Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.

To identify molecularly defined subgroups in multiple myeloma, gene expression profiling was performed on purified CD138 ؉ plasma cells of 320 newly diagnosed myeloma patients included in the DutchBelgian/German HOVON-65/GMMG-HD4 trial. Hierarchical clustering identified 10 subgroups; 6 corresponded to clusters described in the University of Arkansas for Medical Science (UAMS) classification, CD-1 (n ‫؍‬ 13, 4.1%), CD-2 (n ‫؍‬ 34, 1.6%), MF (n ‫؍‬ 32, 1.0%), MS (n ‫؍‬ 33, 1.3%), proliferation-associated genes (n ‫؍‬ 15, 4.7%), and hyperdiploid (n ‫؍‬ 77, 24.1%). Moreover, the UAMS low percentage of bone disease cluster was identified as a subcluster of the MF cluster (n ‫؍‬ 15, 4.7%). One subgroup (n ‫؍‬ 39, 12.2%) showed a myeloid signature. Three novel subgroups were defined, including a subgroup of 37 patients (11.6%) characterized by high expression of genes involved in the nuclear factor kappa lightchain-enhancer of activated B cells pathway, which include TNFAIP3 and CD40. Another subgroup of 22 patients (6.9%) was characterized by distinct overexpression of cancer testis antigens without overexpression of proliferation genes. The third novel cluster of 9 patients (2.8%) showed upregulation of protein tyrosine phosphatases PRL-3 and PTPRZ1 as well as SOCS3. To conclude, in addition to 7 clusters described in the UAMS classification, we identified 3 novel subsets of multiple myeloma that may represent unique diagnostic entities.

The translocation (6;9) is associated with a specific subtype of acute myeloid leukemia (AML). Previously, it was found that breakpoints on chromosome 9 are clustered in one of the introns of a large gene named Cain (can). cDNA probes derived from the 3' part of can detect an aberrant, leukemia-specific 5.5-kb transcript in bone marrow cells from t(6;9) AML patients. cDNA cloning of this mRNA revealed that it is a fusion of sequences encoded on chromosome 6 and 3' can. A novel gene on chromosome 6 which was named dek was isolated. In dek the t(6;9) breakpoints also occur in one intron. As a result the dek-can fusion gene, present Defined karyotypic aberrations are associated with specific subtypes of leukemia. Detailed molecular characterization of these aberrations may identify genes involved in leukemogenesis and in the precise regulation of proliferation and differentiation in the hematopoietic system. Translocations are the best-studied chromosomal abnormalities. As the result of a translocation, the function or activity of oncogenes located at or near the translocation breakpoint is altered. In myeloid leukemia three translocation breakpoints have been cloned and analyzed at the molecular level.The two best studied, t(9;22) in chronic myeloid leukemia (27, 43) and t(15;17) in acute promyelocytic leukemia (2,8,12), result in the formation of chimeric genes that encode fusion proteins. In chronic myeloid leukemia this is a BCR-ABL protein that has an enhanced tyrosine kinase activity (34, 49) directly responsible for its in vivo tumorigenic potential (14,25). In acute promyelocytic leukemia a PMLRARa fusion protein that represents an altered transcription factor (16, 33) is found.The third translocation is the t(6;9) (p23;q34), found in a specific subtype of acute myeloid leukemia (AML) (1,39,41). This leukemia is characterized by a poor prognosis, affects young adults, and is classified mostly as M2 or M4 and rarely as Ml (according to the French-American-British classification of AML). A region on chromosome 9 situated 360 kb downstream of the c-abl gene was cloned and analyzed. It was found that breakpoints were clustered in a region of 8 kb in five patients, four with t(6;9) AML and one with acute undifferentiated leukemia (AUL) (47). Through cDNA cloning this region could be identified as one of the introns of a large gene (>100 kb) encoding a 7-kb transcript. This intron was named icb-9; the intron containing the breakpoints on chromosome 9 and situated in the middle of * Corresponding author. a gene named Cain (can). The 3' part of can is translocated to the 6p-chromosome, and only 3' can probes detect an additional, leukemia-specific 5.5-kb transcript in bone marrow cells from t(6;9) AML patients. No additional transcripts were detected with 5' can probes. The breakpoint region on chromosome 6p23 was isolated from a genomic XEMBL3 library constructed of bone marrow DNA from one of the t(6;9) patients. An area of 40 kb of chromosome 6 DNA was cloned in overlapping phages. Southern blot analysis sh...

