WO2013012648A1

WO2013012648A1 - Gdf15 in diagnostic and therapeutic applications

Info

Publication number: WO2013012648A1
Application number: PCT/US2012/046351
Authority: WO
Inventors: Rita NAHTA
Original assignee: Emory University
Priority date: 2011-07-15
Filing date: 2012-07-12
Publication date: 2013-01-24

Abstract

The disclosure relates to compositions and methods for diagnostic and therapeutic purposes. In certain embodiments, the disclosure relates to pharmaceutical compositions and methods that interfere with GDF15 signaling or suppress GDF15 expression. In other embodiments, the disclosure relates to methods of determining therapeutic treatments after analyzing GDF15 expression.

Description

GDF15 IN DIAGNOSTIC AND THERAPEUTIC APPLICATIONS

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grant K01CA118174 awarded by the NIH. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application 61/508,335 filed July 15, 2011, hereby incorporated by reference in its entirety.

FIELD

BACKGROUND

Trastuzumab (Herceptin) is a recombinant humanized monoclonal antibody directed to the HER2 extracellular domain. HER2 is a member of the ErbB family of receptor tyrosine kinases (RTKs). Breast cancers that over-express HER2 carry a particularly poor prognosis and comprise approximately 20% of all metastatic breast cancer cases. The current standard of care for this subtype of breast cancer is a regiment that contains trastuzumab. Primary and acquired resistance to trastuzumab occurs in the majority of patients. The general strategy in cancers that are non-responsive to

trastuzumab is to begin treatment with the dual EGFR/HER2 kinase inhibitor lapatinib. In trastuzumab-pre-treated cancers, clinical trial data indicates that the response rates to single-agent lapatinib and lapatinib plus capecitabine were still only 14% and 24% effective, respectively. See Cameron et al. (2008), Breast Cancer Res Treat 112: 533-543. Hence, more effective treatment options are needed to improve survival of patients with HER2-overexpressing breast cancer.

Published reports have implicated increased phosphatidylinositol-3 kinase (PI3K) signaling as a potential mechanism of trastuzumab resistance. The tumor suppressor phosphatase and tensin homolog (PTEN) is a phosphatase that counteracts the

phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 2004;6: 117-2. Pharmacologic inhibition of PI3K improves trastuzumab sensitivity in cells that have acquired resistance. See Ozbay et al., (2010), Cancer Chemother Pharmacol 65: 697-706. Growth differentiation factor 15 (GDF15) has been associated clinically with disease progression and resistance to chemotherapy in breast, prostate, ovarian, and colorectal cancer. See Brown et al, (2003), Clin Cancer Res 9: 2642-2650. Park et al, (2010) BMB reports, 43(2): 91-96, disclose that GDF15 transactivates ErbB family receptors via the activation of Src in SK-BR-3 human breast cancer cells. The preceding is not an admission that any of the references cited herein are prior art.

SUMMARY

In certain embodiments, the disclosure relates to methods of determining a chemotherapy regiment comprising assaying a sample from a subject diagnosed with a cancer for elevated GDF15 expression and correlating elevated GDF15 expression in the sample with a resistance to a chemotherapy comprising a HER2 antibody. In a typical embodiment, the HER2 antibody is trastuzumab. In certain embodiments, the subject is diagnosed with breast cancer. In certain embodiments, the subject is diagnosed to over- express HER2. In certain embodiments, the subject was previously administered a HER2 antibody. In certain embodiments, GDF15 measurements are recorded, e.g., in an electronic format on a computer. Typically, the methods comprise the step of reporting GDF15 expression measurement to the subject or a medical professional or representative thereof.

In certain embodiments, the disclosure relates to methods of treating cancer comprising administering a pharmaceutical composition comprising an HER2 antibody in combination with an agent that down regulates extracellular mediated GDF15 effects to a subject diagnosed to over-express HER2. Typically, the agent is an antibody or aptamers of GDF15, PI3K, TGF beta, Sire, or mTOR. The agent may be any GDF15 inhibitor, a PI3K inhibitor, TGF beta receptor inhibitor, Src inhibitor, or mTOR inhibitor. The agent may be a nucleic acid interrupts expression of GDF15 transcription, a siR A of GDF15 or a recombinant vector that encodes a nucleic acid interrupts expression of GDF15 transcription.

In certain embodiments, the disclosure relates to pharmaceutical compositions comprising an antibody of GDF15 optionally comprising a second active ingredient wherein the second active ingredient may be a PI3K inhibitor, TGF beta receptor inhibitor, Src inhibitor mTOR inhibitor, or an HER2 antibody.

In certain embodiments, the disclosure relates to methods of treating cancer comprising administering a pharmaceutical composition comprising an antibody of GDF15 to a subject diagnosed with a HER2 over expressing tumor. Optionally the antibody of GDF15 is administered in combination with a second active ingredient such as a PI3K inhibitor, TGF beta receptor inhibitor, Src inhibitor, mTOR inhibitor, or an HER2 antibody.

In certain embodiments, the disclosure relates to methods of determining a chemotherapy treatment comprising assaying a sample from a subject diagnosed with cancer for elevated GDF15 expression, determining that GDF15 expression in the sample is elevated, and administering an agent that down regulates extracellular mediated GDF15 effects.

In some embodiments, the disclosure relates to pharmaceutical compositions comprising an antibody or aptamer of GDF15, or a nucleic acid that interrupts GDF15 transcription.

In some embodiments, the disclosure relates to methods of treating cancer comprising administering a pharmaceutical composition comprising an antibody, aptamer, or nucleic acid that interrupts GDF15 transcription to a subject diagnosed with cancer. In certain embodiments, the method further comprises the step of administering a second chemotherapeutic agent. In certain embodiments, the second agent is a HER2 antibody.

In some embodiments, the disclosure relates to pharmaceutical compositions comprising a nucleic acid that interrupts expression of GDF15 transcription. In certain embodiments, the nucleic acid is a siRNA of GDF15. In certain embodiments, the nucleic acid is a siRNA of p38.

In some embodiments, the disclosure relates to the use of antibodies, aptamers, or siNA that interfere with GDF15 signaling or reduce GDF15 expression for the production of a medicament useful for the treatment of cancer. In certain embodiments, the assaying comprises the steps of measuring GDF15 in the sample, providing a detected amount of GDF15, and comparing the detected amount of GDF15 to an amount of GDF15 typically found in a sample of a normal subject, e.g., one responsive to a HER2 antibody therapy. In certain embodiments, the assaying comprises the steps of detecting GDF15 gene amplification in the sample, and comparing the detected amount with an amount typically found in a sample of a normal subject, e.g., a subject responsive to a HER2 antibody therapy. In certain embodiments, the assaying comprises the steps of detecting expression of GDF15 mRNA in the sample, and comparing the detected amount of GDF15 mRNA with an amount of GDF15 mRNA typically found in a sample of a normal subject, e.g., a subject responsive to a HER2 antibody therapy. In certain embodiments, the assaying comprises sequencing the mRNA or combining the sample with oligonucleotides that hybridize to GDF15 mRNA or GDF gene. In certain embodiments, assaying comprises moving the sample through a separation medium and detecting GDF 15 or GDF 15 mRNA. In certain embodiments, GDF15 or GDF15 nucleic acid expression levels are more than 2, 3, 4, 5, 10, 20, 50, or 100 times normal.

In certain embodiments, the disclosure relates to diagnosing GDF 15 over- expression with a nucleic acid probe for GDF 15 gene expression or mRNA expression that is the complement of GDF15 mRNA (SEQ ID NO: 2), e.g., SEQ ID NO: 10,

TGAGGTCTAAGGCTCTC AA CGCCTTTGCGA or SEQ ID NO : 11 , GCGGTCT TCACGCCGACCCTAG GCCGCCGGT

GGACGTGGACGCATAGAGAGCCCGGCGGG. These sequences may be conjugated to an appropriate maker such as a fluorescent dye.

In certain embodiments, the disclosure relates to diagnosing GDF 15 over- expression using antibodies with epitopes to SEQ ID NO: 12, ARNGDHCPLGPR; SEQ

ID NO: 13 LEDLGWADWVLSPR; SEQ ID NO: 14, NMHAQIKTSLHRLKPDTV; and

SEQ ID NO: 15, QKTDTGVSL. These sequences may be conjugated to an appropriate maker such as a fluorescent dye.

In certain embodiments, the disclosure relates to probes to detect for GDF 15 over- expression that detect allelic variants. Typically, the nucleotide sequence of a nucleic acid probe will be replaced to appropriately hybridize with the proper allelic variant.

Antibodies or aptamers can be prepared to recognize a resultant change in the amino acid sequence. In certain embodiments, the disclosure relates to collecting a sample of cells from the breast of a woman diagnosed with breast cancer detecting over-expression of HER2 and detecting over-expression of GDF15. In certain embodiments, the cells are obtained from a tumor.

In certain embodiments, the assaying comprises, combining the sample and markers with affinity for a GDF15; measuring markers in the marker bound sample; and comparing the markers in the marker bound sample to those typically found with non- elevated GDF15 expression. In certain embodiments, the markers are antibodies or aptamers for GDF15 protein. In certain embodiments, the markers are fluorescent.

In some embodiments, the disclosure relates to methods of determining a chemotherapy treatment comprising assaying a sample from a subject diagnosed with cancer for elevated GDF15 expression, determining that GDF15 expression in the sample is not elevated, and administering chemotherapeutic regiment comprising a HER2 antibody therapy.

In some embodiment, the disclosure relates to methods of determining a chemotherapy treatment comprising assaying a sample from a subject diagnosed with cancer for elevated GDF15 expression, determining that GDF15 expression in the sample is elevated, and administering an PI3K inhibitor and/or an mTOR inhibitor. In certain embodiments, the method further comprises the step of and administering

chemotherapeutic regiment comprising a HER2 antibody therapy

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows data suggesting HER2-overexpressing trastuzumab-resistant breast cancer cells express increased levels of GDF15. (A) Total RNA was extracted from BT474 parental (Par), resistant clone 2 (c2) and clone 3 (c3), and SKBR3 parental (Par) and resistant clone 3 (c3) cells. RNA was converted to cDNA and analyzed by real-time PCR for GDF15 transcript level. Results are reported as fold increase in GDF15 transcript level versus parental counterpart. Values were normalized to RPLPO housekeeping ribosomal gene transcript levels as internal control. Each sample was run in triplicate per experiment, and 3 independent experiments were performed on separate occasions to ensure reproducibility. Values represent the average of the 3 separate experiments; error bars represent standard error between the triplicate experiments. P-values were calculated by student's t-test for each resistant line versus the parental line; *p<0.05, **p<0.005. GDF15 levels were also determined by (B) ELISA using total protein lysates excluding media, or (C) ELISA using cell culture media only. Error bars represent standard deviation between triplicates. P-values were calculated by student's t-test for each resistant line versus the parental line; *p<0.05, **p<0.005. Results were confirmed on three separate occasions. Acquired resistant cells showed statistically significant increase in both the endogenous and secreted forms of GDF15 versus sensitive lines. (D) Secreted GDF15 was measured by ELISA in the cell culture media of HCC1419, HCC1954, MDA-MB-453, and MDA-MB-361 cells, which have primary resistance to trastuzumab, versus BT474 and SKBR3 trastuzumab-sensitive cells. Error bars represent standard deviation between triplicates. P-values were calculated by student's t-test for each resistant line versus BT474; **p<0.005. Results were confirmed on three separate occasions. Cells with primary trastuzumab resistance showed higher levels of secreted GDF15 versus trastuzumab-sensitive cells.

