Guangyu An

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.

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.

Survival of Patients with Gastrointestinal Cancers Can Be Predicted by a Surrogate microRNA Signature for Cancer Stem-like Cells Marked by DCLK1 Kinase.

Doublecortin-like kinase 1 (DCLK1) is a gastrointestinal (GI) tuft cell kinase that has been investigated as a biomarker of cancer stem-like cells in colon and pancreatic cancers. However, its utility as a biomarker may be limited in principle by signal instability and dilution in heterogeneous tumors, where the proliferation of diverse tumor cell lineages obscures the direct measurement of DCLK1 activity. To address this issue, we explored the definition of a miRNA signature as a surrogate biomarker for DCLK1 in cancer stem-like cells. Utilizing RNA/miRNA-sequencing datasets from the Cancer Genome Atlas, we identified a surrogate 15-miRNA expression signature for DCLK1 activity across several GI cancers, including colon, pancreatic, and stomach cancers. Notably, Cox regression and Kaplan-Meier analysis demonstrated that this signature could predict the survival of patients with these cancers. Moreover, we identified patient subgroups that predicted the clinical utility of this DCLK1 surrogate biomarker. Our findings greatly strengthen the clinical significance for DCLK1 expression across GI cancers. Further, they provide an initial guidepost toward the development of improved prognostic biomarkers or companion biomarkers for DCLK1-targeted therapies to eradicate cancer stem-like cells in these malignancies. Cancer Res; 76(14); 4090-9. ©2016 AACR.

Role of a Genetic Variant on the 15q25.1 Lung Cancer Susceptibility Locus in Smoking-Associated Nasopharyngeal Carcinoma

Background The 15q25.1 lung cancer susceptibility locus, containing CHRNA5, could modify lung cancer susceptibility and multiple smoking related phenotypes. However, no studies have investigated the association between CHRNA5 rs3841324, which has been proven to have the highest association with CHRNA5 mRNA expression, and the risk of other smoking-associated cancers, except lung cancer. In the current study we examined the association between rs3841324 and susceptibility to smoking-associated nasopharyngeal carcinoma (NPC). Methods In this case-control study we genotyped the CHRNA5 rs3841324 polymorphism with 400 NPC cases and 491 healthy controls who were Han Chinese and frequency-matched by age (±5 years), gender, and alcohol consumption. Univariate and multivariate logistic regression analyses were used to calculate the odds ratio (OR) and 95% confidence intervals (95% CI). Results We found that individuals with CHRNA5 rs3841324 combined variant genotypes (ins/del+del/del) had a >1.5-fold elevated risk for NPC than those with the ins/ins genotype (adjusted ORu200a=u200a1.52; 95% CI, 1.16–2.00), especially among ever smokers (adjusted ORu200a=u200a2.07; 95% CI, 1.23–3.48). The combined variant genotypes acted jointly with cigarette smoking to contribute to a 4.35-fold increased NPC risk (adjusted ORu200a=u200a4.35; 95% CI, 2.57–7.38). There was a dose-response relationship between deletion alleles and NPC susceptibility (trend test, Pu200a=u200a0.011). Conclusions Our results suggest that genetic variants on the 15q25.1 lung cancer susceptibility locus may influence susceptibility to NPC, particularly for smoking-associated NPC. Such work may be helpful to facilitate an understanding of the etiology of smoking-associated cancers and improve prevention efforts.

