17-DMAG Targets the NF-κB Family of Proteins to Induce Apoptosis in CLL: Clinical Implications of Hsp90 Inhibition

Chronic lymphocytic leukemia (CLL) is a disease of accumulating B-cell lymphocytes for which there is currently no curative therapy. One attractive therapeutic target currently being explored in CLL is Hsp90, a chaperone which stabilizes various client proteins (Akt, Raf, ZAP-70) which are important for survival of CLL cells. Interfering with Hsp90 protein binding to these chaperone proteins leads to their rapid degradation. Our group and others have demonstrated that 17-allylamino-17-demethoxy-geldanamycin (17-AAG) depletes only select chaperone proteins and promotes only modest cytotoxicity in CLL. 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) is a novel Hsp90 inhibitor with improved solubility, bioavailability and cytotoxicity in other cancer cell lines as compared to 17-AAG. We demonstrate that 17-DMAG more potently induces caspase dependent apoptosis of primary CLL cells, as compared to 17-AAG, but does not mediate apoptosis of normal T-cells and NK-cells. In primary CLL cells, 17-DMAG treatment leads to cellular depletion of a broader range of Hsp90 client proteins (Akt, Cdk9, ZAP-70 and IKK). IKK is the activating kinase of the NF-kB family of transcription factors. To validate the down-stream significance of this we used EMSA to confirm that NF-kB is constitutively active in CLL as compared to normal B-cells. We demonstrate that 17-DMAG effectively reduces NF-kB DNA binding and thereby decreases subsequent transcription of known NF-kB target genes (Mcl-1 and XIAP). In addition to the direct role of NF-kB on target genes, NF-kB inhibition indirectly increases Bim transcription through regulation of the FoxO3a transcription factor. Furthermore, the reciprocal regulation transcription of Bcl-2 family members Mcl-1 and Bim are early events that initiate membrane depolarization and activation of the caspase cascade, leading to 17-DMAG induced cytotoxicity. siRNA experiments to elucidate the direct importance of Bim to 17-DMAG treatment are ongoing at this time. In conclusion, our data demonstrate that the Hsp90 inhibitor 17-DMAG represents a novel multi-targeted inhibitor of several critical kinases, transcription factors and anti-apoptosis genes relevant to CLL survival. Given its oral formulation, which allows administration of 17-DMAG by continuous dosing and uninterrupted inhibition of Hsp90, initiation of phase II clinical trials in CLL that include detailed pharmacodynamic studies monitoring NF-kB target genes are indicated.

This work is supported by the Leukemia and Lymphoma Society of America and the D. Warren Brown Foundation.