Heather Leu

A Pan-BCL2 Inhibitor Renders Bone-Marrow-Resident Human Leukemia Stem Cells Sensitive to Tyrosine Kinase Inhibition

Leukemia stem cells (LSCs) play a pivotal role in the resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) and its progression to blast crisis (BC), in part, through the alternative splicing of self-renewal and survival genes. To elucidate splice-isoform regulators of human BC LSC maintenance, we performed whole-transcriptome RNA sequencing, splice-isoform-specific quantitative RT-PCR (qRT-PCR), nanoproteomics, stromal coculture, and BC LSC xenotransplantation analyses. Cumulatively, these studies show that the alternative splicing of multiple prosurvival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSCS that are quiescent in the marrow niche and that contribute to therapeutic resistance. Notably, sabutoclax, a pan-BCL2 inhibitor, renders marrow-niche-resident BC LSCs sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice-isoform expression in BC LSC maintenance and suggest that the combinatorial inhibition of prosurvival BCL2 family proteins and BCR-ABL may eliminate dormant LSCs and obviate resistance.

A novel patient-derived intra-femoral xenograft model of bone metastatic prostate cancer that recapitulates mixed osteolytic and osteoblastic lesions

Prostate cancer metastasizes to bone in the majority of patients with advanced disease leading to painfully debilitating fractures, spinal compression and rapid decline. In addition, prostate cancer bone metastases often become resistant to standard therapies including androgen deprivation, radiation and chemotherapy. There are currently few models to elucidate mechanisms of interaction between the bone microenvironment and prostate cancer. It is, thus, essential to develop new patient-derived, orthotopic models. Here we report the development and characterization of PCSD1 (Prostate Cancer San Diego 1), a novel patient-derived intra-femoral xenograft model of prostate bone metastatic cancer that recapitulates mixed osteolytic and osteoblastic lesions.MethodsA femoral bone metastasis of prostate cancer was removed during hemiarthroplasty and transplanted into Rag2-/-;γc-/- mice either intra-femorally or sub-cutaneously. Xenograft tumors that developed were analyzed for prostate cancer biomarker expression using RT-PCR and immunohistochemistry. Osteoblastic, osteolytic and mixed lesion formation was measured using micro-computed tomography (microCT).ResultsPCSD1 cells isolated directly from the patient formed tumors in all mice that were transplanted intra-femorally or sub-cutaneously into Rag2-/-;γc-/- mice. Xenograft tumors expressed human prostate specific antigen (PSA) in RT-PCR and immunohistochemical analyses. PCSD1 tumors also expressed AR, NKX3.1, Keratins 8 and 18, and AMACR. Histologic and microCT analyses revealed that intra-femoral PCSD1 xenograft tumors formed mixed osteolytic and osteoblastic lesions. PCSD1 tumors have been serially passaged in mice as xenografts intra-femorally or sub-cutaneously as well as grown in culture.ConclusionsPCSD1 xenografts tumors were characterized as advanced, luminal epithelial prostate cancer from a bone metastasis using RT-PCR and immunohistochemical biomarker analyses. PCSD1 intra-femoral xenografts formed mixed osteoblastic/osteolytic lesions that closely resembled the bone lesions in the patient. PCSD1 is a new primary prostate cancer bone metastasis-derived xenograft model to study metastatic disease in the bone and to develop novel therapies for inhibiting prostate cancer growth in the bone-niche.

Alu-dependent RNA editing of GLI1 promotes malignant regeneration in multiple myeloma

Despite novel therapies, relapse of multiple myeloma (MM) is virtually inevitable. Amplification of chromosome 1q, which harbors the inflammation-responsive RNA editase adenosine deaminase acting on RNA (ADAR)1 gene, occurs in 30–50% of MM patients and portends a poor prognosis. Since adenosine-to-inosine RNA editing has recently emerged as a driver of cancer progression, genomic amplification combined with inflammatory cytokine activation of ADAR1 could stimulate MM progression and therapeutic resistance. Here, we report that high ADAR1 RNA expression correlates with reduced patient survival rates in the MMRF CoMMpass data set. Expression of wild-type, but not mutant, ADAR1 enhances Alu-dependent editing and transcriptional activity of GLI1, a Hedgehog (Hh) pathway transcriptional activator and self-renewal agonist, and promotes immunomodulatory drug resistance in vitro. Finally, ADAR1 knockdown reduces regeneration of high-risk MM in serially transplantable patient-derived xenografts. These data demonstrate that ADAR1 promotes malignant regeneration of MM and if selectively inhibited may obviate progression and relapse.The treatment of multiple myeloma is challenging due to high relapse rates. Here the authors show that expression of ADAR1 correlates with poor patient outcomes, and that ADAR1-mediated editing of GLI1 is a mechanism relevant in the context of multiple myeloma progression and drug resistance.

