SUMMARY Missense mutations in the C-terminal B30.2 domain of pyrin cause familial Mediterranean fever (FMF), the most common Mendelian autoinflammatory disease. However, it remains controversial as to whether FMF is due to the loss of an inhibitor of inflammation or to the activity of a proinflammatory molecule. We generated both pyrin-deficient mice and “knockin” mice harboring mutant human B30.2 domains. Homozygous knockin, but not pyrin-deficient, mice exhibited spontaneous bone marrow-dependent inflammation similar to but more severe than human FMF. Caspase-1 was constitutively activated in knockin macrophages and active IL-1β was secreted when stimulated with lipopolysaccharide alone, which is also observed in FMF patients. The inflammatory phenotype of knockin mice was completely ablated by crossing with IL-1 receptor-deficient or adapter molecule ASC-deficient mice, but not NLRP3-deficient mice. Thus, our data provide evidence for a heretofore unrecognized ASC-dependent NLRP3-independent inflammasome in which gain-of-function pyrin mutations cause autoinflammatory disease.

T he heterodimeric CBF transcription factor genes (CBFA2 (also known as AML1) and CBFB) are the most common translocation targets in human acute myeloid leukaemia (AML), accounting for 30% of AML cases 1 . Approximately onehalf are attributable to a chromosome 16 inversion, inv(16)(p13; q22), found con-

CBFb-SMMHC is expressed from the inv(16) chromosome in M4Eo AML. Mice lacking CBF subunits or expressing the CBFb-SMMHC or AML1-ETO oncoproteins failed to develop de®nitive hematopoiesis. To investigate these e ects on hematopoiesis, we expressed CBFb-SMMHC from the metallothionein promoter, in both 32D cl3 myeloid cells and Ba/F3 B-lymphoid cells. Addition of zinc increased CBFb-SMMHC levels more than tenfold, with higher levels evident in Ba/F3 lines. Levels obtained in 32D cl3 cells were similar to those of endogenous CBFb. Indirect immuno¯uorescence revealed zinc-inducible speckled, nuclear staining in Ba/F3 cells and di use nuclear staining in 32D cl3 cells. CBFb-SMMHC reduced endogenous CBF DNA-binding ®vefold in both cell types, increased cell generation time 1.9-fold, on average, in 32D cl3 cells and 1.5-fold in Ba/ F3 cells and decreased tritiated thymidine incorporation into DNA correspondingly. CBFb-SMMHC increased the proportion of cells in G1 1.7-fold, on average, in 32D cl3 and Ba/F3 cells, and decreased the proportion of cells in S phase by a similar degree. CBFb-SMMHC induced a marked increase in hypophosphorylated Rb, but did not alter IL-3 Receptor a or b subunit levels. Neither apoptosis nor 32D di erentiation was induced by zinc in IL-3 in these lines. Induction of CBFb-SMMHC in 32D cl3 cells did not inhibit their di erentiation to neutrophils or their expression of myeloperoxidase mRNA in G-CSF, and did not produce an eosinophilic phenotype. Additional, proliferative genetic changes in M4eo AMLs might potentiate inhibition of di erentiation by CBFb-SMMHC by allowing its increased expression.

It is known that CBFB-MYH11, the fusion gene generated by inversion of chromosome 16 in human acute myeloid leukemia, is causative for oncogenic transformation. However, the mechanism by which CBFB-MYH11 initiates leukemogenesis is not clear. Previously published reports showed that CBFB-MYH11 dominantly inhibits RUNX1 and CBFB, and such inhibition has been suggested as the mechanism for leukemogenesis. Here we show that Cbfb-MYH11 caused Cbfb/ Runx1 repression-independent defects in both primitive and definitive hematopoiesis. During primitive hematopoiesis, Cbfb-MYH11 delayed differentiation characterized by sustained expression of Gata2, Il1rl1, and Csf2rb, a phenotype not found in Cbfb and Runx1 knockout mice. Expression of Cbfb-MYH11 in the bone marrow induced the accumulation of abnormal progenitor-like cells expressing Csf2rb in preleukemic mice. The expression of all 3 genes was detected in most human and murine CBFB-MYH11 ؉ leukemia samples. Interestingly, Cbfb-MYH11 ϩ preleukemic progenitors and leukemiainitiating cells did not express Csf2rb, although the majority of leukemia cells in our Cbfb-MYH11 knockin mice were Csf2rb ؉ . Therefore Csf2rb can be used as a negative selection marker to enrich preleukemic progenitor cells and leukemiainitiating cells from Cbfb-MYH11 mice. These results suggest that Cbfb/Runx1 repression-independent activities contribute to leukemogenesis by Cbfb-