METHOD FOR INHIBITING CANCER USING ARSENIC TRIOXH)E Cross-Reference to Related Applications

This application claims priority to U.S. S.N. 11/549,347 filed October 13, 2006.

Field of Invention

This invention relates to methods of inhibiting cancer by affecting expression, translation, and biological activity of cancers overexpressing or dependent on cyclin Dl using arsenic trioxide.

Background of the Invention

Mantle cell lymphoma (MCL) is a well-defined subtype of B cell lymphoma in the World Health Organization classification, and accounts for approximately 3-10% of all non-Hodgkin lymphomas. The chromosomal aberration t(l I;14)(ql3;q32) can be found in practically all cases of MCL. The translocation results in juxtaposition of the immunoglobulin heavy chain joining region on chromosome 14 to the cyclin Dl gene on chromosome 11. The molecular consequence of the translocation is to place cyclin Dl under the control of the immunoglobulin heavy chain gene enhancer, leading to over- expression of the cyclin Dl protein.

Although MCL accounts for approximately 3 - 8% of B -cell lymphomas, it is difficult to manage. Initial treatment with rituximab plus combination chemotherapy or purine analogues results in complete remission (CR) rates varying from 34 - 87%. However, relapses occur in most patients with prolonged follow up. Treatment options for relapsed patients are limited. Several approaches have been adopted, including the use of the proteasome inhibitor bortezomib, thalidomide and the mammalian target of rapamycin (mTOR) inhibitor temsirolimus. The overall response (OR) rates of these agents varied from 38 - 81%, but the CR rate was only 3 - 31%. Therefore, there is an urgent need to define effective treatment strategies for MCL.

It is an object of this invention to provide agents and methods for treating cancers such as MCL and other cancers over-expressing cyclin Dl.

It is another object of this invention to provide methods, strategies, doses, and dosing schedules for the administration OfAs ₂ O ₃ in the clinical inhibition of cancers over-expressing cyclin Dl .

Summary of the Invention

It has been discovered that As ₂ O ₃ suppresses cyclin Dl and initiates down-regulation of cyclin Dl by activating GSK-3β, which phosphorylates cyclin Dl. Activation of IKKb leads to phosphorylation of cyclin Dl, which is ubiquitinated. Ubiquitinated cyclin Dl is degraded in the proteasome. This is the basis for the discovery that MCL and other cancers over-expressing cyclin Dl can be treated with As ₂ θ ₃ , preferably oral As ₂ O ₃ .

Brief Description of Drawings

Figure IA is a line graph showing As ₂ O ₃ (concentration in micromolar) percent induced apoptosis in MCL cells, based on a MTT test of Jeko-1 and Granta-519 cells treated for 72 hours with As ₂ O ₃ . There was a dose and time dependent suppression of cellular proliferation. Viability significantly decreased at or above 1 μM As ₂ O ₃ as compared with baseline (one-way ANOVA with Dunnett's post-tests, p < 0.05) (triplicate experiments) Figure IB is a scatter plot of popidium iodide versus annexin expression in cells treated with As ₂ O ₃ . There was a significant increase in apoptotic cells after As ₂ O ₃ treatment. (#: apoptotic cells that were annexin V positive and popidium iodide negative).

Figures 2 A and 2B show down-regulation of cyclin Dl by As ₂ O ₃ treatment. Figure 2A: As ₂ O ₃ (4 μM) induced a time dependent down-regulation of cyclin Dl in Jeko-1 and Granta-519 cells. Triplicate experiments and a representative Western blot demonstrate significant decrease in cyclin Dl level after 2 hours (one-way ANOVA with Dunnett's post-tests, p < 0.05). Figure 2B: As ₂ O ₃ (treatment for 8 hours) induced a dose dependent down-regulation of cyclin Dl in Jeko-1 and Granta-519 cells. Triplicate experiments demonstrate

significant decrease in cyclin Dl level at or above 2 μM (one-way ANOVA with Dunnett's post-tests, p < 0.05).

Figure 3 shows dephosphorylation of retinoblastoma (RB) by As ₂ O ₃ treatment in MCL lines. As ₂ O ₃ treatment resulted in dephosphorylation of RB (significant decrease of phosphor-Rb Ser-795 at or more that 8 hours OfAs ₂ O ₃ treatment, triplicate experiments, one-way ANOVA with Dunnett's post-tests, p < 0.05).

