GSK3 inhibitor enhances gemtuzumab ozogamicin-induced apoptosis in primary human leukemia cells by overcoming multiple mechanisms of resistance.

In acute myeloid leukemia (AML), the heterogeneity of genetic and epigenetic characteristics makes treatment difficult. The prognosis for AML is therefore poor, and there is an urgent need for new treatments for this condition. Gemtuzumab ozogamicin (GO), the first antibody-drug conjugate (ADC), targets the CD33 antigen expressed in over 90% of AML cases. GO therefore has the potential to counter the heterogeneity of AML patients. However, a major clinical problem is that drug resistance to GO diminishes its effect over time. Here, we report that the inhibition of glycogen synthase kinase 3 (GSK3) alone overcomes several forms of GO resistance at concentrations without antileukemic effects. The GSK3 inhibitors tested significantly enhanced the cytotoxic effect of GO in AML cell lines. We elucidated four mechanisms of enhancement: (1) increased expression of CD33, the target antigen of GO; (2) activation of a lysosomal function essential for hydrolysis of the GO linker; (3) reduced expression of MDR1 that eliminates calicheamicin, the payload of GO; and (4) reduced expression of the anti-apoptotic factor Bcl-2. A similar combination effect was observed against patient-derived primary AML cells. Combining GO with GSK3 inhibitors may be efficacious in treating heterogeneous AML.

An mTORC1/2 dual inhibitor, AZD2014, acts as a lysosomal function activator and enhances gemtuzumab ozogamicin-induced apoptosis in primary human leukemia cells.

Gemtuzumab ozogamicin (GO), an anti-CD33 antibody linked to calicheamicin via an acid-labile linker, is the first antibody-drug conjugate (ADC). The acidic environment inside lysosomes of target cells is an important intracellular determinant of the cytocidal action of GO, as the linker is hydrolyzed under acidic conditions. However, lysosomal activity in acute myeloid leukemia (AML) blasts in GO therapy has been insufficiently evaluated. It has been suggested that lysosome activity is suppressed in AML due to hyperactivation of the phosphoinositide 3-kinase/Akt pathway. We therefore hypothesized that agents which activate lysosomal function would potentiate the cytotoxicity of GO. Here, we found that a clinically useful mTORC1/2 dual inhibitor, AZD2014, reduced pH in the acidic organelles, including lysosomes, as shown by increased LysoTracker fluorescent intensity, and synergistically enhanced the cytotoxic effect of GO in primary leukemia cells. GO-induced cytotoxicity appeared to be enhanced with the increase in lysosomal activity by AZD2014. These results indicate that AZD2014 activated lysosomal function in primary leukemia cells, which in turn enhanced the cytotoxicity of GO. Enhancement of lysosomal activity may represent a new therapeutic strategy in the treatment of GO and other ADCs, particularly in cases with low lysosomal activity.

An mTORC1/2 kinase inhibitor enhances the cytotoxicity of gemtuzumab ozogamicin by activation of lysosomal function.

Gemtuzumab ozogamicin (GO), the first antibody-drug conjugate (ADC), has attracted the interest of hematologists because more than 90% of acute myeloid leukemia (AML) blasts express its target, CD33. Although GO and subsequently developed ADCs depend on lysosomes for activation, lysosome number and activity in tumor cells has not been well elucidated. In this study, we investigated whether an mTORC1/2 kinase inhibitor, PP242, which was reported to activate lysosomal function, potentiates the cytotoxicity of GO in AML cells. Eight AML cell lines (U937, THP-1, SKM-1, SKK-1, SKNO-1, HL-60, MARIMO and KO52) were treated with GO and PP242. The cytotoxic effect of GO was enhanced by concurrent treatment with a non-cytotoxic concentration (500 nM) of PP242 in most cell lines, except MARIMO and KO52 cells. We then used LysoTracker to label acidic lysosomes in U937, THP-1, SKM-1, MARIMO and KO52 cells. LysoTracker fluorescence was dramatically increased by treatment with PP242 in U937, THP-1 and SKM-1 cells, and the intensified fluorescence was retained with PP242 + GO. In contrast, PP242 did not induce a significant increase in fluorescence in MARIMO cells, consistent with the lack of combinatory cytotoxicity. LysoTracker fluorescence was also increased by PP242 in KO52 cells, which have been reported to strongly express multidrug resistance (MDR). Further, PP242 suppressed GO-induced Chk1 activation and G2/M cell cycle arrest, which in turn triggered cell cycle promotion and cell death. These results indicate that inhibition of mTORC1/2 kinase by PP242 enhanced the cytotoxicity of GO by increasing lysosomal compartments and promoting the cell cycle via suppression of GO-induced Chk1 activation. This combination may represent an attractive new therapeutic strategy for the treatment of leukemia.

