Related Receptor Tyrosine Kinases Research Articles

LncRNA PVT1 promotes cells proliferation via PI3K–AKT–mTOR Pathway in gastrointestinal stromal tumor

Gastrointestinal stromal tumor (GIST) is one of the most common mesenchymal tumors of the gastrointestinal tract by activating mutations in the related receptor tyrosine kinases KIT and PDGFRA. Few reliable biomarkers have been established despite illustration of the molecular mechanisms of GISTs development. Accumulating evidence has demonstrated that long non-coding RNA (lncRNAs) plasmacytoma variant translocation 1 (lncRNA PVT1) is a tumor regulator in many cancers. The goal of our research was to investigate the level and biological function of PVT1 in GIST-882 and GIST-T1 cells. The relative expression of PVT1 was higher in the GISTs cells. Down-regulation of PVT1 has shown that suppressed the proliferation and migration abilities of GISTs cells, and significantly stimulated cell apoptosis. Furthermore, western blot assay confirmed that knockdown of PVT1 obviously reduced the expression of CCND1, CCNB1, CDK6, PIK3CA, p-AKT, Rictor, mSIN1 and mLST8. The results implicated that PTV1 may regulate the progression of GISTs cells through PI3K/AKT/mTOR pathway. In conclusion, our study provides the evidence that PVT1 plays a vital oncogenic role in GISTs cells, and is a potential prognostic biomarker of recurrence after surgical procedures of GISTs. This highlights the possible of PVT1 serve as a therapeutic target for GISTs treatment.

Kinome Array Profiling of Patient-Derived Pancreatic Ductal Adenocarcinoma Identifies Differentially Active Protein Tyrosine Kinases.

Pancreatic cancer remains one of the most difficult malignancies to treat. Minimal improvements in patient outcomes and persistently abysmal patient survival rates underscore the great need for new treatment strategies. Currently, there is intense interest in therapeutic strategies that target tyrosine protein kinases. Here, we employed kinome arrays and bioinformatic pipelines capable of identifying differentially active protein tyrosine kinases in different patient-derived pancreatic ductal adenocarcinoma (PDAC) cell lines and wild-type pancreatic tissue to investigate the unique kinomic networks of PDAC samples and posit novel target kinases for pancreatic cancer therapy. Consistent with previously described reports, the resultant peptide-based kinome array profiles identified increased protein tyrosine kinase activity in pancreatic cancer for the following kinases: epidermal growth factor receptor (EGFR), fms related receptor tyrosine kinase 4/vascular endothelial growth factor receptor 3 (FLT4/VEGFR-3), insulin receptor (INSR), ephrin receptor A2 (EPHA2), platelet derived growth factor receptor alpha (PDGFRA), SRC proto-oncogene kinase (SRC), and tyrosine kinase non receptor 2 (TNK2). Furthermore, this study identified increased activity for protein tyrosine kinases with limited prior evidence of differential activity in pancreatic cancer. These protein tyrosine kinases include B lymphoid kinase (BLK), Fyn-related kinase (FRK), Lck/Yes-related novel kinase (LYN), FYN proto-oncogene kinase (FYN), lymphocyte cell-specific kinase (LCK), tec protein kinase (TEC), hemopoietic cell kinase (HCK), ABL proto-oncogene 2 kinase (ABL2), discoidin domain receptor 1 kinase (DDR1), and ephrin receptor A8 kinase (EPHA8). Together, these results support the utility of peptide array kinomic analyses in the generation of potential candidate kinases for future pancreatic cancer therapeutic development.

FLT3 and LSD1 Inhibitor Combinations Synergistically Repress Growth of FLT3-Mutant Acute Myeloid Leukemia Via Blockage of MYC Function

