DCLK1 and DNA Damage Response

*Janani Panneerselvam, Dongfeng Qu, Courtney Houchen, Michael Bronze and Parthasarathy Chandrakesan*

## **Abstract**

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.

**Keywords:** DNA damage, DDR, ATM, ATR, DCLK1

## **1. Introduction**

DNA damage exists in all cellular organisms, and DNA, the genetic material in each living cell is the fundamental unit of life and its integrity and stability are essential to life [1]. However, DNA is not passive; rather, it is a chemical unit subject to be attacked from a range of endogenous and environmental damaging agents. The endogenous damages are the damage caused by reactive oxygen species or metabolic byproducts, and DNA metabolization; exogenous damages are caused by external agents, like radiations, toxins, chemicals, and microorganisms [2]. In response to the DNA damage, cells rapidly recruit a sophisticated network which is called DNA damage-response (DDR) systems. DDR systems include DNA repair mechanisms, damage tolerance processes, and cell-cycle checkpoint pathways [3]. Failure of DDR causes genomic instability which results in various diseases including immune deficiency, neurological degeneration, premature aging, and severe cancer susceptibility [2, 4]. Indeed, great progress has been made towards understanding the mechanisms of DDR in homeostasis, carcinogenesis and cancer advancement but much remains to delineate how the DDR network systems are regulated. Furthermore, how the DDR network is formed and how it is fine-tuned by upstream and downstream mediators or signaling pathways that support the homeostasis or disease progression required to understand. While the rapid activation of DDR against the

DNA damage is expected, it is unclear how and who activates or gives the instruction to DDR network systems? Gaining knowledge about DDR and its regulators will not only enhance our understanding of DDR functions but will undoubtedly giving us opportunities to better manage human diseases. Although, very few studies reported that protein kinases and DNA adaptor molecules or DNA regulators may influence or send signals to DDR after DNA gets damaged [5, 6]. DCLK1 is a member of the protein kinase superfamily and the doublecortin family, that belongs to the group of microtubule-associated proteins [7]. Our novel findings that DCLK1 regulate DNA damage response and cell survival following genotoxic injury opens many windows of how DDR is regulated [8]. In this chapter we will highlight the functional role of DCLK1 in injury, DDR and cell survival, which will lead us to a better understanding of DCLK1 expression in helping genomic stability in normal and neoplastic cells.

## **2. DNA damage, DNA damage response, and DNA repair**

DNA is the source of genetic information in all living cells, its integrity and fidelity are essential to life. Because DNA is not passive, it is a chemical entity subject to be assaulted from various reactive agents, causing DNA damage [9]. DNA damage can be subdivided into two types: (1) endogenous damage caused by reactive oxygen species (ROS) that are derived from metabolic byproducts and (2) exogenous damage caused by radiation (UV, X-ray, gamma), hydrolysis, plant toxins, and viruses, chemical toxins [9, 10]. Most of the DNA damage can be repaired by the host systems called the DNA damage response (DDR) and DNA repair systems. Such systems also face failure and not 100% efficient, which resulted in either cell death or cell survival with un-repaired DNA causing mutation and eventually cancer [11]. In some cases, the un-repaired DNA damage accumulates in non-replicating cells, such as neurons or myocytes of adult mammals, and can cause aging [12]. The DDR is a sophisticated cellular network, which constantly monitors the integrity of the genome, in response to DNA damage [13]. Once the DDR gets activated it rapidly recruit downstream protein sensors and adaptors establishing the sensing, activating repair, tolerating damage and apoptosis (**Figure 1**). 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) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [13, 14]. The ATM pathway for homologous recombination (HR) repair is activated after a double-stranded break. The ATR pathway for nonhomologous end-joining (NHEJ) is associated with single-stranded DNA and stalled DNA replication forks. ATM pathway is a higher-fidelity repair pathway than the ATR. For lesions repaired by the HR, double-strand breaks (DSBs) are detected and processed by the MRE11–RAD50–NBS1 (MRN) complex [15, 16]. For lesions repaired by the NHEJ, DNA breaks are detected and the process by the ATR interacting protein (ATRIP) complex. ATM and ATR transduce the most upstream DDR signal by phosphorylating the checkpoint kinases CHK1/CHK2 and the tumor suppressor protein p53, which resulted in cell cycle arrest to allow time for DNA repair. The main function of DNA-PK activated under the ATM/ATR pathway is to induce cell cycle arrest and DNA repair [17]. DNA repair is a vital cellular process required for the maintenance of genomic integrity and fidelity [18]. Living cells employ several DNA repair pathways for distinct types of DNA damage. There are five major DNA repair pathways: (1) mismatch repair (MMR), (2) nucleotide excision repair (NER), (3) base excision repair (BER), (4) homologous recombinational repair (HR), and (5) non-homologous end joining (NHEJ) [19, 20]. MMR's primary

**79**

**3. DCLK1**

**Figure 1.**

*DCLK1 and DNA Damage Response*

*DOI: http://dx.doi.org/10.5772/intechopen.92327*

function is to remove base mismatches and small insertion and deletion loops which is introduced during replication. The NER pathway is a multistep process that serves to repair a variety of DNA damage, including DNA lesions caused by UV radiation, toxic chemicals, or chemotherapeutic drugs that form huge DNA adducts. BER primarily repairs non-bulky lesions produced by alkylation, oxidation or deamination of bases. The BER pathway deals with base damage, the most common insult to cellular DNA. DSBs can be repaired by either HR or NHEJ. HR uses a homologous DNA template and is highly accurate, whereas NHEJ rejoins the broken ends without using a template and is often accompanied by loss of some nucleotides. Direct reversal of DNA damage is one repair mechanism used to restore

*caused by different sources and the cellular response to DNA damage.*

*DNA damage, DNA damage response, and repair. Graphical illustration demonstrating the DNA damage* 

damaged DNA without using excision, resynthesis, and ligation [21, 22].

The human doublecortin (DCX) gene family comprises members that share the tubulin-binding domain and known to have limited functions in microtubuleassociated regulation and neuronal-regulation [23]. One of the best known and most interesting members of this DCX family is doublecortin-like kinase 1 (DCLK1 also known as DCAMKL1), a gene encoding for a protein that is 70% identical to doublecortin in the microtubule-binding N-terminal domain. However, unlike doublecortin, the DCKL1 gene also encodes for a serine–threonine kinase C-terminal domain that is similar to Ca2+/calmodulin-dependent protein kinase II but lacks a canonical calmodulin-binding site [24, 25]. DCLK1 gene also encodes

**Figure 1.**

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

**2. DNA damage, DNA damage response, and DNA repair**

DNA is the source of genetic information in all living cells, its integrity and fidelity are essential to life. Because DNA is not passive, it is a chemical entity subject to be assaulted from various reactive agents, causing DNA damage [9]. DNA damage can be subdivided into two types: (1) endogenous damage caused by reactive oxygen species (ROS) that are derived from metabolic byproducts and (2) exogenous damage caused by radiation (UV, X-ray, gamma), hydrolysis, plant toxins, and viruses, chemical toxins [9, 10]. Most of the DNA damage can be repaired by the host systems called the DNA damage response (DDR) and DNA repair systems. Such systems also face failure and not 100% efficient, which resulted in either cell death or cell survival with un-repaired DNA causing mutation and eventually cancer [11]. In some cases, the un-repaired DNA damage accumulates in non-replicating cells, such as neurons or myocytes of adult mammals, and can cause aging [12]. The DDR is a sophisticated cellular network, which constantly monitors the integrity of the genome, in response to DNA damage [13]. Once the DDR gets activated it rapidly recruit downstream protein sensors and adaptors establishing the sensing, activating repair, tolerating damage and apoptosis (**Figure 1**). 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) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [13, 14]. The ATM pathway for homologous recombination (HR) repair is activated after a double-stranded break. The ATR pathway for nonhomologous end-joining (NHEJ) is associated with single-stranded DNA and stalled DNA replication forks. ATM pathway is a higher-fidelity repair pathway than the ATR. For lesions repaired by the HR, double-strand breaks (DSBs) are detected and processed by the MRE11–RAD50–NBS1 (MRN) complex [15, 16]. For lesions repaired by the NHEJ, DNA breaks are detected and the process by the ATR interacting protein (ATRIP) complex. ATM and ATR transduce the most upstream DDR signal by phosphorylating the checkpoint kinases CHK1/CHK2 and the tumor suppressor protein p53, which resulted in cell cycle arrest to allow time for DNA repair. The main function of DNA-PK activated under the ATM/ATR pathway is to induce cell cycle arrest and DNA repair [17]. DNA repair is a vital cellular process required for the maintenance of genomic integrity and fidelity [18]. Living cells employ several DNA repair pathways for distinct types of DNA damage. There are five major DNA repair pathways: (1) mismatch repair (MMR), (2) nucleotide excision repair (NER), (3) base excision repair (BER), (4) homologous recombinational repair (HR), and (5) non-homologous end joining (NHEJ) [19, 20]. MMR's primary

and neoplastic cells.

