RepSox slows decay of CD34+ acute myeloid leukemia cells and decreases T cell immunoglobulin mucin-3 expression.

Despite initial response to therapy, most acute myeloid leukemia (AML) patients relapse. To eliminate relapse-causing leukemic stem/progenitor cells (LPCs), patient-specific immune therapies may be required. In vitro cellular engineering may require increasing the "stemness" or immunogenicity of tumor cells and activating or restoring cancer-impaired immune-effector and antigen-presenting cells. Leukapheresis samples provide the cells needed to engineer therapies: LPCs to be targeted, normal hematopoietic stem cells to be spared, and cancer-impaired immune cells to be repaired and activated. This study sought to advance development of LPC-targeted therapies by exploring nongenetic ways to slow the decay and to increase the immunogenicity of primary CD34(+) AML cells. CD34(+) AML cells generally displayed more colony-forming and aldehyde dehydrogenase activity than CD34(-) AML cells. Along with exposure to bone marrow stromal cells and low (1%-5%) oxygen, culture with RepSox (a reprogramming tool and inhibitor of transforming growth factor-ß receptor 1) consistently slowed decline of CD34(+) AML and myelodysplastic syndrome (MDS) cells. RepSox-treated AML cells displayed higher CD34, CXCL12, and MYC mRNA levels than dimethyl sulfoxide-treated controls. RepSox also accelerated loss of T cell immunoglobulin mucin-3 (Tim-3), an immune checkpoint receptor that impairs antitumor immunity, from the surface of AML and MDS cells. Our results suggest RepSox may reduce Tim-3 expression by inhibiting transforming growth factor-ß signaling and slow decay of CD34(+) AML cells by increasing CXCL12 and MYC, two factors that inhibit AML cell differentiation. By prolonging survival of CD34(+) AML cells and reducing Tim-3, RepSox may promote in vitro immune cell activation and advance development of LPC-targeted therapies.

c-MYC Amplification in Acute Myelogenous Leukemia Evolving from Double Minutes (dmins) to Homogeneously Staining Region (hsr).

A bone marrow biopsy of a 68-year-old woman revealed 59% blasts and immature monocytes, consistent with acute myeloid leukemia (AML) with monocytic features. Occasional hypolobated megakaryocytes and decreased iron stores were also present. A peripheral blood sample showed 7% blasts in addition to monocytosis, macrocytic anemia and thrombocytopenia. Molecular testing was negative for FLT3-ITD, NPM1 and CEBPA. Fluorescence in situ hybridization (FISH) probes were negative for t(8;21), t(15;17), inversion 16 and 11q23 rearrangements. The karyotype was 46,XX,del(20)(q11.2q13.1),~50dmin[3]/47,idem,+4[13]/47,idem,+22[2]/46,XX[2]. FISH confirmed that the double minutes were c-MYC positive with cryptic deletion of the c-MYC FISH signal on one of the chromosome 8s. Two months post-diagnosis, 57% of our patient's cells were still positive for c-MYC amplification; however, by four months only 8% of cells were positive for c-MYC amplification. After seven months, the patient's karyotype had the 20q deletion, X chromosome loss and a ring chromosome that consisted of a homogeneously staining region (hsr) containing c-MYC amplification. This case demonstrates that gene amplification in the form of double minutes can transform into a more stable hsr.

Patterns of BCR/ABL Gene Rearrangements in Chronic Myeloid Leukemia with Complex t(9;22) Using Fluorescence In Situ Hybridization (FISH).

Chronic myeloid leukemia (CML) is characterized by the reciprocal translocation t(9;22)(q34;q11.2) which fuses the ABL1 oncogene on chromosome 9 with the BCR gene on chromosome 22. It is the BCR/ABL protein that drives the neoplasm and the ABL/BCR is not necessary for the disease. In the majority of CML cases, the BCR/ABL fusion gene is cytogenetically recognizable as a small derivative chromosome 22(der 22), which is known as the Philadelphia (Ph) chromosome. However, approximately 2-10% of patients with CML involve cryptic or complex variant translocations with deletions on the der(9) and/or der(22) occuring in roughly 10-15% of CML cases. Fluorescence in situ hybridization (FISH) analysis can help identify deletions and complex or cryptic rearrangements. Various BCR/ABL FISH probes are available, which include dual color single fusion, dual color extra signal (ES), dual color dual fusion and tri color dual fusion probes. To test the utility of these probes, six patients diagnosed with CML carrying different complex variant Ph translocations were studied by G-banding and FISH analysis using the BCR/ABL ES, BCR/ABL dual color dual fusion, and BCR/ABL tricolor probes. There are differences among the probes in their ability to detect variant rearrangements, with or without accompanying chromoso me 9 and/or 22 deletions, and low level disease.

Chromosomal changes in high- and low-invasive mouse lung adenocarcinoma cell strains derived from early passage mouse lung adenocarcinoma cell strains.

