Characterization of All Possible Single‐Nucleotide Change Caused Amino Acid Substitutions in the Kinase Domain of Bruton Tyrosine Kinase

Knowledge about features distinguishing deleterious and neutral variations is crucial for interpretation of novel variants. Bruton tyrosine kinase (BTK) contains the highest number of unique disease‐causing variations among the human protein kinases, still it is just 10% of all the possible single‐nucleotide substitution‐caused amino acid variations (SNAVs). In the BTK kinase domain (BTK‐KD) can appear altogether 1,495 SNAVs. We investigated them all with bioinformatic and protein structure analysis methods. Most disease‐causing variations affect conserved and buried residues disturbing protein stability. Minority of exposed residues is conserved, but strongly tied to pathogenicity. Sixty‐seven percent of variations are predicted to be harmful. In 39% of the residues, all the variants are likely harmful, whereas in 10% of sites, all the substitutions are tolerated. Results indicate the importance of the entire kinase domain, involvement in numerous interactions, and intricate functional regulation by conformational change. These results can be extended to other protein kinases and organisms.     

BTK inhibitors synergise with 5-FU to treat drug-resistant TP53-null colon cancers

Colorectal cancer (CRC) is the fourth cause of death from cancer worldwide mainly due to the high incidence of drug-resistance. During a screen for new actionable targets in drug-resistant tumours we recently identified p65BTK – a novel oncogenic isoform of Bruton's tyrosine kinase. Studying three different cohorts of patients here we show that p65BTK expression correlates with histotype and cancer progression. Using drug-resistant TP53-null colon cancer cells as a model we demonstrated that p65BTK silencing or chemical inhibition overcame the 5-fluorouracil resistance of CRC cell lines and patient-derived organoids and significantly reduced the growth of xenografted tumours. Mechanistically, we show that blocking p65BTK in drug-resistant cells abolished a 5-FU-elicited TGFB1 protective response and triggered E2F-dependent apoptosis. Taken together, our data demonstrated that targeting p65BTK restores the apoptotic response to chemotherapy of drug-resistant CRCs and gives a proof-of-concept for suggesting the use of BTK inhibitors in combination with 5-FU as a novel therapeutic approach in CRC patients. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Selective role of PKCbeta enzymatic function in regulating cell survival mediated by B cell antigen receptor cross-linking

Cross-linking of the B cell antigen receptor (BCR) results in the activation of several protein tyrosine kinases leading to phospholipase C-gamma2-dependent phospholipid hydrolysis and Ca2+ mobilization, followed by activation of the protein kinase C (PKC) family members. Sustained Ca2+ release in B lymphocytes is dependent on the membrane localization and activation of the protein tyrosine kinase BTK. Ca2+ release is a tightly regulated process involving BTK membrane localization through its phosphorylation by PKCbeta. A selective role of PKCbeta in B cell signaling was first revealed by the characterization of PKCbeta knockout mice, which displayed decreased B cell proliferation in response to various mitogenic stimuli. However, it is not clear whether the B cell defects displayed by the PKCbeta knockout mice are due a B cell developmental defect or the scaffolding function of PKCbeta, resulting in a defect in the recruitment or formation of signal transducing complex molecules. Thus, in this report we investigated the effects of pharmacologic inhibition of the catalytic function of PKCbeta on B cell survival and growth. Treatment of Daudi B lymphoma cell line with a selective PKCbeta inhibitor, LY333531, inhibited anti-IgM-induced phosphorylation of BTK on Ser180 in a concentration-dependent manner, which was concomitant with an increase in BTK activation, and Ca2+ mobilization. In primary splenic B cells, LY333531 inhibited BCR-induced B cell proliferation, but did not affect basal or LPS-induced proliferation. Finally, LY333531 treatment resulted in the induction of apoptosis of anti-IgM-activated B cells, which corroborated with their inability to up-regulate pro-survival factors, Bcl-X(L) and Bcl-2. These results support the important and selective role of the PKCbeta enzymatic function in controlling Ca2+ release during BCR signaling leading to B lymphocyte survival and growth.     

