Publications by authors named "Nicholas P D Liau"

12 Publications

  • Page 1 of 1

ARAF mutations confer resistance to the RAF inhibitor belvarafenib in melanoma.

Nature 2021 06 5;594(7863):418-423. Epub 2021 May 5.

Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea.

Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAF-mutant melanoma, they are ineffective in non-BRAF mutant cells. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAF- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAF- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors.
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http://dx.doi.org/10.1038/s41586-021-03515-1DOI Listing
June 2021

Dimerization Induced by C-Terminal 14-3-3 Binding Is Sufficient for BRAF Kinase Activation.

Biochemistry 2020 10 2;59(41):3982-3992. Epub 2020 Oct 2.

The Ras-RAF-MEK-ERK signaling axis, commonly mutated in human cancers, is highly regulated to prevent aberrant signaling in healthy cells. One of the pathway modulators, 14-3-3, a constitutive dimer, induces RAF dimerization and activation by binding to a phosphorylated motif C-terminal to the RAF kinase domain. Recent work has suggested that a C-terminal "DTS" region in BRAF is necessary for this 14-3-3-mediated activation. We show that the catalytic activity and ATP binding affinity of the BRAF:14-3-3 complex is insensitive to the presence or absence of the DTS, while the ATP sites of both BRAF molecules are identical and available for binding. We also present a crystal structure of the apo BRAF:14-3-3 complex showing that the DTS is not required to attain the catalytically active conformation of BRAF. Rather, BRAF dimerization induced by 14-3-3 is the key step in activation, allowing the active BRAF:14-3-3 tetramer to achieve catalytic activity comparable to the constitutively active oncogenic BRAF V600E mutant.
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http://dx.doi.org/10.1021/acs.biochem.0c00517DOI Listing
October 2020

Negative regulation of RAF kinase activity by ATP is overcome by 14-3-3-induced dimerization.

Nat Struct Mol Biol 2020 02 27;27(2):134-141. Epub 2020 Jan 27.

Department of Structural Biology, Genentech Inc., South San Francisco, USA.

The RAS-RAF-MEK-ERK signaling axis is frequently activated in human cancers. Physiological concentrations of ATP prevent formation of RAF kinase-domain (RAF) dimers that are critical for activity. Here we present a 2.9-Å-resolution crystal structure of human BRAF in complex with MEK and the ATP analog AMP-PCP, revealing interactions between BRAF and ATP that induce an inactive, monomeric conformation of BRAF. We also determine how 14-3-3 relieves the negative regulatory effect of ATP through a 2.5-Å-resolution crystal structure of the BRAF-14-3-3 complex, in which dimeric 14-3-3 enforces a dimeric BRAF assembly to increase BRAF activity. Our data suggest that most oncogenic BRAF mutations alter interactions with ATP and counteract the negative effects of ATP binding by lowering the threshold for RAF dimerization and pathway activation. Our study establishes a framework for rationalizing oncogenic BRAF mutations and provides new avenues for improved RAF-inhibitor discovery.
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http://dx.doi.org/10.1038/s41594-019-0365-0DOI Listing
February 2020

Enzymatic Characterization of Wild-Type and Mutant Janus Kinase 1.

Cancers (Basel) 2019 Nov 1;11(11). Epub 2019 Nov 1.

Walter and Eliza Hall Institute, 1G Royal Parade, Parkville 3052, VIC, Australia.

Janus kinases (JAKs) are found constitutively associated with cytokine receptors and are present in an inactive state prior to cytokine exposure. Activating mutations of JAKs are causative for a number of leukemias, lymphomas, and myeloproliferative diseases. In particular, the JAK2 mutant is found in most human cases of polycythemia vera, a disease characterized by over-production of erythrocytes. The V617F mutation is found in the pseudokinase domain of JAK2 and it leads to cytokine-independent activation of the kinase, as does the orthologous mutation in other JAK-family members. The mechanism whereby this mutation hyperactivates these kinases is not well understood, primarily due to the fact that the full-length JAK proteins are difficult to produce for structural and kinetic studies. Here we have overcome this limitation to perform a series of enzymatic analyses on full-length JAK1 and its constitutively active mutant form (JAK1). Consistent with previous studies, we show that the presence of the pseudokinase domain leads to a dramatic decrease in enzymatic activity with no further decrease from the presence of the FERM or SH2 domains. However, we find that the mutant kinase, in vitro, is indistinguishable from the wild-type enzyme in every measurable parameter tested: K (ATP), K (substrate), k, receptor binding, thermal stability, activation rate, dephosphorylation rate, and inhibitor affinity. These results show that the V658F mutation does not enhance the intrinsic enzymatic activity of JAK. Rather this data is more consistent with a model in which there are cellular processes and interactions that prevent JAK from being activated in the absence of cytokine and it is these constraints that are affected by disease-causing mutations.
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http://dx.doi.org/10.3390/cancers11111701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896158PMC
November 2019

Accumulation of JAK activation loop phosphorylation is linked to type I JAK inhibitor withdrawal syndrome in myelofibrosis.