The incidence of adenocarcinoma of the lung is increasing in the United States, however, the difficulties in obtaining lung cancer families and representative samples of early to late stages of the disease have lead to the study of mouse models for lung cancer. We used Spectral Karyotyping (SKY), mapping with fluorescently labeled genomic clones (FISH), comparative genomic hybridization (CGH) arrays, gene expression arrays, Western immunoblot and real time polymerase chain reaction (PCR) to analyze nine pairs of high-invasive and low-invasive tumor cell strains derived from early passage mouse lung adenocarcinoma cells to detect molecular changes associated with tumor invasion. The duplication of chromosomes 1 and 15 and deletion of chromosome 8 were significantly associated with a high-invasive phenotype. The duplication of chromosome 1 at band C4 and E1/2-H1 were the most significant chromosomal changes in the high-invasive cell strains. Mapping with FISH and CGH array further narrowed the minimum region of duplication of chromosome 1 to 71-82 centimorgans (cM). Expression array analysis and confirmation by real time PCR demonstrated increased expression of COX-2, Translin (TB-RBP), DYRK3, NUCKS and Tubulin-alpha4 genes in the high-invasive cell strains. Elevated expression and copy number of these genes, which are involved in inflammation, cell movement, proliferation, inhibition of apoptosis and telomere elongation, were associated with an invasive phenotype. Similar linkage groups are altered in invasive human lung adenocarcinoma, implying that the mouse is a valid genetic model for the study of the progression of human lung adenocarcinoma.

Increased gene copy number of the transcription factor E2F1 in malignant melanoma.

Translocations and unique chromosome break points in melanoma will aid in the identification of the genes that are important in the neoplastic process. We have previously shown a unique translocation in malignant melanoma cells der(12)t(12;20). The transcription factor E2F1 maps to 20q11. Increased expression of E2F has been associated with the autonomous growth of melanoma cells, however, the molecular basis has not yet been elucidated. To this end, we investigated E2F1 gene copy number and structure in human melanoma cell lines and metastatic melanoma cases. Fluorescent in situ hybridization (FISH) analysis using a specific E2F1 probe indicated increased E2F1 gene copies in melanoma cell lines compared to normal melanocytes. We also observed increased copies of the E2F1 gene in lymph node metastases of melanoma. In addition, Western blot analysis demonstrated increased E2F1 protein levels in 8 out of 9 melanoma cell lines relative to normal melanocytes. Inhibition of E2F1 expression with RNAi also reduced melanoma cell growth. Our results suggest that the release of E2F activity by elevated E2F1 gene copy numbers may play a functional role in melanoma growth.

Comparison of established cell lines at different passages by karyotype and comparative genomic hybridization.

Two established cancer cell lines, MCF-7 and Ishikawa, were both obtained directly from a cell repository and through another laboratory. The karyotypes from the two MCF-7 cell lines had up to 83 chromosomes and similarities for chromosomal gain and structural abnormalities. The two Ishikawa cell lines had up to 60 chromosomes with only a missing X as the common chromosome abnormality. CGH studies were performed by co-hybridizing the two Ishikawa or MCF-7 cell lines to normal metaphases. The differences seen between the two MCF-7 cell cultures reflect changes due to passage number and culture conditions. For Ishikawa, DNA polymorphic data and mutation studies suggest that the two cell lines are not derived from the same established tumor cell line. Our study shows the utilization of CGH in comparing cell lines originating from the same specimen. Our study also demonstrates the necessity for periodically evaluating cell lines to confirm their origin.

Specific chromosomal aberrations in mouse lung adenocarcinoma cell lines detected by spectral karyotyping: a comparison with human lung adenocarcinoma.

Although adenocarcinoma is rapidly becoming the most common form of lung cancer in the United States, the difficulty in obtaining lung cancer families and representative samples of the various stages of adenocarcinoma progression has led to intense study of mouse models. As a powerful approach to delineating molecular changes, we have analyzed 15 early-passage mouse cell lines by spectral karyotyping. Entire copies of chromosomes 1, 2, 6, 12, 15, and 19 were gained, and entire copies of chromosomes 4, 7, 8, and 14 were lost. Significant gains of portions of chromosome 1 (93% of the tumor cell lines analyzed), chromosome 2 (53%), chromosome 6 (73%), chromosome 7 (80%), chromosome 12 (47%), and chromosome 15 (73%) and partial loss of chromosome 4 (87%), chromosome 7 (80%), chromosome 8 (53%), chromosome 10 (33%), and chromosome 14 (33%) were observed. Recurrent translocations included 10:del(10)(A1::C1), t(4;8)(C4;A1), and der (1;12)(C2;C2). The minimal regions of chromosomal alteration, 1G1, 2F1, 4C4, 6D, 7F1, 8B3, and 12C2, contain putative susceptibility genes for mouse lung adenocarcinoma. Chromosomal regions containing susceptibility genes linked to tumor size were frequently amplified, whereas regions with susceptibility loci linked to tumor multiplicity were deleted. Similar linkage groups are altered in human lung adenocarcinoma, implying that the mouse is a valid genetic model for the study of human lung adenocarcinoma susceptibility.