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  Pharmaceutical Patents  

 

Title:  Molecular determinants of myeloma bone disease and uses thereof
United States Patent: 
7,642,238
Issued: 
January 5, 2010

Inventors:
 Shaughnessy; John D. (Little Rock, AR)
Appl. No.:
  11/176,739
Filed:
 July 7, 2005


 

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Abstract

To identify molecular determinants of lytic bone disease in multiple myeloma, the expression profiles of .about.12,000 genes in CD138-enriched plasma cells from newly diagnosed multiple myeloma patients exhibiting no radiological evidence of lytic lesions (n=28) were compared to those with .gtoreq.3 lytic lesions (n=47). Two secreted WNT signaling antagonists, soluble frizzled related protein 3 (SFRP-3/FRZB) and the human homologue of Dickkopf-1 (DKK1), were expressed in 40 of 47 with lytic bone lesions, but only 16 of 28 lacking bone lesions (P<0.05). DKK1 and FRZB were not expressed in plasma cells from 45 normal bone marrow donors or 10 Waldenstrom's macroglobulinemia, a related plasma cells malignancy that lacks bone disease. These data indicate that these factors are important mediators of multiple myeloma bone disease, and inhibitors of these proteins may be used to block bone disease.

Description of the Invention

SUMMARY OF THE INVENTION

To identify the molecular determinants of lytic bone disease, the expression profiles of .about.12,000 genes in CD138-enriched plasma cells from newly diagnosed multiple myeloma exhibiting no radiological evidence of lytic lesions (n=28) were compared to those with .gtoreq.3 lytic lesions (n=47). Consistent with a critical role of WNT signaling in osteoblast differentiation, two secreted WNT signaling antagonists, soluble frizzled related protein 3 (SFRP-3/FRZB) and the human homologue of Dickkopf-1 (DKK-1), were expressed in 40 of 47 with lytic bone lesions, but only 16 of 28 lacking bone lesions (P<0.05). Immunohistochemistry showed high levels of DKK-1 and FRZB in plasma cells from cases with high gene expression. Importantly, DKK-1 and FRZB were not expressed in plasma cells from 45 normal bone marrow donors or 10 Waldenstrom's macroglobulinemia, a related plasma cells malignancy that lacks bone disease.

Serum derived from multiple myeloma patients with high DKK-1 blocked both Wnt signaling and osteoblast differentiation in vitro. Importantly, pre-incubation of the serum with DKK-1 and FRZB antibodies inhibited this function. Consistent with a key role for JUN in controlling DKK-1 expression and in turn apoptosis, plasma cells derived from extramedullary disease as well as primary refractory disease had low expression of JUN and DKK-1.

Multiple myeloma plasma cells showed a massive up-regulation of DKK-1 and FRZB gene expression after in vivo treatment. DKK-1 and FRZB can be upregulated in multiple myeloma plasma cells after treatment of patients with genotoxic drugs used to treat the disease, thus furthering a role for DKK-1 in multiple myeloma cell apoptosis. Primary multiple myeloma cells co-cultured with in vitro derived osteoclasts (OC) lacked apoptosis and that this was tightly correlated with the down-regulation of JUN, FOS, FOSB, and DKK-1.

Results disclosed in the present invention indicate that blocking the production and/or secretion of DKK-1 and FRZB may prevent or reverse bone loss in multiple myeloma patients. Further applications may include using DKK-1 and FRZB inhibitors to prevent bone loss in the general population. Additionally, Wnt signaling has recently been shown to be critical for the self renewal capacity of hematopoietic stem cells. Furthermore, a bone marrow niche required for hepatic stellate cell proliferation is formed by mature osteoblasts. The block to Wnt signaling by DKK1 and FRZB could directly and indirectly impair hepatic stellate cell (HSC) proliferation and thus may partly account for the immunosuppression and anemia seen in multiple myeloma. Thus, blocking DKK1 and/or FRZB may also prevent or reverse the defect in hematopoiesis seen in most patients with myeloma.

The present invention is directed to a method of determining the potential of developing bone disease in a multiple myeloma patient. Such a method comprises the step of examining the expression level of WNT signaling antagonist, where increased expression of the antagonist compared to that in the normal individual indicates that the patient has the potential of developing bone disease.

The present invention is also directed to a method of treating bone disease in a multiple myeloma patient. This method comprises the step of inhibiting the expression of a WNT signaling antagonist.

The present invention is further directed to a method of preventing bone loss in an individual. This method comprises the step of inhibiting the expression of a WNT signaling antagonist.

