scholarly journals DNAJB1-PRKACA fusion kinase interacts with β-catenin and the liver regenerative response to drive fibrolamellar hepatocellular carcinoma

2017 ◽  
Author(s):  
Edward R. Kastenhuber ◽  
Gadi Lalazar ◽  
Shauna L. Houlihan ◽  
Darjus F. Tschaharganeh ◽  
Timour Baslan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Gene Fusion ◽  
Kinase Inhibitors ◽  
Liver Tumors ◽  
Tissue Injury ◽  
Kinase Domain ◽  
Wild Type ◽  
Fibrolamellar Hepatocellular Carcinoma ◽  
Genetic Event ◽  
Genetic And Environmental Factors

AbstractA segmental deletion resulting in DNAJB1-PRKACA gene fusion is now recognized as the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. Here, we implement CRISPR/Cas9 genome editing and transposon-mediated somatic gene transfer to demonstrate that expression of both the endogenous fusion protein or a chimeric cDNA leads to the formation of indolent liver tumors in mice that closely resemble human FL-HCC. Notably, overexpression of the wild type PRKACA was unable to fully recapitulate the oncogenic activity of DNAJB1-PRKACA, implying that FL-HCC does not simply result from enhanced PRKACA expression. Tumorigenesis was significantly enhanced by genetic activation of β-catenin, an observation supported by evidence of recurrent Wnt pathway mutations in human FL-HCC, as well as treatment with hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), which causes tissue injury, inflammation and fibrosis. Our study validates the DNAJB1-PRKACA fusion kinase as an oncogenic driver and candidate drug target for FL-HCC and establishes a practical model for preclinical studies to identify strategies to treat this disease.SignificanceEfforts to understand and treat FL-HCC have been confounded by a lack of models that accurately reflect the genetics and biology of the disease. Here, we demonstrate that the Dnajb1-Prkaca gene fusion drives tumorigenesis in mice, and that fusion to DNAJB1 drives FL-HCC initiation more effectively than wild type PRKACA overexpression. The requirement of the PRKACA kinase domain in tumor initiation establishes the potential utility of kinase inhibitors targeting the fusion. By identifying genetic and environmental factors that can enhance the consistency and aggressiveness of disease progression, we reveal biological characteristics of the disease and advance a robust platform for future pre-clinical studies.

scholarly journals DNAJB1–PRKACA fusion kinase interacts with β-catenin and the liver regenerative response to drive fibrolamellar hepatocellular carcinoma

2017 ◽  
Vol 114 (50) ◽  
pp. 13076-13084 ◽  
Author(s):  
Edward R. Kastenhuber ◽  
Gadi Lalazar ◽  
Shauna L. Houlihan ◽  
Darjus F. Tschaharganeh ◽  
Timour Baslan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Liver Tumors ◽  
Tissue Injury ◽  
Fibrolamellar Hepatocellular Carcinoma ◽  
Genetic Event ◽  
Candidate Drug ◽  
Somatic Gene ◽  
Oncogenic Driver ◽  
Practical Model ◽  
Somatic Gene Transfer

A segmental deletion resulting in DNAJB1–PRKACA gene fusion is now recognized as the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. Here we implement CRISPR-Cas9 genome editing and transposon-mediated somatic gene transfer to demonstrate that expression of either the endogenous fusion protein or a chimeric cDNA leads to the formation of indolent liver tumors in mice that closely resemble human FL-HCC. Notably, overexpression of the wild-type PRKACA was unable to fully recapitulate the oncogenic activity of DNAJB1–PRKACA, implying that FL-HCC does not simply result from enhanced PRKACA expression. Tumorigenesis was significantly enhanced by genetic activation of β-catenin, an observation supported by evidence of recurrent Wnt pathway mutations in human FL-HCC, as well as treatment with the hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which causes tissue injury, inflammation, and fibrosis. Our study validates the DNAJB1–PRKACA fusion kinase as an oncogenic driver and candidate drug target for FL-HCC, and establishes a practical model for preclinical studies to identify strategies to treat this disease.

scholarly journals The dynamic switch mechanism that leads to activation of LRRK2 is embedded in the DFGψ motif in the kinase domain

