Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.
Despite the relevant therapeutic progresses obtained with imatinib, clinical resistance to this drug has emerged and reemerged after cytogenetic remission in a group of patients with chronic myeloid leukemia (CML). Therefore, novel treatment strategies are needed. In this study, we evaluated the anti-CML activity and mechanisms of action of LQB-118, a pterocarpanquinone structurally related to lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone]. LQB-118 treatment resulted in an important reduction of cell viability in cell lines derived from CML, both the vincristine-sensitive K562 cell line, and the resistant K562-Lucena (a cell line overexpressing P-glycoprotein). In agreement with these results, the induction of caspase-3 activation by this compound indicated that a significant rate of apoptosis was taking place. In these cell lines, apoptosis induced by LQB-118 was accompanied by a reduction of P-glycoprotein, survivin, and XIAP expression. Moreover, this effect was not restricted to cell lines as LQB-118 produced significant apoptosis rate in cells from CML patients exhibiting multifactorial drug resistance phenotype such as P-glycoprotein, MRP1 and p53 overexpression. The data suggest that LQB-118 has a potent anti-CML activity that can overcome multifactorial drug resistance mechanisms, making this compound a promising new anti-CML agent.
LQB 118 is a pterocarpanquinone compound synthesized by our group. It has already been shown that it acts against different leukemia cell lines. However, little is known about the pathway through which this compound induces the death of these cells. In this work, we analyzed the cell death process induced by LQB 118 in K562, a chronic myeloid leukemia cell line, and in Jurkat, a lymphoblastic acute leukemia cell line. For this, we carried out a cell viability assay by MTT, an apoptosis/necrosis assay through the annexin/propidium iodide label, cell cycle by flow cytometry, assessed changes in the mitochondrial membrane potential using DiOC6(3), cytoplasmic calcium analysis by Fluo-3-AM, and a caspase-9 and caspase-12 activity assay. We found that LQB 118 induced apoptosis in both cell lines, measuring caspase-12 and caspase-9 activation, phosphatidylserine externalization, and DNA fragmentation. The compound induced an increase in cytoplasmic calcium on both cell lines. However, the compound could only induce mitochondrial membrane depolarization on K562 cells. Our data show that LQB 118 may have potential therapeutic value for leukemia, being able to overcome multiple resistance mechanisms.
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