Protein Tyrosine Kinase Inhibitor Research Articles

Structural basis of acquired resistance to selpercatinib and pralsetinib mediated by non-gatekeeper RET mutations

Background:Selpercatinib (LOXO-292) and pralsetinib (BLU-667) are highly potent RET-selective protein tyrosine kinase inhibitors (TKIs) for treating advanced RET-altered thyroid cancers and non-small-cell lung cancer (NSCLC). It is critical to analyze RET mutants resistant to these drugs and unravel the molecular basis to improve patient outcomes.Patients and methods:Cell-free DNAs (cfDNAs) were analyzed in a RET-mutant medullary thyroid cancer (MTC) patient and a CCDC6-RET fusion NSCLC patient who had dramatic response to selpercatinib and later developed resistance. Selpercatinib-resistant RET mutants were identified and cross-profiled with pralsetinib in cell cultures. Crystal structures of RET-selpercatinib and RET-pralsetinib complexes were determined based on high-resolution diffraction data collected with synchrotron radiation.Results:RETG810C/S mutations at the solvent front and RETY806C/N mutation at the hinge region were found in cfDNAs of an MTC patient with RETM918T/V804M/L, who initially responded to selpercatinib and developed resistance. RETG810C mutant was detected in cfDNAs of a CCDC6-RET-fusion NSCLC patient who developed acquired resistance to selpercatinib. Five RET kinase domain mutations at three non-gatekeeper residues were identified from 39 selpercatinib-resistant cell lines. All five selpercatinib-resistant RET mutants were cross-resistant to pralsetinib. X-ray crystal structures of the RET-selpercatinib and RET-pralsetinib complexes reveal that, unlike other TKIs, these two RET TKIs anchor one end in the front cleft and wrap around the gate wall to access the back cleft.Conclusions:RET mutations at the solvent front and the hinge are resistant to both drugs. Selpercatinib and pralsetinib use an unconventional mode to bind RET that avoids the interference from gatekeeper mutations but is vulnerable to non-gatekeeper mutations.

Preclinical Evaluation of ALLO-605, an Allogeneic BCMA Turbocar TTM Cell Therapy for the Treatment of Multiple Myeloma

Chimeric antigen receptor (CAR) T cells have demonstrated unprecedented efficacy in heavily pretreated relapsed and/or refractory multiple myeloma (MM) patients but may require further engineering to achieve their greatest potential. Enhancing cell expansion and persistence by providing cytokine support has been shown to improve the long-term antitumor activity of adoptively transferred CAR T cells in preclinical models. Combining CAR T cells with systemically-administered cytokines or cytokine mimetics can however result in toxicities and adverse events. Alternatively, cytokine signaling can be provided in a constitutive, CAR T cell-intrinsic fashion, by exogenous expression of a Constitutively Active Chimeric Cytokine Receptor (CACCR). CACCRs can be engineered by combining a membrane-tethered dimerization and JAK-binding domain derived from the thrombopoietin receptor (TpoR) fused to an intracellular signaling domain derived from a cytokine receptor. We investigated the impact of CACCR expression on the phenotype, functionality, persistence, and safety profile of allogeneic CAR T cells targeting BCMA to produce a second generation allogeneic BCMA "TurboCAR T™" candidate (ALLO-605). BCMA CAR T cells were generated from healthy donor T cells by lentiviral transduction with a CAR construct followed by genetic inactivation of the TRAC and CD52 loci using TALEN® gene editing. Allogeneic BCMA TurboCAR™ T cells, engineered for stoichiometric expression of the CAR and a CACCR via a self-cleaving peptide, were produced similarly. Constitutive expression of the CACCR during manufacturing had no negative effects on CAR T cell phenotype or yield and resulted in a product with over 60% stem cell memory/central memory T cells. In vitro, BCMA TurboCAR T™ cells showed enhanced cytokine secretion, polyfunctionality and improved serial killing activity. In a disseminated mouse model of multiple myeloma, BCMA TurboCAR T™ cells exhibited at least a 2-fold increase in peak expansion and enhanced survival and persistence compared to BCMA CAR T cells, resulting in prolonged antitumor responses and delaying relapses. Despite this enhanced persistence, we found that exposure to target cells was absolutely required for the expansion and long-term activity of BCMA TurboCAR T™ cells and no evidence of target- and cytokine-independent proliferation was observed. Since adoptive cell therapies have the potential to elicit toxicities in some patients, two alternative approaches to modulate BCMA TurboCAR T™ cell activity were investigated. First, we tested the ability of a CD20-based off-switch incorporated within the CAR to sensitize cells to rituximab and found effective depletion of BCMA TurboCAR T™ cells by complement or effector cells in vitro and in vivo in the presence of the antibody. In addition, we confirmed rapid inhibition of BCMA TurboCAR T™ cells by the protein tyrosine kinase inhibitor dasatinib, which has been shown to interfere with LCK activity. The prolonged persistence and antitumor responses seen in preclinical models along with a favorable safety profile of BCMA TurboCAR™ T cells support clinical investigation of ALLO-605 in relapsed or refractory multiple myeloma. Disclosures Sommer: Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Lin:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Sutton:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Bentley:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Yoon:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Au:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Vargas-Inchaustegui:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Cheng:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Van Blarcom:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Panowski:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Sasu:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company.

