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Targeting substrate-site in Jak2 kinase prevents emergence of genetic resistance.

Kesarwani M, Huber E, Kincaid Z, Evelyn CR, Biesiada J, Rance M, Thapa MB, Shah NP, Meller J, Zheng Y, Azam M - Sci Rep (2015)

Bottom Line: In vitro binding assays using purified proteins showed strong affinity for the substrate-binding site (Kd = 20 nM) while affinity for the ATP site was poor (Kd = ~8 μM).Our studies demonstrate that mutations affecting the substrate-binding pocket encode a catalytically incompetent kinase, thereby preventing emergence of resistant variants.Most importantly, our data suggest that in order to develop resistance-free kinase inhibitors, the next-generation drug design should target the substrate-binding site.

View Article: PubMed Central - PubMed

Affiliation: Cincinnati Children's Hospital Medical Center, Cancer Blood Disease Institute, Divisions of Experimental Hematology and Cancer Pathology, Cincinnati, Ohio, 45229 USA.

ABSTRACT
Emergence of genetic resistance against kinase inhibitors poses a great challenge for durable therapeutic response. Here, we report a novel mechanism of JAK2 kinase inhibition by fedratinib (TG101348) that prevents emergence of genetic resistance. Using in vitro drug screening, we identified 211 amino-acid substitutions conferring resistance to ruxolitinib (INCB018424) and cross-resistance to the JAK2 inhibitors AZD1480, CYT-387 and lestaurtinib. In contrast, these resistant variants were fully sensitive to fedratinib. Structural modeling, coupled with mutagenesis and biochemical studies, revealed dual binding sites for fedratinib. In vitro binding assays using purified proteins showed strong affinity for the substrate-binding site (Kd = 20 nM) while affinity for the ATP site was poor (Kd = ~8 μM). Our studies demonstrate that mutations affecting the substrate-binding pocket encode a catalytically incompetent kinase, thereby preventing emergence of resistant variants. Most importantly, our data suggest that in order to develop resistance-free kinase inhibitors, the next-generation drug design should target the substrate-binding site.

No MeSH data available.


JAK2-V617F/L983F confers resistance in vivo.One million BaF3 cells expressing luciferase and a single JAK2-V617F variant were transplanted via tail vein in syngeneic Balb/c mice. After 3 days, mice were injected twice daily with ruxolitinib (100 mg/kg) for two weeks. Leukemic burden were measured by monitoring the bioluminiscence—7 days (left panel) and 14 days (right panel). As expected, JAK2-V617F/L983F conferred resistance, JAK2-V617F and JAK2-V617F/Y931C are responsive to ruxolitinib treatment.
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f9: JAK2-V617F/L983F confers resistance in vivo.One million BaF3 cells expressing luciferase and a single JAK2-V617F variant were transplanted via tail vein in syngeneic Balb/c mice. After 3 days, mice were injected twice daily with ruxolitinib (100 mg/kg) for two weeks. Leukemic burden were measured by monitoring the bioluminiscence—7 days (left panel) and 14 days (right panel). As expected, JAK2-V617F/L983F conferred resistance, JAK2-V617F and JAK2-V617F/Y931C are responsive to ruxolitinib treatment.

Mentions: The two most frequent mutations from our in vitro screen (i.e., Y931C and L983F) conferred high-grade resistance to all tested ATP-site inhibitors. To test whether they confer resistance in vivo, as a proof of concept, we transplanted Balb/c mice with 1 million BaF3 cells expressing luciferase and JAK2-V617F variants. As expected, twice daily administration of 100 mg/kg ruxolitinib eradicated the JAK2-V617F- and JAK2-V617F/Y931C-expressing cells (Fig. 9). On the other hand, a similar treatment was ineffective against JAK2-V617F/L983F (Fig. 9), thus demonstrating that this mutation confers resistance in vivo.


Targeting substrate-site in Jak2 kinase prevents emergence of genetic resistance.

Kesarwani M, Huber E, Kincaid Z, Evelyn CR, Biesiada J, Rance M, Thapa MB, Shah NP, Meller J, Zheng Y, Azam M - Sci Rep (2015)

JAK2-V617F/L983F confers resistance in vivo.One million BaF3 cells expressing luciferase and a single JAK2-V617F variant were transplanted via tail vein in syngeneic Balb/c mice. After 3 days, mice were injected twice daily with ruxolitinib (100 mg/kg) for two weeks. Leukemic burden were measured by monitoring the bioluminiscence—7 days (left panel) and 14 days (right panel). As expected, JAK2-V617F/L983F conferred resistance, JAK2-V617F and JAK2-V617F/Y931C are responsive to ruxolitinib treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588578&req=5

f9: JAK2-V617F/L983F confers resistance in vivo.One million BaF3 cells expressing luciferase and a single JAK2-V617F variant were transplanted via tail vein in syngeneic Balb/c mice. After 3 days, mice were injected twice daily with ruxolitinib (100 mg/kg) for two weeks. Leukemic burden were measured by monitoring the bioluminiscence—7 days (left panel) and 14 days (right panel). As expected, JAK2-V617F/L983F conferred resistance, JAK2-V617F and JAK2-V617F/Y931C are responsive to ruxolitinib treatment.
Mentions: The two most frequent mutations from our in vitro screen (i.e., Y931C and L983F) conferred high-grade resistance to all tested ATP-site inhibitors. To test whether they confer resistance in vivo, as a proof of concept, we transplanted Balb/c mice with 1 million BaF3 cells expressing luciferase and JAK2-V617F variants. As expected, twice daily administration of 100 mg/kg ruxolitinib eradicated the JAK2-V617F- and JAK2-V617F/Y931C-expressing cells (Fig. 9). On the other hand, a similar treatment was ineffective against JAK2-V617F/L983F (Fig. 9), thus demonstrating that this mutation confers resistance in vivo.

Bottom Line: In vitro binding assays using purified proteins showed strong affinity for the substrate-binding site (Kd = 20 nM) while affinity for the ATP site was poor (Kd = ~8 μM).Our studies demonstrate that mutations affecting the substrate-binding pocket encode a catalytically incompetent kinase, thereby preventing emergence of resistant variants.Most importantly, our data suggest that in order to develop resistance-free kinase inhibitors, the next-generation drug design should target the substrate-binding site.

View Article: PubMed Central - PubMed

Affiliation: Cincinnati Children's Hospital Medical Center, Cancer Blood Disease Institute, Divisions of Experimental Hematology and Cancer Pathology, Cincinnati, Ohio, 45229 USA.

ABSTRACT
Emergence of genetic resistance against kinase inhibitors poses a great challenge for durable therapeutic response. Here, we report a novel mechanism of JAK2 kinase inhibition by fedratinib (TG101348) that prevents emergence of genetic resistance. Using in vitro drug screening, we identified 211 amino-acid substitutions conferring resistance to ruxolitinib (INCB018424) and cross-resistance to the JAK2 inhibitors AZD1480, CYT-387 and lestaurtinib. In contrast, these resistant variants were fully sensitive to fedratinib. Structural modeling, coupled with mutagenesis and biochemical studies, revealed dual binding sites for fedratinib. In vitro binding assays using purified proteins showed strong affinity for the substrate-binding site (Kd = 20 nM) while affinity for the ATP site was poor (Kd = ~8 μM). Our studies demonstrate that mutations affecting the substrate-binding pocket encode a catalytically incompetent kinase, thereby preventing emergence of resistant variants. Most importantly, our data suggest that in order to develop resistance-free kinase inhibitors, the next-generation drug design should target the substrate-binding site.

No MeSH data available.