Abstract 3351: Aberrant RNA editing of GLI1 promotes malignant regeneration in multiple myeloma

Introduction: Despite novel therapies, most of multiple myeloma (MM) patients relapse as a result of clonal evolution in inflammatory microenvironments. Adenosine-to-inosine (A-to-I) RNA editing, driven by inflammatory cytokine-responsive adenosine deaminase acting on RNA1 (ADAR1), promotes cancer progression by enhancing survival and self-renewal of malignant progenitor cells. Amplifications of chromosome 1q21, containing IL-6R and ADAR1 loci, occur frequently in high-risk MM patients, who frequently develop secondary plasma cell leukemia (PCL) and have shorter survival. While increased IL-6 signaling has been linked to relapse and A-to-I editing contributes to therapeutic resistance in a broad array of malignancies, the role of ADAR1 in MM pathogenesis has not been elucidated. This study aimed to investigate whether pro-inflammatory cues in MM activate ADAR1 editing thereby promoting malignant regeneration. Procedures: Publicly available primary patient datasets were analyzed and validated in a separate cohort of biobanked primary samples and human myeloma cell lines. Lentiviral vector-mediated activation or knockdown of ADAR1, or treatment with extrinsic pro-inflammatory stimuli, was utilized to probe the functional impact of RNA editing activity in MM models. Site-specific qPCR was used to quantify RNA editing in specific cancer stem cell-associated loci. Functional effects of ADAR1 activity were assessed in in vitro survival and self-renewal assays, and in novel in vivo PCL xenografts. Results: Patients harboring 1q21 amplification showed significant and stage-dependent increases in ADAR1 expression. In a set of separate primary PCL samples, aberrant RNA editing in the coding region of the Hedgehog (Hh) pathway transcription factor GLI1 was observed in high ADAR1-expressing samples. Notably, increased GLI1 editing, previously reported to have increased capacity to activate its transcriptional targets, was detected in serially transplantable, patient-derived xenograft models. Furthermore, abolition of ADAR1 editase activity impaired GLI1 editing. Lastly, in vitro pro-inflammatory IL-6 stimulation, or continuous exposure to the immunomodulatory drug lenalidomide led to increased ADAR1 mRNA and protein levels, with a concomitant induction of RNA editing activity. Conclusions: In MM, 1q21 amplification has been linked to progression. We provide new evidence linking expression and activity of ADAR1, located on 1q21, and disease stage. Because ADAR1 induces transcript recoding, A-to-I editing could contribute to the marked transcriptomic diversity typical of advanced MM. While the Hh pathway has been linked to cancer stem cell generation in human MM, here we identified a primate-specific mechanism of Hh pathway activation in MM through RNA editing-dependent stabilization of GLI1. Together, both genetic and microenvironmental factors modulate epitranscriptomic deregulation of cancer stem cell pathways in MM. Citation Format: Elisa Lazzari, Nathaniel Delos Santos, Christina Wu, Heather Leu, Gabriel Pineda, Shawn Ali, Caitlin Costello, Mark Minden, Raffaella Chiaramonte, Leslie Crews, Catriona Jamieson. Aberrant RNA editing of GLI1 promotes malignant regeneration in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3351. doi:10.1158/1538-7445.AM2017-3351

Abstract 2414: ADAR1-dependent RNA editing is a mechanism of therapeutic resistance in human plasma cell malignancies

