Bone Marrow Tyrosine Kinase Research Articles

The Maverick trial: A phase 2 study of abivertinib in patients (pts) with metastatic castration resistant prostate cancer (mCRPC).

TPS5106 Background: Abivertinib is a potent, small molecule third-generation tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR), Bruton tyrosine kinase (BTK) receptor, and bone marrow tyrosine kinase gene on chromosome X (BMX). Abivertinib irreversibly binds to the BTK receptor, preventing the phosphorylation of the receptor, and also BMX. The adrenal-permissive HSD3B1(1245C) allele, which is present in upwards of 50% of men with prostate cancer, encodes a 3βHSD1 enzyme missense that up-regulates the rate-limiting step of androgen biosynthesis from extragonadal precursor steroids and promotes poor clinical outcomes. 3βHSD1 must be phosphorylated by BMX for androgen biosynthesis. Preclinical studies suggest BMX inhibition blocks 3βHSD1, androgen biosynthesis and CRPC in xenograft models. These provide the rationale for use of the combination of abivertinib and abiraterone to block residual androgen synthesis that escapes abiraterone inhibition. Methods: The Maverick Trial is a multicenter, open-label phase 2 study within the US Dept of Defense Prostate Cancer Clinical Trials Consortium (DOD PCCTC) of abivertinib with abiraterone in pts with mCRPC harboring the adrenal-permissive HSD3B1(1245C) allele (germline heterozygous or homozygous). HSD3B1 status will be confirmed centrally prior to enrollment via CLIA testing at the Cleveland Clinic. The study includes two cohorts: abiraterone-naïve and abiraterone-progressing. There will be cap of 50% of accrual in each cohort for pts heterozygous for the HSD3B1(1245C) allele. An interim futility analysis is embedded in each cohort, and toxicity rates will be continuously monitored for early stoppage according to predefined rules. Pts receive continuous treatment with abivertinib 200 mg BID with abiraterone 1000 mg QD + prednisone 5 mg BID until radiographic progression, unacceptable toxicity, intercurrent illness, or other reason, such as subject withdrawal. The primary endpoint is 6-month rPFS defined as percent of subjects alive and without progression by RECIST version 1.1 for measurable disease and PCWG3 criteria for bone metastases. Secondary and exploratory endpoints include objective response rate, duration of response, PSA progression, pharmacokinetics, pharmacodynamic tissue changes, and mechanisms of response and resistance to therapy via sequential tissue and blood sampling. The study is currently open at 1 site and will activate at 9 additional sites. It is sponsored by Sorrento Therapeutics, Inc, and managed by the Prostate Cancer Clinical Trials Consortium. Trial Registration: NCT05361915 Funding Source: Sorrento Therapeutics, Inc, the trial’s sponsor who is funding the trial. Clinical trial information: NCT05361915 .

Abstract 1770: CD28-Y218 phosphorylation in CAR-T cells is mediated by the IL-2-inducible T-cell kinase (ITK)

