- Email: [email protected]
413 Targeting HdmX as a Therapeutic Approach for Cancer
Poster Session – Signal Transduction Modulators 410 POSTER Circumventing Paradoxical Drug-induced Activation of the MAPK Pathway with Next Generation B-RAF V600E Inhibitors J.M. Vernier1 , D. Matthews2 , P. Pearson3 , S. Hershenson4 , J. Freddo5 , A.J. Giaccia6 , R. Tabibiazar7 , P.M. O’Connor8 . 1 Ruga Corporation, Chemistry, Palo Alto California, USA; 2 Ruga Corporation, Crystallography, Palo Alto California, USA; 3 Ruga Corporation, Preclinical Development, Palo Alto California, USA; 4 Ruga Corporation, Pharmaceutics, Palo Alto California, USA; 5 Ruga Corporation, Clinical Development, Palo Alto California, USA; 6 Stanford University School of Medicine, Department of Radiation Oncology, Stanford California, USA; 7 Ruga Corporation, CEO, Palo Alto California, USA; 8 Ruga Corporation, Research & Development, Palo Alto California, USA B-RAF is commonly mutated in a wide variety of different tumors including melanoma, thyroid cancer, ovarian cancer and colorectal cancer. The most common mutation (V600E) constitutively activates kinase activity and drives the growth and survival of cells harboring this mutation. First generation B-RAF inhibitors have demonstrated significant activity in patients with metastatic melanoma expressing B-RAF V600E mutations (e.g., PLX4032, Vemurafinib). However, several laboratories have noted drug-induced adaptation of the MAP kinase pathway leading to paradoxical activation of the downstream kinase ERK in cells with wild-type B-RAF. This druginduced adaptation is most notable in cells harboring activated RAS, and could underlie both the accelerated growth of RAS activated tumor cells in vitro and in vivo as well as the promotion of secondary lesions (e.g., keratoacanthomas and squamous cell carcinomas), seen in patients on first generation B-RAF inhibitor therapy. Using a structure-based drug design approach we set out to design novel agents capable of potently and selectively inhibiting B-RAF V600E while lacking drug-induced activation of the MAP kinase pathway. Leveraging crystallography we evolved several series of novel pyridine-containing compounds that were highly potent and selective to the RAF family kinases while avoiding significant inhibition of other kinases including the type III receptor-tyrosine kinases (e.g., VEGFR2, PDGFR). Assessment of these compounds in A375 (B-RAF V600E) melanoma cells showed as expected dose-dependent inhibition of pERK and concomitant growth inhibition. However, in contrast to PLX4032, that induced paradoxical activation of the MAP kinase pathway in HCT-116 (K-RAS G13D) colorectal cancer cells, these compounds showed no significant pERK induction or indeed in some cases a reduction in basal pERK levels. These next generation B-RAF inhibitors lacking drug-induced adaptation of the MAP kinase pathway will be described in anticipation of future clinical studies targeting cancer patients exhibiting activation of the MAP kinase pathway.
Thursday 8 November 2012 125 activate downstream TGFb signaling through upregulation of p-Smad3 and TGFb transcriptional targets. Upon further exploration, we also find that the miR-106b-25 cluster is sufficient to induce EMT-like features as well as an increase in the TIC population, and that these miRNA are required downstream of Six1 to induce these phenotypes. Lastly, we also demonstrate a significant correlation of Six1 and miR-106b expression in human breast cancers, and further show that high expression of miR-106b or miR-93 correlates to shortened time to relapse in breast cancer patients. All together, we find that the Six1-mediated increase in the miR-106b-25 cluster may not only be involved in facilitating the oncogenic switch of TGFb, but also aids in the activation of this pathway, thus contributing to the pro-metastatic phenotypes found with Six1 overexpression. Because it is difficult to target transcription factors like Six1 directly, our data points to a new therapeutic target for Six1 expressing breast cancers (miR-106b-25), which may also be used to predict which patients should benefit, rather than be harmed, by TGFb inhibitors which are currently in clinical trials. 412 POSTER Antitumor Activity of BM-1197, a Dual Inhibitor of Bcl-2 and Bcl-xL, in Small Cell Lung Cancer Tumors L. Bai1 , J. Chen1 , D. McEachern1 , L. Liu1 , H. Zhou1 , A. Aguilar1 , C.Y. Yang1 , J.L. Meagher2 , J.A. Stuckey2 , S. Wang1 . 