Activation Of Multiple Kinases Research Articles

A plasma membrane-associated form of the androgen receptor enhances nuclear androgen signaling in osteoblasts and prostate cancer cells.

Androgen binding to the androgen receptor (AR) in the cytoplasm induces the AR to translocate to the nucleus, where it regulates the expression of target genes. Here, we found that androgens rapidly activated a plasma membrane-associated signaling node that enhanced nuclear AR functions. In murine primary osteoblasts, dihydrotestosterone (DHT) binding to a membrane-associated form of AR stimulated plasma membrane-associated protein kinase G type 2 (PKG2), leading to the activation of multiple kinases, including ERK. Phosphorylation of AR at Ser515 by ERK increased the nuclear accumulation and binding of AR to the promoter of Ctnnb1, which encodes the transcription factor β-catenin. In male mouse osteoblasts and human prostate cancer cells, DHT induced the expression of Ctnnb1 and CTNN1B, respectively, as well as β-catenin target genes, stimulating the proliferation, survival, and differentiation of osteoblasts and the proliferation of prostate cancer cells in a PKG2-dependent fashion. Because β-catenin is a master regulator of skeletal homeostasis, these results explain the reported male-specific osteoporotic phenotype of mice lacking PKG2 in osteoblasts and imply that PKG2-dependent AR signaling is essential for maintaining bone mass in vivo. Our results suggest that widely used pharmacological PKG activators, such as sildenafil, could be beneficial for male and estrogen-deficient female patients with osteoporosis but detrimental in patients with prostate cancer.

Abstract C096: Targeting Syndecan1 to overcome acquired resistant to KRAS inhibitor in gastrointestinal cancer

Abstract Development of treatment resistance is a major impediment to effective cancer therapy. KRASG12C inhibitors have been recently approved by FDA and specific inhibitors targeting additional KRAS mutations are under clinical and preclinical development. These KRAS oncogene-specific inhibitors (KRASi) represent a promising new targeted therapy for KRAS-driven cancer types. However, as is the case for most targeted therapies, majority of tumors that initially respond to KRASG12C inhibitor quickly develop resistant disease. Unfortunately, the underlying mechanisms of KRASi resistance remain to be fully elucidated. We employed various in vitro and in vivo preclinical model systems to explore the development of KRASi resistance. Our study demonstrated that the YAP1- Syndecan 1 (SDC1) axis as the major driver for the bypass of KRAS-dependence and targeting YAP1-SDC1 will overcome the acquired resistance to KRAS targeted therapy. We previously identified the membrane proteoglycan SDC1 as a KRAS downstream surrogate whose cell surface localization is tightly controlled by oncogenic KRAS signaling and is critical for KRAS-driven tumor development. Here, our analysis of KRAS driven genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) and human pancreatic or colorectal cancer (CRC) cells with KRASG12C mutation discovered that, while cell surface SDC1 is reduced upon acute genetic or pharmacological of oncogenic KRAS, plasma membrane SDC1 is recovered in tumor cells that become resistant to KRAS inhibition (KRAS-bypass cells). Surface localization of SDC1 leads to the activation of multiple receptor tyrosine kinases (RTKs), resulting in the resistance to KRAS blockade. We further identified that the recovery of plasma membrane SDC1 in KRAS-bypass cells is driven by YAP1 oncogene, which mediates the transcription suppression of ARF6 GAPs, leading to the activation of ARF6, a small GTPase required for SDC1 membrane recycling. Genetic depletion of YAP1 or SDC1, as well as pharmacological inhibition of YAP1-SDC1 axis abolishes the tumorigenic activity of KRAS-bypass tumor cells and sensitizes KRAS-driven tumors to KRASi. Moreover, YAP1 and surface SDC1 are accumulated in CRC PDX models with acquired resistance to KRASG12C-inhibitor. Importantly, pharmacological targeting YAP1 significantly suppresses surface SDC1 in KRASi-resistant PDXs and sensitizes the tumors to KRASG12C-inhibitor. Taken together, our research identifies new actionable therapeutic targets and approaches to overcome resistance to KRAS inhibition, thus implicating the therapeutic potential to greatly improve the clinical outcome of patients with KRAS mutated cancers. Citation Format: Mitsunobu Takeda, Alexey Sorokin, Madelaine Theardy, Oluwadara Coker, Scott Kopetz, Haoqiang Ying, Giulio Draetta, Ronald A. DePinho, Wantong Yao. Targeting Syndecan1 to overcome acquired resistant to KRAS inhibitor in gastrointestinal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C096.