Figure 2 shows data suggesting increased GDF15 expression reduces trastuzumab- mediated growth inhibition. (A) SKBR3 and BT474 cells were treated with vehicle control (C), 20 μg/mL trastuzumab (T), or pre-treated with 10 ng/mL GDF15 for 48h followed by 20 μg/mL trastuzumab plus 10 ng/mL GDF15 for an additional 72 h (G+T); media plus treatments were changed each day. Cells were counted by trypan blue exclusion; cell count is shown as a percentage of the control group per cell line. Each sample was run in triplicate cultures per experiment; experiments were performed on 3 independent occasions for reproducibility. Error bars represent standard error between the 3

independent experiments. P-values were determined by student's t-test for GDF15 plus trastuzumab (G+T) group versus trastuzumab alone (T); *p<0.05. Stimulation with recombinant human GDF15 significantly reduced trastuzumab-mediated growth inhibition. (B) SKBR3 and BT474 clones stably transfected with pCMV empty vector control or pCMV-myc-GDF15 were analyzed by real-time PCR for GDF15 transcript level. Results are reported as fold increase in GDF15 transcript level versus stable control clone per line. Values were normalized to RPLPO housekeeping ribosomal gene transcript levels as internal control. Error bars represent standard deviation between triplicates. (C) Stable clones were treated with 20 μg/mL trastuzumab (Tr) for 72h. Cells were counted by trypan blue exclusion; cell count is shown as a percentage of control untreated cells (C) per clone. Error bars represent standard error between duplicate experiments, each run in triplicate. P-values were determined by student's t-test for trastuzumab-treated cells versus untreated cells per clone; *p<0.05. Trastuzumab significantly reduced growth of stable control clones; no significant response to trastuzumab was measured in stable GDF15- overexpressing SKBR3 clones. Stable GDF15 BT474 clone 3 showed statistically significant reduction in growth, but less than control BT474 clone cells.

Figure 3 shows data suggesting GDF 15 -mediated activation of HER2 signaling reduces trastuzumab sensitivity. (A) upper panel: BT474 cells were treated with 2 ng/mL recombinant human GDF 15 for 10, 30, or 60 min or with the corresponding volume of vehicle control for 60 min. Total protein lysates were Western blotted for phosphorylated and total HER2, Akt, and Erkl/2. lower panel: BT474 cells were treated with 2 ng/mL vehicle control for 0, 10, 30, or 60 min, or with 2 ng/mL recombinant human GDF15 for 0, 10, 30, or 60 min. Total protein lysates were Western blotted for phosphorylated and total Erkl/2. (B) BT474 cells were treated with vehicle control (HC1-BSA plus DMSO), 2 ng/mL GDF 15 for 10 or 30 min, or 2 ng/mL GDF 15 plus 5 μΜ AG879 for 30 min. Total protein lysates were Western blotted for phosphorylated and total HER2, Akt, and Erkl/2. (C) BT474 cells were treated with vehicle control (HC1-BSA plus DMSO), 2 ng/mL GDF 15 for 10 or 30 min, or 2 ng/mL GDF 15 plus 1 μΜ lapatinib for 30 min. Total protein lysates were Western blotted for phosphorylated and total Akt and Erkl/2. (D) BT474 cells were treated with vehicle control (C), 20 μg/mL trastuzumab (T), pre -treated with 2 ng/mL GDF 15 for 48h followed by 20 μg/mL trastuzumab plus 2 ng/mL GDF 15 for an additional 72 h (GDF+T), or pre-treated with 2 ng/mL GDF 15 for 48h followed by 20 μg/mL trastuzumab plus 2 ng/mL GDF 15 plus 5 μΜ AG879 for an additional 72 h (GDF+T+AG); media plus treatments were hanged each day. Cells were counted by trypan blue exclusion; cell count is shown as a percentage of the control group per cell line. Error bars represent standard error between duplicate experiments, each run in triplicate. . P-values were determined by student's t-test for GDF+T versus T alone, and for GDF+T+AG versus GDF+T; *p<0.05, **p<0.005. (E) BT474 cells were treated with vehicle control (C), 20 μg/mL trastuzumab (T), 1 μΜ lapatinib (L), pretreated with 2 ng/mL GDF 15 for 48h followed by 20 μg/mL trastuzumab plus 2 ng/mL GDF 15 for an additional 72 h (G+T), pre-treated with 2 ng/mL GDF 15 for 48h followed by 1 μΜ lapatinib plus 2 ng/mL GDF 15 for an additional 72 h (G+L), or pre-treated with 2 ng/mL GDF 15 for 48h followed by 20 μg/mL trastuzumab plus 1 μΜ lapatinib plus 2 ng/mL GDF 15 for an additional 72 h (G+T+L); media plus treatments were changed each day. Cells were counted by trypan blue exclusion; cell count is shown as a percentage of the control group per cell line. Error bars represent standard deviation between triplicates. P- values were determined by student's ttest for G+T+L versus G+T; *p<0.05. (F) Stable SKBR3 control and GDF 15 clones were treated with 100 nM lapatinib (Lap) or corresponding dose of DMSO control (C) for 48h. Proliferation was measured by MTS assay, and is shown as a percentage of control per clone. Error bars represent standard error between duplicate experiments, each run in triplicate. . P-values were determined by student's t-test for lapatinib-treated cells versus control per clone; *p<0.05, **p<0.005. Stable GDF15-overexpressing clones retained sensitivity to lapatinib.

Figure 4 shows data suggesting TGF beta receptor-dependent Src phosphorylation contributes to GDF15- mediated resistance. (A) BT474 cells were treated with vehicle control (HC1-BSA plus DMSO), 2 ng/niL GDF15 for 10 or 30 min, or 2 ng/mL GDF15 plus 5 μΜ SB431542 for 30 min. Total protein lysates were Western blotted for phosphorylated and total Smad2. (B) BT474 cells were treated with vehicle control (C) (HC1-BSA plus DMSO), 20 μg/mL trastuzumab (T), 2 ng/mL GDF15 (G), trastuzumab plus GDF15 (TG), 5 μΜ SB431542 (S), trastuzumab plus SB431542 (TS), or trastuzumab plus GDF15 plus SB431542 (TGS) for 30 min. Total protein lysates were Western blotted for phosphorylated and total Src. (C) BT474 cells were treated with vehicle control (C) (HC1-BS A plus DMSO), 20 μg/mL trastuzumab (T), 2 ng/mL GDF 15 (G), trastuzumab plus GDF 15 (TG), 1 μΜ lapatinib (L), trastuzumab plus lapatinib (TL), or trastuzumab plus GDF 15 plus lapatinib (TGL) for 30 min. Total protein lysates were Western blotted for phosphorylated and total Src. (D) BT474 control and GDF 15 stable clones 2 and 3 were treated with vehicle control (C) (HC1-BSA plus DMSO), 20 μg/mL trastuzumab (T), 5 μΜ SB431542 (S), trastuzumab plus SB431542 (TS), 1 μΜ PP2 (P), or trastuzumab plus PP2 (TP) for 72 h; media plus treatments were changed each day. Cells were counted by trypan blue exclusion; cell count is shown as a percentage of the control group per cell line. Error bars represent standard deviation between triplicates. P-values were determined by student's t-test for each group versus T alone; *p<0.05.

Figure 5 shows data suggesting GDF 15 knockdown increases trastuzumab sensitivity in cells with acquired or primary trastuzumab resistance. (A) BT-HRc3 and SK- HRc3 cells were infected with lentiviral control shRNA (shC) or GDF 15 shRNA (shGDF). After 72 h, 20 μg/mL trastuzumab (Tras) was added to cultures for an additional 72 h or cells were left untreated. Cell culture media was examined using GDF 15 -specific ELISA to confirm knockdown of GDF15. Error bars represent standard deviation between triplicates. (B) BT-parental, BT-HRc3, SK-parental, and SK-HRc3 cells were infected with lentiviral control shRNA (shC) or GDF 15 shRNA (shGDF). After 72 h, 20 μg/mL trastuzumab (Tras) was added to cultures for an additional 72 h or cells were left untreated. Cells were then counted by trypan blue exclusion; growth is shown as a percentage of shC-infected cells per line. Treatments were done in triplicate, with error bars representing standard deviation between replicates. Results were confirmed in duplicate experiments. P-values were determined by student's t-test; *p<0.05, **p<0.005. (C) HCC1419 cells were infected with lentiviral control shRNA (shC) or GDF15 shRNA (shGDF); knockdown of GDF15 was confirmed by real-time PCR. Results are reported as fold change in GDF15 transcript level versus control shRNA. Values were normalized to RPLPO housekeeping ribosomal gene transcript levels as internal control. Error bars represent standard deviation between triplicates. Cells were infected with control shRNA or GDF15 shRNA, and after 72 h, treated with 20 μg/mL trastuzumab (Tras) for an additional 72 h or left untreated. Cells were counted by trypan blue exclusion; growth is shown as a percentage of shC-infected cells. Treatments were done in triplicate, with error bars representing standard deviation between replicates. Results were confirmed in duplicate experiments. P-values were determined by student's t-test; *p<0.05, **p<0.005.

Figure 6 illustrates a proposed mechanism of GDF 15 -mediated trastuzumab resistance. Although embodiments of the disclosure are not intended to be limited by any particular mechanism it is believed that GDF 15 activates TGF beta receptor-Src-HER2 signaling crosstalk as a novel mechanism of trastuzumab resistance. GDF 15 appears to activate TGF beta receptor, as measured by phosphorylation of the TGF beta receptor substrate Smad2. GDF15 activates Src in a TGF beta receptor-dependent manner, which subsequently induces phosphorylation of HER2 and abrogates the growth inhibitory effects of the HER2 -targeted antibody trastuzumab. Inhibition of the HER2 kinase by lapatinib restores sensitivity to trastuzumab in models of GDF 15 -mediated trastuzumab resistance. DETAILED DESCRIPTION

It has been discovered that GDF 15 -mediated HER2 phosphorylation reduces sensitivity to trastuzumab in a TGF beta receptor-dependent manner. Data herein shows that trastuzumab- resistant cell lines express increased levels of GDF 15. Further, increased exposure to recombinant human GDF 15 or stable over-expression of GDF 15 reduced the sensitivity of HER2- overexpressing breast cancer cells to trastuzumab.

