Tumor Treating Fields Alter the Kinomic Landscape in Glioblastoma Revealing Therapeutic Vulnerabilities.

Treatment for the deadly brain tumor glioblastoma (GBM) has been improved through the non-invasive addition of alternating electric fields, called tumor treating fields (TTFields). Improving both progression-free and overall survival, TTFields are currently approved for treatment of recurrent GBMs as a monotherapy and in the adjuvant setting alongside TMZ for newly diagnosed GBMs. These TTFields are known to inhibit mitosis, but the full molecular impact of TTFields remains undetermined. Therefore, we sought to understand the ability of TTFields to disrupt the growth patterns of and induce kinomic landscape shifts in TMZ-sensitive and -resistant GBM cells. We determined that TTFields significantly decreased the growth of TMZ-sensitive and -resistant cells. Kinomic profiling predicted kinases that were induced or repressed by TTFields, suggesting possible therapy-specific vulnerabilities. Serving as a potential pro-survival mechanism for TTFields, kinomics predicted the increased activity of platelet-derived growth-factor receptor alpha (PDGFRα). We demonstrated that the addition of the PDGFR inhibitor, crenolanib, to TTFields further reduced cell growth in comparison to either treatment alone. Collectively, our data suggest the efficacy of TTFields in vitro and identify common signaling responses to TTFields in TMZ-sensitive and -resistant populations, which may support more personalized medicine approaches.

Inhibition of Erb-B2 Receptor Tyrosine Kinase 3 and Associated Regulatory Pathways Potently Impairs Malignant Peripheral Nerve Sheath Tumor Proliferation and Survival.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, currently untreatable Schwann cell-derived neoplasms with hyperactive mitogen-activated protein kinase and mammalian target of rapamycin signaling pathways. To identify potential therapeutic targets, previous studies used genome-scale shRNA screens that implicated the neuregulin-1 receptor erb-B2 receptor tyrosine kinase 3 (erbB3) in MPNST proliferation and/or survival. The current study shows that erbB3 is commonly expressed in MPNSTs and MPNST cell lines and that erbB3 knockdown inhibits MPNST proliferation and survival. Kinomic and microarray analyses of Schwann and MPNST cells implicate Src- and erbB3-mediated calmodulin-regulated signaling as key pathways. Consistent with this, inhibition of upstream (canertinib, sapitinib, saracatinib, and calmodulin) and parallel (AZD1208) signaling pathways involving mitogen-activated protein kinase and mammalian target of rapamycin reduced MPNST proliferation and survival. ErbB inhibitors (canertinib and sapitinib) or erbB3 knockdown in combination with Src (saracatinib), calmodulin [trifluoperazine (TFP)], or proviral integration site of Moloney murine leukemia kinase (AZD1208) inhibition even more effectively reduces proliferation and survival. Drug inhibition enhances an unstudied calmodulin-dependent protein kinase IIα phosphorylation site in an Src-dependent manner. The Src family kinase inhibitor saracatinib reduces both basal and TFP-induced erbB3 and calmodulin-dependent protein kinase IIα phosphorylation. Src inhibition (saracatinib), like erbB3 knockdown, prevents these phosphorylation events; and when combined with TFP, it even more effectively reduces proliferation and survival compared with monotherapy. These findings implicate erbB3, calmodulin, proviral integration site of Moloney murine leukemia kinases, and Src family members as important therapeutic targets in MPNSTs and demonstrate that combinatorial therapies targeting critical MPNST signaling pathways are more effective.

Fibroblast GSK-3α Promotes Fibrosis via RAF-MEK-ERK Pathway in the Injured Heart.