LUBAC is required for RIG-I sensing of RNA viruses.

The ability of cells to mount an interferon response to virus infections depends on intracellular nucleic acid sensing pattern recognition receptors (PRRs). RIG-I is an intracellular PRR that binds short double-stranded viral RNAs to trigger MAVS-dependent signalling. The RIG-I/MAVS signalling complex requires the coordinated activity of multiple kinases and E3 ubiquitin ligases to activate the transcription factors that drive type I and type III interferon production from infected cells. The linear ubiquitin chain assembly complex (LUBAC) regulates the activity of multiple receptor signalling pathways in both ligase-dependent and -independent ways. Here, we show that the three proteins that constitute LUBAC have separate functions in regulating RIG-I signalling. Both HOIP, the E3 ligase capable of generating M1-ubiquitin chains, and LUBAC accessory protein HOIL-1 are required for viral RNA sensing by RIG-I. The third LUBAC component, SHARPIN, is not required for RIG-I signalling. These data cement the role of LUBAC as a positive regulator of RIG-I signalling and as an important component of antiviral innate immune responses.

Abstract 2013: Integrated proteomic analysis identifies four distinct subtypes of high-risk neuroblastoma

Abstract Neuroblastoma is the most common solid extracranial tumor in children. Despite intensive multi-modal therapy, 5-year survival rates for patients with high-risk disease remain at approximately 50%. Neuroblastomas typically have low mutational burden and harbor few recurrent mutations making the identification of new therapeutic targets challenging. To characterize the proteomic landscape of high-risk neuroblastoma, we performed liquid chromatography-mass spectrometry-based deep expression proteomics and phosphoproteomics on 30 neuroblastoma tumors from 26 patients with high-risk disease. Our integrated analysis identified four distinct proteomic subgroups of high-risk neuroblastoma that were not otherwise apparent based on clinical or genomic features. The four subgroups were named based on their defining proteomic signature: C1-Mixed, C2-Neuronal, C3-Functional MYCN and C4-Stromal. Only one third of C3-Functional MYCN tumors had amplification of MYCN. Segmental chromosomal losses and gains were enriched in, but not exclusive to, C1-Mixed and C3-Functional MYCN tumors. The activities of multiple kinases including CDK2, CDK7 and MEK2 differed significantly between subgroups. C3-Functional MYCN and C4-Stromal tumors were enriched for immune and stromal cells, respectively. Focal adhesion signaling was specifically upregulated in C4-Stromal tumors suggesting increased extracellular matrix interactions in this tumor subgroup. C2-Neuronal tumors were enriched for axon guidance and neurotrophin signaling pathways. Rho family GTPase signaling was also evident in multiple tumor subgroups. C1-Mixed and C3-Functional MYCN tumors had elevated expression of RNA processing proteins which was associated with increased alternative splicing. Splicing analysis also identified multiple novel protein coding splice events that were shared amongst multiple neuroblastoma tumors and outliers compared to both the Genotype-Tissue Expression (GTEx) dataset and a panel of commercial reference tissues. Protein domain analysis of these novel splice variants suggested that these novel protein isoforms may have aberrant functions that contribute to tumorigenesis. In conclusion, phosphoproteomic analysis can identify candidate pathways for the development of new therapies for patients with high-risk neuroblastoma. Citation Format: Kristin L. Leskoske, Sara A. Byron, Seema Plaisier, Apurva M. Hegde, Krystine Garcia-Mansfield, Ritin Sharma, Genevieve Bergendahl, Abhinav Nagulapally, William Ferguson, Jaqueline Kraveka, Javier Oesterheld, William P. Hendricks, Giselle L. Saulnier Sholler, Jeffrey M. Trent, Patrick Pirrotte. Integrated proteomic analysis identifies four distinct subtypes of high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2013.

Multiplexing the Quantitation of MAP Kinase Activities Using Differential Sensing.

Protein kinases are therapeutic targets for many human diseases, but the lack of user-friendly quantitative assays limits the ability to follow the activities of numerous kinases at once (multiplexing). To develop such an assay, we report an array of sulfonamido-oxine (SOX)-labeled peptides showing cross-reactivity to different mitogen-activated protein kinases (MAPKs) for use in a differential sensing scheme. We first verified using linear discriminant analysis that the array could differentiate MAPK isoforms. Then, using principal component analysis, the array was optimized based on the discrimination imparted by each SOX-peptide. Next, the activity of individual MAPK families in ternary mixtures was quantified by support vector machine regression. Finally, we multiplexed the quantification of three MAPK families using partial least squares regression in A549 cell lysates, which has possible interference from other kinase classes. Thus, our method simultaneously quantifies the activity of multiple kinases. The technique could be applied to other protein kinase families and the monitoring of diseases.