GDF 15 -mediated trastuzumab resistance involves phosphorylation of HER2, as pharmacologic inhibition of HER2 overcame the resistance conferred by GDF 15. TGF beta receptor signaling was activated by GDF15, which induced phosphorylation of Src. GDF15- mediated phosphorylation of HER2 is Src-dependent . Inhibition of TGF beta receptor or Src overcame GDF 15 -mediated trastuzumab resistance. Knockdown of GDF 15 improved sensitivity to trastuzumab in models of acquired trastuzumab

resistance. Although it is not intended that embodiments of the disclosure be limited by any particular mechanism, these results support GDF15-mediated activation of TGF beta receptor-Src- HER2 signaling crosstalk as a novel mechanism of trastuzumab resistance (Figure 6).

Expression of GDF 15 was increased in HER2-overexpressing breast cancer cells that displayed either acquired or primary resistance. This was a fairly generalized finding, as resistant cells showed higher levels of endogenous and secreted GDF 15 versus SKBR3 and BT474 trastuzumab-sensitive cells, although levels varied from clone to clone. Real-time PCR confirmed that GDF15 transcript levels were increased, suggesting that GDF15 is up-regulated in resistant cancers at the transcriptional level or possibly that GDF 15 rriRNA stability is altered in resistant cancers. Multiple transcription factor binding sites are present in the GDF 15 promoter, including response elements for AP-1, Spl, p53, and EGR. Cell lines used in this study are p53 mutant, suggesting that the mechanism of GDF15 up-regulation is likely to be p53- independent. However, other members of the p53 family may contribute to GDF 15 transcription.

Trastuzumab-sensitive cells stimulated with GDF15 or stably transfected with a

GDF 15 expression construct showed reduced response to trastuzumab, suggesting that GDF 15 directly contributes to the development of trastuzumab resistance. Stimulation with recombinant human GDF 15 partially reduced trastuzumab sensitivity, while endogenous GDF 15

overexpression (stable transfection) appeared to induce a stronger (almost complete) resistance phenotype. The recombinant cytokine is a purified form of the secreted form of GDF 15, whereas stable transfection incorporates the endogenous full-length precursor which can then be cleaved into the secreted form. Thus, it is possible that the precursor form possess additional activity beyond that of the secreted form, and that increased transcription of GDF 15, not just increased release of the secreted form, promotes resistance. This would be consistent with our findings that GDF 15 is increased at the transcript level in cells with acquired or primary trastuzumab resistance.

Further, knockdown of GDF 15 restored trastuzumab sensitivity to SKBR3 -derived resistant cells, and reduced survival of BT474-derived trastuzumab -resistant cells. Interestingly, attempts to develop stable GDF 15 shRNA clones were unsuccessful, as long-term GDF 15 knockdown killed trastuzumab -resistant cells in culture. These data indicate that long-term knockdown of GDF 15 is incompatible with survival of acquired trastuzumab-resistant cells, suggesting that resistant cells may have developed dependence upon GDF15. Thus, inhibition of GDF15 is a potential strategy for treating breast cancers that have progressed on trastuzumab- based therapy.

Data indicated that the mechanism by which GDF 15 promotes trastuzumab resistance involves activation of HER2 signaling. In our study, GDF 15 stimulated phosphorylation of HER2, Akt, and Erkl/2. Pharmacologic inhibition of HER2 kinase blocked GDF 15 -stimulated Akt and Erkl/2 signaling, indicating that GDF15-mediated PI3K and MAPK activation occur downstream of HER2 activation. However, since the major induction of Erkl/2 phosphorylation preceded Her2 phosphorylation, it is likely that there is also a HER2-independent mechanism contributing to GDF 15 -mediated Erkl/2 phosphorylation.

Since GDF 15 is structurally similar to TGF beta, whether GDF 15 stimulated

phosphorylation of the TGF beta receptor substrate Smad2, and whether TGF beta receptor signaling contributes to GDF 15 -mediated trastuzumab resistance was examined. Pharmacologic inhibition of TGF beta receptor type II blocked GDF 15 -mediated Smad2 phosphorylation and reduced GDF15-mediated trastuzumab resistance.

Finally, since GDF 15 -mediated phosphorylation of HER2 involved Src, the role of Src in

GDF 15 -mediated resistance was examined. GDF15-mediated Src phosphorylation in HER2- overexpressing cells was blocked by TGF beta receptor inhibition but not by HER2 kinase inhibition. Thus, GDF 15 appears to activate TGF beta receptor, which in turn activates Src, which induces phosphorylation of HER2, abrogating the growth inhibitory effects of HER2- targeted trastuzumab.

Growth differentiation factor 15 (GDF15)

GDF 15 is sometimes referred to as macrophage inhibitory cytokine- 1 (MIC-1), placental transforming growth factor-β (PTGF-β), placental bone morphogenetic protein (PLAB), prostate-derived factor (PDF), or non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1). Expression in certain cells results in the proteolytic cleavage of the propeptide and secretion of a cysteine -rich dimeric protein. The human propeptide GDF 15 amino acid sequence is SEQ ID NO 1 : 1 MPGQELRTVN GSQMLLVLLV LSWLPHGGAL SLAEASRASF PGPSELHSED SRFRELRKRY 61 EDLLTRLRAN QSWEDSNTDL VPAPAVRILT PEVRLGSGGH LHLRISRAAL PEGLPEASRL 121 HRALFRLSPT ASRSWDVTRP LRRQLSLARP QAPALHLRLS PPPSQSDQLL AESSSARPQL 181 ELHLRPQAAR GRRRARARNG DHCPLGPGRC CRLHTVRASL EDLGWADWVL SPREVQVTMC 241 IGACPSQFRA ANMHAQIKTS LHRLKPDTVP APCCVPASYN PMVLIQKTDT GVSLQTYDDL 301 LAKDCHCI. Cleavage at amino acid 197 allows dimerization into the mature protein.

Human GDF 15 mRNA is SEQ ID NO 2: 1 AGTCCCAGCT CAGAGCCGCA ACCTGCACAG CCATGCCCGG GCAAGAACTC AGGACGGTGA 61

ATGGCTCTCA GATGCTCCTG GTGTTGCTGG TGCTCTCGTG GCTGCCGCAT GGGGGCGCCC 121 TGTCTCTGGC CGAGGCGAGC CGCGCAAGTT TCCCGGGACC CTCAGAGTTG CACTCCGAAG 181 ACTCCAGATT CCGAGAGTTG CGGAAACGCT ACGAGGACCT GCTAACCAGG CTGCGGGCCA

241 ACCAGAGCTG GGAAGATTCG AACACCGACC TCGTCCCGGC

CCCTGCAGTC CGGATACTCA 301 CGCCAGAAGT GCGGCTGGGA

TCCGGCGGCC ACCTGCACCT GCGTATCTCT CGGGCCGCCC 361

TTCCCGAGGG GCTCCCCGAG GCCTCCCGCC TTCACCGGGC TCTGTTCCGG CTGTCCCCGA 421 CGGCGTCAAG GTCGTGGGAC GTGACACGAC

CGCTGCGGCG TCAGCTCAGC CTTGCAAGAC 481 CCCAGGCGCC

CGCGCTGCAC CTGCGACTGT CGCCGCCGCC GTCGCAGTCG GACCAACTGC

541 TGGC AGAATC TTCGTCCGCA CGGCCCC AGC TGGAGTTGC A

CTTGCGGCCG CAAGCCGCCA 601 GGGGGCGCCG CAGAGCGCGT

GCGCGCAACG GGGACCACTG TCCGCTCGGG CCCGGGCGTT 661

GCTGCCGTCT GCACACGGTC CGCGCGTCGC TGGAAGACCT GGGCTGGGCC GATTGGGTGC 721 TGTCGCCACG GGAGGTGCAA GTGACCATGT

GCATCGGCGC GTGCCCGAGC CAGT 781 CGGCAAACAT GCACGCGCAG ATCAAGACGA GCCTGCACCG CCTGAAGCCC GACACGGTGC 841

CAGCGCCCTG CTGCGTGCCC GCCAGCTACA ATCCCATGGT GCTCATTCAA AAGACCGACA 901 CCGGGGTGTC GCTCCAGACC TATGATGACT

TGTTAGCCAA AGACTGCCAC TGCATATGAG 961 CAGTCCTGGT

CCTTCCACTG TGCACCTGCG CGGAGGACGC GACCTCAGTT GTCCTGCCCT

1021 GTGGAATGGG CTCAAGGTTC CTGAGACACC CGATTCCTGC

CCAAACAGCT GTATT 1081 AAGTCTGTTA TTTATTATTA ATTTATTGGG GTGACCTTCT TGGGGACTCG GGGGCTGGTC 1141 TGATGGAACT

GTGTATTTAT TTAAAACTCT GGTGATAAAA ATAAAGCTGT CTGAACTGTT

1201 AAAAAAAAAA AAAAAAAAAA.

The term "GDF15" is not intended to be limited to any protein with a particular sequence or glycosylation pattern. Minor modifications of the recombinant GDF15 primary amino acid sequence may result in proteins which have substantially equivalent activity as compared to the GDF15 polypeptide. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as the biological activity of GDF15 still exists. Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its biological activity. This can lead to the development of a smaller active molecule. For example, one may remove amino or carboxyl terminal amino acids which are not required for GDF15 biological activity.

U.S. Patent no. 7,514,221 provides that a number of allelic variants of GDF15 (referred to as MIC-1) exist, which show amino acid sequence differences at positions 9, 48 and 202. Amino acid position 202 corresponds to position 6 of the mature form of the protein (i.e. with the leader sequence having been removed through cleavage). In some of the identified variants, the normal histidine (H) residue at position 202 (or "H6") is substituted with aspartic acid (D). This is due to a single nucleotide substitution within the gene such that a cytosine (C) at position 604 is substituted by a guanosine (G). In certain embodiments, the disclosure relates to detecting these allelic variants using appropriately complimentary oligonucleotide probes.

Diagnosing HER2 Expression

HER2 diagnosis is typically done on protein level by immunohistochemistry (IH) and/or on DNA level by (Fluorescence)-in-situ-Hybridization (ISH, FISH). See Bofin et al., Am J Clin Pathol 2004;122: 110-119. The HER2 gene is located on chromosome 17 and encodes the pi 85 HER2 protein. In certain breast cancer tumors, the HER2 gene amplifies as part of the malignant transformation.

