WO2007147118A1

WO2007147118A1 - Infection of cells with myxoma virus requires akt activation

Info

Publication number: WO2007147118A1
Application number: PCT/US2007/071349
Authority: WO
Inventors: John W. Barrett; Grant Mcfadden
Original assignee: Robarts Research Institute
Priority date: 2006-06-16
Filing date: 2007-06-15
Publication date: 2007-12-21

Abstract

Decribed herein is a method of screening for, or increasing, the permissiveness of cells to oncolytic therapy with myxoma virus. This disclosure further describes the relationship between the activation state of Akt and relative permissiveness of cancer cells to myxoma virus infection and replication. Cellular Akt activation is increased by contacting Akt- containing cells with a medicament that increases endogenous Akt activation or transducing Akt-containing cells with a vector that expresses exogenous Akt in the cells. Cancer is treated by increasing Akt activation in cancer cells and administering a myxoma virus.

Description

INFECTION OF CELLS WITH MYXOMA VIRUS REQUIRES AKT ACTIVATION

FIELD OF THE INVENTION

Described is a method of screening the permissiveness of cells to myxoma virus oncolytic therapy based upon their endogenous levels of activated (phosphorylated) Akt and methods to increase the permissiveness of cancer cells to oncolytic therapy with myxoma virus based upon activation of endogenous Akt or introduction of exogenous Akt.

BACKGROUND OF THE INVENTION

Myxoma virus (MV) is a rabbit specific poxvirus that causes a lethal disease called myxomatosis in European rabbits {Oryctolagus cuniculus). MV encodes a wide complement of immune evasion molecules, including an ankyrin-repeat host range protein called M-T5. M-T5 acts to prevent apoptosis during infection of rabbit T lymphocytes and is a virulence factor for disease progression in infected rabbits. It has recently been demonstrated that M-T5 also acts to protect MV infected cells from cell cycle arrest through interactions with cullin-1, which is involved in regulation of p27 through ubiquitin dependent proteasome degradation pathway. However, several aspects of M-T5 indicate that it exerts its host range functions in pathways above and beyond selective degradation.

In particular, MV exhibits potent oncolytic activity for human cancer and M-T5 is a critical determinant of MV tropism in human cancer cells. To investigate the tropism of MV in human tumor cells the cellular pathways of significance in human cancer were examined. Akt, or protein kinase B (PKB), is a serine/threonine kinase that plays a central role in the regulation of cellular processes including proliferation, programmed cell death, angiogenesis, and metabolism. Akt is activated by a variety of stimuli, including growth factors, protein phosphatase inhibitors, and cellular stress in a phosphatidylinositol 3-kinase (PI3K)- dependent manner. The kinase activity of Akt contributes to the control of cell transformation and oncogenic activity and Akt activation is frequently dysregulated in human cancer cells. Aktl has an N-terminus pleckstrin homology domain, a central kinase domain and a C terminus regulatory domain and contains two phosphorylation sites, Thr-308 in the kinase domain and Ser-473 in the regulatory domain. While phosphorylation of Thr-308 activates Aktl , full activation requires both Thr-308 and Ser-473 to be simultaneously phosphorylated.

SUMMARY OF THE INVENTION

This invention provides a method of increasing permissiveness of a cell to myxoma virus infection, comprising activation of endogenous Akt or transducing a cell with a vector that expresses exogenous Akt in the cell. When applied to cancer cells, this method increases the susceptibility of the cancer cell to infection by a myxoma virus and replication of myxoma virus in the cell.

This invention provides a method of treating a subject having cancer, comprising administering to the subject: (a) an activator of endogenous Akt or a vector that infects and expresses exogenous Akt in cancer cells in the subject; and (b) a myxoma virus, in a combined amount effective to treat the subject. This invention provides a method of treating a subject having cancer, comprising: (a) activation of Akt within cancer cells from the subject ex vivo with a medicament that activates endogenous Akt or by transducing the cells with a vector that expresses exogenous Akt in the cells (Akt-activated cells); (b) administering a myxoma virus to the Akt-activated cells; and (c) returning the Akt-activated cells to the subject. This invention provides a method of treating a subject having cancer, comprising: (a) determining in accordance with the method of claim 1 whether cancer cells from the subject have Akt activation, and if said cancer cells have Akt activation, then (b) treating the subject with a myxoma virus in an amount effective to treat the subject. These methods are useful for treating any mammalian subject having cancer, preferably a human subject.

This invention provides a method of identifying cells susceptible to infection with a myxoma virus, comprising assaying a test cell for Akt activation, wherein the activation of Akt in the cell indicates susceptibility to myxoma virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings in which:

Figure 1 shows representative western blots demonstrating the activation state of Akt, as measured based upon phosphorylation at Serine 473 and Threonine 308, in Type I, Type II, and Type III human cancer cells before and following infection with wild type myxoma (vMyxlac) virus and an M-T5 gene knockout myxoma (vMyxT5KO) virus, which renders this knockout virus host range defective. This figure also demonstrates the ability of MV to increase Akt phosphorylation at Serine 473 in a manner dependent upon the product of the M-T5 gene. Type III human cancer cells demonstrate little to no activation of Akt both before and after exposure to MV.

