DCLK1 Monoclonal Antibody-Based CAR-T Cells as a Novel Treatment Strategy against Human Colorectal Cancers.

CAR-T (chimeric antigen receptor T cells) immunotherapy is effective in many hematological cancers; however, efficacy in solid tumors is disappointing. Doublecortin-like kinase 1 (DCLK1) labels tumor stem cells (TSCs) in genetic mouse models of colorectal cancer (CRC). Here, we describe a novel CAR-T targeting DCLK1 (CBT-511; with our proprietary DCLK1 single-chain antibody variable fragment) as a treatment strategy to eradicate CRC TSCs. The cell surface expression of DCLK1 and cytotoxicity of CBT-511 were assessed in CRC cells (HT29, HCT116, and LoVo). LoVo-derived tumor xenografts in NOD Scid gamma (NSGTM)mice were treated with CBT-511 or mock CAR-T cells. Adherent CRC cells express surface DCLK1 (two-dimensional, 2D). A 4.5-fold increase in surface DCLK1 was observed when HT29 cells were grown as spheroids (three-dimensional, 3D). CBT-511 induced cytotoxicity (2D; p < 0.0001), and increased Interferon gamma (IFN-γ) release in CRC cells (2D) compared to mock CAR-T (p < 0.0001). Moreover, an even greater increase in IFN-γ release was observed when cells were grown in 3D. CBT-511 reduced tumor growth by approximately 50 percent compared to mock CAR-T. These data suggest that CRC cells with increased clonogenic capacity express increased surface DCLK1. A DCLK1-targeted CAR-T can induce cytotoxicity in vitro and inhibit xenograft growth in vivo.

Dclk1, a tumor stem cell marker, regulates pro-survival signaling and self-renewal of intestinal tumor cells.

BACKGROUND: More than 80% of intestinal neoplasia is associated with the adenomatous polyposis coli (APC) mutation. Doublecortin-like kinase 1 (Dclk1), a kinase protein, is overexpressed in colorectal cancer and specifically marks tumor stem cells (TSCs) that self-renew and increased the tumor progeny in Apc Min/+ mice. However, the role of Dclk1 expression and its contribution to regulating pro-survival signaling for tumor progression in Apc mutant cancer is poorly understood. METHODS: We analyzed DCLK1 and pro-survival signaling gene expression datasets of 329 specimens from TCGA Colon Adenocarcinoma Cancer Data. The network of DCLK1 and pro-survival signaling was analyzed utilizing the GeneMANIA database. We examined the expression levels of Dclk1 and other stem cell-associated markers, pro-survival signaling pathways, cell self-renewal in the isolated intestinal epithelial cells of Apc Min/+ mice with high-grade dysplasia and adenocarcinoma. To determine the functional role of Dclk1 for tumor progression, we knocked down Dclk1 and determined the pro-survival signaling pathways and stemness. We used siRNA technology to gene silence pro-survival signaling in colon cancer cells in vitro. We utilized FACS, IHC, western blot, RT-PCR, and clonogenic (self-renewal) assays. RESULTS: We found a correlation between DCLK1 and pro-survival signaling expression. The expression of Dclk1 and stem cell-associated markers Lgr5, Bmi1, and Musashi1 were significantly higher in the intestinal epithelial cells of Apc Min/+ mice than in wild-type controls. Intestinal epithelial cells of Apc Min/+ mice showed increased expression of pro-survival signaling, pluripotency and self-renewal ability. Furthermore, the enteroids formed from the intestinal Dclk1+ cells of Apc Min/+ mice display higher pluripotency and pro-survival signaling. Dclk1 knockdown in Apc Min/+ mice attenuates intestinal adenomas and adenocarcinoma, and decreases pro-survival signaling and self-renewal. Knocking down RELA and NOTCH1 pro-survival signaling and DCLK1 in HT29 and DLD1 colon cancer cells in vitro reduced the tumor cells' ability to self-renew and survive. CONCLUSION: Our results indicate that Dclk1 is essential in advancing intestinal tumorigenesis. Knocking down Dclk1 decreases tumor stemness and progression and is thus predicted to regulate pro-survival signaling and tumor cell pluripotency. This study provides a strong rationale to target Dclk1 as a treatment strategy for colorectal cancer.

Intestinal tuft cells regulate the ATM mediated DNA Damage response via Dclk1 dependent mechanism for crypt restitution following radiation injury.

