COMPOUNDS FOR USE IN THE TREATMENT OF LEUKEMIA

Abstract

The present invention belongs to the field of cancer therapy and relates to a composition or product comprising at least one retinoid compound, at least one arsenic compound and at least one proteasome inhibitor, for use in the treatment of acute myeloid leukemia (AML) where the tumor cells are positive for the FLT3-ITD mutation.

Claims

1. A method for the treatment of acute myeloid leukemia in a patient having tumor cells which are positive for the FLT3-ITD mutation, comprising administering: a) a retinoid compound and/or pharmaceutically acceptable salts thereof; b) a arsenic compound; and c) a proteasome inhibitor; to a patient in need thereof.

2. (canceled)

3. The method according to claim 1, wherein the retinoid compound is all-trans retinoic acid (ATRA) and/or a pharmaceutically acceptable salt thereof.

4. The method of claim 1, wherein the arsenic compound is arsenic trioxide (ATA).

5. The method of claim 1, wherein the proteasome inhibitor is selected from the group consisting of Bortezomib, Carfilzomib, Oprozomib, Ixazomib and Marizomib.

6. The method of claim 1, wherein a) the retinoid compound is all-trans retinoic acid (ATRA) and/or derivatives thereof and/or pharmaceutically acceptable salts thereof; b) the arsenic compound is arsenic trioxide (ATO); and c) the proteasome inhibitor is Bortezomib.

7. (canceled)

8. The method of claim 1, further comprising administering an agent with redox properties.

9. The method of claim 1, wherein said method is used in sequence, or in combination, with other anticancer therapies.

10. The method according to claim 9, wherein the other anticancer therapies include idarubicin, daunorubicin, cytarabine, the anti-CD33 monoclonal antibody gemtuzumab ozogamicin and/or specific inhibitors of the FLT3 tyrosine kinase receptor.

11. The method of claim 1, wherein a), b) and c) are used in doses at which they show low toxicity when used alone.

12. The method of claim 1, wherein the retinoid compound and/or pharmaceutically acceptable salt thereof; the arsenic compound; and the proteasome inhibitor are administered as a combined preparation.

13. The method of claim 1, wherein the retinoid compound and/or pharmaceutically acceptable salt thereof; the arsenic compound; and the proteasome inhibitor are administered concurrently.

14. The method of claim 1, wherein the retinoid compound and/or pharmaceutically acceptable salt thereof; the arsenic compound; and the proteasome inhibitor are administered sequentially.

15. The method of claim 5, wherein the proteasome inhibitor is Bortezomib.

16. The method of claim 8, wherein the agent with redox properties is ascorbic acid, ascorbate, dehydroascorbic acid (DHA) or melatonin.

Description

[0056] The present invention will now be illustrated by way of non-limiting examples with reference to the following figures.

[0057] FIG. 1. Cells of the MV-4-11 cell line were treated with 10 nM ATRA (R), 1.5 nM Btz (B) and 500 nM ATO, alone or in combination as indicated. A After 72 hours from the treatment, cell death was assessed by the propidium iodide (PI) exclusion method, as measured by flow cytometer (n=6±SEM, one way ANOVA statistical analysis). Cells of the MOLM-13 cell line were treated and analysed in the same manner, except for the concentration of Btz, which in this case is 2.25 nM (n=6±SEM, one way ANOVA statistical analysis). B The morphological analysis of the treated cells confirms cell death especially in RBA-treated cells.

[0058] FIG. 2. Leukemic cells from two FLT3-ITD+ AML patients were isolated, amplified in culture, and treated with the indicated drugs for 7 days (C: untreated Control. R: 10 nM Retinoic acid, B: 3 nM Bortezomib, A: 500 nM Arsenic trioxide). The upper panels show the density of live cells at the end of the treatment, whereas the lower panels show the percentage of dead cells, as assessed by flow cytometry analysis. Treatment with the RBA combination can be seen to strongly slow down cell proliferation and induce death.