DNA damage is expected, it is unclear how and who activates or gives the instruction to DDR network systems? Gaining knowledge about DDR and its regulators will not only enhance our understanding of DDR functions but will undoubtedly giving us opportunities to better manage human diseases. Although, very few studies reported that protein kinases and DNA adaptor molecules or DNA regulators may influence or send signals to DDR after DNA gets damaged [5, 6]. DCLK1 is a member of the protein kinase superfamily and the doublecortin family, that belongs to the group of microtubule-associated proteins [7]. Our novel findings that DCLK1 regulate DNA damage response and cell survival following genotoxic injury opens many windows of how DDR is regulated [8]. In this chapter we will highlight the functional role of DCLK1 in injury, DDR and cell survival, which will lead us to a better understanding of DCLK1 expression in helping genomic stability in normal

**78**

*DNA damage, DNA damage response, and repair. Graphical illustration demonstrating the DNA damage caused by different sources and the cellular response to DNA damage.*

function is to remove base mismatches and small insertion and deletion loops which is introduced during replication. The NER pathway is a multistep process that serves to repair a variety of DNA damage, including DNA lesions caused by UV radiation, toxic chemicals, or chemotherapeutic drugs that form huge DNA adducts. BER primarily repairs non-bulky lesions produced by alkylation, oxidation or deamination of bases. The BER pathway deals with base damage, the most common insult to cellular DNA. DSBs can be repaired by either HR or NHEJ. HR uses a homologous DNA template and is highly accurate, whereas NHEJ rejoins the broken ends without using a template and is often accompanied by loss of some nucleotides. Direct reversal of DNA damage is one repair mechanism used to restore damaged DNA without using excision, resynthesis, and ligation [21, 22].

## **3. DCLK1**

The human doublecortin (DCX) gene family comprises members that share the tubulin-binding domain and known to have limited functions in microtubuleassociated regulation and neuronal-regulation [23]. One of the best known and most interesting members of this DCX family is doublecortin-like kinase 1 (DCLK1 also known as DCAMKL1), a gene encoding for a protein that is 70% identical to doublecortin in the microtubule-binding N-terminal domain. However, unlike doublecortin, the DCKL1 gene also encodes for a serine–threonine kinase C-terminal domain that is similar to Ca2+/calmodulin-dependent protein kinase II but lacks a canonical calmodulin-binding site [24, 25]. DCLK1 gene also encodes

#### **Figure 2.**

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

for a serine/proline-rich domain in between the doublecortin and the protein kinase domains, which mediates multiple protein–protein interactions. In humans, DCLK1 consists of four primary isoforms with a shared kinase domain-driven from two promoter regions termed α and β (**Figure 2**) [26–28]. The α-promoter drives the expression of isoforms termed α-long (isoform 2) and α-short (isoform 1) which contain an N-terminal microtubule-binding region with high homology to DCX. Importantly, the α-promoter isoforms are specifically expressed in the DCLK1+ tuft cells that eventually give rise to tumor stem cells following relevant mutagenesis in the colon and pancreatic cancer [29–31]. The β-promoter drives the expression of two isoforms termed β-long (isoform 4) and β-short (isoform 3) that can be used to predict survival in colon cancer [32]. Although these isoforms likely play a significant role in tumorigenesis through their kinase activity, there is no evidence that they are functionally involved in the regulation of DDR, until our first report to demonstrate its direct interaction with ATM.

## **4. DCLK1 and DDR following injury and inflammation**

Cell survival after severe injury requires highly coordinated complex interplay between the diverse molecular signaling responses to repair the injury [15, 33]. We discussed three fundamental standards about the critical role of DCLK1 in intestinal epithelial cell survival after severe genotoxic injury: (1) how intestinal epithelial cells respond to severe DNA damage because intestinal epithelial cells are the most affected cells after bone marrow during radiotherapy or accidental or incidental radiation exposure [34] and (2) how DCLK1 a kinase protein expression

**81**

**Figure 3.**

*radiation injury.*

*DCLK1 and DNA Damage Response*

*DOI: http://dx.doi.org/10.5772/intechopen.92327*

play key role in injury response, because DCLK1 expressing cells survive high dose radiation and DSS-induced inflammation [29]. It is reported that the deletion of DCLK1 (VillinCre;DCLK1f/f mice) in the intestinal epithelial cells does not confer a significant deleterious phenotype in adult mice, compared with their wild-type littermates [35]. However, after 24 h of 12 Gy total body irradiation (TBI), none of the intestinal epithelial-specific DCLK1 knockout mice survived longer than 5 days [35]. The best-known primary defense mechanism against the genotoxic injury-induced DNA damage is the DDR, which repairs the damaged DNA and increased the survival of intestinal epithelial cells [36]. Indeed studies demonstrated that deficient DDR has been suggested to increase intestinal epithelial death and loss of survival [37]. During the early event of DNA damage, the ATM-H2AX axis gets activated, generating gamma-H2AX and other adaptors, providing a stage for efficient homologous recombinant repair [38]. Recently, ATM knockout or loss of Rad50 and Mre11 was reported to increase intestinal injury and lethality [39, 40]. But how these DDR signaling pathways were regulated following radiation injury is not well known. Chandrakesan et al. reported that the absence of DCLK1 expression in the intestinal epithelial cells abrogated the activation and expression of ATM, gamma-H2AX, and downstream adopter proteins BRCA1, Rad50, and MRE11 in the intestinal epithelial cells 24 h post-TBI [8]. Furthermore, it is reported that this reduction persisted up to 3.5 days post-TBI. It is suggested that there is a profound defect in intestinal DDR in DCLK1 knock-out mice, which

*DCLK1 and DDR. Graphical illustration demonstrating the regulatory role of DCLK1 in DDR following* 

## *DCLK1 and DNA Damage Response DOI: http://dx.doi.org/10.5772/intechopen.92327*

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

for a serine/proline-rich domain in between the doublecortin and the protein kinase domains, which mediates multiple protein–protein interactions. In humans, DCLK1 consists of four primary isoforms with a shared kinase domain-driven from two promoter regions termed α and β (**Figure 2**) [26–28]. The α-promoter drives the expression of isoforms termed α-long (isoform 2) and α-short (isoform 1) which contain an N-terminal microtubule-binding region with high homology to DCX. Importantly, the α-promoter isoforms are specifically expressed in the DCLK1+ tuft cells that eventually give rise to tumor stem cells following relevant mutagenesis in the colon and pancreatic cancer [29–31]. The β-promoter drives the expression of two isoforms termed β-long (isoform 4) and β-short (isoform 3) that can be used to predict survival in colon cancer [32]. Although these isoforms likely play a significant role in tumorigenesis through their kinase activity, there is no evidence that they are functionally involved in the regulation of DDR, until our first report

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

to demonstrate its direct interaction with ATM.

