Dpep is a cell-penetrating peptide that targets transcription factors ATF5, CEBPB and CEBPD to selectively suppress growth and survival of diverse tumor cell types in vitro and in vivo. Due to these actions and its apparent safety, the peptide has potential as a cancer therapeutic. How Dpep might be combined with other anti-cancer agents to achieve synergistic efficacy and to overcome possible peptide resistance has not been assessed in depth. Based on prior work indicating that Dpep promotes apoptotic cancer cell death and up-regulates multiple pro-apoptotic and tumor suppressor genes, we studied combinations of Dpep with ABT-263, a pro-apoptotic BCL2 family inhibitor, and decitabine, a hypomethylating drug. Combining Dpep with each agent alone or together synergistically suppressed the growth of a range of solid and liquid tumor cell types. Moreover, the combinations synergistically inhibited the growth of cells lines that were selected either in vivo or in vitro for Dpep resistance. Finally, we tested the combination of Dpep with ABT-263 in a mouse melanoma xenograft model. The combination more effectively inhibited tumor growth than either agent alone and, in contrast to vehicle or ABT-263, produced a 40% durable survival rate. Taken together, these observations highlight potential drug partners for the therapeutic development of Dpep.

Natural killer (NK) cells are an important innate defense against malignancies, and exogenous sources of NK cells have been developed as anti-cancer agents. Nevertheless, the apparent limitations of NK cells in clearing cancers have suggested that their efficacy might be augmented by combination with other treatments. We have developed cell-penetrating peptides that target the transcription factors ATF5, CEBPB, and CEBPD and that promote apoptotic cancer cell death both in vitro and in vivo without apparent toxicity to non-transformed cells. We report here that one such peptide, Dpep, significantly sensitizes a variety of tumor cell types to the cytotoxic activity of the NK cell line, NK-92MI. Such sensitization requires pre-exposure of tumor cells to Dpep and does not appear due to effects of Dpep on NK cells themselves. Our findings suggest that Dpep acts in this context to lower the apoptotic threshold of tumor cells to NK cell toxicity. Additionally, while Dpep pre-treatment does not prevent tumor cells from causing NK cell “inactivation”, it sensitizes cancer cells to repeated rounds of exposure to fresh NK cells. These findings thus indicate that Dpep pre-treatment is an effective strategy to sensitize cancer cells to the cytotoxic actions of NK cells.

We have designed cell-penetrating peptides that target the leucine zipper transcription factors ATF5, CEBPB and CEBPD and that promote apoptotic death of a wide range of cancer cell types, but not normal cells, in vitro and in vivo. Though such peptides have the potential for clinical application, their mechanisms of action are not fully understood. Here, we show that one such peptide, Dpep, compromises glucose uptake and glycolysis in a cell context-dependent manner (in about two-thirds of cancer lines assessed). These actions are dependent on induction of tumor suppressor TXNIP (thioredoxin-interacting protein) mRNA and protein. Knockdown studies show that TXNIP significantly contributes to apoptotic death in those cancer cells in which it is induced by Dpep. The metabolic actions of Dpep on glycolysis led us to explore combinations of Dpep with clinically approved drugs metformin and atovaquone that inhibit oxidative phosphorylation and that are in trials for cancer treatment. Dpep showed additive to synergistic activities in all lines tested. In summary, we find that Dpep induces TXNIP in a cell context-dependent manner that in turn suppresses glucose uptake and glycolysis and contributes to apoptotic death of a range of cancer cells.

Glioblastoma multiforme (GBM) is one of the most aggressive forms of brain tumor, characterized by a daunting prognosis with a life expectancy hovering around 12–16 months. Despite a century of relentless research, only a select few drugs have received approval for brain tumor treatment, largely due to the formidable barrier posed by the blood–brain barrier. The current standard of care involves a multifaceted approach combining surgery, irradiation, and chemotherapy. However, recurrence often occurs within months despite these interventions. The formidable challenges of drug delivery to the brain and overcoming therapeutic resistance have become focal points in the treatment of brain tumors and are deemed essential to overcoming tumor recurrence. In recent years, a promising wave of advanced treatments has emerged, offering a glimpse of hope to overcome the limitations of existing therapies. This review aims to highlight cutting-edge technologies in the current and ongoing stages of development, providing patients with valuable insights to guide their choices in brain tumor treatment.