Internal Tandem Duplication mutations of Fms Related Receptor Tyrosine Kinase 3 (FLT3), known as FLT3-ITD mutations, are associated with poor prognosis in Acute Myeloid Leukemia (AML). The clinical efficacy of inhibiting FLT3 in AML is limited by the rapid development of drug resistance and relapse, underscoring a need for more potent and durable treatment strategies. The early persistence of leukemic blasts during FLT3 inhibition is a key driver of resistance. We find that in combination, inhibitors of Lysine Specific Demethylase 1 (LSD1) potentiate the activity of FLT3 inhibitors, driving synergistic cell death. This novel therapeutic approach has the potential to drive deeper therapeutic responses in FLT3-Mutant AML, delaying or preventing the development of resistance. LSD1 is a dynamic DNA-associated protein that functions as a chromatin modifier and transcription factor. LSD1 removes methylation on both lysine 4 of histone H3 (H3K4), associated with transcriptional activation, and lysine 9 (H3K9), associated with transcriptional repression. Additionally, LSD1 has been reported to function as a transcription factor independent of its catalytic demethylase function. LSD1 inhibition reduces cell proliferation in several cancer types. In AML specifically, inhibition of LSD1 has been reported to activate enhancers associated with genes that promote differentiation. We hypothesized that combining LSD1 inhibition with FLT3 inhibition in FLT3-ITD AML would result in synergistic effects on cell viability through reactivating differentiation pathways and more strongly blocking proliferation. In this study, we aimed to examine the efficacy, transcriptional effects, and changes in chromatin dynamics when combining LSD1 inhibition with FLT3 inhibition in a FLT3-ITD mutant cell line and patient samples. We used matrix combination screening to determine that combining the FLT3 inhibitor Quizartinib with LSD1 inhibitors (GSK-2879552 or ORY-1001) synergistically represses cell viability in the FLT3-ITD mutant MOLM-13 cell line and in multiple primary AML samples. RNA-seq followed by Gene Set Enrichment Analysis revealed that combining LSD1 and FLT3 inhibition synergistically represses target genes of the oncogenic transcription factor MYC. This finding was corroborated through high-throughput genome-wide profiling of histone marks, using the recently developed technique Cleavage Under Targets and Tagmentation (CUT&Tag). Specifically, we discovered several promoter regions in which acetylation of lysine 27 of Histone H3 (H3K27Ac), associated with transcriptional activation, was repressed by combining LSD1 and FLT3 inhibition. The genes associated with these regions were strongly enriched for known MYC target genes. Through additional genomic profiling methods including ChIP-seq and ATAC-seq, we have established potential roles for several DNA-binding transcription factors including CEBPA, RUNX1, STAT5, and LSD1 itself, that may mediate repression of MYC function resulting from combining LSD1 and FLT3 inhibition. Together, our work establishes LSD1 and FLT3 inhibitor combinations as a promising treatment strategy in FLT3-ITD AML. Importantly, this study identifies combined FLT3 and LSD1 inhibition as an effective strategy to indirectly target MYC function, as MYC is often referred to as an "undruggable" target. Furthermore, it has the potential to drive deeper molecular responses in FLT3-mutant AML, decreasing the likelihood of treatment resistance. Disclosures Druker: Bristol-Myers Squibb: Research Funding; Blueprint Medicines: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Millipore (formerly Upstate Biotechnology): Patents & Royalties; Pfizer: Research Funding; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Patient True Talks: Consultancy; Oregon Health & Science University: Patents & Royalties; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; MolecularMD (acquired by ICON): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Iterion Therapeutics (formerly Beta Cat Pharmaceuticals): Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics Inc. (formerly Lorus): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; GRAIL: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; Leukemia & Lymphoma Society: Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Dana-Farber Cancer Institute: Patents & Royalties; EnLiven: Consultancy, Research Funding. Maxson:Gilead Sciences: Research Funding; Ionis Pharmaceuticals: Other: Joint oversight committee for a collaboration between OHSU and Ionis Pharmaceuticals.

Immune prognostic risk score model in acute myeloid leukemia with normal karyotype.

Acute myeloid leukemia with normal karyotype (NK-AML) is a group of diseases with high heterogeneity and immunological processes are significantly associated with its initiation and development. The implication of the immunogenomic landscape in the prognosis of patients with NK-AML has remained largely elusive. In the present study, the expression profiles of immune-related genes (IRGs) were examined and their association with overall survival (OS) was determined in 60 patients with NK-AML from The Cancer Genome Atlas dataset and 104 patients from the Gene Expression Omnibus (GEO) dataset no. GSE71014. Univariate Cox regression analysis was used to identify 42 and 203 IRGs in the two respective cohorts, which were significantly associated with OS in NK-AML. A risk model was constructed based on the regression coefficient and expression values of nine survival-associated IRGs shared between the two datasets [zinc finger CCCH-type containing, antiviral 1 like; transferrin receptor; suppressor of cytokine signaling 1; ELAV like RNA binding protein 1; roundabout guidance receptor 3; unc-93 homolog B1, Toll-like receptor signaling regulator; protein tyrosine phosphatase non-receptor type 6; interleukin 2 receptor subunit alpha (IL2RA) and IL3RA]. Using this risk model, patients with NK-AML may be divided into high- and low-risk groups in prognostic predictions. The area under the receiver operating characteristic curve for predicting OS was 0.793. The prognostic role of this risk model was successfully verified in another independent cohort (GEO dataset no. GSE71014). The prognostic risk score was positively associated with age and fms related receptor tyrosine kinase 3 mutation and correlated with infiltration by T regulatory cells. In conclusion, the results of the present study provided an IRG score model for prognostic stratification of adult patients with NK-AML, as well as further insight into the implication of IRGs in NK-AML that may lead to the development of novel immunotherapy approaches for this disease.

