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Targeted-sequencing analysis of PDX and PairTree predicted mutational population clusters

dPDX and dRI-PDX leukemia-initiating cell frequencies

Disease recurrence causes significant mortality in B-progenitor acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples shows relapse often arising from minor diagnosis subclones. However, why therapy eradicates some subclones while others survive and progress to relapse remains obscure. Elucidation of mechanisms underlying these differing fates requires functional analysis of isolated subclones. Here, large-scale limiting dilution xenografting of diagnosis and relapse samples, combined with targeted sequencing, identified and isolated minor diagnosis subclones that initiate an evolutionary trajectory toward relapse [termed diagnosis Relapse Initiating clones (dRI)]. Compared with other diagnosis subclones, dRIs were drug-tolerant with distinct engraftment and metabolic properties. Transcriptionally, dRIs displayed enrichment for chromatin remodeling, mitochondrial metabolism, proteostasis programs, and an increase in stemness pathways. The isolation and characterization of dRI subclones reveals new avenues for eradicating dRI cells by targeting their distinct metabolic and transcriptional pathways before further evolution renders them fully therapy-resistant.Significance:Isolation and characterization of subclones from diagnosis samples of patients with B-ALL who relapsed showed that relapse-fated subclones had increased drug tolerance and distinct metabolic and survival transcriptional programs compared with other diagnosis subclones. This study provides strategies to identify and target clinically relevant subclones before further evolution toward relapse.See related video: https://vimeo.com/442838617See related article by E. Waanders et al.

Patient characteristics and patient genomic analysis (WES and SNP 6.0)

Leukemia-initiating cell frequencies of paired diagnosis and relapse patient samples

RNA-sequencing data from Patient 9 PDX

RNA-sequencing, pathway enrichment (GSEA) reports and GSVA results (including gene list HSC vs B) of PDX and paired patient samples

PDX targeted-sequencing tissue concordance

Summary Acute lymphoblastic leukemia (ALL) dissemination to the central nervous system (CNS) is a challenging clinical problem whose underlying mechanisms are poorly understood. Here, we show that primary human ALL samples injected into the femora of immunodeficient mice migrate to the skull and vertebral bone marrow and provoke bone lesions that enable passage into the subarachnoid space. Treatment of leukemia xenografted mice with a biologic antagonist of receptor activator of nuclear factor κB ligand (RANKL) blocks this entry route. In addition to erosion of cranial and vertebral bone, samples from individuals with B-ALL also penetrate the blood-cerebrospinal fluid barrier of recipient mice. Co-administration of C-X-C chemokine receptor 4 (CXCR4) and RANKL antagonists attenuate both identified routes of entry. Our findings suggest that targeted RANKL and CXCR4 pathway inhibitors could attenuate routes of leukemia blast CNS invasion and provide benefit for B-ALL-affected individuals., Graphical abstract, Highlights • Primary human B-ALL cells use multiple independent transit mechanisms to invade the CNS • B-ALL migrate to skull or vertebral bone marrow in patient-derived xenograft (PDX) mice • B-ALL blasts provoke bone lesions through which they enter the subarachnoid space • RANKL and CXCR4 antagonists attenuate B-ALL entry into CNS in PDX mice, Rajakumar et al. show that inhibitors targeting immune receptors attenuate primary human B cell leukemia (B-ALL) cells transplanted into mice from invading the central nervous system (CNS). The findings in this study support clinical efforts to block these specific mechanisms of CNS invasion in B-ALL-affected individuals with targeted therapies.

Although most children survive B cell acute lymphoblastic leukemia (B-ALL), they frequently experience long-term, treatment-related health problems, including osteopenia and osteonecrosis. Because some children present with fractures at ALL diagnosis, we considered the possibility that leukemic B cells contribute directly to bone pathology. To identify potential mechanisms of B-ALL–driven bone destruction, we examined the p53−/−; Rag2−/−; Prkdcscid/scid triple mutant (TM) mice and p53−/−; Prkdcscid/scid double mutant (DM) mouse models of spontaneous B-ALL. In contrast to DM animals, leukemic TM mice displayed brittle bones, and the TM leukemic cells overexpressed Rankl, encoding receptor activator of nuclear factor κB ligand. RANKL is a key regulator of osteoclast differentiation and bone loss. Transfer of TM leukemic cells into immunodeficient recipient mice caused trabecular bone loss. To determine whether human B-ALL can exert similar effects, we evaluated primary human B-ALL blasts isolated at diagnosis for RANKL expression and their impact on bone pathology after their transplantation into NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ (NSG) recipient mice. Primary B-ALL cells conferred bone destruction evident in increased multinucleated osteoclasts, trabecular bone loss, destruction of the metaphyseal growth plate, and reduction in adipocyte mass in these patient-derived xenografts (PDXs). Treating PDX mice with the RANKL antagonist recombinant osteoprotegerin–Fc (rOPG-Fc) protected the bone from B-ALL–induced destruction even under conditions of heavy tumor burden. Our data demonstrate a critical role of the RANK-RANKL axis in causing B-ALL–mediated bone pathology and provide preclinical support for RANKL-targeted therapy trials to reduce acute and long-term bone destruction in these patients.

