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The phosphoinosityl-3-kinase (PI3K) pathway is frequently constitutively active in blast cells from acute myeloid leukemia (AML) patients. RNA and protein from all four catalytic isoforms of PI3K (p110α, β, γ, and δ) were expressed in 38 AML samples, which also showed expression of phosphorylated Akt Ser473, indicating PI3K activation. Initial treatment of 12 AML samples with inhibitors targeting each of the four isoforms demonstrated that p110α and δ inhibition are more effective in killing AML blast colony-forming cells (CFC) than p110β or γ inhibition. In subsequent experiments, AML CFC from 46 patient samples were treated with the p110α and δ selective inhibitors, PI3Kα inhibitor 2 or PCN5603, and dose-dependent progenitor kill and inhibition of phosphorylated Akt Ser473 expression was observed. AML samples were more sensitive to PI3Kα inhibitor 2 and PCN5603 killing than normal bone marrow or normal peripheral blood CFC (median IC(50) for AML and normal CFCs treated with PI3Kα inhibitor 2, 1.8 and 4.3 μM, respectively, and for PCN5603, 1.9 and 6.2 μM, respectively). Furthermore, treatment of AML cells with PCN5603 also decreased survival of more primitive leukemia progenitors identified in long-term culture (AML long-term culture initiating cells), while less toxicity toward normal bone marrow long-term culture initiating cells was observed. Selective inhibition of the p110α and δ isoforms of PI3K kills AML progenitors while causing relative sparing of analogous normal cells., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the possibility of improved selective killing of acute myeloid leukemia (AML) cells with CPX-351 (a liposomal formulation of cytarabine and daunorubicin). CPX-351 and the same molar ratio of free drugs were compared for cytotoxicity against colony-forming cells (CFCs) and subpopulations of cells enriched for primitive progenitors from AML patients and normal granulocyte colony-stimulating factor-mobilized peripheral blood (PB) and bone marrow (BM) donors., Materials and Methods: AML blasts (n = 13) and normal PB and BM cells (n = 7) were incubated for 24 hours in various concentrations of CPX-351 or free drugs before plating in CFC assay or staining with anti-CD34 and anti-CD38 antibodies, Annexin-V, and propidium iodide followed by fluorescence-activated cell sorting analysis. High performance liquid chromatography was used to measure intracellular daunorubicin accumulation., Results: AML blasts and progenitors from patients who achieved complete remission were more sensitive to both CPX-351 and free drugs than the same cells from patients with chemotherapy refractory leukemia. However, AML CFCs and CD34(+)CD38(-) AML blasts (enriched for candidate leukemia stem cells) from the same patient showed similar sensitivity to the liposomal or free drug formulations. In contrast, CFCs and CD34(+)CD38(-) cells from normal PB and BM were fivefold more sensitive to the free drugs than to CPX-351. Consistent with these observations, preferential intracellular accumulation of CPX-351 in AML over normal cells was observed, while there was little difference in drug uptake between AML and normal cells with the free drug cocktail., Conclusions: CPX-351, as compared to free cytarabine:daunorubicin, shows enhanced selective in vitro cytotoxicity for AML rather than normal progenitors., (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Objective: Dysregulation of signaling pathways leading to enhanced cell proliferation and resistance to apoptosis is frequent in acute myeloid leukemia (AML). The effectiveness of inhibiting two such pathways, the phosphatidylinosityl-3-kinase pathway via the intermediate integrin-linked kinase (ILK), and FMS-like tyrosine kinase-3 (FLT-3) signaling pathway in killing AML cells was studied., Materials and Methods: AML colony-forming cell (CFC) assays were used to determine the effects of a small molecule inhibitor of both ILK and FLT-3 (QLT0267) on poor prognosis primary AML sample viability. Kinase assays and Western blots were used to analyze effects of the compound on target molecules., Results: In 31/36 AML blast samples p-Akt was detected indicating phosphatidylinosityl-3-kinase activation. ILK was ubiquitously and FLT-3 abundantly expressed. Downregulation of ILK in the AML cell line TF-1 using small interfering RNA caused >or= 50% CFC death, suggesting ILK inhibition might also be toxic to primary AML cells. In vitro kinase assays on three AML samples showed inhibition of both ILK and FLT-3 by QLT0267. Treatment of AML patient blast cells (n=27) with QLT0267, caused a dose- and time-dependent downregulation of p-Akt and kill of AML-CFC with AML samples containing FLT-3 mutations being more sensitive to QLT0267 than those without. AML samples were more sensitive to QLT0267 killing than normal bone marrow (IC(50)=3 microM, vs 10 microM for AML-CFC and normal CFC, respectively, n=5)., Conclusion: Combined inhibition of ILK and FLT-3 with a small molecule kinase inhibitor can achieve selective targeting of AML rather than normal hematopoietic progenitors.