BACKGROUND: Heart failure is the leading cause of mortality, morbidity, and health care expenditures worldwide. Numerous studies have implicated GSK-3 (glycogen synthase kinase-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms seem to play overlapping, unique and even opposing functions in the heart. Previously, we have shown that of the 2 isoforms of GSK-3, cardiac fibroblast GSK-3ß acts as a negative regulator of myocardial fibrosis in the ischemic heart. However, the role of cardiac fibroblast-GSK-3α in the pathogenesis of cardiac diseases is completely unknown. METHODS: To define the role of cardiac fibroblast-GSK-3α in myocardial fibrosis and heart failure, GSK-3α was deleted from fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or Postn-promoter-driven Cre recombinase. Control and GSK-3α KO mice were subjected to cardiac injury and heart parameters were evaluated. The fibroblast kinome mapping was carried out to delineate molecular mechanism followed by in vivo and in vitro analysis. RESULTS: Fibroblast-specific GSK-3α deletion restricted fibrotic remodeling and preserved function of the injured heart. We observed reductions in cell migration, collagen gel contraction, α-SMA protein levels, and expression of ECM genes in TGFß1-treated KO fibroblasts, indicating that GSK-3α is required for myofibroblast transformation. Surprisingly, GSK-3α deletion did not affect SMAD3 activation, suggesting the profibrotic role of GSK-3α is SMAD3 independent. The molecular studies confirmed decreased ERK signaling in GSK-3α-KO CFs. Conversely, adenovirus-mediated expression of a constitutively active form of GSK-3α (Ad-GSK-3αS21A) in fibroblasts increased ERK activation and expression of fibrogenic proteins. Importantly, this effect was abolished by ERK inhibition. CONCLUSIONS: GSK-3α-mediated MEK-ERK activation is a critical profibrotic signaling circuit in the injured heart, which operates independently of the canonical TGF-ß1-SMAD3 pathway. Therefore, strategies to inhibit the GSK-3α-MEK-ERK signaling circuit could prevent adverse fibrosis in diseased hearts.

Selective suppression of melanoma lacking IFN-γ pathway by JAK inhibition depends on T cells and host TNF signaling.

Therapeutic resistance to immune checkpoint blockers (ICBs) in melanoma patients is a pressing issue, of which tumor loss of IFN-γ signaling genes is a major underlying mechanism. However, strategies of overcoming this resistance mechanism have been largely elusive. Moreover, given the indispensable role of tumor-infiltrating T cells (TILs) in ICBs, little is known about how tumor-intrinsic loss of IFN-γ signaling (IFNγR1KO) impacts TILs. Here, we report that IFNγR1KO melanomas have reduced infiltration and function of TILs. IFNγR1KO melanomas harbor a network of constitutively active protein tyrosine kinases centered on activated JAK1/2. Mechanistically, JAK1/2 activation is mediated by augmented mTOR. Importantly, JAK1/2 inhibition with Ruxolitinib selectively suppresses the growth of IFNγR1KO but not scrambled control melanomas, depending on T cells and host TNF. Together, our results reveal an important role of tumor-intrinsic IFN-γ signaling in shaping TILs and manifest a targeted therapy to bypass ICB resistance of melanomas defective of IFN-γ signaling.

An in vivo model of glioblastoma radiation resistance identifies long noncoding RNAs and targetable kinases.

Key molecular regulators of acquired radiation resistance in recurrent glioblastoma (GBM) are largely unknown, with a dearth of accurate preclinical models. To address this, we generated 8 GBM patient-derived xenograft (PDX) models of acquired radiation therapy-selected (RTS) resistance compared with same-patient, treatment-naive (radiation-sensitive, unselected; RTU) PDXs. These likely unique models mimic the longitudinal evolution of patient recurrent tumors following serial radiation therapy. Indeed, while whole-exome sequencing showed retention of major genomic alterations in the RTS lines, we did detect a chromosome 12q14 amplification that was associated with clinical GBM recurrence in 2 RTS models. A potentially novel bioinformatics pipeline was applied to analyze phenotypic, transcriptomic, and kinomic alterations, which identified long noncoding RNAs (lncRNAs) and targetable, PDX-specific kinases. We observed differential transcriptional enrichment of DNA damage repair pathways in our RTS models, which correlated with several lncRNAs. Global kinomic profiling separated RTU and RTS models, but pairwise analyses indicated that there are multiple molecular routes to acquired radiation resistance. RTS model-specific kinases were identified and targeted with clinically relevant small molecule inhibitors. This cohort of in vivo RTS patient-derived models will enable future preclinical therapeutic testing to help overcome the treatment resistance seen in patients with GBM.

Serine-Threonine Kinase Receptor Associate Protein (STRAP) confers an aggressive phenotype in neuroblastoma via regulation of Focal Adhesion Kinase (FAK).