Fibrinogen-Like Protein 2-Associated Transcriptional and Histopathological Features of Immunological Preeclampsia.

Preeclampsia is a multifactorial hypertensive disorder of pregnancy, with variable presentation in both maternal and fetal factors, such that no treatment or marker is currently universal to all cases. Here, we demonstrate that the prothrombinase and immunomodulatory secreted factor FGL-2 (fibrinogen-like protein 2) is differentially expressed across previously characterized gene expression clusters containing clinically relevant disease subtypes. FGL2 is low in a cluster consistent with the traditional paradigm of the pathology of preeclampsia (canonical preeclampsia) and high in a cluster exhibiting evidence of immune activation (immunological preeclampsia). We show that it is part of an immunoregulatory gene module integral to the transcriptional profile and placental pathology specific to immunological preeclampsia. We determine that FGL2 associates positively with chronic inflammation lesions of the placenta while associating negatively with maternal vascular malperfusion lesions. The transcriptional profiles of maternal vascular malperfusion lesions show downregulation of FGL2 and upregulation of previously investigated preeclampsia biomarkers, such as FLT1 (Fms Related Receptor Tyrosine Kinase 1) and ENG (endoglin). Conversely, the profiles of chronic inflammation lesions show an interesting downregulation of these genes, but an upregulation of FGL2 and of FGL2-correlated immunoregulatory genes, suggesting it is upregulated downstream of major inflammatory mediators such as TNF (tumor necrosis factor)-α and IFN (interferon)-γ, hallmarks of the immunological preeclampsia subtype. This work, overall, demonstrates that FGL-2 expression levels in the term placenta reflect the unique pathophysiology that leads to immunological preeclampsia, leading to its potential as a subtype-specific biomarker.

SRC-Family Kinases in Acute Myeloid Leukaemia and Mastocytosis.

Protein tyrosine kinases have been recognized as important actors of cell transformation and cancer progression, since their discovery as products of viral oncogenes. SRC-family kinases (SFKs) play crucial roles in normal hematopoiesis. Not surprisingly, they are hyperactivated and are essential for membrane receptor downstream signaling in hematological malignancies such as acute myeloid leukemia (AML) and mastocytosis. The precise roles of SFKs are difficult to delineate due to the number of substrates, the functional redundancy among members, and the use of tools that are not selective. Yet, a large num ber of studies have accumulated evidence to support that SFKs are rational therapeutic targets in AML and mastocytosis. These two pathologies are regulated by two related receptor tyrosine kinases, which are well known in the field of hematology: FLT3 and KIT. FLT3 is one of the most frequently mutated genes in AML, while KIT oncogenic mutations occur in 80–90% of mastocytosis. Studies on oncogenic FLT3 and KIT signaling have shed light on specific roles for members of the SFK family. This review highlights the central roles of SFKs in AML and mastocytosis, and their interconnection with FLT3 and KIT oncoproteins.

Assessment of Predictive Biomarkers of the Response to Pazopanib Based on an Integrative Analysis of High-grade Soft-tissue Sarcomas: Analysis of a Tumor Sample from a Responder and Patients with Other Soft-tissue Sarcomas.

