Doublecortin‐like kinase 1 is a therapeutic target in squamous cell carcinoma

Doublecortin like kinase 1 (DCLK1) plays a crucial role in several cancers including colon and pancreatic adenocarcinomas. However, its role in squamous cell carcinoma (SCC) remains unknown. To this end, we examined DCLK1 expression in head and neck SCC (HNSCC) and anal SCC (ASCC). We found that DCLK1 is elevated in patient SCC tissue, which correlated with cancer progression and poorer overall survival. Furthermore, DCLK1 expression is significantly elevated in human papilloma virus negative HNSCC, which are typically aggressive with poor responses to therapy. To understand the role of DCLK1 in tumorigenesis, we used specific shRNA to suppress DCLK1 expression. This significantly reduced tumor growth, spheroid formation, and migration of HNSCC cancer cells. To further the translational relevance of our studies, we sought to identify a selective DCLK1 inhibitor. Current attempts to target DCLK1 using pharmacologic approaches have relied on nonspecific suppression of DCLK1 kinase activity. Here, we demonstrate that DiFiD (3,5‐bis [2,4‐difluorobenzylidene]−4‐piperidone) binds to DCLK1 with high selectivity. Moreover, DiFiD mediated suppression of DCLK1 led to G2/M arrest and apoptosis and significantly suppressed tumor growth of HNSCC xenografts and ASCC patient derived xenografts, supporting that DCLK1 is critical for SCC growth.


Funding information
Thomas P. O'Sullivan IV and Marina O'Sullivan Family Fund; NIH Office of the Director significantly reduced tumor growth, spheroid formation, and migration of HNSCC cancer cells. To further the translational relevance of our studies, we sought to identify a selective DCLK1 inhibitor. Current attempts to target DCLK1 using pharmacologic approaches have relied on nonspecific suppression of DCLK1 kinase activity. Here, we demonstrate that DiFiD (3,5-bis [2,4-difluorobenzylidene]−4-piperidone) binds to DCLK1 with high selectivity. Moreover, DiFiD mediated suppression of DCLK1 led to G2/M arrest and apoptosis and significantly suppressed tumor growth of HNSCC xenografts and ASCC patient derived xenografts, supporting that DCLK1 is critical for SCC growth. Etiologic factors associated with HNSCC include tobacco and alcohol abuse, and human papilloma virus (HPV) infection. Despite therapeutic advances, HNSCC remains difficult to treat; due, in part, to latestage tumor presentation, as well as high rates of local recurrence and distant metastases that contribute to poor 5-year survival. 2,3 Anal SCC (ASCC) is a rare cancer, which like HNSCC is associated with HPV infection, and its incidence is increasing. Accordingly, the identification of novel targets and therapeutics is needed to improve SCC treatment outcomes. There remains a paucity of preclinical descriptions of ASCC biology or potential drug targets.
Recently, doublecortin like kinase 1 (DCLK1) was found to be highly expressed in salivary gland tumors and is associated with low overall and disease-free survival. 4 DCLK1, encodes a member of the doublecortin family and protein kinase superfamily. 5,6 DCLK1 contains two N-terminal doublecortin domains and a C-terminal kinase domain homologous to Ca 2+ /calmodulin-dependent kinases (CamK). Doublecortin domains participate in microtubule polymerization, whereas the serine/threonine protein kinase domain and a serine/proline-rich domain, mediate multiple protein−protein interactions. The activity of each domain is independent of one another. 7 DCLK1 is expressed in various cell types including neurons, osteoblasts, and tuft cells in the colon. Initial reports by our group 5,6,8,9 and that of multiple laboratories, [10][11][12] have reported DCLK1 expression is observed in a small percentage of cells (<5%) in colon and pancreatic adenocarcinomas. Further, it has been established as a marker of tumor-initiating cells. 13 The role of DCLK1 in SCCs remains unknown.
Presently, we determine that DCLK1 expression is upregulated in SCC. Further, DCLK1 regulates HNSCC proliferation, invasion, and migration. We also report for the first time that DiFiD (3,5-bis [2,4-difluorobenzylidene]−4-piperidone), which has shown efficacy in preclinical models of pancreatic cancer, binds to and inhibits DCLK1 activity and demonstrated antitumor effects in SCC. 14 These studies demonstrate the potential therapeutic value of targeting DCLK1 in SCC. were used in this study. 15

| Cellular thermal shift assay (CETSA)
The ability of DiFiD to interact with and stabilize DCLK1 in cells was determined using CETSA. 22 Briefly, cells (8 × 10 6 ) were treated with media containing DMSO or DiFiD (5 μM) for 4 h. After treatment, the cells were aliquoted into PCR tubes and exposed to a temperature gradient. Subsequently, cells were lysed using three repeated freezethaw cycles in liquid nitrogen followed by centrifugation. The resultant lysates were then utilized in downstream western blot analyses.