Enhancement of carotenogenesis regulated by phosphorylation signaling in the aerial microalga Coelastrella sp. KGU-Y002

To investigate the roles of phosphorylation signaling toward carotenogenesis in microalgae, we cultured the aerial microalga Coelastrella sp. KGU-Y002 with inhibitors against protein kinase or phosphatase and evaluated carotenoid accumulation using a carotenoid assay. When the algal cells were cultured for 7 days at 60 μmol photons m−2 s−1 in nitrogen-deficient medium with an inhibitor of protein serine/threonine kinase, the c-jun terminal kinase inhibitor increased the carotenoid content to 2.0-fold that in the control. Furthermore, the addition of the inhibitor suppressed algal growth. Incubation with a protein tyrosine kinase inhibitor increased carotenoid accumulation by up to 2.0-fold and did not affect algal growth. Broad inhibitors against protein serine/threonine and tyrosine phosphatase promoted carotenoid accumulation by up to 1.6- and 3.8-fold without suppressing the growth mechanism. Carotenogenesis in microalgae could be enhanced by the general involvement of both phosphorylation and dephosphorylation mechanisms in serine/threonine- and tyrosine-type phosphorylation signaling.

Research progress of anti-angiogenic drugs in the treatment of small cell lung cancer

Small cell lung cancer (SCLC), a special type of lung cancer, is a highly malignant neuroendocrine tumor with strong invasiveness and rapid progression. SCLC is sensitive to radiotherapy and chemotherapy, so radiotherapy and chemotherapy have been the main first-line treatment of SCLC. However, it is easy to develop drug resistance after treatment. Therefore, the study of anti-angiogenic therapy has attracted more and more attention. At present, anti-angiogenic drugs mainly focus on four categories: monoclonal antibodies (such as bevacizumab), endogenous angiogenesis inhibitors (such as endostar), anti-angiogenic fusion protein (such as aflibercept) and small molecular tyrosine kinase inhibitors (such as anlotinib). There are still some bottlenecks in the research and clinical application of antiangiogenic drugs. It is the right direction to explore better combination therapy and effective dual-field and multi-target drugs.

In Vitro Evolution Reveals a Single Mutation as Sole Source of Src-Family Kinase C-Helix-out Inhibitor Resistance.

