Sergio Iadevaia

Identification of Optimal Drug Combinations Targeting Cellular Networks: Integrating Phospho-Proteomics and Computational Network Analysis

Targeted therapeutics hold tremendous promise in inhibiting cancer cell proliferation. However, targeting proteins individually can be compensated for by bypass mechanisms and activation of regulatory loops. Designing optimal therapeutic combinations must therefore take into consideration the complex dynamic networks in the cell. In this study, we analyzed the insulin-like growth factor (IGF-1) signaling network in the MDA-MB231 breast cancer cell line. We used reverse-phase protein array to measure the transient changes in the phosphorylation of proteins after IGF-1 stimulation. We developed a computational procedure that integrated mass action modeling with particle swarm optimization to train the model against the experimental data and infer the unknown model parameters. The trained model was used to predict how targeting individual signaling proteins altered the rest of the network and identify drug combinations that minimally increased phosphorylation of other proteins elsewhere in the network. Experimental testing of the modeling predictions showed that optimal drug combinations inhibited cell signaling and proliferation, whereas nonoptimal combination of inhibitors increased phosphorylation of nontargeted proteins and rescued cells from cell death. The integrative approach described here is useful for generating experimental intervention strategies that could optimize drug combinations and discover novel pharmacologic targets for cancer therapy.

Kinome siRNA-phosphoproteomic screen identifies networks regulating AKT signaling.

To identify regulators of intracellular signaling, we targeted 541 kinases and kinase-related molecules with small interfering RNAs (siRNAs), and determined their effects on signaling with a functional proteomics reverse-phase protein array (RPPA) platform assessing 42 phospho and total proteins. The kinome-wide screen demonstrated a strong inverse correlation between phosphorylation of AKT and mitogen-activated protein kinase (MAPK) with 115 genes that, when targeted by siRNAs, demonstrated opposite effects on MAPK and AKT phosphorylation. Network-based analysis identified the MAPK subnetwork of genes along with p70S6K and FRAP1 as the most prominent targets that increased phosphorylation of AKT, a key regulator of cell survival. The regulatory loops induced by the MAPK pathway are dependent on tuberous sclerosis complex 2 but demonstrate a lesser dependence on p70S6K than the previously identified FRAP1 feedback loop. The siRNA screen also revealed novel bi-directionality in the AKT and GSK3 (Glycogen synthase kinase 3) interaction, whereby genetic ablation of GSK3 significantly blocks AKT phosphorylation, an unexpected observation as GSK3 has only been predicted to be downstream of AKT. This method uncovered novel modulators of AKT phosphorylation and facilitated the mapping of regulatory loops.

Proteomic Characterization of Breast Cancer Xenografts Identifies Early and Late Bevacizumab-Induced Responses and Predicts Effective Drug Combinations

Purpose: Neoangiogenesis is an important feature in tumor growth and progression, and combining chemotherapy and antiangiogenic drugs have shown clinical efficacy. However, as treatment-induced resistance often develops, our goal was to identify pathways indicating response and/or evolving resistance to treatment and inhibit these pathways to optimize the treatment strategies. Experimental Design: To identify markers of response and/or resistance, reverse-phase protein array (RPPA) was used to characterize treatment-induced changes in a bevacizumab-responsive and a nonresponsive human breast cancer xenograft. Results were combined with bioinformatic modeling to predict druggable targets for optimization of the treatment. Results: RPPA analysis showed that both tumor models responded to bevacizumab with an early (day 3) upregulation of growth factor receptors and downstream signaling pathways, with persistent mTOR signaling until the end of the in vivo experiment. Adding doxorubicin to bevacizumab showed significant and superior growth inhibition of basal-like tumors, whereas no additive effect was seen in the luminal-like model. The combination treatment corresponded to a continuous late attenuation of mTOR signaling in the basal-like model, whereas the inhibition was temporary in the luminal-like model. Integrating the bevacizumab-induced dynamic changes in protein levels with bioinformatic modeling predicted inhibition of phosphoinositide 3-kinase (PI3K) pathway to increase the efficacy of bevacizumab monotherapy. In vivo experiments combining bevacizumab and the PI3K/mTOR inhibitor BEZ235 confirmed their significant and additive growth-inhibitory effect in the basal-like model. Conclusions: Treatment with bevacizumab caused compensatory upregulation of several signaling pathways. Targeting such pathways increased the efficacy of antiangiogenic therapy. Clin Cancer Res; 20(2); 404–12. ©2013 AACR.