2019 ◽  
Vol 116 (30) ◽  
pp. 14979-14988 ◽  
Author(s):  
Sven H. Schmidt ◽  
Matthias J. Knape ◽  
Daniela Boassa ◽  
Natascha Mumdey ◽  
Alexandr P. Kornev ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Regulatory Mechanism ◽  
Kinase Domain ◽  
Leucine Rich Repeat ◽  
Wild Type ◽  
Conformational Switch ◽  
Multidomain Protein ◽  
Switch Mechanism ◽  
And Function

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein, and LRRK2 mutants are recognized risk factors for Parkinson’s disease (PD). Although the precise mechanisms that control LRRK2 regulation and function are unclear, the importance of the kinase domain is strongly implicated, since 2 of the 5 most common familial LRRK2 mutations (G2019S and I2020T) are localized to the conserved DFGψ motif in the kinase core, and kinase inhibitors are under development. Combining the concept of regulatory (R) and catalytic (C) spines with kinetic and cell-based assays, we discovered a major regulatory mechanism embedded within the kinase domain and show that the DFG motif serves as a conformational switch that drives LRRK2 activation. LRRK2 is quite unusual in that the highly conserved Phe in the DFGψ motif, which is 1 of the 4 R-spine residues, is replaced with tyrosine (DY2018GI). A Y2018F mutation creates a hyperactive phenotype similar to the familial mutation G2019S. The hydroxyl moiety of Y2018 thus serves as a “brake” that stabilizes an inactive conformation; simply removing it destroys a key hydrogen-bonding node. Y2018F, like the pathogenic mutant I2020T, spontaneously forms LRRK2-decorated microtubules in cells, while the wild type and G2019S require kinase inhibitors to form filaments. We also explored 3 different mechanisms that create kinase-dead pseudokinases, including D2017A, which further emphasizes the highly synergistic role of key hydrophobic and hydrophilic/charged residues in the assembly of active LRRK2. We thus hypothesize that LRRK2 harbors a classical protein kinase switch mechanism that drives the dynamic activation of full-length LRRK2.

scholarly journals Persistent hepatocyte apoptosis promotes tumorigenesis from diethylnitrosamine-transformed hepatocytes through increased oxidative stress, independent of compensatory liver regeneration

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yasutoshi Nozaki ◽  
Hayato Hikita ◽  
Satoshi Tanaka ◽  
Kenji Fukumoto ◽  
Makiko Urabe ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hepatocellular Carcinoma ◽  
Chronic Hepatitis ◽  
Liver Regeneration ◽  
Liver Tumors ◽  
Tumor Formation ◽  
Transformed Cells ◽  
Wild Type ◽  
Hepatocyte Apoptosis ◽  
Chronic Liver Injury

AbstractHepatocellular carcinoma highly occurs in chronic hepatitis livers, where hepatocyte apoptosis is frequently detected. Apoptosis is a mechanism that eliminates mutated cells. Hepatocyte apoptosis induces compensatory liver regeneration, which is believed to contribute to tumor formation. Hepatocyte-specific Mcl-1 knockout mice (Mcl-1Δhep mice) developed persistent hepatocyte apoptosis and compensatory liver regeneration with increased oxidative stress in adulthood but had not yet developed hepatocyte apoptosis at the age of 2 weeks. When diethylnitrosamine (DEN) was administered to 2-week-old Mcl-1Δhep mice, multiple liver tumors were formed at 4 months, while wild-type mice did not develop any tumors. These tumors contained the B-Raf V637E mutation, indicating that DEN-initiated tumorigenesis was promoted by persistent hepatocyte apoptosis. When N-acetyl-L-cysteine was given from 6 weeks of age, DEN-administered Mcl-1Δhep mice had reduced oxidative stress and suppressed tumorigenesis in the liver but showed no changes in hepatocyte apoptosis or proliferation. In conclusion, enhanced tumor formation from DEN-transformed hepatocytes by persistent hepatocyte apoptosis is mediated by increased oxidative stress, independent of compensatory liver regeneration. For patients with livers harboring transformed cells, the control of oxidative stress may suppress hepatocarcinogenesis based on chronic liver injury.