Typically in immunohistochemical applications, the HER2 protein is first detected in the tissue by a primary antibody with a high level of specificity for the antigen. This primary antibody is in turn detected by an appropriate species-specific biotinylated secondary antibody. An enzyme conjugated to streptavidin is then applied which binds the biotin-labeled secondary antibody. Finally, the location of the primary antibody is visualized by the application of a colorimetric chromogen that precipitates in the presence of the streptavidin conjugated-enzyme. Results are interpreted using a light microscope.

The PathVysion™ HER-2 DNA probe is an FDA approved kit designed to detect amplification of the HER-2/neu gene via fluorescence in situ hybridization (FISH) in formalin- fixed, paraffin-embedded human breast cancer tissue specimens. The kit contains two fluorescently labeled nucleic acid probes specific for the HER2 amplicon and the alpha satellite sequence at the centromeric region of chromosome 17 (CEN-17). Results are expressed as a ratio of HER2 gene copies per number of chromosome 17 copies. Unlabeled blocking sequences are also included to suppress hybridization to loci that are common to other chromosomes. Diagnosing HER2 Antibody Therapy Resistance

The present application reveals a role for GDF15 as a mediator and biomarker of trastuzumab resistance. Microarray analysis identified growth differentiation factor 15 (GDF15) as being highly over-expressed in cell culture models of acquired trastuzumab resistance. Enzyme-linked immunosorbent assay (ELISA) confirmed over-expression of GDF15 in cells with acquired resistance as well as in cells with primary resistance to trastuzumab. In addition, exposure of trastuzumab-sensitive cells to recombinant human GDF15 (rhGDF15) conferred resistance to trastuzumab supporting a causative role of GDF15 in trastuzumab resistance. PI3K and p38 signaling were activated by rhGDF15, and pharmacologic inhibition of PI3K, mTOR, or p38MAPK abrogated rhGDF 15- mediated trastuzumab resistance. Knockdown of p38MAPK improved trastuzumab sensitivity of primary resistant cells, implicating p38MAPK signaling as an additional mechanism of trastuzumab resistance. Knockdown of GDF15 improved sensitivity of resistant cells to trastuzumab.

HEPv2-overexpressing breast cancer cells that have primary or acquired resistance to trastuzumab have increased expression of the cytokine GDF15. Exposure of trastuzumab-sensitive cells to GDF15 resulted in development of resistance and activation of PI3K and p38MAPK signaling. Inhibition of PI3K and p38MAPK signaling blocked GDF 15 -mediated trastuzumab resistance. Finally, knockdown of GDF15 improved trastuzumab sensitivity in cells with primary or acquired resistance.

GDF 15 over-expression was found in multiple models of primary and acquired trastuzumab resistance. PI3K signaling is an accepted mechanism leading to trastuzumab resistance, although the molecular changes leading to PI3K activation in resistant cancers is not well-established. Our data suggest GDF 15 is a mediator of PI3K activation. In addition, p38MAPK signaling has not previously been reported as a mechanism of trastuzumab resistance. However, data shows increased phosphorylation of p38 in resistant cancers, with p38 inhibition improving trastuzumab response. GDF 15 over- expression is a potential mechanism by which p38 is activated in trastuzumab-resistant breast cancers.

Stimulation of SKBR3 and BT474 cells with rhGDF 15 did not change levels of phospho-HEPv2 or phospho-Erkl/2 in comparison to cells stimulated with the vehicle control. The experimental results are consistent with the concept that increased GDF 15 expression is associated with advanced, drug-resistant cancer. Thus, elevated GDF 15 can serve as a prognostic marker for advanced or metastatic cancers. GDF 15 exposure did not abrogate responses to docetaxel or the dual EGFR/HER2 kinase inhibitor lapatinib, which suggests that GDF 15 has a highly specific inhibitory role for the anti-cancer activity of trastuzumab. Resistance to trastuzumab and lapatinib often occur simultaneously. Our data suggests that GDF 15 confers resistance to trastuzumab, but not to lapatinib. Thus, GDF 15 appears to be a valuable target for restoring sensitivity to trastuzumab in HER2-overexpressing breast cancer.

Cell viability was assessed and compared to cells treated with rhGDF15 plus trastuzumab alone. LY294002, a PI3K inhibitor and rapamycin and mTOR inhibitor disrupted rhGDF 15 -mediated trastuzumab resistance in a statistically significant manner in both cell lines, implicating a role for PBK/mTOR signaling in GDF 15 -mediated trastuzumab resistance. Rapamycin partially disrupted resistance in contrast to LY294002, which achieved an almost complete disruption of GDF 15 -mediated resistance. Thus, PI3K signaling appears to be important for GDF 15 -mediated resistance. MEK inhibition, on the other hand, did not reduce the ability of rhGDF 15 to confer trastuzumab resistance, consistent with lack of activation of p-Erkl/2 by rhGDF 15.

In some embodiments, the disclosure relates to methods of determining a chemotherapy treatment comprising assaying a sample from a subject diagnosed with cancer for elevated GDF 15 expression, determining that GDF 15 expression in the sample is elevated, and administering a tyrosine kinase inhibitor and/or a TGF beta receptor inhibitor and/or a Src inhibitor, mTOR inhibitor a PI3K inhibitor and/or an mTOR inhibitor and/or a taxane. In certain embodiments, the subject is diagnosed with HER2 overexpressing breast cancer. In certain embodiment, the tyrosine kinase inhibitor, PI3K inhibitor, mTOR inhibitor and/or a taxane is administered in combination with a HER2 antibody. In certain embodiments, the method further comprises administering a granulocyte -macrophage colony-stimulating factor vaccine.

Trastuzumab is a representative HER2 antibody. Representative tyrosine kinase inhibitors are axitinib, bosutinib, cediranib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, neratinib, nilotinib, semaxanib, sunitinib, toceranib, tyrphostins, vandetanib, and vatalanib. Representative PI3K inhibitors include

wortmannin, 2-morpholin-4-yl-8-phenylchromen-4-one, SF1126 (a RGDS-conjugated prodrug of2-morpholin-4-yl-8-phenylchromen-4-one , GDC-0941, PF-04691502, PX- 866, XL147, XL765, CAL-26, 3BKM120, BEZ235 (2-methyl-2-(4-(3-methyl-2-oxo-8- (quinolin-3 -yl)-2,3 -dihydro- 1 H-imidazo [4,5 -c] quinolin- 1 -yl)phenyl)propanenitrile) and BGT226. A number of PI3K inhibitors are described in U.S. Patent No. 7,767,669. Representative mTOR inhibitors include everolimus, deforolimus, temsirolimus, XL765, RAD001. Representative taxanes include docetaxel and paclitaxel.

Examples of TGF beta receptor inhibitors include SB-431542, SB-505124, SM-16, SD-208, LY2109761 3-[6-(2-Morpholin-4-yl-ethoxy)-naphthalen-l-yl]-2-pyridin-2-yl-5,6- dihydro-4H-pyrrolo[l,2-b]pyrazole and derivatives provided for in Li et al, J. Med.

Chem., 2008, 51 (7), pp 2302-2306, hereby incorporated by reference, (Z)-N-ethyl-N- methyl-2-oxo-3-(phenyl((4-(piperidin- 1 -ylmethyl)phenyl)amino) methylene)indoline-6- carboxamide, (Z)-3-[(4-Dimethylaminomethyl-aminophenyl)-phenyl-methylene]-2-oxo- 2,3-dihydro-lH-indole-6-carboxylic acid ethylamide and derivatives provided for in Roth et al, J. Med. Chem. 2010, 53, 7287-7295, hereby incorporated by reference. Examples of Src inhibitors include dasatinib, (4-((2-cyclopentyl-9-ethyl-9H-purin-6- yl)amino)phenyl)dipropylphosphine oxide and derivatives disclosed in Summy et al, Mol Cancer Ther 2005;4(12): 1900-11, hereby incorporated by reference.

Nucleic Acid Interference of GDF15 Expression

In certain embodiments, the disclosure relates to compounds, compositions, and methods useful for modulating GDF15 expression using short interfering nucleic acid (siNA) molecules. In particular, the instant disclosure features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of GDF.

RNA interference refers to the process of sequence specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) sometimes referred to as post-transcriptional gene silencing or RNA silencing. The presence of long dsRNAs in cells is thought to stimulate the activity of a ribonuclease III enzyme referred to as Dicer. Dicer is thought to be involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs). Short interfering RNAs derived from Dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control. The RNAi response is thought to feature an endonuclease complex containing a siRNA, commonly referred to as an RNA- induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex. In addition, RNA interference is thought to involve small RNA (e.g., micro-RNA or miRNA) mediated gene silencing, presumably though cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences. As such, siNA molecules can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Elbashir et al, 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21 -nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates has revealed certain preferences for siRNA length, structure, chemical composition, and sequence that mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are typical when using two 2- nucleotide 3'-terminal nucleotide overhangs. Substitution of 3'-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated. Other studies have indicated that a 5 '-phosphate on the target-complementary strand of an siRNA duplex is beneficial for siRNA activity and that ATP is utilized to maintain the 5 '-phosphate moiety on the siRNA. siRNA molecules lacking a 5'-phosphate are active when introduced exogenously.

A siNA can be unmodified or chemically-modified. A siNA can be chemically synthesized, expressed from a vector or enzymatically synthesized. Various chemically- modified synthetic short interfering nucleic acid (siNA) molecules are capable of modulating GDF15 expression or activity in cells by RNA interference (RNAi).

In one embodiment, the disclosure relates to a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a GDF15, wherein said siNA molecule comprises about 15 to about 35 base pairs.

In some embodiments, the disclosure relates to methods of treating a subject diagnosed with cancer by administering a pharmaceutical composition with a double stranded nucleic acid with one strand comprising SEQ ID NO: 4

CTCCAGACCTATGATGACTTGTTAGCCAA. In certain embodiments, the disclosure relates to treating a subject with cancer comprising administering the pharmaceutical composition in combination with a HER2 antibody. In some embodiments, the disclosure relates to methods of treating a subject diagnosed with cancer by administering a pharmaceutical composition with a nucleic acid that is a single strand hairpin with a sense strand having SEQ ID NO: 5

CGAGAGTTGCGGAAACGCT. In other embodiments, the disclosure relates to a pharmaceutical composition comprising a nucleic acid having SEQ ID NO: 6

TGCTGTTGACAGTGAGCGACCCAAACAGCTGTATTTATAT TAGTGAAGCCACAGATGTAATATAAATACAGCTGTTTGGG CTGCCTACTGCCTCGGA. In certain embodiments, the disclosure relates to treating a subject with cancer comprising administering the pharmaceutical composition in combination with a HER2 antibody.