Soft-tissue sarcomas are a rare group of malignant tumors that usually are treated with surgical excision and radiation therapy, but recently, pazopanib, an oral tyrosine kinase inhibitor, has been used in patients with metastases who do not respond to standard chemotherapy regimens. Based on patients with advanced soft-tissue sarcomas who had received prior chemotherapy, several clinical studies have reported the survival and sensitivity (approximately 5% to 10% sensitive) of patients with soft-tissue sarcomas treated with pazopanib. Recently, next-generation sequencing (NGS) technologies have been used to provide a wide genetic information and to develop personalized medicine in cancer treatment. However, there are few reports and no genetic analyses of patients with soft-tissue sarcomas who had a complete response (CR) to pazopanib. We described the clinicopathologic features of a patient with a rare, advanced soft-tissue sarcoma who achieved a CR to pazopanib treatment. Furthermore, integrative analyses using NGS and arrays were performed to elucidate characteristic alterations, including gene mutations, copy number changes, and protein expression that were associated with response to pazopanib. Additionally, functional analyses consisting of in vitro and in vivo assays were also performed to elucidate whether the identified alterations were associated with oncogenic abilities and drug responses. In a sample from a 70-year-old woman with an advanced soft-tissue sarcoma treated for 1 month with 800 mg of oral pazopanib daily, CT scans demonstrated a CR to treatment. To our knowledge, there have been no patients with soft-tissue sarcomas among several clinical trials of pazopanib that have achieved a CR and therefore, our patient is considered to be extremely rare. We performed an integrative analysis including whole-exome sequencing, transcriptome sequencing, and phosphorylation profiling of receptor tyrosine kinases (RTK) using tumor samples from a patient with a CR matched to normal samples. From here on we will refer to this patient as having a CR, although a short term high-grade partial response may be more accurate. These analyses were performed using NGS and the phosphoreceptor tyrosine kinase (phospho-RTK) array. As a validation study, we also performed target sequencing using three samples from patients with long-term stable disease and two samples from patients with progressive disease who responded to pazopanib treatment. In addition, characteristic gene alterations that were identified according to the response to pazopanib in one patient with a CR, in three patients with long-term stable disease, and in 27 patients with high-grade soft-tissue sarcomas with different histologic subtypes and different responses to pazopanib were verified by quantitative real-time polymerase chain reaction. We conducted a focus formation assay to evaluate the transforming activities of these genomic alterations. In the patient with a CR to pazopanib, we identified several somatic mutations including Fms related receptor tyrosine kinase 1 (FLT1) p.G38S, platelet-derived growth factor receptor alpha (PDGFRA) p.T83S, and platelet-derived growth factor receptor beta (PDGFRB) exon 13 skipping. Amplification at chromosome 12q13-14 encompassing GLI family zinc finger 1 (GLI1) and cyclin-dependent kinase-4 (CDK4) was also detected. Furthermore, an elevated PDGFRB phosphorylation level was observed in the tumor. In target sequencing analyses in five patients, one of three patients with long-term stable disease had 12q13-14 amplification. The mRNA expression of GLI1, CDK4, and pazopanib targets including PDGFRA, PDGFRB, vascular endothelial growth factor receptor (VEGFR)1-3, and stem cell factor receptor (KIT) in samples from the patient with a CR, and 27 patients with high-grade soft-tissue sarcomas was verified. The expression of GLI1 was characteristically increased in the patient with a CR and in those with long-term stable disease relative to other patients with soft-tissue sarcomas. Overexpression of GLI1 showed strong transforming potential in 3T3 cells. Moreover, the overexpression of GLI1 upregulated the expression of the PDGFRB protein and promoted phosphorylation, which was dose-dependently inhibited by pazopanib. However, inhibition of GLI1-induced transformation by pazopanib was limited in the focus formation assay; therefore, mechanisms other than PDGFRB activation may contribute to transformation. We identified several gene alterations that might be associated with a CR and long-term stable disease in patients who received pazopanib for advanced soft-tissue sarcomas. We therefore believe that this distinct molecular profile warrants further investigation to identify predictive biomarkers of the response to pazopanib. Our findings identify molecular mechanisms that possibly explain the high sensitivity of soft-tissue sarcomas to pazopanib and may lead to the development of predictive biomarkers and novel therapies in patients with this and other types of soft-tissue sarcomas.

