Ildiko Grandal

The RAG-1/2 endonuclease causes genomic instability and controls CNS complications of lymphoblastic leukemia in p53/Prkdc-deficient mice

Double-strand DNA breaks (DSB) induce chromosomal translocations and gene amplification in cell culture, but mechanisms by which DSB cause genomic instability in vivo are poorly understood. We show that RAG-1/2-induced DSB cause IgH/c-Myc translocations in leukemic pro-B cells from p53/Prkdc-deficient mice. Strikingly, these translocations were complex, clonally heterogeneous and amplified. We observed reiterated IgH/c-Myc fusions on dicentric chromosomes, suggesting that amplification occurred by repeated cycles of bridge, breakage and fusion. Leukemogenesis was not mitigated in RAG-2/p53/Prkdc-deficient mice, but leukemic pro-B cells lacked IgH/c-Myc translocations. Thus, global genomic instability conferred by p53/Prkdc disruption efficiently transforms pro-B cells lacking RAG-1/2-induced DSB. Unexpectedly, RAG-2/p53/Prkdc-deficient mice also developed leptomeningeal leukemia, providing a novel spontaneous model for this frequent complication of human lymphoblastic malignancies.

Irradiation Promotes V(D)J Joining and RAG-Dependent Neoplastic Transformation in SCID T-Cell Precursors

ABSTRACT Defects in the nonhomologous end-joining (NHEJ) pathway of double-stranded DNA break repair severely impair V(D)J joining and selectively predispose mice to the development of lymphoid neoplasia. This connection was first noted in mice with the severe combined immune deficient (SCID) mutation in the DNA-dependent protein kinase (DNA-PK). SCID mice spontaneously develop thymic lymphoma with low incidence and long latency. However, we and others showed that low-dose irradiation of SCID mice dramatically increases the frequency and decreases the latency of thymic lymphomagenesis, but irradiation does not promote the development of other tumors. We have used this model to explore the mechanistic basis by which defects in NHEJ confer selective and profound susceptibility to lymphoid oncogenesis. Here, we show that radiation quantitatively and qualitatively improves V(D)J joining in SCID cells, in the absence of T-cell receptor-mediated cellular selection. Furthermore, we show that the lymphocyte-specific endonuclease encoded by the recombinase-activating genes (RAG-1 and RAG-2) is required for radiation-induced thymic lymphomagenesis in SCID mice. Collectively, these data suggest that irradiation induces a DNA-PK-independent NHEJ pathway that facilitates V(D)J joining, but also promotes oncogenic misjoining of RAG-1/2-induced breaks in SCID T-cell precursors.

Therapeutic Potential of Spleen Tyrosine Kinase Inhibition for Treating High-Risk Precursor B Cell Acute Lymphoblastic Leukemia

Pre-BCR–independent activation of spleen tyrosine kinase plays a pathogenic role in high-risk B cell acute lymphoblastic leukemia. Targeting the “SYK-ness” in B-ALL Intensive chemotherapy in B cell acute lymphoblastic leukemia (B-ALL) provides improved outcomes for children and achieves remission in adults, but patients in both groups relapse, resulting in low survival rates. The discovery of aberrant signaling pathways in cancers has spurred development of targeted kinase inhibitors. In a mouse model of spontaneous B-ALL, Perova et al. demonstrate that aberrant activation of the spleen tyrosine kinase (SYK) was required for leukemic cell growth. SYK pathway activity and B-ALL cell survival were sensitive to two inhibitors of SYK signaling. Like the mouse model, the authors show that primary pediatric and adult human B-ALL samples exhibited basal SYK activation. Phosphorylation of SYK and its targets and leukemic cell proliferation were attenuated by in vitro treatment of the human leukemic cells with SYK inhibitors. Xenotransplantation of poor-prognosis primary human B-ALL samples into immunodeficient mice resulted in extensive bone marrow engraftment and dissemination to spleen, liver, and central nervous system. In vivo treatment of the transplanted animals with SYK inhibitors reduced human leukemia burden in these tissues. Thus, SYK activation regulates key signal transduction pathways of abnormal growth in multiple subtypes of B-ALL, suggesting that small-molecule SYK inhibitors may be promising agents for treating poor-prognosis and relapsed B-ALL. Intensified and central nervous system (CNS)–directed chemotherapy has improved outcomes for pediatric B cell acute lymphoblastic leukemia (B-ALL) but confers treatment-related morbidities. Moreover, many patients suffer relapses, underscoring the need to develop new molecular targeted B-ALL therapies. Using a mouse model, we show that leukemic B cells require pre–B cell receptor (pre-BCR)–independent spleen tyrosine kinase (SYK) signaling in vivo for survival and proliferation. In diagnostic samples from human pediatric and adult B-ALL patients, SYK and downstream targets were phosphorylated regardless of pre-BCR expression or genetic subtype. Two small-molecule SYK inhibitors, fostamatinib and BAY61-3606, attenuated the growth of 69 B-ALL samples in vitro, including high-risk (HR) subtypes. Orally administered fostamatinib reduced heavy disease burden after xenotransplantation of HR B-ALL samples into immunodeficient mice and decreased leukemia dissemination into spleen, liver, kidneys, and the CNS of recipient mice. Thus, SYK activation sustains the growth of multiple HR B-ALL subtypes, suggesting that SYK inhibitors may improve outcomes for HR and relapsed B-ALL.