Introduction Multiple myeloma (MM) is a plasma cell malignancy that accounts for more than 10% of all blood cancers and may progress to plasma cell leukemia (PCL). Despite treatment, virtually all patients become unresponsive to treatment. RNA editing is a post-transcriptional pre-mRNA processing activity that represents an unexplored potential source of clonal molecular heterogeneity contributing to therapeutic resistance. In particular, adenosine deaminase acting on RNA (ADAR) 1, which exists in two isoforms, one constitutive and one inflammation-responsive, has been associated with disease progression and cancer stem cell (CSC) maintenance. The aim of this study was to investigate whether enhanced ADAR1 expression and activity contributed to therapeutic resistance of MM and PCL. Procedures 1) ADAR Quantification: Whole gene and isoform-specific qRT-PCR was used to detect ADAR1 expression in PCL and MM primary samples and in human MM cell lines (HMCL). 2) RNA Editing Detection: We developed a RNA editing site-specific qPCR (RESS-qPCR) assay to detect RNA editing in cancer stem-cell associated transcripts. 3) Therapeutic Resistance Assay. A MM cell line was exposed to lenalidomide continuously in vitro to establish a model of therapeutic resistance. 4) Development of a humanized PCL mouse model: We established novel in vivo PCL primagrafts by intrahepatic transplantation of primary total mononuclear cells into neonatal RAG2-/-gc-/- mice. Results Approximately, 30% of MM patients in the MM Genomic Initiative dataset harbor copy number amplifications of the ADAR locus on chromosome 1q21, which portends a poor prognosis. We observed significantly increased ADAR1 expression in primary PCL samples and aberrant RNA editing of the stem cell transcription factor GLI1 and the DNA cytidine deaminase APOBEC3D. Notably, high-ADAR1-expressing PCL cells successfully engrafted in RAG2-/-gc-/- mice. As the inflammation-responsive isoform of ADAR1 was upregulated in primary samples, we sought to explore the effects of the anti-MM agent and immunomodulatory drug lenalidomide on ADAR1 expression and activity. Continuous in vitro exposure to lenalidomide led to increased ADAR1 mRNA and protein level and a potent induction of RNA editing activity. Increased RNA editing was detected in several cancer and stem cell-associated transcripts, including GLI1, APOBEC3D, AZIN1 and MDM2. Notably, this aberrant RNA editing activity was associated with increased self-renewal capacity in vitro and a cancer stem cell phenotype. Conclusions ADAR1 overexpression and deregulated RNA editing represents a unique source of RNA and proteomic diversity, and may confer a survival and self-renewal advantage to MM cells. This research identifies ADAR1 as a new diagnostic and therapeutic target in MM, and establishes a robust humanized PCL primagraft model for future pre-clinical testing of ADAR1 modulatory agents. Citation Format: Elisa Lazzari, Leslie A. Crews, Christina Wu, Heather Leu, Shawn Ali, Raffaella Chiaramonte, Mark Minden, Caitlin Costello, Catriona H.M. Jamieson. ADAR1-dependent RNA editing is a mechanism of therapeutic resistance in human plasma cell malignancies. [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 2414.

Abstract A43: Novel prostate cancer patient-derived xenograft models of bone metastatic castrate-resistant prostate cancer

Prostate cancer metastasis to bone occurs in 50-90% of men with advanced disease for which there is no cure. Bone metastasis leads to debilitating fractures and severe bone pain. It is associated with disease progression, therapy resistance, poor prognosis, and rapid decline. Androgen ablation therapy is standard of care for advanced prostate cancer, however, the role of androgens in bone metastatic prostate cancer is not understood. The effects of anti-androgens as seen on bone scans can also be inconsistent with the biochemical PSA response. There are few pre-clinical models to understand the interaction between the bone microenvironment and prostate cancer. It is essential to understand the unique interaction of prostate cancer with the bone environment and to develop novel therapies that target these pathways. Here we report the development of novel patient-derived intra-femoral xenograft models of prostate bone metastatic cancer. METHODS: Surgical prostate cancer bone metastasis specimens were transplanted by direct injection into the femurs of Rag2-/-γc-/- mice or sub-cutaneously into the right flank. Patient-derived xenograft (PDX) tumors that grew out were analyzed for prostate cancer biomarker expression using quantitative RT-PCR and immunohistochemistry. Bone lesion formation was measured using micro-computed tomography (μCT). RESULTS: Prostate cancer surgical bone metastasis specimens have been collected from which we have established new serially transplantable, prostate cancer bone metastasis xenograft models – PCSD1, PCSD4 and PCSD5. PCSD1 (Prostate Cancer San Diego 1) was molecularly characterized as advanced, luminal epithelial-type prostate cancer. PCSD1 intra-femoral xenografts formed mixed osteoblastic/osteolytic lesions that closely mimicked those of the patient. Treatment with the anti-androgen, bicalutamide, did not inhibit intra-femoral PCSD1 xenograft growth although there was a decrease in PSA as seen in some patients treated with anti-androgen who had discordant PSA and bone scan tests. CONCLUSION: PCSD1, PCSD4 and PCSD5 are new patient-derived prostate cancer bone metastasis-derived xenograft models. PCSD1 xenograft model closely recapitulates the mixed osteolytic/osteoblastic bone metastatic lesions seen in patients, and we are using it to develop novel therapies for inhibiting prostate cancer growth in the bone-niche. Citation Format: Christina Jamieson, Christina Wu, Amy Strasner, Jason R. Woo, Michelle Muldong, Young B. Jeong, Michael A. Liss, Omer Raheem, Tomonori Yamaguchi, Heather Leu, Deborah Marshall, Sheldon Morris, Nicholas A. Cacalano, Koichi Masuda, Catriona H.M. Jamieson, Anna A. Kulidjian, Christopher J. Kane. Novel prostate cancer patient-derived xenograft models of bone metastatic castrate-resistant prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A43.