The Arf/p53 protein module, which induces apoptosis, down-regulates histone H2AX to allow normal cells to survive in the presence of anti-cancer drugs.

BACKGROUND: It is unclear how DNA-damaging agents target cancer cells over normal somatic cells. RESULTS: Arf/p53-dependent down-regulation of H2AX enables normal cells to survive after DNA damage. CONCLUSION: Transformed cells, which harbor mutations in either Arf or p53, are more sensitive to DNA-damaging agents. SIGNIFICANCE: Cellular transformation renders cells more susceptible to some DNA-damaging agents. Anti-cancer drugs generally target cancer cells rather than normal somatic cells. However, the factors that determine this differential sensitivity are poorly understood. Here we show that Arf/p53-dependent down-regulation of H2AX induced the selective survival of normal cells after drug treatment, resulting in the preferential targeting of cancer cells. Treatment with camptothecin, a topoisomerase I inhibitor, caused normal cells to down-regulate H2AX and become quiescent, a process mediated by both Arf and p53. In contrast, transformed cells that harbor mutations in either Arf or p53 do not down-regulate H2AX and are more sensitive to drugs unless they have developed drug resistance. Such transformation-associated changes in H2AX expression rendered cancer cells more susceptible to drug-induced damage (by two orders of magnitude). Thus, the expression of H2AX and γH2AX (phosphorylated form of H2AX at Ser-139) is a critical factor that determines drug sensitivity and should be considered when administering chemotherapy.

Mechanism of the alkali degradation of (6-4) photoproduct-containing DNA.

The (6-4) photoproduct is one of the major damaged bases produced by ultraviolet light in DNA. This lesion is known to be alkali-labile, and strand breaks occur at its sites when UV-irradiated DNA is treated with hot alkali. We have analyzed the product obtained by the alkali treatment of a dinucleoside monophosphate containing the (6-4) photoproduct, by HPLC, NMR spectroscopy, and mass spectrometry. We previously found that the N3-C4 bond of the 5' component was hydrolyzed by a mild alkali treatment, and the present study revealed that the following reaction was the hydrolysis of the glycosidic bond at the 3' component. The sugar moiety of this component was lost, even when a 3'-flanking nucleotide was not present. Glycosidic bond hydrolysis was also observed for a dimer and a trimer containing 5-methyl-2-pyrimidinone, which was used as an analog of the 3' component of the (6-4) photoproduct, and its mechanism was elucidated. Finally, the alkali treatment of a tetramer, d(GT(6-4)TC), yielded 2'-deoxycytidine 5'-monophosphate, while 2'-deoxyguanosine 3'-monophosphate was not detected. This result demonstrated the hydrolysis of the glycosidic bond at the 3' component of the (6-4) photoproduct and the subsequent strand break by ß-elimination. It was also shown that the glycosidic bond at the 3' component of the Dewar valence isomer was more alkali-labile than that of the (6-4) photoproduct.

Onset of quiescence following p53 mediated down-regulation of H2AX in normal cells.

Normal cells, both in vivo and in vitro, become quiescent after serial cell proliferation. During this process, cells can develop immortality with genomic instability, although the mechanisms by which this is regulated are unclear. Here, we show that a growth-arrested cellular status is produced by the down-regulation of histone H2AX in normal cells. Normal mouse embryonic fibroblast cells preserve an H2AX diminished quiescent status through p53 regulation and stable-diploidy maintenance. However, such quiescence is abrogated under continuous growth stimulation, inducing DNA replication stress. Because DNA replication stress-associated lesions are cryptogenic and capable of mediating chromosome-bridge formation and cytokinesis failure, this results in tetraploidization. Arf/p53 module-mutation is induced during tetraploidization with the resulting H2AX recovery and immortality acquisition. Thus, although cellular homeostasis is preserved under quiescence with stable diploidy, tetraploidization induced under growth stimulation disrupts the homeostasis and triggers immortality acquisition.

Lesion bypass by S. cerevisiae Pol ζ alone.

DNA polymerase zeta (Pol ζ) participates in translesion synthesis (TLS) of DNA adducts that stall replication fork progression. Previous studies have led to the suggestion that the primary role of Pol ζ in TLS is to extend primers created when another DNA polymerase inserts nucleotides opposite lesions. Here we test the non-exclusive possibility that Pol ζ can sometimes perform TLS in the absence of any other polymerase. To do so, we quantified the efficiency with which S. cerevisiae Pol ζ bypasses abasic sites, cis-syn cyclobutane pyrimidine dimers and (6-4) photoproducts. In reactions containing dNTP concentrations that mimic those induced by DNA damage, a Pol ζ derivative with phenylalanine substituted for leucine 979 at the polymerase active site bypasses all three lesions at efficiencies between 27 and 73%. Wild-type Pol ζ also bypasses these lesions, with efficiencies that are lower and depend on the sequence context in which the lesion resides. The results are consistent with the hypothesis that, in addition to extending aberrant termini created by other DNA polymerases, Pol ζ has the potential to be the sole DNA polymerase involved in TLS.