Identification of Gαi3 as a promising target for osteosarcoma treatment.

Sustained activation of multiple receptor tyrosine kinases (RTKs) simultaneously is vital for tumorigenesis and progression of osteosarcoma (OS). Gαi proteins recruitment to various RTKs mediates downstream oncogenic signaling activation. The expression, functions and underlying mechanisms of Gαi3 in human OS were examined. Expression of Gαi3 is robustly elevated in human OS tissues and is correlated with a poor overall survival. In patient-derived primary OS cells and immortalized lines (MG63 and U2OS), Gαi3 depletion, by shRNA and CRISPR/Cas9 strategies, robustly suppressed cell viability, proliferation and migration, while provoking G1-S arrest and apoptosis activation. Conversely, Gαi3 overexpressing ectopically can accelerate proliferation and migration of OS cells. In OS cells, Gαi3 immunoprecipitated with VEGFR2, FGFR, PGDFR and EGFR, mediating downstream cascade transduction. Akt-mTOR activation in primary OS cells was potently inhibited by Gαi3 shRNA, knockout or dominant negative mutation, but augmented after Gαi3 overexpression. In vivo studies showed that Gαi3 shRNA AAV (adeno-associated viruses) intratumoral injection largely inhibited the growth of subcutaneous xenografts of primary OS cells. Moreover, the growth of the Gαi3-knockout primary OS xenografts was much slower than that of OS xenografts with empty vector. In Gαi3-depleted OS xenografts tissues, Gαi3 downregulation and Akt-mTOR inactivation were detected. Taken together, overexpressed Gαi3 mediates RTK-Akt signaling to drive OS progression.

Enhancing the Therapeutic Efficacy of KRASG12C Inhibitors in Lung Adenocarcinoma Cell Models by Cotargeting the MAPK Pathway or HSP90.

Background KRASG12C inhibitors have shown promising efficacy in early clinical trials, but drug resistance compromises their long-term benefits. Therefore, it is critical to understand the mechanisms of drug resistance and to design appropriate combinatory treatments to improve efficacy. Methods To understand the comprehensive mechanisms of drug resistance, we treated lung cancer cells with KRASG12C inhibitors for different periods and performed transcriptional profiling and signaling analysis to identify critical factors and pathways that drive drug tolerance and resistance. We also evaluated several drug combinations in vitro and in vivo to identify potentially effective therapeutics. Results We found that the feedback activation of multiple receptor tyrosine kinases (RTKs) may have cooperatively induced intrinsic and adaptive resistance to KRASG12C inhibitors. Notably, continuous KRAS inhibition induced a multidrug-resistant phenotype, implying that upfront combinatory treatment might be required to treat this group of patients. We also demonstrated that concurrently targeting multiple nodes in the RTK/RAS/RAF/MEK/ERK axis improved the efficacy of KRASG12C inhibitors, mainly by suppressing the reactivation of the mitogen-activated protein kinase (MAPK) pathway. Moreover, the combined use of HSP90 and KRASG12C inhibitors effectively induced tumor regression in lung adenocarcinoma models in vitro and in vivo. Conclusion Together, our findings revealed mechanisms underlying KRASG12C inhibitors resistance and provided novel candidate combinatory strategies to improve their anticancer activity.