Crypt epithelial survival and regeneration after injury require highly coordinated complex interplay between resident stem cells and diverse cell types. The function of Dclk1 expressing tuft cells regulating intestinal epithelial DNA damage response for cell survival/self-renewal after radiation-induced injury is unclear. Intestinal epithelial cells (IECs) were isolated and purified and utilized for experimental analysis. We found that small intestinal crypts of VillinCre;Dclk1f/f mice were hypoplastic and more apoptotic 24 h post-total body irradiation, a time when stem cell survival is p53-independent. Injury-induced ATM mediated DNA damage response, pro-survival genes, stem cell markers, and self-renewal ability for survival and restitution were reduced in the isolated intestinal epithelial cells. An even greater reduction in these signaling pathways was observed 3.5 days post-TBI, when peak crypt regeneration occurs. We found that interaction with Dclk1 is critical for ATM and COX2 activation in response to injury. We determined that Dclk1 expressing tuft cells regulate the whole intestinal epithelial cells following injury through paracrine mechanism. These findings suggest that intestinal tuft cells play an important role in regulating the ATM mediated DNA damage response, for epithelial cell survival/self-renewal via a Dclk1 dependent mechanism, and these processes are indispensable for restitution and function after severe radiation-induced injury.

Plasma DCLK1 is a marker of hepatocellular carcinoma (HCC): Targeting DCLK1 prevents HCC tumor xenograft growth via a microRNA-dependent mechanism.

Tumor stem cell marker Doublecortin-like kinase1 (DCLK1) is upregulated in several solid tumors. The role of DCLK1 in hepatocellular carcinoma (HCC) is unclear. We immunostained tissues from human livers with HCC, cirrhosis controls (CC), and non-cirrhosis controls (NCC) for DCLK1. Western blot and ELISA analyses for DCLK1 were performed with stored plasma samples. We observed increased immunoreactive DCLK1 in epithelia and stroma in HCC and CCs compared with NCCs, and observed a marked increase in plasma DCLK1 from patients with HCC compared with CC and NCC. Analysis of the Cancer Genome Atlas' HCC dataset revealed that DCLK1 is overexpressed in HCC tumors relative to adjacent normal tissues. High DCLK1-expressing cells had more epithelial-mesenchymal transition (EMT). Various tumor suppressor miRNAs were also downregulated in HCC tumors. We evaluated the effects of DCLK1 knockdown on Huh7.5-derived tumor xenograft growth. This was associated with growth arrest and a marked downregulation of cMYC, and EMT transcription factors ZEB1, ZEB2, SNAIL, and SLUG via let-7a and miR-200 miRNA-dependent mechanisms. Furthermore, upregulation of miR-143/145, a corresponding decrease in pluripotency factors OCT4, NANOG, KLF4, and LIN28, and a reduction of let-7a, miR-143/145, and miR-200-specific luciferase activity was observed. These findings suggest that the detection of elevated plasma DCLK1 may provide a cost-effective, less invasive tool for confirmation of clinical signs of cirrhosis, and a potential companion diagnostic marker for patients with cirrhosis and HCC. Our results support evaluating DCLK1 as a biomarker for detection and as a therapeutic target for eradicating HCC.

Regulation of miRNAs by agents targeting the tumor stem cell markers DCLK1, MSI1, LGR5, and BMI1.

Gastrointestinal cancers such as colorectal, pancreatic, liver, gastric, and esophageal, are the most common forms of malignant cancers. MicroRNAs (miRNA) play important role in regulating gastrointestinal cancer progress either as potent oncogenes or tumor suppressors. In this report, we will discuss the importance of several tumor suppressors involved in colon or pancreatic cancer. Some recent studies on tumor stem cells and regulation of these miRNAs via agents targeting the tumor stem cell markers doublecortin-like kinase 1 (DCLK1), Musashi-1 (MSI1), polycomb protein BMI1, and WNT genes (LGR5 and ASCL2) will also be discussed. Agents such as siRNA/shRNA, small molecule kinase inhibitors, and general herbal drugs (curcumin) targeting these tumor stem cell markers and tumor suppressor miRNAs could be the perfect therapeutic agents for the treatment of these cancers.

Dclk1+ small intestinal epithelial tuft cells display the hallmarks of quiescence and self-renewal.