Figures 4A, 4B and 4C show As ₂ O ₃ treatment induced phosphorylation of cyclin Dl and GSK-3. Figure 4A. Cell lysates immunoblotted with anti- phospho-cyclin Dl (Thr-286). As ₂ O ₃ treatment led to significantly increased phosphor-cyclin Dl (triplicate experiments, one-way ANOVA with Dunnett's post-tests, p < 0.05). Figure 4B. Cell lysates immunoblotted with anti-phospho- cyclin GSK-3 β (Try-216). As ₂ O ₃ treatment led to significantly increased phosphor-GSK-3β (triplicate experiments, one-way ANOVA with Dunnett's post-tests, p < 0.05). Figure 4C. Pre-incubation with 6-bromoindirubin-3 '- oxime (BIO; 10 μM) before As ₂ O ₃ treatment (4 μM, 8 hour, 37°C) prevented cyclin Dl down-regulation, showing that GSK-3b was involved. Result a significant reduction of cyclin Dl as compared with control (triplicate experiments, one-way ANOVA with Dunnett's post-tests,/? < 0.05). Figures 5A and 5B show that IKK was involved in As ₂ O ₃ -induced down-regulation of cyclin Dl. Figure 5 A. As ₂ O ₃ treatment (4 μM for 2 hours) led to a significant increase in phosphor-IKKα/β (Ser - 176/180) (triplicate experiments, one-way ANOVA with Dunnett's post-tests,/? < 0.05). Figure 5B. Pre-incubation with the IKK inhibitor BMS (10 mM, 30 minutes) successfully prevented As ₂ θ ₃ -induced cyclin Dl down-regulation (triplicate experiments, one-way ANOVA with Dunnett's post-tests, p < 0.05).

Figure 6 shows As ₂ O ₃ induced ubiquitination of cyclin Dl in MCL. Cell lysates were immunoprecipitation with anti ubiquitin (Ub) or anti-cyclin Dl antibody. The immunoprecipitates and the crude lysates were immunoblotted with anti-cyclin Dl and anti-ubiquitin antisera. As ₂ O ₃ induced a significant increase in binding between cyclin Dl and ubiquitin (increase in ubiquitination

from 30 minutes to 2 hours after As ₂ O ₃ treatment as compared to the baseline, triplicate experiments, one-way ANOVA with Duπnett's post-tests, p < 0.05).

Figures 7A and 7B show As ₂ O ₃ -induced cyclin Dl degradation involved the proteasome but not the lysosome in MCL. Figure 7A. Pre- incubation with the proteasome inhibitors MGl 32 (MG, 30 μM), bortezomib (bort, 10 μg/ml) and lactacystin (lact, 10 μM) successfully prevented As ₂ O ₃ induced cyclin Dl degradation. Figure 7B. Pre-incubation with the lysosomal inhibitor ammonium chloride (NH ₄ Cl, 2.5 mM) was ineffective in preventing As ₂ O ₃ -induced cyclin Dl degradation. Figure 8 is a schematic diagram showing the proposed mechanism of degradation of cyclin Dl mediated by As ₂ O ₃ .

Detailed Description of the Preferred Embodiments I. Arsenic Trioxide Formulations

Arsenic Trioxide Arsenic trioxide is available from a number of different suppliers.

Arsenic trioxide is an amphoteric oxide which is known for its acidic properties. It dissolves readily in alkaline solutions to give arsenites. It is much less soluble in acids, but will dissolve in hydrochloric acid to give arsenic trichloride or related species. It reacts with oxidizing agents such as ozone, hydrogen peroxide and nitric acid to give arsenic pentoxide, As ₂ O ₅ . It is also readily reduced to arsenic, and arsine (AsH ₃ ) may also be formed.

Arsenic trioxide has many uses including as: a starting material for arsenic-based pesticides; a starting material for arsenic-based pharmaceuticals, such as a neosalvarsan, a synthetic organoarsenic antibiotic; a decolorizing agent for glasses and enamels, a wood preservative, and a cytostatic in the treatment of refractory promyelocytic (M3) subtype of acute myeloid leukemia.

An oral arsenic trioxide (As ₂ O ₃ ) is highly efficacious for relapsed acute promyelocytic leukemia. Oral As ₂ O ₃ causes a smaller prolongation of QT intervals, and therefore is a much safer drug for treating leukemia.

Formulations

The following delivery systems, which employ a number of routinely used pharmaceutical carriers, are only representative of the many embodiments envisioned for administering the instant compositions. Parenteral Formulations

Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA's). Implantable systems include rods and discs, and can contain excipients such as PLGA and polycaprylactone.

Enteral Formulations

Oral delivery systems include solid dosage forms such as tablets (e.g, compressed tablets, sugar-coated tablets, film-coated tablets, and enteric coated tablets), capsules (e.g., hard or soft gelatin or non-gelatin capsules), blisters, and cachets. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc). The solid dosage forms can be coated using coatings and techniques well known in the art.

