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DCLK1-isoform2 staining is positively and negatively correlated with CD8 T cell staining. Fixed tumor tissues with CD8+ T-cell high ("T-cell high",) CD8+ T-cell mild ("T-cell mild") and CD8+ T-cell low ("T-cell low") from an autochthonous mouse model of pancreatic adenocarcinoma were stained for Dclk1. Bar graph representing the percentage positive staining intensity of DCLK1 between the tissue samples, quantified using ImageJ.

DCLK1-isoform2 expression increased in PDAC. A) TSVdb webtool were utilized to analyze TCGA-PAAD dataset to identify the expression of different isoforms of DCLK1 in human pancreatic adenocarcinoma. B) DCLK1-isoform2 mRNA is highly expressed in pancreatic adenocarcinoma compared to adjacent solid pancreatic normal tissue in the TCGA-PAAD dataset analyzed from TSVdb webtool. DCLK1-isoform1 showed a marginal increase in pancreatic adenocarcinoma, whereas DCLK1-isoform 3&4 were not altered.

Dclk1+ tumor cells and myeloid cells increased in the PDAC tissue samples compared to pre-neoplastic pancreatic tissues, while the CD8+ T-cell showed a mere change in their population. Fixed pancreatic tissues from 5 weeks and 20-week old KPCY mice were stained for Dclk1, CD11B, and CD8. Bar graph representing the percentage positive staining intensity between the tissue samples, quantified using ImageJ. All quantitative data are expressed as means {plus minus} SD and P values of

Tumor-associated M2-macrophages are one of the most abundant immunosuppressive cell types in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). However, the molecular mechanisms responsible for the generation of M2-macrophages are unclear. Here, we demonstrated that overexpression of DCLK1-isoform2 in AsPC1 and MIA PaCa2 cells resulted in the polarization of M1-macrophages toward an M2 phenotype via secreted chemokines/cytokines. These M2-macrophages enhanced parental PDAC cell migration, invasion, and self-renewal, and this was associated with increased expression of Snail and Slug. We observed distinct expression of Dclk-isoform2, marked infiltration of M2-macrophages, and a marginal increase of CD8+ T cells in 20-week-old KPCY mice pancreas compared with 5 weeks old. Utilizing an autochthonous mouse model of pancreatic adenocarcinoma, we observed distinct immunoreactive Dclk1 and arginase1 in tissues where CD8+ T-cell infiltration was low and observed a paucity of DCLK1 and arginase1 staining where CD8+ T-cell infiltration was high. Finally, we found that DCLK1-isoform2 tumor-educated M2-macrophages inhibit CD8+ T-cell proliferation and granzyme-B activation. Inhibition of DCLK1 in an organoid coculture system enhanced CD8+ T-cell activation and associated organoid death. We conclude that DCLK1-isoform2 is a novel initiator of alternate macrophage activation that contributes to the immunosuppression observed in the PDAC TME. These data suggest that tumor DCLK1-isoform2 may be an attractive target for PDAC therapy, either alone or in conjunction with immunotherapeutic strategies.

Correlation between the average number of Dclk1+ cells and M2 macrophages in the Dclk1 high and low PDAC. A. Fixed tumor tissues with CD8+ T-cell high (Dclk1 low) and CD8+ T-cell low (Dclk1 high) from an autochthonous mouse model of pancreatic adenocarcinoma were stained for Dclk1 and Arginase 1. Bar graph representing the average number of Dclk1+ cells and M2 macrophages in Dclk1 low and Dclk1 high expressing PDAC. Number of cells were quantified using ImageJ. All quantitative data are expressed as means {plus minus} SD and P values of

DCLK1-isoforms. Graphical illustration demonstrating the length of each isoforms and shared protein kinase domain between DCLK1 isoforms referenced in UniProt; www.uniprot.org/uniprot/O15075. DCX1=Doublecortin1; DCX2=Doublecortin2; P/S=Pro/Ser rich domain.

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.

A separate file containging supplemental figure legends as requested by the editorial office

Supplemental material containing a flowchart for how the 15-miRNA signature was generated, a table of pertinent patient clinical data, and a forest plot of hazard ratios of high-risk colon, pancreatic, and stomach cancer patients with high miRNA signatures.

Tumor-associated M2-macrophages are one of the most abundant immunosuppressive cell types in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). However, the molecular mechanisms responsible for the generation of M2-macrophages are unclear. Here, we demonstrated that overexpression of DCLK1-isoform2 in AsPC1 and MIA PaCa2 cells resulted in the polarization of M1-macrophages toward an M2 phenotype via secreted chemokines/cytokines. These M2-macrophages enhanced parental PDAC cell migration, invasion, and self-renewal, and this was associated with increased expression of Snail and Slug. We observed distinct expression of Dclk-isoform2, marked infiltration of M2-macrophages, and a marginal increase of CD8+ T cells in 20-week-old KPCY mice pancreas compared with 5 weeks old. Utilizing an autochthonous mouse model of pancreatic adenocarcinoma, we observed distinct immunoreactive Dclk1 and arginase1 in tissues where CD8+ T-cell infiltration was low and observed a paucity of DCLK1 and arginase1 staining where CD8+ T-cell infiltration was high. Finally, we found that DCLK1-isoform2 tumor-educated M2-macrophages inhibit CD8+ T-cell proliferation and granzyme-B activation. Inhibition of DCLK1 in an organoid coculture system enhanced CD8+ T-cell activation and associated organoid death. We conclude that DCLK1-isoform2 is a novel initiator of alternate macrophage activation that contributes to the immunosuppression observed in the PDAC TME. These data suggest that tumor DCLK1-isoform2 may be an attractive target for PDAC therapy, either alone or in conjunction with immunotherapeutic strategies.

