WO2008115470A2 - Hox-gene expression as a biomarker for igf-1r therapeutics - Google Patents

Abstract

This invention provides methods for treating or preventing the onset of a tumor in a subject, wherein the tumor is determined to overexpress a Hox gene, including administering to the subject a therapeutically or prophylactically effective amount of an insulin-like growth factor-1 receptor (IGF-1R) antagonist. The invention also provides methods for determining whether a tumor in a subject is amenable to treatment with an IGF-1R antagonist including determining whether the tumor overexpresses a Hox gene, wherein overexpression of the Hox gene indicates that the tumor is amenable to treatment with an IGF-1R inhibitor.

Description

HOX-GENE EXPRESSION AS A BIOMARKER FOR IGF lR THERAPEUTICS

RELATED APPLICATION DATA

The present application claims the benefit of U.S. Patent Application Serial No. 60/918,593, filed March 16, 2007, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION The present invention relates to the use of an insulin-like growth factor receptor antagonist to treat tumors that overexpress a Hox gene.

BACKGROUND OF THE INVENTION The cellular microenvironment and cancer The development, maintenance and/or progression of a cancer require a tumor microenvironment and "tumor stromal cells" that support the proliferation and survival of the original malignant lesion. This phenomenon has been best characterized in leukemias. Normal hematopoietic development depends upon interactions with the bone marrow microenvironment (Bertrand et al., 2000; Gibson, 2002), which comprises a complex mixture of growth factors, extracellular matrix and stromal cells that provide extrinsic signals that regulate the growth, differentiation and survival of hematopoietic precursors. Acute leukemia is often characterized by the clonal expansion of precursors of any one of several hematopoietic developmental stages. However, the precise role of the bone marrow microenvironment in the promotion of leukemic cell proliferation and survival is not fully characterized. Successful culture of freshly isolated leukemic blasts generally includes the presence of stromal cell feeder layers, suggesting that stromal cells play a role in modulating proliferation and survival of acute leukemic cells (Manabe et al., 1994). Accordingly, sub-types of B-lineage leukemia have been shown to localize to distinct stromal cell/microenvironmental regions of the bone marrow (Vega et al., 2002). Loss of responsiveness to microenvironmental cues, such as growth factor or stromal cell-derived signals, is a step in leukemic progression (Gibson, 2002; LeBien, 2000). As leukemic cells lose the need for stromal cell and microenvironmental support for survival, there is no longer any selective pressure to retain those cells in discreet compartments of the bone marrow. Thus, these cells can survive and continue to grow through the metastatic process. Much of what is known today regarding hematopoiesis, leukemia and interactions with the bone marrow microenvironment has been shown to be similar in other malignancies. Thus, as has been observed with loss of stromal cell dependency and the progression of leukemia, advanced metastatic disease in solid rumors such as prostate or breast tumors correlates with a loss of dependency upon the tumor microenvironment for proliferation and survival (Kim et al., 2005; Alberti, 2006). This is associated with drug resistance and poor prognosis, and has important clinical correlates for treatment design and outcome prediction. For example, in prostate cancer, the 5-year survival rate for advanced metastatic disease is only 34%, but for disease localized within the prostate the 5-year survival rate is greater than 90% (American Cancer Society, 2006). Role of Hox genes in cancer and in regulation of growth and survival pathways

Many subtypes of leukemia, such as those bearing translocations of the mixed lineage leukemia gene (MLL), typically exhibit an ability to proliferate and survive under more stringent growth conditions, as compared with other subtypes (Kersey et al., 1998). Interestingly, MLL leukemias characteristically exhibit overexpression of HoxA9

(Armstrong et al., 2002; Yeoh et al., 2002; Hess, 2004; Basecke et al., 2006). In mouse models of MLL-oncogenesis, HoxA9 has been shown to be necessary for leukemogenesis and in human cell line models, lack of HoxA9 in the presence of functional MLL and AF4 genes correlates with the retention of growth factor- and stromal cell-dependent growth (Bertrand et al., 2003; 2001 ; Ayton et al., 2003). Thus, HoxA9 overexpression appears to be linked to loss of growth factor/stromal cell responsiveness.

In humans, there are at least 39 Hox genes organized into four loci located on 4 different chromosomes spanning 100 kb each (Apiou et al., 1996; Acampora et al., 1989). Based on position and sequence similarity, Hox genes can be classified into paralog groups that span each Hox gene cluster. For example, HoxAl, HoxBl and HoxDl form paralog group 1 and HoxAl shares more sequence homology with these genes than with other HoxA genes, such as HoxA2 (Apiou et al., 1996). During development Hox gene expression occurs in a temporal and spatial fashion, with 3' Hox genes (paralog groups 1 to 4) being expressed earlier in anterior regions followed by more 5' Hox gene expression in the posterior (Apiou et al., 1996). Interestingly, there is evidence for a similar 3' to 5' expression pattern in hematopoietic cell differentiation (Magli et al., 1997). HoxA and HoxB cluster gene expression is found in CD34⁺ subsets and HoxB genes are expressed in peripheral B- and T-cells (Sauvageau et al., 1994). There is evidence too for lineage-specific Hox gene expression such as the restriction of HoxAlO expression to myeloid cells (Lawrence et al., 1995).

HoxA9 in particular appears to be involved in normal hematopoiesis and in leukemogenesis (Grier et al., 2005; Abramovich et al., 2005). In normal hematopoietic development, HoxA9 expression occurs within the CD34⁺ compartment and decreases with developmental maturity (Sauvageau et al., 1994). Overexpression of HoxA9 is one of several key "signature" genes that correlate well with certain subtypes of leukemia, such as those bearing MLL translocations (Armstrong et al., 2002; Yeoh et al., 2002; Hess et al., 2004). Some studies have suggested that increased HoxA9 gene expression is the key step in A/LZ-mediated leukemogenesis and may also have a role in modulating the phenotype of the leukemia (Bertrand et al., 2003; 2001; Ayton et al., 2003; Kumar et al., 2004). HoxA9 is also involved in translocations with the nucleoporin 98 gene (NUP98) that are highly leukemogenic. Overexpression of HoxA9 is also commonly found in a variety of other acute leukemias, and has been reported to result in proliferative expansion of hematopoietic stem/progenitor cells at the expense of mature compartments (Thorsteinsdottir et al., 2002).

Different Hox genes play a critical role in hematopoiesis, and abnormal expression of individual Hox genes is associated with the onset of lymphoid malignancies (Magli et al., 1997; Cillo et al., 2001). HoxA4, HoxA5, HoxA7, and HoxA9 are often considered to be pivotal Hox genes required for transformation, and an increasing body of literature indicates that HoxA9 in particular is involved in normal hematopoiesis and in leukemogenesis (Grier et al., 2005; Abramovich et al., 2005). In multiple myeloma, altered Hox gene expression was observed in greater than 9% of the patients studied, although studies of Hox genes in this malignancy are few (Hudlebusch et al., 2004). Altered Hox gene expression has also been reported in small cell lung cancer (SCLC) (Grier et al., 2005; Tiherio et al., 1994; Lechner et al., 2001; 2002). HoxA9 and HoxA7 were among those Hox genes whose expression was frequently found in SCLC. Aberrant HoxC8 expression is frequently associated with prostate tumors (Kikugawa et al., 2006; Miller at al., 2003; Waltregny et al., 2002), and altered Hox gene expression is also found in breast and colon tumors (Grier et al., 2005). Whereas these studies are not as extensive as those performed in hematological malignancy, they do point towards a fundamental role for dysregulated Hox gene expression in the malignant expansion of certain solid tumors and multiple myeloma.

The exact molecular pathways regulated by Hox gene expression in hematopoietic cell growth and differentiation have not been fully elucidated, but a variety of studies indicate that altered Hox expression can influence differentiation and growth factor responses (Magli et al., 1997). For example, expression of HoxB8 in a mouse myeloid cell line has been shown to block IL-6-induced differentiation (Blatt et al., 1992). HoxB7 is expressed in human bone marrow upon GM-CSF stimulation and reduced HoxB7 expression inhibits formation of GM-CSF-induced colony formation (LiIl et al., 1995). HoxA9^{" "} mice exhibit a reduction in peripheral lymphocytes, myeloid progenitors and pre-B cells (Lawrence et al., 1997). These mice also have a profound reduction in GM- CSF responses. Prior to the studies disclosed herein, however, it was not clear whether altered growth factor responses in the presence of abnormal Hox gene expression was a direct effect or part of a larger pathway(s). HoxA5 and HoxAlO have recently been shown to bind to the forkhead related transcription factor (FKHR) resulting in increased expression of insulin-like growth factor binding protein-1 (IGFBP-I) (Foucher et al.,

2002; Kim et al., 2003). IGFBP-I participates in regulating the availability of insulin-like growth factor- 1 (IGF-I) (Baxter, 2000). High serum levels of IGF-I have been shown to correlate with childhood leukemia, and there is documentation of a correlation between high infant birth weight and infant acute lymphocytic leukemia (ALL) that also correlates with high levels of IGF-I (Vorwerk et al., 2002; Ross et al., 1996). Overexpression of the insulin-like growth factor- 1 receptor (IGF-IR) can relieve hematopoietic cells of cytokine dependency, provided IGF-I is present (McCubrey et al., 1991). Altogether, these studies provide evidence for a direct mechanism of growth regulation by Hox genes.

Involvement of IGF-I receptor in cell proliferative diseases Many research and clinical studies have implicated the IGF-IR and its ligands

(IGFs) in the development, maintenance, and progression of cancer. In tumor cells, overexpression of the receptor, often in concert with overexpression of IGF ligands, leads to potentiation of IGF signals and result in enhanced cell proliferation and survival. IGF- 1 and IGF-2 have been shown to be strong mitogens for a wide variety of cancer cell lines including prostate (Nickerson et al., 2001; Hellawell et al., 2002), breast (Gooch et al., 1999), lung, colon (Hassan and Macaulay, 2002), stomach, leukemia, pancreas, brain, myeloma (Ge and Rudikoff, 2000), melanoma (All-Ericsson et al., 2002), and ovary (reviewed by Macaulay, 1992), and this effect is mediated through the IGF-IR. High circulating levels of IGF-I in serum have been associated with an increased risk of breast, prostate, and colon cancer (Pollak, 2000). In a mouse model of colon cancer, increases in circulating IGF-I levels in vivo led to a significant increase in the incidence of tumor growth and metastasis (Wu et al., 2002). Constitutive expression of IGF-I in epidermal basal cells of transgenic mice has been shown to promote spontaneous tumor formation (DiGiovanni et al., 2000; BoI et al., 1997). Over-expression of IGF-2 in cell lines and tumors occurs with high frequency and may result from loss of genomic imprinting of the IGF-2 gene (Yaginuma et al., 1997). Receptor over-expression has been demonstrated in many diverse human tumor types including lung (Quinn et al., 1996), breast (Cullen et al., 1990; Peyrat and Bonneterre, 1992; Lee and Yee, 1995), sarcoma (van Valen et al., 1992); Scotlandi et al., 1996), prostate (Nickerson et al., 2001), and colon (Hassan and Macaulay, 2002). In addition, highly metastatic cancer cells have been shown to possess higher expression of IGF-2 and IGF-IR than tumor cells that are less prone to metastasize (Guerra et al., 1996). A critical role of the IGF-IR in cell proliferation and transformation was demonstrated in experiments of IGF-IR knockout derived mouse embryo fibroblasts. These primary cells grow at significantly reduced rates in culture medium containing 10% serum and fail to transform by a variety of oncogenes including SV40 Large T (Sell et al., 1994)). Recently, it was demonstrated that resistance to the drug Herceptin in some forms of breast cancer may be due to activation of IGF-IR signaling in those cancers (Lu et al., 2001). Over-expression or activation of IGF-IR may therefore not only be a major determinant in tumorigenicity, but also in tumor cell drug resistance.

Activation of the IGF system has also been implicated in several pathological conditions besides cancer, including acromegaly (Drange and Melmed, 1999), retinal neovascularization (Smith et al., 1999), and psoriasis (Wraight et al., 2000). In the latter study, an antisense oligonucleotide preparation targeting the IGF-IR was effective in significantly inhibiting the hyperproliferation of epidermal cells in human psoriatic skin grafts in a mouse model, suggesting that anti-IGF- IR therapies may be an effective treatment for this chronic disorder.

A variety of strategies has been developed to inhibit the IGF-IR signaling pathway in cells, including use of antibodies directed against human IGF-IR (Artega and Osborne, 1989; Scotlandi et al., 1998; Furlanetto et al., 1993), inhibitory peptides targeting the IGF-IR (Pietrzkowski et al., 1992; Haylor et al., 2000; Reiss et al., 1999), small molecule IGF-IR inhibitors, antisense oligonucleotides in vitro and in an experimental mouse model to inhibit hyperproliferation of epidermal cells in psoriasis (Wraight et al., 2000), overexpression of dominant-negative mutants of the IGF-IR in tumor cell lines (Scotlandi et al., 2002; Seely et al., 2002), and a soluble form of the IGF- IR (D'Ambrosio et al., 1996).

Whereas studies on the association of Hox with transformation has provided evidence for regulation of cell growth and survival pathways by Hox genes, almost nothing is known about events downstream of Hox gene expression that specifically promote tumor cell proliferation and survival. The data disclosed herein illuminate the pathways specifically regulated by Hox gene overexpression and which promote tumor cell proliferation and survival. These pathways provide attractive targets for therapeutics to treat cancers that overexpress Hox genes.

Full details for the various publications cited throughout this application are provided at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated in their entireties by reference into this application. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention.

SUMMARY OF THE INVENTION

Embodiments of the present invention provide a method for treating a tumor in a subject, which tumor is determined to overexpress a Hox gene, comprising administering to the subject a therapeutically effective amount of an insulin-like growth factor- 1 receptor (IGF-IR) antagonist. In different embodiments, the antagonist may be, but is not limited to, an antibody or functional derivative thereof that binds immunospecifically to IGF-IR, a small molecule IGF-IR antagonist, a small interfering RNA (siRNA), an antisense nucleic acid, an insulin-like growth factor (IGF) mimetic, a ribozyme, a triple helix-forming nucleic acid, a dominant negative mutant, or a soluble form of IGF-IR. According to some embodiments, the tumor is cancerous tumor, a benign tumor, a benign growth, or a benign neoplasm.

Embodiments of the invention further provide use of an insulin-like growth factor- 1 receptor (IGF-IR) antagonist for the preparation of a medicament for the treatment of a tumor in a subject, which tumor is determined to overexpress a Hox gene.

Embodiments of the invention also provide a method for determining whether a tumor in a subject is amenable to treatment with an IGF-IR antagonist comprising determining whether the tumor overexpresses a Hox gene, wherein overexpression of the Hox gene indicates that the tumor is amenable to treatment with an IGF-IR inhibitor.

Embodiments of the invention provide a method of identifying an agent effective for treating a tumor, wherein the tumor is determined to overexpress a Hox gene, comprising determining whether the agent inhibits insulin-like growth factor- 1 receptor (IGF-IR) expression, wherein an agent that inhibits IGF-IR expression is effective for treating the tumor.

Embodiments of the invention further provide kits for use in treating a tumor in a subject comprising a packaging material containing therein an agent identified as effective for treating the tumor, and a label providing instructions for administering the agent to the subject. Embodiments of the invention also include kits for inhibiting tumor growth.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Retroviral transduction of BLIN-2 cells to generate stably transfected inducible BLIN-2/HoxA9:ER cells. A, Retroviral construct. HoxA9 was fused in frame with the hormone binding domain of the estrogen receptor and the V5 epitope tag. The construct is based on an IRES-GFP backbone so that transduced cells can be rapidly identified based on GFP expression. B, Post-sort analysis of transduced BLIN-2 cells. BLIN-2 cells were transduced with HoxA9:ER or MigRl (empty GFP vector) retroviruses packaged with an amphotropic coat. Transduced cells were purified by fluorescence-activated cell-sorting (FACS) based on GFP expression. The percentages of GFP-positive BLIN-2/HoxA9:ER and control BLIN-2/MigRl cells post-sort are shown. Parental BLIN-2 cells are included as a negative control for GFP expression. Figure 2. Illustration of HoxA9:ER inducible system. HoxA9 is fused to the hormone binding domain of the estrogen receptor (ER HB). Retrovirally transduced HoxA9 protein is always expressed. In the absence (top panel) of the estrogen analogue, 4HT, heat shock proteins (Hsp) bind to HoxA9, preventing nuclear localization. In the presence of 4HT (bottom panel), Hsp are competed off the ER HB and HoxA9 is translocated to the nucleus where it can function to bind HoxA9 target promoters.

Figure 3. Western analysis of 4HT-mediated induction of HoxA9 in stably transduced BLIN-2/HoxA9:ER cells. Total cellular protein (W = whole cell lysate) was prepared from BLIN-2/MigRl cells (lanes 1 and 2), and cytoplasmic (C) and nuclear (N) extracts were prepared from BLIN-2/HoxA9:ER cells (lanes 3 - 6). Cells grown in the presence or absence of 1 μM 4HT and 20 μg of protein was subjected to western blotting. Actin serves as a loading control for cytoplasmic and total protein, and Pax5 for nuclear protein.

Figure 4. HoxA9:ER binds a consensus HoxA9 DNA binding sequence in BLIN- 2/HoxA9:ER cells. EMSA was used to assess DNA binding of HoxA9:ER. 10 μg of total protein from BLIN-2/MigRl or BLIN-2/HoxA9:ER cells was incubated with 4 ng of a labeled consensus binding sequence for HoxA9 (lanes 2 - 9) in the presence or absence of 250X excess unlabeled probe (Competitor) (lanes 3, 5, 7, and 9) and 1 μg of anti-HoxA9 antibody (lanes 4, 5, 8, and 9). The top arrow marks the migration of the HoxA9:ER/DNA complex (lane 6).

Figure 5. Induction of HoxA9:ER with 4HT alters the growth characteristics of BLIN-2/HoxA9:ER cells in the absence of stromal cell support. Proliferation of BLIN- 2/MigRl and BLIN-2/HoxA9:ER cells was evaluated by a quantitative colorimetric assay used to detect cell survival and proliferation. Cells were cultured for 5 days in media alone or media containing 1 μM 4HT. At the indicated time points cells were assayed for relative proliferation (expressed as absorbance at 492 nm).

Figure 6. c-Myb levels are increased in the presence of HoxA9:ER activation. A, Western blot analysis of total cellular protein from parental BLIN -2 and BLIN- 2/HoxA9:ER cells grown in the presence of 1 μM 4HT. 20 μg of total cellular protein was resolved by SDS-PAGE and transferred to a PVDF membrane. Membranes were probed with antibodies against HoxA9 and c-Myb (β-tubulin serves as a loading control). B, Cytoplasmic and Nuclear fractions were prepared from BLIN-2 and BLIN- 2/HoxA9:ER cells treated with 1 μM 4HT and western analysis was performed as described above. Actin serves as a loading control for cytoplasmic protein. Pax5 and p70 both serve as nuclear fraction loading controls.

