Receptor Tyrosine Kinase Mutations Research Articles

Functional Investigation of IGF1R Mutations in Multiple Myeloma.

High expression of the receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF1R) and RTK mutations are associated with high-risk/worse prognosis in multiple myeloma (MM). Combining the pIGF1R/pINSR inhibitor linsitinib with the proteasome inhibitor (PI) bortezomib seemed promising in a clinical trial, but IGF1R expression was not associated with therapy response. Because the oncogenic impact of IGF1R mutations is so far unknown, we investigated the functional impact of IGF1R mutations on survival signaling, viability/proliferation and survival response to therapy. We transfected four human myeloma cell lines (HMCLs) with IGF1RWT, IGF1RD1146N and IGF1RN1129S (Sleeping Beauty), generated CRISPR-Cas9 IGF1R knockouts in the HMCLs U-266 (IGF1RWT) and L-363 (IGF1RD1146N) and tested the anti-MM activity of linsitinib alone and in combination with the second-generation PI carfilzomib in seven HMCLs. IGF1R knockout entailed reduced proliferation. Upon IGF1R overexpression, survival signaling was moderately increased in all HCMLs and slightly affected by IGF1RN1129S in one HMCL, whereby the viability remained unaffected. Expression of IGF1RD1146N reduced pIGF1R-Y1135, especially under serum reduction, but did not impact downstream signaling. Linsitinib and carfilzomib showed enhanced anti-myeloma activity in six out of seven HMCL irrespective of the IGF1R mutation status. In conclusion, IGF1R mutations can impact IGF1R activation and/or downstream signaling, and a combination of linsitinib with carfilzomib might be a suitable therapeutic approach for MM patients potentially responsive to IGF1R blockade.

Loss-of-function mutations of the TIE1 receptor tyrosine kinase cause late-onset primary lymphedema.

Primary lymphedema (PL), characterized by tissue swelling, fat accumulation and fibrosis, results from defective lymphatic vessels or valves caused by mutations in genes involved in development, maturation and function of the lymphatic vascular system. Pathogenic variants in various genes have been identified in about 30% of PL cases. By screening of a cohort of 755 individuals with PL, we identified two TIE1 (tyrosine kinase with immunoglobulin- and epidermal growth factor-like domains 1) missense variants and one truncating variant, all predicted to be pathogenic by bioinformatic algorithms. The TIE1 receptor, in complex with TIE2, binds angiopoietins to regulate the formation and remodelling of blood and lymphatic vessels. The premature stop codon mutant encoded an inactive truncated extracellular TIE1 fragment with decreased mRNA stability and the amino acid substitutions led to decreased TIE1 signaling activity. By reproducing the two missense variants in mouse Tie1 via CRISPR-Cas9, we showed that both cause edema and are lethal in homozygous mice. Thus, our results indicate that TIE1 loss-of-function variants can cause lymphatic dysfunction in patients. Together with our earlier demonstration that ANGPT2 loss-of-function mutations can also cause PL, our results emphasize the important role of the ANGPT2-TIE1 pathway in lymphatic function.

Brefeldin A and M-COPA block the export of RTKs from the endoplasmic reticulum via simultaneous inactivation of ARF1, ARF4, and ARF5

Normal receptor tyrosine kinases (RTKs) need to reach the plasma membrane (PM) for ligand-induced activation, whereas its cancer-causing mutants can be activated before reaching the PM in organelles, such as the Golgi/trans-Golgi network (TGN). Inhibitors of protein export from the endoplasmic reticulum (ER), such as brefeldin A (BFA) and 2-methylcoprophilinamide (M-COPA), can suppress the activation of mutant RTKs in cancer cells, suggesting that RTK mutants cannot initiate signaling in the ER. BFA and M-COPA block the function of ADP-ribosylation factors (ARFs) that play a crucial role in ER–Golgi protein trafficking. However, among ARF family proteins, the specific ARFs inhibited by BFA or M-COPA, that is, the ARFs involved in RTKs transport from the ER, remain unclear. In this study, we showed that M-COPA blocked the export of not only KIT but also PDGFRA/EGFR/MET RTKs from the ER. ER-retained RTKs could not fully transduce anti-apoptotic signals, thereby leading to cancer cell apoptosis. Moreover, single knockdown of ARF1, ARF3, ARF4, ARF5, or ARF6 could not block ER export of RTKs, indicating that BFA/M-COPA treatment cannot be mimicked by knockdown of only one ARF member. Interestingly, simultaneous transfection of ARF1, ARF4, and ARF5 siRNAs mirrored the effect of BFA/M-COPA treatment. Consistent with these results, in vitro pulldown assays showed that BFA/M-COPA blocked the function of ARF1, ARF4, and ARF5. Taken together, these results suggest that BFA/M-COPA targets at least ARF1, ARF4, and ARF5; in other words, RTKs require the simultaneous activation of ARF1, ARF4, and ARF5 for their ER export.