Identifying early-stage cancer patients at risk for progression is a major goal of biomarker research. This report describes a novel 19-gene signature (19-GCS) that predicts stage I lung adenocarcinoma (LAC) recurrence and response to therapy and performs comparably in pancreatic adenocarcinoma (PAC), which shares LAC molecular traits. Kaplan-Meier, Cox regression, and cross-validation analyses were used to build the signature from training, test, and validation sets comprising 831 stage I LAC transcriptomes from multiple independent data sets. A statistical analysis was performed using the R language. Pathway and gene set enrichment were used to identify underlying mechanisms. 19-GCS strongly predicts overall survival and recurrence-free survival in stage I LAC (P=0.002 and P

Genome integrity is constantly monitored by sophisticated cellular networks, collectively termed as the DNA damage response (DDR). The DDR is a signaling network that includes cell cycle checkpoints and DNA repair and damage tolerance pathways. Failure of the DDR or associated events causes various diseases, including cancer. DDR is primarily mediated by phosphatidylinositol-3-kinase-like protein kinase (PIKKs) family members ataxia-telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related protein (ATR). However, one of the many unanswered questions regarding these signal-transduction pathways is: how does the cell turn the DDR signals on? There was no conclusive demonstration of the involvement of a specific sensory kinase in DDR signals until our recent research on the DCLK1 role in regulating ATM after genotoxic injury. Currently, various studies are demonstrating the importance of DCLK1 in DNA damage response. Here, we discuss the novel insights into the role of DCLK1 in DNA damage response.

Simple Summary Doublecortin-like kinase 1 (DCLK1) is a tumor stem cell marker in colon, pancreatic, and potentially other cancers that has received wide attention recently. Aside from its role as a tuft cell marker in normal tissue and as a tumor stem cell marker in cancer, previous studies have demonstrated that silencing DCLK1 functionally reduces stemness, epithelial mesenchymal transition (EMT), and tumorigenesis in cancers. More recently, DCLK1′s role in regulating the inflammatory, pre-cancer, and tumor microenvironment including its ability to modulate immune cell mechanisms has started to come into focus. Importantly, clinically viable therapeutic means of targeting DCLK1 have finally become available in the form of kinase inhibitors, monoclonal antibodies, and chimeric antigen receptor T cells (CAR-T). Herein, we comprehensively review the mechanistic role of DCLK1 in the tumor microenvironment, assess the potential for targeting DCLK1 in colon, pancreatic and renal cancer. Abstract Microtubule-associated doublecortin-like kinase 1 (DCLK1) is an accepted marker of tuft cells (TCs) and several kinds of cancer stem cells (CSCs), and emerging evidence suggests that DCLK1-positive TCs participate in the initiation and formation of inflammation-associated cancer. DCLK1-expressing CSCs regulate multiple biological processes in cancer, promote resistance to therapy, and are associated with metastasis. In solid tumor cancers, tumor epithelia, immune cells, cancer-associated fibroblasts, endothelial cells and blood vessels, extracellular matrix, and hypoxia all support a CSC phenotype characterized by drug resistance, recurrence, and metastasis. Recently, studies have shown that DCLK1-positive CSCs are associated with epithelial-mesenchymal transition, angiogenesis, and immune checkpoint. Emerging data concerning targeting DCLK1 with small molecular inhibitors, monoclonal antibodies, and chimeric antigen receptor T-cells shows promising effects on inhibiting tumor growth and regulating the tumor immune microenvironment. Overall, DCLK1 is reaching maturity as an anti-cancer target and therapies directed against it may have potential against CSCs directly, in remodeling the tumor microenvironment, and as immunotherapies.

Tuft cells (TCs) are an isolated chemosensory cell type found in the gastrointestinal and respiratory tract epithelium, which can be recognized by their tufted/teardrop appearance, large microvilli...

