PTKs are enzymes that transfer the y-phosphate group of ATP to tyrosine residues on target substrate proteins. They are a large superfamily of enzymes comprising both transmembrane-spanning receptors with an intrinsic tyrosine kinase activity in their cytoplasmic domains and subfamilies of cytoplasmic tyrosine kinases such as (JAK) families.
Tyrosine phosphorylation is a covalent modificaiton of proteins. It provides a rapid mechanism of modifying the enzymic activity of target proteins. In addition, it can modify the ability of target proteins to act as adaptor molecules to recruit other signaling molecules. For example, the tyrosine phosphorylation of receptors or signaling molecules creates docking sites that allow protein-protein interactions leading to recruitment of downstream signal transducers. Signal transducers are recruited to these phosphorylated tyrosines by protein-protein interactions domains, called WSH2 domains, contained with the sequence of many siganl transducers. SH2 domains comprise about 100 amino acids and spcifically recognize a phosphotyrosime plus the 3 amino acides immediatley C-terminal to that phosphotyrosine.
Tyrosine phosphorylation is reversible by the action of protein tyrosine phosphatases.
Types of Protein Tyrosine Kinases:
Bruton’s Tyrosine Kinase (BTK): is a member of the Tec family of non-receptor tyrosine kinases that is critically important for the growth, differentiation and activation of B cells, myeloid cells and mast cells. The BTK gene is located at cytogenetic band Xq21.22-q22 and includes 19 exons, spanning 37 kb, encoding the full lenght BTK protein. BTK is essential to B cell recetpor (BCR signaling and in knockout mouse models, its mutations has a B cell specific phenotype. The centrol role of BTK in B cell funciton is underscored by the human disease X-linked agammaglobulinemia or Bruton’s agammaaglobulinemia, which is caused by loss of fucntion mutations in BTK. These mutations result in the vitual absence of all B cells and immunoglboulins, leading to recurrent bacterial infections. (Maher Albitar, Neogenomics, US Patent No: 10,253,370).
BTK protein and mRNA are significantly overexpressed in chronic lymphocytic leukemia (CLL) compared with normal B cells. BTK activity is involved in the disease progression of B cell malignancies, such as Non-Hodgkin’s Lymphomas, such as chronic lymphocytic leukemia (CLL), mantel cell leukemia (MCL), follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) and muyltiple myeloma (MM). (Maher Albitar, Neogenomics, US Patent No: 10,253,370).
BTK is activated by membrane localizaiton stimulated by PIP3 (phosphatidlinositol-3,4,5-triphosphate) generation and bonding to the BTK pleckstrin homology (PH) domain, and transphosphorylation of Try-551 by Src family kinases. Activated BTK is involved in the phosphorlation of a number of signlaing molecuels involved int he PLCy (phospholipase c gamma), NJK (c-Jun NH2 temrinal kinase) and p38 MAPK pathways, leading to Ca2+ mobilization, mRNA stabilization and the induction of NF-kB and AP-1 transcription factors. BTK activity is negatively regulated by a number of proteins including inhibitor of BTK (IBTK), Sab and c-Cbl. During antigenic challenge, the classical NF-kB pathway is strongly activated by B cell receptor signaling, via formation of a “CBM” signaling complex consisting of CARD11, MALT1, and BCL10. The CBM lies downstream of PLCy activaiton of BTK. The CBM pathway is pathologically altered in several lymphoma subtypes; mutations in CARD11 have been found to constituively activate down-stream NK-kB signaling. (Maher Albitar, Neogenomics, US Patent No: 10,253,370).
Ibrutinib (Unbryvuca) is a potent covalent kinase inhibitor that targets BTK, binding coavlaently to Cys-481 in the activate site of BTK, resulting in inhibition of kianse activity with IC50 0.5 nM. It is a small molecule drug that inhibits B-cell prolifreration and survival by irreversibly binding the protein Bruton’s tyrosine Kinase (BTK) which inhbitors the B-cell receptor pathway, which is often aberrantly active in B cell cancers. BTK inhibitors like ibrutinib have demonstrated high clinical response rates and durable remissions in patients with chronic lymphocytic leukemia (CLL) including refractory patients to conventional therapy or patients with tumor protein p53 (TP53) mutations. Patients who develop resistance to ibrutinib therapy typically have mutations in either BTK or phospholipase c y 2 (PLCy2). Mutations in BTK at the C481S hotspot alter the BTK binding stie rendering it reversible to binding ibrutinib resulting in ineffective therapetuic resutls. (Maher Albitar, Neogenomics, US Patent No: 10,253,370).
Maher Albitar, Neogenomics, (US Patent No: 10,253,370) discloses a method for screening and/or monitoring a patient for a BTK inhibitor resistant mutation which includes isolating DNA from a sample selected from bone marrow aspirate, fresh peripheral blood and tissue, performing PCR on the isoalted DNA to produce amplified DNA while blocking amplification of wild type DNA in a portion of the isolated DNA that encodes a BTK polypeptide and a portion of the isolated DNA that encodes a PLCy2 polypeptide, sequencing the amplified DNA in an automated sequencer and analyzing and output of the automated sequencer to identify mutations in the sequence. In some embodimetns, amplification of WT DNA is blocked by a synthetic nucleotide comprising a locked nucleic acid (LNA) ofr a bridged nucleic acid (BNA). The locked nucleic acids (LNA) or bridged nucleic acids (BNA) are used to block amplificaton of WT DNA in a poriton of the isoalted DNA that encodes a BTK polypeptide and a portion of the isoalted DNA that encoes a PLCy2 polypeptide. The ethod improves sensitivity of NGS analysis by enriching the mutant DNA and reducing the relative ratio of the WT DNA in the anlyzed sample through selective sequencing. In an exemplary emobdiment, a locked nucleic acid probe that is identical to the WT is sued to block the WT DNA amplificaiton while the mutant DNA is enriched for sequencing using amplion-based NGS procedure.