Understanding the role of cross-arm binding efficiency in the activity of monoclonal and multispecific therapeutic antibodies

Antibodies are essential components of the adaptive immune system that provide protection from extracellular pathogens and aberrant cells in the host. Immunoglobulins G, which have been adapted for therapeutic use due to their exquisite specificity of target recognition, are bivalent homodimers composed of two antigen binding Fab arms and an immune cell recruiting Fc module. In recent years significant progress has been made in optimizing properties of both Fab and Fc components to derive antibodies with improved affinity, stability, and effector function. However, systematic analyses of the efficiency with which antibodies crosslink their targets have lagged, despite the well-recognized importance of this cross-arm binding for optimal antigen engagement. Such an understanding is particularly relevant given the variety of next-generation multispecific antibody scaffolds under development. In this manuscript we attempt to fill this gap by presenting a framework for analysis and optimization of antibody cross-arm engagement. We illustrate the power of this integrated approach by presenting case studies for rational multispecific antibody design based on quantitative assessment of the interplay between antibody valency, target expression, and cross-arm binding efficiency. We conclude that optimal design parameters for cross-arm binding strongly depend on the biological context of the disease, and that cross-arm binding efficiency needs to be considered for successful application of multispecific antibodies.

Mapping Network Motif Tunability and Robustness in the Design of Synthetic Signaling Circuits

Cellular networks are highly dynamic in their function, yet evolutionarily conserved in their core network motifs or topologies. Understanding functional tunability and robustness of network motifs to small perturbations in function and structure is vital to our ability to synthesize controllable circuits. In establishing core sets of network motifs, we selected topologies that are overrepresented in mammalian networks, including the linear, feedback, feed-forward, and bifan circuits. Static and dynamic tunability of network motifs were defined as the motif ability to respectively attain steady-state or transient outputs in response to pre-defined input stimuli. Detailed computational analysis suggested that static tunability is insensitive to the circuit topology, since all of the motifs displayed similar ability to attain predefined steady-state outputs in response to constant inputs. Dynamic tunability, in contrast, was tightly dependent on circuit topology, with some motifs performing superiorly in achieving observed time-course outputs. Finally, we mapped dynamic tunability onto motif topologies to determine robustness of motif structures to changes in topology and identify design principles for the rational assembly of robust synthetic networks.

Abstract CT237: Preclinical characterization and first-in-human study of MM-141, a dual antibody inhibitor of IGF-1R and ErbB3

