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Kinase domain mutations confer resistance to novel inhibitors targeting JAK2V617F in myeloproliferative neoplasms.

Deshpande A, Reddy MM, Schade GO, Ray A, Chowdary TK, Griffin JD, Sattler M - Leukemia (2011)

Bottom Line: Cells containing mutations had a 9- to 33-fold higher EC(50) for ruxolitinib compared with native JAK2V617F.Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387.Surprisingly, introduction of the 'gatekeeper' mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (fourfold increase in EC(50)).

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

ABSTRACT
The transforming JAK2V617F kinase is frequently associated with myeloproliferative neoplasms and thought to be instrumental for the overproduction of myeloid lineage cells. Several small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would be predicted to have potential clinical relevance and associated with drug resistance to the JAK2 inhibitor ruxolitinib (INCB018424). Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would confer resistance to ruxolitinib, using a BaF3 cell-based assay. We identified five different non-synonymous point mutations that conferred drug resistance. Cells containing mutations had a 9- to 33-fold higher EC(50) for ruxolitinib compared with native JAK2V617F. Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387. Surprisingly, introduction of the 'gatekeeper' mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (fourfold increase in EC(50)). These results suggest that JAK2 inhibitors currently in clinical trials may be prone to resistance as a result of point mutations and caution should be exercised when administering these drugs.

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Related in: MedlinePlus

Structural analysis of JAK2V617F kinase domain mutationsA, cartoon and transparent surface representation of ruxolitinib-docked JAK2 kinase domain (A and B) (left) and JAK2 with location of point mutations that lead to drug resistance (right). N-terminal lobe (salmon), C-terminal lobe (grey), glycine loop (purple), activation loop (blue) and hinge region (red) form the boundaries for the binding site of ruxolitinib (stick representation in yellow (carbon) and blue (nitrogen)). The I960V sidechain (purple) is buried within the protein interior. B, enlarged ruxolitinib binding pocket with secondary structure elements (cartoon) and the interactions of the sidechains (labeled sticks) with the inhibitor. Hydrogen-bonds between the inhibitor and the protein are indicated as dotted yellow lines (one hydrogen-bond between backbone of Y931 and L932; and two hydrogen-bonds with R980 and N981 and pyrrolopyrimidine ring of the inhibitor; additional hydrogen bonds are with water molecules (cyan spheres)). Mutated amino acids are labeled red (right panels). C, surface electrostatic potential representation of the native (left) and G935R (right) containing JAK2 JH1 domain with ruxolitinib. Charged surfaces are displayed in shades of blue (positive), red (negative) and white (non-polar).
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Figure 1: Structural analysis of JAK2V617F kinase domain mutationsA, cartoon and transparent surface representation of ruxolitinib-docked JAK2 kinase domain (A and B) (left) and JAK2 with location of point mutations that lead to drug resistance (right). N-terminal lobe (salmon), C-terminal lobe (grey), glycine loop (purple), activation loop (blue) and hinge region (red) form the boundaries for the binding site of ruxolitinib (stick representation in yellow (carbon) and blue (nitrogen)). The I960V sidechain (purple) is buried within the protein interior. B, enlarged ruxolitinib binding pocket with secondary structure elements (cartoon) and the interactions of the sidechains (labeled sticks) with the inhibitor. Hydrogen-bonds between the inhibitor and the protein are indicated as dotted yellow lines (one hydrogen-bond between backbone of Y931 and L932; and two hydrogen-bonds with R980 and N981 and pyrrolopyrimidine ring of the inhibitor; additional hydrogen bonds are with water molecules (cyan spheres)). Mutated amino acids are labeled red (right panels). C, surface electrostatic potential representation of the native (left) and G935R (right) containing JAK2 JH1 domain with ruxolitinib. Charged surfaces are displayed in shades of blue (positive), red (negative) and white (non-polar).

