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CDK10 expression in CRC based on a meta-analysis of independent gene expression profiling studies [7].

Effect of CDK10 expression on transcript levels of CDKs. CDK2, CDK4, CDK5, CDK6 and CDK9 expression in (A) MIP101 and (B) RKO CDK10 WT and DN overexpressing cells. Results represents n = 3, means {plus minus} SD.

Legends of Figures S1-S6

NOTCH1 is recurrently mutated in chronic lymphocytic leukemia (CLL), most commonly as a 2-bp frameshift deletion (c.7541_7542delCT). This mutated allele encodes a truncated form of the receptor (p.P2514Rfs∗4) lacking the C-terminal proline, glutamic acid, serine, and threonine (PEST) degradation domain that increases NOTCH1 signaling duration. NOTCH1 mutation has been associated with poor clinical outcomes in CLL. We validated a highly sensitive and quantitative droplet digital PCR assay for the NOTCH1 delCT mutation, which was anticipated to perform well compared with Sanger sequencing and allele-specific PCR. Performance characteristics of this assay were tested on 126 samples from an unselected CLL cohort and a separate cohort of 85 samples from patients with trisomy 12 CLL. The delCT mutation was detected at allele frequencies as low as 0.024%; 25% of unselected cases and 55% of trisomy 12 cases were positive at the 0.024% detection threshold. Mutational burdens ≥1% were significantly associated with shorter overall survival (OS) in patients with trisomy 12+ disease in multivariate analysis (median OS, 9.1 versus 13 years, with hazard ratio of 2.34; P = 0.031). Mutational burdens1% correlated with shorter OS in univariate, but not multivariate, analyses. These results suggest that droplet digital PCR testing for NOTCH1 delCT mutation may aid in risk stratification and/or disease monitoring in certain subsets of patients with CLL.

T-cell acute lymphoblastic leukemia (T-ALL) can affect any age group; however, disease outcome is worse in older patients. Aging has been described to be accompanied by an increase in the body's proinflammatory status, a phenomenon referred as "inflammaging". By analysing two T-ALL transcriptome datasets, we have found that inflammation pathways are upregulated in older patients, whereas younger patients present upregulation of cell cycle pathways, suggesting that inflammaging is also recapitulated in T-ALL. Here, we hypothesize that T-ALL oncogenes can cooperate with specific cytokines/stimuli and their signaling pathways to create distinct leukemic phenotypes. To test this, human cord blood CD34+ cells were transduced with different T-ALL oncogenes and grown in co-culture with stromal cells under different pro-inflammatory conditions. We observed that most of the transduced cells had their cell growth negatively affected by the proinflammatory conditions, although some oncogene combinations showed more tolerance to these conditions than others. Activated NOTCH1+TLX3 transduced cells represented the only gene combination that showed significantly higher cell growth in the presence of interferon-gamma (IFNγ) when compared to non-transduced cells in the same culture. IL-15 and IFNγ potentiated differentiation arrest observed with certain oncogene combinations. Normal uncommitted T-cell progenitors (CD7+CD5+CD44+CD1a-) responded to IL-15 via STAT5 and AKT phosphorylation, and this response was enhanced by chronic treatment with IL-15. Our results suggest that proinflammatory conditions can alter the effect of certain oncogenes on T-cell progenitors and influence leukemogenesis, potentially leading to distinct biological behaviors of resulting leukemias. These findings may lead to identification of cytokine dependencies associated with particular T-ALL genetic subtypes.