Oral liquid dosage forms include solutions, syrups, suspensions, emulsions, elixirs (e.g., hydroalcoholic solutions), and powders for reconstitutable delivery systems. The formulations can contain one or more carriers or excipients, such as suspending agents (e.g., gums, zanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG, glycerin, and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), emulsifiers, preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, chelating agents (e.g., EDTA), flavorants, colorants, and combinations thereof. The compositions can be formulated as a food or

beverage (e.g., a shake) containing buffer salts, flavoring agents, coloring agents, sweetening agents, and combinations thereof.

Topical Formulations

Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).

Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer. II. Methods of Treatment

Cyclin Dl is a D-type cyclin critically involved in the control of the cell cycle. It assembles with its catalytic partners cyclin-dependent kinase 4 (CDK4) and CDK6 to form an active holoenzyme complex, which controls Gl progression and Gl/S transition. The active holoenzyme complex phosphorylates the retinoblastoma protein RB. Phosphorylated RB releases the E2F family of transcription factors from inhibition, enabling E2Fs to coordinately regulate genes necessary for DNA replication and hence progression into S phase. Over-expression of cyclin Dl is demonstrable in many cancers, including cancers of the digestive tract, cancers of the female genital tract, and malignant lymphomas.

Owing to its important influence on the cell cycle, cyclin Dl expression is carefully regulated. Cyclin Dl gene mRNA and transcription appears to be constant through the cell cycle. However, a decline in cyclin Dl level occurs during S phase, which has been attributed to its increased proteasomal

degradation. Cyclin Dl phosphorylation at a threonine residue 286 (Thr-286) positively regulates its proteasomal degradation. Thr-286 phosphorylation is mediated by glycogen synthase kinase-3β (GSK-3β). In addition to targeting cyclin Dl to proteosomes, GSK-3β -induced Thr-286 phosphorylation also promotes cyclin Dl nuclear export, by increasing the binding of cyclin Dl to a nuclear exportin CRMl. IkappaB kinase (IKK) alpha, IKKα, associates with and phosphorylates cyclin Dl also at Thr-286, thereby participating in the subcellular localization and turnover of cyclin Dl.

As ₂ O ₃ induced apoptosis in MCL lines at 2 - 4 μM, which is within the plasma levels achieved after As ₂ O ₃ therapy. As ₂ O ₃ induces a dose and time dependent suppression of cyclin Dl. The suppression of cyclin Dl restores RB to a hypophosphorylated state, in parallel with a change in cell cycle. These biologic changes are consistent with the apoptosis observed upon As ₂ O ₃ treatment. The down-regulation of cyclin Dl mediated by As ₂ O ₃ occurs at a post- transcriptional level since cyclin Dl is under the transcriptional control of the immunoglobulin heavy chain gene enhancer in MCL, which is unlikely to be affected by As ₂ O ₃ . Furthermore, in physiologic conditions, the control of cyclin Dl during the cell cycle is also mediated in part via alteration in the stability of cyclin Dl. This process is controlled by phosphorylation of cyclin Dl at Thr- 286, a process mediated by GSK-3β. GSK-3β is itself tightly regulated. Mitogens inactivate GSK-3β by a pathway involving Ras, phosphatidylinositol 3 kinase (PBK), and protein kinaseB/Akt. Ras activates PI3K, which in turn activates Akt. Akt inactivates GSK-3β by phosphorylating it at serine residue 9. This removes the inhibition of GSK-3β on cyclin Dl, allowing cyclin Dl to accumulate and thus activate cell cycling. GSK-3β can also be activated by phosphorylation at a tyrosine residue 216 (Try-216) in the kinase domain. As ₂ O ₃ -mediates an increase of GSK-3β Try-216 phosphorylation. The end result of As ₂ O ₃ -mediated increase in GSK-3β Try-216 phosphorylation is the increase in cyclin Dl Thr-286 phosphorylation, a key step in its degradation.

The IKK complex is the major regulatory component in the NK-κB pathway. It comprises the catalytic subunits IKKα and IKKβ, and a regulatory subunit IKKγ/NEMO. IKKα has been shown to phosphorylate cyclin Dl at Thr- 286, the same site targeted by GSK-3b. IKKα needs to be activated by phosphorylation at a serine residue 176 (Ser-176) before participating in the regulation of NF-κB by phosphorylating IKB. IKKα Ser-176 phosphorylation is mediated by NK-κB inducing kinase (NIK). As ₂ O ₃ -induces an increase in IKK phosphorylation. As ₂ θ ₃ -mediates an increase in physical interaction between IKK and cyclin Dl, as shown in immunoprecipitation experiments. An IKK specific inhibitor BMS-345541 alleviated As ₂ O ₃ -induced cyclin Dl down- regulation. These results indicate that IKK is also an effector OfAs ₂ O ₃ treatment.