Inhibitors of BTK and ITK: State of the New Drugs for Cancer,Autoimmunity and Inflammatory Diseases

BTK and ITK are cytoplasmic tyrosine kinases of crucial importance for B and T cell development, with loss‐of‐function mutations causing X‐linked agammaglobulinemia and susceptibility to severe, frequently lethal, Epstein–Barr virus infection, respectively. Over the last few years, considerable efforts have been made in order to develop small‐molecule inhibitors for these kinases to treat lymphocyte malignancies, autoimmunity or allergy/hypersensitivity. The rationale is that even if complete lack of BTK or ITK during development causes severe immunodeficiency, inactivation after birth may result in a less severe phenotype. Moreover, therapy can be transient or only partially block the activity of BTK or ITK. Furthermore, a drug‐induced B cell deficiency is treatable by gamma globulin substitution therapy. The newly developed BTK inhibitor PCI‐32765, recently renamed Ibrutinib, has already entered several clinical trials for various forms of non‐Hodgkin lymphoma as well as for multiple myeloma. Experimental animal studies have demonstrated highly promising treatment effects also in autoimmunity. ITK inhibitors are still under the early developmental phase, but it can be expected that such drugs will also become very useful. In this study, we present BTK and ITK with their signalling pathways and review the development of the corresponding inhibitors.     

Ack1 tyrosine kinase activation correlates with pancreatic cancer progression

Pancreatic cancer is a significant cause of cancer mortality worldwide as the disease has advanced significantly in patients before symptoms are evident. The signal transduction pathways that promote this rapid progression are not well understood. Ack1 or TNK2, an ubiquitously expressed oncogenic non-receptor tyrosine kinase, integrates signals from ligand-activated receptor tyrosine kinases to modulate intracellular signaling cascades. In the present study, we investigated the Ack1 activation profile in a pancreatic cancer tumor microarray, and observed that expression levels of activated Ack1 and pTyr284-Ack1 positively correlated with the severity of disease progression and inversely correlated with the survival of patients with pancreatic cancer. To explore the mechanisms by which Ack1 promotes tumor progression, we investigated the role of AKT/PKB, an oncogene and Ack1-interacting protein. Ack1 activates AKT directly in pancreatic and other cancer cell lines by phosphorylating AKT at Tyr176 to promote cell survival. In addition, the Ack1 inhibitor AIM-100 not only inhibited Ack1 activation but also suppressed AKT tyrosine phosphorylation, leading to cell cycle arrest in the G1 phase. This effect resulted in a significant decrease in the proliferation of pancreatic cancer cells and induction of apoptosis. Collectively, our data indicate that activated Ack1 could be a prognostic marker for ascertaining early or advanced pancreatic cancer. Thus, Ack1 inhibitors hold promise for therapeutic intervention to inhibit pancreatic tumor growth.     

Genistein和乳腺癌干细胞PTKs信号转导通路的研究进展

饮食中的genistein在降低亚洲人乳腺癌的发病率上有很重要的作用。最新研究表明genistein抑制乳腺癌细胞生长是通过调节细胞的增殖、分化和凋亡等相关信号转导通路来完成的。乳腺癌干细胞可能是乳腺癌发生耐药、复发及转移的根源。近年来发现乳腺癌干细胞相关信号转导通路复杂且涉及面广泛,其中蛋白酪氨酸激酶（proteintyrosine kinases,PTKs）信号转导通路在癌干细胞中的异常过表达,是引起癌基因转化、肿瘤生长的重要因素之一。因此,Genistein和乳腺癌干细胞相关的PTKs信号转导通路之间的关系也越来越受到关注,对寻找更为有效的乳腺癌靶向治疗具有重要意义。     

Tyrosine kinase - Role and significance in Cancer

Tyrosine kinases are important mediators of the signaling cascade, determining key roles in diverse biological processes like growth, differentiation, metabolism and apoptosis in response to external and internal stimuli. Recent advances have implicated the role of tyrosine kinases in the pathophysiology of cancer. Though their activity is tightly regulated in normal cells, they may acquire transforming functions due to mutation(s), overexpression and autocrine paracrine stimulation, leading to malignancy. Constitutive oncogenic activation in cancer cells can be blocked by selective tyrosine kinase inhibitors and thus considered as a promising approach for innovative genome based therapeutics. The modes of oncogenic activation and the different approaches for tyrosine kinase inhibition, like small molecule inhibitors, monoclonal antibodies, heat shock proteins, immunoconjugates, antisense and peptide drugs are reviewed in light of the important molecules. As angiogenesis is a major event in cancer growth and proliferation, tyrosine kinase inhibitors as a target for anti-angiogenesis can be aptly applied as a new mode of cancer therapy. The review concludes with a discussion on the application of modern techniques and knowledge of the kinome as means to gear up the tyrosine kinase drug discovery process.     