[0059] FIG. 3. A Cells of the MV-4-11 and MOLM-13 cell lines were treated as in FIG. 1A for 48 hours and the RNA was analysed by qRT-PCR to assess the expression of the UPR target genes, BiP, spliced XBP1 and CHOP (n=3±SEM, one way ANOVA statistical analysis). B Analysis of the expression and localization of the ER chaperones calnexin (CNX) and calreticulin (CRT), in green, by immunofluorescence confocal microscopy, in MV-4-11 and MOLM-13 cells exposed to treatments as in FIG. 1A. The DNA is highlighted in blue by the TO-PRO-3 dye. White arrows indicate where CNX distribution is particularly changed. C MOLM-13 cells were treated as in FIG. 1A for 24 or 48 hours. At the end of the treatment, expression of the active, lipidated form of LC3 was assessed by Western blot analysis (upper panel). The lower panel depicts the ratio between the Western blot signals from the LC3-II form (active) and the LC3-I form (inactive). D The same cells analysed by Western blot in C were also assessed by confocal microscopy analysis to highlight the LC3 dots (in green); the DNA is highlighted in blue by the Hoechst dye.

[0060] FIG. 4. A Analysis of the expression and localization of the Nrf-2 protein (in green), by immunofluorescence confocal microscopy, in MOLM-13 cells exposed to RBA treatment for 48 hours. The DNA is highlighted in blue by the Hoechst dye. B Flow cytometry analysis of Nrf-2 expression in the nuclei of MOLM-13 cells treated with the RBA combination for 48 hours by flow cytometry. The left panels show DNA staining, through which it is also possible to assess cell distribution in the various phases of the cell cycle and the presence of numerous cells in sub-G1, therefore apoptosis, after treatment with RBA. The right panels depict the fluorescence due to the anti-Nrf2 antibody, and the percentages indicate the amount of cells with the highest levels of fluorescence. C Western blot assessment of the translocation of the Nrf-2 protein from the cytosol to the nucleus in MOLM-13 cells exposed to RBA treatment for 48 hours. Cells were treated with H.sub.2O.sub.2 (100 μM for 30 minutes) or Tunicamycin (Tm, 350 nG/ml 0/N) as positive controls for Nrf-2 activation. D qRT-PCR analysis of the expression of the HMOX gene in MV-4-11 and MOLM-13 cells treated as in FIG. 1A for 24 hours (n=3±SEM, one way ANOVA statistical analysis). E Flow cytometry analysis of ROS levels in MV-4-11 and MOLM-13 cells treated with RBA for 48 hours. F MV-4-11 and MOLM-13 cells were treated as described in FIG. 1A in the presence (NAC) or absence (nil) of the reducing agent N-acetylcysteine (NAC). After 48 hours from the treatment, cell death was assessed by the propidium iodide (PI) exclusion method, as measured by flow cytometer (n=3±SEM, one way ANOVA statistical analysis). G Assessment of ROS levels, shown as the ratio to the nil control, in MOLM-13 cells treated with RBA in the presence or absence of NAC for 48 hours.

[0061] FIG. 5 Wt mice were treated for three weeks with 70 mg/kg trans-retinoic acid (R), 0.5 mg/kg Bortezomib (B) and 3 mg/kg arsenic trioxide (A). A The treatment did not significantly affect the animals' growth and weight, nor was it found to alter their behaviour. B Post-mortem analysis of the organs normally most affected by this type of drug showed no signs of toxicity.

