Notch1-induced T-ALL Research Articles

Abstract 3928: Therapeutic targeting of NOTCH1 and neddylation pathway in T cell acute lymphoblastic leukemia

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) is hematologic tumor characterized by the diffuse infiltration of the bone marrow by malignant hematopoietic cells expressing immature T cell markers. Although T-ALL currently has better cure rates primarily due to multiagent or intensified chemotherapy, the prognosis for patients who are resistant or develop relapse to therapy remains very poor. Aberrant Notch homolog-1 (NOTCH1) signaling is a major driver of T-ALL pathogenesis as more than 60% of T-ALL cases harbor activating mutations in the NOTCH1 gene. Gamma Secretase Inhibitors (GSIs) which effectively block the activation of oncogenic protein NOTCH1 are potential candidates for the treatment of T-ALL. However, the clinical application of GSIs is hampered by severe gastrointestinal toxicity due to the inhibition of NOTCH1 signaling in the gut. Here we demonstrate that combination therapy of GSIs and a small molecule inhibitor of the neddylation pathway circumvents the GSI-induced gut toxicity in vitro and in vivo. Genome-wide CRISPR loss-of-function screen in LS174T adenocarcinoma cells revealed neddylation pathway as a main regulator of massive goblet cell differentiation upon NOTCH1 inhibition. Genetic and pharmacologic inhibition of the neddylation pathway in LS174T cells rescued GSI-induced differentiation and cell death. Mechanistically, neddylation inhibition increases the protein stability of Hairy and enhancer of split-1 (HES1), a known regulator of absorptive and secretory cell fate decisions. Combination treatment of GSI and neddylation inhibitor in C57/BL6 mice showed a profound decrease in the number of goblet cells and maintained HES1 protein levels in the intestinal epithelium compared to GSI treatment alone. Remarkably, combined treatment of GSI and neddylation inhibitor in NOTCH1-induced T-ALL mice showed leukemia regression and improved overall survival without any associated gut toxicity. Overall, these results substantiate the potential of targeting NOTCH1 and neddylation pathway in the treatment of NOTCH1-induced T-ALL. Citation Format: Carla Bertulfo, Pablo Perez-Duran, Teresa Palomero, Adolfo Ferrando. Therapeutic targeting of NOTCH1 and neddylation pathway in T cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3928.