Chronic liver injury is a risk factor for cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms that regulate the decision between normal injury repair and neoplastic initiation are unclear. Doublecortin-like kinase 1 (DCLK1), a tumor stem cell marker, is induced during cirrhosis and HCC. Here, we demonstrate that DCLK1-overexpressing primary human hepatocytes formed spheroids in suspension cultures. Spheroids derived from DCLK1-overexpressing hepatoma cells showed high level expression of active β-catenin, α-fetoprotein, and SOX9, suggesting that DCLK1 overexpression induces clonogenicity and dedifferentiated phenotypes in hepatoma cells. DCLK1 overexpression in hepatoma cells also increased phosphorylation of GSK-3β at Ser9. This was associated with an induction of a 48-kDa active β-catenin with a preserved hypophosphorylated N-terminus that interacted with nuclear TCF-4 resulting in luciferase reporter activity and cyclin D1 expression. DCLK1 downregulation inhibited 48-kDa β-catenin expression. The proteasome inhibitor bortezomib did not block the 48-kDa β-catenin, instead, caused a threefold accumulation, suggesting a proteasome-independent mechanism. Liver tissues from patients with cirrhosis and HCC revealed epithelial co-staining of DCLK1 and active β-catenin, and cleaved E-cadherin. Repopulated DCLK1-overexpressing primary human hepatocytes in humanized FRG mouse livers demonstrated active β-catenin. In conclusion, DCLK1 regulates oncogenic signaling and clonogenicity of hepatocytes by a novel non-canonical/atypical β-catenin-dependent mechanism.

Immunotherapy that has proven efficacy in several solid cancers plays a partial role in improving clinical outcomes of advanced gastrointestinal (GI) cancers. There is an unmet need to find new immune-related therapeutic targets. Doublecortin-like kinase 1 (DCLK1) marks tuft cells which are recognized as cancer-initiating cells and regulators of the type II immune response, and has been studied for its role in many cancers including colon and gastric cancers, but its role in tumor immunity remains unexplored. In the current study, we analyzed colon and gastric cancer RNA sequencing data from 283 and 415 patients, respectively, from The Cancer Genome Atlas (TCGA). High DCLK1 expression predicted the worse clinical outcomes in colon and gastric cancer patients and correlated with increased immune and stromal components. Further analysis indicated that DCLK1 was strongly linked to infiltration of multiple immune cell types, especially TAMs and Treg, and strongly correlated with increased CD8+ T cell inhibitors TGFB1 and CXCL12 and their receptors, suggesting it may contribute to TAM-mediated inhibition of CD8+ T cells. Interestingly, we found that DCLK1 was a prognostic biomarker in left-sided colon cancer, which has worse outcomes and demonstrates a reduced response to existing immunotherapies. In conclusion, our results demonstrate that DCLK1 is linked with functional regulation of the tumor microenvironment and may have potential as a prognostic biomarker and adjuvant target to promote immunotherapy sensitivity in colon and gastric cancer patients.

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 &lt, 0.0001), and increased Interferon gamma (IFN-&gamma, ) release in CRC cells (2D) compared to mock CAR-T (p &lt, 0.0001). Moreover, an even greater increase in IFN-&gamma, 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.

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.

Oncogenic KRAS mutation plays a key role in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis with nearly 95% of PDAC harboring mutation-activated KRAS, which has been considered an undruggable target. Doublecortin-like kinase 1 (DCLK1) is often overexpressed in pancreatic cancer, and recent studies indicate that DCLK1+ PDAC cells can initiate pancreatic tumorigenesis. In this study, we investigate whether overexpressing DCLK1 activates RAS and promotes tumorigenesis, metastasis, and drug resistance. Human pancreatic cancer cells (AsPC-1 and MiaPaCa-2) were infected with lentivirus and selected to create stable DCLK1 isoform 2 (alpha-long, AL) overexpressing lines. The invasive potential of these cells relative to vector control was compared using Matrigel coated transwell assay. KRAS activation and interaction were determined by a pull-down assay and coimmunoprecipitation. Gemcitabine, mTOR (Everolimus), PI3K (LY-294002), and BCL-2 (ABT-199) inhibitors were used to evaluate drug resistance downstream of KRAS activation. Immunostaining of a PDAC tissue microarray was performed to detect DCLK1 alpha- and beta-long expression. Analysis of gene expression in human PDAC was performed using the TCGA PAAD dataset. The effects of targeting DCLK1 were studied using xenograft and Pdx1CreKrasG12DTrp53R172H/+ (KPC) mouse models. Overexpression of DCLK1-AL drives a more than 2-fold increase in invasion and drug resistance and increased the activation of KRAS. Evidence from TCGA PAAD demonstrated that human PDACs expressing high levels of DCLK1 correlate with activated PI3K/AKT/MTOR-pathway signaling suggesting greater KRAS activity. High DCLK1 expression in normal adjacent tissue of PDAC correlated with poor survival and anti-DCLK1 mAb inhibited pancreatic tumor growth in vivo in mouse models.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. Chronic hepatitis C virus (HCV) infection causes induction of several tumors/cancer stem cell (CSC) markers and is known to be a major risk factor for development of HCC. Therefore, drugs that simultaneously target viral replication and CSC properties are needed for a risk-free treatment of advanced stage liver diseases, including HCC. Here, we demonstrated that (Z)-3,5,4′-trimethoxystilbene (Z-TMS) exhibits potent antitumor and anti-HCV activities without exhibiting cytotoxicity to human hepatocytes in vitro or in mice livers. Diethylnitrosamine (DEN)/carbon tetrachloride (CCl4) extensively induced expression of DCLK1 (a CSC marker) in the livers of C57BL/6 mice following hepatic injury. Z-TMS exhibited hepatoprotective effects against DEN/CCl4-induced injury by reducing DCLK1 expression and improving histologic outcomes. The drug caused bundling of DCLK1 with microtubules and blocked cell-cycle progression at G2–M phase in hepatoma cells via downregulation of CDK1, induction of p21cip1/waf1 expression, and inhibition of Akt (Ser473) phosphorylation. Z-TMS also inhibited proliferation of erlotinib-resistant lung adenocarcinoma cells (H1975) bearing the T790M EGFR mutation, most likely by promoting autophagy and nuclear fragmentation. In conclusion, Z-TMS appears to be a unique therapeutic agent targeting HCV and concurrently eliminating cells with neoplastic potential during chronic liver diseases, including HCC. It may also be a valuable drug for targeting drug-resistant carcinomas and cancers of the lungs, pancreas, colon, and intestine, in which DCLK1 is involved in tumorigenesis. Cancer Res; 76(16); 4887–96. ©2016 AACR.