Figure 7. HoxA9:ER induces expression of the insulin-like growth factor receptor- 1. A, RT-PCR amplification of BLIN-2/MigRl and BLIN-2/HoxA9:ER cells cultured in the presence of 1 μM 4HT. RS4;11 (Stong et al., 1985) is a well characterized t(4;l 1) cell line that expresses endogenous HoxA9. "- RT" means sample prepared without reverse transcriptase. B, Western analysis of parental BLIN-2 and BLIN- 2/HoxA9:ER cells stimulated with 1 μM 4HT for 24 h. β-tubulin serves as a control for equal protein loading. C, FACS analysis of BLIN-2 and BLIN-2/HoxA9:ER cells stimulated with 1 μM 4HT for 24h. Cells were centrifuged for 5 min and resuspended with mouse anti-IGF-lR, and goat anti-mouse IgG-FITC was used as a secondary antibody to evaluate surface expression of IGF-IR. Dotted lines represent isotype- matched negative controls; solid lines represent anti-IGF-lR stained cells. Figure 8. Loss of endogenous HoxA9 expression results in loss of IGF-IR expression. RS4;11 cells were mock transfected (lane C) or transfected with the indicated amount of HoxA9 siRNA followed by RT-PCR analysis to detect HoxA9 and IGF-IR expression. Loss of HoxA9 expression correlated with loss of IGF-IR expression. Expression of the B-lineage specific gene, MB-I, and expression of the MLL/ AF4 fusion protein was not altered in response to loss of HoxA9 expression. The negative image of an ethidium bromide gel is shown. "- RT" means sample prepared without reverse transcriptase. "H₂O" means samples without a cDNA template.

Figure 9. Inhibition of IGF-IR signaling reduces BLIN-2/HoxA9:ER proliferation in the presence of stromal cell support. BLIN-2/MigRl (left panel) and BLIN-2/HoxA9:ER cells (right panel) were cultured on stromal cell feeder layers for 10 days in the presence of 1 μM 4HT and treated with 1 μg/ml of the IGF-IR inhibitor, AGl 024. Proliferation was determined by colorimetric assay and is presented as absorbance at 492 nm. All experiments were performed in triplicate. * denotes statistical significance (p < 0.0001). Figure 10. Inhibition of IGF-IR signaling reduces BLIN-2/HoxA9:ER proliferation in the absence of stromal cell support. BLIN-2/HoxA9:ER cells were treated with 1 μM 4HT, (empty bars) or with 1 μM 4HT and 1 μg/ml of AG 1024 (solid bars). Cells were cultured in the absence of stromal cell support for 5 days. Proliferation was determined by colorimetric assay and is presented as fold increase over the initial input number. * denotes statistical significance (p < 0.0001).

Figure 11. Blocking of the IGF-I receptor with monoclonal antibody inhibits proliferation of BLIN-2/HoxA9:ER cells. Parental BLIN-2 (left panel) and BLIN- 2/HoxA9:ER cells (right panel) were treated with 1 μM 4HT to induce activity of

HoxA9:ER and grown in the presence or absence of the anti-IGF-lR antibody, A12 (15 μg). Proliferation was determined as described. * denotes statistical significance (p < 0.02).

Figure 12. Blocking IGF-IR signaling reduces the proliferation of RS4;11. The t(4; 11 ) cell line, RS4; 11 , was cultured in the presence or absence of 15 μg A12 monoclonal antibody (left panel) and 50 ng of IGF-I (right panel). Proliferation was determined as described above. * denotes statistical significance (p < 0.05).

Figure 13. Model of HoxA9-induced regulation of IGF-IR and IGF-I expression. In this model, HoxA9 induces expression of the c-Myb transcription factor which in turn increases IGF-IR expression. Because c-Myb also promotes lGF-1 expression, an autocrine loop may be established that ultimately leads to stromal cell/growth factor- independent growth.

Figure 14. IGF-I Gene Expression. RT-PCR was performed to access IGF-I gene expression in the indicated cells. H2O means no cDNA template. Some cells were treated for 24 hours with 4HT (+4HT) prior to analysis. A Southern blot of the PCR products separated on a 1.5% agarose gel is shown.

Figure 15. Al 2 inhibits IGF-IR phosphorylation in BLIN-2/HoxA9:ER cells. A) BLIN-2, BLIN-2/MigRl, and BLIN-2/HoxA9:ER cells were cultured in the presence of 4HT (4HT), IGF-I (IGF-I), 4HT and IGF-I (4HT + IGF-I), or 4HT, IGF-I and Al 2 (A12). Total protein was isolated and immunoprecipitation (IP) of the IGF-lRα subunit of IGF-IR was performed followed by western blot analysis of phosphorylated tyrosine (P- Tyr) residues on the IGF- lRβ chain. B) Phosphorylated IGF-IR was detected by ELISA. Cells were cultured as described above and ELISA was performed using 40 μg of total cellular protein. Plates were read at 450 nm and results are presented as fold increase in IGF-IR phosphorylation relative to untreated controls.

Figure 16. Signaling through HoxA9 induced IGF-IR protects BLIN- 2:HoxA9:ER cells from apoptosis. Cells were culture in the presence of 4-HT and IGF-I, and in the presence or absence of AG 1024, off of stromal cell support for 24 hr. Western blot analysis was performed using anti-cleaved PARP antibody. PARP cleavage is a hallmark of apoptosis. β-tubulin serves as a loading control.

DETAILED DESCRIPTION

The present invention relates to the use of IGF-IR antagonists to treat a tumor that overexpresses a Hox gene. Several reports have indicated that aberrant Hox expression can perturb normal cell development and are involved in tumorigenesis. However, prior to the present study, relatively little was known regarding the pathways activated by Hox overexpression that directly contribute to the proliferation and survival of tumor cells. Data presented herein indicate that Hox overexpression promotes proliferation and survival of cells, in the absence of growth factor and stromal cell support, through the activation of specific downstream signaling pathways involving increased c-Myb expression and increased surface expression of IGF-IR. These and other data demonstrating the inhibition of cell proliferation using IGF-IR inhibitors suggest that Hox overexpression promotes tumor cell growth through increased expression of the IGF- IR via induction of the c-Myb transcription factor, making inhibition of IGF-IR an attractive target for therapeutics aimed at treating tumors that overexpress one or more Hox genes.

Accordingly, the invention provides a method for treating a tumor in a subject, wherein the tumor is determined to overexpress a Hox gene, comprising administering to the subject a therapeutically effective amount of an IGF-IR antagonist. This invention also provides a method for inhibiting in a subject the onset of a tumor, wherein a pre- tumorous cell is determined to overexpress a Hox gene, comprising administering to the subject a prophylactically effective amount of an IGF-IR antagonist. The IGF-IR antagonist may operate indirectly by inhibiting Hox or c-Myb gene expression. Hence, the IGF-IR antagonist includes an inhibitor of Hox or c-Myb gene expression. Thus, this invention further provides a method for treating a tumor in a subject, wherein the tumor is determined to overexpress a Hox gene, comprising administering to the subject a therapeutically effective amount of an agent that inhibits Hox or c-Myb gene expression. Additionally, the invention provides a method for inhibiting in a subject the onset of a tumor, wherein a pre-tumorous cell is determined to overexpress a Hox gene, comprising administering to the subject a prophylactically effective amount of an agent that inhibits Hox or c-Myb gene expression. The invention further provides methods of decreasing and/or inhibiting cancer and/or tumor cell proliferation comprising administering to a subject or contacting a target of interest an effective amount of an agent that inhibits Hox or c-Myb gene expression.

In any of the methods described herein, the tumor is cancerous tumor, a benign tumor, a benign growth, or a benign neoplasm.

As used herein, a "subject" shall mean any animal or artificially modified animal. Animals include, but are not limited to, humans, non-human primates, dogs, cats, rabbits, ferrets, and rodents such as mice, rats and guinea pigs. Artificially modified animals include, but are not limited to, SCID mice with human immune systems. In particular embodiments of this invention, the subject is a human. Moreover, the subjects may be male or female and may be of any race or ethnicity, including, but not limited to, Caucasian, African-American, African, Asian, Hispanic, Indian, etc. The subjects may be of any age, including newborn, neonate, infant, child, adolescent, adult, and geriatric. Animal subjects, particularly mammalian subjects such as canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g. rats and mice), lagomorphs, primates (including non-human primates), etc., may further be subjects for veterinary medicine and/or pharmaceutical and veterinary drug development and screening purposes. Hox gene overexpression induces cell transformation via induction of IGF-IR

Little is known about the identity of the pathways activated by Hox gene overexpression that directly contribute to the proliferation and survival of tumor cells. Experiments were therefore conducted to investigate whether Hox overexpression promotes growth factor/stromal cell-independent growth of pre-cancer cells through the activation of specific downstream signaling pathways.

These experiments, described in detail in the Examples set forth below, focused on the effects of HoxA9 overexpression in pre-leukemic cells. HoxA9 is one of the most frequently overexpressed Hox genes in leukemia, and several studies have indicated that aberrant HoxA9 expression can perturb normal hematopoiesis resulting in developmental defects in both myeloid and lymphoid lineages. Depending upon the availability of co- factors (namely Meisla), HoxA9 has been shown to be transforming, though it also appears likely that HoxA9 may act to promote tumor cell proliferation and survival in situations where other abnormalities are likely the initial transforming event, such as MLL translocations. Aberrant expression of many other Hox genes has also been demonstrated in malignancies other than leukemia. Hox genes are remarkably similar and bind very similar consensus sequences in DNA. Regulation of specificity among DNA binding targets of various Hox genes is poorly understood, and to date it appears that most of the specificity comes from temporal expression of different Hox genes. Accordingly, one skilled in the art would recognize that the HoxASVleukemia model system described herein is applicable to the expression of Hox genes other than HoxA9 and to malignancies other than leukemia, including solid tumors and multiple myeloma, and particularly those in which HoxA locus genes have been implicated, such as breast cancer and small cell lung cancer.

To investigate the mechanisms by which HoxA9 overexpression promotes growth factor/stromal cell-independent growth of B-cell precursor acute lymphoblastic leukemic (pre-B ALL) cells, an inducible model of HoxA9 expression was developed using a stromal cell-dependent pre-B ALL cell line, BLIN -2, that permits the identification of HoxA9-mediated effects on cell proliferation and survival in the biologically relevant context of the bone marrow/stromal cell microenvironment. BLIN-2 is a pre-B ALL cell line that lacks expression of HoxA genes and requires viable stromal cell contact for proliferation and survival. In the stable BLIN-2/HoxA9:ER cell line, a cDNA encoding HoxA9 was fused to a polynucleotide encoding the hormone binding domain of the estrogen receptor (ER) and was stably integrated into the BLIN-2 genome via retroviral transduction. HoxA9 activity was induced upon addition of the estrogen analog 4- hydroxytamoxifen (4HT) to the culture medium through stabilization of the HoxA9:ER protein and its translocation from the cytosol to the nucleus. The ability of HoxA9:ER to bind its target DNA sequence was confirmed via electrophoretic mobility shift assays (EMSA).

Using the BLIN-2/HoxA9:ER inducible model of HoxA9 activity, it was observed that induction of HoxA9 resulted in (1) increased cell proliferation in the presence or absence of stromal cell support; (2) prolonged cell survival due to increased apoptotic resistance in the absence of stromal cell support as compared with parental BLIN-2 cells or BLIN-2 cells transduced with an empty vector (BLIN-2/MigRl); (3) the appearance of detectable surface expression of insulin-like growth factor- 1 receptor (IGF-IR), which correlated with (4) increased expression of the transcription factor c-Myb. An increase in c-Myb expression has been reported to promote both IGF-IR and IGF-I expression. In addition, inhibition of IGF-IR signaling with two inhibitors that have very distinct modes of action, mAb Al 2 and the small molecule antagonist AG 1024, abrogated the HoxA9- induced proliferative and survival effects. Treatment of BLIN-2/HoxA9:ER cells with the IGF-IR small molecule inhibitor, AGl 024, blocked the HoxA9-mediated survival and proliferation of these cells in the absence of stromal support, whereas AG 1024 treatment had little effect on parental BLIN-2 cells or BLIN-2/MigRl cells bearing an empty vector. Use of an anti-IGF-lR antibody that blocks IGF-IR signaling also inhibited HoxA9:ER- mediated proliferative effects. Moreover, treatment of a leukemic cell line, RS4;11, that expresses high levels of endogenous HoxA9, with mAb Al 2 inhibited cell proliferation.

These data collectively indicate that IGF-IR is a downstream target of HoxA9 expression and that increased expression of IGF-IR accounts for the observed biological effects on proliferation and cell survival in leukemic cells overexpressing HoxA9. hi support of this conclusion, it was found that in a small sampling of B-ALL cells lines, all those that expressed HoxA9 also expressed IGF-IR, whereas those in which HoxA9 was not expressed, IGF-IR was frequently not expressed (data not shown). The data support a model in which overexpression of HoxA9 promotes leukemic cell growth through induction of the c-Myb transcription factor which in turn increases expression of the IGF- IR receptor. This model validates the concept of targeting the IGF-IR receptor as a therapeutic for the treatment of leukemia and other cancers that are associated with increased Hox gene expression. Thus, the overexpression of HoxA9 and other Hox family genes provides a good biomarker for cancers that can be treated by therapeutic targeting of the IGF-IR using IGF-IR antagonists.

Structure and function of IGF-IR The IGF-IR is a ubiquitous transmembrane tyrosine kinase receptor that is essential for normal fetal and post-natal growth and development. IGF-IR can stimulate cell proliferation and differentiation, changes in cell size, and can protect cells from apoptosis. It has also been considered to be quasi-obligatory for cell transformation (reviewed in Adams et al., 2000; Baserga, 2000). The IGF-IR is located on the cell surface of most cell types and serves as the signaling molecule for the growth factors,

IGF-I and insulin-like growth factor-2 (IGF-2). IGF-IR also binds insulin, albeit at three orders of magnitude lower affinity than it binds to IGFs. IGF-IR is a pre-formed hetero- tetramer containing two alpha and two beta chains covalently linked by disulfide bonds. The receptor subunits are synthesized as part of a single polypeptide chain of 180 kD, which is proteolytically processed into alpha (130 kD) and beta (95 kD) subunits. The entire alpha chain is extracellular and contains the site for ligand binding. The beta chain possesses the transmembrane domain, the tyrosine kinase catalytic domain, and a C- terminal extension that is necessary for cell differentiation and transformation, but is dispensable for mitogen signaling and protection from apoptosis.

The IGF-IR is highly similar to the insulin receptor (IR), particularly within the beta chain (70% sequence homology). Because of this homology, recent studies have demonstrated that these receptors can form hybrids containing one IR dimer and one IGF- IR dimer (Pandini et al., 1999). The formation of hybrids occurs in both normal and transformed cells and the hybrid content is dependent upon the concentration of the two homodimer receptors (IR and IGF-IR) within the cell. In one study of 39 breast cancer specimens, although both IR and IGF-IR were over-expressed in all tumor samples, hybrid receptor content consistently exceeded the levels of both homo-receptors by approximately 3-fold (Pandini et al., 1999). Though hybrid receptors are composed of IR and IGF-IR pairs, the hybrids bind selectively to IGFs, with affinity similar to that of IGF-IR, and only weakly bind insulin (Siddle and Soos, 1999). These hybrids therefore can bind IGFs and transduce signals in both normal and transformed cells. A second IGF receptor, IGF-2R, or mannose-6-phosphate (M6P) receptor, also binds IGF-II ligand with high affinity, but lacks tyrosine kinase activity (Oates et al., 1998). Because it results in the degradation of IGF-2, it is considered a sink for IGF-2, antagonizing the growth promoting effects of this ligand. Loss of the IGF-2R in tumor cells can enhance growth potential through release of its antagonistic effect on the binding of IGF-2 with the IGF- 1 R (Byrd et al., 1999).

Endocrine expression of IGF-I is regulated primarily by growth hormone and produced in the liver, but recent evidence suggests that many other tissue types are also capable of expressing IGF-I. This ligand is therefore subjected to endocrine and paracrine regulation, as well as autocrine in the case of many types of tumor cells (Yu and Rohan, 2000).

Six IGF binding proteins (IGFBPs) with specific binding affinities for the IGFs have been identified in serum (Yu and Rohan, 2000). IGFBPs can either enhance or inhibit the action of IGFs, as determined by the molecular structures of the binding proteins as a result of post-translational modifications. Their primary roles are for transport of IGFs, protection of IGFs from proteolytic degradation, and regulation of the interaction of IGFs with IGF-IR. Only about 1% of serum IGF-I is present as free ligand, the remainder is associated with IGFBPs (Yu and Rohan, 2000). Upon binding of ligand (IGFs), the IGF-IR undergoes autophosphorylation at conserved tyrosine residues within the catalytic domain of the beta chain. Subsequent phosphorylation of additional tyrosine residues within the beta chain provides docking sites for the recruitment of downstream molecules critical to the signaling cascade. The principle pathways for transduction of the IGF signal are mitogen-activated protein kinase (MAPK) and phosphatidylinosifol 3-kinase (PI3K) (reviewed in Blakesley et al., 1999). The MAPK pathway is primarily responsible for the mitogenic signal elicited following stimulation by IGFs, and PBK is responsible for the IGF-dependent induction of anti- apoptotic or survival processes.

A key role of IGF-IR signaling is its anti-apoptotic or survival function. Activated IGF-IR signals PDK and downstream phosphorylation of Akt, or protein kinase B. Akt can effectively block, through phosphorylation, molecules such as BAD, which are essential for the initiation of programmed cell death, and inhibit initiation of apoptosis (Datta et al., 1997). Apoptosis is an important cellular mechanism that is critical to normal developmental processes (Oppenheim, 1991)). It is a key mechanism for eliminating severely damaged cells and reducing the potential persistence of mutagenic lesions that may promote tumorigenesis. To this end, it has been demonstrated that activation of IGF signaling can promote the formation of spontaneous tumors in a mouse transgenic model (DiGiovanni et al., 2000). Furthermore, IGF over-expression can rescue cells from chemotherapy-induced cell death and may be an important factor in tumor cell drug resistance (Gooch et al., 1999). Consequently, modulation of the IGF signaling pathway has been shown to increase the sensitivity of tumor cells to chemotherapeutic agents (Benini et al., 2001).