Abstract SY04-03: Identification of SIRT1 as a novel therapeutic target in T-ALL

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive hematological malignancy that affects both children and adults. Still, 20%-50% of patients show primary resistance or relapse after treatment, and ultimately die from their disease. Aberrant NOTCH1 signaling has a major role in the pathogenesis of TALL, as more than 60% of T-ALL cases harbor activating mutations in the NOTCH1 gene. In this context, small-molecule γ-secretase inhibitors (GSIs), which effectively block NOTCH1 activation via inhibition of a critical intramembrane proteolytic cleavage required for NOTCH1 signaling, are being tested in clinical trials for the treatment of relapsed and refractory T-ALL. However, the clinical development of anti-NOTCH1 therapies in T-ALL has been hampered by limited and delayed therapeutic response to these drugs, underscoring the need to identify novel therapeutic targets and to develop more effective drugs for the treatment of this disease. We previously demonstrated the importance of NOTCH1-driven metabolic pathways in the response to anti-NOTCH1 therapies (gamma-secretase inhibitors, GSIs). Moreover, epigenetic plasticity has also been proposed to mediate resistance to GSIs. Thus, we postulated that central regulators that control both the metabolic and epigenetic status of cells could act as master regulators of NOTCH1-induced transformation. Indeed, our results have identified the SIRT1 histone deacetylase, a central epigenetic and metabolic regulator, as a key player in T-ALL. Analyses of gene expression profiling data from T-ALL patients revealed a significant upregulation of SIRT1 in TALL. Consistently, SIRT1 protein levels are significantly upregulated in T-ALL cells as compared to normal human thymus. Moreover, through the integration of GSI-washout experiments, epigenetic profiling and CRISPR/Cas9-induced experiments, we have identified a distal enhancer of Sirt1 that is bound and controlled by NOTCH1, which might help explain the broad upregulation of Sirt1 observed in T-ALL patients. Next, and to formally test the effects of Sirt1 on T-cell transformation, we generated NOTCH1-driven primary T-ALLs from different Sirt1 genetic backgrounds. In this context, our results demonstrate that Sirt1 genetic overexpression leads to accelerated kinetics of NOTCH1-induced T-ALL and promotes resistance to GSI treatment in T-ALL in vivo in a deacetylase-dependent manner. Conversely, germinal loss of Sirt1 leads to delayed T-ALL development and reduced disease penetrance. Moreover, pharmacological inhibition of SIRT1 with EX-527 shows anti-leukemic and synergistic effects with NOTCH1 inhibition in T-ALL cell lines in vitro. Finally, genetic deletion of Sirt1 in already established primary isogenic Sirt1 conditional knockout leukemias leads to significant and highly synergistic anti-leukemic effects with GSI treatment in vivo. Mechanistically, acetyl-proteomics analyses revealed that acute deletion of Sirt1 consistently leads to hyperacetylation of Kat7 and Brd1, which are both part of a histone acetyltransferase complex. Indeed, Sirt1 loss results in global epigenetic changes including decreased levels of H4K12ac, which is a Kat7-target mark. Moreover, gene expression profiling analyses upon Sirt1 loss in leukemia in vivo revealed broad transcriptional changes. Gene-set enrichment analyses revealed that the transcriptional signature upon Sirt1 loss significantly correlates with the one obtained upon Kat7 loss, overall suggesting that Sirt1 loss leads to hyperacetylation of Kat7, which might be less active. Finally, our gene expression analyses also revealed a marked block in mTOR signaling, suggesting leukemia cells suffer a metabolic crisis upon Sirt1 loss. Consistently, acute deletion of Sirt1 results in prominent global metabolic changes in glycolysis, glutaminolysis and TCA, with concomitant activation of AMPK, resulting in markedly cytotoxic effects. Overall, our results reveal an oncogenic role for Sirt1 in T-ALL generation and progression, demonstrate that Sirt1 contributes to mediate resistance to anti-NOTCH1 therapies, identify a novel Notch1-Sirt1-Kat7 link and uncover Sirt1 as a novel therapeutic target for the treatment of T-ALL. Citation Format: Daniel Herranz. Identification of SIRT1 as a novel therapeutic target in T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY04-03.

A novel and highly effective mitochondrial uncoupling drug in T-cell leukemia

AbstractT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy. Despite recent advances in treatments with intensified chemotherapy regimens, relapse rates and associated morbidities remain high. In this context, metabolic dependencies have emerged as a druggable opportunity for the treatment of leukemia. Here, we tested the antileukemic effects of MB1-47, a newly developed mitochondrial uncoupling compound. MB1-47 treatment in T-ALL cells robustly inhibited cell proliferation via both cytostatic and cytotoxic effects as a result of compromised mitochondrial energy and metabolite depletion, which severely impaired nucleotide biosynthesis. Mechanistically, acute treatment with MB1-47 in primary leukemias promoted adenosine monophosphate–activated serine/threonine protein kinase (AMPK) activation and downregulation of mammalian target of rapamycin (mTOR) signaling, stalling anabolic pathways that support leukemic cell survival. Indeed, MB1-47 treatment in mice harboring either murine NOTCH1-induced primary leukemias or human T-ALL patient-derived xenografts (PDXs) led to potent antileukemic effects with a significant extension in survival without overlapping toxicities. Overall, our findings demonstrate a critical role for mitochondrial oxidative phosphorylation in T-ALL and uncover MB1-47–driven mitochondrial uncoupling as a novel therapeutic strategy for the treatment of this disease.