BACKGROUND: Serine-threonine kinase receptor associated protein (STRAP), a scaffolding protein, is upregulated in many solid tumors. As such, we hypothesized that STRAP may be overexpressed in neuroblastoma tumors and may play a role in neuroblastoma tumor progression. METHODS: We examined two publicly available neuroblastoma patient databases, GSE49710 (n = 498) and GSE49711 (n = 498), to investigate STRAP expression in human specimens. SK-N-AS and SK-N-BE(2) human neuroblastoma cell lines were stably transfected with STRAP overexpression (OE) plasmid, and their resulting phenotype studied. PamChip® kinomic peptide microarray evaluated the effects of STRAP overexpression on kinase activation. RESULTS: In human specimens, higher STRAP expression correlated with high-risk disease, unfavorable histology, and decreased overall neuroblastoma patient survival. STRAP OE in neuroblastoma cell lines led to increased proliferation, growth, supported a stem-like phenotype and activated downstream FAK targets. When FAK was targeted with the small molecule FAK inhibitor, PF-573,228, STRAP OE neuroblastoma cells had significantly decreased growth compared to control empty vector cells. CONCLUSION: Increased STRAP expression in neuroblastoma was associated with unfavorable tumor characteristics. STRAP OE resulted in increased kinomic activity of FAK. These findings suggest that the poorer outcomes in neuroblastoma tumors associated with STRAP overexpression may be secondary to FAK activation.

Cardiomyocyte stromal interaction molecule 1 is a key regulator of Ca2+ -dependent kinase and phosphatase activity in the mouse heart.

Stromal interaction molecule 1 (STIM1) is a major regulator of store-operated calcium entry in non-excitable cells. Recent studies have suggested that STIM1 plays a role in pathological hypertrophy; however, the physiological role of STIM1 in the heart is not well understood. We have shown that mice with a cardiomyocyte deletion of STIM1 (cr STIM1-/- ) develop ER stress, mitochondrial, and metabolic abnormalities, and dilated cardiomyopathy. However, the specific signaling pathways and kinases regulated by STIM1 are largely unknown. Therefore, we used a discovery-based kinomics approach to identify kinases differentially regulated by STIM1. Twelve-week male control and cr STIM1-/- mice were injected with saline or phenylephrine (PE, 15 mg/kg, s.c, 15 min), and hearts obtained for analysis of the Serine/threonine kinome. Primary analysis was performed using BioNavigator 6.0 (PamGene), using scoring from the Kinexus PhosphoNET database and GeneGo network modeling, and confirmed using standard immunoblotting. Kinomics revealed significantly lower PKG and protein kinase C (PKC) signaling in the hearts of the cr STIM1-/- in comparison to control hearts, confirmed by immunoblotting for the calcium-dependent PKC isoform PKCα and its downstream target MARCKS. Similar reductions in cr STIM1-/- hearts were found for the kinases: MEK1/2, AMPK, and PDPK1, and in the activity of the Ca2+ -dependent phosphatase, calcineurin. Electrocardiogram analysis also revealed that cr STIM1-/- mice have significantly lower HR and prolonged QT interval. In conclusion, we have shown several calcium-dependent kinases and phosphatases are regulated by STIM1 in the adult mouse heart. This has important implications in understanding how STIM1 contributes to the regulation of cardiac physiology and pathophysiology.

Mesangioproliferative Kidney Diseases and Platelet-Derived Growth Factor-Mediated AXL Phosphorylation.