CSIG-31. MULTI-OMICS TO EDGE INTO PRECISION MEDICINE FOR DIPG

Abstract DIPG is an incurable pediatric brain tumor with 80% of patients harboring H3F3A (H3.3) mutation that substitutes methionine for lysine at position 27 (K27M), resulting in global depletion of H3.3K27 me3 (trimethylation). These histone mutations modify the epigenome and alter oncogenic transcription, causing oncogenic insults to progenitor cells in early neurodevelopment (1). To determine the reprogramming pathways in the cell context of H3.3K27M tumors, we conducted LC-MS based proteomic and phosphoproteomic analysis on seven patient-derived DIPG cell lines. Three normal neuronal stem cell lines were included as non-tumor brain cells for comparison. Pathway analysis identified 29 pathways that are significantly altered in DIPG compared to normal brain cells at both the protein abundance and phosphosite level. Notably, AKT and MAPK associated PI3K signaling, VEGF signaling, mTOR signaling, and HIF1a signaling were differentially active in H3.3K27M tumors compared to healthy control cell lines. We saw significantly higher activity of multiple kinases involved in axon guidance and cytoskeletal remodeling in DIPG, such as PTK2B, DYRK2, TTBK2 and MARK2. This is the first time to report an increased abundance and kinase activity of PYK2 protein (coded by PTK2B), a close homologue of FAK and its associated signaling in DIPG. PYK2 has been proposed to act in concert with Src to link Gi- or Gq-coupled receptors with the mitogen-activated protein (MAP) kinase signaling pathway (2). Because of the shared signaling across kinase pathways, targeting activated PYK2 in DIPG may complement inhibitors of other dysregulated signaling networks in DIPG such as MAPK2, VEGFR, PI3K and Src. Our data also found that IL13RA2 was upregulated in DIPG. We conclude that for H3 K27M DIPG tumors, campaigns to target PYK2, MAPK2, VEGFR, PI3K, Src and IL13Ra2 using small molecules that traverse the blood brain barrier loom as promising opportunities for drug development.

Abstract 318: Multi-omics to edge into precision medicine for DIPG

Abstract Effective targeted cancer therapies are limited due to lack of our understanding of disease pathogenesis at the molecular level. Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brain tumor with 80% of DIPGs harboring H3F3A (H3.3) mutation that lead to a substitution of methionine for lysine at position 27 (K27M). The tumors bearing H3.3K27M mutation arise throughout the midline structures with global depletion of H3.3K27 me3 (trimethylation). These histone mutations modify the epigenome and alter oncogenic transcription, causing oncogenic insults to progenitor cells in early neurodevelopment (PMID: 30453529). To determine the reprogramming pathways in the cell context of H3.3K27M tumors, we conducted liquid chromatography-mass spectrometry-based proteomic and phosphoproteomic analysis on seven patient-derived DIPG cell lines that can be stably passaged in serum-free neural stem cell media and displayed distinct morphologies, growth rates and chromosome abnormalities (PMID: 21368213). Three normal neuronal stem cell lines were included as non-tumor brain cells for comparison. Pathway analysis identified 29 pathways that are significantly altered in DIPG compared to normal brain cells at both the protein abundance and phosphosite level. Notably, AKT and MAPK associated PI3K signaling, VEGF signaling, mTOR signaling, and HIF1a signaling were differentially active in H3.3K27M tumors compared to healthy control cell lines. We saw significantly higher activity of multiple kinases involved in axon guidance and cytoskeletal remodeling in DIPG, such as PTK2B, DYRK2, TTBK2 and MARK2. This is the first time to report an increased abundance and kinase activity of Pyk2 protein (coded by PTK2B), a close homologue of FAK and its associated signaling in DIPG. Overexpression and autophosphorylation of Pyk2 are required to stimulate glioma cell migration (PMIDs: 15967096, 18648907). Pyk2 has also been proposed to act in concert with Src to link Gi- or Gq-coupled receptors with the mitogen-activated protein (MAP) kinase signaling pathway (PMID:12960403). Because of the shared signaling across kinase pathways, targeting activated Pyk2 in DIPG may complement inhibitors of other dysregulated signaling networks in DIPG such as MAPK2, VEGFR, PI3K and Src. Our data also found that IL13RA2 was upregulated in DIPG; others have shown that increased expression of IL13RA2 is associated with poor prognosis in GBM (PMIDs: 32913543, 30366424). We conclude that for H3 K27M DIPG tumors, campaigns to target Pyk2, MAPK2, VEGFR, PI3K, Src and IL13Ra2 using small molecules that traverse the blood brain barrier loom as promising opportunities for drug development. Citation Format: Nanyun Tang, Kristin Leskoske, Krystine Garcia-Mansfield, Ritin Sharma, Hannah Tolson, Patrick Pirrotte, Michael E. Berens. Multi-omics to edge into precision medicine for DIPG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 318.

The Caveolin-1 Scaffolding Domain Peptide Reverses Aging-Associated Deleterious Changes in Multiple Organs.