Abstract 3723: SIRT1 is a novel therapeutic target in T-ALL

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a NOTCH1-driven aggressive disease that requires treatment with intensified chemotherapy. Still, 20-50% of patients relapse and ultimately die, highlighting the need to identify novel therapeutic targets. We previously demonstrated the importance of NOTCH1-driven metabolic pathways in the response to anti-NOTCH1 therapies (gamma-secretase inhibitors, GSIs), and identified both glutaminolysis and autophagy as therapeutic targets that are highly synergistic with NOTCH1 inhibition in T-ALL in vivo. Moreover, epigenetic plasticity has also been proposed to mediate resistance to GSIs. Thus, we postulated that central regulators that control both the metabolic and epigenetic status of cells could act as master regulators of NOTCH1-induced transformation and might lead us to the identification of novel therapeutic targets in this disease. In this context, our results have identified the SIRT1 histone deacetylase, a central epigenetic and metabolic regulator, as a key player in T-ALL. Analyses of gene expression profiling data from T-ALL patients revealed a significant upregulation of SIRT1 in T-ALL patients. Consistently, SIRT1 protein levels are significantly upregulated in T-ALL cells as compared to normal human thymus. Next, and to formally test the effects of Sirt1 on T-cell transformation, we generated NOTCH1-driven primary T-ALLs from different Sirt1 genetic backgrounds. In this context, our results demonstrate that Sirt1 genetic overexpression leads to accelerated kinetics of NOTCH1-induced T-ALL and promotes resistance to GSI treatment in T-ALL in vivo in a deacetylase-dependent manner. Conversely, germinal loss of Sirt1 leads to delayed T-ALL development and reduced disease penetrance. Moreover, pharmacological inhibition of SIRT1 with Tenovin-6 shows anti-leukemic and synergistic effects with NOTCH1 inhibition in T-ALL cell lines in vitro. Finally, genetic deletion of Sirt1 in already established primary isogenic Sirt1 conditional knockout leukemias leads to significant and highly synergistic anti-leukemic effects with GSI treatment in vivo, resulting in complete cure of 10% of mice. Mechanistically, acute deletion of Sirt1 leads to broad transcriptional changes characterized by a block in mTOR signaling, suggesting leukemia cells suffer a metabolic crisis upon Sirt1 loss. Consistently, acute deletion of Sirt1 results in prominent global metabolic changes in glycolysis, TCA cycle and fatty acid oxidation pathways. Specifically, Sirt1 loss results in a significant glycolytic block with concomitant activation of AMPK, resulting in markedly cytotoxic effects. Overall, our results reveal SIRT1 as a critical major player in T-ALL; unveil an oncogenic role for Sirt1 in T-ALL generation and progression; demonstrate that Sirt1 mediates resistance to anti-NOTCH1 therapies; and uncover Sirt1 as a novel therapeutic target for the treatment of T-ALL. Citation Format: Olga Lancho, Victoria da Silva-Diz, Shirley Luo, Amarthya Singh, Hossein Khiabanian, Daniel Herranz. SIRT1 is a novel therapeutic target in T-ALL [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3723.

IL-7R is essential for leukemia-initiating cell activity of T-cell acute lymphoblastic leukemia

AbstractT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from the dysregulation of signaling pathways that control intrathymic T-cell development. Relapse rates are still significant, and prognosis is particularly bleak for relapsed patients. Therefore, development of novel therapies specifically targeting pathways controlling leukemia-initiating cell (LIC) activity is mandatory for fighting refractory T-ALL. The interleukin-7 receptor (IL-7R) is a crucial T-cell developmental pathway that is commonly expressed in T-ALL and has been implicated in leukemia progression; however, the significance of IL-7R/IL-7 signaling in T-ALL pathogenesis and its contribution to disease relapse remain unknown. To directly explore whether IL-7R targeting may be therapeutically efficient against T-ALL relapse, we focused on a known Notch1-induced T-ALL model, because a majority of T-ALL patients harbor activating mutations in NOTCH1, which is a transcriptional regulator of IL-7R expression. Using loss-of-function approaches, we show that Il7r-deficient, but not wild-type, mouse hematopoietic progenitors transduced with constitutively active Notch1 failed to generate leukemia upon transplantation into immunodeficient mice, thus providing formal evidence that IL-7R function is essential for Notch1-induced T-cell leukemogenesis. Moreover, we demonstrate that IL-7R expression is an early functional biomarker of T-ALL cells with LIC potential and report that impaired IL-7R signaling hampers engraftment and progression of patient-derived T-ALL xenografts. Notably, we show that IL-7R–dependent LIC activity and leukemia progression can be extended to human B-cell acute lymphoblastic leukemia (B-ALL). These results have important therapeutic implications, highlighting the relevance that targeting normal IL-7R signaling may have in future therapeutic interventions, particularly for preventing T-ALL (and B-ALL) relapse.

