**5. Emerging kinase targets**

pharmacogenetics, the tumor's genomics and/or proteomics, and the tumor's response to drug exposure. Patient pharmacogenetics refers to the patients genetics that are relevant to effects on pharmacokinetics of drugs, for example, polymorphisms in drug metabolizing enzymes. Tumor genomics and/or proteomics refers to specific genetic abnormalities that effect specific gene expression and is thought to contribute to tumor development or maintenance. The application of any or all of these pieces of information may improve the outcome of pharma‐

The application of pharmacogenomics to personalized therapy can be exemplified by appli‐ cation to tamoxifen therapy for the breast cancer patient. Tamoxifen acts as an anti-estrogen on breast tissue and is used for treatment of estrogen receptor positive (ER+) breast cancer. It has been determined that tamoxifen is converted in vivo to 4-hydroxytamoxifen, which is a much more active anti-estrogen agent. It was subsequently found that this transformation is accomplished by the drug metabolizing enzymes cytochrome P-450 (CYP) isofroms 2D6 and 2C19 [31]. Patients that were receiving tamoxifen therapy and concurrently receiving selective serotonin reuptake inhibitor (SSRI) drugs, known to inhibit CYP 2D6, experienced poor outcomes because of the failure to convert tamoxifen to 4-hydroxtamoxifen. A significant percentage of the population carry a genetic polymorphism in the CYP 2D6 gene which results in poor metabolism with respect for the CYP 2D6 isoform and will not receive the full benefit of tamoxifen therapy [32]. Consequently a readily available genomic test is available for patients that can identify those who are not good candidates for tamoxifen therapy, because

they have a specific 2D6 polymorphism, so that they can be directed to alternatives.

33% of non-responders also had EGFR amplification.

Advances in genomics and proteomics have enabled selection of patients that may benefit from targeted therapies for certain tumor types. Analysis of tumor cells on the protein level using immunohistochemistry (IHC) can identify cells that express relevant protein targets. An example of the application of this approach is the detection of c-erbB2 (Her2/Neu) receptor in breast cancer patients which can direct them toward trastuzumab therapy. Specific gene mutations in tumor cells may be detected using techniques of RT-PCR, DNA microarray, and fluorescence in situ hybridization (FISH) that can be used to direct patients to therapy that may be beneficial. For example, a study demonstrated the utility of screening non-small cell lung cancer patients for amplification of the epidermal growth factor receptor (EGFR). Patients with amplified EGFR receiving an EGFR kinase inhibitor (gefitinib) had longer median progression free survival (PFS) and overall survival (OS), compared to those who did not have amplified EGFR [33, 34]. Furthermore, of those patients who responded to gefitinib therapy 77% had EGFR gene amplification, whereas only 33% of non-responders had EGFR gene amplification. This study demonstrates the utility of identifying patients with mutations that drive tumor growth and survival and matching them with appropriate targeted therapy. The study also highlights the shortcoming of single gene determination directed therapy in that

A potentially more invasive and perhaps technically challenging approach is to directly test samples of the patient's tumor against available drugs. This approach of using personalized xenografts to direct patient therapy was demonstrated in a patient with advanced pancreatic patient [35]. After initial surgery metastases were discovered and adjuvant gemcitabine

cotherapy for the patient.

24 Updates on Cancer Treatment

Because kinase enzymes are involved in numerous biological processes it is not surprising that many of them have become therapeutic targets for various disease sates including cancers. As noted above, drug development of kinase inhibitors for oncology indications has accelerated in the last decade with no sign of slowing. This acceleration has been spurred by emergent technologies and advances in molecular and systems biology, proteomics, and genomics. The continued advancement of understanding of the molecular changes that occur in the devel‐ opment of a cancer has helped identify likely therapeutic targets. Some of the more recently identified targets include the BTK, CDK8, and DNA-PK.

Although a Bruton's tyrosine kinase (BTK) inhibitor (ibrutinib) was recently approved for clinical use, BTK is a relatively new kinase target. BTK plays a role in B-cell receptor signaling, proliferation, differentiation, and survival. Ibrutinib works by irreversible inhibition of BTK through covalent modification of the enzyme. In a clinical trial in mantle cell lymphoma (MCL) patients, who had three prior therapies, an overall response rate of 69% and a progression free survival of 13.9 months were observed. Ibrutinib is approved for second line treatment of MCL and chronic lymphocytic leukemia (CLL) [38].


**Table 2.** US FDA Cleared or Approved In Vitro Companion Diagnostic Tools for Kinase Inhibitors.

Cyclin dependent kinase 8 (CDK8) was recently identified as a colorectal cancer oncogene [39]. It was discovered that 47% of colorectal cancer samples demonstrated copy number gain of the chromosomal region carrying the CDK8 gene. It has also been shown that 76% of colorectal cancers showed positive expression of nuclear CDK8, and that increased positive expression rate correlated with increased clinical stage [40]. Additionally, expression of CDK8 trans‐ formed NIH 3T3 cells, however a kinase inactive version did not. Experimental knockdown of CDK8, in colorectal cancer cell lines with high CDK8 expression levels, induced a decrease in cell proliferation. While most CDKs regulate cell cycle progression CDK8 and some others play a role in transcription regulation. Assembly of CDK8 with other key protein partners forms a mediator complex which can activate β-catenin dependent transcription. Furthermore it was shown that CDK8 function represses E2F1 activity, a known negative regulator of βcatenin [41]. Of note is the fact that knockdown of CDK8 in some colorectal cancer cell lines does not completely diminish β-catenin levels, which suggests that other genetic determinants of resistance must be identified to exclude patients that would not benefit from a future CDK8 targeted therapeutic.