Targeting of the IGF-IR to treat Hox-overexpressing cancers

The experimental results disclosed herein indicate that the administration of IGF- IR antagonists to a patient would be beneficial in the treatment of cancers that exhibit elevated Hox gene expression. As used herein, an "IGF-IR antagonist" is any substance that blocks or impedes the signaling mediated by the IGF-IR, and comprises IGF-IR inhibitors such as antibodies, small molecule antagonists and insulin-like growth factor (IGF) mimetics that bind directly to IGF-IR and reduce the receptor's activity or concentration on a cell surface, as well as other substances that indirectly reduce IGF-IR activity by, for example, binding to ligands of IGF-IR or inhibiting expression of IGF-IR RNA. IGF-IR expression can also be downregulated indirectly by inhibiting Hox or c- Myb gene expression, as demonstrated in Example 5.

Hox overexpression has been detected in leukemias and a variety of other cancers, including SCLC, breast cancer, prostate cancer, and multiple myeloma. For example, approximately 30 to 40% of acute myelogenous leukemia (AML) and ALL exhibit elevated Hox gene expression (Look, 1997). An IGF-lR-based therapeutic strategy may be of particular and immediate benefit in treating leukemia that bear translocations of the MLL gene. 10% of all acute leukemias have MLL translocations and 80% of infant ALL have MLL translocations. The presence of MLL translocations is associated with an extremely poor prognosis and there are no therapeutic options for this subset. Overexpression of HoxA genes, including HoxA9, is a hallmark of leukemia bearing MLL-translocations and is found in nearly 100% of these patients (Hess et al., 2004; Basecke et al., 2006; and references cited therein). Overexpression of Hox genes is also found in AML bearing MLL partial tandem duplications (Basecke et al., 2006; Dorrance et al., 2006). Interestingly, in one well characterized exception to this (Bertrand et al., 2003; 2001), the BLIN-3 cell line derived from a MLL/AF4 patient lacked HoxA gene expression and also lacked IGF-IR expression.

Assays for detecting Hox overexpression

As used herein, a tumor cell that "overexpresses" a Hox gene refers to a cell that produces in its nucleus an increased amount of functional protein encoded by the Hox gene relative to an untransformed cell that does not overexpress the Hox gene. The encoded Hox protein itself may also be said to be overexpressed. In various embodiments, the level of Hox protein in the nucleus of a tumor cell is at least 2-fold higher, in some embodiments, at least 5-fold higher, and in other embodiments at least 10-fold higher, than the level of Hox protein in the nucleus of an untransformed cell known to be not overexpressing the Hox gene. The increased level of Hox protein in the nucleus of a Hox-overexpressing cell may be due, for example, to increased transcription of the Hox gene, or to increased translocation of Hox protein from the cytoplasm to the nucleus.

In certain embodiments of the present methods, the Hox gene overexpressed in a tumor is HoxA4, HoxA5, HoxA7, HoxA8, HoxA9, HoxAlO, HoxB7, HoxB8, HoxC8 gene, or any combination thereof. In some embodiments, the Hox gene is a HoxA9 gene. Various assays may be used to determine whether a tumor overexpresses a Hox gene. For example, one assay comprises measuring the level of Hox protein in a nuclear fraction of a tumor cell and comparing said level with the level of Hox protein in a nuclear fraction of an untransformed cell known to be not overexpressing the Hox gene, wherein an at least 2-fold higher level of Hox protein in the nuclear fraction of the tumor cell indicates that the tumor overexpresses the Hox gene. Another method comprises measuring the level of Hox RNA level in a tumor cell by, for example, northern blot or microarray analysis, and comparing said level with the level of Hox RNA in an untransformed cell known to be not overexpressing the Hox gene, wherein an at least 2- fold higher level of Hox RNA in the tumor cell indicates that the tumor overexpresses the Hox gene.

The detection of Hox protein in a biological sample can be accomplished by any of a number of methods well known in the art. Exemplary diagnostic methods for the detection of Hox proteins can involve, for example, immunoassays wherein Hox proteins are detected by their interaction with a Hox-specific antibody. In addition, reagents other than antibodies, such as, for example, polypeptides that bind specifically to Hox proteins can be used in assays to detect the level of protein expression. Immunoassays useful in the practice of the invention include, but are not limited to, assay systems using techniques such as western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitin reaction, gel diffusion precipitin reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assays, fluorescent immunoassay, and protein A immunoassay.

An immunoassay for detecting expression of a Hox protein typically comprises incubating the biological sample, such as the nuclear fraction from cells of a tumor, with an anti-Hox protein antibody under conditions such that an immunospecific antigen- antibody binding reaction occurs, and detecting or measuring the amount of any immunospecific binding by the antibody. Such binding of antibody can be used, for example, to detect the presence and increased production of a Hox protein, wherein the detection of increased production of a Hox protein is an indication of Hox overexpression. The level of Hox protein in the biological sample is compared to norms established for the level of Hox protein in, for example, a nuclear fraction of an untransformed cell known to be not overexpressing the Hox gene. In certain embodiments of the present screening assays, the biological sample, is brought in contact with a solid phase support or carrier, such as nitrocellulose, for the purpose of immobilizing any proteins present in the sample. The support is then washed with suitable buffers followed by treatment with detectably labeled anti-Hox protein antibody. The solid phase support is then washed with the buffer a second time to remove unbound antibody. The amount of bound antibody on the solid support is then determined according to well known methods. Those skilled in the art will be able to determine optional assay conditions for each determination by employing routine experimentation.

Hox-specific antibodies may be detectably labeled, for example, by linking the antibody to an enzyme, such as for use in an enzyme immunoassay (EIA). See, e.g.,

Voller et al. (1978); Butler (1981). The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected and quantified, for example, by spectrophotometric or fluorimetric means. Enzymes that can be used to detectably label the antibody include, but are not limited to, horseradish peroxidase and alkaline phosphatase. Detection can also be accomplished by colorimetric methods that employ a chromogenic substrate for the enzyme.

Detection of Hox antibodies may also be accomplished using a variety of other methods. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect Hox protein expression through the use of a radioimmunoassay (RIA). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

The antibody may also be labeled with a fluorescent compound. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin and fluorescamine. Likewise, a bioluminescent compound, e g. , luciferin, luciferase or aequorin, may be used to label the Hox antibody.

In certain embodiments of the invention, the levels of a Hox protein in biological sample can be analyzed by two-dimensional gel electrophoresis. Methods of two- dimensional electrophoresis are known to those skilled in the art. Biological samples, such as nuclear fractions from tumor tissue, are loaded onto electrophoretic gels for separation in the first dimension by isoelectric focusing which separates proteins based on charge. A number of first-dimension gel preparations may be utilized including tube gels for carrier ampholytes-based separations or gels strips for immobilized gradients-based separations. After first-dimensional separation, proteins are transferred onto the second dimension gel, following an equilibration procedure and separated using SDS-PAGE which separates the proteins based on molecular weight. When comparing biological samples derived from different subjects, multiple gels are prepared from individual biological samples (including samples from untransformed cell controls). Following separation, the proteins are transferred from the two-dimensional gels onto membranes commonly used for western blotting. The techniques of western blotting and subsequent visualization of proteins are also well known in the art. See, e.g., Sambrook et al (1989). Antibodies that bind to Hox proteins are utilized in an incubation step, as in the procedure of western blot analysis. A second labeled antibody specific for the first antibody may be utilized to visualize proteins that reacted with the first antibody.

Assays for measuring cell proliferation

As disclosed herein, overexpression of Hox genes can induce IGF-IR expression and thereby cause an increase in cell proliferation. In contrast, decreasing Hox gene expression may downregulate IGF-IR expression and reduce cell proliferation.

Accordingly, the present invention provides methods for identifying modulators of Hox activity based on cell proliferation assays. For example, Hox expressing cells may be grown in a 96-well plate and exposed to varying concentrations of a test substance for 4- 24 h followed by measurement of cell proliferation. Cells that may be utilized in the proliferation assays of the invention include cells over-expressing a Hox gene, wherein said overexpression results in an increase in cell proliferation. Such cells include cells that naturally overexpress a Hox gene as well as cells genetically engineered to overexpress a Hox gene.

Methods of measuring cell proliferation are well known in the art and most commonly include determining DNA synthesis characteristic of cell replication. There are numerous methods in the art for measuring DNA synthesis, any of which may be used according to the invention. For example, DNA synthesis may be determined using a radioactive label ([³H]thymidine) or labeled nucleotide analogues (BrdU) for detection by immunofluorescence. Alternatively, the rate of proliferation can be measured using any of a number of commercial colorimetric kits, such as the MTT assay. Additionally, the cells may be assayed to determine whether there are changes in levels, or modification, of proteins known to be associated with cell proliferation. Such proteins include, for example, cyclin Dl, CDK4 or pi 07. The efficacy of the test compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. A control assay can also be performed to provide a baseline for comparison. IGF-IR antagonists

An IGF-IR antagonist includes, but is not limited to, an antibody that binds to IGF-IR, an antibody that binds to an IGF-IR ligand, an IGF mimetic, a small molecule IGF-IR antagonist, a protein, a polypeptide, a small interfering RNA (siRNA), an antisense nucleic acid, a ribozyme, a triple helix-forming nucleic acid, a dominant negative mutant, or a soluble form of IGF- 1 R. IGF- 1 R antagonists also comprise both extracellular and intracellular antagonists. Extracellular IGF-IR antagonists are typically substances that reduce or block receptor-ligand interactions, and can also function to down-regulate the concentration of cell surface receptor. Examples of extracellular IGF- IR antagonists include antibodies and other proteins or polypeptides that bind to IGF-IR, as well as antibodies or other proteins or polypeptides specific for an IGF-IR ligand. In an embodiment of the invention, an IGF-IR inhibitor binds to IGF-IR and blocks ligand binding. In another embodiment, the IGF-IR inhibitor binds to IGF-IR and promotes reduction in the level of IGF-IR on a cell surface. In a further embodiment, the IGF-IR inhibitor binds to IGF-IR and inhibits IGF-lR-mediated signal transduction. Antibody antagonists of IGF-IR

In certain embodiments of the invention, the IGF-IR antagonist is an antibody or a functional derivative or fragment thereof that binds immunospecifϊcally to IGF-IR. As used herein, an "antibody" shall include, without limitation, an immunoglobulin molecule that recognizes an antigen and comprises two heavy chains and two light chains. The immunoglobulin molecule may derive from any of the commonly known classes, including, but not limited to, IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those in the art and include, but are not limited to, human IgGl, IgG2, IgG3 and IgG4. "Antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or nonhuman antibodies; wholly synthetic antibodies; and single chain antibodies. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man. Methods for humanizing antibodies are well known to those skilled in the art. The term "antibody" in its broadest usage also includes, without limitation, an antigen-binding fragment or portion of any of the aforementioned immunoglobulin molecules, including a monovalent and a divalent fragment or portion.

Antibodies are attractive therapeutics chiefly because, inter alia, they (1) can possess high selectivity for a particular protein antigen, (2) are capable of exhibiting high affinity binding to the antigen, (3) possess long half-lives in vivo, and, since they are natural immune products, should (4) exhibit low in vivo toxicity (Park and Smolen, 2001). Repeated application of antibodies derived from non-human sources, e.g. , a mouse, may, however, elicit a directed immune response against the therapeutic antibody itself, thereby neutralizing the antibody's effectiveness. Thus, fully human antibodies offer the greatest potential for success as human therapeutics since they would likely be less immunogenic than murine or chimeric antibodies in humans, and should function similarly to naturally occurring immuno-responsive antibodies.

Naturally occurring antibodies typically have two identical heavy chains and two identical light chains, with each light chain covalently linked to a heavy chain by an interchain disulfide bond and multiple disulfide bonds further link the two heavy chains to one another. Individual chains can fold into domains having similar sizes (110-125 amino acids) and structures, but different functions. The light chain can comprise one variable domain (V_L) and/or one constant domain (C_L). The heavy chain can also comprise one variable domain (V_H) and/or, depending on the class or isotype of antibody, three or four constant domains (C_HI , CH2, C_H3 and CH4). In humans, the isotypes are IgA, IgD, IgE, IgG, and IgM, with IgA and IgG further subdivided into subclasses or subtypes (IgAi_-2 and IgGi_-4). Generally, the variable domains show considerable amino acid sequence variability from one antibody to the next, particularly at the location of the antigen-binding site. Three regions, called hypervariable or complementarity- determining regions (CDRs), are found in each of V_L and V_H, and are supported by less variable regions called frameworks (FWs).

Portions of an antibody that retain antigen binding function and specificity include Fv (Fragment variable), the portion of an antibody consisting of V_L and V_H domains and constituting the antigen-binding site; Fab (Fragment, antigen binding), the monovalent fragments of the antibody produced by papain digestion consisting of V_L-C_L and VH-C_HI domains; F(ab')₂, a divalent antigen binding fragment produced by pepsin digestion that retains the antibody hinge region by which two heavy chains are normally linked, as well as intact interchain disulfide bonds; and Fab', produced when the disulfide bonds of an F(ab')₂ are reduced and the heavy chains are separated. Single chain Fv (scFv) is an antibody fragment containing a V_L domain and a V_H domain on one polypeptide chain, wherein the N-terminus of one domain and the C-terminus of the other domain are joined by a flexible linker. Because they are divalent, intact antibodies and F(ab')₂ fragments have higher avidity for antigen than the monovalent Fab or Fab' fragments.

Antibody formats have also been developed which retain binding specificity, but have other characteristics that may be desirable, including for example, bispecificity, multivalence (more than two binding sites), and compact size (e.g., binding domains alone).

Single chain antibodies lack some or all of the constant domains of the whole antibodies from which they are derived and, therefore, may have certain advantages over whole antibodies, including greater permeability, lower likelihood of provoking an unwanted immune response in a recipient, diminished undesired interactions between heavy-chain constant regions and other biological molecules. Multiple single chain antibodies, each single chain having one V_H and one V_L domain covalently linked by a first peptide linker, can be covalently linked by one or more peptide linkers to form a multivalent single chain antibody, which can be monospecific or multispecific. Two single chain antibodies can be combined to form a diabody, also known as a bivalent dimer. Diabodies have two chains and two binding sites, and can be monospecific or bispecific. Three single chain antibodies can be combined to form triabodies, also known as trivalent trimers. Triabodies are constructed with the amino acid terminus of a V_L or V_H domain directly fused to the carboxyl terminus of a V_L or V_H domain, i.e., without any linker sequence. Triabodies can be monospecific, bispecific or trispecific.

Thus, antibody inhibitors and fragments thereof that block IGF-IR activity include, but are not limited to, naturally occurring antibodies, bivalent fragments such as (Fab')₂, monovalent fragments such as Fab, single chain antibodies, single chain Fv (scFv), single domain antibodies, multivalent single chain antibodies, diabodies, triabodies, and the like that bind specifically with antigens.

Antibodies of the invention and functional derivatives thereof may be obtained by methods known in the art. These methods include, for example, the immunological methods described by Kohler and Milstein (1975) and Campbell (1985), as well as by the recombinant DNA methods such as described by Huse et al. (1989). The antibodies can also be obtained from phage display libraries bearing combinations of V_H and V_L domains in the form of scFv or Fab. The V_H and V_L domains can be encoded by nucleotides that are synthetic, partially synthetic, or naturally derived. In certain embodiments, phage display libraries bearing human antibody fragments are employed. Other sources of human antibodies are transgenic mice engineered to express human immunoglobulin genes. Antibody fragments can be produced by cleaving a whole antibody or by expressing DNA that encodes the fragment. Fragments of antibodies may be prepared by methods described by Lamoyi and Nisonoff (1983) and Parham (1983). Such fragments may contain one or both Fab fragments or the F(ab')₂ fragment, or may also contain single-chain fragment variable region antibodies, i.e., scFv, diabodies, or other antibody fragments.

The properties and preparation of various derivatives and functional fragments of antibodies are described in more detail in International Publication No. WO 2005/016970, the entire contents of which are incorporated herein by reference.

Antibodies typically bind with a dissociation constant (K_<i) of 10^"5 to 10^'11 M^"1 or better. Any K_d greater than 10^"4 M^'1 is generally considered to indicate nonspecific binding. The lower the value of the K_d, the stronger the binding strength between an antigenic determinant and the antibody binding site. In certain embodiments of this invention, the IGF-IR inhibitor is an antibody that binds to IGF-IR with a K_d that is less than about 10^" M^"1, in some embodiments than about 3 x 10^"10 M^"1, in some embodiments less than about 10^"10 M^"1, or in some embodiments less than about 3 x 10^~π M^"1.

In various embodiments of any of the methods described herein, the anti-IGF-lR antibody is a monoclonal antibody. "Monoclonal antibodies," also designated mAbs, are antibody molecules whose primary sequences are essentially identical and which exhibit the same antigenic specificity. Monoclonal antibodies may be produced by hybridoma, recombinant, transgenic or other techniques known to those skilled in the art. In other embodiments, the antibody is a human, humanized or chimeric antibody. In particular embodiments, the antibody is a human antibody. A "human" antibody shall mean an antibody wherein all of the amino acids correspond to amino acids in human immunoglobulin molecules. "Fully human" and "human" are used synonymously. A "humanized" antibody shall mean an antibody wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind a given antigen. Typically, in a "humanized" antibody, the CDRs (but not the framework regions) from an antibody from a non-human species are transferred from the heavy and light variable domains of the non-human antibody into human heavy and light variable domains. A "chimeric" antibody shall mean a recombinant antibody that contains whole variable domains, including the CDRs and framework regions, of an antibody from one species (e.g., a mouse), and the constant domains of an antibody molecule from a different species (e.g., a human). "Human", "humanized" and "chimeric" antibodies retain an antigenic specificity similar to that of the original antibody.

In various embodiments of the invention, the antibodies bind to the external domain of IGF-IR and inhibit binding of IGF-I or IGF-2 to IGF-IR. Inhibition can be determined, for example, by a direct binding assay using purified or membrane bound receptor. In certain embodiments, the antibodies or fragments thereof bind to IGF-IR at least as strongly as do the IGF-I and IGF-2 natural ligands of IGF-IR.

In other embodiments, the antibodies neutralize IGF-IR. Binding of a ligand, e.g., IGF-I or IGF-2, to an external, extracellular domain of IGF-IR stimulates autophosphorylation of the beta subunit and phosphorylation of IGF-IR substrates, including MAPK, Akt, and IRS-I. Neutralization of IGF-IR includes inhibition, diminution, inactivation and/or disruption of one or more of these activities normally associated with signal transduction. Neutralization of IGF-IR includes inhibition of IGF- IR / IR heterodimers as well as IGF-IR homodimers. Thus, neutralizing IGF-IR has various effects, including, but not limited to, inhibition, diminution, inactivation and/or disruption of growth (proliferation and differentiation), angiogenesis (blood vessel recruitment, invasion, and metastasis), and cell motility and metastasis (cell adhesion and invasiveness). One measure of IGF-IR neutralization is inhibition of the tyrosine kinase activity of the receptor which can be measured using well-known methods; see, e.g., Panek et al., (1997) and Batley et al. (1998). Antibodies of the invention cause a decrease in tyrosine phosphorylation of IGF-IR of at least about 75%, in some embodiments at least about 85%, and in some embodiments at least about 90% in cells that respond to ligand.