Supplementary Figure 14 shows that CP-d/n-ATF5 induces anoikis of neuroblastoma cell lines

Depletion of ATF5 induces anoikis and decreases metastasis of neuroblastoma cells. A and B, Viability of BE(2)-C and SK-N-DZ suspension cells in poly-HEMA–coated plates, expressing Dox-inducible shATF5-1 or shATF5-2 at different timepoints after Dox treatment. Mean ± SD. C and D, Quantification of anoikis of BE(2)-C and SK-N-DZ suspension cells cultured as in A and B at different timepoints after Dox addition. Mean ± SD. E, Quantification of whole-body bioluminescence flux (photons/second) in mice 24 hours after intracardiac injection of BE(2)-C-shATF5-2 cells, + Dox (n = 9); −Dox, (n = 9). Mice were maintained on drinking water containing Dox (2 mg/mL) from 3 days before injection to the time of euthanasia. F, Bioluminescent images at 24 hours after intracardiac injection under conditions described in E. G, Quantification of bioluminescence of blood from mice collected 12 and 24 hours after intracardiac injection under conditions described in E. H, Quantification of apoptosis of BE(2)-C-shATF5-2 CTCs isolated from mice 12 hours after intracardiac injection and treatment ± Dox (see E and Materials and Methods), + Dox (n = 5), −Dox, (n = 5). I, RT-PCR analyses of ATF5 and ACTB in circulating BE(2)-C-shATF5-2 cells isolated after 12 hours. J, Time course of whole-body bioluminescence flux in mice treated as in E. + Dox (n = 8); −Dox, (n = 9). The mice were monitored for metastatic spread by bioluminescence and euthanized at day 31. K, Quantification of total flux (photons/second) by ex vivo liver bioluminescence at the time of euthanasia (day 31). L, Quantification of bioluminescence in bone marrow homogenates at the time of euthanasia (day 31). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Supplementary figure legend unmarked

Supplementary Figure 9 shows that BMF knockdown rescues ATF5 loss-induced reduction of anchorage-independent cell viability

Supplementary Figure 4 shows that decreased ATF5 expression does not alter the invasiveness of BE(2)-C cells in vitro

Supplementary Figure 18 shows that CP-d/n-ATF5 treatment decreased viability and induced anoikis of SK-N-DZ CTCs

Supplementary Figure S8 shows that BMF overexpression reduces the anchorage-independent viability of BE(2)-C and SK-N-DZ cells.

Supplementary Figure 5 shows that overexpression of ATF5 promotes anoikis resistance of CHLA-255 in vitro

Supplementary Figure 13 shows that CP-d/n-ATF5 reduces the anchorage-independent viability of neuroblastoma cell lines