1 University of Michigan, Comprehensive Cancer Center, Ann Arbor, USA; 2 University of Michigan, Life Science Institute, Ann Arbor, USA Bcl-2 family proteins are central regulators of apoptosis. Overexpression of anti-apoptotic Bcl-2 and Bcl-xL proteins is frequently found in small cell lung cancer (SCLC) where high Bcl-2 expression is also associated with poor prognosis. Thus targeting Bcl-2 and Bcl-xL may provide therapeutic benefit in SCLC. Using a computational structure-based design strategy, we have developed a new class of Bcl-2 and Bcl-xL dual inhibitor BM1197, which binds to Bcl-2 and Bcl-xL with Ki <1 nM. Cell viability assays revealed that BM-1197 exerts potent growth-inhibitory activities in a panel of SCLC cell lines with IC50 values in the nanomolar range. The antitumor activity of BM-1197 was evaluated using mouse xenograft models of human SCLC cell lines. BM-1197, at 10 mg/kg daily IV dosing for 5 days, achieved long-lasting complete tumor regression in multiple SCLC xenograft models without affecting body weights. Remarkably, a single dose of BM-1197 at 15 mg/kg achieved complete and sustained tumor regression in NCIH146 and NCI-H1963 xenograft models. Our preliminary results suggest the therapeutic potential of this new class of Bcl-2/Bcl-xL dual inhibitor in human SCLC. 413 Targeting HdmX as a Therapeutic Approach for Cancer
POSTER
411 POSTER The miR-106b-25 Cluster as a Potential Target for Six1-mediated Breast Cancer Metastasis, and a Molecular Marker of the TGFb Paradox
D. Wald1 , M. Agarwal2 , A. Chakrabarti2 . 1 Case Western Reserve University, Cleveland, USA; 2 Invenio Therapeutics, Research, Cleveland, USA
A.L. Smith1 , R. Iwanaga1 , D.J. Drasin1 , D.S. Micalizzi1 , R.L. Vartuli1 , H.L. Ford1 . 1 University of Colorado Anschutz Medical Campus, Pharmacology, Denver Colorado, USA
It is well accepted that loss of the p53 signaling pathway, either by mutation or loss of upstream or downstream signaling components, occurs in the vast majority of human cancers. Loss of p53 eliminates a number of barriers that prevent transformation, including protective apoptotic or arrest signals (depending upon cell type) and repression of genes involved in angiogenesis and metastasis. Hdm2 and HdmX coordinately regulate the stability and function of p53 in normal cells; however, each is overexpressed in subsets of many different types of malignancy. In fact, HdmX overexpression was reported to be as high as 20% in breast, colon and lung cancers. Recently, several compounds such as Nutlin have been reported that disrupt the p53–Hdm2 interaction and lead to p53-dependent toxicity in tumor cell lines and xenograft tumors in mice. Unfortunately Hdm2-p53 inhibitors such as Nutlin-3 efficiently induce apoptosis and diminishes long term survival of human cells transformed in vitro by Hdm2 but not HdmX. The resistance of cells overexpressing HdmX to Nutlin3 is due to its inability to disrupt the p53–HdmX interaction, resulting in continued suppression of p53 activity. In order to identify novel inhibitors capable of disrupting the p53–HdmX interaction a cell-based readout suitable for high throughput screening of compounds that activate a p53dependent luciferase reporter in HdmX-overexpressing MCF7 cells was established. These cells are highly refractory to the Hdm2 inhibitor Nutlin3 due to high levels of HdmX expression. Over 20,000 small molecules were screened and 6 compounds were identified that reproducibly activated p53-dependent luciferase activity 5-fold or greater. From these hits, one compound, CLTX1, shows specificity in targeting HdmX and synergizes with Hdm2 inhibition. We determined that CLTX1 is specific for HdmX as opposed to Hdm2 using an in vitro transformation model in which p53 was suppressed by HdmX overexpression, Hdm2 overexpression, or an shRNA targeting p53. CLTX1 induced significant p53-dependent apoptosis in HdmX-expressing cells, but not cells in which p53 is inactivated by Hdm2
Previous work in our laboratory has demonstrated a critical role for the homeoprotein Six1 in breast cancer metastasis. Our data has shown that Six1 controls metastatic spread through an upregulation of TGFb signaling, leading to an EMT-like transformation and an increase in the tumor initiating cell (TIC) population. TGFb is a multifunctional cytokine, which can function as either a tumor suppressor or a tumor promoter, making this pathway a difficult target in cancer therapy. Additionally, our data shows that the presence of Six1 in cells will implement the switch of TGFb from tumor suppressive to tumor promotional. To further understand the mechanism by which Six1 mediates metastasis through TGFb signaling, we explored miRNAs that may be involved in this process. Through a miRNA microarray and further validation studies, we found that Six1 upregulates a cluster of three miRNAs, the miR-106b-25 cluster, which is comprised of miR-106b, miR-93, and miR25. Interestingly, this cluster has already been implicated in the impairment of TGFb growth suppression through repression of the cell cycle inhibitor, p21, and proapoptotic factor, Bim. Therefore suggesting that upregulation of these miRNA by Six1 may help mediate the switch in TGFb signaling. Indeed, preliminary results in NMuMG (normal mouse mammary gland) cells and BT549 breast cancer cells demonstrates that inhibition of the miR-106b25 miRNAs in the context of Six1 overexpression restores TGFb-mediated growth inhibition. In addition to its role in TGFb growth suppression, we have also recently identified that this same cluster of miRNA is capable of activating the tumor promotional functions of TGFb. We have shown that these miRNA bind and repress the TGFb inhibitor, Smad7, and
