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. 2010 Jul 8;116(1):45-53.
doi: 10.1182/blood-2010-01-263756. Epub 2010 Mar 29.

17-DMAG targets the nuclear factor-kappaB family of proteins to induce apoptosis in chronic lymphocytic leukemia: clinical implications of HSP90 inhibition

Erin Hertlein  1 , Amy J Wagner, Jeffrey Jones, Thomas S Lin, Kami J Maddocks, William H Towns 3rd, Virginia M Goettl, Xiaoli Zhang, David Jarjoura, Chelsey A Raymond, Derek A West, Carlo M Croce, John C Byrd, Amy J Johnson
Affiliations

17-DMAG targets the nuclear factor-kappaB family of proteins to induce apoptosis in chronic lymphocytic leukemia: clinical implications of HSP90 inhibition

Erin Hertlein et al. Blood. .

Abstract

The HSP90 client chaperone interaction stabilizes several important enzymes and antiapoptotic proteins, and pharmacologic inhibition of HSP90 results in rapid client protein degradation. Therefore, HSP90 inhibition is an attractive therapeutic approach when this protein is active, a phenotype commonly observed in transformed but not normal cells. However, preclinical studies with HSP90 inhibitors such as 17-AAG demonstrated depletion of only a subset of client proteins and very modest tumor cytotoxicity in chronic lymphocytic leukemia (CLL) cells. Herein, we describe another HSP90 inhibitor, 17-DMAG, which is cytotoxic to CLL but not normal lymphocytes. Treatment with 17-DMAG leads to depletion of the HSP90 client protein IKK, resulting in diminished NF-kappaB p50/p65 DNA binding, decreased NF-kappaB target gene transcription, and caspase-dependent apoptosis. Furthermore, treatment with 17-DMAG significantly decreased the white blood cell count and prolonged the survival in a TCL1-SCID transplant mouse model. The ability of 17-DMAG to function as an NF-kappaB inhibitor is of great interest clinically, as few currently available CLL drugs target this transcription factor. Therefore, the effect of 17-DMAG on NF-kappaB signaling pathways represents a novel therapy warranting further clinical pursuit in this and other B-cell lymphoproliferative disorders.