Understanding cancer cell drug resistance to protein-tyrosine kinase inhibitors, which often arises from acquired mutations in the target kinase, is central to the development of more durable therapies. Experimental systems that reveal potential paths to resistance for a given inhibitor and kinase target have an important role in preclinical development of kinase inhibitor drugs. Here, we employed a codon mutagenesis strategy to define the mutational landscape of acquired resistance in HCK, a member of the SRC tyrosine kinase family and therapeutic target in acute myeloid leukemia (AML). Using PCR-based saturation mutagenesis, we created a cDNA library designed to replace each codon in the HCK open reading frame with all possible codons. This HCK mutant library was used to transform Rat-2 fibroblasts, followed by selection for resistant colonies with A-419259, a pyrrolopyrimidine HCK inhibitor and drug lead for AML. X-ray crystallography has shown that A-419259 binding induces outward rotation of the kinase domain αC-helix, a conformation incompatible with phosphotransfer. Remarkably, only a single resistance mutation evolved during A-419259 selection: histidine substitution for threonine at the gatekeeper position in the kinase domain. Deep sequencing confirmed representation of nearly all other missense mutations across the entire HCK open reading frame. This observation suggests that A-419259 and other C-helix-out Src-family kinase inhibitors may have a narrow path to acquired resistance in the context of AML cases where Hck is an oncogenic driver.

Tyrosine Kinase Inhibitors Reduce NMDA NR1 Subunit Expression, Nuclear Translocation, and Behavioral Pain Measures in Experimental Arthritis.

In the lumbar spinal cord dorsal horn, release of afferent nerve glutamate activates the neurons that relay information about injury pain. Here, we examined the effects of protein tyrosine kinase (PTK) inhibition on NMDA receptor NR1 subunit protein expression and subcellular localization in an acute experimental arthritis model. PTK inhibitors genistein and lavendustin A reduced cellular histological translocation of NMDA NR1 in the spinal cord occurring after the inflammatory insult and the nociceptive behavioral responses to heat. The PTK inhibitors were administered into lumbar spinal cord by microdialysis, and secondary heat hyperalgesia was determined using the Hargreaves test. NMDA NR1 cellular protein expression and nuclear translocation were determined by immunocytochemical localization with light and electron microscopy, as well as with Western blot analysis utilizing both C- and N-terminal antibodies. Genistein and lavendustin A (but not inactive lavendustin B or diadzein) effectively reduced (i) pain related behavior, (ii) NMDA NR1 subunit expression increases in spinal cord, and (iii) the shift of NR1 from a cell membrane to a nuclear localization. Genistein pre-treatment reduced these events that occur in vivo within 4 h after inflammatory insult to the knee joint with kaolin and carrageenan (k/c). Cycloheximide reduced glutamate activated upregulation of NR1 content confirming synthesis of new protein in response to the inflammatory insult. In addition to this in vivo data, genistein or staurosporin inhibited upregulation of NMDA NR1 protein and nuclear translocation in vitro after treatment of human neuroblastoma clonal cell cultures (SH-SY5Y) with glutamate or NMDA (4 h). These studies provide evidence that inflammatory activation of peripheral nerves initiates increase in NMDA NR1 in the spinal cord coincident with development of pain related behaviors through glutamate non-receptor, PTK dependent cascades.

Inhibitory effect of genistein and PTP1B on grasshopper Oedaleus asiaticus development

Protein tyrosine kinase (PTKs) and protein tyrosine phosphatase (PTPs) collectively regulate the insect insulin-signaling pathway (ISP), cell growth, metabolism, proliferation, differentiation, cell communication and monitoring of immune responses. We studied the inhibitory response of the grasshopper Oedaleus asiaticus Bey-Bienko to treat with the flavonoid genistein (PTK inhibitor), the enzyme PTP1B-IN-1 (PTP1B inhibitor) and control (dimethyl sulfoxide (DMSO)). Mean survival rates were not significantly different amongst the three treatments, but growth rate, body mass, and overall biological performance were reduced significantly following treatment with genistein. On comparison, treatment with PTP1B-IN-1 was not significantly different from the control. Relative gene expression of INSR, IRS, PI3K, PDK, and Akt was substantially lower (P < 0.05) following treatment with genistein compared to PTP1B-IN-1 and control. Similarly, gene expression and protein phosphorylation levels of the PTK5, PTP1B → FOXO cascade, indicators of stress response in the ISP, were also reduced in response to genistein treatments. The activity of the protective enzymes (POD and CAT) was upregulated by FOXO in response to PTP1B-IN-1. Our results demonstrated that genistein negatively regulates the ISP pathway, with a consequent effect on growth and development of O. asiaticus, as higher energy was required for detoxification. Given the negative effect of genistein on O. asiaticus physiology and development, the compound could be used in a biopesticide formulation for insect pest control in grasslands and other crop plants as a potential application to grasshopper biological control.

