**3.1 Current therapeutics used to treat cancers with constitutive NF-κB activity**

The NF-κB pathway is widely considered an attractive therapeutic target in a broad range of cancers. Yet, despite the efforts to develop NF-κB inhibitors, none has been clinically approved. This is largely due to immune-related toxicities associated with global NF-κB suppression [51]. Furthermore, the high complexity of the NF-κB signaling network presents another unique challenge for developing specific NF-κB inhibitors. To further complicate matters, some standard anticancer agents can inadvertently activate the NF-κB pathway via induction of proinflammatory cytokines such as IL-1β and TNF-α and cellular stressors such as reactive oxygen species (ROS), or by activating DNA-repair mechanisms [52]. Finally, constitutive NF-κB activity can also be achieved via secreted cytokines and chemokines from inflammatory cells within the tumor microenvironment [52]. Taken together, consideration of all these factors is imperative when strategizing the development of the most effective and least toxic anticancer agents.

Currently, the use of NF-κB inhibitors has mainly been combined with other agents [47, 50]. Some of these combinatorial therapies have shown promising effectiveness and have been made it as far as the clinical trial phase. For example, combination of irinotecan with the proteasome and NF-κB inhibitor bortezomib was shown to increase sensitivity of colon cancer cells to irinotecan [53]. A separate study showed that bortezomib could sensitize non-small cell lung cancer (NSCLC) cells to sodium butyrate, which acts to inhibit histone deacetylases [54]. Moreover, several clinical trials testing the efficacy of inhibitors against IKK to target solid tumors have been undertaken. For example, perturbation of IKKβ with the inhibitory ML120B led to synergistic enhancement of vincristine cytotoxicity in lymphoma. These results implicate IKK disruption using inhibitors as a useful adjunct therapy with standard chemotherapeutics. Other attempted trials using IKK inhibitors, such as CHS-828, EB-1627, and IMD-1041, as single or combinatorial agents unfortunately produced toxicity concerns for patients [55–57].

Other examples of combinatorial therapies include the use of NF-κB inhibitors Bay11-7082 and sulfasalazine in combination with more commonly used

**53**

*Phosphorylation of NF-κB in Cancer*

regulate NF-κB in various cancers.

combinatorial therapies.

kinases themselves.

**3.2 Benefits and pitfalls for targeting kinases in cancers**

*DOI: http://dx.doi.org/10.5772/intechopen.83650*

chemotherapeutics such as 5-fluorouracil and cisplatin to synergistically reduce colon cancer cell growth [58]. Other indirect means of targeting NF-κB such as inhibition of upstream kinases have also shown promise. For instance, one study using pancreatic cancer cells showed that inhibition of GSK3β by a small molecule inhibitor reduced phosphorylation of p65 at S536 resulting in decreased NF-κB activity and cell growth [59]. Another study with the chemical compound ursolic acid showed reduced p65 phosphorylation via inhibition of IKKβ, which impaired overall cell growth in leukemia cell lines [60]. Other studies with proteasome inhibitors, including Tosyl phenylalanyl chloromethyl ketone (TPCK) and Tosyl-Llysyl-chloromethane hydrochloride (TLCK), demonstrated that not only do these inhibitors target IKKβ, but they were also able to reduce overall phosphorylation levels of NF-κB [61, 62]. In summary, these studies suggest there may be many benefits to targeting hyperactive NF-κB signaling and, in particular, the kinases that

The development of small-molecule kinase inhibitors for the treatment of cancer has continued to be of intense interest. Notably, many inhibitors have received FDA approval with approximately another 150 are in preclinical and clinical phase trials. Despite these important advances, many factors have confounded the clinical efficacy of these kinase-targeted drugs including the challenges of tumor heterogeneity and microenvironment as well as the emergence of mutations that confer drug resistance. Another major challenge of kinase inhibition is that of the development of adverse side effects. Some classic examples of this include dermatologic complications and cardiotoxicity associated with inhibition of EGFR and vascular endothelial growth factor receptor (VEGFR), respectively. Furthermore, there is an urgent need to develop relevant models of resistance in response to kinase inhibitors in efforts to overcome this resistance via potential synergistic

Another critical issue facing clinical trial design with kinase-targeted agents is that of determining the types of tumors that are most likely to respond to specific kinase inhibitors and thus identify the subsets of patients who will likely benefit from these treatments. To combat this issue, many studies have been dedicated to identifying certain "kinase dependencies" in cancer cells that would make them more susceptible to inhibition. These so-called dependencies are primarily based on the existence of constitutively activate kinases achieved by gene mutation, amplification, or fusion. Among the potential approaches to identifying signatures of kinase dependency are proteomic profiling, next-generation sequencing and various applications utilizing phospho-specific antibodies against numerous specific kinase substrates. Additional mechanisms of kinase dependency include impairment of the function of phosphatases, the negative regulators of phosphorylation as is the case with mutations in the phosphatase and tensin homolog (PTEN) tumor suppressor gene. The consequence of *PTEN* loss is signal propagation through downstream kinases such as Akt. Moreover, growing evidence from isogenic human and mouse models also suggests that this type of indirect avenue of kinase dependency may be analogous to direct, activating mutations in the

Finally, there are also cases in which the beneficial effect of a kinase inhibitor is counteracted by an additional genetic lesion in a compensatory signaling pathway. Therefore, studies to identify such secondary events are urgently needed. Taken together, these evidences underscore the critical need to optimize the use of kinase inhibitors against cancers by continued detailed molecular characterization

### *Phosphorylation of NF-κB in Cancer DOI: http://dx.doi.org/10.5772/intechopen.83650*

*Adenosine Triphosphate in Health and Disease*

melphalan- or doxorubicin-resistant cells [50].

of the most effective and least toxic anticancer agents.