In some embodiments, the disclosure relates to methods of treating a subject diagnosed with cancer by administering a pharmaceutical composition with a nucleic acid that is a single strand hairpin with a sense strand having SEQ ID NO: 7,

CTGATGGAACTGTGTATTT. In other embodiments, the disclosure relates to a pharmaceutical composition comprising a nucleic acid comprising SEQ ID NO: 8 TGCTGTTGACAGTGAGCGATCTGATGGAACTGTGT

ATTTATAGTGAAGCCACAGATGTATAAATACACAGTTCCATCAGACTGCCTAC TGCCTCGGA. In certain embodiments, the disclosure relates to treating a subject with cancer comprising administering the pharmaceutical composition in combination with a HER2 antibody.

In some embodiments, the disclosure relates to nucleic acids obtained by endo- ribonuclease prepared siRNA (esiRNA). A representative endo-ribonuclease is naturally isolated or recombinant bacterial RNase III. Upon purification, one uses the enzyme to generate esiRNAs. One can generate double stranded RNA of GDF15 mRNA by in vitro transcription. See. Yang et al, (2002), Proc. Natl. Acad. Sci. USA 99(15): 9942-9947. One uses the RNase III to digest the transcripts into smaller fragments. One runs the digested RNA molecules on a gel and RNA duplexes of 15-30 nucleotides are isolated.

In some embodiments, the disclosure relates to methods of treating a subject diagnosed with cancer by administering a pharmaceutical composition with a

heterogeneous mixture of siNAs that are homologous to the GDF15 mRNA sequence or fragment thereof. In certain embodiments, the fragments have greater than 150 or 200 nucleotides. In certain embodiments, the mixture is obtained by digesting a double stranded RNA having SEQ ID NO 9:

GAGGTGCAAGTGACCATGTGCATCGGCGCGTGCCCGAGCCAGTTCCGGGCGG CAAACATGCACGCGCAGATCAAGACGAGCCTGCACCGCCTGAAGCCCGACAC GGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTC AAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAA GACTGCCACTGCATATGAGCAGTCCTGGTCCTTCCACTGTGCACC.

In certain embodiment, nucleic acids disclosed herein are expressed in a recombinant vector in vivo contained in the pharmaceutical product. Representative recombinant vectors include plasmids, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and lentiviral vectors.

Synthesis of Nucleic Acid Molecules

Small nucleic acid motifs ("small" refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery. The structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure. Exemplary molecules of the instant disclosure are chemically synthesized, and others can similarly be synthesized.

One synthesizes oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides) using protocols known in the art as, for example, described in U.S. Patent No. 6,001,311. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5 '-end and phosphoramidites at the 3 '-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 micro mol scale protocol with a 2.5 min coupling step for 2'-0-methylated nucleotides and a 45 second coupling step for 2'-deoxy nucleotides or 2'-deoxy-2'-fluoro nucleotides. Alternatively, syntheses at the 0.2 micro mol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess of 2'-0-methyl phosphoramidite and a 105-fold excess of S- ethyl tetrazole can be used in each coupling cycle of 2'-0-methyl residues relative to polymer-bound 5'-hydroxyl. A 22-fold excess of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole mop can be used in each coupling cycle of deoxy residues relative to polymer-bound 5'-hydroxyl. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PerSeptive

Biosystems, Inc.). S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the

introduction of phosphorothioate linkages, Beaucage reagent (3H-l,2-benzodithiol-3-one 1,1 -dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows: the polymer-bound trityl-on oligonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40%> aqueous methylamine (1 mL) at 65 degrees for 10 minutes. After cooling to -20 degrees, the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H20/3: l : l, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligonucleotide, are dried.

Alternatively, the nucleic acid molecules can be synthesized separately and joined together post-synthetically, for example, by ligation or by hybridization following synthesis and/or deprotection.

An siNA molecule can also be assembled from two distinct nucleic acid strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the R A molecule.

The nucleic acid molecules can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-0-methyl, 2'-H). siNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography and re-suspended in water.

Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency. See e.g., U.S. Patent No. 5,652,094, U.S. Patent No. 5,334,711, and U.S. Patent No. 6,300,074. All of the above references describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired. In one embodiment, nucleic acid molecules include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in nucleic acid molecules results in both enhanced affinity and specificity to nucleic acid targets, complementary sequences, or template strands. In another embodiment, nucleic acid molecules include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA "locked nucleic acid" nucleotides such as a 2',4'-C methylene bicyclo nucleotide (see for example U.S. Patent No. 6,639,059, U.S. Patent No. 6,670,461, U.S. Patent No. 7,053,207).

In another embodiment, the disclosure features conjugates and/or complexes of siNA molecules. Such conjugates and/or complexes can be used to facilitate delivery of siNA molecules into a biological system, such as a cell. The conjugates and complexes provided may impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid molecules into a number of cell types originating from different tissues, in the presence or absence of serum (see U.S. Patent No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.

In yet another embodiment, siNA molecules having chemical modifications that maintain or enhance enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and/or in vivo the activity should not be significantly lowered.

In another aspect a siNA molecule comprises one or more 5' and/or a 3'-cap structure, for example on only the sense siNA strand, the antisense siNA strand, or both siNA strands.

A "cap structure" refers to chemical modifications, which have been incorporated at either terminus of the oligonucleotide. See, for example, Adamic et al., U.S. Patent No. 5,998,203. These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5 '-terminus (5 '-cap) or at the 3 '-terminal (3 '-cap) or may be present on both termini. In non-limiting examples, the 5'-cap includes, but is not limited to, glyceryl, inverted deoxy abasic residue (moiety); 4',5 '-methylene nucleotide; l-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1,5- anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3 '-3'- inverted nucleotide moiety; 3 '-3 '-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate;

hexylphosphate; aminohexyl phosphate; 3'-phosphate; 3'-phosphorothioate;

phosphorodithioate; or bridging or non-bridging methylphosphonate moiety.

Non-limiting examples of the 3'-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety), 4',5'-methylene nucleotide; l-(beta-D- erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; l,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1 ,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5 '-5 '-inverted nucleotide moiety; 5 '-5 '-inverted abasic moiety; 5'-phosphoramidate; 5'-phosphorothioate; 1 ,4-butanediol phosphate; 5'- amino; bridging and/or non-bridging 5'-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925).

By the term "non-nucleotide" is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the 1 '-position.

In one embodiment, the disclosure features modified siNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate,

phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.

Pharmaceutical Compositions of Nucleic Acid Molecules

The following protocols can be utilized for the delivery of nucleic acid molecules.

A siNA molecule can be adapted for use to prevent or treat cancers and other proliferative conditions and/or any other trait, disease or condition that is related to or will respond to the levels of GDP 15 in a cell or tissue, alone or in combination with other therapies. For example, a siNA molecule can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. U.S. Patent No. 6,395,713 and U.S. Patent No. 5,616,490 further describe general methods for delivery of nucleic acid molecules. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by

iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example U.S. Patent No. 7,141,540 and U.S. Patent No. 7,060,498), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example U.S. Patent No. 6,447,796), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (U.S. Patent No. 7,067,632). In another embodiment, the nucleic acid molecules can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine-polyethyleneglycol- N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N- acetylgalactosamine (PEI-PEG-triGAL) derivatives.

In one embodiment, a siNA molecule is complexed with membrane disruptive agents such as those described in U.S. Patent No. 6,835,393. In another embodiment, the membrane disruptive agent or agents and the siNA molecule are also complexed with a cationic lipid or helper lipid molecule, such as those lipids described in U.S. Patent No. 6,235,310.

Embodiments of the disclosure feature a pharmaceutical composition comprising one or more nucleic acid(s) in an acceptable carrier, such as a stabilizer, buffer, and the like. The oligonucleotides can be administered (e.g., RNA, DNA or protein) and introduced into a subject by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions can also be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions, suspensions for administration by injection, and the other compositions known in the art.

Embodiments of the disclosure also feature the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing the circulation and accumulation of in target tissues. The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA. See U.S. Patent No. 5,820,873 and U.S. Patent No. 5,753,613. Long-circulating liposomes are also likely to protect from nuclease degradation.

Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such

compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic

pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl- methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as

polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti -oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.

In one embodiment, a siNA molecule is designed or formulated to specifically target endothelial cells or tumor cells. For example, various formulations and conjugates can be utilized to specifically target endothelial cells or tumor cells, including PEI-PEG- folate, PEI-PEG-RGD, PEI-PEG-biotin, PEI-PEG-cholesterol, and other conjugates known in the art that enable specific targeting to endothelial cells and/or tumor cells.

Alternatively, certain siNA molecules can be expressed within cells from eukaryotic promoters. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/R A vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by an enzymatic nucleic acid. See U.S. Patent No. 5,795,778, and U.S. Patent No. 5,837,542.

In certain embodiments, the disclosure relates to RNA molecules expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, lentivirus, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another embodiment, pol III based constructs are used to express nucleic acid molecules (see for example U.S. Patent No. 5,902,880 and U.S. Patent No. 6, 146,886). The recombinant vectors capable of expressing the siNA molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by

administration to target cells ex -planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.

In certain embodiments, the disclosure relates to an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the instant disclosure. The expression vector can encode one or both strands of a siNA duplex, or a single self- complementary strand that self hybridizes into an siNA duplex. The nucleic acid sequences encoding the siNA molecules of the instant disclosure can be operably linked in a manner that allows expression of the siNA molecule.

In certain embodiments, the disclosure relates to an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siNA molecules of the instant disclosure, wherein said sequence is operably linked to said initiation region and said termination region in a manner that allows expression and/or delivery of the siNA molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5' side or the 3'-side of the sequence encoding the siNA; and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells. More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as siNA in cells. See U.S. Patent No. 5,624,803 and U.S. Patent No. 5,672,501. The above siNA transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors.

Antibodies

In some embodiments, the disclosure relates to pharmaceutical compositions comprising GDF15 antibodies and methods of administering these antibodies to treat cancer patients optionally in combination with HER2 antibodies. Fairlie et al.,

Biochemistry, 2001, 40 (1), pp 65-73, discloses method of generating GDF15 antibodies.

Numerous methods known to those skilled in the art are available for obtaining antibodies or antigen-binding fragments thereof. For example, antibodies can be produced using recombinant DNA methods (U.S. Patent No. 4,816,567). Monoclonal antibodies may also be produced by generation of hybridomas in accordance with known methods. Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance analysis, to identify one or more hybridomas that produce an antibody that specifically binds with a specified antigen. Any form of the specified antigen may be used as the immunogen, e.g., recombinant antigen, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptide thereof.

One exemplary method of making antibodies includes screening protein expression libraries, e.g., phage or ribosome display libraries. Phage display is described, for example, in U.S. Patent No. 5,223,409.

In addition to the use of display libraries, the specified antigen can be used to immunize a non-human animal, e.g., a rodent, e.g., a mouse, hamster, or rat. In one embodiment, the non-human animal includes at least a part of a human immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using the hybridoma technology, antigen-specific monoclonal antibodies derived from the genes with the desired specificity may be produced and selected. U.S. Patent No. 7,064,244.

