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Reactive oxygen species (ROS), a byproduct of cellular metabolism, damage intracellular macromolecules and, when present in excess, can promote normal hematopoietic stem cell differentiation and exhaustion. However, mechanisms that regulate the amount of ROS in leukemia-initiating cells (LICs) and the biological role of ROS in these cells are largely unknown. We show here that the ROS(low) subset of CD44(+) cells in T cell acute lymphoblastic leukemia (T-ALL), a malignancy of immature T cell progenitors, is highly enriched in the most aggressive LICs and that ROS accumulation is restrained by downregulation of protein kinase C θ (PKC-θ). Notably, primary mouse T-ALLs lacking PKC-θ show improved LIC activity, whereas enforced PKC-θ expression in both mouse and human primary T-ALLs compromised LIC activity. We also show that PKC-θ is regulated by a new pathway in which NOTCH1 induces runt-related transcription factor 3 (RUNX3), RUNX3 represses RUNX1 and RUNX1 induces PKC-θ. NOTCH1, which is frequently activated by mutation in T-ALL and required for LIC activity in both mouse and human models, thus acts to repress PKC-θ. These results reveal key functional roles for PKC-θ and ROS in T-ALL and suggest that aggressive biological behavior in vivo could be limited by therapeutic strategies that promote PKC-θ expression or activity, or the accumulation of ROS.

Human acute T-cell lymphoblastic leukemias and lymphomas (T-ALL) are commonly associated with gain-of-function mutations in Notch1 that contribute to T-ALL induction and maintenance. Starting from an expression-profiling screen, we identified c-myc as a direct target of Notch1 in Notch-dependent T-ALL cell lines, in which Notch accounts for the majority of c-myc expression. In functional assays, inhibitors of c-myc interfere with the progrowth effects of activated Notch1, and enforced expression of c-myc rescues multiple Notch1-dependent T-ALL cell lines from Notch withdrawal. The existence of a Notch1–c-myc signaling axis was bolstered further by experiments using c-myc-dependent murine T-ALL cells, which are rescued from withdrawal of c-myc by retroviral transduction of activated Notch1. This Notch1-mediated rescue is associated with the up-regulation of endogenous murine c-myc and its downstream transcriptional targets, and the acquisition of sensitivity to Notch pathway inhibitors. Additionally, we show that primary murine thymocytes at the DN3 stage of development depend on ligand-induced Notch signaling to maintain c-myc expression. Together, these data implicate c-myc as a developmentally regulated direct downstream target of Notch1 that contributes to the growth of T-ALL cells.

Polymorphisms in the major histocompatibility complex (MHC) and non-MHC genes were recently reported to be associated with persistent hepatitis B virus (HBV) infection and host response to hepatitis B vaccine in Asian populations. We aimed to confirm the associations in Chinese population and develop a non-invasive screening method for the risk loci.We genotyped 2 risk alleles on the MHC loci, HLA-DPA1 (rs3077) and HLA-DPB1 (rs9277535), and 1 risk allele near a non-MHC gene, FOXP1 (rs6789153) using high-resolution melting curve analysis. With minimal processing steps and time, salivary DNA was extracted with a modified protocol of a blood kit. We compared the genotyping fidelity between peripheral blood DNA and salivary DNA.Both rs3077 and rs9277535, but not rs6789153, are significantly associated with CHB in Chinese population (p-value0.001). High genotype concordance between different sources of genomic DNA was obtained.Genotyping salivary DNA using our modified methods provides a non-invasive fast screening for host susceptibility loci. The transmission mechanism of hepatitis B can now be modified by adding genetic susceptibility to the traditional vertical transmission model of hepatitis B.

The present study examined the psychometric properties of the Eating Disorder Inventory (EDI) among Chinese adolescent girls in Hong Kong. We also attempted to provide normative data for the EDI among 12- to 18-year-old Chinese girls in Hong Kong. Two thousand two hundred and fifty-six high school girls between the age of 12 and 18 years participated in this study. Subjects completed the questionnaire package anonymously at their schools. Findings indicate that the internal consistency estimates for the eight EDI scales were in the acceptable range, with alpha ranging from 0.68 for Perfectionism to 0.87 for Body Dissatisfaction. With the exception of a few items, the item-total correlations were also acceptable for the eight scales. Findings of the principal components analysis support the original eight clinically derived EDI constructs. Group comparison analyses reveal significant age differences and race differences in several EDI scales. EDI percentile scores are presented for girls 12–13 and 14–18 years of age in the present study. This study provides further empirical support to the transcultural validity of the EDI. It also highlights the importance of establishing age- and race-specific norms for the inventory.