Abstract 5103: RNA editing enzyme induces accelerated cell cycle in normal hematopoiesis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We have previously shown that inflammation-responsive RNA editase ADAR1 (ademosine deaminases acting on dsRNA) is a contribution factor in CML (chronic myeloid leukemia) disease progression. Whole transcriptome sequencing (RNA-Seq) data on primary CML chronic phase and CML blast crisis patient demonstrated over-representation of inflammatory IFN-pathways involved in hematological development. The resulting ADAR1 up-regulation plays important roles in both stem cell differentiation and self-renewal, as indicated by in vitro colony-formation assay and in vivo CML xenotransplantation mouse model. Though we have established ADAR1-mediated RNA editing as a novel theraputic target for treating CML, we do not yet understand the underlying mechanism of RNA editases involvement in normal hematopoietic stem cells self-renewal and differentiation. In our new study, we describe ADAR1s role in cell cycle regulation of normal hematopoietic stem cell and its molecular editing targets. Normal cord bloods or aged bone marrow samples (CD34+) were lentivirally transduced with either backbone or ADAR1-overexpression vector. The cell cycle status of progenitors was analyzed by FACS and immunofluorescence. The expressions of genes in various cell cycle stages were analyzed by qRT-PCR. CML BC (CD34+) was transduced with lenti-shADAR1 and transplanted into immunodeficient mice. After 20 weeks, hematopoietic organs were harvested and analyzed for cell cycle status by FACS and serial transplantation. Our results demonstrated that ADAR1 accelerates G0 to G1 phase transition in normal hematopoietic stem cells, coupled with increased cell size and elevated expression of Ki67. The expanded population maintains stemness without any significant increase in differentiation. qRT-PCR microarray of cell cycle genes indicates that p21 expression level is reduced by >70% when ADAR1 is overexpressed. Moreover, shRNA knockdown of ADAR1 in CML BC sample shows a reduction of engraftment in bone marrow and spleen, and an enrichment of G0 population in the remaining cells. A decrease of self-renewal capacity as demonstrated by serial engraftment suggests the residual LSC failed to propagate. Our finding suggests carefully regulated A-to-I editing by ADAR1 is essential for the maintenance of proper cell grow and proliferation in HSC. It is plausible that the elevated expression level of ADAR1 observed in CML BC LSC contributes to false regulation of cell cycle that leads to the expansion of malignant leukemia stem cells. Citation Format: Qingfei Jiang, Marianna Zipeto, Angela Cournt, Heather Leu, Leslie Crews, Sheldon Morris, Catriona Jamieson. RNA editing enzyme induces accelerated cell cycle in normal hematopoiesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5103. doi:10.1158/1538-7445.AM2014-5103

Abstract 1011: NOTCH1 signaling is essential for leukemia initiating cell self-renewal in T-ALL