Disease recurrence causes significant mortality in B-progenitor acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples shows relapse often arising from minor diagnosis subclones. However, why therapy eradicates some subclones while others survive and progress to relapse remains obscure. Elucidation of mechanisms underlying these differing fates requires functional analysis of isolated subclones. Here, large-scale limiting dilution xenografting of diagnosis and relapse samples, combined with targeted sequencing, identified and isolated minor diagnosis subclones that initiate an evolutionary trajectory toward relapse [termed diagnosis Relapse Initiating clones (dRI)]. Compared with other diagnosis subclones, dRIs were drug-tolerant with distinct engraftment and metabolic properties. Transcriptionally, dRIs displayed enrichment for chromatin remodeling, mitochondrial metabolism, proteostasis programs, and an increase in stemness pathways. The isolation and characterization of dRI subclones reveals new avenues for eradicating dRI cells by targeting their distinct metabolic and transcriptional pathways before further evolution renders them fully therapy-resistant. Significance: Isolation and characterization of subclones from diagnosis samples of patients with B-ALL who relapsed showed that relapse-fated subclones had increased drug tolerance and distinct metabolic and survival transcriptional programs compared with other diagnosis subclones. This study provides strategies to identify and target clinically relevant subclones before further evolution toward relapse. See related video: https://vimeo.com/442838617 See related article by E. Waanders et al .

Without prophylactic therapy, B-cell Acute Lymphoblastic Leukemia (B-ALL) spreads to the leptomeninges of the central nervous system (CNS) in up to 70% of patients. CNS involvement is more common in certain high risk B-ALL subgroups, including patients with KMT2A (MLL)-translocations, and disease relapse in the CNS carries a poor prognosis. The genetic determinants and biology of B-ALL dissemination to the CNS are poorly defined and therefore therapies targeting the drivers of CNS disease are lacking. Whereas B-ALL exhibits significant subclonal diversity that contributes to functional heterogeneity and disease relapse, recent reports suggest similar clonal composition of bone marrow (BM) and CNS disease, with the potential for CNS dissemination being a universal property of B-ALL cells (Williams et al. 2016, Bartram et al. 2018). Furthermore, functional studies of leptomeningeal disease have focused on the invasion of B-ALL cells into the CNS but limited studies have addressed the selection of genetic clones with the ability to grow within the subarachnoid space. To better define the evolutionary history and biology of leptomeningeal B-ALL we performed targeted DNA, SNP copy number, RNA sequencing, and functional analysis on cells isolated from matched BM and CNS tissue of patient derived xenografts (PDX) generated from a cohort of paired diagnosis and relapse samples from 14 pediatric and adult B-ALL patients of varying cytogenetics. The majority of primary patient samples yielded CNS disease 20 weeks after intrafemoral injection into NSG mice. CNS disease burden was higher in PDXs derived from relapsed B-ALL samples. Human B-ALL cells isolated from the CNS of PDXs retained competence to repopulate disease in the BM, spleen, and CNS upon serial transplantation. Targeted DNA sequencing results analyzed using a Bayesian clustering method revealed different genetic clonal composition between matched BM and CNS cells in approximately half of the xenografts. PDXs from relapse samples were more likely to show genetic discordance between the BM and CNS. Copy number analysis also confirmed frequent genetic discordance between cells isolated from the BM and CNS from individual PDXs. Interestingly, in one patient all PDXs generated from the relapse sample displayed chromosome 6p and 17p hemi-deletions that were unique to the CNS. In total, PDXs from four patients showed recurrent enrichment of specific lesions in CNS-engrafting cells, suggesting that transit to and/or survival within the subarachnoid space can be the product of selection for genetic clones with increased CNS tropism. RNA-seq of matched BM and CNS cells derived from 45 of the primary PDXs demonstrated that CNS-isolated cells were transcriptionally distinct from their matched BM. These differences were most pronounced in samples from patients with MLL-AF4 translocations, whose CNS isolated cells grouped together in multi-dimensional scaling. Using GSEA, the most highly CNS-enriched gene sets in MLL samples were related to mRNA translation initiation and polypeptide elongation. Translation-related gene sets are similarly enriched in the blasts of MLL B-ALL patients with CNS disease in the COG 9906 study. CNS-isolated cells from PDXs of MLL patients exhibited altered rates of protein synthesis compared to matched BM-isolated cells. Treatment of PDXs with the clinically-approved translational inhibitor omacetaxine mepesuccinate (OMA) effectively decreased rates of translation in CNS-engrafting cells. Moreover, OMA reduced leukemia burden nearly 4-fold in PDXs bearing established CNS infiltration generated from two MLL patients. Our data represent an advance in the understanding of B-ALL CNS disease. We present a rich resource of genomic and transcriptomic data from xenografts spanning multiple B-ALL subgroups across diagnosis and relapse and have identified selection for genetically and biologically distinct clones in the CNS, contrary to the current model. Furthermore, we demonstrate that in MLL patients, dysregulation of protein synthesis occurs at CNS dissemination and targeting this process is a novel therapeutic paradigm that may benefit patients with CNS disease. Disclosures Mullighan: Cancer Prevention and Research Institute of Texas: Consultancy; Pfizer: Honoraria, Research Funding, Speakers Bureau; Loxo Oncology: Research Funding; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding.