Another measure of IGF-IR neutralization is inhibition of phosphorylation of downstream substrates of IGF-IR. Accordingly, the level of phosphorylation of MAPK, Akt, or IRS-I can be measured. The decrease in substrate phosphorylation is at least about 50%, in some embodiments at least about 65%, and in some embodiments at least about 80%.

In addition, well known methods for detection of protein expression can be utilized to determine IGF-IR neutralization, wherein the proteins being measured are regulated by IGF-IR tyrosine kinase activity. These methods include immunohistochemistry (IHC) for detection of protein expression, fluorescence in situ hybridization (FISH) for detection of gene amplification, competitive radioligand binding assays, solid matrix blotting techniques such as western blots, and enzyme-linked immunosorbent assay (ELISA). See, e.g., Rubin Grandis et al. (1996); Shimizu et al. (1994); Sauter et al. (1996); Collins (1995); Radinsky et al. (1995); Petrides et al. (1990); Hoffmann et al. (1997); Wikstrand et al. (1995). In vivo assays can also be utilized to determine IGF-IR neutralization. For example, receptor tyrosine kinase inhibition can be observed by mitogenic assays using cell lines stimulated with receptor ligand in the presence and absence of inhibitor. Another method involves testing for inhibition of growth of IGF-I R-expressing tumor cells or cells transfected to express IGF-IR. Inhibition can also be observed using tumor models, for example, human tumor cells injected into a mouse. The present invention is not limited by any particular mechanism of IGF-IR neutralization.

In further embodiments of the invention, the antibodies down-modulate IGF-IR. The amount of IGF-IR present on the surface of a cell depends on receptor protein production, internalization, and degradation. The amount of IGF-IR present on the surface of a cell can be measured indirectly, by detecting internalization of the receptor or of a molecule bound to the receptor. Another way to determine down-modulation is to directly measure the amount of the receptor present on the cell following treatment with an anti-IGF-lR antibody or other substance, for example, by FACS analysis of cells stained for surface expression of IGF-IR. Cell surface IGF-IR can also be detected and measured using a different antibody that is specific for IGF-IR and that does not block or compete with binding of the antibody being tested. See Burtrum et al. (2003).

Treatment of an IGF-lR-expressing cell with an antibody of the invention results in reduction of cell surface IGF-IR. In some embodiments, the reduction is at least about 70%, in some embodiments at least about 80%, and in some embodiments at least about 90% in response to treatment with an antibody of the invention. A significant decrease can be observed in as little as four hours.

Another measure of down-modulation is reduction of the total receptor protein present in a cell, and reflects degradation of internal receptors. Accordingly, treatment of cancer cells with antibodies of the invention results in a reduction in total cellular IGF- IR. In a particular embodiment, the reduction is at least about 70%, in some embodiments at least about 80%, and in some embodiments at least about 90%.

The binding characteristics of antibodies used in the present invention may have been improved by direct mutation, methods of affinity maturation, or chain shuffling. For example, affinity and specificity may be modified or improved by mutating CDRs and screening for antigen binding sites having the desired characteristics. See, e.g., Yang et al. (1995).

Non-limiting examples of anti-IGF-lR antibodies that can be used according to the invention include Al 2 and 2F8 (described below), antibodies that compete with A12 and/or 2F8 for binding to IGF-IR, the XenoMouse®-derived human antibody CP- 751,871 (Cohen et al., 2005), humanized antibody EM164 (Maloney et al., 2003), humanized antibody h7C10 (Goetsch et al., 2005), AMG-479 (Amgen), and scFv-Fc- IGF-IR (Sachdev et al., 2003). Small molecule antagonists of IGF-IR

Another means of blocking IGF-lR-mediated signal transduction other than using anti-IGF-lR antibodies is via small molecule antagonists of IGF-IR. As used herein, a "small molecule" refers to a small organic compound, such as any of a heterocycle, peptide, saccharide, steroid, and the like, in some embodiments having a molecular weight of less than about 2000 daltons, in some embodiments less than about 1000. daltons, and in some embodiments less than about 500 daltons. Such compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Small molecule antagonists include, but are not limited to, small molecules that bind to block the ATP binding domain, substrate binding domain, or catalytic domain of IGF-IR. In addition to blocking IGF-IR activity directly, small molecules can be inhibitors of other components of the IGF-IR signal transduction pathway. In certain embodiments, a small molecule inhibitor binds to the ligand binding domain of IGF- 1 R and blocks receptor activation by an IGF-IR ligand.

The design and synthesis of small molecule antagonists that bind to, and inhibit, various components of the IGF-IR signal transduction pathway may be facilitated by experimental approaches that are well known in the art, including traditional medicinal chemistry and the newer technology of combinatorial chemistry, both of which may be supported by computer-assisted molecular modeling. Combinatorial chemistry involves automated synthesis of a variety of novel agents by assembling them using different combinations of chemical building blocks. The use of this technique greatly accelerates the process of generating agents. The resulting libraries of compounds are used to screen for agents ("lead agents") that demonstrate a sufficient level of binding to molecules of interest. By using combinatorial chemistry it is possible to synthesize "focused" libraries of agents anticipated to be highly biased toward the target molecule.

Once lead agents are identified, a variety of homologs and analogs are prepared to facilitate an understanding of the relationship between chemical structure, binding affinity for the target molecule, and biological or functional activity. These studies define structure activity relationships (SARs) which are then used to design drugs with improved potency, selectivity and pharmacokinetic properties. Combinatorial chemistry is also used to rapidly generate a variety of structures for lead optimization. Traditional medicinal chemistry, which involves the synthesis of agents one at a time, is also used for further refinement and to generate agents not synthesizable by automated techniques.

Numerous non-peptidyl small molecules are available from a variety of commercial sources for screening for agents having desired functional properties. Small molecule libraries can be screened for inhibitory activity using high-throughput biochemical, enzymatic, or cell based assays. For example, ChemDiv (San Diego, CA) provides high throughput hit hunting services involving assay development and screening of up to 1 million small molecules. Based on structural information on a target or protein domain of interest, a set of templates can be designed followed by the chemical synthesis of individual molecules in a medicinal chemistry fashion. The assays can be formulated to detect, for example, the ability of a test agent to inhibit binding of IGF-IR to IGF-IR ligands or substrate IRS-I, or to inhibit the formation of functional receptors from IGF- lR dimers.

Known small molecule antagonists of IGF-IR include, for example, the IGF-IR selective kinase inhibitors NVP-AEW541 (Garcia-Echeverria et al., 2004) and NVP- ADW742 (Mitsiades et al., 2004), INSM- 18 (Insmed Incorporated) that is reported to selectively inhibit IGF-IR and HER2, and the tyrosine kinase inhibitor tryphostins AGl 024 and AGl 034 (Parrizas et al., 1997) that inhibit phosphorylation by blocking substrate binding and have a significantly lower IC₅₀ for inhibition of IGF-IR phosphorylation than for IR phosphorylation. The cyclolignan derivative picropodophyllin (PPP) is another IGF-IR antagonist that inhibits IGF-IR phosphorylation without interfering with IR activity (Girnita et al., 2004). Other small molecule IGF-IR antagonists include the benzimidazol derivatives BMS-536924 (Wittman et al., 2005) and BMS-554417 (Haluska et al., 2006) that inhibit IGF-IR and IR almost equipotently. In addition, inhibitory peptides targeting the IGF-IR have been generated that possess antiproliferative activity in vitro and in vivo (Pietrzkowski et al., 1992; Haylor et al. 2000).

For compounds that inhibit receptors in addition to IGF-IR, it should be noted that IC50 values measured in vitro in direct binding assays may not reflect IC₅₀ values measured ex vivo or in vivo (i.e., in intact cells or organisms). For example, where it is desired to avoid inhibition of IR, a compound that inhibits IR in vitro may not significantly affect the activity of the receptor when used in vivo at a concentration that effectively inhibits IGF-IR.

In certain embodiments of any of the present methods, the small molecule IGF-IR antagonist is AG1024, AG1034, NVP-AEW541, NVP-ADW742, picropodophyllin (PPP), BMS-536924, or BMS-554417. In some embodiments, the small molecule IGF-IR antagonist is AGl 024.

In other embodiments of the invention, the IGF-IR antagonist is a small molecule that binds to the ligand binding domain of IGF-IR and blocks binding of an IGF-IR ligand. This small molecule may be a low molecular weight natural or synthetic product or metabolite, or an element of a combinatorial chemistry library. Small interfering RNA (siRNA)

Innate RNA-mediated mechanisms can regulate mRNA stability, message translation, and chromatin organization (Mello and Conte, 2004). Furthermore, exogenously introduced long double-stranded RNA (dsRNA) is an effective tool for gene silencing in a variety of lower organisms. However, in mammals, long dsRNAs elicit highly toxic responses that are related to the effects of viral infection and interferon production (Williams, 1997). To avoid this, Elbashir and colleagues (Elbashir et al., 2001) initiated the use of siRNAs composed of 19-mer duplexes with 5' phosphates and 2-base 3' overhangs on each strand, which selectively degrade targeted mRNAs upon introduction into cells.

The action of interfering dsRNA in mammals usually involves two enzymatic steps. First, Dicer, an RNase Ill-type enzyme, cleaves dsRNA to 21— 23-mer siRNA segments. Then, RNA-induced silencing complex (RISC) unwinds the RNA duplex, pairs one strand with a complementary region in a cognate mRNA, and initiates cleavage at a site 10 nucleotides upstream of the 5' end of the siRNA strand (Harmon, 2002). Short, chemically synthesized siRNAs in the 19-22-mer range do not require the Dicer step and can enter the RISC machinery directly. It should be noted that either strand of an RNA duplex can potentially be loaded onto the RISC complex, but the composition of the oligonucleotide can affect the choice of strands. Thus, to attain selective degradation of a particular mRNA target, the duplex should favor loading of the antisense strand component by having relatively weak base pairing at its 5' end (Khvorova, 2003). Exogenous siRNAs can be provided as synthesized oligonucleotides or expressed from plasmid or viral vectors (Paddison and Hannon, 2003). In the latter case, precursor molecules are usually expressed as short hairpin RNAs (shRNAs) containing loops of 4-8 nucleotides and stems of 17-30 nucleotides; these are then cleaved by Dicer to form functional siRNAs.

The present invention provides a double stranded siRNA comprising a sense RNA strand and a complementary antisense RNA strand that downregulates expression of a targeted gene via RNA interference, wherein (a) each strand of the siRNA molecule is independently about 17 to about 30 nucleotides in length, in some embodiments about 19 to about 25 nucleotides in length, (b) the antisense strand of the siRNA comprises an oligonucleotide having sufficient sequence complementarity to an mRNA of the targeted gene for the siRNA molecule to direct cleavage of the mRNA via RNA interference, and (c) the targeted gene is a Hox, c-Myb, or IGF-IR gene. In some embodiments, each strand of the siRNA comprises at least about 14 to 24 nucleotides that are complementary to the nucleotides of the other strand. Generally, a target sequence on the target mRNA can be selected from a given cDNA sequence corresponding to the target mRNA, in some embodiments, beginning 50 to 100 nt downstream from the translation initiation codon. The target sequence can, however, be located in the 5' or 3 untranslated regions, or in the region less than 50 to 100 nt downstream from the initiation codon. The sense and antisense strands of the siRNA may comprise two complementary, single-stranded RNAs or may comprise a single RNA in which two complementary portions are base-paired and are covalently linked by a single-stranded hairpin loop. siRNAs may be obtained using a number of techniques well known to those of skill in the art. For example, the siRNA can be chemically synthesized or recombinantly produced using methods known in the art, such as the Drosophila in vitro system described in U.S. Patent Publication No. 2002/0086356. The siRNA can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Alternatively, the siRNA is expressed from recombinant circular or linear DNA plasmids using a suitable promoter, including, for example, the U6, Hl RNA pol III, or the cytomegalovirus promoter. In certain embodiments, the target gene is a Hox gene or a IGF-I R gene. In further embodiments, the Hox gene is a HoxA4, HoxA5, HoxA7, HoxA8, HoxA9, HoxAlO, HoxB7, HoxB8, or HoxC8 gene. In some embodiments, the Hox gene is a HoxA9 gene. In some embodiments of a siRNA targeting the HoxA9 gene, the sense RNA strand comprises 5'-UCAACAAAGACCGAGCAAAUU-S ' (SEQ ID NO: 1) and the antisense RNA strand comprises 5 ' - UUUGCUCGGUCUUUGUUGAUU-3 ' (SEQ ID NO:2). This siRNA targets the HoxA9 gene sequence 5'- AATCAAC AAAGACCGAGC AAA-3' (SEQ ID NO:3).

This invention also provides a pharmaceutical composition comprising any of the siRNAs disclosed herein and a pharmaceutically acceptable carrier. Additionally, this invention provides a method for inhibiting IGF-IR expression in a tumor by downregulating HoxA9 gene expression. A siRNA may be used for downregulating Hox gene expression. In some embodiments, the nucleotide sequences of the sense and antisense strands of a siRNA designed to inhibit expression of a HoxA9 gene are SEQ ID NO:1 SEQ ID NO.2, respectively.

Antisense-mediated inhibition of IGF-lR-mediated signaling

The ability of antisense oligonucleotides to suppress gene expression was discovered more than 25 years ago (Zamecnik and Stephenson, 1978). Antisense nucleic acid molecules interact with complementary strands of nucleic acids in targeted genes, mRNA or pre-RNA and thereby inhibit the expression of these genes or RNAs by a variety of mechanisms including disrupting intron splicing, polyadenylation, export from the nucleus, RNA stability, and protein translation (Sazani and KoIe, 2003). Many antisense-mediated strategies for gene inhibition have been developed and can be broadly categorized into enzyme-dependent antisense or steric blocking antisense. Enzyme- dependent antisense includes forms dependent on RNase H activity to degrade target mRNA, including single-stranded oligodeoxynucleotides, mRNA, phosphorothioate antisense oligonucleotides, and double stranded RNA molecules that act via the RNAi/siRNA pathway. Steric blocking antisense interferes with gene expression or other mRNA-dependent cellular processes by binding to a target sequence of mRNA and getting in the way of other processes. Steric blocking antisense includes use of 2'-0 alkyl (usually in chimeras with RNase-H dependent antisense), peptide nucleic acid (PNA), locked nucleic acid (LNA) and morpholino antisense. The two most powerful and widely used antisense strategies are the degradation of mRNA or pre-mRNA via RNase H and the alteration of splicing via targeting aberrant splice junctions. RNase H recognizes

DNA/RNA heteroduplexes and cleaves the RNA approximately midway between the 5' and 3' ends of the DNA oligonucleotide.

The expression of receptor tyrosine kinases and other proteins critical for IGF signaling can be inhibited using antisense techniques. Inhibition of IGF-IR by antisense oligonucleotides is exemplified in Wraight et al. (2000).

Other antagonists of IGF-lR-mediated signaling

Other means of inhibiting IGF-IR mediated signal transduction include, but are not limited to, IGF-I or IGF-2 mimetics that bind to but do not activate the receptor, and expression of genes or polynucleotides that reduce IGF-IR levels or activity such as triple helix inhibitors and dominant negative IGF-IR mutants. In certain embodiments of the present invention, the IGF-IR antagonists bind to a ligand of IGF-IR. Examples of such antagonists include, but are not limited to, antibodies and soluble IGF-IR fragments that bind to IGF-I or IGF-2. A synthetic peptide sequence from the C-terminus of IGF-IR has been shown to induce apoptosis and significantly inhibit tumor growth. See Reiss et al. (1999). In other embodiments, the IGF-IR antagonist is a mimetic of an IGF-IR ligand that binds to, but does not activate, the receptor. It has been demonstrated that overexpression of any of several dominant-negative mutants of the IGF-IR in tumor cell lines compete with wild-type IGF-IR for ligand and effectively inhibit tumor cell growth in vitro and in vivo (Scotland! et al., 2002; Seely et al., 2002). Additionally, a soluble form of the IGF-IR has been demonstrated to inhibit tumor growth in vivo. See D'Ambrosio et al. (1996). In further embodiments, the IGF-IR antagonist blocks interaction of IGF-IR with its substrate IRS-I.

Cancers amenable to treatment with an IGF-IR antagonist

The present methods are applicable to treatment of a variety of tumors. Tumor as used herein refers to an abnormal growth of cells or tissues. Tumors can be malignant or benign. As used herein, malignant tumors include cancerous growth denoted as an uncontrolled growth of tissue that has the potential to spread to adjacent or distant sites of the body. Tumors can include, but are not limited to, a leukemia, a lymphoma, a multiple myeloma, or a solid tumor. In certain embodiments, cells of the leukemia harbor a mixed lineage leukemia gene (MLL) translocation or a MLL partial tandem duplication. In other embodiments, the solid tumor includes, but is not limited to, a SCLC, a prostate cancer, a breast cancer, a colorectal cancer, an ovarian cancer, a neuroblastoma, a central nervous system tumor, a glioblastoma multiforme, or a melanoma. Moreover, tumors affected by the methods of the present invention further include osteosarcomas, angiosarcomas, fibrosarcomas and other sarcomas, sinus tumors, uretal, bladder, prostate and other genitourinary cancers, esophageal and stomach cancers and other gastrointestinal cancers, lung cancers, pancreatic cancers, liver cancers, kidney cancers, endocrine cancers, skin cancers, melanomas, angiomas, and peripheral nervous (PNS) system tumors, malignant or benign, including gliomas and neuroblastomas.

The instant invention also provides a method for determining whether a tumor in a subject is amenable to treatment with an IGF-IR antagonist comprising determining whether the tumor overexpresses a Hox gene, wherein overexpression of the Hox gene indicates that the tumor is amenable to treatment with an IGF-IR inhibitor. The tumor may be a cancerous tumor, a benign tumor, a benign growth, or a benign neoplasm or any of those listed above. In particular embodiments of this invention, the subject is a human. In certain embodiments, the Hox gene is a HoxA4, HoxA5, HoxA7, HoxA8, HoxA9, HoxAlO, HoxB7, HoxBδ, HoxC8 gene, or any combination thereof. In particular embodiments, the Hox gene is a HoxA9 gene. In further embodiments, the tumor is a leukemia, a lymphoma, a multiple myeloma, or a solid tumor. The tumor may be a leukemia that harbors a mixed lineage leukemia gene (MLL) translocation or a MLL partial tandem duplication. The solid tumor may be, but is not limited to, a small cell lung cancer (SCLC), a prostate cancer, a breast cancer, a colorectal cancer, an ovarian cancer, a neuroblastoma, a central nervous system tumor, a glioblastoma multiforme, or a melanoma.

