33 research outputs found
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Selective Akt Inhibitors Synergize with Tyrosine Kinase Inhibitors and Effectively Override Stroma-Associated Cytoprotection of Mutant FLT3-Positive AML Cells
Objectives: Tyrosine kinase inhibitor (TKI)-treated acute myeloid leukemia (AML) patients commonly show rapid and significant peripheral blood blast cell reduction, however a marginal decrease in bone marrow blasts. This suggests a protective environment and highlights the demand for a better understanding of stromal:leukemia cell communication. As a strategy to improve clinical efficacy, we searched for novel agents capable of potentiating the stroma-diminished effects of TKI treatment of mutant FLT3-expressing cells. Methods: We designed a combinatorial high throughput drug screen using well-characterized kinase inhibitor-focused libraries to identify novel kinase inhibitors capable of overriding stromal-mediated resistance to TKIs, such as PKC412 and AC220. Standard liquid culture proliferation assays, cell cycle and apoptosis analysis, and immunoblotting were carried out with cell lines or primary AML to validate putative candidates from the screen and characterize the mechanism(s) underlying observed synergy. Results and Conclusions Our study led to the observation of synergy between selective Akt inhibitors and FLT3 inhibitors against mutant FLT3-positive AML in either the absence or presence of stroma. Our findings are consistent with evidence that Akt activation is characteristic of mutant FLT3-transformed cells, as well as observed residual Akt activity following FLT3 inhibitor treatment. In conclusion, our study highlights the potential importance of Akt as a signaling factor in leukemia survival, and supports the use of the co-culture chemical screen to identify agents able to potentiate TKI anti-leukemia activity in a cytoprotective microenvironment
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Acute myeloid leukemia cells require 6-phosphogluconate dehydrogenase for cell growth and NADPH-dependent metabolic reprogramming
Acute myeloid leukemia (AML) cells are highly dependent on glycolytic pathways to generate metabolic energy and support cell growth, hinting at specific, targetable vulnerabilities as potential novel targets for drug development. Elevated levels of NADPH, a central metabolic factor involved in redox reactions, are common in myeloid leukemia cells, but the significance or biochemical basis underlying this increase is unknown. Using a small molecule analog that efficiently inhibits NADPH-producing enzymes, we found that AML cells require NADPH homeostasis for cell growth. We also found that inhibiting NADPH production through knockdown of 6-phosphogluconate dehydrogenase (6PGD) within the pentose phosphate pathway was sufficient to reduce cell growth and lactate production, a measure of metabolic reprogramming. Further, inhibition of 6PGD activity reduced NADH levels and enzymatic activity of the oxidized NADH-dependent sirtuin-1. Targeting 6PGD and NADPH production was sufficient to block growth of AML cell lines resistant to the chemotherapeutics daunorubicin and cytarabine. Importantly, stromal cell-mediated resistance to targeted inhibition of oncogenic FLT3 kinase activity by quizartinib was circumvented by 6PGD knockdown. Overall, these data suggest that the dependency of AML cells on NADPH to permit increased glycolytic flux creates a potential vulnerability of possible therapeutic benefit, since much of the enhanced production of NADPH is dependent on the activity of a single enzyme, 6PGD
Akt and p38 MAPK synergize with tyrosine kinase inhibitors and effectively override stroma-associated cytoprotection of mutant FLT3-positive AML cells
Tyrosine kinase inhibitor (TKI)-treated acute myeloid leukemia (AML) patients commonly show rapid and significant peripheral blood blast cell reduction, however a marginal decrease in bone marrow blasts. This suggests a protective environment and highlights the need for a greater understanding of stromal:leukemia cell communication. To address this, we used a novel adherent stroma-based co-culture system as the foundation for a chemical screen designed to identify novel kinase inhibitors capable of potentiating the apoptosis-inducing effects of TKIs against adherent stroma-protected mutant FLT3-positive cells. Screenings led to the identification of selective inhibitors of Akt and MAPK, mediators of signaling pathways implicated in TKI exposure-induced drug resistance, and found to be activated in stromal cells in direct contact with leukemia. Although constitutive MAPK and Akt activation is characteristic of mutant FLT3-transformed cells, our screen suggests that Akt and MAPK may play an unexpected role in stromal protection of TKI-treated cells. In addition, it is common to observe residual Akt and MAPK activity following FLT3 inhibitor treatment. Our findings highlight the potential importance of these signaling factors in leukemia survival and stromal protection of TKI-treated leukemia, and support the use of the co-culture chemical screen to identify agents able to potentiate TKI anti-leukemia activity.
Key words: acute myeloid leukemia, FLT3 inhibitor, multi-targeted kinase inhibitor, PKC412, AC220, Akt, p38 MAPK, stromal-mediated chemoresistance, synerg