To date, no discrete genetic signature has been defined for isolated Dclk1+ tuft cells within the small intestine. Furthermore, recent reports on the functional significance of Dclk1+ cells in the small intestine have been inconsistent. These cells have been proposed to be fully differentiated cells, reserve stem cells, and tumor stem cells. In order to elucidate the potential function of Dclk1+ cells, we FACS-sorted Dclk1+ cells from mouse small intestinal epithelium using transgenic mice expressing YFP under the control of the Dclk1 promoter (Dclk1-CreER;Rosa26-YFP). Analysis of sorted YFP+ cells demonstrated marked enrichment (~6000 fold) for Dclk1 mRNA compared with YFP- cells. Dclk1+ population display ~6 fold enrichment for the putative quiescent stem cell marker Bmi1. We observed significantly greater expression of pluripotency genes, pro-survival genes, and quiescence markers in the Dclk1+ population. A significant increase in self-renewal capability (14-fold) was observed in in vitro isolated Dclk1+ cells. The unique genetic report presented in this manuscript suggests that Dclk1+ cells may maintain quiescence, pluripotency, and metabolic activity for survival/longevity. Functionally, these reserve characteristics manifest in vitro, with Dclk1+ cells exhibiting greater ability to self-renew. These findings indicate that quiescent stem-like functionality is a feature of Dclk1-expressing tuft cells.

Dietary Pectin Increases Intestinal Crypt Stem Cell Survival following Radiation Injury.

Gastrointestinal (GI) mucosal damage is a devastating adverse effect of radiation therapy. We have recently reported that expression of Dclk1, a Tuft cell and tumor stem cell (TSC) marker, 24h after high dose total-body gamma-IR (TBI) can be used as a surrogate marker for crypt survival. Dietary pectin has been demonstrated to possess chemopreventive properties, whereas its radioprotective property has not been studied. The aim of this study was to determine the effects of dietary pectin on ionizing radiation (IR)-induced intestinal stem cell (ISC) deletion, crypt and overall survival following lethal TBI. C57BL/6 mice received a 6% pectin diet and 0.5% pectin drinking water (pre-IR mice received pectin one week before TBI until death; post-IR mice received pectin after TBI until death). Animals were exposed to TBI (14 Gy) and euthanized at 24 and 84h post-IR to assess ISC deletion and crypt survival respectively. Animals were also subjected to overall survival studies following TBI. In pre-IR treatment group, we observed a three-fold increase in ISC/crypt survival, a two-fold increase in Dclk1+ stem cells, increased overall survival (median 10d vs. 7d), and increased expression of Dclk1, Msi1, Lgr5, Bmi1, and Notch1 (in small intestine) post-TBI in pectin treated mice compared to controls. We also observed increased survival of mice treated with pectin (post-IR) compared to controls. Dietary pectin is a radioprotective agent; prevents IR-induced deletion of potential reserve ISCs; facilitates crypt regeneration; and ultimately promotes overall survival. Given the anti-cancer activity of pectin, our data support a potential role for dietary pectin as an agent that can be administered to patients receiving radiation therapy to protect against radiation-induces mucositis.

Ablation of Doublecortin-Like Kinase 1 in the Colonic Epithelium Exacerbates Dextran Sulfate Sodium-Induced Colitis.

Doublecortin-like kinase 1 (Dclk1), a microtubule-associated kinase, marks the fifth lineage of intestinal epithelial cells called tuft cells that function as tumor stem cells in Apc mutant models of colon cancer. In order to determine the role of Dclk1 in dextran sulfate sodium (DSS) induced colonic inflammation both intestinal epithelial specific Dclk1 deficient (VillinCre;Dclk1f/f) and control (Dclk1f/f) mice were fed 3% DSS in drinking water for 9 days, allowed to recover for 2 days, and killed. The clinical and histological features of DSS-induced colitis were scored and immunohistochemical, gene expression, pro-inflammatory cytokines/chemokines, and immunoblotting analyses were used to examine epithelial barrier integrity, inflammation, and stem and tuft cell features. In DSS-induced colitis, VillinCre;Dclk1f/f mice demonstrated exacerbated injury including higher clinical colitis scores, increased epithelial barrier permeability, higher levels of pro-inflammatory cytokines and chemokines, decreased levels of Lgr5, and dysregulated Wnt/b-Catenin pathway genes. These results suggest that Dclk1 plays an important role in regulating colonic inflammatory response and colonic epithelial integrity.