Mentions: The crystal structure for JAK2-bound ruxolitinib is not available and we therefore performed docking simulations of this drug onto the monomer JAK2 structure, extracted from the crystal structure of the JAK2-CMP6 complex. Published structures of JAK2 bound to CMP6 3 and CP690,550 4 provide important clues on the mode of binding and interactions between the related JAK2 inhibitors and the protein. Both CMP6 and CP690,550 bind in the ATP-binding pocket of JAK2. With this in mind, we set the parameters to preferentially simulate ruxolitinib docking positions in the CMP6 and CP690,550 binding pocket on JAK2. The best scoring docking pose, with least estimated free energy of binding (-9.05 kCal/mol), best estimated inhibition constant (KI of 231.83nM) and highest interaction interface area (567.6 Å2), was used for the inhibitor-JAK2 interface analysis. Ruxolitinib snugly fits into the ATP-binding pocket of JAK2 similar to CMP6 and CP690,550, with the cyclopentyl and pyrazol rings tightly fitting in the deep hydrophobic groove (Figure 1A). JAK2-ruxolitinib interaction interface buries most of the surface area of the inhibitor. The inhibitor is held in the pocket by polar contacts between cyclopentyl ring and mainchain atoms in the hinge region (between Y931 and L932), and also pyrrolopyrimidine moiety with N981 sidechain. Ruxolitinib may also form hydrogen bonds with water molecules in the pocket. Ruxolitinib makes extensive hydrophobic interactions with several residues that line the binding pocket, similar to what was observed for CMP6 and CP690,550. A880, L855, V863 and M929 hold the inhibitor tight from the top and L932 from the hinge region holds it from the side. Further, V911 and L983 provide hydrophobic interactions from the bottom (Figure 1B, left panel). The pyrazol ring of ruxolitinib is in a distance to have π–π interaction with the Y931 ring. Most mutations that were identified in our screen are either interacting residues with ruxolitinib or in proximity of the binding pocket (Figure 1B, right panels) and hence are likely to alter the inhibitor binding. Y931 seems to be a critical residue for inhibitor-protein interaction as its side chain and mainchain atoms have interactions with the inhibitor. The Y931C mutation might disrupt the π–π interaction between tyrosine ring and the inhibitor ring structure, thus weakening the inhibitor binding and resulting in easy expulsion from the pocket. The G935R mutation pushes a large charged sidechain towards the mouth of the hydrophobic cavity (Figure 1B, right), which results in a strong positive charge at the corner of the binding pocket, compared to the native protein (Figure 1C). The exact mechanism by which the R938L (Figure 1B, right) and I960V mutations may effect the inhibitor binding cannot easily be explained based on our computational analysis of the structure, but these two residues lie near the binding pocket (R938L at the end of the hinge region and I960V in close proximity of the binding pocket). The E985K mutation could bring the sidechain very close to the inhibitor-binding site and result in charge repulsion of the inhibitor.


Kinase domain mutations confer resistance to novel inhibitors targeting JAK2V617F in myeloproliferative neoplasms.

Deshpande A, Reddy MM, Schade GO, Ray A, Chowdary TK, Griffin JD, Sattler M - Leukemia (2011)