MuLV-related endogenous retroviral elements and Flt3 participate in aberrant end-joining events that promote B-cell leukemogenesis

During V(D)J recombination of immunoglobulin genes, p53 and nonhomologous end-joining (NHEJ) suppress aberrant rejoining of DNA double-strand breaks induced by recombinase-activating genes (Rags)-1/2, thus maintaining genomic stability and limiting malignant transformation during B-cell development. However, Rag deficiency does not prevent B-cell leukemogenesis in p53/NHEJ mutant mice, revealing that p53 and NHEJ also suppress Rag-independent mechanisms of B-cell leukemogenesis. Using several cytogenomic approaches, we identified a novel class of activating mutations in Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase important for normal hematopoiesis in Rag/p53/NHEJ triple-mutant (TM) B-cell leukemias. These mutant Flt3 alleles were created by complex genomic rearrangements with Moloney leukemia virus (MuLV)-related endogenous retroviral (ERV) elements, generating ERV-Flt3 fusion genes encoding an N-terminally truncated mutant form of Flt3 (trFlt3) that was transcribed from ERV long terminal repeats. trFlt3 protein lacked most of the Flt3 extracellular domain and induced ligand-independent STAT5 phosphorylation and proliferation of hematopoietic progenitor cells. Furthermore, expression of trFlt3 in p53/NHEJ mutant hematopoietic progenitor cells promoted development of clinically aggressive B-cell leukemia. Thus, repetitive MuLV-related ERV sequences can participate in aberrant end-joining events that promote development of aggressive B-cell leukemia.

Abstract 1397: A novel ligand-independent Flt3 allele drives RANKL expression in a murine model of B-precursor acute lymphoblastic leukemia with CNS dissemination