The protein tyrosine kinase inhibitor, Genistein, delays intervertebral disc degeneration in rats by inhibiting the p38 pathway-mediated inflammatory response

The treatment for intervertebral disc degeneration (IDD) has drawn great attention and recent studies have revealed that the p38 MAPK pathway is a potential therapeutic target for delaying the degeneration of intervertebral discs. In this study, we analyzed a nature-derived protein tyrosine kinase inhibitor, Genistein, and its function in delaying IDD in rats both in vitro and in vivo via the p38 MAPK pathway. Nucleus pulposus cells treated with Genistein showed better function compared with untreated cells. Further study revealed that Genistein could play a protective role in IDD by inhibiting phosphorylation of p38, consequently inhibiting the p38 pathway-mediated inflammatory response. The rat IDD model also demonstrated that Genistein could effectively delay the degeneration of intervertebral disc tissue. The current study reveals new biological functions of Genistein, further demonstrates the effects of the p38 MAPK pathway on intervertebral disc degeneration, and deepens our understanding of the treatment and prevention of IDD.

RET kinase alterations in targeted cancer therapy.

The rearranged during transfection (RET) gene encodes a protein tyrosine kinase. RET alterations by point mutations and gene fusions were found in diverse cancers. RET fusions allow abnormal expression and activation of the oncogenic kinase, whereas only a few of RET point mutations found in human cancers are known oncogenic drivers. Earlier studies of RET-targeted therapy utilized multi-targeted protein tyrosine kinase inhibitors (TKIs) with RET inhibitor activity. These multi-targeted TKIs often led to high-grade adverse events and were subject to resistance caused by the gatekeeper mutations. Recently, two potent and selective RET TKIs, pralsetinib (BLU-667) and selpercatinib (LOXO-292), were developed. High response rates to these selective RET inhibitors across multiple forms of RET alterations in different types of cancers were observed in clinical trials, demonstrating the RET dependence in human cancers harboring these RET lesions. Pralsetinib and selpercatinib were effective in inhibiting RETV804L/M gatekeeper mutants. However, adaptive mutations that cause resistance to pralsetinib or selpercatinib at the solvent front RETG810 residue have been found, pointing to the need for the development of the next-generation of RET TKIs.

Synthesis and anticancer activity of benzotriazole derivatives

AbstractA series of benzotriazole (BTA) derivatives were synthesized as tyrosine protein kinase inhibitors using fragment‐based design strategy. All desired compounds were synthesized with the reaction of benzotriazole, chloroacetonitrile and aromatic aldehyde using Ultrasonic‐Microwave method and characterized by IR, 1H and 13C‐NMR, mass spectrometry (MS) and elemental analysis. The anticancer activity of these compounds was evaluated by CCK‐8 method against carcinoma VX2, lung cancer A549, stomach cancer cell lines MKN45 and MGC in vitro. The results showed that all compounds showed good antiproliferative activity. In particular, compound 2.1 showed the most prominent inhibition of VX2 cell lines with IC50 of 3.80 ± 0.75 μM. Compound 2.2 exhibited highly potent anticancer activity of stomach MGC cell lines with IC50 of 3.72 ± 0.11 μM. A549 and MKN45 cell lines were sensitive to compound 2.5 with IC50 of 5.47 ± 1.11 and 3.04 ± 0.02 μM, respectively.