Figure 2 shows that Akt phosphorylation (activation) following MV infection is insensitive to pharmacological inhibitors of PI3K and Akt. The resistance to pharmacological inhibiton of Akt phosphorylation increased with the time of MV infection.

Figure 3 shows that M-T5 physically interacts with either exogenously introduced Akt (A) or endogenously expressed Akt (B).

Figure 4 demonstrates the necessity of Akt activation for MV infection and the formation of foci in MV infected human cancer cells. This figure also shows that Akt activation is required following infection with MV for the expression of the late viral gene, Serpl, without affecting the expression of the early viral gene, M-T7.

Figure 5 exhibits the relationship between endogenous levels of phosphorylated Akt and the relative permissiveness of human cancer cells to infection with either wild type (vMyxlac) of T5KO (vMyxT5K0) MV. Figure 6 demonstrates that both Type I and Type II human cancer cell lines require functionally active Akt signaling to be permissive to MV infection and foci formation. Exogenous introduction of dominant negative Akt inhibits myxoma infection and foci formation in Type I and Type II human cancer cells.

Figure 7 demonstrates the ability of MV to infect and replicate within Type I and Type II human cancer cells as seen by expression of both early and late gene products within these cells. While MV is able to enter Type III cells, there is no evidence of late myxoma gene product expression in these cells and thus they have also been termed, "Abortive".

Figure 8 shows that expression of constitutively active Akt in non-permissive human Type III cancer cells renders the cells susceptible to MV infection and replication.

DETAILED DESCRIPTION OF THE INVENTION

Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

In the method of this invention for increasing the susceptibility of a cancer cell to myxoma virus infection comprising activation of endogenous Akt within said cell, any medicament that results in an increase in endogenous Akt activation can be used. This activation of endogenous Akt can be a result of administering a medicament to cancer cells that specifically causes the activation of Akt (i.e. Lithium Chloride, Insulin-like Growth Factor 1) or administration of a medicament that specifically inhibits the ability of an Akt inactivator (i.e. Rapamycin), the end result of which is increased Akt activation within the cell. Endogenous Akt activation can be performed either ex vivo or in vivo.

Three mammalian Akt isoforms have been characterized and are identified as Aktl, Akt2, and Akt3. All three isoforms share the same structural organization with a conserved N- terminus pleckstrin homology domain, a central kinase domain and a C terminus regulatory domain. In the literature "Akt" sometimes refers to the Aktl isoform and sometimes refers to all isoforms of Akt. As used herein "Akt" refers to any and all forms of Akt and is not limited to Aktl.

In the method of this invention for increasing the susceptibility of a cancer cell to myxoma virus infection by transducing a cell with a vector that expresses exogenous Akt in the cell, any conventional type of vector can be used. Examples of suitable vectors include a plasmid or a virus. For in vivo transduction a virus is preferred, for example a myxoma virus. When a myxoma virus is to be administered as well, it is preferred that the myxoma virus itself also serve as the vector to introduce exogenous Akt into the cells. The transduction can be performed either ex vivo or in vivo. In vivo transduction can be performed in a human or in a non-human mammal.

Although the method of this invention can increase myxoma virus infection in all types of cells, it is most useful in cases where the cell has low levels of Akt activation prior to activation of endogenous Akt or expression of exogenous Akt within the cell. In a more specific embodiment the cell has substantially no Akt activation prior to activation of endogenous Akt or expression of exogenous Akt within the cell. As used herein "Akt activation" in a cell refers to cells having a low level of Akt activation or a high level of Akt activation, as those terms as defined herein. As used herein "low level(s) of Akt activation" means cells that are permissive to myxoma virus that is positive for M-T5 but not to M-T5 knockout myxoma virus. Such cells are also referred to herein as Type II cells. "Substantially no Akt activation" refers to cells that are non-permissive to both M-T5 positive myxoma virus and M-T5 knockout myxoma virus. Such cells are also referred to herein as Type III cells. The cells used in this method can be normal cells, but preferably are cancer cells, still more preferably human cancer cells. As used herein "high level(s) of Akt activation" means cells that are permissive for both M-T5 positive and M-T5 knockout myxoma viruses (also referred to herein as Type I cells). Alternatively, the activation level of a cell or group of cells can be ascertained by determining the phosphorylation level of Akt in the cells. Examples of cells that have high levels of Akt activation include normal cell lines RK- 13 (kidney, rabbit), BGMK (kidney, primate), HEK293 (kidney, human), and the cancer cell lines HOS (osteocaroma), Caki- 1 (renal cancer), and PC3 (prostate cancer). Examples of cells that have low levels of Akt activation include HCTl 16 (colon cancer), 786-0 (renal cancer), ACHN (renal cancer), SK-OV-3 (ovarian cancer) and U373 (glioma). Examples of cells that have substantially no Akt activation include MCF-7 (breast cancer), COLO205 (colon cancer), MDA-MB435 (breast cancer) and SK-MEL5 (melanoma).