| Drug affinity responsive target stability (DARTS)
The ability of DiFiD to interact with and stabilize DCLK1 in cells was studied by using DARTS. 23   was administered for 16 weeks to C3H mice (n = 20) using a previously reported protocol. 24 Mice were then given sterile drinking water for 3 weeks, at which time animals were killed and tongues excised.

| Animal studies
Immunohistochemistry was then used to assess DCLK1 expression.
Five-week-old female Foxn1/nude mice (Charles River Laboratory) were injected with 1 × 10 6 HN5 or FaDu cells in the flank. One week following implantation, mice were randomized into two groups STANDING ET AL. | 147 with 7 mice per group in the HN5 injected mice and 10 mice per group in the FaDu treated mice. Animals were treated with either vehicle control (2.5% DMSO in water) or DiFiD (2 mg/kg body weight), administered intraperitoneally daily for 15 days. Tumor growth was measured every 2−3 days by a blinded observer measuring tumor diameters using vernier calipers and volume was calculated (Tumor volume = longest dimension × shortest dimension 2 x 0.52) as previously described. 25 At the end of treatment, animals were euthanized, and the tumors were collected, weighed, and processed for downstream analytical assays.
Additionally, we established a patient-derived xenograft from an anal SCC that metastasized to the liver.  Immunofluorescence revealed DCLK1 to be expressed in HN5 spheroids (Supporting Information: Figure S1C). Therefore, HN5 and FaDu were used for subsequent studies. These data suggest that DCLK1 is elevated in HNSCC compared to normal oral mucosa and is associated with tumor progression. Further, ASCC patient samples (n = 17) were stained for DCLK1 expression. DCLK1 was more significantly expressed in ASCC tumor compared to normal anal mucosa (Supporting Information: Figure S1D and E). We observed increased nuclear staining along the tumor invasive front (Supporting Information: Figure S1D).

| DCLK1 suppression inhibits HNSCC growth
To evaluate the antitumor efficacy of targeting DCLK1 in HNSCC, we attenuated DCLK1 levels using two shRNA constructs, sh3 and sh4. DCLK1 mRNA and protein were significantly reduced (Figure 2A suppression may lead to cell cycle arrest and apoptosis.