GATA3-Controlled Nucleosome Eviction Drives MYC Enhancer Activity in T-cell Development and Leukemia.

Long-range enhancers govern the temporal and spatial control of gene expression; however, the mechanisms that regulate enhancer activity during normal and malignant development remain poorly understood. Here, we demonstrate a role for aberrant chromatin accessibility in the regulation of MYC expression in T-cell lymphoblastic leukemia (T-ALL). Central to this process, the NOTCH1-MYC enhancer (N-Me), a long-range T cell-specific MYC enhancer, shows dynamic changes in chromatin accessibility during T-cell specification and maturation and an aberrant high degree of chromatin accessibility in mouse and human T-ALL cells. Mechanistically, we demonstrate that GATA3-driven nucleosome eviction dynamically modulates N-Me enhancer activity and is strictly required for NOTCH1-induced T-ALL initiation and maintenance. These results directly implicate aberrant regulation of chromatin accessibility at oncogenic enhancers as a mechanism of leukemic transformation. SIGNIFICANCE: MYC is a major effector of NOTCH1 oncogenic programs in T-ALL. Here, we show a major role for GATA3-mediated enhancer nucleosome eviction as a driver of MYC expression and leukemic transformation. These results support the role of aberrant chromatin accessibility and consequent oncogenic MYC enhancer activation in NOTCH1-induced T-ALL.This article is highlighted in the In This Issue feature, p. 1631.

Hedgehog pathway mutations drive oncogenic transformation in high-risk T-cell acute lymphoblastic leukemia.

The role of Hedgehog signaling in normal and malignant T-cell development is controversial. Recently, Hedgehog pathway mutations have been described in T-ALL, but whether mutational activation of Hedgehog signaling drives T-cell transformation is unknown, hindering the rationale for therapeutic intervention. Here, we show that Hedgehog pathway mutations predict chemotherapy resistance in human T-ALL, and drive oncogenic transformation in a zebrafish model of the disease. We found Hedgehog pathway mutations in 16% of 109 childhood T-ALL cases, most commonly affecting its negative regulator PTCH1. Hedgehog mutations were associated with resistance to induction chemotherapy (P = 0.009). Transduction of wild-type PTCH1 into PTCH1-mutant T-ALL cells induced apoptosis (P = 0.005), a phenotype that was reversed by downstream Hedgehog pathway activation (P = 0.007). Transduction of most mutant PTCH1, SUFU and GLI alleles into mammalian cells induced aberrant regulation of Hedgehog signaling, indicating that these mutations are pathogenic. Using a CRISPR/Cas9 system for lineage-restricted gene disruption in transgenic zebrafish, we found that ptch1 mutations accelerated the onset of notch1-induced T-ALL (P = 0.0001), and pharmacologic Hedgehog pathway inhibition had therapeutic activity. Thus, Hedgehog-activating mutations are driver oncogenic alterations in high-risk T-ALL, providing a molecular rationale for targeted therapy in this disease.

Leukemia-Associated Macrophages Educated By Organ-Specific Leukemic Microenvironment Acquire Diverse Functions and Gene-Expression Signatures