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Leukemia initiating cells (LIC) contribute to therapeutic resistance through mutations in cellular self-renewal and survival pathways. NOTCH1 mutations are common in T-cell acute lymphoblastic leukemia (T-ALL). However, the role of NOTCH1 activation in human LIC propagation has not been established. Pediatric T-ALL serially transplantable LIC were found to be enriched in the CD34+CD4− and CD34+CD7− fractions of newly diagnosed patient samples. More recently, a CD7+CD1a− glucocorticoid resistant LIC population, capable of engrafting leukemia in NOD/SCID IL2R gamma null (NSG) mice, was identified in primary adult T-ALL. To identify the molecularly characterized potential LIC populations in pediatric T-ALL without proceeding in vitro culture and examine the role of NOTCH1 activation in LIC propagation. 12 pediatric T-ALL samples were sequenced for NOTCH1 mutation examination. Humanized LIC mouse models were established and dosed with either NOTCH1 mAb or IgG1 mAb control at 10 mg/kg intraperitoneally every 4 days for 6 doses. Mice were sacrificed one day after the last dose, and hematopoietic organs were collected for FACS analysis. To further define the LIC populations in pediatric T-ALL, CD34+CD38+CD2+CD7+Lin− and CD34+CD38+CD2+CD7−Lin− cells were isolated from T-ALL primary patients’ blood by FACS sorting and transplanted into neonatal RAG2−/−γc−/− mice to determine their leukemic engraftment potential. Serial transplantations were done for testing the LIC self-renewal capacity. Mouse hematopoietic organs were collected for FACS analysis, mouse brains were sectioned for human cells examination by immunohistochemistry. NOTCH1 and its downstream gene expressions were examined by q-RT-PCR between the T-ALL CD34+ and CD34− populations. Six of 12 pediatric T-ALL patient samples were found NOTCH1 mutation. Mice transplanted with CD34+ and CD34+CD2+CD7+ or CD34+CD2+CD7− cells developed a T-ALL-like disease characterized by pale BM and enlarged spleen, thymus and liver. Human CD34+ enriched cells from NOTCH1 mutated T-ALL maintained leukemic engraftment while an equivalent number of CD34+ cells from NOTCH1 wild type T-ALL did not. T-ALL CD34+ progenitors from NOTCH1 mutated T-ALL have a significant higher engraftment in BM when compared with those from NOTCH1 wild type T-ALL. CD34+CD2+CD7+ and CD34+CD2+CD7− populations are more prominent in NOTCH1 mutated samples. Both the human CD34+ and CD34+CD2+CD7+ populations were significantly reduced in BM when treated with hN1 mAb in vivo. NOTCH1 and its downstream genes expression were significantly reduced in NOTCH1 mutated CD34+ cells when compared with CD34− cells. Human T-ALL LIC have enhanced NOTCH1 expression; CD34+CD2+CD7+ and CD34+CD2+CD7− subpopulations are enriched for LIC activity in pediatric T-ALL; A selective hN1 mAb inhibits human T-ALL LIC survival and self-renewal in vivo. 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 1011. doi:1538-7445.AM2012-1011

Abstract 5217: RNA editing enzyme ADAR1 drives leukemia stem cell differentiation and self-renewal in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is the first cancer that was shown to originate from a genetic abnormality - the Philadelphia chromosome translocation, and production of its constitutively active protein tyrosine kinase product, BCR-ABL. The disease progresses slowly from chronic phase to accelerated phase, and later transforms to blast crisis (BC) stage. Cancer stem cells (CSCs) are a subset of tumor cells that have acquired certain treatment-resistant stem cell properties. High levels of RNA editing are associated with a primitive transcriptional program typical of human embryonic stem cells, and RNA editing plays an important role in both embryonic hematopoietic cell fate determination and in maintenance of normal hematopoiesis. Human RNA editing occurs primarily in secondary structures created by Alu retroelements and is carried out by enzymes such as the adenosine deaminase acting on RNA (ADAR) family. Among these, ADAR1 was also recently shown to be required for normal hematopoiesis by suppressing interferon-induced apoptosis. Our research focuses on dissecting the role of ADAR-mediated RNA editing in normal human hematopoietic progenitor cell development compared with malignant editing programs that may be activated in leukemia stem cells (LSC) during the progression of human CML. Our data demonstrates that BC LSC harbor increased levels of the interferon-responsive ADAR1 p150 isoform compared with chronic phase (CP) progenitors and normal cord blood progenitors. Expression of this isoform also exhibits a positive correlation with BCR-ABL expression levels - an effect which is specific to BC progenitors, suggesting that ADAR1 expression correlates with disease progression from CP to BC. In vitro hematopoietic progenitor assays with normal cord blood progenitors and CP samples transduced with lentiviral vectors overexpressing human ADAR1 reveals a significant shift in cell differentiation fate towards granulocyte-macrophage progenitor (GMP) colonies, which has been shown to be the initiating LSC population in CML Correspondingly, a progression towards erythroid lineage was observed in BC CML LSC transduced with lentiviral vectors expressing shRNA targeting ADAR1. Further qRT-PCR analyses revealed that the mechanism through which ADAR1 drives LSC and HSC differentiation towards myelopoiesis involves regulation of PU.1, which in turn inhibits GATA1 expression. Moreover, in vivo studies in a robust humanized CML mouse model showed a significant decrease in LSC serial transplantation potential of lentiviral shADAR1-transduced BC progenitors transplanted into neonatal RAG2-/-γc-/- mice. Together, these data support a crucial role for ADAR1 in cell fate determination and self-renewal potential of hematopoietic stem cells in both normal human progenitors and in malignant LSC that drive disease progression and therapeutic resistance. 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 5217. doi:1538-7445.AM2012-5217