The methods of this invention for treating a subject having cancer are useful for treating any subject. In a more specific embodiment of this invention the subject is a mammal, either a human or a non-human mammalian subject.

In the method of this invention for treating a subject having cancer, comprising: (a) administering to cancer cells from the subject ex vivo a medicament that increases endogenous Akt activation in the cells; or transducing cancer cells from the subject ex vivo with a vector that expresses exogenous Akt in the cells; (b) administering a myxoma virus to the transduced cells; and (c) returning the transduced cells to the subject, step (b) can be performed either before or after step (c).

The method of this invention for identifying cells susceptible to infection with a myxoma virus, is applicable to normal cells as well as cancer cells. In a specific embodiment the cancer cell is a human cancer cell.

A number of viruses have developed strategies to survive for long periods in the host and the PI3K-Akt signaling pathway, in particular, has attracted much interest due to its central role in the regulation of apoptotic inhibition. Activation of the PI3K- Akt signaling pathway is believed to contribute to increased cell survival, and seems to be a common strategy for many viruses to manipulate cell survival pathways until the virus life cycle is complete. Some viruses inhibit apoptosis, and others accelerate it, depending on the biological strategy of that particular virus. Infection with some viruses, such as respiratory syncytial virus (RSV), immediately induces activation of Akt through PDK-NFkB mediated antiapoptotic signaling at a very early stage of infection. In contrast, other viruses, such as hepatitis B, hepatitis C, and flaviviruses, encode gene products that directly induce PDK-Akt phosphorylation, which contributes to an antiapoptosis strategy. In this study, several human cancer cells that exhibit activated Akt with LY294002 and AKT inhibitor IV were treated, and after MV infection, no apoptosis was detected with or without the inhibitors. This finding demonstrates MV infection induced activation of Akt in a manner independent of PI3K activation and was resistant to inhibitors upstream of Akt in a time-dependent manner (Fig. 2). Also, inhibition of Akt activation by expression of DN- Akt effectively prevented productive MV infection. In contrast, human cytomegalovirus (HCMV) induces PBK activation throughout the course of infection of quiescent fibroblasts (26). HCMV infection caused immediate PI3K activation at 15-30 min after infection, and PI3K inhibitor could thus inhibit virus replication.

The M-T5 host range factor of MV does not share significant similarity with any other nonviral proteins, although it is related to other poxvirus host range proteins, many of which have been defined by their ability to mediate viral tropism. M-T5 possesses seven ankyrin- repeat domains within the N-terminal and central regions of the protein. The ankyrin repeat, a 33 -residue sequence motif, has been found in many proteins from viruses, bacteria, and eukaryotes are thought to mediate specific protein- protein interactions. Recently the E3 ligase complex member cullin was identified as a binding partner of M-T5 and demonstrated that the M-T5/cull complex functions to regulate cell cycle through p27 degradation pathway in MV-infected cells including human tumor cells. Here cellular Akt is identified as a second, additional binding partner of M-T5 that regulates Akt signaling and is an important tropism factor for MV infection in human cancer type II cells. In the present study, no M-T5 binding to either the p85 or pi 10 subunits of PI3K was detected. Because the induction of Akt by M- T5 is insensitive to PI3K/Akt inhibitors (Fig. 2), it is postulated that M-T5 operates directly in conjunction with Akt. Experiments are underway to explore the role of downstream targets of Akt. Although it cannot yet be deduced whether M-T5 alters availability of Akt to kinases or phosphatases, it is noted that the activation of Ser-473 in the presence of M-T5 is particularly dramatic (Fig. IB). Thus, M-T5 is a good candidate to regulate Akt activation via either the Akt kinase, mTOR, or the Akt phosphatase, PHLPP.

MV has been shown to exhibit tropism for many human cancer cells and to be a potent oncolytic therapeutic for human gliomas in a murine xenograft model. The results in this study suggest that Akt manipulation may allow the oncolytic capacity of this virus to extend to an even broader spectrum of human cancer cells. EXAMPLES

Materials and Methods

Cell Culture. Established cell lines used in this study included rabbit kidney (RKl 3) cells, baby green monkey kidney (BGMK) cells, and human embryonic kidney (HEK) 293 epithelial cells. Human cancer cells including human osteosarcoma (HOS) cells, glioma

(U373) cells human renal cancer lines (786-0, Caki-1, ACHN), prostate cancer (DU145, PC3) cells, breast cancer (MDA-MB435, MCF-7, T47D) cells, colon cancer (HCTl 16, COLO205) cells, ovarian cancer (SK-OV3, OVC AR5) cells, and melanoma (SK-MEL5) cells were obtained from the NCI-60 reference collection. All cells were grown in DMEM supplemented with 10% FBS (Sigma), 100 units of penicillin per ml, and 100 μg of streptomycin per ml (Invitrogen).