Macrophages are important components of microenvironments and participate diverse physiological and pathological processes. Though CD68+macrophages have been demonstrated a potentially clinical marker to predict adverse prognosis in lymphoma, the role of macrophages in leukemia have not been fully elucidated. Most recently, we reported leukemia-associated macrophages (LAMs), which showed leukemia-promoting effects, in Notch1-induced mouse T-ALL leukemic microenvironment. In the present study, we studied the characteristics of LAMs from BM and spleen in MLL-AF9 induced mouse AML model, and compared the gene expression diversities among LAMs from BM and SP of AML and T-ALL models.LAMs were sorted from leukemic BM and spleen in the middle stage of AML, control macrophages were sorted from normal mouse. Then LAMs were cocultured with AML cells for 24, 48 and 72hrs. Cell count, BrdU incorporation assay and Annexin V assay were used to detect the proliferation and apoptosis of leukemia cells, respectively. Significant increase of BrdU positive and decrease of apoptotic AML cells were detected in leukemia cells cocultured with LAMs than control Mϕ. Latex-beads uptaken assay and transwell assay were used to detect the phagocytosis and migration of LAMs. Significant decrease of phagocytosis and increase of migration ability were detected in LAMs than control Mϕ. Furthermore, T cell coculture assay revealed that spleen LAMs suppress the CD25 and CD69 expression in both CD4+ and CD8+T cells but increase induction of Treg cells than BM LAMs. Moreover SP LAMs had a significantly higher proliferation promoting effect on leukemia cells than BM LAMs. To explore the diverse effect among LAMs from BM and spleen, we compared the transcriptional data of LAMs from BM and spleen from AML and T-ALL. The ordered list algorithm revealed the similarity score in LAM samples with same microenvironment (991.8) is higher than those with same type of leukemia (709.1), also the expression pattern analysis revealed that LAMs from leukemic BM and spleen are much more different, the phenotypic gene validated by qRT-PCR revealed that the BM LAMs are more M1-like while the spleen LAMs are more M2-like.Our results suggested that the diverse response of LAMs to leukemia are largely determined by organ-specific leukemic microenvironment.The work was supported by the Grants 81370634, 81570153, 81300376 from the National Natural Science Foundation of China (NSFC). DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-181: KLF4 suppresses T-cell acute lymphoblastic leukemia by inhibiting the stress kinase MAP2K7 pathway

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with the highest incidence of relapse of any pediatric ALL. A minimal two-hit model of leukemogenesis suggests that an initial genetic driver transforms hematopoietic progenitor cells into LICs, whereas a secondary genetic alteration would endow LICs with proliferative and survival advantages. Although most T-ALL patients exhibit activating mutations in NOTCH1, the cooperating genetic events required to accelerate onset of leukemia and worsen disease progression are largely unknown. Here, we show that low levels of the transcription factor KLF4 in children with T-ALL were associated with methylation of its promoter. Consistent with a tumor suppressor function, loss of KLF4 accelerated the development of NOTCH1-induced T-ALL in mice by enhancing the G1-to-S transition and promoting the expansion of leukemia-initiating cells that are responsible for chemoresistance and relapses. Mechanistically, KLF4 represses the gene encoding the kinase MAP2K7, and thus loss of KLF4 activates MAP2K7 and downstream effector JNK both in murine model of T-ALL and lymphoblasts from pediatric patients with T-ALL. Furthermore, pharmacological inhibition of JNK reduced leukemia burden in a xenograft model of human T-ALL and small molecule inhibitors exhibited anti-leukemic properties in patient-derived xenograft cells. In summary, our findings demonstrate a novel tumor suppressor function of KLF4 in a T-ALL mouse model and in pediatric T-ALL and support a model of leukemia inhibition by repression of the stress kinase MAP2K7 and its downstream targets JNK, c-JUN, and ATF2. In addition, our study provides proof-of-principle pre-clinical data supporting JNK inhibition as a potential targeted therapy for T-ALL and prompts future studies in high-risk T-ALL patients with refractory and relapsed disease. Citation Format: Ye Shen, Chun Shik Park, Koramit Suppipat, Toni-Ann Mistretta, Monica Puppi, Terzah Horton, Karen Rabin, Daniel Lacorazza. KLF4 suppresses T-cell acute lymphoblastic leukemia by inhibiting the stress kinase MAP2K7 pathway. [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-181.

Effect of PDK1 on Notch1-Induced Mouse T-cell Acute Lymphoblastic Leukemia

To explore the role of PDK1 in T-ALL development through establishing the Notch1-induced T-ALL mouse model by using Mx1-cre; LoxP system to knock-out PDK1. Cell cycle and apoptosis of leukemic cells were detected by flow cytometry, and relative expression of tumor-related genes and transcription factors of leukemic cells were determined by quantitative real-time PCR. Notch1-induced T-ALL mouse model with inducible knock-out of PDK1 was established successfully. Compared to T-ALL control mouse model, PDK1 knock-out mice showed a significant longer survival time (P<0.01). There was no difference of cell cycle between control and PDK1 knock-out mice, and the apoptosis rate of leukemic cells in PDK1 knock-out mice was higher than that of control mice (P<0.001). PDK1 knock-out resulted in decreased expression of tumor-related genes and transcription factors, such as c-Myc and NF-κB (P<0.01), and increased expression level of P53 (P<0.01). PDK1 knock-out can inhibit the development of T-ALL, and its mechanism may be the leukemia progression inhibited by regulating the apoptosis and expression of multiple related genes and transcription factors.

