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. Author manuscript; available in PMC: 2009 Apr 1.
Published in final edited form as: Mol Cancer Ther. 2008 Apr;7(4):769–778. doi: 10.1158/1535-7163.MCT-08-0130

Small molecules targeting Histone H4 as potential therapeutics for chronic myelogenous leukemia

C James Chou 1, Michelle E Farkas 2, Sherry M Tsai 2, David Alvarez 1,†, Peter B Dervan 2,*, Joel M Gottesfeld 1,*
PMCID: PMC2376757  NIHMSID: NIHMS47642  PMID: 18413791

Abstract

We recently identified a polyamide-chlorambucil conjugate, 1R-Chl, that alkylates and down-regulates transcription of the human histone H4c gene, and inhibits the growth of several cancer cell lines in vitro and in a murine SW620 xenograft model, without apparent animal toxicity. In this study, we analyzed the effects of 1R-Chl in the chronic myelogenous leukemia cell line K562, and identified another polyamide conjugate, 6R-Chl, which targets H4 genes and elicits a similar cellular response. Other polyamide conjugates that do not target the H4 gene do not elicit this response. In a murine model, both 1R-Chl and 6R-Chl were found to be highly effective in blocking K562 xenograft growth with high dose tolerance. Unlike conventional and distamycin-based alkylators, little or no cyto- and animal toxicities were observed in mg/kg dosage ranges. These results suggest that these polyamide alkylators may be a viable treatment alternative for chronic myelogenous leukemia.

Keywords: chlorambucil, polyamide, cancer, growth-arrest, K562 cells

Introduction

Therapies targeting specific genes or gene products are a major aim of modern cancer biology and intervention (1–4). Notable recent successes in the field include both mononclonal antibodies (5, 6) and small molecules that target enzymes and receptors that are over-expressed or mutated in various cancers (3, 7, 8). Notwithstanding these important developments, DNA alkylating agents remain the most common drugs for treatment of several solid and hematological malignancies (9–11). Myelotoxicity and concomitant cytotoxicity due to limited DNA sequence selectivity is often the dose-limiting factor for use of these compounds in humans. Alkylating agents based on the minor groove binder distamycin A, including Tallimustine and Brostallicin, have shown improvements over non-conjuated DNA alkylating agents in terms of affinity and specificity at the nucleotide level (12–15). However, these compounds possess only limited specificity (16), and myelotoxcity is still the dose-limiting factor in establishing an effective chemotherapy (12, 13).

Pyrrole-imidazole (Py-Im) polyamides are a class of sequence-specific DNA- binding small molecules that have been shown to have high binding specificity and affinity, with some molecules even exceeding the binding affinities of transcription factors (17). Many polyamides are cell permeable and readily localize to the nuclei of cultured cells (18–21). Polyamides are effective inhibitors of RNA transcription when they disrupt essential protein-DNA interactions at promoter and enhancer elements (17, 22–25), and they may also modulate gene expression by modifying chromatin structure (26–28). Linked with DNA-alkylating agents, such as chlorambucil (Chl) (29) or CC-1065/CBI derivatives (30, 31), polyamide conjugates react covalently with DNA at specific sites and inhibit transcription by stalling RNA polymerase during elongation.

Recently, α-diaminobutyric acid-linked hairpin polyamide-Chl conjugates have been shown to bind and alkylate DNA sequences both in vitro and in cell culture models, with good sequence specificity (32–34). Initial screens of various hairpin polyamide-Chl conjugates have shown the polyamide 1R-Chl (Fig. 1A, top) to be an inhibitor of cell proliferation in various cancer cell lines with no apparent cytotoxicity and little or no murine animal toxicity (32, 33, 35). This molecule binds within the coding region of the histone H4c gene both in vitro and in SW620 human colon carcinoma cells, and down-regulates H4c transcription. Polyamides with similar pyrrole and imidazole compositions targeted to different DNA sequences failed to alkylate the coding region of the histone H4c gene and were found to be inactive in both cell culture and a SW620 xenograft cancer model (32).

Fig. 1.

Fig. 1

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DNA sequence of the coding region of the human histone H4c gene and chemical structures of 1R-Chl and 6R-Chl. A. Chemical structures of 1R-Chl (top) and 6R-Chl (bottom), which target the DNA sequences 5′-WGGWGW-3′ and 5′-WGWGCW-3′, respectively (where W = A or T). Imidazole rings are shown in bold. B. DNA sequence of the coding region of the human histone H4c gene. Potential binding sites for 1R-Chl and 6R-Chl are in bold. The alkylation sites of 1R-Chl and 6R-Chl as verified by LM-PCR are italic-bold and underlined, respectively.