Mechanistic studies in human cancer have relied heavily on established cell lines and genetically engineered mouse models, but these are limited by in vitro adaptation and species context issues, respectively. More recent efforts have utilized patient-derived xenografts (PDX); however, as an experimental model these are hampered by their variable genetic background, logistic challenges in establishing and distributing diverse collections, and the fact they cannot be independently reproduced. We report here a completely synthetic, efficient, and highly reproducible means for generating T-cell acute lymphoblastic leukemia (T-ALL) de novo by lentiviral transduction of normal CD34+ human cord blood (CB) derived hematopoietic progenitors with a combination of known T-ALL oncogenes. Transduced CB cells exhibit differentiation arrest and multi-log expansion when cultured in vitro on OP9-DL1 feeders, and generate serially transplantable, aggressive leukemia when injected into immunodeficient NSG mice with latencies as short as 80 days (median 161 days, range 79-321 days). RNA-seq analysis of synthetic CB leukemias confirmed their reproducibility and similarity to PDX tumors, while whole exome sequencing revealed ongoing clonal evolution in vivo with acquisition of secondary mutations that are seen recurrently in natural human disease. The in vitro component of this synthetic system affords direct access to "pre-leukemia" cells undergoing the very first molecular changes as they are redirected from normal to malignant developmental trajectories. Accordingly, we performed RNA-seq and modified histone ChIP-seq on nascently transduced CB cells harvested from the first 2-3 weeks in culture. We identified coordinate upregulation of multiple anterior HOXB genes (HOXB2-B5) with contiguous H3K27 demethylation/acetylation as a striking feature in these early pre-leukemia cells. Interestingly, we also found coordinate upregulation of these same HOXB genes in a cohort of 264 patient T-ALLs (COG TARGET study) and that they defined a subset of patients with significantly poorer event-free survival (Log-rank p-value = 0.0132). Patients in the "HOXB high" subgroup are distinct from those with ETP-ALL, but are enriched within TAL1, NKX2-1, and "unknown" transcription factor genetic subgroups. We further show by shRNA-mediated knockdown that HOXB gene expression confers growth advantage in nascently transduced CB cells, established synthetic CB leukemias, and a subset of established human T-ALL cell lines. Of note, while there is prior literature on the role of HOXA genes in AML and T-ALL, and of HOXB genes in normal HSC expansion, this is the first report to our knowledge of a role for HOXB genes in human T-ALL despite over 2 decades of studies relying mostly on mouse leukemia and cell line models. The synthetic approach we have taken here allows investigation of both early and late events in human leukemogenesis and delivers an efficient and reproducible experimental platform that can support functional testing of individual genetic variants necessary for precision medicine efforts and targeted drug screening/validation. Further, since all tumors including PDXs continue to evolve during serial propagation in vivo, synthetic tumors represent perhaps the only means by which we can explore early events in cellular transformation and segregate their biology from confounding effects of multiple and varied secondary events that accumulate in highly "evolved" samples. Disclosures Steidl: Seattle Genetics: Consultancy; Tioma: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

Summary Acute leukemias are aggressive malignancies of developmentally arrested hematopoietic progenitors. We sought here to explore the possibility that changes in hematopoietic stem/progenitor cells during development might alter the biology of leukemias arising from this tissue compartment. Using a mouse model of acute T cell leukemia, we found that leukemias generated from fetal liver (FL) and adult bone marrow (BM) differed dramatically in their leukemia stem cell activity with FL leukemias showing markedly reduced serial transplantability as compared to BM leukemias. We present evidence that this difference is due to NOTCH1-driven autocrine IGF1 signaling, which is active in FL cells but restrained in BM cells by EZH2-dependent H3K27 trimethylation. Further, we confirmed this mechanism is operative in human disease and show that enforced IGF1 signaling effectively limits leukemia stem cell activity. These findings demonstrate that resurrecting dormant fetal programs in adult cells may represent an alternate therapeutic approach in human cancer.

Cyclin-dependent kinase 10 (CDK10), a CDC2-related kinase, is highly expressed in colorectal cancer. Its role in the pathogenesis of colorectal cancer is unknown. This study examines the function of CDK10 in colorectal cancer, and demonstrates its role in suppressing apoptosis and in promoting tumor growth in vitro and in vivo. Modulation of CDK10 expression in colorectal cancer cell lines demonstrates that CDK10 promotes cell growth, reduces chemosensitivity and inhibits apoptosis by upregulating the expression of Bcl-2. This effect appears to depend on its kinase activity, as kinase-defective mutant colorectal cancer cell lines have an exaggerated apoptotic response and reduced proliferative capacity. In vivo, inhibiting CDK10 in colorectal cancer following intratumoral injections of lentivirus-mediated CDK10 siRNA in a patient-derived xenograft mouse model demonstrated its efficacy in suppressing tumor growth. Furthermore, using a tissue microarray of human colorectal cancer tissues, the potential for CDK10 to be a prognostic biomarker in colorectal cancer was explored. In tumors of individuals with colorectal cancer, high expression of CDK10 correlates with earlier relapse and shorter overall survival. The findings of this study indicate that CDK10 plays a role in the pathogenesis in colorectal cancer and may be a potential therapeutic target for treatment. Mol Cancer Ther; 16(10); 2292–303. ©2017 AACR.