Sci Adv 2018 11 28;4(11):eaat3834. Epub 2018 Nov 28.

Centre for Cancer Biology, SA Pathology and University of South Australia, Adelaide, South Australia, Australia.

Treatment of patients with myelofibrosis with the type I JAK (Janus kinase) inhibitor ruxolitinib paradoxically induces JAK2 activation loop phosphorylation and is associated with a life-threatening cytokine-rebound syndrome if rapidly withdrawn. We developed a time-dependent assay to mimic ruxolitinib withdrawal in primary JAK2 and CALR mutant myelofibrosis patient samples and observed notable activation of spontaneous STAT signaling in JAK2 samples after drug washout. Accumulation of ruxolitinib-induced JAK2 phosphorylation was dose dependent and correlated with rebound signaling and the presence of a JAK2 mutation. Ruxolitinib prevented dephosphorylation of a cryptic site involving Tyr in JAK2 blocking ubiquitination and degradation. In contrast, a type II JAK inhibitor, CHZ868, did not induce JAK2 phosphorylation, was not associated with withdrawal signaling, and was superior in the eradication of flow-purified JAK2 mutant CD34 progenitors after drug washout. Type I inhibitor-induced loop phosphorylation may act as a pathogenic signaling node released upon drug withdrawal, especially in JAK2 patients.
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http://dx.doi.org/10.1126/sciadv.aat3834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261652PMC
November 2018

The molecular basis of JAK/STAT inhibition by SOCS1.

Nat Commun 2018 04 19;9(1):1558. Epub 2018 Apr 19.

Walter and Eliza Hall Institute, 1G Royal Parade, Parkville, VIC, 3052, Australia.

The SOCS family of proteins are negative-feedback inhibitors of signalling induced by cytokines that act via the JAK/STAT pathway. SOCS proteins can act as ubiquitin ligases by recruiting Cullin5 to ubiquitinate signalling components; however, SOCS1, the most potent member of the family, can also inhibit JAK directly. Here we determine the structural basis of both these modes of inhibition. Due to alterations within the SOCS box domain, SOCS1 has a compromised ability to recruit Cullin5; however, it is a direct, potent and selective inhibitor of JAK catalytic activity. The kinase inhibitory region of SOCS1 targets the substrate binding groove of JAK with high specificity and thereby blocks any subsequent phosphorylation. SOCS1 is a potent inhibitor of the interferon gamma (IFNγ) pathway, however, it does not bind the IFNγ receptor, making its mode-of-action distinct from SOCS3. These findings reveal the mechanism used by SOCS1 to inhibit signalling by inflammatory cytokines.
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http://dx.doi.org/10.1038/s41467-018-04013-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908791PMC
April 2018

Expression and Purification of JAK1 and SOCS1 for Structural and Biochemical Studies.

Methods Mol Biol 2018 ;1725:267-280

The Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, VIC, Australia.

Interferon gamma (IFNγ) is a potent inflammatory and immune cytokine. IFNγ signals via the interferon gamma receptor (IFNGR), which is constitutively bound to Janus Kinase (JAK) 1 and JAK2 via its intracellular domain. These two JAK proteins then initiate the inflammatory signaling cascade. The most potent inhibitor of IFNγ signaling is Suppressor of Cytokine Signaling 1 (SOCS1). SOCS1 negatively regulates IFNγ signaling pathway (and other pathways) by directly inhibiting JAKs. Here, we describe a protocol for the recombinant production and purification of the JAK1 kinase domain and its inhibitor SOCS1, for structural and biochemical studies.
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http://dx.doi.org/10.1007/978-1-4939-7568-6_21DOI Listing
January 2019

Purification of SOCS (Suppressor of Cytokine Signaling) SH2 Domains for Structural and Functional Studies.

Methods Mol Biol 2017 ;1555:173-182

The Walter and Eliza Hall Institute of Medical Research, 1G Royal Pde, Parkville, Melbourne, 3052, VIC, Australia.

Src Homology 2 (SH2) domains are protein domains which have a high binding affinity for specific amino acid sequences containing a phosphorylated tyrosine residue. The Suppressors of Cytokine Signaling (SOCS) proteins use an SH2 domain to bind to components of certain cytokine signaling pathways to downregulate the signaling cascade. The recombinantly produced SH2 domains of various SOCS proteins have been used to undertake structural and functional studies elucidating the method of how such targeting occurs. Here, we describe the protocol for the recombinant production and purification of SOCS SH2 domains, with an emphasis on SOCS3.
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http://dx.doi.org/10.1007/978-1-4939-6762-9_10DOI Listing
February 2018

JAK1 Takes a FERM Hold of Type II Cytokine Receptors.