Objective: Overexpression of members of the adenosine triphosphate binding cassette (ABC) transporter superfamily has been implicated in multidrug resistance in cancer, but results in acute myeloid leukemia (AML) have been inconsistent. We investigated the expression and activity of ABC transporters in patient total blasts and subpopulations along the leukemic stem cell hierarchy., Materials and Methods: Using quantitative reverse transcriptase polymerase chain reaction, we measured expression of the ABC transporter superfamily in the blast cells from AML patients prior to chemotherapy. In addition, we measured ex vivo daunorubicin resistance of subpopulations with or without ABC inhibitors., Results: In the total blasts, no consistent difference was observed in 18 patients achieving complete remission (CR) and 13 patients who were refractory to induction chemotherapy (NR). However, among the subpopulation of CD34(+)CD38(-) AML cells (candidate "leukemic stem cells"), elevated expression of MDR1 and/or BCRP1, two ABC transporters associated with drug resistance, was found in 8 of 10 NR patients as compared to 0 of 7 CR patients. No such association was observed in the more differentiated CD34(+)CD38(+) or CD34(-) subpopulations. There was no significant difference in MRP1 expression between CR and NR patient samples in any of the subpopulations examined. The increased expression of MDR1 and BCRP1 in leukemic cells correlated with increased cellular daunorubicin resistance, which could be reversed by the ABC transporter inhibitors verapamil and PSC-833., Conclusion: Expression of MDR1 and BCRP1 in leukemic stem cells correlates with chemotherapy response both at the cellular level and in AML patients.

In previous studies, we demonstrated that the diphtheria toxin-urokinase fusion protein DTAT was selectively toxic to acute myeloid leukemia (AML) cell lines overexpressing the CD87 urokinase receptor. In the present study, we analyzed the sensitivity of patient leukemic progenitors to DTAT and correlated the sensitivity with CD87 expression. We isolated leukemic blasts by density gradient centrifugation and performed immunophenotyping by flow cytometry and blast sensitivity measurements by inhibition of cell proliferation and colony formation in semisolid media. We found CD87 overexpression in 18 (25%) of 71 patient leukemic blast samples, including 18 (28%) of 64 myeloid malignancies and 0 (0%) of 7 lymphoid malignancies. DTAT was toxic to patient leukemic blasts by both proliferation inhibition (IC50 85% inhibition by 10 nM DTAT in 11/41 evaluable samples). Only AML and chronic myeloid leukemia (CML) blast crisis blasts (18/61 [30%]) were sensitive to DTAT by the proliferation inhibition assay. Lymphoid leukemia and chronic phase CML/chronic myelomonocytic leukemia (CMML) progenitors were insensitive to DTAT by the proliferation inhibition assay (n = 7 and n = 3, respectively). Similarly, normal marrow progenitors were insensitive to DTAT by both proliferation inhibition (n = 2) and colony inhibition (n = 5) assays. The DTAT toxicity measured by both proliferation inhibition assay and colony inhibition assay correlated with CD87 density (p < 0.0001 and p = 0.001, respectively). DTAT toxicity results were similar for leukemic blasts measured by either of the two assays (p = 0.0002). This study provides the first evidence that a urokinase receptor targeted diphtheria fusion protein is toxic to patient AML blasts. The work also suggests that blast proliferation assays yield similar responses to leukemia colony-forming cell colony assays.

Objective: To study the clonality of cytogenetically normal progenitors detected in the peripheral blood (PB) of acute myeloid leukemia (AML) patients., Methods: Five female patients with cytogenetically abnormal, newly-diagnosed AML who were heterozygous for informative alleles of the androgen receptor (AR) gene were studied using the human androgen receptor allele (HUMARA) assay., Results: PB mononuclear cells and bone marrow (BM) fibroblasts from these patients were monoclonal and polyclonal, respectively. Both cytogenetically normal and abnormal colony-forming cells (CFC) were detected from 3 AML samples and the HUMARA assay determined that most of these CFC were part of the leukemic clone. The fourth sample generated colonies that were 100% normal by cytogenetics and polyclonal by HUMARA. In contrast, 5-week-old long-term culture (LTC)-derived colonies were 100% cytogenetically normal by FISH and polyclonal by HUMARA in 4 of the 5 samples. The fifth sample, which showed a small number of karyotypically abnormal LTC-derived colonies, nevertheless showed amplification of the "leukemia-associated" AR allele in 46/50 LTC-derived colonies as well as all 40 directly clonogenic cells tested., Conclusions: Thus in 4 of 5 AML samples tested, both cytogenetics and the HUMARA assay indicate that a substantial number of normal, polyclonal hematopoietic progenitors often persist in AML PB at diagnosis in spite of the predominance of malignant blasts and the severe cytopenias of normal mature blood cells that are typically seen clinically.