Materials and Methods

Cell Lines, Cultures and Treatments

[0062] The MV4-11 (cat. no.: CRL-9591) and MOLM-13 (cat. no.: ACC 554) cell lines used were purchased from ATCC (Manassas, Va., USA) and DSMZ (Branuschweig, Germany), respectively, and kept in suspension culture in RPMI 1640 (Gibco, ThermoFisher Scientific, Waltham, Mass., USA) supplemented with penicillin (50 U/1110/streptomycin (50 μg/ml) (Gibco, ThermoFisher Scientific, Waltham, Mass., USA), 2 mM L-glutamine (Gibco, ThermoFisher Scientific, Waltham, Mass., USA) and 10% fetal bovine serum (FBS) (Gibco, ThermoFisher Scientific, Waltham, Mass., USA), in an incubator at 37° C., in a 5% CO2 humid atmosphere. Cells were treated as indicated with 10 nM retinoic acid (RA, Sigma-Aldrich, St. Louis, Mo.) and/or 500 nM arsenic trioxide (ATO, Sigma-Aldrich, St. Louis, Mo.) and/or 1.5 or 2.5 nM Bortezomib (Btz, Med Chem Express, NJ, USA) as indicated in the figure. N-acetylcysteine, at a concentration of 20 mM (NAC, Sigma-Aldrich, St. Louis, Mo.) was added 24 hours before starting treatment and again when adding RA, ATO and Btz. Equivalent amounts of DMSO were added in order to have the same DMSO concentration in all samples, including the control. Primary cells were isolated at the Tor Vergata Polyclinic from FLT3-ITD+ AML patients, with prior informed consent and according to the protocol approved by the PTV Ethics Committee. These were cultured for 7 days in Stem Cell Spam leukemic cell expansion medium (STEMCELL technologies, UK) according to the manufacturer's instructions and treated with 10 nM retinoic acid (RA, Sigma-Aldrich, St. Louis, Mo.) and/or 500 nM arsenic trioxide (ATO, Sigma-Aldrich, St. Louis, Mo.) and/or 3 nM Bortezomib (Btz, Med Chem Express, NJ, USA) as shown in the figure, in the same medium.

[0063] Death, Measurement of Reactive Oxygen Species (ROS) and Cell Differentiation

[0064] Cell counts were performed by optical microscope counting in a Burker chamber, excluding dead cells by Trypan Blue staining. Cell death was assessed by flow cytometry (Cytoflex, Beckman Coulter) after staining the cells with 2.5 μg/ml propidium iodide (Sigma-Aldrich, St. Louis, Mo., USA), a dye that can only penetrate dead cells. Reactive oxygen species (ROS) were measured by flow cytometry after incubation with the ROS-specific dye CM-H2DCFDA (Thermo Fisher Scientific, Waltham, Mass., USA) at a concentration of 2 μM, following the manufacturer's instructions. Nrf-2 fluorescence in the nuclei was assessed by flow cytometry after isolating the nuclei as described for the preparation of the cytosolic lysate for the Western blot. Then, instead of being lysed, the nuclei were fixed with 4% paraformaldehyde for 7 min, permeabilized with 0.1% TritonX100 in PBS/1% BSA for 5 min and stained with the primary anti-Nrf-2 antibodies (rabbit monoclonal, (D1Z9C) XP® Rabbit mAb #12721, Cell Signaling Technologies, Danvers, Mass., USA) and then with an Alexa Fluor-488 secondary anti-rabbit IgG antibody (Molecular Probes, ThermoFisher Scientific, Waltham, Mass., USA). The DNA was stained with the Sytox Blue dye (molecular Probes, ThermoFisher Scientific, Waltham, Mass., USA). Cell differentiation was assessed by morphological analysis of cell preparations by cytospin (Shandon, Thermo Fisher Scientific, Waltham, Mass., USA), obtained by centrifuging approximately 250,000 cells per slide, which were then stained by the Wright-Giemsa method (Sigma-Aldrich). The preparations were analysed under a Zeiss Axioskop 2 microscope and the images were acquired with an AxioCam HRc camera and analysed using the Axiovision 4.8 software (Zeiss, Oberkochen, Germany).

[0065] Immunofluorescence.

