Acquired FANCA dysfunction and cytogenetic instability in adult acute myelogenous leukemia by M. William Lensch, Marc Tischkowitz, Tracy A. Christianson, Carol A. Reifsteck, S. Ashley Speckhart, Petra M. Jakobs, Michael E. O'Dwyer, Susan B. Olson, Michelle M. Le Beau, Shirley V. Hodgson, Christopher G. Mathew, Richard A. Larson, and Grover C. Bagby Blood Volume 102(1):7-16 July 1, 2003 ©2003 by American Society of Hematology

UoC-M1 is sensitive to chromosomal breakage and radial formation following MMC treatment. UoC-M1 is sensitive to chromosomal breakage and radial formation following MMC treatment. Arrows indicate representative chromosomal anomalies in UoC-M1: (A) single chromatid break and (B) complex chromosomal rearrangement with at least 2 chromosomal breaks and one end-to-end fusion (radial). Such findings in clastogen-treated cells represent the clinical diagnostic laboratory standard for Fanconi anemia. UoC-M1 also demonstrated occasional, low-level chromosomal breakage without clastogen treatment, although most of the untreated cells had no evidence of instability. No radial forms or cells with multiple chromosomal breaks were ever noted in untreated specimens (of more than 500 total metaphases analyzed), although cells with single breaks were occasionally noted (maximum of 20% in an untreated specimen that when clastogen treated in a simultaneously prepared, parallel flask demonstrated 26% breakage with up to 3 breaks per cell and 38% radial formation; data not shown). M. William Lensch et al. Blood 2003;102:7-16 ©2003 by American Society of Hematology

A lymphoblastoid cell line from the same patient is resistant to MMC A lymphoblastoid cell line from the same patient is resistant to MMC. The myeloid cell line (UoC-M1) was exposed to MMC as was the lymphoblastoid cell line (4081) derived from the same patient. A lymphoblastoid cell line from the same patient is resistant to MMC. The myeloid cell line (UoC-M1) was exposed to MMC as was the lymphoblastoid cell line (4081) derived from the same patient. Although the UoC-M1 cell line consistently demonstrated radial formation in excess of 20% (mean = 29%) in multiple experiments (left bar), the 4081 cell line was resistant (P = .003) to chromosomal breaks and radial formation after exposure to MMC (right bar). Treatment with DEB showed a similar difference. Error bars represent ± SD. *P = .003; M indicates myeloid; L, lymphoid. M. William Lensch et al. Blood 2003;102:7-16 ©2003 by American Society of Hematology

Fanconi anemia gene transcripts are not reduced in UoC-M1. Fanconi anemia gene transcripts are not reduced in UoC-M1. QRT-PCR reactions were performed in at least triplicate. Error bars represent standard deviations. Fold values were obtained by externally standardizing against identical amplifications in Mo7e and by internally standardizing against 18S RNA in each cell line. Compared with Mo7e, UoC-M1 does not exhibit reduced levels of mRNA for FANCA, FANCC, FANCD2, FANCE, and FANCG. An approximate 2-fold reduction in FANCF was observed. This finding was attributed to monosomy 11p in UoC-M1,10 as FANCF is encoded on chromosome 11.27 M. William Lensch et al. Blood 2003;102:7-16 ©2003 by American Society of Hematology

Nuclear FA proteins are reduced in UoC-M1 cells. Nuclear FA proteins are reduced in UoC-M1 cells. Immunoblots of cytoplasmic and nuclear components were probed with antisera for the FANCA, FANCC, and FANCG proteins. Compared with the human, cervical carcinoma cell line HeLa (lane 1) and the factor-dependent, myeloid cell line Mo7e (lane 2), UoC-M1 (lane 3) has a mild reduction in the levels of cytoplasmic FANCA, FANCC, and FANCG proteins (rows 1, 4, and 5, respectively). Nuclear fractions (lanes 4-6) demonstrated a reduction of FANCC in UoC-M1 (row 4, lane 6) and a nearly complete absence of nuclear FANCA (row 1, lane 6) and FANCG proteins (row 5, lane 6). Purity of nuclear fractions was confirmed by reprobing the filters with antisera against beta-tubulin (rows 2 and 6) and topoisomerase II (rows 3 and 7). M. William Lensch et al. Blood 2003;102:7-16 ©2003 by American Society of Hematology

