Complementary activities of DOT1L and Menin inhibitors in MLL-rearranged leukemia.

Chromosomal rearrangements of the mixed lineage leukemia (MLL/KMT2A) gene leading to oncogenic MLL-fusion proteins occur in ~10% of acute leukemias and are associated with poor clinical outcomes, emphasizing the need for new treatment modalities. Inhibition of the DOT1-like histone H3K79 methyltransferase (DOT1L) is a specific therapeutic approach for such leukemias that is currently being tested in clinical trials. However, in most MLL-rearranged leukemia models responses to DOT1L inhibitors are limited. Here, we performed deep-coverage short hairpin RNA sensitizer screens in DOT1L inhibitor-treated MLL-rearranged leukemia cell lines and discovered that targeting additional nodes of MLL complexes concomitantly with DOT1L inhibition bears great potential for superior therapeutic results. Most notably, combination of a DOT1L inhibitor with an inhibitor of the MLL-Menin interaction markedly enhanced induction of differentiation and cell killing in various MLL disease models including primary leukemia cells, while sparing normal hematopoiesis and leukemias without MLL rearrangements. Gene expression analysis on human and murine leukemic cells revealed that target genes of MLL-fusion proteins and MYC were suppressed more profoundly upon combination treatment. Our findings provide a strong rationale for a novel targeted combination therapy that is expected to improve therapeutic outcomes in patients with MLL-rearranged leukemia.

Using in silico transcriptomics to search for tumor-associated antigens for immunotherapy.

Immunotherapy approaches to fight cancer are based on the principle of mounting an immune response against a self-antigen expressed by the tumor cells. In order to reduce potential autoimmunity side-effects, the antigens used should be as tumor-specific as possible. A complementary approach to experimental tumor antigen discovery is to screen the human genome in silico, particularly the databases of "Expressed Sequence Tags" (ESTs), in search of tumor-specific and tumor-associated antigens. The public databases currently provide a massive amount of ESTs from several hundreds of cDNA tissue libraries, including tumoral tissues from various types. We describe a novel method of EST database screening that allows new potential tumor-associated genes to be efficiently selected. The resulting list of candidates is enriched in known genes, described as being expressed in tumor cells.

The ectodomain of the Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients.

Mutations in Notch3 cause CADASIL (cerebral autosomal dominant adult onset arteriopathy), which leads to stroke and dementia in humans. CADASIL arteriopathy is characterized by major alterations of vascular smooth muscle cells and the presence of specific granular osmiophilic deposits. Patients carry highly stereotyped mutations that lead to an odd number of cysteine residues within EGF-like repeats of the Notch3 receptor extracellular domain. Such mutations may alter the processing or the trafficking of this receptor, or may favor its oligomerization. In this study, we examined the Notch3 expression pattern in normal tissues and investigated the consequences of mutations on Notch3 expression in transfected cells and CADASIL brains. In normal tissues, Notch3 expression is restricted to vascular smooth muscle cells. Notch3 undergoes a proteolytic cleavage leading to a 210-kDa extracellular fragment and a 97-kDa intracellular fragment. In CADASIL brains, we found evidence of a dramatic and selective accumulation of the 210-kDa Notch3 cleavage product. Notch3 accumulates at the cytoplasmic membrane of vascular smooth muscle cells, in close vicinity to but not within the granular osmiophilic material. These results strongly suggest that CADASIL mutations specifically impair the clearance of the Notch3 ectodomain, but not the cytosolic domain, from the cell surface.

Demethylation of DNA by purified chick embryo 5-methylcytosine-DNA glycosylase requires both protein and RNA.

We have previously purified and characterized a 5-methylcytosine (5-MeC)-DNA glycosylase from 12 day old chick embryos [Jost,J.P. et al. (1995) J. Biol. Chem. 270, 9734-9739]. The activity of the purified enzyme is abolished upon treatment with proteinase K and ribonuclease A. RNA copurifies with 5-MeC-DNA glycosylase activity throughout all chromatographic steps and preparative gel electrophoresis. RNA with a length of approximately 300-500 nucleotides was isolated from the gel purified enzyme. Upon extensive treatment with proteinase K, the gel eluted and labeled RNA did not show any significant change in molecular mass. The purified RNA incubated alone or in the presence of Mg2+and deoxyribonucleotide phosphates had no 5-MeC-DNA glycosylase or demethylating activities. However, activity of 5-MeC-DNA glycosylase could be restored when the purified RNA was incubated with the inactive protein, free of RNA.