**4. DCLK1 and DDR following injury and inflammation**

Cell survival after severe injury requires highly coordinated complex interplay between the diverse molecular signaling responses to repair the injury [15, 33]. We discussed three fundamental standards about the critical role of DCLK1 in intestinal epithelial cell survival after severe genotoxic injury: (1) how intestinal epithelial cells respond to severe DNA damage because intestinal epithelial cells are the most affected cells after bone marrow during radiotherapy or accidental or incidental radiation exposure [34] and (2) how DCLK1 a kinase protein expression

**80**

**Figure 2.**

play key role in injury response, because DCLK1 expressing cells survive high dose radiation and DSS-induced inflammation [29]. It is reported that the deletion of DCLK1 (VillinCre;DCLK1f/f mice) in the intestinal epithelial cells does not confer a significant deleterious phenotype in adult mice, compared with their wild-type littermates [35]. However, after 24 h of 12 Gy total body irradiation (TBI), none of the intestinal epithelial-specific DCLK1 knockout mice survived longer than 5 days [35]. The best-known primary defense mechanism against the genotoxic injury-induced DNA damage is the DDR, which repairs the damaged DNA and increased the survival of intestinal epithelial cells [36]. Indeed studies demonstrated that deficient DDR has been suggested to increase intestinal epithelial death and loss of survival [37]. During the early event of DNA damage, the ATM-H2AX axis gets activated, generating gamma-H2AX and other adaptors, providing a stage for efficient homologous recombinant repair [38]. Recently, ATM knockout or loss of Rad50 and Mre11 was reported to increase intestinal injury and lethality [39, 40]. But how these DDR signaling pathways were regulated following radiation injury is not well known. Chandrakesan et al. reported that the absence of DCLK1 expression in the intestinal epithelial cells abrogated the activation and expression of ATM, gamma-H2AX, and downstream adopter proteins BRCA1, Rad50, and MRE11 in the intestinal epithelial cells 24 h post-TBI [8]. Furthermore, it is reported that this reduction persisted up to 3.5 days post-TBI. It is suggested that there is a profound defect in intestinal DDR in DCLK1 knock-out mice, which

**Figure 3.**

*DCLK1 and DDR. Graphical illustration demonstrating the regulatory role of DCLK1 in DDR following radiation injury.*

might contribute to defective epithelial survival and overall survival. Interestingly they established that phosphorylation of ATM which is critical for its activation is reduced in the intestinal epithelial cells of DCLK1 knock-out mice, under physiological conditions, and discovered that DCLK1 can directly interact with ATM for its activation. ATM activation during and or after radiation injury directly depends on the ratio of DCLK1-ATM interaction [8]. Furthermore, DCLK1 knockdown and overexpression experiments with the YAMC cell line in vitro established that DCLK1 interaction is important for ATM activation. It is the first study to establish a direct link between DCLK1 and ATM mediated DDR, for the survival of cell in response to severe genotoxic injury (**Figure 3**).

## **5. DCLK1 in the regulation of DDR in cancers**

A faulty DDR system can initiate cancer development [41]. Cancer cells with a DDR deficiency are profoundly dependent on remaining DDR [42, 43], for example in the case of ATM deficiency cancer cell relies on the ATR pathway. Therefore, DDR inhibition in cancers exploits these defects by inhibiting the remaining DDR system, and which in turn causes cancer cell death. Indeed the healthy cells are not vulnerable to DDR targeted therapies because normal cells can have higher expression of DDR only if they exposed to injury [44, 45]. Most cancer cells depend on their enhanced DDR activation for their survival, mainly activation of ATM and ATR pathways, and associated, CHK2, histone H2AX, and p53 [46, 47].

The present conventional radiotherapy and chemotherapies including platinumbased therapies are used to kill cancer cells by inducing DNA damage. A huge problem that arises when using conventional therapies is the development of resistance by these cancer cells whose DDR repair the genomic instability, which causes conventional therapies to fail [48, 49]. Cancer cells as a short-term solution can bypass the DNA damage caused by chemotherapeutic agents by a mechanism known as translesion synthesis [50, 51]. Cancer cells with high DNA damage tolerance allow DNA replication to proceed in the presence of DNA damage include the convergence of adjacent replicons, re-priming of DNA synthesis downstream of lesions on the leading strand and discontinuous synthesis of Okazaki fragments on the lagging DNA strand [52, 53]. Given the fundamental role of DDR in the gain of chemo-resistance, the novel strategies of combination therapies including DDR targeted therapies will be effective [41]. Recent regulatory approval of olaparib (Lynparza), a poly (ADP ribose) polymerase (PARP) inhibitor, which inhibits PARP enzyme activity and forms severe DSBs [54]. In cancers, PARP inhibitor increases genomic instability that results in tumor cell death [55, 56]. Although, the pharmacological inhibitors of PARP have shown promising results in preclinical studies and in clinical trials, the gain of resistance in cancer cells to PARP inhibitors, is inevitable [57]. However, the combination of PARP inhibitors with other DDR agents including ATR inhibitors, CHK1 inhibitors, ATM inhibitors, and DNA-PKs inhibitors, or with chemotherapeutic agents are novel strategies currently investigated to overcome resistance to PARP inhibitors [57] (**Table 1**—[58]). However, while the DDR targeted therapies are expected to cause DNA damage in tumor cells, it is unclear how these DDR networks are regulated in cancer cells? DDR regulators in cancers are reported recently, (1) MORC2 (MORC Family CW-Type Zinc Finger 2) is required for DNA damage-induced PAR production and PAR-dependent DNA repair signaling cascades and stimulates chromatin remodeling [59, 60]. Inhibition of MORC2 in breast cancer cells impaired DDR and sensitize cancer cells to PARP inhibitors. (2) MYB is an oncogene that plays an important role in regulating DDR in ER+ breast cancers and inhibition of MYB induces DNA

**83**

*DCLK1 and DNA Damage Response*

**ClinicalTrials. gov identifier**

*DOI: http://dx.doi.org/10.5772/intechopen.92327*

NCT02797964 A Phase 1 Trial of SRA737 in Subjects with Advanced Cancer

NCT02797977 A Phase 1 Trial of SRA737 in Combination

Advanced Cancer

NCT02516813 Phase 1 Trial of MSC2490484A, an Inhibitor

Small Cell Lung Cancer

NCT02660034 The Safety, Pharmacokinetics and Antitumor

Novel Anti Cancer Agents

Chemotherapy. (SOLO-1)

NCT03057145 Combination Study of Prexasertib and

NCT03308942 Phase 2, Multi-Arm Study of Niraparib

Tumors

Tumors

NCT02264678 Ascending Doses of AZD6738 in

NCT01844986 Olaparib Maintenance Monotherapy in

NCT02282020 Olaparib Treatment in Relapsed Germline

NCT02446704 Study of Olaparib and Temozolomide

Chemotherapy

NCT02789332 Assessing the Efficacy of Paclitaxel and

(GeparOla)

NCT02264678 Ascending Doses of AZD6738 in

with Gemcitabine Plus Cisplatin or Gemcitabine Alone in Subjects with

Olaparib in Patients with Advanced Solid

of a DNA-dependent Protein Kinase, in Combination with Radiotherapy

Administered Alone and in Combination with PD-1 Inhibitor in Patients with Non-

Activity of BGB-A317 in Combination with BGB-290 in Subjects with Advanced Solid

Combination with Chemotherapy and/or

Patients with BRCA Mutated Ovarian Cancer Following First Line Platinum Based

Breast Cancer Susceptibility Gene (BRCA) Mutated Ovarian Cancer Patients Who Have Progressed at Least 6 Months After Last Platinum Treatment and Have Received at Least 2 Prior Platinum Treatments (SOLO3)

in Patients With Recurrent Small Cell Lung Cancer Following Failure of Prior

Olaparib in Comparison to Paclitaxel/ Carboplatin Followed by Epirubicin/ Cyclophosphamide as Neoadjuvant Chemotherapy in Patients with HER2 negative Early Breast Cancer and Homologous Recombination Deficiency

Combination With Chemotherapy and/or

Drug: administration of AZD6738 in combination with carboplatin Drug: administration of AZD6738 Drug: administration of AZD6738 in combination with olaparib Drug: administration of AZD6738 in combination with MEDI4736

Novel Anti Cancer Agents

**Title Phase Drug target**

I CHK1 inhibitor

I CHK1 inhibitor

I CHK1 inhibitor

I DNA-PK inhibitor Radiotherapy

II PARP Inhibitor

I PARP Inhibitor

I ATR Inhibitor

III PARP inhibitor

III PARP inhibitor

I PARP inhibitor

II PARP inhibitor

I/II PARP inhibitor

Chemotherapy

Chemotherapy

Chemotherapy

PARP inhibitor

PD-1 Inhibitor

PD-1 Inhibitor

Chemotherapy PDL-1 Inhibitor *Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

response to severe genotoxic injury (**Figure 3**).