RAF and MEK Inhibitors in Non-Small Cell Lung Cancer.

Lung cancer, despite recent advancements in survival rates, represents a significant global health burden. Non-small cell lung cancer (NSCLC), the most prevalent type, is driven largely by activating mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) and receptor tyrosine kinases (RTKs), and less in v-RAF murine sarcoma viral oncogene homolog B (BRAF) and mitogen-activated protein-kinase kinase (MEK), all key components of the RTK-RAS-mitogen-activated protein kinase (MAPK) pathway. Learning from melanoma, the identification of BRAFV600E substitution in NSCLC provided the rationale for the investigation of RAF and MEK inhibition as a therapeutic strategy. The regulatory approval of two RAF-MEK inhibitor combinations, dabrafenib-trametinib, in 2017, and encorafenib-binimetinib, in 2023, signifies a breakthrough for the management of BRAFV600E-mutant NSCLC patients. However, the almost universal emergence of acquired resistance limits their clinical benefit. New RAF and MEK inhibitors, with distinct biochemical characteristics, are in preclinical and clinical development. In this review, we aim to provide valuable insights into the current state of RAF and MEK inhibition in the management of NSCLC, fostering a deeper understanding of the potential impact on patient outcomes.

Abstract 5949: The clinical dual KRASG12C inhibitor FMC-376 has demonstrated potential as both a monotherapy and in combination for the treatment of patients with KRASG12C mutation positive NSCLC

Abstract KRAS, a membrane-associated guanosine triphosphatase (GTPase), is the most frequently mutated oncogene in cancer with activating mutations occurring in approximately 25% of NSCLC. Of these, KRASG12C occurs in approximately 14% of adenocarcinomas and in 0.5 to 4% of squamous NSCLCs. This mutation impairs GTPase activity and GTP-hydrolysis leading to an increase in the active, GTP-bound (ON), state. KRAS was long considered undruggable until the recent discovery of inhibitors that bind the GDP-bound, inactive (OFF) state of KRASG12C. The most clinically advanced of these first-generation molecules have demonstrated clinical response rates of 37-43% and 6 to 7-month progression-free survival in patients with KRASG12C positive NSCLC. While significant, a majority of cancers do not respond, and acquired resistance is common. FMC-376 is a next-generation dual inhibitor of both the ON and OFF states of KRASG12Cwith the potential to deliver improved outcomes as both a monotherapy and in combination, for patients with KRASG12C positive NSCLC. FMC-376 was discovered through the FrontierTM platform, which integrates chemoproteomics, machine-learning, and covalent fragment-based drug discovery. The potential for FMC-376 in the treatment of patients with KRASG12C mutant NSCLC has been assessed in a broad panel of NSCLC PDX models representing settings of sotorasib/adagrasib resistance, in a model of NSCLC CNS-metastasis and in combination with an immune checkpoint inhibitor. The dual ON + OFF state mechanism of action of FMC-376 is highly active across a panel of NSCLC CDX and PDX models, including those that carry co-mutations that are associated with both primary and acquired resistance to the OFF-state inhibitors adagrasib and sotorasib in NSCLC. These drivers of resistance include activating mutations of receptor tyrosine kinases (e.g. EGFR, MET, etc.), co-mutations including KEAP1 and adaptive resistance due to release of negative feedback control. Evaluation of FMC-376 in models where EGFR signaling is induced by EGF demonstrated rapid and durable target engagement in contrast to both sotorasib and adagrasib which show decreased effectiveness after EGF stimulation. As a result, FMC-376 is highly effective in NSCLC PDX models that have EGFR and MET upregulation. In addition, 27-42% of patients with KRASG12C positive NSCLC present with CNS metastasis. FMC-376 is highly active in an intracranial NSCLC model of CNS-metastasis, resulting in tumor regression. Furthermore, FMC-376 has demonstrated activity in combination with an anti-PD-1 mAb in a KRASG12C mutant mouse syngeneic tumor model. Together, these data demonstrate the potential of FMC-376, a next-generation clinical stage dual inhibitor of ON and OFF state KRASG12C, for the treatment of patients with KRASG12C mutant NSCLC as well as other KRASG12C positive tumors. Citation Format: Yan Wang, Allison Roberts, Philamer Calses, Richard M. Neve, Jocelyn Staunton, Kevin R. Webster. The clinical dual KRASG12C inhibitor FMC-376 has demonstrated potential as both a monotherapy and in combination for the treatment of patients with KRASG12C mutation positive NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5949.