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Figures

Figure 1
Figure 1
Apoptosis is significantly increased by 17-DMAG. (A) Left: Viability of CLL patient cells (n = 6) treated with vehicle control or increasing doses of 17-DMAG for 24 and 48 hours determined by annexinV/PI (Ann/PI) staining. Ann/PI double-negative cells are considered live cells. Right: Viability of CLL patient cells (n = 7) treated with vehicle control or increasing doses of 17-DMAG or 1μM 17-AAG for 24 and 48 hours determined by MTT assay. (B) Viability of normal B cells (n = 12), T cells (n = 8), and NK cells (n = 8) treated with vehicle control or increasing doses of 17-DMAG for 24 and 48 hours determined by Ann/PI. (C) Normal T cells (i) and B cells (ii) (n = 3) either unstimulated, or stimulated with CD3 (T cells) or CpG (B cells) concurrently or 3 hours before treatment with vehicle control or 1μM 17-DMAG for 24 and 48 hours. Viability is determined by Ann/PI staining, and Ann/PI double-negative cells are considered live cells. The T-cell and B-cell activation is demonstrated by increased CD69 (iii) and CD40 (iv) mean fluorescence intensity (MFI) surface staining, respectively.
Figure 2
Figure 2
17-DMAG–mediated cytotoxicity is caspase dependent. (A) Representative JC-1 flow diagram of CLL patient cells treated with vehicle or 1μM 17-DMAG for 24 hours. The percentage of cells containing intact mitochondria and depolarized mitochondria are indicated. The data are shown gated on live cells by forward and side scatter properties (top row) or ungated (bottom row). (B) Analysis of mitochondrial membrane potential in vehicle control or 1μM 17-DMAG–treated CLL at 8 and 24 hours by JC-1 staining (n = 6). Values shown in this experiment were calculated as a decrease in the JC-1 aggregate relative to the vehicle control. (C) Western blot analysis for PARP in whole cell lysate prepared from CLL patient cells treated with vehicle control or 1μM 17-DMAG for 24 hours in the presence or absence of 100μM caspase inhibitor z-VAD-fmk. Blots are probed with actin as a loading control. Results shown are representative of at least 6 patient samples. (D) Viability of CLL patient cells (n = 12) treated with vehicle control or 1μM 17-DMAG for 24 hours in the presence or absence of 100μM caspase inhibitor z-VAD-fmk determined by Ann/PI staining.
Figure 3
Figure 3
17-DMAG down-regulates NF-κB signaling through IKKα and IKKβ. Western blot analysis for AKT, IKKα, and IKKβ in whole cell lysate prepared from CLL patient cells treated with vehicle control or increasing doses of 17-DMAG for 24 hours. Blots are probed with actin as a loading control. Results shown are representative of 8 patient samples.
Figure 4
Figure 4
17-DMAG regulates NF-κB activity and target gene transcription. (A) Western blot analysis for IκBα and phosphorylated IκBα (P-IκBα) in cytoplasmic cell lysate and p65 and p50 in nuclear cell lysates prepared from CLL patient cells treated with vehicle control or 1μM 17-DMAG for 24 hours, 20 ng/mL TNF-α for 30 minutes, or 10μM Bay-11 for 1 hour. Blots are probed with actin or BRG1 as a loading control. Results shown are representative of at least 6 patient samples. (B) Electrophoretic mobility shift assay analysis with nuclear extract prepared from cells treated with vehicle control or 1μM 17-DMAG for 24 hours, 20 ng/mL TNF-α for 30 minutes, or 10μM Bay-11 for 1 hour using a radiolabeled oligonucleotide containing a consensus NF-κB-binding site. Antibody shifts are performed with nuclear extract prepared from vehicle control sample incubated with antibodies specific to the p65 or p50 subunits of NF-κB. The p65/p50 and p50/p50 complexes are indicated. Results shown are representative of 6 CLL samples. (C) Real-time PCR for MCL1 and BCL2 (n = 5 and n = 11, respectively) after 24 hours 1μM 17-DMAG treatment. Data are normalized to 18S transcript and represented as fold change in expression of 17-DMAG treated relative to the vehicle control. ○ represents individual patient samples. Bar represents the average of all patient samples. (D) Western blot analysis for MCL1 and BCL2 in cytoplasmic cell lysate prepared from CLL patient cells grown treated with vehicle control or 1μM 17-DMAG for 24 hours. Blots are probed with actin as a loading control. Results shown are representative of at least 6 patient samples. (E) Real-time PCR for MCL1 (n = 6) in CLL patient cells treated with vehicle control or 1μM 17-DMAG for 24 hours in the presence or absence of 100μM caspase inhibitor z-VAD-fmk. Data are normalized to TBP or 18S transcript and represented as fold change in expression of 17-DMAG treated relative to the vehicle control. ○ represents individual patient samples. Bar represents the average of all patient samples.
Figure 5
Figure 5
17-DMAG prevents CD40L- and CpG-induced NF-κB activity in CLL and prevents CD40L-mediated viability. (A) Western blot analysis for MCL1 in cytoplasmic cell lysate and p65 in nuclear cell lysate prepared from CLL patient cells treated with vehicle control, or 1μM 17-DMAG for 0, 4, 8, or 24 hours, followed by stimulation with 500 ng/mL CD40L for 1 hour. Blots shown are representative of 6 patient samples, and actin and BRG1 were probed as loading controls. (B) Western blot analysis for MCL1 in cytoplasmic cell lysate and p65 in nuclear cell lysate prepared from CLL patient cells treated with vehicle control, or 1μM 17-DMAG for 0, 4, 8, 16, or 24 hours, followed by stimulation with 3.2μM CpG oligodeoxynucleotides for 3 hours. Blots shown are representative of 6 patient samples, and blots are stripped and probed with actin and BRG1 as loading controls. (C) Viability by Ann/PI at 24 hours in CLL patient cells treated with vehicle control or 17-DMAG with and without 1 μg/mL CD40L.
Figure 6
Figure 6
17-DMAG prolongs survival in a mouse model of CLL. (A) Survival curve for TCL1-SCID mice treated with 10 mg/kg 17-DMAG treatment or vehicle control (n = 10/group). (B) White blood cell count (WBC) in 17-DMAG and vehicle control treated TCL1-SCID mice (n = 20/group). Count is determined at day 55 after initiation of treatment by hematoxylin and eosin-stained peripheral blood smear.
Figure 7
Figure 7
The proposed effect of 17-DMAG on NF-κB activity. Diagram of NF-κB signaling through the alternative and classic pathways. Classic signaling occurs after stimulation of IKKα/IKKβ heterodimers with inducers, such as TNF-α, interleukin-1, lipopolysaccharide, and CpG oligodeoxynucleotide, to activate p65/p50 complexes. Alternative signaling occurs after stimulation of IKKα homodimers with inducers, such as CD40L and BAFF, to activate RelB/p52 complexes. The proposed level of 17-DMAG intervention with NF-κB signaling is through the down-regulation of IKKα and IKKβ protein levels.
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References

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