The D-type cyclin-dependent kinases CDK4 and CDK6 are complexed with many small cellular proteins (p14, plS, p16, plS, and p20). We have isolated cDNA sequences corresponding to the MTS2 genomic fragment that encodes the CDK4-and CDK6-associated p14 protein. By use of a yeast interaction screen to search for CDK6-interacting proteins, we have also identified an 18-kD human protein, p18, that is a homolog of the cyclin D-CDK4 inhibitors p16 {INK4A/MTS1) and p14 (MTS2/INK4B). Both in vivo and in vitro, p18 interacts strongly with CDK6, weakly with CDK4, and exhibits no detectable interaction with the other known CDKs. Recombinant p18 inhibits the kinase activity of cyclin D-CDK6. Distinct from the p21/p27 family of CDK inhibitors that form ternary complexes with cyclin-CDKs, only binary complexes of p14, p16, and p18 were found in association with CDK4 and/or CDK6. Ectopic expression of p18 or p16 suppresses cell growth with a correlated dependence on endogenous wild-type pRb.[Key Words: Cyclin-dependent kinase inhibitors; cell cycle; CDK4 and CDK6 interacting proteins]

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IntroductionThe myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem-cell disorders characterized by cytopenias and frequent leukemic progression. MDS constitutes a prototype of age-related malignancy, with a prevalence in the United States that may be more than 100 000. 1 Its incidence in the United States, estimated to be more than 10 000 yearly, is likely to further increase due to the greater life expectancy of the general population (http://www.census.gov/).Chromosomal aberrations can be detected by metaphase cytogenetics (MC) in approximately 50% of MDS patients and are responsible for some of the observed clinical diversity. Based on the experience that certain chromosomal lesions have a major impact on survival in MDS, 2-5 cytogenetic results were included in The International Prognostic Scoring System (IPSS), the most commonly applied prognostic algorithm for MDS. Moreover, recent studies demonstrate that MDS patients with certain cytogenetic abnormalities may be candidates for targeted therapies. For example, lenalidomide results in a high remission rate in MDS patients with 5q-abnormalities. 6,7 High-resolution single nucleotide polymorphisms arrays (SNP-A) can be applied in karyotypic analysis. SNP-A-based karyotyping does not depend upon the availability of live, dividing cells, and consequently can yield results when routine MC is not informative. Moreover, due to the higher resolution of SNP-A as compared with MC, smaller, previously cryptic deletions and duplications can be detected. A major advantage of this technology over MC is its ability to identify loss of heterozygosity (LOH) that occurs without concurrent changes in the gene copy number (CN). Such defects are consistent with acquired uniparental disomy (UPD) and can be attributed to errors in mitotic recombination occurring in somatic cells. Acquired segmental UPD is being increasingly recognized in a variety of neoplasms. 8,9 UPD has been described in chronic lymphocytic leukemia 10 and polycythemia vera as a mechanism leading to homozygosity for the Jak2 mutation. 11 Recently, an extensive study of acute lymphoblastic leukemia using SNP-A revealed chromosomal deletions and amplifications, many of them involving genes encoding principal regulators of B-lymphocyte development. 12 SNP-A also has been used for detecting genomic lesions in smaller case series of myeloma, 13 leukemias, 14-16 and lymphoma. 17 Initially using 50K arrays, we have demonstrated the potential diagnostic value of this technology, in a smaller cohort of myelodysplastic syndrome (MDS) patients. 18 This preliminary study demonstrated frequent detection of UPD in MDS. Subsequent larger studies limited to low-risk MDS showed similar results. 19 MDS is a particularly suitable target for demonstrating the use of SNP-A, as acquired cytogenetic abnormalities are relatively frequent and mostly unbalanced. 20 Using this disease as a model, we tested the hypothesis that high-density SNP-A can complement routine MC and enhance its diagnostic re...

as a candidate TSG on chromosome 7. In patients with chromosome deletion at the FZD9 locus, aberrant methylation of the remaining allele was associated with the poorest clinical outcome. These results indicate that aberrant methylation can cooperate with chromosome deletions to silence TSG. However, the ubiquity, extent, and correlation with disease progression suggest that aberrant DNA methylation is the dominant mechanism for TSG silencing and clonal variation in MDS evolution to AML. (Blood.

Two types of acquired loss of heterozygosity are possible in

We hypothesized that specific molecular mutations are important biomarkers for response to DNA methyltransferase inhibitors (DNMT inhibitors) and may have prognostic value in patients with myelodysplastic syndromes (MDS). Mutational analysis was performed in 92 patients with MDS and related disorders who received 5-azacytidine (n=55), decitabine (n=26) or both (n=11). Mutational status was correlated with overall response rate (ORR), progression-free survival (PFS) and overall survival (OS) by univariate and multivariate analysis. Risk stratification models were created. TET2, DNMT3A, IDH1/IDH2, ASXL1, CBL, RAS and SF3B1 mutations were found in 18, 9, 8, 26, 3, 2 and 13% of patients, respectively. In multivariate analysis, TET2(MUT) and/or DNMT3A(MUT) (P=0.03), platelets > or = 100 × 10(9)/l (P=0.007) and WBC<3.0 × 10(9)/l (P=0.03) were independent predictors of better response. TET2(MUT) and/or DNMT3A(MUT) (P=0.04) status was also independently prognostic for improved PFS, as were good or intermediate cytogenetic risk (P<0.0001), age<60 (P=0.0001), treatment with both 5-azacytidine and decitabine (P=0.02) and hemoglobin > or = 10 g/dl (P=0.01). Better OS was associated with ASXL1(WT) (P=0.008) and SF3B1(MUT) (P=0.01), and, similar to PFS, cytogenetic risk (P=0.0002), age (P=0.02) and hemoglobin (P=0.04). These data support the role of molecular mutations as predictive biomarkers for response and survival in MDS patients treated with DNMT inhibitors.

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