Viruses and Infections. The viruses used in this study included vMyxlac, vMyxT5KO, and vMyxgfp. All viruses were propagated and titrated by focus formation on BGMK cells.

Antibodies and Reagents. Generation of polyclonal anti M-T5 anti-serum, mouse monoclonal antibody of anti-Serp 1 , and rabbit polyclonal antibody of T-7 have been described. Rabbit polyclonal phospho-Akt (Thr-308), mouse monoclonal phospho-Akt (Ser-473) (587Fl 1) antibody, and polyclonal Akt antibody that detects total levels of endogenous Aktl, Akt2, and Akt3 proteins were obtained from Cell Signaling Technology. Mouse monoclonal antibody against the epitope of haemagglutinin (HA) protein of human influenza virus (clone 12CA5) (Roche) and a peptide epitope directed against the 11-aa gene (T7-Tag; Novagen), a goat polyclonal antibody directed against the C terminus of actin (C-1 1) of human origin

(Santa Cruz Biotechnology), and horseradish peroxidase-coupled goat antimouse or goat anti- rabbit secondary antibodies were obtained from Jackson ImmunoResearch. PI3K-AKT kinase inhibitors (LY294002 and AKT inhibitor IV) and [_-32P]ATP were purchased from Calbiochem and PerkinElmer Life Sciences, respectively. Histone H2B was used as exogenous substrate (Roche Applied Science).

Plasmids. M-T5 was amplified from MV genomic DNA by PCR using primers, which incorporated T7 Tag sequences into the expression vector pcDNA3 (pcDNA3T7-MT5). The CMV-based expression constructs encoding wild-typeHA-Aktl, constitutively active HA- Myr-Aktl have been described. Dominant negative, kinase-dead, mutant HA-DN-Aktl cassette was prepared by point mutation method. HA-tagged PBK, including both subunits (pi 10 and p85) have been described. Cellular Bad was amplified by RT PCR from HEK293 cells and cloned into HA-pcDNA3 vector (HA-Bad).

Immunoprecipation and Immunoblotting. HEK293 cells were cotransfected by the calcium phosphate method (Clontech) for 24 h with plasmids HA-Aktl, HA-tagged PI3K, including both subunits, (pi 10 and p85), and HA-Bad together with pcDNA3T7-MT5 or the vector alone, respectively. Immunoprecipitations were performed as described. Immunocomplexes were resolved by SDS-PAGE and then analyzed by Western blotting with appropriate antibodies. Immunoreactive proteins were detected by chemiluminescence (PerkinElmer). Loading of equal amounts of protein (50 μg per lane) from each sample was confirmed by detection of the housekeeping gene actin.

Transfection, PI3K, and Akt Kinase Inhibitor Treatments. Cells were seeded in six-well plates at a density of 5 X 10⁵ cells per well in complete growth medium with 10% FCS. Transfections were performed with LipofectAMINE 2000 (Invitrogen) in accordance with the manufacturer's instructions. 786-0 or MDAMB435 cells were transfected withHA-DN-Aktl , HA-Myr-Aktl plasmid, or pcDNA3 alone (4 μg). Transfection efficiency was determined by expression of a GFP vector and found to be 90-95% efficient. For inhibition experiments, cells were serumstarved overnight and treated with PI3K and Akt kinase inhibitors LY29004 (50 μM) or Akt kinase IV (10 μM) for 1 h, then infected with vMyxlac (MOI of 5) for 1 h. After removal of the inoculum, the same inhibitor was added to cells and grown in complete growth medium supplemented with 10% FBS. The cells were collected at various time points. The lysate was used for detection with appropriate antibodies.

In Vitro Kinase Assay. Protein kinase assays were performed as described. The proteins were separated on SDS-PAGE gels. Each experiment was repeated three times, and the relative amounts of incorporated radioactivity were determined by autoradiography. Measurements of Akt Inhibition or Activation After Virus Infection. Human renal cancer cells (786-0) were transfected with HADN-Akt or the empty vector pcDNA3 vector for 12 h. Cells were then infected with vMyxgfp virus for 24 and 48 h at several MOIs. The infected supernatants were used for detection of the expression of M-T7 (early MV protein) and Serpl (late MV protein) by immunoblotting. The fluorescent foci were enumerated under fluorescent microscopy at 24 and 48 hpi. Cells were harvested and fixed with 1% formaldehyde, and fluorescent green cell numbers were counted by flow cytometry. To measure the effect of activation of Akt kinase on virus infection, MDAMB435 cells were transfected with constitutively active Akt (HA-Myr-Akt) (4 μg) or vector alone for 12 h and then infected with vMyxgfp for 24, 48, and 72 h at MOI 0.01, 0.1, and 1, respectively. Foci numbers were counted under a Leica fluorescent microscope as a measure of virus spread.