Abstract CD28-derived co-stimulation modules are frequently used as part of the signaling domain of chimeric antigen receptors (CAR). These modules contain well characterized tyrosine-containing moieties (YMNM and PYAP) that become phosphorylated upon CAR activation, and result in IL-2 secretion and T cell expansion. Previous work by our group has shown that an additional tyrosine residue, located in the C-terminus of CD28 (Y218), was also phosphorylated upon CAR activation. The kinase responsible for this phosphorylation remains unknown, but in silico studies point towards the IL-2-inducible T-cell kinase (ITK), the protein-tyrosine kinase TEC and the bone marrow tyrosine kinase (BMX). In this work we sought to elucidate the molecular mechanism and functional relevance of CD28-Y218 phosphorylation in CAR-T cells.To analyze the kinetics of Y218 phosphorylation, prostate stem cell antigen (PSCA)-specific second-generation CAR-T cells were stimulated through co-culture with HPAC pancreatic cancer cells for 0, 1, 10, 30 and 60 minutes. Phosphorylation of CD28 Y218, Zap70, PLCγ and Vav1 was analyzed by Western blotting. To determine the role of ITK in CD28-218 phosphorylation: 1) PSCA-specific CAR-T cells were treated with ITK inhibitors 10 μM (BMS-509744 or Ibrutinib) for 24h and 2) ITK-deficient Jurkat cells expressing a PSCA-specific CAR were cocultured with HPAC cells for 0 or 10 min. To determine the functional relevance of this phosphorylation event, we generated mutant CARs where the tyrosine residue was replaced with a non-phosphorylatable amino acid (Y218F) and evaluated its effects in vivo and in vitro. CAR-T cell cytotoxicity was monitored using the xCELLigence Real Time Cytotoxicity Assay (RTCA) and cytokine production was assessed following overnight co-culture with target cells using the ELLA system (Biotechne, CA, USA). To evaluate the in vivo antitumor efficacy of PSCA-specific CAR-T cells, NSG mice were injected (s.c.) with 1 HPAC cells and infused 14 days later with 5 CAR-T cells (i.v.). Tumor growth was monitored for 35 days.Our results show that phosphorylation of CD28-Y218 occurs after the phosphorylation of early TCR signaling mediators, reaching maximum levels around 10-30 min post-stimulation. Pharmacological inhibition and genomic ablation of ITK impairs CD28-Y218 phosphorylation, suggesting an essential role of ITK in this signaling event. We observed that mutation of the Y218 residue did not affect CAR expression, in vitro cytotoxicity, or INFγ production. However, this mutation was associated with reduced IL-2 and TNFα secretion. In vivo, this mutation completely abrogated the therapeutic effect of CAR-T cells in a pancreatic cancer model. In conclusion, these results indicate that ITK is required for CD28-Y218 phosphorylation, a signaling event that plays an important role in CAR-T cell function, with direct impact on IL-2 secretion and antitumor efficacy. Citation Format: Elena Martinez-Planes, Miguel G. Fontela, Cecilia Ramello, Daniel Abate-Daga. CD28-Y218 phosphorylation in CAR-T cells is mediated by the IL-2-inducible T-cell kinase (ITK) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1770.

Use of <i>Escherichia coli</i> expression system for analyzing kinase motifs

Sixty-eight human tyrosine kinases were cloned into the Escherichia coli expression vector pET21a(+) and 48 of them were successfully expressed in the BL21(DE3) strain. Among them, 18 kinases were activated in the bacterial cells by autophosphorylation and they phosphorylated endogenous proteins. Since E. coli expression systems do not usually enable post-translational modifications, the in vivo phosphorylation should be due to the tyrosine kinase expressed. In this study, we attempted to use the bacterial expression system as a site for the kinase reaction to determine the substrate sequence preference of a certain kinase, namely, a phosphorylation motif. As a first example, phosphoproteomic analysis of E. coli expressing active bone marrow tyrosine kinase gene in chromosome X protein (BMX) was performed to extract the phosphorylation motifs. The motifs were compared with that obtained from Kinapple, the database of in vitro human kinome profiling, and common features rich in acidic amino acids were found. Furthermore, the phosphorylation state of expressed BMX was analyzed. Phos-tag SDS-PAGE in conjunction with alanine scans of the phosphorylation sites found by mass spectrometry revealed that Y224, Y234, and Y566 are predominantly phosphorylated. Moreover, phosphorylation at S453 and T572 was implicated in activation in E. coli, whereas these sites did not affect activity in 293 cells. These results suggest that, although the bacterial system does not fully reflect the physiological properties of the kinase, it is a simple experimental method that can infer the substrate preference of tyrosine kinases similar to that observed with substrates of human origin.

Discovery of a Novel Class of Covalent Dual Inhibitors Targeting the Protein Kinases BMX and BTK.