Background: MM-141 is a tetravalent bi-specific monoclonal antibody that binds IGF-1R and ErbB3, oncogenic receptors commonly co-expressed in solid tumors. In preclinical models, MM-141 blocks both ligand-dependent and -independent PI3K/AKT/mTOR signaling initiated through IGF-1R and ErbB3 complexes and potentiates the activity of gemcitabine, paclitaxel, nab-paclitaxel, docetaxel, irinotecan, tamoxifen, and everolimus. A multi-arm phase I study is ongoing, with continuing patient enrollment in Arm B (MM-141 in combination with everolimus). Monotherapy Arm A and combination Arm C (MM-141 with nab-paclitaxel and gemcitabine) are completed. Methods: Tumor expression of IGF-1R and ErbB3 was measured by immunohistochemistry. In vitro expression and degradation of IGF-1R and ErbB3 in pancreatic cell line models post-treatment were measured by immunoblotting and ubiquitination, respectively. The phase I dose-escalation study evaluated safety, tolerability, pharmacokinetic (PK), and pharmacodynamic (PD) properties of MM-141 as monotherapy (Arm A, n = 15) and in combination with everolimus (Arm B) or with nab-paclitaxel and gemcitabine (Arm C, n = 11). Pre- and post-treatment biopsies were acquired where mandated. Patients in the monotherapy Arm A received MM-141 at 6, 12, 20 mg/kg weekly or 40 mg/kg biweekly. Patients in the dose-escalation portion of Arm C received MM-141 at a weekly dose of 12 or 20 mg/kg in combination with weekly nab-paclitaxel (125 mg/m2) and gemcitabine (1000 mg/m2) on a schedule of 3 weeks on, 1 week off. Enrollment in Arm B (MM-141 in combination with everolimus) is ongoing. Patient serum free IGF-1 levels were detected using an in-house developed CLIA validated ELISA-based assay. Results: Here we report common co-expression of IGF-1R and ErbB3 in solid tumors. In stage IV metastatic pancreatic cancer, co-expression of IGF-1R and ErbB3 was associated with decreased patient survival. In preclinical models, increased expression of IGF-1R and ErbB3 desensitized tumors to gemcitabine and paclitaxel. However, co-treatment with MM-141 reversed this acquired resistance through blockade of growth factor binding and induction of IGF-1R and ErbB3 degradation. In the monotherapy arm of a phase I study, no dose-limiting toxicities were observed at any of the studied dose levels. The safety, tolerability, PK and PD profile of MM-141 support 2.8g bi-weekly MM-141 phase II recommended dose. The analysis of pre- and post-treatment biopsies confirmed that levels of IGF-1R and ErbB3 were decreased following MM-141 administration. In Arm C, the observed safety profile of MM-141 in combination with nab-paclitaxel and gemcitabine was comparable to expected toxicities reported with the chemotherapy combination when used alone. Retrospective analysis of serum free IGF-1 levels in breast cancer patients (Arm B) demonstrated that patients with elevated levels of this potential biomarker remained on study longer and received a greater number of doses of MM-141. Conclusion: These data support continued development of MM-141 in biomarker-selected patient populations and the upcoming phase II study of MM-141 in combination with nab-paclitaxel and gemcitabine in front-line metastatic pancreatic cancer patients with detectable free IGF-1 serum levels. Citation Format: Alexey A. Lugovskoy, Michel Curley, Jason Baum, Sharlene Adams, Sergio Iadevaia, Victoria Rimkunas, Adam Camblin, Lin Nie, Gege Tan, Bryan Johnson, Sara Mathews, Kerry Horgan, Chrystal U. Louis, Akos G. Czibere, Monica Arnedos, Jean-Charles Soria, Rastilav Bahleda, Anthony Shields, Patricia M. LoRusso, Mansoor Saleh, Steven J. Isakoff. Preclinical characterization and first-in-human study of MM-141, a dual antibody inhibitor of IGF-1R and ErbB3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT237. doi:10.1158/1538-7445.AM2015-CT237

Dual Inhibition of IGF-1R and ErbB3 Enhances the Activity of Gemcitabine and Nab-Paclitaxel in Preclinical Models of Pancreatic Cancer

Purpose: Insulin-like growth factor receptor 1 (IGF-1R) is critically involved in pancreatic cancer pathophysiology, promoting cancer cell survival and therapeutic resistance. Assessment of IGF-1R inhibitors in combination with standard-of-care chemotherapy, however, failed to demonstrate significant clinical benefit. The aim of this work is to unravel mechanisms of resistance to IGF-1R inhibition in pancreatic cancer and develop novel strategies to improve the activity of standard-of-care therapies. Experimental Design: Growth factor screening in pancreatic cancer cell lines was performed to identify activators of prosurvival PI3K/AKT signaling. The prevalence of activating growth factors and their receptors was assessed in pancreatic cancer patient samples. Effects of a bispecific IGF-1R and ErbB3 targeting antibody on receptor expression, signaling, cancer cell viability and apoptosis, spheroid growth, and in vivo chemotherapy activity in pancreatic cancer xenograft models were determined. Results: Growth factor screening in pancreatic cancer cells revealed insulin-like growth factor 1 (IGF-1) and heregulin (HRG) as the most potent AKT activators. Both growth factors reduced pancreatic cancer cell sensitivity to gemcitabine or paclitaxel in spheroid growth assays. Istiratumab (MM-141), a novel bispecific antibody that blocks IGF-1R and ErbB3, restored the activity of paclitaxel and gemcitabine in the presence of IGF-1 and HRG in vitro. Dual IGF-1R/ErbB3 blocking enhanced chemosensitivity through inhibition of AKT phosphorylation and promotion of IGF-1R and ErbB3 degradation. Addition of istiratumab to gemcitabine and nab-paclitaxel improved chemotherapy activity in vivo. Conclusions: Our findings suggest a critical role for the HRG/ErbB3 axis and support the clinical exploration of dual IGF-1R/ErbB3 blocking in pancreatic cancer. Clin Cancer Res; 24(12); 2873–85. ©2018 AACR.