Results

Endogenous Levels of Phosphorylated Akt of Human Cancer Cells Is an Indicator of Susceptibility to MV Infection.

Human tumor cells were previously screened for either wild-type [vMyxlac (expressing β- gal) and vMyxgfp (expressing EGFP)] or a host range defective (vMyxT5KO) MV infection. Two phenomena regarding MV infection of human cancer cells lines have been demonstrated. First, human tumor cell lines exhibited one of three phenotypes after infection. Some tumor lines could support a productive infection by both wild-type or host range defective MV. A second group generated abortive, or nonproductive infection regardless of whether the infecting virus was wild type or the host range mutant. Finally, a proportion of tumour lines were described as restrictive. These cancer cells supported wild-type virus infection but were resistant to infection by the host range mutant (vMyxT5KO). The second phenomenon described was that expression of one specific MV host range gene, M-T5, is necessary for infection of restrictive cancer cell lines but dispensable for infection of supportive tumor cell lines. The cancer origin or tissue type of the tumor cell line could not explain this observation. However, when a survey was made of possible oncogenic proteins that might differ among the lines it was noticed that human cancer cell lines could be grouped into one of three types based on the levels of endogenous phosphorylated Akt, and that this grouping correlated with the permissiveness to MV infection (Table 1).

All screened tumor lines expressed detectable levels of Akt but exhibited either high or low levels of endogenous phosphorylated Akt, or did not express any detectable phospho-Akt (Table 1). High levels of endogenous phospho-Akt were associated with type I cells, low or very low levels of phospho-Akt were detected in type II cells, and type III cells did not express any detectable endogenous phospho-Akt (Table 1 and Fig. 5/4, which is published as supporting information on the PNAS web site). Significantly, this classification matched perfectly with the ability of MV or vMyxT5KO to infect each specific cell line. Type I cells were infected by either MV or vMyxT5K0, type II cells supported MV infection but were not permissive for vMyxT5KO infection, and type III cells did not support productive infection by either virus (Table 1).

As an example of type I cells, endogenous phospho-Akt (both P- Akt Thr-308 and P- Akt Ser- 473) is highly elevated in HOS cells (Fig. XA, lanes 1 and 7) and infection with either vMyxlac or vMyxT5KO, both of which result in a productive infection (Fig. 5B), does not result in any further elevated levels of phosphorylated Akt (Fig. XA and Table 1). This finding suggests that certain cancer cell lines have sufficiently high levels of constitutively activated phospho-Akt at both Ser-473 and Thr-308 so as to support productive MV infection regardless of the expression of M-T5 and that infection does not induce an increase in the measurable levels of activated Akt.

In contrast, endogenous levels of Ser-473 phosphorylated Akt in 786-0 cells (type II) was very low, whereas Thr-308 P-Akt was detectably phosphorylated (Fig. XB, lanes 1 and 8). However, infection of 786-0 cells with vMyxlac dramatically induced Akt phosphorylation at P-Ser-473 and increased the level of P-Thr-308, but when 786-0 cells were infected with vMyxT5KO, the endogenous levels of phospho-Akt were relatively unchanged at both sites (Fig. XB). These results were reproducible in other type II tumor cell lines, including HCTl 16, ACHN, U373, and SK-OV-3 (Table 1), but total Akt protein levels were unchanged in any cell after vMyxlac and vMyxT5KO infection (Fig. 1 and data not shown). This result suggests that M-T5 expression can activate Akt under conditions of MV infection in a cell line that is abortive for vMyxT5KO infection. Therefore, it was postulated that activation of Akt is necessary to support productive MV infection in human tumor cell lines and that M-T5 has a role in activating Akt during MV infection (Fig. 5Q. This finding is consistent with the observation that MV failed to infect type II cells, such as 786-0, in the absence of M-T5 expression.

Finally, infection of type III cells such as MDA-MB435 cells, a breast cancer cell line that is abortive for MV infection, exhibits undetectable endogenous phosphorylated Akt levels (Fig. 1 C, lanes 1 and 7) and MV infection does not induce any measurable increased levels of activated Akt at either Ser-473 or Thr-308 (Fig. IQ. Nevertheless, the total Akt protein level was unchanged, similar to that observed for 786-0 and HOS cells. MDA-MB435 cells do not support productive infection by either vMyxlac or vMyxT5KO (Fig. 5D), and it is concluded that when expression of M-T5 is unable to induce the activation of Akt, the cancer cells remains nonpermissive (Table 1). Thus, the level of phosphorylated Akt, whether endogenous or virus-induced, is predictive for which cancer cell lines will support productive MV infection (Table 1).