T-ALL pathogenesis requires a NOTCH1-driven MYC enhancer.

A NOTCH1-bound MYC enhancer is necessary for normal T-cell development and NOTCH1-induced T-ALL.

Histone Demethylases Have Divergent Functions in T-ALL

Abstract The H3K27 demethylases UTX and JMJD3 have opposing roles in regulating NOTCH1-induced T-ALL growth.

Expression of P2X family receptors in peritoneal macrophages of mouse with acute T lymphoblastic leukemia

This study was aimed at exploring the expression pattern of P2X family receptors (P2XR) in peritoneal macrophages and their relationship with the activation states of macrophages in Notch1-induced mouse T-ALL model. After establishment of the leukemia model, F4/80(+) peritoneal macrophages, F4/80(+)CD206(+) M2-like and F4/80(+)CD206(-) M1-like peritoneal macrophages were sorted by flow cytometry based on F4/80 and CD206 surface markers. The expression of P2XR in each cell population was detected by real time RT-PCR. The results showed that macrophages,M1-like and M2-like macrophages moderately expressed P2XR except for P2X5R. The expression of P2XR varied with the development of leukemia. The expression of P2X1R and P2X7R in peritoneal macrophages increased steadily; the expression of P2X2R and P2X3R decreased at late stage of leukemia;the expression of P2X4R slightly decreased at intermediate stage;the expression of P2X6R kept unchanged. At intermediate stage of leukemia, the expression of P2XR in M1-like and M2-like peritoneal macrophages varied. M1-like macrophages expressed higher level of P2X1R than M2-like macrophages, whereas M2-like macrophages expressed higher level of P2X7R than M1-like macrophages, which suggested that the expression of P2XR were related to the activation states. It is concluded that the expression of P2XR in peritoneal macrophages from leukemia mice is related to the progression of leukemia and the activation states of macrophages, which lay a foundation for further studying the role of macrophages in the development of leukemia.

Functional expression of P2X family receptors in macrophages is affected by microenvironment in mouse T cell acute lymphoblastic leukemia

Nucleotides are important players in intercellular signaling communication network. P2X family receptors (P2XRs) are ATP-gated plasma membrane ion channels with diverse biological functions. Macrophages are important components in the microenvironment of hematopoiesis participating in both physiological and pathological processes. However, the role of P2XRs in macrophages in leukemia has not been established. Here we investigated expression pattern and functions of P2XRs in macrophages from bone marrow (BM) and spleen of Notch1-induced T-ALL mice. Real-time PCR showed that P2XRs except P2X5R were expressed in BM and spleen macrophages. Furthermore, with the development of leukemia, the expression of P2X7R increased in both BM and spleen macrophages whereas expression of P2X1R increased in spleen macrophages. Live cell imaging recoding the Ca2+ response demonstrated that P2X7R expressed in macrophages was functional. TUNEL and electron microscopy analysis found that apoptotic macrophages were frequently observed in BM and spleen at late stage of leukemia, which was partly contributed by the activation of overexpressed P2X7R. Our results suggested that the intercellular communication mediated by nucleotides might orchestrate in the pathological process of leukemia and could be a potential target for the treatment of leukemia.

Notching Up MYC Gives a LIC

In a recent issue of Cell, King etal. (2013) show that mutations in the ubiquitin ligase subcomponent FBXW7 increase MYC protein levels and expand the number of leukemia-initiating cells (LICs) in Notch1-induced T-ALL. Genetic and pharmacological inhibition of MYC abolishes LIC activity with broader therapeutic implications.