The t(12;21) translocation is present in up to 30% of childhood B-cell acute lymphoblastic leukemias and fuses a potential dimerization motif from the ets-related factor TEL to the N terminus of AML1. The t(12;21) translocation encodes a 93-kDa fusion protein that localizes to a high-salt-and detergent-resistant nuclear compartment. This protein binds the enhancer core motif, TGTGGT, and interacts with the AML-1-binding protein, core-binding factor beta. Although TEL/AML-1B retains the C-terminal domain of AML-1B that is required for transactivation of the T-cell receptor beta enhancer, it fails to activate transcription but rather inhibits the basal activity of this enhancer. TEL/AML-1B efficiently interferes with AML-1B-dependent transactivation of the T-cell receptor beta enhancer, and coexpression of wild-type TEL does not reverse this inhibition. The N-terminal TEL helix-loop-helix domain is essential for TEL/AML-1B-mediated repression. Thus, the t(12;21) fusion protein dominantly interferes with AML-1B-dependent transcription, suggesting that the inhibition of expression of AML-1 target genes is critical for B-cell leukemogenesis.AML1 is one of the most frequently mutated genes in human leukemia and is targeted either directly or indirectly in t(8;21), t(3;21), t(12;21), and inv(16). t(8;21) and inv(16) are present in up to 30% of de novo acute myeloid leukemias (AML) and target the two components of the AML-1 transcription factor complex, AML-1 and core-binding factor beta (CBF␤). In the t(8;21) translocation, AML-1 sequences including the DNA binding domain are fused to ETO (MTG8), a gene encoding the human homolog of the Drosophila gene nervy (7,9,33,35). inv(16) fuses the non-DNA-binding factor CBF␤ to a smooth muscle myosin heavy-chain gene, MHY11 (25). t(3;21) is rare in de novo AML but is found in therapy-related AML and myelodysplasias and fuses AML-1 to three different genes, including the gene encoding the Evi-1 transcription regulator (32, 34, 36, 37, 44, 45). t(12;21) is present in up to 30% of childhood B-cell acute lymphoblastic leukemias and fuses a potential dimerization motif from the ets-related factor TEL to the N terminus of AML1 (11,12,42,43,47). Thus, AML-1 is altered in both myeloid and B-cell acute leukemias.Two independent lines of investigation identified AML1 as an important regulator of transcription. The cloning of the t(8;21) breakpoint led to the isolation of the AML-1 cDNA clone and the realization that AML-1 could be the human homolog of a Drosophila pair-rule protein encoded by runt (5, 7). Recombinant AML-1 proteins were used to demonstrate that the runt homology domain of AML-1 is responsible for

Interactions within the hematopoietic niche in the BM microenvironment are essential for maintenance of the stem cell pool. In addition, this niche is thought to serve as a sanctuary site for malignant progenitors during chemotherapy. Therapy resistance induced by interactions with the BM microenvironment is a major drawback in the treatment of hematologic malignancies and bone-metastasizing solid tumors. To date, studying these interactions was hampered by the lack of adequate in vivo models that simulate the human situation. In the present study, we describe a unique human-mouse hybrid model that allows engraftment and outgrowth of normal and malignant hematopoietic progenitors by implementing a technology for generating a human bone environment. Using luciferase gene marking of patient-derived multiple myeloma cells and bioluminescent imaging, we were able to follow pMM cells outgrowth and to visualize the effect of treatment. Therapeutic interventions in this model IntroductionIn the BM, specialized microenvironments such as hematopoietic niches regulate hematopoiesis. Within these niches, hematopoietic stem cells (HSCs) are present in a complex network consisting of mesenchymal stromal cells (MSCs), osteoblasts, osteoclasts, endothelial cells, and adipocytes embedded in an extracellular matrix. The bidirectional interactions with the hematopoietic niche are essential for HSC maintenance and function. [1][2][3][4] The BM niche is also thought to serve as a sanctuary site for leukemic stem cells (LSCs), which, in addition to their immortalizing genetic events, highly depend on interaction with the microenvironment to survive and proliferate. 5,6 Although the majority of leukemias initially respond to therapeutic intervention, relapse rates are high. [7][8][9] There is increasing evidence that the tumor niche plays a crucial role in the survival and drug resistance of LSCs. Interactions with the niche provide signals protecting the LSCs from apoptosis and eventually leading to the selection and outgrowth of a resistant cell. 10-13 Therefore, it is apparent that the hematopoietic niche plays an important role in hematopoietic development and in chemotherapy resistance of BM-localized leukemic and solid tumors.Although our understanding of how the BM niche regulates HSC self-renewal and confers therapy resistance has advanced greatly over the past years, most of this knowledge is based on genetic loss-of-function or gain-of-function murine models. 1,2,10,11,14 However, these murine models do not simulate human physiology and much of the constituents of the human hematopoietic niche remain largely unclear. [14][15][16] This emphasizes the need for more suitable models that recapitulate the human BM microenvironment and, very importantly, facilitate the engraftment and outgrowth of normal HSCs and patient-derived tumor cells within these protected sites.In the present study, we describe a unique humanized model that implements a novel scaffold-based technology for generating a human bone environment in RAG 2 Ϫ/Ϫ ...