126 Thursday 8 November 2012 overexpression or shRNA. Further biochemical analysis demonstrated that unlike doxorubicin, which causes DNA damage mediated phosphorylation and stabilization of p53 and phosphorylation of H2AX, CLTX1 is a potent HdmX-specific inhibitor capable of rapidly stabilizing p53 in a DNA damage independent manner. An in vitro ELISA using recombinant p53, HdmX or Hdm2 demonstrated that CLTX1 disrupted HdmX-p53, but not Hdm2– p53 interactions. Moreover, while CLTX1 exhibits some anti-cancer activity alone when used to treat wild-type p53 expressing cell lines, particularly those that do not overexpress Hdm2, we have observed that the combined inhibition of HdmX (with CLTX1) and Hdm2 (with Nutlin 3) synergize to induce apoptosis. Overall, CLTX1 is a promising new anti-cancer agent that is capable of targeting cancer cells through the induction of p53 in a non-genotoxic manner. 414 POSTER The Anti-tumor Activity of the ERBB3 Inhibitory Antibody AV-203 in Patient Derived Tumor Explant Models K. Meetze1 , S. Tyler1 , K. Clark1 , E. Mazsa1 , A. Delpero1 , J. Gyuris1 , S. Vincent1 . 1 AVEO Pharmaceuticals Inc., Drug Discovery, Cambridge MA, USA The ERBB3 receptor is broadly expressed on cancers of the head and neck, lung, breast, ovaries, prostate, colon, pancreas, and gastrointestinal tract. Its expression is often linked to poor prognosis. In addition, it has been implicated in the development of resistance to other receptor-targeted tyrosine kinase inhibitors and radiotherapy. ERBB3 lacks detectable tyrosine kinase activity and its activation requires the association with RTK partners, such asHER2, EGFR or MET. The ERBB3 ligand, Neuregulin 1 (NRG1), promotes the engagement of ERBB3 in heterodimer complexes. AV-203 is a humanized IgG1 antibody directed against ERBB3. AV-203 potently inhibits the ligand-dependent or ligand-independent activation of ERBB3. AV-203 was shown to inhibit the binding of NRG1 to ERBB3 and promote ERBB3 degradation. The tumor growth inhibitory activity of AV-203 was demonstrated in numerous xenograft models representing major human carcinomas (breast, lung, kidney and pancreas). AVEO has developed a collection of patient derived tumor explant models from over 12 tissue types through serial propagation of the tumor material in immunecompromised mice. The anti-tumor activity of AV-203 was tested in a broad panel of these tumors, including lung, breast, ovarian, colon and head and neck tumors, all expressing varying levels of ERBB3. Anti-tumor activity was demonstrated in these tumors across the tissue types and ranged within each tumor type from lack of response to durable tumor growth inhibition. The variations in tumor growth will be used to identify potential mechanisms of response and resistance to AV-203. These potential biomarkers will be further evaluated using tumor materials from patients enrolled in AV-203 clinical trials with known responses to AV-203 treatment. AV-203 is currently in Phase I development for the treatment of solid tumors. 415 POSTER Investigating the Role of HER4 in Relation to Trastuzumab Treatment and Resistance in HER2 Positive Breast Cancer S. Mohd Nafi1 , A. Kong1 , M. Gijsen1 , G. Kramer-Marek2 , J. Capala2 . 1 University of Oxford, Epidermal Growth Factor Group (Weatherall Institute of Molecular Medicine), Oxford, United Kingdom; 2 National Cancer Institute, Radiation Oncology Branch (Centre for Cancer Reseach), Maryland, USA Background: Trastuzumab resistance imposes a crucial limitation to the successful treatment of HER2 positive breast cancer. The role of HER4 in relation to prognosis of various subtypes of breast cancer is controversial. Furthermore, its role in relation to Trastuzumab treatment and resistance in HER2 positive breast cancer has not been reported. The aim of study is to investigate the role of HER4 in relation to Trastuzumab treatment and resistance in HER2 positive breast cancer. Methods: The level of HER4 was assessed in HER2 positive breast cancer cell lines (SKBR3 and BT474) compared to the HER2 negative cell line (MCF-7). The cells were treated with different doses and durations of Trastuzumab and the level of HER4 protein and mRNA were analysed using western blot and RT-PCR. HER4 localisation after Trastuzumab treatment was determined by confocal microscopy and nuclear fractionation. In addition, the level of HER4 expression and its localisation were also compared in the Trastuzumab sensitive and resistant SKBR3 and BT474 cells. The response of Trastuzumab in HER4 cleavage was also studied using a Gamma-secretase inhibitor and Neratinib. The effect of HER4 was further validated using siRNA methodology in both sensitive and Trastuzumab resistant cells. HER4 expression and its localization were also assessed by immunohistochemistry in BT474 xenograft samples treated with Trastuzumab in comparison to control.