[0066] After being deposited on a slide via cytospin, the cells were fixed with 4% paraformaldehyde for 7 min, permeabilized with 0.1% TritonX100 in PBS/1% BSA for 5 min and stained with the primary anti-FLT3 (rabbit monoclonal, FLT3 (8F2), #3462, Cell Signaling Technologies, Danvers, Mass., USA) or anti-calnexin (CNX, rabbit polyclonal AB22595, AbCam, Cambridge, UK) or anti-calreticulin (CRT, rabbit polyclonal AB2907, AbCam, Cambridge, UK) or anti-LC3 (LC3B Antibody #2775) antibodies, and then with an Alexa Fluor-488 secondary anti-rabbit IgG antibody Molecular Probes, ThermoFisher Scientific, Waltham, Mass., USA); the DNA was detected with the TOPRO-3 (Molecular Probes, ThermoFisher Scientific, Waltham, Mass., USA) or Hoechst dye (Molecular Probes, ThermoFisher Scientific, Waltham, Mass., USA) and the slides were mounted with the Vectashield medium (Vector Laboratories, Burlingame, Calif., USA). The images were acquired with a Leica confocal laser scanning microscope TCS SP2 at 40× magnification or with a Zeiss LSM900 at 63× magnification. The images were analysed using the Leica Confocal software (Leica, Milan, Italy) or the Zeiss Zen Blue software.

[0067] RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)

[0068] Total RNA was extracted using the TRIzol RNA Isolation System (Invitrogen) according to the protocol provided by the manufacturer. The reverse transcription was carried out using the High Capacity RNA-to-cDNA kit (Applied Biosystems) and the obtained cDNA was amplified by quantitative real time PCR (qRT-PCR) with the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) using the following primers: CHOP_for 5′-GAGTCCGCAGCAGGTGC-3′ (SEQ ID NO:1), CHOP_rev 5′-TGTGACCTCTGCTGGTTCTG-3′(SEQ ID NO:2); sXBP1_for 5′-GAGTCCGCAGCAGGTGC-3′(SEQ ID NO:3), sXBP1_rev 5′-TCCTTCTGGGTAGACCTCTGGGAG-3′(SEQ ID NO:4); BiP_for 5′-TAGCGTATGGTGCTGCTGTC-3′(SEQ ID NO:5), BiP_rev 5′-TTTGTCAGGGGTCTTTCACC-3′(SEQ ID NO:6); H3_for 5′-GTGAAGAAACCTCATCGTTACAGGCCTGGT3′ (SEQ ID NO:7), H3_rev5′CTGCAAAGCACCAATAGCTGCACTCTGGAA-3′(SEQ ID NO:8). Data analysis was performed using the ΔΔCt method with the histone 3 (H3) gene as a normalizer. Amplification of the HMOX gene was carried out by using the PrimeTime Std qPCR Assay (Integrated DNA Technologies, Skokie, Ill., USA) consisting of the primers: for: -TCATGAGGAACTTTCAGAAGGG-rev (SEQ ID NO:9): -TGCGCTCAATCTCCTCCT-(SEQ ID NO:10) and of the probe (/56-FAM/AAGGTCGGA/ZEN/GTCAACGGATTTGGTC/3IABkFQ/(SEQ ID NO:11)). In this case, the normalizer used was the housekeeping gene GAPDH: for: -ACATCGCTCAGACACCATG-rev (SEQ ID NO:12): -TGTAGTTGAGGTCAATGAAGGG-(SEQ ID NO:13) and the probe used was (/56 FAM/AAGGTCGGA/ZEN/GTCAACGGATTTGGTC/3IABkFQ/). These reactions were carried out by using the TaqMan Universal PCR Master Mix reagent (Applied Biosystems, ThermoFisher Scientific, Waltham, Mass., USA).