RATIONALE & OBJECTIVE: Immunoglobulin A nephropathy (IgAN) is a common glomerular disease, with mesangial cell proliferation as a major feature. There is no disease-specific treatment. Platelet-derived growth factor (PDGF) contributes to the pathogenesis of IgAN. To better understand its pathogenic mechanisms, we assessed PDGF-mediated AXL phosphorylation in human mesangial cells and kidney tissue biopsy specimens. STUDY DESIGN: Immunostaining using human kidney biopsy specimens and in vitro studies using primary human mesangial cells. SETTING & PARTICIPANTS: Phosphorylation of AXL was assessed in cultured mesangial cells and 10 kidney-biopsy specimens from 5 patients with IgAN, 3 with minimal change disease, 1 with membranous nephropathy, and 1 with mesangioproliferative glomerulonephritis (GN). PREDICTOR: Glomerular staining for phospho-AXL in kidney biopsy specimens of patients with mesangioproliferative diseases. OUTCOMES: Phosphorylated AXL detected in biopsy tissues of patients with IgAN and mesangioproliferative GN and in cultured mesangial cells stimulated with PDGF. ANALYTIC APPROACH: t test, Mann-Whitney test, and analysis of variance were used to assess the significance of mesangial cell proliferative changes. RESULTS: Immunohistochemical staining revealed enhanced phosphorylation of glomerular AXL in IgAN and mesangioproliferative GN, but not in minimal change disease and membranous nephropathy. Confocal-microscopy immunofluorescence analysis indicated that mesangial cells rather than endothelial cells or podocytes expressed phospho-AXL. Kinomic profiling of primary mesangial cells treated with PDGF revealed activation of several protein-tyrosine kinases, including AXL. Immunoprecipitation experiments indicated association of AXL and PDGF receptor proteins. An AXL-specific inhibitor (bemcentinib) partially blocked PDGF-induced cellular proliferation and reduced phosphorylation of AXL and PDGF receptor and the downstream signals (AKT1 and ERK1/2). LIMITATIONS: Small number of kidney biopsy specimens to correlate the activation of AXL with disease severity. CONCLUSIONS: PDGF-mediated signaling in mesangial cells involves transactivation of AXL. Finding appropriate inhibitors to block PDGF-mediated transactivation of AXL may provide new therapeutic options for mesangioproliferative kidney diseases such as IgAN.

Serine-Threonine Kinase Receptor-Associated Protein (STRAP) Knockout Decreases the Malignant Phenotype in Neuroblastoma Cell Lines.

Background: Serine-threonine kinase receptor-associated protein (STRAP) plays an important role in neural development but also in tumor growth. Neuroblastoma, a tumor of neural crest origin, is the most common extracranial solid malignancy of childhood and it continues to carry a poor prognosis. The recent discovery of the role of STRAP in another pediatric solid tumor, osteosarcoma, and the known function of STRAP in neural development, led us to investigate the role of STRAP in neuroblastoma tumorigenesis. Methods: STRAP protein expression was abrogated in two human neuroblastoma cell lines, SK-N-AS and SK-N-BE(2), using transient knockdown with siRNA, stable knockdown with shRNA lentiviral transfection, and CRISPR-Cas9 genetic knockout. STRAP knockdown and knockout cells were examined for phenotypic alterations in vitro and tumor growth in vivo. Results: Cell proliferation, motility, and growth were significantly decreased in STRAP knockout compared to wild-type cells. Indicators of stemness, including mRNA abundance of common stem cell markers Oct4, Nanog, and Nestin, the percentage of cells expressing CD133 on their surface, and the ability to form tumorspheres were significantly decreased in the STRAP KO cells. In vivo, STRAP knockout cells formed tumors less readily than wild-type tumor cells. Conclusion: These novel findings demonstrated that STRAP plays a role in tumorigenesis and maintenance of neuroblastoma stemness.

PIM kinases mediate resistance to cisplatin chemotherapy in hepatoblastoma.

Despite increasing incidence, treatment for hepatoblastoma has not changed significantly over the past 20 years. Chemotherapeutic strategies continue to rely on cisplatin, as it remains the most active single agent against hepatoblastoma. However, chemoresistance remains a significant challenge with 54-80% of patients developing resistance to chemotherapy after 4-5 cycles of treatment. Stem cell-like cancer cells (SCLCCs) are a subset of cells thought to play a role in chemoresistance and disease recurrence. We have previously demonstrated that Proviral Integration site for Moloney murine leukemia virus (PIM) kinases, specifically PIM3, play a role in hepatoblastoma cell proliferation and tumor growth and maintain the SCLCC phenotype. Here, we describe the development of a cisplatin-resistant hepatoblastoma xenograft model of the human HuH6 cell line and a patient-derived xenograft, COA67. We provide evidence that these cisplatin-resistant cells are enriched for SCLCCs and express PIM3 at higher levels than cisplatin-naïve cells. We demonstrate that PIM inhibition with AZD1208 sensitizes cisplatin-resistant hepatoblastoma cells to cisplatin, enhances cisplatin-mediated apoptosis, and decreases the SCLCC phenotype seen with cisplatin resistance. Together, these findings indicate that PIM inhibition may be a promising adjunct in the treatment of hepatoblastoma to effectively target SCLCCs and potentially decrease chemoresistance and subsequent disease relapse.

