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AKT signaling as a novel factor associated with in vitro resistance of human AML to gemtuzumab ozogamicin.

Rosen DB, Harrington KH, Cordeiro JA, Leung LY, Putta S, Lacayo N, Laszlo GS, Gudgeon CJ, Hogge DE, Hawtin RE, Cesano A, Walter RB - PLoS ONE (2013)

Bottom Line: Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells.Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ(1) with particularly pronounced effects in otherwise GO or free calicheamicin-γ(1)-resistant cells.The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ(1) and, by extrapolation, other DNA damage-based therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Nodality Inc., South San Francisco, California, United States of America.

ABSTRACT
Gemtuzumab ozogamicin (GO), an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ(1) derivative, induces remissions and improves survival in a subset of patients with acute myeloid leukemia (AML). As the mechanisms underlying GO and calicheamicin-γ(1) resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP) assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ(1) with particularly pronounced effects in otherwise GO or free calicheamicin-γ(1)-resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ(1). Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ(1) and, by extrapolation, other DNA damage-based therapeutics.

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Growth factor/cytokine-induced signaling in primary AML specimens.Plots of signaling log2-fold responses: FLT3L induced p-S6 (X-axis) vs. SCF induced p-AKT (top) or SCF induced p-S6 (bottom). Samples are coded by in vitro GO response (sensitive: green symbols; resistant: red symbols).
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pone-0053518-g004: Growth factor/cytokine-induced signaling in primary AML specimens.Plots of signaling log2-fold responses: FLT3L induced p-S6 (X-axis) vs. SCF induced p-AKT (top) or SCF induced p-S6 (bottom). Samples are coded by in vitro GO response (sensitive: green symbols; resistant: red symbols).

Mentions: To identify downstream pathways that might affect GO cytotoxicity, we functionally assessed PI3K/AKT/mTOR, JAK/STAT, and Ras/Raf/MEK signaling pathways using SCNP assays; for details regarding modulators and assessed targets, please see Supporting Table S2. Initial experiments in AML cell lines indicated that stem cell factor (SCF)-modulated PI3K pathway activity (as measured by levels of SCF-induced p-AKT and p-S6) was elevated in the GO-resistant GDM-1 but not the GO-sensitive U937 cells (p-AKT: mean log2-fold increase of 1.98 in GDM-1 cells vs. −0.11 in U937 cells; for p-S6: mean log2-fold increase of 1.00 for GDM-1 cells vs. −0.03 for U937 cells). We then used SCNP assays to assess these signaling pathways in the pediatric AML samples (the adult specimens lacked sufficient material for signaling analyses) using established SCNP methodology for primary human AML [24], [33], [34]. As shown in Figure 4 and Table S3, we found that SCF-modulated PI3K pathway activity was elevated in all 3 GO resistant samples relative to GO sensitive samples (p-AKT: mean log2-fold increase of 3.33±0.58 vs. 0.98±0.69, P = 0.060; for p-S6: mean log2-fold increase of 1.62±0.18 vs. 0.38±0.42, P = 0.054). PI3K pathway activity was similarly elevated in GO resistant samples vs. GO sensitive samples when modulated with FLT3 ligand (FLT3L) (p-S6: mean log2-fold increase of 1.82±0.29 vs. 0.80±0.21, respectively, P = 0.045). Of note, no differences in basal levels p-S6 and p-Akt were observed between GO resistant vs. GO sensitive samples in the absence of SCF or FLT3L (data not shown).


AKT signaling as a novel factor associated with in vitro resistance of human AML to gemtuzumab ozogamicin.

Rosen DB, Harrington KH, Cordeiro JA, Leung LY, Putta S, Lacayo N, Laszlo GS, Gudgeon CJ, Hogge DE, Hawtin RE, Cesano A, Walter RB - PLoS ONE (2013)

Growth factor/cytokine-induced signaling in primary AML specimens.Plots of signaling log2-fold responses: FLT3L induced p-S6 (X-axis) vs. SCF induced p-AKT (top) or SCF induced p-S6 (bottom). Samples are coded by in vitro GO response (sensitive: green symbols; resistant: red symbols).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3539972&req=5

pone-0053518-g004: Growth factor/cytokine-induced signaling in primary AML specimens.Plots of signaling log2-fold responses: FLT3L induced p-S6 (X-axis) vs. SCF induced p-AKT (top) or SCF induced p-S6 (bottom). Samples are coded by in vitro GO response (sensitive: green symbols; resistant: red symbols).
Mentions: To identify downstream pathways that might affect GO cytotoxicity, we functionally assessed PI3K/AKT/mTOR, JAK/STAT, and Ras/Raf/MEK signaling pathways using SCNP assays; for details regarding modulators and assessed targets, please see Supporting Table S2. Initial experiments in AML cell lines indicated that stem cell factor (SCF)-modulated PI3K pathway activity (as measured by levels of SCF-induced p-AKT and p-S6) was elevated in the GO-resistant GDM-1 but not the GO-sensitive U937 cells (p-AKT: mean log2-fold increase of 1.98 in GDM-1 cells vs. −0.11 in U937 cells; for p-S6: mean log2-fold increase of 1.00 for GDM-1 cells vs. −0.03 for U937 cells). We then used SCNP assays to assess these signaling pathways in the pediatric AML samples (the adult specimens lacked sufficient material for signaling analyses) using established SCNP methodology for primary human AML [24], [33], [34]. As shown in Figure 4 and Table S3, we found that SCF-modulated PI3K pathway activity was elevated in all 3 GO resistant samples relative to GO sensitive samples (p-AKT: mean log2-fold increase of 3.33±0.58 vs. 0.98±0.69, P = 0.060; for p-S6: mean log2-fold increase of 1.62±0.18 vs. 0.38±0.42, P = 0.054). PI3K pathway activity was similarly elevated in GO resistant samples vs. GO sensitive samples when modulated with FLT3 ligand (FLT3L) (p-S6: mean log2-fold increase of 1.82±0.29 vs. 0.80±0.21, respectively, P = 0.045). Of note, no differences in basal levels p-S6 and p-Akt were observed between GO resistant vs. GO sensitive samples in the absence of SCF or FLT3L (data not shown).

Bottom Line: Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells.Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ(1) with particularly pronounced effects in otherwise GO or free calicheamicin-γ(1)-resistant cells.The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ(1) and, by extrapolation, other DNA damage-based therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Nodality Inc., South San Francisco, California, United States of America.

ABSTRACT
Gemtuzumab ozogamicin (GO), an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ(1) derivative, induces remissions and improves survival in a subset of patients with acute myeloid leukemia (AML). As the mechanisms underlying GO and calicheamicin-γ(1) resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP) assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ(1) with particularly pronounced effects in otherwise GO or free calicheamicin-γ(1)-resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ(1). Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ(1) and, by extrapolation, other DNA damage-based therapeutics.

Show MeSH
Related in: MedlinePlus