Background: Chronic intestinal inflammation is a major risk factor for the development of CRC and consequently individuals suffering from inflammatory bowel diseases (IBD) are at increased risk. However, the molecular mechanism by which inflammation initiates colonic tumorigenesis in the cell type is unclear and the regulatory factors that mediate the complex interplay between inflammation and the colonic tumor initiation are unknown. Doublecortin-like kinase 1 (Dclk1) is a microtubule-associated kinase, which marks TSCs in colon cancer, and is predominantly expressed in a unique intestinal epithelial cell type termed the tuft cells. Tuft cells are chemosensory cells that regulate host immunity and inflammation and is highly increased in CRC and, ablation of Dclk1 expressing cells in the ApcMin/+ mice completely regressed the tumors without affecting the normal stem cell population. Our aim is to elucidate the molecular mechanism that is responsible for inflammation-associated colon cancer and to determine the role of Dclk1 expressing tuft cells in this process. Experimental Procedure: YMCA non-malignant colonic epithelial cells were used. Loss and gain of Dclk1 expression in the YMCA cells were carried with siRNA transfection and lentivirus overexpression vector infection respectively. Dclk1 expressing cells were selected for stable cell generation. DSS and LPS were used. Cell self-renewal, secondary and tertiary organoid generation, survival, DNA damage, DNA damage response (DDR) and apoptosis were assessed. Protein and mRNA expressions by western-blot and RT-PCR. Results: Here, we report that increased expression of Dclk1 is the key to the generation of tumor-initiating cells under the influence of an inflammatory environment. We observed increased self-renewal of Dclk1+ YMCA cells treated with DSS and further increase in self-renewal was observed with DSS+LPS treatment. Silencing Dclk1 failed to initiate self-renewal under the influence of DSS and LPS treatment. Dclk1+ YMCA cells displayed enhanced inflammatory mediators, survival with colony formation and increased survival pathways (AKT/mTOR/Beta-catenin/NFkB pathways) and DDR (ATM/ATR) with DSS or LPS treatment. However, the DNA damage and cell apoptosis were not increased in vector control cells and significantly increased in Dclk1 knockdown cells after DSS or LPS treatment. Conclusion: Our data demonstrate that the increased expression of Dclk1 is associated with tumor-initiating features, and survival ability in an inflammatory environment, which is important to understand the cell type and inflammation associated mechanisms in tumor initiation. Based on these findings we proposed that specific targeting of Dclk1-expressing colonic crypt epithelial cells has the potential to result in ablation of tumor initiation. Citation Format: Parthasarathy Chandrakesan, Janani Panneerselvam, Dongfeng Qu, Chinthalapally Rao, Michael Bronze, Courtney Houchen. Dclk1 expressing crypt epithelial cells become tumor initiating stem cells in an inflammation associated environment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3829.