**5. DCLK1 in the regulation of DDR in cancers**

might contribute to defective epithelial survival and overall survival. Interestingly they established that phosphorylation of ATM which is critical for its activation is reduced in the intestinal epithelial cells of DCLK1 knock-out mice, under physiological conditions, and discovered that DCLK1 can directly interact with ATM for its activation. ATM activation during and or after radiation injury directly depends on the ratio of DCLK1-ATM interaction [8]. Furthermore, DCLK1 knockdown and overexpression experiments with the YAMC cell line in vitro established that DCLK1 interaction is important for ATM activation. It is the first study to establish a direct link between DCLK1 and ATM mediated DDR, for the survival of cell in

A faulty DDR system can initiate cancer development [41]. Cancer cells with a DDR deficiency are profoundly dependent on remaining DDR [42, 43], for example in the case of ATM deficiency cancer cell relies on the ATR pathway. Therefore, DDR inhibition in cancers exploits these defects by inhibiting the remaining DDR system, and which in turn causes cancer cell death. Indeed the healthy cells are not vulnerable to DDR targeted therapies because normal cells can have higher expression of DDR only if they exposed to injury [44, 45]. Most cancer cells depend on their enhanced DDR activation for their survival, mainly activation of ATM and

The present conventional radiotherapy and chemotherapies including platinum-

ATR pathways, and associated, CHK2, histone H2AX, and p53 [46, 47].

based therapies are used to kill cancer cells by inducing DNA damage. A huge problem that arises when using conventional therapies is the development of resistance by these cancer cells whose DDR repair the genomic instability, which causes conventional therapies to fail [48, 49]. Cancer cells as a short-term solution can bypass the DNA damage caused by chemotherapeutic agents by a mechanism known as translesion synthesis [50, 51]. Cancer cells with high DNA damage tolerance allow DNA replication to proceed in the presence of DNA damage include the convergence of adjacent replicons, re-priming of DNA synthesis downstream of lesions on the leading strand and discontinuous synthesis of Okazaki fragments on the lagging DNA strand [52, 53]. Given the fundamental role of DDR in the gain of chemo-resistance, the novel strategies of combination therapies including DDR targeted therapies will be effective [41]. Recent regulatory approval of olaparib (Lynparza), a poly (ADP ribose) polymerase (PARP) inhibitor, which inhibits PARP enzyme activity and forms severe DSBs [54]. In cancers, PARP inhibitor increases genomic instability that results in tumor cell death [55, 56]. Although, the pharmacological inhibitors of PARP have shown promising results in preclinical studies and in clinical trials, the gain of resistance in cancer cells to PARP inhibitors, is inevitable [57]. However, the combination of PARP inhibitors with other DDR agents including ATR inhibitors, CHK1 inhibitors, ATM inhibitors, and DNA-PKs inhibitors, or with chemotherapeutic agents are novel strategies currently investigated to overcome resistance to PARP inhibitors [57] (**Table 1**—[58]). However, while the DDR targeted therapies are expected to cause DNA damage in tumor cells, it is unclear how these DDR networks are regulated in cancer cells? DDR regulators in cancers are reported recently, (1) MORC2 (MORC Family CW-Type Zinc Finger 2) is required for DNA damage-induced PAR production and PAR-dependent DNA repair signaling cascades and stimulates chromatin remodeling [59, 60]. Inhibition of MORC2 in breast cancer cells impaired DDR and sensitize cancer cells to PARP inhibitors. (2) MYB is an oncogene that plays an important role in regulating DDR in ER+ breast cancers and inhibition of MYB induces DNA

**82**



#### **Table 1.**

*Ongoing DDR inhibitor trials.*

damage and tumor cell death [61]. (3) IKKα directly activates ATM via BRAF regulates DNA damage and inhibition of IKKα induces DNA damage associated cell death in colon cancer [62]. Although these signaling molecules are involved in the regulation of DDR in cancers, their mechanism and therapeutic efficiency are yet to develop.

DCLK1, a protein kinase is overexpressed in various tumor cancers [63–65]. DCLK1 plays a critical role in injury response for repair via regulating DDR [8]. However, recently the role of DCLK1 in the regulation of DDR in cancers has established by many investigators [66–68]. In an in vitro mechanistic study, it is reported that DCLK1 caused chromatin instability, and chromatin rearrangement in colon, lung, and breast cancer cell lines, which drives the advancement of cancer cells for progression and this function is independent of its kinase activity [68]. In another study, it is reported that DCLK1 regulates the phosphorylation of CHK1 in pancreatic cancer cells. Inhibition of DCLK1 enhanced the sensitivity to gemcitabine treatment [67]. In a parallel study, it is shown that DCLK1 by regulating the phosphorylation of CHK1 enhances the sensitivity of 5-FU in colon cancer [69]. Taken together these reports suggest that DCLK1 plays a critical role in the regulation of DDR for cancer cell survival and progression. Novel therapies in the combination of targeting DCLK1 along with chemotherapeutic agents or targeting DCLK1 plus targeting an ATM or ATR with chemotherapeutic agents will be beneficial for the most effective treatment against cancers particularly the resistant cancers.

## **6. DCLK1 and radiation mitigators**

Radiation therapy has been used for the treatment of a wide range of malignancies, especially cancers. Radiation not only kills cancer cells, but it also kills/ affects normal healthy cells. Exposure of normal tissues to a substantial amount of radiation may cause both acute and chronic damage that can result in adverse effects for intended treatment [70, 71]. For example, radiation enteritis (RE) is an

**85**

**7. Conclusion**

*DCLK1 and DNA Damage Response*

*DOI: http://dx.doi.org/10.5772/intechopen.92327*

allowing more intense therapies [75].

ameliorate radiation-induced adverse effects.

intestinal inflammatory process that occurs in response to radiotherapy [72]. It is a major health concern characterized by abdominal pain, diarrhea, and rectal bleeding [73]. It can be complicated by translocation of gut bacteria into the circulation due to the loss of intestinal epithelial cells, disruption of intraepithelial tight junctions, and loss of regenerative ability resulting in severe impairment of gut function and even death. Relatively little is known about the mechanisms underlying the intestinal epithelial injury repair, cell survival and crypt regeneration in RE. Besides the severe side effects resulted in gastrointestinal mucosal damage, ionizing radiation also impairs the bone marrow-derived hematopoietic cells and immune response, which causes a significant increase in morbidity and mortality [74]. Prevention and amelioration of radiation-induced adverse effects would improve the quality of life for patients and would help cancer curability by

There are three types of chemical/biological agents used to interfere with radiation effects. Agents used before or at the time of radiation treatment are called radioprotectors, whereas agents used post-treatment are called radiation mitigators, agents used to ameliorate established normal tissue toxicity are considered treatment [76]. Currently, Amifostine is the only radioprotector in clinical use, and a few radiation mitigators been used [76]. DCLK1 can be a novel target for radiation mitigators for action, as it is mentioned above, deletion of DCLK1 within intestinal epithelial cells results in the premature death of mice following severe radiation injury, suggesting that DCLK1 is a major mediator of the crypt epithelial survival to severe genotoxic injury via a DDR-ATM mediated mechanism [8]. Recently, singlecell analysis in the intestine has revealed that the DCLK1 expressing epithelial cells in the intestine is the primary source of Cox1 (Ptgs1) and Cox2 (Ptgs2) for PGE2 synthesis [77]. PGE2 increases the survival of murine intestinal stem cells when given before photon radiation [78, 79]. It is reported that the treatment of dimethyl-PGE2 to the intestinal epithelial cells increased the survival of the colonic epithelial cells by enhancing DCLK1 expression and reduced the DNA damage [8]. Qu et al. reported that Notch signaling in the intestinal epithelium prevents the death of epithelial cells expressing DCLK1 following radiation injury [80]. Also, dietary pectin has been demonstrated to increase intestinal crypt stem cell survival following radiation injury via a DCLK1 [81]. Kantara et al. have reported that a novel regenerative peptide TP508 can significantly increase survival and delay mortality by mitigating radiation-induced intestinal and colonic toxicity, and its mechanism of action via upregulating the expression of DCLK1 in the intestinal epithelial cells which are responsible for maintaining and regenerating intestinal crypts [82]. In summary, DCLK1 could be a potential radiation mitigator by regulating DDR to

It is becoming clear that DCLK1 contributes to DNA damage response and repair via direct and indirect mechanisms that are distinct from its role as a stem cell marker. A long-standing question of how DDR is regulated in response to DNA damage is now getting a new clarity. Furthermore, (i) DCLK1 and ATM direct interaction for ATM activation following DSBs and (ii) radiation mitigators enhance the survival of cells following DSBs via a DCLK1 dependent mechanism, which expects that DCLK1 can be a potential target for radiation mitigators in radiotherapy. Finally, in the expanding field of DDR, it is important to consider how DCLK1 is involved in the repair of DNA in cancer and homeostatic injure conditions. This will allow clinical and non-clinical researchers and practitioners to