miR-31 Displays Subtype Specificity in Lung Cancer.

miRNA rarely possess pan-oncogenic or tumor-suppressive properties. Most miRNAs function under tissue-specific contexts, acting as either tumor suppressors in one tissue, promoting oncogenesis in another, or having no apparent role in the regulation of processes associated with the hallmarks of cancer. What has been less clear is the role of miRNAs within cell types of the same tissue and the ability within each cell type to contribute to oncogenesis. In this study, we characterize the role of one such tissue-specific miRNA, miR-31, recently identified as the most oncogenic miRNA in lung adenocarcinoma, across the histologic spectrum of human lung cancer. Compared with normal lung tissue, miR-31 was overexpressed in patient lung adenocarcinoma, squamous cell carcinoma, and large-cell neuroendocrine carcinoma, but not small-cell carcinoma or carcinoids. miR-31 promoted tumor growth in mice of xenografted human adenocarcinoma and squamous cell carcinoma cell lines, but not in large- or small-cell carcinoma lines. While miR-31 did not promote primary tumor growth of large- and small-cell carcinoma, it did promote spontaneous metastasis. Mechanistically, miR-31 altered distinct cellular signaling programs within each histologic subtype, resulting in distinct phenotypic differences. This is the first report distinguishing diverse functional roles for this miRNA across the spectrum of lung cancers and suggests that miR-31 has broad clinical value in human lung malignancy. SIGNIFICANCE: These findings demonstrate the oncogenic properties of miR-31 in specific subtypes of lung cancer and highlight it as a potential therapeutic target in these subtypes. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/1942/F1.large.jpg.

Fibroblast growth factor receptor facilitates recurrence of minimal residual disease following trastuzumab emtansine therapy.

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) that efficiently delivers a highly potent microtubule inhibitor to HER2 overexpressing cells. Herein, we utilize HER2 transformed human mammary epithelial cells (HME2) to demonstrate in vitro and in vivo response and recurrence upon T-DM1 treatment. Continuous in vitro dosing of HME2 cells with T-DM1 failed to produce a spontaneously resistant cell line. However, induction of epithelial-mesenchymal transition (EMT) via pretreatment with TGF-ß1 was capable of promoting emergence of T-DM1-resistant (TDM1R) cells. Flow cytometric analyses indicated that induction of EMT decreased trastuzumab binding, prior to overt loss of HER2 expression in TDM1R cells. Kinome analyses of TDM1R cells indicated increased phosphorylation of ErbB1, ErbB4, and FGFR1. TDM1R cells failed to respond to the ErbB kinase inhibitors lapatinib and afatinib, but they acquired sensitivity to FIIN4, a covalent FGFR kinase inhibitor. In vivo, minimal residual disease (MRD) remained detectable via bioluminescent imaging following T-DM1-induced tumor regression. Upon cessation of the ADC, relapse occurred and secondary tumors were resistant to additional rounds of T-DM1. These recurrent tumors could be inhibited by FIIN4. Moreover, ectopic overexpression of FGFR1 was sufficient to enhance tumor growth, diminish trastuzumab binding, and promote recurrence following T-DM1-induced MRD. Finally, patient-derived xenografts from a HER2+ breast cancer patient who had progressed on trastuzumab failed to respond to T-DM1, but tumor growth was significantly inhibited by FIIN4. Overall, our studies strongly support therapeutic combination of TDM1 with FGFR-targeted agents in HER2+ breast cancer.

Combining PARP and DNA-PK Inhibitors With Irradiation Inhibits HPV-Negative Head and Neck Cancer Squamous Carcinoma Growth.