Human gastric cancer kinase profile and prognostic significance of MKK4 kinase

Alterations of protein tyrosine kinase are often associated with uncontrolled cell growth and tumor progression. Knowledge of the overall expression pattern of tyrosine kinases should prove beneficial in understanding the signaling pathways involved in gastric cancer oncogenesis and in providing possible biomarkers for gastric cancer progression. To establish a general tyrosine-kinase expression profile, degenerated polymerase chain reaction primers designed from the consensus catalytic kinase motifs were used to amplify protein tyrosine kinase molecules from gastric cancer tissues. We observed more than 50 tyrosine and serine/threonine kinases from matching pairs of gastric cancer tissue and normal mucosa. Based on this new kinase profile information, we selected the MKK4 gene for further immunohistochemical studies. Statistical analysis of MKK4 protein expression and clinicopathological features indicated that MKK4 kinase expression could serve as a significant prognostic factor for relapse-free survival and for overall survival. We demonstrated a simple and sensitive method for establishing protein tyrosine-kinase expression profiles of human gastric cancer tissues as well as for discovering novel and useful clinical biomarkers from such kinase expression profiles.     

The SH3 Domain of Bruton's Tyrosine Kinase Interacts with Vav, Sam68 and EWS

Bruton tyrosine kinase (BTK) is a cytoplasmic protein tyrosine kinase which controls crucial steps of differentiation of B lymphocytes. Mutations affecting either the PH, SH3, SH2 or kinase domain of BTK all give rise to X linked agammaglobulinaemia (XLA) in humans. In this study, the authors report that the BTK-SH3 domain binds to a set of proteins expressed in pro-B, pre-B and B cell lines. Three of them were characterized as Vav, Sam68 and EWS. The authors show that a Pro → Leu substitution in a region of the SH3 domain, which is deleted in an XLA patient, is sufficient to abolish BTK-SH3 binding potential. The authors also report that several of the BTK-SH3 binding proteins, including Sam68, EWS and Vav, are tyrosine phosphorylated in conditions that also promote BTK kinase activity. For EWS and Sam68 this tyrosine phosphorylation was cell cycle dependent.     

Sorafenib paradoxically activates the RAS/RAF/ERK pathway in polyclonal human NK cells during expansion and thereby enhances effector functions in a dose‐ and time‐dependent manner

Natural killer (NK) cells play a major role in host immunity against leukaemia and lymphoma. However, clinical trials applying NK cells have not been as efficient as hoped for. Patients treated with rapidly accelerated fibrosarcoma (RAF) inhibitors exhibit increased tumour infiltration by immune cells, suggesting that a combination of RAF inhibitors with immunotherapy might be beneficial. As mitogen‐activated protein kinases (MAPKs) such as raf‐1 proto‐oncogene, serine/threonine kinase (CRAF) regulate NK cell functions, we performed an in‐vitro investigation on the potential of clinically relevant short‐acting tyrosine kinase inhibitors (TKIs) as potential adjuvants for NK cell therapy: NK cells from healthy human blood donors were thus treated with sorafenib, sunitinib or the pan‐RAF inhibitor ZM336372 during ex‐vivo expansion. Functional outcomes assessed after washout of the drugs included cytokine production, degranulation, cytotoxicity, apoptosis induction and signal transduction with/without target cell contact. Paradoxically, sorafenib enhanced NK cell effector functions in a time‐ and dose‐dependent manner by raising the steady‐state activation level. Of note, this did not lead to NK cell exhaustion, but enhanced activity against target cells such as K562 or Daudis mediated via the RAS/RAF/extracellular‐regulated kinase (ERK) pathway, but not via protein kinase B (AKT). Our data will pave the path to develop a rationale for the considered use of RAF inhibitors such as sorafenib for pre‐activation in NK cell‐based adoptive immune therapy.     

Clinical potential of targeting Bruton's tyrosine kinase

Targeting Bruton's tyrosine kinase (BTK) with a small-molecule inhibitor may be useful in treatment of BTK-expressing malignancies because of the antiapoptotic function of BTK in cancer cells. Furthermore, BTK inhibitors also exhibit antithrombotic properties, which may be desirable in the context of the increased risk of thromboembolic complications in cancer patients. This review will focus on the role of BTK in drug resistance in cancer, thromboembolism, and various pathologic immune responses, such as graft-versus-host disease. The therapeutic potential of targeting BTK is illustrated by discussion of the biologic activity profile of the rationally designed BTK inhibitor LFM-A13.     