As ₂ O ₃ -mediated cyclin Dl Thr-286 phosphorylation increases its ubiquitination. The time course of ubiquitination is commensurate with the timing of the biologic functions OfAs ₂ O ₃ on the MCL lines. After As ₂ O ₃ treatment, increased ubiquitination is first detected at 30 minutes and continues to increase. At two hours, significant down-regulation of cyclin Dl is first observed, which is associated with a parallel hypophosphorylation of RB. Significant activation of caspase 3 is observed at four hours. These sequence of events are consistent with cyclin Dl down-regulation initiated by Thr-286 phosphorylation.

Cyclin Dl is a cytosolic and nuclear protein. Therefore, polyubiquitination is involved, which targets the protein to degrade in proteasomes. Inhibition of proteasomes successfully prevented As ₂ O ₃ -induced down-regulation of cyclin Dl. Inhibition of lysosomes, the site of degradation of monoubiquitinated proteins, does not interfere with As ₂ O ₃ -induced down- regulation of cyclin Dl. These results confirm that As ₂ O ₃ down-regulated cyclin Dl by promoting its proteasomal degradation.

Arsenic trioxide can be used for the treatment of cancers that are dependent on cyclin Dl for proliferation, survival, metastasis and differentiation. Patients with cancers that overexpress cyclin D can be treated with

As ₂ O ₃ . Mantle cell lymphoma is a cancer characterized by overexpression of

cyclin D, as are cancers of the digestive tract, cancers of the female genital tract, and malignant lymphomas.

The dose of oral As ₂ O ₃ is typically adjusted according to age and kidney function. In one embodiment, the dose range OfAs ₂ O ₃ varies from 1 to 10 mg, typically about 5 to 10 mg.

The present invention will be further understood by reference to the following non-limiting examples.

Examples

Example 1: In vitro studies show AS ₂ O3 is effective in treatment of MCL by targeting cyclin Dl. Materials and Methods

Cell lines. The MCL lines Jeko-1 and Granta-519 were obtained from German Collection of Microorganisms and Cell Cultures (ACC 553 and ACC 342, Braunschweig, Germany). Jeko-1 cells were cultured in RPMI 1640 with 20% fetal bovine serum (FBS), and Granta-519 cells in DMEM with 10% FBS; both with 50 units/ml penicillin and 50 μg/ml streptomycin, at 5% CO ₂ .

Reagents and antibodies. Reagents and antibodies used included cell culture reagents (Invitrogen, Carlsbad, CA, USA); kinase inhibitors and their inactive analogues (Calbiochem, Darmstadt, Germany); antiserum to phospho- GSK3 (tyrosine 216, Try-216) (Upstate, Lake Placid, NY, USA); antisera to cyclin Dl, phospho-cyclin Dl (Thr-286), GSK3β, phospho-GSK3β (Tyr-216), IKB kinase (IKK)α/β, phospho-IKKα/β (serine 176/180, Ser-176/180), RB and phospho-RB (serine 795, Ser-795), caspase-3 and β-actin (Cell Signaling Technology, Beverly, MA, USA); protein G-agarose (Upstate); ECL kit (Amersham, Piscataway, NJ, USA); cell proliferation kit I (MTT) (Roche Applied Science, Indianapolis, IN, USA); annexin V-FITC Kit (Beckman Coulter, Fullerton, CA, USA); and RNeasy Kit and One-Step RT-PCR Kit (Qiagen, Valencia, CA, USA).

Cell viability assays. Cells were seeded on 96-well microplates at 2 x 10 ⁴ /well in 100 ml growth medium containing different concentration OfAs ₂ O ₃ as indicated at 37 ⁰ C for 72 hours. MTT labeling reagent (10 μl, 5 mg/ml)

(Roche Applied Science, Indianapolis, IN, USA) was added to each well at 37 ⁰ C for 4 hours, followed by 100 μl solubilization at 37 ⁰ C overnight. Solubilized fomarzan crystals were quantified spectophotometrically at 590 nm with a microplate ELISA reader. Apoptosis assay. Cells were seeded at 1 x 10 ⁶ /ml in different concentrations OfAs ₂ O ₃ as indicated at 37 ⁰ C for 24 hours, harvested, rinsed in ice-cold phosphate buffered saline (PBS), and resuspended in 500 μl binding buffer containing annexin V- FITC and propidium iodide (PI) (Beckman Coulter, Fullerton, CA, USA) for 20 minutes on ice. The percentages of apoptotic cells (annexin-V positive, PI negative) were determined on a flow cytometer (Epics, Beckman Coulter) with appropriate color compensation.