#### *DCLK1 and DNA Damage Response DOI: http://dx.doi.org/10.5772/intechopen.92327*

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

NCT00535353 AZD2281 and Irinotecan in Treating Patients

Colorectal Cancer

NCT00515866 Study to Assess the Safety & Tolerability

NCT01460888 Radiotherapy & Olaparib in COmbination

NCT02308072 Phase I Study of Olaparib Combined with

(ROCOCO)

(ORCA-2)

*conventional chemotherapy or radiotherapy in cancer patients.*

NCT00678132 AZD2281 and Cisplatin Plus Gemcitabine to Treat Solid Tumor Cancers

with Locally Advanced or Metastatic

of a PARP Inhibitor in Combination with Gemcitabine in Pancreatic Cancer

Cisplatin-based Chemoradiotherapy to Treat Locally Advanced Head and Neck Cancer

*Selected clinical studies involving DDR inhibitors as a single agent or adjuvant therapy in combination with* 

for Carcinoma of the Oesophagus

**ClinicalTrials. gov identifier**

damage and tumor cell death [61]. (3) IKKα directly activates ATM via BRAF regulates DNA damage and inhibition of IKKα induces DNA damage associated cell death in colon cancer [62]. Although these signaling molecules are involved in the regulation of DDR in cancers, their mechanism and therapeutic efficiency are yet to

**Title Phase Drug target**

I PARP inhibitor

I PARP inhibitor

I PARP inhibitor

I PARP inhibitor Radiotherapy

I PARP inhibitor

Chemotherapy

Chemotherapy

Chemotherapy

Chemoradiotherapy

DCLK1, a protein kinase is overexpressed in various tumor cancers [63–65]. DCLK1 plays a critical role in injury response for repair via regulating DDR [8]. However, recently the role of DCLK1 in the regulation of DDR in cancers has established by many investigators [66–68]. In an in vitro mechanistic study, it is reported that DCLK1 caused chromatin instability, and chromatin rearrangement in colon, lung, and breast cancer cell lines, which drives the advancement of cancer cells for progression and this function is independent of its kinase activity [68]. In another study, it is reported that DCLK1 regulates the phosphorylation of CHK1 in pancreatic cancer cells. Inhibition of DCLK1 enhanced the sensitivity to gemcitabine treatment [67]. In a parallel study, it is shown that DCLK1 by regulating the phosphorylation of CHK1 enhances the sensitivity of 5-FU in colon cancer [69]. Taken together these reports suggest that DCLK1 plays a critical role in the regulation of DDR for cancer cell survival and progression. Novel therapies in the combination of targeting DCLK1 along with chemotherapeutic agents or targeting DCLK1 plus targeting an ATM or ATR with chemotherapeutic agents will be beneficial for the

most effective treatment against cancers particularly the resistant cancers.

Radiation therapy has been used for the treatment of a wide range of malignancies, especially cancers. Radiation not only kills cancer cells, but it also kills/ affects normal healthy cells. Exposure of normal tissues to a substantial amount of radiation may cause both acute and chronic damage that can result in adverse effects for intended treatment [70, 71]. For example, radiation enteritis (RE) is an

**6. DCLK1 and radiation mitigators**

**84**

develop.

**Table 1.**

*Ongoing DDR inhibitor trials.*

intestinal inflammatory process that occurs in response to radiotherapy [72]. It is a major health concern characterized by abdominal pain, diarrhea, and rectal bleeding [73]. It can be complicated by translocation of gut bacteria into the circulation due to the loss of intestinal epithelial cells, disruption of intraepithelial tight junctions, and loss of regenerative ability resulting in severe impairment of gut function and even death. Relatively little is known about the mechanisms underlying the intestinal epithelial injury repair, cell survival and crypt regeneration in RE. Besides the severe side effects resulted in gastrointestinal mucosal damage, ionizing radiation also impairs the bone marrow-derived hematopoietic cells and immune response, which causes a significant increase in morbidity and mortality [74]. Prevention and amelioration of radiation-induced adverse effects would improve the quality of life for patients and would help cancer curability by allowing more intense therapies [75].

There are three types of chemical/biological agents used to interfere with radiation effects. Agents used before or at the time of radiation treatment are called radioprotectors, whereas agents used post-treatment are called radiation mitigators, agents used to ameliorate established normal tissue toxicity are considered treatment [76]. Currently, Amifostine is the only radioprotector in clinical use, and a few radiation mitigators been used [76]. DCLK1 can be a novel target for radiation mitigators for action, as it is mentioned above, deletion of DCLK1 within intestinal epithelial cells results in the premature death of mice following severe radiation injury, suggesting that DCLK1 is a major mediator of the crypt epithelial survival to severe genotoxic injury via a DDR-ATM mediated mechanism [8]. Recently, singlecell analysis in the intestine has revealed that the DCLK1 expressing epithelial cells in the intestine is the primary source of Cox1 (Ptgs1) and Cox2 (Ptgs2) for PGE2 synthesis [77]. PGE2 increases the survival of murine intestinal stem cells when given before photon radiation [78, 79]. It is reported that the treatment of dimethyl-PGE2 to the intestinal epithelial cells increased the survival of the colonic epithelial cells by enhancing DCLK1 expression and reduced the DNA damage [8]. Qu et al. reported that Notch signaling in the intestinal epithelium prevents the death of epithelial cells expressing DCLK1 following radiation injury [80]. Also, dietary pectin has been demonstrated to increase intestinal crypt stem cell survival following radiation injury via a DCLK1 [81]. Kantara et al. have reported that a novel regenerative peptide TP508 can significantly increase survival and delay mortality by mitigating radiation-induced intestinal and colonic toxicity, and its mechanism of action via upregulating the expression of DCLK1 in the intestinal epithelial cells which are responsible for maintaining and regenerating intestinal crypts [82]. In summary, DCLK1 could be a potential radiation mitigator by regulating DDR to ameliorate radiation-induced adverse effects.

## **7. Conclusion**

It is becoming clear that DCLK1 contributes to DNA damage response and repair via direct and indirect mechanisms that are distinct from its role as a stem cell marker. A long-standing question of how DDR is regulated in response to DNA damage is now getting a new clarity. Furthermore, (i) DCLK1 and ATM direct interaction for ATM activation following DSBs and (ii) radiation mitigators enhance the survival of cells following DSBs via a DCLK1 dependent mechanism, which expects that DCLK1 can be a potential target for radiation mitigators in radiotherapy. Finally, in the expanding field of DDR, it is important to consider how DCLK1 is involved in the repair of DNA in cancer and homeostatic injure conditions. This will allow clinical and non-clinical researchers and practitioners to avoid possible issues with DCLK1 therapeutics, such as enhanced cancer survival and cancer advancement with DCLK1 dependent mitigators during radiotherapy, and, more excitingly, inhibition of DCLK1 along with DDR following chemotherapy or radiotherapy in cancers will lead the way to develop novel strategies for the effective treatment of cancer.

## **Acknowledgements**

This work was supported by DOD grant # W81XWH-18-1-0457 (PI: Chandrakesan).

## **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Janani Panneerselvam, Dongfeng Qu, Courtney Houchen, Michael Bronze and Parthasarathy Chandrakesan\* Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

\*Address all correspondence to: parthasarathy-chandrakesan@ouhsc.edu

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**87**

*DCLK1 and DNA Damage Response*

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Mechanisms of DNA damage, repair, and mutagenesis. Environmental

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[3] Derks KW, Hoeijmakers JH, Pothof J. The DNA damage response: The omics era and its impact. DNA Repair (Amst). 2014;**19**:214-220

[4] Ribezzo F, Shiloh Y, Schumacher B. Systemic DNA damage responses in aging and diseases. Seminars in Cancer

*DOI: http://dx.doi.org/10.5772/intechopen.92327*

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[10] Gupta D, Lin B, Cowan A, Heinen CD. ATR-Chk1 activation mitigates replication stress caused by mismatch repair-dependent processing of DNA damage. Proceedings of the National Academy of Sciences of the United States of America.