Immunotherapy that has proved efficacy in several solid cancers plays a partial role in improving clinical outcomes of advanced gastrointestinal (GI) cancers. There is an unmet need to find new immune-related therapeutic targets. Doublecortin-like kinase 1 (DCLK1) marks tuft cells which are recognized as cancer initiating cells and regulators of the type II immune response, and has been studied for its role in many cancers including colon and gastric cancers, but its role in tumor immunity remains unexplored. In the current study, we analyzed colon and gastric cancer RNA sequencing data from 283 and 415 patients respectively from The Cancer Genome Atlas (TCGA). High DCLK1 expression predicted the worse clinical outcomes in colon and gastric cancer patients and correlated with increased immune and stromal components. Further analysis indicated that DCLK1was strongly linked to infiltration of multiple immune cell types, especially TAMs and Tregs, and strongly correlated with increased CD8+ T cell inhibitors TGFB1 and CXCL12 and their receptors, suggesting it may contribute to TAM-mediated inhibition of CD8+ T cells. Interestingly, we found that DCLK1 was a prognostic biomarker in left-sided colon cancer which has worse outcomes and demonstrates a reduced response to existing immunotherapies. In conclusion, our results demonstrate that DCLK1 is linked with functional regulation of the tumor microenvironment and may have potential as a prognostic biomarker and adjuvant target to promote immunotherapy sensitivity in colon and gastric cancer patients. Citation Format: Dongfeng Qu, Xiangyan Wu, Nathaniel Weygant, Jun Peng, Courtney Houchen. Cancer stem cell marker DCLK1 correlates with tumorigenic immune infiltrates in the colon and gastric adenocarcinoma microenvironments [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1702.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by defective intestinal barrier integrity toward the microbiota and epithelial damage. Double cortin-like kinase 1 (Dclk1), a marker of intestinal tuft cells, can regulate tissue regenerative responses, but its role in epithelial repair during bacterial-dependent chronic colitis is unclear. We addressed this question using our recently developed mouse model of spontaneous microbiota-dependent colitis induced by mucin-type O-glycan deficiency (DKO), which recapitulates most features of human UC. We generated DKO mice lacking intestinal epithelial Dclk1 (DKO;Dclk1(ΔIEC)) and analyzed colitis onset and severity using clinical and histologic indices, immune responses by qPCR and immunostaining, and epithelial responses using proliferation markers and organoid culture. We found 3–4-week-old DKO;Dclk1(ΔIEC) mice developed worsened spontaneous colitis characterized by reduced body weight, loose stool, severe colon thickening, epithelial lesions, and inflammatory cell infiltrates compared with DKO mice. The primary defect was an impaired epithelial proliferative response during inflammation. Dclk1 deficiency also reduced inflammation-induced proliferation and growth of colon organoids ex vivo. Mechanistically, Dclk1 expression was important for inflammation-induced Cox2 expression and prostaglandin E2 (PGE2) production in vivo, and PGE2 rescued proliferative defects in Dclk1-deficient colonic organoids. Although tuft cells were expanded in both DKO and DKO;Dclk1(ΔIEC) relative to WT mice, loss of Dclk1 was associated with reduced tuft cell activation (i.e., proliferation) during inflammation. Similar results were found in DKO vs. DKO;Dclk1(ΔIEC) mice at 3–6 months of age. Our results support that tuft cells, via Dclk1, are important responders to bacterial-induced colitis by enhancing epithelial repair responses, which in turn limits bacterial infiltration into the mucosa.

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.

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. 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. 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. 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.

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.

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.

Background: Lung cancer is the deadliest cancer in both men and women. About 85% of lung cancers are non-small cell lung cancer (NSCLC) and almost 25% of all types of NSCLC contain KRAS mutations, which remain as an undruggable challenge. Doublecortin-like kinase 1 (DCLK1) regulate many key oncogenes including KRAS in various solid cancers is overexpressed in lung cancer, raising the possibility to selectively target DCLK1 expression to combat the undruggable KRAS mutant NSCLC and its drug resistance. Experimental Procedure: H460 and A549 (KRAS mutant NSCLC cell lines) and H1299 (WT KRAS NSCLC cell line) were used. Cisplatin resistance KRAS mutant NSCLC cells (CR-A549) were generated. Transfection of siRNA against DCLK1 in NSCLC cells and CR-A549 cells were carried out. Tumor cell apoptosis, DNA damage, DNA damage response (DDR) and tumor cell self-renewal were assessed. Protein and mRNA expressions by western-blot and RT-PCR, apoptosis by FACS, cell death by COMET assay and self-renewal by clonogenic assay. Results: Here, we report that increased expression of DCLK1 is the key to develop chemoresistance and self-renewal in KRAS mutant NSCLC. We observed increased immunostaining for DCLK1 in patient lung adenocarcinoma compared to normal lung tissue. We observed an increased expression of DCLK1 protein and mRNA in KRAS mutant NSCLC cells compared to WT KRAS NSCLC cells. To investigate the role of DCLK1 in KRAS mutant NSCLC tumor progression, we knockdown DCLK1 in KRAS mutant NSCLC cells, which resulted in reduced cell proliferation/survival and self-renewal. We generated KRAS mutant NSCLC cisplatin resistance cells (CR-A549) and found that CR-A549 cells acquired higher DCLK1 expression with enhanced self-renewal capacity and display ~9fold higher IC50 for cisplatin treatment compared to parental cells. However, silencing DCLK1 in CR-A549 cells reversed the tumor cell resistance to cisplatin and increased cell death and reduced self-renewal. To further investigate mechanistically, we examine the expression of DDR signaling in CR-A549 cells. We observed an increased expression of ATR-DDR in CR-A549 cells. Furthermore, DCLK1 knockdown reduced the expression of ATR and treatment of siATR plus cisplatin (5µM) to CR-A549 cells demonstrated similar effects observed in the treatment of siDCLK1 plus cisplatin (5uM), which resulted in increased sensitivity of CR-A549 cells to cisplatin, and thus increased cell death and complete abrogation of self-renewal capacity, suggesting that DCLK1 mediated cisplatin resistance occurs via ATR-DDR dependent mechanism. Conclusion: Our data demonstrate that the increased expression of DCLK1 is associated with enhanced cancer stem cell-like features, ATR-DDR signaling and chemoresistance in KRAS mutant NSCLC. Targeting DCLK1 alone or in combination with cisplatin may represent a novel therapeutic strategy for the effective treatment of undruggable KRAS mutant NSCLC. Citation Format: Courtney Houchen, Janani Panneerselvam, Priyanga Mohandoss, Nathaniel Weygant, Randal May, Dongfeng Qu, Naushad Ali, Timothy Wang, Chinthalapally Rao, Michael Bronze, Parthasarathy Chandrakesan. DCLK1 regulates ATR-DNA damage response for KRAS mutant lung cancer drug resistance and stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2119.