Poster Session – Signal Transduction Modulators Results: The basal level of HER4 in both SKBR3 and BT474 is comparable to the expression in HER2 negative-MCF7 cells. In dose responsive manner, Trastuzumab treatment upregulated HER4 mRNA and protein expression. Moreover, at higher dosages, more HER4 m80 was found, indicating that Trastuzumab also promotes HER4 cleavage. In SKBR3, acute Trastuzumab treatment increased nuclear intensity HER4 and caused co-localisation of HER4 and HER2 at the cytoplasm. In Trastuzumabresistant cell lines, more HER4 was detected in the nucleus compared to the sensitive SKBR3. However, there was a decrease in nuclear HER4 localisation upon overnight withdrawal of Trastuzumab in resistant cell lines. In addition, knockdown of HER4 increased the sensitivity to Trastuzumab in SKBR3 and BT474 cells. There was also an additive inhibition effect on cell viability by Trastuzumab with a Gamma-secretase inhibitor that prevents HER4 cleavage, confirming the importance of HER4 in Trastuzumab treatment. Furthermore, combination Trastuzumab with Neratinib, a tyrosine kinase inhibitor to HER4 also showed the similar effect on cell viability. We further showed that there was increased HER4 expression and nuclear localisation in BT474 xenograft samples treated with Trastuzumab compared to control. Conclusions: This preliminary data suggest an important role of HER4 in relation to Trastuzumab treatment and resistance in HER2 positive breast cancer although further validation is required. The role of HER4 expression and nuclear HER4 in relation to prognosis and resistance to Trastuzumab in HER2 positive breast cancer patients will be further established by carrying out further IHC experiments in human TMAs samples from retrospective samples and prospective trials. 416 POSTER XGFR* is a Novel Tri-functional Anti-EGFR/IGF-1R Antibody Blocking Key Tumor Signaling Pathways and Activating Host Immune Functions for Targeted Treatment of Pancreatic Cancer T. Friess1 , J.M. Schanzer1 , E. Ruge1 , N. Dimoudis1 , S. Hoves1 , C.H. Ooi1 , T. Otz2 , M. Bacac2 , M. Weisser1 , K. Wartha1 . 1 Roche Diagnostics GmbH, pRED, Penzberg, Germany; 2 Roche Glycart AG, pRED, Schlieren, Switzerland A common characteristic of many cancers is elevated signaling via IGF1R and EGFR tyrosine kinases as a common characteristic thus enabling IGF-1R and EGFR signaling predominantly through the PI3K and MAPK pathways. Furthermore, activation of this dual signaling pathway has been reported as an important mechanism for tumor escape / resistance after anti-EGFR or anti-HER2 directed treatments resulting in a poor prognosis for affected patients. However, phase II clinical trials in unselected CRC patients treated with a combination of anti-EGFR and anti-IGF-1R antibodies have been disappointing so far. Here we present XGFR*, a novel tri-functional bispecific antibody targeting EGFR and IGF-1R signal transduction pathways in combination with increased immune-effector functions (ADCC) due to glycoengineering of the Fc region. The novel bispecific antibody was engineered using the knob-into-hole technology for heterodimerization of an anti-EGFR Mab (GA201) and a one-arm single chain Fab of an IgG1 affinity matured IGF-1R Mab (F13B5). ADCC properties of XGFR* have been significantly enhanced by reducing Fc-fucosylation using GlycoMab® technology. In vitro testing of EGFR/IGF-1R phosphorylation, receptor down-regulation, inhibition of 3D proliferation and ADCC induction revealed superior efficacy of XGFR* compared to the combination of an anti-EGFR antibody with an anti-IGF-1R antibody. XGFR* displayed strong anti-tumor efficacy in several in vivo preclinical xenograft models dependent on tumor growth inhibition by blocking receptor signaling. In addition XGFR* also exhibited superior efficacy in preclinical orthotopic ADCC competent models when compared to an EGFR/IGF-1R antibody combination. In contrast to CRC (colorectal carcinoma), PaC (pancreatic carcinoma) derived cell lines responded strongly to EGFR/IGF-1R signaling inhibition. Furthermore elevated of EGFR/IGF-1R (co-)expression levels were observed in PaC cell lines and primary PaC tissue compared to CRC. Consequently, these observations suggest that PaC might be a promising indication for XGFR* as EGFR/IGF-1R signaling pathways are drivers of PaC tumors and expression levels of EGFR/IGF-1R are high, favoring induction of ADCC. In conclusion, our novel tri-functional bispecific XGFR* antibody, optimized for targeting EGFR and IGF-1R simultaneously as well as induction of ADCC should be a promising new therapeutic candidate for the treatment of PaC patients.
