Tanespimycin Reverses Bortezomib-Induced Inhibition of Granulopoiesis.

Tanespimycin, an Hsp90 inhibitor, is in phase 3 clinical trials with bortezomib for the treatment of multiple myeloma (MM). Neutropenia and thrombocytopenia are commonly observed during bortezomib treatment in patients with MM. However, in a phase 1/2 study of tanespimycin + bortezomib in patients with MM, the incidence and severity of neutropenia was low (Richardson ASCO 2009). Here we present the in vitro effects of tanespimycin and bortezomib on hematopoiesis and granulopoiesis in cell culture systems using mononuclear cells from healthy subjects and cryopreserved hematopoietic stem cells. METHODS: Mononuclear cells were separated from fresh whole healthy human bone marrow cells and cultured for up to 14 days in suspension in the presence of tanespimycin (1 pM–300 μM) and bortezomib (1 nM–300 μM). Committed hematopoietic progenitor growth (CFC-GEMM [granulocyte/erythroid/macrophage/megakaryocyte], BFU-E [erythroid], GM-CFC [granulocyte/macrophage], MkCFC [megakaryocyte]) of cultured cells was assessed using intracellular ATP bioluminescence in the HemoGenix HALO^® system, which couples specific hematopoietic lineage growth conditions with a surrogate for cell count. In addition, cryopreserved healthy human hematopoietic stem cells were cultured in semisolid methylcellulose and colonies (CFC-GEMM, CFU-E, BFU-E) were microscopically enumerated. RESULTS: When bortezomib and tanespimycin were given alone in suspension cultures, IC₅₀ values for bortezomib-induced cytotoxicity and tanespimycin-induced cytotoxicity were approximately 1 nM to 2 nM and 100 nM, respectively, for all hematopoietic lineages. In semisolid methylcellulose cultures, 1 nM to 10 nM bortezomib induced a concentration-dependent inhibition of granulocyte-macrophage colony formation from 2% to 46% and of erythroid colony formation from 8% to 35%. While tanespimycin alone at a concentration of 10 nM had little or no effect on erythropoiesis or granulocytopoiesis, when bortezomib (1 nM or 3 nM) and tanespimycin (10 nM) were cocultured, tanespimycin completely reversed bortezomib-induced inhibition of erythropoiesis and granulopoiesis. More severe erythropoietic inhibition at 10 nM bortezomib was not reversed by tanespimycin, while granulopoietic inhibition was mitigated approximately 23%. At concentrations of melphalan (1 μM) and camptothecin (3 nM) that inhibit in vitro hematopoiesis approximately 50% and 90%, respectively, 10 nM tanespimycin had no effect. CONCLUSION: At clinically relevant drug concentrations in hematopoietic cultures, tanespimycin reverses bortezomib-induced inhibition of granulopoiesis, consistent with the low frequency of neutropenia observed in clinical trials of tanespimycin + bortezomib. This suggests tanespimycin may prevent bortezomib-induced apoptosis of granulocyte-macrophage progenitors.

Flint:Bristol-Myers Squibb: Employment, Equity Ownership. Oberdoerster:Bristol-Myers Squibb: Employment. Price:Bristol-Myers Squibb: Employment. Foster:Bristol-Myers Squibb: Employment. Gemzik:Bristol-Myers Squibb: Employment. Berman:Bristol-Myers Squibb: Employment.