SUPPL FIG 3: A) Relative DEPTOR expression in MMCLs and hepatocellular carcinoma (HCC) cell lines in a representative experiment (upper panel) and means+/-SD of 3 experiments (lower bar graph); B) Relative sensitivity of U266 and FR4 MMCLs vs DEPTOR-expressing JHH7, HUH-1 or HIH7 HCCs; data are means +/- SD (n=3); Asterix demonstrates significant (p<0.05) decreased cell survival in MMCLs vs HCC cells ; C) MMCLs or HUH-1 HCC cells following shRNA transfection with control (scramble) or DEPTOR-targeted lentivirus; D) Percent apoptosis at 3 or 4 days following DEPTOR silencing in MMCLs or HUH-1 HCC; Data are means +/- SD, n=4 following transfection with control shRNA (scr) or shRNA targeting 2 separate sequences of DEPTOR.

SUPPL FIG 6: PBLs obtained from 3 normal individuals and incubated with increasing concentrations of NSC 126405 for 72 hrs. Surviving viable cells enumerated. Data are % of control (no drug B), mean+/-SD, n=3.

Abstract To assess the role of the serum and glucocorticoid-regulated kinase (SGK) kinase in multiple myeloma, we ectopically expressed wild type or a phosphomimetic version of SGK into multiple myeloma cell lines. These cells were specifically resistant to the ER stress inducers tunicamycin, thapsigargin, and bortezomib. In contrast, there was no alteration of sensitivity to dexamethasone, serum starvation, or mTORC inhibitors. Mining of genomic data from a public database indicated that low baseline SGK expression in multiple myeloma patients correlated with enhanced ability to undergo a complete response to subsequent bortezomib treatment and a longer time to progression and overall survival following treatment. SGK overexpressing multiple myeloma cells were also relatively resistant to bortezomib in a murine xenograft model. Parental/control multiple myeloma cells demonstrated a rapid upregulation of SGK expression and activity (phosphorylation of NDRG-1) during exposure to bortezomib and an SGK inhibitor significantly enhanced bortezomib-induced apoptosis in cell lines and primary multiple myeloma cells. In addition, a multiple myeloma cell line selected for bortezomib resistance demonstrated enhanced SGK expression and SGK activity. Mechanistically, SGK overexpression constrained an ER stress–induced JNK proapoptotic pathway and experiments with a SEK mutant supported the notion that SGK's protection against bortezomib was mediated via its phosphorylation of SEK (MAP2K4) which abated SEK/JNK signaling. These data support a role for SGK inhibitors in the clinical setting for myeloma patients receiving treatment with ER stress inducers like bortezomib. Implications: Enhanced SGK expression and activity in multiple myeloma cells contributes to resistance to ER stress, including bortezomib challenge. Mol Cancer Res; 14(4); 397–407. ©2016 AACR.

Abstract DEPTOR is a 48 kDa protein that binds to mTOR and inhibits this kinase in TORC1 and TORC2 complexes. Overexpression of DEPTOR specifically occurs in a model of multiple myeloma. Its silencing in multiple myeloma cells is sufficient to induce cytotoxicity, suggesting that DEPTOR is a potential therapeutic target. mTORC1 paralysis protects multiple myeloma cells against DEPTOR silencing, implicating mTORC1 in the critical role of DEPTOR in multiple myeloma cell viability. Building on this foundation, we interrogated a small-molecule library for compounds that prevent DEPTOR binding to mTOR in a yeast-two-hybrid assay. One compound was identified that also prevented DEPTOR–mTOR binding in human myeloma cells, with subsequent activation of mTORC1 and mTORC2. In a surface plasmon resonance (SPR) assay, the compound bound to recombinant DEPTOR but not to mTOR. The drug also prevented binding of recombinant DEPTOR to mTOR in the SPR assay. Remarkably, although activating TORC1 and TORC2, the compound induced apoptosis and cell-cycle arrest in multiple myeloma cell lines and prevented outgrowth of human multiple myeloma cells in immunodeficient mice. In vitro cytotoxicity against multiple myeloma cell lines was directly correlated with DEPTOR protein expression and was mediated, in part, by the activation of TORC1 and induction of p21 expression. Additional cytotoxicity was seen against primary multiple myeloma cells, whereas normal hematopoietic colony formation was unaffected. These results further support DEPTOR as a viable therapeutic target in multiple myeloma and suggest an effective strategy of preventing binding of DEPTOR to mTOR. Cancer Res; 76(19); 5822–31. ©2016 AACR.