B-cell acute lymphoblastic leukemia (B-ALL) readily disseminates to the leptomeninges of the central nervous system (CNS). CNS involvement is more frequent in certain poor prognosis subgroups including patients with MLL-AF4 translocations, and late CNS relapse is often lethal. The biology and clonal history of CNS leukemia are poorly defined and consequently therapies exploiting drivers of metastasis are lacking. To characterize leptomeningeal leukemia we performed targeted DNA sequencing, SNP copy number analysis, RNA sequencing, and functional analysis on cells isolated from the bone marrow (BM) and CNS of xenografts generated from a cohort of paired diagnosis and relapse samples from 14 B-ALL patients. The majority of patient samples disseminated to the CNS following intrafemoral injection into irradiated NSG mice, with greater CNS involvement in xenografts derived from relapse patient samples. Secondary transplantation of both BM- and CNS-purified cells demonstrated their capacity to re-engraft BM, CNS, and spleen. Targeted-sequencing results were analyzed using a Bayesian clustering method to determine the clonal composition of matched BM and CNS, demonstrating discordance in subclonal prevalence in nearly half the xenografts tested. Xenografts derived from two patient samples demonstrated recurrent enrichment of a particular subclone in the CNS versus BM. Similarly, copy number analysis identified frequent discordance between BM and CNS tissues within individual mice. All xenografts from one patient exhibited chromosome 6p and 17p hemi-deletions that were exclusive to CNS cells. While these data suggest that individual B-ALLs harbor subclones with CNS tropism, there were no recurrently enriched single nucleotide mutations or copy number alterations across all patients. RNA-sequencing of 45 BM and CNS pairs from primary xenografts demonstrated that CNS-isolated cells were consistently distinct from their matched BM. GSEA analysis of xenografts generated from patients with MLL-AF4 translocations (MLL) (n=2 patients, 26 mice), identified CNS cell enrichment of gene sets related to mRNA translation and nascent peptide elongation compared to BM. MLL-CNS cells exhibited altered rates of protein synthesis compared to BM cells from the same mouse. The clinically-approved translation inhibitor omacetaxine mepesuccinate effectively diminished protein translation rates of CNS isolated cells and reduced CNS engraftment by four fold in xenografts derived from two MLL-AF4 patients. These data demonstrate that the CNS microenvironment selects for the outgrowth of B-ALL cells with genetically and/or biologically distinct properties. Moreover, we demonstrate that in MLL-AF4 patients, altered protein synthesis occurs in CNS dissemination and that targeting this process may clinically benefit patients with CNS disease. Citation Format: Robert J. Vanner, Stephanie M. Dobson, Ildiko Grandal, Olga Gan, Jessica McLeod, James Kennedy, Veroniqu Voisin, Abdellatif Daghrach, Erwin M. Schoof, Cynthia Guidos, Jayne Danska, Esme Waanders, Mark Minden, Charles G. Mullighan, John E. Dick. Genetic profiling of central nervous system dissemination of B-acute lymphoblastic leukemia reveals clonal selection and therapeutic vulnerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5173.