Distinct Effects of SCF and Hes1 On Normal Hematopoietic Stem and Progenitor Cells During Leukemic Cell Expansion in a T-ALL Mouse Model

Abstract 1216During leukemia development, emerging leukemic cells out-compete normal hematopoietic cells and become predominant in the body. How hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) respond to the growth of leukemic cell population is an important, yet less investigated area. Our previous study demonstrated differential effects of a leukemic environment on normal HSCs and HPCs in the Notch1-induced T-ALL mouse model (Hu X, et al. Blood 2009). We found that normal HSCs were better preserved in the leukemic bone marrow in part due to increased quiescence of the HSCs and in contrast, HPCs were exhausted during the expansion of leukemic cells. Our current work is aimed to further explore the molecular mechanisms concerning the distinct impacts of leukemic environment on normal HSCs and HPCs in the T-ALL mouse model.Given the previous report by others showing that increased secretion of stem cell factor (SCF) by myeloid leukemia cells played an important role in inducing normal HSCs/HPCs out of their niche and thus allowing leukemic cells to occupy the niche in the human-NOD/SCID xeno-graft model (Sipkins DA et al, Science 2008), we first examined the expression of SCF by ELISA, Western blot and real-time RT PCR in both normal hematopoietic and leukemic cell fractions in the Notch1-induced T-ALL mouse model as previously reported. We found that while expression of SCF in peripheral blood (PB) or bone marrow (BM) was increased in the leukemic mice, both mRNA and protein levels of SCF in normal hematopoietic cells were higher than that in leukemic cells, thereby suggesting that elevated SCF might be mainly secreted by non-leukemic cells in the leukemic hosts of our model. Further assessments on the role of SCF in leukemogenesis with the mice specifically deficient in SCF in different niche cell types are currently under investigation in our laboratory.In order to define potential mediators in HSCs in response to leukemic cell growth, a microarray study on normal HSCs isolated from T-ALL leukemic mice and the control mice was conducted. Gene expression profiling showed significantly differed expression of 169 genes (127 up and 42 down). Especially, real-time RT PCR confirmed an increase of Hes1, p21, Fbxw11, IL-18R1 and Itgb3, and a decrease of CXCR4 and Mmp2. Interestingly, the expression of Hes1 and its target gene, p21 were elevated in normal HSCs but not in HPCs, letting us to hypothesize that Hes1 might be in part mediate the different responses of HSCs and HPCs to the T-ALL leukemic environment. To test this hypothesis, we ectopically expressed Hes1 in normal hematopoietic cells and then examined their functions under the leukemic condition. BM cells from B6.SJL mice were transduced with either MSCV-Hes1-IRES-GFP or control MSCV-GFP vector. After transduction, Hes1-GFP+or control-GFP+cells were co-transplanted with the Notch1-induced T-ALL cells into lethally irradiated C57BL/6J recipients. The engrafted cells from the leukemic BM were analyzed and Hes1-GFP+or control-GFP+cells were sorted for functional assessments. Interestingly, although over-expression of Hes1 inhibited the growth of colony forming cell (CFC) in vitro, it could potentiate the long-term repopulating cells by maintaining more cells in the quiescent (G0) state in vivo. Taken together, our current study supports a role of Hes1 in mediating the distinct responses of normal HSCs and HPCs to the T-ALL leukemic environment. Disclosures:No relevant conflicts of interest to declare.

Bortezomib suppresses the growth of leukemia cells with Notch1 overexpression in vivo and in vitro

Bortezomib has been widely used in the treatment of various cancers; however, its exact mechanisms of action are not fully understood, particularly in acute T lymphoblast leukemia (T-ALL). Here, we visualize the anti-leukemia effect of bortezomib in both human T-ALL cell line and animal models. In vitro study, a human T-ALL cell line bearing Notch1 mutations, MOLT-4, was treated with bortezomib. At clinically achievable concentrations, bortezomib inhibited cell growth by inducing G1 phase arrest and apoptosis with a dose-dependent manner. A murine tumor xenograft model was achieved by subcutaneous injection of MOLT-4 cells for in vivo study. Administration of bortezomib significantly reduced tumor mass volume when compared with controls. Of note, bortezomib inhibited growth of leukemia cells in a Notch1-induced murine T-ALL model, and the life span of leukemia-bearing mice was markedly increased. Further studies revealed that bortezomib led to inhibited expression of Notch1 target genes. Taken together, our results demonstrate that bortezomib shows significant anti-leukemia effect in T-ALL bearing Notch1 mutations in vitro and in vivo. The present study provides evidence that bortezomib might be a candidate therapeutic reagent in the treatment of T-ALL.