Novel Pyrimidines as Multitarget Protein Tyrosine Kinase Inhibitors for the Treatment of Idiopathic Pulmonary Fibrosis (IPF).

A new class of pyrimidine derivatives were identified as potent protein tyrosine kinase (PTK) inhibitors for the treatment of idiopathic pulmonary fibrosis (IPF). Most of these small-molecule inhibitors displayed strong enzymatic activity against BTK and JAK3 kinases at concentrations lower than 10 nM. The representative compound N-(3-((5-chloro-2-(4-((1-morpholino)acetylamino)phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (6 a) also exhibited high inhibitory potency toward both BTK and JAK kinase families, as well as ErbB4, at a concentration of 10 nM, achieving rates of inhibition higher than 57 %. Additionally, in vivo biological evaluations showed that 6 a can remarkably decrease the severity of IPF disease. All these investigations suggested that the multi-PTK inhibitor 6 a may serve as a promising agent for the treatment of IPF.

Nanoformulations of small molecule protein tyrosine kinases inhibitors potentiate targeted cancer therapy.

Protein tyrosine kinases (PTKs) are closely related to tumor development and usually participate in apoptosis, DNA repair, and cell proliferation by activating signaling pathways. Therefore, PTKs have become the most promising targets for cancer therapy. In recent years, a large number of studies on the mechanism of tyrosine kinase activation have indicated that tyrosine kinase inhibitors (TKIs) have important clinical significance and application prospects as targeted anticancer drugs because they can effectively block certain cellular signaling pathways, inhibit tumor metastases and reduce tumor proliferation. Although the increasing emergence of anticancer drug resistance limits the clinical application of TKIs, emerging nanotechnology has made it possible to solve this problem. In this work, the state-of-art of small molecule protein tyrosine kinase inhibitors and the applications of drug delivery systems for TKIs are reviewed, and the potentials and challenges for future research of small molecule TKIs are addressed.

Protein tyrosine kinase inhibitors

Protein tyrosine kinase inhibitors

The Role of Pazopanib in Non-Clear Cell Renal Cell Carcinoma: A Systematic Review.

Pazopanib is a protein tyrosine kinase inhibitor that limits tumor growth through angiogenesis inhibition. The use of other protein tyrosine kinase inhibitors, specifically sunitinib, within non-clear cell renal cell carcinoma (nccRCC) has led to increased survival with a decreased adverse event profile. The data for the treatment of nccRCC is limited, with most studies evaluating the use of sunitinib. Therefore, the evaluation of pazopanib is of particular clinical interest in the treatment of nccRCC. The objective of this systematic review was to assess the efficacy and safety of pazopanib for nccRCC. PubMed (1946 to April 2019) and Embase (1947 to April 2019) were queried using the search term combination: protein tyrosine kinase inhibitor or pazopanib and non clear cell renal cell carcinoma or non-clear cell renal cell carcinoma. Studies evaluating clinical outcomes of pazopanib for nccRCC were included, represented by 3 retrospective cohort studies and 1 single-arm, open-label prospective study. In patients with advanced or metastatic nccRCC, treatment with pazopanib resulted in positive effects for multiple markers of efficacy, including progression-free survival, overall survival, and objective response rates. The median duration of follow-up ranged from 11.8 months to 24.4 months. Pazopanib was well-tolerated in most studies. The most commonly reported adverse events were fatigue, diarrhea, and hypertension. Pazopanib appears to be an effective and safe option for the treatment of advanced or metastatic nccRCC. Future investigation with larger randomized controlled trials is warranted to further define the role of pazopanib in patients with nccRCC.

