A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers

Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interaction-based neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2–interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence–specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death

Perifosine and CCI 779 co-operate to induce cell death and decrease proliferation in PTEN-intact and PTEN-deficient PDGF-driven murine glioblastoma.

Platelet derived growth factor receptor (PDGFR) activity is deregulated in human GBM due to amplification and rearrangement of the PDGFR-alpha gene locus or overexpression of the PDGF ligand, resulting in the activation of downstream kinases such as phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). Aberrant PDGFR signaling is observed in approximately 25-30% of human GBMs, which are frequently molecularly classified as the proneural subclass. It would be valuable to understand how PDGFR driven GBMs respond to Akt and mTOR inhibition.Using genetically engineered PTEN-intact and PTEN-deficient PDGF-driven mouse models of GBM that closely mimic the histology and genetics of the human PDGF subgroup, we investigated the effect of inhibiting Akt and mTOR alone or in combination in vitro and in vivo. We used perifosine and CCI-779 to inhibit Akt and mTOR, respectively. Here, we show in vitro data demonstrating that the most effective inhibition of Akt and mTOR activity in both PTEN-intact and PTEN-null primary glioma cell cultures is obtained when using both inhibitors in combination. We next investigated if the effects we observed in culture could be duplicated in vivo by treating mice with gliomas for 5 days. The in vivo treatments with the combination of CCI-779 and perifosine resulted in decreased Akt and mTOR signaling, which correlated to decreased proliferation and increased cell death independent of PTEN status, as monitored by immunoblot analysis, histology and MRI.These findings underline the importance of simultaneously targeting Akt and mTOR to achieve significant down-regulation of the PI3K pathway and support the rationale for testing the perifosine and CCI-779 combination in the human PDGF-subgroup of GBM

<i>In vivo</i> imaging of therapeutic response using contrast enhanced diffusion weighted MRI.

<p>(A) MR gadolinium (Gd) contrast-enhanced T1-weighted images and ADC color overlay maps demonstrate effects of <i>in vivo</i> effects of treatment with obtained from representative control, perifosine, CCI-779 and combination (perifosine + CCI-779) treated glioma mice at 5-7 days post-initiation of therapy. (B) Plot of mean MRI-determined tumor volumes based upon Gd-contrast enhanced regions versus time post-treatment initiation for control, perifosine, CCI-779 and combination (perifosine + CCI-779) treated mice. (Error bars ± SEM). (C) Plot of percent change of mean ADC values versus time post-treatment initiation for control, perifosine, CCI-779 and combination (perifosine + CCI-779) treated mice. (Error bars ± SEM).</p

PDGF-B-driven glimoas treated with the combination of perifosine and CCI-779 undergo cell death and have decreased proliferation.

<p>Images of H&E, IHC with anti-p-S6RP, anti-PCNA, anti-Ki67, and TUNEL of (A) PTEN +/+ and (B) PTEN -/- GBMs after glioma-bearing mice were treated <i>in vivo</i> for 5 days with either vehicle, 30 mg/Kg perifosine, 40 mg/Kg CCI-779, or a combination of 30 mg/Kg perifosine with 40 mg/Kg CCI-779. PCNA and Ki67 images are 200x with the black bar indicating 100 microns. The graph on the right shows quantification of Ki67 and TUNEL staining for 3-4 independent tumors. *,**, and *** represent significance determined by ANOVA analysis for p>0.05, p>0.01, and p>0.001.</p

Combing perifosine and CCI-779 effectively decreases the levels of pAKT and pS6RP of gliomas treated <i>in vivo</i>.

<p>Immunoblot analysis of (A) PTEN +/+ and (B) PTEN -/- tumors after mice with GBMs were treated for 5 days with either vehicle, 30 mg/kg perifosine, 40 mg/kg CCI-779, or a combination of 30 mg/Kg perifosine with 40 mg/Kg CCI-779. The right panel shows the quantification of the changes in pAkt and pS6RP based on comparing the average of each group to the vehicle treated tumors (n = 3 mice per group).</p

Combing perifosine and CCI-779 effectively inhibits both the Akt and mTOR pathways in primary glioma cultures treated <i>in vitro</i>.

<p>(A) A representative immunoblot of PTEN +/+ primary GBM cell cultures after 4 hours with no treatment (NT), vehicle (V), 30 µM perifosine (P), 1 nM CCI-779 (C) or combination of 30 µM perifosine and 1 nM CCI-779 (P+C). (B) Quantification of the changes in pAkt and pS6RP based on three independent primary PTEN +/+ GBM cell cultures, with 20 µg protein per well. (C) A representative immunblot of PTEN -/- primary GBM cell cultures after 4 hours with no treatment (NT), vehicle (V), 30 µM perifosine (P), 1 nM CCI-779 (C) or combination of 30 µM perifosine and 1 nM CCI-779 (P+C). (D) Quantification of the change in pAKT and pS6RP based on three independent primary PTEN -/- GBM cultures, with 20 µg protein per well.</p

Generation of PTEN wt and PTEN null gliomas using the RCAS/tv-a system.

<p>(A) Four-to-six week old <i>nestin-tv-a/ink4a-arf-/-/pten^fl/fl</i> mice are stereotactically injected with DF-1 cells expressing the RCAS-PDGF-B virus and from high-grade gliomas with PTEN intact. (B) Four-to-six week old <i>nestin-tv-a/ink4a-arf-/-/pten^fl/fl</i> mice are stereotactically injected with DF-1 cells expressing the RCAS-PDGF-B virus and DF-1 cells expressing RCAS-Cre virus and from high-grade tumors with PTEN deleted.</p