Abstract 974: A selective Notch1 mAb targets leukemia progenitor cells in T-ALL

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Difficulties in maintaining primary cultures of leukemia cells hampered efforts to investigate the biology of human T cell acute lymphoblastic leukemia (T-ALL) underscoring the need for a direct transplantation model to characterize leukemia progenitor cells (LPC) in vivo and as a paradigm for screening candidate drugs that inhibit self-renewal pathways active in T-ALL. Approximately, 50% of patients with T-ALL harbor NOTCH1 activating mutations that promote therapeutic resistance providing the impetus for developing selective NOTCH1-inhibitory therapeutic strategies. To investigate 1) whether a select subclone of T-ALL cells harbor a greater capacity to propagate disease in vivo than other clones, 2) to establish a humanized T-ALL LPC mouse model and 3) to test whether a selective NOTCH1-NRR/Fc (hN1) mAb inhibits LPC survival and self-renewal. Experimental Procedures: To facilitate non-invasive in vivo monitoring of leukemic engraftment, lentiviral luciferase transduced LPC were intrahepatically transplanted into neonatal immune deficient mice to establish humanized T-ALL LPC mouse models. These models were treated with hN1 mAb or a control IgG1 mAb at the dose of 10 mg/kg every 4 days for 21 days, and another group was treated with mouse IgG1 isotype control at the same dosing plan. Mice were sacrificed one day after the last dose. Thymus, spleen, liver and bone marrow (BM) were collected and analyzed by FACS. Some BM were sectioned for CD45, NOTCH1 and active Caspase 3 examination by immunohistochemistry. Results: Human CD34+ enriched cells maintained leukemic engraftment while an equivalent number of Lin+ cells did not. T-ALL CD34+ progenitors from 32 T-ALL LPC models established with NOTCH1 mutated T-ALL have a significant higher engraftment in BM when compared with those from 14 T-ALL LPC models established with Non-NOTCH1 mutated T-ALL. Human CD45+CD34+CD2+ population in serial transplant recipients was more prominent in NOTCH1 mutated samples. Human CD34+ populations were significantly reduced in both BM (p < 0.01, Student t test) and spleen (p < 0.05, Student t test) when the T-ALL LPC treated with hN1 mAb, while CD45+ populations were also significantly reduced in both BM and spleen (p < 0.05, Student t test). NOTCH1 expression level in human CD34+ cells in NOTCH1 mutated T-ALL was markedly reduced after hN1 mAb treatment when compared with IgG1 mAb treatment. NOTCH1 and CD45 positive cells were significantly reduced, while apoptosis was remarkably increased in the BM treated with therapeutic hN1 mAb when compared with those treated with IgG1 mAb. Intracellular domain of Notch1 was significantly reduced in the BM treated with hN1 mAb when compared with those treated with IgG1 mAb. Conclusions: 1. Human T-ALL LPC have enhanced NOTCH1 expression. 2. Human self-renewing T-ALL LPC are enriched in the CD45+CD34+CD2+ population. 3. A selective hN1 mAb inhibits human T-ALL LPC survival and self-renewal in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 974. doi:10.1158/1538-7445.AM2011-974