Induction of DNA strand breaks is critical to predict the cytotoxicity of gemtuzumab ozogamicin against leukemic cells

Abstract

Gemtuzumab ozogamicin (GO) consists of the CD33 antibody linked to calicheamicin. The binding of GO to the CD33 antigen on leukemic cells results in internalization followed by the release of calicheamicin, thereby inducing DNA strand breaks. We hypothesized that the induction of DNA strand breaks would be a surrogate marker of GO cytotoxcity. Here, two GO-resistant variants (HL/GO-CSA [225-fold], HL/GO [200-fold]) were established by serially incubating human leukemia HL-60 cells with GO with or without a P-glycoprotein (P-gp) inhibitor, cyclosporine A, respectively. The CD33 positivity was reduced in both variants. The HL/GO-CSA cells showed an increased multidrug resistance protein-1 (MRP1) transcript, and an MRP1 inhibitor partially reversed GO resistance. The HL/GO cells had neither P-gp nor MRP1 overexpression. Microarray analysis and Western blotting indicated elevated levels of DNA repair-associated proteins in both variants. Two other leukemic subclones, showing either P-gp or MRP1 overexpression, were also GO-resistant. Using single cell gel electrophoresis analysis, it was determined that GO-induced DNA strand breaks increased dose-dependently in HL-60 cells, whereas the number of breaks was reduced in the GO-resistant cell lines. The induction of DNA strand breaks was correlated with GO sensitivity among these cell lines. The CD33 positivity and the expression levels of transporters were not proportional to drug sensitivity. Using primary leukemic cells, the induction of DNA strand breaks appeared to be associated with GO sensitivity. Thus, GO-induced DNA strand breaks as the final output of the mechanism of action would be critical to predict GO cytotoxicity. (Cancer Sci 2012; 103: 1722-1729 T o improve therapeutic outcomes for patients with AML, new treatment regimes and agents are needed.(1-4) Gemtuzumab ozogamicin (Mylotarg; Wyeth-Ayers Research, Pearl River, NY, USA) is a humanized mAb directed against the CD33 surface antigen that is conjugated to a derivative of the cytotoxic antibiotic calicheamicin. (5) CD33 is an antigen normally expressed on early myeloid progenitor cells in normal bone marrow and on leukemic blasts in 90% of all newly diagnosed AML but not on normal stem cells.(6) Because CD33 is specific to leukemic cells, GO is an attractive targeted agent that could improve the clinical outcome of AML chemotherapy without increasing toxicity. Gemtuzumab ozogamicin received marketing approval from the US Food and Drug Administration under accelerated approval regulations for the treatment of patients with CD33-positive AML who are in the first relapse, are 60 years of age or older, and who are not considered candidates for cytotoxic chemotherapy.(7) After approval, however, the Southwest Oncology Group compared GO plus standard induction therapy versus standard induction therapy alone, and found that there was no difference in disease-free survival between the two treatment regimes.(8) One major reason why the study was negative for GO's additional efficacy was that the dose of DNR was ...

Early changes in rpS6 phosphorylation and BH3 profiling predict response to chemotherapy in AML cells

“…Data from Fig 1 suggests early changes in rpS6 phosphorylation may be used as a predictor of chemotherapeutic response in AML cells. To test this hypothesis, 11 AML cell lines were subjected to 48 hour dose response assays with the following agents: FLT3 inhibitors (AC220 and sorafenib),[ 29 , 30 ] DNA double strand break (DSB) inducers (etoposide, GO and vosaroxin),[ 31 – 33 ] the standard-of-care drug cytarabine and the hsp90 inhibitor 17-AAG. [ 34 ]…”

Unlocking the potential of anti-CD33 therapy in adult and childhood acute myeloid leukemia

“…They saw upregulation of DNA repair proteins at the gene and protein level (XRCC5, PARP1, RPA, GADD45A) compared to sensitive cell lines providing support for DNA repair pathways in resistance to GO. 72 They also provide evidence to suggest that CD33 expression levels, drug efflux transporters and upregulation of DNA repair pathways all contribute in part to the development of GO resistance (Figure 3). GO attaches to CD33 on cell surface and the complex is internalised, where the bifunctional linker is hydrolysed within the lysosome.…”

“…61 In the acidic conditions of the lysosome, the bifunctional linker is hydrolysed and the free CalichDMH is reduced to a diradical species, that binds to the minor groove of DNA, causing single and double-strand DNA breaks and a strong DNA damage response, with phosphorylation of the minor histone 2A variant (H2AX) and increased activation of DNA-dependent protein kinase (DNA-PK). 60,72 The IgG4 hP67.6 antibody alone is not associated with cell death but acts as the vehicle for drug delivery. Human cells exposed to GO in vitro, undergo cell cycle arrest at G2/M phase and cells either undergo apoptosis or can escape cell death through DNA repair.…”

“…74,61,70 Members of the ATP-binding cassette (ABC) superfamily of transporter proteins, P-glycoprotein (Pgp/MDR1) and multi-drug resistance protein 1 (MRP-1) are thought to reduce sensitivity and DNA damage by efflux of CalichDMH out of the cell. 72 Yamauchi et al, created GO-resistant cell lines by incubating the human cell line HL-60 with increasing doses of GO over an 8 month time frame followed by limited dilution cell clone selection. They saw upregulation of DNA repair proteins at the gene and protein level (XRCC5, PARP1, RPA, GADD45A) compared to sensitive cell lines providing support for DNA repair pathways in resistance to GO.…”

Antibody-based therapy of acute myeloid leukemia with gemtuzumab ozogamicin

“…Conceptually, the intracellular load of activated calicheamicin-γ 1 I is impacted by the amount of GO uptake, the efficacy of toxin release from the antibody and subsequent activation via cellular thiols, as well as toxin inactivation/metabolism or expulsion. However, while induction of DNA damage appears to be a prerequisite for GO-induced cytotoxicity (105, 106), it is not sufficient, indicating that the toxicity of the calicheamicin-γ 1 I moiety is modulated by the cell’s ability to repair DNA damage and the activity of downstream pro- and anti-apoptotic pathways. Overall, the sensitivity to the toxic moiety varies over 100,000-fold between individual primary AML cells samples (107), an observation that emphasizes the importance of patient-specific factors for the clinical efficacy of GO.…”