Notch-driven expression of IGF1R promotes the growth, viability, and transplantability of T-ALL cells., T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer of immature T cells that often shows aberrant activation of Notch1 and PI3K–Akt pathways. Although mutations that activate PI3K–Akt signaling have previously been identified, the relative contribution of growth factor-dependent activation is unclear. We show here that pharmacologic inhibition or genetic deletion of insulin-like growth factor 1 receptor (IGF1R) blocks the growth and viability of T-ALL cells, whereas moderate diminution of IGF1R signaling compromises leukemia-initiating cell (LIC) activity as defined by transplantability in syngeneic/congenic secondary recipients. Furthermore, IGF1R is a Notch1 target, and Notch1 signaling is required to maintain IGF1R expression at high levels in T-ALL cells. These findings suggest effects of Notch on LIC activity may be mediated in part by enhancing the responsiveness of T-ALL cells to ambient growth factors, and provide strong rationale for use of IGF1R inhibitors to improve initial response to therapy and to achieve long-term cure of patients with T-ALL.

Abstract 12 T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cell progenitors which affects both children and adults. Current therapies achieve cure in about 80% of pediatric patients, but only 40% of adults survive beyond 5 years. Disease relapse may be attributed to ineffective targeting of leukemia stem cells (LSCs) due to their presumed quiescence, resistance to apoptosis, enhanced expression of DNA repair enzymes and drug efflux pumps, and localization to protective or inaccessible niches. More efficient targeting of LSCs in this disease thus could offer great potential for improving patient outcomes. Recent interest in the role of reactive oxygen species (ROS) in cancer stem cell biology has been sparked by studies demonstrating that low ROS levels promote clonogenicity and radioresistance of breast cancer stem cells and that parthenolide specifically induces apoptosis of LSCs in AML and blast crisis CML by increasing levels of ROS in these cells. In T-ALL, activation of NOTCH1 signaling, either by mutation of NOTCH1 itself (50-60% of cases) or its negative regulator FBW7 (15% of cases), contributes to malignant behavior by blocking differentiation, promoting cellular proliferation/survival, and enhancing self-renewal. Similar gain-of-function Notch1 mutations are also observed to occur spontaneously in several murine T-ALL models at high frequency. To examine ROS levels in T-ALL LSCs, we first generated murine disease by retroviral transduction of bone marrow with a constitutively activated form of NOTCH1 termed “ΔE”. We utilized limiting dilution transplantation analyses of different cellular phenotypes isolated from primary ΔE leukemias to demonstrate that the CD44+ subset of CD4+ CD8+ (DP) cells were enriched for LSCs and exhibited lower ROS levels as compared to the CD44- DP subset and bulk cells. Since ROS levels were heterogeneous within the CD44+ DP subset, we combined these markers with DC-FDA staining for ROS and demonstrated as few as 100 ROSlow CD44+ DP cells are able to reproduce disease in 100% of recipients whereas 100,000 bulk cells are required to achieve the same effect. Because protein kinase C theta (PKCθ) was recently identified as an important mediator of ROS production in T-cells during activation-induced cell death and expression of PKCθ mRNA could be detected broadly in primary human and mouse T-ALL samples, we reasoned that this signaling intermediate might be a regulator of ROS levels in malignant T-ALL LSCs. In fact, using a combination of viral expression and knock-down approaches in both human and mouse T-ALLs as well as mouse PKCθnull ΔE leukemias, we demonstrate that PKCθ is a primary regulator ROS production in T-ALL. Moreover, low PKCθ/ROS levels correlate with resistance to chemotherapy and irradiation, and promote the competitive in vivo growth of T-ALL cells. Interestingly, we also find that Notch signaling represses PKCθ expression which in turn results in reduced ROS levels. Our results identify PKCθ as a novel target of Notch signaling in LSCs whose key malignant properties are highly dependent on low ROS levels. These findings provide mechanistic basis for rational design of LSC-targeted therapies in T-ALL. Disclosures: No relevant conflicts of interest to declare.