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.

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.

Survival rates for pediatric B-Cell Acute Lymphoblastic Leukemia (B-ALL) have improved dramatically over the past 40 years approaching a current long-term survival rate of 85%. However childhood B-ALL patients continue to confront co-morbidities and their long-term consequences. For example, osteopenia and osteoporosis associated fractures are a common complication of pediatric leukemia at diagnosis, during treatment and in long-term B-ALL survivors. The STeroid-associated Osteoporosis in the Pediatric Population (STOPP) study reported that at ALL diagnosis, 16% of children and adolescents present with bone pain, vertebral compression and low vertebral Bone Mineral Density (BMD) scores, with the greatest incidence of vertebral fractures (VF) seen in the first year following diagnosis (J Clin Endocrinol Metab. 2015, 100:3408-17). Glucocorticoid treatment further elevated fracture risk in this population. These data underscore the need to identify molecular mechanism by which leukemic cells contribute to bone loss, and provide targeted therapies to limit these effects. Our laboratory previously showed that Rag2-/- p53-/- Prkdcscid/scid triple mutant (TM) and p53-/- Prkdcscid/scid double mutant (DM) mice develop spontaneous B-ALL, but only TM animals exhibit dissemination of leukemic blasts to the leptomeninges of the CNS, a poor prognosis feature observed in pediatric and adult ALL patients. We observed that TM leukemic mice also displayed fragile vertebral bones. Using comparative transcriptome analysis, we found that RANKL (Receptor Activator of the Nuclear factor-kB Ligand), a Tumor Necrosis Factor (TNF) superfamily member ligand and a key regulator of B cell and osteoclast differentiation, was expressed at greater levels in TM compared to the DM leukemia cells. RANKL binds to its receptor RANK, which is expressed in osteoclast precursor cells. RANK-RANKL interaction induces signaling in the osteoclast precursors and drives their differentiation into mature bone resorbing osteoclasts (Proc. Natl. Acad. Sci. 1999, 96:3540-3545). Upon adoptive leukemia cell transfer into immune deficient mice, RANKL+ TM but not DM cells caused decreased vertebral trabecular bone density in the recipients. Treatment with the recombinant RANKL antagonist protein Osteoprotegerin (OPG-Fc) inhibited the growth and dissemination of RANKL+TM leukemic cells and attenuated bone destruction in the recipient mice. These data suggested that TM mouse leukemia cells cause bone loss in the absence of glucocorticoid or other chemotherapy agents. We then examined the potential role of RANKL in osteoporosis associated with human B-ALL. RANKL mRNA was expressed by a majority of primary human adult and pediatric B-ALL. To determine whether primary patient B-ALL can cause bone loss, we transplanted RANKL+ human B-ALL samples of multiple cytogenetic high-risk subgroups (Complex, hypo-diploid and Mixed Lineage Leukemia (MLL) rearranged) into NOD.SCID.gC-/-(NSG) recipient mice. Micro-CT imaging and bone density measures in the xenotransplant recipients revealed extensive vertebral trabecular bone destruction. Immuno-histological analysis of the human B-ALL recipient mice demonstrated extensive osteoporotic damage of the long bones and marked RANKL protein expression in the long bones of mice harboring extensive human B-ALL cell burden compared to NSG control mice. To determine whether RANKL-RANK interaction was required for the B-ALL mediated bone destruction, cohorts of NSG mice engrafted with human B-ALL were treated with recombinant OPG-Fc compared to a matched Fc control protein. OPG-Fc treatment did not attenuate leukemia cell expansion and bone marrow burden, but despite bulky disease, the treatment conferred robust protection from bone destruction suggesting that RANKL was a critical mediator of this clinical complication. Our data demonstrate a central role of the RANK-RANKL axis in B-ALL-mediated bone disease and identify an actionable therapeutic target to reduce acute and long-term morbidity. Denosumab, an anti-RANKL antibody has been approved for the treatment of bone metastasis by solid tumors and for post-menopausal osteoporosis. Our pre-clinical studies suggest that Denosumab and other agents that inhibit the RANK-RANKL pathway may be efficacious in patients with B-ALL associated bone degeneration. Disclosures No relevant conflicts of interest to declare.