Cell Cycle Analysis. Cells were seeded at 1 x 10 ⁵ /ml in different concentrations OfAs ₂ O ₃ as indicated at 37 ⁰ C for 8 hours, harvested, washed in ice-cold PBS, resuspended in 500 μl PBS, stained with PI for 10 minutes on ice. Cell cycle was determined by flow cytometry (Epics, Beckman Coulter).

Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) for cyclin Dl. Cells were seeded at a density of 1 x 10 ⁶ /ml in different concentrations OfAs ₂ O ₃ at 37°C for 8 hours, washed with PBS buffer and lysed with RTL buffer. RNA was extracted with an RNeasy Kit, followed by cDNA synthesis and a 30-cycle PCR with a One-Step RT-PCR Kit with the forward primer 5'-CTG GCC ATG AAC TAC CTG GA-3' and the reverse primer 5'-GTC ACA CTT GAT CAC TCT GG-3'. Cycling conditions were denaturation (1 minute at 94 ⁰ C, first cycle 5 minutes), annealing (2 minutes at 50 ⁰ C) and extension (3 minutes at 72 ⁰ C, last cycle 10 minutes). Western Blotting Analysis. Cells were seeded at a density of 1 x 10 ⁶ /ml overnight. Where applicable, cells were pre-treated with various inhibitors for 30 minutes, and then incubated with 4 μM As ₂ O ₃ for different time periods as indicated. Cells were lysed in lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM EDTA, 40 mM NaP ₂ O ₇ , pH 7.5, 1% Triton X-IOO, 4 μg/ml aprotinin, 1 mM dithiothreitol, 200 μM Na ₃ VO ₄ , 0.7 μg/ml pepstatin, 100 μM phenylmethylsulfonyl fluoride, and 2 μg/ml leupeptin). Clarified lysates were

resolved on 12% SDS-phenylmethylsulfonyl fluoride and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk, washed, incubated with the appropriate antibodies followed by horseradish peroxidase-conjugated secondary antisera. Immuno-reactive bands were visualized by chemiluminescence with the ECL kit, detected on X-ray films and quantified by densitometric scanning (Eagle Eye II still video system, Stratagene, La Jolla, CA, USA).

Coimmunoprecipitation Assays. Cells were seeded at 1 x 10 ⁶ /ml overnight, treated with 4 μM As ₂ O ₃ at 37°C for different time periods as indicated, and lysed in lysis buffer. Cell lysates were incubated with an anti- cyclin Dl, anti-ubiquitin, anti-calpain 2 or anti-IKKα/β antibodies (4 μg/sample) at 4°C for 1 hour, followed by incubation with 30 μl of protein G-agarose (50% slurry) at 4°C for another 2 hour, hnmunoprecipitates were washed four times with 400 μl lysis buffer, resuspended in 50 μl lysis buffer and 10 ml 6X sample buffer and boiled for 5 minutes, hnmunoprecipitates were then analysed by Western blot analysis. Results

As ₂ O ₃ induced dose and time dependent apoptosis in MCL cells. The MTT test showed that As ₂ O ₃ induced a dose-dependent cytotoxicity in Jeko-1 and Granta-519 cells. Flow cytometric analysis showed that As ₂ O ₃ treatment led to induction of apoptosis. Western blot analysis showed that caspase 3 activation was involved in As ₂ O ₃ -induced apoptosis.

Figures IA and IB are graphs showingAs ₂ O ₃ (concentration in microM) percent induced apoptosis in MCL cells measured using a MTT test of Jeko-1 and Granta-519 cells treated for 72 hours with As ₂ O ₃ . There was a dose and time dependent suppression of cellular proliferation. Viability significantly decreased at or above 1 μM As ₂ O ₃ as compared with baseline (one-way ANOVA with Dunnett's post-tests, p < 0.05) (triplicate experiments). (. Significant increase in apoptotic cells after As ₂ O ₃ treatment. #: apoptotic cells that were annexin V positive and popidium iodide negative). Western Blotting showed activation of

caspase 3 by As ₂ O ₃ treatment, 0, 1.5 and 2.5 microM. Cleaved caspase 3 were detectable four hours after As ₂ O ₃ treatment.

