A novel tescalcin-sodium/hydrogen exchange axis underlying sorafenib resistance in FLT3-ITD+ AML.

Internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) in acute myeloid leukemia (AML) is associated with inferior clinical prognosis. Sorafenib is effective in clearing leukemic blasts in chemorefractory FLT3-ITD(+) AML, but leukemia progression invariably occurs. Mechanisms of drug resistance are not completely understood. We hypothesized that a gene encoding tescalcin (TESC), known to be upregulated at leukemia progression during continuous sorafenib treatment and activate an Na(+)/H(+) exchanger type-1 (NHE1), may underlie tyrosine kinase inhibitor resistance. TESC was highly expressed in FLT3-ITD(+) AML lines MOLM-13 and MV4-11, and its knockdown by small-interfering RNA lowered intracellular pH (pHi) and induced apoptosis. The results were recapitulated by treatment with an NHE1 inhibitor, 5-(N,N-hexamethylene) amiloride (HMA). Induction of sorafenib resistance in the MOLM-13 cell line (M13-RE) significantly increased its sensitivity to HMA. The later also enhanced suppression of FLT3 signaling by sorafenib in otherwise resistant cell lines. HMA treatment of MOLM-13 and MV4-11 as well as primary FLT3-ITD(+) AML cells significantly reduced leukemia initiation in anti-CD122-primed NOD/SCID mouse xenotransplantation. These observations provided novel information about the pathogenetic role of a TESC-NHE1-pHi axis in mediating sorafenib resistance in AML.

Functions of flt3 in zebrafish hematopoiesis and its relevance to human acute myeloid leukemia.

FMS-like tyrosine kinase 3 (FLT3) is expressed in human hematopoietic stem and progenitor cells (HSPCs) but its role during embryogenesis is unclear. In acute myeloid leukemia (AML), internal tandem duplication (ITD) of FLT3 at the juxtamembrane (JMD) and tyrosine kinase (TKD) domains (FLT3-ITD(+)) occurs in 30% of patients and is associated with inferior clinical prognosis. TKD mutations (FLT3-TKD(+)) occur in 5% of cases. We made use of zebrafish to examine the role of flt3 in developmental hematopoiesis and model human FLT3-ITD(+) and FLT3-TKD(+) AML. Zebrafish flt3 JMD and TKD were remarkably similar to their mammalian orthologs. Morpholino knockdown significantly reduced the expression of l-plastin (pan-leukocyte), csf1r, and mpeg1 (macrophage) as well as that of c-myb (definitive HSPCs), lck, and rag1 (T-lymphocyte). Expressing human FLT3-ITD in zebrafish embryos resulted in expansion and clustering of myeloid cells (pu.1(+), mpo(+), and cebpα(+)) which were ameliorated by AC220 and associated with stat5, erk1/2, and akt phosphorylation. Human FLT3-TKD (D835Y) induced significant, albeit modest, myeloid expansion resistant to AC220. This study provides novel insight into the role of flt3 during hematopoiesis and establishes a zebrafish model of FLT3-ITD(+) and FLT3-TKD(+) AML that may facilitate high-throughput screening of novel and personalized agents.

Sorafenib treatment of FLT3-ITD(+) acute myeloid leukemia: favorable initial outcome and mechanisms of subsequent nonresponsiveness associated with the emergence of a D835 mutation.

Internal tandem duplication (ITD) of the fms-related tyrosine kinase-3 (FLT3) gene occurs in 30% of acute myeloid leukemias (AMLs) and confers a poor prognosis. Thirteen relapsed or chemo-refractory FLT3-ITD(+) AML patients were treated with sorafenib (200-400 mg twice daily). Twelve patients showed clearance or near clearance of bone marrow myeloblasts after 27 (range 21-84) days with evidence of differentiation of leukemia cells. The sorafenib response was lost in most patients after 72 (range 54-287) days but the FLT3 and downstream effectors remained suppressed. Gene expression profiling showed that leukemia cells that have become sorafenib resistant expressed several genes including ALDH1A1, JAK3, and MMP15, whose functions were unknown in AML. Nonobese diabetic/severe combined immunodeficiency mice transplanted with leukemia cells from patients before and during sorafenib resistance recapitulated the clinical results. Both ITD and tyrosine kinase domain mutations at D835 were identified in leukemia initiating cells (LICs) from samples before sorafenib treatment. LICs bearing the D835 mutant have expanded during sorafenib treatment and dominated during the subsequent clinical resistance. These results suggest that sorafenib have selected more aggressive sorafenib-resistant subclones carrying both FLT3-ITD and D835 mutations, and might provide important leads to further improvement of treatment outcome with FLT3 inhibitors.