The present invention is still further directed to a method of controlling bone loss in an individual. This method comprises the step of inhibiting the expression of the DKK1 gene (accession number NM 012242; SEQ ID NO: 1 or the activity of the protein expressed by the DKK1 gene.

The present invention is also directed to a method of controlling bone loss in an individual. Such a method comprises the step of administering to the individual a pharmacological inhibitor of DKK1 protein.

The present invention is further directed to a method of preventing bone resorption and increasing bone formation in an individual. Such a method comprises blocking the activity of DKK1. This blocking of the DKK1 activity increases osteoblast numbers and reduces osteoclast activity, thereby preventing bone resorption and increasing bone formation in the individual.

The present invention is also directed to a method of inhibiting tumor growth in bone of an individual. Such a method comprises the step of blocking the activity of DKK1.

The present invention is also directed to a method of screening for a compound that controls bone loss and inhibits human myeloma growth. Such a method comprises engrafting human myeloma cells in a rabbit bone implanted in a SCID-rab mouse. This is followed by administration of a candidate compound to the mouse. Subsequently, bone mineral density of the implanted bone and level of serum human monoclonal immunoglobulin in the mouse is compared with a control mouse that has not received the compound. An increase in the bone mineral density and a decrease in the level of the serum immunoglobulin in the treated mouse compared to the control mouse indicates that the compound controls bone loss and inhibits human myeloma growth.

DETAILED DESCRIPTION OF THE INVENTION

The present invention demonstrates that the secreted WNT signaling antagonists DKK-1 and FRZB mediate bone destruction seen in multiple myeloma. These data strongly implicate these factors in causing osteoblast anergy and contributing to multiple myeloma bone disease by suppressing the normal compensatory bone production that follows bone loss.

The role of multiple myeloma plasma cells in stimulating osteoclast activity has been intensely investigated and several key links established. Data presented herein provide for the first time evidence of a possible mechanistic explanation of osteoblast dysfunction in multiple myeloma. These are significant observations in that inhibition of WNT signaling causes defects in osteoblast function. The secreted DKK-1 and FRZB could account for both the systemic osteoporosis seen in multiple myeloma as well as the exaggerated local bone destruction proximal to plasma cells foci.

Importantly, DKK-1 and FRZB act to inhibit WNT signaling through independent mechanisms, indicating that their co-expression may have synergistic effects. Thus, these genes could be used to predict extent of bone disease and future risk of developing bone disease. Moreover, inhibitors of these proteins could be used to block bone disease. It is also possible that these factors play a role in osteoporosis in the general population.

WNT Signaling Pathway

Wnt genes comprise a large family of secreted polypeptides that are expressed in spatially and tissue-restricted patterns during vertebrate embryonic development. Mutational analysis in mice has shown the importance of Wnts in controlling diverse developmental processes such as patterning of the body axis, central nervous system and limbs, and the regulation of inductive events during organogenesis. The Wnt family of secreted growth factors initiates signaling via the Frizzled (Fz) receptor and its coreceptor, LDL receptor-related protein 5 or 6 (LPR5 or LRP6), presumably through Fz-LPR5/LRP6 complex formation induced by Wnt.

Secreted antagonists of Wnt include Frizzled (Fz)-related proteins (FRPs), Cerberus, Wnt inhibitory factor (WIF) and Dickkopf (DKK). Frizzled (Fz)-related proteins, Cerberus and Wnt inhibitory factor have all been shown to act by binding and sequestering Wnt. Unlike Wnt antagonists which exert their effects by molecular mimicry of Fz or Wnt sequestration through other mechanisms, Dickkopf-1 (DKK-1) specifically inhibits canonical Wnt signalling by binding to the LPR5/LRP6 component of the receptor complex.

DKK-1 is a head inducer secreted from the vertebrate head organizer and induces anterior development by antagonizing Wnt signaling. DKK-1 is a high-affinity ligand for LRP6 and inhibits Wnt signaling by preventing Fz-LRP6 complex formation induced by Wnt. DKK-1 binds neither Wnt nor Fz, nor does it affect Wnt-Fz interaction. DKK-1 function in head induction and Wnt signaling inhibition strictly correlates with its ability to bind LPR5/LRP6 and to disrupt the Fz-LPR5/LRP6 association. LPR5/LRP6 function and DKK-1 inhibition appear to be specific for the Wnt/Fz beta-catenin pathway. These findings thus reveal a novel mechanism for Wnt signal modulation.

