DNA index (DI) and percentages of cells in S and G2/M phase were determined in Feulgen stained nuclei of blasts from 31 cases of childhood ALL at diagnosis. In 6 cases the results of DNA analysis and ...cytogenetics were concordant showing hyperdiploidy. Two other cases with normal karyotype were revealed as DNA aneuploid with image analysis. Cases with cytogenetic abnormalities like translocation, deletion or presence of single or double supernumerary chromosomes had DI within normal ranges. Nine ALL cases (29%) were found to be DNA aneuploid--8 hyperdiploid and 1 hypodiploid. The percentages of cells in S and G2/M phase for blasts from bone marrow (mean 17.6%) were significantly higher than those estimated in the peripheral blood (mean 1.57%). We conclude that analysis by image cytometry can detect aneuploid DNA content even in cases, which showed a normal karyotype and provides new information concerning the biological aspects of leukemic blasts.
Burkitt lymphoma is characterized by deregulation of MYC, but the contribution of other genetic mutations to the disease is largely unknown. Here, we describe the first completely sequenced genome ...from a Burkitt lymphoma tumor and germline DNA from the same affected individual. We further sequenced the exomes of 59 Burkitt lymphoma tumors and compared them to sequenced exomes from 94 diffuse large B-cell lymphoma (DLBCL) tumors. We identified 70 genes that were recurrently mutated in Burkitt lymphomas, including ID3, GNA13, RET, PIK3R1 and the SWI/SNF genes ARID1A and SMARCA4. Our data implicate a number of genes in cancer for the first time, including CCT6B, SALL3, FTCD and PC. ID3 mutations occurred in 34% of Burkitt lymphomas and not in DLBCLs. We show experimentally that ID3 mutations promote cell cycle progression and proliferation. Our work thus elucidates commonly occurring gene-coding mutations in Burkitt lymphoma and implicates ID3 as a new tumor suppressor gene.
The increasing characterization of childhood acute lymphoblastic leukemia (ALL) has led to the identification of multiple molecular targets but has yet to translate into more effective targeted ...therapies, particularly for high-risk, relapsed T-cell ALL. Searching for master regulators controlling multiple signaling pathways in T-ALL, we investigated the multifunctional protein redox factor-1 (Ref-1/APE1), which acts as a signaling "node" by exerting redox regulatory control of transcription factors important in leukemia. Leukemia patients' transcriptome databases showed increased expression in T-ALL of Ref-1 and other genes of the Ref-1/SET interactome. Validation studies demonstrated that Ref-1 is expressed in high-risk leukemia T cells, including in patient biopsies. Ref-1 redox function is active in leukemia T cells, regulating the Ref-1 target NF-κB, and inhibited by the redox-selective Ref-1 inhibitor E3330. Ref-1 expression is not regulated by Notch signaling, but is upregulated by glucocorticoid treatment. E3330 disrupted Ref-1 redox activity in functional studies and resulted in marked inhibition of leukemia cell viability, including T-ALL lines representing different genotypes and risk groups. Potent leukemia cell inhibition was seen in primary cells from ALL patients, relapsed and glucocorticoid-resistant T-ALL cells, and cells from a murine model of Notch-induced leukemia. Ref-1 redox inhibition triggered leukemia cell apoptosis and downregulation of survival genes regulated by Ref-1 targets. For the first time, this work identifies Ref-1 as a novel molecular effector in T-ALL and demonstrates that Ref-1 redox inhibition results in potent inhibition of leukemia T cells, including relapsed T-ALL. These data also support E3330 as a specific Ref-1 small-molecule inhibitor for leukemia.
.
Hepatosplenic T-cell lymphoma (HSTL) is a rare and lethal lymphoma; the genetic drivers of this disease are unknown. Through whole-exome sequencing of 68 HSTLs, we define recurrently mutated driver ...genes and copy-number alterations in the disease. Chromatin-modifying genes, including
, and
, were commonly mutated in HSTL, affecting 62% of cases. HSTLs manifest frequent mutations in
(31%),
(9%), and
(9%), for which there currently exist potential targeted therapies. In addition, we noted less frequent events in
, and
was the most frequently silenced gene in HSTL. We experimentally demonstrated that
acts as a tumor suppressor gene. In addition, we found that mutations in
and
activate critical signaling pathways important to cell survival in HSTL. Our work thus defines the genetic landscape of HSTL and implicates gene mutations linked to HSTL pathogenesis and potential treatment targets.