In another embodiment, a monoclonal antibody is obtained from the non-human animal, and then modified, e.g., humanized, deimmunized, chimeric, may be produced using recombinant DNA techniques known in the art. A variety of approaches for making chimeric antibodies have been described. See, e.g., U.S. Patent No. 4,816,567 and U.S. Patent No. 4,816,397. Humanized antibodies may also be produced, for example, using transgenic mice that express human heavy and light chain genes, but are incapable of expressing the endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR-grafting method that may be used to prepare the humanized antibodies described herein (U.S. Patent No. 5,225,539). All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR, or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.

Humanized antibodies or fragments thereof can be generated by replacing sequences of the Fv variable domain that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for generating humanized antibodies or fragments thereof are provided by U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,761; U.S. Patent No. 5,693,762; U.S. Patent No.

5,859,205; and U.S. Patent No. 6,407,213. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable domains from at least one of a heavy or light chain. Such nucleic acids may be obtained from a hybridoma producing an antibody against a predetermined target, as described above, as well as from other sources. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

In certain embodiments, a humanized antibody is optimized by the introduction of conservative substitutions, consensus sequence substitutions, germline substitutions and/or backmutations. An antibody or fragment thereof may also be modified by specific deletion of human T cell epitopes or "deimmunization" by the methods disclosed in U.S. Patent No. 7,125,689 and U.S. Patent No. 7,264,806. Briefly, the heavy and light chain variable domains of an antibody can be analyzed for peptides that bind to MHC Class II; these peptides represent potential T-cell epitopes. For detection of potential T-cell epitopes, a computer modeling approach termed "peptide threading" can be applied, and in addition a database of human MHC class II binding peptides can be searched for motifs present in the VH and VL sequences. These motifs bind to any of the 18 major MHC class II DR allotypes, and thus constitute potential T cell epitopes. Potential T-cell epitopes detected can be eliminated by substituting small numbers of amino acid residues in the variable domains, or preferably, by single amino acid substitutions. Typically,

conservative substitutions are made. Often, but not exclusively, an amino acid common to a position in human germline antibody sequences may be used. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences. These sequences can be used as a source of human sequence, e.g., for framework regions and CDRs. Consensus human framework regions can also be used, e.g., as described in U.S. Patent No. 6,300,064.

Aptamers

In certain embodiments, aptamers are contemplated as inhibitors of GDF15.

Oligonucleotides can be developed to target GDF15. SELEX ("Systematic Evolution of Ligands by Exponential Enrichment") is a combinatorial chemistry technique for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to a target. Standard details on generating aptamers can be found in U.S. Patent No.

5,475,096, and U.S. Patent No. 5,270,163.

The SELEX process provides a class of products which are referred to as nucleic acid ligands or aptamers, each having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX process is based on the fact that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets. The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.

The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. Patent No. 5,707,796 describes the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. Patent No. 5,763,177 and U.S. Patent No. 6,011,577 describe a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. Patent No. 5,580,737 describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, which can be non-peptidic, termed Counter-SELEX. U.S. Patent No. 5,567,588 describes a SELEX-based method which achieves efficient partitioning between oligonucleotides having high and low affinity for a target molecule.

The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples include U.S. Patent No. 5,660,985 and U.S. Patent No. 5,580,737.

The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Patent No. 5,637,459 and U.S. Patent No. 5,683,867. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.

The term aptamer is intended to include those nucleic acids assembled at least partially or completely, from the non-natural L-nucleotides. Methods for the preparation of such nucleic acids are described in U.S. Patent No. 6,605,713. Combination Therapies

The anti-cancer treatment defined herein may be applied as a sole therapy or may involve, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include one or more of the following categories of anti -tumour agents:

(i) antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as alkylating agents (for example cis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulfan and

nitrosoureas); antimetabolites (for example antifolates such as fluoropyrimidines like 5- fluorouracil and gemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea); antitumour antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol and taxotere); and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin); and proteosome inhibitors (for example bortezomib [Velcade®]); and the agent anegrilide [Agrylin®]; and the agent alpha- interferon

(ii) cytostatic agents such as antioestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptor down regulators (for example fulvestrant), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5 -reductase such as finasteride;

(iii) agents which inhibit cancer cell invasion (for example metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function);

(iv) inhibitors of growth factor function, for example such inhibitors include growth factor antibodies, growth factor receptor antibodies (for example the anti-Her2 antibody trastuzumab and the anti- epidermal growth factor receptor (EGFR) antibody, cetuximab) , farnesyl transferase inhibitors, tyrosine kinase inhibitors and serine/threonine kinase inhibitors, for example inhibitors of the epidermal growth factor family (for example EGFR family tyrosine kinase inhibitors such as: N-(3-chloro-4-fluorophenyl)-7- methoxy-6-(3-morpholinopropoxy)quinazolin-4-a mine (gefitinib), N-(3-ethynylphenyl)- 6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib), and 6-acrylamido-N-(3-chloro-4- fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine (CI 1033), for example inhibitors of the platelet-derived growth factor family and for example inhibitors of the hepatocyte growth factor family, for example inhibitors of phosphotidylinositol 3 -kinase (PI3K) and for example inhibitors of mitogen activated protein kinase kinase (MEKl/2) and for example inhibitors of protein kinase B (PKB/Akt), for example inhibitors of Src tyrosine kinase family and/or Abelson (Abl) tyrosine kinase family such as dasatinib (BMS-354825) and imatinib mesylate (Gleevec™); and any agents that modify STAT signalling;

(v) antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, (for example the anti-vascular endothelial cell growth factor antibody bevacizumab [Avastin™]) and compounds that work by other mechanisms (for example linomide, inhibitors of integrin οσνβ3 function and angiostatin);

(vi) vascular damaging agents such as Combretastatin A4;

(vii) antisense therapies, for example those which are directed to the targets listed above, such as an anti-ras antisense; and

(viii) immunotherapy approaches, including for example ex -vivo and in-vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte -macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine - transfected tumour cell lines and approaches using anti-idiotypic antibodies, and approaches using the immunomodulatory drugs thalidomide and lenalidomide

[Revlimid®].

Terms

As used herein a "sample" refers to a composition taken from or originating from a subject. Examples of samples include cell samples, blood samples, tissue samples, hair samples, and urine or excrement samples.

A "subject" refers to any animal such as a human patient, livestock or a domestic pet.

As used herein "cancer" refers any of various cellular diseases with malignant neoplasms characterized by the proliferation of cells. Most cancers form a tumor but some, like leukemia, do not. It is not intended that the diseased cells must actually invade surrounding tissue and metastasize to new body sites. Cancer can involve any tissue of the body and have many different forms in each body area. Within the context of certain embodiments, whether "cancer is reduced" may be identified by a variety of diagnostic manners known to one skill in the art including, but not limited to, observation the reduction in size or number of tumor masses or if an increase of apoptosis of cancer cells observed, e.g., if more than a 5 % increase in apoptosis of cancer cells is observed. It may also be identified by a change in relevant biomarker or gene expression profile, such as HER2 for breast cancer.

Breast cancer is commonly diagnosed using a "triple test," i.e., clinical breast examination, mammography, and fine needle aspiration and cytology. Fine needle aspiration and cytology (FNAC) involves extracting a small portion of fluid from a lump. Clear fluid makes the lump unlikely to be cancerous. Bloody fluid may be sent off for inspection under a microscope for cancerous cells. Other options for biopsy include core biopsy, where a section of the breast lump is removed, and an excisional biopsy, where the entire lump is removed.

Some breast cancers require the hormones estrogen and progesterone to grow. After surgery these cancers are typically treated with drugs that interfere with hormones, such as tamoxifen, and with drugs that shut off the production of estrogen in the ovaries or elsewhere. After surgery, low-risk, hormone-sensitive breast cancers may be treated with hormone therapy and radiation. Another breast cancers regimen is cyclophosphamide plus doxorubicin (Adriamycin), referred to as CA. Sometimes a taxane, such as docetaxel, is added, and the regime is then referred to as CAT. An alternative treatment is

cyclophosphamide, methotrexate, and fluorouracil (CMF). Therapeutic antibodies, such as trastuzumab (Herceptin), are typically used for cancer cells that over express the HER2. It is contemplated that methods disclosed herein may be used in combination with any of the regiments described above.

As used herein, the term "marker" is used broadly to encompass a variety of types of molecules which are detectable through spectral properties (e.g. fluorescent markers or "fluorophores") or through functional properties (e.g. affinity markers). A representative affinity marker includes biotin, which is a ligand for avidin and streptavidin. An epitope marker or "epitope tag" is a marker functioning as a binding site for antibody. Since chimeric receptor proteins and antibodies can be produced recombinantly, receptor ligands are effective affinity markers. "Nucleotide" as used herein, and as recognized in the art, includes natural bases (standard), and modified bases well known in the art. Such bases are generally located at the Γ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, and non-standard nucleotides. See, for example, U.S. Patent No. 5,652,094. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5- alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6- azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), and others. By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at position or their equivalents.

As used herein, the term "nucleic acid molecule" refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA. The term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N-6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil,

dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1-methylpseudouracil, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3 -methyl cytosine, 5 -methyl cytosine, N6-methyladenine, 7-methylguanine, 5 -methylaminomethyluracil, 5 -methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queuosine, 2-thiocytosine, and 2,6-diaminopurine.

The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full- length or fragments are retained. The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non- translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non- coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

The term "short interfering nucleic acid", "siNA", "short interfering RNA", "siRNA", "short interfering nucleic acid molecule", "short interfering oligonucleotide molecule", or "chemically-modified short interfering nucleic acid molecule" as used herein refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication, for example by mediating RNA interference "RNAi" or gene silencing in a sequence-specific manner. For example the siNA can be a double- stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is

complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.

"Chromatography" refers to processes used to purify individual components from mixtures by passing a mixture contained in a "mobile phase" through a "stationary phase," which separates the analyte to be measured from other components in the mixture. A "separation medium" refers to the stationary phase or adsorbent. In certain embodiments, the disclosure relates to analysis of samples using chromatographic processes.

"Recombinant vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant

polynucleotide.

Ion exchange chromatography, liquid chromatography, normal-phase (NP) and reversed-phase chromatography (RP), affinity chromatography, and expanded bed adsorption (EBA) chromatograph all use separation mediums. In ion exchange

chromatography, the separation medium is typically an ion exchange resin that carries charged functional groups which interact with oppositely charged groups of the compound to be retained. In affinity chromatography, the separation medium is typically a gel matrix, often of agarose, typically coupled with metals or molecules that bind with markers or tags such antigens, antibodies, enzymes, substrates, receptors, and ligands. Methods utilizing antibodies or antigens (epitopes) coupled to the separation medium is typically referred to as immunoaffinity chromatography and the separation medium is referred to as an immunoabsorbant.