To confirm that MV infection induced the activation of Akt kinase activity in permissive cells, an in vitro kinase assay was performed. Type II 786-0 cells were mock-infected or infected with either vMyxlac or vMyxT5K0, collected at 4 h postinfection (hpi), and immunoprecipitated with anti-Akt antibody. The immunoprecipitates were subjected to an in vitro kinase assay using histone H2B as the substrate (Fig. ID). This result indicates that Akt kinase activity can be increased in 786-0 cells by infection with vMyxlac, but not with vMyxT5KO, which is consistent with immunoblotting analysis in the same cell line. This result is reproducible in other type II cells such as ACHN, SK-OV3, and U373 (data not shown). Taken together, these data indicate that phosphorylation of Akt is predictive for productive MV infection in specific human cancer cells.

Table 1. Akt Activation Correlates With Permissiveness for Myxoma Virus o

O O Endogenous Endogenous p-Akt p-Akt Permissive Permissive in Cell Line Cell Origin Akt p-Akt (vMyxlac) (vMyxT5KO) (vMyxlac) (vMyxT5KO)

Controls RK-13 Kidney (rabbit) + high + + + +

H U Kidney

BGMK (primate) + high + + + +

HEK293 Kidney human) + high + + + +

Type I HOS Osteocarcoma + high + + + +

Caki-1 Renal Cancer + high + + + +

Prostate

PC3 Cancer + high + + + +

Type II HCTl 16 Colon Cancer + low + - + -

Renal

786-0 Cancer + low + +

Renal

ACHN Cancer + low + - + -

SK-OV-3 Ovarian Cancer + low + - + -

U373 Glioma + low + - + -

Type III MCF-7 Breast Cancer + None - - - -

Colon

COLO205 Cancer + None - - -

MDA-

MB435 Breast Cancer + None - - - -

SK-MEL5 Melanoma + None - - - -

90 Note: + indicates that endogenous Akt protein or phospho-Akt are detectable by immunoblotting: or that cells were permissive to MV infection; - represents undetectable endogenous phosphorylated Akt Ser473 or Thr308, or that cells were non-permissive to infection by MV. o o

Akt Phosphorylation Is Resistant to PBK/ Akt Drug Inhibitors After MV Infection.

Type II cells exhibited the largest change in activated levels of Akt after MV infection (see Fig. IB); therefore, the human renal cancer cells 786-0 were used as a representative type II cell to test whether drugs that inhibit PI3K/Akt could block Akt phosphorylation activated by infection of MV. LY294002 is a potent and specific inhibitor of PBK and blocks downstream pathways of PBK, including Akt activation. In contrast, Akt inhibitor IV was designed to target an ATP-binding site of a kinase immediately upstream of Akt, but downstream of PBK, thereby specifically inhibiting Akt phosphorylation. The most likely candidate for inhibition is PDKl ; however, this remains to be confirmed conclusively. Human renal cancer (786-0) cells were pretreated with LY294002 (50 μM) or Akt inhibitor IV (10 μM) for 1 h and then infected with vMyxlac (multiplicity of infection, MOI, of 5). At various times after infection, vMyxlac-infected cells were collected and whole cell lysates were prepared. The results show that treatment of uninfected 786-0 cells with LY294002 or Akt inhibitor IV blocked phosphorylation of Akt (Fig. 2, lanes 1-3). However, treatment with the inhibitors did not appear to significantly inhibit the increased phosphorylation of Akt at Ser-473 or Thr- 308 after MV infection (Fig. 2). There was some reduction early in the infection (0-4 hpi) when compared to untreated control samples (Fig. 2 Upper). This finding suggests that activation of Akt depends on MV infection and is under the control of the expression of M- T5 protein, and when either PBK or downstream activation of Akt are blocked by LY294002 or Akt inhibitor IV (Fig. 2 Lower), then wild-type MV is still robustly capable of activating Akt. Therefore, it is concluded that specific Akt inhibitors are unable to significantly block Akt activation after infection by vMyxlac in time-dependent manner, and that M-T5 acts downstream of these inhibitors.

Inhibition of the PBK pathway with LY294002 or Akt inhibitor IV did not induce cell death as measured by trypan blue staining when type II cells were infected with vMyxlac. In particular, when levels of phospho-Bad, caspase 3, and poly(ADP ribose polymerase) (PARP) were tested, they were also unchanged. Together with data shown in Fig. 1 , it is proposed that M-T5 plays a direct role in activation of Akt phosphorylation after infection in type II cells. Then tests were done to determine whether M-T5 activates Akt through protein- protein interaction. M-T5 Interacts with Akt Under Both Transfection and Virus Infection Conditions.

