A number of "suicide genes" have been developed as safety switches for gene therapy vectors or as potential inducible cytotoxic agents for hyperproliferative disorders, such as cancer or restenosis. However, most of these approaches have relied on foreign proteins, such as HSV thymidine kinase, that primarily target rapidly dividing cells. In contrast, novel artificial death switches based on chemical inducers of dimerization (CIDs) and endogenous proapoptotic molecules function efficiently in both dividing and nondividing cells. In this approach, lipid-permeable, nontoxic CIDs are used to conditionally cross-link target proteins that are fused to CID-binding domains (CBDs), thus activating signaling cascades leading to apoptosis. In previous reports, CID-regulated Fas and caspases 1, 3, 8, and 9 were described. Since the maximum efficacy of these artificial death switches requires low basal and high specific activity, we have optimized these death switches for three parameters: (1) extent of oligomerization, (2) spacing between CBDs and target proteins, and (3) intracellular localization. We describe improved conditional Fas and caspase 1, 3, 8, and 9 alleles that function at subnanomolar levels of the CID AP1903 to trigger apoptosis. Further, we demonstrate for the first time that oligomerization of the death effector domain of the Fas-associated protein, FADD, is sufficient to trigger apoptosis, suggesting that the primary function of FADD, like that of Apaf-1, is oligomerization of associated caspases. Finally, we demonstrate that nuclear-targeted caspases 1, 3, and 8 can trigger apoptosis efficiently, implying that the cleavage of nuclear targets is sufficient for apoptosis.

EGFR blocking antibodies are approved for the treatment of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Although ERBB3 signaling has been proposed to limit the effectiveness of EGFR inhibitors, the underlying molecular mechanisms are not fully understood. To gain insight into these mechanisms, we generated potent blocking antibodies against ERBB3 (REGN1400) and EGFR (REGN955). We show that EGFR and ERBB3 are coactivated in multiple HNSCC cell lines and that combined blockade of EGFR and ERBB3 inhibits growth of these cell lines more effectively than blockade of either receptor alone. Blockade of EGFR with REGN955 strongly inhibited activation of ERK in HNSCC cell lines, whereas blockade of ERBB3 with REGN1400 strongly inhibited activation of Akt; only the combination of the 2 antibodies blocked both of these essential downstream pathways. We used a HER2 blocking antibody to show that ERBB3 phosphorylation in HNSCC and colorectal cancer cells is strictly dependent on association with HER2, but not EGFR, and that neuregulin 1 activates ERBB3/HER2 signaling to reverse the effect of EGFR blockade on colorectal cancer cell growth. Finally, although REGN1400 and REGN955 as single agents slowed the growth of HNSCC and colorectal cancer xenografts, the combination of REGN1400 plus REGN955 caused significant tumor regression. Our results indicate that activation of the Akt survival pathway by ERBB3/HER2 limits the effectiveness of EGFR inhibition, suggesting that REGN1400, which is currently in a phase I clinical trial, could provide benefit when combined with EGFR blocking antibodies. Mol Cancer Ther; 13(5); 1345-55. Ó2014 AACR.

<p>PDF - 78K, Effect of REGN1400 and REGN955 on ligand-induced signaling by ERBB3 and EGFR, respectively.</p>

<div>Abstract<p>EGFR blocking antibodies are approved for the treatment of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Although ERBB3 signaling has been proposed to limit the effectiveness of EGFR inhibitors, the underlying molecular mechanisms are not fully understood. To gain insight into these mechanisms, we generated potent blocking antibodies against ERBB3 (REGN1400) and EGFR (REGN955). We show that EGFR and ERBB3 are coactivated in multiple HNSCC cell lines and that combined blockade of EGFR and ERBB3 inhibits growth of these cell lines more effectively than blockade of either receptor alone. Blockade of EGFR with REGN955 strongly inhibited activation of ERK in HNSCC cell lines, whereas blockade of ERBB3 with REGN1400 strongly inhibited activation of Akt; only the combination of the 2 antibodies blocked both of these essential downstream pathways. We used a HER2 blocking antibody to show that ERBB3 phosphorylation in HNSCC and colorectal cancer cells is strictly dependent on association with HER2, but not EGFR, and that neuregulin 1 activates ERBB3/HER2 signaling to reverse the effect of EGFR blockade on colorectal cancer cell growth. Finally, although REGN1400 and REGN955 as single agents slowed the growth of HNSCC and colorectal cancer xenografts, the combination of REGN1400 plus REGN955 caused significant tumor regression. Our results indicate that activation of the Akt survival pathway by ERBB3/HER2 limits the effectiveness of EGFR inhibition, suggesting that REGN1400, which is currently in a phase I clinical trial, could provide benefit when combined with EGFR blocking antibodies. <i>Mol Cancer Ther; 13(5); 1345–55. ©2014 AACR</i>.</p></div>

<div>Abstract<p>EGFR blocking antibodies are approved for the treatment of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Although ERBB3 signaling has been proposed to limit the effectiveness of EGFR inhibitors, the underlying molecular mechanisms are not fully understood. To gain insight into these mechanisms, we generated potent blocking antibodies against ERBB3 (REGN1400) and EGFR (REGN955). We show that EGFR and ERBB3 are coactivated in multiple HNSCC cell lines and that combined blockade of EGFR and ERBB3 inhibits growth of these cell lines more effectively than blockade of either receptor alone. Blockade of EGFR with REGN955 strongly inhibited activation of ERK in HNSCC cell lines, whereas blockade of ERBB3 with REGN1400 strongly inhibited activation of Akt; only the combination of the 2 antibodies blocked both of these essential downstream pathways. We used a HER2 blocking antibody to show that ERBB3 phosphorylation in HNSCC and colorectal cancer cells is strictly dependent on association with HER2, but not EGFR, and that neuregulin 1 activates ERBB3/HER2 signaling to reverse the effect of EGFR blockade on colorectal cancer cell growth. Finally, although REGN1400 and REGN955 as single agents slowed the growth of HNSCC and colorectal cancer xenografts, the combination of REGN1400 plus REGN955 caused significant tumor regression. Our results indicate that activation of the Akt survival pathway by ERBB3/HER2 limits the effectiveness of EGFR inhibition, suggesting that REGN1400, which is currently in a phase I clinical trial, could provide benefit when combined with EGFR blocking antibodies. <i>Mol Cancer Ther; 13(5); 1345–55. ©2014 AACR</i>.</p></div>