Studies suggest a two-hit mechanism for the observed cellular effects of 1R-Chl: down-regulation of histone gene transcription causes nucleosome depletion, followed by widespread alkylation of open chromatin, which elicits cell cycle arrest through the DNA repair pathway (35). While our initial results point to the histone H4c gene as the major target of 1R-Chl, microarray studies in the SW620 cancer cell line indicate that the mRNA levels of several other genes are also affected (32). Thus, down-regulation of other genes may be involved in the cellular response to 1R-Chl.

In the present study we extend our analysis to the well-established chronic myelogenous leukemia (CML) cell line K562. If histone H4 genes are the primary targets of 1R-Chl that lead to a block in cancer cell proliferation (35), other polyamide-Chl conjugates targeting H4 genes would be predicted to elicit the same cellular response. We describe the synthesis and characterization of a small library of constitutional isomers of 1R-Chl. These molecules have the same chemical composition as 1R-Chl but would be expected to bind different DNA sequences.

One conjugate, 6R-Chl (Fig. 1A, bottom), which targets sites adjacent to and overlapping the binding site for 1R-Chl (Fig. 1B) in the H4c gene was found to have biological properties similar to 1R-Chl in both K562 cell culture and in a mouse xenograft model established with K562 cells. Other polyamide-Chl alkylators that did not bind within the H4c gene or down-regulate histone H4 expression had no effect on cell proliferation. Microarray analysis in K562 cells reveals that the histone H4 genes H4c and H4j/k are down-regulated by 1R-Chl treatment. Transcripts for the H4k and H4j genes cannot be distinguished due to similarity in sequence. In addition, we explored the pharmacokinetic properties of 1R-Chl, the results of which point to this class of molecules as potential human cancer therapeutics.

Materials and Methods

Synthesis and characterization of pyrrole-imidazole polyamides

Pyrrole-imidazole (Py-Im) polyamides were synthesized by standard solid phase methods (36), using α-(R)- or α(S)-2,4-diaminobutyric acid as the hairpin turn unit (33). Polyamide-Chl conjugates were generated as previously described (29), whereby the carboxylic acid of Chl (Sigma-Aldrich, WI) is activated and coupled to the free amine of the hairpin turn. The identity and purity of the compounds were established by mass spectrometry analysis (MALDI-TOF-MS and ES-MS) and analytical HPLC, respectively.

Binding affinities of the parent polyamides (which lack chlorambucil) for target match and mismatch sites were determined by quantitative DNase I footprinting (37), using a radiolabeled DNA fragment of the plasmid pMFST6 (Supplementary Fig. 1A). The plasmid was constructed by annealing the oligonucleotide pair: 5′-AGCTGTAGTATCTATAGTGCTTAC-TATCTATAGTCCTTACTATCTATAGCTCTTACTATCTATAGCTGTTACTATCTATACT-ATCTAC-3′and 5′-CATCATAGATATCACGAATGATAGATATCAGGAATGATAGAT-ATCGAGAATATAGATATCGACAATGATAGATAGATAGATGCTAG-3′. Annealed oligonucleotides were ligated into the BamHI/HindIII restriction fragment of pUC19 using T4 DNA ligase, and the plasmid was transformed into Escherichia coli JM109 competent cells. Ampicillin-resistant white colonies were selected from 25 mL Luria-Bertani agar plates containing 50 mg/mL ampicillin treated with XGAL and isopropyl-β-D-thiogalactopyranoside (IPTG) solutions and grown overnight at 37 °C. Cells were harvested the following day, and purification of the plasmid was performed with a Wizard Plus Midiprep DNA purification kit (Promega). Alkylation experiments performed on the pMFST6 insert were performed as previously described (29). Alkylation experiments were also performed on a 240 bp region of the H4c mRNA-coding sequence, which was amplified from genomic DNA with the following PCR primers: 5′-GTGCTAAGCGCCATCGTAAG-3′ and 5′-CCCTGACGTTTTAGGGCATA-3′. These experiments were conducted using 10 ng of the PCR product incubated for 16 h at 37°C in 100 μL of 20 mM NaCl, 10 mM Tris-Cl, pH 7.4, with each of the polyamide-Chl conjugates at 10, 100 and 1000 nM concentration, followed by thermal cleavage and primer extension labeling, as described (32).