Cyclin Dl was down-regulated in MCL by As ₂ C> ₃ . To determine the molecular mechanisms of As ₂ O ₃ -induced apoptosis in MCL, the expression of cyclin Dl was examined. Western blot analysis showed that As ₂ O ₃ -induced a time and dose dependent suppression of cyclin Dl in both Jeko-1 and Granta- 519 cell lines. Treatment with As ₂ O ₃ at 4 μM led to suppression of cyclin Dl, first detectable at 2 hours and almost complete at 8 - 12 hours As ₂ O ₃ suppression of cyclin Dl was also dose-dependent. Triplicate experiments demonstrate significant decrease in cyclin Dl level after 2 hours (one-way ANOVA with Dunnett's post-tests, p < 0.05) Triplicate experiments demonstrate significant decrease in cyclin Dl level at or above 2 μM (one-way ANOVA with Dunnett's post-tests, p < 0.05). Semi-quantitative polymerase chain reaction showing that cyclin Dl gene transcription was unaffected by As ₂ O ₃ treatment.

As ₂ O ₃ induced down-regulation of cyclin Dl disrupted its signaling. To investigate if cyclin Dl down-regulation is biologically relevant, RB phosphorylation was investigated. As ₂ O ₃ treatment led to a time dependent decrease in RB phosphorylation, which occurred at a similar time-frame as compared with cyclin Dl down-regulation. Cell cycle analysis by flow cytometry showed that there was an increase in the proportion of apoptotic cells. Down-regulation of cyclin Dl by As ₂ O ₃ was post-transcriptional. RT- PCR showed that cyclin-Dl gene transcription was unaffected by As ₂ O ₃ treatment of up to 8 μM, suggesting that the down-regulation of cyclin Dl was post-transcriptional.

As ₂ O ₃ -induced cyclin Dl down-regulation was related to GSK3β activation. Western blot analysis showed that As ₂ O ₃ treatment resulted in significant increases in cyclin Dl phosphorylation at Thr-286, a prerequisite for cyclin Dl degradation. Cyclin Dl phosphorylation by GSK-3β requires prior activation of GSK-3 β by phosphorylation at Tyr-216. As ₂ O ₃ treatment significantly increased GSK-3 β Tyr-216 phosphorylation, indicating that GSK-

3β might mediate As ₂ O ₃ -induced cyclin Dl phosphorylation and hence degradation. To confirm the role of GSK-3β as a mediator OfAs ₂ O ₃ , Jeko-1 cells were pre-incubated with the GSK-3β inhibitor 6-bromoindirubin-3'-oxime (BIO; 10 μM) before As ₂ O ₃ treatment. The results showed that BIO successfully prevented As ₂ O ₃ -induced down-regulation of cyclin Dl . Collectively, these observations indicate that As ₂ O ₃ down-regulated cyclin Dl post- transcriptionally, probably by increasing its degradation.

As2θ3-induced cyclin Dl down-regulation was also dependent on IKKα/β. To determine if IKK was involved in As ₂ O ₃ -induced down-regulation of cyclin Dl, IKKα/β phosphorylation at Ser-178/180 was examined. As ₂ O ₃ significantly increased IKKα/β Ser-178/180 phosphorylation, which was required for activation of IKKα/β (Figure 5A). Pre-treatment with the IKKα/β inhibitor BMS-345541 (BMS; 10 μM) significantly prevented As ₂ θ ₃ -induced cyclin Dl down-regulation, suggesting that IKKα/β was a molecular mediator OfAs ₂ O ₃ (Figure 5B). Immunoprecipitation with an anti-IKKα/β antibody showed that cyclin Dl bound IKKα/β. Similarly, when cyclin Dl was immunoprecipitated, IKKα/β was also confirmed to co-immunoprecipitate. These results confirmed that As ₂ O ₃ activated IKKα/β, which participated in the down-regulation of cyclin Dl. AS ₂ O ₃ promoted cyclin Dl ubiquitination. To study if As ₂ O ₃ -induced cyclin Dl down-regulation was mediated via ubiquitination, immunoprecipitation experiments were performed on lysates from Jeko-1 cells treated with As ₂ O ₃ . hrimunoprecipitation with an anti-ubiquitin antibody showed a time-dependent increase in bound cyclin Dl (Figure 6). Similarly, lysates immunoprecipitated with an anti-cyclin Dl antibody also showed a time dependent increase in bound ύbiquitin. These results showed that As ₂ O ₃ promoted cyclin Dl ubiquitination, confirming that As ₂ O ₃ -induced GSK-3β and IKKα/β activation was biologically relevant.

As ₂ O ₃ induced cyclin Dl degradation in 26S and 2OS proteasomes but not lysosomes. Pre-incubation of Jeko-1 cells with the 26S and 2OS

proteosome inhibitors MGl 32 (30 μM), bortezimab (10 μg/ml) and lactacystin (10 μM) attenuated As ₂ O ₃ -induced cyclin Dl down-regulation (Figure 7A). However, pre-incubation with the lysosomal inhibitor ammonium chloride (NH ₄ Cl) had no effect on As ₂ O ₃ -induced down-regulation of cyclin Dl (Figure 7B). The results confirmed that As ₂ O ₃ down-regulated cyclin Dl by promoting its ubiquitination, hence targeting it to the proteosome for degradation.