[0069] Western Blot

[0070] The cells were lysed in two steps in order to obtain a cytosolic lysate and a nuclear lysate. For the cytosolic lysate, they were incubated for 15 min on ice in a buffer composed of 150 mM NaCl, 10 mM Hepes, 0.25% Sodium Deoxycholate, 1% NP40, 0.1% SDS, and the supernatant was recovered after centrifugation for 5 min at 300×g. The residual nuclear fraction was extracted by incubation with a buffer composed of 150 mM NaCl, 10 mM Hepes and 2% SDS, and sonicated. 40 μg of each lysate were subjected to SDS-PAGE, after boiling for 5 min with 50 mM DDT. After transferring to nitrocellulose, the proteins were stained with anti-Nrf2 antibody (rabbit monoclonal, (D1Z9C) XP® Rabbit mAb #12721, Cell Signaling Technologies, Danvers, Mass., USA) and images of the blots were obtained by ChemiDoc XRS+, using the Image Lab software (Bio-Rad, Hercules, Calif., USA). For LC3 expression analysis, the cytosolic and nuclear lysates were combined to obtain a total lysate, and the anti-LC3 antibody (LC3B Antibody #2775) was used.

[0071] Toxicity Analysis of the RBA Combination

[0072] Wt C56BL/6 mice were treated with 70 mg/kg R, 0.5 mg/kg B and 3 mg/kg A for three weeks according to the following regimen. Retinoic acid: administered via 10 mg subcutaneous pellets, which delivers a daily amount of 0.5 mg for 21 days. Bortezomib: administered intraperitoneally, 0.5 mg/Kg (in 100 μl saline) for 3 weeks (every 4 days). ATO: administered intraperitoneally, 3 mg/Kg (in 100 μl saline) for 3 weeks (at 5-day cycles of administration followed by 2 days without administration).

Example 1

[0073] Results

[0074] The experimental data obtained by the present authors demonstrate that the combination of ATRA, Btz and ATO, at doses at which they show low toxicity when used alone, induces a high mortality rate in the FLT3-ITD.sup.+ MV-4-11 AML human line; the greatest effect was obtained by using the triple combination which induces the same mortality pattern as that obtained with the combination of ATRA, Tm and ATO. The data was confirmed in a second FLT3-ITD.sup.+ line, the MOLM-13 line (FIGS. 1a and b). The same combination has been found to be effective in stopping proliferation and inducing cell death in FLT3-ITD+ AML cells isolated from the bone marrow of patients at diagnosis and cultured and treated ex vivo, as shown in FIG. 2. The present authors verified the working hypothesis by checking for the presence of ER stress following the combined treatments. The working hypothesis predicts that the RBA combination generates an impaired cellular protein homeostasis (also called proteostasis). In fact, the proteasome inhibitor Bortezomib (B), by inhibiting the degradation of the improperly folded proteins, causes the retention thereof in the endoplasmic reticulum (ER) with an increase in the stress of the latter and consequent activation of the Unfolded Protein Response (UPR). Arsenic trioxide (A) is a known oxidizing agent and therefore increases the oxidative stress of the cell. However, these cellular responses are activated entirely or in part according to the general state of cellular proteostasis, on the maintenance of which numerous, widely interconnected responses to cellular stresses (UPR, oxidative stress response, heat shock response, autophagy, ubiquitin-proteasome system . . . ) intervene, the activation of which is linked to threshold stress values. Therefore, we tested the activation of UPR following treatment with single drugs and the various combinations of drugs. UPR is an adaptive response aimed at recovering cellular homeostasis, but if the stress is too strong or prolonged it leads to cellular apoptosis. Of the many genes activated during UPR, some favour the adaptive response (such as BiP and sXBP-1), whereas others cause apoptosis (CHOP). Our experiments show, more clearly in MOLM-13 than in MV-4-11, that the treatments, used in double and triple combinations, cause a reduction in the expression of the genes involved in the adaptive response, without significantly altering the expression of those involved in the pro-apoptotic response (FIG. 3A). This result suggests that the impairment of the proteostasis obtained with the various combinations does not activate the adaptive UPR, consistent with the high rate of cell death.

