A Novel Mutational Mechanism Drives Over-Expression of Ligand-Independent FLT3 in a Murine Model of B-Precursor ALL

Abstract 640

To identify dysregulated transcriptional and signaling networks that contribute to the development of acute lymphoblastic leukemia (ALL), our laboratory has generated and characterized p53^−/−Prkdc^scid/scid double mutant (DM) mice and Rag-2^−/−p53^−/−Prkdc^scid/scid triple mutant (TM) mice. Both strains develop CD19⁺ B-precursor ALL by 8–16 weeks of age, but with distinct cytogenetic abnormalities and clinical sequelae. Interestingly, TM but not DM leukemic mice exhibited dissemination of leukemic blasts to the leptomeninges of the central nervous system (CNS), a characteristic of many poor prognosis pediatric and adult ALL. Using genome-wide expression profiling, we found that TM leukemic blasts over-express FMS-like tyrosine kinase 3 (Flt3), a class III receptor tyrosine kinase, relative to normal early B cell progenitors and DM leukemias. This finding was unexpected, since Flt3 is normally repressed by PAX5 upon B cell commitment and CD19 expression. High Flt3 is also typical of infant ALL, an aggressive malignancy characterized by CNS dissemination of leukemic blasts. To identify mechanisms of aberrant Flt3 expression in TM ALLs, we performed array comparative genomic hybridization and cytogenetic studies and observed recurrent chromosome 5 polyploidy or amplification of the telomeric region of chromosome 5 containing Flt3. Molecular studies revealed that TM but not DM leukemias express a truncated form of FLT3 encoded by aberrant Flt3 transcripts in which the 5' exons that encode the extracellular ligand-binding region are replaced by endogenous retroviral long terminal repeat (ERV-LTR) elements. Strikingly, we only detected ERV-Flt3 chimeric transcripts and truncated FLT3 in TM ALL that displayed CNS invasion, suggesting involvement of FLT3 signaling in this poorly understood pathologic process. The chimeric transcripts appear to arise from genomic rearrangements that insert ERV-LTR elements into Flt3 intron 9, driving expression of Flt3 exons 10–24 from ERV-LTR enhancer and promoter elements. In silico translation of the ERV-Flt3 chimeric transcripts suggested that they encode a truncated ligand-independent, but membrane tethered mutant form of FLT3.

To probe mechanisms by which this novel FLT3 mutation subverts normal B cell development and promotes leukemogenesis, we used 8–10 color flow cytometry to characterize hemato-lymphoid and B cell precursor subsets in leukemic TM mice. Abnormal CD19⁺ lymphoblasts were abundant in bone marrow from leukemic TM mice, where we also observed increased numbers of more primitive CD19⁻ FLT3⁺ hemato-lymphoid precursors. The CD19⁺ and lineage-negative (Lin⁻) CD19⁻ bone marrow fractions both showed aberrant growth factor-independent proliferation in vitro, that was abrogated by FLT3 inhibitors. Transplantation studies showed that Lin⁻ CD19⁻ FLT3⁺ cells preferentially gave rise to leukemic CD19⁺ blasts in vivo, indicating that TM ALLs are organized as a hierarchy. Importantly, we detected ERV-Flt3 chimeric transcripts in sorted Lin⁻ CD19⁻ FLT3⁺ and Lin⁻ CD19⁻ FLT3⁻ progenitors from leukemic TM mice. Thus, the genomic rearrangements that generate chimeric ERV-Flt3 transcripts occur in primitive hemato-lymphoid progenitors prior to Pax5 expression and B cell commitment. Collectively, we have identified a novel mutational mechanism that drives over-expression of ligand-independent, constitutively active FLT3 in primitive hemato-lymphoid progenitors, and prevents PAX5-mediated Flt3 repression upon B cell commitment, ultimately leading to leukemic transformation of B cell precursors. Current studies are focused on defining mechanisms by which aberrant FLT3 signaling promotes CNS dissemination of leukemic blasts.