Su Chu

Effective Targeting of Quiescent Chronic Myelogenous Leukemia Stem Cells by Histone Deacetylase Inhibitors in Combination with Imatinib Mesylate

Imatinib mesylate (IM) induces remission in chronic myelogenous leukemia (CML) patients but does not eliminate leukemia stem cells (LSCs), which remain a potential source of relapse. Here we investigated the ability of HDAC inhibitors (HDACis) to target CML stem cells. Treatment with HDACis combined with IM effectively induced apoptosis in quiescent CML progenitors resistant to elimination by IM alone, and eliminated CML stem cells capable of engrafting immunodeficient mice. In vivo administration of HDACis with IM markedly diminished LSCs in a transgenic mouse model of CML. The interaction of IM and HDACis inhibited genes regulating hematopoietic stem cell maintenance and survival. HDACi treatment represents an effective strategy to target LSCs in CML patients receiving tyrosine kinase inhibitors.

Persistence of leukemia stem cells in chronic myelogenous leukemia patients in prolonged remission with imatinib treatment

Imatinib mesylate treatment markedly reduces the burden of leukemia cells in chronic myelogenous leukemia (CML) patients. However, patients remain at risk for relapse on discontinuing treatment. We have previously shown that residual BCR-ABL(+) progenitors can be detected in CML patients within the first 2 years of imatinib treatment. However, reduced rates of relapse and continued decline of BCR-ABL levels with prolonged treatment, together with the ability of selected patients to maintain remission after discontinuing treatment, led us to investigate whether prolonged imatinib exposure resulted in reduction or elimination of BCR-ABL(+) stem cells. We evaluated BCR-ABL expression in CD34(+)CD38(+) (38(+)) committed progenitors and CD34(+)CD38(-) (38(-)) stem/primitive progenitor cells in samples from CML patients on imatinib treatment for at least 4 years with cytogenetic and molecular response. High levels of BCR-ABL expression were maintained over time in the 38(-) stem cell fraction. The absolute frequency of BCR-ABL(+) cells as determined by limiting dilution analysis was consistently higher in 38(-) compared with 38(+) cells. Transplantation into NOD/SCID-IL2Rγ-chain knockout mice demonstrated that BCR-ABL(+) cells had long-term in vivo repopulating capacity. These results directly demonstrate that BCR-ABL(+) stem cells persist in CML patients despite prolonged treatment with imatinib, and support ongoing efforts to target this population.

Effects of dasatinib on SRC kinase activity and downstream intracellular signaling in primitive chronic myelogenous leukemia hematopoietic cells.

Bcr-Abl tyrosine kinase inhibitors (TKI) are effective in inducing remissions in chronic myelogenous leukemia (CML) patients but do not eliminate primitive CML hematopoietic cells. There is a need to identify mechanisms that contribute to retention of CML progenitors. Src family tyrosine kinases have been identified as potential mediators of Bcr-Abl-induced leukemogenesis. Dasatinib (BMS-354825) is a potent dual Abl/Src kinase inhibitor approved for clinical use in CML patients. We evaluated Src activity in primitive human CML progenitors from different stages of disease and investigated effects of Dasatinib on Src activity and downstream signaling pathways. P-Src expression was increased in CD34+ cells and CD34+CD38- cells in all phases of CML. Dasatinib showed potent Src inhibitory activity in CML progenitors, inhibiting both Bcr-Abl-dependent and -independent Src activity. In contrast, Imatinib inhibited only Bcr-Abl-dependent Src activity. Dasatinib inhibited P-mitogen-activated protein kinase (MAPK), P-Akt, and P-STAT5 levels in CML progenitors in the absence of growth factors but not in the presence of growth factors. A marked increase in P-MAPK levels seen in the presence of growth factors with Imatinib was much less prominent with Dasatinib. Dasatinib significantly suppressed CML colony-forming cells and long-term culture-initiating cells but did not significantly alter the level of apoptosis-regulating proteins in CML CD34+ cells. Our results indicate that Dasatinib, in addition to potent anti-Bcr-Abl kinase activity, effectively inhibits Src kinase activity and downstream signaling pathways in CML progenitors but does not induce a strong proapoptotic response. These observations argue against a prominent role for Src kinases in persistence of primitive CML cells in TKI-treated patients.