Abstract 2001: Autocrine HGF maturation as a targetable regulatory step in cetuximab resistance in colorectal cancer

Abstract Although amplifications and mutations in receptor tyrosine kinases (RTKs) act as bona fide oncogenes, in most cancers, RTKs maintain moderate expression and remain wild-type. Consequently, cognate ligands control many facets of tumorigenesis, including resistance to anti-RTK therapies. Herein, we show that the ligands for the RTKs MET and RON, HGF and HGFL, respectively, are synthesized as inactive precursors that are activated by cellular proteases. Our newly generated HGF/HGFL protease inhibitors could overcome both de novo and acquired cetuximab resistance in colorectal cancer (CRC). Conversely, HGF overexpression was necessary and sufficient to induce cetuximab resistance and loss of polarity. Moreover, HGF-induced cetuximab resistance could be overcome by the downstream MET inhibitor, crizotinib, and upstream protease inhibitors. Additionally, HAI-1, an endogenous inhibitor of HGF proteases, (i) was downregulated in CRC, (ii) exhibited increased genomic methylation that correlated with poor prognosis, (iii) HAI-1 expression correlated with cetuximab response in a panel of cancer cell lines, and (iv) exogenous addition of recombinant HAI-1 overcame cetuximab resistance in CC-HGF cells. Thus, we describe a targetable, autocrine HAI-1/Protease/HGF/MET axis in cetuximab resistance in CRC. Citation Format: Vivian Truong Jones, Ramona Graves-Deal, Zheng Cao, Galina Bogatcheva, Marisol A. Ramirez, Sarah J. Harmych, James N. Higginbotham, Claudia C. Wahoski, Neeraj Joshi, Joseph T. Roland, Gregory D. Ayers, Qi Liu, Robert J. Coffey, James W. Janetka, Bhuminder Singh. Autocrine HGF maturation as a targetable regulatory step in cetuximab resistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2001.

EGFR signaling and pharmacology in oncology revealed with innovative BRET-based biosensors

Mutations of receptor tyrosine kinases (RTKs) are associated with the development of many cancers by modifying receptor signaling and contributing to drug resistance in clinical settings. We present enhanced bystander bioluminescence resonance energy transfer-based biosensors providing new insights into RTK biology and pharmacology critical for the development of more effective RTK-targeting drugs. Distinct SH2-specific effector biosensors allow for real-time and spatiotemporal monitoring of signal transduction pathways engaged upon RTK activation. Using EGFR as a model, we demonstrate the capacity of these biosensors to differentiate unique signaling signatures, with EGF and Epiregulin ligands displaying differences in efficacy, potency, and responses within different cellular compartments. We further demonstrate that EGFR single point mutations found in Glioblastoma or non-small cell lung cancer, impact the constitutive activity of EGFR and response to tyrosine kinase inhibitor. The BRET-based biosensors are compatible with microscopy, and more importantly characterize the next generation of therapeutics directed against RTKs.

Inhibition of autocrine HGF maturation overcomes cetuximab resistance in colorectal cancer

Although amplifications and mutations in receptor tyrosine kinases (RTKs) act as bona fide oncogenes, in most cancers, RTKs maintain moderate expression and remain wild-type. Consequently, cognate ligands control many facets of tumorigenesis, including resistance to anti-RTK therapies. Herein, we show that the ligands for the RTKs MET and RON, HGF and HGFL, respectively, are synthesized as inactive precursors that are activated by cellular proteases. Our newly generated HGF/HGFL protease inhibitors could overcome both de novo and acquired cetuximab resistance in colorectal cancer (CRC). Conversely, HGF overexpression was necessary and sufficient to induce cetuximab resistance and loss of polarity. Moreover, HGF-induced cetuximab resistance could be overcome by the downstream MET inhibitor, crizotinib, and upstream protease inhibitors. Additionally, HAI-1, an endogenous inhibitor of HGF proteases, (i) was downregulated in CRC, (ii) exhibited increased genomic methylation that correlated with poor prognosis, (iii) HAI-1 expression correlated with cetuximab response in a panel of cancer cell lines, and (iv) exogenous addition of recombinant HAI-1 overcame cetuximab resistance in CC-HGF cells. Thus, we describe a targetable, autocrine HAI-1/Protease/HGF/MET axis in cetuximab resistance in CRC.