| DiFiD binds to DCLK1 and inhibits its activity
The small molecule, 3,5-bis (2,4-difluorobenzylidene)-4-piperidone (DiFiD) was previously shown to suppress the growth of pancreatic cancer cells in vitro and in vivo. 14 However, the precise target for DiFiD remained unknown. DCLK1 is implicated in pancreatic and colorectal cancer progression. Therefore, we first determined compound−protein interaction. Molecular docking predicted DiFiD interacts with DCLK1 by forming hydrogen bonds with aspartic acid 533, with a binding energy of −7.9 kcal/mol, as shown in ribbon and surface views ( Figure 3A,B,C,D).
Additionally, the binding kinetics of DiFiD and DCLK1 were studied via SPR. We observed dissociation constants of K D1 = 71 nM and K D2 = 902 nM ( Figure 3E). Taken together this data shows that DiFiD binds DCLK1 with high affinity. To confirm that DCLK1 is a binding target of DiFiD in the cells, we performed CETSA to assess protein stability following thermal denaturation. FaDu cells were treated with 5 µM DiFiD at 37°C for 4 h. Cells were aliquoted into equal volumes, subjected to a thermal gradient and DCLK1 expression was then evaluated by western blot. Thermal denaturation of DCLK1 occurred at 54°C in the DMSO treated control group, which increased to 58°C in the presence of DiFiD ( Figure 3F). To further validate DiFiD binding with DCLK1, we performed the DARTS assay. Briefly, FaDu cell lysates were incubated with DMSO or DiFiD (5 µM) for 30 min. We then incubated cells with increasing protein:pronase ratio. We observed that DiFiD protected DCLK1 from protease-mediated degradation, as DCLK1 expression was extended to 1:1/800 protein: pronase ratio compared to 1:1/1600 in the DMSO control arm ( Figure 3G). Altogether, these data suggest that DiFiD binds to DCLK1.
To confirm DiFiD specifically interacts with the kinase domain of DCLK1, we performed CETSA analyses. For this, we stably expressed the N-terminal DCX domain (lacking the kinase domain) in HNSCC cells (Supporting Information: Figure S3A and S3B). To detect DCX domain fragments in FaDu cells, we utilized DCLK1 antibody produced using residues near the N-terminus. This specifically recognizes DCLK1 protein isoforms containing 82 kDa DCX sequences. We found that cells expressing the predicted 51 kDa N-terminal DCX domain did not exhibit thermal stabilization when treated with DiFiD (Supporting Information: Figure S3C). This suggests that DiFiD does not stabilize DCLK1 lacking the kinase domain. Therefore, we conclude that DiFiD preferentially binds to the kinase domain of DCLK1, further confirming molecular docking predictions.
DCLK1 belongs to the family of kinases with homology to calmodulin kinases, but it does not depend on Ca2+/calmodulin for its kinase activity. 27 Since DiFiD interacts with the kinase domain, we next assessed the effect of DiFiD on DCLK1 kinase activity by performing an in vitro kinase assay. Here, we incubated recombinant DCLK1 with GS peptide as substrate to assess the ability of DCLK1 to phosphorylate the peptide. 18,19 Along with DiFiD, we also tested the ability of EF24 (IKK inhibitor) and KN62, a pan-CaMKII inhibitor.
We observed greater inhibition of DCLK1-mediated ATP consumption following incubation with DiFiD, than with either EF24 or KN-62 ( Figure 3H). To determine the specificity of DiFiD to DCLK1, we also performed studies with CaMKIIα, CaMKIIβ, and CaMKIV. We observed that DiFiD did not affect the activities of these CaMK proteins. These data demonstrate a higher selectivity and specificity of DiFiD for DCLK1.
Since DiFiD has favorable binding to DCLK1, inhibits its kinase activity, and is relatively ineffective at reducing Het1A proliferation (Supporting Information: Figure 4B), we hypothesized that cells expressing lower levels of DCLK1 would be resistant to DiFiD. Consequently, we performed hexosaminidase assays with cells where DCLK1 was knocked down using specific shRNA.
There was an increase in their respective half maximal inhibitory concentration (IC 50 ) values from 1.3 μM to greater than 11 μM compared to control cells ( Figure 3I). These data demonstrate DiFiD activity is dependent upon the presence of DCLK1, and DiFiD has poor activity against cells with low DCLK1 expression.
These data further suggest that DCLK1 is a highly specific direct target of DiFiD.

| DiFiD demonstrates potent cytotoxicity in HNSCC in vitro
Since DiFiD inhibits DCLK1 activity, we sought to determine the effect of DiFiD on HNSCC cancer cell viability. We observed that DiFiD inhibits HNSCC viability in a dose and time-dependent manner ( Figure 4A and Supporting Information: Figure S3A). We identified an effective dose to assess the mechanism of action. The IC 50 was measured by hexosaminidase assay and observed within 48 h at concentrations of 750 nM and 1.5 µM in HN5 and FaDu cell lines, respectively. In addition, we tested toxicity of DiFiD on an immortalized noncancerous cell line, Het1A 28 ( Figure 1G). We observed that the IC 50 for Het1A at 48 h was 9 µM, a 6−12-fold increase compared to FaDu and HN5 cells, respectively (Supporting Information: Figure S4B). In addition, we observed that DiFiD attenuates spheroid growth in both HN5 and FaDu cell lines when treated at IC 50 concentrations, suggesting that it affects anchorage independent growth ( Figure 4B). We next determined the effect of  Figure 4E,F, p < 0.01). These data suggest that DiFiD has potent cytotoxic effects on HNSCC that are long lasting. Taken together, these data confirmed that DiFiD mediated suppression of DCLK1 activity contributes to mitotic catastrophe of HNSCC.

| DiFiD has antitumor effects in vivo
To determine the in vivo antitumor activity of DiFiD, we treated treatment arm (n = 10, p < 0.05, Figure 6C and Supporting Information: Figure 6E). Lastly, the antitumor effect of DiFiD was tested in an ASCC patient-derived xenograft model. DiFiD treatment significantly reduced PDX tumor growth as evidenced by tumor volume and weight of ASCC PDX tumors when compared to the control group (p < 0.05, Figure 6D and p < 0.001, Figure 6E). These preclinical data indicate that DiFiD is a promising, well-tolerated, therapeutic agent for the management of SCC.
To elucidate the molecular mechanism whereby DiFiD exerts its antitumor effects on HNSCC, we analyzed xenograft tumors using  Figure 6G) and DCLK1 ( Figure 6G and Supporting Information: Figure 6H), further substantiating our observations in vivo. Taken together, these studies demonstrate that DiFiD inhibits tumor growth by targeting DCLK1 and inducing cellular apoptosis.

