FOXO transcription factor activity is partially retained in quiescent CML stem cells and induced by tyrosine kinase inhibitors in CML progenitor cells

Abstract Bcr-Abl tyrosine kinase supports CML cell survival in part by regulating antiapoptotic Bcl-2 proteins such as Bcl-xL and Mcl-1. Tyrosine kinase inhibition, the front-line therapy for patients with chronic phase CML, is less effective in blast crisis (BC) patients and inactive against quiescent CML stem/progenitor cells. We reported that ABT-737, a dual Bcl-2/Bcl-xL inhibitor, induces apoptosis in BC CML cells including CD34+quiescent CML cells. ABT-199, a potent Bcl-2 specific inhibitor, has entered clinical trials for various hematological malignancies. We hypothesized that cooperative targeting of antiapoptotic Bcl-2 proteins using a combination of ABT-199 and tyrosine kinase inhibitors (TKIs) would exert enhanced activity against BC CML and CML stem/progenitor cells. Cells from patients (n=4) with TKI-resistant BC CML were treated with ABT-199, TKIs, and combinations. Although exerting low activity by itself, ABT-199 in combination with TKIs synergistically induced apoptosis (CI<0.1) in bulk and CD34+38- cells from these patients regardless of their previous clinical responses to TKIs. The combinations had minimal activity against normal CD34+cells (n=3). Mechanistic studies demonstrated that nilotinib inhibited the expression of Bcl-xL and Mcl-1 mRNA and protein, even in cells from TKI (including nilotinib) resistant patients. Individual inhibition of Bcl-xL or Mcl-1, and even more so inhibition of both, by siRNAs increased the sensitivity of cells to ABT-199, suggesting that cooperative inhibition of Bcl-2 by ABT-199 and Bcl-xL/Mcl-1 by TKIs contributes to the synergy. To evaluate the effect of these combinations on TKI-insensitive quiescent stem/progenitor CML cells, BC CML patient cells were stained with the cell division-tracking dye carboxyfluorescein succinimidyl ester (CFSE) and then co-cultured with human bone marrow (BM)-derived mesenchymal stromal cells (MSCs). Once proliferating and quiescent cells were distinguishable by flow cytometry, cells were treated with ABT-199, TKIs, and their combinations for 48 hours with or without MSC co-culture. Apoptosis was measured in proliferating and quiescent progenitor cells, defined as the percentage of annexin V positivity in CD34+CFSEdim and CD34+CFSEbright cells, respectively. ABT-199 as a single agent decreased viability of CML cells cultured alone or co-cultured with MSCs in both proliferating (IC50=191±103nM and 194±64nM, respectively) and quiescent (IC50=221±75nM and 205±123nM, respectively) CD34+ CML cells. Combinations of ABT-199 with TKIs, including imatinib, nilotinib, dasatinib, or ponatinib, synergistically induced death (CI<0.2) and decreased the number of viable cells in proliferating as well as quiescent CD34+progenitor cell populations (n=6). All 6 patients were resistant to TKIs, and 4 had mutations in the BCR-ABL gene, including three with the T315I mutation. To further test the ability of ABT-199 and TKI combinations to eradicate CML stem cells, we used an inducible transgenic CML mouse model in which the BCR-ABL gene is expressed under control of a tet-regulated enhancer of the murine stem cell leukemia (Scl) gene, allowing targeted BCR-ABL expression in stem/progenitor cells. Once BM cells from transgenic Scl-tTa-BCR-ABL/GFP mice were engrafted in wild type recipient mice, the mice were treated with ABT-199, nilotinib, or both. At the end of a 3-week treatment period, each single agent alone, and even more so with the combinations, significantly decreased blood total GFP+ WBC (12.9±1.4, 5.2±0.3, 6.1±0.4, and 1.6±0.3 x106/ml in controls, ABT-199, nilotinib, and combination, respectively) and neutrophils (1.43±0.03, 0.49±0.06, 0.32±0.03, and 0.25±0.05 x106/ml in the respective groups). ABT-199 (P=0.02), and more so with the combination (P<0.01) but not nilotinib alone (P=0.29), significantly decreased BM GFP+ LSK cells (12.0±1.2, 6.8±0.6, 9.5±1.6, and 2.2±0.2 x103 cells in the respective groups). The in vivo experiments are ongoing. Conclusions: ABT-199 and TKIs cooperatively target antiapoptotic Bcl-2 family proteins. This combination is highly effective in killing bulk and CD34+38- CML cells and quiescent CD34+ CML stem/progenitor cells from BC CML patients in vitro and in suppressing leukemia and leukemia stem cells in vivo. This strategy has the potential to eradicate BC CML cells and CML stem/progenitor cells, neither of which are effectively targeted by TKIs alone. Disclosures Carter: AbbVie, Inc.: Research Funding. Leverson:AbbVie, Inc.: Employment. Konopleva:AbbVie, Inc: clinic trial Other.

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Resistance to tyrosine kinase inhibitors and novel insights into genomic instability of chronic myeloid leukemia stem cells

Chronic Myeloid Leukemia ◽

10.3109/9781842145784.009 ◽

2013 ◽

pp. 108-117

Keyword(s):

Stem Cells ◽

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Genomic Instability ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Leukemia Stem Cells

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

Blood ◽

10.1182/blood-2006-10-054098 ◽

2007 ◽

Vol 110 (2) ◽

pp. 678-685 ◽

Cited By ~ 108

Author(s):

Cong Peng ◽

Julia Brain ◽

Yiguo Hu ◽

Ami Goodrich ◽

Linghong Kong ◽

...

