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Title:  Strategy for leukemia therapy
United States Patent: 
7,238,525
Issued: 
July 3, 2007

Inventors: 
Wang; Jean Y. J. (San Diego, CA), Vigneri; Paolo (Catania, IT)
Assignee: 
The Regents of the University of California (Oakland, CA)
Appl. No.: 
10/312,918
Filed: 
June 29, 2001
PCT Filed: 
June 29, 2001
PCT No.: 
PCT/US01/20602
371(c)(1),(2),(4) Date: 
December 27, 2002
PCT Pub. No.: 
WO02/00024
PCT Pub. Date: 
January 03, 2002


 

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Abstract

The chimeric Bcr-Abl oncoprotein is the molecular hallmark of chronic myelogenous leukemia (CML). In the cytoplasm, the protein transduces a growth signal that is responsible for overexpansion of cells. In the nucleus, the protein induces apoptosis. The invention is a method of treating cancer/killing Bcr-Abl expressing cells by inducing the translocation of Bcr-Abl to the nucleus to activate the apoptotic pathway in cancer cells.

SUMMARY OF THE INVENTION

The invention is a method for the treatment of cancers expressing the bcr-abl oncogene product by localizing an active Bcr-Abl to the nucleus to induce apoptosis. Endogenous Bcr-Abl may be accumulated in the nucleus by use of drugs that alter the localization of the protein in the cancer cell by administration of a Bcr-Abl kinase inhibitor (e.g. STI571; PD173955 or PD180970 both from Parke-Davis) and leptomycin B (LMB) (Schaumberg et al., 1984). The kinase inhibitor encourages Bcr-Abl translocation into the nucleus. Functional block of the protien is reversed by clearance or breakdown of the kinase inhibitor while the nuclear localization of the protein is maintained by LMB. Upon reactivation of the nuclear pool of Bcr-Abl, apoptosis is induced. Such a therapeutic protocol can be used in vivo for the treatment of leukemias or ex vivo to purge bone marrow to allow for autologous bone marrow transplantation.

Alternatively the coding sequence for a mutant form of bcr-abl lacking a functional nuclear export signal (NES) (SEQ ID NO 1, 2) (Taagepera et al., 1998) can be transferred into a solid tumor by use of a nucleic acid expression cassette. Methods of transfer of "suicide genes" to solid tumors followed by treatment with drugs to kill the tumor cells is known to those skilled in the art (see U.S. Pat. No. 6,066,624, incorporated herein by reference). A number of gene transfer methods are known including, but are not limited to, adenoviral viral vectors, (e.g. U.S. Pat. No. 6,080,578, incorporated herein by reference), adenovirus associated vectors (e.g. U.S. Pat. No. 5,989,540, incorporated herein by reference) or retroviral vectors (e.g. U.S. Pat. No. 6,051,427, incorporated herein by reference). Alternatively, nucleic acid can be delivered by direct injection of DNA into tumor either alone or encapsulated in liposomes or other DNA transfer reagent (e.g. U.S. Pat. No. 5,976,567, incorporated herein by reference). Translocation of the NES-defective Bcr-Abl to the nucleus is driven by administration of a kinase inhibitor. Apoptosis is promoted in cells containing the nuclear localized Bcr-Abl by washout of the kinase inhibitor to allow for activation of the protein. The rapid clearance of STI571 allows for the activation of the nuclear Bcr-Abl which induces apoptosis.

In another embodiment, the coding sequence for the NES-defective Bcr-Abl is fused in frame to the coding sequence of at least one heterologous nuclear localization signal (NLS) (e.g. SV40 large T antigen, nucleoplasmin) for transfer into a solid tumor by any of the methods listed above. The presence of the NLS abrogates the need for therapy with a kinase inhibitor or LMB. Upon translation of the protein, it is translocated into the nucleus where it activates apoptotic pathways.