Cell lines and cell viability assays

The human CML lymphoblast cell line K562 (purchased from ATCC), which contains the b3a2 Bcr-Abl translocation, was used in this study. Cells were grown and maintained in RPMI 1640 medium containing 10% FBS, under standard mammalian cell culture conditions as recommended by the ATCC. Direct phase contrast microscopic visualization was used to monitor the effects of polyamide-Chl conjugates on cell growth rates and cell morphology. Promega CellTiter 96 Aqueous One Solution Cell Proliferation assay (Promega, WI; utilizing [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS) conversion to formazan to examine mitochondrial activity), Trypan blue exclusion (Vi-Cell XR viability analyzer, Beckman Coulter, CA), and Annexin V-FITC/propidium iodide (PI) apoptosis staining (BD Pharmingen, CA) were used to determine cell proliferation (EC50), viability, and initiation of apoptosis, respectively.

Cell cycle analysis

The effects of polyamide-Chl conjugates on cell cycle progression were monitored by flow cytometry analysis in the Scripps FACS core facility. Polyamide-treated cells (250 nM of polyamide in culture media for 24 h) were collected by centrifugation (200X g for 5 min). Cell pellets were re-suspended in 500 iL of PBS and fixed with addition of 4.5 mL of pre-chilled 70% ethanol, stained with propidium iodide (50 μg/mL), and analyzed for DNA content, reflecting the fraction of cells at each point in the cell cycle (Go/G1, S, and G2/M). Cells with less than a 2C DNA content are indicative of DNA fragmentation and apoptosis.

Ligation-mediated PCR

K562 CML cells were incubated in culture medium for 24 h with each of the polyamide-Chl conjugates at a 250 nM concentration, followed by digestion of purified DNA with DraI, thermal cleavage and ligation-mediated PCR, using the primers for the H4c gene listed above, as previously described (32).

Real-time quantitative RT-PCR

RNA from polyamide-treated cells was extracted using the Absolutely RNA® Miniprep kit (Stratagene, CA). RT-PCR was performed using iScript One-Step TR-PCR kit with SYBR green (Bio-Rad Laboratories, CA) in accordance with the manufacturer’s instructions. Levels of H4c, H4k/j, SMG1, RPL13, HNRPD, RAB35, and H2AFY transcripts were quantified by amplifying a segment of their respective mRNAs with appropriate primer sets (Supplementary Table 1). The reverse-transcription reaction was carried out at 50°C for 10 min, followed by iTaq hot-start DNA polymerase activation by heating at 95°C for 15 min. Three-step cycling was performed: denaturation – 15 s at 95°C, annealing – 30 s at 55°C, and extension – 30 s at 72°C, for 45 cycles. All gene expression levels were normalized by parallel amplification and quantification of mRNA levels for the housekeeping gene glyceraldehyde-3-phospahte dehydrogenase (GAPDH) mRNA, as an endogenous reference with the following primers: 5′-GAGTCAACGGATTTGGTCGT-3′ and 5′-GAGGTCAATGAGGGGTCAT-3′.

SDS-PAGE and Western blot analysis

Equal numbers of cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% deoxycholate, 1 mM EDTA) with 1 X complete mini protease inhibitor cocktail (Roche Diagnostics, IN) for 30 min at 4°C followed by 15 s sonication pulses (Branson Sonifier-150 at 3 watts). Cell lysates were then centrifuged at 14,000 rpm (12,000 g) for 15 min and the supernatants removed and combined with LDS sample loading buffer (Invitrogen, CA). SDS-PAGE was performed with the Invitrogen NuPAGE system using 4–12% Bis-Tris gels and MES running buffer (Invitrogen, CA). Electrophoresis was carried out at 200 V for 45 min, and gel contents were transferred at 30 V for 1 h to a 0.2 μm nitrocellulose membrane. Membranes were then blocked with 5% bovine serum albumin for 1 h at 4°C and probed with histone H4 or GAPDH (Abcam, CA) primary antibodies. Protein-antibody complexes were then visualized by enhanced chemiluminescence using Amersham ECL system (GE Healthcare, UK), with either anti-rabbit or anti-goat horseradish peroxidase (HRP) conjugated secondary antibodies (Santa Cruz Biotechnology, CA).

Xenograft studies of 1R-Chl, 6R-Chl, and 1S-Chl

Female athymic nude mice were purchased from The Scripps Research Institute Division of Animal Resources. Experimental protocols were approved by the Scripps Institutional Animal Welfare Committee. K562 cells were suspended to 50 million cells/mL in Matrigel™ (BD Biosciences, CA), 0.2 mL of which was subcutaneously injected into the rear left flank of each mouse (6–8 wks of age). Mice were monitored and tumor sizes measured daily; tumor volumes were calculated as ½ length × (width)2. Tumors were staged for 7 to 14 d to enter growth phase. At this point compounds were administered at a dose of 7.5mg/kg per injection, via the tail vein three times over a five-day period.