[11] Basu B, Yap TA, Molife LR, de Bono JS. Targeting the DNA damage response in oncology: Past, present and future perspectives. Current Opinion in

Oncology. 2012;**24**(3):316-324

[12] Pan MR, Li K, Lin SY, Hung WC. Connecting the dots: From DNA damage and repair to aging. International Journal of Molecular Sciences. 2016;**17**(5):685

[13] Petsalaki E, Zachos G. DNA damage response proteins regulating mitotic cell division: Double agents preserving genome stability. The FEBS Journal. 6 February 2020. DOI: 10.1111/febs.15240

[14] Scully R, Panday A, Elango R, Willis NA. DNA double-strand break repairpathway choice in somatic mammalian cells. Nature Reviews. Molecular Cell

[15] Hoeijmakers JH. DNA damage, aging, and cancer. The New England Journal of Medicine. 2009;**361**(15):1475-1485

[16] Maynard S, Schurman SH, Harboe C, de Souza-Pinto NC, Bohr VA. Base excision repair of oxidative DNA

damage and association with cancer and aging. Carcinogenesis. 2009;**30**(1):2-10

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*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

The authors declare no conflict of interest.

the effective treatment of cancer.

**Acknowledgements**

**Conflict of interest**

Chandrakesan).

avoid possible issues with DCLK1 therapeutics, such as enhanced cancer survival and cancer advancement with DCLK1 dependent mitigators during radiotherapy, and, more excitingly, inhibition of DCLK1 along with DDR following chemotherapy or radiotherapy in cancers will lead the way to develop novel strategies for

This work was supported by DOD grant # W81XWH-18-1-0457 (PI:

**86**

**Author details**

Parthasarathy Chandrakesan\*

provided the original work is properly cited.

Oklahoma City, OK, USA

Janani Panneerselvam, Dongfeng Qu, Courtney Houchen, Michael Bronze and

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Department of Medicine, University of Oklahoma Health Sciences Center,

\*Address all correspondence to: parthasarathy-chandrakesan@ouhsc.edu

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[7] Lin PT, Gleeson JG, Corbo JC, Flanagan L, Walsh CA. DCAMKL1 encodes a protein kinase with homology to doublecortin that regulates microtubule polymerization. The Journal of Neuroscience. 2000;**20**(24):9152-9161

[8] Chandrakesan P, May R, Weygant N, Qu D, Berry WL, Sureban SM, et al. Intestinal tuft cells regulate the ATM mediated DNA damage response via Dclk1 dependent mechanism for crypt restitution following radiation injury. Scientific Reports. 2016;**6**:37667

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[13] Petsalaki E, Zachos G. DNA damage response proteins regulating mitotic cell division: Double agents preserving genome stability. The FEBS Journal. 6 February 2020. DOI: 10.1111/febs.15240

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[21] Eker AP, Quayle C, Chaves I, van der Horst GT. DNA repair in mammalian cells: Direct DNA damage reversal: Elegant solutions for nasty problems. Cellular and Molecular Life Sciences. 2009;**66**(6):968-980

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*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

et al. Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and

pancreatic cancer through inhibition of doublecortin-like kinase 1. Molecular

[28] Omori Y, Suzuki M, Ozaki K, Harada Y, Nakamura Y, Takahashi E, et al. Expression and chromosomal localization of KIAA0369, a putative

kinase structurally related to Doublecortin. Journal of Human Genetics. 1998;**43**(3):169-177

[29] Westphalen CB, Asfaha S, Hayakawa Y, Takemoto Y, Lukin DJ, Nuber AH, et al. Long-lived intestinal tuft cells serve as colon cancer-

initiating cells. The Journal of Clinical Investigation. 2014;**124**(3):1283-1295

[30] Nakanishi Y, Seno H, Fukuoka A, Ueo T, Yamaga Y, Maruno T, et al. Dclk1 distinguishes between tumor and normal stem cells in the intestine. Nature Genetics. 2013;**45**(1):98-103

[31] Westphalen CB, Takemoto Y, Tanaka T, Macchini M, Jiang Z,

2016;**18**(4):441-455

2015;**5**:14983

2005;**126**(1):71-77

[32] O'Connell MR, Sarkar S, Luthra GK, Okugawa Y, Toiyama Y, Gajjar AH, et al. Epigenetic changes and alternate promoter usage by human colon cancers for expressing DCLK1-isoforms: Clinical implications. Scientific Reports.

[33] Hasty P. The impact of DNA damage, genetic mutation and cellular responses on cancer prevention, longevity and aging: Observations in humans and mice. Mechanisms of Ageing and Development.

[34] DiCarlo AL, Tamarat R, Rios CI, Benderitter M, Czarniecki CW,

Renz BW, et al. Dclk1 defines quiescent pancreatic progenitors that promote injury-induced regeneration and tumorigenesis. Cell Stem Cell.

Cancer. 2014;**13**:103

[18] Sirbu BM, Cortez D. DNA damage response: Three levels of DNA repair regulation. Cold Spring Harbor Perspectives in Biology.

[19] Fleck O, Nielsen O. DNA repair. Journal of Cell Science. 2004;**117**

[20] Marti TM, Fleck O. DNA repair nucleases. Cellular and Molecular Life

[21] Eker AP, Quayle C, Chaves I, van der Horst GT. DNA repair in mammalian cells: Direct DNA damage reversal: Elegant solutions for nasty problems. Cellular and Molecular Life Sciences.

[22] Mishina Y, Duguid EM, He C. Direct reversal of DNA alkylation damage. Chemical Reviews. 2006;**106**(2):215-232

Sciences. 2004;**61**(3):336-354

2009;**66**(6):968-980

[23] Matsumoto N, Pilz DT,

pattern of human DCAMKL1 (KIAA0369), a homologue of DCX (XLIS). Genomics. 1999;**56**(2):179-183

2002;**277**(20):17696-17705

2004;**120**(2):103-114

[25] Engels BM, Schouten TG, van Dullemen J, Gosens I, Vreugdenhil E. Functional differences between two DCLK splice variants. Brain Research. Molecular Brain Research.

[26] Sossey-Alaoui K, Srivastava AK.

DCAMKL1, a brain-specific transmembrane protein on 13q12.3 that is similar to doublecortin (DCX).

Genomics. 1999;**56**(1):121-126

[27] Weygant N, Qu D, Berry WL, May R, Chandrakesan P, Owen DB,

Ledbetter DH. Genomic structure, chromosomal mapping, and expression

[24] Burgess HA, Reiner O. Alternative splice variants of doublecortin-like kinase are differentially expressed and have different kinase activities. The Journal of Biological Chemistry.

2013;**5**(8):a012724

(Pt 4):515-517

**88**

Allio TC, et al. Cellular therapies for treatment of radiation injury: Report from a NIH/NIAID and IRSN Workshop. Radiation Research. 2017;**188**(2):e54-e75

[35] May R, Qu D, Weygant N, Chandrakesan P, Ali N, Lightfoot SA, et al. Brief report: Dclk1 deletion in tuft cells results in impaired epithelial repair after radiation injury. Stem Cells. 2014;**32**(3):822-827

[36] Yu J. Intestinal stem cell injury and protection during cancer therapy. Translational Cancer Research. 2013;**2**(5):384-396

[37] Kenyon J, Gerson SL. The role of DNA damage repair in aging of adult stem cells. Nucleic Acids Research. 2007;**35**(22):7557-7565

[38] Lee JH, Paull TT. Activation and regulation of ATM kinase activity in response to DNA double-strand breaks. Oncogene. 2007;**26**(56):7741-7748

[39] Ch'ang HJ, Maj JG, Paris F, Xing HR, Zhang J, Truman JP, et al. ATM regulates target switching to escalating doses of radiation in the intestines. Nature Medicine. 2005;**11**(5):484-490

[40] Rotolo JA, Mesicek J, Maj J, Truman JP, Haimovitz-Friedman A, Kolesnick R, et al. Regulation of ceramide synthase-mediated crypt epithelium apoptosis by DNA damage repair enzymes. Cancer Research. 2010;**70**(3):957-967

[41] Broustas CG, Lieberman HB. DNA damage response genes and the development of cancer metastasis. Radiation Research. 2014;**181**(2):111-130

[42] Sun S, Osterman MD, Li M. Tissue specificity of DNA damage response and tumorigenesis. Cancer Biology & Medicine. 2019;**16**(3):396-414

[43] Biss M, Xiao W. Selective tumor killing based on specific DNA-damage response deficiencies. Cancer Biology & Therapy. 2012;**13**(5):239-246

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[54] Cerrato A, Morra F, Celetti A. Use of poly ADP-ribose polymerase [PARP] inhibitors in cancer cells bearing DDR defects: The rationale for their inclusion in the clinic. Journal of Experimental & Clinical Cancer Research. 2016;**35**(1):179