NTRK2 expression in gastrointestinal stromal tumors with a special emphasis on the clinicopathological and prognostic impacts

Gastrointestinal stromal tumors (GISTs) are typically characterized by activating mutations of the KIT proto-oncogene receptor tyrosine kinase (KIT) or platelet-derived growth factor receptor alpha (PDGFRA). Recently, the neurotrophic tyrosine receptor kinase (NTRK) fusion was reported in a small subset of wild-type GIST. We examined trk IHC and NTRK gene expressions in GIST. Pan-trk immunohistochemistry (IHC) was positive in 25 (all 16 duodenal and 9 out of 16 small intestinal GISTs) of 139 cases, and all pan-trk positive cases showed diffuse and strong expression of c-kit. Interestingly, all of these cases showed only trkB but not trkA/trkC expression. Cap analysis of gene expression (CAGE) analysis identified increased number of genes whose promoters were activated in pan-trk/trkB positive GISTs. Imbalanced expression of NTRK2, which suggests the presence of NTRK2 fusion, was not observed in any of trkB positive GISTs, despite higher mRNA expression. TrkB expression was found in duodenal GISTs and more than half of small intestinal GISTs, and this subset of cases showed poor prognosis. However, there was not clear difference in clinical outcomes according to the trkB expression status in small intestinal GISTs. These findings may provide a possible hypothesis for trkB overexpression contributing to the tumorigenesis and aggressive clinical outcome in GISTs of duodenal origin.

Abstract C010: A comprehensive predictive biomarker analysis for MEK Inhibitors in Patient-derived Xenograft (PDX) models

Abstract Introduction: The RAS-RAF-MEK-ERK pathway has been extensively studied for its prominent role in cancer development. Activating mutations in receptor tyrosine kinases, RAS, or RAF, are commonly observed in cancer. Pathway activation caused by these mutations is mediated through MEK kinases, making MEK inhibition an attractive therapeutic strategy. Although several in vitro studies have been carried out to identify predictive biomarkers for sensitivity to MEK inhibition, the results have not been validated in vivo. Methods: Cryopreserved or fresh PDX tumor tissues (~2–3 mm in diameter) were transplanted into NOD/SCID or BALB/c nude mice subcutaneously. Trametinib was administered orally daily at 0.3mg - 1mg/kg of body weight, starting when tumor size reached 100-300 mm3. Sequencing reads were aligned to both human and mouse reference genomes. Based on which alignment yielded fewer mismatches, they were subsequently sorted into human or mouse groups, representing cancer and stromal transcriptomes, respectively. Ambiguous readings were discarded. Transcript abundance was quantified using the Kallisto software tool. Nucleotide variants in human genes were detected using the GATK best practice workflow. Results: We systematically analyzed tumor growth data collected from 62 PDX models in 9 cancer types (colorectal, head and neck, esophageal, kidney, melanoma, lung, gastric, ovarian, and pancreatic) treated with Trametinib, an allosteric MEK1/2 inhibitor. The responses to Trametinib were classified into 3 categories based on the amount of tumor growth inhibition (TGI) achieved. About 20% of these models were sensitive to Trametinib treatment (TGI > 70% compared to vehicle), 35% were resistant (TGI ≤ 30% compared to vehicle), and the remaining showed a partial response (30% < TGI ≤ 70% compared to vehicle). Cross-group comparisons of PDX transcriptomic and mutation profiles allowed identification of novel biomarkers as well as validation of biomarkers previously reported in in vitro studies. Conclusions: The different sensitivity to MEK inhibition observed in PDX models mirrors the patient heterogeneity observed in clinic. Thus, our results highlight the need to identify the appropriate genetic background using predictive biomarkers when applying targeted therapies. Citation Format: Yanghui Sheng, Wubin Qian, Jingjing Wang, Sheng Guo. A comprehensive predictive biomarker analysis for MEK Inhibitors in Patient-derived Xenograft (PDX) models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C010.

Transient TKI-resistant CD44+pBAD+ blasts undergo intrinsic homeostatic adaptation to promote the survival of acute myeloid leukemia in vitro.