scholarly journals Intercellular crosstalk regulating ARRB2/RARRES1 is involved in transition from fibrosis to cancer

2021 ◽  
Author(s):  
Robert Schierwagen ◽  
Peter Dietrich ◽  
Judith Heinzen ◽  
Sabine Klein ◽  
Frank E. Uschner ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Retinoic Acid ◽  
Liver Tumors ◽  
Retinoic Acid Receptor ◽  
Cancer Development ◽  
Wild Type ◽  
Chronic Liver Injury ◽  
Deficient Mice

AbstractProgressive fibrogenesis in chronic liver injury is often associated with cancer development. Beta-arrestin-2 (ARRB2) is a regulator of the profibrotic Angiotensin II type 1 receptor (AGTR1). The role of ARRB2 in liver fibrosis and in the transition from fibrosis to cancer is not fully understood and was investigated in this study.This study demonstrates that upregulation of the retinoic acid receptor responder 1 (RARRES1) in HSC mediated by ARRB2 leads to fibrosis. This process is driven by exosomal ARRB2 transfer to HSC, major fibrosis contributors, from injured hepatocytes, which highly express ARRB2. By contrast, downregulation of RARRES1 in hepatocytes induces malignant transformation and hepatocellular carcinoma (HCC) development. Consequently, Arrb2-deficient mice show higher number and size of liver tumors than wild-type mice in a hepatocellular carcinoma model with fibrosis. The identified relationship between ARRB2 and RARRES1 was observed in at least two species, including human cells and tissues in fibrosis and HCC and has a predictive value for survival in cancer patients. This study describes the discovery of a novel molecular pathway mediating the transition from fibrosis to cancer offering potential diagnostics and therapeutics.

Random Mutagenesis Reveals Residues of JAK2 Critical in Evading Inhibition by Tyrosine Kinase Inhibitors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3717-3717
Author(s):  
Michael Marit ◽  
Manprit Chohan ◽  
Natasha Matthew ◽  
Kai Huang ◽  
Dwayne Barber
Keyword(s):  
Crystal Structure ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Random Mutagenesis ◽  
Jak2 V617f ◽  
Kinase Domain ◽  
Jak Inhibitor ◽  
Wild Type ◽  
Kinase Domain Mutations

Abstract Mutation and activation of JAK2 is a common event in Myeloproliferative Disease as JAK2 V617F and related deletion mutations are observed in Polycythemia Vera, Essential Thrombocythemia and Primary Myelofibrosis. In addition, the TEL-JAK2 chromosomal translocation is a rare event in Acute Myeloid Leukemia and Chronic Myelomonocytic Leukemia. We reasoned that activated alleles of JAK2 would develop resistance to JAK inhibitors in a clinical setting, similar to the development of Imatinib resistance in BCR-ABL-mediated Chronic Myeloid Leukemia. The objective of this study was to develop a random mutagenesis screen to isolate mutations of JAK2 resistant to tyrosine kinase inhibitors. We selected JAK Inhibitor-1 for this study, since the crystal structure of this inhibitor bound to the JAK2 JH1 kinase domain has been reported and would allow for mapping of confirmed mutations. TEL-JAK2(5–12) and JAK2 V617F were subcloned into retroviral expression vectors and random libraries of mutations were generated by transformation into XL-1 Red strain of E. coli, a strain defective in pathways of DNA repair. High titer retroviral supernatants were generated and used to transduce Ba/F3 (for TEL-JAK2) or Ba/F3-EPO-R (for JAK2 V617F). Control experiments were performed with “wild type” versions of each JAK2 allele. Inhibitor-resistant clones were identified and DNA sequencing was performed to identify JAK2 JH1 kinase domain mutations that confer resistance to inhibitor. We have restricted the analysis of JAK inhibitor-resistant mutants to those that map within the kinase domain of JAK2 for purposes of this study. We have confirmed that E864K, V881A, N909K, G935R, R975G confer resistance to JAK inhibitor-1 in growth assays. In addition, M929I (analogous to BCR-ABL T315I) mediates resistance to JAK inhibitor-1, relative to wild-type activated JAK2 alleles. All mutations result in increased phosphorylation of STAT5, Akt and Erk in the presence of inhibitor. We are currently testing the catalytic activity of each mutant to determine whether JAK2 kinase domain mutations have similar enzymatic activity or whether mutation also affects catalysis. We have mapped each mutation within the JAK2 JH1 crystal structure and models for how each mutant affects inhibitor binding will be presented. Importantly, many of these residues are highly conserved in other JAK tyrosine kinases and within BCR-ABL. We are extending these observations by testing clinically relevant inhibitors in our screen. At the conclusion of this study we will be able to identify common residues critical for resistance by JAK2 inhibitors and unique residues that are inhibitor-specific. Random mutagenesis screening offers an excellent strategy to identify JAK2 residues that may be relevant in the clinic and also serve in enhancing our knowledge regarding JAK kinase activation and regulation.

