Using a global phosphoproteome screen, we have recently identified heat shock proteins (HSPs) to be crucial effectors to stabilize KIT oncoprotein function in the presence of tyrosine kinase inhibitors in mutant-KIT leukemia models (Kampa-Schittenhelm et al., ASH annual meeting 2010). We now show that HSP60 and (p)HSP90 are frequently expressed in acute leukemia, arguing for an integrative function of HSPs to stabilize leukemia-driving oncoproteins. Importantly, HSPs are highly expressed in leukemia stem cells (LSCs), which provides a rationale for therapy insensitivity. Targeting HSPs may therefore be an attractive strategy to target the LSC clone.
Intracellular protein levels of (p)HSPs in leukemic blasts were studied by flow cytometry. Three dimensional detection of the putative CD34+/CD38-/HSP+ leukemia stem/progenitor cell fraction was performed using -FITC, -PE and APC-coupled secondary antibodies using standard techniques. Cellular proliferation and induction of apoptosis in leukemia cells treated with the HSP90 inhibitor IPI-504 was determined by XTT- and annexin V-based assays. Immunoblot experiments to assess IPI-504 interaction on the chaperone level were set up using standard protocols including a LICOR imaging system.
Of 16 evaluated patients with AML, 50% and 56% demonstrated significant HSP90 and HSP60 expression levels (with a threshold level set as > 3× of the relative mean fluorescence compared to IgG controls), respectively. Interestingly, HSP expression was predominantly seen in patients with core–binding factor leukemia (associated with KIT mutations). Moreover, five evaluable freshly harvested patient samples were used to assess the putative CD34+/CD38-/HSP90 and HSP60 population by flow cytometry. Tantalizingly, all tested CD34+/CD38- samples revealed high co-expression of HSPs. Consequently, an HSP90 inhibitor (IPI-504) potently inhibited cellular proliferation in in vitro leukemia models as well as freshly harvested leukemia cells at highly variable doses starting in low nanomolar ranges up to lower micromolar concentrations with regard to IC50s. Several cell lines with defined leukemia-driving oncogenes (MOLM14 – FLT3 ITD, HMC1.1 – KIT V560G, Kasumi1 – KIT N822H) were treated with IPI-504, and degradation of FLT3 and KIT was confirmed by immunoblotting. Consequently, oncogene-degradation led to induction of apoptosis in the nanomolar range which was confirmed in ex vivo blasts. Noteworthy, treating isolated freshly harvested CD34+/CD38- progenitor cells lead to potent reduction of the viable cell fraction.
HSPs are frequently expressed in acute leukemias, including LSCs, arguing for a function as mechanism to protect and uphold LSC function in the presence of cell stress such as antileukemic treatment. Targeting the HSP-expressing stem cell pool may be an attractive novel therapeutic strategy in acute leukemia.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.