Effect of Mutational Inactivation of Tyrosine Kinase Activity on BCR/ABL-Induced Abnormalities in Cell Growth and Adhesion in Human Hematopoietic Progenitors

Chronic myelogenous leukemia (CML) results from transformation of a primitive hematopoietic cell by the BCR/ABL gene. The specific BCR/ABL signaling mechanisms responsible for transformation of primitive human hematopoietic cells are not well defined. Previous studies have suggested that constitutively activated tyrosine kinase activity plays an important role for in abnormal proliferation of CML progenitors but has not clearly defined its role in abnormal adhesion and migration. We established a human progenitor model of CML by ectopic expression of BCR/ABL in normal CD34+ cells using retrovirus-mediated gene transfer. CD34+ cells expressing BCR/ABL demonstrated several features characteristic of primary CML progenitors including increased proliferation in committed and primitive progenitor culture, reduced adhesion to fibronectin, and reduced chemotaxis to stroma-derived factor-1α. We expressed a kinase-inactive BCR/ABL gene to directly investigate the role of kinase activity in abnormal progenitor function. Abnormalities in proliferation were completely reversed, whereas defects in adhesion and migration were significantly improved but not completely reversed in cells expressing a kinase-inactive BCR/ABL. Furthermore, the BCR/ABL kinase inhibitor imatinib mesylate markedly inhibited proliferation of BCR/ABL-expressing progenitors but did not fully correct the adhesion and migration defects. Expression of BCR/ABL genes with deletions of either the COOH-terminal actin binding or proline-rich domains resulted in enhanced adhesion and chemotaxis compared with wild-type BCR/ABL but did not affect progenitor proliferation. We conclude that abnormal kinase activity is essential for abnormal proliferation and survival of CML progenitors but that abnormal adhesion and migration result from both kinase-dependent and -independent mechanisms.

SIRT1 activation by a c-MYC oncogenic network promotes the maintenance and drug resistance of human FLT3-ITD acute myeloid leukemia stem cells.

The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and is associated with poor prognosis. In such patients, FLT3 tyrosine kinase inhibitors (TKIs) are only partially effective and do not eliminate the leukemia stem cells (LSCs) that are assumed to be the source of treatment failure. Here, we show that the NAD-dependent SIRT1 deacetylase is selectively overexpressed in primary human FLT3-ITD AML LSCs. This SIRT1 overexpression is related to enhanced expression of the USP22 deubiquitinase induced by c-MYC, leading to reduced SIRT1 ubiquitination and enhanced stability. Inhibition of SIRT1 expression or activity reduced the growth of FLT3-ITD AML LSCs and significantly enhanced TKI-mediated killing of the cells. Therefore, these results identify a c-MYC-related network that enhances SIRT1 protein expression in human FLT3-ITD AML LSCs and contributes to their maintenance. Inhibition of this oncogenic network could be an attractive approach for targeting FLT3-ITD AML LSCs to improve treatment outcomes.

Bortezomib induces apoptosis in primitive chronic myeloid leukemia cells including LTC-IC and NOD/SCID repopulating cells.

Chronic myeloid leukemia (CML) is treated effectively with tyrosine kinase inhibitors (TKIs); however, 2 key problems remain-the insensitivity of CML stem and progenitor cells to TKIs and the emergence of TKI-resistant BCR-ABL mutations. BCR-ABL activity is associated with increased proteasome activity and proteasome inhibitors (PIs) are cytotoxic against CML cell lines. We demonstrate that bortezomib is antiproliferative and induces apoptosis in chronic phase (CP) CD34+ CML cells at clinically achievable concentrations. We also show that bortezomib targets primitive CML cells, with effects on CD34+38(-), long-term culture-initiating (LTC-IC) and nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells. Bortezomib is not selective for CML cells and induces apoptosis in normal CD34+38(-) cells. The effects against CML cells are seen when bortezomib is used alone and in combination with dasatinib. Bortezomib causes proteasome but not BCR-ABL inhibition and is also effective in inhibiting proteasome activity and inducing apoptosis in cell lines expressing BCR-ABL mutations, including T315I. By targeting both TKI-insensitive stem and progenitor cells and TKI-resistant BCR-ABL mutations, we believe that bortezomib offers a potential therapeutic option in CML. Because of known toxicities, including myelosuppression, the likely initial clinical application of bortezomib in CML would be in resistant and advanced disease.

MicroRNA-486 regulates normal erythropoiesis and enhances growth and modulates drug response in CML progenitors

MicroRNAs (miRNAs) are key regulators of hematopoietic cell differentiation and may contribute to altered growth of leukemic stem cells. Using microarray-based miRNA profiling, we found that miRNA 486 (miR-486) is significantly upregulated in chronic myeloid leukemia (CML) compared with normal CD34(+) cells, particularly in the megakaryocyte-erythroid progenitor population. miR-486-5p expression increased during erythroid differentiation of both CML and normal CD34(+) cells. Ectopic miR-486-5p expression enhanced in vitro erythroid differentiation of normal CD34(+) cells, whereas miR-486-5p inhibition suppressed normal CD34(+) cell growth in vitro and in vivo and inhibited erythroid differentiation and erythroid cell survival. The effects of miR-486-5p on hematopoietic cell growth and survival are mediated at least in part via regulation of AKT signaling and FOXO1 expression. Using gene expression and bioinformatics analysis, together with functional screening, we identified several novel miR-486-5p target genes that may modulate erythroid differentiation. We further show that increased miR-486-5p expression in CML progenitors is related to both kinase-dependent and kinase-independent mechanisms. Inhibition of miR-486-5p reduced CML progenitor growth and enhanced apoptosis following imatinib treatment. In conclusion, our studies reveal a novel role for miR-486-5p in regulating normal hematopoiesis and of BCR-ABL-induced miR-486-5p overexpression in modulating CML progenitor growth, survival, and drug sensitivity.