[0075] Accordingly, the distribution of two important ER chaperones, calnexin (CNX) and calreticulin (CRT), is significantly modified, particularly in RBA-treated samples, suggesting major damage to the ER (FIG. 3B), thus confirming the authors' hypothesis.

[0076] In a context of proteostasis impairment, another adaptive response that can intervene is autophagy, which is known to compensate for the inhibition of the proteasomal degradation pathway. However, the evaluation of autophagy activation in MOLM-13 cells treated with the RBA combination showed that this is not the case since, although a slight increase in LC3 activity is observed after 48 hours in culture in the control samples and in those treated with R, this is less evident in cells treated with the various combinations, in particular with BA and RBA. The probable reason why an increase in autophagy is observed in the other samples after 48 hours in culture is the high proliferation rate of these cells, which consequently rapidly deplete the nutrients present in the medium (FIGS. 3C and D).

Example 2

[0077] Based on studies previously published by the authors of the invention with the combination of trans-retinoic acid, Tunicamycin and arsenic trioxide (RTA), wherein ER stress was induced by the glycosylation inhibitor Tunicamycin, RBA treatment was expected to induce oxidative stress. Accordingly, oxidative stress induction was assessed in the course of the present invention. Indeed, immunofluorescence tests show that the Nrf-2 protein, i.e., the master gene that regulates the response aimed at contrasting the oxidative stress, translocates to the cell nucleus, and is then activated, following RBA treatment in MOLM-13 cells (FIGS. 4A, B and C). In accordance with the activation of the oxidative stress response triggered by Nrf-2, both MV-4-11 and MOLM-13 cells show a strong increase in the expression of the HMOX gene, an Nrf-2 target gene, in particular following the combined RBA treatment (FIG. 4D). Nrf-2 pathway activation indicates the presence of high levels of oxidative stress: in fact, the RBA combination increases the amount of reactive oxygen species (ROS) in both MV-4-11 and MOLM-13 cells (FIG. 4E). Like autophagy, the UPR is also not activated but, on the contrary, the expression of its target genes is reduced, whereas a high level of oxidative stress is observed; this leads to hold the latter probably responsible for the toxic effects of the RBA combination. We therefore incubated MV-4-11 and MOLM-13 cells with a reducing agent, N-acetylcysteine (NAC) in order to increase the reducing power of the cells, before and during treatment with the RBA combination, and this was sufficient to completely eliminate the toxic effect in MOLM-13 cells and to partially eliminate it in MV-4-11 cells (FIG. 4F). This difference is probably due to the fact that MV-4-11 cells are sensitive to R even when it is used alone; this result indicates that the toxicity of R alone is independent of the generation of oxidative stress, whereas, on the other hand, the toxicity caused by the synergy with B and A depends on it, since the mortality rate of cells treated with RBA, in the presence of NAC, is comparable to that of cells treated with R alone. The ability of NAC to decrease the oxidative stress level has been demonstrated in MOLM-13 cells in which, in the presence of NAC, the amount of ROS remains the same as in control cells even after treatment with RBA (FIG. 4G). The use of NAC demonstrates that the toxicity of the RBA combination is due to the generation of oxidative stress, which is linked to proteostasis impairment. In fact, as mentioned above, the various adaptive responses required to maintain the proteostasis are closely related to each other, and the ER stress is known to increase the oxidative stress, and vice versa.

Example 3

[0078] Preclinical toxicity studies were performed in wt murine models. As regards toxicity, the results lead to the conclusion that the combination, as proposed in the patent application, is not harmful to animals. In fact, no changes in the behaviour, feeding or mobility of the animals treated with the RBA combination compared to the control, nor changes in body weight are observed (FIG. 5A). Seven days after the end of the treatment, the animals were sacrificed to assess the macroscopic and microscopic state of several organs, including pancreas and liver, which could be more sensitive to any toxicity of the treatment. Both macroscopic and microscopic examinations showed no differences between the control animals and those treated with the RBA combination (FIG. 5B).

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