Adult Acute Myeloid Leukemia Cells Research Articles

Abstract 5220: Identifying AML-specific key targeted drugs using high-throughput drug sensitivity and resistance testing profiles of AML cells.

Abstract Introduction: Conventional cytotoxic chemotherapy regimens for adult acute myeloid leukemia (AML) are effective in curing less than 50% of the patients, and there is a major need for targeted drugs with better anti-cancer selectivity. Here, our aim was to i) identify potential clinically used or emerging cancer drugs by quantitative drug sensitivity and resistance testing (DSRT) of 16 AML cell lines ii) compare the cell line data with results obtained from tested 24 ex vivo AML patient specimens iii) identify genomic correlations potentially explaining drug responsiveness. Methods: The cancer pharmacopeia-wide drug collection is composed of 119 FDA approved and 90 investigational chemical compounds including cytotoxic agents and cell signaling molecule inhibitors. Each drug was tested over a 10,000-fold concentration range and that has generated quantitative five point dose-response curves. AML cells were plated in 384 well plates (where the drugs were pre-printed using an acoustic nano-dispensing technology, Labcyte®) and incubated in standard cell culture conditions. Cell viability was measured by Cell Titer Glow® luminescence assays. Analysis of dose response curves using Dotmatics® software resulted in IC50 values. Moreover, the genomic profiles of the AML cell lines were determined by microarrays and/or next-gen sequencing data for further integration with drug responses. Results: Comprehensive data analysis of 16 AML cell lines indicated that specific targeted drugs were selectively killing AML cells. The data analysis revealed relatively strong responses for MEK inhibitors in most AML cell lines (e.g. refametinib 87%, trametinib 82%, selumetinib 75%) while 21% of ex vivo AML patient samples were sensitive to these MEK inhibitors. In case of rapalog sensitivity, 80% of AML cell lines (e.g. temsirolimus 82%, everolimus 71%, sirolimus 81%) and 25% of ex vivo AML patient cases were responsive to the mTOR inhibitors. The AML cell lines carrying FLT3-ITD mutations were extremely sensitive to FLT3 inhibitors (e.g. quizartinib, lestaurtinib, tandutinib, and sorafenib) but very few responses to FLT3 inhibitors were observed in AML patients carrying an ITD mutation in the FLT3 kinase. Summary: Systematic DSRT profiling of AML cell lines illustrates drug sensitivity patterns to classify the cell lines as sensitive or resistant to specific classes of drugs. mTOR and MEK inhibitors were among the most effective inhibitors for most cell lines and also in some ex vivo patient cases suggesting that these drugs may have potential as therapeutic agents in AML. Also, bioinformatics predictions can be used to identify key synergistic combinations of tested drugs for effective AML therapy. Further integration of molecular profiles and functional responses of AML cell lines will help provide better understanding of drug efficacy based on known genetic background of the disease. Citation Format: Disha Malani, Astrid Murumägi, Tea Pemovska, Bhagwan Yadav, Evgeny Kulesskiy, Jing Tang, John Patrick Mpindi, Maija Wolf, Riikka Karjalainen, Tero Aittokallio, Caroline Heckman, Kimmo Porkka, Krister Wennerberg, Olli Kallioniemi. Identifying AML-specific key targeted drugs using high-throughput drug sensitivity and resistance testing profiles of AML cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5220. doi:10.1158/1538-7445.AM2013-5220

Abstract 895: Quantitative drug sensitivity and resistance testing (DSRT) of primary ex vivo AML blasts highlights mTOR and MEK as potential key molecular driver signals of therapeutic significance