**3. p65 modifying kinases as potential therapeutic targets**

**3.1 Current therapeutics used to treat cancers with constitutive NF-κB activity**

The NF-κB pathway is widely considered an attractive therapeutic target in a broad range of cancers. Yet, despite the efforts to develop NF-κB inhibitors, none has been clinically approved. This is largely due to immune-related toxicities associated with global NF-κB suppression [51]. Furthermore, the high complexity of the NF-κB signaling network presents another unique challenge for developing specific NF-κB inhibitors. To further complicate matters, some standard anticancer agents can inadvertently activate the NF-κB pathway via induction of proinflammatory cytokines such as IL-1β and TNF-α and cellular stressors such as reactive oxygen species (ROS), or by activating DNA-repair mechanisms [52]. Finally, constitutive NF-κB activity can also be achieved via secreted cytokines and chemokines from inflammatory cells within the tumor microenvironment [52]. Taken together, consideration of all these factors is imperative when strategizing the development

Currently, the use of NF-κB inhibitors has mainly been combined with other agents [47, 50]. Some of these combinatorial therapies have shown promising effectiveness and have been made it as far as the clinical trial phase. For example, combination of irinotecan with the proteasome and NF-κB inhibitor bortezomib was shown to increase sensitivity of colon cancer cells to irinotecan [53]. A separate study showed that bortezomib could sensitize non-small cell lung cancer (NSCLC) cells to sodium butyrate, which acts to inhibit histone deacetylases [54]. Moreover, several clinical trials testing the efficacy of inhibitors against IKK to target solid tumors have been undertaken. For example, perturbation of IKKβ with the inhibitory ML120B led to synergistic enhancement of vincristine cytotoxicity in lymphoma. These results implicate IKK disruption using inhibitors as a useful adjunct therapy with standard chemotherapeutics. Other attempted trials using IKK inhibitors, such as CHS-828, EB-1627, and IMD-1041, as single or combinatorial agents unfortunately produced toxicity concerns for patients

Other examples of combinatorial therapies include the use of NF-κB inhibitors Bay11-7082 and sulfasalazine in combination with more commonly used

chemoresistance have been extensively reviewed by others such as Li, Sethi, and Godwin et al., which will not be further discussed in this chapter [45, 46]. However, the specific contribution of dysregulated p65 phosphorylation to chemoresistance is less well understood and requires further exploration. Nonetheless, a few reports suggest that upstream kinases involved in chemoresistance can modulate p65 phosphorylation levels in this context. For instance, siRNA-mediated depletion of IKKα in HT1080 human fibrosarcoma cells was shown to decrease phosphorylation of p65 in response to doxorubicin, thus severely impairing the ability of doxorubicin to initiate NF-κB DNA-binding activity. These findings suggest that IKKα plays a critical role in NF-κB-mediated chemoresistance in response to doxorubicin and potentially serves as a therapeutic target for improving chemotherapeutic response [47]. Other studies have shown that p65, in a hyperphosphorylated state, can be correlated with resistance to thymidylate synthases and irinotecan in stomach and colon cancers, respectively [44, 48, 49]. Doxorubicin resistance in lung cancer has also been correlated with p65 S536 phosphorylation states [47]. Additionally, multiple myelomas have exhibited increased p65 S536 phosphorylation within

**52**

[55–57].

chemotherapeutics such as 5-fluorouracil and cisplatin to synergistically reduce colon cancer cell growth [58]. Other indirect means of targeting NF-κB such as inhibition of upstream kinases have also shown promise. For instance, one study using pancreatic cancer cells showed that inhibition of GSK3β by a small molecule inhibitor reduced phosphorylation of p65 at S536 resulting in decreased NF-κB activity and cell growth [59]. Another study with the chemical compound ursolic acid showed reduced p65 phosphorylation via inhibition of IKKβ, which impaired overall cell growth in leukemia cell lines [60]. Other studies with proteasome inhibitors, including Tosyl phenylalanyl chloromethyl ketone (TPCK) and Tosyl-Llysyl-chloromethane hydrochloride (TLCK), demonstrated that not only do these inhibitors target IKKβ, but they were also able to reduce overall phosphorylation levels of NF-κB [61, 62]. In summary, these studies suggest there may be many benefits to targeting hyperactive NF-κB signaling and, in particular, the kinases that regulate NF-κB in various cancers.