Pharmacokinetic properties of 1R-Chl and 1S-Chl

Pharmacokinetic (PK) studies were performed in normal Balb/c mice in order to determine plasma levels of the compounds 1R-Chl and 1S-Chl over time, and to calculate their constant of elimination (kel), half-life (t1/2), and volume of distribution (Vd). 48 female Balb/c mice were divided into three groups of 16 mice each. Group 1 received 100 μL (100 nmol) of 1mM 1R-Chl, group 2 received the same amount of 1S-Chl, and group 3 received an equal volume of PBS (control) via I.V. bolus injection. 0.5 mL of blood was collected from each animal at specific time-points (0, 5, 15, 30 min and 1, 4, 8 and 24 h) in heparinized tubes, then allowed to coagulate at ambient temperature for 24 h, and centrifuged at 3,100 rpm for 10 min to separate the serum from the clot.

Concentrations of 1R-Chl and 1S-Chl in the serum were determined by mass spectrometry (see below). Each time-point had two animals per group. Data collected was plotted as concentration (y-axis) vs. time (x-axis), and subjected to first order kinetic analysis in order to calculate the PK parameters. From this graph, t1/2was calculated, and from the natural logarithm (ln) of the concentration vs. time, kel (slope) and Vd (dose/anti-ln y-intercept) were determined (y-intercept represents the theoretical concentration at t = 0). Vd for humans was predicted by multiplying the Vd by 70 kg (average human weight).

1R-Chl distribution in the mouse body was analyzed as follows: mice were injected with 500 nmol of 1R-Chl or PBS and euthanized after either 2 h or 24 h; organs were fixed in formalin. A small piece from each major organ was weighed, homogenized in PBS buffer, and sonicated by two pulses of 15 s each. 1R-Chl concentration was determined by mass spectrometry (see below).

Mass spectrometry determination of 1R-Chl and 1S-Chl

A control polyamide-Chl conjugate, with the sequence ImImPyIm- (R)Chlγ-PyPyPyPy-β-Dp, was used as an internal standard for concentration determination by mass spectrometry. The molecular mass of this molecule differs from 1R-Chl and 1S-Chl by 79.1 amu. From each sample, a 200 iL serum aliquot was taken to which 2 iL of 100 pmol/iL control polyamide solution was added along with 800 iL of chilled 100 % methanol. Samples were vigorously vortexed for 1 min and then incubated for 15 min at 4ºC. They were subsequently centrifuged at 12,000 rpm for 10 min, and the supernatant was harvested and concentrated to 50 iL by methanol extraction. Standards with concentrations from 100 to 12,500 fmol/iL of 1R-Chl or 1S-Chl were prepared in serum and extracted by the above procedure. Concentrations were determined by electrospray mass spectrometry with an Agilent® 1100 single quadrapole instrument coupled to an Agilent®1100 liquid chromatography system, and a 50 mm × 2.0 mm C18 column for injection and separation.

Results

Only polyamides targeting histone H4 genes are potent inhibitors of K562 cell proliferation

To investigate the effects of Py-Im polyamide-Chl conjugates on K562 cells, we synthesized a small library of constitutional isomers of 1R-Chl, along with its stereoisomer, 1S-Chl (Fig. 2A). All molecules targeted the general DNA sequence 5′-WGNNNW-3′ (where W = A or T; N = A, C, G, or T); the binding site for 1R-Chl is 5′-WGGWGW-3′. Binding affinities for non-alkylating parent molecules were determined via quantitative DNase I footprinting on plasmid inserts containing match sites (Fig. 2A). It has been previously shown that attachment of the Chl moiety does not appear to affect DNA binding properties (29). Affinities for 1R, 3R, 5R, and 1S have been previously reported (33–35); polyamides 4R and 6R were analyzed on plasmid insert pMFST6 (Supplementary Fig. 1B). All polyamides were found to bind their expected match sites.

Fig. 2.