WNT Signaling and Osteoblast Differentiation

Recent studies have shown that the Wnt signaling pathway is critical for osteoblast differentiation and function. Mice with a targeted disruption in the gene for low-density lipoprotein receptor-related protein 5 (LRP5) developed a low bone mass phenotype. LRP5 is expressed in osteoblasts and is required for optimal Wnt signaling in osteoblasts. In vivo and in vitro analyses indicated that this phenotype becomes evident postnatally, and it was secondary to decreased osteoblast proliferation and function in a Cbfa1-independent manner.

In human, mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG). Osteoporosis-pseudoglioma syndrome carriers have reduced bone mass when compared to age- and gender-matched controls.

Importantly, separate and distinct mutations in LRP result in a high bone mass phenotype. In contrast to the osteoporosis-pseudoglioma mutations, the high bone mass traits are gain of function mutations. Markers of bone resorption were normal in the affected subjects, whereas markers of bone formation such as osteocalcin were markedly elevated. Levels of fibronectin, a known target of signaling by Wnt, were also elevated. In vitro studies showed that the normal inhibition of Wnt signaling by Dickkopf-1 (DKK-1) was defective in the presence of the mutation and that this resulted in increased signaling due to unopposed Wnt activity. These findings demonstrated the role of altered LRP5 function in high bone mass and point to DKK as a potential target for the prevention or treatment of osteoporosis.

WNT Signaling and Bone Disease in Multiple Myeloma

Indirect evidence of a role of DKK-1 in osteoblast function has been provided by identification of gain of function mutations in LRP-5 being linked to a high bone mass phenotype. In addition, targeted disruption of secreted frizzled-related protein (SFRP-1), a homologue of FRZB (SFRP-3), leads to decreased osteoblast and osteocyte apoptosis and increased trabecular bone formation.

A quantitative trait loci (QTL) influencing bone mass has been localized to the LRP-5 region, suggesting that the population at large have different risk of developing osteoporosis. It is conceivable that multiple myeloma bone disease may be influenced by the combined effects of DKK-1/FRZB expression with an inherited predisposition to low bone mass conferred by inherited LRP-5 alleles. Multiple myeloma cases may be genotyped for LRP-5 allele variations and correlate this information with bone disease, and DKK-1 and FRZB expression.

Monoclonal gammopathy of undetermined significance (MGUS), a plasma cell dyscrasia that is predisposed to develop into multiple myeloma, is differentiated from multiple myeloma by the lack of obvious bone disease. The significance of discovering DKK-1 and/or FRZB expression in a third of monoclonal gammopathy of undetermined significance is unclear but could suggest that these cases may be at higher risk for developing multiple myeloma. As with multiple myeloma, this predisposition may also be related to inherited LRP5 alleles. Alternatively, these monoclonal gammopathy of undetermined significance cases could have underlying preclinical bone disease that is not yet apparent by radiological scans.

Data presented herein suggests a model for how DKK-1 expression by multiple myeloma plasma cells can be linked to multiple myeloma disease growth control and bone destruction and how these two phenomena can be integrated by one molecule. In the model, primary multiple myeloma express high levels of DKK and these levels can be increased with drug therapies used to treat the disease. High levels of DKK-1 likely induce apoptosis of multiple myeloma cells and could explain the relatively slow progression of the disease in its early phase as cell growth is tempered by high rate of DKK-1 induced apoptosis. However, as the disease progresses there is an osteoclast-induced reduction in JUN and DKK-1 that eventually develops into a constitutive loss of JUN and DKK-1 expression as seen in extramedullary disease.

Thus, if one were to view DKK-1 expression from the perspective of the multiple myeloma plasma cells, high levels of DKK-1 expression could be seen as positive feature of the disease. However, with the mesenchymal cell lineage being exquisitely sensitive to DKK-1 induced apoptosis, the high levels of this secreted product likely has a double edge to it in that it also induces massive programmed cell death of osteoblast precursors and possibly even mesenchymal stem cells. It is expected that high levels of DKK-1 early in the disease could lead to a permanent loss of mesenchymal stem cells, a notion supported by the observed lack of bone repair after remission induction or during disease progression when osteoclasts likely suppress DKK-1 secretion by multiple myeloma plasma cells. Thus, exploitation of this knowledge might lead to the development of new therapies for multiple myeloma that accentuate DKK-1's effects on multiple myeloma plasma cells, but at the same time prevent DKK's bone damaging effects on osteoblast or their precursors.

Furthermore, the present invention also demonstrated that blocking of DKK1 activity in primary human myeloma-bearing SCID-rab mice was associated with increased osteoblast numbers and reduced osteoclast activity. These effects resulted in prevention of bone resorption, increased bone formation and most importantly inhibition of tumor burden. These in vivo data confirmed that DKK1 is critical factor involved in myeloma bone disease and tumor progression. Thus, therapeutic approaches to inhibit DKK1 activity in patients with myeloma will not only improve skeletal complications and quality of life but also help control myeloma.