This method overcomes a number of the problems with the use of STI571 as a single chemotherapeutic agent. First, the cancer cells are killed and not simply maintained in a quiescent state. This overcomes difficulties of maintaining rigorous therapeutic schedules for indefinite periods of time, increasing the quality of life for patients as well as decreasing the chances of the development of resistance. Such a therapy can be useful in blast crisis as only a portion of the Bcr-Abl must be translocated into the nucleus to induce apoptosis. The severe adverse events seen in the study with patients in the blast crisis stage of the disease would not likely be seen with less than a week of drug treatment. Steady state blood levels of 1 .mu.M STI571 and 10 nm LMB, effective doses of the drugs in culture, can be reached in two to three days. STI571 is rapidly cleared in a predictable manner with the time line dependent on the amount of drug given to the patient. If the Bcr-Abl is the endogenous protein from the cell, the patient is maintained on LMB for 2 to 3 days after combined treatment with a kinase inhibitor and LMB to retain the protein in the nucleus. Similar time considerations would be appropriate for LMB to purge bone marrow ex vivo. Ex vivo, effective doses of kinase inhibitor and LMB can be reached essentially instantaneously. In mouse bone marrow, clearance of at least 98% of the Bcr-Abl expressing bone marrow cells was acheived by treament of the cells for 12 hours with STI571 with LMB added for the last 8 hours of STI571 treatment. Maintaining the cells in LMB alone for an additional period of time after treatment with the drugs together can increase the rate of cell killing as with the tissue culture cells. If the Bcr-Abl is a nuclear export defective mutant transferred to the tumor by gene therapy, the patient is treated with kinase inhibitor alone for up to a week to encourage the entry of the protein into the nucleus. If an NLS supplemented NES-defective Bcr-Abl construct is used, no treatment with drugs is required. Upon completion of the first round of chemotherapy, the patient is monitored and the regimen may be repeated as needed.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

The cytoplasmic Bcr-Abl tyrosine kinase is a potent inhibitor of apoptosis (McGahon et al., 1994). The anti-apoptotic activity of Bcr-Abl contributes to the development of CML and the resistance of CML cells to chemotherapy (Warmuth et al., 1999). Evidence is presented that the Bcr-Abl tyrosine kinase can be converted into an activator of apoptosis by allowing it to function inside the nucleus. Apoptosis induced by the nuclear Bcr-Abl cannot be suppressed by the cytoplasmic Bcr-Abl, because nuclear entrapment of a fraction of the total Bcr-Abl is sufficient to kill cells. Because the nuclear Bcr-Abl can kill cells, cytoplasmic retention of this activated tyrosine kinase is required for cell transformation. That the Bcr-Abl kinase can induce apoptosis from the nucleus is consistent with the role of the nuclear c-Abl tyrosine kinase in the activation of cell death (Gong et al., 1999; Wang, 2000).

The discovery of STI571 has raised the prospect of a CML treatment with increased efficacy and limited side effects (Drucker and Lyndon, 2000). STI571 has shown efficacy in phase I and phase 11 clinical trials on CML patients in the chronic phase (Goldman, 2000). It is already evident, however, that STI571 does not have a long-term efficacy on CML patients in the acute phase (Vastag, 2000). Moreover, prolonged exposure to STI571, CML cells could develop resistance to this drug (Gambacourti, et al., 2000; le Coutre et al., 2000; Mahon et al., 2000; Weisberg and Griffin, 2000). LMB is a potent inhibitor of cell proliferation; however, its therapeutic application is limited by neuronal toxicity observed in a phase I clinical trial (Newlands et al., 1996). Though LMB is toxic to cells irrespective of Bcr-Abl expression, its effect is reversible after drug removal. Experiments on mouse bone marrow cells suggest that the combined treatment with LMB and STI571 is useful to purge explanted bone marrow of CML cells. This purging strategy allows autologous bone marrow transplantation to become a therapeutic option for CML.

Previous studies have shown that STI571 could induce apoptosis of CML cell lines in culture (Beran et al., 1998; Deininger et al., 1997). Herein is disclosed an alternative mechanism to induce apoptosis through STI571. When STI571 is combined with an inhibitor of nuclear export, it causes Bcr-Abl to be trapped in the nucleus. When the nuclear Bcr-Abl recovers its kinase activity, through the removal or metabolic decay of STI571, apoptosis is activated. These results indicate an interesting approach to treating CML, by trapping Bcr-Abl in the nucleus of leukemic cells and thus converting Bcr-Abl into a terminator of this disease.