The nonreceptor tyrosine TEC kinases are key regulators of the immune system and play a crucial role in the pathogenesis of diverse hematological malignancies. In contrast to the substantial efforts in inhibitor development for Bruton’s tyrosine kinase (BTK), specific inhibitors of the other TEC kinases, including the bone marrow tyrosine kinase on chromosome X (BMX), remain sparse. Here we present a novel class of dual BMX/BTK inhibitors, which were designed from irreversible inhibitors of Janus kinase (JAK) 3 targeting a cysteine located within the solvent-exposed front region of the ATP binding pocket. Structure-guided design exploiting the differences in the gatekeeper residues enabled the achievement of high selectivity over JAK3 and certain other kinases harboring a sterically demanding residue at this position. The most active compounds inhibited BMX and BTK with apparent IC50 values in the single digit nanomolar range or below showing moderate selectivity within the TEC family and potent cellular target engagement. These compounds represent an important first step towards selective chemical probes for the protein kinase BMX.

A scalable insect cell-based production process of the human recombinant BMX for in-vitro covalent ligand high-throughput screening.

Bone Marrow Tyrosine kinase in the chromosome X (BMX) is a TEC family kinase associated with numerous pathological pathways in cancer cells. Covalent inhibition of BMX activity holds promise as a therapeutic approach against cancer. To screen for potent and selective covalent BMX inhibitors, large quantities of highly pure BMX are normally required which is challenging with the currently available production and purification processes. Here, we developed a scalable production process for the human recombinant BMX (hrBMX) using the insect cell-baculovirus expression vector system. Comparable expression levels were obtained in small-scale shake flasks (13mL) and in stirred-tank bioreactors (STB, 5 L). A two-step chromatographic-based process was implemented, reducing purification times by 75% when compared to traditional processes, while maintaining hrBMX stability. The final production yield was 24mg of purified hrBMX per litter of cell culture, with a purity of > 99%. Product quality was assessed and confirmed through a series of biochemical and biophysical assays, including circular dichroism and dynamic light scattering. Overall, the platform herein developed was capable of generating 100mg purified hrBMX from 5L STB in just 34days, thus having the potential to assist in-vitro covalent ligand high-throughput screening for BMX activity inhibition.

BMX activates Wnt/β-catenin signaling pathway to promote cell proliferation and migration in breast cancer.

Breast cancer has become a dangerous killer for the female, which seriously threatened women's life, leading to huge pressures to society. The present study assessed the mechanism underlying the involvement of bone marrow tyrosine kinase on chromosome X (BMX) in breast cancer development. The expression of BMX was examined by qPCR and immunohistochemistry. The effect of BMX on cell proliferation and migration was detected by Clone formation assay and Transwell assay. In vitro study, the correlation of BMX with Wnt/β-catenin pathway was explored by western blot and TOP/FOP flash assay. In the present study, we found that BMX was up-regulated in breast cancer, which was associated with the tumor differentiation and TNM stage. Oncogenic BMX enhanced the ability of breast cancer cell proliferation and migration. Furthermore, BMX could up-regulate the protein expression levels of p-β-catenin (Y142), p-β-catenin(Y654) and inhibit the expression level of p-β-catenin (S33/37), thus activating Wnt/β-catenin pathway in MCF-7 and MDA-MB-231 cells. In addition, we revealed that BMX promoted GSK3β phosphorylation, which suppressed the degradation of β-catenin. In this study, we identified that BMX-activated Wnt/β-catenin signaling pathway, playing an oncogenic role in breast cancer, suggesting that BMX could become a potential treatment target of breast cancer.

Nuclear localization of the tyrosine kinase BMX mediates VEGFR2 expression.