Acute myeloid leukemia (AML) patients have frequent mutations in FMS-like receptor tyrosine kinase 3 (FLT3-mut AML), who respond poorly to salvage chemotherapies and targeted therapies such as tyrosine kinase inhibitors (TKIs). Disease relapse is a common reason of treatment failures in FLT3-mut AML patients, but its intracellular refractory mechanism remains to be discovered. In this study, we designed serial in vitro time-course studies to investigate the biomarkers of TKI-resistant blasts and their survival mechanism. First, we found that a group of transient TKI-resistant blasts were CD44+Phosphorylated-BAD (pBAD)+ and that they could initiate the regrowth of blast clusters in vitro. Notably, TKI-treatments upregulated the compensation pathways to promote PIM2/3-mediated phosphorylation of BAD to initiate the blast survival. Next, we discovered a novel process of intracellular adaptive responses in these transient TKI-resistant blasts, including upregulated JAK/STAT signaling pathways for PIM2/3 expressions and activated SOCS1/SOCS3/PIAS2 inhibitory pathways to down-regulate redundant signal transduction and kinase phosphorylation to regain intracellular homeostasis. Finally, we found that the combination of TKIs with TYK2/STAT4 pathways-driven inhibitors could effectively treat FLT3-mut AML in vitro. In summary, our findings reveal that TKI-treatment can activate a JAK/STAT-PIM2/3 axis-mediated signaling pathways to promote the survival of CD44+pBAD+blasts in vitro. Disrupting these TKIs-activated redundant pathways and blast homeostasis could be a novel therapeutic strategy to treat FLT3-mut AML and prevent disease relapse in vivo.

Novel Mechanisms of Venetoclax Resistance in Acute Myeloid Leukemia Based on Genomic Rearrangements

Introduction: Venetoclax (VEN), a BCL-2 inhibitor, effectively induces apoptosis in acute myeloid leukemia (AML) cells. Resistance to VEN in the treatment of AML can occur through various mechanisms. Some of the identified resistance mechanisms include: expansion or gain of clones with activation of the receptor tyrosine kinase (RTK) pathway; disruption of TP53 function, a tumor suppressor gene known to play a critical role in cell cycle regulation and apoptosis; selection of AML populations with monocytic differentiation, possibly altering cellular characteristics and response to treatment; overexpression of the MCL1 gene, which encodes for a protein that can counteract the pro-apoptotic effects of venetoclax; switch to lipid metabolism, an adaptive response by leukemia cells that may confer resistance to venetoclax; mutations in BCL-2 and BAX genes, which could affect the activity of these key regulators of apoptosis. While these mechanisms have been extensively studied, the role of rearrangements, often combined with genomic events, has not been fully explored. The INTHEMA protocol (NCT04298892) aimed to identify new VEN resistance mechanisms in patients with relapsed/refractory AML treated with VEN in combination with hypomethylating agents and to provide addressable molecular pathways useful for restoring drug sensitivity or offer novel therapeutic opportunities. Methods: Informed consent was obtained from 21 consecutive patients. Samples were collected from these patients with relapse or refractory disease after VEN + hypomethylating agent treatment. TruSight RNA Pan-Cancer analysis was performed to investigate fusion genes (using capture technology covering 1385 genes), RNA expression, and expressed mutations for each sample. Results: Among the 21 patients, 17 (81%) showed at least one putative mechanism of VEN resistance (as illustrated in the figure). These mechanisms included co-occurring mutations and genomic rearrangements. RTK mutations were found in 14 patients (67%), with specific mutations observed in NRAS, PTPN11, CBL, FLT3, JAK2, cKIT, EPHB6, FGFR and PDGFRb. One patient had a BAX mutation (p.G67R; BH3 domain), three patients exhibited a switch to a monocytic phenotype, and two patients had marker chromosomes. Gene fusions were identified in eight patients (38%): in particular, we discovered a cryptic rearrangement of MECOM (TBL1XR1-MECOM and ETV6_MECOM) in 2 patients, a novel BCL11B fusion ( BCL11B-GSDMC), a CCND2-ETV6 BIRC6-LTBP1, the reciprocal NUP98-NSD1, the canonical CBFB-MYH11 and additional transcripts with less defined roles ( PSPC1-ZMYM2, PER1-DDX5, ELL-SUGP2). Interestingly, most of the genes involved in traslocations, with a potential driver role in therapy resistance ( MECOM, ETV6, CCND2, BCL11B, NUP98) also showed high expression levels in patients harboring the traslocation. Unsupervised clustering of RNA expression data revealed two main patient groups. The first group (24%) demonstrated an “activating-like” signature, characterized by overexpression of CSF3R and IKZF, suggesting a potential target for tyrosine kinase inhibitors. The second group exhibited a “self-renewal like” signature, suggesting a possible benefit from histone deacetylase inhibitors, as previously published. Patients with gene fusions were predominantly clustered within the “self-renewal like” group, particularly in a subgroup (subgroup 4) showing overexpression of HOXA genes. Conclusions: Through deep transcriptomic characterization combined with conventional diagnostics, this analysis uncovered novel mechanisms of VEN resistance while confirming established ones. The distinct gene expression patterns may help tailor targeted therapies, with patients showing the “activating-like” signature potentially benefiting from tyrosine kinase inhibitors and those with the “self-renewal like” signature possibly responding well to histone deacetylase inhibitors. Furthermore, HOXA gene overexpression presents an exciting therapeutic opportunity for selected patients. Further research and clinical validation are needed to translate these findings into actionable clinical strategies.

