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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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Effects of Star 27 on D. rario (zebrafish) WT morphology and myelopoiesis, and FLT3-ITD AML context.Effect of Star 27 on zebrafish normal myelopoiesis and the FLT3/ITD-induced myeloid cells expansion. (A–D) The effect of Star 27 on zebrafish embryonic development at 3 dpf, showing no noticeable morphological defects up to 10 μM. (E–I) The effect of PKC412 on zebrafish embryonic development at 3 dpf, showing substantial pericardial defects beginning at 500 nM (data not shown), and tail curvature and length defects beginning at 1 μM and 2.5 μM, respectively vs. vehicle. (J, K, M) The effect of 10 μM Star 27 treatment on mpo+ myeloid cells development in the posterior blood island (PBI) at 30 hpf, showing no statistically significant change. (J, L, M) The effect of 10 μM PKC412 treatment on mpo+ myeloid cells development in the PBI at 30 hpf, showing a statistically significant granulogenesis/myelosuppression. (N–P) Three categories of mpo transcription (N, normal; O, intermediate; P, severe) were defined based on the WISH results (three experiments). (Q) The rescue effect of 10 μM Star 27 treatment on FLT3/ITD-induced mpo+ myeloid cells expansion at 30 hpf, showing rescue of normal phenotype approaching that seen for AC220 (He et al., 2014). Scale bar equals 500 μm. Blue arrows indicate the pericardial edema, green arrows indicate tail shortening and curving, and red arrows indicate the FLT3-ITD AML mpo+ myeloid cells expansion. PKC412 treatment on FLT3/ITD-induced mpo+ myeloid cells expansion at 30 hpf, showing an efficaciousness similar to Star 27, consistent with Figures 1–3. For all experiments: Zebrafish embryos were collected and kept in standard E3 medium at 28°C. Different concentrations of either drug were added to the E3 medium from 6 hr post fertilization (hpf) to 3 days post fertilization (dpf). Embryos treated with DMSO or 10 μM drug from 6 to 30 hpf were collected for mpo whole mount in situ hybridization (WISH) analysis. 80 ng plasmid DNA containing FLT3/ITD sequence was microinjected into one-cell stage embryos, and the uninjected embryos were used as control. FLT3/ITD-injected embryos were treated with DMSO or 10 μM drug from 6 to 30 hpf. Embryos were collected at 30 hpf for mpo WISH analysis. **p <0.01.DOI:http://dx.doi.org/10.7554/eLife.03445.006
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fig4: Effects of Star 27 on D. rario (zebrafish) WT morphology and myelopoiesis, and FLT3-ITD AML context.Effect of Star 27 on zebrafish normal myelopoiesis and the FLT3/ITD-induced myeloid cells expansion. (A–D) The effect of Star 27 on zebrafish embryonic development at 3 dpf, showing no noticeable morphological defects up to 10 μM. (E–I) The effect of PKC412 on zebrafish embryonic development at 3 dpf, showing substantial pericardial defects beginning at 500 nM (data not shown), and tail curvature and length defects beginning at 1 μM and 2.5 μM, respectively vs. vehicle. (J, K, M) The effect of 10 μM Star 27 treatment on mpo+ myeloid cells development in the posterior blood island (PBI) at 30 hpf, showing no statistically significant change. (J, L, M) The effect of 10 μM PKC412 treatment on mpo+ myeloid cells development in the PBI at 30 hpf, showing a statistically significant granulogenesis/myelosuppression. (N–P) Three categories of mpo transcription (N, normal; O, intermediate; P, severe) were defined based on the WISH results (three experiments). (Q) The rescue effect of 10 μM Star 27 treatment on FLT3/ITD-induced mpo+ myeloid cells expansion at 30 hpf, showing rescue of normal phenotype approaching that seen for AC220 (He et al., 2014). Scale bar equals 500 μm. Blue arrows indicate the pericardial edema, green arrows indicate tail shortening and curving, and red arrows indicate the FLT3-ITD AML mpo+ myeloid cells expansion. PKC412 treatment on FLT3/ITD-induced mpo+ myeloid cells expansion at 30 hpf, showing an efficaciousness similar to Star 27, consistent with Figures 1–3. For all experiments: Zebrafish embryos were collected and kept in standard E3 medium at 28°C. Different concentrations of either drug were added to the E3 medium from 6 hr post fertilization (hpf) to 3 days post fertilization (dpf). Embryos treated with DMSO or 10 μM drug from 6 to 30 hpf were collected for mpo whole mount in situ hybridization (WISH) analysis. 80 ng plasmid DNA containing FLT3/ITD sequence was microinjected into one-cell stage embryos, and the uninjected embryos were used as control. FLT3/ITD-injected embryos were treated with DMSO or 10 μM drug from 6 to 30 hpf. Embryos were collected at 30 hpf for mpo WISH analysis. **p <0.01.DOI:http://dx.doi.org/10.7554/eLife.03445.006

Mentions: We treated embryos with Star 27 up to 10 μM and studied the effects on WT morphology at 3 days post fertilization (dpf), finding no change in heart and tail morphology (tail length and lack of curvature, see Figure 4A–D). In contrast, PKC412 showed substantial morphological defects to the heart at 1 μM vs vehicle (Figure 4E–I), tail length and tail curvature at 1 μM and 2.5 μM (see Figure 4E,G, respectively).10.7554/eLife.03445.006Figure 4.Effects of Star 27 on D. rario (zebrafish) WT morphology and myelopoiesis, and FLT3-ITD AML context.