Novel targeted agents to inhibit DNA repair pathways to sensitize tumors to irradiation (IR) are being investigated as an alternative to chemoradiation for locally advanced human papilloma virus negative (HPV-negative) head and neck squamous cell carcinoma (HNSCC). Two well-characterized targets that, when inhibited, exhibit potent IR sensitization are PARP1 and DNA-PKcs. However, their cooperation in sensitizing HPV-negative HNSCC to IR remains to be explored given that PARP1 and DNA-Pk CS bind to unresected stalled DNA replication forks and cooperate to recruit XRCC1 to facilitate double-strand break repair. Here, we show that the combination of the DNA-PK inhibitor NU7441 and the PARP inhibitor olaparib significantly decrease proliferation (61-78%) compared to no reduction with either agent alone (p < 0.001) in both SCC1 and SCC6 cell lines. Adding IR to the combination further decreased cell proliferation (91-92%, p < 0.001) in SCC1 and SCC6. Similar results were observed using long-term colony formation assays [dose enhancement ratio (DER) 2.3-3.2 at 4Gy, p < 0.05]. Reduced cell survival was attributed to increased apoptosis and G2/M cell cycle arrest. Kinomic analysis using tyrosine (PTK) and serine/threonine (STK) arrays reveals that combination treatment results in the most potent inhibition of kinases involved in the CDK and ERK pathways compared to either agent alone. In vivo, a significant delay of tumor growth was observed in UM-SCC1 xenografts receiving IR with olaparib and/or NU7441, which was similar to the cisplatin-IR group. Both regimens were less toxic than cisplatin-IR as assessed by loss of mouse body weight. Taken together, these results demonstrate that the combination of NU7441 and olaparib with IR enhances HPV-negative HNSCC inhibition in both cell culture and in mice, suggesting a potential innovative combination for effectively treating patients with HPV-negative HNSCC.

A cell-penetrating MARCKS mimetic selectively triggers cytolytic death in glioblastoma.

Glioblastoma (GBM) is an aggressive malignancy with limited effectiveness of standard of care therapies including surgery, radiation, and temozolomide chemotherapy necessitating novel therapeutics. Unfortunately, GBMs also harbor several signaling alterations that protect them from traditional therapies that rely on apoptotic programmed cell death. Because almost all GBM tumors have dysregulated phosphoinositide signaling as part of that process, we hypothesized that peptide mimetics derived from the phospholipid binding domain of Myristoylated alanine-rich C-kinase substrate (MARCKS) could serve as a novel GBM therapeutic. Using molecularly classified patient-derived xenograft (PDX) lines, cultured in stem-cell conditions, we demonstrate that cell permeable MARCKS effector domain (ED) peptides potently target all GBM molecular classes while sparing normal human astrocytes. Cell death mechanistic testing revealed that these peptides produce rapid cytotoxicity in GBM that overcomes caspase inhibition. Moreover, we identify a GBM-selective cytolytic death mechanism involving plasma membrane targeting and intracellular calcium accumulation. Despite limited relative partitioning to the brain, tail-vein peptide injection revealed tumor targeting in intracranially implanted GBM PDX. These results indicate that MARCKS ED peptide therapeutics may overcome traditional GBM resistance mechanisms, supporting further development of similar agents.

Leukemia Inhibitory Factor Signaling Enhances Production of Galactose-Deficient IgA1 in IgA Nephropathy.

OBJECTIVES: IgA nephropathy (IgAN) is thought to involve an autoimmune process wherein galactose-deficient IgA1 (Gd-IgA1), recognized as autoantigen by autoantibodies, forms pathogenic immune complexes. Mounting evidence has implicated abnormal activation of some protein-tyrosine kinases (PTKs) in IgAN. Furthermore, genome-wide association studies (GWAS) of IgAN provided insight into disease pathobiology and genetics. A GWAS locus on chromosome 22q12 contains genes encoding leukemia inhibitory factor (LIF) and oncostatin M, interleukin (IL)-6-related cytokines implicated in mucosal immunity and inflammation. We have previously shown that IL-6 mediates overproduction of Gd-IgA1 through aberrant STAT3 activation. Here, we show that LIF enhanced production of Gd-IgA1 in IgA1-secreting cells of patients with IgAN and provide initial analyses of LIF signaling. METHODS: We characterized LIF signaling that is involved in the overproduction of Gd-IgA1, using IgA1-secreting cell lines derived from peripheral blood of patients with IgAN and healthy controls (HC). We used global PTK activity profiling, immunoblotting, lectin ELISA, and siRNA knock-down. RESULTS: LIF stimulation did not significantly affect production of total IgA1 in IgA1-secreting cells from patients with IgAN or HC. However, LIF increased production of Gd-IgA1, but only in the cells from patients with IgAN. LIF stimulation enhanced phosphorylation of STAT1 in IgA1-secreting cells from patients with IgAN to a higher degree than in the cells from HC. siRNA knock-down of STAT1 blocked LIF-mediated overproduction of Gd-IgA1. Unexpectedly, this abnormal phosphorylation of STAT1 in IgA1-secreting cells from patients with IgAN was not mediated by JAK, but rather involved activation of Src-family PTKs (SFKs). CONCLUSION: Abnormal LIF/STAT1 signaling represents another pathway potentially leading to overproduction of Gd-IgA1 in IgAN, providing possible explanation for the phenotype associated with chromosome 22q12 GWAS locus. Abnormal LIF/STAT1 signaling and the associated SFKs may represent potential diagnostic and/or therapeutic targets in IgAN.