Keyword(s):

Stem Cells ◽

Heat Shock ◽

Heat Shock Protein ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Kinase Inhibitors ◽

Heat Shock Protein 90 ◽

Hsp90 Inhibitor ◽

Myelogenous Leukemia ◽

Leukemia Stem Cells

Abstract Development of kinase domain mutations is a major drug-resistance mechanism for tyrosine kinase inhibitors (TKIs) in cancer therapy. A particularly challenging example is found in Philadelphia chromosome–positive chronic myelogenous leukemia (CML) where all available kinase inhibitors in clinic are ineffective against the BCR-ABL mutant, T315I. As an alternative approach to kinase inhibition, an orally administered heat shock protein 90 (Hsp90) inhibitor, IPI-504, was evaluated in a murine model of CML. Treatment with IPI-504 resulted in BCR-ABL protein degradation, decreased numbers of leukemia stem cells, and prolonged survival of leukemic mice bearing the T315I mutation. Hsp90 inhibition more potently suppressed T315I-expressing leukemia clones relative to the wild-type (WT) clones in mice. Combination treatment with IPI-504 and imatinib was more effective than either treatment alone in prolonging survival of mice simultaneously bearing both WT and T315I leukemic cells. These results provide a rationale for use of an Hsp90 inhibitor as a first-line treatment in CML by inhibiting leukemia stem cells and preventing the emergence of imatinib-resistant clones in patients. Rather than inhibiting kinase activity, elimination of mutant kinases provides a new therapeutic strategy for treating BCR-ABL–induced leukemia as well as other cancers resistant to treatment with tyrosine kinase inhibitors.

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Unexpected Role for Dosage Compensation in the Control of Dauer Arrest, Insulin-Like Signaling, and FoxO Transcription Factor Activity in Caenorhabditis elegans

Genetics ◽

10.1534/genetics.113.149948 ◽

2013 ◽

Vol 194 (3) ◽

pp. 619-629 ◽

Cited By ~ 11

Author(s):

Kathleen J. Dumas ◽

Colin E. Delaney ◽

Stephane Flibotte ◽

Donald G. Moerman ◽

Gyorgyi Csankovszki ◽

...

Keyword(s):

Transcription Factor ◽

Caenorhabditis Elegans ◽

Dosage Compensation ◽

Transcription Factor Activity ◽

Factor Activity ◽

Foxo Transcription Factor

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Role of New Tyrosine Kinase Inhibitors in Osteoblastogenesis

Blood ◽

10.1182/blood.v112.11.4751.4751 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4751-4751

Author(s):

Daniele Tibullo ◽

Cesarina Giallongo ◽

Piera La Cava ◽

Provvidenza Guagliardo ◽

Maide Cavalli ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Tyrosine Kinase ◽

Osteogenic Differentiation ◽

Tyrosine Kinase Inhibitors ◽

Kinase Inhibitors ◽

Osteogenic Medium ◽

Standard Medium ◽

Alizarin Red

Abstract It has been reported that imatinib mesylate (IM) may affect bone tissue remodeling mainly by both an inhibitory activity on osteoclastogenesis and an induction of osteoblastogenesis. Dasatinib (DA) and Nilotinib (NI) are new generation tyrosine kinase inhibitors presently approved for chronic myeloid leukemia patients after imatinib failure. We therefore evaluated possible effects of DA and NI on osteoblatic differentiation of Mesenchymal Stem Cells derived from bone marrow (BM-MSCs). BM-MSCs are multipotent non-haematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, skeletal myocytes and nervous cells. Mesenchymal stem cells (hBM-MSCs) were obtained from bone marrow samples of normal healthy adult bone marrow donors, isolated by density gradient (mononuclear fraction) and cultured either in standard medium (SM) or in osteogenic medium (OM) (0.2 mM ascorbic acid, 0.1 μm dexamethasone and 10 mM β-glycerophosphate) with or without DA 2nM or NI 100nM. Osteogenic differentiation of hBM-MSCs was evaluated by changes in morphology, presence of mineralized nodules (evidenced by Alizarin red) and expression of osteoblast-associated genes such as osteocalcin (OCN), RUNX2 and Bone morphogenetic protein (BMP-2) evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed by Scion Image. After 21days of culture, in comparison to control cultures, hBM-MSCs placed in OM, DA, NI and DA+OM, NI+OM exhibited changes in cell morphology from a spindle-shaped fibroblastic appearance to a rounder more cuboidal shape and the cells formed an extensive network of dense multilayered nodules (extracellular mineralization). Table I indicates mRNA expression of osteogenic markers in different culture conditions and shows that both DA and NI alone or in combination with OM, increase RUNX2, OCN, and BMP-2 expression. SM DA NI OM DA + OM NI + OM SM= standard medium, OM= osteogenic medium, DA= dasatinib, NI= nilotinib In summary, our data show that both DA and NI, as already reported IM, may induce osteogenic differentiation of mesenchymal cells thus indicating that they potentially favour osteoblastogenesis. RUNX2 1,59 0,20 2,09 0,16 4,2 0,31 2,86 0,25 4,41 0,41 4,18 0,24 OCN 2,57 0,28 3,2 0,14 3,14 0,09 3,59 0,17 3,6 0,28 3,62 0,25 BMP-2 1,55 0,19 2,27 0,17 4,16 0,27 2,84 0,28 4,43 0,30 4,21 0,30

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