Limits...
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.

Show MeSH

Related in: MedlinePlus

Relationship between CD33 level and GO-induced DNA damage and cytotoxicity.(A) Correlation between CD33 expression and GO-induced DNA damage, as quantified by measurement of γH2AX levels 6 hours after drug exposure. (B) Correlation between CD33 expression and GO-induced cytotoxicity after 48 hours of drug exposure. Samples resistant to GO (as defined by <15% GO-induced apoptosis/cell death 24 hours after drug exposure) are shown in bold. Note: the CD33-QDot605 MFI for AML-11 was converted from CD33 PE MFI, as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3539972&req=5

pone-0053518-g002: Relationship between CD33 level and GO-induced DNA damage and cytotoxicity.(A) Correlation between CD33 expression and GO-induced DNA damage, as quantified by measurement of γH2AX levels 6 hours after drug exposure. (B) Correlation between CD33 expression and GO-induced cytotoxicity after 48 hours of drug exposure. Samples resistant to GO (as defined by <15% GO-induced apoptosis/cell death 24 hours after drug exposure) are shown in bold. Note: the CD33-QDot605 MFI for AML-11 was converted from CD33 PE MFI, as described in Materials and Methods.

Mentions: The postulated mechanism of action of GO implies a pivotal role of drug-induced DNA damage and intact downstream pro- and anti-apoptotic pathway activity [5]. As depicted in Figure 1, our SCNP assay focused on functional, quantitative measurements of DNA damage pathways as well as PI3K/AKT, MEK/ERK, and JAK/STAT signaling pathways in leukemic cells in response to in vitro stimulation of diagnostic specimens from 6 pediatric and 6 adult patients containing >78% blast cells in all cases (Table 1; AML-01– AML-12). First, the in vitro GO sensitivity of AML cells in these specimens was characterized. At the clinically relevant concentration of 30 ng/mL [30], GO treatment resulted in an induced apoptotic/dead cell fraction averaging 19.3±5.2% and 43.8±7.1% after continuous exposure for 24 and 48 hours, respectively (Table 2). Using an arbitrary cut-off for the induced apoptotic/dead cell fraction of 15% at 24 hours, 5 specimens were classified as “GO sensitive”, whereas 7 were classified as “GO resistant”. The lack of apoptotic response in these GO resistant samples was likely directly due to insufficient intracellular GO concentrations rather than the consequence of dysfunctional apoptotic pathways, as parallel experiments with staurosporine, a classic activator of the mitochondrial apoptosis pathway [31], demonstrated robust apoptosis not only in GO-sensitive but also in most (5/6 tested) GO resistant samples (Figure S2), establishing that the apoptotic machinery was generally intact in the tested AML specimens. Of note, among all patient specimens, there was no correlation between CD33 expression levels and GO-induced cytotoxicity at 24 hours (r = 0.181 [95% confidence interval: −0.439–0.684], P = 0.574) or 48 hours (r = 0.266 [−0.397–0.747], P = 0.429). Importantly, GO failed to induce significant cytotoxicity after 24 or 48 hours in 2 cases (AML-07 and AML-08) with bright CD33 expression, indicating the presence of relevant resistance mechanisms that are unrelated to target antigen expression (Figure 2).


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)

Relationship between CD33 level and GO-induced DNA damage and cytotoxicity.(A) Correlation between CD33 expression and GO-induced DNA damage, as quantified by measurement of γH2AX levels 6 hours after drug exposure. (B) Correlation between CD33 expression and GO-induced cytotoxicity after 48 hours of drug exposure. Samples resistant to GO (as defined by <15% GO-induced apoptosis/cell death 24 hours after drug exposure) are shown in bold. Note: the CD33-QDot605 MFI for AML-11 was converted from CD33 PE MFI, as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0053518-g002: Relationship between CD33 level and GO-induced DNA damage and cytotoxicity.(A) Correlation between CD33 expression and GO-induced DNA damage, as quantified by measurement of γH2AX levels 6 hours after drug exposure. (B) Correlation between CD33 expression and GO-induced cytotoxicity after 48 hours of drug exposure. Samples resistant to GO (as defined by <15% GO-induced apoptosis/cell death 24 hours after drug exposure) are shown in bold. Note: the CD33-QDot605 MFI for AML-11 was converted from CD33 PE MFI, as described in Materials and Methods.
Mentions: The postulated mechanism of action of GO implies a pivotal role of drug-induced DNA damage and intact downstream pro- and anti-apoptotic pathway activity [5]. As depicted in Figure 1, our SCNP assay focused on functional, quantitative measurements of DNA damage pathways as well as PI3K/AKT, MEK/ERK, and JAK/STAT signaling pathways in leukemic cells in response to in vitro stimulation of diagnostic specimens from 6 pediatric and 6 adult patients containing >78% blast cells in all cases (Table 1; AML-01– AML-12). First, the in vitro GO sensitivity of AML cells in these specimens was characterized. At the clinically relevant concentration of 30 ng/mL [30], GO treatment resulted in an induced apoptotic/dead cell fraction averaging 19.3±5.2% and 43.8±7.1% after continuous exposure for 24 and 48 hours, respectively (Table 2). Using an arbitrary cut-off for the induced apoptotic/dead cell fraction of 15% at 24 hours, 5 specimens were classified as “GO sensitive”, whereas 7 were classified as “GO resistant”. The lack of apoptotic response in these GO resistant samples was likely directly due to insufficient intracellular GO concentrations rather than the consequence of dysfunctional apoptotic pathways, as parallel experiments with staurosporine, a classic activator of the mitochondrial apoptosis pathway [31], demonstrated robust apoptosis not only in GO-sensitive but also in most (5/6 tested) GO resistant samples (Figure S2), establishing that the apoptotic machinery was generally intact in the tested AML specimens. Of note, among all patient specimens, there was no correlation between CD33 expression levels and GO-induced cytotoxicity at 24 hours (r = 0.181 [95% confidence interval: −0.439–0.684], P = 0.574) or 48 hours (r = 0.266 [−0.397–0.747], P = 0.429). Importantly, GO failed to induce significant cytotoxicity after 24 or 48 hours in 2 cases (AML-07 and AML-08) with bright CD33 expression, indicating the presence of relevant resistance mechanisms that are unrelated to target antigen expression (Figure 2).

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