[55] Ceccaldi R, Liu JC, Amunugama R, Hajdu I, Primack B, Petalcorin MI, et al. Homologous-recombinationdeficient tumours are dependent on Poltheta-mediated repair. Nature. 2015;**518**(7538):258-262

[56] Mateos-Gomez PA, Gong F, Nair N, Miller KM, Lazzerini-Denchi E, Sfeir A. Mammalian polymerase theta promotes alternative NHEJ and suppresses recombination. Nature. 2015;**518**(7538):254-257

[57] Francica P, Rottenberg S. Mechanisms of PARP inhibitor resistance in cancer and insights into the DNA damage response. Genome Medicine. 2018;**10**(1):101

[58] Minchom A, Aversa C, Lopez J. Dancing with the DNA damage response: Next-generation anti-cancer therapeutic strategies. Therapeutic Advances in Medical Oncology. 2018;**10**:1758835918786658

[59] Liu J, Zhang Q, Ruan B, Chen W, Zheng J, Xu B, et al. MORC2 regulates C/EBPalpha-mediated cell differentiation via sumoylation. Cell Death and Differentiation. 2019;**26**(10):1905-1917

[60] Li DQ, Nair SS, Ohshiro K, Kumar A, Nair VS, Pakala SB, et al. MORC2 signaling integrates

phosphorylation-dependent, ATPasecoupled chromatin remodeling during the DNA damage response. Cell Reports. 2012;**2**(6):1657-1669

[61] Yang RM, Nanayakkara D, Kalimutho M, Mitra P, Khanna KK, Dray E, et al. MYB regulates the DNA damage response and components of the homology-directed repair pathway in human estrogen receptorpositive breast cancer cells. Oncogene. 2019;**38**(26):5239-5249

[62] Colomer C, Margalef P, Villanueva A, Vert A, Pecharroman I, Sole L, et al. IKKalpha kinase regulates the DNA damage response and drives chemo-resistance in cancer. Molecular Cell. 2019;**75**(4):669-682

[63] Chandrakesan P, Panneerselvam J, Qu D, Weygant N, May R, Bronze MS, et al. Regulatory roles of Dclk1 in epithelial mesenchymal transition and cancer stem cells. Journal of Carcinogenesis & Mutagenesis. 2016;**7**(2):257

[64] Chandrakesan P, Weygant N, May R, Qu D, Chinthalapally HR, Sureban SM, et al. DCLK1 facilitates intestinal tumor growth via enhancing pluripotency and epithelial mesenchymal transition. Oncotarget. 2014;**5**(19):9269-9280

[65] Chandrakesan P, Yao J, Qu D, May R, Weygant N, Ge Y, et al. Dclk1, a tumor stem cell marker, regulates prosurvival signaling and self-renewal of intestinal tumor cells. Molecular Cancer. 2017;**16**(1):30

[66] Mohammadi C, Najafi R. DCLK1 as a promising marker for radioresistance in colorectal cancer. Journal of Gastrointestinal Cancer. 17 August 2019. DOI: 10.1007/s12029-019-00292-z

[67] Kawamura D, Takemoto Y, Nishimoto A, Ueno K, Hosoyama T, Shirasawa B, et al. Enhancement of

**91**

*DCLK1 and DNA Damage Response*

cytotoxic effects of gemcitabine by Dclk1 inhibition through suppression of Chk1 phosphorylation in human pancreatic cancer cells. Oncology Reports. 2017;**38**(5):3238-3244

[68] Lu Y, Maruyama J, Kuwata K, Fukuda H, Iwasa H, Arimoto-

2018;**16**:130-137

2018;**38**(11):6225-6230

2016;**6**(2):185-206

Matsuzaki K, et al. Doublecortin-like kinase 1 compromises DNA repair and induces chromosomal instability. Biochemistry and Biophysics Reports.

[69] Suehiro Y, Takemoto Y, Nishimoto A, Ueno K, Shirasawa B, Tanaka T, et al. Dclk1 inhibition cancels 5-FU-induced cell-cycle arrest and decreases cell survival in colorectal cancer. Anticancer Research.

[70] Bray FN, Simmons BJ, Wolfson AH, Nouri K. Acute and chronic cutaneous

reactions to ionizing radiation therapy. Dermatology and Therapy.

[71] Lam SY, Peppelenbosch MP, Fuhler GM. Prediction and treatment of radiation enteropathy: Can intestinal bugs lead the way? Clinical Cancer Research. 2019;**25**(21):6280-6282

[72] Harb AH, Abou Fadel C,

[73] Huang Y, Guo F, Yao D,

[74] Dainiak N. Hematologic

2016;**204**(2):335-343

2002;**30**(6):513-528

2012;**85**(1012):313-330

Sharara AI. Radiation enteritis. Current Gastroenterology Reports. 2014;**16**(5):383

Li Y, Li J. Surgery for chronic radiation enteritis: Outcome and risk factors. The Journal of Surgical Research.

consequences of exposure to ionizing radiation. Experimental Hematology.

[75] Koukourakis MI. Radiation damage and radioprotectants: New concepts in the era of molecular medicine. The British Journal of Radiology.

*DOI: http://dx.doi.org/10.5772/intechopen.92327*

[76] Citrin D, Cotrim AP, Hyodo F, Baum BJ, Krishna MC, Mitchell JB. Radioprotectors and mitigators of radiation-induced normal tissue injury. The Oncologist. 2010;**15**(4):360-371

[77] Haber AL, Biton M, Rogel N,

333-339

Herbst RH, Shekhar K, Smillie C, et al. A single-cell survey of the small intestinal epithelium. Nature. 2017;**551**(7680):

[78] Hanson WR. Radiation protection of murine intestine by WR-2721, 16,16-dimethyl prostaglandin E2, and the combination of both agents. Radiation Research. 1987;**111**(2):361-373

[79] Hanson WR, Grdina DJ. Radiationinduced DNA single-strand breaks in the intestinal mucosal cells of mice treated with the radioprotectors WR-2721 or 16-16 dimethyl

prostaglandin E2. International Journal of Radiation Biology and Related Studies in Physics, Chemistry, and

Medicine. 1987;**52**(1):67-76

2014;**306**(5):G404-G411

[82] Kantara C, Moya SM,

2015;**95**(11):1222-1233

Houchen CW, Umar S, Ullrich RL, Singh P, et al. Novel regenerative peptide TP508 mitigates radiation-induced gastrointestinal damage by activating stem cells and preserving crypt integrity. Laboratory Investigation.

[81] Sureban SM, May R, Qu D, Chandrakesan P, Weygant N, Ali N, et al. Dietary pectin increases intestinal crypt stem cell survival following radiation injury. PLoS One. 2015;**10**(8):e0135561

[80] Qu D, May R, Sureban SM, Weygant N, Chandrakesan P, Ali N, et al. Inhibition of notch signaling reduces the number of surviving Dclk1+ reserve crypt epithelial stem cells following radiation injury. American Journal of Physiology. Gastrointestinal and Liver Physiology.

## *DCLK1 and DNA Damage Response DOI: http://dx.doi.org/10.5772/intechopen.92327*

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

phosphorylation-dependent, ATPasecoupled chromatin remodeling during the DNA damage response. Cell Reports.

[61] Yang RM, Nanayakkara D, Kalimutho M, Mitra P, Khanna KK, Dray E, et al. MYB regulates the DNA damage response and components of the homology-directed repair pathway in human estrogen receptorpositive breast cancer cells. Oncogene.

2012;**2**(6):1657-1669

2019;**38**(26):5239-5249

[62] Colomer C, Margalef P,

Cell. 2019;**75**(4):669-682

2016;**7**(2):257

Villanueva A, Vert A, Pecharroman I, Sole L, et al. IKKalpha kinase regulates the DNA damage response and drives chemo-resistance in cancer. Molecular

[63] Chandrakesan P, Panneerselvam J, Qu D, Weygant N, May R, Bronze MS, et al. Regulatory roles of Dclk1 in epithelial mesenchymal transition and cancer stem cells. Journal of Carcinogenesis & Mutagenesis.

[64] Chandrakesan P, Weygant N, May R, Qu D, Chinthalapally HR, Sureban SM, et al. DCLK1 facilitates

[65] Chandrakesan P, Yao J, Qu D, May R, Weygant N, Ge Y, et al. Dclk1, a tumor stem cell marker, regulates prosurvival signaling and self-renewal of intestinal tumor cells. Molecular Cancer.

enhancing pluripotency and epithelial mesenchymal transition. Oncotarget.