DNA-dependent protein kinase (DNA-PK) is another emerging oncology kinase target. Being that the development of cancer is due to genomic instability that results in the acquiring of genetic mutations that drive tumor formation, it should be no surprise that tumor cells may have defective DNA repair pathways that result in a mutator phenotype. It is no wonder that the first class of anti-cancer drugs were DNA alkylating agents that cause irreparable damage to DNA of tumor cells, although with severe side effects and risk of secondary tumors arising from DNA damaged normal cells. A less devastating targeted approach to induce irreparable DNA damage in tumor cells, while sparing normal cells, is to target still operating DNA repair pathways that are critical to the tumor cells survival. In other words disable a DNA repair pathway that will synergize with an already disabled pathway. For example, DNA-PK is critical for repairing double strand DNA breaks through the non-homologous end joining (NHEJ) pathway. A second pathway for repairing double strand breaks is the homologous recombination pathway, which is impaired in tumor cells with mutations in genes such as BRCA1 and BRCA2 as well as others. Targeting tumors with impaired homologous recombi‐ nation by inhibiting the NHEJ pathway with DNA-PK inhibitors should, in theory, cause activation of cell death pathways as double strand DNA-breaks build-up in the cells [42, 43]. Normal cells should be able to withstand the assault due to both pathways being operable.

## **6. Summary**

survival of 13.9 months were observed. Ibrutinib is approved for second line treatment of MCL

afatinib Therascreen EGFR RGQ PCR Kit Detects EGFR exon 19 deletions and

dabrafenib and tramatenib THxID BRAF Kit Detection of BRAF(V600E) and (V600K)

erlotinib Cobas EGFR Mutation Test Detects EGFR exon 19 deletions and

imatinib mesylate DAKO C-KIT PharmDx Quantitative c-Kit detection in GIST

vemurafenib COBAS 4800 BRAF V600 Mutation TestDetection of BRAF (V600E) mutation in

Cyclin dependent kinase 8 (CDK8) was recently identified as a colorectal cancer oncogene [39]. It was discovered that 47% of colorectal cancer samples demonstrated copy number gain of the chromosomal region carrying the CDK8 gene. It has also been shown that 76% of colorectal cancers showed positive expression of nuclear CDK8, and that increased positive expression

**Table 2.** US FDA Cleared or Approved In Vitro Companion Diagnostic Tools for Kinase Inhibitors.

exon 21 substitution mutations. Intended for selection of NSCLC patients who may benefit from afatinib

Prescription use only, intended for selection of NSCLC patients eligible for

mutations in melanoma tissue. Intended to aid selection of melanoma patients who may benefit from treatment with dabrafenib and/or

exon 21 substitution mutations. Intended for selection of patients with metastatic NSCLC who may benefit

and normal tissues. Intended to aid selection of GIST patients who may be eligible for imatinib mesylate

melanoma. Intended to aid selection of melanoma patients who may benefit

from vemurafenib therapy.

from afatinib therapy.

Detection of ALK gene rearrangements.

crizotinib treatment.

trametinib.

treatment.

therapy.

**Drug (generic name) Diagnostic Product Diagnostic Product Use**

and chronic lymphocytic leukemia (CLL) [38].

26 Updates on Cancer Treatment

crizotinib VTSIS ALK Break Apart FISH Probe Kit

> Kinase inhibitors have enormous potential to facilitate improved outcomes for some cancer patients especially in the context of personalized medicine. The personalized approach that implements genomic analysis to identify potential driver kinases in patient tumor samples will enable matching the patient with the best kinase inhibitor for the best outcome. The identifi‐ cation of drug resistant mutations in the targeted kinase will be critical in order to avoid treatment that is not likely to be beneficial to the patient. Also critical, is the identification of relevant polymorphisms in drug metabolizing enzymes and transporters that may affect pharmacokinetics of the kinase inhibitor, in order to assure adjustments are made to achieve optimum drug exposure. These considerations along with proper management of side effects can maximize patient benefit from targeted personalized cancer therapy with kinase inhibitors.

#### **Nomenclature**

ALL-acute lymphoblastic leukemia; ASM-aggressive systemic mastocytosis; CLL-chronic lymphocytic leukemia; CML-chronic myeloid leukemia; CRC-colorectal cancer; DTC-differ‐ entiated thyroid carcinoma; GIST-gastrointestinal stromal tumor; IPF-idiopathic pulmonary fibrosis; MTC-medullary carcinoma of the thyroid; NSCLC-non-small-cell lung cancer; RCCrenal cell carcinoma.
