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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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Effect of AKT inhibition on GO-induced cytotoxicity in human AML cell lines in vitro.Various doses of the allosteric AKT inhibitor, MK-2206, were incubated with increasing concentrations of GO in (A) HL-60, (B) NB4, (C) TF-1, and (D) KG-1 cells. For KG-1 cells, conditions including drug efflux inhibitor PK11195 are also shown. After 3 days, viability (left-side panel) and cell numbers (right-side panel) was determined by flow cytometry. *P<0.05 as compared to medium alone; **P<0.01 as compared to medium alone; ***P<0.001 as compared to medium alone; ****P<0.0001 as compared to medium alone.
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pone-0053518-g005: Effect of AKT inhibition on GO-induced cytotoxicity in human AML cell lines in vitro.Various doses of the allosteric AKT inhibitor, MK-2206, were incubated with increasing concentrations of GO in (A) HL-60, (B) NB4, (C) TF-1, and (D) KG-1 cells. For KG-1 cells, conditions including drug efflux inhibitor PK11195 are also shown. After 3 days, viability (left-side panel) and cell numbers (right-side panel) was determined by flow cytometry. *P<0.05 as compared to medium alone; **P<0.01 as compared to medium alone; ***P<0.001 as compared to medium alone; ****P<0.0001 as compared to medium alone.

Mentions: Taken together, these data suggest a potential role of PI3K/AKT signaling in the mechanism of resistance to GO or, by extension, calicheamicin-γ1-induced cytotoxicity, thus raising the possibility of using inhibitors of this signaling pathway as a novel means to increase the sensitivity of AML cells to calicheamicin-γ1-based therapies. To test this hypothesis, we used MK-2206, an investigational small molecule allosteric inhibitor of AKT currently in Phase 2 clinical development for the treatment of various human malignancies including AML. Consistent with previous investigations on solid tumor cancer as well as acute lymphoblastic leukemia cells [35]–[39], treatment with MK-2206 (1 µM for 6 hours) indeed resulted in effective inhibition of S473 AKT phosphorylation while leaving total AKT levels unchanged in human AML cell lines, confirming target inhibition (data not shown). We initially assessed the effect of MK-2206 on GO and calicheamicin-γ1-cytotoxicity in various human AML cell lines, which are relatively sensitive (HL-60, NB4) or resistant (TF-1, KG-1) to single agent GO or calicheamicin-γ1. As shown in Figure 5, MK-2206 dose-dependently increased in vitro GO cytotoxicity in all 4 cell lines, with most marked effects noted in the GO-resistant TF-1 and KG-1 cell lines, suggesting that inhibition of PI3K/AKT survival signaling can sensitize otherwise resistant AML cells to GO-induced cytotoxicity. Of note, in KG-1 cells, which have active drug efflux, MK-2206 significantly sensitized the cells to GO cytotoxicity both in the absence as well as the presence of a drug efflux reversal agent, PK11195, supporting the relevance of PI3K/AKT pathway activation as a limiting factor for GO cytotoxicity even after inhibition of drug efflux. Consistent with the notion that GO-induced cytotoxic effects are primarily due to the calicheamicin-γ1 moiety rather than the anti-CD33 antibody, comparable results were obtained when the effect of MK-2206 on cytotoxicity induced by free calicheamicin-γ1 was assessed in these cell lines (Figure S5). Finally, to begin investigating the effects of AKT inhibition on GO and calicheamicin-γ1-based treatment of primary AML, we treated 3 AML specimens with various doses of calicheamicin-γ1 in the presence or absence of a relatively non-toxic dose of MK-2206 (1 µM); limited cell numbers precluded the simultaneous testing of GO in these assays. As shown in Figure 6, the extent of calicheamicin-γ1-induced cytotoxicity varied between these 3 specimens; nevertheless, in all 3 cases, MK-2206 enhanced the cytotoxic effects of calicheamicin-γ1.