In one embodiment of the present invention, there is provided a method of determining the potential of developing a bone disease in a multiple myeloma patient by examining the expression level of WNT signaling antagonist. Increased expression of the antagonist compared to that in normal individual would indicate that the patient has the potential of developing bone disease. Preferably, the WNT signaling antagonist is soluble frizzled related protein 3 (SFRP-3/FRZB) or the human homologue of Dickkopf-1 (DKK1). In general, the expression levels of these proteins can be determined at the nucleic acid or protein level.

In another embodiment, there is provided a method of treating bone disease in a multiple myeloma patient by inhibiting the expression of WNT signaling antagonist. Preferably, the WNT signaling antagonist is soluble frizzled related protein 3 (SFRP-3/FRZB) or the human homologue of Dickkopf-1 (DKK1). In general, the expression of these antagonists can be inhibited at the nucleic acid or protein level or in any other fashion readily know to those having ordinary skill in this art.

In yet another embodiment, there is provided a method of preventing bone loss in an individual by inhibiting the expression of WNT signaling antagonist. Preferably, the WNT signaling antagonist is soluble frizzled related protein 3 (SFRP-3/FRZB) or the human homologue of Dickkopf-1 (DKK1). In general, the expression of these antagonists can be inhibited at the nucleic acid or protein level or in any other fashion readily know to those having ordinary skill in this art.

In yet another embodiment, there is provided a method of controlling bone loss in an individual, comprising the step of inhibiting the expression of the DKK1 gene (accession number NM 012242: SEQ ID NO: 1 or the activity of the protein expressed by the DKK1 gene. The DKK1 gene expression is inhibited by any method known to a person having ordinary skill in this art including, e.g., anti-sense oligonucleotides or by anti-DKK1 antibodies or soluble LRP receptors.

In yet another embodiment, there is provided a method of controlling bone loss in an individual, comprising the step of administering to the individual a pharmacological inhibitor of DKK1 protein. Generally, this method would be useful where the individual has a disease such as multiple myeloma, osteoporosis, post-menopausal osteoporosis or malignancy-related bone loss. Generally, the malignancy-related bone loss is caused by breast cancer metastasis to the bone or prostate cancer metastasis to the bone.

In another embodiment of the present invention, there is provided a method of preventing bone resorption and increasing bone formation in an individual, comprising: blocking the activity of DKK1, where the blocking increases osteoblast numbers and reduces osteoclast activity, thereby preventing bone resorption and increasing bone formation in the individual. Generally, the activity of DKK1 is blocked by administering anti-DKK1 antibodies, DKK1 anti-sense oligonucleotides or small molecule inhibitor to the individual. Additionally, an individual who will benefit from this method is the same as described supra.

In yet another embodiment of the present invention, there is a method of inhibiting tumor growth in bone of an individual, the method comprising the step of blocking the activity of DKK1. Generally, the DKK1 activity is blocked by administering anti-DKK1 antibodies, DKK1 anti-sense oligonucleotides or small molecule inhibitor to the individual. Moreover, an individual who will benefit from such a method although not limited to includes one who has multiple myeloma, metastatic breast cancer or prostate cancer.

In another embodiment of the present invention, there is a method of screening for a compound that controls bone loss and inhibits human myeloma cell growth, comprising: engrafting human myeloma cells in a rabbit bone implanted in a SCID-rab mouse, administering the compound to the mouse; and comparing bone mineral density of the implanted bone and level of serum human monoclonal immunoglobulin in the mouse with a control SCID-rab mouse that has not received the compound, where an increase in the bone mineral density and a decrease in the level of serum immunoglobulin in the treated mouse compared to the control mouse indicates that the compound controls bone loss and inhibits human myeloma growth. Generally, the compound is an inhibitor of WNT signaling antagonist. Specifically, the WNT signaling antagonist is human homologue of Dickkopf-1 (DKK1) or soluble frizzled related protein 3 (SFRP-3/FRZB).
 

Claim 1 of 4 Claims

1. A method of controlling bone loss characterized by increased DKK1 expression or activity in an individual comprising the steps of: administering to an individual in need thereof an amount of an anti-DKK1 antibody that is pharmacologically effective to inhibit the expression of the DKK1 gene of SEQ ID NO: 1 or the activity of the Dkk1 protein expressed by the DKK1 gene.

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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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