Inflammatory and oncogenic roles of a tumor stem cell marker doublecortin-like kinase (DCLK1) in virus-induced chronic liver diseases.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality worldwide. We previously showed that a tumor/cancer stem cell (CSC) marker, doublecortin-like kinase (DCLK1) positively regulates hepatitis C virus (HCV) replication, and promotes tumor growth in colon and pancreas. Here, we employed transcriptome analysis, RNA interference, tumor xenografts, patient's liver tissues and hepatospheroids to investigate DCLK1-regulated inflammation and tumorigenesis in the liver. Our studies unveiled novel DCLK1-controlled feed-forward signaling cascades involving calprotectin subunit S100A9 and NFκB activation as a driver of inflammation. Validation of transcriptome data suggests that DCLK1 co-expression with HCV induces BRM/SMARCA2 of SW1/SNF1 chromatin remodeling complexes. Frequently observed lymphoid aggregates including hepatic epithelial and stromal cells of internodular septa extensively express DCLK1 and S100A9. The DCLK1 overexpression also correlates with increased levels of S100A9, c-Myc, and BRM levels in HCV/HBV-positive patients with cirrhosis and HCC. DCLK1 silencing inhibits S100A9 expression and hepatoma cell migration. Normal human hepatocytes (NHH)-derived spheroids exhibit CSC properties. These results provide new insights into the molecular mechanism of the hepatitis B/C-virus induced liver inflammation and tumorigenesis via DCLK1-controlled networks. Thus, DCLK1 appears to be a novel therapeutic target for the treatment of inflammatory diseases and HCC.

Doublecortin-like kinase 1 is elevated serologically in pancreatic ductal adenocarcinoma and widely expressed on circulating tumor cells.

Doublecortin-like kinase 1 (DCLK1) is a putative pancreatic stem cell marker and is upregulated in pancreatic cancer, colorectal cancer, and many other solid tumors. It marks tumor stem cells in mouse models of intestinal neoplasia. Here we sought to determine whether DCLK1 protein can be detected in the bloodstream and if its levels in archived serum samples could be quantitatively assessed in pancreatic cancer patients. DCLK1 specific ELISA, western blotting, and immunohistochemical analyses were used to determine expression levels in the serum and staining intensity in archived tumor tissues of pancreatic ductal adenocarcinoma (PDAC) patients and in pancreatic cancer mouse models. DCLK1 levels in the serum were elevated in early stages of PDAC (stages I and II) compared to healthy volunteers (normal controls). No differences were observed between stages III/IV and normal controls. In resected surgical tissues, DCLK1 expression intensity in the stromal cells was significantly higher than that observed in tumor epithelial cells. Circulating tumor cells were isolated from KPCY mice and approximately 52% of these cells were positive for Dclk1 staining. Dclk1 levels in the serum of KPC mice were also elevated. We have previously demonstrated that DCLK1 plays a potential role in regulating epithelial mesenchymal transition (EMT). Given the increasingly recognized role of EMT derived stem cells in cancer progression and metastasis, we hypothesize that DCLK1 may contribute to the metastatic process. Taken together, our results suggest that DCLK1 serum levels and DCLK1 positive circulating tumor cells should be further assessed for their potential diagnostic and prognostic significance.

DCLK1 is a broadly dysregulated target against epithelial-mesenchymal transition, focal adhesion, and stemness in clear cell renal carcinoma.

Renal clear cell carcinoma (RCC) is the most common type of kidney cancer and the 8th most common cancer overall in the US. RCC survival rates drop precipitously with regional and distant spread and recent studies have demonstrated that RCC presents an epithelial-mesenchymal transition (EMT) phenotype linked to increased recurrence and decreased survival. EMT is a key characteristic of tumor stem cells (TSCs) along with chemo-resistance and radio-resistance, which are also phenotypic of RCC. Targeting these factors is key to increasing the survival of RCC patients. Doublecortin-like kinase 1 (DCLK1) marks TSCs in pancreatic and colorectal cancer and regulates EMT and stemness. Analysis of the Cancer Genome Atlas' RCC dataset revealed that DCLK1 is overexpressed and dysregulated on the mRNA and epigenetic level in more than 93% of RCC tumors relative to adjacent normal tissue. Immunohistochemistry using α-DCLK1 antibody confirmed overexpression and demonstrated a major increase in immunoreactivity in stage II-III tumors compared to normal kidney and stage I tumors. Small-interfering RNA (siRNA) mediated knockdown of DCLK1 resulted in decreased expression of EMT and pluripotency factors and significantly reduced invasion, migration, focal adhesion, drug-resistance, and clonogenic capacity. These findings suggest that DCLK1 is a novel, overexpressed factor in RCC progression that may be targeted to suppress EMT, metastasis, and stemness in early-stage and advanced RCC to increase patient survival. Moreover, the possibility that DCLK1 may mark a population of tumor stem-like cells in RCC should be further investigated in light of these findings.