Vascular endothelial growth factor receptors (VEGFRs) are major contributors to angiogenesis and lymphangiogenesis through the binding of VEGF ligands. We have previously shown that the bone marrow tyrosine kinase BMX is critical for inflammatory angiogenesis via its direct transactivation of VEGFR2. In the present study, we show that siRNA‐mediated silencing of BMX led to a significant decrease in the total levels of VEGFR2 mRNA and protein, without affecting their stability, in human endothelial cells (ECs). Interestingly, BMX was detected in the nuclei of ECs, and the SH3 domain of BMX was necessary for its nuclear localization. Luciferase assays showed a significant decrease in the Vegfr2 (kdr) gene promoter activity in ECs after BMX silencing, indicating that BMX is necessary for Vegfr2 transcription. In addition, we found that wild‐type BMX, but not a catalytic inactive mutant BMX‐K445R, promoted Vegfr2 promoter activity and VEGF‐induced EC migration and tube sprouting. Mechanistically, we show that the enhancement of Vegfr2 promoter activity by BMX was mediated by Sp1, a transcription factor critical for the Vegfr2 promoter. Loss of BMX significantly reduced Sp1 binding to the Vegfr2 promoter as assayed by chromatin immunoprecipitation assays. Wild‐type BMX, but not a kinase‐inactive form of BMX, associated with and potentially phosphorylated Sp1. Moreover, a nuclear‐targeted BMX (NLS‐BMX), but not cytoplasm‐localized form (NES‐BMX), bound to Sp1 and augmented VEGFR2 expression. In conclusion, we uncovered a novel function of nuclear‐localized BMX in regulating VEGFR2 expression and angiogenesis, suggesting that BMX is a therapeutic target for angiogenesis‐related diseases.

Voluntary Physical Exercise Improves Subsequent Motor and Cognitive Impairments in a Rat Model of Parkinson's Disease.

Background: Parkinson’s disease (PD) is typically characterized by impairment of motor function. Gait disturbances similar to those observed in patients with PD can be observed in animals after injection of neurotoxin 6-hydroxydopamine (6-OHDA) to induce unilateral nigrostriatal dopamine depletion. Exercise has been shown to be a promising non-pharmacological approach to reduce the risk of neurodegenerative disease. Methods: In this study, we investigated the long-term effects of voluntary running wheel exercise on gait phenotypes, depression, cognitive, rotational behaviors as well as histology in a 6-OHDA-lesioned rat model of PD. Results: We observed that, when compared with the non-exercise controls, five-week voluntary exercise alleviated and postponed the 6-OHDA-induced gait deficits, including a significantly improved walking speed, step/stride length, base of support and print length. In addition, we found that the non-motor functions, such as novel object recognition and forced swim test, were also ameliorated by voluntary exercise. However, the rotational behavior of the exercise group did not show significant differences when compared with the non-exercise group. Conclusions: We first analyzed the detailed spatiotemporal changes of gait pattern to investigate the potential benefits after long-term exercise in the rat model of PD, which could be useful for future objective assessment of locomotor function in PD or other neurological animal models. Furthermore, these results suggest that short-term voluntary exercise is sufficient to alleviate cognition deficits and depressive behavior in 6-OHDA lesioned rats and long-term treatment reduces the progression of motor symptoms and elevates tyrosine hydroxylase (TH), Brain-derived neurotrophic factor (BDNF), bone marrow tyrosine kinase in chromosome X (BMX) protein expression level without affecting dopaminergic (DA) neuron loss in this PD rat model.

Relationship of mechanical impact magnitude to neurologic dysfunction severity in a rat traumatic brain injury model.

ObjectiveTraumatic brain injury (TBI) is a major brain injury type commonly caused by traffic accidents, falls, violence, or sports injuries. To obtain mechanistic insights about TBI, experimental animal models such as weight-drop-induced TBI in rats have been developed to mimic closed-head injury in humans. However, the relationship between the mechanical impact level and neurological severity following weight-drop-induced TBI remains uncertain. In this study, we comprehensively investigated the relationship between physical impact and graded severity at various weight-drop heights.ApproachThe acceleration, impact force, and displacement during the impact were accurately measured using an accelerometer, a pressure sensor, and a high-speed camera, respectively. In addition, the longitudinal changes in neurological deficits and balance function were investigated at 1, 4, and 7 days post TBI lesion. The inflammatory expression markers tested by Western blot analysis, including glial fibrillary acidic protein, beta-amyloid precursor protein, and bone marrow tyrosine kinase gene in chromosome X, in the frontal cortex, hippocampus, and corpus callosum were investigated at 1 and 7 days post-lesion.ResultsGradations in impact pressure produced progressive degrees of injury severity in the neurological score and balance function. Western blot analysis demonstrated that all inflammatory expression markers were increased at 1 and 7 days post-impact injury when compared to the sham control rats. The severity of neurologic dysfunction and induction in inflammatory markers strongly correlated with the graded mechanical impact levels.ConclusionsWe conclude that the weight-drop-induced TBI model can produce graded brain injury and induction of neurobehavioral deficits and may have translational relevance to developing therapeutic strategies for TBI.

