The mutational oncoprint of recurrent cytogenetic abnormalities in adult patients with de novo acute myeloid leukemia.

Recurrent chromosomal abnormalities and gene mutations detected at the time of diagnosis of acute myeloid leukemia (AML) are associated with particular disease features, treatment response and survival of AML patients, and are used to denote specific disease entities in the World Health Organization classification of myeloid neoplasms and acute leukemia. However, large studies that integrate cytogenetic and comprehensive mutational information are scarce. We created a comprehensive oncoprint of mutations associated with recurrent cytogenetic findings by combining the information on mutational patterns of 80 cancer- and leukemia-associated genes with cytogenetic findings in 1603 adult patients with de novo AML. We show unique differences in the mutational profiles among major cytogenetic subsets, identify novel associations between recurrent cytogenetic abnormalities and both specific gene mutations and gene functional groups, and reveal differences in cytogenetic and mutational features between patients younger than 60 years and those aged 60 years or older. The identified associations between cytogenetic and molecular genetic data may help guide mutation testing in AML, and result in more focused application of targeted therapy in patients with de novo AML.

Mutations in the CCND1 and CCND2 genes are frequent events in adult patients with t(8;21)(q22;q22) acute myeloid leukemia.

Core-binding factor acute myeloid leukemia (CBF-AML) is defined by the presence of either t(8;21)(q22;q22)/RUNX1-RUNX1T1 or inv(16)(p13.1q22)/t(16;16)(p13.1;q22)/CBFB-MYH11. The resulting fusion genes require a 'second hit' to initiate leukemogenesis. Mutation assessment of 177 adults with CBF-AML, including 68 with t(8;21) and 109 with inv(16)/t(16;16), identified not only mutations well known in CBF-AML but also mutations in the CCND1 and CCND2 genes, which represent novel frequent molecular alterations in AML with t(8;21). Altogether, CCND1 (n=2) and CCND2 (n=8) mutations were detected in 10 (15%) patients with t(8;21) in our cohort. A single CCND2 mutation was also found in 1 (0.9%) patient with inv(16). In contrast, CCND1 and CCND2 mutations were detected in only 11 (0.77%) of 1426 non-CBF-AML patients. All CCND2 mutations cluster around the highly conserved amino-acid residue threonine 280 (Thr280). We show that Thr280Ala-mutated CCND2 leads to increased phosphorylation of the retinoblastoma protein, thereby causing significant cell cycle changes and increased proliferation of AML cell lines. The identification of CCND1 and CCND2 mutations as frequent mutational events in t(8;21) AML may provide further justification for cell cycle-directed therapy in this disease.

ABCC4 Is a Determinant of Cytarabine-Induced Cytotoxicity and Myelosuppression.

Resistance to cytarabine remains a major challenge in the treatment of acute myeloid leukemia (AML). Based on previous studies implicating ABCC4/MRP4 in the transport of nucleosides, we hypothesized that cytarabine is sensitive to ABCC4-mediated efflux, thereby decreasing its cytotoxic response against AML blasts. The uptake of cytarabine and its monophosphate metabolite was found to be facilitated in ABCC4-expressing vesicles and intracellular retention was significantly impaired by overexpression of human ABCC4 or mouse Abcc4 (P < 0.05). ABCC4 was expressed highly in AML primary blasts and cell lines, and cytotoxicity of cytarabine in cells was increased in the presence of the ABCC4 inhibitors MK571 or sorafenib, as well as after ABCC4 siRNA. In Abcc4-null mice, cytarabine-induced hematological toxicity was enhanced and ex vivo colony-forming assays showed that Abcc4-deficiency sensitized myeloid progenitors to cytarabine. Collectively, these studies demonstrate that ABCC4 plays a protective role against cytarabine-mediated insults in leukemic and host myeloid cells.

Inherited variation in OATP1B1 is associated with treatment outcome in acute myeloid leukemia.