Studies demonstrated that the kinase defective Bcr-Abl (Bcr-Abl-KD) entered the nucleus spontaneously; therefore, STI571 was tested to determine if it could stimulate the nuclear import of wild-type Bcr-Abl through inactivation of the kinase. Abl.sup.-/- fibroblast were transfected using the calcium phosphate method, well known to those skilled in the art, with plasmids encoding Bcr-Abl. Cells were treated with STI571 alone or with a combination of STI571 and LMB. Treatment with STI571 alone did not result in the nuclear accumulation of Bcr-Abl. However, the combined treatment with STI571 and LMB led to the accumulation of Bcr-Abl in the nucleus.

To quantify the stimulatory effect of STI571 on the nuclear import of Bcr-Abl without the influence of LMB, the NES of Bcr-Abl was inactivated by mutating a critical leucine residue (L1064A) (SEQ ID NO 1, 2) (Taagepera, et al., 1998). The NES mutation affects neither the kinase activity nor the interaction of Bcr-Abl with STI571, which binds to the tyrosine kinase domain (Schindler et al., 2000). When expressed in Abl.sup.-/- cells, BCR-Abl-NES was localized to the cytoplasm (FIG. 2a, see Original Patent). Following incubation with STI571, BCR-Abl-NES became detectable in the nucleus (FIG. 2b). STI571 did not alter the steady-state levels of BCR-Abl-NES (FIG. 2c, see Original Patent). The fraction of BCR-Abl-NES found in the nucleus was dependent on the concentration of STI571 and increased with time (FIG. 2d, see Original Patent). The amount of BCR-Abl-NES in the nucleus was 5, 10 and 20%, respectively, after a 12-hour incubation with 0.1, 1 and 10 M of STI571. Between 12 and 18 hours, the amount of nuclear Bcr-Abl-NES continued to increase with 0.1 and 1 .mu.M of STI571, but approached a plateau with 10 .mu.M of STI571 (FIG. 2d). The in vivo IC.sub.50 for the inhibition of Bcr-Abl tyrosine kinase is 0.25 0.4 .mu.M (Drucker et al., 1996). Thus, the nuclear accumulation of Bcr-Abl-NES could be correlated with the inhibition of its kinase activity. The nuclear fraction of Bcr-Abl-NES at 24 hours of treatment was between 25 35%. The nuclear fraction of Bcr-Abl-KD treated with LMB alone, or Bcr-Abl treated with both LMB and STI571 was also between 25 35%. These results indicate that mechanisms other than the kinase activity also mediate the cytoplasmic retention of Bcr-Abl.

It is known that activation of the nuclear Abl tyrosine kinase by DNA damage can contribute to the induction of apoptosis (Gong et al., 1999; Wang, 2000). Similarly, when translocated into the nucleus, Bcr-Abl kinase can activate apoptosis. The mere expression of Bcr-Abl does not cause apoptosis (as measured by TUNEL assay). However, trapping Bcr-Abl in the nucleus by combined treatment with STI571 and LMB caused apoptosis in 20% of Bcr-Abl expressing cells, which was not significantly different from the 10% of untransfected cells (Table 2, see Original Patent). Maintenance of Bcr-Abl in the inactivated form did not stimulate apoptosis. However, removal of the inhibitor while maintaining the Bcr-Abl in the nucleus strongly stimulated apoptosis. Following incubation with both STI571 and LMB to trap Bcr-Abl in the nucleus, cells were washed extensively and placed in fresh media with LMB alone to allow the recovery of Bcr-Abl kinase activity in the nucleus. With this protocol, between 70 80% of the Bcr-Abl expressing cells were positive for TUNEL staining, whereas the apoptosis rate remained at the 10% level with untransfected cells (Table 2). By expressing Bcr-Abl-KD and treating cells with LMB alone, it was confirmed that an active nuclear Bcr-Abl kinase was required to induce apoptosis. Despite its nuclear accumulation, Bcr-Abl-KD did not induce apoptosis (Table 2).

The inhibition of nuclear export by LMB has a cytotoxic effect of its own. To rule out that Bcr-Abl-induced apoptosis is dependent entirely on LMB, BCR-Abl-NES, which could be trapped in the nucleus by treatment with STI571 alone was tested for its ability to induce apoptosis (Table 2). The nuclear accumulation and re-activation of BCR-Abl-NES was able to cause apoptosis, in that 60% of the BCR-Abl-NES expressing cells became TUNEL-positive as compared with only 3% of the untransfected cells (Table 2). This result demonstrates that the nuclear Bcr-Abl kinase induces apoptosis in the absence of LMB.