Fig. 2

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Analysis of a polyamide library. A. Shown in the table are the following: ball-and-stick model for the structure of each polyamide (closed circles are imidazoles, open circles are pyrroles, diamonds are β-alanines, semicircle with positive sign is dimethylaminopropylamine, and X indicates a free amino group in the parent polyamide or Chl in the conjugates); targeted match binding sites for the polyamides, where W = A or T, (names of plasmid constructs on which footprinting and alkylation assays were performed are shown in parentheses); equilibrium association constants of the parent polyamides as determined by quantitative DNase I footprinting (standard deviations are in parentheses); the effective concentration 50 (EC50) against K562 cell proliferation was determined by MTS assay (standard deviations are in parentheses); effects of polyamide-Chl conjugates on cell morphology (enlargement) and growth. B. Effect of polyamides on growth and viability of K562 cells. Cells were incubated with the indicated polyamides at 250 nM for 3 d. Only 1R-Chl and 6R-Chl affected cell proliferation. C. Phase microscopy images illustrate that 1R-Chl and 6R-Chl induce an increase in cell volume at 250nM after 3 d. Treatment with all other polyamide conjugates and chlorambucil seem similar to control and do not show similar cell enlargement (Supplementary Fig. 4A). D. G2/M cell cycle arrest is observed for 1R-Chl and 6R-Chl after 24 h of treatment with 250 nM polyamide (top). 6R-Chl caused a greater G2/M cycle arrest than 1R-Chl; all other polyamide-treated cells appear similar to control and do not show any significant cycle perturbation (Supplementary Fig. 4B). AnnexinV/PI staining of 1R-Chl and 6R-Chl treated cells after 3 and 6 d. Treatment of cells with 250 nM 1R-Chl or 6R-Chl resulted in increased AnnexinV-positive and PI-negative populations and AnnexinV-positive and PI-positive populations. Treatments with other polyamide conjugates are similar to control and do not result in an effect (Supplementary Fig. 4C).

The ability of each polyamide-Chl conjugate to alkylate nucleotides adjacent to its match binding sites on synthetic DNA constructs was also evaluated. Alkylation profiles of polyamides 1R-Chl, 3R-Chl, 5R-Chl, and 1S-Chl have been previously reported (33–35); polyamides 4R-Chl and 6R-Chl were analyzed on plasmid insert pMFST6 (Supplementary Fig. 1C). All polyamides alkylated DNA in the nanomolar concentration range. H4c gene-specific DNA alkylation activities were monitored with the polyamide-Chl conjugates on a PCR product derived from the H4c gene. Significantly, only 1R-Chl and 6R-Chl have binding sites within the H4c gene and only these compounds effectively alkylate the H4c PCR product in vitro (Supplementary Fig. 2). 6R-Chl targets the DNA sequence 5′-WGWGCW-3′; the sequence 5′-TGTGCT-3′ is found both adjacent to and overlapping the binding site for 1R-Chl on the top strand of the H4c and H4k/j genes (5′-AGGTGT-3′, Fig. 1B).

Polyamide-Chl DNA alkylators were then tested for effects on growth and morphology of K562 cells. Only cells treated with either 1R-Chl or 6R-Chl exhibited growth inhibition at 250 nM; all others were inactive against K562 proliferation at the same concentration. The control polyamide 5R-Chl has EC50 of 4.2 μM, and 3R-Chl, 4R-Chl, and 1S-Chl have EC50 s > 10 μM (Fig. 2A and 2B; Supplementary Fig. 3). This reflects a minimum 16-fold difference in EC50 values for the active polyamide-conjugates relative to the controls. 1R-Chl and 6R-Chl also caused an enlargement of cell volume at 250 nM and induced G2/M cell cycle arrest (Fig. 2C and 2D). Following treatment with 1R-Chl or 6R-Chl for 3 to 6 d, an increase in the number of apoptotic cells was observed via annexin V-FITC/propidium iodine FACS analysis (Fig. 2D). Other polyamide-Chl conjugates had no effect on apoptosis (Supplementary Fig. 4).

Alkylation of the H4 genes by 1R-Chl and 6R-Chl in K562 cells

Ligation-mediated PCR (LM-PCR) (38) was used to determine sites of alkylation by 1R-Chl and 6R-Chl on the H4c gene in K562 cells. Cells were incubated with each of the hairpin polyamide-Chl conjugates for 24 h at a concentration shown to cause growth arrest with 1R-Chl and 6R-Chl (250 nM). After purification of genomic DNA and digestion with the restriction enzyme DraI, the DNA was heated to induce strand breakage at sites of alkylation (29). LM-PCR with H4c gene-specific primers demonstrated that both 1R-Chl and 6R-Chl alkylate the H4c gene in cultured K562 cells. Consistent with in vitro alkylation results, other molecules in the library failed to alkylate the H4c gene in cell culture (Fig. 3).

Fig. 3.

Fig. 3

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Alkylation of the H4c gene in K562 cells. Cells were incubated with the indicated polyamide-Chl conjugates for 24 h, followed by DNA purification and LM-PCR. The radiolabeled primer interrogates alkylation events on the bottom, coding strand of DNA. “0” indicates a no polyamide control, and “G” denotes a guanine-only sequencing reaction performed on a PCR product from the H4c gene, followed by LM-PCR. Binding sites for 1R-Chl (bold) and 6R-Chl (underlined) are shown at left. Both molecules alkylate the adenine at the 5′ end of their respective binding site. Non-specific background bands appear in all lanes and are unrelated to the polyamides.

