REPORT AND RECOMMENDATIONS OF THE PANEL TO ASSESS THE NIH

1. REPORT AND RECOMMENDATIONS OF THE PANEL TO ASSESS THE NIH INVESTMENT IN RESEARCH ON GENE THERAPY Stuart H. Orkin, M.D. Arno G. Motulsky, M.D. Co-chairs December 7, 1995 “Because clinical experience is still so limited, it is not possible to exclude longterm adverse effects of gene transfer therapy, such as might arise from mutations when viral sequences randomly integrate at critical sites in the genome of somatic cells. It must be noted that multiple integration events resulting from repeated administration of large doses of retroviruses theoretically pose a risk for leukemic transformation. Only longitudinal clinical followup of treated patients can provide data on the long term safety of gene therapy protocols.”

2. Leukemia: Derangement of Blood Cell Development

3. pluripotential multipotential committed mature cells stem cells progenitors precursors Hematopoiesis monocyte/ m macrophage n neutrophil e eosinophil Specified Mesoderm Mesoderm b mast cell M HSC platelet E erythrocyte T T-lymphocyte B B-lymphocyte

4. } Leukemia genes: • discovered at translocation breakpoints • either deregulated or expressed as fusions • some present together (eg tel-runx1) • some interact as proteins (eg scl and Lmo2) • required for formation, survival, or maintenance of HSCs Transcription Factor Requirements Stem cell class Runx-1* Scl/tal-1* Lmo-2* Mll* Tel* GATA-2

5. Mechanisms of translocation-associated leukemia 1. Deregulated expression: • c-myc (Burkitt’s lymphoma) • SCL/tal-1, Lmo2 (T-ALL) 2. Expression of chimeric fusion protein: • bcr-abl (CML) • PML-RAR (APML • TEL-x, MLL-x, AML1-x (multiple partners, various leukemias)

6. LMO2 Gene and T-cell Acute Lymphocytic Leukemia 1. LMO2 gene (formally Rbtn2) discovered at breakpoint of t(11:14)(p13;q11) chromosomal translocation in T-ALL. 2. Translocation brings LMOs gene (11p13) under control of TCRd locus (14q11). Thus, regulation of LMO2 expression is deranged but protein product is unaltered. 3. LMO2 is a LIM-only protein. LIM domain mediates protein-protein interaction and does not bind DNA. 4. LMO2 participates in gene regulation through physical interaction with critical proteins, specifically SCL/tal-1, another T-cell oncoprotein involved in translocations and leukemia.

7. LMO2 Gene and T-cell Acute Lymphocytic Leukemia 5. Expressed in hematopoietic stem cells, red cell precursors, vascular cells. 6. LMO2 is essential for all hematopoietic and some aspects of angiogenesis. 7. Phenotype of LMO2 loss is identical to loss of its partner protein (SCL/tal-1). 8. Transgenic expression of LMO2 (and LMO1) in T-cells leads to T-cell leukemia after long latency. Inhibition of differentiation (accumulation of immature CD4-, CD8-, CD25+, CD44+ cells; DN T-cells) precedes leukemia. Leukemia enhanced by coexpression of partner SCL/tal-1. 9. LMO2 often expressed in human T-cell ALL, even in absence of recognizable chromosomal translocation. Frequently expressed in lyl+ ALL (lyl is close relative of SCL/tal-1)

8. pluripotential multipotential committed mature cells stem cells progenitors precursors Block to Hematopoiesis in Absence of LMO2 monocyte/ m macrophage LMO2required n neutrophil e eosinophil Specified Mesoderm Mesoderm b mast cell M HSC platelet E erythrocyte T T-lymphocyte B B-lymphocyte

9. 1. Essential: all heme 2. T-ALL 1. Essential: all heme 2. T-ALL 1. Essential: E, Meg, Eos 2. M7 meg leukemia 1. Essential: B-cells 2. -/-: lymphomas Components of an Hematopoietic Complex Ldb1 Lmo2 SCL/tal GATA-1 E2A E-box (7-9 bp) GATA

10. Leukemogenesis by LMO2 Differentiation Block TRANSGENIC TUMORS CD4-, CD8- CD25+, CD44+ CLP preT DN DP SP CD3+, CD4+ or CD8+ RAG+ Onset of TCR rearrangement TRANSLOCATION AND T-ALL (After Rabbitts)

11. LMO2 and Human T-Cell ALL Misexpression of LMO2 in T-cell ALL is sometimes due to chromosomal translocation involving LMO2 locus itself. Frequently LMO2 (and other T-cell oncoproteins) are misexpressed in the absence of recognizable translocations.

12. Expression of LMO2 in T-cell ALL Ferrando et al, Cancer Cell 2002

13. Expression of LMO2 in Lyl+ T-ALL Ferrando et al, Cancer Cell 2002

14. Retroviral Insertional Mutagenesis retrovirus Leukemia/lymphoma Susceptible mouse strain • Consequences of retroviral integration: Abnormal expression of neighboring gene due to dominant influence of retroviral regulatory sequences or interference with regulatory elements of the gene itself.

15. Retroviral Insertional Mutagenesis retrovirus Leukemia/lymphoma Susceptible mouse strain Recent genome-wide findings: At least 152 loci tagged (Suzuki et al, Nat. Genet. 2002). 747 unique sequences; 17 previously identified common integration sites and 37 new common sites (Lund et al, Nat. Genet. 2002). Thus, there are numerous potential sites for insertional leukemogenesis

16. Concluding Comments LMO2 gene is a bona fide target for T-cell leukemogenesis either through chromosomal translocation or secondary to changes in the regulatory network. Long latency of LMO2-mediated leukemia in mouse experiments suggests that additional genetic events are required for onset of leukemia. Could a block to differentiation be complemented by expression of gene that confers proliferative or survival advantage? LMO2 is representative of genes that are required for hematopoietic stem cell (HSC) formation and also enriched in HSCs. Are loci of stem cell expressed genes more accessible than the “average” gene to retroviral integrations?