Aging is a progressive, multifactorial, degenerative process in which deleterious changes occur in the biochemistry and function of organs. We showed that angiotensin II (AngII)-induced pathologies in the heart and kidney of young (3-month-old) mice are suppressed by the caveolin-1 scaffolding domain (CSD) peptide. Because AngII mediates many aging-associated changes, we explored whether CSD could reverse pre-existing pathologies and improve organ function in aged mice. Using 18-month-old mice (similar to 60-year-old humans), we found that >5-fold increases in leakage of serum proteins and >2-fold increases in fibrosis are associated with aging in the heart, kidney, and brain. Because tyrosine phosphorylation of cell junction proteins leads to the loss of microvascular barrier function, we analyzed the activation of the receptor tyrosine kinase PDGFR and the nonreceptor tyrosine kinases c-Src and Pyk2. We observed 5-fold activation of PDGFR and 2- to 3-fold activation of c-Src and Pyk2 in aged mice. Treatment with CSD for 4 weeks reversed these pathologic changes (microvascular leakage, fibrosis, kinase activation) in all organs almost down to the levels in healthy, young mice. In studies of heart function, CSD reduced the aging-associated increase in cardiomyocyte cross-sectional area and enhanced ventricular compliance in that echocardiographic studies demonstrated improved ejection fraction and fractional shortening and reduced isovolumic relation time. These results suggest that versions of CSD may be developed as treatments for aging-associated diseases in human patients based on the concept that CSD inhibits tyrosine kinases, leading to the inhibition of microvascular leakage and associated fibrosis, thereby improving organ function. SIGNIFICANCE STATEMENT: The caveolin-1 scaffolding domain (CSD) peptide reverses aging-associated fibrosis, microvascular leakage, and organ dysfunction in the heart, kidneys, and brain via a mechanism that involves the suppression of the activity of multiple tyrosine kinases, suggesting that CSD can be developed as a treatment for a wide range of diseases found primarily in the aged.

Focal adhesion kinase inhibition synergizes with nab-paclitaxel to target pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a very lethal disease, with minimal therapeutic options. Aberrant tyrosine kinase activity influences tumor growth and is regulated by phosphorylation. We investigated phosphorylated kinases as target in PDAC.MethodsMass spectrometry-based phosphotyrosine proteomic analysis on PDAC cell lines was used to evaluate active kinases. Pathway analysis and inferred kinase activity analysis was performed to identify novel targets. Subsequently, we investigated targeting of focal adhesion kinase (FAK) in vitro with drug perturbations in combination with chemotherapeutics used against PDAC. Tyrosine phosphoproteomics upon treatment was performed to evaluate signaling. An orthotopic model of PDAC was used to evaluate the combination of defactinib with nab-paclitaxel.ResultsPDAC cell lines portrayed high activity of multiple receptor tyrosine kinases to various degree. The non-receptor kinase, FAK, was identified in all cell lines by our phosphotyrosine proteomic screen and pathway analysis. Targeting of this kinase with defactinib validated reduced phosphorylation profiles. Additionally, FAK inhibition had anti-proliferative and anti-migratory effects. Combination with (nab-)paclitaxel had a synergistic effect on cell proliferation in vitro and reduced tumor growth in vivo.ConclusionsOur study shows high phosphorylation of several oncogenic receptor tyrosine kinases in PDAC cells and validated FAK inhibition as potential synergistic target with Nab-paclitaxel against this devastating disease.

Comprehensive Biology and Genetics Compendium of Wilms Tumor Cell Lines with Different WT1 Mutations.

Simple SummaryWilms tumor is a childhood kidney tumor arising from embryonal cells. Wilms tumors are heterogeneous with several distinct subgroups that differ in their response to treatment. The genetic basis for these diverse forms of Wilms tumor is not fully understood. One subgroup of Wilms tumors is associated with mutations in the WT1 gene, encoding a transcription factor with a role in early kidney differentiation. Patients with WT1 mutant Wilms tumor may harbor germline mutations in this gene. Cell lines from Wilms tumors are notoriously difficult to establish and only few exist. We developed a method to cultivate cells from the WT1 mutant subtype of Wilms tumors and have established 11 cell lines with different mutations in WT1 to date. These cells will be instrumental to study the biology and genetics ultimately to develop precision treatmentsPurpose: WT1 mutant Wilms tumors represent a distinct subgroup, frequently associated with CTNNB1 mutations. The genetic basis for the development of this subtype is currently not fully understood. Methods: Live WT1 mutant Wilms tumors were collected during surgery of patients and cell cultures established in mesenchymal stem cell medium. They were studied for mutations in WT1 and CTNNB1, their differentiation capacity and protein activation status. Four cell lines were immortalized with a triple mutant ts SV40 largeT antigen and Telomerase. Results: 11 cell lines were established from Wilms tumors of nine patients, including a left and right tumor from the same patient and a primary and second tumor from another patient. Six patients had germ line and three were tumor specific mutations. All cell lines harbored only mutant or deleted WT1 genes. CTNNB1 was wild type in three, all others carried mutations affecting amino acid S45. They had variable and limited capacities for mesenchymal differentiation, a high migratory capacity and a low invasive potential. All cells showed an activation of multiple receptor tyrosine kinases and downstream signaling pathways. Conclusions: These cell lines represent an important new tool to study WT1 mutant Wilms tumors, potentially leading to new treatment approaches.