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cell progenitors in which we described activating mutations of Notch1 to occur in over 50% of cases. As well, others have identified loss-of-function mutations in Sel10/Fbw7 to occur in 8–16% of cases, which also enhance Notch signaling. Notably, inhibition of Notch signaling in these cells induces growth arrest and in some cases apoptosis as well. Subsequent studies have characterized c-myc as a critical downstream target of Notch signaling in this context. More recently, mutations in PTEN (occurring in 17% of cases) were shown to potentiate PI3K/Akt signaling and proposed to confer resistance to Notch signaling inhibition, but then impose “addiction” to PI3K/Akt. Most leukemias likely derive cooperative growth/survival signals from Notch and PI3K/Akt pathways, as evidenced by synergistic effects of gamma-secretase inhibitors (GSI) which block Notch signaling and rapamycin which blocks mTOR downstream of PI3K/Akt. In mouse models, combined activation of c-myc and b-catenin with inactivation of PTEN elicited T-ALL which was devoid of Notch mutations, suggesting Notch signaling coordinately activates c-myc, Wnt, and PI3K/Akt signaling pathways. In studying the mechanism for PI3K/Akt activation in T-ALL with activated Notch signaling, we examined insulin-like growth factor receptor-1 (IGF1R) as a candidate upstream initiator of PI3K/Akt activation. We observed IGF1R expression consistently in T-ALL cells from primary human leukemias and cell lines, as well as in primary mouse leukemias derived experimentally by retroviral transduction of marrow with activated forms of Notch1. In all cases, inhibition of IGF1R signaling either with blocking antibody or small molecule kinase inhibitors resulted in growth suppression and apoptosis of leukemia cells. We further observed that inhibition of Notch signaling either by small molecule GSI or transduction with a dominant negative coactivator, Mastermind, resulted in a 2–3 fold decrease in IGF1R expression. Given that the PI3K/Akt/mTOR pathway is known to be important for growth/survival of T-ALL leukemia cells, we hypothesized that Notch might be potentiating activation of this pathway by upregulating IGF1R expression. In fact, we found leukemia cells with active Notch and higher IGF1R levels showed 20-fold greater sensitivity to IGF-1 ligand induced Akt activation as compared to leukemia cells with inactive Notch and lower IGF1R levels. This enhanced signaling output cannot be explained by Notch suppression of PTEN as this effect was noted in both PTEN wild-type and PTEN null leukemia cells. Additionally, cells with wild type PTEN demonstrated only minimal if any changes in PTEN protein levels after Notch inhibition as measured by a highly quantitative flow cytometry assay. In sum, we have identified IGF-1 signaling as being critical to growth and survival of T-ALL leukemia cells, and provide evidence that Notch may potentiate PI3K/Akt signaling by upregulating expression of IGF1R. These data are of immediate clinical relevance as several IGF1R inhibitors are currently in Phase 3 clinical trial and hopefully will provide additional therapeutic options to refractory/relapsed patients and/or in combination with front-line therapy in newly diagnosed patients.

Abstract 3649 T-cell acute lymphoblastic leukemia (T-ALL) is a lethal cancer of immature T cells that often shows aberrant activation of NOTCH1 and PI3K/Akt pathways. Although PI3K/Akt signaling is potentiated by PTEN mutations which occur in up to 20% of cases, upstream signals which initiate PI3K/Akt activation remain unclear. IGF1R is a receptor tyrosine kinase upstream of PI3K/Akt and Ras/Raf/MAPK signaling pathways with known roles in cancer cell growth and is expressed broadly in primary human T-ALL cells and established cell lines. We thus considered that IGF1R may represent a biologically relevant upstream activator of the PI3K/Akt pathway in T-ALL. Consistent with this idea, we demonstrate that treatment of human and mouse T-ALL cells with small molecule IGF1R kinase inhibitors and IGF1R blocking antibodies resulted in growth arrest of bulk cells. As well, genetic deletion of IGF1R by inducible Cre expression in mouse T-cell leukemias also leads to growth arrest and apoptosis of bulk cells. IGF1R and PI3K/Akt/mTOR signaling have also been described to promote self-renewal of normal and cancer stem cells, and thus we addressed whether IGF1R may play a role in leukemia stem cell (LSC) activity in T-ALL. Since outgrowth of bulk leukemia cells is required to detect LSC activity in vivo, we were not able to address IGF1R function in LSCs by genetic deletion; however, we elected to take advantage of a well-characterized hypomorphic allele of IGF1R carrying an integrated neo cassette within the second intron. IGF1Rneo/neo mice express full-length IGF1R protein, but at reduced levels (30-60% of wild-type). Despite this reduced expression level, we were able to generate lethal T-cell leukemias with 100% penetrance in primary recipients of IGF1Rneo/neo bone marrow transduced with activated NOTCH1 retrovirus, similar to what is observed with IGF1R+/+ bone marrow. Upon IV or intra-medullary injection into secondary congenic recipients, however, IGF1Rneo/neo leukemias exhibited a striking transplantation defect. Whereas IGF1R+/+ leukemias are consistently serially transplantable, 5 of 16 primary IGF1Rneo/neo leukemias failed to reconstitute disease in any secondary recipients, 5 produced disease in only a subset of secondary recipients, and 6 produced disease in all secondary recipients. Furthermore, treatment of IGF1R+/+ leukemia cells in vitro with IGF1R inhibitor prior to injection into secondary recipients prevented their transplantability, whereas treatment with PI3K and ERK inhibitors did not. Finally, given that inhibition of Notch signaling in human T-ALL cells compromises their ability to recapitulate disease in NOD/Scid recipients, we wondered whether this effect could be mediated by downregulation of IGF1R. In fact, we observed inhibition of Notch signaling, either by small molecule gamma-secretase inhibitor or transduction with dominant negative Mastermind, to result in a 2–3 fold decrease in IGF1R protein expression, corresponding to a 5 to 20-fold decrease in response to stimulation by IGF-1 ligand. Thus, these studies demonstrate high-level IGF1R signaling is required for LSC activity in T-ALL, and that Notch signaling may promote LSC function by supporting IGF1R expression. IGF1R inhibitors may therefore prove clinically useful in preventing disease relapse by compromising LSC self-renewal. Disclosures: Carboni: Bristol-Myers Squibb: Employment. Gottardis:Bristol-Myers Squibb: Employment.