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Typical separation mediums for liquid column chromatography include silica gel, alumina, and cellulose powder. Liquid chromatography can be carried out under a relatively high pressure is referred to as high performance liquid chromatography (HPLC). HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases. The technique in which the stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) is called normal phase liquid chromatography (NPLC) and the opposite (e.g. water-methanol mixture as the mobile phase and C 18 = octadecylsilyl as the stationary phase) is called reversed phase liquid chromatography (RPLC).

EXPERIMENTAL

Materials

Trastuzumab was purchased from the Winship Cancer Institute pharmacy and dissolved in sterile water at a stock concentration of 20 mg/ml. LY294002 PI3K inhibitor (EMD Biosciences; Gibbstown, NJ) was dissolved in DMSO to a final concentration of lOmM. PD0325901 MEK inhibitor (Cayman Chemical; Ann Arbor, MI) was dissolved in DMSO to a final concentration of 2mM. Rapamycin mTOR inhibitor (Sigma- Aldrich; St. Louis, MO) was supplied as a 2.74mM solution in DMSO. SB203580 p38MAPK inhibitor (Sigma-Aldrich) was dissolved in DMSO. Recombinant human GDF15 (rhGDF15; R&D Systems; Minneapolis, MN) was dissolved in 4mM HC1. Control shRNA in pCMV6 plasmid, GDF 15 -specific shRNA-204 in pCMV6 plasmid, and empty pCMV6 vector were purchased from Origene (Rockville, MD).

Cell culture

SKBR3, BT474, HCC1419, HCC1954, MDA-MB-361, and MDA-MB-453 HER2-overexpressing breast cancer cells (all purchased from American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. Trastuzumab- resistant cells were derived from SKBR3 and BT474 by maintaining cells in 4 μg/ml trastuzumab for 3 months, at which point surviving pools and clones were selected; all SKBR3- and BT474-derived resistant cells are routinely maintained on 4 μg/ml trastuzumab, and trastuzumab is removed from cultures for 24h prior to performing experiments.

Microarray analysis.

Total RNA was extracted from triplicate cultures of each cell line (RNeasy;

Qiagen, Germantown, MD). RNA was then taken to the Emory University Microarray Core Facility where RNA integrity was confirmed using an Agilent 2100 Bioanalyzer. RNA was labeled using TotalPrep RNA (Ambion; Austin, TX), and hybridized onto Illumina Human Ref-8 v3 Expression BeadChip for analysis of approximately 24,500 well-annotated transcripts. Triplicate cultures were run for each cell line in order to assess the reproducibility of microarray results; for each set of triplicates, r2=0.99, confirming reproducibility of results. Transcripts that were differentially expressed in each resistant pool versus parental cell line were determined using significance analysis of microarrays (SAM), with false discovery rate (FDR) less than 1%.

ELISA

To quantify the amount of GDF 15 released into the media from cells, human

GDF 15 immunoassay (R&D Systems) was used according to the manufacturer's directions. Briefly, sample media was incubated in GDF 15 antibody-coated microplate for 2 hours which was washed 4X and then incubated with GDF 15 antibody conjugated to horseradish peroxidase for 1 hour. After washing 4X the wells were incubated with color reagent (hydrogen peroxide-chromogen mix) for 30 minutes, at which point the stop solution was added. Optical density of each well was determined using a microplate reader set to 450nm. The concentrations were calculated according to the standards supplied with the kit by creating a four parameter logistic curve-fit.

Cell viability and proliferation assays

For viability assays, cells were plated at 3 x 105 per well in 6-well plate format and pre-treated with GDF15 +/- PI3K inhibitor LY294002, mTOR inhibitor rapamycin, MEK inhibitor PD0325901, or p38MAPK inhibitor SB203580 for 48 hours. Control cultures were treated with 4mM HC1 (solvent for rhGDF15) +/- DMSO (solvent for all kinase inhibitor drugs). Trastuzumab (20 μg/mL) was added for an additional 72 hours. Media, rhGDF15, and drugs were renewed every day. Alternatively, cells were transfected with lOOnM p38 siRNA or control siRNA for 24h, and then treated with 20 μg/mL trastuzumab for 72h. Cell survival was measured by trypan blue exclusion assay, in which cells were trypsinized, stained with trypan blue and viable cells were counted under a microscope. For proliferation, 3000 cells were plated per well in 96-well format, and either untreated or treated with 20 μg/mL trastuzumab, which is dissolved in sterile water. Six replicates were run per group. After 6 days of treatment, proliferation was measured by MTS assay as directed by the manufacturer (Promega) protocol.

Anchorage-independent growth. Cells were plated at 15 x 105 in 6-well plate format in lml matrigel (BD Biosciences; Franklin Lakes, NJ) diluted 3: 1 (media:matrigel). The matrigel-cell suspension was allowed to solidify for 2 hours at 37C°. Then 2ml of media containing trastuzumab alone or together with rhGDF15 (lOOng/ml or lOOOng/ml) was added to each well. Media was changed twice a week for approximately 4 weeks.

Photographs were taken with an Olympus 1X50 inverted microscope at 4X magnification. Matrigel was then digested using dispase (BD Biosciences), and viable cells were counted by trypan blue exclusion. Trans fection

Cells were plated at 3 x 105 in 6-well plate format in antibiotic-free media. The next day, cells were transfected with either lOOnM p38 siRNA, lOOnM control siRNA, 1 μg empty vector, 1 μg control shRNA, or 1 μg GDF15 shRNA 204 (Origene) using Lipofectamine 2000 (Invitrogen; Carlsbad, CA). After 48h, cells were lysed for protein for Western blotting or ELIS A. Alternatively, after 24h transfection, cells were treated with trastuzumab for an additional 72h, after which cells were counted by trypan blue exclusion. Western blotting

Cells were lysed in RIPA buffer (Cell Signaling; Danvers, MA) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). Total protein extracts (50 μg) were run on SDS-PAGE and blotted onto nitrocellulose. Blots were probed overnight using the following antibodies at indicated dilutions. HER2 monoclonal Ab-3 (1 : 1000), p- Thrl 80/Tyrl 82 p38 (1 : 1000), and total p38 polyclonal (1 : 1000) were from EMD

Chemicals; from Cell Signaling, p-T308Akt (monoclonal C31E5E) used at 1 :200, and polyclonal antibodies against Akt (1 : 1000), p-Thr202/Tyr204 p42/p44 Erkl/2 (1 : 1000), total p42/p44 Erkl/2 (1 : 1000); from Santa Cruz, polyclonal p-Tyrl248 HER2 (1 : 1000); and β-actin monoclonal AC-15 (Sigma-Aldrich) at 1 : 10,000. Secondary antibodies were chosen according to species of origin of the primary antibody. Protein bands were detected using the Odyssey Imaging System (Li-Cor Biosciences; Lincoln, NE).

Example 1 : Trastuzumab-resistant breast cancer cell lines show increased GDF15 expression Gene microarray analysis was performed on parental SKBR3 and BT474 HER2- overexpressing breast cancer cell lines, a trastuzumab-resistant pool of cells derived from SKBR3, and a trastuzumab-resistant pool of cells derived from BT474. Total RNA was isolated from each cell line in triplicate to ensure reproducibility. For each set of triplicates, r2=0.99, confirming reproducibility of results. Samples were hybridized onto Illumina Human Ref-8 v3 Expression BeadChip containing approximately 24,500 well-annotated transcripts. Differentially expressed transcripts in each resistant pool versus the corresponding parental line were identified using significance analysis of microarrays (SAM) with a false discovery rate less than 1%. For BT474 resistant pool vs. BT474 parental, 1903 genes were differentially regulated (up or down) by 1.5- fold or more; in SKBR3 resistant cells vs. SKBR3 parental, 3207 genes were differentially regulated by 1.5-fold or more. There were 690 genes that were up-regulated by 1.5- fold or more in BT474 resistant cells vs. parental cells, and 1417 genes up-regulated in SKBR3 resistant vs.

parental cells. Among these up-regulated genes, only 218 were up-regulated by 1.5- fold or more in both SKBR3 resistant and BT474 resistant vs. parental cells.

The most highly over-expressed transcript in the resistant pools was PPPlRlB/Darpp-32, which was previously shown to be over-expressed in trastuzumab-resistant cells. The second most highly over-expressed transcript in resistant cells versus parental cells was GDF15 (also called macrophage inhibitory cytokine 1, or MIC-1), which was over-expressed an average of 26-fold in resistant vs. parental cells. To confirm microarray data, real-time PCR analysis of GDF15 transcript levels was performed in BT474 and SKBR3 acquired trastuzumab-resistant cells versus their parental counterparts (Figure 1 A). These results were consistent with microarray data, showing increased GDF15 transcript expression in cells with acquired resistance versus the parental, sensitive cell lines. Since GDF15 is believed to be bioactive as a secreted cytokine, we performed ELISAs for both the endogenous and secreted forms of GDF15 protein in parental and trastuzumab-resistant cells. GDF 15 -specific ELISA (R&D Systems, Inc., Minneapolis, MN) performed on whole cell protein lysates (excluding media) showed that endogenous GDF 15 expression was elevated in BT474 resistant cells by 3- to 20-fold versus BT474 parental cells (depending on the resistant clone), and 50- to 400-fold in SKBR3 resistant clones versus SKBR3 parental cells (Figure IB). In addition, media collected from cells showed that the concentration of secreted GDF15 was 4- fold higher in BT474 resistant clones vs. BT474 parental and 76- to 172- fold higher in SKBR3 resistant cells vs. SKBR3 parental (Figure 1C). Thus, both the secreted and endogenous forms of GDF15 protein were increased in trastuzumab-resistant cells versus trastuzumab-sensitive parental cells. In addition to models of acquired trastuzumab resistance, we examined GDF 15 secretion by ELISA in HER2-overexpressing cell lines that have primary (intrinsic) resistance to trastuzumab. The level of secreted GDF15 in the cell culture media from primary trastuzumabresistant HCC1419, HCC1954, MDA453, and MDA361 cell lines was significantly higher than the level of secreted GDF 15 in SKBR3 and BT474 cells (Figure ID). Thus, multiple human cell line models of acquired and primary trastuzumab resistance showed increased expression of GDF15 relative to trastuzumab-sensitive breast cancer cell lines.

