Role of Activated Cdc42-Associated Kinase 1 (ACK1/TNK2)-Inhibitors in Precision Oncology

Activated Cdc42-associated kinase 1 (ACK1) is an intracellular non-receptor tyrosine kinase referred to as TNK2, which is considered as an oncogene and therapeutic target in various cancers including breast cancer, non-small-cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), and many others. Oncogenic non-receptor tyrosine kinase mutations occur either due to point mutations, duplications or insertions and deletions, or by involving in the development of a fusion gene resulting from a chromosomal rearrangement. ACK1 is involved with multiple signaling pathways of tumor progression. With these signaling networks, ACK1 participates in cell survival, invasion, migration, and tumorigenesis that are strongly related to the prognosis and clinicopathology of cancers. Previous studies predicted that ACK1 is a carcinogenic factor and blockage of ACK1 inhibits cancer cell survival, proliferation, migration, and radiation resistance. FDA has approved many multi-kinase inhibitors as therapeutic drugs that show good inhibitory activity not against ACK1 but also towards multiple targets. As ACK1 is a key target for other neurological diseases, inflammation, and immunological diseases also, so the studies on these inhibitors not only provide potential strategies for the treatment of cancers that require simultaneous targeting of multiple targets but also can be used in drug repurposing for other diseases.


Introduction
Tyrosine kinases are enzyme family member which interpose the movement of the phosphate group to tyrosine residues of target protein, thus transmitting signals from the cell surface to cytoplasmic proteins and the nucleus to regulate physiological processes. TKs are divided in two sub groups: receptor and non-receptor proteins. Receptor tyrosine kinases (RTKs) include Platelet-derived growth factor receptors (PDGFR), Fibroblast growth factor receptor (FGFR), Epidermal growth factor receptor (EGFR), and Insulin receptor (IR). The Non-receptor TKs (NRTK) are divided in 9 sub-families based on the sequence similarities which included Abl, FES, JAK, ACK, SYK, TEC, FAK, SRC, and CSK. Activated Cdc42-associated kinase 1 (ACK1/TNK2) (PDB code-6VQM) is a non-receptor tyrosine kinase, which belongs to VIII tyrosine kinase family. There are seven different types of ACKs as, ACK1/TNK2, ACK2, DACK, TNK1, ARK1, DPR2 and KOS1 [1]. ACK1  [6].
ACK1 phosphorylates and activates key survival-promoting kinase receptors on different tyrosine residues and eliminates tumor suppressors through similar mechanisms, resulting in cell survival, proliferation, and migration. ACK1 can interact with several components of vesicle dynamics in cell endocytosis and trafficking. ACK1 plays an important role in promoting extrinsic apoptosis, intervene in mechanically-induced inhibition of growth and weaken mitogenic signals to avert the abnormal growth of tissues.
The physiological roles of ACK1 include both the cancer and the normal tissues. In cancer, ACK1 participates in the regulation of many signaling pathways and exerts corresponding physiological functions, which include proliferation, differentiation, survival, apoptosis, migration, and epidermal-mesenchymal transition (EMT) and influences several important cellular processes. ACK1 is frequently overexpressed in various aggressive tumors also. It was found that ACK1 is a molecular component of the signaling cascade of neurotrophins. It is highly expressed in human brain and plays important physiological function in inflammation and immune system. There are three ways to activate ACK1, which are RTK interaction, somatic cell missense mutation, and gene amplification. In previous studies, mutations in ACK1 genes have been observed in 21 kinds of cancers. 131 missense mutations, 39 nonsense mutations, and 3 fusion mutations are found in different regions of ACK1 [6]. The gene amplification of ACK1 is also observed in approximately 20 types of cancers. In cancers ACK1 is a key drug target of approximately 24 types of cancers as Metastatic Colorectal Cancer, Breast Cancer, Leukemia, Prostate Cancer, Melanoma, Gastric cancer, Lung cancer and many more. In one of RNA sequencing studies on Non-small Cell lung cancer (NSCLC) it was found that silencing of ACK1 upregulated several immune pathways as T cell receptor signaling, PI3K-Akt, Ras signaling pathways, MAPK, cAMP, Wnt signaling pathways. It was observed that ACK1 gene copy numbers were inversely linked with the infiltration levels of B cell, CD8+ T cell, CD4+ T cell, macrophage, neutrophil, and dendritic cells in NSCLC [25]. Studies showed that many ACK1 tyrosine kinase signaling proteins in many tumor cells are activated repeatedly in breast cancers and the expression of ACK1 is positively correlated to the disease