Flavopiridol Induces Apoptosis of Normal Lymphoid Cells, Causes Immunosuppression, and Has Potent Antitumor Activity In Vivo Against Human Leukemia and Lymphoma Xenografts

AbstractFlavopiridol is a novel semisynthetic flavone derivative of the alkaloid rohitukine. Flavopiridol is known to inhibit potently the activity of multiple cyclin-dependent kinases. We have assessed its effects on normal and malignant cells in preclinical animal models of localized and disseminated human hematopoietic neoplasms. Flavopiridol, when administered as daily bolus intravenous (IV) injections, produced selective apoptosis of cells in the thymus, spleen, and lymph nodes, resulting in atrophy of these organs. With the exception of the intestinal crypts, apoptosis or tissue damage was absent in all other organs investigated (kidneys, liver, lungs, bone/bone marrow, muscle, and heart). Flavopiridol had a marked apoptotic effect documented by DNA nick-end labeling, or DNA agarose gels in xenografts of human hematopoietic tumors HL-60, SUDHL-4, and Nalm/6. After treatment with 7.5 mg/kg flavopiridol bolus IV or intraperitoneal on each of 5 consecutive days, 11 out of 12 advanced stage subcutaneous (s.c.) human HL-60 xenografts underwent complete regressions, and animals remained disease-free several months after one course of flavopiridol treatment. SUDHL-4 s.c. lymphomas treated with flavopiridol at 7.5 mg/kg bolus IV for 5 days underwent either major (two out of eight mice) or complete (four out of eight mice) regression, with two animals remaining disease-free for more than 60 days. The overall growth delay was 73.2%. The acquired immunodeficiency syndrome–associated lymphoma AS283 showed no significant response when flavopiridol was used in advanced s.c. tumors, but when treatment was initiated in early stages, there was a complete regression of the early tumors, and a significant overall growth delay (>84%). When flavopiridol was used in severe combined immunodeficient mice bearing disseminated human acute lymphoblastic leukemia Nalm/6 cells, there was 15-day prolongation in survival (P = .0089). We conclude that flavopiridol greatly influences apoptosis in both normal and malignant hematopoietic tissues. This activity was manifested in our study as a potent antileukemia or antilymphoma effect in human tumor xenografts, which was dose and schedule dependent. These findings provide compelling evidence for the use of flavopiridol in human hematologic malignancies.

Loss of ACK1 Upregulates EGFR and Mediates Resistance to BRAF Inhibition

Targeted BRAF(V600E) suppression by selective BRAF inhibitors (BRAFis; e.g., vemurafenib and dabrafenib) has led to a sea change in the treatment of metastatic melanoma. Despite frequent upfront responses, acquired resistance has compromised long-term applicability. Among the various mechanisms of resistance, activation of multiple receptor tyrosine kinases is a known critical factor that contributes to vemurafenib resistance⁠. EGFR activation has been recurrently identified in a set of vemurafenib-resistant melanomas, but little is known about how EGFR, or possibly other receptor tyrosine kinases, becomes activated. Here, we report that ACK1, a protein kinase that modulates EGFR turnover, is downregulated in vemurafenib-resistant melanoma cells. We also found that ACK1 depletion with short hairpin RNA decreased EGFR degradation when activated by epidermal growth factor, increased EGFR protein expression, and conferred resistance to BRAFis both invitro and invivo. Vemurafenib resistance mediated by ACK1 inhibition can be reversed by the EGFR inhibitor gefitinib. Our data indicate that ACK1 loss may be a post-transcriptional mechanism that increases EGFR signaling and contributes to drug resistance.