Abstract Identification of signaling pathways that are required for the growth and differentiation block of cells from adult acute myeloid leukemia (AML) is urgently required to facilitate development of novel therapies. Here, we describe an approach to functionally determine molecular drivers of AML by quantitative drug sensitivity and resistance testing (DSRT) of AML blast cells in primary culture ex vivo. The selection of drugs covered the entire cancer pharmacopeia and much of the pipeline of drugs under development in the industry: 120 FDA approved small molecular cancer drugs and 120 emerging drugs, investigational compounds and signal transduction inhibitors. All compounds were tested over a 10,000-fold concentration range to generate quantitative and reliable dose-response data. In addition, whole exome and transcriptome sequencing and phophoproteomic profiling were also performed to derive a comprehensive understanding of the molecular AML-related aberrations on an individual basis. Comparison of 17 AML patient samples and 3 healthy bone marrow control samples based on ex vivo drug responses identified several classes of approved and investigational drugs that showed selective anti-AML activities: mTOR inhibitors (e.g. temsirolomus, everolimus, sirolimus), MEK inhibitors (e.g. AS703026, GSK1120212, RDEA119, selumetinib), tyrosine kinase inhibitors (e.g. dasatinib, ponatinib, sunitinib), Bcl-2 inhibitors (navitoclax) and HSP90 inhibitors (e.g. BIIB021, NVP-AUY922, tanespimycin). In particular, the rapamycin class of mTOR inhibitors and allosteric MEK inhibitors stood out as effective and selective inhibitors in 8/17 (47%) and 9/17 (52%) of the patients, respectively. Simultaneous data from other targeted inhibitors made it possible to dissect the critical steps in signaling and therapeutic efficacy. For example, PI3K and Akt inhibitors were not effective in these patients, suggesting that the mTOR dependency is mediated through a PI3K-Akt-independent pathway. Similarly, the dependency of MEK signaling appears to be through a Ras-Raf-independent pathway since Raf inhibitors were not effective. In conclusion, the DSRT platform allows us to derive quantitative data on the ex vivo drug response profiles of AML cells from individual patients. This information could be used as a diagnostic tool to optimize personalized therapies in the future. Our data demonstrate that mTOR and MEK signaling and the associated inhibitors are the most promising leads for improved AML therapeutics. This analysis also demonstrates gaps in our current understanding of the redundancy of key cancer cell signaling pathways and proves the significant value of data from experimental drug response testing ex vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 895. doi:1538-7445.AM2012-895

No Influence of 3435C>T ABCB1 (MDR1) Gene Polymorphism on Risk of Adult Acute Myeloid Leukemia and P-glycoprotein Expression in Blast Cells

Inherited differences in xenobiotic transport and metabolism may play an important role in the development of adult acute myeloid leukemia (AML) and response to the chemotherapy. An ATP-binding cassette (ABC) family transporter P-glycoprotein (P-gp or ABCB1), encoded by ABCB1 (MDR1) gene, is involved in the protection against xenobiotics and multi-drug resistance. The aim of this study was to investigate the potential involvement of the ABCB1 gene exon 26 3435C>T single nucleotide polymorphism (SNP) in the genetic susceptibility to AML and regulation of P-gp expression and activity in AML cells. A total of 180 adult AML patients and 180 sex-matched controls were genotyped using PCR-RFLP method. Moreover, in 40 AML patients ABCB1 gene expression was studied by real-time RT-PCR and P-gp expression and activity were assessed by flow cytometry assays. The prevalence of 3435C>T ABCB1 polymorphism was similar in patient and control cohorts (P = 0.16). Furthermore, the carriers of different ABCB1 genotypes did not differ significantly according to ABCB1 gene expression (P = 0.99), P-gp expression (P = 0.42) and P-gp activity (P = 0.83) in leukemic cells. The authors conclude that isolated 3435C>T ABCB1 SNP is not a major factor of the genetic susceptibility to adult AML, and that genotyping of this polymorphism does not allow predicting P-gp expression or activity in AML cells.

Immunohistochemistry predicts nucleophosmin (NPM) mutations in acute myeloid leukemia

AbstractNucleophosmin (NPM) exon-12 mutations occur in 50% to 60% of adult acute myeloid leukemia (AML) with normal karyotype and are predictors of favorable prognosis. We evaluated bone marrow or peripheral blood samples from 450 adult patients with AML of the GIMEMA (Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto)/AML12 EORTC (European Organization for Research and Treatment of Cancer) trial to (1) search for new exon-12 NPM mutations; (2) determine whether NPM immunostaining on paraffin-embedded biopsies predicts NPM mutations; and (3) investigate altered nucleocytoplasmic NPM traffic in primary AML cells. Fourteen NPM mutations, including 8 new variants, were identified. All 200 AML cases expressing cytoplasmic NPM (NPMc+ AML) carried NPM mutations. None of the 250 cases with nucleus-restricted NPM (NPMc– AML) was mutated. At the C-terminus, NPM leukemic mutants carried mutations of only tryptophan 290 or of both tryptophans 288 and 290 and a new nuclear export signal (NES) motif, which appear to underlie their nuclear export. The specific Crm1/exportin-1 inhibitor leptomycin-B relocated NPM mutants from cytoplasm to nucleus of primary NPMc+ AML cells, demonstrating that nuclear export is NES dependent. NPM mutants bound and recruited wild-type NPM into leukemic cell cytoplasm. Because alterations at C-terminus of leukemic NPM mutants are similar, immunohistochemistry detects all exon-12 NPM mutations and is a valuable, inexpensive tool in the diagnostic-prognostic work-up of patients with AML with normal karyotype.