Representation of various exogenous and endogenous agents activating DNA damage checkpoints in cancers.
Chromatin alterations can also activate DNA damage signaling pathways. Activated checkpoint kinases, ATM or ATR arrest the cells at a specific stage in the cell cycle and allow time for repair. DNA double strand breaks due to ionizing radiation may be repaired either by the homologous recombinational repair pathway (HRR) or the non-homologous end joining pathway (NHEJ). Eukaryotic cells face a many situations, which lead to unstable genomic states, aberrant activity of the end joining proteins and mutations in the DNA and histone modifying enzymes. Small molecule inhibitors can be a therapeutic option to restore genome stability and also inhibiting tumor growth by radio sensitization. Adapted from Mahajan and Mahajan [24].
As ACK1 is highly expressed in many cancers and play a major role in tumor occurence, targeting ACK1 gives a promising strategy for tumor treatment. Interestingly, increased Cdc42-dependent Ack1 phosphorylation has been observed in cells depleted of dynamin, and in these cells, ACK1 showed enhanced binding of both endocytic and ubiquitylated proteins [37]. ACK1 has shown potential to overcome drug resistance and provide novel possibilities of drug combination schemes for targeted therapies in cancer treatment. Kinase Inhibitors as a major drug class were emerged after the FDA approval of imtinib in 2001. Till now there are 71 small-molecule FDA approved kinase inhibitors (SMKIs) and additional 16 SMKIs which are approved by other government authorities. In oncology, 110 novel kinases as a target are explored, for which 45 targets of approved kinase inhibitors are developed so far [38]. Small molecule inhibitors are discovered, designed and synthesized by researchers to target ACK1. Various methods as fragment-based drug design, high-throughput screening, repurposing, and skeleton transitions are used for this purpose. Many inhibitors exhibited favorable pharmacokinetic activities and good anticancer activity, which can be used for clinical treatment of cancers. These drugs can be divided as (a) Selective Inhibitors, (b) Multikinase Inhibitors, and (c) Combination Drugs.
The chemical structures of few selective drugs are given in Figure 4. Compound 1 having IC 50 (24 nM), is used to suppress pan cancer cells [39] through PTEN/ AKT/mTOR signaling pathways [40]. Compound 2 and 3 has hindrance activity for ACK1. It was observed that the ACK1 inhibitory ability was not higher in Compound 4. Compound 5 is also a suitable drug with good pharmacokinetic properties. Compound 6 is a fragment based drug design with low water solubility. Though Compound 7, 8, 9, 10 have low pharmacokinetic activities, they can be used to provide reference to develop novel inhibitors for mutations in ACK1 tumors. Many other studied inhibitors are Pyrrolo [2,3-d]pyrimidine, Pyrazolopyrimidine, Imidazopyrazine and their derivatives [6].
As these drugs alone are not enough for the survival, so advances are made on the combination therapies for effective cancer treatment. The studied inhibitors showed better results when used in combination with other drugs.

