LAUSR

Chromosomal translocations resulting in NUP98 fusion genes have been associated with a wide spectrum of hematologic malignancies, including MDS, AML, T-ALL, and B cell precursor (BCP) ALL. Based on gene expression profiles and murine transplantation experiments, it is thought that NUP98 fusions can confer aberrant self-renewal potential to hematopoietic cells. Approximately 90% of mice that express a NUP98-PHF23 (NP23) fusion in the hematopoietic compartment, under the control of Vav1 regulatory elements develop AML and/or T-ALL. However, approximately 10% of NP23 mice develop an aggressive acute lymphoblastic leukemia of B1-lymphocyte progenitor origin (pro B-1 ALL). Whole exome sequencing demonstrated that all NP23 pro-B1-ALL had acquired somatic frameshift mutations of the BCL6 co-repressor (Bcor) gene, and most had acquired mutations in the Jak/Stat pathway. To determine whether experimentally engineered Bcor mutations would lead to pro B-1 ALL, we used CRISPR-Cas9 to introduce Bcor indel mutations into NP23 hematopoietic stem and progenitor cells through the use of Bcor single guide RNAs (Bcor sgRNA). Recipient mice transplanted with NP23 bone marrow (BM) or fetal liver (FL) cells that had been transduced with a Bcor sgRNA developed pro B-1 ALL, characterized by a B-1 progenitor immunophenotype, clonal Igh gene rearrangement, and Bcor indel mutation, whereas control recipients did not. In addition, similar to some human BCP ALL, the Bcor sgRNA/NP23 murine pro B-1 ALL had acquired somatic mutations in Jak kinase genes. A distinct subset of pediatric BCP ALL are characterized by rearrangement and overexpression of the CRLF2 gene (designated CRLF2r); the CRLF2 gene is the receptor for thymic stromal lymphopoietin (TSLP), a cytokine that plays a role in normal progenitor B1 cell development. The NP23 pro-B1 ALL are similar to CRLF2r BCP ALL in terms of a preferential V heavy chain (VH) usage, gene expression profile, and propensity for acquired JAK/STAT pathways mutations. JAK inhibitors (ruxolitinib and tofacitinib) induced apoptosis and inhibited the growth of pro B-1 ALL cell lines established from Bcor sgRNA/NP23 recipients, at clinically achievable concentrations (10-100 nM). Taken together, these findings demonstrate that a CRISPR-induced Bcor frameshift collaborates with an NP23 transgene to predispose B-1 progenitors to leukemic transformation. These two events are unlikely to be sufficient for leukemic transformation, as we detected spontaneous Jak pathway mutations that were required for continued growth of the leukemic cells. This constellation of mutations (NP23 expression leading to increased stem cell self-renewal, Bcor frameshift leading to impaired B cell differentiation, and Jak pathway mutations leading to dysregulated proliferation) is similar to that seen in human BCP ALL patients, and suggests that the NP23/Bcor mutant mice and cell lines will be a useful model for human pro-B1 ALL. Aplan: NIH Office of Technolgy Transfer: Employment, Patents & Royalties: NUP98-HOXD13 mice.