Thursday 8 November 2012 125 activate downstream TGFb signaling through upregulation of p-Smad3 and TGFb transcriptional targets. Upon further exploration, we also find that the miR-106b-25 cluster is sufficient to induce EMT-like features as well as an increase in the TIC population, and that these miRNA are required downstream of Six1 to induce these phenotypes. Lastly, we also demonstrate a significant correlation of Six1 and miR-106b expression in human breast cancers, and further show that high expression of miR-106b or miR-93 correlates to shortened time to relapse in breast cancer patients. All together, we find that the Six1-mediated increase in the miR-106b-25 cluster may not only be involved in facilitating the oncogenic switch of TGFb, but also aids in the activation of this pathway, thus contributing to the pro-metastatic phenotypes found with Six1 overexpression. Because it is difficult to target transcription factors like Six1 directly, our data points to a new therapeutic target for Six1 expressing breast cancers (miR-106b-25), which may also be used to predict which patients should benefit, rather than be harmed, by TGFb inhibitors which are currently in clinical trials. 412 POSTER Antitumor Activity of BM-1197, a Dual Inhibitor of Bcl-2 and Bcl-xL, in Small Cell Lung Cancer Tumors L. Bai1 , J. Chen1 , D. McEachern1 , L. Liu1 , H. Zhou1 , A. Aguilar1 , C.Y. Yang1 , J.L. Meagher2 , J.A. Stuckey2 , S. Wang1 . 1 University of Michigan, Comprehensive Cancer Center, Ann Arbor, USA; 2 University of Michigan, Life Science Institute, Ann Arbor, USA Bcl-2 family proteins are central regulators of apoptosis. Overexpression of anti-apoptotic Bcl-2 and Bcl-xL proteins is frequently found in small cell lung cancer (SCLC) where high Bcl-2 expression is also associated with poor prognosis. Thus targeting Bcl-2 and Bcl-xL may provide therapeutic benefit in SCLC. Using a computational structure-based design strategy, we have developed a new class of Bcl-2 and Bcl-xL dual inhibitor BM1197, which binds to Bcl-2 and Bcl-xL with Ki <1 nM. Cell viability assays revealed that BM-1197 exerts potent growth-inhibitory activities in a panel of SCLC cell lines with IC50 values in the nanomolar range. The antitumor activity of BM-1197 was evaluated using mouse xenograft models of human SCLC cell lines. BM-1197, at 10 mg/kg daily IV dosing for 5 days, achieved long-lasting complete tumor regression in multiple SCLC xenograft models without affecting body weights. Remarkably, a single dose of BM-1197 at 15 mg/kg achieved complete and sustained tumor regression in NCIH146 and NCI-H1963 xenograft models. Our preliminary results suggest the therapeutic potential of this new class of Bcl-2/Bcl-xL dual inhibitor in human SCLC. 413 Targeting HdmX as a Therapeutic Approach for Cancer
POSTER
411 POSTER The miR-106b-25 Cluster as a Potential Target for Six1-mediated Breast Cancer Metastasis, and a Molecular Marker of the TGFb Paradox
D. Wald1 , M. Agarwal2 , A. Chakrabarti2 . 1 Case Western Reserve University, Cleveland, USA; 2 Invenio Therapeutics, Research, Cleveland, USA
A.L. Smith1 , R. Iwanaga1 , D.J. Drasin1 , D.S. Micalizzi1 , R.L. Vartuli1 , H.L. Ford1 . 1 University of Colorado Anschutz Medical Campus, Pharmacology, Denver Colorado, USA