RAS/BRAF Circulating Tumor DNA Mutations as a Predictor of Response to First-Line Chemotherapy in Metastatic Colorectal Cancer Patients

Background Since circulating tumor DNA (ctDNA) offers clear advantages as a minimally invasive method for tumor monitoring compared with tumor tissue, we aimed to evaluate genotyping ctDNA using a next-generation sequencing- (NGS-) based panel to identify the prognostic value of mutation status in metastatic colorectal cancer (mCRC) patients with primary tumor resected and with subsequent lines of treatment in this study. Methods 76 mCRC patients treated in Beijing Chao-Yang Hospital from 2011 to 2017 were enrolled. Genotyping of RAS/BRAF in tumor tissue and ctDNA was determined by ARMS PCR and with a 40-gene panel using NGS, respectively. Patient clinicopathologic features and RAS/BRAF gene mutation status were evaluated by survival analysis for disease-free survival (DFS) and progression-free survival (PFS). Results Among 76 patients, KRAS distributions were not significantly correlated with any clinicopathologic features. The concordance between tumor tissue and ctDNA KRAS mutation was 81.25%. Mutations of RAS/BRAF had no significant impact on DFS after surgery (hazard ratio (HR), 1.205; 95% CI, 0.618 to 2.349; P = 0.5837) but prognosticated poorer PFS in subsequent first-line therapy (HR, 3.351; 95% CI, 1.172 to 9.576; P = 0.024). Conclusion ctDNA was comparable with tumor tissue for mutation detection. RAS/BRAF mutations detected in ctDNA predict a worse PFS in mCRC patients with first-line chemotherapy. Our results provide support for the prognostic value of RAS/BRAF ctDNA mutation detection in mCRC patients.

Abstract 577: Systemic delivery of CBT-15G DCLK1-targeted monoclonal antibody dramatically decreases tumorigenesis in a xenograft model of pancreatic cancer

Introduction Pancreatic cancer is the 4th leading cause of cancer mortality in the US. Treatments for pancreatic cancer are desperately needed as current drugs lead to little or no improvement in patient survival. Doublecortin-like kinase 1 (DCLK1) marks putative stem cells in colon and pancreatic cancers and regulates important oncogenic processes including epithelial-mesenchymal transition, which supports metastasis. DCLK19s extracellular domain is a novel target for therapeutic monoclonal antibodies (mABs) that has not previously been pursued. Materials and Methods Development of a DCLK1-targeted mAB: Balb/c mice were immunized with proprietary KLH-linked peptides targeting the DCLK1 extracellular domain. After immunization, mice were killed and spleens were taken and used to generate hybridoma cell lines, which were subcloned to the monoclonal level. Hybridoma media containing DCLK1-mAB were screened for their ability to bind immunogen peptide and DCLK1 purified protein, and the best clone was selected for further production (CBT-15G). CBT-15G was produced on a large scale in roller bottles, purified using a MEP column, and dialyzed into PBS. Xenograft study: 0.5×106 SW1990 human pancreatic cancer cells suspend in matrigel were injected into the flanks of 8-week old athymic nude mice and allowed to grow until reaching an average tumor volume of 100 mm3. Xenografts were treated with intraperitoneal CBT-15G mAB or isotype control at 25 mg/kg twice per week for 4 weeks. Tumor volume measurements were taken every other day using calipers. 30 days from the start of injections mice were killed and tumors excised, measured, weighed, and lysed for downstream analysis. Changes in volume and weight were analyzed statistically using Graphpad Prism 6.0. Results CBT-15G dramatically decreases tumorigenesis in SW1990 xenografts via a DCLK1-based mechanism of action: Biweekly injections of CBT-15G mAB significantly inhibited SW1990 pancreatic cancer xenograft growth over a period of 4 weeks (p Conclusion These findings for the first time demonstrate the systemic in vivo efficacy of DCLK1-targeted mABs against pancreatic cancer. As viable treatments for pancreatic cancer are currently severely lacking, CBT-15G DCLK1-targeted therapy should be investigated further as a potential treatment. Citation Format: Nathaniel Weygant, Dongfeng Qu, Randal May, Parthasarathy Chandrakesan, Yang Ge, Chelsea D. Ryan, Guangyu An, Michael J. Schlosser, Edwin Bannerman-Menson, Courtney W. Houchen. Systemic delivery of CBT-15G DCLK1-targeted monoclonal antibody dramatically decreases tumorigenesis in a xenograft model of pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 577.