BCR-tyrosine 177 plays an essential role in Ras and Akt activation and in human hematopoietic progenitor transformation in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) results from the transformation of a primitive hematopoietic cell by the BCR/ABL gene. BCR/ABL signaling has been studied in cell lines and murine models, but the transforming effects of BCR/ABL are highly dependent on cellular context, and mechanisms responsible for the transformation of primitive human hematopoietic cells remain poorly understood. Current targeted therapies fail to eliminate malignant CML progenitors, and improved understanding of crucial molecular mechanisms of progenitor transformation may facilitate the development of improved therapeutic approaches. We investigated the role of BCR/ABL tyrosine 177 (BCR/ABL-Y177) in CML progenitor transformation by comparing the effects of expression of Y177-mutated BCR/ABL, wild-type BCR/ABL, or green fluorescent protein alone on normal CD34(+) cells. We show that BCR/ABL-Y177 plays a critical role in CML progenitor expansion, proliferation, and survival. BCR/ABL expression results in enhanced Ras and Akt activity but reduced mitogen-activated protein kinase activity in human hematopoietic cells, which is reversed by BCR/ABL-Y177 mutation. Blocking BCR/ABL-Y177-mediated signaling enhances targeting of CML progenitors by imatinib mesylate. Our studies indicate that BCR/ABL-Y177 plays an essential role in Ras and Akt activation and in human hematopoietic progenitor transformation in CML.

Setbp1 promotes the self-renewal of murine myeloid progenitors via activation of Hoxa9 and Hoxa10

Acquisition of self-renewal capability by myeloid progenitors to become leukemic stem cells during myeloid leukemia development is poorly understood. Here, we show that Setbp1 overexpression efficiently confers self-renewal capability to myeloid progenitors in vitro, causing their immortalization in the presence of stem cell factor and IL-3. Self-renewal after immortalization requires continuous Setbp1 expression. We also found that Hoxa9 and Hoxa10 mRNA are present at dramatically higher levels in Setbp1-immortalized cells compared with other immortalized cells, and are induced shortly after Setbp1 expression in primary myeloid progenitors. Suppression of either gene in Setbp1-immortalized cells drastically reduces their colony-forming capability. Interestingly, Setbp1 protein associates with Hoxa9 and Hoxa10 promoters in chromatin immunoprecipitation assays in these cells, suggesting that both are direct transcriptional targets of Setbp1. Setbp1 also promotes self-renewal of myeloid progenitors in vivo as its coexpression with BCR/ABL transforms primary mouse myeloid progenitors, generating aggressive leukemias in recipient mice resembling chronic myelogenous leukemia (CML) myeloid blast crisis. Increased SETBP1 mRNA levels were also detected in a subset of CML advanced phase/blast crisis patients with high levels of HOXA9 and HOXA10 expression. Thus, Setbp1 activation represents a novel mechanism conferring self-renewal capability to myeloid progenitors in myeloid leukemia development.

Inhibition of Grb2 expression demonstrates an important role in BCR-ABL-mediated MAPK activation and transformation of primary human hematopoietic cells

Chronic myeloid leukemia (CML) results from the expression of the BCR/ABL oncogene in a primitive hematopoietic cell. However, BCR/ABL-activated signaling mechanisms are dependent on the cellular context in which it is expressed, and mechanisms underlying primitive human hematopoietic cell transformation by BCR–ABL are not well understood. Our previous studies have shown that BCR/ABL-Y177 has an essential role in Ras activation and human hematopoietic progenitor transformation in CML. The adapter protein growth factor receptor-binding protein-2 (Grb2) can bind phosphorylated BCR/ABL-Y177, induce Grb2-SoS complex formation and activate Ras signaling. We investigated the role of Grb2 in CML progenitor transformation by cotransducing human CD34+ cells with lentivirus vectors expressing short hairpin RNA to Grb2 and retrovirus vectors expressing BCR/ABL. We show that Grb2 knockdown significantly inhibits proliferation and survival of BCR–ABL-expressing CD34+ cells, but not control CD34+ cells. Grb2 knockdown reduced mitogen-activated protein kinase (MAPK) activity in BCR–ABL-expressing hematopoietic cells. We conclude that inhibition of Grb2 expression demonstrates an important role in BCR–ABL-mediated MAPK activation and transformation of primary human hematopoietic cells.These results support further investigation of downstream effectors of Grb2-mediated signals and targeting of Grb2 interactions in the treatment of CML.