Widespread Aberrant Alternative Splicing despite Molecular Remission in Chronic Myeloid Leukaemia Patients

Vast transcriptomics and epigenomics changes are characteristic of human cancers, including leukaemia. At remission, we assume that these changes normalise so that omics-profiles resemble those of healthy individuals. However, an in-depth transcriptomic and epigenomic analysis of cancer remission has not been undertaken. A striking exemplar of targeted remission induction occurs in chronic myeloid leukaemia (CML) following tyrosine kinase inhibitor (TKI) therapy. Using RNA sequencing and whole-genome bisulfite sequencing, we profiled samples from chronic-phase CML patients at diagnosis and remission and compared these to healthy donors. Remarkably, our analyses revealed that abnormal splicing distinguishes remission samples from normal controls. This phenomenon is independent of the TKI drug used and in striking contrast to the normalisation of gene expression and DNA methylation patterns. Most remarkable are the high intron retention (IR) levels that even exceed those observed in the diagnosis samples. Increased IR affects cell cycle regulators at diagnosis and splicing regulators at remission. We show that aberrant splicing in CML is associated with reduced expression of specific splicing factors, histone modifications and reduced DNA methylation. Our results provide novel insights into the changing transcriptomic and epigenomic landscapes of CML patients during remission. The conceptually unanticipated observation of widespread aberrant alternative splicing after remission induction warrants further exploration. These results have broad implications for studying CML relapse and treating minimal residual disease

The new allosteric inhibitor ABL001 is susceptible to resistance mediated by ABCB1 and ABCG2 overexpression in vitro

ABL001, which binds the myristate-binding pocket of the Bcr-Abl kinase domain, is in phase I clinical trials as monotherapy and in combination with imatinib, nilotinib and dasatinib for the treatment of patients with refractory CML or Ph+ ALL. ABL001 sensitivity was evaluated in ABL001 naïve BCR-ABL1+ cell lines K562 (negligible ABCB1/ABCG2 expression), K562-Dox (ABCB1-overexpressing) and K562-ABCG2 (transduced ABCG2 overexpression) with results demonstrating ABL001 efflux by both transporters. K562-Dox and K562-ABCG2 cells demonstrated increased LD50ABL001 vs. K562 control cells: 256 and 299 nM respectively vs. 23 nM, p<0.001. Sensitivity was completely restored with specific inhibitors cyclosporine (ABCB1) and Ko143 (ABCG2): K562-Dox LD50ABL001+Cyclosporine=13 nM, K562-ABCG2 LD50ABL001+Ko143=15 nM (p<0.001). When ABL001 resistance was modelled in vitro, ABCB1 and ABCG2 overexpression was integral in the development of ABL001 resistance. In K562 ABL001-resistant cells, ABCG2 expression increased prior to BCR-ABL1 overexpression and remained high (up to 7.6-fold greater levels in resistant vs. control cells, p<0.001). K562-Dox ABL001-resistant cells had increased ABCB1 expression (2.1-fold vs. control cells p=0.0033). U812 ABL001-resistant cells overexpressed ABCB1 (5.4-fold increase, p<0.001) and ABCG2 (6-fold increase, p<0.001) before emergence of a novel myristate-binding pocket mutation (F497L). In all three cell lines, ABL001 resistance was reversible upon chemical inhibition of ABCB1, ABCG2 or both (p<0.001). When K562 ABL001-resistant cells were treated with ABL001 in combination with clinically achievable doses of either imatinib or nilotinib, reversal of the resistance phenotype was also observed (p<0.01), potentially through inhibition of ABCG2. Overexpression of efflux transporters will likely be an important pathway for ABL001 resistance in the clinical setting. Given the lack of evidence for ABCG2-mediated transport of nilotinib or imatinib at clinically relevant concentrations, our data provide a strong rationale for using ABL001 in combination with either TKI

Solute Carrier Family 29A1 Mediates In Vitro Resistance to Azacitidine in Acute Myeloid Leukemia Cell Lines

Azacitidine (AZA) is commonly used hypomethylating agent for higher risk myelodysplastic syndromes and acute myeloid leukemia (AML). Although some patients achieve remission, eventually most patients fail AZA therapy. Comprehensive analysis of intracellular uptake and retention (IUR) of carbon-labeled AZA (14C-AZA), gene expression, transporter pump activity with or without inhibitors, and cytotoxicity in naïve and resistant cell lines provided insight into the mechanism of AZA resistance. AML cell lines were exposed to increasing concentrations of AZA to create resistant clones. 14C-AZA IUR was significantly lower in MOLM-13- (1.65 ± 0.08 ng vs. 5.79 ± 0.18 ng; p p 14C-AZA IUR progressively reduced with downregulation of SLC29A1 expression in MOLM-13- and SKM-1-resistant cells. Furthermore, nitrobenzyl mercaptopurine riboside, an SLC29A inhibitor, reduced 14C-AZA IUR in MOLM-13 (5.79 ± 0.18 vs. 2.07 ± 0.23, p p = 0.0002) and reduced efficacy of AZA. As the expression of cellular efflux pumps such as ABCB1 and ABCG2 did not change in AZA-resistant cells, they are unlikely contribute to AZA resistance. Therefore, the current study provides a causal link between in vitro AZA resistance and downregulation of cellular influx transporter SLC29A1

