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Overcoming myelosuppression due to synthetic lethal toxicity for FLT3-targeted acute myeloid leukemia therapy.

Warkentin AA, Lopez MS, Lasater EA, Lin K, He BL, Leung AY, Smith CC, Shah NP, Shokat KM - Elife (2014)

Bottom Line: Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML).We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27.As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.

ABSTRACT
Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML). Clinically active investigational FLT3 inhibitors can achieve complete remissions but their utility has been hampered by acquired resistance and myelosuppression attributed to a 'synthetic lethal toxicity' arising from simultaneous inhibition of FLT3 and KIT. We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27. Star 27 maintains potency against FLT3 in proliferation assays of FLT3-transformed cells compared with KIT-transformed cells, shows no toxicity towards normal human hematopoiesis at concentrations that inhibit primary FLT3-mutant AML blast growth, and is active against mutations that confer resistance to clinical inhibitors. As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity.

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Heatmap of cellular EC50 values for leading clinical therapies (S = Sorafenib, A = AC220, P = Ponatinib, PKC412) and Star 27 against a panel of AML-relevant human- and mouse-derived drug-resistant cell lines.(A) (adapted from Guo et al., 2007; Kampa-Shittenhelm et al., 2013; Smith et al., 2013). Log10-scale fold resistance between Ba/F3 FLT3 ITD and drug-induced mutations (ITD + KD double mutant) shown via heat map. Point mutations corresponding to each drug-relevant resistance shown. Star 27 maintains potency between FLT3 ITD and resistance mutants comparable to PKC412. Mutations derived independently from both saturating mutagenesis and patient-derived samples [Smith et al., 2012]. Replicates shown are the result of at least two or three attempts, each in triplicate, and error ranges represent the standard error of the mean. (B and C) Crenolanib's effect on p-KIT inhibition and of colony growth in normal BM. (B) Crenolanib inhibits p-KIT in HMC1.1 cells at 12 nM IC50. This level of inhibition translates to downstream kinases, with inhibition of p-AKT (S473) and p-S6 (S235/S236). (C) Crenolanib potently inhibits normal BM colony formation at <63 nM and ca. 63 nM IC50's of CFU and BFU colonies, respectively.DOI:http://dx.doi.org/10.7554/eLife.03445.008
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fig6: Heatmap of cellular EC50 values for leading clinical therapies (S = Sorafenib, A = AC220, P = Ponatinib, PKC412) and Star 27 against a panel of AML-relevant human- and mouse-derived drug-resistant cell lines.(A) (adapted from Guo et al., 2007; Kampa-Shittenhelm et al., 2013; Smith et al., 2013). Log10-scale fold resistance between Ba/F3 FLT3 ITD and drug-induced mutations (ITD + KD double mutant) shown via heat map. Point mutations corresponding to each drug-relevant resistance shown. Star 27 maintains potency between FLT3 ITD and resistance mutants comparable to PKC412. Mutations derived independently from both saturating mutagenesis and patient-derived samples [Smith et al., 2012]. Replicates shown are the result of at least two or three attempts, each in triplicate, and error ranges represent the standard error of the mean. (B and C) Crenolanib's effect on p-KIT inhibition and of colony growth in normal BM. (B) Crenolanib inhibits p-KIT in HMC1.1 cells at 12 nM IC50. This level of inhibition translates to downstream kinases, with inhibition of p-AKT (S473) and p-S6 (S235/S236). (C) Crenolanib potently inhibits normal BM colony formation at <63 nM and ca. 63 nM IC50's of CFU and BFU colonies, respectively.DOI:http://dx.doi.org/10.7554/eLife.03445.008

Mentions: Mutations that confer drug resistance to existing Type II FLT3 inhibitors AC220 and sorafenib commonly prevent the kinase from efficiently adopting an inactive conformation required for drug binding. Type I FLT3 inhibitors, such as PKC412 and Star 27, are predicted to be less vulnerable to such mutations. We tested both inhibitors against a panel of 17 cell lines containing different FLT3 TKI mutations with varying resistance to the leading clinical candidate therapies, sorafenib, ponatinib, and AC220 (Figure 6A). IC50 values are presented as log10-fold resistance in a color-coded heat map to highlight the range of offset. Using published IC50 values for sorafenib, AC220, and ponatinib for comparison (Guo et al., 2007; Kampa-Shittenhelm et al., 2013; Smith et al., 2013), we observed equipotent values for PKC412 against most KD point mutants. Intriguingly, Star 27 maintained similar potency against most mutants compared to FLT3-ITD alone.10.7554/eLife.03445.008Figure 6.Heatmap of cellular EC50 values for leading clinical therapies (S = Sorafenib, A = AC220, P = Ponatinib, PKC412) and Star 27 against a panel of AML-relevant human- and mouse-derived drug-resistant cell lines.