Alternative splice variants of DCLK1 mark cancer stem cells, promote self-renewal and drug-resistance, and can be targeted to inhibit tumorigenesis in kidney cancer

Renal cell carcinoma (RCC) is a common and devastating disease characterized by a hypoxic microenvironment, epithelial‐mesenchymal transition and potent resistance to therapy evidencing the presence of cancer stem cells (CSCs). Various CSC markers have been studied in RCC, but overall there is limited data on their role and most markers studied have been relatively nonspecific. Doublecortin‐like kinase 1 (DCLK1) is a validated CSC marker in the gastrointestinal tract and evidence for an equivalent role in other cancers is accumulating. We used bioinformatics, immunohistochemistry, flow cytometry, spheroid self‐renewal and chemoresistance assays in combination with overexpression and siRNA‐knockdown to study the stem cell‐supportive role of DCLK1 alternative splice variants (DCLK1 ASVs) in RCC. To target tumor cells expressing DCLK1 ASVs directly, we developed a novel monoclonal antibody (CBT‐15) and delivered it systemically to RCC tumor xenografts. DCLK1 ASVs were overexpressed, enriched together with CSC markers and predictive of overall and recurrence‐free survival in RCC patients. In vitro, DCLK1 ASVs were able to directly stimulate essential molecular and functional characteristics of renal CSCs including expression of aldehyde dehydrogenase, self‐renewal and resistance to FDA‐approved receptor tyrosine kinase and mTOR inhibitors, while targeted downregulation of DCLK1 reversed these characteristics. Finally, targeting DCLK1 ASV‐positive cells with the novel CBT‐15 monoclonal antibody blocked RCC tumorigenesis in vivo. These findings establish DCLK1 as a CSC marker with implications for therapy, disease progression and survival in RCC and demonstrate the therapeutic value of DCLK1‐targeted monoclonal antibodies against renal CSCs.

Abstract 3888: DCLK1 is part of an EMT feedback loop and promotes colorectal cancer cell invasion and drug resistance

Colorectal cancer (CRC) is the third leading cause of cancer death in the U.S., with only a 6% 5-yr survival rate for stage IV disease. Its spread and acquisition of resistance to chemotherapy, which are fueled by the epithelial-mesenchymal transition (EMT) process and supported by tumor stem cells (TSCs), are major challenges to improving patient outcomes. New therapies that target stemness and EMT are desperately needed to prevent or delay metastasis and improve patient survival. Recently doublecortin-like kinase 1 (DCLK1) has been definitively proven to mark TSCs in CRC by two independent groups. Previous studies have demonstrated that DCLK1 is a prognostic factor in CRC and that targeted downregulation or inhibition of DCLK1 results in decreased CRC proliferation, migration, invasion, and other anti-oncogenic effects. However, the effect of overexpression of DCLK1 and its kinase active mutant on CRC has not been assessed. In this study, we investigate the correlative role of EMT and DCLK1 expression in CRC progress. Human colon cancer cells (HCT116) were infected with Lentivirus containing wild type DCLK1 or mutant DCLK1R326C cDNA sequences to overexpress DCLK1, DCLK1R326C or green fluorescent protein (GFP) cDNA sequence as control. The expressing levels of DCLK1 and EMT factors were analyzed by western blotting. The proliferative and invasive potential of these cells were compared using a MTT assay for proliferation, wound healing assay for migration, and Matrigel coated transwell assay for invasion. Knockdown of either ZEB1 or DCLK1 by specific siRNA in HCT116 cells was performed. The effects of siDCLK1 on 5-FU were performed in both HCT116 and SW480 cells using a Caspase 3/7 activity assay. Analysis of human CRC was performed using TCGA COADREAD dataset. Here we report that compared to GFP control cells, HCT116-DCLK1 and HCT116-DCLK1R326C cells exhibited a more than 20% increase in proliferation, approximately 30% increase in migration, and a 2-fold increase (p Citation Format: Dongfeng Qu, Nathaniel Weygant, William L. Berry, Randal May, Parthasarathy Chandrakesan, James J. Tomasek, Sripathi M. Sureban, Courtney Houchen, Yang Ge, Jiannan Yao, Guangyu An, Edwin Bannerman-Menson. DCLK1 is part of an EMT feedback loop and promotes colorectal cancer cell invasion and drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3888. doi:10.1158/1538-7445.AM2017-3888