Role of bone marrow tyrosine kinase on chromosome X in the production of pro-inflammatory cytokines from mouse mononuclear-macrophages RAW264.7 induced by endotoxin/lipopolysaccharide and its mechanism

Role of bone marrow tyrosine kinase on chromosome X in the production of pro-inflammatory cytokines from mouse mononuclear-macrophages RAW264.7 induced by endotoxin/lipopolysaccharide and its mechanism

Glucose-Dependent Insulinotropic Polypeptide Ameliorates Mild Traumatic Brain Injury-Induced Cognitive and Sensorimotor Deficits and Neuroinflammation in Rats.

Mild traumatic brain injury (mTBI) is a major public health issue, representing 75–90% of all cases of TBI. In clinical settings, mTBI, which is defined as a Glascow Coma Scale (GCS) score of 13–15, can lead to various physical, cognitive, emotional, and psychological-related symptoms. To date, there are no pharmaceutical-based therapies to manage the development of the pathological deficits associated with mTBI. In this study, the neurotrophic and neuroprotective properties of glucose-dependent insulinotropic polypeptide (GIP), an incretin similar to glucagon-like peptide-1 (GLP-1), was investigated after its steady-state subcutaneous administration, focusing on behavior after mTBI in an in vivo animal model. The mTBI rat model was generated by a mild controlled cortical impact (mCCI) and used to evaluate the therapeutic potential of GIP. We used the Morris water maze and novel object recognition tests, which are tasks for spatial and recognition memory, respectively, to identify the putative therapeutic effects of GIP on cognitive function. Further, beam walking and the adhesive removal tests were used to evaluate locomotor activity and somatosensory functions in rats with and without GIP administration after mCCI lesion. Lastly, we used immunohistochemical (IHC) staining and Western blot analyses to evaluate the inflammatory markers, glial fibrillary acidic protein (GFAP), amyloid-β precursor protein (APP), and bone marrow tyrosine kinase gene in chromosome X (BMX) in animals with mTBI. GIP was well tolerated and ameliorated mTBI-induced memory impairments, poor balance, and sensorimotor deficits after initiation in the post-injury period. In addition, GIP mitigated mTBI-induced neuroinflammatory changes on GFAP, APP, and BMX protein levels. These findings suggest GIP has significant benefits in managing mTBI-related symptoms and represents a novel strategy for mTBI treatment.

Abstract 3380: Direct contact with perivascular tumor cells enhances integrin αvβ3 signaling and migration of endothelial cells

Abstract The secretion of soluble pro-angiogenic factors by tumor cells and stromal cells in the perivascular niche promotes the aggressive angiogenesis that is typical of glioblastoma (GBM), which requires endothelial cell (EC) migration. Here, we show that angiogenesis can also be promoted by a direct interaction between brain tumor cells, including tumor cells with cancer stem-like properties (CSCs), and ECs. The direct interaction between CSCs and ECs was mediated by binding of integrin αvβ3 expressed on ECs to the RGD-peptide in L1CAM expressed on CSCs. Using in vitro assays, we found that this interaction increased network formation and migration-associated signaling events in ECs, including activation of integrin αvβ3, FAK, bone marrow tyrosine kinase on chromosome X (BMX), p130CAS, ERK and JNK. Comparison of the effects of co-culturing CSCs with ECs versus the effects of conditioned media from CSCs co-cultured with ECs indicated that upstream and downstream effector activation was not attributed to a secreted factor(s). Activation of αvβ3 and BMX was required for migration stimulated by direct binding but not for migration stimulated by soluble factors. RGD-peptide treatment of mice with established intracerebral GBM xenografts significantly reduced the percentage of Sox2-positive tumor cells (consistent with CSCs) in close proximity to ECs, decreased integrin αvβ3 and BMX activation and p130CAS phosphorylation in the ECs, and reduced the vessel surface area. These results show that direct interactions between CSCs and ECs have potent effects on EC migration and reveal a previously unrecognized aspect of the regulation of angiogenesis in GBM that can impact therapeutic anti-angiogenic targeting. Citation Format: Monica Burgett, Justin Lathia, Patrick Roth, Amy Nowacki, Elena Pugacheva, Ping Huang, Amit Vasanji, Li Meizhang, Tatiana Byzova, Tom Mikkelsen, Shideng Bao, Jeremy Rich, Michael Weller, Candece Gladson. Direct contact with perivascular tumor cells enhances integrin αvβ3 signaling and migration of endothelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3380.