Therapeutically actionable PAK4 is amplified, overexpressed, and involved in bladder cancer progression.

Muscle-invasive bladder carcinomas (MIBCs) are aggressive genitourinary malignancies. Metastatic urothelial carcinoma of the bladder is generally incurable by current chemotherapy and leads to early mortality. Recent studies have identified molecular subtypes of MIBCs with different sensitivities to frontline therapy, suggesting tumor heterogeneity. We have performed multi-omic profiling of the kinome in bladder cancer patients with the goal of identify therapeutic targets. Our analyses revealed amplification, overexpression, and elevated kinase activity of P21 (RAC1) activated kinase 4 (PAK4) in a subset of Bladder cancer (BLCA). Using bladder cancer cells, we confirmed the role of PAK4 in BLCA cell proliferation and invasion. Furthermore, we observed that a PAK4 inhibitor was effective in curtailing growth of BLCA cells. Transcriptomic analyses identified elevated expression of another kinase, protein tyrosine kinase 6 (PTK6), upon treatment with a PAK4 inhibitor and RNA interference of PAK4. Treatment with a combination of kinase inhibitors (vandetanib and dasatinib) showed enhanced sensitivity compared with either drug alone. Thus, PAK4 may be therapeutically actionable for a subset of MIBC patients with amplified and/or overexpressed PAK4 in their tumors. Our results also indicate that combined inhibition of PAK4 and PTK6 may overcome resistance to PAK4. These observations warrant clinical investigations with selected BLCA patients.

Insulin receptor substrates differentially exacerbate insulin-mediated left ventricular remodeling.

Pressure overload (PO) cardiac hypertrophy and heart failure are associated with generalized insulin resistance and hyperinsulinemia, which may exacerbate left ventricular (LV) remodeling. While PO activates insulin receptor tyrosine kinase activity that is transduced by insulin receptor substrate 1 (IRS1), the present study tested the hypothesis that IRS1 and IRS2 have divergent effects on PO-induced LV remodeling. We therefore subjected mice with cardiomyocyte-restricted deficiency of IRS1 (CIRS1KO) or IRS2 (CIRS2KO) to PO induced by transverse aortic constriction (TAC). In WT mice, TAC-induced LV hypertrophy was associated with hyperactivation of IRS1 and Akt1, but not IRS2 and Akt2. CIRS1KO hearts were resistant to cardiac hypertrophy and heart failure in concert with attenuated Akt1 activation. In contrast, CIRS2KO hearts following TAC developed more severe LV dysfunction than WT controls, and this was prevented by haploinsufficiency of Akt1. Failing human hearts exhibited isoform-specific IRS1 and Akt1 activation, while IRS2 and Akt2 activation were unchanged. Kinomic profiling identified IRS1 as a potential regulator of cardioprotective protein kinase G-mediated signaling. In addition, gene expression profiling revealed that IRS1 signaling may promote a proinflammatory response following PO. Together, these data identify IRS1 and Akt1 as critical signaling nodes that mediate LV remodeling in both mice and humans.

The effects of focal adhesion kinase and platelet-derived growth factor receptor beta inhibition in a patient-derived xenograft model of primary and metastatic Wilms tumor.