[Influence of rosiglitazone and all-trans-retinoic acid on angiogenesis and growth of myeloma xenograft in nude mice].

OBJECTIVE: To observe the effect of rosiglitazone (RGZ) and all-trans-retinoic acid (ATRA) on the growth of myeloma xenograft in nude mice and to explore the influence of RGZ and ATRA on VEGF expression and angiogenesis in the tumor. METHODS: VEGF gene expression in myeloma cell line U266 cells was analyzed by semi-quantitative RT-PCR after incubation with RGZ, ATRA, or RGZ + ATRA for 24 h. Myeloma xenograft was established by subcutaneous injection of 10(7) U266 cells in the scapula area of 4-week old nude mice. 7 days later, the nude mice were administered with RGZ, ATRA or RGZ + ATRA, respectively, by intraperitoneal injection once every day for 21 days. The control mice were given equal volume of normal saline instead of the drug. On the 21(st) day of treatment, the mice were sacrificed and the tumors were taken off, and the tumor volume and weight were measured. The tumors were examined by histopathology with HE staining, and microvessel density (MVD), CD34 and VEGF expression in the tumors were analyzed by immunohistochemical staining. RESULTS: VEGF mRNA was highly expressed in U266 cells and was decreased in a dose-dependent manner after incubation with RGZ. The VEGF mRNA level was further more decreased after RGZ + ATRA treatment. Xenografts of U266 cells were developed in all nude mice. The volume and weight of xenografts in the RGZ group were (785 ± 262) mm(3) and (1748 ± 365) mg, respectively, significantly lower than those of the control group (both P < 0.01). More significant inhibition was in the RGZ + ATRA group, (154 ± 89) mm(3) and (626 ± 102) mg, respectively, both were P < 0.05 vs. the RGZ group. RGZ inhibited the angiogenesis in U266 xenografts and immunohistochemical staining showed that the tumor MVD and VEGF expression were significantly decreased by RGZ treatment, and further more inhibited in the RGZ + ATRA group. VEGF protein was expressed in all xenografts in the nude mice. Its immunohistochemical staining intensity was 2.20 ± 0.40 in the control group, significantly higher than that of 1.48 ± 0.37 in the RGZ group (P < 0.01), and that of RGZ + ATRA group was 0.58 ± 0.26, further significantly lower than that of the RGZ group (P < 0.01). CD34 was expressed in all xenografts, most highly in the control group and lowest in the RGZ + ATRA group. The microvessel density (MVD) was highest in the control group (56.4 ± 15.2), significantly lower in the RGZ group (44.6 ± 11.2) (P < 0.05), and lowest in the RGZ + ATRA group (21.5 ± 8.6, P < 0.01). CONCLUSIONS: The growth of myeloma cells can also be inhibited by RGZ and ATRA in nude mice in vivo. In addition to differentiation and apoptosis induction, RGZ can inhibit the formation of myeloma xenograft probably also through the downregulation of VEGF expression and subsequent angiogenesis.

A comparative study of bone marrow and peripheral blood CD34+ myeloblasts in acute myeloid leukaemia.

To examine the differences between primitive bone marrow (BM) and peripheral blood (PB) myeloblasts in acute myeloid leukaemia (AML), we compared CD34(+) myeloblasts of paired BM and PB samples from 14 AML patients in terms of surface phenotype, homing and engraftment in a xenogeneic transplantation model, and gene expression, based on microarray studies and quantitative polymerase chain reaction. While there was no significant difference in surface phenotypes between these two populations, in vivo assay showed significantly better homing potential of PB CD34(+) cells than BM CD34(+) cells. Significant correlation between homing and engraftment in AML samples was also noted. In addition, gene expression profiling of CD34(+) cells from five paired BM and PB leukaemic samples showed that genes involved in G-protein and prostaglandin signalling, chemotaxis and stress response, cell proliferation and apoptosis were down-regulated in PB CD34(+) myeloblasts. These data suggested that circulating primitive myeloblasts in AML are functionally different from those residing in the marrow compartment, and such differences may be partly regulated by the BM microenvironment.