Melanoma is the deadliest form of all skin cancer, and the incidence of melanoma has been rising for the last 30 years. There are only a few reported biomarkers for prediction and prognosis for melanoma, therefore, there is an unmet medical need to identify predictive and prognostic biomarkers. Doublecortin-like kinase 1 (DCLK1) marks a population of tumor-initiating cells in colonic and pancreatic cancers, and it is overexpressed in many solid tumors. Our preliminary studies suggest that DCLK1 is overexpressed in serum samples of melanoma patients and it maybe a novel biomarker for melanoma. In this study we examined DCLK1 expression in melanoma tumor tissues and further evaluated whether its levels in serum could be quantitatively assessed in melanoma patients before and after therapy treatment. Immunohistochemistry (IHC) analysis was performed to detect DCLK1 in a tissue microarray with 60 tumor and normal adjacent tissues (NAT) from melanoma patients. ELISA assay were performed to detect DCLK1 levels in the serum samples of melanoma patients before and after treatment. Western blotting was also performed to detect DCLK1 protein in the serum samples of melanoma patients. Additionally, analysis of DCLK1 and correlative gene expressions was performed using TCGA Melanoma dataset. Here we report that the intensity of DCLK1 staining in melanoma tumor tissues are significantly increased compared to NAT. DCLK1 levels in the serum were elevated in melanoma patients (n=65) compared to healthy volunteers. Patients with higher levels of serum DCLK1 had reduced overall survival compared to patients with lower levels of serum DCLK1. We also compared DCLK1 levels in 10 patients for whom pre- and post- treatment samples were available. Among these patients, seven had elevated DCLK1 before treatment and had a significantly decrease (about 30-50%) in serum DCLK after treatment. Three patients had no change in DCLK1 levels after treatment. Interestingly, the patients with decrease level of DCLK1 post-treatment demonstrated significant clinical responses to therapy, however, the patients with no change in DCLK1 level post therapy showed no clinical improvement (p Citation Format: Dongfeng Qu, Nathaniel Weygant, Parthasarathy Chandrakesan, Kamille Pitts, Randal May, Adam Asch, Alexandra Ikeguchi, Courtney Houchen. DCLK1 is upregulated in melanoma and it is a novel predictive marker for survival and response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4034.

Multiple Myeloma (MM) is characterized by the clonal expansion of malignant plasma cells in the bone marrow, and is the second most common hematological cancer. Cellular therapies including chimeric antigen T receptor cell (CAR-T) have emerged as a promising therapeutic strategy to combat this devastating disease. MM are derived from a rare population of cancer stem cells (CSC) that are responsible for initiation, progression, resistance to conventional therapy and recurrence. To date there have been no CAR-T therapies directed at CSC-specific antigens. In this report, we describe the effects of a novel CAR-T targeting the CSC marker DCLK1 on MM cells. Using mouse models, it has been demonstrated that DCLK1 marks the cell of origin of colon and pancreatic cancers. DCLK1 regulates epithelial to mesenchymal transition (EMT), and its upregulation is associated with poor overall survival in many cancer subtypes. Inhibition of DCLK1 activity following RNA silencing resulted in tumor xenograft growth arrest. These features lead us to explore the therapeutic potential of targeting DCLK1+ cells in MM. In order to create a novel DCLK1 CAR, we generated a new humanized DCLK1 monoclonal antibody, clone CBT-15 #8. This antibody recognizes a small extra cellular C-terminal epitope on human isoform 2 and 4 of DCLK1. Novel DCLK1 CAR-T cells containing the clone were generated and detected with anti-human FAB antibody by flow cytometry. Furthermore, we observed cell surface expression of DCLK1 on >93 percent of MM Cells (RPMI8226) by flow cytometry. The CAR-T cells had significantly higher cytotoxicity in MM (RPMI8226 and MM1S) cells than in leukemia K562 cells. The CAR-T cells secreted significantly higher levels of IFN-γ in vitro than T cells (p=0.0036). Further studies are underway to determine its efficacy in inhibiting tumor xenografts in vivo. This report is the first to link the TSC marker DCLK1 to MM and may represent a novel therapeutic strategy for MM and other hematologic malignancies. Citation Format: Sripathi M. Sureban, Robert Berahovich, Hua Zhou, Shirley Xu, Lijun Wu, Vita Golubovskaya, Dongfeng Qu, Randal May, Edwin Bannerman-Menson, Courtney W. Houchen. DCLK1 monoclonal antibody-based CAR-T cells as novel treatment strategy against multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1154.