RT-PCR and Western blot anaylses of 1R-Chl in K562 Cells

Quantitative real-time PCR was used to verify the results of the expression arrays for several significant genes from K562 microarray studies (Gene Expression Omnibus accession number GSE8832). The expression of histone H4 mRNAs H4c and H4k/j were decreased more than 60% compared to untreated control cells (Fig. 4A). Identical DNA binding and alkylation sites for 1R-Chl are present in the H4c, H4k, and H4j genes. Western blot analysis for total histone H4 protein in whole cell lysate shows significant down-regulation of total H4 protein levels after 24 h of incubation with 1R-Chl (Fig. 4B). Surprisingly, several of the most down-regulated and highly significant genes, based on p-values and extent of down-regulation in microarray analysis (i.e. SMG1, RPL13, HNRPD, RAB35, and H2AFY) appeared not to be significantly affected by 1R-Chl when analyzed by real-time qRT-PCR (Supplementary Fig. 5).

Fig. 4.

Fig. 4

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Effect of polyamides on histone H4c and H4k/j transcript expression and Western blot analysis. A. Chart showing polyamide effect on H4c and H4k/j expression; only 1R-Chl and 6R-Chl caused gene down-regulation. B. K562 cells were treated with 1R-Chl and 6R-Chl, at the indicated concentrations for 24 h, and histone H4 and GAPDH protein levels were assayed by Western blot analysis (top left and bottom right, respectively). After incubating with the secondary antibody, the membrane was washed 3 times and soaked in ECL Western Blotting reagents for 30 sec. The membrane was visualized by autoradiography films after 5–30 sec exposure (5 sec for H4 and 30 sec for GAPDH). Analysis of treatment with 1S-Chl and 3R-Chl (top right and bottom left, respectively) did not show significant decreases in histone H4 levels.

1R-Chl and 6R-Chl arrest the growth of K562 xenografts

K562 cells were injected into athymic nude mice, and tumors were staged to >100 mm3 (approximately 7 to 14 d post injection of cells). In a first experiment, mice were intravenously injected with three doses of 1R-Chl, 1S-Chl, or PBS vehicle control (Fig. 5A, left); for a second experiment mice were injected with 1R-Chl, 6R-Chl, or PBS vehicle (Fig. 5A, right). The molecule 1S-Chl was used as a control polyamide; although it targets the same sequence as 1R-Chl, the opposite stereochemistry of its turn unit results in a greatly reduced ability to bind and alkylate DNA (32, 33). The dosage routine was 7.5 mg/kg per injection and the polyamides were administered on treatment days 0, 2, and 5. The treatment dose of 1R-Chl was determined based on the LD20 of Tallimustine and Brostallicin, both of which have shown severe dose limiting toxicities in the murine model and during phase II clinical trails (9, 39).

Fig 5.

Fig 5

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Murine K562 xenograft studies. A. Athymic nude mice were injected with K562 cells and tumors were allowed to develop for either 7 d (left) or 14 d (right), after which mice were treated with the indicated polyamides; tumor volumes were determined 14 d later. B. Representative photographs of 1R-Chl, 6R-Chl, 1S-Chl, and PBS-treated animals. C. mRNA was isolated from tumor xenografts after treatment with 1R-Chl and 1S-Chl, 24 h after the last injection. Real-time qRT-PCR showed down-regulation of H4c and H4k/j transcripts in vivo by 1R-Chl; 1S-Chl did not significantly down-regulate either H4c or H4k/j. D. Kaplan-Meier survival plot showing extended lifespan of 1R-Chl and 6R-Chl treated mice. The life extensions by both 1R-Chl and 6R-Chl are approximately 2 wks with p = 4.44 × 10−7 and 4.64 × 10−5, respectively.

Mice treated with 1R-Chl and 6R-Chl showed immediate growth regression of the tumor xenografts, while 1S-Chl and PBS vehicle had no effect on tumor growth (Fig. 5A and 5B). Importantly, all mice treated with either 1R-Chl or 6R-Chl appeared healthy with minimal weight loss, while all 1S-Chl and PBS vehicle treated mice showed signs of wasting with significant weight loss due to exponential tumor growth. No obvious toxicity was associated with polyamide treatment. Based on the T/C% (tumor volume analysis) and LCK values (tumor growth delay; Log cell kill), 1R-Chl and 6R-Chl treatments are highly effective: according to the National Cancer Institute standards, T/C < 42% and LCK > 0.7 are minimal levels for activity (39). For the two experiments conducted, the T/C% for 1R-Chl treated mice versus untreated control were 24.4% and 12.52%, and LCK was 1.41 and 1.57, respectively. For 6R-Chl the T/C% was 11.30% and LCK was 1.63. In contrast, treatment with 1S-Chl gives results outside of the therapeutic range (T/C% = 78.13 and LCK = 0.20).