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)

Effect of AKT inhibition on GO-induced cytotoxicity in human AML cell lines in vitro.Various doses of the allosteric AKT inhibitor, MK-2206, were incubated with increasing concentrations of GO in (A) HL-60, (B) NB4, (C) TF-1, and (D) KG-1 cells. For KG-1 cells, conditions including drug efflux inhibitor PK11195 are also shown. After 3 days, viability (left-side panel) and cell numbers (right-side panel) was determined by flow cytometry. *P<0.05 as compared to medium alone; **P<0.01 as compared to medium alone; ***P<0.001 as compared to medium alone; ****P<0.0001 as compared to medium alone.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3539972&req=5

pone-0053518-g005: Effect of AKT inhibition on GO-induced cytotoxicity in human AML cell lines in vitro.Various doses of the allosteric AKT inhibitor, MK-2206, were incubated with increasing concentrations of GO in (A) HL-60, (B) NB4, (C) TF-1, and (D) KG-1 cells. For KG-1 cells, conditions including drug efflux inhibitor PK11195 are also shown. After 3 days, viability (left-side panel) and cell numbers (right-side panel) was determined by flow cytometry. *P<0.05 as compared to medium alone; **P<0.01 as compared to medium alone; ***P<0.001 as compared to medium alone; ****P<0.0001 as compared to medium alone.
Mentions: Taken together, these data suggest a potential role of PI3K/AKT signaling in the mechanism of resistance to GO or, by extension, calicheamicin-γ1-induced cytotoxicity, thus raising the possibility of using inhibitors of this signaling pathway as a novel means to increase the sensitivity of AML cells to calicheamicin-γ1-based therapies. To test this hypothesis, we used MK-2206, an investigational small molecule allosteric inhibitor of AKT currently in Phase 2 clinical development for the treatment of various human malignancies including AML. Consistent with previous investigations on solid tumor cancer as well as acute lymphoblastic leukemia cells [35]–[39], treatment with MK-2206 (1 µM for 6 hours) indeed resulted in effective inhibition of S473 AKT phosphorylation while leaving total AKT levels unchanged in human AML cell lines, confirming target inhibition (data not shown). We initially assessed the effect of MK-2206 on GO and calicheamicin-γ1-cytotoxicity in various human AML cell lines, which are relatively sensitive (HL-60, NB4) or resistant (TF-1, KG-1) to single agent GO or calicheamicin-γ1. As shown in Figure 5, MK-2206 dose-dependently increased in vitro GO cytotoxicity in all 4 cell lines, with most marked effects noted in the GO-resistant TF-1 and KG-1 cell lines, suggesting that inhibition of PI3K/AKT survival signaling can sensitize otherwise resistant AML cells to GO-induced cytotoxicity. Of note, in KG-1 cells, which have active drug efflux, MK-2206 significantly sensitized the cells to GO cytotoxicity both in the absence as well as the presence of a drug efflux reversal agent, PK11195, supporting the relevance of PI3K/AKT pathway activation as a limiting factor for GO cytotoxicity even after inhibition of drug efflux. Consistent with the notion that GO-induced cytotoxic effects are primarily due to the calicheamicin-γ1 moiety rather than the anti-CD33 antibody, comparable results were obtained when the effect of MK-2206 on cytotoxicity induced by free calicheamicin-γ1 was assessed in these cell lines (Figure S5). Finally, to begin investigating the effects of AKT inhibition on GO and calicheamicin-γ1-based treatment of primary AML, we treated 3 AML specimens with various doses of calicheamicin-γ1 in the presence or absence of a relatively non-toxic dose of MK-2206 (1 µM); limited cell numbers precluded the simultaneous testing of GO in these assays. As shown in Figure 6, the extent of calicheamicin-γ1-induced cytotoxicity varied between these 3 specimens; nevertheless, in all 3 cases, MK-2206 enhanced the cytotoxic effects of calicheamicin-γ1.

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