Adverse outcomes for chronic myeloid leukemia patients with splenomegaly and low in vivo kinase inhibition on imatinib

Abstract Variability in the molecular response to frontline tyrosine kinase inhibitor (TKI) therapy in chronic myeloid leukemia may be partially driven by differences in the level of kinase inhibition induced. We measured in vivo BCR::ABL1 kinase inhibition (IVKI) in circulating mononuclear cells after 7 days of therapy. In 173 patients on imatinib 600 mg/day, 23% had low IVKI (<11% reduction in kinase activity from baseline); this was associated with higher rates of early molecular response (EMR) failure; lower rates of major molecular response (MMR), and MR4.5 by 36 months, compared to high IVKI patients. Low IVKI was more common (39%) in patients with large spleens (≥10 cm by palpation). Notably 55% of patients with large spleens and low IVKI experienced EMR failure whereas the EMR failure rate in patients with large spleens and high IVKI was only 12% (p = 0.014). Furthermore, patients with large spleen and low IVKI had a higher incidence of blast crisis, inferior MMR, MR4.5, and event-free survival compared to patients with large spleen and high IVKI and remaining patients. In nilotinib-treated patients (n = 73), only 4% had low IVKI. The combination of low IVKI and large spleen is associated with markedly inferior outcomes and interventions in this setting warrant further studies

Functional activity of the OCT-1 protein is predictive of long-term outcome in patients with chronic-phase chronic myeloid leukemia treated with Imatinib

Purpose: Organic cation transporter-1 (OCT-1) activity (OA), a measure of the OCT-1–mediated influx of imatinib into CML mononuclear cells (MNCs), is predictive of major molecular response (MMR) at 12 and 24 months in patients with untreated CML. We now report the impact of OA on loss of response, disease transformation, and survival after 5 years of imatinib. Patients and Methods: OA is defined as the difference in intracellular concentration of carbon-14–imatinib with and without OCT-1 inhibition. OA was measured in blood from 56 patients with untreated chronic-phase CML. Results: More patients who had high OA (ie, > median OA value) achieved MMR by 60 months compared with patients who had low OA (89% v 55%; P = .007). A low OA was associated with a significantly lower overall survival (87% v 96%; P = .028) and event-free survival (EFS; 48% v 74%; P = .03) as well as a higher kinase domain mutation rate (21% v 4%; P = .047). These differences were highly significant in patients who averaged less than 600 mg/d of imatinib in the first 12 months but were not significant in patients averaging ≥ 600 mg/d. Patients with very low OA (ie, quartile 1) were the only group who developed leukemic transformation (21% in quartile 1 v 0% in all other quartiles; P = .002). Conclusion: Measurement of OA pretherapy is a predictor for the long-term risk of resistance and transformation in patients with imatinib-treated CML. Early dose-intensity may reduce the negative prognostic impact of low OA. We propose that OA could be used to individualize dosage strategies for patients with CML to maximize molecular response and optimize long-term outcome.Deborah L. White, Phuong Dang, Jane Engler, Amity Frede, Stephanie Zrim, Michael Osborn, Verity A. Saunders, Paul W. Manley, and Timothy P. Hughe

BCR-ABL1 genomic DNA PCR response kinetics during first-line imatinib treatment of chronic myeloid leukemia

Accurate quantification of minimal residual disease (MRD) during treatment of chronic myeloid leukemia (CML) guides clinical decisions. The conventional MRD method, RQ-PCR for BCR-ABL1 mRNA, reflects a composite of the number of circulating leukemic cells and the BCR-ABL1 transcripts per cell. BCR-ABL1 genomic DNA only reflects leukemic cell number. We used both methods in parallel to determine the relative contribution of the leukemic cell number to molecular response. BCR-ABL1 DNA PCR and RQ-PCR were monitored up to 24 months in 516 paired samples from 59 newly-diagnosed patients treated with first-line imatinib in the TIDEL-II study. In the first three months of treatment, BCR-ABL1 mRNA values declined more rapidly than DNA. By six months, the two measures aligned closely. The expression of BCR-ABL1 mRNA was normalized to cell number to generate an expression ratio. The expression of e13a2 BCR-ABL1 was lower than that of e14a2 transcripts at multiple time points during treatment. BCR-ABL1 DNA was quantifiable in 48% of samples with undetectable BCR-ABL1 mRNA, resulting in MRD being quantifiable for an additional 5-18 months (median 12 months). These parallel studies show for the first time that the rapid decline in BCR-ABL1 mRNA over the first three months of treatment is due to a reduction in both cell number and transcript level per cell, whereas beyond three months, falling levels of BCR-ABL1 mRNA are proportional to the depletion of leukemic cells