Agents for treating a tumor that overexpresses a Hox gene

This invention further provides a method of identifying an agent effective for treating a tumor, wherein the tumor is determined to overexpress a Hox gene, comprising determining whether the agent antagonizes IGF-IR expression, wherein an agent that antagonizes IGF-IR expression is effective for treating the tumor. The tumor may be a cancerous tumor, a benign tumor, a benign growth, or a benign neoplasm or any of those listed above. In different embodiments of this method, the Hox gene is a HoxA4, HoxA5, HoxA7, HoxA8, HoxA9, HoxAlO, HoxB7, HoxB8, HoxC8 gene, or any combination thereof. In particular embodiments, the Hox gene is a HoxA9 gene. The tumor may be a leukemia, a lymphoma, a multiple myeloma, or a solid tumor including, but not limited to, a SCLC, a prostate cancer, a breast cancer, a colorectal cancer, an ovarian cancer, a neuroblastoma, a central nervous system tumor, a glioblastoma multiforme, or a melanoma. In addition, the agent may be any of the different types of substances described herein that directly or indirectly reduce IGF-IR expression or antagonize IGF- IR activity. Examples of agents that indirectly reduce IGF-IR expression include agents that inhibit expression of Hox or c-Myb genes. In certain embodiments, the inhibitor of Hox gene expression is a siRNA. In preferred embodiments, the siRNA targets a HoxA9 gene, wherein the sense RNA strand comprises 5'-UCAACAAAGACCGAGCAAAUU- 3 ' (SEQ ID NO: 1 ) and the antisense RNA strand comprises 5 '- UUUGCUCGGUCUUUGUUGAUU-B' (SEQ ID NO:2). The invention also provides a composition comprising an agent identified by the instant methods and a carrier. Pharmaceutically acceptable carriers are well known in the art and are described below. Also provided are kits for use in treating a tumor in a subject comprising a packaging material containing therein an agent identified as effective for treating the tumor, and a label providing instructions for administering the agent to the subject.

Therapeutic regimens

"Treat, " "treating" and "treatment" as used herein refer to an action resulting in a reduction in the severity of the subject's condition or at least the condition is partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the condition and/or prevention or delay of the onset of the condition.

According to the present invention, treating a tumor in a subject comprises administering to the subject a therapeutically effective amount of an IGF-IR antagonist. As used herein, a "therapeutically effective amount" an IGF-IR antagonist is any amount of the IGF-IR antagonist that, when used alone or in combination with an anti-neoplastic agent, promotes cancer regression in a subject. In particular embodiments, a therapeutically effective amount of an IGF-IR antagonist promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression"means that administering an effective amount of IGF-IR antagonist, alone or in combination with an anti-neoplastic agent, results in a reduction in size, or necrosis, of the tumor. In other embodiments of the invention, tumor regression may be observed and continue for a period of at least about 20 days, in some embodiments at least about 40 days, or in some embodiments at least about 60 days. Tumor regression may be measured as an average across a group of subjects undergoing a particular treatment regimen, or may be measured by the number of subjects in a treatment group in which tumors regress. A "therapeutically effective amount" also refers to a target serum concentration shown to be effective in promoting cancer regression in a subject. Determining the therapeutically effective amount of an IGF-IR antagonist is within the ordinary skill of the art and requires no more than routine experimentation.

As used herein, a "prophylactically effective amount" of an IGF-IR antagonist is any amount of the IGF-IR antagonist that, when administered alone or in combination with an anti-neoplastic agent to a subject at risk of developing a cancer, inhibits the development of the cancer. In some embodiments, the prophylactically effective amount prevents the development of the cancer entirely. "Inhibiting" the onset of a cancer means either lessening the likelihood of the cancer's onset, or preventing the onset of the disorder entirely.

The anti-cancer agent regimens utilized according to the invention include any regimen believed to be optimally suitable for the treatment of the patient's neoplastic condition. One of skill in the art would understand that dosages and frequency of treatment depend on numerous factors, including, for example, the type and the pharmacological and pharmacokinetic properties of the IGF-IR antagonist(s) used, the type and severity of the cancer being treated, the tolerance of the individual patient, and the route of administration of the antagonist(s). Different malignancies can require use of specific IGF-IR antagonist(s), optionally in combination with specific anti-neoplastic agents, which will be determined on a patient by patient basis. To achieve saturatable pharmacokinetics the loading dose of an anti-IGF-lR antibody can range, for example, from about 10 to about 1000 mg/m², preferably from about 200 to about 400 mg/m². This can be followed by several additional daily or weekly dosages ranging, for example, from about 200 to about 400 mg/m . (For conversions between mg/kg and mg/m for humans and other mammals, see Freireich et al., 1966.) The patient is monitored for undesirable side effects such as, for example, local injection site irritation or increase in blood pressure, and the treatment is stopped if such side effects are severe. Depending on the desired outcome, saturation kinetics may not be desired.

Any suitable method or route can be used to administer IGF-IR antagonists of the invention, and optionally, to co-administer one or more other anti-neoplastic agents and/or antagonists of other receptors in a combination therapy. "Administering" an IGF- IR antagonist to a subject shall mean delivering the antagonist to the subject using any of the various methods and delivery systems known to those skilled in the art. Administering can be performed, for example, topically, orally, intravenously, intramuscularly, subcutaneously, intraperitoneally or parenterally. An agent or composition may also be administered in an aerosol, such as for pulmonary and/or intranasal delivery. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. The present invention is not limited, however, to any particular method, route or frequency of administration. It is understood that an IGF-IR antagonist of the invention, where used prophylactically or therapeutically to prevent or treat a cancer in a subject, will be administered in the form of a composition additionally comprising a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, one or more of 0.01-0. IM and in some embodiments 0.05M phosphate buffer, phosphate-buffered saline (PBS), or 0.9% saline. Such carriers also include aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives and other additives, such as, for example, antimicrobials, antioxidants and chelating agents, which enhance the shelf life and/or effectiveness of the active ingredients. The instant compositions can, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to a subject.

The IGF-IR antagonists can be used in vivo and in vitro for investigative, or diagnostic methods, which are well known in the art. The diagnostic methods include use of kits containing IGF-IR antagonists of the present invention. Combination therapy

In the experiments described herein (see Examples below), cells overexpressing HoxA9 were not completely killed upon inhibition of IGF-IR, suggesting that other Hox- induced factors may also be involved in cell survival and proliferation. Evidence for the involvement of such factors stems from the fact that while HoxA9 induction in BLIN-2 prolongs cell proliferation and survival in the absence of stromal cell support, the establishment of stromal cell-independent clones was not achieved even in the presence of both HoxA9 induction and IGF-I . This suggests that inhibition of IGF-IR in combination with other specific pathway inhibitors or chemotherapeutic agents may yield the most effective therapeutic results. For example, leukemic cells bearing MLL translocations are well known for being resistant to a variety of cell stress conditions (e.g., serum deprivation) and chemotherapeutic drugs (Kersey et al., 1998). IGF-IR inhibition may sensitize these cells to other agents and be of immediate benefit to patients.

In certain embodiments of the invention, one or more IGF-IR antagonists is concurrently administered with one or more other anti-cancer agents, treatments, behavioral modifications, or surgical interventions. Any suitable anti-neoplastic agent can be used, such as a chemotherapeutic agent, radiation or combinations thereof.

Combination therapies are disclosed in, for example, U.S. Patent No. 6,217,866 (anti- EGFR antibodies in combination with anti-neoplastic agents) and International Publication No. WO 99/60023 (anti-EGFR antibodies in combination with radiation). In certain embodiments, the IGF-IR antagonist with or without other drugs is part of a comprehensive treatment for cancer, including modifications in diet, behavioral modification (e.g., cessation of smoking), radiation treatment, and/or surgical intervention.

"Concurrently administered", with respect to the administration of two or more therapies to a subject means that each therapeutic agent or method is administered to the subject within the same treatment time period as is each other therapeutic agent or method. The therapies can be administered together, at the same time and in the same or different compositions or via the same or different routes of administration. Alternatively, each therapy is administered via a dosing regimen (e.g., frequency, route and amount) different from that by which each other therapy is administered. For example, the first of two concurrently administered agents (e.g., an anti-IGF-lR antibody) may be administered via intravenous injection at two-week intervals for a 6-month treatment time period, whereas during that same 6-month period, the second concurrently administered agent (e.g., a chemotherapeutic agent) is orally administered twice per day. In some embodiments of the invention, chemotherapy is administered together with or subsequent to antibody therapy. In other embodiments, an anti-IGF-lR antibody is administered between 1 and 30 days, in some embodiments 3 and 20 days, and in some embodiments between 5 and 12 days, before commencing radiation therapy.

The anti-neoplastic agent can be an alkylating agent or an anti-metabolite. Examples of alkylating agents include, but are not limited to, cisplatin, cyclophosphamide, melphalan, and dacarbazine. Examples of anti -metabolites include, but are not limited to, doxorubicin, daunorubicin, and paclitaxel, gemcitabine, and topoisomerase inhibitors, irinotecan (CPT-11), aminocamptothecin, camptothecin, DX- 895 If, and topotecan (inhibitors of topoisomerase I), and etoposide (VP- 16) and teniposide (VM-26) (inhibitors of topoisomerase II). When the anti-neoplastic agent is radiation, the source of the radiation can be either external (external beam radiation therapy - EBRT) or internal (brachytherapy - BT) to the patient being treated. The dose of anti-neoplastic agent administered depends on numerous factors, including, for example, the type of agent, the type and severity of tumor being treated, and the route of administration of the agent. The present invention is not, however, limited to any particular dose.

The anti-neoplastic agents that are presently known in the art or are being evaluated can be grouped into a variety of classes including, for example, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones, and anti-angiogenesis agents.

Topoisomerase inhibitors have been shown to be particularly effective antineoplastic agents when used in combination with antibodies that bind to IGF-IR. See WO 2005/016970. Accordingly, some embodiments of the invention include methods in which a topoisomerase inhibitor is administered in combination with an antibody that binds to IGF-IR. The inhibitors can be inhibitors of topoisomerase I or topoisomerase II. Other substances are currently being evaluated with respect to topoisomerase inhibitory activity and effectiveness as anti-neoplastic agents. In some embodiments, the topoisomerase inhibitor is irinotecan (CPT-11). The antibodies used in combination are antibodies of the invention that bind to IGF-IR and have at least one of the following properties: (i) inhibit binding of IGF-I or IGF-2 to IGF-IR; (ii) neutralize activation of IGF-IR by IGF-I or IGF-2; (iii) reduce IGF-IR surface receptor; and bind to IGF-IR with a K_d of about 1 x 10^"10 M^"1 or less. In other embodiments, the antibodies to be used in combination with a topoisomerase inhibitor have the characteristics of the human anti- IGF-IR antibodies set forth above.

Anti-IGF-1R antagonists of the invention can be co-administered with antagonists that neutralize other receptors involved in tumor growth or angiogenesis. In certain embodiments of the invention, an anti-IGF-lR antibody is co-administered with a receptor antagonist that binds specifically to epidermal growth factor receptor (EGFR). In some embodiments, antigen-binding proteins that bind to the extracellular domain of EGFR and block binding of one or more of its ligands and/or neutralize ligand-induced activation of EGFR are employed. An EGFR antagonist can be an antibody that binds to EGFR or a ligand of EGFR and inhibits binding of EGFR to its ligand. Ligands for EGFR include, for example, epidermal growth factor (EGF), transforming growth factor α (TGF-α), amphiregulin, heparin-binding EGF (HB-EGF) and betacellulin. EGF and TGF-α are thought to be the main endogenous ligands that result in EGFR-mediated stimulation, although TGF-α has been shown to be more potent in promoting angiogenesis. It should be appreciated that the EGFR antagonist can bind externally to the extracellular portion of EGFR, which can or can not inhibit binding of the ligand, or internally to the tyrosine kinase domain. Examples of EGFR antagonists that bind EGFR include, without limitation, biological molecules, such as antibodies (and functional equivalents thereof) specific for EGFR, and small molecules, such as synthetic kinase inhibitors that act directly on the cytoplasmic domain of EGFR.

Another example of such a receptor involved in tumor growth or angiogenesis is a vascular endothelial growth factor (VEGF) receptor (VEGFR). In various embodiments of the present invention, an anti-IGF-lR antagonist is used in combination with a VEGFR antagonist. In certain embodiments, an anti-IGF-lR antibody is used in combination with a receptor antagonist that binds specifically to VEGFR-I /FIt-I receptor. In certain other embodiments, an anti-IGF-lR antibody is used in combination with a receptor antagonist that binds specifically to VEGFR-2/KDR receptor. Antigen-binding proteins that bind to the extracellular domain of VEGFR-I or VEGFR-2 and block binding by their ligands (VEGFR-2 is stimulated most strongly by VEGF; VEGFR-I is stimulated most strongly by placental growth factor (PlGF), but also by VEGF) and/or neutralize ligand-induced activation are employed. For example, IMC-1121 is a human antibody that binds to and neutralizes VEGFR-2. See International Publication No. WO 03/075840). Another example is mAb 6.12, a scFv that binds to soluble and cell surface-expressed VEGFR-I. ScFv 6.12 comprises the V_L and V_H domains of mouse monoclonal antibody MAb 6.12. A hybridoma cell line producing MAb 6.12 has been deposited as ATCC No. PTA-3344 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the regulations thereunder (Budapest Treaty).

Other examples of growth factor receptors involved in tumorigenesis are the receptors for platelet-derived growth factor (PDGFR), nerve growth factor (NGFR), and fibroblast growth factor (FGFR).

In various other embodiments, the IGF-IR antibody is administered in combination with one or more suitable adjuvants, including, for example, cytokines (IL- 10 and IL- 13, for example) or other immune stimulators, such as, but not limited to, chemokines, tumor-associated antigens, and peptides. The administration of an anti-IGF- IR antagonist may by itself, however, be therapeutically effective to promote cancer regression, or prophylactically effective to prevent or slow cancer development. The present invention also includes kits for inhibiting tumor growth comprising a therapeutically effective amount of an IGF-IR antagonist. The kits can further contain an anti-neoplastic agent or a suitable antagonist of, for example, another growth factor receptor involved in tumorigenesis or angiogenesis (e.g., EGFR, VEGFR-I /FIt-I, VEGFR-2, PDGFR, NGFR, FGFR, and the like, as described above) for concurrent administration with the IGF-IR antagonist. Examples of suitable anti-neoplastic agents in the context of the present invention have been described herein. The kits of the present invention can further comprise an adjuvant, examples of which have also been described above.

Of course, it is to be understood and expected that variations in the principles of invention herein disclosed can be made by one skilled in the art and it is intended that such modifications are to be included within the scope of the present invention. The following Examples further illustrate the invention, but should not be construed to limit the scope of the invention in any way. Detailed descriptions of conventional methods, such as those employed in the construction of vectors and plasmids, the insertion of genes encoding polypeptides into such vectors and plasmids, the introduction of plasmids into host cells, the expression and determination thereof of genes and gene products, and immunological techniques can be obtained from numerous publications, including Sambrook et al. (1989) and Coligan et al. (1994). All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention described herein. EXAMPLE 1 Generation of an inducible Hox gene expression system

B-ALL cell lines and stromal cell co-culture

BLIN-2 is a B-cell precursor acute lymphoblastic leukemic (pre-B ALL) cell line that requires viable stromal cell contact for optimal proliferation and growth (Shah et al., 1998). This cell line model of B-ALL permits the study of pathways that promote the growth and survival of ALL in the biologically relevant context of the stromal cell microenvironment (Shah et al., 1998; Bertrand et al., 2005; Spengeman et al., 2005; Shah et al., 2004). The apoptotic characteristics of BLIN-2 are well documented (Shah et al., 1998; Bertrand et al., 2005; Spengeman et al., 2005; Shah et al., 2004). BLIN-2 cells were routinely cultured in serum free conditions in XVIVO-10 medium (BioWhittaker, Cambridge, MA) in the presence of stromal cell monolayers.

A strain of untransformed human foreskin fibroblasts was used as a source of stromal cells. Growth of BLIN-2 in the presence of these fibroblasts was indistinguishable from that when freshly isolated bone marrow stromal cells are used

(Shah et al., 1998; Bertrand et al., 2005; Spengeman et al., 2005; Shah et al., 2004). The skin fibroblasts (referred to herein as stromal cells) promote the differentiation of CD34⁺ progenitors into IgM⁺ B-lineage cells similar to freshly isolated bone marrow stromal cells (Kurosaka et al., 1999). The fibroblasts were not transformed and were maintained at a low passage number (less than 15) in the present experiments.

Generation of an inducible HoxA9 retroviral construct

The cDNA for mouse HoxA9 (Thorsteinsdottir et al., 2002; kind gift of G. Sauvangeau, University of Montreal) was fused in frame with the hormone binding domain of the human estrogen receptor and with a V5 epitope tag. Mouse HoxA9 shares greater than 90% amino acid identity with human HoxA9. This fusion cDNA was then inserted into the multi-cloning site of the IRES-GFP vector MigRl (Pear et al., 1998; kind gift of W. Pear, University of Pennsylvania). This generated a HoxA9:ER:V5-IRES-GFP retroviral vector. Fig. IA illustrates the features of this construct.

Generation of inducible Hox A9 -transduced BLIN-2 cells HoxA9:ER retrovirus was packaged with an amphotropic coat via co-transfection in 293 cells with pCL-Ampho (Naviaux et al., 1996; Pear et al., 1993) by calcium phosphate precipitation. Supernatants_, containing viral particle were collected 48 h post- transfection and used to transduce BLIN-2 cells. Briefly, BLIN-2 cells were removed from stromal cell layers and incubated with a 1 :1 mixture of viral supernatant and XVIVO-10 growth medium in the presence of 2 μg/ml of polybrene for 6 h. Next, cells were resuspended in fresh medium and plated in the presence of stromal cell layers. Transduced BLIN-2 cells were analyzed 24 h later for GFP expression via flow cytometry. Once the transduced cells had been sufficiently expanded, BLIN- 2/HoxA9:ER cells were FACS purified to greater than 90% (Fig. IB). Expression of HoxA9 was confirmed by western blot analysis (Fig. 3). A similar strategy was used to generate BLIN-2 cells bearing an empty GFP vector (BLIN-2/MigRl) as a control. Parental BLIN-2 cells or empty vector control BLIN-2/MigRl cells do not exhibit HoxA9 expression. BLIN-2/HoxA9:ER cells were maintained in serum-free culture conditions in the presence of stromal cell layers in phenol red-free XVIVO-10 medium.