Rare primitive progenitors among the malignant cells from most patients with AML include AML long-term culture-initiating cells (AML LTC-IC) and NOD/SCID mouse leukemia-initiating cells (NOD/SL-IC). To evaluate the feasibility of genetic modification of these progenitors for gene marking and/ or gene therapy strategies, cells from patients with newly-diagnosed AML were cocultured with retroviral producer cells and then placed in colony (AML-CFC) assays, LTC, and injected intravenously into NOD/SCID mice. Southern blotting demonstrated transfer of the neo(r) gene to 30% to 80% of leukemic blasts when cells were cultured for 48 hours in the presence of IL-3 and steel factor (SF) prior to 48-hour coculture with viral producers. Three of six retrovirally-infected AML samples showed both engraftment in NOD/SCID mice and the presence of the neo(r) transgene in mouse tissues 8-15 weeks after injection of transduced cells. Thirteen weeks after injection of one of these samples, >80% of cells from mouse bone marrow were the progeny of two retrovirally-transduced AML progenitors. Four of the remaining five samples showed markedly reduced ability to engraft in mice after retroviral infection. Subsequent experiments demonstrated that the loss of engraftment potential took place within 24 hours of culture initiation in the absence of retroviral producers and regardless of the cytokines present. Interestingly, the majority of AML-CFC or AML LTC-IC survived the 24-hour culture period. A retroviral vector containing the murine cell surface marker heat stable antigen (HSA), which allows purification of transduced cells on immunomagnetic columns, was used to obtain an enriched population of gene-modified AML cells following an infection protocol that eliminated the 48 hours of prestimulation in IL-3 and SF and reduced coculture with viral producers to 10-36 hours. These modifications failed to improve engraftment of the infected cells. In addition, in these experiments more than 10 hours of cocultivation with viral producer cells was necessary to achieve gene transfer and expression in AML LTC-IC. These data demonstrate that although retroviral-mediated gene transfer can be achieved to AML progenitors, including NOD/SL-IC, improved culture conditions will be required before substantial numbers of such transduced primitive progenitors can be obtained. In addition, the difference in the ability of AML LTC-IC and NOD/SL-IC to survive ex vivo suggests that these assays may detect different populations of cells or that changes are induced in vitro in primitive cells which can only be detected in the mouse assay.

Interleukin (IL)-6 and IL-3, both alone and in combination, stimulate hematopoietic cells in short-term in vitro assays and in vivo. To study their ability to influence hematopoiesis in a system that mimics many features of the marrow microenvironment, long-term cultures (LTC) were produced by co-cultivating normal human marrow cells on feeder layers of murine marrow-derived stromal cells (M2-10B4 cells) genetically engineered to produce human IL-6 and/or IL-3. Feeders stably producing 20 ng/ml IL-6 slightly increased the output of clonogenic progenitors in these LTC but did not change the production of mature (total nonadherent) cells as compared to control cultures. Feeders producing 50 ng/ml IL-3 increased both clonogenic progenitor output (approximately threefold) and the output of mature cells (six-fold) as compared to controls. Feeders producing both factors also increased the output of both progenitors and mature cells. At the time of the weekly half-medium change when primitive clonogenic progenitors in the adherent layer are quiescent, such progenitors were actively cycling in all cultures with factor-producing feeders, as shown by [3H]thymidine suicide assays. Similarly, three sequential daily additions of 20 ng/ml of IL-6 also stimulated the quiescent progenitors to enter S-phase 2 days later, although single doses of recombinant IL-6 as high as 100 ng/ml failed to do so. The combined presence of IL-6- and IL-3-producing feeders, but neither alone, was also able to enhance more than twofold the maintenance and early differentiation of cells capable of generating clonogenic cells for at least a further 5 weeks in secondary LTC. Thus, the provision of a continuous source of IL-6 or IL-3 to primitive hematopoietic cells even in the LTC system can enhance late events in the hierarchy of hematopoietic cell differentiation, but a combination of the two factors is required to stimulate early multipotent progenitors.