Analysis of resistance mechanisms to ALK kinase inhibitors in ALK+ NSCLC patients.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 7504-7504 ◽  
Author(s):  
Robert Charles Doebele ◽  
Dara L Aisner ◽  
Anh T Le ◽  
Eamon M Berge ◽  
Amanda B. Pilling ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Copy Number ◽  
Gene Fusion ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Copy Number Gain ◽  
Kinase Domain ◽  
Anaplastic Lymphoma ◽  
Alk Inhibitor ◽  
Nsclc Patients

7504 Background: Patients with anaplastic lymphoma kinase (ALK) gene fusions derive significant clinical benefit from crizotinib, an ALK inhibitor; moreover, next generation ALK kinase inhibitors are in development. Unfortunately, drug resistance develops after initial benefit (acquired) or occurs in patients who never derive a benefit (intrinsic). This study aimed to define molecular mechanisms of resistance to ALK kinase inhibitors in ALK+ non-small cell lung cancer (NSCLC) patients. Methods: 30 ALK+ crizotinib-treated NSCLC patients experienced radiologic disease progression, of whom 7 progressed only in the CNS. Of the 23 patients with extra-CNS progression, a biopsy was attempted on 19. One of the patients without tissue post-crizotinib then proceeded to a second generation ALK inhibitor and tissue was obtained post progression on this drug. We performed molecular analysis and initiated cell lines from tumor tissue for these 19 patients. Results: 15 patients had material evaluable for molecular analysis. Six patients (40%) developed secondary mutations in the kinase domain of ALK. Two novel mutations were identified in samples from ALK+ NSCLC patients, F1174C and D1203N. Two patients each demonstrated G1269A or L1196M mutations. Two patients each, one with a resistance mutation, exhibited new onset ALK copy number gain (CNG). Four patients demonstrated the presence of another oncogenic driver (1 with EGFR mutation; 3 with KRAS mutation) with or without a persistent ALK gene rearrangement. One patient lacked an ALK gene fusion on progression biopsy, but had no identifiable alternate oncogene alteration. 3 patients retained ALK positivity with no identifiable resistance mechanism. Data on additional patients and an in vitro model of copy number gain will be presented. Conclusions: ALK kinase inhibitor resistance in ALK+ NSCLC occurs through a diverse array of kinase domain mutations, ALK gene fusion CNG, and emergence of second (same cell) or separate (different cell) oncogenic drivers. In order to overcome resistance it will be important to differentiate patients that preserve ALK dominance (secondary mutations and CNG) versus those that have diminished ALK dominance (separate or second oncogenic drivers).

Characterization of a novel mutation in fibrolamellar hepatocellular carcinoma.