Targeted therapy for gastrointestinal and pancreatic neuroendocrine tumors

Extensive investigation of neuroendocrine tumors (NETs) has only started in recent years. The increased attention to this issue is due to the more frequent detection of NETs as a result of diagnostic methods improvement.The limited effectiveness of chemotherapy for well-differentiated advanced metastatic NETs of gastrointestinal tract (GIT) and pancreas demonstrates the need for development of the new and more effective treatment options.Recent studies on specific biological features of NETs have led to the development of the new targeted therapies which take into account high vascularization and overexpression of specific growth factors and related tyrosine kinase receptors. Thus, studying the mTOR TSC2, PTEN and PIK3CA signaling pathways opens up the new opportunities in the treatment of gastrointestinal or pancreatic NETs, especially in case of inoperable or metastatic tumors. Targeted therapy, which specifically inhibits growth factor receptors and related signaling pathways, is a promising approach to drug therapy for patients with gastrointestinal or pancreatic NETs.This review summarizes the state of the art and prospects for using targeted therapy, and describes clinical studies in the treatment of gastrointestinal and pancreatic NETs.

Preclinical Activity of HER2-Selective Tyrosine Kinase Inhibitor Tucatinib as a Single Agent or in Combination with Trastuzumab or Docetaxel in Solid Tumor Models.

HER2 is a transmembrane tyrosine kinase receptor that mediates cell growth, differentiation, and survival. HER2 is overexpressed in approximately 20% of breast cancers and in subsets of gastric, colorectal, and esophageal cancers. Both antibody and small-molecule drugs that target HER2 and block its tyrosine kinase activity are effective in treating HER2-driven cancers. In this article, we describe the preclinical properties of tucatinib, an orally available, reversible HER2-targeted small-molecule tyrosine kinase inhibitor. In both biochemical and cell signaling experiments, tucatinib inhibits HER2 kinase activity with single-digit nanomolar potency and provides exceptional selectivity for HER2 compared with the related receptor tyrosine kinase EGFR, with a >1,000-fold enhancement in potency for HER2 in cell signaling assays. Tucatinib potently inhibits signal transduction downstream of HER2 and HER3 through the MAPK and PI3K/AKT pathways and is selectively cytotoxic in HER2-amplified breast cancer cell lines in vitro. In vivo, tucatinib is active in multiple HER2+ tumor models as a single agent and shows enhanced antitumor activity in combination with trastuzumab or docetaxel, resulting in improved rates of partial and complete tumor regression. These preclinical data, taken together with the phase-I tucatinib clinical trial results demonstrating preliminary safety and activity, establish the unique pharmacologic properties of tucatinib and underscore the rationale for investigating its utility in HER2+ cancers. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/4/976/F1.large.jpg.

Enhancer Domains in Gastrointestinal Stromal Tumor Regulate KIT Expression and Are Targetable by BET Bromodomain Inhibition.

Gastrointestinal stromal tumor (GIST) is a mesenchymal neoplasm characterized by activating mutations in the related receptor tyrosine kinases KIT and PDGFRA. GIST relies on expression of these unamplified receptor tyrosine kinase (RTK) genes through a large enhancer domain, resulting in high expression levels of the oncogene required for tumor growth. Although kinase inhibition is an effective therapy for many patients with GIST, disease progression from kinase-resistant mutations is common and no other effective classes of systemic therapy exist. In this study, we identify regulatory regions of the KIT enhancer essential for KIT gene expression and GIST cell viability. Given the dependence of GIST upon enhancer-driven expression of RTKs, we hypothesized that the enhancer domains could be therapeutically targeted by a BET bromodomain inhibitor (BBI). Treatment of GIST cells with BBIs led to cell-cycle arrest, apoptosis, and cell death, with unique sensitivity in GIST cells arising from attenuation of the KIT enhancer domain and reduced KIT gene expression. BBI treatment in KIT-dependent GIST cells produced genome-wide changes in the H3K27ac enhancer landscape and gene expression program, which was also seen with direct KIT inhibition using a tyrosine kinase inhibitor (TKI). Combination treatment with BBI and TKI led to superior cytotoxic effects in vitro and in vivo, with BBI preventing tumor growth in TKI-resistant xenografts. Resistance to select BBI in GIST was attributable to drug efflux pumps. These results define a therapeutic vulnerability and clinical strategy for targeting oncogenic kinase dependency in GIST. SIGNIFICANCE: Expression and activity of mutant KIT is essential for driving the majority of GIST neoplasms, which can be therapeutically targeted using BET bromodomain inhibitors.