Direct contact with perivascular tumor cells enhances integrin αvβ3 signaling and migration of endothelial cells.

The secretion of soluble pro-angiogenic factors by tumor cells and stromal cells in the perivascular niche promotes the aggressive angiogenesis that is typical of glioblastoma (GBM). Here, we show that angiogenesis also can be promoted by a direct interaction between brain tumor cells, including tumor cells with cancer stem-like properties (CSCs), and endothelial cells (ECs). As shown in vitro, this direct interaction is mediated by binding of integrin αvβ3 expressed on ECs to the RGD-peptide in L1CAM expressed on CSCs. It promotes both EC network formation and enhances directed migration toward basic fibroblast growth factor. Activation of αvβ3 and bone marrow tyrosine kinase on chromosome X (BMX) is required for migration stimulated by direct binding but not for migration stimulated by soluble factors. RGD-peptide treatment of mice with established intracerebral GBM xenografts significantly reduced the percentage of Sox2-positive tumor cells and CSCs in close proximity to ECs, decreased integrin αvβ3 and BMX activation and p130CAS phosphorylation in the ECs, and reduced the vessel surface area. These results reveal a previously unrecognized aspect of the regulation of angiogenesis in GBM that can impact therapeutic anti-angiogenic targeting.

Current understanding of tyrosine kinase BMX in inflammation and its inhibitors.

Tec family kinases, which include tyrosine kinase expressed in hepatocellular carcinoma (TEC), Bruton’s tyrosine kinase (BTK), interleukin (IL)-2-inducible T-cell kinase (ITK), tyrosine-protein kinase (TXK), and bone marrow tyrosine kinase on chromosome X (BMX), are the second largest group of non-receptor tyrosine kinases and have a highly conserved carboxyl-terminal kinase domain. BMX was identified in human bone marrow cells, and was demonstrated to have been expressed in myeloid hematopoietic lineages cells, endothelial cells, and several types of cancers. Significant progress in this area during the last decade revealed an important role for BMX in inflammation and oncologic disorders. This review focuses on BMX biology, its role in inflammation and possible signaling pathways, and the potential of selective BMX inhibitors.

The Influence ofBMXGene Polymorphisms on Clinical Symptoms after Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is one of the most common neurological disorders. Most patients diagnosed with mTBI could fully recover, but 15% of patients suffer from persistent symptoms. In recent studies, genetic factors were found to be associated with recovery and clinical outcomes after TBI. In addition, results from our previous research have demonstrated that the bone marrow tyrosine kinase gene in chromosome X (BMX), a member of the Tec family of kinases, is highly expressed in rats with TBI. Therefore, our aim in this study was to identify the association between genetic polymorphisms of BMX and clinical symptoms following mTBI. Four tagging single nucleotide polymorphisms (tSNPs) of BMX with minimum allele frequency (MAF) >1% were selected from the HapMap Han Chinese database. Among these polymorphisms, rs16979956 was found to be associated with the Beck anxiety inventory (BAI) and dizziness handicap inventory (DHI) scores within the first week after head injury. Additionally, another SNP, rs35697037, showed a significant correlation with dizziness symptoms. These findings suggested that polymorphisms of the BMX gene could be a potential predictor of clinical symptoms following mTBI.