Exmaple 2 GDF 15 reduces response of HER2-overexpressing breast cancer cells to trastuzumab Next, whether increased GDF 15 expression results in reduced sensitivity to trastuzumab was examined. Exposure of HER2-overexpressing SKBR3 or BT474 breast cancer cells to a physiologically relevant concentration of recombinant human GDF 15 (lOng/mL) blocked growth inhibition of a clinically relevant concentration of trastuzumab (20μg/mL) (Figure 2A). In addition, stable GDF15-overexpressing clones were developed from the SKBR3 and BT474 cell lines. Real-time PCR confirmed increased expression of GDF15 in stable clones versus empty vector control clones in both SKBR3 and BT474 cells (Figure 2B). To determine if stable GDF 15 over-expression reduced response to trastuzumab, we treated GDF 15 stable clones or control empty vector clones with 20μg/mL trastuzumab for 72 h. Control SKBR3 and BT474 clones showed significant reduction in growth upon treatment with trastuzumab (Figure 2C). In contrast, GDF15 stable clones showed reduced sensitivity to trastuzumab. These results suggest that increased exposure to exogenous GDF 15 or increased expression of endogenous GDF 15 reduces the sensitivity of HER2-overexpressing breast cancer cells to trastuzumab. Exmaple 3. GDF15-mediated phosphorylation of HER2 reduces trastuzumab sensitivity

GDF15/MIC-1 stimulates phosphorylation of HER2 in SKBR3 HER2 overexpressing breast cancer cells. Stimulation of BT474 cells with recombinant human GDF 15 induced phosphorylation of HER2 and downstream Akt and rapid, but transient phosphorylation of Erkl/2 (Figure 3A). Tyrosine kinase inhibition of HER2 using tyrphostin AG879 reduced GDF15- mediated phosphorylation of HER2, Akt, and Erkl/2 (Figure 3B). Similarly, the dual EGFR/HER2 kinase inhibitor lapatinib blocked GDF15-mediated phosphorylation of Akt and Erkl/2 (Figure 3C). These results suggest that GDF15-mediated activation of Akt and Erkl/2 occurs downstream of HER2 activation.

It was determined if phosphorylation of HER2 is the mechanism by which GDF 15 promotes trastuzumab resistance. BT474 cells were treated with trastuzumab in the absence or presence of recombinant human GDF15. Again, GDF 15 blocked the ability of trastuzumab to inhibit growth (Figure 3D). Inhibition of HER2 kinase using AG879 restored trastuzumab sensitivity to GDF15-stimulated cells. Further, the dual EGFR/HER2 kinase inhibitor lapatinib inhibited survival of BT474 cells even in the presence of GDF15 stimulation (Figure 3E).

Cotreatment with lapatinib plus trastuzumab blocked survival of HER2-overexpressing breast cancer cells exposed to GDF 15. Similarly, lapatinib inhibited proliferation of stable GDF15- overexpressing SKBR3 clones at a level comparable to control clone cells (Figure 3F).

Collectively, these results indicate that GDF 15 -mediated trastuzumab resistance is dependent upon phosphorylation of HER2, as HER2 kinase inhibition overcomes GDF 15 -mediated resistance.

Example 4 TGF beta receptor-dependent Src phosphorylation contributes to GDF 15 -mediated resistance

GDF15 induces Src-dependent phosphorylation of HER2. In addition GDF15 shares structural homology with TGF beta. Thus, whether TGF beta receptor and Src signaling are required for GDF15-mediated trastuzumab resistance was examined. Stimulation of BT474 cells with recombinant human GDF 15 induced phosphorylation of Smad2 (Figure 4A). Inhibition of TGF beta receptor type II using SB431542 resulted in reduced GDF 15-mediated Smad2 phosphorylation. Thus, GDF 15 activated TGF beta receptor signaling in HER2-overexpressing breast cancer cells. GDF 15 also induced phosphorylation of Src in BT474 cells (Figure 4B). Addition of trastuzumab reduced GDF 15-mediated Src phosphorylation (Figures 4B and 4C). Inhibition of TGF beta receptor with SB431542 further reduced GDF15-mediated Src phosphorylation (Figure 4B). However, lapatinib did not block GDF 15-mediated Src

phosphorylation (Figure 4C), suggesting that GDF 15 -mediated HER2 phosphorylation does not regulate Src phosphorylation. These results indicate that GDF 15 activates TGF beta receptor signaling, which in turn induces Src phosphorylation. Next, survival of BT474 control and GDF15 stable clones in response to trastuzumab combined with TGF beta receptor inhibitor SB431542 or Src inhibitor PP2 was examined.

Trastuzumab-mediated growth inhibition of BT474 control clone cells was not affected by TGF beta receptor inhibition or Src inhibition (Figure 4D). In contrast, trastuzumab sensitivity was increased in GDF15-overexpressing stable clone cells when co-treated with SB431542 or PP2. These results suggest that TGF beta receptor and Src signaling are involved in GDF 15 -mediated trastuzumab resistance, as pharmacologic inhibition of TGF beta receptor or Src abrogated GDF 15 -mediated resistance. Example 5 GDF 15 knockdown increases trastuzumab sensitivity

To determine whether inhibition of GDF 15 improves trastuzumab sensitivity, GDF 15 expression and measured response to trastuzumab in resistant cells was knocked down. BT474 parental and trastuzumab-resistant clone 3 (BT-HRc3) cells and SKBR3 parental and trastuzumab- resistant clone 3 (SK-HRc3) cells were infected with GDF15-specific shRNA in a lentiviral backbone or with corresponding control shRNA in the same lentiviral vector backbone. After 72 h, cells were either untreated or treated with trastuzumab for an additional 72 h. GDF 15 -specific ELISA performed on cell culture media showed 60-90% knockdown of GDF15 in infected cells compared to non-infected cells (Figure 5 A); GDF 15 knockdown was maintained in the presence of trastuzumab. Knockdown of GDF 15 alone reduced growth of SK-HRc3 by 20% and BTHRc3 cells by 50% (Figure 5B). In SK-HRc3 cells, GDF15 knockdown significantly increased trastuzumab sensitivity, with growth inhibition increasing from 20% with trastuzumab alone to 50% when combined with shGDF15. Similarly, BT-HRc3 cells showed significantly reduced cell survival with combination shGDF15 and trastuzumab versus trastuzumab alone; the majority of growth inhibition in BT-HRc3 cells appeared to be due to knockdown of GDF15. These experiments suggest that inhibition of GDF 15 expression reduces cell survival and improves trastuzumab sensitivity in cells that have acquired trastuzumab resistance. In addition, HCC1419 cells, which show primary resistance to trastuzumab and secrete high levels of GDF 15, showed improved sensitivity to trastuzumab with knockdown of GDF 15 versus control shRNA (Figure 5C). Hence, GDF 15 knockdown is a potential strategy for improving response to trastuzumab in HER2-overexpressing breast cancer cells that have acquired resistance to trastuzumab or are trastuzumab -naive but fail to respond to single agent trastuzumab.

Claims

CLAIMS What is claimed:

1. A method of determining a chemotherapy regiment comprising assaying a sample from a subject diagnosed with cancer for elevated GDF15 expression and correlating elevated GDF15 expression in the sample with a resistance to a chemotherapy comprising a HER2 antibody.

2. The method of Claim 1, wherein the HER2 antibody is trastuzumab.

3. The method of Claim 1, wherein the subject is diagnosed with breast cancer.

4. The method of Claim 1, wherein the subject is diagnosed to overexpress HER2.

5. The method of Claim 1, further comprising the step of reporting GDF15 expression to the subject or a medical professional.

6. The method of Claim 1, wherein the assaying comprises the steps of measuring GDF15 in the sample, providing a detect amount of GDF15, and comparing the detected amount of GDF15 to an amount of GDF15 typically found in a normal subject.

7. The method of Claim 1, wherein the assaying comprises the steps of detecting expression of GDF15 mRNA in the sample, and comparing the detected amount of GDF15 mRNA with an amount of GDF15 mRNA typically found in a normal subject.

8. The method of Claim 1, wherein the assaying comprises, combining the sample with oligonucleotides that hybridize to GDF15 mRNA and comparing the detected amount of GDF15 mRNA with an amount of GDF15 mRNA typically found in a normal subject.

9. The method of Claim 1, wherein assaying comprises moving the sample through separation medium and detecting GDF15 protein or mRNA.

10. The method of Claim 1, wherein the assaying comprises, combining the sample and markers with affinity for a GDF15; measuring markers in the marker bound sample; and comparing the markers in the marker bound sample to those typically found with non- elevated GDF15 expression.

11. The method of Claim 10, wherein the markers are antibodies or aptamers for GDF15 protein.

12. The method of Claim 10, wherein the markers are fluorescent.

13. A method of treating cancer comprising administering a pharmaceutical composition comprising an HER2 antibody in combination with an agent that down regulates extracellular mediated GDF15 effects to a subject diagnosed with a HER2 over expressing tumor.

14. The method of Claim 13, wherein the agent is an antibody of GDF15.

15. The method of Claim 13, wherein the agent is a Src inhibitor, TGF beta receptor inhibitor, PI3K inhibitor, or mTOR inhibitor.

16. The method of Claim 14, wherein the agent is an aptamers GDF 15.

17. The method of Claim 14, wherein the agent is a nucleic acid interrupts expression of GDF 15 transcription.

18. The method of Claim 17, wherein the nucleic acid is siRNA of GDF15.

19. The method of Claim 14, wherein the agent is a recombinant vector that encodes a nucleic acid interrupts expression of GDF 15 transcription.

20. The method of Claim 18, wherein the nucleic acid is siRNA of GDF15.

21. A pharmaceutical composition comprising an antibody of GDF 15.

22. A pharmaceutical composition of Claim 21, further comprising a second active ingredient.

23. The pharmaceutical composition of Claim 22, wherein the second active ingredient is a PI3K inhibitor, mTOR inhibitor, Src inhibitor, TGF beta receptor inhibitor, or an HER2 antibody.

24. A method of treating cancer comprising administering a pharmaceutical composition comprising an antibody of GDF15 to a subject diagnosed with a HER2 over expressing tumor.

25. The method of Claim 24, wherein the antibody of GDF15 is administered in combination with a second active ingredient.

26. The method of Claim 25, wherein the second active ingredient is a PI3K inhibitor, mTOR inhibitor, Src inhibitor, TGF beta receptor inhibitor, or an HER2 antibody.

27. A method of determining a chemotherapy treatment comprising assaying a sample from a subject diagnosed with cancer for elevated GDF15 expression, determining that GDF15 expression in the sample is not elevated, and administering chemotherapeutic regiment comprising a HER2 antibody therapy.

28. A method of determining a chemotherapy treatment comprising assaying a sample from a subject diagnosed with cancer for elevated GDF15 expression, determining that GDF15 expression in the sample is elevated, and administering an agent that down regulates extracellular mediated GDF15 effects.

29. The method of Claim 28, wherein the agent is a GDF15 antibody, Src inhibitor, TGF beta receptor inhibitor, a PI3K inhibitor and/or a mTOR inhibitor.