Next-generation NAMPT inhibitors identified by sequential high-throughput phenotypic chemical and functional genomic screens.

Phenotypic high-throughput chemical screens allow for discovery of small molecules that modulate complex phenotypes and provide lead compounds for novel therapies; however, identification of the mechanistically relevant targets remains a major experimental challenge. We report the application of sequential unbiased high-throughput chemical and ultracomplex small hairpin RNA (shRNA) screens to identify a distinctive class of inhibitors that target nicotinamide phosphoribosyl transferase (NAMPT), a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide, a crucial cofactor in many biochemical processes. The lead compound STF-118804 is a highly specific NAMPT inhibitor, improves survival in an orthotopic xenotransplant model of high-risk acute lymphoblastic leukemia, and targets leukemia stem cells. Tandem high-throughput screening using chemical and ultracomplex shRNA libraries, therefore, provides a rapid chemical genetics approach for seamless progression from small-molecule lead identification to target discovery and validation.

Runx1 dose-dependently regulates endochondral ossification during skeletal development and fracture healing.

Runx1 is expressed in skeletal elements, but its role in fracture repair has not been analyzed. We created mice with a hypomorphic Runx1 allele (Runx1(L148A) ) and generated Runx1(L148A/-) mice in which >50% of Runx1 activity was abrogated. Runx1(L148A/-) mice were viable but runted. Their growth plates had extended proliferating and hypertrophic zones, and the percentages of Sox9-, Runx2-, and Runx3-positive cells were decreased. Femoral fracture experiments revealed delayed cartilaginous callus formation, and the expression of chondrogenic markers was decreased. Conditional ablation of Runx1 in the mesenchymal progenitor cells of the limb with Prx1-Cre conferred no obvious limb phenotype; however, cartilaginous callus formation was delayed following fracture. Embryonic limb bud-derived mesenchymal cells showed delayed chondrogenesis when the Runx1 allele was deleted ex vivo with adenoviral-expressed Cre. Collectively, our data suggest that Runx1 is required for commitment and differentiation of chondroprogenitor cells into the chondrogenic lineage.

Hierarchical maintenance of MLL myeloid leukemia stem cells employs a transcriptional program shared with embryonic rather than adult stem cells.

The genetic programs that promote retention of self-renewing leukemia stem cells (LSCs) at the apex of cellular hierarchies in acute myeloid leukemia (AML) are not known. In a mouse model of human AML, LSCs exhibit variable frequencies that correlate with the initiating MLL oncogene and are maintained in a self-renewing state by a transcriptional subprogram more akin to that of embryonic stem cells (ESCs) than to that of adult stem cells. The transcription/chromatin regulatory factors Myb, Hmgb3, and Cbx5 are critical components of the program and suffice for Hoxa/Meis-independent immortalization of myeloid progenitors when coexpressed, establishing the cooperative and essential role of an ESC-like LSC maintenance program ancillary to the leukemia-initiating MLL/Hox/Meis program. Enriched expression of LSC maintenance and ESC-like program genes in normal myeloid progenitors and poor-prognosis human malignancies links the frequency of aberrantly self-renewing progenitor-like cancer stem cells (CSCs) to prognosis in human cancer.

Disease mutations in RUNX1 and RUNX2 create nonfunctional, dominant-negative, or hypomorphic alleles.

Monoallelic RUNX1 mutations cause familial platelet disorder with predisposition for acute myelogenous leukemia (FPD/AML). Sporadic mono- and biallelic mutations are found at high frequencies in AML M0, in radiation-associated and therapy-related myelodysplastic syndrome and AML, and in isolated cases of AML M2, M5a, M3 relapse, and chronic myelogenous leukemia in blast phase. Mutations in RUNX2 cause the inherited skeletal disorder cleidocranial dysplasia (CCD). Most hematopoietic missense mutations in Runx1 involve DNA-contacting residues in the Runt domain, whereas the majority of CCD mutations in Runx2 are predicted to impair CBFbeta binding or the Runt domain structure. We introduced different classes of missense mutations into Runx1 and characterized their effects on DNA and CBFbeta binding by the Runt domain, and on Runx1 function in vivo. Mutations involving DNA-contacting residues severely inactivate Runx1 function, whereas mutations that affect CBFbeta binding but not DNA binding result in hypomorphic alleles. We conclude that hypomorphic RUNX2 alleles can cause CCD, whereas hematopoietic disease requires more severely inactivating RUNX1 mutations.

Runx1 is expressed in adult mouse hematopoietic stem cells and differentiating myeloid and lymphoid cells, but not in maturing erythroid cells.

The transcription factor Runx1 marks all functional hematopoietic stem cells (HSCs) in the embryo and is required for their generation. Mutations in Runx1 are found in approximately 25% of acute leukemias and in familial platelet disorder, suggesting a role for Runx1 in adult hematopoiesis as well. A comprehensive analysis of Runx1 expression in adult hematopoiesis is lacking. Here we show that Runx1 is expressed in functional HSCs in the adult mouse, as well as in cells with spleen colony-forming unit (CFU) and culture CFU capacities. Additionally, we document Runx1 expression in all hematopoietic lineages at the single cell level. Runx1 is expressed in the majority of myeloid cells and in a smaller proportion of lymphoid cells. Runx1 expression substantially decreases during erythroid differentiation. We also document effects of reduced Runx1 levels on adult hematopoiesis.

Energetic contribution of residues in the Runx1 Runt domain to DNA binding.

Core-binding factors (CBFs) are a small family of heterodimeric transcription factors that play critical roles in hematopoiesis and in the development of bone, stomach epithelium, and proprioceptive neurons. Mutations in CBF genes are found in leukemias, bone disorders, and gastric cancer. CBFs consist of a DNA-binding CBF alpha subunit and a non-DNA-binding CBF beta subunit. DNA binding and heterodimerization with CBF beta are mediated by the Runt domain in CBF alpha. Here we report an alanine-scanning mutagenesis study of the Runt domain that targeted amino acids identified by structural studies to reside at the DNA or CBF beta interface, as well as amino acids mutated in human disease. We determined the energy contributed by each of the DNA-contacting residues in the Runt domain to DNA binding both in the absence and presence of CBF beta. We propose mechanisms by which mutations in the Runt domain found in hematopoietic and bone disorders affect its affinity for DNA.