Current cancer therapies are directed at molecular markers or dominant pathways present in the bulk of neoplastic cells. However, recent studies have identified many genetically distinct subclones co-existing within a single neoplasm. In over 50% of patients with relapsed acute lymphoblastic leukemia (ALL), the genetic clones present at relapse are not the dominant clone present at diagnosis, but have evolved from a minor or ancestral clone (Mullighan et al., Science, 2008). Previous work has shown that this subclonal diversity in B-ALL exists at the level of the leukemia-initiating cells (L-IC) capable of generating patient derived xenografts (Notta et al., Nature, 2011). In order to investigate the functional consequences of genetic clonal evolution during disease progression, we performed in-depth genomic and functional analysis of 14 paired diagnosis/relapse samples from adult and pediatric B-ALL patients of varying cytogenetics. Patient samples were subjected to whole exome sequencing (WES), SNP analysis and RNA sequencing. Diagnosis-specific, relapse-specific, and shared variants at both clonal and subclonal frequencies were identified. Limiting dilution analysis by transplantation of CD19+ leukemic blasts into 870 immune-deficient mice (xenografts) identified no significant trend in enrichment in L-IC frequency between paired patient samples with a median frequency of 1 in 2691. Despite similar frequencies of L-IC, functional differences within identically sourced patient xenografts were observed, including increased leukemic dissemination of relapse cells to distal sites such as the central nervous system (CNS), differences in engraftment levels and differences in immunophenotypes. Targeted-sequencing and copy number analysis of the xenografts, in comparison to the patient sample from which they were derived, has uncovered clonal variation and the unequivocal identification of minor subclones ancestral to the relapse in xenografts transplanted with the diagnostic sample from 8 patients. Some of these subclones are rare and were not captured through standard WES analysis of the patient samples, highlighting the value of xenografting to functionally identify and viably isolate subclones for further study. Interrogation of the therapeutic responses of the ‘relapse-like’ diagnosis subclones in secondary xenografts displayed differential resistance to standard chemotherapeutic agents (vincristine and L-asparaginase) pre-existing in the patient diagnosis samples prior to treatment. Furthermore, investigation of different sites of leukemic infiltration in the xenografts provided evidence of distinct clonal selection in the CNS, a known site of disease relapse, in comparison to the bone marrow. Using this data we can begin to draw the evolutionary paths to relapse. We have shown evidence that minor subclones at diagnosis, ancestral to the relapsing clone, possess functional advantages over other diagnostic clones. Overall, this work provides a substantial advance in connecting genetic diversity to functional consequences, thereby furthering our understanding of the heterogeneity identified in B-ALL and its contributions to therapy failure and disease recurrence. Citation Format: Stephanie M. Dobson, Robert Vanner, Esmé Waanders, Olga I. Gan, Jessica McLeod, Ildiko Grandal, Debbie Payne-Turner, Michael Edmonson, Zhaohui Gu, Xioatu Ma, Yiping Fan, Pankaj Gupta, Sagi Abelson, Michael Rusch, Ying Shao, Scott Olsen, Geoffrey Neale, John Easton, Cynthia J. Guidos, Jayne S. Danska, Jinghui Zhang, Mark D. Minden, Charles G. Mullighan, John E. Dick. Evolving functional heterogeneity in B-acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-341.

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.

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

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

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−/−Prkdcscid/scid double mutant (DM) mice and Rag-2−/−p53−/−Prkdcscid/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. Disclosures: No relevant conflicts of interest to declare.