Overall model. An overall model of the action OfAs ₂ O ₃ on MCL is shown in Figure 8. Example 2: Clinical study of oral- As ₂ O ₃ in the treatment of patients with refractory and relapsed MCL that over-expressed cyclin Dl. Materials and Methods

Patients. Consenting patients with relapsed or refractory B-cell lymphomas, and an ECOG performance status of < 2 were recruited. AU patients gave informed consent, and the treatment was approved by the institute review board of Queen Mary Hospital.

Treatment. Treatment was initiated with oral-As ₂ O ₃ (10 mg/day for patients below 70 years old with normal renal function; 5 mg/day for patients over 70 years old, or with impaired renal function), ascorbic acid (AA, 1 g/day) and chlorambucil (4 mg/day) as outpatients until disease response or progression was documented. In patients with bulky disease, debulking with VPP

(vincristine 2 mg/day x 1, prednisolone 30 mg/day x 14 and procarabzine 50 - 100 mg/day x 14) was used. After maximum response was achieved, chlorambucil was taken off and a maintenance regimen OfAs ₂ O ₃ (5 - 10 mg/day) and AA (1 g/day) was given for two weeks every 2 months for a planned two years. Responses were classified according to standard NCI criteria, and monitored by regular physical examination, marrow and blood assessment, and computerized tomographic scans.

Results

Characteristics of patients with MCL. Table 1 shows the results of the clinical use of oral-As ₂ O ₃ in patients with refractory or relapsed mantle cell lymphoma that over-expressed cyclin Dl. The results showed an overall

response rate of 64%. Four patients achieved complete remission (CR), whereas two patients achieved complete remission unconfirmed. Of the fourteen patients treated (Table 1), eleven had advanced relapses (R) (R2, n=5; R3, n=4; R4, n=2). Three patients treated in Rl had advanced age (76, 77 and 90 years). All but two patients had received an anthracycline based multi-agent chemotherapy. Other previous treatment included rituximab (n=8), autologous hematopoietic stem cell transplantation (HSCT) (n=3), and bortezomib (n=l). Other poor prognostic indicators included marrow infiltration (n=l 1) and extensive extranodal involvement (n=9), so that 12/14 (86%) cases had stage IV disease. The median time from initial diagnosis to As ₂ O ₃ treatment was 33 (8 — 85) months.

Cable 1. Clinicopathologic features and treatment outcome of 14 patients with relapsed or refractory MCL

Initial disease Current relapse stage sites Previous treatment Time *No Sites Total As ₂ O ₃ response Outcome and survival

[ M / 69 HI Colon, abdomen FND x 6, COPP x 6 56 m 2 Cervical 140 mg CR off Rx, 28 m+.

M/ 63 IV BM, generalized LN R-CEOP x 6, IMVP x 6 11 m 2 BM, cervical 160 mg CR on Rx, 13 m+.

S M/ 65 IV BM, mesentery, generalized FND x 7, IMVP x 2, R-DHAP 85 m 3 Eye 120 mg CR on Rx, 17 m+.

LN x 8

I F/ 77 IV Pleura, generalized LN CIb 33 m 1 Groin, jaw 140 mg CR R2 at 16 m, CR again with As ₂ O ₃ + CIb

) M/ 70 πi Generalized LN COPP x2, IMVP x 6, CIb 85 m 4 Cervical, abdomen 250 mg CRu R5 at 20 m, on As ₂ O ₃

+ CIb

) M/ 76 rv BM, generalized LN CEOP x 7 19 m 1 BM, leukemic, eyes, 210 mg* CRu on Rx, 8 m+ generalized LN

1 M/ 58 IV BM, generalized LN CEOP x 6, R-ESHAP x 6 18 m 2 Generalized skin 140 mg PR On Rx, 3 m+

\ M / 81 IV BM, leukemic, liver, spleen CHOP x 6, ChIVPP x 2 18 m 2 BM, LN, liver, 300 mg* PR died at 16 m spleen, leukemic

) M / 51 IV Generalized LN, spleen, BM, CVAD x 7, CEOP x 2, 25 m 4 BM, LN, scalp NA* Static on Rx, 8 m-t- scalp, eye R-DHAP x 3, Thai

.0 F/ 76 IV General LN, BM, scalp R-COPP x 6 12 m 2 BM, LN 160 mg* PR died at 6 m