DCLK1 is detectable in plasma of patients with Barrett's esophagus and esophageal adenocarcinoma.

BACKGROUND: Doublecortin-like kinase 1 (DCLK1), a putative tumor stem cell marker has been shown to be highly expressed in the stromal and epithelial compartments in colon and pancreatic cancer as well as Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). AIM: To prospectively investigate whether the immunohistochemical expression of DCLK1 was associated with detectable DCLK1 plasma expression in patients with existing BE and EAC. METHODS: Immunohistochemistry was performed on paraffin-embedded sections using DCLK1 antibody and scored based on staining intensity and tissue involvement. Purified human plasma samples were subjected to Western blot and ELISA analysis. RESULTS: Forty (40) patients were enrolled: 10 controls (normal endoscopy) and 30 with BE/EAC (13 nondysplastic BE [NDBE], 9 dysplastic BE [DBE] and 8 EAC). Mean epithelial DCLK1 staining was as follows: controls = 0.11, NDBE = 3.83, DBE = 6.0, EAC = 7.17. Mean stromal DCLK1 staining was as follows: NDBE = 5.83, DBE = 5.375, EAC = 10.83. DCLK1 was detected by plasma Western blot in 1 control and in all patients with BE/EAC p < 0.0005. Plasma DCLK1 was elevated by ELISA in EAC compared to other groups, p < 0.05. CONCLUSIONS: Increased expression of DCLK1 was observed in the epithelium, stroma and plasma of patients with BE/EAC. Furthermore, the presence of detectable DCLK1 in plasma of BE/EAC patients may provide a less invasive, detection tool in those patients as well as represent a novel molecular marker distinguishing between normal esophageal mucosa and BE or EAC.

DCLK1 facilitates intestinal tumor growth via enhancing pluripotency and epithelial mesenchymal transition.

Doublecortin-like kinase 1 (Dclk1) is overexpressed in many cancers including colorectal cancer (CRC) andit specifically marks intestinal tumor stem cells. However, the role of Dclk1 in intestinal tumorigenesis in Apc mutant conditions is still poorly understood. We demonstrate that Dclk1 expression and Dclk1+ cells are significantly increased in the intestinal epithelium of elderly ApcMin/+ mice compared to young ApcMin/+ mice and wild type mice. Intestinal epithelial cells of ApcMin/+ mice demonstrate increased pluripotency, self-renewing ability, and EMT. Furthermore, miRNAs are dysregulated, expression of onco-miRNAs are significantly increased with decreased tumor suppressor miRNAs. In support of these findings, knockdown of Dclk1 in elderly ApcMin/+ mice attenuates intestinal adenomas and adenocarcinoma by decreasing pluripotency, EMT and onco-miRNAs indicating that Dclk1 overexpression facilitates intestinal tumorigenesis. Knocking down Dclk1 weakens Dclk1-dependent intestinal processes for tumorigenesis. This study demonstrates that Dclk1 is critically involved in facilitating intestinal tumorigenesis by enhancing pluripotency and EMT factors in Apc mutant intestinal tumors and it also provides a potential therapeutic target for the treatment of colorectal cancer.

Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1.