Insulin-like growth factor 1 receptor (IGF1R) is a prevalent signaling pathway in human cancer that supports cell growth/survival and thus contributes to aggressive biological behavior. Much work has gone into development of IGF1R inhibitors; however, candidate agents including small molecule tyrosine kinase inhibitors and blocking antibodies have yet to fulfill their promise clinically. Understanding cellular features that define sensitivity versus resistance are important for effective patient selection and anticipation of outgrowth of a resistant clone. We previously identified an important role for IGF signaling in T-cell acute lymphoblastic leukemia (T-ALL) relying primarily upon genetically defined mouse models. We present here an assessment of IGF1R dependence in human T-ALL using a broad panel of 27 established cell lines that capture a spectrum of the genetic variation that might be encountered in clinical practice. We observed that a subset of cell lines are sensitive to IGF1R inhibition and are characterized by high levels of surface IGF1R expression and PTEN positivity. Interestingly, lentiviral expression or knock-down of PTEN in PTEN-negative/positive cell lines, respectively, had limited effects on their response to IGF1R inhibition, suggesting that PTEN contributes to, but does not define IGF dependence. Additionally, we characterize downstream PI3K/AKT signaling as dominant over RAS/RAF/MEK/ERK in mediating growth and/or survival in this context. Finally, we demonstrate that IGF and interleukin-7 (IL-7) fulfill non-overlapping roles in supporting T-ALL growth. These findings are significant in that they reveal cellular features and downstream mechanisms that may determine the response of an individual patient's tumor to IGF1R inhibitor therapy.

To identify microRNAs regulated by oncogenic Notch signaling, we performed microarray-based miRNA profiling of T-cell acute lymphoblastic leukemia (T-ALL) cells before and after treatment with γ-secretase inhibitor (GSI) to block Notch signaling. We show miR-223 levels increase after GSI treatment suggesting that active Notch signaling represses miR-223 expression. We also demonstrate that insulin-like growth factor-1 receptor (IGF1R) is regulated by miR-223 in this context, but observe no apparent effects on cell growth by overexpression or knock-down of miR-223 alone. We conclude that miR-223 contributes to IGF1R regulation, but may act in concert with other genes and/or microRNAs to alter T-ALL biology.

Abstract 3537 Acute lymphoblastic leukemia (ALL) is the most common malignancy diagnosed in children, accounting for 30% of cancer diagnoses in patients under the age of 15 years. Of these cases, roughly 15% derive from the T-cell lineage (T-ALL). Steady improvements in treatment protocols have recently led to survival rates near 80%; however, the remaining 20% have uniformly poor outcomes. Thus far, the therapeutic strategies implemented have focused on intensifying multiagent chemotherapy. As undesirable toxicity arises with additional intensification of standard chemotherapy agents, more targeted molecular agents for T-ALL are urgently needed to further improve outcomes of this disease. Preclinical studies have largely relied on a relatively small number of established T-ALL cell lines that have been maintained in culture for long periods of time, as well as primary T-ALLs xenografted in immunodeficient mice. Recently, research efforts have focused on the development of robust in vitro culture models for efficient testing and validation of novel therapeutics on primary human T-ALL blasts. A relatively simple and efficient in vitro culturing system would in part eliminate the concerns of using cell lines that might not reflect the biology of a typical disease in patients, and mitigate the costly and time-consuming endeavor of xenografting T-ALL blasts. Moreover, personalized medicine initiatives would be dramatically improved by the ability to assess drug sensitivity profiles on patients' own tumor cells in direct in vitro culture assays. Several groups have devised methods for in vitro culture of patient T-ALL cells, the most successful of which have utilized stromal feeders. These methods have relied also upon serum-containing medium and thus have yielded variable results due in part to lot-to-lot variation in serum quality. Using a defined, serum-free media based on the WIT formulation initially described for in vitro propagation of normal and transformed human mammary epithelial cells, we have now optimized culture conditions required for primary human T-ALL samples. We demonstrate this serum-free culture system to consistently outperform serum-containing medium, supporting expansion of primary T-ALL samples 2- to 200-fold within 3 weeks of initial explantation from either xenografted mice or patient biopsy material and with only limited cell death. When carried for longer periods of time (up to 6 weeks), cultures maintained at higher cell densities attained maximal expansions of 40- to 100-fold while those maintained at lower cell densities expanded up to 10,000-fold. Importantly, T-cell receptor γ heteroduplex analysis confirmed expansion of the original clone throughout the culture period and flow cytometric analysis confirmed cultures to be composed of immature human T-lineage cells. Of note, cultures tended to regress after 30–40 days in vitro, suggesting that long-term renewing cells are not supported under these conditions. The use of defined, serum-free medium in this culture system has facilitated systematic characterization of growth factor requirements. We show that supplemental IL-7 and insulin/IGF1 are critical for short-term culture expansion, whereas SCF and Flt3L are dispensable. This culture system will also enable us to assess other “stem” supporting factors and genes (by lentiviral transduction) for their contribution to long-term culture maintenance. Finally, we demonstrate the facility of this culture system for testing of novel therapeutic compounds. By allowing direct study of primary samples in vitro, this culture system will advance basic mechanistic studies in T-ALL biology as well as preclinical studies aimed at developing individually tailored therapy protocols. Disclosures: Ince: Stemgent (2008–10): Consultancy, TAI receives royalties for WIT-P, -I, and -T media Other.