24 h following the final 1R-Chl treatment, K562 xenograft tumors were dissected and their mRNA was isolated for qRT-PCR analysis. Both H4c and H4k/j transcripts were observed to be down-regulated in tumors from 1R-Chl treated mice. Analysis of tumor samples from 1S-Chl and PBS treated mice revealed no effect on histone H4c/k/j mRNA levels (Fig. 5C), consistent with in vitro results.

A Kaplan-Meier survival plot for mice treated with 1R-Chl or 6R-Chl versus PBS shows a significant extension in lifespan, even after only one dosing regimen (Fig. 5D). We expect that prolonged administration of these polyamide-Chl conjugates would extend lifespan even further, and will be determined in future studies.

Pharmacokinetic parameters and pharmacotoxicity of 1R-Chl

To predict the behavior and likely dosage regime of the polyamides in humans, pharmacokinetic parameters for 1R-Chl and 1S-Chl were determined in Balb/c mice. Mass spectrometry was used to determine polyamide concentration in serum following injection of 1R-Chl or 1S-Chl. Both compounds appeared to exhibit first-order decay kinetics after a brief lag time (Fig. 6A), indicating that the rate of elimination from serum is proportional to the concentration of the compound in serum at a particular time-point. From these results, we determined that both 1R-Chl and 1S-Chl have t1/2 values of ~2 h. The constant of elimination (kel) was determined by plotting the natural logarithm of 1R-Chl serum concentration vs. time (during the clearance phase), and calculating the slope of the best fit straight line; kel was found to be ~0.45 h−1. The concentration of the compound achieved in blood at t = 0 was determined by extrapolating the line to the y-intercept. Volume of distribution (Vd) was calculated by dividing the dose injected by this theoretical initial concentration (t0). 1R-Chl was predicted to have a Vd of ~0.48 L/kg, corresponding to ~33.8 L for a 70 kg human (Fig. 6B). PK parameters for 1S-Chl were also determined and are similar to those of 1R-Chl: Kel ~ 0.55 h−1; Vd ~0.68 L/kg; Vd ~ 47.62 for a 70 kg human.

Fig. 6.

Fig. 6

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Pharmacokinetic parameters and biodistribution of 1R-Chl. A. Concentration (in μg/mL, y-axis) of 1R-Chl (rhombus) and 1S-Chl (squares) in serum over time. Mice were injected with either 1R-Chl or 1S-Chl (100 nmol, equivalent to 7.5 mg/kg) and blood extracted at indicated time-points. Amounts of polyamide were determined by mass spectrometry, and half-lives (t1/2) for 1R-Chl and 1S-Chl calculated. B. Graph depicting the natural logarithm (ln) of 1R-Chl (rhombus) and 1S-Chl (squares) concentration versus.time. The y-intercept represents the theoretical concentration at t = 0, where the constant of elimination and the apparent volume of distribution were calculated. C. Graph depicting the biodistribution of 1R-Chl. Mice were treated with 1R-Chl (500 nmol = 37.5 mg/kg), and euthanized either 2 h (black bars) or 24 h (white bars) post-injection. Major organs were harvested, and a weighed portion of each homogenized and sonicated. 1R-Chl concentration was determined by mass spectrometry.

The distribution of 1R-Chl in various mouse organs was determined by mass spectrometry (Fig. 6C). Following a 500 nmol dose, equal amounts of 1R-Chl were detected in the brain, muscle, stomach, liver, heart and kidney; it is likely that 1R-Chl crosses the blood-brain barrier. Extensive amounts of 1R-Chl were found in the lung, spleen, small intestine, and pancreas after 2 h post-injection and at t1/2, as well as 24 h post-injection, at which point 1R-Chl is undetectable in serum (Fig. 6A). These results indicate that 1R-Chl can be readily eliminated from serum, and persist in peripheral organs after tissue entry.

Discussion

Histone genes as primary targets of polyamide-Chl conjugates leading to growth inhibition of cancer cells

In the library of polyamide-Chl conjugates described herein, 1R-Chl and 6R-Chl are the only molecules expected to bind and alkylate within histone H4c/k/j genes. Sites of alkylation within the H4c gene are on the template strand, at the 5′ ends of the 1R-Chl and 6R-Chl targeted sequences (Figs. 1 and 3, and Supplementary Fig. 2). Alkylation of this strand would be predicted to block transcription elongation by RNA polymerase II. Reporter gene assays have shown that alkylation of the template strand produces a more pronounced effect on transcription inhibition than on the non-coding strand (40). Real-time qRT-PCR and Western blot analyses confirm that K562 cells treated with 1R-Chl or 6R-Chl (250 nM for 24 h) exhibit down-regulation of H4c and H4k/j transcripts and total histone H4 protein levels. Other polyamide-Chl conjugates do not alkylate H4 genes in vitro or in living cells and neither affect H4c and H4k/j mRNA and histone H4 levels, nor inhibit K562 cell growth.