Response to the drug regimen was further tested in a CML cell line, K562, and a non-CML myeloid leukemic cell line, HL60. Treatment of cells with a combination of STI571 and LMB for 8 hours, followed by treatment of the cells with LMB alone for 4 hours induced apoptosis in the K562 cells, but not in the HL60 cell line, further confirming the role of Bcr-Abl in the killing of the cells. Additionally, the studies demonstrated that the constitutively expressed Bcr-Abl, rather than just transiently expressed Bcr-Abl, may participate in the cell killing process induced by STI571 and LMB.

STI571 is more effective in preventing cells from dividing than it is in killing them. Upon removal of the drug, cells are able to proliferate again. To determine the efficacy and the specificity of cell killing by the nuclear Bcr-Abl, the long-term survival of drug-treated cells was investigated. The murine pro-B cell line TonB, which can be induced to express p210 Bcr-Abl through a doxycycline-regulated promoter was used (Klucher et al., 1998). The uninduced TonB and the induced TonB210 (expressing Bcr-Abl) cells were treated for a total of 48 hours with either LMB or STI571 alone, or both drugs combined. At the end of the 48-hour period, cells were washed extensively, placed in fresh media and the number of live cells was counted every 48 hours for a total of 14 to 16 days (FIGS. 3a c).

TonB cells that were not induced to express Bcr-Abl did not exhibit any reaction to STI571 (FIG. 3a). Treatment of TonB cells with LMB alone or with LMB plus STI571 caused a reduction in cell numbers (FIG. 3a). LMB causes an irreversible inactivation of Crm1/exportin-1 (Kudo et al., 1999); however, cells can recover from LMB, through the de novo synthesis of Crm1/exportin-1. Indeed, TonB cells resumed growth several days after the removal of LMB (compare FIG. 3a). Because TonB cells are dependent on IL-3 for survival and proliferation (Klucher et al., 1998), IL-3 was included in these experiments.

TonB210 cells induced to express Bcr-Abl became IL-3-independent (Klucher et al., 1998). The drug treatments were performed on TonB210 cells with and without IL-3. Without IL-3, TonB210 cells were sensitive to treatment with STI571 (FIG. 3b). Treatment with LMB alone for 48 hours also caused a decrease in cell numbers. Nevertheless, TonB210 cells could resume growth following the removal of LMB or STI571 (FIG. 3b). In contrast, the combined treatment with STI571 and LMB for 48 hours caused the complete loss of viable cells after the removal of drugs (FIG. 3b). With IL-3, TonB210 cells were not sensitive to treatment with STI571 (FIG. 3c). The inclusion of IL-3 also promoted the recovery of TonB210 cells following the removal of LMB (compare FIGS. 3b and c). However, even with the continuous presence of IL-3, treatment with a combination of STI571 and LMB caused a complete eradication of the TonB210 population following drug removal (FIG. 3c). In combination with LMB, concentrations of STI571 between 1 to 10 .mu.M were similarly effective in mediating the complete killing of TonB210.

Irreversible killing by the combined action of STI571 and LMB in the blast crisis cell line K562 was observed (FIG. 3d). Similar to the murine TonB210 cells, K562 cells showed initial death when STI571 was present, but this was consistently followed by recovery after the removal of STI571 (FIG. 3d). A similar initial decline in cell number was observed and recovery when K562 cells were treated with LMB alone (FIG. 3d). Again, treatment with both STI571 and LMB resulted in the complete loss of viable K562 cells by six days after drug removal.

The precipitous decline in viable TonB210 or K562 cells occurred several days after the removal of both drugs (FIGS. 3b d). One reason for this delayed killing could be the time required to recover the Bcr-Abl kinase activity in the nucleus. The tyrosine-phosphorylated proteins in total cell lysates were examined at the end of drug treatments and then before their precipitous death. TonB cells showed a low level of tyrosine phosphorylation, which was not affected by the drug treatments. In TonB210 and K562 cells, the overall levels of tyrosine-phosphorylated proteins decreased following incubation with STI571 alone or STI571 plus LMB. Before cell death, the levels of tyrosine phosphorylation returned to those of untreated cells. Thus, the precipitous death could be correlated with the recovery of Bcr-Abl kinase activity.