Using broad interrogation of clinically relevant drug absorption, distribution, metabolism, and excretion (ADME) genes on the DMET platform, we identified a genetic variant in SLCO1B1 (rs2291075; c.597C>T), encoding the transporter OATP1B1, associated with event-free (P = 0.006, hazard ratio = 1.74) and overall survival (P = 0.012, hazard ratio = 1.85) in children with de novo acute myeloid leukemia (AML). Lack of SLCO1B1 expression in leukemic blasts suggested the association might be due to an inherited rather than a somatic effect. rs2291075 was in strong linkage with known functional variants rs2306283 (c.388A>G) and rs4149056 (c.521T>C). Functional studies in vitro determined that four AML-directed chemotherapeutics (cytarabine, daunorubicin, etoposide, and mitoxantrone) are substrates for OATP1B1 and the mouse ortholog Oatp1b2. In vivo pharmacokinetic studies using Oatp1b2-deficient mice further confirmed our results. Collectively, these findings demonstrate an important role for OATP1B1 in the systemic pharmacokinetics of multiple drugs used in the treatment of AML and suggest that inherited variability in host transporter function influences the effectiveness of therapy.

OATP1B1 polymorphism as a determinant of erythromycin disposition.

Previous studies have demonstrated that the pharmacokinetic profile of erythromycin, a probe for CYP3A4 activity, is affected by inhibitors or inducers of hepatic solute carriers. We hypothesized that these interactions are mediated by OATP1B1 (gene symbol, SLCO1B1), a polypeptide expressed on the basolateral surface of hepatocytes. Using stably transfected Flp-In T-Rex293 cells, erythromycin was found to be a substrate for OATP1B1*1A (wild type) with a Michaelis-Menten constant of ~13 µmol/l, and that its transport was reduced by ~50% in cells expressing OATP1B1*5 (V174A). Deficiency of the ortholog transporter Oatp1b2 in mice was associated with a 52% decrease in the metabolic rate of erythromycin (P = 0.000043). In line with these observations, in humans the c.521T>C variant in SLCO1B1 (rs4149056), encoding OATP1B1*5, was associated with a decline in erythromycin metabolism (P = 0.0072). These results suggest that impairment of OATP1B1 function can alter erythromycin metabolism, independent of changes in CYP3A4 activity.

Effect of ABCC2 (MRP2) transport function on erythromycin metabolism.

The macrolide antiobiotic erythromycin undergoes extensive hepatic metabolism and is commonly used as a probe for cytochrome P450 (CYP) 3A4 activity. By means of a transporter screen, erythromycin was identified as a substrate for the transporter ABCC2 (MRP2) and its murine ortholog, Abcc2. Because these proteins are highly expressed on the biliary surface of hepatocytes, we hypothesized that impaired Abcc2 function may influence the rate of hepatobiliary excretion and thereby enhance erythromycin metabolism. Using Abcc2 knockout mice, we found that Abcc2 deficiency was associated with a significant increase in erythromycin metabolism, whereas murine Cyp3a protein expression and microsomal Cyp3a activity were not affected. Next, in a cohort of 108 human subjects, we observed that homozygosity for a common reduced-function variant in ABCC2 (rs717620) was also linked to an increase in erythromycin metabolism but was not correlated with the clearance of midazolam. These results suggest that impaired ABCC2 function can alter erythromycin metabolism, independent of changes in CYP3A4 activity.

Pharmacogenetic pathway analysis of docetaxel elimination.

The purpose of this study was to evaluate the affinity of docetaxel for 14 transporter proteins and assess the functional significance of 17 variants in five genes involved in drug elimination. Among the transfected models investigated, OATP1B3 (SLCO1B3) was identified as the most efficient influx transporter for docetaxel. None of the observed genotypes (SLCO1B3, ABCB1, and ABCC2) was related with docetaxel clearance in 92 white patients (P > 0.17). However, the simultaneous presence of the CYP3A4*1B and CYP3A5*1A alleles was associated with a 64% increase in docetaxel clearance (P = 0.0015), independent of both sex and CYP3A activity (as determined using the erythromycin breath test). This haplotype was also associated with increased midazolam clearance in another population (P = 0.0198). An analysis of the CYP3A locus among CEPH-HapMap samples revealed that CYP3A4*1B is present exclusively among a subset of CYP3A5 expressors. Therefore, future studies should first stratify the population on the basis of CYP3A5 genotype and then compare CYP3A activity between individuals with and without the CYP3A4*1B allele.