A Functional Genomics Review of Non-Small-Cell Lung Cancer in Never Smokers.

There is currently a dearth of information regarding lung cancer in never smokers (LCINS). Additionally, there is a difference in somatic mutations, tumour mutational burden, and chromosomal aberrations between smokers and never smokers (NS), insinuating a different disease entity in LCINS. A better understanding of actionable driver alterations prevalent in LCINS and the genomic landscape will contribute to identifying new molecular targets of relevance for NS that will drastically improve outcomes. Differences in treatment outcomes between NS and smokers, as well as sexes, with NSCLC suggest unique tumour characteristics. Epidermal growth factor receptor (EGFR) tyrosine kinase mutations and echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) gene rearrangements are more common in NS and have been associated with chemotherapy resistance. Moreover, NS are less likely to benefit from immune mediators including PD-L1. Unravelling the genomic and epigenomic underpinnings of LCINS will aid in the development of not only novel targeted therapies but also more refined approaches. This review encompasses driver genes and pathways involved in the pathogenesis of LCINS and a deeper exploration of the genomic landscape and tumour microenvironment. We highlight the dire need to define the genetic and environmental aspects entailing the development of lung cancer in NS.

MET exon 14 skipping mutation is a hepatocyte growth factor (HGF)-dependent oncogenic driver in vitro and in humanized HGF knock-in mice.

Exon skipping mutations of the MET receptor tyrosine kinase (METex14), increasingly reported in cancers, occur in 3-4% of non-small cell lung cancer (NSCLC). Only 50% of patients have a beneficial response to treatment with MET-tyrosine kinase inhibitors (TKIs), underlying the need to understand the mechanism of METex14 oncogenicity and sensitivity to TKIs. Whether METex14 is a driver mutation and whether it requires hepatocyte growth factor (HGF) for its oncogenicity in a range of in vitro functions and in vivo has not been fully elucidated from previous preclinical models. Using CRISPR/Cas9, we developed a METex14/WT isogenic model in non-transformed human lung cells, and report that the METex14 single alteration was sufficient to drive MET-dependent in vitro anchorage-independent survival and motility and in vivo tumorigenesis, sensitising tumours to MET-TKIs. However, we also show that human HGF (hHGF) is required, as demonstrated in vivo using a humanized HGF knock-in strain of mice and further detected in tumor cells of METex14 NSCLC patient samples. Our results also suggest that METex14 oncogenicity is not a consequence of an escape from degradation in our cell model. Thus, we developed a valuable model for preclinical studies and present results that have potential clinical implication.

Current perspectives on the management of patients with advanced RET-driven thyroid cancer in Europe.

The incidence of thyroid cancer is increasing worldwide with the disease burden in Europe second only to that in Asia. In the last several decades, molecular pathways central to the pathogenesis of thyroid cancer have revealed a spectrum of targetable kinases/kinase receptors and oncogenic drivers characteristic of each histologic subtype, such as differentiated thyroid cancer, including papillary, follicular, and medullary thyroid cancer. Oncogenic alterations identified include B-Raf proto-oncogene (BRAF) fusions and mutations, neurotrophic tyrosine receptor kinase (NTRK) gene fusions, and rearranged during transfection (RET) receptor tyrosine kinase fusion and mutations. Multikinase inhibitors (MKIs) targeting RET in addition to multiple other kinases, such as sorafenib, lenvatinib and cabozantinib, have shown favourable activity in advanced radioiodine-refractory differentiated thyroid cancer or RET-altered medullary thyroid cancer; however, the clinical utility of MKI RET inhibition is limited by off-target toxicity resulting in high rates of dose reduction and drug discontinuation. Newer and selective RET inhibitors, selpercatinib and pralsetinib, have demonstrated potent efficacy and favourable toxicity profiles in clinical trials in the treatment of RET-driven advanced thyroid cancer and are now a therapeutic option in some clinical settings. Importantly, the optimal benefits of available specific targeted treatments for advanced RET-driven thyroid cancer require genetic testing. Prior to the initiation of systemic therapy, and in treatment-naïve patients, RET inhibitors may be offered as first-line therapy if a RET alteration is found, supported by a multidisciplinary team approach.