Recent studies have shown that infection of mouse bone marrow cells with a Bcr-Abl retrovirus can cause a CML-like syndrome following transplantation of infected cells into syngeneic mice (Daley et al., 1990; Li et al., 1999; Pear et al., 1998; Zhang et al., 1998). As an ex vivo verification of the results obtained with cell lines, STI571 and LMB were tested for their ability to kill Bcr-Abl-infected primary bone marrow cells. Mouse bone marrow cells were collected 5 days after 5-fluoracil (5-FU) injection. Cells were infected with a retrovirus that expresses the p210 Bcr-Abl and the green fluorescent protein (GFP) from a single bi-cistronic RNA (Zhang et al., 1998). The Bcr-Abl-infected cells could therefore be followed as GFP.sup.+ cells. Mixed populations of GFP.sup.+ and GFP.sup.- bone marrow cells were treated with STI and LMB either alone or in combination for a total of 12 hours, washed and cultured the cells for 60 hours. The percentage of GFP.sup.+ cells were determined by FACS (FIG. 4). Before treatment (BT), 10 12% of the population was positive for GFP. In the untreated populations (NT), the GFP.sup.+ cells remained at 10 12%. Treatment with LMB (L) or STI571 (S) alone resulted in a moderate increase in the percent of GFP.sup.+ cells. However, the combined treatment with LMB and STI571 (L+S) reduced the GFP.sup.+ cells to a level of 1 2%. These experiments demonstrate that the treatment regimen is useful for the killing of Bcr-Abl cells ex vivo as well as in vitro.

A similar experiment was performed with bone marrow cells isolated from mice that had not been injected with 5-FU (FIG. 4b, see Original Patent). Before treatment, 18 20% of the cells were positive for GFP (BT). Treatment with LMB or STI571 alone had no significant effect on the percentage of GFP.sup.+ cells (FIG. 4b). The combined treatment with LMB and STI571 again reduced the GFP.sup.+ cells to 1 2% (FIG. 4b, see Original Patent). The total number of GFP.sup.- and GFP.sup.+ cells were determined in these populations (FIG. 4c). Without drug treatment, both GFP.sup.- and GFP.sup.+ cells increased by 40 50% relative to the starting populations (FIG. 4c, NT, see Original Patent). LMB exhibited a toxic effect by reducing both the GFP.sup.- and GFP.sup.+ cells to a similar level (FIG. 4c, L). STI571 was also toxic to both the GFP.sup.- and GFP.sup.+ cells (FIG. 4c, S). The mouse bone marrow cells were cultured in media containing the stem-cell factor, which acts through the c-Kit receptor tyrosine kinase. Because STI571 can inhibit the c-Kit tyrosine kinase (Heinrich et al., 2000), the sensitivity of GFP.sup.- cells to STI571 could be due to the interference of c-Kit function. The combined treatment with STI571 and LMB was somewhat more toxic to the GFP.sup.- cells then either drug alone (FIG. 4c, see Original Patent). However, the combined treatment with STI571 and LMB removed the majority (>97%) of the GFP.sup.+ cells (FIG. c, see Original Patent). Thus, primary bone marrow cells that had been infected with a retrovirus to express the Bcr-Abl tyrosine kinase could be preferentially eliminated by the combined treatment with LMB and STI571.
 


Claim 1 of 6 Claims

1. A method of inducing apoptosis in leukemia cells expressing Bcr-Abl, the method comprising: (a) providing an in vitro cell culture comprising (i) leukemia cells expressing Bcr-Abl and (ii) a first cell culture medium; (b) administering to the cell culture (i) Bcr-Abl tyrosine kinase activity inhibitor STI-571, and (ii) leptomycin B (LMB), in amounts sufficient to induce accumulation of Bcr-Abl in nuclei of the leukemia cells expressing Bcr-Abl; and (c) replacing the cell culture medium of (b) with a second cell culture medium comprising LMB in an amount sufficient for induction of apoptosis in leukemia cells having Bcr-Abl accumulated in nuclei, thereby inducing apoptosis in the leukemia cells expressing Bcr-Abl.
 

 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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