To confirm any physical association between M-T5 and Akt, HEK293 cells were cotransfected with tagged M-T5 (pcDNA3T7-MT5) and tagged Akt (pcDNA3HA-Aktl). Twenty-four hours after transfection, immunpcomplexes were precipitated with anti-T7 to pull down T7-MT5 fusions, and any complexes that were immunoblotted with anti- hemagglutinin (HA) antibody. When reciprocal immunoprecipitations were performed, T7 tagged M-T5 was detected in the HA- Akt immunoprecipitates (Fig. IA Upper), and HA- Akt was coimmunoprecipitated by anti-T7 antibody (Fig. ?>A Lower). These reciprocal coimmunoprecipitations confirmed that M-T5 forms complexes with Akt (Fig. 3). To confirm the ability of M-T5 to also interact with endogenous untagged human Akt after MV infection, type II 786-0 cells infected with vMyxlac at an MOI of 5 were examined. Twelve hpi, the cells were collected and lysed, and immune complexes were precipitated with an anti-Akt antibody. Immunoblotting with anti-T5 antiserum indicated that expression of T5 protein was able to coprecipitate with endogenous Akt from MV-infected human cancer cells in vivo (Fig. W Upper).

Also, additional coimmunoprecipitations from cells transfected with plasmids that express both M-T5 and HA-tagged PI3K, including both subunits (pi 10 and p85) or HA-Bad, as well as by virus infected cell coimmunoprecipitation, confirmed that M-T5 was unable to bind to exogenous or endogenous p85 or pi 10 or the downstream regulator Bad under either transfection or infection conditions. Therefore, these data suggest that M-T5 is able to specifically interact with Akt and that this interaction is a requirement for enhancement of Akt activation.

Activation of Akt Is Required for MV Infection.

As shown in Fig. 1, Akt becomes activated following vMyxlac infection in type II cells. I has also been demonstrated that the Akt specific inhibitor IV, which acts immediately upstream of Akt, does not block Akt activation after MV infection. Therefore, 786-0 cells were transfected with a dominant negative Akt cassette (HA-DN-Akt) for 12 h and then infected with MV (vMyxgfp) at three MOIs to measure any inhibitory effect of Akt activation by DN- Akt expression upon MV infection. It was observed that DN- Akt can dramatically inhibit MV infection of 786-0 cells (Fig. 4 and Fig. 6, which is published as supporting information on the PNAS web site). Transfection of DN-Akt followed by infection of vMyxgfp caused a significant decrease in the number of GFP expressing cells at 24 and 48 h at various MOIs (Figs. AA and 6A). 786-0 cells transfected with DN-Akt and then infected with vMyxgfp for 24 or 48 h (MOI of 1) produced significantly fewer foci than the control transfections/infections (Fig. 4A) and reduced viral titres (Fig. 6B). Similar results were obtained with other type II cells (ACHN and SK-OV3) that were transfected with DN-Akt and then infected with MV. These data demonstrate that inhibition of Akt signaling interferes with MV infection and spread. Therefore, Akt is a determinant of productive MV infection in type II human cancer cells and likely also explains the M-T5 independence of MV replication in type I cells, in which Akt is already constitutively activated. However, if Akt activation is blocked in type I cells by transfection of DN-Akt, then MV replication is also reduced, thus confirming that activated Akt is necessary for productive MV infection (Fig. 6C).

Inhibition of Akt Signaling Significantly Reduces MV Replication at the Level of Late Viral Gene Expression.

To further confirm the influence of direct inhibition of Akt on MV replication, the expression levels of representative MV early (M-T7) and late (Serp-1) viral genes after infection of 786- 0 cells with vMyxgfp (MOI of 1) were assessed. The efficiency of transfection for 786-0 cells was 90-95%, which was monitored by GFP vector transfection. 786-0 cells were transfected with DN-Akt or control vector for 12 h and then infected with vMyxgfp. Transfection of DN- Akt did not affect the expression of the early gene, M-T7; however, there was a significant decrease in expression of the viral late gene, Serpl (Fig. 4B). These data indicate that inhibition of Akt activation has little affect on early viral gene expression but reduced late viral gene expression, suggesting that inhibition of Akt signaling resulted in a late stage block to MV replication. This observation is consistent with the data obtained in Figs. 4A and 6 and, taken together, indicate that activation of Akt is essential for completing the full MV replication cycle and that M-T5 is critical through its interaction with Akt. These findings were also reproduced in other type II cells (ACHN and SK-OV3). Thus, if Akt activation is blocked or M-T5 expression is ablated, then MV cannot productively infect type II cancer cells. Transient Expression of Constitutively Active Aktl Facilitates MV Infection of Nonpermissive Cancer Cells.

