treatment and evofosfamide is warranted in patients with locally advanced pancreatic cancer.

In Vitro and In Vivo Comparison of Gemcitabine and the Gemcitabine Analog 1-(2'-deoxy-2'-fluoroarabinofuranosyl) Cytosine (FAC) in Human Orthotopic and Genetically Modified Mouse Pancreatic Cancer Models.

PURPOSE: Although gemcitabine is a mainstay of pancreatic cancer therapy, it is only moderately effective, and it would be desirable to measure drug uptake in patients. 1-(2'-deoxy-2'-fluoroarabinofuranosyl) cytosine (FAC), is an analog of gemcitabine, and when labeled with F-18, it may be a potential surrogate PET tracer for the drug. PROCEDURES: [18F]FAC was synthesized to a radiochemical purity of >96 %. The human tumor lines AsPC1, BxPC3, Capan-1, Panc1, and MiaPaca2 were grown orthotopically in nude mice. KPC mice that conditionally express oncogenic K-ras and p53 mutations in pancreatic tissue were also used. The intra-tumoral distributions of [14C]gemcitabine and [18F]FAC were mapped with autoradiography. The inter-tumor correlation between [14C]gemcitabine and [18F]FAC was established in the orthotopic tumors. Expression of the equilibrative and concentrative nucleoside transporters (ENT, CNT) in vitro was detected by western blotting. Drug uptake was characterized in vitro using [3H]gemcitabine and the effect of transporter inhibition on gemcitabine and FAC uptake was investigated. The relative affinity of cells for gemcitabine and FAC was tested in competition assays. The cell lines differed in sensitivity to transport inhibitors and in competition studies. There was a good in vivo correlation between the total uptake of [18F]FAC and [14C]gemcitabine, measured across all orthotopic tumors. Using the KPC and BxPC3 models, we found that [14C]gemcitabine and [18F]FAC were largely co-localized. CONCLUSIONS: In the lines examined here, [18F]FAC uptake correlates well with gemcitabine in vivo, supporting the notion that [18F]FAC can serve as a PET radiotracer surrogate to determine the uptake and distribution of gemcitabine within pancreatic tumors.

Distinct Levels of Radioresistance in Lgr5+ Colonic Epithelial Stem Cells versus Lgr5+ Small Intestinal Stem Cells.

Although small and large intestines possess seemingly similar Wnt-driven leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)+ adult epithelial stem cells, we report here that the two organs exhibit distinct mechanisms of tissue response to ionizing radiation. Employing Lgr5-lacZ transgenic mice and Lgr5 in situ hybridization, we found colonic epithelial stem cells (CESC) markedly more radioresistant in vivo than small intestinal crypt base columnar stem cells (CBC; D0 = 6.0 ± 0.3 Gy vs. 1.3 ± 0.1, respectively; P < 0.01). Accordingly, CESCs survived 30 Gy exposure, while CBCs were completely depleted after 15 Gy. EdU incorporation studies indicated that after 19 Gy, CBCs exited growth arrest at 12 hours, resuming normal mitotic activity despite 60% of this population displaying residual γH2AX foci, indicative of persistent unrepaired DNA damage. Checkpoint recovery before complete double-strand break (DSB) repair represents the sine qua non of a newly defined potentially lethal pathophysiology termed checkpoint adaptation. In the small intestinal mucosa, checkpoint adaptation resulted in CBCs succumbing to an 8-fold increase in the incidence of highly lethal chromosomal aberrations and mitotic catastrophe by 48 hours postradiation. In contrast, Lgr5+ CESCs displayed delayed checkpoint recovery at 48 hours post-19 Gy, coordinated with complete DSB repair and regeneration of colonic mucosa originating, at least in part, from surviving CESCs. The discovery that small intestinal CBCs succumb to checkpoint adaptation is the first demonstration that this aberrant cell-cycle response may drive mammalian tissue radiosensitivity. Cancer Res; 77(8); 2124-33. ©2017 AACR.