Co-Treatment with a Novel Heat Shock Protein (hsp) 90 Inhibitor IPI504 and the Histone Deacetylase Inhibitor (HDI) Vorinostat (Suberoylanilide Hydroxamic Acid, SAHA): A Highly Active Combination Against Wild Type or Mutant Bcr-Abl-T315I or FLT-3-ITD-Containing Human Leukemia Cells.
Authors: Fiskus Warren , Bali Purva , Pranpat Michael , Balasis Maria , Kumaraswamy Sandhya , Boyapalle Sandhya , Rocha Kathy , Tong Jeffery , Richon Victoria , Normant Emmanuel , Bhalla Kapil 
Published: October 2019

Abstract
Hsp90 is an ATP-dependent molecular chaperone, which helps in folding its client proteins, e.g., Bcr-Abl, FLT-3, c-Raf and Akt, into active conformation. Geldanamycin analogue, 17-AAG (Kosan Biosciences Inc., Hayward, CA) inhibits the chaperone function of hsp90, which promotes polyubiquitylation and proteasomal degradation of the misfolded hsp90 client proteins. We recently reported that, by inhibiting the activity of histone deacetylase 6, the hydroxamate HDIs such as vorinostat (Merck & Co., Inc.) induce acetylation and inhibition of hsp90, thus also causing the depletion of its client proteins. In the present studies, we determined the anti-leukemia effects of the novel, highly soluble, hsp90 antagonist IPI504 (Infinity Pharmaceuticals), which, in vitro and in vivo, interconverts with 17-AAG, ± vorinostat, against human cultured or primary, wild type or mutant Bcr-Abl or mutant FLT-3 containing acute leukemia cells. Treatment with IPI504 (0.5 to 2.0 μM) for 24 to 48 hours, in a dose dependent manner, induced apoptosis of WT Bcr-Abl-expressing K562 and LAMA-84 cells. This was associated with attenuation of the levels of Bcr-Abl, pCrkL, pSTAT5, c-Raf and pAkt. In a dose dependent manner (50 to 500 nM for 48 hours), IPI504 also induced apoptosis of FLT-3 internal tandem duplication (ITD)-containing human acute leukemia MV4-11 cells, which was associated with attenuation of the levels of FLT-3, pAkt, pSTAT5, pERK1/2. Notably, treatment with IPI504 induced similar level of apoptosis of mouse bone marrow BaF3 cells, which had been transformed and rendered IL-3 independent for growth by ectopic expression of WT Bcr-Abl, its P-loop (Bcr-Abl-E255K) or highly imatinib mesylate (IM) resistant, contact-inhibition (Bcr-Abl-T315I) point mutant. This was also associated with attenuation of the levels of WT and mutant Bcr-Abl-E255K or Bcr-Abl-T315I. In previous studies we had demonstrated that treatment with vorinostat depletes WT and mutant Bcr-Abl levels and induces apoptosis of expressing human leukemia cells. Therefore, we determined the effect of the co-treatment of IPI504 (1.0 μM) and vorinostat (1.0 μM) against cultured or primary human CML cells. Co-treatment with IPI504 and vorinostat induced significantly more apoptosis of K562 and MV4-11 cells, which was associated with more depletion of WT-Bcr-Abl and FLT-3-ITD levels in K562 and MV4-11 cells, respectively. Notably, co-treatment with IPI504 and vorinostat, versus treatment with either agent alone, also induced more apoptosis of primary CML cells (4 samples) derived from patients with IM-resistant CML, including a sample of cells documented to have Bcr-Abl-T315I mutation. Additionally, as compared to treatment with either agent alone, the combination of IPI504 and vorinostat also induced more apoptosis of primary AML cells (4 samples), including two samples that contained FLT-3-ITD. These findings demonstrate that the combination of IPI504 with vorinostat exerts a high level of in vitro activity against FLT-3-ITD-containing acute leukemia, as well as against highly IM-resistant mutant Bcr-Abl-expressing leukemia cells.