Abstract 4630 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer of immature T cells that often shows aberrant activation of the Notch1 signaling pathway. Several studies have utilized mRNA expression profiling to identify downstream mediators of oncogenic Notch signaling in this context. Since microRNAs (miRNAs) have in recent years been shown to play important roles in hematological maliganancy, we performed a microarray-based screen for Notch-dependent miRNA expression in T-ALL. Jurkat and P12-Ichikawa cell lines were treated with gamma-secretase inhibitor to block Notch signaling vs. DMSO control for 4 days and profiled using Exigon miRCURY LNA miRNA microarrays. Surprisingly few miRNAs were found to be regulated by this approach; however, one of the hits, miR-223, showed consistent upregulation after gamma-secretase treatment in Jurkat cells and 5 additional human T-ALL cell lines assessed by miRNA qPCR. This observation was unique to human T-ALL as murine models of T-ALL showed no evidence for Notch-dependent miR-223 expression. Given that canonical Notch signaling results in transcriptional activation, our observation that Notch signaling is associated with reduced miR-223 expression suggests an intermediary repressor may be involved. miR-223 has been reported to play an important role in normal granulopoiesis, to be expressed relatively highly in T-ALL with myeloid-like gene features, and most recently to accelerate Notch-mediated T-cell leukemogenesis. To explore potential functional consequences for Notch-dependent miR-223 repression in T-ALL, candidate miR-223 targets identified by TargetScan software were analyzed with Ingenuity Pathway Analysis software, which indicated IGF-1, insulin receptor, PTEN, and ERK5 signaling pathways as the top hits. We recently reported IGF1R signaling to be important for growth and viability of bulk T-ALL cells as well as for leukemia-initiating cell activity. Additionally, we reported that Notch signaling directly upregulates IGF1R transcription by binding to an intronic enhancer which is present between exons 21/22 in the human, but not mouse IGF1R locus. As miR-223 has previously been reported to target IGF1R mRNA and reduce its translation, we hypothesized that Notch signaling may also upregulate net IGF1R protein expression by repressing miR-223. To test this hypothesis, we transduced several human T-ALL cell lines with miR-223 retrovirus and observed a modest decrease in total IGF1R protein levels by western blot; however, no significant change was observed in surface IGF1R levels as assessed by flow cytometry. Addtionally, knockdown of miR-223 by lentiviral expression miR-223 target sequences (miR-223 “sponge”) resulted in modestly increased total IGF1R protein levels, but again showed no demonstrable effect on surface IGF1R levels. Of note, we also observed no apparent effect of either overexpression or knockdown of miR-223 on bulk cell growth or viability. We interpret these findings to suggest that Notch signaling does not have major effects on the miR transcriptome, and that up- or down-modulation of miR-223 in established T-ALL cells does not have significant effects on overall cell growth/viability. Further studies will be required to determine if miR-223 may act in concert with other Notch target genes to modulate cell physiology. Disclosures: No relevant conflicts of interest to declare.

