Cytotoxic Properties of a DEPTOR-mTOR Inhibitor in Multiple Myeloma Cells

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.

Figure 1. Identification of an inhibitor preventing DEPTOR/mTOR binding

A) Structure of NSC 126405; B&C) OPM-2 cells treated +/− drug for 6 hrs followed by immunoprecipitation of mTOR (in ‘B’) or DEPTOR (in ‘C’) and immunoblotting; D)MMCLs incubated with NSC 126405 for 6 hrs followed by immunoblot; E) 8226 cells treated +/− drug (1 uM) for 2, 4 or 8 hrs followed by immunoblot assay; F) 8226 cells treated +/− drug followed by immunoblot assay for 4E-BP1 phosphorylation; G) Surface plasmon resonance assay- Increasing concentrations of NSC 126405 (100uM curve shown in figure)passed over immobilized DEPTOR, mTOR or mLST8 to test binding partner of drug (left panel). In right panel, NSC 126405 (100uM), recombinant DEPTOR (5ug/ml) or combination of drug + DEPTOR passed over immobilized mTOR to assess binding.

Figure 2. Cytotoxicity of NSC 126405

A) MTT assay of 8226 cells treated for 24–96 hrs with NSC 126405; B) DEPTOR protein expression in MMCLs (top panel) and relative DEPTOR/tubulin expression (mean+/−SD, n=3–7) assayed by immunoblot and densitometric analysis (bottom panel). Relative DEPTOR expression of 8226 cells arbitrarily designated ‘1’. C) Example of an MTT assay testing 6 MMCLs; D) Correlation between NSC 126405 sensitivity and relative DEPTOR expression. IC₅₀ in uM determined for all MMCLs exposed to drug for 72 hrs and is presented as means of 3–6 individual experiments. Relative DEPTOR expression is taken from figure 2B and represents mean DEPTOR/tubulin expression, n=3–7; E) Cell cycle distribution in MMCLs exposed +/− NSC 126405 (2 uM) for 48 hrs. Data are mean+/−SD, n=3. NSC 126405 caused a significant alteration (p<0.05) in G1, S and G2/M distribution; F) Percent apoptosis in MM cell lines exposed to drug for 72 hrs. Data are mean+/−SD, n=3;

Figure 3. Effect of NSC 126405 on AKT

A) MM cell lines exposed to NSC 126405 for 6 hrs followed by immunoblot assay; B) Dox-inducible DEPTOR shRNA cells treated with DMSO (control), NSC 126405 (2 uM) for 6 hrs or doxycycline for 48 hrs to induce DEPTOR shRNA; C) Dox-inducible DEPTOR shRNA cells treated with dox for 48 hrs or NSC 126405 (2uM) for increasing intervals followed by immunoblot assay; D) TORC2 in vitro kinase assay. Left panel=8226 lysate obtained and TORC2 complex immunoprecipitated by anti-RICTOR antibody (or IgG control) followed by immunoblot detection of mTOR and RICTOR. Middle panel=8226 cells treated with 0, 1 or 2 uM NSC 126405 for 6 hrs, after which TORC2 complex was immunoprecipitated by anti-RICTOR antibody and tested for its phosphorylation of AKT substrate on S473. Right panel shows input; E) MMCLs treated with NSC 126405 for 6 hrs followed by immunoblot assay. F) 8226 cells treated +/− NSC 126405 for 6 hrs, followed by immunoblot assay.

Figure 4. Effect of RAPTOR and p21 shRNA

A) 8226 cells expressing shRNA targeting a scrambled sequence (control) or two separate sequences of RAPTOR, followed by immunoblot assay; B) MTT assay of control (SCR-control) or RAPTOR-silenced 8226 MM cells (shRaptor1 and 2) challenged with NSC 126405 for 48 or 72 hrs. Data are mean+/−SD, n=3. * denotes significant difference (p<0.05) from SCR control cells. Apoptosis (mean+/−SD) is shown above 2 & 4 uM bars in the 72 hrs assay; C) 8226 cells expressing shRNA targeting scrambled sequence or two separate sequences of p21, followed by immunoblot assay; D) Cell cycle distribution of control (SCR) or p21-silenced MM cells (Shp21-1 & Shp21-2) exposed to 0, 1 or 2 uM NSC 126405 for 48 hrs. * denotes significant difference (p<0.05) from control (0 uM).

Figure 5. Effect of NSC 126405 in mice

A) Weights (means, n=5 mice/group) of mice treated +/− NSC 126405 with daily IP injections x 7 days; B) RBC and WBC counts in mice (5 mice/group) treated with daily IP injections (7 days) of 0, 10 or 20 mg/kg NSC 126405. Data are % of control. * denotes significant difference (p<0.05) vs control; C) Immunodeficienct mice (9 mice/group) challenged with 8226 cells SQ and, when tumors became palpable, treatment initiated with daily IP injections of vehicle control or NSC 126405 for 11 days. Data represent tumor volume, mean+/−SEM. * denotes significant differences (p<0.05) from control. D) Three tumors excised at random from treatment groups of figure 5C and probed for expression of phorphorylated 4E-BP1 and p21. E) Mice challenged with CD45+ 8226 MM cells by IV injection (n=6) and either treated with vehicle or NSC 126405 (20 mg/kg) by IP daily injection x 14. Mice then sacrificed and CD45+ MM cells enumerated by flow cytometry. Our anti-human CD45 antibody did not stain any murine marrow cells in control marrow.

Figure 6. Effect of NSC 126405 on human cells

A) Survival assay of CD138-isolated primary cells from patients (n=17) exposed to NSC 126405 for 48 hrs. Data are percent surviving cells vs control (0 uM), mean+/−SD. B) Flow cytometric analysis of apoptosis in 4 primary specimens incubated with NSC 126405 for 48 hrs. Data are mean+/−SD; C) Normal hematopoietic colony formation in cells exposed to NSC 126405 for 14 days. Data represent percent of control (0 uM) mean+/−SD, n=4; D) Comparison of IC₅₀ for NSC 126405 between primary samples containing IgH translocations (n=7) vs those without IgH translocations (n=10). Data are means+/−SEM; E) Flow cytometry assay for 4E-BP1 phosphorylation in a primary MM specimen.