Retroviral and Chemical Mutagenesis Identifies Pax5 as a Tumor Suppressor in B-Progenitor Acute Lymphoblastic Leukemia

Recent genome-wide analyses of acute lymphoblastic leukemia (ALL) have identified genetic alterations targeting the B lymphoid transcription factor PAX5 in over 30% of B-progenitor ALL (Nature 2007;446:758; Nature 2008;453:110). The PAX5 mutations include deletions, focal internal amplification, sequence mutations and translocations that result in PAX5 haploinsufficiency or generate PAX5 mutants with impaired DNA-binding or transactivating activitiy. In almost all B-ALL cases, the mutations are predicted to result in attenuation, but not complete abrogation of PAX5 activity, suggesting that PAX5 is a haploinsufficient tumor suppressor in B-ALL. To test this hypothesis, we have performed mutagenesis screens of mice heterozygous for a Pax5 null allele ( Pax5 +/−; Cell 1994;79:901). In a chemical mutagenesis screen, thymectomized C57BL6/Sv129 Pax5 +/− (N=25) and wild type ( Pax5 +/+; N=20) mice received a single 100mg/kg dose of the alkylating agent N-ethyl-S-nitrosourea (ENU) at 4–5 weeks of age. Pax5 +/− animals exhibited a markedly increased frequency of leukemia in comparison to wild type animals. After one year, 24 of 25 Pax5 +/ − animals developed leukemia (median latency 246 days), compared to 3 of 20 Pax5 +/+ animals ( P <0.0001, Fig. 1). Furthermore, 20 of 22 evaluable Pax5 +/− tumors were of B-progenitor cell lineage, in contrast to the Pax5+/+ tumors which were either myeloid (N=2) or T-lymphoid (N=1). Moreover, the Pax5 +/ − tumors exhibited a range of maturation from pro-B (B220+Cd19-) to pre-B (B220+Cd19+), suggesting the acquisition of additional lesions targeting the B-cell developmental pathway in the less mature tumors. In a complementary retroviral mutagenesis study, thymectomized Pax5 +/ −(N=32) and Pax5 +/+ (N=47) mice received a single dose of Moloney murine leukemia virus at 1–2 days after birth. Pax5 +/− mice displayed a higher penetrance and reduced latency of leukemia than Pax5 +/+ mice (97% of Pax5 +/− mice developed leukemia with median latency of 192 days, v. 66% of Pax5 +/+ mice, latency 299 days, P <0.0001). Again, a significantly higher proportion of Pax5 +/− tumors (23 of 29 evaluable) were B-progenitor cell lineage than the Pax5 +/+ tumors (12 of 26; P =0.01; Fig. 2), and the B-lineage tumors exhibited a spectrum of maturation from pro- to pre-B cell. Cloning and capillary sequencing of retroviral integration sites identified recurring insertions targeting the DNA polymerase gene Poln , the tumor suppressor Wwox , the tyrosine phosphatase Ptprc , the cyclin gene Ccnd3 , and orthologs of genes targeted by DNA copy number alterations in human B-ALL including Tbl1xr1 . Together, these data indicate that a decrease in the normal transcriptional level of Pax5 directly contributes to the development of B-progenitor ALL, consistent with Pax5 functioning as a haploinsufficient tumor suppressor. Next-generation sequencing to comprehensively identify all genes targeted by retroviral insertions, coupled with genome-wide copy number analysis, are in progress and should provide invaluable information on the range of mutations that cooperate with Pax5 haploinsufficincy to induce ALL. ![Fig. 1 ][1] Fig. 1 ENU mutagenesis ![Fig. 2 ][1] Fig. 2 B-cell tumors in MoMuLV infected mice [1]: pending:yes