SUPPL FIG 2: A) 8226 cells treated with or w/o NSC 126405 for 6 hrs, followed by immunoprecipitation of mTOR (or control IgG). Immunoprecipitates immunoblotted for expression of mTOR and DEPTOR. Input shown for DEPTOR, mTOR and tubulin expression. B) 8226 cells treated with DMSO, pp242 or NSC 126405 and then tested for 4E-BP1 phosphorylation by flow cytometry. C) Summarized data from all 3 treatment groups

PDF file - 2312K, Heat map and table of metabolites of significantly altered metabolites in AICAr-treated samples for rapamycin treated samples.

Supplementary Fig. S2 from Effect of autophagy on multiple myeloma cell viability

SUPPL FIG 4: A) 8226 cells treated +/- NSC 126405 for 18 hrs followed by immunoblot assays. Fold BIM increase is determined by densitometry, mean of 3 experiments; B) Control (shSCR) or RAPTOR-silenced 8226 cells (shRAPTOR) treated +/- NSC 126405 for 18 hrs followed by immunoblot assays. Fold BIM increase is mean of 3 experiments.

PDF file - 145K, AICAr does not activate AMPK in 8226 cells or OPM2 cells and does not inhibit TORC1.

Suppl fig 4: Lysate harvested from tumors of mice challenged with EV- or SGK-transfected tumors and treated with 0, 0.5 or 1 mg/kg Bortezomib; Lysates are combined from 3 mice/group and harvested 1 week after initiating treatment (mice had received 2 treatments); IKB-alpha then immunoprecipitated from lysates and immunoblotted for IKB as well as ubiquitin;

Suppl fig 7: A: Examples of IHC staining of normal tissues for DEPTOR expression following daily injection of DMSO or drug 3g (10mg/kg x 21 days); B) Immunoblot analysis of heart tissue for DEPTOR expression

When mTOR inhibitor rapalogs prevent cap-dependent translation of cell-cycle proteins like c-myc, continuing tumor cell growth depends on cap-independent translation, which is mediated by internal ribosome entry sites (IRESes) located in the 5′-UTR (untranslated region) of transcripts. To investigate if rapalog-induced activation of MNK kinases had a role in such IRES activity, we studied multiple myeloma (MM) cells. Rapamycin (RAP)-activated MNK1 kinase activity in MM cell lines and primary specimens by a mitogen-activated protein kinase-dependent mechanism. Pharmacological inhibition of MNK activity or genetic silencing of MNK1 prevented a rapalog-induced upregulation of c-myc IRES activity. Although RAP, used alone, had little effect on myc protein expression, when combined with a MNK inhibitor, myc protein expression was abrogated. In contrast, there was no inhibition of myc RNA, consistent with an effect on myc translation. In a RAP-resistant MM cell lines as well as a resistant primary MM specimen, co-exposure to a MNK inhibitor or MNK1 knockdown significantly sensitized cells for RAP-induced cytoreduction. Studies in MNK-null murine embryonic fibroblasts additionally supported a role for MNK kinases in RAP-induced myc IRES stimulation. These results indicate that MNK kinase activity has a critical role in the fail-safe mechanism of IRES-dependent translation when mTOR is inhibited. As kinase activity also regulated sensitivity to RAP, the data also provide a rationale for therapeutically targeting MNK kinases for combined treatment with mTOR inhibitors.

Suppl fig 5: SGK protein stability in 8226 cells treated +/- bortezomib (20nM); Data are Mean+/-SD, n=3.

PDF file - 64K, Chemical structure of bortezomib, melphalan and metformin.

Suppl fig 1: A) OPM2 cells transfected with empty vector (EV) wild type SGK (sgk) or phosphomimetic SGK (sgks422d) followed by immunoblot assay; B) OPM-2 cells transfected with EV (black columns) or wild type SGK (white columns) and treated with boretzomib (Bort) for 24 hrs followed by MTT assays. Data are percent survival, mean+/-SE, n=3. Apoptosis also assessed and shown above bars as mean % apoptosis, n=3; SGK-transfected cells show significant resistance (p<0.05) to all concentrations of bortezomib; C) OPM-2 cells treated for 24 hrs with thapsigargin (thaps) or tunicamycin (tun) followed by apoptosis assay. Data are percent apoptosis above control, means+/-SE, n=3. Black columns are EV-transfected OPM-2 cells and white columns are SGK-transfected cells. * denotes significant (p<0.05) decreased apoptosis vs control EV cells.

Supplemental figure 8: Representative sections of liver obtained from control (DMSO) mice or mice injected IP daily with drug 3g at 20mg/kg. Magnification=100x