We report the first systematic application of whole-exome sequencing to define the genetic basis of HSTL, a rare but lethal disease. Our work defines
as a tumor suppressor gene in HSTL and implicates genes including
and
in the disease.
.
A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in ...any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-β pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions.
Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier ...to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole-exome sequencing and transcriptome sequencing in a cohort of 1,001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.
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•Exome sequencing in 1,001 DLBCL patients comprehensively identifies 150 driver genes•Unbiased CRISPR screen in DLBCL cell lines identifies essential oncogenes•Integrative analysis connects genomics, CRISPR hits, and clinical outcome•A genomic risk model of survival outperforms existing risk-assessment methods
An integrative analysis in 1,001 newly diagnosed DLBCL patients identifies 150 genetic drivers with functional characterization using an unbiased CRISPR screen in DLBCL cell lines and connects with clinical outcome.
The flexibility in the structure of calmodulin (CaM) allows its binding to over 300 target proteins in the cell. To investigate the structure-function relationship of CaM, we combined methods of ...computer simulation and experiments based on circular dichroism (CD) to investigate the structural characteristics of CaM that influence its target recognition in crowded cell-like conditions. We developed a unique multiscale solution of charges computed from quantum chemistry, together with protein reconstruction, coarse-grained molecular simulations, and statistical physics, to represent the charge distribution in the transition from apoCaM to holoCaM upon calcium binding. Computationally, we found that increased levels of macromolecular crowding, in addition to calcium binding and ionic strength typical of that found inside cells, can impact the conformation, helicity and the EF hand orientation of CaM. Because EF hand orientation impacts the affinity of calcium binding and the specificity of CaM's target selection, our results may provide unique insight into understanding the promiscuous behavior of calmodulin in target selection inside cells.
Fanconi anemia (FA) is a disease of genomic instability and cancer. In addition to DNA damage repair, FA pathway proteins are now known to be critical for maintaining faithful chromosome segregation ...during mitosis. While impaired DNA damage repair has been studied extensively in FA-associated carcinogenesis
in vivo
, the oncogenic contribution of mitotic abnormalities secondary to FA pathway deficiency remains incompletely understood. To examine the role of mitotic dysregulation in FA pathway deficient malignancies, we genetically exacerbated the baseline mitotic defect in
Fancc-/-
mice by introducing heterozygosity of the key spindle assembly checkpoint regulator
Mad2
.
Fancc-/-;Mad2+/-
mice were viable, but died from acute myeloid leukemia (AML), thus recapitulating the high risk of myeloid malignancies in FA patients better than
Fancc-/-
mice. We utilized hematopoietic stem cell transplantation to propagate
Fancc-/-; Mad2+/-
AML in irradiated healthy mice to model
FANCC
-deficient AMLs arising in the non-FA population. Compared to cells from
Fancc-/-
mice, those from
Fancc-/-;Mad2+/-
mice demonstrated an increase in mitotic errors but equivalent DNA cross-linker hypersensitivity, indicating that the cancer phenotype of
Fancc-/-;Mad2+/-
mice results from error-prone cell division and not exacerbation of the DNA damage repair defect. We found that FANCC enhances targeting of endogenous MAD2 to prometaphase kinetochores, suggesting a mechanism for how FANCC-dependent regulation of the spindle assembly checkpoint prevents chromosome mis-segregation. Whole-exome sequencing revealed similarities between human FA-associated myelodysplastic syndrome (MDS)/AML and the AML that developed in
Fancc-/-; Mad2+/-
mice. Together, these data illuminate the role of mitotic dysregulation in FA-pathway deficient malignancies
in vivo
, show how FANCC adjusts the spindle assembly checkpoint rheostat by regulating MAD2 kinetochore targeting in cell cycle-dependent manner, and establish two new mouse models for preclinical studies of AML.
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