HDAC3 functions as a positive regulator in Notch signal transduction.

Aberrant Notch signaling plays a pivotal role in T-cell acute lymphoblastic leukemia (T-ALL) and chronic lymphocytic leukemia (CLL). Amplitude and duration of the Notch response is controlled by ubiquitin-dependent proteasomal degradation of the Notch1 intracellular domain (NICD1), a hallmark of the leukemogenic process. Here, we show that HDAC3 controls NICD1 acetylation levels directly affecting NICD1 protein stability. Either genetic loss-of-function of HDAC3 or nanomolar concentrations of HDAC inhibitor apicidin lead to downregulation of Notch target genes accompanied by a local reduction of histone acetylation. Importantly, an HDAC3-insensitive NICD1 mutant is more stable but biologically less active. Collectively, these data show a new HDAC3- and acetylation-dependent mechanism that may be exploited to treat Notch1-dependent leukemias.

Oxidative stress as candidate therapeutic target to overcome microenvironmental protection of CLL.

Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental non-malignant cells for survival. We compared the transcriptomes of primary CLL cells cocultured or not with protective bone marrow stromal cells (BMSCs) and found that oxidative phosphorylation, mitochondrial function, and hypoxic signaling undergo most significant dysregulation in non-protected CLL cells, with the changes peaking at 6-8 h, directly before induction of apoptosis. A subset of CLL patients displayed a gene expression signature resembling that of cocultured CLL cells and had significantly worse progression-free and overall survival. To identify drugs blocking BMSC-mediated support, we compared the relevant transcriptomic changes to the Connectivity Map database. Correlation was found with the transcriptomic signatures of the cardiac glycoside ouabain and of the ipecac alkaloids emetine and cephaeline. These compounds were highly active against protected primary CLL cells (relative IC50's 287, 190, and 35 nM, respectively) and acted by repressing HIF-1α and disturbing intracellular redox homeostasis. We tested emetine in a murine model of CLL and observed decreased CLL cells in peripheral blood, spleen, and bone marrow, recovery of hematological parameters and doubling of median survival (31.5 vs. 15 days, P = 0.0001). Pathways regulating redox homeostasis are thus therapeutically targetable mediators of microenvironmental support in CLL cells.

Dissecting the Prognostic Significance and Functional Role of Progranulin in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is known for its strong dependency on the tumor microenvironment. We found progranulin (GRN), a protein that has been linked to inflammation and cancer, to be upregulated in the serum of CLL patients compared to healthy controls, and increased GRN levels to be associated with an increased hazard for disease progression and death. This raised the question of whether GRN is a functional driver of CLL. We observed that recombinant GRN did not directly affect viability, activation, or proliferation of primary CLL cells in vitro. However, GRN secretion was induced in co-cultures of CLL cells with stromal cells that enhanced CLL cell survival. Gene expression profiling and protein analyses revealed that primary mesenchymal stromal cells (MSCs) in co-culture with CLL cells acquire a cancer-associated fibroblast-like phenotype. Despite its upregulation in the co-cultures, GRN treatment of MSCs did not mimic this effect. To test the relevance of GRN for CLL in vivo, we made use of the Eµ-TCL1 CLL mouse model. As we detected strong GRN expression in myeloid cells, we performed adoptive transfer of Eµ-TCL1 leukemia cells to bone marrow chimeric Grn-/- mice that lack GRN in hematopoietic cells. Thereby, we observed that CLL-like disease developed comparable in Grn-/- chimeras and respective control mice. In conclusion, serum GRN is found to be strongly upregulated in CLL, which indicates potential use as a prognostic marker, but there is no evidence that elevated GRN functionally drives the disease.

FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL.

NOTCH1 is mutated in 10% of chronic lymphocytic leukemia (CLL) patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately one-half of CLL cases even in the absence of NOTCH1 mutations. Hence, there appear to be additional factors responsible for the impairment of NOTCH1 degradation. E3-ubiquitin ligase F-box and WD40 repeat domain containing-7 (FBXW7), a negative regulator of NOTCH1, is mutated in 2% to 6% of CLL patients. The functional consequences of these mutations in CLL are unknown. We found heterozygous FBXW7 mutations in 36 of 905 (4%) untreated CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain; 10% of mutations occurred in the first exon of the α-isoform. To identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in CLL cell line HG-3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9). Homozygous truncation of FBXW7 resulted in an increase of activated NOTCH1 intracellular domain (NICD) and c-MYC protein levels as well as elevated hypoxia-inducible factor 1-α activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates. This differential binding was confirmed via coimmunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary CLL cells harboring FBXW7 mutations, activated NICD levels were increased and remained stable upon translation inhibition. FBXW7 mutations coincided with an increase in NOTCH1 target gene expression and explain a proportion of patients characterized by dysregulated NOTCH1 signaling.