Abstract 3340: Doublecortin-like kinase 1 marks cancer stem-like cells and modulates drug-resistance, self-renewal, and tumorigenesis in renal cancer

Introduction Doublecortin-like kinase 1 (DCLK1), is a tumor stem cell-specific marker, expressed in many cancers and highly correlated to cancer initiation, EMT and progression. We recently reported DCLK19s epigenetic dysregulation and specific overexpression in clear cell renal carcinoma (RCC). In this study, we assessed DCLK19s value as a therapeutic target in RCC progression. Materials and Methods RCC cell line Caki-2 was infected with Lentivirus to overexpress DCLK1. Moreover, DCLK1 gene expression was knocked down in ACHN and Caki-2 cell lines using small interfering RNA. Gene and protein expression levels were measured by real time RT-PCR and Western blotting respectively. Proliferation and drug resistance was determined by MTT assay. Cell cycle analysis and sorting of DCLK1+/DCLK1- cells were completed by FACS. Clonogenicity was evaluated using colony formation assay in matrigel. Results Overexpression of DCLK1 increases Caki-2 cell proliferation, but despite DCLK19s microtubule localization, there was no evidence of altered cell cycle status. Aldehyde dehydrogenase (ALDH), one of only a few known stem cell markers in RCC, was highly upregulated (>5-fold) by DCLK1 overexpression and downregulated (2-fold) by knockdown of DCLK1 in RCC cells. FACS results also indicated the existence of small ALDH and DCLK1 double-positive populations in both Caki-2 (1.51%) and ACHN (0.54%) RCC cells. Furthermore, there was approximately 7% more ALDH+ cells in DCLK1 overexpressing Caki-2 cells compared to control. In further support of molecular hallmarks, we observed increased expression of HIF-1α and Vimentin mRNA and protein as well as enhanced clonogenic capacity of ACHN and Caki-2 RCC cell lines in matrigel at baseline and after 72 h treatment with 0.5 μM Sunitinib. Immunocytochemistry of the spheroids suggested that DCLK1 enhances colony formation ability by triggering expression of pluripotency factors NANOG and POU5F1/OCT4. Since DCLK1 is known to have an extracellular domain we performed FACS sorting to determine if we could isolate a viable DCLK1+ population from the ACHN cell line. ACHN-DCLK1+ cells had >1.5 fold higher relative colony number and larger sphere size in three-dimensional clonogenic assay compared to ACHN-DCLK1- cells. Finally, we performed MTT assays demonstrating that overexpression of DCLK1 caused Caki-2 cells to become resistant to Sunitinib, Everolimus, and Temsirolimus compared to controls after 48 h treatment. Converesely, knockdown of DCLK1 sensitized both ACHN and Caki-2 cells to Sunitinib and Sorafenib after 48 hours treatment. Conclusion DCLK1 modulates proliferation, stemness, tumorigenesis and resistance to FDA-approved drugs, and marks a population of stem-like cells in RCC. These findings suggest that targeting DCLK1 may be a novel primary or adjuvant therapeutic strategy in RCC. Citation Format: Yang Ge, Nathaniel Weygant, Dongfeng Qu, Randal May, William L. Berry, Parthasarathy Chandrakesan, James J. Tomasek, Guangyu An, Courtney W. Houchen. Doublecortin-like kinase 1 marks cancer stem-like cells and modulates drug-resistance, self-renewal, and tumorigenesis in renal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3340.