Previous studies involving 1R-Chl have shown that down-regulation of the H4c gene in SW620 cells is required for growth inhibition, but changes in H4k/j transcription were not observed (32). Further inspection of Affymetrix microarray data for SW620 cells revealed that H4k/j genes are not highly expressed in SW620 cells relative to H4c. However, K562 cells have similar H4c and H4k/j expression levels as confirmed by real-time qRT-PCR. The primary targets of down-regulation by 1R-Chl in K562 cells were the histone H4 genes, H4c and H4j/k. The only gene down-regulated by 1R-Chl in both K562 and SW620 cells is H4c, indicating it to be the key target that elicits growth effects. Furthermore, only cultured cell lines that express the histone H4c gene at high levels have been shown to be affected by 1R-Chl (32, 35).

Polyamide-Chl conjugates block tumor progression with little animal toxicity

Polyamides 1R-Chl and 6R-Chl are the first sequence-specific DNA alkylators reported to show little animal toxicity at mid to high mg/kg range with three consecutive injections every other day over 5 d. This dosing regimen is highly effective in blocking the growth of established tumors (Fig. 5). No behavior or significant weight changes were observed during experiments with normal Balb/c or xenograft mice. In comparison, Tallimustine and Brostallicin, both of which are micromolar DNA alkylators targeting AT-rich sequences, have shown significant animal toxicity at 4mg/kg and 0.8 mg/kg respectively (9, 39).

We have shown here that 1R-Chl possesses desirable PK parameters: it has a t1/2of ~ 2 hours – enough time to allow the drug to effectively reach its target while minimizing prolonged presence in the blood stream, which can result in toxic effects. The constant of elimination of 1R-Chl is relatively low (~0.45/h) compared to those of most anti-cancer drugs, indicating that should a steady concentration of the drug be necessary to achieve efficacy, a regime of constant infusion may be considered. The kel of 1R-Chl is similar to that of Capecitabine (0.5/h), a non-alkylating anti-cancer drug used to treat breast cancer. Also, Vd > 30 for 1R-Chl, indicating that it is very well distributed in the body, an observation which is consistant with the presence of the molecule in the brain, muscle, stomach, liver, heart, and kidney in our biodistribution analysis (Fig. 6C). The PK parameters for 1R-Chl are close to those of some commonly used chemotherapeutic drugs, including both alkylating agents and other types of compounds.

Polyamide-Chl conjugates as potential therapeutics for CML and other forms of cancer

The development of the selective p210 Bcr-Abl tyrosine kinase inhibitor Gleevec (STI 571, Imatinib mesylate, Novartis) as a directed therapeutic for CML has been a major advance in cancer therapy (41, 42). However, many Gleevec patients acquire resistance mutations to the drug or acquisition of Bcr-Abl-independent genetic abnormalities during the course of treatment (43–46). Thus, development of additional therapeutic approaches for CML and other maligancies is worthwhile. Here we describe two sequence-specific DNA alkylators, 1R-Chl and 6R-Chl, that are capable of inhibiting K562 CML cell growth both in vitro and in vivo. 1R-Chl and 6R-Chl treatments are also highly effective in blocking K562 xenograft growth, with high dose tolerance in the murine model. Based on these observations and the finding that 1R-Chl blocks SW620 xenograft growth in nude mice (32), and the growth of two other xenograft models (Calu-1 lung cancer cells and 22Rv1 prostate cancer cells, data not shown), polyamide-Chl conjugates appear to be promising cancer therapeutics. The inhibitory mechanism of 1R-Chl and 6R-Chl in K562 cells is likely to be independent of Bcr-Abl because no significant down-regulation of Bcr-Abl transcripts was observed in either Affymatrix genechip analysis or real time qRT-PCR (data not shown). These results suggest that 1R-Chl maybe an effective treatment alternative for CML or may be useful in combination with Gleevec.

Supplementary Material

SupFigandTab
SupLegends

Acknowledgments

This work was supported by grants from the National Cancer Institute (CA107311) and the Department of Defense (CM043013) to J.M.G. and P.B.D. M.E.F. is supported by a predoctoral NIH NRSA training grant. We thank the staff of The Scripps Research Institute Department of Animal Resources, and The Scripps Mass Spectrometry and DNA Microarray facilities for technical assistance.

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Supplementary Materials

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