Abstract 1209: Istiratumab (MM-141), a bispecific antibody targeting IGF-1R and ErbB3, inhibits pro-survival signaling in vitro and potentiates the activity of standard of care chemotherapy in vivo in ovarian cancer models

Insulin-like growth factor receptor 1 (IGF-1R) signaling has been implicated in the pathogenesis of ovarian cancer. However, clinical trials evaluating monospecific IGF-1R inhibitors have demonstrated limited clinical efficacy. Our data indicate that ErbB3, a member of the ErbB receptor tyrosine kinase family, can activate pro-survival AKT signaling in response to IGF-1R blockade and may represent a potential escape route in the development of resistance to therapy. Istiratumab (MM-141), an IGF-1R and ErbB3 directed bispecific antibody, inhibits ligand activation of these signaling pathways and degrades IGF-1R and ErbB3 receptor-containing complexes, leading to inhibition of downstream pro-survival signaling. Here we tested the activity of istiratumab, alone and in combination with chemotherapy, in in vitro and in vivo models of ovarian cancer. Anti-proliferative activity of istiratumab monotherapy was evaluated in a panel of ovarian cancer cell lines in vitro. The effects of istiratumab and the ligands IGF-1 and heregulin on IGF-1R- and ErbB3-mediated survival signaling were tested by ELISA and immunoblotting. Co-treatment assays with istiratumab and chemotherapy investigated mechanisms of synergy and additivity. Anti-tumor activity of istiratumab, alone and in combination with chemotherapy, was tested in in vivo ovarian xenograft tumor models. Our results indicated that istiratumab monotherapy inhibits ovarian cancer cell line proliferation in vitro. In addition, istiratumab blocked ligand-mediated resistance to chemotherapy. Co-treatment of istiratumab, ligands or chemotherapy indicated a strong correlation between drug activity and IGF-1R expression. Furthermore, co-treatment of chemotherapies and ligands potentiated AKT activation, which was inhibited by istiratumab. In vivo studies showed that istiratumab potentiates the activity of chemotherapy in ovarian xenograft tumor models. Our findings demonstrate that co-inhibition of IGF-1R and ErbB3 signaling with istiratumab can potentiate standard of care chemotherapies in ovarian tumor models and warrant further investigation of istiratumab as a potential therapy for ovarian cancer patients. Citation Format: Michael D. Curley, Gege Tan, Isabel Yannatos, Adam Camblin, Sergio Iadevaia, Chrystal Louis, Alexey Lugovskoy. Istiratumab (MM-141), a bispecific antibody targeting IGF-1R and ErbB3, inhibits pro-survival signaling in vitro and potentiates the activity of standard of care chemotherapy in vivo in ovarian cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1209.

Abstract LB-243: The ErbB3-targeting antibody MM-121 (seribantumab) reverses heregulin-driven resistance to multiple chemotherapies on tumor cell growth

Purpose: Heregulin-mediated activation of the human epidermal growth factor receptor 3 (HER3/ErbB3) is required for the growth and survival of many epithelial cancers. This signaling pathway is also emerging as a mechanism of resistance to targeted agents and chemotherapies. MM-121 (seribantumab) is an investigational human monoclonal anti-ErbB3 antibody that has previously been shown to effectively block ligand-dependent activation of ErbB3 in a range of tumors, and has demonstrated clinical activity in biomarker positive patients in several Phase II trials. The purpose of this study was to examine in three indications of interest, the ability of heregulin to induce resistance to standard chemotherapies and the reversal of this effect by MM-121. Such systematic evaluation of different combinations can serve as a guide for the future clinical development of MM-121. Methods: To assess the effect of heregulin and MM-121 on chemotherapies in cancer cells, we conducted a high throughput proliferation screen in 3D cultures. A panel of 60 cell lines of relevant clinical indications (ovarian, breast and lung cancer) was selected and tested for the sensitivity to respective standard-of-care chemotherapies in the absence or presence of exogenously added heregulin. Using these data, we analyzed the rescuing capacity of heregulin and the MM-121 combination9s sensitivity, and selected representative combinations for in vivo models. Results: We show that in a large panel of cancer cell lines the presence of heregulin can induce resistance to multiple chemotherapies with very different mechanisms of action. The combination of MM-121 with any one of these chemotherapies can reverse the heregulin-meditated rescue and provide an additive treatment effect at therapeutically relevant doses achieved in the clinic. These results were further validated in xenograft mouse models of all three indications, using representative chemotherapies and doses. In addition, biomarker analysis revealed that ErbB3 receptor levels largely determine responsiveness to heregulin and MM-121. Conclusions: MM-121 is an anti-ErbB3 antibody designed to block ligand-mediated signaling, and currently in clinical development. The results presented here demonstrate the role of heregulin in reducing the sensitivity of tumors to standard-of-care chemotherapies, and the effect of ErbB3 pathway inhibition across indications. Citation Format: Kristina Masson, Viara Grantcharova, Olga Burenkova, Marisa Wainszelbaum, Sergio Iadevaia, Sharlene Adams, Andreas Raue, Akos Czibere, Birgit Schoeberl, Gavin MacBeath. The ErbB3-targeting antibody MM-121 (seribantumab) reverses heregulin-driven resistance to multiple chemotherapies on tumor cell growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-243. doi:10.1158/1538-7445.AM2015-LB-243