It is well accepted that loss of the p53 signaling pathway, either by mutation or loss of upstream or downstream signaling components, occurs in the vast majority of human cancers. Loss of p53 eliminates a number of barriers that prevent transformation, including protective apoptotic or arrest signals (depending upon cell type) and repression of genes involved in angiogenesis and metastasis. Hdm2 and HdmX coordinately regulate the stability and function of p53 in normal cells; however, each is overexpressed in subsets of many different types of malignancy. In fact, HdmX overexpression was reported to be as high as 20% in breast, colon and lung cancers. Recently, several compounds such as Nutlin have been reported that disrupt the p53–Hdm2 interaction and lead to p53-dependent toxicity in tumor cell lines and xenograft tumors in mice. Unfortunately Hdm2-p53 inhibitors such as Nutlin-3 efficiently induce apoptosis and diminishes long term survival of human cells transformed in vitro by Hdm2 but not HdmX. The resistance of cells overexpressing HdmX to Nutlin3 is due to its inability to disrupt the p53–HdmX interaction, resulting in continued suppression of p53 activity. In order to identify novel inhibitors capable of disrupting the p53–HdmX interaction a cell-based readout suitable for high throughput screening of compounds that activate a p53dependent luciferase reporter in HdmX-overexpressing MCF7 cells was established. These cells are highly refractory to the Hdm2 inhibitor Nutlin3 due to high levels of HdmX expression. Over 20,000 small molecules were screened and 6 compounds were identified that reproducibly activated p53-dependent luciferase activity 5-fold or greater. From these hits, one compound, CLTX1, shows specificity in targeting HdmX and synergizes with Hdm2 inhibition. We determined that CLTX1 is specific for HdmX as opposed to Hdm2 using an in vitro transformation model in which p53 was suppressed by HdmX overexpression, Hdm2 overexpression, or an shRNA targeting p53. CLTX1 induced significant p53-dependent apoptosis in HdmX-expressing cells, but not cells in which p53 is inactivated by Hdm2
Previous work in our laboratory has demonstrated a critical role for the homeoprotein Six1 in breast cancer metastasis. Our data has shown that Six1 controls metastatic spread through an upregulation of TGFb signaling, leading to an EMT-like transformation and an increase in the tumor initiating cell (TIC) population. TGFb is a multifunctional cytokine, which can function as either a tumor suppressor or a tumor promoter, making this pathway a difficult target in cancer therapy. Additionally, our data shows that the presence of Six1 in cells will implement the switch of TGFb from tumor suppressive to tumor promotional. To further understand the mechanism by which Six1 mediates metastasis through TGFb signaling, we explored miRNAs that may be involved in this process. Through a miRNA microarray and further validation studies, we found that Six1 upregulates a cluster of three miRNAs, the miR-106b-25 cluster, which is comprised of miR-106b, miR-93, and miR25. Interestingly, this cluster has already been implicated in the impairment of TGFb growth suppression through repression of the cell cycle inhibitor, p21, and proapoptotic factor, Bim. Therefore suggesting that upregulation of these miRNA by Six1 may help mediate the switch in TGFb signaling. Indeed, preliminary results in NMuMG (normal mouse mammary gland) cells and BT549 breast cancer cells demonstrates that inhibition of the miR-106b25 miRNAs in the context of Six1 overexpression restores TGFb-mediated growth inhibition. In addition to its role in TGFb growth suppression, we have also recently identified that this same cluster of miRNA is capable of activating the tumor promotional functions of TGFb. We have shown that these miRNA bind and repress the TGFb inhibitor, Smad7, and
126 Thursday 8 November 2012 overexpression or shRNA. Further biochemical analysis demonstrated that unlike doxorubicin, which causes DNA damage mediated phosphorylation and stabilization of p53 and phosphorylation of H2AX, CLTX1 is a potent HdmX-specific inhibitor capable of rapidly stabilizing p53 in a DNA damage independent manner. An in vitro ELISA using recombinant p53, HdmX or Hdm2 demonstrated that CLTX1 disrupted HdmX-p53, but not Hdm2– p53 interactions. Moreover, while CLTX1 exhibits some anti-cancer activity alone when used to treat wild-type p53 expressing cell lines, particularly those that do not overexpress Hdm2, we have observed that the combined inhibition of HdmX (with CLTX1) and Hdm2 (with Nutlin 3) synergize to induce apoptosis. Overall, CLTX1 is a promising new anti-cancer agent that is capable of targeting cancer cells through the induction of p53 in a non-genotoxic manner. 