Inhibition of heat shock protein 90 prolongs survival of mice with BCR-ABL-T315I–induced leukemia and suppresses leukemic stem cells

Abstract

IntroductionThe human Philadelphia chromosome (Ph) arises from a translocation between chromosomes 9 and 22 [t(9;22)(q34;q11)]. 1 The resulting chimeric BCR-ABL oncogene encodes a constitutively activated, oncogenic tyrosine kinase that induces chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). The BCR-ABL TKI, imatinib mesylate, induces a complete hematologic and cytogenetic response in the majority of chronicphase CML patients, 2 but is unable to completely eradicate BCR-ABL-expressing leukemic cells, 3,4 suggesting that leukemia stem cells are not eliminated. Over time, patients frequently become drug resistant and develop progressive disease despite continued treatment. [5][6][7][8][9][10][11] Resistance is predominantly due to emergence of kinase domain mutations. Three newly developed BCR-ABL kinase inhibitors-dasatinib, 12 AP23464, 13 and AMN107 14 -inhibit most of imatinib-resistant BCR-ABL mutants at biochemical and cellular levels, but are ineffective against the BCR-ABL-T315I mutant. 15,16 New approaches are needed to treat drug-resistant forms of CML as well as BCR-ABL-induced B-ALL, a leukemia that does not respond well to available TKIs. 15,16 Heat shock protein 90 (Hsp90) is a highly conserved, constitutively expressed molecular chaperone that facilitates folding of client proteins such as BCR-ABL, and affects the stability of these proteins. [17][18][19][20][21] When BCR-ABL contains resistance-conferring mutations, it becomes even more dependent on Hsp90 in vitro. 20 We therefore evaluated the therapeutic effect of Hsp90 inhibition by using a novel water-soluble inhibitor, 22 in drug-resistant animal models of leukemia induced by BCR-ABL-WT and T315I. Materials and methods Cell linesThe 32D myeloid cell line was grown in RPMI 1640 medium containing 10% FCS and 10% WEHI medium. The BaF/3 pre-B-cell line was grown in RPMI 1640 medium containing 10% FCS, 10% WEHI medium, and 50 M 2-mercaptoethanol. To generate the BCR-ABL-expressing 32D or BaF/3 line, the cells were transduced with the BCR-ABL-WT-or BCR-ABL-T315I-IRES-GFP-MSCV retrovirus, and the BCR-ABL-expressing cells were selected by GFP sorting by fluorescence-activated cell sorter (FACS). HistologyThe lungs from the placebo-or drug-treated mice were fixed in Bouin fixative (Fisher Scientific, Pittsburgh, PA) for 24 hours at room temperature, followed by an overnight rinse in water. Ten-m sections were stained with hematoxylin and eosin (H&E) and observed by a model DMRE compound microscope (Leica, Heidelberg, Germany). All sections were imaged with a 2.5 ϫ PH1 objective (NPLan, NA 0.25) and 10 ϫ PH1 objective (NPLan, NA 0.40). All images were imported into MetaMorph software (Molecular Devices, Downingtown, PA) as a series of tagged image files. All images were then constructed in Adobe Photoshop 6.0 (Adobe, San Jose, CA). Antibodies and Western blot analysisAntibodies against c-ABL, Hsp90, Hsp70, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Protein lysates were prepared by lysing cells...

Bag1 directly routes immature BCR-ABL for proteasomal degradation

“…Previous reports from others showed that imatinib enhanced the cytotoxic effect of Hsp90 inhibitors. 29,38 We have also found a similar combinatory effect (see the effects at a fixed dose of GA in For personal use only. on May 12, 2018.…”

“…T315I was found to bind Bag1 more strongly than the wt, which correlates with the sensitivity of Hsp90 inhibitors for BCR-ABL degradation and CML mice with the mutant. 8,29 These informations suggest that imatinib binding induces structural maturation of BCR-ABL to which Bag1 can no longer bind, and the T315I mutant is presumed to be less tightly bound to Hsp90 than the wt. The ABL kinase domain bound to imatinib may escape its degradation for the time being.…”

“…The imatinib binding site mutant T315I mutant degrades more readily with and CML mice expressing the mutant respond better to Hsp90 inhibitors than the wt BCR-ABL. 8,29 T315I was found to bind Bag1 more strongly than the wt ( Figure 4A). The imatinib-bound ABL kinase domain has been shown to display a specific inactive conformation.…”

Hsp90 inhibitor as a sensitizer of cancer cells to different therapies (Review)

“…Elimination of mutant BCR-ABL (T315I) kinase seems to be more effective therapeutic strategy for treating BCR-ABL-induced leukemia than the inhibition of BCR-ABL (T315I) kinase activity. Indeed, combination treatment with Hsp90 inhibitor IPI-504 and imatinib was more effective than either treatment alone in prolonging survival of mice simultaneously bearing both wild-type and T315I leukemic cells (131). Simultaneous exposure of BaF3 cells expressing BCR-ABL mutants including T315I to AUY922 and nilotinib was more effective at reducing the outgrowth of resistant cell clones as well as at survival prolongation of mice transplanted with mixture of BaF3 cells expressing wild-type BCR-ABL and mutant forms (132).…”

Targeting survival cascades induced by activation of Ras/Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways for effective leukemia therapy

“…122 Novel Hsp 90 inhibitors, such as IPI-504 will decrease the expression of the mutant BCR-ABL protein, although it should be kept in mind that the Hsp 90 inhibitors affect many proteins including Raf and Akt. 123 Combinations of MEK and dasatinib inhibitors may be effective in the treatment of imatinib-resistant patients harboring certain BCR-ABL mutations (for example, E225K, M351T). Unfortunately, this combination was not effective against imatinib-resistant cells with the T315I mutation.…”