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Survival rates for pediatric acute lymphoblastic leukemia (ALL) have improved dramatically, but outcomes for the 15% who relapse and for adults with ALL remain poor. Up to 40% of pediatric ALL patients require central nervous system (CNS) prophylaxis treatments that pose significant risk. We previously showed that p53−/− Rag-2−/− Prkdcscid;scid triple mutant (TM) mice spontaneously develop early B-cell ALL that disseminates to the CNS. We used this model to investigate molecular mechanisms that drive CNS dissemination of leukemic B-cells. Array comparative genomic hybridization revealed that TM ALLs have recurrent polyploidy or partial gains of telomeric chromosome 5 containing the Fms-like tyrosine kinase 3 (Flt3) locus. FLT3 is a type III receptor tyrosine kinase normally expressed on multi-potent hematopoietic progenitors. Small molecule FLT3 inhibitors abrogated proliferation of leukemic TM blasts in vitro, suggesting that Flt3 is a leukemic driver. During normal B-cell development, Flt3 is repressed by the PAX5 transcription factor, which also induces expression of B-lineage genes. Although TM ALLs expressed many genes indicative of B-lineage commitment, they ectopically over-expressed a truncated form of Flt3 (trFlt3) driven by an endogenous retrovirus long terminal repeat. The trFlt3 allele lacked most of the extracellular ligand-binding domain, but retained the trans-membrane region. Retroviral transduction of trFlt3 into BaF3 hematopoietic progenitor cells rendered their growth independent of interleukin-3. Phospho-flow cytometric profiling studies demonstrated that the signaling properties of trFLT3 are similar to those of FLT3-ITD, but are distinct from ligand-dependent FLT3 signaling. Thus, genomic rearrangements make Flt3 resistant to PAX5 repression and allow ectopic ligand-independent FLT3 signaling in TM B-ALL. Gene set enrichment analysis was used to identify genes downstream of Flt3 as potential drivers of B-ALL CNS dissemination. TM B-ALL blasts (but not murine B-ALL blasts lacking Flt3 expression) expressed RANKL, a key regulator of osteoclast differentiation and normal B-cell development. Cell surface expression of RANKL was absolutely correlated with the presence of trFLT3 protein, and RANKL cell surface expression was down-regulated by FLT3 inhibitors, suggesting that RANKL expression was controlled by trFLT3 signaling. We also found that interleukin-7, an important cytokine in B-cell development, up-regulates RANKL in normal pro-B cells. We are currently investigating if a RANKL antagonist can inhibit CNS dissemination of TM B-ALL expressing RANKL. Our studies have identified a novel Flt3 mutant allele with unusual signaling properties as a leukemic driver of early B-ALL, and provide a model for in vivo testing of the role of RANKL in mediating CNS dissemination of leukemic B-cell progenitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1397. doi:1538-7445.AM2012-1397

Abstract 867: Therapeutic potential of small molecule SYK inhibitors for treatment of primary B cell acute lymphoblastic leukemia

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. Intensified and central nervous system (CNS)-directed chemotherapy has significantly improved outcomes for pediatric patients but are associated with late-effect morbidities. Moreover, ∼20% pediatric and a higher frequency of adult patients suffer relapses that are often fatal. Thus there is a need to develop therapies that target signaling abnormalities in B-ALL, which may reduce complications of CNS leukemia and decrease long-term morbidities. Rationale: Using a p53-/- SCID mouse model of B-ALL we observed pre-B cell receptor (pre-BCR)-independent activation of the spleen tyrosine kinase (SYK) and found that it was crucial for the proliferation and survival of these leukemias. We then asked whether abnormal SYK activation occurs in human B-ALL and whether these cells are sensitive to small molecule SYK inhibitors. Methods: Viably frozen diagnostic B-ALL samples from children (n=54) and adults (n=42) tested for sensitivity to SYK inhibitors R406 (Astra-Zeneca) and BAY61-3606 in a short-term in vitro proliferation assay. Phospho-flow cytometry was also performed to quantify phosphorylation of SYK and other signaling proteins in B-ALL samples. The R406 pro-drug (Fostamatinib: Fosta) was used in a xenotransplant assay to determine therapeutic potential of SYK inhibition in vivo. Results: Phospho-flow cytometry profiling of primary B-ALL samples revealed prominent phosphorylation of SYK (Y348) and downstream signaling proteins that was decreased by SYK inhibitors. Furthermore, SYK inhibitors significantly attenuated proliferation of pre-BCR-negative and pre-BCR-positive B-ALL samples indicating that SYK was required for their survival and proliferation. In contrast, FLT3 or SRC inhibitors did not inhibit proliferation of pediatric and adult B-ALL samples. Importantly, siRNA-mediated SYK knockdown also reduced proliferation of B-ALL cell lines. Therefore, we tested the therapeutic potential of SYK inhibition using xenotransplantion. NOD.SCID.gamma C-/- (NSG) mice were injected intrafemorally with primary B-ALL samples (n=9) and fed chow containing either vehicle (AIN-76A diet) or Fosta (AIN-76A diet with 2g Fosta/kg). Leukemia burden was assessed 4-8 weeks post-transplantation. Mice given the Fosta diet had significantly reduced numbers of leukemic blasts in their injected femurs, other bones, spleens and CNS as compared to vehicle-treated mice. In addition, Fosta treatment reduced spleen, liver and kidney weight in ALL-transplanted mice. Conclusion: SYK signaling is vital to B cell acute lymphoblastic leukemia survival; small molecule SYK inhibitors have therapeutic potential in poor-prognosis and relapsed B-ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 867. doi:1538-7445.AM2012-867