Induction ofHoxA9:ER nuclear localization in BLIN-2 cells In the BLIN-2/HoxA9:ER cell system, HoxA9:ER: V5 is constitutively expressed.

In the absence of β-estradiol or 4-hydoxytamoxifen (4HT), heat shock proteins occupy the hormone binding domain preventing the transduced HoxA9 from entering the nucleus and promotes degradation of the protein via the proteasome pathway. However, in the presence of β-estradiol or 4HT, the heat shock proteins are outcompeted and HoxA9 enters the nucleus where it can exert its effect as a transcription factor. This strategy has been employed successfully by many groups in the study of oncogenes and signaling molecules (McMahon, 2001; Sykes et al., 2003; McCubrey et al., 2004; Samuels et al., 1994) and has been previously used successfully by us to study EGFR signaling in BLIN- 2 cells (Spengeman et al., 2005). An illustration of the strategy is presented in Fig. 2. BLIN-2/MigRl and BLIN-2/HoxA9:ER cells were cultured in the presence or absence of 4HT and analyzed for HoxA9:ER protein expression. Little HoxA9:ER was observed in BLIN-2/HoxA9:ER cells cultured without 4HT. However, upon culture with 4HT, HoxA9:ER was readily detected in the nuclear protein fraction (Fig. 3). Pax5 is a B-cell-specific transcription factor and was included as a control for a nuclear specific protein. Actin is expressed in the cytoplasm only. These data confirm the stabilization and nuclear localization of the HoxA9:ER fusion protein in BLIN-2/HoxA9:ER cells treated with the estrogen analog 4HT. HoxA9:ER binds to a HoxA9 DNA consensus binding sequence

Electrophoretic mobility shift assay (EMSA) analysis was performed to verify that HoxA9:ER was capable of binding to the HoxA9 DNA consensus binding sequence. Protein lysates from BLIN-2/MigRl and BLIN-2/HoxA9:ER cells were incubated with a digoxigenin-labeled probe and resolved on a non-denaturing polyacrylamide gel (Fig. 4). A shifted complex was observed when BLIN-2/HoxA9:ER lysate was incubated with the labeled probe (lane 6). This was competed away with excess unlabeled probe (lane 7). Pre-incubation of the lysate with anti-HoxA9 antibody resulted in a reduction in the intensity of the shifted band (lane 8), indicating that HoxA9 is part of this complex. No mobility shift was detected in lanes 2-5 using lysate from BLIN-2/MigRl which lacks expression of endogenous HoxA9. These data indicate that the HoxA9:ER fusion protein is capable of binding the HoxA9 DNA binding sequence.

EXAMPLE 2 Effect of HoxA9:ER induction on cell proliferation and survival

BLIN-2/HoxA9:ER cells exhibit increased stromal cell-independent proliferation and survival

To examine the effects of HoxA9:ER transcriptional activity on the proliferation and survival of BLIN-2/MigRl and BLIN-2/HoxA9:ER, cells were cultured in the absence of stromal cell support with or without 1 μM 4HT for 5 days and proliferation was determined by an MTS, soluble tetrazolium salt-based colorimetric assay which measures mitochondrial dehydrogenase activity as a surrogate for cell number (Fig. 5). The BLIN-2/MigRl cells showed little change in relative proliferation over the course of the experiment in the presence or absence of 4HT. However, the BLIN-2/HoxA9:ER cells, in both the presence and absence of inducer, showed steady increases in relative proliferation up to day three. After day three, BLIN-2/HoxA9:ER cells cultured in the presence of 4HT continued to exhibit increased proliferation through day five, whereas the untreated cells ceased to proliferate. Thus, HoxA9:ER expression call promote stromal cell-independent growth of B-ALL cells. EXAMPLE 3 Effect of HoxA9:ER induction on c-Myb protein levels

HoxA9 activity alters protein levels of c-Myb

It has recently been reported that the cellular proto-oncogene c-Myb is a direct downstream target of HoxA9, and the HoxA9 cofactor Meisl (Hess et al., 2006). To evaluate c-Myb expression in the BLIN-2/HoxA9:ER cell line, western analysis was performed on proteins prepared from parental BLIN-2 and BLIN-2/HoxA9:ER cells grown in the presence or absence of 4HT (Fig. 6). Increased c-Myb protein levels were observed in whole cell lysates from 4HT-treated BLIN-2/HoxA9:ER cells, indicating that c-Myb is a potential downstream target of HoxA9:ER in BLIN-2/HoxA9:ER cells. To further confirm these results, cytoplasmic and nuclear protein fractions were prepared from parental and BLIN-2/HoxA9:ER cells stimulated with 4HT and also analyzed for c- Myb expression (Fig. 6B). Upon 4HT stimulation, large amounts of HoxA9:ER were detected in the nuclear fraction only. C-Myb levels were increased in 4HT-treated cells relative to parental BLIN-2, indicating that nuclear localization of HoxA9:ER resulted in increased c-Myb protein levels.

EXAMPLE 4 Effect of HoxA9:ER induction on surface expression of IGF-IR HoxA9 activation induces surface expression of IGF-IR c-Myb has been shown to participate in the regulation of the IGF-IR (Reiss et al., 1991; 1992; Travali et al., 1991). Therefore, BLIN-2/HoxA9:ER cells were examined for expression of IGF-IR by RT-PCR and western blot analysis. BLIN-2/HoxA9:ER cells were treated with 4HT for 24 h and cDNA was prepared from total mRNA. RT-PCR was performed to detect HoxA9 and IGF-IR expression. IGF-IR was detected in BLIN- 2/HoxA9:ER cells treated with 4HT. No IGF-IR transcript was detected in untreated BLIN-2/HoxA9:ER cells or BLIN-2/MigRl empty vector control cells (Fig. 7A). The RS4;11 cell line bears an MLL chromosomal translocation and expresses both HoxA9 (Stong et al., 1985; Quentmeier et al., 2004) and IGF-IR (data presented herein). Interestingly, there was more HoxA9 mRNA detected in BLIN-2/HoxA9:ER cells treated with 4HT, which suggests that the transcript was stabilized by a positive feedback mechanism. Western analysis confirmed the presence of IGF-IR protein in 4HT-treated BLIN-2/HoxA9:ER cells, while no IGF-IR was detected in parental controls cells (Fig. 7B). Next, to determine if IGF-IR surface expression could be detected on the BLIN- 2/HoxA9:ER cells, these cells were examined by flow cytometry (Fig. 7C). Surface IGF- 1 R could be detected on BLIN-2/HoxA9:ER cells but not on the parental BLIN-2 cells. Collectively, these results suggest that HoxA9 activity increases the cell surface expression of IGF-IR.

EXAMPLE 5 Effect of inhibition of endogenous HoxA9 expression on IGF-IR expression.

Targeted inhibition ofHoxA9 expression using siRNA

HoxA9 siRNA was generated by Ambion (Austin, TX). The nucleotide sequences for the HoxA9 siRNA were: 5'-UCAACAAAGACCGAGCAAAUU-S' (sense) (SEQ ID NO:1) and 5'- UUUGCUCGGUCUUUGUUGAUU-S' (antisense) (SEQ ID NO:2). The HoxA9 siRNA was designed to target the host HoxA9 sequence 5'- AATCAACAAAGACCGAGCAAA-3' (SEQ ID NO:3). Cells were transiently transfected or mock transfected with siRNA using an AMAXA Nucleofector (Cologne, Germany), according to the manufacturer's instructions. RS4;11 cells were transfected with increasing concentrations of HoxA9 siRNA followed by RT-PCR analysis 24 h to monitor expression of the indicated genes.

Loss of endogenous HoxA9 expression reduces IGF-IR expression in MLL/AF4 positive leukemia

Acute leukemia bearing chromosomal translocations of the MLL gene typically exhibit overexpression of HoxA9 (Armstrong et al., 2002; Hess, 2004). To test if loss of endogenous HoxA9 expression would also result in reduced expression of IFG-IR,

RS4;11 cells were transfected with increasing concentrations of HoxA9 siRNA followed by RT-PCR analysis for the indicated genes 24 h later. As shown in Fig. 8, abrogation of HoxA9 gene expression with siRNA resulted in a loss of IGF-IR and c-myb expression. In contrast, expression of the B-lineage-specific gene, mb-1, and the MLL/ AF4 fusion gene was not affected by the inhibition of HoxA9 gene expression. Thus, not only does HoxA9 overexpression cause increased expression of IGF-IR, but inhibition of HoxA9 gene expression also downregulates IGF-IR expression. Accordingly, HoxA9 expression may be targeted as a means of reducing IGF-IR expression and thereby treating cancer.

EXAMPLE 6 Isolation and functional characterization of anti-IGF-lR mAbs, A12 and 2F8

Isolation of mAbs

In order to isolate high affinity antibodies to human IGF-IR, a recombinant extracellular portion of human IGF-IR was used to screen a human naϊve (non- immunized) bacteriophage Fab library containing 3.7 x 10¹⁰ unique clones (de Haard et al., 1999). After two rounds of selection, the diversity of the anti-IGF-lR Fab clones obtained was assessed by restriction enzyme analysis. Fab fragments encoded by plasmids from individual clones exhibiting positive binding to IGF-IR and a unique DNA profile were expressed in a nonsuppressor Escherichia coli host, HB2151, and purified from the periplasmic fraction by affinity chromatography on a Protein G column (Amersham Pharmacia Biotech).

Candidate binding Fab clones were screened for competitive blocking of radiolabeled human IGF-I ligand to immobilized IGF-IR. Only one Fab clone, 2F8, exhibited greater than 50% inhibition of control radiolabeled ligand binding, with an IC₅₀ of approximately 200 nM, and it was selected for conversion to full length IgGl format (Burtrum et al., 2003). Fab 2F8 sequencing determined that this Fab possessed a lambda light chain constant region. This antibody was determined to bind to the IGF-IR with an affinity of 0.5 - 1 nM (0.5 - 1 x 10^"9 M).

In order to improve the affinity of this antibody, a second generation Fab phage library was generated in which the 2F8 heavy chain was conserved and the light chain was varied to a diversity of greater than 10⁸ unique species by light chain shuffling

(Chames et al., 2002). Four rounds of screening of this library for binding to the human IGF-IR for enrichment of high affinity binding Fabs led to the isolation of a Fab designated Al 2 that was shown to possess a lambda light chain constant region (Burtrum et al., 2003). Amino acid sequence comparison of 2F8 and Al 2 light chains determined that the two variable regions differed by a total of 1 1 amino acids, nine of the changes being present within CDR regions, with the majority (6 amino acid residues) occurring within CDR3.

Binding affinity, avidity and specificity ofmAbs Al 2 and 2F8

A comparison of the two antibody (full IgG) affinities for human IGF-IR and their ligand blocking activity is shown in Table 1. Binding results were determined by human IGF-IR ELISA and represent the concentration of titered antibody necessary to achieve 50% binding relative to saturation. Blocking results represent the level of antibody necessary to inhibit 50% binding of ¹²⁵I-IGF-I ligand to immobilized human IGF-IR. Affinity was determined by BIAcore analysis according to manufacturer's specifications (Pharmacia BIACORE 3000). Soluble IGF-IR was immobilized on the sensor chips and antibody binding kinetics were determined.

The antibody changes incurred in the 2F8 light chain to generate antibody Al 2 effected a significantly higher affinity of A12, compared to 2F8, for IGF-IR. Concomitantly, this increase effected a 6- to 7-fold greater binding ability of Al 2 for the receptor, as determined by ELISA, and at least a three-fold increase in blocking activity of ligand for immobilized receptor. Al 2 also blocked binding of radiolabeled IGF-I ligand to immobilized IGF-IR, and possessed similar blocking activity to cold IGF-I, with an IC₅₀ of approximately 1 nM (0.15 μg/ml), and greater ligand blocking activity than 2F8 or IGF-2 (IC₅₀ = 6 nM).

The isolation of 2F8 and A 12, the full amino acid sequences of their heavy and light chains, the generation of fully human IgGl anti-IGF-lR antibodies from the Fab clones, and the functional characterization of the antibodies are described in more detail in Burtrum et al. (2003) and International Publication No. WO 2005/016970.

Table 1. Antibody binding characteristics

Antibody Binding (ED₅₀) (nM) Blocking (EC₅₀) (nM) Affinity (M^"')

K_d = 6.5 x 10 ^TTT

2F8 2.0 3 - 6 K_0n = 2.8 x lθ^b

Koff = 1.8 x l0^"

K_D = 4.1 x l0

A12 0.3 0.6 - 1 K_0n - 7.2 x lO⁵

K_off = 3.0 x lO ^5

IGF-IR shares considerable structural homology with the insulin receptor (IR). To demonstrate the selectivity of Al 2 for IGF-IR, the antibody was tested in human IR binding and blocking assays. Al 2 was titered onto immobilized IR from a concentration of 1 μM. A commercial anti-human IR antibody was used as a positive control for binding to IR. At a concentration of up to at least 1 μM, there was no detection of bound Al 2 to IR. See Fig. 3 A in Provisional Application No. 60/861,827, filed November 11, 2006, entitled "Insulin-Like Growth Factor- 1 Receptor Antagonists For Modulation Of Weight And Liposity." The ED₅₀ for binding of Al 2 to human IGF-IR is 0.3 nM, indicating selectivity of Al 2 for IGF-IR in comparison to IR of greater than 3, 000-fold. Accordingly, Al 2 did not block the binding of insulin to IR even at 100 nM antibody concentration. See Fig. 3B in Application No. 60/861,827. In this assay, cold insulin effectively competed with an IC₅₀ of approximately 0.5 nM whereas the commercial anti- IR blocking antibody, 47-9, showed modest activity (50% maximal inhibition) and cold IGF-I competed only at high concentrations.

To test for species cross-reactivity to mouse, a recombinant mouse IGF-IR (ml GF- IR) was expressed and a binding analysis was performed. This experiment indicated that Al 2 recognized and bound to immobilized recombinant mIGF-lR in ELISA with an ED₅₀ of 0.3-0.5 nM. See Fig. 4 in Application No. 60/861 ,827. For comparison, the human IGF-IR binding ELISA was repeated with this sample of A12, resulting in an ED₅₀ of 0.3-0.5 nM, consistent with previous data. These results indicate that Al 2 fully cross-reacts with mIGF-lR and binds with similar kinetics to human IGF- IR, suggesting that Al 2 can be used in mice to model the effects of blocking IGF-IR in human patients. EXAMPLE 7 Effect of inhibition of IGF-IR on proliferation and survival of HoxA9:ER cells

Small molecule IGF-IR antagonist decreases proliferation and survival of HoxA9:ER cells To determine if IGF-IR signaling was participating in the enhanced proliferation and survival of BLIN-2/HoxA9:ER cells, BLIN-2/MigRl and BLIN-2/HoxA9:ER cells were separately co-cultured for 10 days on stromal cell feeder layers with 1 μM 4HT in the presence or absence of the IGF-IR tyrosine kinase inhibitor, AGl 024 (1 μg/ml). BLIN-2/HoxA9:ER cells grown in the presence of 4HT showed a 2-fold increase in proliferation compared with BLIN-2/MigRl control cells (Fig. 9). BLIN-2/MigRl cells exhibited a slight decrease in proliferation when treated with AG 1024 at both days 5 and 10. However, by day 5 there was a nearly 1.5-fold decrease in the proliferation of BLIN- 2/HoxA9:ER cells treated with AGl 024. By day 10, AG 1024 treated cells showed a 2- fold reduction in proliferation, compared to cells treated with 4HT alone (Fig. 9). Next, BLIN-2/HoxA9:ER cells were cultured off of stromal support in the presence of 4HT and AG 1024, or left untreated. Relative proliferation was determined at days 2 and 5. BLIN-2/HoxA9:ER cells treated with AGl 024 showed a significant decrease in proliferation at days 2 and 5, with a more pronounced effect at day 5 (Fig. 10). IGF-IR mAb antagonist decreases proliferation and survival ofHoxA9:ER cells

A series of separate and independent experiments were performed to confirm the effects of IGF-IR inhibition on the proliferation and survival BLIN-2/HoxA9:ER by treating them with the αIGF-lR mAb, A 12. Al 2 is an IGF-IR specific monoclonal antibody which possesses high affinity for the receptor and blocks ligand binding. See Example 6. The binding of Al 2 to IGF-IR results in rapid internalization and degradation of the receptor, thus inhibiting IGF-IR signaling and reducing cell surface receptor levels (Burtrum et al., 2003). In addition, Al 2 has been demonstrated to inhibit the growth of various cancer cell lines, including multiple myelomas, and shown strong antitumor activity in nude mouse models (Burtrum et al., 2003; Wu et al., 2006; Bertrand et al., 2006). To determine if specific inhibition of IGF- 1 R would inhibit the proliferative effects of HoxA9:ER in BLIN-2 cells, 4HT-treated BLIN-2 and BLIN-2/HoxA9:ER cells were cultured in the presence of 15 μg of Al 2 niAb (Fig. 11). Al 2 treatment had no effect on the proliferation of parental BLIN-2 cells. BLIN-2/HoxA9:ER cells demonstrated increased proliferation in the presence of 4HT, compared to the parental cell line, which was significantly inhibited by Al 2 antibody treatment. These results demonstrate that HoxA9-mediated expression of IGF-IR is responsible for the increased proliferative capacity of BLIN-2/HoxA9:ER cells, and blocking signaling through the IGF-IR receptor by two independent mechanisms inhibited growth in the presence or absence of stromal cell support.

EXAMPLE 8

Effect of inhibition of IGF-IR signaling on proliferation of ML£-positive leukemia cells

Blocking IGF-IR signaling in RS4J1 cells inhibits proliferation

To test if blocking IGF-IR would be effective in MZX-positive leukemia cells expressing endogenous HoxA9, RS4;11 cells were cultured in the presence or absence of

A12 mAb. Growth of RS4;11 cells was inhibited with A12 antibody treatment (Fig. 12).

However, IGF-I treatment abrogated the effects of the Al 2 antibody and resulted in increased proliferation of both Al 2 treated and untreated cells.

The above data collectively indicate that IGF-IR is a downstream target of HoxA9 expression and that increased expression of IGF-IR accounts for the observed biological effects on proliferation and cell survival in leukemic cells overexpressing HoxA9.

