TY - JOUR
T1 - AML-326 Novel FLT3-ALK2 Dual Inhibitor TP-0184 Inhibits Leukemia Growth by Targeting Serine Biosynthesis in FLT3-ITD-Positive AML Cells
AU - Tyagi, Anudishi
AU - Ly, Stanley
AU - Yuan, Bin
AU - El-Dana, Fouad
AU - Jaggupilli, Appalaraju
AU - Foulks, Jason
AU - Warner, Steven
AU - Battula, V. Lokesh
N1 - Publisher Copyright:
© 2022 Elsevier Inc.
PY - 2022/10
Y1 - 2022/10
N2 - Context: FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) inhibitors have been studied; however, molecular factors contributing to this resistance are unknown. Objective: i) To investigate the clinical significance of BMP type 1 receptor ALK2 in AML patients with FLT3-ITD mutations. ii) To elucidate the therapeutic effect of FLT3-ALK2 dual inhibitor TP-0184 on leukemia growth. Methods: To determine whether ALK2 is a potential target in FLT3-mutated AML patients, we analyzed OHSU and TCGA datasets. We treated AML cell lines with FLT3-WT or -ITD mutations with ALK2 inhibitors and measured their effect on cell cycle and proliferation. To determine the mechanism of TP-0184, we measured the activation of FLT3 downstream signaling by using western blotting and RNA sequencing. Further, we performed kinase profiling to understand the binding specificity of TP-0184 with 11 different FLT3 mutants. Finally, we investigated the effect of TP-0184 on AML growth in vivo using FLT3-mutated PDX and xenograft models. Results: Analysis of AML datasets showed that ALK2 is significantly upregulated in AML patients with FLT3 mutations compared to those with WT-FLT3 (P<0.00001) and predicts poor overall survival (P=0.05). Treatment of FLT3-WT and -mutated AML cell lines with the ALK2 inhibitors LDN-212854 and TP-0184 resulted in significant inhibition of FLT3-ITD—mutant cell growth at low concentrations (IC50<25 nM), while WT-FLT3 cells were affected only at high concentrations (IC50>100 nM). Interestingly, TP-0184 induced G1/G0 arrest in AML cell lines with FLT-ITD mutations but had minimal to no effect in FLT3-WT AML cells. Further, we observed that treatment with TP-0184 in AML cell lines significantly inhibited multiple signaling proteins downstream of FLT3, such as p-STAT5 and p-ERK, as well as p-PI3K, p-mTOR, and p-S6K. Gene expression analysis revealed that treatment with TP-0184 in FLT3-ITD cell lines significantly downregulated the serine biosynthesis pathway. Lastly, treatment with TP-0184 significantly prolonged the survival of FLT3-ITD-mutated AML-bearing mice (P<0.0001). Conclusions: Our data indicate that ALK2 is a prognostic marker in FLT3-mutated AML. TP-0184 inhibits cell proliferation by inhibiting FLT3 downstream signaling pathways in AML cells. Kinase assays confirmed that TP-0184 is a highly specific FLT3-ALK2 dual inhibitor. TP-0184 inhibits AML growth in FLT3-mutated PDX and xenograft models.
AB - Context: FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) inhibitors have been studied; however, molecular factors contributing to this resistance are unknown. Objective: i) To investigate the clinical significance of BMP type 1 receptor ALK2 in AML patients with FLT3-ITD mutations. ii) To elucidate the therapeutic effect of FLT3-ALK2 dual inhibitor TP-0184 on leukemia growth. Methods: To determine whether ALK2 is a potential target in FLT3-mutated AML patients, we analyzed OHSU and TCGA datasets. We treated AML cell lines with FLT3-WT or -ITD mutations with ALK2 inhibitors and measured their effect on cell cycle and proliferation. To determine the mechanism of TP-0184, we measured the activation of FLT3 downstream signaling by using western blotting and RNA sequencing. Further, we performed kinase profiling to understand the binding specificity of TP-0184 with 11 different FLT3 mutants. Finally, we investigated the effect of TP-0184 on AML growth in vivo using FLT3-mutated PDX and xenograft models. Results: Analysis of AML datasets showed that ALK2 is significantly upregulated in AML patients with FLT3 mutations compared to those with WT-FLT3 (P<0.00001) and predicts poor overall survival (P=0.05). Treatment of FLT3-WT and -mutated AML cell lines with the ALK2 inhibitors LDN-212854 and TP-0184 resulted in significant inhibition of FLT3-ITD—mutant cell growth at low concentrations (IC50<25 nM), while WT-FLT3 cells were affected only at high concentrations (IC50>100 nM). Interestingly, TP-0184 induced G1/G0 arrest in AML cell lines with FLT-ITD mutations but had minimal to no effect in FLT3-WT AML cells. Further, we observed that treatment with TP-0184 in AML cell lines significantly inhibited multiple signaling proteins downstream of FLT3, such as p-STAT5 and p-ERK, as well as p-PI3K, p-mTOR, and p-S6K. Gene expression analysis revealed that treatment with TP-0184 in FLT3-ITD cell lines significantly downregulated the serine biosynthesis pathway. Lastly, treatment with TP-0184 significantly prolonged the survival of FLT3-ITD-mutated AML-bearing mice (P<0.0001). Conclusions: Our data indicate that ALK2 is a prognostic marker in FLT3-mutated AML. TP-0184 inhibits cell proliferation by inhibiting FLT3 downstream signaling pathways in AML cells. Kinase assays confirmed that TP-0184 is a highly specific FLT3-ALK2 dual inhibitor. TP-0184 inhibits AML growth in FLT3-mutated PDX and xenograft models.
KW - ACVR1
KW - ALK2
KW - AML
KW - BMP signaling
KW - FLT3-ITD
KW - TP-0184
UR - http://www.scopus.com/inward/record.url?scp=85138192670&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85138192670&partnerID=8YFLogxK
U2 - 10.1016/S2152-2650(22)01266-6
DO - 10.1016/S2152-2650(22)01266-6
M3 - Article
C2 - 36163811
AN - SCOPUS:85138192670
SN - 2152-2650
VL - 22
SP - S236
JO - Clinical Lymphoma, Myeloma and Leukemia
JF - Clinical Lymphoma, Myeloma and Leukemia
ER -