414 POSTER The Anti-tumor Activity of the ERBB3 Inhibitory Antibody AV-203 in Patient Derived Tumor Explant Models K. Meetze1 , S. Tyler1 , K. Clark1 , E. Mazsa1 , A. Delpero1 , J. Gyuris1 , S. Vincent1 . 1 AVEO Pharmaceuticals Inc., Drug Discovery, Cambridge MA, USA The ERBB3 receptor is broadly expressed on cancers of the head and neck, lung, breast, ovaries, prostate, colon, pancreas, and gastrointestinal tract. Its expression is often linked to poor prognosis. In addition, it has been implicated in the development of resistance to other receptor-targeted tyrosine kinase inhibitors and radiotherapy. ERBB3 lacks detectable tyrosine kinase activity and its activation requires the association with RTK partners, such asHER2, EGFR or MET. The ERBB3 ligand, Neuregulin 1 (NRG1), promotes the engagement of ERBB3 in heterodimer complexes. AV-203 is a humanized IgG1 antibody directed against ERBB3. AV-203 potently inhibits the ligand-dependent or ligand-independent activation of ERBB3. AV-203 was shown to inhibit the binding of NRG1 to ERBB3 and promote ERBB3 degradation. The tumor growth inhibitory activity of AV-203 was demonstrated in numerous xenograft models representing major human carcinomas (breast, lung, kidney and pancreas). AVEO has developed a collection of patient derived tumor explant models from over 12 tissue types through serial propagation of the tumor material in immunecompromised mice. The anti-tumor activity of AV-203 was tested in a broad panel of these tumors, including lung, breast, ovarian, colon and head and neck tumors, all expressing varying levels of ERBB3. Anti-tumor activity was demonstrated in these tumors across the tissue types and ranged within each tumor type from lack of response to durable tumor growth inhibition. The variations in tumor growth will be used to identify potential mechanisms of response and resistance to AV-203. These potential biomarkers will be further evaluated using tumor materials from patients enrolled in AV-203 clinical trials with known responses to AV-203 treatment. AV-203 is currently in Phase I development for the treatment of solid tumors. 415 POSTER Investigating the Role of HER4 in Relation to Trastuzumab Treatment and Resistance in HER2 Positive Breast Cancer S. Mohd Nafi1 , A. Kong1 , M. Gijsen1 , G. Kramer-Marek2 , J. Capala2 . 1 University of Oxford, Epidermal Growth Factor Group (Weatherall Institute of Molecular Medicine), Oxford, United Kingdom; 2 National Cancer Institute, Radiation Oncology Branch (Centre for Cancer Reseach), Maryland, USA Background: Trastuzumab resistance imposes a crucial limitation to the successful treatment of HER2 positive breast cancer. The role of HER4 in relation to prognosis of various subtypes of breast cancer is controversial. Furthermore, its role in relation to Trastuzumab treatment and resistance in HER2 positive breast cancer has not been reported. The aim of study is to investigate the role of HER4 in relation to Trastuzumab treatment and resistance in HER2 positive breast cancer. Methods: The level of HER4 was assessed in HER2 positive breast cancer cell lines (SKBR3 and BT474) compared to the HER2 negative cell line (MCF-7). The cells were treated with different doses and durations of Trastuzumab and the level of HER4 protein and mRNA were analysed using western blot and RT-PCR. HER4 localisation after Trastuzumab treatment was determined by confocal microscopy and nuclear fractionation. In addition, the level of HER4 expression and its localisation were also compared in the Trastuzumab sensitive and resistant SKBR3 and BT474 cells. The response of Trastuzumab in HER4 cleavage was also studied using a Gamma-secretase inhibitor and Neratinib. The effect of HER4 was further validated using siRNA methodology in both sensitive and Trastuzumab resistant cells. HER4 expression and its localization were also assessed by immunohistochemistry in BT474 xenograft samples treated with Trastuzumab in comparison to control.
