Experimental Procedures

Cell Purification and Culture
Cells were cultured according to standard protocols (Mauro et al., 2011; Piva et al., 2008). BMSCs and peripheral blood mononuclear cells (PBMCs) were purified from MM patients or the blood of healthy volunteers, respectively, as reported in Piva et al. (2008). CD138+ cells were purified from the bone marrow aspirates of patients using CD138 MicroBeads (Miltenyi Biotech). Patients were recruited at the Hematology Division of “Ospedale San Giovanni Battista” (Turin, Italy) and the Haematology Clinic at Imperial College Healthcare NHS Trust, under the approval of the Ethics Committee of “Ospedale San Giovanni Battista” (VMP-VMPT trial 163) and the London Harrow Research Ethics Committee (11/LO/1628), respectively. Written consent was documented for all subjects. Additional details are provided in Supplemental Experimental Procedures.

Biochemical Assays
Quantitative RT-PCR (qRT-PCR) assays were carried out using the TaqMan Gene Expression Assays kit (Applied Biosystems). The kinase profiling of DTP3 across 142 human kinases was outsourced. Western blots, coimmunoprecipitations, and kinase assays were performed as described previously (Papa et al., 2004, 2008; Mauro et al., 2011). Additional details are provided in Supplemental Experimental Procedures.

Cellular Assays
Lentiviral infections were performed using pLentiLox.3.7, as described by Mauro et al. (2011). Enhanced GFP (eGFP)+ cells were purified, when necessary, by fluorescence-activated cell sorting. [3H]Thymidine incorporation and trypan blue exclusion assays were performed using standard methods (Mauro et al., 2011). IC50 values were defined as the mean concentration of compound inducing 50% inhibition of cell viability relative to the viability in the untreated cultures. Apoptosis analyses were performed using propidium iodide (PI) staining, as described by Mauro et al. (2011). Additional details are provided in Supplemental Experimental Procedures.

Peptide Synthesis and ELISA
Proteins were purified as described by Tornatore et al. (2008). The combinatorial L-tetrapeptide libraries and individual peptides were synthesized as reported by Sandomenico et al. (2012). The methods used to assess peptide identity and purity and deconvolute the tetrapeptide libraries and the ELISA GADD45β/MKK7 competition assays were described previously (Sandomenico et al., 2012; Tornatore et al., 2008). IC50 values were defined as the mean concentration of peptide inducing 50% inhibition of GADD45β binding to MKK7 relative to the binding measured in the absence of peptide. Additional details are provided in Supplemental Experimental Procedures.

DTP3 Binding Assays
The stoichiometry and KD value of the DTP3/MKK7 interaction were determined by tryptophan fluorescence quenching analysis, after fitting the fluorescence data with a nonlinear regression algorithm, as described by Williamson (2013). Additional details are provided in Supplemental Experimental Procedures, along with a description of the methods used for CD and MALDI-TOF mass spectrometry analyses and the pull-down of MKK7 from cells with DTP3.

Pharmacophore Analyses and Modeling
See Supplemental Experimental Procedures.

Pharmacokinetic Analyses
The pharmacokinetic analyses of DTP3 and z-DTP2 were outsourced. Additional details are provided in Supplemental Experimental Procedures.

Animal Studies
Mice were housed in the animal facilities at Hammersmith. All experiments were conducted under Procedure Project License (PPL) 70/6874, after approval by the Imperial College Ethical Review Process and the Home Office. For the plasmacytoma model, nonobese diabetic/severe combined immunodeficiency mice (NOD.CB17-Prkdcscid/IcrCrl; Charles River) were injected subcutaneously with 1.0 × 107 U266 or KMS-11 MM cells and then randomized into treatment groups and treated by infusion, as shown. Tumor volumes were measured as described by Mauro et al. (2011). For the orthotopic MM model, mice of the same strain were sublethally irradiated and then injected intravenously with 1.0 × 107 KMS-12-BM MM cells, as described by Rabin et al. (2007). Mice were then randomized into treatment groups and treated by infusion for 8 weeks, as shown. Animals were monitored daily and euthanized on day 161 of treatment start or when they reached any of the end points in the PPL. Additional details are provided in Supplemental Experimental Procedures.

Statistical Analyses
See Supplemental Experimental Procedures.