2015 ◽  
Vol 33 (3_suppl) ◽  
pp. 284-284
Author(s):  
Kevin M Riggle ◽  
Heidi Kenerson ◽  
Machiko Kazami ◽  
Renay Bauer ◽  
Raymond Sze Yeung ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Normal Liver ◽  
Kinase Assay ◽  
Mutant Protein ◽  
Intrinsic Activity ◽  
Chimeric Transcript ◽  
Wild Type ◽  
Fibrolamellar Hepatocellular Carcinoma ◽  
Mutant Transcript ◽  
Elevated Expression

284 Background: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a subtype of HCC occurring in children and young adults in the absence of underlying liver disease. Currently, there is no effective therapy for unresectable or metastatic FL-HCC. Recent genomic analysis identified a consistent mutation in FL-HCC involving a deletion on chromosome 19 resulting in a chimeric transcript containing the 5’-region of a heat shock protein (DNAJB1) fused to the catalytic subunit of protein kinase A (PRKACA). We sought to characterize the resultant protein and its effects on PKA activity in human FL-HCC. Methods: We prepared tissue lysates from four snap-frozen FL-HCC samples with paired, non-tumor liver as well as adult HCCs. PKA activity was determined via a radioactive kinase assay in the presence and absence of cAMP, a PKA activator. RNA was extracted using TRIZOL, and used for qRT-PCR. Triple immuno-fluorescent labeling was performed using antibodies to PRKACA, PKA RIIα, and a nuclear marker, DRAQ5. Results: We found that expression of the chimeric transcript was increased 40-fold in FL-HCC compared to normal liver, and was significantly higher than that of wild-type DNAJB1 and PRKACA in FL-HCC. The corresponding mutant protein was highly expressed in the tumors and was unique to FL-HCC. Basal PKA activities from freshly lysed tumors and paired livers were not significantly different, but cAMP-stimulated PKA activity was significantly higher in FL-HCC tumors when compared to normal liver. Using multi-color immuno-fluorescence we detected mutant DNAJB1-PRKACA within the nucleus, while wild-type PRKACA localizes to the cytoplasm. Conclusions: The expression of DNAJB1-PRKACA is unique to FL-HCC and not found in classic forms of HCC. Markedly elevated expression of the mutant transcript is indicative of dysregulated DNAJB1 promoter activity. PKA activity in FL-HCCs remains cAMP-dependent, but is greatly enhanced in the presence of cAMP; this could reflect an altered intrinsic activity of the mutant protein and/or elevated expression. Further, the mutant protein showed aberrant localization to the nucleus. These findings suggest that DNAJB1-PRKACA in FL-HCC leads to over-activation of PKA, which may contribute to tumor development.

scholarly journals Identification of Flavonoids as Putative ROS-1 Kinase Inhibitors Using Pharmacophore Modeling for NSCLC Therapeutics

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2114
Author(s):  
Shraddha Parate ◽  
Vikas Kumar ◽  
Jong Chan Hong ◽  
Keun Woo Lee
Keyword(s):  
Kinase Inhibitors ◽  
Binding Free Energy ◽  
Pharmacophore Model ◽  
Kinase Domain ◽  
Pharmacophore Modeling ◽  
Free Energy Calculations ◽  
Wild Type ◽  
Small Cell Lung ◽  
Poisson Boltzmann ◽  
Dynamics Simulations

Non-small cell lung cancer (NSCLC) is a lethal non-immunogenic malignancy and proto-oncogene ROS-1 tyrosine kinase is one of its clinically relevant oncogenic markers. The ROS-1 inhibitor, crizotinib, demonstrated resistance due to the Gly2032Arg mutation. To curtail this resistance, researchers developed lorlatinib against the mutated kinase. In the present study, a receptor-ligand pharmacophore model exploiting the key features of lorlatinib binding with ROS-1 was exploited to identify inhibitors against the wild-type (WT) and the mutant (MT) kinase domain. The developed model was utilized to virtually screen the TimTec flavonoids database and the retrieved drug-like hits were subjected for docking with the WT and MT ROS-1 kinase. A total of 10 flavonoids displayed higher docking scores than lorlatinib. Subsequent molecular dynamics simulations of the acquired flavonoids with WT and MT ROS-1 revealed no steric clashes with the Arg2032 (MT ROS-1). The binding free energy calculations computed via molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) demonstrated one flavonoid (Hit) with better energy than lorlatinib in binding with WT and MT ROS-1. The Hit compound was observed to bind in the ROS-1 selectivity pocket comprised of residues from the β-3 sheet and DFG-motif. The identified Hit from this investigation could act as a potent WT and MT ROS-1 inhibitor.

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