Abstract C169: MM-141, a bispecific antibody inhibitor of PI3K/AKT/mTOR, attenuates tumor growth and potentiates everolimus in mouse models of anti-hormonal therapy-resistant ER/PR+ breast cancer.

Purpose: PI3K/AKT/mTOR network adaptation through the upregulation of receptor tyrosine kinases signaling and subsequent re-activation of AKT is a common resistance mechanism to chemotherapies and small molecule inhibitors of mammalian target of rapamycin (mTOR). In order to block this feedback loop, MM-141, a tetravalent bispecific antibody directed at IGF-1R and ErbB3, was co-administered in combination with everolimus in various preclinical models of cancer. As everolimus is an approved therapy for women with advanced hormone receptor-positive breast cancer after failure of treatment with aromatase inhibitor, we have specifically focused on the analysis of a MM-141/everolimus combination in a hormone refractory breast cancer mouse model. Experimental Procedures: Estrogen-supplemented mice bearing BT-474-M3 ER+/PR+ breast cancer xenografts were implanted with tamoxifen-releasing pellets. Following the development of resistance to tamoxifen, these mice were randomized into pharmacodynamics and efficacy assessment sub-groups, and treated with everolimus, MM-141 or the combination. In pharmacodynamics assessment subgroups, the tumors were harvested 24 hours following the last MM-141 dose and profiled for a panel of molecular markers of activation of PI3K/AKT/mTOR pathway. In addition we have assessed the activity of MM-141, everolimus or the combination in cell culture on a broad panel of breast cancer cell lines using CellTiter-Glo® Luminescent Cell Viability Assay. Data Summary: We have demonstrated that MM-141 has significant anti-tumor activity in the ER+/PR+ BT-474-M3 breast cancer xenograft model and strongly potentiates the activity of everolimus in a tamoxifen-resistant setting. Pharmacodynamic profiling illustrates that the combination treatment achieves sustained inhibition of PI3K/AKT/mTOR signaling. MM-141 mediates the resistance to everolimus by inhibiting IGF-1R, ErbB3, and IRS-1 and controlling the re-activation of AKT. Further, MM-141 is able to inhibit the proliferation of a panel of breast cancer cell lines in vitro. This inhibition is increased upon co-treatment of the cells with MM-141 and everolimus. Conclusions: Our in vivo preclinical studies show that MM-141 combines synergistically with the mTOR inhibitor everolimus and this combination is active in reversing acquired resistance to the anti-hormonal therapy tamoxifen. This potentiation of everolimus by MM-141 is tied to its ability to reverse the re-activation of AKT signaling induced by this mTOR blocker. We demonstrate that this potentiation phenomenon is common in a wide panel of breast cancer cell lines. Overall, our results suggest that MM-141 could be a potentially valuable addition to the breast cancer treatment regimens comprising an mTOR inhibitor and an anti-hormonal therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C169. Citation Format: Sharlene Adams, Jason Baum, Breanne Sparta, Victoria Rimkunas, Jian Tang, Emily Pace, Shoshana Rosenthal, Adam Camblin, Sergio Iadevaia, Akos Czibere, Ulrik Nielsen, Alexey Lugovskoy. MM-141, a bispecific antibody inhibitor of PI3K/AKT/mTOR, attenuates tumor growth and potentiates everolimus in mouse models of anti-hormonal therapy-resistant ER/PR+ breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C169.