Overcoming myelosuppression due to synthetic lethal toxicity for FLT3-targeted acute myeloid leukemia therapy.

Warkentin AA, Lopez MS, Lasater EA, Lin K, He BL, Leung AY, Smith CC, Shah NP, Shokat KM - Elife (2014)

Heatmap of cellular EC50 values for leading clinical therapies (S = Sorafenib, A = AC220, P = Ponatinib, PKC412) and Star 27 against a panel of AML-relevant human- and mouse-derived drug-resistant cell lines.(A) (adapted from Guo et al., 2007; Kampa-Shittenhelm et al., 2013; Smith et al., 2013). Log10-scale fold resistance between Ba/F3 FLT3 ITD and drug-induced mutations (ITD + KD double mutant) shown via heat map. Point mutations corresponding to each drug-relevant resistance shown. Star 27 maintains potency between FLT3 ITD and resistance mutants comparable to PKC412. Mutations derived independently from both saturating mutagenesis and patient-derived samples [Smith et al., 2012]. Replicates shown are the result of at least two or three attempts, each in triplicate, and error ranges represent the standard error of the mean. (B and C) Crenolanib's effect on p-KIT inhibition and of colony growth in normal BM. (B) Crenolanib inhibits p-KIT in HMC1.1 cells at 12 nM IC50. This level of inhibition translates to downstream kinases, with inhibition of p-AKT (S473) and p-S6 (S235/S236). (C) Crenolanib potently inhibits normal BM colony formation at <63 nM and ca. 63 nM IC50's of CFU and BFU colonies, respectively.DOI:http://dx.doi.org/10.7554/eLife.03445.008
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4307180&req=5

fig6: Heatmap of cellular EC50 values for leading clinical therapies (S = Sorafenib, A = AC220, P = Ponatinib, PKC412) and Star 27 against a panel of AML-relevant human- and mouse-derived drug-resistant cell lines.(A) (adapted from Guo et al., 2007; Kampa-Shittenhelm et al., 2013; Smith et al., 2013). Log10-scale fold resistance between Ba/F3 FLT3 ITD and drug-induced mutations (ITD + KD double mutant) shown via heat map. Point mutations corresponding to each drug-relevant resistance shown. Star 27 maintains potency between FLT3 ITD and resistance mutants comparable to PKC412. Mutations derived independently from both saturating mutagenesis and patient-derived samples [Smith et al., 2012]. Replicates shown are the result of at least two or three attempts, each in triplicate, and error ranges represent the standard error of the mean. (B and C) Crenolanib's effect on p-KIT inhibition and of colony growth in normal BM. (B) Crenolanib inhibits p-KIT in HMC1.1 cells at 12 nM IC50. This level of inhibition translates to downstream kinases, with inhibition of p-AKT (S473) and p-S6 (S235/S236). (C) Crenolanib potently inhibits normal BM colony formation at <63 nM and ca. 63 nM IC50's of CFU and BFU colonies, respectively.DOI:http://dx.doi.org/10.7554/eLife.03445.008
Mentions: Mutations that confer drug resistance to existing Type II FLT3 inhibitors AC220 and sorafenib commonly prevent the kinase from efficiently adopting an inactive conformation required for drug binding. Type I FLT3 inhibitors, such as PKC412 and Star 27, are predicted to be less vulnerable to such mutations. We tested both inhibitors against a panel of 17 cell lines containing different FLT3 TKI mutations with varying resistance to the leading clinical candidate therapies, sorafenib, ponatinib, and AC220 (Figure 6A). IC50 values are presented as log10-fold resistance in a color-coded heat map to highlight the range of offset. Using published IC50 values for sorafenib, AC220, and ponatinib for comparison (Guo et al., 2007; Kampa-Shittenhelm et al., 2013; Smith et al., 2013), we observed equipotent values for PKC412 against most KD point mutants. Intriguingly, Star 27 maintained similar potency against most mutants compared to FLT3-ITD alone.10.7554/eLife.03445.008Figure 6.Heatmap of cellular EC50 values for leading clinical therapies (S = Sorafenib, A = AC220, P = Ponatinib, PKC412) and Star 27 against a panel of AML-relevant human- and mouse-derived drug-resistant cell lines.

Bottom Line: Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML).We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27.As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.

ABSTRACT
Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML). Clinically active investigational FLT3 inhibitors can achieve complete remissions but their utility has been hampered by acquired resistance and myelosuppression attributed to a 'synthetic lethal toxicity' arising from simultaneous inhibition of FLT3 and KIT. We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27. Star 27 maintains potency against FLT3 in proliferation assays of FLT3-transformed cells compared with KIT-transformed cells, shows no toxicity towards normal human hematopoiesis at concentrations that inhibit primary FLT3-mutant AML blast growth, and is active against mutations that confer resistance to clinical inhibitors. As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity.

Show MeSH
Related in: MedlinePlus