Dale B. Watkins

Tyrosine kinase inhibitor resistance in chronic myeloid leukemia cell lines: investigating resistance pathways

Abstract There are three currently identified secondary resistance mechanisms observed in patients with chronic myeloid leukemia (CML) receiving tyrosine kinase inhibitors (TKIs). These are BCR–ABL kinase domain (KD) mutations, increased BCR–ABL expression, and overexpression of drug-efflux proteins (ABCB1 and ABCG2). To investigate the interplay between these three modes of resistance, three CML blast crisis cell lines (K562, its ABCB1-overexpressing variant K562 Dox, and KU812) were cultured in gradually increasing concentrations of imatinib to 2 μM, or dasatinib to 200 nM. Eight imatinib- and two dasatinib-resistant cell lines were established. Two imatinib-resistant K562 lines both had increased BCR–ABL expression as the apparent mode of resistance. However, when a dasatinib-resistant K562 culture was generated we observed gradually increasing BCR–ABL expression which peaked prior to identification of the T315I mutation. BCR–ABL overexpression followed by mutation development was observed in a further 4/10 cell lines, each with different KD mutations. In contrast, three imatinib-resistant K562 Dox lines exhibited only a further increase in ABCB1 expression. All TKI-resistant cell lines generated had increased IC50 (dose of drug required to reduce phosphorylation of the adaptor protein p-Crkl by 50%) to imatinib, dasatinib, and nilotinib, regardless of which TKI was used to induce resistance. This suggests that currently available TKIs share the same susceptibilities to drug resistance.

OCT1 and imatinib transport in CML: is it clinically relevant?

Imatinib is a highly effective therapy for chronic phase-chronic myeloid leukaemia (CP-CML) patients; however, responses to frontline imatinib are variable. The human organic cation transporter 1 (OCT1; SLC22A1) has been reported to be the main influx transporter involved in imatinib uptake into CML cells. Furthermore, variation in the efficiency of imatinib influx via OCT1 has been demonstrated to result in the inter-patient variation observed in primary response to imatinib. Although studies have questioned the role of OCT1 in imatinib influx, these have been largely performed in non-clinical settings. Measuring both OCT1 mRNA levels and the functional activity of OCT1 in primary leukaemic cells has been demonstrated to predict molecular response and outcome in imatinib-treated CP-CML patients in several independent studies. Here, the role of OCT1 and OCT1 genetic variants in imatinib uptake and response prediction is summarised and data generated from model systems assessing the role of OCT1 in imatinib transport is discussed.

Low GFI1 expression in white blood cells of CP-CML patients at diagnosis is strongly associated with subsequent blastic transformation.

Low GFI1 expression in white blood cells of CP–CML patients at diagnosis is strongly associated with subsequent blastic transformation

Reverse Transcription with Random Pentadecamer Primers Improves the Detection Limit of a Quantitative PCR Assay for BCR-ABL Transcripts in Chronic Myeloid Leukemia: Implications for Defining Sensitivity in Minimal Residual Disease

BACKGROUND Real-time quantitative reverse transcription PCR (RQ-PCR) assay for BCR-ABL is used to monitor treatment response in chronic myeloid leukemia (CML). BCR-ABL transcript levels decline over several years of imatinib treatment, and increasing numbers of patients have BCR-ABL transcripts at or below the limit of detection. More sensitive PCR methods are required to assess whether these patients have a long-term continuing decline in residual disease. METHODS We used random pentadecamer (R15) primers for reverse transcription in RQ-PCR and compared the results with our established method that uses random hexamers. An increase in assay sensitivity would be detected as an increase in the number of BCR-ABL transcripts. RESULTS BCR-ABL transcripts increased by 86% with R15 primers. We used R15 primers to retest 19 samples from selected CML patients who had no BCR-ABL transcripts recently detectable with hexamer primers and detected BCR-ABL transcripts in 68% of the samples. Use of R15 primers showed variable increases in the transcripts for control genes BCR (breakpoint cluster region), ABL1 (c-abl oncogene 1, receptor tyrosine kinase), and GUSB (glucuronidase, beta), depending on the gene examined. The reported BCR-ABL/control gene ratio was affected, and the estimated detection limit of the assay, which was based on increased control gene copy number, was different for each control gene. CONCLUSIONS This simple modification to the reverse transcription methodology improved the detection limit of the RQ-PCR assay for BCR-ABL transcripts. In the field of CML, these results have important implications for defining the detection limit of an assay when the BCR-ABL transcript is undetectable. Random pentadecamer primers may also be useful in other reverse transcription PCR assays for which the abundance of the target RNA is low.

NPM1 mutations occur rarely or not at all in chronic myeloid leukaemia patients in chronic phase or blast crisis.

NPM1 mutations occur rarely or not at all in chronic myeloid leukaemia patients in chronic phase or blast crisis

TGF-α and IL-6 plasma levels selectively identify CML patients who fail to achieve an early molecular response or progress in the first year of therapy

Early molecular response (EMR, BCR-ABL1 (IS)⩽10% at 3 months) is a strong predictor of outcome in imatinib-treated chronic phase chronic myeloid leukemia (CP-CML) patients, but for patients who transform early, 3 months may be too late for effective therapeutic intervention. Here, we employed multiplex cytokine profiling of plasma samples to test newly diagnosed CP-CML patients who subsequently received imatinib treatment. A wide range of pro-inflammatory and angiogenesis-promoting cytokines, chemokines and growth factors were elevated in the plasma of CML patients compared with that of healthy donors. Most of these normalized after tyrosine kinase inhibitor treatment while others remained high in remission samples. Importantly, we identified TGF-α and IL-6 as novel biomarkers with high diagnostic plasma levels strongly predictive of subsequent failure to achieve EMR and deep molecular response, as well as transformation to blast crisis and event-free survival. Interestingly, high TGF-α alone can also delineate a poor response group raising the possibility of a pathogenic role. This suggests that the incorporation of these simple measurements to the diagnostic work-up of CP-CML patients may enable therapy intensity to be individualized early according to the cytokine-risk profile of the patient.

Elevated PTPN2 expression is associated with inferior molecular response in de-novo chronic myeloid leukaemia patients.

Elevated PTPN2 expression is associated with inferior molecular response in de-novo chronic myeloid leukaemia patients