Genome-wide profiling of patient-derived glioblastoma stem-like cells reveals recurrent genetic and transcriptomic signatures associated with brain tumors

PurposePatient-derived cancer cell lines can be very useful to investigate genetic as well as epigenetic mechanisms of transformation and to test new drugs. In this multi-centric study, we performed genomic and transcriptomic characterization of a large set of patient-derived glioblastoma (GBM) stem-like cells (GSCs).Methods94 (80 I surgery/14 II surgery) and 53 (42 I surgery/11 II surgery) GSCs lines underwent whole exome and trascriptome analysis, respectively.ResultsExome sequencing revealed TP53 as the main mutated gene (41/94 samples, 44%), followed by PTEN (33/94, 35%), RB1 (16/94, 17%) and NF1 (15/94, 16%), among other genes associated to brain tumors. One GSC sample bearing a BRAF p.V600E mutation showed sensitivity in vitro to a BRAF inhibitor. Gene Ontology and Reactome analysis uncovered several biological processes mostly associated to gliogenesis and glial cell differentiation, S − adenosylmethionine metabolic process, mismatch repair and methylation. Comparison of I and II surgery samples disclosed a similar distribution of mutated genes, with an overrepresentation of mutations in mismatch repair, cell cycle, p53 and methylation pathways in I surgery samples, and of mutations in receptor tyrosine kinase and MAPK signaling pathways in II surgery samples. Unsupervised hierarchical clustering of RNA-seq data produced 3 clusters characterized by distinctive sets of up-regulated genes and signaling pathways.ConclusionThe availability of a large set of fully molecularly characterized GCSs represents a valuable public resource to support the advancement of precision oncology for the treatment of GBM.

Novel receptor tyrosine kinase mutations in rare paranasal sinus cancers and their potential functional implications

Amaç: Paranazal sinüs kanserleri oldukça nadir görülen heterojen bir hastalık grubudur. Maksiler sinüs skuamoz hücreli karsinomu, paranazal sinüs kanserlerinin anatomik ve histolojik olarak en yaygın alt tipidir. Bu kanserin genetik profiline dair bilginin sınırlı olması, hastaların hedefli tedavi seçeneklerinden yararlanamamasına neden olmaktadır. Çalışmamızda bu nadir kanserdeki reseptör tirozin kinaz mutasyonlarının tanımlanması ve mutasyonların olası fonksiyonel etkilerinin tahmin edilmesi amaçlanmıştır. Gereç ve Yöntem: Bu amaçla 30 olgunun tümörüne ait FFPE dokulardan DNA izolasyonu gerçekleştirildi, olguların mutasyon profili yeni nesil sekanslama yöntemi ve biyoinformatik değerlendirme ile belirlendi. Belirlenen patojenik/ olası patojenik varyantların fonksiyonel etkileri farklı in silico araçlar yardımıyla tahminlendi. Bulgular: Olgularının tamamında en az bir adet patojenik/olası patojenik KIT, PDFGRA ve RETmutasyonu belirlendi. KIT geninin katalitik bölgesindeki mutasyonların kinaz aktivitesini arttıracağı tahmin edildi. PDFGRA genindeki p.P567P ve p.D1074D mutasyonları, 30 olgunun tamamında ve SRA veritabanından elde edilen normal dokulara ait okumaların tümünde belirlendi. Sonuç: Reseptör tirozin kinaz mutasyonlarının paranazal sinüs kanserlerinde de önemli rol oynayabileceğinin belirlenmiş olması özellikle artmış kinaz aktivitesini hedefleyen tedavi yaklaşımlarını bu olguların erişimine sunma potansiyeli taşıması bakımından oldukça önemlidir.