[66] Mohammadi C, Najafi R. DCLK1 as a promising marker for radioresistance

Gastrointestinal Cancer. 17 August 2019. DOI: 10.1007/s12029-019-00292-z

in colorectal cancer. Journal of

[67] Kawamura D, Takemoto Y, Nishimoto A, Ueno K, Hosoyama T, Shirasawa B, et al. Enhancement of

intestinal tumor growth via

2014;**5**(19):9269-9280

2017;**16**(1):30

photoproducts during eukaryotic chromosomal DNA replication. Molecular Cell. 2013;**52**(4):566-573

[53] Heller RC, Marians KJ. Nonreplicative helicases at the replication

[54] Cerrato A, Morra F, Celetti A. Use of poly ADP-ribose polymerase [PARP] inhibitors in cancer cells bearing DDR defects: The rationale for their inclusion in the clinic. Journal of Experimental

[55] Ceccaldi R, Liu JC, Amunugama R, Hajdu I, Primack B, Petalcorin MI, et al. Homologous-recombinationdeficient tumours are dependent on Poltheta-mediated repair. Nature.

fork. DNA Repair (Amst).

& Clinical Cancer Research.

2015;**518**(7538):258-262

2015;**518**(7538):254-257

Medicine. 2018;**10**(1):101

[58] Minchom A, Aversa C, Lopez J. Dancing with the DNA damage response: Next-generation anti-cancer therapeutic strategies. Therapeutic Advances in Medical Oncology. 2018;**10**:1758835918786658

[59] Liu J, Zhang Q, Ruan B, Chen W,

Zheng J, Xu B, et al. MORC2 regulates C/EBPalpha-mediated cell differentiation via sumoylation. Cell Death and Differentiation.

[60] Li DQ, Nair SS, Ohshiro K, Kumar A, Nair VS, Pakala SB, et al. MORC2 signaling integrates

2019;**26**(10):1905-1917

[57] Francica P, Rottenberg S. Mechanisms of PARP inhibitor resistance in cancer and insights into the DNA damage response. Genome

[56] Mateos-Gomez PA, Gong F,

Sfeir A. Mammalian polymerase theta promotes alternative NHEJ and suppresses recombination. Nature.

Nair N, Miller KM, Lazzerini-Denchi E,

2007;**6**(7):945-952

2016;**35**(1):179

**90**

cytotoxic effects of gemcitabine by Dclk1 inhibition through suppression of Chk1 phosphorylation in human pancreatic cancer cells. Oncology Reports. 2017;**38**(5):3238-3244

[68] Lu Y, Maruyama J, Kuwata K, Fukuda H, Iwasa H, Arimoto-Matsuzaki K, et al. Doublecortin-like kinase 1 compromises DNA repair and induces chromosomal instability. Biochemistry and Biophysics Reports. 2018;**16**:130-137

[69] Suehiro Y, Takemoto Y, Nishimoto A, Ueno K, Shirasawa B, Tanaka T, et al. Dclk1 inhibition cancels 5-FU-induced cell-cycle arrest and decreases cell survival in colorectal cancer. Anticancer Research. 2018;**38**(11):6225-6230

[70] Bray FN, Simmons BJ, Wolfson AH, Nouri K. Acute and chronic cutaneous reactions to ionizing radiation therapy. Dermatology and Therapy. 2016;**6**(2):185-206

[71] Lam SY, Peppelenbosch MP, Fuhler GM. Prediction and treatment of radiation enteropathy: Can intestinal bugs lead the way? Clinical Cancer Research. 2019;**25**(21):6280-6282

[72] Harb AH, Abou Fadel C, Sharara AI. Radiation enteritis. Current Gastroenterology Reports. 2014;**16**(5):383

[73] Huang Y, Guo F, Yao D, Li Y, Li J. Surgery for chronic radiation enteritis: Outcome and risk factors. The Journal of Surgical Research. 2016;**204**(2):335-343

[74] Dainiak N. Hematologic consequences of exposure to ionizing radiation. Experimental Hematology. 2002;**30**(6):513-528

[75] Koukourakis MI. Radiation damage and radioprotectants: New concepts in the era of molecular medicine. The British Journal of Radiology. 2012;**85**(1012):313-330

[76] Citrin D, Cotrim AP, Hyodo F, Baum BJ, Krishna MC, Mitchell JB. Radioprotectors and mitigators of radiation-induced normal tissue injury. The Oncologist. 2010;**15**(4):360-371

[77] Haber AL, Biton M, Rogel N, Herbst RH, Shekhar K, Smillie C, et al. A single-cell survey of the small intestinal epithelium. Nature. 2017;**551**(7680): 333-339

[78] Hanson WR. Radiation protection of murine intestine by WR-2721, 16,16-dimethyl prostaglandin E2, and the combination of both agents. Radiation Research. 1987;**111**(2):361-373

[79] Hanson WR, Grdina DJ. Radiationinduced DNA single-strand breaks in the intestinal mucosal cells of mice treated with the radioprotectors WR-2721 or 16-16 dimethyl prostaglandin E2. International Journal of Radiation Biology and Related Studies in Physics, Chemistry, and Medicine. 1987;**52**(1):67-76

[80] Qu D, May R, Sureban SM, Weygant N, Chandrakesan P, Ali N, et al. Inhibition of notch signaling reduces the number of surviving Dclk1+ reserve crypt epithelial stem cells following radiation injury. American Journal of Physiology. Gastrointestinal and Liver Physiology. 2014;**306**(5):G404-G411

[81] Sureban SM, May R, Qu D, Chandrakesan P, Weygant N, Ali N, et al. Dietary pectin increases intestinal crypt stem cell survival following radiation injury. PLoS One. 2015;**10**(8):e0135561

[82] Kantara C, Moya SM, Houchen CW, Umar S, Ullrich RL, Singh P, et al. Novel regenerative peptide TP508 mitigates radiation-induced gastrointestinal damage by activating stem cells and preserving crypt integrity. Laboratory Investigation. 2015;**95**(11):1222-1233

**93**

**Chapter 6**

**Abstract**

ascorbate).

carnosine

**1. Introduction**

Oxidative Stress and Vanadium

*Gabriela Guerrero-Palomo, María Eugenia Cervantes-Valencia,* 

Air pollution is a worldwide health problem, and metals are one of the various air pollutants to which living creatures are exposed. The pollution by metals such as: lead, cadmium, manganese, and vanadium have a common mechanism of action: the production of oxidative stress in the cell. Oxidative stress favors the production of free radicals, which damage biomolecules such as: DNA, proteins, lipids, and carbohydrates; these free radicals produce changes that are observed in different organs and systems. Vanadium is a transition element delivered into the atmosphere by the combustion of fossil fuels as oxides and adhered to the PM enters into the respiratory system, then crosses the alveolar wall and enters into the systemic circulation. In this chapter, we will review the oxidative stress induced by vanadium—as a common mechanism of metal pollutants—; in addition, we will review the protective effect of the antioxidants (carnosine and

**Keywords:** air pollution, metals, vanadium, oxidative stress damage, ascorbate,

in the induction of toxic effects reported in the literature [3].

Air pollution is a worldwide concern because of the health problems associated with its uncontrolled emissions that affect all the biological systems. Within the wide range of pollutants, the suspended particles or particulate matter (PM) are of particular interest, which became more important since IARC listed them as carcinogens. The toxicity of PM is the consequence of the elements adhered to its surface [1]. An example of this are the particles generated by the combustion of fossil fuels and its derivatives, these particles usually consist of a carbon core on which complex mixtures of compounds are adhered, such as: polyaromatic hydrocarbons, toxins, sulfates, nitrates, and especially transition metals like vanadium, manganese, chromium, among others [2]. Metals are considered to play an important role

*Marcela Rojas-Lemus, Patricia Bizarro-Nevares,* 

*Nelly López-Valdez, Adriana González-Villalva,* 

*Otto Tavera-Cabrera, Norma Rivera-Fernández,* 

*and Teresa Fortoul-van der Goes*

*Brenda Casarrubias-Tabarez, Martha Ustarroz-Cano,* 

*Armando Rodríguez-Zepeda, Francisco Pasos-Nájera* 

## **Chapter 6**
