Junia V. Melo

Expression of interferon regulatory factor (IRF) genes and response to interferon-α in chronic myeloid leukaemia

Interferon regulatory factors (IRF) 1 and 2 are DNA-binding proteins which control interferon (IFN) gene expression. IRF1 functions as an activator for IFN and IFN-inducible genes, whereas IRF2 represses the action of IRF1. Expression of the two regulatory genes is itself IFN-inducible. Because therapeutic responses of chronic myeloid leukaemia (CML) patients to IFN-α may be determined by intracellular levels of these two mutually antagonistic transcription factors, we have devised a competitive reverse-transcription polymerase chain reaction (RT-PCR) assay which provides an estimate of the ratio of IRF1 to IRF2 expression in a given cell population. Analysis of peripheral blood leucocytes from 25 normal individuals showed that the IRF1:IRF2 ratio varied between 1.13 and 2.30 (mean ± s.d. 1.49 ± 0.33). Similar values were obtained for normal bone marrow specimens, with no significant difference between CD34+ and CD34− cells. In contrast, the IRF1:IRF2 ratio in leucocytes from CML patients showed a much wider variation (0.53–5.11). Eleven out of 130 patients in chronic phase had ratios above the normal range, whereas none of the 33 blast crisis samples had a ratio >2.5. Analysis of diagnostic specimens in 59 CML patients treated subsequently with IFN-α showed a high IRF1:IRF2 ratio of 5.11 in one of two patients who became complete responders; all the 53 patients with minimal or no cytogenetic response had ratios below 2.5. In a separate series of 97 CML patients studied after IFN-α therapy a highly significant correlation was found between the IRF1:IRF2 ratio and both the cytogenetic and the molecular response (ie low concentration of BCR-ABL transcripts) to treatment: 53 out of 115 prospectively analysed samples of good cytogenetic responders had ratios above 2.0, as opposed to only 13 out of 91 samples from poor responders (Pu2009<u20090.0001; χ2 test). We conclude that a high ratio of IRF1/IRF2 expression may be associated with good cytogenetic and molecular response to IFN- α in CML.

Nilotinib-mediated inhibition of ABCB1 increases intracellular concentration of dasatinib in CML cells: implications for combination TKI therapy

Nilotinib-mediated inhibition of ABCB1 increases intracellular concentration of dasatinib in CML cells: implications for combination TKI therapy

Antileukemic Activity of Lysophosphatidic Acid Acyltransferase-β Inhibitor CT32228 in Chronic Myelogenous Leukemia Sensitive and Resistant to Imatinib

Purpose: Lysophosphatidic acid acyltransferase (LPAAT)-β catalyzes the conversion of lysophosphatidic acid to phosphatidic acid, an essential component of several signaling pathways, including the Ras/mitogen-activated protein kinase pathway. Inhibition of LPAAT-β induces growth arrest and apoptosis in cancer cell lines, implicating LPAAT-β as a potential drug target in neoplasia. Experimental Design: In this study, we investigated the effects of CT32228, a specific LPAAT-β inhibitor, on BCR-ABL-transformed cell lines and primary cells from patients with chronic myelogenous leukemia. Results: CT32228 had antiproliferative activity against BCR-ABL-positive cell lines in the nanomolar dose range, evidenced by cell cycle arrest in G2-M and induction of apoptosis. Treatment of K562 cells with CT32228 led to inhibition of extracellular signal-regulated kinase 1/2 phosphorylation, consistent with inhibition of mitogen-activated protein kinase signaling. Importantly, CT32228 was highly active in cell lines resistant to the Bcr-Abl kinase inhibitor imatinib. Combination of CT32228 with imatinib produced additive inhibition of proliferation in cell lines with residual sensitivity toward imatinib. In short-term cultures in the absence of growth factors, CT32228 preferentially inhibited the growth of granulocyte-macrophage colony-forming units from chronic myelogenous leukemia patients compared with healthy controls. Conclusion: These data establish LPAAT-β as a potential drug target for the treatment of BCR-ABL-positive leukemias.

Rapid automated detection of ABL kinase domain mutations in imatinib-resistant patients

ABL tyrosine kinase inhibitor (TKI), imatinib is used for BCR-ABL(+) leukemias. We developed an automatic method utilizing guanine-quenching probes (QP) to detect 17 kinds of mutations frequently observed in imatinib-resistance. Results were obtained from 100μL of whole blood within 90min by this method. Detected mutations were almost identical between QP method and direct sequencing. Furthermore, the mutation-biased PCR (MBP) was added to the QP method to increase sensitivity, resulting earlier detection of T315I mutation which was insensitive to any ABL TKIs. Thus, the QP and MBP-QP may become useful methods for the management of ABL TKI-treated patients.

Clonal instability preceding lymphoid blastic transformation of chronic myeloid leukemia

We have sought the presence of rearrangements of the immunoglobulin heavy chain gene locus in 13 patients with chronic myeloid leukemia (CML) in lymphoid blastic transformation (L-BT) using the polymerase chain reaction (PCR). The lymphoid nature of the transformation was confirmed by immunophenotyping and/or Southern blot hybridization with a JH probe. Clonal rearrangements were detected in 85% of cases and two or more rearrangements were visible in 64% of informative cases. The pattern of VH gene family utilization revealed an apparent reduction in VH 4 family gene usage but otherwise reflected the known proportion of each gene family in the germline repertoire. In six cases the third complementary determining regions (CDR3) of the predominant blast crisis clone/s were sequenced revealing minimal evidence of somatic mutation. No clonal changes were detected in the chronic phase leukemia cells collected more than 6 months before the onset of L-BT in three of these patients. Of the other three patients studied in chronic phase from 1 to 6 months before L-BT, two showed clonal rearrangements which differed in size from those present at L-BT. In one patient a VH3 to VH5–DH–JH substitution had occurred at least 3 months prior to L-BT. In the other patient, however, the sequence of the rearrangement present 5 months prior to L-BT was unrelated to the rearrangements at the time of L-BT indicating a pattern of clonal succession. We conclude that: (1) IgH gene rearrangements are detectable in the majority of patients with L-BT using PCR and the lymphoid lineage of blastic CML is most readily confirmed using consensus primers to the framework 3 region; (2) somatic mutation is uncommon; and (3) B lymphoid clones distinct from those identified later may be detected before overt lymphoid BT. The identification of such ‘abortive’ clones is evidence for clonal instability before the onset of transformation and might have prognostic value.