Overexpression of HoxA9 induces expression of the c-Myb transcription factor which results in increased IGF-IR expression. Because c-Myb also promotes IGF-I expression, an autocrine loop may be established that ultimately leads to stromal cell/growth factor- independent growth. This regulatory cascade is illustrated in Fig. 13. At present, it has not been determined whether IGF-I is also induced in the BLIN-2/HoxA9:ER system. The findings disclosed herein provide a mechanistic pathway for the role of Hox overexpression generally in cancer development and progression. Hox gene expression serves as a biomarker that identifies cancers that may be amenable to treatment with therapeutics targeting the IGF-IR, and the disclosed data provide a sound rationale for targeting IGF-IR in cancers that overexpress Hox genes. REFERENCES

U.S. Patent No. 6,217,866, issued April 17, 2001 to Schlessinger et al. U.S. Patent Publication No. 2002/0086356 Al, published July 4, 2002. PCT International Publication No. WO 99/60023, published November 25, 1999 (ImClone Systems Inc.).

PCT International Publication No. WO 03/075840, published August 18, 2003 (ImClone Systems Inc.).

PCT International Publication No. WO 2005/016970, published February 24, 2005 (ImClone Systems Inc.). Adams TE, Epa VC, Garrett TP, Ward CW (2000) Structure and function of the type 1 insulin-like growth factor receptor. Cell MoI Life Sci 57: 1050-1093.

Abramovich C, Humphries RK (2005) Hox regulation of normal and leukemic hematopoietic stem cells. Curr Opin Heml2: 210-216.

Acampora D, D'Esposito M, Faiella A, Pannese M, Migliaccio E et al. (1989) The human HOX gene family. Nucleic Acids Res. 17: 10385-10402.

Alberti C (2006) Prostate cancer progression and the surrounding microenvironment. Int J Biol Markers 21: 88-95.

All-Ericsson C, Girnita L, Seregard S, Bartolazzi A, Jager MJ, Larsson O (2002) Insulin-like growth factor- 1 receptor in uveal melanoma: a predictor for metastatic disease and a potential therapeutic target. Invest Ophthalmol Vis Sci 43: 1-8.

American Cancer Society (2006) Cancer Facts and Figures. Atlanta, American Cancer Society.

Apiou F, Flagiello D, Cillo C, Malfoy B, Poupon MF, Dutrillaux B (1996) Fine mapping of human HOX gene clusters. Cytogenet Cell Genet 73: 114-115. Armstrong SA, Staunton JE, Silverman LB, Pieters R, den Boer ML et al. (2002)

MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nature Genet 1 : 41-47.

Arteaga CL, Osbome CK (1989) Growth inhibition of human breast cancer cells in vitro with an antibody against the type I somatomedin receptor. Cancer Res 49: 6237- 6241.

Ayton PM, Cleary ML (2003) Transformation of myeloid progenitors by MLL oncoproteins is dependent on Hoxa7 and Hoxa9. Genes Dev 17: 2298-307.

Basecke J, Whelan JT, Griesinger F, Bertrand FE (2006) The MLL partial tandem duplication in acute myeloid leukaemia. Br J Haematol 135: 438-449.

Baserga R (2000) The contradictions of the insulin-like growth factor 1 receptor. Oncogene 19: 5574-5581.

Batley BL, Doherty AM, Hamby JM, Lu GH, Keller P et al. (1998) Inhibition of FGF- 1 receptor tyrosine kinase activity by PD 161570, a new protein-tyrosine kinase inhibitor. Life Sci 62: 143-150.

Baxter RC (2000) Insulin-like growth factor (IGF)-binding proteins: interactions with TGFs and intrinsic bioactivities. Am J Physiol Endocrinol Metal 278: E967-976.

Benini S, Manara MC, Baldini N, Cerisano V, Massimo Serra et al. (2001) Inhibition of insulin-like growth factor I receptor increases the antitumor activity of doxorubicin and vincristine against Ewing's sarcoma cells. Clinical Cancer Res 7: 1790- 97.

Bertrand FE, Eckfeldt CE, Fink JR, Lysholm AS, Pribyl JA, Shah N, LeBien TW (2000) Microenvironmental influences on human B-cell development. Immunol Rev 175: 175-186.

Bertrand FE, Spengeman JD, Shah N, LeBien TW (2003) B-cell development in the presence of the MLL/ AF4 oncoprotein proceeds in the absence of HOX A7 and HOX A9 expression. Leukemia 17: 2454-2459.

Bertrand FE, Spengeman JD, Shelton JG, McCubrey JA (2005) Inhibition of PI3K, mTOR and MEK signaling pathways promotes rapid apoptosis in B-lineage ALL in the presence of stromal cell support. Leukemia 19: 98-102.

Bertrand FE, Steelman LS, Chappell WH, Abrams SL, Shelton JG et al. (2006) Synergy between an IGF-IR antibody and Raf/MEK/ERK and PI3K/Akt/mTOR pathway inhibitors in suppressing IGF-lR-mediated growth in hematopoietic cells. Leukemia. 20: 1254-1260.

Bertrand FE, Vogtenhuber C, Shah N, LeBien TW (2001) Pro-B cell to pre-B cell development in B-lineage acute lymphoblastic leukemia expressing the MLL/AF4 fusion protein. Blood 98: 3398-3405.

Blakesley VA, Butler AA, Koval AP, Okubo Y, LeRoith D (1999) IGF-I receptor function. In The IGF System: Molecular Biology, Physiology and Clinical Applications (Rosenfeld RG, Roberts CT, Jr, eds.), pp. 143-163, Humana Press, Totowa, NJ.

Blatt C, Loten J, Sachs L (1992) Inhibition of specific pathways of myeloid cell differentiation by an activated HOX 2.4 homeobox gene. Cell Growth Differ 3: 671-676. BoI DK, Kiguchi K, Gimenez-Conti I, Rupp T, DiGiovanni J (1997) Overexpression of insulin-like growth factor- 1 induces hyperplasia, dermal abnormalities, and spontaneous tumor formation in transgenic mice. Oncogene 14: 1725-1734.

Burtrum D, Zhu Z, Lu D, Anderson DM, Prewett M et al. (2003) A fully human monoclonal antibody to the insulin-like growth factor I receptor blocks ligand-dependent signaling and inhibits human tumor growth in vivo. Cancer Res 63: 8912-8921.

Butler JE (1981) The amplified ELISA: principles of and applications for the comparative quantitation of class and subclass antibodies and the distribution of antibodies and antigens in biochemical separates. Meth Enzymol 73: 482-523. Byrd JC, Devi GR, de Souza AT, Jirtle RL, MacDonald RG ( 1999) Disruption of ligand binding to the insulin-like growth factor Il/mannose 6-phosphate receptor by cancer-associated missense mutations. J Biol Chem 274: 24408-24416.

Campbell AM (1985) Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas, Burdon et al., Eds., Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam, 256 pp.

Chames P, Willemsen RA, Rojas G, Dieckmann D, Rem L et al. (2002) TCR-like human antibodies expressed on human CTLs mediate antibody affinity-dependent cytolytic activity. J Immunol 169: 1110-1118. Cillo C, Candle M, Faiella A, Boncinelli E (2001) Homeohox genes in normal and malignant cells. J Cell Phys 188: 161-169.

Cohen BD, Baker DA, Soderstrom C, Tkalcevic G, Rossi AM et al. (2005) Combination therapy enhances the inhibition of tumor growth with the fully human antitype 1 insulin-like growth factor receptor monoclonal antibody CP-751,871. Clin Cancer Res 1 1 : 2063-2073.

Coligan J, Kruisbeek A, Margulies D, Shevach E, Strober W, eds. (1994) Current Protocols in Immunology, Wiley & Sons, Inc., New York.

Collins VP (1995) Gene amplification in human gliomas. Glia 15: 289-296.

Cullen KJ, Yee D, Sly WS, Perdue J, Hampton B, Lippman ME, Rosen N (1990) Insulin-like growth factor receptor expression and function in human breast cancer. Cancer Res 50: 48-53.

D'Ambrosio et al., Cancer Res. 56:4013-20 (1996).

Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME (1997) Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91 : 231-241. de Haard HJ, van Neer N, Reurs A, Hufton SE, Roovers RC et al. (1999) A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J. Biol. Chem. 274: 18218-18230.

DiGiovanni J, BoI DK, Wilker E, Beltran L, Carbajal S et al. (2000) Constitutive expression of insulin-like growth factor- 1 in epidermal basal cells of transgenic mice leads to spontaneous tumor promotion. Cancer Res 60: 1561-70.

Dorrance AM, Liu S, Yuan W, Becknell B, Arnoczky KJ et al. (2006) MLL partial tandem duplication induces aberrant Hox expression in vivo via specific epigenetic alterations. J Clin Invest 116: 2707-2716.

Drange MR, Melmed S (1999) IGFs in the Evaluation of Acromegaly. In The IGF System: Molecular Biology, Physiology, and Clinical Applications (Rosenfeld RG, Roberts CT, Jr, eds.), pp. 699-720, Humana Press, Totowa, NJ. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001 )

Duplexes of 21 -nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411 : 494-498.

Foucher I, Volovitch M, Frain M, Kim J.I, Souberbielle TC et al. (2002) HOXa5 overexpression correlates with IGFBPI upregulation and postnatal dwarfism: evidence for an interaction between HOXa5 and Forkhead box transcription factors. Development 129: 4065-4074.

Freireich EJ, Gehan EA, Rail DP, Schmidt LH, Skipper HE (1966) Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man. Cancer Chemother Rep 50: 219-244. Furlanetto RW, Harwell SE, Baggs RB (1993) Effects of insulin-like growth factor receptor inhibition on human melanomas in culture and in athymic mice. Cancer Res 53: 2522-2526.

Garcia-Echeverria C, Pearson MA, Marti A, Meyer T, Mestan J et al. (2004) In vivo antitumor activity of NVP-AEW541-A novel, potent, and selective inhibitor of the IGF-IR kinase. Cancer Cell 5: 231-239.

Ge NL, Rudikoff S (2001) Insulin-like growth factor I is a dual effector of multiple myeloma cell growth. Blood 96: 2856-2861.

Gibson LF (2002) Survival of B lineage leukemic cells: signals from the bone marrow microenvironment. Leuk Lymphoma 43: 19-27.

Girnita A, Girnita L, del Prete F, Bartolazzi A, Larsson O, Axelson M (2004) Cyclolignans as inhibitors of the insulin-like growth factor- 1 receptor and malignant cell growth. Cancer Res 64: 236-242. Goetsch L, Gonzalez A, Leger O, Beck A, Pauwels PJ, Haeuw JF, Corvaia N

(2005) A recombinant humanized anti-insulin-like growth factor receptor type I antibody (h7C10) enhances the antitumor activity of vinorelbine and anti-epidermal growth factor receptor therapy against human cancer xenografts. Int J Cancer 113 : 316-328.

Gooch JL, Van Den Berg CL, Yee D (1999) Insulin-like growth factor (IGF)-I rescues breast cancer cells from chemotherapy-induced cell death — proliferative and anti-apoptotic effects. Breast Cancer Res. Treat. 56: 1-10.

Grier DG, Thompson A, Kwasniewska A, McGonigle GJ, Halliday HL, Lappin TR (2005) The pathophysiology of HOX genes and their role in cancer. J Pathol 205: 154-171. Guerra FK., Eijan AM, Puricelli L, Alonso DF, BaI de Kier Joffe E et al. (1996)

Varying patterns of expression of insulin-like growth factors I and II and their receptors in murine mammary adenocarcinomas of different metastasizing ability. Int J Cancer 65: 812-820.

Haluska P, Carboni JM, Loegering DA, Lee FY, Wittman M et al. (2006) In vitro and in vivo antitumor effects of the dual insulin-like growth factor-I/insulin receptor inhibitor, BMS-554417. Cancer Res 66: 362-371.

Harmon GJ (2002) RNA interference. Nature 418: 244-251.

Hassan AB, Macaulay VM (2002) The insulin-like growth factor system as a therapeutic target in colorectal cancer. Ann Oncol 13: 349-356. Haylor J, Hickling H, El Eter E, Moir A, Oldroyd S, Hardisty C, El Nahas AM

(2000) JB3, an IGF-I receptor antagonist, inhibits early renal growth in diabetic and uninephrectomized rats. J Am Soc Nephrol 11 : 2027-2035.

Hellawell GO, Turner GD, Davies DR, Poulsom R, Brewster SF, Macaulay VM (2002) Expression of the type 1 insulin-like growth factor receptor is up-regulated in primary prostate cancer and commonly persists in metastatic disease. Cancer Res 62: 2942-2950.

Hess JL (2004) Mechanisms of transformation by MLL. Crit Rev Eukaryot Gene Expr 14: 235-254. Hess JI, Bittner CB, Zeisig DT, Bach C, Fuchs U et al. (2006) c-Myb is an essential downstream target for homeobox mediated transformation of hematopoietic cells. Blood 108: 297-304.

Hoffmann T, Hafner D, Ballo H, Haas I, Bier H (1997) Antitumor activity of anti- epidermal growth factor receptor monoclonal antibodies and cisplatin in ten human head and neck squamous cell carcinoma lines. Anticancer Res 17: 4419-4426.

Hudlebusch HR, Lodahl M, Johnsen HE, Rasmussen T (2004) Expression of HOXA genes in patients with multiple myeloma. Leu Lymph 45: 1215-1217.

Huse WD, Sastry L, Iverson SA, Kang AS, Alting-Mees M et al. (1989) Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246: 1275-1281.

Kersey JH, Wang D, Oberto M (1998) Resistance of t(4;l 1) (MLL- AF4 fusion gene) leukemias to stress-induced cell death: possible mechanism for extensive extramedullary accumulation of cells and poor prognosis. Leukemia 12: 1561-1564. Kikugawa T, Kinugasa Y, Shiraishi K, Nanba D, Nakashiro K et al. (2006) PLZF regulates Pbxl transcription and Pbx-I-HoxC8 complex leads to androgen independent prostate cancer proliferation. Prostate 66: 1092-1099.

Kim JB, Stein R, O'Hare MJ (2005) Tumor-stromal interactions in breast cancer: the role of stroma in tumorigenesis. Tumor Biol 26: 173- 185. Kim JJ, Taylor HS, Akbas GE, Foucher I, Trembleau A et al. (2003) Regulation of insulin-like growth factor binding protein- 1 promoter activity by FKHR and HOXAlO in primate endometrial cells. Biol Reprod 68: 24-30.

Khvorova A, Reynolds A, Jayasena SD (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115: 209-216. Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-497.

Kumar AR, Hudson WA, Chen W, Nishiuchi R, Yao Q, Kersey JH (2004) HoxA9 influences the phenotpe but not the incidence of MLL/ AF9 fusion gene leukemia. Blood 103: 1823-1828. Kurosaka D, LeBien TW, Pribyl JA (1999) Comparative studies of different stromal cell mieroenvironments in support of human B-cell development. Exp Hematol 27: 1271-1281.

Lamoyi E, Nisonoff A (1983) Preparation of F(ab')₂ fragments from mouse IgG of various subclasses. J Immunol Methods 56: 235-243.

Lawrence HJ, Helgason CD, Sauvageau G, Fong S, Izon DJ et al. (1997) Mice bearing a targeted interruption of the homeobox gene HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood 89: 1922-1930. Lawrence HJ, Sauvageau G, Ahmadi N, Lopez AR, LeBcau MM et al. (1995)

Stage- and lineage-specific expression of the HOXAlO homeobox gene in normal and leukemic hematopoietic cells. Exp Hematol 23: 1160-1166.

LeBien, TW (2000) Fates of human B-cell precursors. Blood 96: 9-23.

Lechner JF, Fugaro JM, Wong Y, Pass HI, Harris CC, Belinsky SA (2001) Perspective: cell differentiation theory may advance early detection of and therapy for lung cancer. Radiat Res 155: 235-238.

Lechner JF, Wang Y, Siddiq F, Fugaro JM, WaIi A et al. (2002) Human lung cancer cells and tissues partially recapitulate the homeobox gene expression profile of the embryonic lung. Lung Cancer 37:41-47. Lee AV, Yee D (1995) Insulin-like growth factors and breast cancer. Biomed

Pharmacother 49: 415-421.

LiIl MC, Fuller JF, Herzig R, Crooks GM, Casson JC (1995) The role of the homeobox gene, HOX B7, in human myelomonocytic differentiation. Blood 85: 692-697.

Look AT (1997) Oncogenic transcription factors in the human acute leukemias. Science 278: 1059-1064.

Lu Y, Zi X, Zhao Y, Mascarenhas D, Pollak M (2001) Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin). J Natl Cancer Inst (Bethesda) 93: 1852-1857.

Macaulay VM (1992) Insulin-like growth factors and cancer. Br J Cancer 65: 311-320.

Magli MC, Largman C, Lawrence HJ (1997) Effects of HOX homeobox genes in blood cell differentiation. J Cell Phys 173: 168-177.

Maloney EK, McLaughlin JL, Dagdigian NE, Garrett LM, Connors KM et al. (2003) An anti-insulin-like growth factor I receptor antibody that is a potent inhibitor of cancer cell proliferation. Cancer Res 63: 5073-5083.

Manabe A, Murti KG, Coustan-Smith E, Kumagai M, Behm FG, Raimondi SC, Campana D (1994) Adhesion-dependent survival of normal and leukemic human B lymphoblasts on bone marrow stromal cells. Blood 83: 758-766. McMahon M (2001) Steroid receptor fusion proteins for conditional activation of Raf-MEK-ERK signaling pathway. Methods Enzymol 332: 401-417.

McCubrey JA, Shelton JG, Steelman LS, Franklin RA, Sreevalsan T, McMahon M (2004) Effects of a conditionally active v-ErbB and EGF-R inhibitor on transformation of N1H-3T3 cells and abrogation of cytokine dependency of hematopoietic cells. Oncogene 23: 7810-7820.

McCubrey JA, Steelman LS, Mayo MW, Algate PA, Dellow RA, Kaleko M (1991) Growth-promoting effects of insulin-like growth factor- 1 (TGF-I) on hematopoietic cells: overexpression of introduced IGF-I receptor abrogates interluekin-3 dependency of murine factor-dependent cell by a ligand-dependent mechanism. Blood 78: 921-929.

Mello CC, Conte D Jr (2004) Revealing the world of RNA interference. Nature 431: 338-342.

Miller GJ, Miller HL van Bokhoven A, Lambert JR, Werahera PN et al. (2003) Aberrant HOXC expression accompanies the malignant phenotype in human prostate. Cancer Res 63: 5879-5888.

Mitsiades CS, Mitsiades NS, McMullan CJ, Poulaki V, Shringarpure R et al (2004) Inhibition of the insulin-like growth factor receptor- 1 tyrosine kinase activity as a therapeutic strategy for multiple myeloma, other hematologic malignancies, and solid tumors. Cancer Cell 5: 221-230.

Naviaux RK, Costanzi E, Haas M, Verma IM (1996) The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J Virol 70: 5701- 5705.