Aggressive therapies for patients with metastatic Wilms tumor (WT) with subsequent severe late effects warrant the search for novel therapies. The role of focal adhesion kinase (FAK), a non-receptor tyrosine kinase important in pediatric solid tumor development and progression, has not been examined in metastatic WT. Using a novel patient-derived xenograft (PDX) of a primary and matched, isogenic, metastatic WT, the hypothesis of the current study was that FAK would contribute to metastatic WT and small molecule inhibition would decrease tumor growth. Immunohistochemical staining, immunoblotting, cell viability and proliferation assays, cell cycle analysis, and cellular motility and attachment-independent growth assays were performed. FAK was present and phosphorylated in both WT PDXs and in the human samples from which they were derived. FAK inhibition decreased cellular survival, proliferation, and cell cycle progression in both PDXs but only significantly decreased migration, invasion, and attachment-independent growth in the primary WT PDX. Kinomic profiling revealed that platelet-derived growth factor receptor beta (PDGFRß) may be affected by FAK inhibition in WT. Pharmacologic inhibition of FAK and PDGFRß was synergistic in primary WT PDX cells. These findings broaden the knowledge of metastatic WT and support further investigations on the potential use of FAK and PDGFRß inhibitors.

Intratumoral spatial heterogeneity of BTK kinomic activity dictates distinct therapeutic response within a single glioblastoma tumor.

OBJECTIVE: Despite significant recent efforts applied toward the development of efficacious therapies for glioblastoma (GBM) through exploration of GBM's genome and transcriptome, curative therapeutic strategies remain highly elusive. As such, novel and effective therapeutics are urgently required. In this study, the authors sought to explore the kinomic landscape of GBM from a previously underutilized approach (i.e., spatial heterogeneity), followed by validation of Bruton's tyrosine kinase (BTK) targeting according to this stepwise kinomic-based novel approach. METHODS: Twelve GBM tumor samples were obtained and characterized histopathologically from 2 patients with GBM. PamStation peptide-array analysis of these tissues was performed to measure the kinomic activity of each sample. The Ivy GBM database was then utilized to determine the intratumoral spatial localization of BTK activity by investigating the expression of BTK-related transcription factors (TFs) within tumors. Genetic inhibition of BTK family members through lentiviral short hairpin RNA (shRNA) knockdown was performed to determine their function in the core-like and edge-like GBM neurosphere models. Finally, the small-molecule inhibitor of BTK, ONO/GS-4059, which is currently under clinical investigation in nonbrain cancers, was applied for pharmacological inhibition of regionally specified newly established GBM edge and core neurosphere models. RESULTS: Kinomic investigation identified two major subclusters of GBM tissues from both patients exhibiting distinct profiles of kinase activity. Comparatively, in these spatially defined subgroups, BTK was the centric kinase differentially expressed. According to the Ivy GBM database, BTK-related TFs were highly expressed in the tumor core, but not in edge counterparts. Short hairpin RNA-mediated gene silencing of BTK in previously established edge- and core-like GBM neurospheres demonstrated increased apoptotic activity with predominance of the sub-G1 phase of core-like neurospheres compared to edge-like neurospheres. Lastly, pharmacological inhibition of BTK by ONO/GS-4059 resulted in growth inhibition of regionally derived GBM core cells and, to a lesser extent, their edge counterparts. CONCLUSIONS: This study identifies significant heterogeneity in kinase activity both within and across distinct GBM tumors. The study findings indicate that BTK activity is elevated in the classically therapy-resistant GBM tumor core. Given these findings, targeting GBM's resistant core through BTK may potentially provide therapeutic benefit for patients with GBM.

Differential escape mechanisms in cetuximab-resistant head and neck cancer cells.

Acquired cetuximab resistance is a challenge for oncologists treating advanced head and neck carcinoma (HNC). While intrinsic cetuximab resistance mechanism in colorectal cancer is known, resistance in HNC is unclear. We established two different cetuximab resistant HNC cell lines by culturing epidermal growth factor (EGFR) expressing UM-SCC-1 and UM-SCC-6 cell lines in the presence of 5 µg/ml cetuximab. We then explored potential mechanisms of resistance. We found that the 2 cell lines developed resistance by different mechanisms. Specifically, we found that UM-SCC-1 resistant cells (UM-SCC-1R) showed enhanced EGF-induced downstream signals while UM-SCC-6 resistant cells (UM-SCC-6R) demonstrated EGF-independent signaling. Global kinase activity (kinomic) profiling revealed unique signaling differences in the two resistant cell lines. However, both of the resistant lines demonstrated increased phospho-serine 727 and total STAT3 expression compared to the parental lines. STAT3 knockdown promoted increased cytotoxicity both in the presence and absence of cetuximab in the resistant lines suggesting that STAT3 may be a common target in cetuximab resistance.