Downregulation of the ubiquitin ligase KBTBD8 prevented epithelial ovarian cancer progression

ObjectivesKelch repeat and BTB domain-containing protein 8, KBTBD8, has been identified as a female fertility factor. However, there have been no reports on the role of KBTBD8 in the progression of epithelial ovarian cancer, EOC. Our study aimed to address this issue.MethodsWe first examine KBTBD8 expression in EOC tissues and cells. Next, we performed RNA sequencing to reveal the overall mechanism. Then we investigated the roles of KBTBD8 in the proliferation, migration, and health status of cultured EOC cells. Finally, we employed tumor xenograft models to evaluate the role of KBTBD8 in vivo.ResultsFirst, KBTBD8 level was significantly higher in EOC tissues and cells. Next, comparative RNA sequencing identified more tumorigenesis-related genes that KBTBD8 might regulate. Then we found that KBTBD8 knockdown significantly decreased EOC cell proliferation, migration, and the activities of multiple tumorigenesis-related kinases. Finally, KBTBD8 knockdown significantly diminished ovarian tumor formation in vivo.ConclusionProper KBTBD8 level is essential for the healthy growth of ovarian somatic cells, such as ovarian epithelial cells. Excessive KBTBD8 might be a significant impetus for EOC progression. KBTBD8 reduction greatly inhibits EOC proliferation and migration.

Small-molecule modulation of the p75 neurotrophin receptor inhibits a wide range of tau molecular pathologies and their sequelae in P301S tauopathy mice

In tauopathies, phosphorylation, acetylation, cleavage and other modifications of tau drive intracellular generation of diverse forms of toxic tau aggregates and associated seeding activity, which have been implicated in subsequent synaptic failure and neurodegeneration. Suppression of this wide range of pathogenic species, seeding and toxicity mechanisms, while preserving the physiological roles of tau, presents a key therapeutic goal. Identification and targeting of signaling networks that influence a broad spectrum of tau pathogenic mechanisms might prevent or reverse synaptic degeneration and modify disease outcomes. The p75 neurotrophin receptor (p75NTR) modulates such networks, including activation of multiple tau kinases, calpain and rhoA-cofilin activity. The orally bioavailable small-molecule p75NTR modulator, LM11A-31, was administered to tauP301S mice for 3 months starting at 6 months of age, when tau pathology was well established. LM11A-31 was found to reduce: excess activation of hippocampal cdk5 and JNK kinases and calpain; excess cofilin phosphorylation, tau phosphorylation, acetylation and cleavage; accumulation of multiple forms of insoluble tau aggregates and filaments; and, microglial activation. Hippocampal extracts from treated mice had substantially reduced tau seeding activity. LM11A-31 treatment also led to a reversal of pyramidal neuron dendritic spine loss, decreased loss of dendritic complexity and improvement in performance of hippocampal behaviors. These studies identify a therapeutically tractable upstream signaling module regulating a wide spectrum of basic mechanisms underlying tauopathies.

Phosphoproteomic analysis identifies phospho-Threonine-17 site of phospholamban important in low molecular weight isoform of fibroblast growth factor 2-induced protection against post-ischemic cardiac dysfunction

RationaleAmong its many biological roles, fibroblast growth factor 2 (FGF2) protects the heart from dysfunction and damage associated with an ischemic attack. Our laboratory demonstrated that its protection against myocardial dysfunction occurs by the low molecular weight (LMW) isoform of FGF2, while the high molecular weight (HMW) isoforms are associated with a worsening in post-ischemic recovery of cardiac function. LMW FGF2-mediated cardioprotection is facilitated by activation of multiple kinases, including PKCalpha, PKCepsilon, and ERK, and inhibition of p38 and JNK. ObjectiveYet, the substrates of those kinases associated with LMW FGF2-induced cardioprotection against myocardial dysfunction remain to be elucidated. Methods and resultsTo identify substrates in LMW FGF2 improvement of post-ischemic cardiac function, mouse hearts expressing only LMW FGF2 were subjected to ischemia-reperfusion (I/R) injury and analyzed by a mass spectrometry (MS)-based quantitative phosphoproteomic strategy. MS analysis identified 50 phosphorylation sites from 7 sarcoendoplasmic reticulum (SR) proteins that were significantly altered in I/R-treated hearts only expressing LMW FGF2 compared to those hearts lacking FGF2. One of those phosphorylated SR proteins identified was phospholamban (PLB), which exhibited rapid, increased phosphorylation at Threonine-17 (Thr17) after I/R in hearts expressing only LMW FGF2; this was further validated using Selected Reaction Monitoring-based MS workflow. To demonstrate a mechanistic role of phospho-Thr17 PLB in LMW FGF2-mediated cardioprotection, hearts only expressing LMW FGF2 and those expressing only LMW FGF2 with a mutant PLB lacking phosphorylatable Thr17 (Thr17Ala PLB) were subjected to I/R. Hearts only expressing LMW FGF2 showed significantly improved recovery of cardiac function following I/R (p < 0.05), and this functional improvement was significantly abrogated in hearts expressing LMW FGF2 and Thr17Ala PLB (p < 0.05). ConclusionThe findings indicate that LMW FGF2 modulates intracellular calcium handling/cycling via regulatory changes in SR proteins essential for recovery from I/R injury, and thereby protects the heart from post-ischemic cardiac dysfunction.