BACKGROUND: Doublecortin-like kinase 1 (DCLK1) is emerging as a tumor specific stem cell marker in colorectal and pancreatic cancer. Previous in vitro and in vivo studies have demonstrated the therapeutic effects of inhibiting DCLK1 with small interfering RNA (siRNA) as well as genetically targeting the DCLK1+ cell for deletion. However, the effects of inhibiting DCLK1 kinase activity have not been studied directly. Therefore, we assessed the effects of inhibiting DCLK1 kinase activity using the novel small molecule kinase inhibitor, LRRK2-IN-1, which demonstrates significant affinity for DCLK1. RESULTS: Here we report that LRRK2-IN-1 demonstrates potent anti-cancer activity including inhibition of cancer cell proliferation, migration, and invasion as well as induction of apoptosis and cell cycle arrest. Additionally we found that it regulates stemness, epithelial-mesenchymal transition, and oncogenic targets on the molecular level. Moreover, we show that LRRK2-IN-1 suppresses DCLK1 kinase activity and downstream DCLK1 effector c-MYC, and demonstrate that DCLK1 kinase activity is a significant factor in resistance to LRRK2-IN-1. CONCLUSIONS: Given DCLK1's tumor stem cell marker status, a strong understanding of its biological role and interactions in gastrointestinal tumors may lead to discoveries that improve patient outcomes. The results of this study suggest that small molecule inhibitors of DCLK1 kinase should be further investigated as they may hold promise as anti-tumor stem cell drugs.

XMD8-92 inhibits pancreatic tumor xenograft growth via a DCLK1-dependent mechanism.

XMD8-92 is a kinase inhibitor with anti-cancer activity against lung and cervical cancers, but its effect on pancreatic ductal adenocarcinoma (PDAC) remains unknown. Doublecortin-like kinase1 (DCLK1) is upregulated in various cancers including PDAC. In this study, we showed that XMD8-92 inhibits AsPC-1 cancer cell proliferation and tumor xenograft growth. XMD8-92 treated tumors demonstrated significant downregulation of DCLK1 and several of its downstream targets (including c-MYC, KRAS, NOTCH1, ZEB1, ZEB2, SNAIL, SLUG, OCT4, SOX2, NANOG, KLF4, LIN28, VEGFR1, and VEGFR2) via upregulation of tumor suppressor miRNAs let-7a, miR-144, miR-200a-c, and miR-143/145; it did not however affect BMK1 downstream genes p21 and p53. These data taken together suggest that XMD8-92 treatment results in inhibition of DCLK1 and downstream oncogenic pathways (EMT, pluripotency, angiogenesis and anti-apoptotic), and is a promising chemotherapeutic agent against PDAC.

Brief report: Dclk1 deletion in tuft cells results in impaired epithelial repair after radiation injury.

The role of Dclk1(+) tuft cells in the replacement of intestinal epithelia and reestablishing the epithelial barrier after severe genotoxic insult is completely unknown. Successful restoration requires precise coordination between the cells within each crypt subunit. While the mechanisms that control this response remain largely uncertain, the radiation model remains an exceptional surrogate for stem cell-associated crypt loss. Following the creation of Dclk1-intestinal-epithelial-deficient Villin-Cre;Dclk1(flox/flox) mice, widespread gene expression changes were detected in isolated intestinal epithelia during homeostasis. While the number of surviving crypts was unaffected, Villin-Cre;Dclk1(flox/flox) mice failed to maintain tight junctions and died at approximately 5 days, where Dclk1(flox/flox) mice lived until day 10 following radiation injury. These findings suggest that Dclk1 plays a functional role critical in the epithelial restorative response.

Inhibition of Notch signaling reduces the number of surviving Dclk1+ reserve crypt epithelial stem cells following radiation injury.

We have previously reported that doublecortin-like kinase 1 (Dclk1) is a putative intestinal stem cell (ISC) marker. In this report, we evaluated the use of Dclk1 as a marker of surviving ISCs in response to treatment with high-dose total body irradiation (TBI). Both apoptotic and mitotic Dclk1(+) cells were observed 24 h post-TBI associated with a corresponding loss of intestinal crypts observed at 84 h post-TBI. Although the Notch signaling pathway plays an important role in regulating proliferation and lineage commitment within the intestine, its role in ISC function in response to severe genotoxic injury is not yet fully understood. We employed the microcolony assay to functionally assess the effects of Notch inhibition with difluorophenacetyl-l-alanyl-S-phenylglycine t-butyl ester (DAPT) on intestinal crypt stem cell survival following severe (>8 Gy) radiation injury. Following treatment with DAPT, we observed a nearly 50% reduction in the number of surviving Dclk1(+) crypt epithelial cells at 24 h after TBI and similar reduction in the number of surviving small intestinal crypts at 84 h. These data indicate that inhibition of Notch signaling decreases ISC survival following radiation injury, suggesting that the Notch signaling pathway plays an important role in ISC-mediated crypt regeneration. These results also suggest that crypt epithelial cell Dclk1 expression can be used as one potential marker to evaluate the early survival of ISCs following severe radiation injury.