Nickerson T, Chang F, Lorimer D, Smeekens SP, Sawyers CL, Pollak M (2001) In vivo progression of LAPC-9 and LNCaP prostate cancer models to androgen independence is associated with increased expression of insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR). Cancer Res 61 : 6276-6280.

Oates AJ, Schumaker LM, Jenkins SB, Pearce AA, DaCosta SAet al. (1998) The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R), a putative breast tumor suppressor gene. Breast Cancer Res Treat 47: 269-81.

Oppenheim RW (1991) Cell death during development of the nervous system. Annu Rev Neurosci 14: 453-501.

Paddison PJ, Harmon GJ (2003) siRNAs and shRNAs: Skeleton keys to the human genome. Curr Opin MoI Ther 5: 217-224.

Pandini G, Vigneri R, Costantino A, Frasca F, Ippolito A et al. (1999) Insulin and insulin-like growth factor-I (IGF-I) receptor overexpression in breast cancers leads to insulin/IGF-I hybrid receptor overexpression: evidence for a second mechanism of IGF-I signaling. Clin Cancer Res 5: 1935-1944.

Panek RL, Lu GH, Klutchko SR, Batley BL, Dahring TK et al. (1997) In vitro pharmacological characterization of PD 166285, a new nanomolar potent and broadly active protein tyrosine kinase inhibitor. J Pharmacol Exp Ther 283: 1433-1444.

Parham P (1983) On the fragmentation of monoclonal IgGl, IgG2a, and IgG2b from BALB/c mice. J Immunoll31: 2895-2902.

Park JW, Smolen JS (2001) Monoclonal antibody therapy. In Advances in Protein Chemistry, VoI 56: Drug Discovery and Design (Scolnick EM, ed.), pp.360-421, Academic Press, San Diego, CA.

Parrizas M, Gazit A, Levitzki A, Wertheimer E, LeRoith D (1997) Insulin-like growth factor- 1 inhibition of apoptosis is associated with increased expression of the bcl- xL gene product. Endocrinology 138: 1427-1433.

Pear WS, Miller JP, Xu L, Pui JC, Soffer B et al. (1998) Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. Blood 92: 3780-3792. Pear WS, Nolan GP, Scott ML, Baltimore D (1993) Production of high-titer helper-free retroviruses by transient transfection. Proc Nail Acad Sci USA 90: 8392-8396.

Petrides PE, Bock S, Bovens J, Hofmann R, Jakse G (1990) Modulation of pro- epidermal growth factor, pro-transforming growth factor alpha and epidermal growth factor receptor gene expression in human renal carcinomas. Cancer Res 50: 3934-39. Peyrat JP, Bonneterre J (1992) Type 1 IGF receptor in human breast diseases.

Breast Cancer Res Treat 22: 59-67.

Pietrzkowski Z, Wernicke D, Porcu P, Jameson BA, Baserga R (1992) Inhibition of cellular proliferation by peptide analogues of insulin-like growth factor 1. Cancer Res 52: 6447-6451. Pollak M (2000) Insulin-like growth factor physiology and cancer risk. Eur J

Cancer 36: 1224-1228.

Quentmeier H, Dirks WC, Macleod RA, Reinhardt J, Zaborski M, Drexler HG (2004) Expression of HOX genes in acute leukemia cell lines with and without MLL translocations. Leuk Lymphoma 45: 567-574.

Quinn KA, Treston AM, Unsworth EJ, Miller MJ, Vos M et al. (1996) Insulin-like growth factor expression in human cancer cell lines. J Biol Chem 271 : 11477-11483.

Radinsky R, Risin S, Fan D, Dong Z, Bielenberg D, Bucana CD, Fidler IJ (1995) Level and function of epidermal growth factor receptor predict the metastatic potential of human colon carcinoma cells. Clin Cancer Res 1 : 19-31.

Reiss K, Yumet G, Shan S, Huang Z, Alnemri E et al. (1999) Synthetic peptide sequence from the C-terminus of the insulin-like growth factor-I receptor that induces apoptosis and inhibition of tumor growth. J Cell Phys 181: 124-35. Reiss K, Ferber A, Travali S, Poreu P, Phillips PD, Baserga R ( 1991 ) The protooncogene c-myb increases the expression of insulin-like growth factor 1 and insulin-like growth factor 1 receptor messenger RNAs by a transcriptional mechanism. Cancer Res 51 : 5997-6000.

Reiss K, Porcu P, Sell C, Pietrzkowski Z, Baserga R (1992) The insulin-like growth factor 1 receptor is required for the proliferation of hemopoietic cells. Oncogene 7: 2243-2248.

Ross JA, Perentesis JP, Robison LL, Davies SM (1996) Big babies and infant leukemia: a role for insulin-like growth factor-1? Cancer Causes Control 7: 553-559.

Rubin Grandis J, Melhem MF, Barnes EL, Tweardy DJ (1996) Quantitative immunohistochemical analysis of transforming growth factor-α and epidermal growth factor receptor in patients with squamous cell carcinoma of the head and neck. Cancer (Phila.) 78: 1284-1292.

Sachdev D, Li SL, Hartell JS, Fujita-Yamaguchi Y, Miller JS, Yee D (2003) A chimeric humanized single-chain antibody against the type I insulin-like growth factor (IGF) receptor renders breast cancer cells refractory to the mitogenic effects of IGF-I. Cancer Res 63: 627-35.

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Samuels ML, Weber MJ, Bishop JM, McMahon M (1993) Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-I protein kinase. MoI Cell Biol 13: 6241- 6252.

Sauter G, Maeda T, Waldman FM, Davis RL, Feuerstein BG (1996) Patterns of epidermal growth factor receptor amplification in malignant gliomas. Am J Path 148: 1047-1053.

Sauvageau G, Lansdorp PM, Eaves CJ, Hogge DE, Dragowska WH et al. (1994) Differential expression of homeobox genes in functionally distinct CD34⁺ subpopulations of human bone marrow cells. Proc. Natl Acad Sci USA 91 : 12223-12227.

Sazani P, KoIe R (2003) Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing. J Clin Invest 112: 481-486.

Scotlandi K, Avnet S, Benini S, Manara MC, Serra M et al. (2002) Expression of an IGF-I receptor dominant negative mutant induces apoptosis, inhibits tumorigenesis and enhances chemosensitivity in Ewing's sarcoma cells. Int J Cancer 101 : 11-16.

Scotlandi K, Benini S, Sarti M, Serra M, Lollini PL et al. (1996) Insulin-like growth factor I receptor-mediated circuit in Ewing's sarcoma/peripheral neuroectodermal tumor: a possible therapeutic target. Cancer Res 56: 4570-4574.

Scotlandi K, Benini S, Nanni P, Lollini PL, Nicoletti G et al. (1998) Blockage of insulin-like growth factor-I receptor inhibits the growth of Ewing's sarcoma in athymic mice. Cancer Res 58: 4127-31.

Seely BL, Samimi G, Webster NJ (2002) Retroviral expression of a kinase- defective IGF-I receptor suppresses growth and causes apoptosis of CHO and U87 cells in- vivo. BMC Cancer 2: 15. Sell C, Dumenil G, Deveaud C, Miura M, Coppola D et al. (1994) Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts. MoI Cell Biol 3604-3612.

Shimizu N, Wang Y, Minoshima S, Ishitoya J (1994) Detection of epidermal growth factor receptor gene amplification in human squamous cell carcinomas using fluorescence in situ hybridization. Jpn J Cancer Res 85: 567-571.

Shah N, Asch RJ, Lysholm AS, LeBien TW (2004) Enhancement of stress- induced apoptosis in B-lineage cells by caspase-9 inhibitor. Blood 104: 2873-2878.

Shah N, Oseth L, LeBien TW (1998) Development of a model for evaluating the interaction between human pre-B Acute lymphoblastic leukemic cells and the bone marrow stromal cell mieroenvironment. Blood 92: 3817-3828.

Siddle, K, Soos M (1999) Alternative IGF-related receptors. In The IGF System: Molecular Biology, Physiology and Clinical Applications (Rosenfeld RG, Roberts CT Jr., eds.), pp. 199-225, Humana Press, Totowa, NJ. Smith LE, Shen W, Perruzzi C, Soker S, Kinose F et al. (1999) Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor- 1 receptor. Nature Med 12: 1390-1395.

Spengeman JD, Green TD, McCubrey JA, Bertrand FE (2005) Activated EGFR promotes the survival of B-lineage acute leukemia in the absence of stromal cells. Cell Cycle 4: 483-487.

Stong RC, Korsmeyer SJ, Parkin JL, Arthur DC, Kersey JH (1985) Human acute leukemia cell line with the t(4;l 1) chromosomal rearrangement exhibits B lineage and monocytic characteristics. Blood 65: 21-31. Sykes DB, Kamps MP (2003) Estrogen-regulated conditional oncoproteins: tools to address open questions in normal myeloid cell function, normal myeloid differentiation, and the genetic basis of differentiation arrest in myeloid leukemia. Leuk Lymphoma 44: 1131-1139.

Thorsteinsdottir U, Mamo A, Kroon E, Jerome L, Bijl (2002) Overexpression of the myeloid leukemia-associated Hoxa9 gene in bone marrow cells induces stem cell expansion. Blood 99: 121-129.

Tiberio C, Barba P, Magli MC, Arvelo F, Le Chevalier T, Poupon MF, Cillo C (1994) Hox gene expression in human small cell lung cancers xenografted into nude mice. Int J Cancer 58: 608-615. Travali S, Reiss K, Ferber A, Petralia S, Mercer WE, Calabretta B, Baserga R

(1991) Constitutively expressed c-myb abrogates the requirement for insulin-like growth factor 1 in 3T3 fibroblasts. MoI Cell Biol 11 : 731-736. van VaI en F, Winkelmann W, Jurgens H (1992) Type I and type II insulin-like growth factor receptors and their function in human Ewing's sarcoma cells. J Cancer Res Clin Oncol 118: 269-275.

Vega F, Mcdeiros LJ, Lang W-H, Mansoor A, Bueso-Ramos C, Jone D (2002) The atonal composition of malignant lymphoid aggregates in bone marrow: variations in architecture and phenotype in different B-cell tumours. Br T Haema 117: 569-576.

Voller A, Bartlett A, Bidwell DE. (1978) Enzyme immunoassays with special reference to ELISA techniques. J Clin Pathol 31 : 507-520.

Vorwerk P, Wex H, Hohmann B, Mohnike K, Schmidt U, Mittler U (2002) Expression of components of the IGF signaling system childhood acute lymphoblastic leukaemia. J Clin Pathol MoI Pathol 55: 40-45. Waltregny D. Alami Y, Clausse N, do Leval J, Castronovo V (2002) Overexpression of the homeobox gene HOXC8 in human prostate cancer correlates with loss of tumor differentiation. Prostate 50: 162-169.

Wikstrand CJ, Hale LP, Batra SK, Hill ML, Humphrey PA et al. (1995) Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas. Cancer Res 55: 3140-3148.

Williams BR (1997) Role of the double-stranded RNA-activated protein kinase (PKR) in cell regulation. Biochem Soc Trans 25: 509-513.

Wittman M, Carboni J, Attar R, Balasubramanian B, Balimane P et al. (2005) Discovery of a (lH-benzoimidazol-2-yl)-lH-pyridin-2-one (BMS-536924) inhibitor of insulin-like growth factor I receptor kinase with in vivo antitumor activity. J Med Chem 48: 5639-5643.

Wraight CJ, White PJ, McKean SC, Fogarty RD, Venables DJ, Liepe IJ, Edmondson SR, Werther GA (2000) Reversal of epidermal hyperproliferation in psoriasis by insulin-like growth factor I receptor antisense oligonucleotides. Nature Biotechnol 18: 521-26.

Wu Y, Yakar S, Zhao L, Hennighausen L, LeRoith D (2002) Circulating insulin- like growth factor-I levels regulate colon cancer growth and metastasis. Cancer Res 62: 1030-1035. Wu KD, Zhou L, Burtrum D, Ludwig DL, Moore MA (2006) Antibody targeting of the insulin-like growth factor I receptor enhances the anti -tumor response of multiple myeloma to chemotherapy through inhibition of tumor proliferation and angiogenesis. Cancer Immunol Immunother [Epub July 11, 2006 ahead of print].

Yaginuma Y, Nishiwaki K, Kitamura S, Hayashi H, Sengoku K, Ishikawa M (1997) Relaxation of insulin-like growth factor-II gene imprinting in human gynecologic tumors. Oncology (Basel) 54: 502-507.

Yang WP, Green K, Pinz-Sweeney S, Briones AT, Burton DR, Barbas CF 3rd. (1995) CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV- 1 antibody into the picomolar range. J MoI Biol 254: 392-403. Yeoh EJ, Ross ME., Shurtleff SA, Williams WK, Patel D et al. (2002)

Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell 1 : 133-143.

Yu H, Rohan T (2000) Role of the insulin-like growth factor family in cancer development and progression. J Natl Cancer Inst 92: 1472-89 (2000).

Zamecnik PC, Stephenson ML (1978) Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci USA 75: 280-284.

Claims

What is claimed is:

1. A method for treating a tumor in a subject, wherein the tumor is determined to overexpress a Hox gene, comprising administering to the subject a therapeutically effective amount of an insulin-like growth factor- 1 receptor (IGF-IR) antagonist.

2. The method of claim 1 , wherein determining whether the tumor overexpresses a Hox gene comprises measuring the level of Hox protein in a nuclear fraction of a tumor cell and comparing said level with the level of Hox protein in a nuclear fraction of a nontransformed cell known to be not overexpressing the Hox gene, wherein an at least 2-fold higher level of Hox protein in the nuclear fraction of the tumor cell indicates that the tumor overexpresses the Hox gene.

3. The method of claim 1 , wherein determining whether the tumor overexpresses a Hox gene comprises measuring the level of Hox RNA level in a tumor cell and comparing said level with the level of Hox RNA in a nontransformed cell known to be not overexpressing the Hox gene, wherein an at least 2-fold higher level of Hox RNA in the tumor cell indicates that the tumor overexpresses the Hox gene.

4. The method of claim 1, wherein the Hox gene is a HoxA4, HoxA5, HoxA7, HoxA8, HoxA9, HoxAlO, HoxB7, HoxB8, HoxC8 gene, or any combination thereof.

5. The method of claim 1, wherein the Hox gene is a HoxA9 gene.

6. The method of claim 1, wherein the IGF-IR antagonist is an antibody or functional derivative thereof that binds specifically to IGF-IR, a small molecule IGF-IR antagonist, an insulin-like growth factor (IGF) mimetic, a small interfering RNA (siRNA), an antisense nucleic acid, a ribozyme, a triple helix-forming nucleic acid, a dominant negative mutant, or a soluble form of IGF-IR.

7. The method of claim 6, wherein the antibody is a monoclonal antibody.

8. The method of claim 7, wherein the monoclonal antibody is a human, humanized or chimeric antibody.

9. The method of claim 6, wherein the antibody is A12, 2F8, CP-751,871, EM 164, h7C10, AMG-479, cFv-Fc-IGF-lR, or an antibody that competes with Al 2 and/or 2F8 for binding to IGF-IR.

10. The method of claim 6, wherein the small molecule IGF-IR antagonist is AGl 024, AGl 034, NVP-AEW541, NVP-ADW742, picropodophyllin (PPP), BMS- 536924, or BMS-554417.

11. The method of claim 6, wherein the small molecule IGF- 1 R antagoni st is AG 1024.

12. The method of claim 1, wherein the tumor is a leukemia, a lymphoma, a multiple myeloma, or a solid tumor.

13. The method of claim 12, wherein cells of the leukemia harbor a mixed lineage leukemia gene (MLL) translocation or a MLL partial tandem duplication.

14. The method of claim 12, wherein the solid tumor is a small cell lung cancer (SCLC), a prostate cancer, a breast cancer, a colorectal cancer, an ovarian cancer, a neuroblastoma, a central nervous system tumor, a glioblastoma multiforme, or a melanoma.

15. The method of claim 1, wherein the IGF-IR antagonist is concurrently administered to the subject with at least one other anti -neoplastic agent.

16. The method of claim 15, wherein the at least one other anti-neoplastic agent is a chemotherapeutic agent, radiation or a combination thereof.

17. A method for inhibiting the onset of a tumor in a subject, wherein a pre- tumor cell is determined to overexpress a Hox gene, comprising administering to the subject a prophylactically effective amount of an insulin- like growth factor- 1 receptor (IGF-IR) antagonist.

18. A method for determining whether a tumor in a subject is amenable to treatment with an insulin-like growth factor- 1 receptor (IGF-IR) antagonist comprising determining whether the tumor overexpresses a Hox gene, wherein overexpression of the Hox gene indicates that the tumor is amenable to treatment with an IGF-IR inhibitor.

19. The method of claim 18, wherein determining whether the tumor overexpresses a Hox gene comprises measuring the level of Hox protein in a nuclear fraction of a tumor cell and comparing said level with the level of Hox protein in a nuclear fraction of a nontransformed cell known to be not overexpressing the Hox gene, wherein an at least 2-fold higher level of Hox protein in the nuclear fraction of the tumor cell indicates that the tumor overexpresses the Hox gene.

20. The method of claim 18, wherein determining whether the tumor overexpresses a Hox gene comprises measuring the level of Hox RNA level in a tumor cell and comparing said level with the level of Hox RNA in a nontransformed cell known to be not overexpressing the Hox gene, wherein an at least 2-fold higher level of Hox RNA in the tumor cell indicates that the tumor overexpresses the Hox gene.

21. A double stranded siRNA comprising a sense RNA strand and a complementary antisense RNA strand that downregulates expression of a targeted gene via RNA interference, wherein (a) each strand of the siRNA molecule is independently about 17 to about 30 nucleotides in length; (b) the antisense strand of the siRNA comprises an oligonucleotide having sufficient sequence complementarity to an mRNA of the targeted gene for the siRNA molecule to direct cleavage of the mRNA via RNA interference; and (c) the targeted gene is a Hox, c-Myb, or IGF-IR gene.

22. The siRNA of claim 21, wherein the Hox gene is a HoxA4, HoxA5, HoxA7, HoxA8, HoxA9, HoxAlO, HoxB7, HoxB8, or HoxC8 gene.

23. The siRNA of claim 21, wherein the Hox gene is a HoxA9 gene.

24. The siRNA of claim 23, wherein the sense RNA strand comprises 5'- UC AACAAAGACCGAGCAAAUU-S' (SEQ ID NO: 1) and the antisense RNA strand comprises 5'- UUUGCUCGGUCUUUGUUGAUU-3' (SEQ ID NO:2).

25. A pharmaceutical composition comprising the siRNA of claim 21 and a pharmaceutically acceptable carrier.