CP-d/n-ATF5 induces anoikis and inhibits neuroblastoma growth and metastasis in vivo. A, Effects of CP-dn-ATF5 on the expression of ATF5 and apoptosis-related BCL-2 proteins in suspension cells. Immunoblot of proapoptotic and antiapoptotic proteins in BE(2)-C and SK-N-DZ suspension cells at 72 hours after CP-d/n-ATF5 treatment. β-Actin was used as a loading control. Densitometric analysis was performed using ImageJ. B, BMF knockdown inhibits CP-d/n-ATF5–induced anoikis of BE(2)-C and SK-N-DZ cells. Adherent cells were treated with or without siBMFs (50 nmol/L) as indicated for 24 hours and then seeded in nonadherent plates. CP-d/n-ATF5 (200 µmol/L) was added, and 72 hours later, anoikis was evaluated. C, Kaplan–Meier analysis of survival of mice bearing BE(2)-C tumors, treated with vehicle (n = 7) or CP-d/n-ATF5 (50 mg/kg; n = 7). Treatment was started 7 days after cell implantation, once daily for the first 3 days, and then twice weekly. P < 0.01, log-rank (Mantel–Cox). D, Quantification of liver bioluminescence flux (photons/second) by ex vivo imaging at the time of euthanasia from mice bearing BE(2)-C tumors described in C. Vehicle (n = 7), CP-d/n-ATF5 (n = 6). E, Quantification of bioluminescence in bone marrow homogenate measured at the time of euthanasia (RLU/# BM cells, RLU: Relative luciferase unit). Vehicle (n = 7), CP-d/n-ATF5 (n = 6). F, Quantification of CTC measured at the time of euthanasia by bioluminescence of blood. Vehicle (n = 7), CP-d/n-ATF5 (n = 6). G, Twelve hours after intracardiac injection of BE(2)-C cells, blood was collected for (left) quantification of CTC by measurement of blood bioluminescence and (right) for measurement of apoptosis in isolated BE(2)-C CTC. Mice were treated with CP-d/n-ATF5 (n = 9) or vehicle (n = 9) immediately after intracardiac injection. H, BE(2)-C cells were injected intracardially and then treated with vehicle (n = 8) or CP-d/n-ATF5 (n = 10). Whole-body bioluminescence flux was then monitored for subsequent tumor cell growth. I, Representative bioluminescence images showing metastatic growth of BE(2)-C in mice treated with vehicle or CP-dn-ATF5 at day 38 of the experiment described in H. J, Quantification of total flux (photons/second) by ex vivo liver bioluminescence at time of euthanasia (day 38). K, Quantification of bioluminescence in bone marrow homogenate at the time of euthanasia (day 38). L, RT-PCR analysis of ATF5, BMF, and ACTB (internal control) in BE(2)-CTC from K. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Supplementary Figure 11 shows that FOXO3 silencing rescues cell viability following ATF5 depletion

Overexpression of ATF5 promotes anoikis resistance in vitro and in vivo. A and B, ATF5 overexpression, by immunoblot, in suspension culture of BE(2)-C and SH-SY5Y cells, 72 hours after transient transfection with pCCL-GFP or pCCL-ATF5. ACTB was used as a loading control. Densitometric analysis was performed using ImageJ. C and D, BE(2)-C and SH-SY5Y suspension cell viability and anoikis at 12, 24, 48, and 72 hours after transfection, respectively. At each timepoint, the comparison is between cells overexpressing ATF5 and control cells expressing GFP (100%). Mean ± SD. E, Quantification of mouse whole-body bioluminescence flux (photons/second) 12 hours after intracardiac injection of BE(2)-C cells overexpressing ATF5 (n = 8) or GFP (n = 7). F, Images showing increased bioluminescence of animals described in E indicate tumor cell survival 12 hours after intracardiac injection. G, Quantification of bioluminescence of blood collected 12 hours after intracardiac injection as in E. H, Quantification of apoptosis of BE(2)-C CTC isolated at 12 hours, GFP (n = 7); ATF5 (n = 7). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Supplementary Figure 17 shows that CP-dn-ATF5 reduces tumor growth and metastasis of SK-N-DZ in vivo

Supplementary materials and methods untracked

Developing novel therapeutics often follows three steps: target identification, design of strategies to suppress target activity and drug development to implement the strategies. In this review, we recount the evidence identifying the basic leucine zipper transcription factors ATF5, CEBPB, and CEBPD as targets for brain and other malignancies. We describe strategies that exploit the structures of the three factors to create inhibitory dominant-negative (DN) mutant forms that selectively suppress growth and survival of cancer cells. We then discuss and compare four peptides (CP-DN-ATF5, Dpep, Bpep and ST101) in which DN sequences are joined with cell-penetrating domains to create drugs that pass through tissue barriers and into cells. The peptide drugs show both efficacy and safety in suppressing growth and in the survival of brain and other cancers in vivo, and ST101 is currently in clinical trials for solid tumors, including GBM. We further consider known mechanisms by which the peptides act and how these have been exploited in rationally designed combination therapies. We additionally discuss lacunae in our knowledge about the peptides that merit further research. Finally, we suggest both short- and long-term directions for creating new generations of drugs targeting ATF5, CEBPB, CEBPD, and other transcription factors for treating brain and other malignancies.

Supplementary Figure 1 shows that ATF5 is expressed in neuroblastoma cell lines.