Abstract 2117: Different sensitivities of four protein tyrosine kinase inhibitors towards drug-resistant RET mutations

Abstract The RET protein tyrosine kinase (PTK) is a clinically validated target of therapy in non-small cell lung cancer (NSCLC) and thyroid cancer. Mutations in the targeted PTKs is a mechanism of drug resistance in cancer therapy with tyrosine kinase inhibitors (TKIs). Mutation-sensitive secondary drugs have been used successfully to overcome acquired drug-resistance to the first line TKIs. Cabozantinib, lenvatinib, vandetanib, and nintedanib are FDA-approved multikinase TKIs with anti-RET activity. Using RET kinase-dependent BaF3/KIF5B-RET cells, we isolated thirteen mutations resistant to one of these TKIs. Cross-analysis of sensitivities of these four TKIs on these drug-resistant RET mutants and the RETM918T mutant, which is found in medullary thyroid carcinoma, revealed different TKI resistance-sensitivity profiles. In particular, the RETM918T mutant was resistant to cabozantinib, lenvatinib, and vandetanib but did not affect the potency of nintedanib. RETL881V was isolated as a vandetanib-resistant mutation. The RETL881V mutation also induced resistance to cabozantinib and lenvatinib but did not affect the nintedanib sensitivity. Examination of the RET-vandetanib co-crystal structure and the chemical structures of vandetanib and nintedanib suggested that a phenyl group in nintedanib, which corresponds to a methoxy group in vandetanib, may form hydrophobic interaction with the shorter side chain of Val881 and thus allows nintedanib to inhibit RETL881V. To test this possibility, we synthesized two nintedanib analogs (Compound 1 and Compound 2) without the phenyl group and tested their activities. The data showed that RETL881V was resistant to Compound 1 and Compound 2, supporting a role of the phenyl group of nintedanib in mediating inhibition of RETL881V. Taken together, we have identified 13 RET mutations that display different resistance-sensitivity profiles against four anti-RET TKIs. Moreover, nintedanib is effective in inhibiting RETM918T and RETL881V mutants that are resistant to the other three TKIs. Citation Format: Xuan Liu, Tao Shen, Qingling Huang, Teng Peng, Frank Hilberg, Jianfeng Cai, Blaine H. Mooers, Jie Wu. Different sensitivities of four protein tyrosine kinase inhibitors towards drug-resistant RET mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2117.

Structural basis of resistance of mutant RET protein-tyrosine kinase to its inhibitors nintedanib and vandetanib

RET is a transmembrane growth factor receptor. Aberrantly activated RET is found in several types of human cancer and is a target for treating RET aberration-associated cancer. Multiple clinically relevant RET protein-tyrosine kinase inhibitors (TKIs) have been identified, but how TKIs bind to RET is unknown except for vandetanib. Nintedanib is a RET TKI that inhibits the vandetanib-resistant RET(G810A) mutant. Here, we determined the X-ray co-crystal structure of RET kinase domain-nintedanib complex to 1.87 Å resolution and a RET(G810A) kinase domain crystal structure to 1.99 Å resolution. We also identified a vandetanib-resistant RET(L881V) mutation previously found in familial medullary thyroid carcinoma. Drug-sensitivity profiling of RET(L881V) revealed that it remains sensitive to nintedanib. The RET-nintedanib co-crystal structure disclosed that Leu-730 in RET engages in hydrophobic interactions with the piperazine, anilino, and phenyl groups of nintedanib, providing a structural basis for explaining that the p.L730V mutation identified in nine independently isolated cell lines resistant to nintedanib. Comparisons of RET-nintedanib, RET(G810A), and RET-vandetanib crystal structures suggested that the solvent-front Ala-810 makes hydrophobic contacts with a methyl group and aniline in nintedanib and blocks water access to two oxygen atoms of vandetanib, resulting in an energetic penalty for burying polar groups. Of note, even though the p.L881V mutation did not affect sensitivity to nintedanib, RET(L881V) was resistant to nintedanib analogs lacking a phenyl group. These results provide structural insights into resistance of RET mutants against the TKIs nintedanib and vandetanib.