| DISCUSSION
SCCs of the head and neck and anus are aggressive malignancies with high rates of local recurrence, distant metastasis, and poor clinical outcomes, including reduced survival. Current standard of care include surgery followed by chemoradiotherapy. However, despite aggressive treatment, the survival rate remains low highlighting a significant unmet medical need in SCC patients. In this article, we demonstrate that DCLK1 is a clinically relevant target for HNSCC and ASCC. DCLK1 is well characterized as a reserve, stress induced stem cell marker in pancreatic and colorectal cancers. 5,6 Using multiple platforms, we demonstrate DCLK1 expression is significantly upregulated in HNSCC compared to normal oral mucosa of patient tissues. Furthermore, high expression of DCLK1 correlates with poor clinical outcomes. These findings agree with a recent report associating high DCLK1 levels with poor HNSCC patient survival. 4 Genetic knockdown of DCLK1 has demonstrated promising findings in neuroblastoma, colorectal, and pancreatic tumors. 29,30 DCLK1 knockdown triggers apoptosis and inhibits proliferation, mitochondrial function, and ATP synthesis in neuroblastoma cells. 30 TCGA analysis has shown DCLK1 to be positively correlated with NOTCH signaling in HNSCC. 31  nonspecific activity, with a comparable affinity toward DCLK1 as much as their target kinases. 35 Therefore, while inhibition of DCLK1 may play a role in the therapeutic activity of these compounds, it is highly valuable to identify a specific DCLK1 inhibitor for future clinical applications that has potent antitumor activity.
Previously, we reported that DiFiD shows antitumor activity toward pancreatic cancer cells. 11 However, the direct target for DiFiD was not elucidated. We used an in vitro kinase assay to demonstrate that DiFiD effectively inhibits DCLK1 kinase activity, but not related CaMK family members, suggesting that the compound is a specific competitive inhibitor of DCLK1. We further confirmed a direct interaction through in vitro binding assays and SPR analysis. The CETSA and DARTs binding assays involved the uptake of the compound by cells before thermal or enzymatic denaturation, respectively. 22 We showed that DCLK1 was robustly stabilized by When we knocked-down DCLK1 in FaDu cells using shRNA, we observed a 10-fold decrease in DiFiD toxicity, further demonstrating the specificity of DiFiD for DCLK1. DiFiD increased cell cycle arrest in the G 2 /M phase resulting in mitotic catastrophe, data consistent with previously published studies. 14,33 This is further supported by our observation of a significant increase in the sub-G 0 population following treatment with DiFiD. Moreover, we observed an upregulation of the G 2 /M associated protein, cyclin B1, and a decrease in CDC2, proteins that are abnormally regulated when cells undergo mitotic catastrophe. 37,38 These data corroborate our previous reported results demonstrating the induction of p21 and a reduction in cyclins A1 and D1 following DiFiD treatment in pancreatic cancer. 14 ASCC has an incidence rate of 0.2−4.4 per 100,000 people per year which has risen worldwide over the last three decades, especially in homosexual men (35 per 100,000 per year) and those with HIV (75−135 per 100,000 per year). 39,40 Despite these rising numbers, the standard of care treatment for this cancer comprised of fluorouracil and mitomycin C has remained essentially unchanged since its inception. 41 The addition of intensity-modulated radiation therapy and cisplatin has shown similar efficacy to fluorouracil and mitomycin C in a large, randomized trial. 42,43 The relative rarity of ASCC presents challenges in conducting pivotal clinical trials. In addition, validated preclinical models of ASCC that accurately replicate clinical observations are limited. Existing preclinical models of ASCC include a cell line derived from a lymph node metastasis, two transgenic mouse models and a xenograft from a single patient. [44][45][46][47] Here we present a patient derived xenograft model and provide preliminary evidence of a promising, new therapeutic target for the management of ASCC.
In our studies, we observed significant antitumor effects in SCC preclinical models following treatment with DiFiD. DCLK1 is expressed in various normal cell types, including neurons, osteoblasts, and colon stem cells, 48 and is involved in physiological processes, including retrograde transport, neuronal migration, and neurogenesis. The data presented in this article suggest that DiFiD is well tolerated at doses that demonstrated antitumor activity, as mice maintained normal weight gain and ambulation. DiFiD tolerance was observed in an earlier study, 14 further supporting the notion that it is well tolerated at the doses used to inhibit cancer growth. Padhye: oversaw production of DiFiD and drug study consultant.

| CONCLUSION
Bernhard Biersack: drug study consultant. Shrikant Anant: interpreted data sets, major contributor in writing manuscript, oversaw study design. Sufi Mary Thomas: interpreted data sets, major contributor in writing manuscript, oversaw study design. All authors read and approved the final manuscript.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.