PI3K and STAT3: A New Alliance

Recent proteomic data have uncovered an interdependence of PI3K and STAT3. In PI3K-tranformed murine cells, STAT3 is phosphorylated on Y705 and activated in a PI3K-dependent manner. Dominant negative STAT3 interferes with PI3K-induced oncogenic transformation. Phosphorylation of STAT3 in PI3K-transformed murine cells is mediated by the TEC kinase BMX. Observations on glioblastoma stem cells reveal similar critical roles for STAT3 and BMX. The new data document an important role of STAT3 in PI3K-driven oncogenic transformation and mark BMX as a promising therapeutic target that could enhance the effectiveness of PI3K inhibitors. The PI3K–TOR and STAT3 signaling pathways represent two distinct regulatory networks. The discovery of a functional link between these pathways is significant for our understanding of PI3K- and STAT3-driven oncogenic mechanisms and identifies the TEC kinase BMX as a new cancer target.

Endothelial-Specific Transgenesis of TNFR2 Promotes Adaptive Arteriogenesis and Angiogenesis

We have previously shown that the tumor necrosis factor receptor 2 (TNFR2) protein is highly upregulated in vascular endothelium in response to ischemia, and a global deletion of TNFR2 in mice blunts ischemia-induced arteriogenesis and angiogenesis. However, the role of endothelial TNFR2 is not defined. In this study, we used endothelial cell (EC)-specific transgenesis of TNFR2 (TNFR2-TG) in mice to determine the in vivo function of TNFR2 in arteriogenesis and angiogenesis. In a femoral artery ligation model, TNFR2-TG mice had enhanced limb perfusion recovery and ischemic reserve capacity. TNFR2-TG mice also exhibited significantly enhanced arteriogenesis in the upper limb, whereas capillary formation and maturation in the lower limb were associated with reduction in cellular apoptosis and increased proliferation. Consistently, ischemia-induced TNFR2-dependent bone marrow tyrosine kinase in chromosome X-vascular endothelial growth factor receptor 2 proangiogenic signaling was augmented in TNFR2-TG mice. To further determine whether EC-expressed TNFR2 is sufficient to mediate ischemia-induced angiogenesis, we crossed TNFR2-TG with TNFR2-deficient mice to generate TNFR2-knockout (KO)/TG mice, in which only vascular ECs express TNFR2. The EC-expressed TNFR2 partially rescued the defects of TNFR2-KO in ischemia-induced angiogenic signaling and flow recovery. These in vivo data support a critical role for endothelial TNFR2 in ischemia-mediated adaptive angiogenesis. Therefore, specific expression and activation of TNFR2 in ECs may provide a novel strategy for the treatment of vascular diseases such as coronary artery disease and peripheral arterial disease.

Abstract B263: Mechanistic, functional, and in vivo efficacy of inhibiting ETK/BMX in cancer models

Abstract Epithelial and endothelial tyrosine kinase (ETK), also known as bone marrow tyrosine kinase gene in chromosome X (BMX), belongs to the Burton's Tyrosine Kinase (BTK) family. ETK is a nonreceptor tyrosine kinases involved in tumorigenicity, adhesion, motility, angiogenesis, proliferation, and differentiation. It is overexpressed in metastatic breast and prostate cancers and is implicated in the neuroendocrine transformation of prostate cells. Significant oncogenes such as Src, focal adhesion kinase (FAK), and phosphatidyl-inositol (PI)-3 kinase are upstream regulators of ETK while several hub proteins, also critical for cancer development and progression, such as AKT, STAT3, and p21 activated kinase 1 (PAK1), are downstream of ETK. Cell-based validation studies also demonstrate that inhibition of ETK diminishes cellular transformation, proliferation, and migration. Such evidence provides substantial reasoning for developing new small molecule inhibitors against ETK. Applying SuperGen's proprietary small molecule drug discovery process, CLIMB™ a series of lead compounds were effectively developed to potently and selectively inhibit ETK. Biochemical data from kinase screens against ETK demonstrated nanomolar potency of SuperGen's inhibitors and good selectivity toward ETK from a panel of 75 kinases. To mechanistically demonstrate ETK inhibition in cell-based models, immunoprecipitations were carried out and phospho-ETK levels were detected. These compounds exhibited a dose-dependent response against phospho-ETK. Functionally, the inhibition of ETK will produce changes in phospho-AKT (S473) levels as well as phospho-STAT3 (Y705) levels downstream of EGF stimulation. Using Luminex bead-based technology and western blot techniques respectively, SuperGen's lead ETK compounds also yielded concentration-dependent responses with EC50 values in the nanomolar region. Given ETK's reported angiogenic role in endothelial cell migration and proliferation, these compounds were evaluated in an in vitro tube formation assay. Likewise, these ETK inhibitors demonstrated potent nanomolar inhibition of endothelial tube formation. Finally, having shown mechanistically and functionally the efficacy of ETK inhibitors through in vitro assays, the compounds were tested with in vivo solid tumor efficacy models in nude mice. All five lead inhibitors effectively decreased tumor burden volume relative to the vehicle treatment group in an AN3CA xenograft model. Taken together, these data suggest SuperGen's small molecule ETK inhibitors represent a potentially new therapeutic avenue for treating patients with solid tumor malignancies in the future. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B263.

