Mti-101 (cyclized HYD1) Binds to CD44 and Induces Necrotic Cell Death in Multiple Myeloma

Abstract 4022

Our laboratory has previously shown that treatment with the novel d-amino acid peptide HYD1 (kikmviswkg) induces necrotic cell death in multiple myeloma (MM) cell lines. Importantly, this novel peptide and similar compounds demonstrate increased activity in specimens obtained from relapsed patients (Nair et al. Mol Cancer Ther. 2009 Aug;8(8):2441-51., Emmons et al. Mol Cancer Ther. 2011 Dec;10(12):2257-66). Due to the intriguing biological activity of our linear peptide, we pursued strategies for increasing the therapeutic potential of this class of compounds. These efforts have lead to a cyclized peptidomimetic, MTI-101, with increased in vitro activity and robust in vivo activity as single agent using two independent myeloma models which consider the bone marrow microenvironment. To further delineate the binding complex, we pursued an unbiased chemical biology approach using biotinylated peptide and LC-MS/MS analysis as an unbiased analytical tool for defining the HYD1 binding complex. Using this approach the adhesion receptor CD44 was determined to be among the adhesion receptors that had the most peptides identified and was confirmed via Western Blot analysis. Furthermore, we were able to show that HYD1 binds to recombinant CD44 through two independent binding assays. In preliminary ELISA-based binding experiments with full length recombinant human CD44, we were able to show that CD44 bound to biotin conjugated HYD1 in a concentration dependent manner. Additionally, we produced recombinant human CD44 ectodomain and utilizing surface plasmon resonance (SPR), we were able to show that the recombinant human CD44 ectodomain bound to biotin conjugated HYD1 and MTI-101 with a high affinity (K_d of approximately 30 nM). We are currently pursuing Isothermal Titration Calorimetry (ITC) binding assays to confirm binding affinities. Finally, using fluorescently tagged HYD1 (the 5(6)-FAM conjugate) as a molecular tool, we were able to demonstrate that overexpression of the standard form of CD44 (CD44s) in the 8226 myeloma cell line resulted in increased binding of our peptide. Paradoxically, increased expression of CD44s and subsequent increased binding of the FAM-conjugated peptide did not correlate with increased cell death. These data suggest that either CD44v expression is mediating cell death or that downstream or lateral signaling within the binding complex is rate limiting for cell death. Moreover, co-immunoprecipitation assays demonstrated that CD44 and the pro-necrotic receptor interacting protein 1 (RIP1) co-localized upon treatment with MTI-101 in NCI-H929 and U266 cells. Thus, we have elucidated a novel signaling pathway linking CD44 to RIP1, which is part of the necroptosome. Further studies using RNA interference are currently being conducted to determine the causative role of RIP1 and RIP3 in mediating HYD1 and MTI-101-induced necrotic cell death. Together, our data begin to elucidate the mechanism of action and definitively support continued development of MTI-101 for the treatment of relapsed myeloma, providing another therapeutic option for this currently incurable disease.