HSP90 Inhibition Has Potent Activity Against T-Cell Acute Lymphoblastic Leukemia (T-ALL) Through Degradation Of TYK2 Kinase

The intensification of therapy for patients with T-cell acute lymphoblastic leukemia (T-ALL) has improved clinical outcomes substantially. However, first-line therapy still continues to fail in approximately 25% of children and in more than 50% of adults, clearly indicating that further therapeutic improvement is urgently needed. Recently, we identified a novel oncogenic pathway that involves aberrant activation of the TYK2 tyrosine kinase and its downstream substrate, STAT1, which ultimately promotes T-ALL cell survival through the upregulation of BCL2 expression (Sanda et al. Cancer Discovery 2013). This finding indicates that in many T-ALL cases, the leukemic cells are dependent upon the TYK2-STAT1-BCL2 pathway for continued survival, suggesting that drugs able to potently inhibit or degrade the TYK2 kinase are likely to provide a therapeutic advantage in patients with T-ALL. However, there are no kinase inhibitors with potent activity to TYK2 under current clinical trials. Heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone that is exploited by cancer cells to support activated oncoproteins including many cancer-associated kinases, and recent reports on the early clinical efficacy of HSP90 inhibitors are encouraging in some tumors. Based on the finding that TYK2 is related to JAK2 and the reports implicating JAK2 as an HSP90 client protein (Weigert et al. JEM 2012 and Marubayashi et al. J Clin Invest 2010), we investigated the therapeutic efficacy of HSP90 inhibition in T-ALL. First, we tested the effect of two HSP90 inhibitors (AUY922 and HSP990) on the cell viability of T-ALL cell lines. The growth of 10 T-ALL lines, which harbor different genetic aberrations, was dramatically reduced in response to treatment with both compounds, with IC₅₀ values of 5-280 nM (AUY922) and 18-221 nM (HSP990) after 72 hours of exposure. Treatment with AUY922 induced significant apoptosis in KOPT-K1, HPB-ALL and Jurkat “TYK2-dependent” T-ALL cell lines, which are sensitive to shRNA knockdown of the TYK2 gene, as indicated by increased levels of Annexin V staining, which was not observed in Loucy and TALL-1 “TYK2-independent” cells. AUY922 also induced strong cell-cycle arrest at the G2/M phase in some of T-ALL cell lines. Western blotting analysis using T-ALL cell lines after exposure to AUY922 demonstrated rapid reduction of the TYK2 kinase and dephosphorylation of STAT1 in concentrations less than 30 nM of AUY922. The decrease of BCL2 expression after AUY922 treatment was seen only in “TYK2-dependent” T-ALL cell lines, not in “TYK2-independent” lines. These results indicate that pharmacological HSP90 inhibition resulted in TYK2 degradation and subsequent downregulation of its downstream pathway, which includes phosphorylation of STAT1 and activation of BCL2, and this effect is critical especially for the survival of “TYK2-dependent” T-ALL cells. To assess the significance of BCL2 on the cell viability of T-ALL cells after HSP90 inhibition, we tested AUY922 on specific Jurkat cell lines overexpressing each of the pro-survival BCL2 family proteins BCL2, BCL_XL, or MCL1. The growth curve after 72 hours of treatment demonstrated that BCL2 overexpression could partially rescue the AUY922-induced decrease of cell viability, but overexpression of BCL_XL or MCL1 had no effect. Immunoprecipitation assay using Jurkat cells overexpressing BCL2 indicated that treatment with AUY922 resulted in increased interaction between overexpressed BCL2 and the pro-apoptotic BH3-only protein, Bim. These results suggest that decreasd BCL2 expression is at least partially responsible for the ability of HSP90 inhibitors to induce apoptosis in T-ALL cells. In conclusion, these results provide preclinical evidence of the therapeutic potential of HSP90 inhibitors against T-ALL, providing a rationale for the evaluation of these inhibitors in clinical trials of patients with T-ALL.