An activated JAK/STAT3 pathway and CD45 expression are associated with sensitivity to Hsp90 inhibitors in multiple myeloma.

Exp Cell Res. 2013 Mar 10;319(5):600-11.

Huiqiong Lin1*, Iryna Kolosenko*, Ann-Charlotte Björklund, Darya Protsyuk, Anders Österborg, Dan Grandér and Katja Pokrovskaja Tamm

 

Department of Oncology-Pathology, Cancer Centre Karolinska (CCK), Karolinska Institutet, and Department of Hematology, Karolinska University Hospital Solna, Stockholm, Sweden

1present address: Department of Plant Sciences, University of California, One Shields Avenue, Davis, CA 95616, USA

*These authors contributed equally to the work

  

Corresponding author:

Katja Pokrovskaja Tamm, PhD

Department of Oncology-Pathology

Cancer Centre Karolinska (CCK)

Karolinska University Hospital Solna

SE-171 76 Stockholm, Sweden.

Tel: +46(8)517 73738 Fax: +46(8) 339031.

E-mail:  katja.pokrovskaja@ki.se

 

Running title: Sensitivity to Hsp90 inhibitors in multiple myeloma

Abbreviations: MM, multiple myeloma; Hsp90, heat shock protein 90 kD; Hsp90-Is, Hsp90 inhibitors; STAT3C, constitutively active STAT3, 17DMAG, 17-Desmethoxy-17-N,N-dimethylaminoethylamino-geldanamycin, HCl.

 

Abstract

Background. Multiple myeloma (MM) is a relatively common B-cell malignancy which still remains incurable. It is characterized by accumulation of a malignant clone of plasma cells in the bone marrow followed by lytic bone damage, abnormalities in hematopoiesis, renal failure.  The bone marrow stroma cells (BMSC) play a crucial role in supporting growth and survival of MM cells by secreting pro-survival cytokines. The molecular chaperone Hsp90 (heat shock protein 90) is required for maintaining protein homeostasis and the activity of many signaling oncoproteins, including members of the JAK/STAT and the PI3K pathways. Therefore, inhibitors of Hsp90 (Hsp90-Is) can contribute to the set of anti-cancer and, particularly, anti-myeloma drugs. When clinically evaluated, however, Hsp90-Is have demonstrated varying activity against MM. In this study, we aimed to determine which signaling pathways account for differential sensitivity to the Hsp90-I 17DMAG in MM.

Methods. Tyrosine phosphatase CD45, required for lymphocyte development and maturation, is differentially expressed in MM cell lines and primary tumors. CD45 expression is induced by IL-6, correlates with an activated JAK/STAT signaling pathway and negatively regulates PI3K/Akt signaling. Nine MM cell lines were characterized for the expression of CD45, the activity of and dependence on the JAK/STAT3 and PI3K pathways using FACS, Western blotting and viability assays. This allowed dividing cell lines into two major groups: CD45+/JAK/STAT-dependent and CD45/PI3K-dependent.  Freshly obtained MM cells from newly diagnosed patients were also characterized for the presence of CD45, pSTAT3 and pAkt expression using immunostainings and FACS. They also fell into two distinct groups: with the prevalence of either high or low CD45 expressers among MM cells.

Results. The CD45+ cell lines with an activated JAK/STAT3 pathway were more sensitive to 17DMAG and underwent prominent apoptosis upon treatment, while the majority of CD45 cell lines, dependent on the activated PI3K pathway, were more resistant to the drug. Among primary myeloma cells, the high CD45 expressers also expressed significantly higher levels of pSTAT3 and underwent more prominent cell death upon ex vivo treatment with 17DMAG, as compared to the low CD45 expressers. Culturing the most resistant cell line, LP1, in the presence of IL-6 resulted in the up-regulation of CD45 and pSTAT3, and sensitized cells to 17DMAG-induced apoptosis, primarily in the induced CD45+ sub-population of the cells. STAT3 activity was efficiently inhibited by Hsp90-Is both in cell lines and in primary cells suggesting the importance of STAT3 inactivation for the pro-apoptotic effects of HSP90-Is. Indeed, overexpression of a constitutively activated variant of STAT3, STAT3C, in U266 MM cell line, protected from 17DMAG-induced cell death. The down-regulation of the STAT3 target gene Mcl-1 at both the mRNA and protein levels following 17DMAG treatment was significantly attenuated in STAT3C-expressing cells. In line with this, transient overexpression of Mcl-1 protected U266 cells from 17DMAG-induced cell death.

Conclusions. The finding that CD45+ MM cells with an IL-6-activated JAK/STAT3 pathway are particularly sensitive to Hsp90-Is as compared to the low CD45 expressers may provide a rational basis for selection of MM patients amenable to Hsp90-I treatment. Our data also suggest that IL6-mediated activation of JAK/STAT3 is highly dependent on Hsp90, and that inhibition of STAT3 by Hsp90-Is is crucial for cell death induced by these drugs. These findings also suggest a potential activity of Hsp90-Is in a broader range of both cancer and other diseases that depend on IL-6 and an elevated activity of JAK/STAT3 pathway.

 

Key words:  multiple myeloma, apoptosis, STAT3, Akt, 17DMAG, HSP90

 

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