The Protective Role of Decorin in Hepatic Metastasis of Colorectal Carcinoma.

Decorin, the prototype member of the small leucine-rich proteoglycan gene family of extracellular matrix (ECM) proteins, acts as a powerful tumor suppressor by inducing the p21Waf1/Cip1 cyclin-dependent kinase inhibitor, as well as through its ability to directly bind and block the action of several tyrosine kinase receptors. Our previous studies suggested that the lack of decorin promotes hepatic carcinogenesis in mice. Based on this, we set out to investigate whether excess decorin may protect against the liver metastases of colon carcinoma. We also analyzed the effect of decorin in tissue microarrays of human colon carcinoma liver metastasis and examined whether the tumor cells can directly influence the decorin production of myofibroblasts. In humans, low levels of decorin in the liver facilitated the development of colon carcinoma metastases in proportion with more aggressive phenotypes, indicating a possible antitumor action of the proteoglycan. In vitro, colon carcinoma cells inhibited decorin expression in LX2 hepatic stellate cells. Moreover, liver-targeted decorin delivery in mice effectively attenuated metastasis formation of colon cancer. Overexpressed decorin reduced the activity of multiple receptor tyrosine kinases (RTKs) including the epidermal growth factor receptor (EGFR), an important player in colorectal cancer (CRC) pathogenesis. Downstream of that, we observed weakened signaling of ERK1/2, PLCγ, Akt/mTOR, STAT and c-Jun pathways, while p38 MAPK/MSK/CREB and AMPK were upregulated culminating in enhanced p53 function. In conclusion, decorin may effectively inhibit metastatic tumor formation in the liver.

Abstract 5038: Limited inhibition of multiple nodes in a driver network blocks metastasis

Abstract High-dose, single-drug treatment is unsuccessful at suppressing metastasis due to cellular heterogeneity and feedback activation. Here we demonstrate that targeting driver network signaling capacity by limited inhibition at multiple nodes is a more effective anti-metastatic strategy. A physiological metastasis suppressor, Raf Kinase Inhibitory Protein, decreases the activity of multiple kinases in the stress-regulated MAP kinase (MAPK) network and reduces the output to metastatic transcription factor BACH1 that upregulates motility-related genes. This effect is mimicked by a low-dose four-drug cocktail (4D-MAPKi) that blocks metastatic colonization in mouse breast cancer models and increases survival. Drug treatment and network modeling show that 1) multi-kinase inhibition is required to suppress signaling output across cells with different MAPK network topologies, and 2) restricting inhibition of individual kinases prevents threshold activation of other compensatory kinase networks. This low-dose multi-drug approach to decrease signaling capacity of a driver network represents a transformative, clinically-relevant strategy for anti-metastatic treatment. Citation Format: Ali Ekrem Yesilkanal, Dongbo Yang, Payal Tiwari, Alan U. Sabino, Jiyoung Lee, Xiao-He Xie, Siqi Sun, Christopher Dann, Ethan Steinberg, Timothy Stuhlmiller, Casey Frankenberger, Elizabeth Goldsmith, Gary L. Johnson, Alexandre F. Ramos, Marsha Rosner. Limited inhibition of multiple nodes in a driver network blocks metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5038.

Cotargeting BET proteins overcomes resistance arising from PI3K/mTOR blockade-induced protumorigenic senescence in colorectal cancer.

Therapeutics targeting the phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway initially produce potent antitumor effects, but resistance frequently occurs. Using a phosphoproteome analysis, we found that colorectal cancer (CRC) cells exhibit resistance against PI3K/mTOR inhibition through feedback activation of multiple receptor tyrosine kinases, and their downstream focal adhesion kinase, Src and extracellular signal-regulated kinases signaling. Unexpectedly, PI3K/mTOR blockade causes senescence, mediated by the activation of the stress kinase p38. The senescent cancer cells induce the secretion of various cytokines and this senescence-associated secretome increases migration and invasion capabilities of CRC cells. We found that cotargeting PI3K/mTOR and bromodomain and extra-terminal domain can suppress activation of many oncogenic kinases involved in resistance to the PI3K/mTOR inhibition, induce cell death in vitro and tumor regression in vivo, and further prolong the survival of xenograft models. Our findings provide a rationale for a novel therapeutic strategy to overcome resistance to the PI3K/mTOR inhibitors in CRC.