Why wild-type MV is unable to induce activation of Akt after infection of type III cells. A cellular block to virus entry and early gene expression might explain the observed failure to replicate. Alternatively, a dysregulation of Akt activation by M-T5 might also explain this apparent abort of MV infection of type III cells. To test these alternative explanations, each cell type was infected with vMyxlac and then assessed for viral gene expression by immunofluorescence (Fig. 7). Type I and II cells exhibited similar patterns of punctate cytoplasmic M-T5 staining; however, there was either decreased M-T5 expression or stability, or possibly aberrant localization in the type III cells, despite the fact that a control early viral protein (M-T7) was expressed normally. This finding suggested that the failure of MV infection in type III was not due to a block to virus entry or early gene expression. It was then reasoned that if phosphorylation of Akt was necessary for MV replication in cells that exhibit very low activated Akt levels (type II cells, Table 1), then expression of a constitutively active Akt cassette (HA-Myr-Akt) in cells that are nonpermissive to infection, and do not exhibit detectable levels of endogenous phosphorylated Akt levels (i.e., type III cells), might convert them from nonpermissive to permissive for MV infection. The highly transfectable human breast cancer cells MDA-MB435 was selected as an example of nonpermissive type III cells (Table 1) to test the hypothesis that constitutive expression of activated Akt could rescue the ability of MV to infect resistant cancer cell lines. A constitutively active Akt expression construct (HA-Myr-Akt) or control vector (pcDNA3) were transfected into MDA-MB435 cells, and 12 hpi they were infected with vMyxgfp at an MOI of 0.01, 0.1, or 1.0. Classic MV foci expressing GFP at 48 hpi from cells expressing

Myr-Akt were observed, which were not detected in control cells that were infected only with MV (Fig. SA). Foci were enumerated under the florescent microscope and the results indicated that transfection of activated Akt followed by infection with MV dramatically increased levels of MV replication (Fig. SB). Similar results were also obtained with another noninfectable type III cancer line (MCF-7). These data indicate that the expression of constitutively active Akt is able to rescue MV infectivity in type III nonpermissive human cancer cells. In contrast, when M-T5 was transfected into type III cells, MV infection (Fig. SC) could not be rescued, suggesting that the block to a productive infection lies in either the inability of M-T5 to bind and activate Akt, or a dysregulation of the Akt signaling pathway itself. These results indicate that activated Akt is necessary for MV infection of cancer cells and nonpermissive cells can be rendered permissive by the ectopic expression of constitutively active Akt.

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Claims

CLAIMSWhat is claimed is:

1. A method of identifying cells susceptible to infection with a myxoma virus, comprising assaying a test cell for Akt activation, wherein Akt activation in the cell indicates susceptibility to myxoma virus infection.

2. The method of claim 1, wherein the cell has a high level of Akt activation.

3. A method of increasing Akt activation in a cell, comprising administering to a cell a medicament that increases endogenous Akt activation.

4. A method of increasing Akt activation in a cell, comprising transducing an Akt- containing cell with a vector that expresses exogenous Akt in the cell.

5. The method of claim 4, wherein the vector is a plasmid.

6. The method of claim 4, wherein the vector is a virus.

7. The method of claim 6, wherein the virus vector is a myxoma virus.

8. The method of claim 4, wherein the transduction is performed ex vivo.

9. The method of claim 4, wherein the transduction is performed in vivo.

10. The method of claim 9, wherein the transduction is performed in a human.

1 1. The method of claim 3 or 4, wherein the cell has low levels of Akt activation prior to the administration or transduction.

12. The method of claim 3 or 4, wherein the cell has substantially no Akt activation prior to the administration or transduction.

13. The method of any one of claims 1 to 12, wherein the cell is a cancer cell.

14. The method of claim 13, wherein the cancer cell is a human cancer cell.

15. A method of increasing the susceptibility of a cancer cell to myxoma virus infection and replication, comprising the method of any one of claims 3 to 12, wherein the cell is cancer cell.

16. The method of claim 15, wherein the cancer cell is a human cancer cell.

17. A method of treating a subject having cancer, comprising administering to the subject: (a) a medicament that increases endogenous Akt activation; or a vector that infects and expresses exogenous Akt in cancer cells in the subject; and (b) a myxoma virus, in a combined amount effective to treat the subject.

18. The method of claim 17, wherein the subject is a human.

19. A method of treating a subject having cancer, comprising: (a) administering to cancer cells from the subject ex vivo a medicament that increases endogenous Akt activation in the cells; or transducing cancer cells from the subject ex vivo with a vector that expresses exogenous Akt in the cells; (b) administering a myxoma virus to the transduced cells; and (c) returning the transduced cells to the subject.

20. The method of claim 19, wherein step (b) is performed before step (c).

21. The method of claim 19, wherein step (c) is performed before step (b).

22. A method of treating a subject having cancer, comprising:

(a) determining in accordance with the method of claim 1 whether cancer cells from the subject have Akt activation, and if said cancer cells have Akt activation, then (b) treating the subject with a myxoma virus in an amount effective to treat the subject.