Structure 2016 06;24(6):840-2

Department of Medical Biology, The Walter and Eliza Hall Institute of Medical Research, University of Melbourne, Parkville, VIC 3052, Australia.

Janus kinases (JAKs) initiate the intracellular signaling cascade triggered by exposure of cells to cytokines and interferons. In order to achieve this, JAKs are bound to the intracellular domain of specific cytokine receptors immediately adjacent to the cell membrane. In this issue of Structure, Ferrao et al. (2016) provide structural details of such an interaction and in doing so, identify for the first time the motif used by type II cytokine receptors to recruit JAK1.
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http://dx.doi.org/10.1016/j.str.2016.05.007DOI Listing
June 2016

CIS is a potent checkpoint in NK cell-mediated tumor immunity.

Nat Immunol 2016 07 23;17(7):816-24. Epub 2016 May 23.

Immunology in Cancer and Infection Laboratory QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia.

The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. We identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity toward tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. Cish(-/-) mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Our data uncover a potent intracellular checkpoint in NK cell-mediated tumor immunity and suggest possibilities for new cancer immunotherapies directed at blocking CIS function.
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http://dx.doi.org/10.1038/ni.3470DOI Listing
July 2016

Mechanistic insights into activation and SOCS3-mediated inhibition of myeloproliferative neoplasm-associated JAK2 mutants from biochemical and structural analyses.

Biochem J 2014 Mar;458(2):395-405

‡School of Medicine, University of Tampere and Tampere University Hospital, Tampere 33014, Finland.

JAK2 (Janus kinase 2) initiates the intracellular signalling cascade downstream of cell surface receptor activation by cognate haemopoietic cytokines, including erythropoietin and thrombopoietin. The pseudokinase domain (JH2) of JAK2 negatively regulates the catalytic activity of the adjacent tyrosine kinase domain (JH1) and mutations within the pseudokinase domain underlie human myeloproliferative neoplasms, including polycythaemia vera and essential thrombocytosis. To date, the mechanism of JH2-mediated inhibition of JH1 kinase activation as well as the susceptibility of pathological mutant JAK2 to inhibition by the physiological negative regulator SOCS3 (suppressor of cytokine signalling 3) have remained unclear. In the present study, using recombinant purified JAK2JH1-JH2 proteins, we demonstrate that, when activated, wild-type and myeloproliferative neoplasm-associated mutants of JAK2 exhibit comparable enzymatic activity and inhibition by SOCS3 in in vitro kinase assays. SAXS (small-angle X-ray scattering) showed that JAK2JH1-JH2 exists in an elongated configuration in solution with no evidence for interaction between JH1 and JH2 domains in cis. Collectively, these data are consistent with a model in which JAK2's pseudokinase domain does not influence the activity of JAK2 once it has been activated. Our data indicate that, in the absence of the N-terminal FERM domain and thus cytokine receptor association, the wild-type and pathological mutants of JAK2 are enzymatically equivalent and equally susceptible to inhibition by SOCS3.
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http://dx.doi.org/10.1042/BJ20131516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4085142PMC
March 2014

SOCS3 binds specific receptor-JAK complexes to control cytokine signaling by direct kinase inhibition.

Nat Struct Mol Biol 2013 Apr 3;20(4):469-76. Epub 2013 Mar 3.

Department of Structural Biology, Walter and Eliza Hall Institute, Parkville, Australia.

The inhibitory protein SOCS3 plays a key part in the immune and hematopoietic systems by regulating signaling induced by specific cytokines. SOCS3 functions by inhibiting the catalytic activity of Janus kinases (JAKs) that initiate signaling within the cell. We determined the crystal structure of a ternary complex between mouse SOCS3, JAK2 (kinase domain) and a fragment of the interleukin-6 receptor β-chain. The structure shows that SOCS3 binds JAK2 and receptor simultaneously, using two opposing surfaces. While the phosphotyrosine-binding groove on the SOCS3 SH2 domain is occupied by receptor, JAK2 binds in a phosphoindependent manner to a noncanonical surface. The kinase-inhibitory region of SOCS3 occludes the substrate-binding groove on JAK2, and biochemical studies show that it blocks substrate association. These studies reveal that SOCS3 targets specific JAK-cytokine receptor pairs and explains the mechanism and specificity of SOCS action.
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http://dx.doi.org/10.1038/nsmb.2519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3618588PMC
April 2013
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