Challenges
Though ACK1 is a therapeutic target for cancers, inflammation, immune and neurological diseases, very few inhibitors have entered the clinical trials. Hence there is urgent need to develop potential inhibitors. The in vivo pharmacokinetic properties of inhibitors also need to be improved. Some inhibitors have limited solubility in water which restricts the studies to be carried out on the animal models only. Due to the large distribution and participation of ACK1 in regulation of many signaling pathways, high specificity and precise positioning of inhibitors to diseased tissues are required, which increases the difficulty in drug designing. So, it is necessary to explore more biological functions of ACK1 and to verify the effectiveness of drugs in vivo and in vitro. As the inhibitors are developed by only limited methods (screening small molecules and fragment libraries), it have weak affinities which makes the selection of drug candidates difficult and time consuming. Further the development of allosteric inhibitors of ACK1 is also difficult as  (7), , benzopyrimidodiaze-pinone derivatives ((9), (10)). Adapted from Aoxue Wang et al. [6].  (6), ceritinib (7), PD158780 (8), vemurafenib (9), ADZ9291 (10), sunitinib (11), flavopiridol (12), and gefitinib (13). Adapted from Srivastava [41].
Role of Activated Cdc42-Associated Kinase 1 (ACK1/TNK2)-Inhibitors in Precision Oncology DOI: http://dx.doi.org /10.5772/intechopen.102343 it need full-length proteins in the biochemical analysis of ACK1, which is a great challenge as these proteins may exhibit aggregation, conformational changes and other phenomena, which are not possible for the in vivo and in vitro studies. In last 10 years, innovative immunochemotherapies have shown promising results in disease control rates but not survival. So, there is an acute need to develop novel drugs that can target dysregulated pathways in malignant tumors. Several functional challenges include the description of genetic abnormalities in the cancer kinomes and the recognition of accurate drivers which are accountable for tumor development. Only the precise analysis of the therapeutic involvement will indicate the clear role of kinases; as a tumor suppressor in non-cancer cells or a tumor mediator in cancer cells.

Application of inhibitors in drug repurposing
In oncology, repurposing of drugs means the reuse of already existing drugs to treat cancer rather than testing new drugs for the existing symptoms with malignancies. Introducing new drugs is a very time-consuming and costly process which requires many pre-clinical trials before its use for the commercial purposes. The existing drugs have a huge potential with untapped agents, which are clinically more relevant for disease treatment. More than 200 existing used off patent drugs have shown some evidence for anti-cancer treatment. Since these FDA approved drugs are not in larger number, it is better to repurpose the existing drugs for therapeutic purposes. These drugs can be repurposed for not only cancer treatment but also in rheumatoid arthritis and other disorders. Interestingly multikinase inhibitors are used to interact simultaneously many targets, these drugs can play an important role in drug repurposing for treatment of different diseases.

Future perspectives
Now it is well established that ACK1 is a promising target for tumor therapy and the clinical studies show that there is a strong correlation between the expression level of activated ACK1 and prognosis and progression of cancers. Six specific inhibitors with high affinity for ACK1 has been identified which showed potential inhibitory activity. Some inhibitors also showed good pharmacokinetics properties in vivo. It has been observed that light-controlled PROTACs degrade specific proteins at certain locations in the body, so novel ACK1 inhibitors could have a local impact on pathologic tissues by light control. Fortunately, immunotherapy has been considered as an alternative tool for cancer patients. The treatment included many checkpoint inhibitors as nivolumab, pembrolizumab, and atezolizumab. Many other inhibitors as dasatinib, nilotinib, bosutinib along with imatinib mesylate has also used as chemotherapeutic agent for treatment in chronic myeloid leukemia (CML) patients. Considering these problems, Allosteric inhibitors, inhibitors targeting different structural domains of ACK1, inhibitors having blocking interactions within proteins, Proteolysis targeting chimeras (PROTACs), Combination therapies and dual-target drug complexes need to be develop in future. Moreover, many ACK1 interacted proteins or substrates need to be identified which can be utilized for precision medicine in cancer patients. The implementation of bioinformatics based methodologies as structure based drug designing can definitely help in drug delivery precision medicine for cancers. Refinement of effective compound screening and profiling technologies, and natural compounds need to be explored to reduce the off-target toxicity. Allosteric and covalent inhibitors, and targeted

Author details
Ruby Srivastava Bioinformatics, CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India *Address all correspondence to: amitruby1@gmail.com degraders such as PROTACs and molecular glues will be the next players of kinase drug discovery in future.