Structural analysis of JAK2V617F kinase domain mutationsA, cartoon and transparent surface representation of ruxolitinib-docked JAK2 kinase domain (A and B) (left) and JAK2 with location of point mutations that lead to drug resistance (right). N-terminal lobe (salmon), C-terminal lobe (grey), glycine loop (purple), activation loop (blue) and hinge region (red) form the boundaries for the binding site of ruxolitinib (stick representation in yellow (carbon) and blue (nitrogen)). The I960V sidechain (purple) is buried within the protein interior. B, enlarged ruxolitinib binding pocket with secondary structure elements (cartoon) and the interactions of the sidechains (labeled sticks) with the inhibitor. Hydrogen-bonds between the inhibitor and the protein are indicated as dotted yellow lines (one hydrogen-bond between backbone of Y931 and L932; and two hydrogen-bonds with R980 and N981 and pyrrolopyrimidine ring of the inhibitor; additional hydrogen bonds are with water molecules (cyan spheres)). Mutated amino acids are labeled red (right panels). C, surface electrostatic potential representation of the native (left) and G935R (right) containing JAK2 JH1 domain with ruxolitinib. Charged surfaces are displayed in shades of blue (positive), red (negative) and white (non-polar).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
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Figure 1: Structural analysis of JAK2V617F kinase domain mutationsA, cartoon and transparent surface representation of ruxolitinib-docked JAK2 kinase domain (A and B) (left) and JAK2 with location of point mutations that lead to drug resistance (right). N-terminal lobe (salmon), C-terminal lobe (grey), glycine loop (purple), activation loop (blue) and hinge region (red) form the boundaries for the binding site of ruxolitinib (stick representation in yellow (carbon) and blue (nitrogen)). The I960V sidechain (purple) is buried within the protein interior. B, enlarged ruxolitinib binding pocket with secondary structure elements (cartoon) and the interactions of the sidechains (labeled sticks) with the inhibitor. Hydrogen-bonds between the inhibitor and the protein are indicated as dotted yellow lines (one hydrogen-bond between backbone of Y931 and L932; and two hydrogen-bonds with R980 and N981 and pyrrolopyrimidine ring of the inhibitor; additional hydrogen bonds are with water molecules (cyan spheres)). Mutated amino acids are labeled red (right panels). C, surface electrostatic potential representation of the native (left) and G935R (right) containing JAK2 JH1 domain with ruxolitinib. Charged surfaces are displayed in shades of blue (positive), red (negative) and white (non-polar).
Mentions: The crystal structure for JAK2-bound ruxolitinib is not available and we therefore performed docking simulations of this drug onto the monomer JAK2 structure, extracted from the crystal structure of the JAK2-CMP6 complex. Published structures of JAK2 bound to CMP6 3 and CP690,550 4 provide important clues on the mode of binding and interactions between the related JAK2 inhibitors and the protein. Both CMP6 and CP690,550 bind in the ATP-binding pocket of JAK2. With this in mind, we set the parameters to preferentially simulate ruxolitinib docking positions in the CMP6 and CP690,550 binding pocket on JAK2. The best scoring docking pose, with least estimated free energy of binding (-9.05 kCal/mol), best estimated inhibition constant (KI of 231.83nM) and highest interaction interface area (567.6 Å2), was used for the inhibitor-JAK2 interface analysis. Ruxolitinib snugly fits into the ATP-binding pocket of JAK2 similar to CMP6 and CP690,550, with the cyclopentyl and pyrazol rings tightly fitting in the deep hydrophobic groove (Figure 1A). JAK2-ruxolitinib interaction interface buries most of the surface area of the inhibitor. The inhibitor is held in the pocket by polar contacts between cyclopentyl ring and mainchain atoms in the hinge region (between Y931 and L932), and also pyrrolopyrimidine moiety with N981 sidechain. Ruxolitinib may also form hydrogen bonds with water molecules in the pocket. Ruxolitinib makes extensive hydrophobic interactions with several residues that line the binding pocket, similar to what was observed for CMP6 and CP690,550. A880, L855, V863 and M929 hold the inhibitor tight from the top and L932 from the hinge region holds it from the side. Further, V911 and L983 provide hydrophobic interactions from the bottom (Figure 1B, left panel). The pyrazol ring of ruxolitinib is in a distance to have π–π interaction with the Y931 ring. Most mutations that were identified in our screen are either interacting residues with ruxolitinib or in proximity of the binding pocket (Figure 1B, right panels) and hence are likely to alter the inhibitor binding. Y931 seems to be a critical residue for inhibitor-protein interaction as its side chain and mainchain atoms have interactions with the inhibitor. The Y931C mutation might disrupt the π–π interaction between tyrosine ring and the inhibitor ring structure, thus weakening the inhibitor binding and resulting in easy expulsion from the pocket. The G935R mutation pushes a large charged sidechain towards the mouth of the hydrophobic cavity (Figure 1B, right), which results in a strong positive charge at the corner of the binding pocket, compared to the native protein (Figure 1C). The exact mechanism by which the R938L (Figure 1B, right) and I960V mutations may effect the inhibitor binding cannot easily be explained based on our computational analysis of the structure, but these two residues lie near the binding pocket (R938L at the end of the hinge region and I960V in close proximity of the binding pocket). The E985K mutation could bring the sidechain very close to the inhibitor-binding site and result in charge repulsion of the inhibitor.

Bottom Line: Cells containing mutations had a 9- to 33-fold higher EC(50) for ruxolitinib compared with native JAK2V617F.Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387.Surprisingly, introduction of the 'gatekeeper' mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (fourfold increase in EC(50)).

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

ABSTRACT
The transforming JAK2V617F kinase is frequently associated with myeloproliferative neoplasms and thought to be instrumental for the overproduction of myeloid lineage cells. Several small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would be predicted to have potential clinical relevance and associated with drug resistance to the JAK2 inhibitor ruxolitinib (INCB018424). Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would confer resistance to ruxolitinib, using a BaF3 cell-based assay. We identified five different non-synonymous point mutations that conferred drug resistance. Cells containing mutations had a 9- to 33-fold higher EC(50) for ruxolitinib compared with native JAK2V617F. Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387. Surprisingly, introduction of the 'gatekeeper' mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (fourfold increase in EC(50)). These results suggest that JAK2 inhibitors currently in clinical trials may be prone to resistance as a result of point mutations and caution should be exercised when administering these drugs.

Show MeSH
Related in: MedlinePlus