Abstract 3092 Poster Board III-29 Introduction Early B cell acute lymphoblastic leukemia (B-ALL) is the most common type of childhood malignancy, characterized by abnormal accumulation and proliferation of progenitor-B or precursor-B (pre-B) cells. Current challenges associated with B-ALL treatment include fatal relapses, treatment-related toxicities and long-term morbidities underscoring a need to develop new targeted therapies aimed at eradicating leukemia cells and their stem cells. To achieve this, a better understanding of molecular mechanisms involved in leukemia initiation and progression is required. Our laboratory developed p53-/- PrkdcSCID/SCID double mutant (DM) strain as a mouse model of early B-ALL. We showed that DM leukemias progress through discrete developmental stages of leukemogenesis despite the absence of a pre-B cell receptor (pre-BCR), a crucial checkpoint in B cell development. Spleen tyrosine kinase (SYK), a key proximal component of pre-BCR signaling, was activated in the DM leukemias despite the absence of pre-BCR and was required for their survival. Approximately 70% of pediatric pre-B-ALLs also do not express pre-BCR, which lead us to investigate SYK signaling in human pre-B-ALL and to test potential therapeutic application of SYK inhibition in these leukemias. Patients and Methods We examined 22 viably frozen primary pediatric pre-B-ALL bone marrow samples to test their responses to SYK inhibition in vitro and in vivo and have investigated the molecular basis for aberrant SYK-mediated signaling in B-ALL. Results Western blot analyses revealed that SYK and BLNK, a dominant target of SYK, were expressed in pre-B-ALL patient samples. The majority of human pre-B ALL samples tested (14/22) displayed significantly attenuated proliferation in the presence of SYK inhibitors suggesting that SYK is necessary for their survival and/or proliferation. Treatment with SYK inhibitor R406 prevented phosphorylation of downstream SYK targets including BLNK and PLC-γ2. We are continuing to study the effects of SYK inhibition using phospho-flow cytometry and genome wide expression arrays. Preliminary data will also be presented on therapeutic efficacy of an orally bioavailable form of R406-mediated SYK inhibition in vivo by xenotransplantation of human leukemias into immuno-deficient mice. Conclusions Understanding the molecular mechanisms of pre-BCR-independent SYK activation involved in proliferation and survival of leukemic blasts may provide a rational basis for development of effective treatment for ALL. Specifically, targeted therapeutic inhibition of SYK signaling may be effective B-ALL treatment that may improve outcomes of current treatment regiments with minimal additional treatment-related toxicity. Disclosures Pine: Rigel Pharmaceuticals: Employment, Equity Ownership. Hitoshi:Rigel Pharmaceuticals: Employment, Equity Ownership.

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.

Despite high survival rates for children with acute lymphoblastic leukemia (ALL), only 40% of adult patients will achieve long-term disease-free survival, and relapses in both pediatric and adult ALL are often fatal. Most current therapies are directed at molecular markers or dominant pathways present in the bulk of neoplastic cells, yet recent studies have identified many genetically distinct subclones co-existing within a single neoplasm. The functional properties and clinical relevance of these neoplastic subclones remain undefined. Genome wide copy number analysis of matched diagnostic and relapse ALL samples identified that in 50% of patients, the clones present at relapse are not the dominant clones at diagnosis, but have evolved from an ancestral pre-leukemic clone (Mullighan et al., 2008). In order to investigate the functional consequences of clonal evolution in disease progression and therapy resistance, we performed limiting dilution analysis of 3 diagnostic and 14 paired diagnostic/relapse samples from adult and pediatric B-ALL patients of varying cytogenetics, by transplantation into immune-deficient mice (xenografts). In one patient, the leukemia-initiating cell (LIC) frequency was 7.65 fold higher in the relapse sample than at diagnosis, while another patient showed the reverse with a 5.81 fold higher LIC frequency in the diagnostic sample. Two patients showed no significant differences in LIC frequency from diagnosis to relapse. LIC frequency varied from 1 in 14.2 to 1 in 4802 CD19+ blast cells. Interestingly, in 50% of the paired patient samples, transplantation of cells from the relapse sample gave rise to greater leukemic dissemination to the spleen and/or central nervous system of recipient mice in comparison to the diagnostic sample, despite similar levels of engraftment in the bone marrow. This data suggests that although the LIC frequency in B-ALL remains high and relatively static between diagnosis and relapse, relapse cells acquire increased invasive properties. To investigate the clonal composition of 3 diagnostic B-ALL samples, we undertook copy number variation (CNV) analysis of xenografts generated at both limiting and high transplanted cell doses. In all 3 samples, we detected subclones in the xenografts that were distinct from the predominant clone in the primary patient sample. We performed network analysis on these subclones and identified differentially enriched pathways, including differential expression of anti-apoptotic and apoptosis regulation pathways, providing evidence of putative functional differences. These results support the existence of functionally diverse subclones within diagnostic samples as well as functional diversity between the subclones present at diagnosis and relapse. Ongoing in depth genomic analysis of the diagnosis/relapse paired samples will add to our understanding of the functional role of the subclones identified at diagnosis in the establishment of disease relapse. In summary, these experiments will provide further insight into the functional heterogeneity present in B-ALL and the drivers of lymphoid leukemogenesis that lead to therapy failure and disease relapse. Disclosures: Danska: Trillium Therapeutics/Stem Cell Therapeutics: Research Funding.