Highlights of This Issue

NF2 is an important tumor suppressor and regulator of physical stresses and loss of NF2 cause NF2-related cancers (NF2 syndrome and mesothelioma). However, there is no proper treatment for these diseases until now. This study by Cho and colleagues revealed that loss of NF2 induces a disturbance in the balance of TβR and facilitates TβR1-mediated oncogenic signaling, which could be blocked by TEW7197, a specific TβR1 kinase inhibitor. These findings support TEW7197 would be efficacious drug candidate for NF2-related diseases.Targeting tumor lactate homeostasis is considered to be an attractive approach for cancer therapy. In a cell based screen, Quanz and colleagues have discovered a novel specific and orally available inhibitor (BAY-8002) of the lactate transporter MCT1. BAY-8002 showed potent inhibition of lactate transport in cancer cells and tumor growth inhibition in vivo. The investigation of resistant cells as well as response to treatment in a large cell panel provide new insight into the metabolic plasticity of tumor cells. Furthermore, the results suggest stratification markers to be potentially pursued in the clinical development of MCT1 inhibitors.The receptor tyrosine kinase (RTK) AXL is overexpressed in many human cancers including head and neck squamous cell carcinoma, triple-negative breast cancer, and non-small cell lung cancer. McDaniel, Cummings and colleagues identified a mechanism of resistance to AXL-targeting agents mediated by MERTK, a related RTK. Endogenous MERTK expression increased after treatment with AXL inhibitors and ectopic overexpression conferred resistance to AXL-targeting agents. Moreover, MERTK inhibitors overcame resistance to AXL inhibition and significantly reduced tumor cell expansion and tumor growth. The synergistic interactions between AXL and MERTK inhibitors suggest that therapeutic co-targeting strategies could be very effective in these malignancies.Delivery of therapeutics to pancreatic ductal adenocarcinomas (PDAC) is challenging due to the physical stromal barriers surrounding these tumors. Lo and colleagues applied tumor-penetrating nanocomplexes (TPNs) to breach the stroma and effectively deliver therapeutic siRNA to PDAC. These particles are self-assembled nanostructures comprised of tandem peptides containing both a tumor-penetrating iRGD domain and a cell-penetrating domain, PEGylated derivatives of these tandem peptides, and siRNA. TPNs delivering anti-Kras siRNA exhibited tumor-penetrating ability in 3D organoids and autochthonous tumors, and slowed PDAC growth in vivo. The modular construction of the TPN platform allows for facile adaptation for future genetic targets.

Pan-HER-targeted approach for cancer therapy: Mechanisms, recent advances and clinical prospect

The Human Epidermal Growth Factor Receptor family is composed of 4 structurally related receptor tyrosine kinases that are involved in many human cancers. The efficacy and safety of HER inhibitors have been compared in a wide range of clinical trials, suggesting the superior inhibitory ability of multiple- HER-targeting blockade compared with single receptor antagonists. However, many patients are currently resistant to current therapeutic treatment and novel strategies are warranted to conquer the resistance. Thus, we performed a critical review to summarize the molecular involvement of HER family receptors in tumour progression, recent anti-HER drug development based on clinical trials, and the potential resistance mechanisms of anti-HER therapy.

CRISPR-assisted receptor deletion reveals distinct roles for ERBB2 and ERBB3 in skin keratinocytes.

While the epidermal growth factor receptor (EGFR) is an established regulator of skin development and homeostasis, the functions of the related tyrosine kinase receptors ERBB2 and ERBB3 in this tissue have only recently been examined. Previously reported, skin-specific deletion of each of these receptors in mice resulted in similar defects in keratinocyte proliferation and migration, resulting in impaired wound healing and tumorigenesis. Because both ERBB2 and ERBB3 are targets for treating an array of cancer types, it is important to examine the consequences of receptor inhibition in human keratinocytes. Here, we employed the CRISPR/Cas9 technology to generate HaCaT cells (an established human keratinocyte cell line) lacking ERBB2 or ERBB3. HaCaT clones lacking ERBB2 or ERBB3 showed comparable reductions in cell proliferation as assessed by BrdU staining. Apoptosis, in contrast, was reduced in ERBB3-deficient HaCaT cells only. Assessment of cell migration using a wound healing (scratch) assay showed that the closure of the wound gaps was completed by 48 h in mock and in ERBB3 knockout clones. In contrast, this process was considerably delayed in ERBB2 knockout clones, and a complete closure of the gap in the latter cells did not occur before 72 h. In conclusion, both ERBB2 and ERBB3 are essential for normal proliferation of skin keratinocytes, but in contrast to ERBB3, ERBB2 is essential for migration of human keratinocytes. These observations might bear significance to patient adverse effects of therapeutic agents targeting ERBB2 and ERBB3.