Poster Session – Signal Transduction Modulators Results: The basal level of HER4 in both SKBR3 and BT474 is comparable to the expression in HER2 negative-MCF7 cells. In dose responsive manner, Trastuzumab treatment upregulated HER4 mRNA and protein expression. Moreover, at higher dosages, more HER4 m80 was found, indicating that Trastuzumab also promotes HER4 cleavage. In SKBR3, acute Trastuzumab treatment increased nuclear intensity HER4 and caused co-localisation of HER4 and HER2 at the cytoplasm. In Trastuzumabresistant cell lines, more HER4 was detected in the nucleus compared to the sensitive SKBR3. However, there was a decrease in nuclear HER4 localisation upon overnight withdrawal of Trastuzumab in resistant cell lines. In addition, knockdown of HER4 increased the sensitivity to Trastuzumab in SKBR3 and BT474 cells. There was also an additive inhibition effect on cell viability by Trastuzumab with a Gamma-secretase inhibitor that prevents HER4 cleavage, confirming the importance of HER4 in Trastuzumab treatment. Furthermore, combination Trastuzumab with Neratinib, a tyrosine kinase inhibitor to HER4 also showed the similar effect on cell viability. We further showed that there was increased HER4 expression and nuclear localisation in BT474 xenograft samples treated with Trastuzumab compared to control. Conclusions: This preliminary data suggest an important role of HER4 in relation to Trastuzumab treatment and resistance in HER2 positive breast cancer although further validation is required. The role of HER4 expression and nuclear HER4 in relation to prognosis and resistance to Trastuzumab in HER2 positive breast cancer patients will be further established by carrying out further IHC experiments in human TMAs samples from retrospective samples and prospective trials. 416 POSTER XGFR* is a Novel Tri-functional Anti-EGFR/IGF-1R Antibody Blocking Key Tumor Signaling Pathways and Activating Host Immune Functions for Targeted Treatment of Pancreatic Cancer T. Friess1 , J.M. Schanzer1 , E. Ruge1 , N. Dimoudis1 , S. Hoves1 , C.H. Ooi1 , T. Otz2 , M. Bacac2 , M. Weisser1 , K. Wartha1 . 1 Roche Diagnostics GmbH, pRED, Penzberg, Germany; 2 Roche Glycart AG, pRED, Schlieren, Switzerland A common characteristic of many cancers is elevated signaling via IGF1R and EGFR tyrosine kinases as a common characteristic thus enabling IGF-1R and EGFR signaling predominantly through the PI3K and MAPK pathways. Furthermore, activation of this dual signaling pathway has been reported as an important mechanism for tumor escape / resistance after anti-EGFR or anti-HER2 directed treatments resulting in a poor prognosis for affected patients. However, phase II clinical trials in unselected CRC patients treated with a combination of anti-EGFR and anti-IGF-1R antibodies have been disappointing so far. Here we present XGFR*, a novel tri-functional bispecific antibody targeting EGFR and IGF-1R signal transduction pathways in combination with increased immune-effector functions (ADCC) due to glycoengineering of the Fc region. The novel bispecific antibody was engineered using the knob-into-hole technology for heterodimerization of an anti-EGFR Mab (GA201) and a one-arm single chain Fab of an IgG1 affinity matured IGF-1R Mab (F13B5). ADCC properties of XGFR* have been significantly enhanced by reducing Fc-fucosylation using GlycoMab® technology. In vitro testing of EGFR/IGF-1R phosphorylation, receptor down-regulation, inhibition of 3D proliferation and ADCC induction revealed superior efficacy of XGFR* compared to the combination of an anti-EGFR antibody with an anti-IGF-1R antibody. XGFR* displayed strong anti-tumor efficacy in several in vivo preclinical xenograft models dependent on tumor growth inhibition by blocking receptor signaling. In addition XGFR* also exhibited superior efficacy in preclinical orthotopic ADCC competent models when compared to an EGFR/IGF-1R antibody combination. In contrast to CRC (colorectal carcinoma), PaC (pancreatic carcinoma) derived cell lines responded strongly to EGFR/IGF-1R signaling inhibition. Furthermore elevated of EGFR/IGF-1R (co-)expression levels were observed in PaC cell lines and primary PaC tissue compared to CRC. Consequently, these observations suggest that PaC might be a promising indication for XGFR* as EGFR/IGF-1R signaling pathways are drivers of PaC tumors and expression levels of EGFR/IGF-1R are high, favoring induction of ADCC. In conclusion, our novel tri-functional bispecific XGFR* antibody, optimized for targeting EGFR and IGF-1R simultaneously as well as induction of ADCC should be a promising new therapeutic candidate for the treatment of PaC patients.