PB2328 CMV‐SPECIFIC T‐CELL RECONSTITUTION AS A KEY FACTOR FOR IDENTIFICATION OF CANDIDATES FOR CMV‐SPECIFIC LYMPHOCYTES INFUSION

Background:Cytomegalovirus (CMV) is the opportunistic infection that persists in healthy individuals asymptomatically and reactivates in immunodeficient host, e.g. after allo‐HSCT. The main cause of viral reactivation is the lack of CMV‐specific T cells. The adoptive cellular therapy with CMV‐specific T cells seems very attractive clinical option to accelerate virus‐specific T‐cell reconstitution after allo‐HSCT. Here we report the preliminary data about CMV‐specific T‐cell reconstitution in allo‐HSCT patients.Aims:To identify a group of patients after allo‐HSCT who are in high‐risk of CMV reactivation and could be eligible for prophylaxis with CMV‐specific T cells.Methods:To detect CMV‐specific CD8+ cells we utilized MHC I tetramers loaded with the two immunodominant epitopes of viral pp65 protein: NLVPMVATV (NLV) and TPRVTGGGAM (TPR) presented in HLA‐A∗02 and ‐B∗07 respectively.We analyzed CD3+CD8+ cytotoxic T‐lymphocytes (CTLs) in peripheral blood and bone marrow of 7 patients on day +30 after allo‐HSCT. All patients were CMV‐seropositive. 5 patients were HLA‐A∗02 positive and 2 HLA‐B∗07 positive.Leukocyte cell suspension (5∗106 white blood cells per test) after preliminary red blood cells lysing with Lysing Buffer (BD Pharm Lyse™) were incubated with the protein tyrosine kinase inhibitor dasatinib for 1 hr at 37°С to increase surface expression of both TCR and CD8. NLV/TPR‐ specific cytotoxic T‐lymphocytes (CTLs) were identified by flow cytometry using PE‐Cy7 labeled anti‐CD45 antibody (Ab), Alexa Fluor 700 labeled anti‐CD3 Ab and PerCP‐Cy5.5 labeled anti‐CD8 Ab (Sony Biotechnology Inc.) and in house produced MHC class I tetramers conjugated with Phycoerythrin and loaded with NLV or TPR peptides. Alexa Fluor™ 750 NHS Ester (Succinimidyl Ester) was used (Invitrogen™) to exclude dead cells. Flow cytometry was performed on a BD FACSCanto™ II Flow Cytometer using BD FACSDiva™ software. Two‐platform method was used to calculate absolute CMV‐specific CD3+CD8+ count in peripheral blood.Results:According to our data, the lowest rates of CMV‐specific CTLs are presented in patients who underwent allo‐HSCT with post‐transplant high‐dose cyclophosphamide (PT‐Cy, 50 mg/kg on +3, +4 day) as GVHD prophylaxis.Results are presented in Table 1.Summary/Conclusion:Clinical option to accelerate virus‐specific T‐cell reconstitution using adoptive cellular therapy with CMV‐specific T cells seems very attractive. Modern technologies make this possible but first we should identify the patients after allo‐HSCT who benefit from this effective but very expensive therapy. Patients with using PT‐Cy as GVHD prophylaxis are the possible candidates for this treatment. Further studies CMV‐specific T‐cell reconstitution will help to expand the group of patients in high risk.image

Ponatinib Inhibits Proliferation and Induces Apoptosis of Liver Cancer Cells, but Its Efficacy Is Compromised by Its Activation on PDK1/Akt/mTOR Signaling.