A novel, somatic, transforming mutation in the extracellular domain of Epidermal Growth Factor Receptor identified in myeloproliferative neoplasm

We describe a novel ERBB1/EGFR somatic mutation (p. C329R; c.985 T > C) identified in a patient with JAK2V617F Polycythaemia Vera (PV). This substitution affects a conserved cysteine residue in EGFR domain 2 and leads to the formation of a ligand-independent covalent receptor dimer, associated with increased transforming potential. Aberrant signalling from the EGFRC329R receptor is cell type-dependent and in the TF1.8 erythroid cell line expression of this mutant suppresses EPO-induced differentiation. Clonal analysis shows that the dominant JAK2V617F-positive clone in this PV patient harbors EGFRC329R, thus this mutation may contribute to clonal expansion. Somatic mutations affecting other ERBB and related receptor tyrosine kinases are observed in myeloproliferative neoplasms (MPN), and we show elevated EGFR levels in MPN samples, consistent with previous reports. Thus activation of this group of receptors, via multiple mechanisms, may contribute to clonal growth and survival of the JAK2V617F disease clone in MPN.

Phase 2 trial of the novel multi-receptor tyrosine kinase inhibitor sitravatinib in well-differentiated/dedifferentiated liposarcoma.

TPS11082 Background: Well-differentiated/dedifferentiated liposarcoma (WD/DD LPS) is a sarcoma subtype of adipocytic origin characterized by amplification of cyclin dependent kinase 4 (CDK4) and MDM2. WD/DD LPS is resistant to chemotherapy and success with CDK4 inhibitors is limited. We recently characterized the landscape of activated receptor tyrosine kinases (RTKs) and intracellular signaling pathways finding marked heterogeneity by sarcoma subtype [Patwardhan et al. Oncotarget 2016;7(4)]. In WD/DD LPS cell lines, phosphorylated (p) IGF1-R, MET and PDGFRb are strongly expressed. Selective siRNA knockdown of expression of 1 or more of these RTKs inhibited growth of WD/DD LPS cell lines. Sitravatinib (S) is a novel inhibitor of a broad panel of related RTKs. We showed that S abrogates expression of p-RTKs, including IGF1-R, MET and PDGFRb, at low nanomolar concentrations and potently inhibits proliferation of WD/DD LPS cell lines, where anti-proliferative effects of S were superior to other RTK inhibitors including imatinib, crizotinib and pazopanib. S suppressed tumor growth in vivo in WD/DD LPS . A phase 1 trial of S in solid tumors showed clinical activity in WD/DD LPS. Recommended phase 2 dose was 150 mg/day. As there are no approved RTK inhibitors for adipocytic sarcomas, and based on these findings, we initiated a phase 2 trial of S in WD/DD LPS. Methods: This is a single-arm open-label multi-center Simon 2 stage phase II trial of S in 29 patients (pts) with advanced WD/DD LPS who failed 1 prior therapy and show disease progression before enrollment. Pts receive S 150 mg orally daily continuously. Primary endpoint is the progression free rate at 12 weeks (PFR12) versus historical controls. The design has power of 85% to show improvement in PFR12 from 20% (inactive) to 40% (active) with α = 0.10. Secondary endpoints are ORR, PFS and safety. A subset of pts undergo baseline and on-treatment biopsies and reverse phase protein array used to measure changes in expression of p-RTKs and signaling pathway proteins with confirmation by immunoblot. Genomic landscape of these tumors will be analyzed by next generation sequencing. The study opened in 1/2017. Clinical trial information: NCT02978859.

Abstract 4157: Heterogeneity of vascular endothelial growth factor receptors 1, 2, and 3 in human non-small cell lung carcinomas