Mitogen-activated protein kinases and apoptosis in PIN

 Mitogen-activated protein (MAP) kinases are key elements of the signalling systems needed to transduce different extracellular messages into cellular responses. At least three parallel MAP kinase pathways have been identified: one, stimulated by serum and growth factors to activate extracellular signal-regulated protein kinases (ERKs) by dual tyrosine and threonine phosphorylation, triggers cell proliferation or differentiation; the other two, induced by a variety of cellular stresses to activate c-jun N-terminal kinases (JNKs) and reactivating kinase (p38/RK), result in growth arrest and induction of apoptosis. Mitogen-activated protein kinase phosphatases (MKPs) inactivate MAP kinases through dephosphorylation and, thus, can modulate the MAP kinase pathways. Expression of JNK-1, ERK-1, p38/RK and MKP-1 proteins was investigated by immunohistochemistry and expression of MKP-1 mRNA by in situ hybridisation in 50 cases of high-grade prostatic intraepithelial neoplasia (PIN), thought to represent the precursor of prostate cancer. The frequency of apoptotic cells was also determined in these cases. Overexpression of the three MAP kinases and MKP-1 mRNA was found in all cases of high-grade PIN compared with normal prostate. Immunoreactivity for MKP-1 protein was found to be as intense as in normal glands in 30% and weaker in 56% of the PIN cases. Fourteen per cent of PIN cases did not stain with MKP-1 antibody. The proportion of apoptosis was significantly higher (P < 0.008) in PIN lesions that did not express MKP-1 protein than in those that did. These results are consistent with our previous demonstration of preferential inhibition of the apoptosis-related kinases by MKP-1 and further support the contention that MKP-1, even in PIN, may shift the balance existing between cell proliferation and death. When expressed, it may inhibiting those pathways that lead to apoptosis. Received: 27 August 1997 / Accepted: 29 December 1997     

胰岛素样生长因子-I受体与肿瘤

胰岛素样生长因子I受体(insulin-like growth factor I receptor,IGF-IR)为跨膜蛋白，是酪氨酸蛋白激酶类受体家族的主要成员之一。IGF-IR在多种恶性肿瘤中均有表达，它可促进肿瘤细胞的增殖、抑制肿瘤细胞的凋亡，在肿瘤的发生发展中起重要作用。现已在多种肿瘤细胞中证实，用抗IGF-IR的抗体或相关药物及以反义IGF-IR RNA封闭IGF-IR来阻断IGF-IR信号转导，可以抑制肿瘤细胞的生长和增殖，促进其凋亡。因此针对IGF-IR靶向治疗将是恶性肿瘤生物治疗值得关注的一个研究方向。     

Clinical Potential of Targeting Bruton's Tyrosine Kinase

Targeting Bruton's tyrosine kinase (BTK) with a small-molecule inhibitor may be useful in treatment of BTK-expressing malignancies because of the antiapoptotic function of BTK in cancer cells. Furthermore, BTK inhibitors also exhibit antithrombotic properties, which may be desirable in the context of the increased risk of thromboembolic complications in cancer patients. This review will focus on the role of BTK in drug resistance in cancer, thromboembolism, and various pathologic immune responses, such as graft-versus-host disease. The therapeutic potential of targeting BTK is illustrated by discussion of the biologic activity profile of the rationally designed BTK inhibitor LFM-A13.     

Targeted therapies in solid tumors: monoclonal antibodies and small molecules

The considerable progress in our understanding of the complex cellular, molecular, and genetic mechanisms underlying tumorigenesis over the last decade has fostered the development of novel and improved targeted therapies in cancer intervention. Because these therapies specifically interfere with signaling pathways essential for tumor cell proliferation, survival, and migration, they may be able to inhibit tumor growth and metastasis effectively, with fewer severe adverse events than seen with current chemotherapeutic interventions that have a narrow therapeutic index and are associated with severe toxic side effects. Monoclonal antibodies and protein tyrosine kinase inhibitors represent two classes among these promising new therapeutic interventions. This review discusses the advantages, limitations, and potential of monoclonal antibodies and protein tyrosine kinase inhibitors, and offers a perspective on the requirements and goals likely to propel the design of additional anticancer agents in the future.     