Co-targeting of ACK1 and KIT triggers additive anti-proliferative and -migration effects in imatinib-resistant gastrointestinal stromal tumors

Most gastrointestinal stromal tumors (GIST) harbor mutated receptor tyrosine kinase (RTK) KIT/PDGFRA, which provides an attractive therapeutic target. However, a majority of GISTs ultimately develop resistance to KIT/PDGFRA inhibitor imatinib, multiple therapeutic targets will be identified as a reasonable strategy in imatinib-resistant GISTs. Biological mechanisms of non-RTK activated CDC42 associated kinase 1 (ACK1) are still unclear, which has been found to be activated in GISTs. In the current report, ACK1 overexpression is demonstrated in GIST cell lines and biopsies. RNA-seq analysis and immunoblotting show that ACK1 expression is dependent on imatinib treatment time in GIST-T1 cell line. The colocalization/complex of KIT and ACK1 in GIST cells are observed, and ACK1 activation is in a partially KIT and CDC42 dependent manner. Treatment with a specific ACK1 inhibitor AIM-100 or ACK1 siRNA, mildly suppresses cell viability, but markedly inhibits cell migration in imatinib sensitive and in imatinib resistant GIST cell lines, which is associated with inactivation of PI3K/AKT/mTOR and RAF/MAPK signaling pathways, and inhibition of epithelial-mesenchymal transition, evidencing upregulation of E-cadherin and downregulation of ZEB1, N-cadherin, vimentin, snail, and/or β-catenin after treatment with AIM-100 or ACK1/CDC42 shRNAs. Combination inhibition of ACK1 and KIT results in additive effects of anti-proliferation and pro-apoptosis as well as cell cycle arrest, and inhibition of invasiveness and migration in vitro and in vivo, compared to either intervention alone through dephosphorylation of KIT downstream intermediates (AKT, S6, and MAPK). Our data suggest that co-targeting of ACK1 and KIT might be a novel therapeutic strategy in imatinib-resistant GIST.

Genomic and transcriptomic insights into the precision treatment of pulmonary enteric adenocarcinoma

BackgroundPulmonary enteric adenocarcinoma (PEAC) is a rare subtype of lung adenocarcinoma. More investigations about precision therapy in PEAC were required to improve the prognosis. Methods: Twenty-four patients with PEAC were enrolled in this study. Tumor tissue samples were available from 17 patients for both DNA and RNA based next-generation sequencing, PD-L1 IHC staining and PCR-based microsatellite instability (MSI) analysis. Results: TP53 (70.6%) and KRAS (47.1%) were the most frequently mutated genes in PEAC. For KRAS mutations, the prevalence of G12D (37.5%) and G12V (37.5%) was higher than G12A (12.5%) and G12C (12.5%). Actionable mutations in receptor tyrosine kinase (including one EGFR and two ALK mutations), PI3K/mTOR, RAS/RAF/MEK, homologous recombination repair (HRR) and cell cycle signaling pathways were identified in 94.1% of patients with PEAC. While PD-L1 expression was observed in 17.6% (3/17) patients, no MSI-H patients were identified. Transcriptomic data showed that two patients with positive PD-L1 expression had relatively high immune infiltration. In addition, prolonged survival was obtained with the treatment of osimertinib, ensartinib, and immunotherapy combined with chemotherapy in two EGFR-mutated, one ALK-rearranged, and one PD-L1 expressed patients, respectively. Conclusion: PEAC is a disease of genetic heterogeneity. The administration of EGFR and ALK inhibitors was effective in patients with PEAC. PD-L1 expression and KRAS mutation type may be used as predictive biomarkers for immunotherapy in PEAC. This study provided both theoretical basis and clinical evidence for PEAC.

Proteome-Wide Detection and Annotation of Receptor Tyrosine Kinases (RTKs): RTK-PRED and the TyReK Database.

Receptor tyrosine kinases (RTKs) form a highly important group of protein receptors of the eukaryotic cell membrane. They control many vital cellular functions and are involved in the regulation of complex signaling networks. Mutations in RTKs have been associated with different types of cancers and other diseases. Although they are very important for proper cell function, they have been experimentally studied in a limited range of eukaryotic species. Currently, there is no available database for RTKs providing information about their function, expression, and interactions. Therefore, the identification of RTKs in multiple organisms, the documentation of their characteristics, and the collection of related information would be very useful. In this paper, we present a novel RTK detection pipeline (RTK-PRED) and the Receptor Tyrosine Kinases Database (TyReK-DB). RTK-PRED combines profile HMMs with transmembrane topology prediction to identify and classify potential RTKs. Proteins of all eukaryotic reference proteomes of the UniProt database were used as input in RTK-PRED leading to a filtered dataset of 20,478 RTKs. Based on the information collected for these RTKs from multiple databases, the relational TyReK database was created.