Abstract The Vascular Endothelial Growth Factor (VEGF) pathway plays a prominent role in the growth and progression of human cancers, including non-small cell lung carcinoma (NSCLC). The key mediators of VEGF signaling are a family of related receptor tyrosine kinases that include VEGFR1, VEGFR2, and VEGFR3. The relative expression levels, activity and cross-talk among these receptors may contribute to clinical response of NSCLC patients to anti-angiogenic therapies. Using a well-annotated tissue microarray (TMA) and robust immunohistochemical (IHC) assays developed, standardized and implemented in our laboratory, we comparatively evaluated expression of the three VEGFRs in archival primary NSCLC tissues (n = 97). VEGFR1 and VEGFR2 were localized both in tumor vessels and cells, while VEGFR3 was only localized in tumor vessels. VEGFR1 immunoreactivity was reported as negative/low, medium, or high, based on intensity and proportion of stained tumor cell by an experienced solid tumor immunopathologist (AN), who was blinded to clinico-pathologic details. VEGFR2 and VEGFR3 positive vessels were counted by manual assessment of each core by the same immunopathologist. For systematic comparative analysis of VEGFR data, IHC expression thresholds were selected based on the 25% and 75% quartiles around the median of the range of counts for VEGFR2 and VEGFR3: 0-2, 3-10 and >10 (VEGFR2+ vascular count) respectively; and 0-1, 2-9 and >9 (VEGFR3+ vascular count) respectively. Based on VEGFR (1,2 and3) expression levels defined above, a set of eight VEGFR staining profiles were identified: Triple VEGFR positive (n = 11, 11.3%), VEGFR1 predominant (22, 22.7%), VEGFR2 predominant (9, 9.3%), VEGFR3 predominant (3, 3.1%), VEGFR1/2 predominant (13, 13.4%), VEGFR1/3 predominant (2, 2.1%), VEGFR2/3 predominant (9, 9.3%), and triple VEGFR negative (28, 28.9%). These new data provide original insights on the tissue distribution, subcellular localization and heterogeneity of expression of VEGFRs in human NSCLC cells and stromal vessels. The proposed human NSCLC sub-classification, based on the observed differential VEGFR 1, 2, 3 expression profiles, has identified various subsets of human NSCLC, especially triple VEGFR +, triple VEGFR -, VEGFR1 predominant and VEGFR 1 / 2 predominant. These profiles are distinct from the VEGF receptor profiles that we reported in a prior study on colorectal carcinomas (Holzer, Nasir et al., AACR 2014), of which VEGFR1/2 predominant subset was 50% and triple VEGFR-negative subset was only 4%. This work suggests distinct patterns of heterogeneity of VEGF receptor profiles in human NSCLC and CRCs. These data also support further evaluation of whether the reported VEGFR profiles correlate with differential sensitivity to therapeutic VEGF/VEGFR pathway inhibition. Citation Format: Timothy R. Holzer, Angie D. Fulford, Leslie O'Neill Reising, Drew M. Nedderman, Laura E. Benjamin, Andrew E. Schade, Aejaz Nasir. Heterogeneity of vascular endothelial growth factor receptors 1, 2, and 3 in human non-small cell lung carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4157. doi:10.1158/1538-7445.AM2015-4157

Signaling by Met and related receptor tyrosine kinases in urothelial carcinoma of the bladder.

e15511 Background: Interim results from a phase II clinical trial of cabozantinib, a multikinase inhibitor of Met and related receptor tyrosine kinases (RTKs) for patients with advanced UC, reveal ...

Crystal Structure of the FLT3 Kinase Domain Bound to the Inhibitor Quizartinib (AC220)

More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an “Abl-like” inactive conformation with the activation loop stabilized in the “DFG-out” orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain of FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.

The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

Inhibition of the insulin-like growth factor 1 receptor by CHM-1 blocks proliferation of glioblastoma multiforme cells

The insulin-like growth factor-1 receptor (IGF-1R) plays a pivotal role in transformation, growth, and survival of glioblastoma multiforme (GBM) cells, and has emerged as a general and promising target for cancer treatment. In this study, we examined the anti-tumor effects of CHM-1, a synthetic 6,7-methylenedioxy substituted 2-phenyl-4-quinolone derivative, on GBM cells in vitro and in vivo. CHM-1 selectively blocked IGF-1R auto-phosphorylation, with an ability to distinguish between IGF-1R and related tyrosine kinase receptors, such as insulin receptor (IR), epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR). Further investigation revealed that, the phosphorylation of ERK1/2 as well as Akt was inhibited in CHM-1 treated GBM8401 cells possibly through the selective blockage of IGF-1R auto-phosphorylation. Our study also showed that p.o. treatment with the hydrophilic dihydrogen phosphate CHM-1P reduced the tumor volumes of the GBM8401 derived tumors in mouse brain and also prolonged the survival rate. The results provided potential opportunities for effective chemotherapy for GBM.