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)

Effects of Star 27 on D. rario (zebrafish) WT morphology and myelopoiesis, and FLT3-ITD AML context.Effect of Star 27 on zebrafish normal myelopoiesis and the FLT3/ITD-induced myeloid cells expansion. (A–D) The effect of Star 27 on zebrafish embryonic development at 3 dpf, showing no noticeable morphological defects up to 10 μM. (E–I) The effect of PKC412 on zebrafish embryonic development at 3 dpf, showing substantial pericardial defects beginning at 500 nM (data not shown), and tail curvature and length defects beginning at 1 μM and 2.5 μM, respectively vs. vehicle. (J, K, M) The effect of 10 μM Star 27 treatment on mpo+ myeloid cells development in the posterior blood island (PBI) at 30 hpf, showing no statistically significant change. (J, L, M) The effect of 10 μM PKC412 treatment on mpo+ myeloid cells development in the PBI at 30 hpf, showing a statistically significant granulogenesis/myelosuppression. (N–P) Three categories of mpo transcription (N, normal; O, intermediate; P, severe) were defined based on the WISH results (three experiments). (Q) The rescue effect of 10 μM Star 27 treatment on FLT3/ITD-induced mpo+ myeloid cells expansion at 30 hpf, showing rescue of normal phenotype approaching that seen for AC220 (He et al., 2014). Scale bar equals 500 μm. Blue arrows indicate the pericardial edema, green arrows indicate tail shortening and curving, and red arrows indicate the FLT3-ITD AML mpo+ myeloid cells expansion. PKC412 treatment on FLT3/ITD-induced mpo+ myeloid cells expansion at 30 hpf, showing an efficaciousness similar to Star 27, consistent with Figures 1–3. For all experiments: Zebrafish embryos were collected and kept in standard E3 medium at 28°C. Different concentrations of either drug were added to the E3 medium from 6 hr post fertilization (hpf) to 3 days post fertilization (dpf). Embryos treated with DMSO or 10 μM drug from 6 to 30 hpf were collected for mpo whole mount in situ hybridization (WISH) analysis. 80 ng plasmid DNA containing FLT3/ITD sequence was microinjected into one-cell stage embryos, and the uninjected embryos were used as control. FLT3/ITD-injected embryos were treated with DMSO or 10 μM drug from 6 to 30 hpf. Embryos were collected at 30 hpf for mpo WISH analysis. **p <0.01.DOI:http://dx.doi.org/10.7554/eLife.03445.006
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fig4: Effects of Star 27 on D. rario (zebrafish) WT morphology and myelopoiesis, and FLT3-ITD AML context.Effect of Star 27 on zebrafish normal myelopoiesis and the FLT3/ITD-induced myeloid cells expansion. (A–D) The effect of Star 27 on zebrafish embryonic development at 3 dpf, showing no noticeable morphological defects up to 10 μM. (E–I) The effect of PKC412 on zebrafish embryonic development at 3 dpf, showing substantial pericardial defects beginning at 500 nM (data not shown), and tail curvature and length defects beginning at 1 μM and 2.5 μM, respectively vs. vehicle. (J, K, M) The effect of 10 μM Star 27 treatment on mpo+ myeloid cells development in the posterior blood island (PBI) at 30 hpf, showing no statistically significant change. (J, L, M) The effect of 10 μM PKC412 treatment on mpo+ myeloid cells development in the PBI at 30 hpf, showing a statistically significant granulogenesis/myelosuppression. (N–P) Three categories of mpo transcription (N, normal; O, intermediate; P, severe) were defined based on the WISH results (three experiments). (Q) The rescue effect of 10 μM Star 27 treatment on FLT3/ITD-induced mpo+ myeloid cells expansion at 30 hpf, showing rescue of normal phenotype approaching that seen for AC220 (He et al., 2014). Scale bar equals 500 μm. Blue arrows indicate the pericardial edema, green arrows indicate tail shortening and curving, and red arrows indicate the FLT3-ITD AML mpo+ myeloid cells expansion. PKC412 treatment on FLT3/ITD-induced mpo+ myeloid cells expansion at 30 hpf, showing an efficaciousness similar to Star 27, consistent with Figures 1–3. For all experiments: Zebrafish embryos were collected and kept in standard E3 medium at 28°C. Different concentrations of either drug were added to the E3 medium from 6 hr post fertilization (hpf) to 3 days post fertilization (dpf). Embryos treated with DMSO or 10 μM drug from 6 to 30 hpf were collected for mpo whole mount in situ hybridization (WISH) analysis. 80 ng plasmid DNA containing FLT3/ITD sequence was microinjected into one-cell stage embryos, and the uninjected embryos were used as control. FLT3/ITD-injected embryos were treated with DMSO or 10 μM drug from 6 to 30 hpf. Embryos were collected at 30 hpf for mpo WISH analysis. **p <0.01.DOI:http://dx.doi.org/10.7554/eLife.03445.006
Mentions: We treated embryos with Star 27 up to 10 μM and studied the effects on WT morphology at 3 days post fertilization (dpf), finding no change in heart and tail morphology (tail length and lack of curvature, see Figure 4A–D). In contrast, PKC412 showed substantial morphological defects to the heart at 1 μM vs vehicle (Figure 4E–I), tail length and tail curvature at 1 μM and 2.5 μM (see Figure 4E,G, respectively).10.7554/eLife.03445.006Figure 4.Effects of Star 27 on D. rario (zebrafish) WT morphology and myelopoiesis, and FLT3-ITD AML context.

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