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.

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.

The identification of functionally relevant subpopulations of therapy-resistant cancer cells is a challenge. These cells, intrinsically resistant to conventional therapy, can cause recurrence. Evidence has suggested that therapy-resistant cancer cells are likely epithelial-mesenchymal transition (EMT) cells and/or stem-like cells called cancer stem cells (CSCs). EMT, a normal embryological process that converts epithelial cells into mesenchymal cells, is frequently activated during cancer development and progression. CSCs are a small subpopulation of cancer cells within a tumor mass that have the ability to self-renew and maintain tumor-initiating capacity by giving rise to heterogeneous lineages of cancer cells that comprise the whole tumor. Although the origin of CSCs and EMT cells remains to be fully explored, a growing body of evidence has indicated that the biology of EMT and CSCs is strongly linked. Doublecortin-like kinase 1 (DCLK1), a cancer stem cell marker, is functionally involved in maintaining cancer stemness and the process of EMT important for cancer initiation, cancer metastasis, and secondary tumor formation. Therefore, targeting these cells may provide new strategies to overcome tumor heterogeneity, therapeutic resistance, and cancer relapse. In this review, we will provide a potential mechanistic link between EMT induction and the emergence of CSCs for the origin and progression of cancer. We will highlight the functional activity of DCLK1 in supporting EMT and cancer cell self-renewal, which will lead us to a better understanding of DCLK1 expression in cancer development and progression, and help us to develop targeted therapies for effective cancer treatment.

Background and Aim: In Barrett's esophagus (BE), the normal esophageal squamous epithelium is replaced with a specialized metaplastic columnar epithelium. BE is a premalignant lesion that can progress to esophageal adenocarcinoma (EAC). Currently, there are no early molecular indicators that would predict progression from BE to EAC. As the only permanent residents of the epithelium, stem cells have been implicated in this metaplastic progression. The aim of the present study was to determine the expression of doublecortin and CaM kinase-like-1 (DCAMKL-1) and other putative gastrointestinal stem cell markers in normal esophageal mucosa (NEM), BE, and EAC. Methods: Human NEM, BE, EAC, and multitissue microarrays were analyzed for DCAMKL-1, and immunohistochemically scored based on staining intensity and tissue involvement, with epithelia and stroma scored separately. Total RNA isolated from BE and paired NEM was subjected to real-time reverse-transcription–polymerase chain reaction analysis for DCAMKL-1, leucine-rich repeat-containing G-protein-coupled receptor (LGR5), and Musashi-1 (Msi-1) mRNA expression. Results: DCAMKL-1 was minimally expressed in squamous NEM, but increased in BE (with and without dysplasia) and EAC tissues. In EAC, we found increased stromal DCAMKL-1 staining compared to adjacent epithelia. Within the submucosa of dysplastic BE tissues, an increase in the endothelial cell expression of DCAMKL-1 was observed. Finally, an upregulation of DCAMKL-1, LGR5, and Msi-1 mRNA was seen in BE compared to squamous NEM. Conclusions: In the present study, we report the progressive increase of DCAMKL-1 expression in BE from dysplasia to EAC. Furthermore, there was an increase in putative stem cell markers DCAMKL-1, LGR5, and Msi-1 mRNA. Taken together, these data suggest that the regulation of resident stem cells might play an important role in the progression of BE to EAC.