1 M/90 IV BM, leukemic CIb 8 m 1 BM, leukemic NA NR died at 4 m

2 M/ 54 IV Generalized LN, BM, gut, CEOP x 6, DHAP x 1, NOPP x 36 m 3 BM, generalized LN, NA Static died at 17 m liver, spleen, leukemic 5, CIb spleen

3 F/ 57 IV Generalized LN, BM, spleen CEOP x 6, DHAP x 4, R-BVP 36 m 3 BM ₅ LN NA NR died at 1 m v X D ^* 2

.4 M / 63 IV Generalized LN, pleura, BM CEOP x 6, AHSCT, R-DHAP x72 m 3 BM, generalized LN NA NR died at 1 m

6, Thai, velcade, FND vl: male, F: female; LN: lymphadenopathy; BM: bone marrow; m: months; R: rituximab; CEOP: cyclophospamide, epirubicin, vincristine, predniolone

⁷ ND: fludarabine, mitoxantrone, dexamethasone; DHAP: cisplatinum, cytosine arabinoside, dexamethasone; Thai: thalidomide

HhIvPP: chlorambucil, vincristine, procarbazine, prednisolone; COPP: cyclophosphamide, vincristine, procarbazine, prednisolone ;

¹ TOPP: mitoxantrone; vincristine, procarbazine, prednisolone; BVP; bleomycin, vinblastine, prednisolone; AHSCT: autologous hematopoietic stem cell ransplantation

DIb: chlorambucil; NA: not available; CR: complete remission; CRu: complete remission (unconfirmed); PR: partial remission; NR; no response

Treatment response. Nine patients responded, giving an OR rate of 64%. Four patients (cases 1 - 4) achieved CR. Two patients (cases 5, 6) achieved unconfirmed CR (CRu). They had become asymptomatic without any detectable superficial diseases. Marrow and peripheral blood involvement was also cleared. However, small residual internal lymph nodes remained. These lymph nodes were negative on gallium scan and had remained static in size. Three patients had partial responses (PR) with > 50% reduction in the size of assessable lymph nodes. Case 6 had bilateral orbital infiltration at relapse that completely resolved after 4 months of oral As ₂ O ₃ treatment and ascorbic acid. Case 8 who was relapsing in leukemic phase with massive splengomegaly showed partial remission after 8 months of treatment with oral As ₂ O ₃ and ascorbic acid as determined by MRI scans. Histological analysis revealed that case 8 had dense marrow infiltration that resolved after 8 months of treatment with oral-As ₂ O ₃ and ascorbic acid.

Outcome. Of the four patients with CR, one had relapsed at 16 months. She achieved a CR3 again with daily As ₂ O ₃ and resumption of chlorambucil. Two patients were still on maintenance As ₂ O ₃ + AA treatment, while one had completed the planned two years of treatment. Of the two patients with CRu, one patient had relapsed at 20 months. He achieved CR5 again with As ₂ O ₃ and chlorambucil therapy. For the three patients with PR, one patient developed progressive disease while on maintenance therapy 12 months later and died of refractory lymphoma. Two defaulted treatment and both relapsed. Toxicity. Significant (W.H.O grade 3 - 4) neutropenia and thrombocytopenia was observed in 7 patients. These patients had previously received multiple chemotherapy, or autologous HSCT. The neutropenia responded to hematopoietic growth factors. No significant sepsis or bleeding were observed. Other side effects included fever (n=7), herpes zoster reactivation (n=3), fluid accumulation (n=2), nausea (n=3) and headache (n=2).

No significant QT prolongation or arrhythmia was observed. Five patients did not report any side effects at all.

As ₂ O ₃ suppresses MCL cell growth by targeting cyclin Dl . As ₂ O ₃ induces the phosphorylation of GSK-3β and IKK. Cyclin Dl over-expression is patho genetically important in a vast diversity of cancers. Oral-As ₂ O ₃ inhibited refractory or relapsed MCL in 14 patients, which over-expressed cyclin Dl, with an overall response in 9 patients (64%). Four patients achieved complete remission, two patients complete remission unconfirmed, and three patients with partial remissions. These results were very good, given that these patients had refractory or relapsed disease.

Taken together, the evidence demonstrates that As ₂ O ₃ decreases cyclin Dl and that the decrease in cyclin Dl was post-transcriptional. As ₂ O ₃ induces GSK-3β and IKK activation and hence phosphorylation of cyclin Dl. Phosphorylated cyclin Dl is degraded in the proteasome. Oral As ₂ O ₃ induces a high response rate clinically in patients with refractory or relapsed MCL, a cancer that over-expresses cyclin Dl.