Ponatinib is a multi-target protein tyrosine kinase inhibitor, and its effects on hepatocellular carcinoma cells have not been previously explored. In the present study, we investigated its effects on hepatocellular carcinoma cell growth and the underlying mechanisms. Toward SK-Hep-1 and SNU-423 cells, ponatinib induces apoptosis by upregulation of cleaved caspase-3 and -7 and promotes cell cycle arrest in the G1 phase by inhibiting CDK4/6/CyclinD1 complex and phosphorylation of retinoblastoma protein. It inhibits the growth-stimulating mitogen-activated protein (MAP) kinase pathway, the phosphorylation of Src on both negative and positive regulation sites, and Jak2 and Stat3 phosphorylation. Surprisingly, it also activates the PDK1, the protein kinase B (Akt), and the mechanistic target of rapamycin (mTOR) signaling pathway. Blocking mTOR signaling strongly sensitizes cells to inhibition by ponatinib and makes ponatinib a much more potent inhibitor of hepatocellular carcinoma cell proliferation. These findings demonstrate that ponatinib exerts both positive and negative effects on hepatocellular cell proliferation, and eliminating its growth-stimulating effects by drug combination or potentially by chemical medication can significantly improve its efficacy as an anti-cancer drug.

A novel miR-365-3p/EHF/keratin 16 axis promotes oral squamous cell carcinoma metastasis, cancer stemness and drug resistance via enhancing \u03b25-integrin/c-met signaling pathway

BackgroundTargeting the c-Met signaling pathway has become a therapeutic strategy in multiple types of cancer. We unveiled a novel c-Met regulating mechanism that could be applied as a modality for oral squamous cell carcinoma (OSCC) therapy.MethodsUpregulation of keratin 16 (KRT16) was found by comparing isogenic pairs of low and high invasive human OSCC lines via microarray analysis. OSCC cells with ectopic expression or silencing of KRT16 were used to scrutinize functional roles and associated molecular mechanisms.ResultsWe observed that high KRT16 expression significantly correlated with poorer pathological differentiation, advanced stages, increased lymph nodes metastasis, and decreased survival rate from several Taiwanese OSCC patient cohorts. We further revealed that miR-365-3p could target ETS homologous factor (EHF), a KRT16 transcription factor, to decrease migration, invasion, metastasis and chemoresistance in OSCC cells via inhibition of KRT16. Under confocal microscopic examination, c-Met was found possibly partially associates with KRT16 through β5-integrin. Colocalization of these three proteins may facilitate c-Met and β5-integrin–mediated signaling in OSCC cells. Depletion of KRT16 led to increased protein degradation of β5-integrin and c-Met through a lysosomal pathway leading to inhibition of their downstream Src/STAT3/FAK/ERK signaling in OSCC cells. Knockdown of KRT16 enhanced chemosensitivity of OSCC towards 5-fluorouracil (5-FU). Various combination of c-Met inhibitor (foretinib), protein tyrosine kinase inhibitor (genistein), β5-integrin antibody, and 5-FU markedly augmented cytotoxic effects in OSCC cells as well as tumor killing effects in vitro and in vivo.ConclusionsOur data indicate that targeting a novel miR-365-3p/EHF/KRT16/β5-integrin/c-Met signaling pathway could improve treatment efficacy in OSCC.

Bifunctional Duocarmycin Analogues as Inhibitors of Protein Tyrosine Kinases.

Bifunctional duocarmycin analogues are highly cytotoxic compounds that have been shown to be irreversible aldehyde dehydrogenase 1 inhibitors. Interestingly, cells with low aldehyde dehydrogenase 1 expression are also sensitive to bifunctional duocarmycin analogues, suggesting the existence of another target. Through in silico approaches, including principal component analysis, structure-similarity search, and docking calculations, protein tyrosine kinases, and especially the vascular endothelial growth factor receptor 2 (VEGFR-2), were predicted as targets of bifunctional duocarmycin analogues. Biochemical validation was performed in vitro, confirming the in silico results. Structural optimization was performed to mainly target VEGFR-2, but not aldehyde dehydrogenase 1. The optimized bifunctional duocarmycin analogue was synthesized. In vitro assays revealed this bifunctional duocarmycin analogue as a strong inhibitor of VEGFR-2, with low residual aldehyde dehydrogenase 1 activity. Altogether, studies revealed bifunctional duocarmycin analogues as a new class of naturally derived compounds that express a very high cytotoxicity to cancer cells overexpressing aldehyde dehydrogenase 1 as well as VEGFR-2.