Characterization of the binding of distamycin A to damaged DNA: a comparison with the DNA recognition of the human DDB protein.

Distamycin A binds to DNA containing the (6-4) photoproduct, a major UV lesion that is recognized by the damaged DNA-binding (DDB) protein in human cells. We analyzed the binding properties of distamycin A and compared the results with those of the DDB protein. Structural change of the DNA duplex was not observed for distamycin A in two types of experiments, whereas the protein induced a large bending of the helix. Although the substrate specificity was different between the drug and the protein, thymine glycol was recognized by both of them, and inhibition of the DDB protein binding to the (6-4) photoproduct-containing DNA by distamycin A was tested.

Binding of distamycin A to UV-damaged DNA.

We have found that distamycin A can bind to DNA duplexes containing the (6-4) photoproduct, one of the major UV lesions in DNA, despite the changes, caused by photoproduct formation, in both the chemical structure of the base moiety and the local tertiary structure of the helix. A 20-mer duplex containing the target site, AATT.AATT, was designed, and then one of the TT sequences was changed to the (6-4) photoproduct. Distamycin binding to the photoproduct-containing duplex was detected by CD spectroscopy, whereas specific binding did not occur when the TT site was changed to a cyclobutane pyrimidine dimer, another type of UV lesion. Distamycin binding was analyzed in detail using 14-mer duplexes. Curve fitting of the CD titration data and induced CD difference spectra revealed that the binding stoichiometry changed from 1:1 to 2:1 with photoproduct formation. Melting curves of the drug-DNA complexes also supported this stoichiometry.

Analysis of distamycin A binding to UV-damaged DNA.

We have found that distamycin A can bind to DNA duplexes containing the (6-4) photoproduct, one of the major UV lesions in DNA, in spite of the changes caused by photoproduct formation in the chemical structure of the base moiety and the local tertiary structure of the duplex. Distamycin binding was analyzed in detail using 14-mer duplexes. Curve fitting of the CD titration data and induced CD difference Spectra revealed that the binding stoichiometry changed from 1:1 to 2:1 with the photoproduct formation. Melting curves of the drug-DNA complexes also supported this stoichiometry.

Identification and characterization of an intermediate in the alkali degradation of (6-4) photoproduct-containing DNA.

The (6-4) photoproduct formed by ultraviolet light is known as an alkali-labile DNA lesion. Strand breaks occur at (6-4) photoproducts when UV-irradiated DNA is treated with hot alkali. We have analyzed the degradation reaction of this photoproduct under alkaline conditions using synthetic oligonucleotides. A tetramer, d(GT(6-4)TC), was prepared, and its degradation in 50 mm KOH at 60 degrees C was monitored by high performance liquid chromatography. A single peak with a UV absorption spectrum similar to that of the starting material was detected after the reaction, and this compound was regarded as an intermediate before the strand break. The formation of this intermediate was compared with intermediates from the degradation of other alkali-labile lesions such as the abasic site, thymine glycol, and 5,6-dihydrothymine. The results strongly suggested that the first step of the alkali degradation of the (6-4) photoproduct was the hydrolysis between the N3 and C4 positions of the 5'-pyrimidine component. Analyses by NMR spectroscopy and mass spectrometry supported the chemical structure of this product. Assays of the complex formation with XPC.HR23B and the translesion synthesis by DNA polymerase eta revealed that the biochemical properties are indistinguishable between the intact and hydrolyzed photoproducts.

Towards artificial repair of UV-damaged DNA: studies on drug binding and alkali hydrolysis.

Properties of the DNA containing the (6-4) photoproduct, one of the major UV-induced lesions, were analyzed. Two basic studies towards artificial recognition and repair of this type of damaged DNA are presented here. One is recognition of the UV-damaged DNA by a minor groove-binding drug. It was found by CD spectroscopy that distamycin could bind DNA duplexes containing the (6-4) photoproduct as effectively as the unmodified DNA, whereas a DNA duplex containing the cyclobutane pyrimidine dimer was not recognized by this drug. The other is a mechanistic study on alkali degradation of this photoproduct. HPLC and NMR analyses revealed that hydrolysis between the N3 and C4 positions of the 5' pyrimidine component occurred first. This intermediate was relatively stable, and further degradation to the strand break required severe conditions like the hot piperidine treatment.