Rearrangement of the TEL gene distinguishes a large subset of children with favorable-prognosis B-precursor leukemia who cannot be identified by standard prognostic features. It may be possible to treat these patients less aggressively without loss of therapeutic efficacy.

Hereditary mutations associated with hematologic malignancies are rare. Heterozygous mutations affecting the hematopoietic transcription factor CBFA2 (also AML1/RUNX1) were recently reported to be associated with familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML, MIM 601399). A new 3-generation family with FPD/AML with a novel CBFA2 mutation is described. In this family, AML was diagnosed in a second-generation male. After allogeneic stem cell transplantation from his human leukocyte antigen-identical sister, a donor-derived, genetically identical leukemia developed in the recipient and the donor. Sequencing analysis identified a G-to-T transition within the CBFA2 gene, which involves codon 198, encoding a conserved aspartic acid within the DNA- IntroductionHereditary mutations are rare in hematologic malignancies. Generally, somatic mutations are associated with leukemogenesis. The hematopoietic transcription factor CBFA2/AML1/RUNX1 is frequently affected in leukemia. Chromosomal translocations (8;21), resulting in a fusion of CBFA2 and ETO, and t(12;21), resulting in a chimerical TEL-CBFA2 gene, are detected in 15% and 25% of acute myeloid leukemia (AML) and childhood acute lymphoid leukemia (ALL), respectively. Recently, mutations in the DNA binding Runt domain of CBFA2 have been reported in hematologic (pre-) malignancies of the myeloid lineage. [1][2][3][4] Familial platelet disorder (FPD) with predisposition to AML (FPD/ AML, MIM 601399) is an autosomal dominant disorder characterized by thrombocytopenia, functional platelet abnormalities, and prolonged bleeding time, and it is associated with predisposition to AML. 5 Recently, Gilliland and colleagues 7 reported haploinsufficiency of CBFA2 in 6 families with familial thrombocytopenia and propensity to the development of AML. Genetic analyses revealed heterozygous nonsense and missense mutations or intragenic deletions in one allele of the CBFA2 gene. These studies support a model for FPD/AML in which haploinsufficiency of CBFA2 is causal in leukemogenesis.We present a newly identified family with FPD/AML, and we describe the clinical, cytogenetic and molecular features of this family harboring a novel missense mutation within the Runt domain. Our data demonstrate the necessity of performing sequence analysis of the CBFA2 gene in families with FPD/AML. Study designMaterials were collected after informed consent of the patients. Leukocytederived genomic DNA was used to amplify exons 3, 4, and 5 and flanking splice sites of the CBFA2 gene by polymerase chain reaction and primers as described. 2 Automated DNA sequence analysis was performed with the ABI Prism dRhodamine Terminator Cycle Sequence Ready Reaction kit (PE Biosystems, Warrington, United Kingdom), and samples were analyzed on an ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Interphase fluorescence in situ hybridization (FISH) was performed using chromosome 21 probe LSI21 (Vysis, Downers Grove, IL). Results and discussionThe pedigree of the family w...

Key Points• Higher expression of the cereblon gene is associated with better outcome in newly diagnosed multiple myeloma patients treated with thalidomide maintenance.