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 8 T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cell progenitors in which activating mutations of NOTCH1 occur in over 50% of cases and loss-of-function mutations in FBW7, which degrades activated NOTCH1, occur in 8-16% of cases. We and others have characterized c-MYC as a critical downstream transcriptional target of Notch signaling in this context, and loss of FBW7 also contributes here as intact FBW7 marks c-MYC protein for proteosomal degradation. Inhibition of Notch signaling in T-ALL cells by various means including small molecule gamma-secretase inhibitors (GSI) causes cell cycle/growth arrest and in some cases apoptosis, providing rationale for NOTCH1 as a therapeutic target. The tumor suppressor PTEN is also mutated or lost in up to 20% of T-ALL cases. PTEN activity converts PIP3 to PIP2, and thus loss of PTEN potentiates PI3K signaling. It was observed among human T-ALL cell lines that PTEN loss correlated with resistance to Notch inhibition with GSI, leading to the idea that constitutive PI3K/AKT signaling relieves T-ALL cells of their “addiction” to oncogenic Notch signaling. Since GSI and other NOTCH1 signaling inhibitors are presently under investigation as targeted therapeutic agents for T-ALL and other cancers including breast, brain, colorectal, pancreatic, and melanoma, this idea that PTEN loss could confer resistance to NOTCH1 inhibition raises concern that patients with PTEN-negative (or presumably PIK3CA-mutated) diseases may fail Notch inhibitor therapy. As the studies linking GSI-resistance/Notch-independence to PTEN loss were limited to established cell lines, we sought to address this issue using a well established and genetically defined mouse retroviral transduction/bone marrow transplantation model. Briefly, we generated primary murine T-cell acute leukemias using mutated NOTCH1 retroviruses on both wild-type and PTEN-null backgrounds and tested these tumors for response to GSI. Unexpectedly, we observed these primary murine leukemias to undergo growth arrest and cell size reduction with GSI treatment on both wild-type and PTEN-null backgrounds. We even observed wild-type background tumors which had lost PTEN spontaneously after either serial transplantation or extended culture in vitro to remain sensitive to GSI. Given that most cases of human T-ALL have also lost expression of p16INK4A/p14ARF tumor suppressors either by deletion or silencing, and since p16INK4A/p14ARF are critical regulators at the G1/S cell cycle checkpoint, we also generated primary murine tumors on a Pten, Ink4a/Arf double-null background. Even when both PTEN and Ink4a/Arf were deleted, we still observed the murine tumors to remain sensitive to GSI. To confirm these findings were not unique to murine tumors, we examined a panel of 13 primary human T-ALL cases including 7 PTEN-positive cases (6 of which were NOTCH1 mutated) and 6 PTEN-negative cases (4 of which were NOTCH1 mutated). We found 11 of these 13 cases to be GSI sensitive (6 PTEN-positive, 5 PTEN-negative) as illustrated by decreased proliferation and reduction in cell size. We found only 2 cases to be GSI resistant, including 1 PTEN-positive and 1 PTEN-negative. Our panel contained only one case with FBW7 mutation; this case was PTEN-positive and GSI-sensitive. Thus, in contrast to previous studies with established cell lines, we find in this study using primary human T-ALL samples and genetically defined primary murine leukemias that PTEN loss indeed does not confer resistance to Notch inhibition. Of note, other groups have postulated a role for FBW7 mutation in conferring resistance to Notch inhibition; however, we encountered only 2 GSI-resistant cases in our panel of primary human samples, neither of which was FBW7 mutated. In generating the murine Notch leukemias, we also had the opportunity to compare disease penetrance/latency and clonality among the various genetic backgrounds. We noted that PTEN loss was associated with accelerated disease onset and multiclonal tumors as compared to wild-type, whereas INK4A/ARF loss alone had no such effect. These findings suggest NOTCH1 activation and PTEN loss may collaborate in leukemia induction; however, technical caveats related to potentially enhanced retroviral transduction efficiency of PTEN-null bone marrow progenitors limit this conclusion, and thus more definitive experiments to address this issue are needed. 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.