UoC-M1 does not monoubiquitinylate FANCD2. UoC-M1 does not monoubiquitinylate FANCD2. A lymphoblastoid cell line control from an FA-C patient (HSC536N) does not exhibit the monoubiquitinylated form of FANCD2 (FANCD2-L), whereas retroviral correction of the FANCC defect in these cells (HSC536N/FANCC) restores FANCD2-L. UoC-M1 is incapable of forming FANCD2-L. The 4081 lymphoblastoid cell line (from the same patient as the UoC-M1 myeloid cell line) demonstrates FANCD2-L that is capable of being immunodepleted with a monoclonal antiubiquitin antibody (lane 1; ub. dep. 4081). Both Mo7e (a factor-dependent myeloid leukemia cell line) and JY (an EBV-transformed lymphoblastoid line from a healthy volunteer) demonstrate cellular resistance to MMC and FANCD2-L. M. William Lensch et al. Blood 2003;102:7-16 ©2003 by American Society of Hematology

Introduction of a FANCA cDNA reduces MMC sensitivity in UoC-M1. Introduction of a FANCA cDNA reduces MMC sensitivity in UoC-M1. MMC resistance was evaluated in transduced UoCM-1 cells using cytogenetic analysis of radial formation after treatment with MMC. Transduction with a FANCA cDNA (UoC-M1/FA) reduced MMC resistance significantly (*P = .002). Neither FANCC nor FANCG transduction (UoC-M1/FC and UoC-M1/FG) significantly altered MMC sensitivity (P = .059 and P = .064, respectively). UoC-M1/FF demonstrated continued MMC sensitivity in 2 independent experiments with 38% and 26% radial formation in each (of 50 metaphases analyzed per experiment). Values shown are means ± SD. M. William Lensch et al. Blood 2003;102:7-16 ©2003 by American Society of Hematology

FANCA transduction enhances FA protein complex stability and restores nuclear FANCA levels. FANCA transduction enhances FA protein complex stability and restores nuclear FANCA levels. (A) Whole cell lysates. All 4 rows depict data obtained from the same filter, cut horizontally at 80 kDa to allow FANCA to be interrogated on the top portion and FANCC (then FANCG after stripping the FANCC signal) on the bottom portion. Retroviral transduction with each of 3 FA cDNA constructs resulted in increased levels of their respective proteins (the results from independently obtained duplicates are shown for each transduced cell line). Transduction with FANCA (UoC-M1/FA) also led to an increase in cellular levels of FANCG, whereas the reverse was not observed (ie, transduction with FANCG [UoC-M1/FG] did not increase cellular levels of FANCA). Row 4 is a constant, nonspecific fragment detected by the FANCG antibody and demonstrates equal loading in all lanes. (B) Nuclear extracts from 3 transduced lines (UoC-M1/FA, UoC-M1/FC, and UoC-M1/FG), and the myeloid cell line Mo7e probed with anti-FANCA (row 1) and anti-FANCG (row 2) antisera. Transduction with FANCA restored nuclear levels of FANCA in UoC-M1/FA (row 1, column 2), whereas transduction with FANCG did not increase nuclear levels of FANCG (UoC-M1/FG) (row 2, column 5). (C) Nuclear levels of FANCA and FANCG are not reduced in the lymphoblastoid cell line 4081. The cell line JY is an EBV-transformed lymphoblastoid cell line established from a healthy adult volunteer and also demonstrates cellular resistance to MMC. Lane 1 is a whole cell extract (WCL) from HeLa cells. M. William Lensch et al. Blood 2003;102:7-16 ©2003 by American Society of Hematology

Acquired dysfunction of FA proteins, a potential progression factor in secondary AML/MDS. Acquired dysfunction of FA proteins, a potential progression factor in secondary AML/MDS. We propose that FANCA dysfunction was acquired by an evolving clone of initiated hematopoietic stem cells. As shown on the right, an early FA-inactivating event would render the cell hypersensitive to apoptotic stimuli, resulting in apoptosis. However, as shown on the left, if a similar event occurred in an already initiated cell, the cell could resist apoptotic cues. The FA defect would then contribute to genetic instability and lead to multiple cytogenetic defects in the evolving clone. HSC indicates hematopoietic stem cell; FA, any FA protein or factor that enhances FA protein function. M. William Lensch et al. Blood 2003;102:7-16 ©2003 by American Society of Hematology