Bmx tyrosine kinase regulates TLR4-induced IL-6 production in human macrophages independently of p38 MAPK and NFκB activity

Chronic inflammation, as seen in conditions such as rheumatoid arthritis and Crohn disease, is in part driven by discordant production of inflammatory cytokines, such as tumor necrosis factor-alpha and interleukin-6 (IL-6). Tyrosine kinase activity is essential to lipopolysaccharide-induced cytokine production in monocytes, and previous studies by us and others have implicated a role for the Tec kinase Bruton's tyrosine kinase (Btk) in inflammatory cytokine production. Here we show that knockdown of Btk using RNA interference results in decreased tumor necrosis factor-alpha, but not IL-6 production. Further investigations into the signaling mechanisms regulating IL-6 production led to the discovery that the Tec kinase bone marrow tyrosine kinase gene in chromosome X (Bmx) regulates Toll-like receptor-induced IL-6 production. Our data further showed that Bmx-dependent super-induction of IL-6 does not involve nuclear factor-kappaB activity. More detailed investigations of pathways downstream of Bmx signaling revealed that Bmx targets the IL-6 3' untranslated region to increase mRNA stabilization via a novel, thus far undefined, p38 mitogen activated protein kinase-independent pathway. These data have important implications for the design of therapeutics targeted against specific cytokines and their regulators in inflammatory disease.

Differential Functions of Tumor Necrosis Factor Receptor 1 and 2 Signaling in Ischemia-Mediated Arteriogenesis and Angiogenesis

We have previously shown that tumor necrosis factor (TNF) acts via its two receptors TNFR1 and TNFR2 to elicit distinct signaling pathways in vascular endothelial cells (ECs). Here we used a femoral artery ligation model to demonstrate that TNFR1-knockout (KO) mice had enhanced, whereas TNFR2-KO had reduced, capacity in clinical recovery, limb perfusion, and ischemic reserve capacity compared with the wild-type mice. Consistently, ischemia-initiated collateral growth (arteriogenesis) in the upper limb and capillary formation and vessel maturation (angiogenesis) in the lower limb were enhanced in TNFR1-KO but were reduced in TNFR2-KO mice. Furthermore, our results suggest that vascular proliferation, but not infiltration of macrophages and lymphocytes, accounted for the phenotypic differences between the TNFR1-KO and TNFR2-KO mice. In wild-type animals TNFR2 protein in vascular endothelium was highly up-regulated in response to ischemia, leading to increased TNFR2-specific signaling as determined by the formation TNFR2-TRAF2 complex and activation of TNFR2-specific kinase Bmx/Etk. In isolated murine ECs, activation of TNFR2 induced nuclear factor-kappaB-dependent reporter gene expression, EC survival, and migration. In contrast, activation of TNFR1 caused inhibition of EC migration and EC apoptosis. These data demonstrate that TNFR1 and TNFR2 play differential roles in ischemia-mediated arteriogenesis and angiogenesis, partly because of their opposite effects on EC survival and migration.