Choline kinase‐α protein and phosphatidylcholine but not phosphocholine are required for breast cancer cell survival

High levels of total choline and phosphocholine (PC) are consistently observed in aggressive cancers. Choline kinase (Chk) catalyzes choline phosphorylation to produce PC in phosphatidylcholine (PtdCho) biosynthesis. PtdCho is the most abundant phospholipid in eukaryotic cell membranes and plays a dual role as the structural component of membranes and as a substrate to produce lipid second messengers such as phosphatidic acid and diacylglycerol. Chk‐α, but not Chk‐β, is overexpressed in various cancers, and is closely associated with tumor progression and invasiveness. We have previously shown that downregulation of mRNA using small interfering RNA (siRNA) against Chk‐α (siRNA‐Chk) or Chk short hairpin RNA, and the resultant decrease of Chk‐α protein levels, significantly reduced proliferation in breast cancer cells and tumors. A novel potent and selective small‐molecule Chk‐α inhibitor, V‐11‐0711, that inhibits the catalytic activity of Chk has recently been developed. Here, we used triple negative MDA‐MB‐231 and SUM149 breast cancer cells to further investigate the role of Chk‐α in cancer, by examining Chk‐α protein levels, cell viability/proliferation, choline phospholipid and lipid metabolism, lipid droplet formation, and apoptosis, following treatment with V‐11‐0711. Under the conditions used in this study, treatment with V‐11‐0711 significantly decreased PC levels but did not reduce cell viability as long as Chk‐α protein and PtdCho levels were not reduced, suggesting that Chk‐α protein and PtdCho, but not PC, may be crucial for breast cancer cell survival. These data also support the approach of antitumor strategies that destabilize Chk‐α protein or downregulate PtdCho in breast cancer treatment. Copyright © 2015 John Wiley & Sons, Ltd.     

Targets for Ibrutinib Beyond B Cell Malignancies

Ibrutinib (Imbruvica^?) is an irreversible, potent inhibitor of Bruton's tyrosine kinase (BTK). Over the last few years, ibrutinib has developed from a promising drug candidate to being approved by FDA for the treatment of three B cell malignancies, a truly remarkable feat. Few, if any medicines are monospecific and ibrutinib is no exception; already during ibrutinib's initial characterization, it was found that it could bind also to other kinases. In this review, we discuss the implications of such interactions, which go beyond the selective effect on BTK in B cell malignancies. In certain cases, the outcome of ibrutinib treatment likely results from the combined inhibition of BTK and other kinases, causing additive or synergistic, effects. Conversely, there are also examples when the clinical outcome seems unrelated to inhibition of BTK. Thus, more specifically, adverse effects such as enhanced bleeding or arrhythmias could potentially be explained by different interactions. We also predict that during long‐term treatment bone homoeostasis might be affected due to the inhibition of osteoclasts. Moreover, the binding of ibrutinib to molecular targets other than BTK or effects on cells other than B cell‐derived malignancies could be beneficial and result in new indications for clinical applications.     

Vascular endothelial growth factor expression promotes the growth of breast cancer brain metastases in nude mice

Patients with breast cancer brain metastases cannot be cured and have a poor prognosis, with a median survival time of six months after diagnosis, despite developments in diagnostic and therapeutic modalities. In large part the progress in understanding the biology of breast cancer brain metastasis has been limited by the lack of suitable cell lines and experimental models. The objective of this study was to develop a reliable experimental model to study the pathogenesis of breast cancer brain metastases, using intra-internal carotid artery injection of breast cancer cells into nude mice. Brain metastasis-selected variant cells were recovered after three cycles of injection into the internal carotid artery of nude mice and harvest of brain metastases, resulting in variants termed MDA-231 BR1, -BR2 and -BR3. The metastasis-selected cells had increased potential for experimental brain metastasis and mice injected with these cells had significantly shorter mean survival than mice injected with the original cell line. Brain metastatic lesions of the selected variants contained significantly more CD31-positive blood vessels than metastases of the non-selected cell line. The variants selected from brain metastases released significantly more VEGF-A and IL-8 into culture supernatants than the original cell line, and more VEGF-A RNA when cultured in normoxic conditions. Mice injected with MDA-231 BR3 into the carotid artery were treated with the VEGF-receptor tyrosine kinase inhibitor PTK787/Z 222584. Oral administration of the inhibitor resulted in a significant decrease in brain tumor burden, reduced CD31-positive vessels in the brain lesions and incidence of PCNA positive tumor cells, and increased apoptosis in the tumor, as measured by TUNEL labeling. We conclude that elevated VEGF expression contributes to the ability of breast cancer cells to form brain metastases. Targeting endothelial cells with a VEGF-receptor specific tyrosine kinase inhibitor reduced angiogenesis and restricted the growth of the brain metastases.