Background: Accurately predicting recurrence in early stage cancer patients may be used to avoid unnecessary therapies with significant side effects or deliver early interventions that can improve outcomes. Both early stage lung (LAc) and pancreatic adenocarcinoma (PDAc) are high-recurrence cancers for which biomarkers that can quantify this risk could benefit patients. In stage I LAc only a small percentage of stage IB patients receive chemotherapy, while more patients may derive additional benefit if identified. In PDAc, patients with the most aggressive stage I-II disease could derive benefit from early aggressive combination chemotherapy if identified. Methods: Training and test sets (n=10) were selected by random sampling RNA-seq expression data into groups of 65% and 35% respectively. Cox proportional hazards and Kaplan-Meier analysis were used to predict RFS and OS. Maximally selected rank statistics were used to designate low and high-risk signature groups. Subgroup analysis was performed using multivariate Cox methods. Pathway analysis was performed with Webgestalt. RNA-seq expression data for LAc and PDAc was obtained from the cancer genome atlas and gene microarray data from NCBI GEO. All statistical analyses and plots were generated in R v3.2. Results/Discussion: An unbiased analysis of RNA-seq data identified a 19-gene consensus signature (19-GCS) that is upregulated in tumors and able to predict LAc stage I RFS. Multivariate analysis demonstrated its independence as a prognostic factor and efficiency in independent validation sets. Pathway analysis identified proliferation and immune surveillance as altered pathways in 19-GCS high-risk (sHR) compared to low-risk (sLR) tumors. In confirmation of this finding Cyclin D was downregulated while Cyclin E/A/B were upregulated in sHR tumors. Moreover, the 19-GCS was correlated with proliferation markers Ki-67 and PCNA and showed universal correlation with G2/M phase-specific genes. The expression of MHC Class I and II subunit markers were lost in sHR tumors as were specific MHC-related genes. Moreover, the expression of tumor-infiltrating immune cell-specific markers were largely lost in sHR tumors. The 19-GCS was better able to predict RFS and OS in PDAc than in LAc, and consistent with PDAc's severity, more patients were identified as high-risk. Gene expression analysis identified the same molecular characteristics in sHR PDAc as in sHR LAc. Analysis of clinical outcomes for both LAc and PDAc demonstrated that sHR predicted progressive disease in response to both primary and followup therapy. Overall, the 19-GCS identifies highly proliferative early stage lung and pancreatic tumors with impaired MHC mechanisms that shield them from immune-mediated destruction. This deadly combination leads to recurrence and poor clinical outcomes that might be avoided if identified early. Citation Format: Nathaniel Weygant, Jiannan Yao, Dongfeng Qu, Parthasarathy Chandrakesan, Guangyu An, Courtney W. Houchen. A multigene recurrence signature identifies highly proliferative tumors that escape immune surveillance in early stage lung and pancreas adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2594.

Melanoma is the deadliest form of skin cancer, and the incidence of melanoma has been rising for the last 40 years. There is a paucity of readily available tools to predict for melanoma progression and response to therapy, leaving an unmet medical need to identify predictive and prognostic biomarkers. Doublecortin-like kinase 1 (DCLK1) is overexpressed and marks a population of tumor-initiating cells in colonic and pancreatic cancers. It regulates key oncogenes, pluripotency factors, angiogenic factors, and epithelial mesenchymal transition (EMT) related transcription factors. In this study, we evaluated whether DCLK1 can be detected in the bloodstream and if its levels in serum samples could be quantitatively assessed in melanoma patients. ELISA assay was performed to detect DCLK1 levels in the serum samples of melanoma patients before and after treatment with either targeted agents or immune checkpoint inhibitors. Western blotting was also performed to detect DCLK1 protein in the serum samples of melanoma patients. Immunohistochemistry (IHC) analysis was performed to detect DCLK1 in the same patients' tumor tissues. Additionally, analysis of DCLK1 and correlative gene expression profiles was performed using TCGA Melanoma dataset. Here we report that the intensity of DCLK1 staining in melanoma tumor tissues is increased compared to normal tissues. DCLK1 levels in the serum were elevated in melanoma patients (n=20) compared to healthy volunteers. Next, we compared DCLK1 levels in 10 patients for whom pre- and post- treatment samples were available. Among these patients, seven had elevated DCLK1 before treatment and had a significant decrease (about 30-50%) in serum DCLK after treatment. Three patients had no change in DCLK1 levels after treatment. The patients with a decrease in their level of DCLK1 post-treatment demonstrated radiologic response to therapy as well, while the patients with no change in DCLK1 level post therapy showed either no improvement or progression (p Citation Format: Dongfeng Qu, Nathaniel Weygant, Parthasarathy Chandrakesan, Kamille Pitts, Randal May, Sripathi Sureban, Adam Asch, Alexandra Ikeguchi, Courtney Houchen. Serum DCLK1 levels are elevated in melanoma patients and it is a novel predictive marker for survival and response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3611.

Endothelial protein C receptor (EPCR) plays an important role in the protein C anticoagulation pathway. Previously, we have reported that EPCR can be shed from the cell surface, and that this is mediated by an unidentified metalloproteinase. In this study, we demonstrate that tumor necrosis factor-alpha converting enzyme/ADAM17 (TACE) is responsible for EPCR shedding. Phorbol-12-myristate 13-acetate (PMA)-stimulated EPCR shedding is reduced by approximately 50% in HEK293 cells transfected with human EPCR cDNA and by 60% in human umbilical vein endothelial cells after transfection of TACE small interfering RNA (siRNA) into these cells. PMA-stimulated EPCR shedding is completely blocked in fibroblasts from TACE-deficient mice transfected with human EPCR cDNA, and restored by transfection of TACE cDNA into this cell line. To characterize the EPCR sequence requirement for shedding, we generated several mutants of EPCR. Replacing amino acids from residue 193 to residue 200 with the FLAG sequence (DYKDDDDK) completely blocks EPCR shedding, whereas a single amino acid substitution in this region has less effect on EPCR shedding.

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.