All-trans retinoic acid can intensify the growth inhibition and differentiation induction effect of rosiglitazone on multiple myeloma cells.

OBJECTIVE: Activation of PPARgamma by its ligands has shown potential anti-neoplastic effects in solid tumors. In this study, we investigate the effects of rosiglitazone (RGZ) alone as well as in combination with all trans-retinoic acid (ATRA) on human myeloma cell lines and try to address its potential mechanism. METHODS: U266, RPMI-8226 and primary myeloma cells from patients were treated with different concentrations of RGZ in the presence or absence of ATRA and various biological responses were studied by the methods of [3H] thymidine incorporation, MTT, cell cycle analysis, Annexin V-PI staining, Wright-Giemsa staining, CD49e expression assay, light chain protein detection, RT-PCR and caspase-3 activity assay. RESULTS: We report that exposure to RGZ induced proliferation inhibition and viability reduction in a dose-dependent manner in both U266 and RPMI-8226 cells. A similar exposure to RGZ also induced cell cycle arrest and cell apoptosis of myeloma cells. A combination of RGZ with ATRA enhanced the effects of RGZ and induced cell cycle arrest and apoptosis more profoundly in both cell lines. RGZ treated cells displayed morphological characteristics of cell differentiation, and more evident signs of differentiation were observed when combined with ATRA. These changes were confirmed by the detection of CD49e expression and light chain protein secretion. Similar cell apoptosis and differentiation were observed when primary CD138+ myeloma cells were treated with RGZ and ATRA. The mRNA expressions of FLIP, XIAP and survivin were detected in both cell lines and the levels decreased significantly after culture with RGZ. The addition of ATRA in culture medium made these changes more apparently. Caspase-3 activity was increased upon exposure to RGZ in both U266 and RPMI-8226 cells while combination of RGZ and ATRA brought out more effective activation of caspase-3. Similar apoptosis and cell differentiation induced by RGZ and ATRA can also be observed in primary CD138+ cells from myeloma patients. CONCLUSION: Concomitant RXRalpha activation by ATRA enhanced the inhibitory effects of RGZ on myeloma cell proliferation, cell cycle, apoptosis and differentiation. Combination of RGZ and ATRA may be a useful therapy for human multiple myeloma.

[Differentiating effect of PPARgamma ligand rosiglitazone and all trans-retinoic acid on myeloma cells and its possible mechanism].

OBJECTIVE: To investigate the effects of PPARgamma ligand (rosiglitazone, RGZ) as well as combined with all trans-retinoic acid (ATRA) on human myeloma cells and try to explore the possible mechanism. METHODS: Human myeloma cell lines U266 and RPMI-8226 cells were treated with RGZ in the presence or absence of ATRA. Cell proliferation was evaluated by [(3)H] thymidine incorporation, cell cycle distribution and CD49e expression were analyzed by flow cytometry, morphology changes were evaluated by Wright-Giemsa staining, and p27(Kip1) and p21(Waf1) expression was detected by Western blotting. RESULTS: The exposure to RGZ induced proliferation inhibition in both cell lines in a dose-dependent manner. After cultured with 5 micromol/L RGZ, the proportion of U266 and RPMI-8226 cells in phase G(0)/G(1) was (45.2 +/- 6.7)% and (40.3 +/- 7.3)%, respectively (P < 0.05). The proportion of the cells in phase G(2)/M and S was (52.2 +/- 7.4)% and (57.4 +/- 9.5)%, respectively (P < 0.05). These changes were more evident when the RGZ concentration was increased to 10 micromol/L. A combination of RGZ with ATRA enhanced the growth inhibition and cell cycle arrest effects of RGZ. The RGZ-treated myeloma cells displayed morphological characteristics of cell differentiation, and more evident signs of differentiation were observed when RGZ was combined with ATRA. These changes were confirmed by the detection of CD49e expression. The expression of p27(Kip1) and p21(Waf1) in myeloma cells was up-regulated by RGZ and this change was more apparent when RGZ was used in combination with ATRA. CONCLUSION: RGZ can induce cell cycle arrest and cell differentiation in myeloma cells which maybe caused by up-regulation of p27(Kip1) and p21(Waf1) expression. ATRA can enhance these effects of RGZ on multiple myeloma cells and combined use of these two drugs may show a synergistic effect on myeloma cells.

All-trans retinoic acid induces proliferation of an irradiated stem cell supporting stromal cell line AFT024.

OBJECTIVE: We have previously shown that all-trans retinoic acid (ATRA) enhanced the maintenance of early human hematopoietic progenitor cells (HPCs) in the presence of an irradiated stromal cell line AFT024. In this study, we examined the effects of ATRA on the stromal cell component with particular reference to cellular proliferation and gene expression. METHODS: Irradiated AFT024 cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum and were incubated with ATRA at 1 mumol/L up to 21 days. The cells were examined in terms of immunostaining for proliferative cell nuclear antigen (PCNA) and BrdU incorporation, apoptosis assay, cell cycle analysis, and gene expression using semiquantitative reverse-transcriptase polymerase chain reaction. RESULTS: In the control experiments, AFT024 cells lost their confluence in culture after 15-Gy irradiation and were arrested in G2/M phase on days 7 and 21. ATRA restored the cellular confluence with an increase in proliferation on day 21 (BrdU incorporation: 20.6-fold; PCNA staining: 51.7-fold) with reversal of cell cycle arrest (S phase: 2.7-fold increase; G2/M phase: 2.0-fold decrease). There was no effect on apoptosis as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. ATRA significantly upregulated the expression of cell cycle genes for checkpoint transition, including cyclin A2, B2, and aurora kinase B, as well as genes associated with a putative role in HPC maintenance, including osteopontin, HoxA5, enhancer of zeste homolog 2, and peroxisome proliferator-activated receptor gamma. CONCLUSION: We concluded that ATRA induced cellular proliferation of irradiated AFT024 cells and expression of a number of genes whose relevance to HPC homeostasis would have to be further examined.

FLT3/internal tandem duplication subclones in acute myeloid leukemia differ in their engraftment potential in NOD/SCID mice.

In this study, we tested if FLT3/internal tandem duplication (ITD) in acute myeloid leukemia (AML) might occur at different hierarchical stages during leukemogenesis. In 56 AML cases, 10 showed FLT3/ITD (single ITD=5; multiple ITD=5). Myeloblasts from seven cases (CD34-selected=4; unselected=3) were transplanted into NOD/SCID mice. Five cases engrafted successfully into 14 mice. Two patients carried single FLT3/ITD subclones, which were maintained during primary and secondary transplantations. In three patients with multiple FLT3/ITD subclones, some subclones persisted or expanded while others diminished upon transplantation. Their different engraftment capabilities in NOD/SCID mice supported the proposition that FLT3/ITD might occur at different stages during leukemogenesis.

Successful engraftment by leukemia initiating cells in adult acute lymphoblastic leukemia after direct intrahepatic injection into unconditioned newborn NOD/SCID mice.

OBJECTIVE: Xenogeneic transplantation has been the gold standard for enumeration of leukemia initiating cells in acute myeloid and lymphoblastic leukemia (ALL). Most transplantation models have required conditioning in which the recipients were either irradiated or treated with chemotherapy prior to injection of human leukemia cells. In this study, we reported an undescribed model in which adult ALL cells were injected into unconditioned newborn nonobese diabetic severe combined immunodeficient mice via an intrahepatic route. MATERIALS AND METHODS: Bone marrow (BM) and peripheral blood were collected from patients with ALL at diagnosis or relapse. CD34(+) selected lymphoblasts or mononuclear cells were transplanted as mentioned previously. Cells were also transplanted into sublethally irradiated adult mice via intravenous route for comparison. Leukemia engraftment was enumerated from mouse BM 6 to 18 weeks after transplantation. Clonality of the engrafting cells was examined based on IGH rearrangement and fluorescent in situ hybridization. RESULTS: Five of 13 ALL samples engrafted into the recipient BM 6 to 18 weeks after transplantation. Engrafted cells recapitulated the immunophenotype and cytogenetic characteristics of the original samples. Engraftment in BM and peripheral blood was significantly correlated. Importantly, there was significant correlation of engraftment between this and the conventional adult nonobese diabetic severe combined immunodeficient mouse model involving irradiation. CONCLUSION: Our results demonstrated that this unconditioned newborn mouse model could be used for enumeration of leukemia initiating cells in ALL and should be further evaluated.