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In mouse mid-gestational embryos, definitive hematopoietic stem progenitor cells are derived directly from a very small proportion of the arterial endothelium. However, the physiological mechanisms restraining excessive endothelial-hematopoietic transition remain elusive. We show here that genetic deletion of Smad4 from the endothelium stage (using Tie2-Cre), but not from embryonic hematopoietic cells (using Vav-Cre), leads to a strikingly augmented emergence of intra-arterial hematopoietic clusters and an enhanced in vitro generation of hematopoietic progenitors, with no increase in the proliferation and survival of hematopoietic cluster cells. This finding indicates a temporally restricted negative effect of Smad4 on the endothelial to hematopoietic progenitor transition. Furthermore, the absence of endothelial Smad4 causes an increased expression of subaortic bone morphogenetic protein 4 and an activation of aortic extracellular signal-regulated kinase, thereby resulting in the excessive generation of blood cells. Collectively, our data for the first time identify a physiological suppressor that functions specifically during the transition of endothelial cells to hematopoietic progenitors and further suggest that endothelial Smad4 is a crucial modulator of the subaortic microenvironment that controls the hematopoietic fate of the aortic endothelium.

Aims: Human histone lysine-specific demethylase 1(LSD1) inhibit gene transcription and thereby block myeloid differentiation, apoptosis and cell cycle in AML. 4SC-202, a novel inhibitor of LSD1, is potential therapeutic agent for treating Myelodysplastic syndrome, while its mechanism of action remains unclear. M ainly method : In the study, LSD1 and HO-1 gene expression were detected in MDS/AML patients. After MDS cell line SKM-1 was treated with 4SC-202, cell viability, apoptosis and cell cycle were detected by CCK-8 or FACS. Lastly, western blotting analyzed that the agent caused the change of the related cellular function protein in SKM-1 cells. K ey findings: We found that LSD1 and HO-1 were overexpression in MDS/AML patients. LSD1 inhibitor 4SC-202 inhibited cell viability. The agent induced apoptosis by up-regulated BAX and cleaved caspase3/9 as well as down-regulated BCL-2, arrested cell cycle in the G2/M phase by down-regulated CDK1 and up-regulated p21, as well as inhibited activation of the NF-κB pathway and decreased HO-1. LSD1 and HO-1 expression were decreased after exposure with the NF-κB inhibitor OBY11-7082. Silencing LSD1 by LSD1 siRNA hardly caused apoptosis in SKM-1 cells, while the combination of Bay11-7082 and silencing LSD1 significantly decreased LSD1 and HO-1 expression and further increased apoptosis. While up-regulated HO-1 significantly limited the 4SC-202-induced down-regulation of BCL-2, up-regulation of cleaved caspase 3/9 and suppressed activation of NF-κB pathway as well as thereby attenuated the agent efficacy. S ignificant : In conclusion, LSD1 inhibitor 4SC-202 induced apoptosis through the NF-κB-mediated HO-1 pathway. LSD1 might be a potential target for the treatment of MDS. Disclosures No relevant conflicts of interest to declare.

Background Multiple myeloma (MM), one of the most prevalent hematological malignancies, is characterized by the proliferation of abnormal plasma cells. The proteasome inhibitor bortezomib is a frontline drug in the treatment of MM; however, most patients will develop acquired resistance to bortezomib, which has been a critical challenge in clinic. Our previous studies have indicated that inhibition of heme oxygenase-1 (HO-1) can attenuate drug resistance to MM. Moveover, emerging evidence suggests that growth arrest-specific gene 6 (Gas6) may regulate tumor microenvironment and serve as a novel target for cancer therapy. However, whether HO-1 mediated-Gas6 production involved in the development of bortezomib resistance in MM remains unclear. Aims The aims to perform the present study were to: 1) assess the association between intracellular HO-1 and Gas6 expression in the bone marrow of CD138+ cells of patients with MM; 2) test the role of HO-1 and Gas6 in the development of bortezomib resistance in MM; 3) explore whether HO-1 mediate Gas6 production and the potential mechanism. Methods Patients with different stages of MM (n=18) and healthy donors (n=18) at Affiliated Hospital of Guizhou Medical University were included in this study. Baseline clinical characteristic data and bone marrow samples were obtained from study participants. CD138+ plasma cells were purified from bone marrow samples. The recombinant lentiviral vector of HO-1 was constructed and subsequent transfected into U266 and RPMI8226 cells. The expressions of mRNA and protein levels were determined by real-time PCR and western blot, respectively. Secreted Gas6 was determined by ELISA. Flow cytometry was used to assess cell apoptosis. Results In human study, the HO-1 mRNA and protein level in CD138+ cells of patients with MM showed a significant increase compared with those in healthy group, respectively (P Conclusion These results demonstrate that inhibition of HO-1 attenuates bortezomib resistance in MM via down-regulating Gas6 production. Targeting these pathological interactions holds promise for improve MM treatment. Disclosures No relevant conflicts of interest to declare.

OBJECTIVE: NR4A1 is a member of the orphan nuclear receptor family, which is involved in biological processes such as cell proliferation, apoptosis, metabolism, and differentiation. The expression of NR4A1 is increased in a variety of solid tumors, which promotes oncogenesis and enhances the viability of tumor cells. However, in hematological malignancies, its expression is significantly lower than normal. Studies have shown that clearance of NR4A1 can lead to the progression of mice to acute myeloid leukemia, reducing the amount of NR4A gene expression can make mice progress to MDS / MPN, and even acute myeloid leukemia. This indicates that NR4A1 is an important tumor suppressor in acute myeloid leukemia. The present study was to investigate the effect of drug-induced NR4A1 expression on apoptosis in acute myeloid leukemia and its related mechanisms. The results suggested that NR4A1 may become a new target for the treatment of acute myeloid leukemia. METHODS: In vitro, on the one hand, bone marrow mononuclear cells from patients with newly diagnosed or relapsed acute myeloid leukemia were extracted as samples, and the expression of NR4A1 mRNA and protein was detected by Real-time PCR and Western blot. Acute myeloid leukemia cell lines HL60 and Kasumi-1 were tested for fenretinide activity and apoptosis, as well as NR4A1 and mitochondrial apoptosis pathway-related protein expression. We used siNR4A1 to knockdown NR4A1 expression and LMB to inhibit nuclear export, the apoptosis rate and apoptosis protein were detected. The mechanism of anti-apoptotic effect of NR4A1 was verified by western blot, immunofluorescence and co-immunoprecipitation. AML mice model were established and fenretinide was injected into the tail veins to observe the effects of NR4A1 expression on survival time, blood routine, bone marrow and important organs. Results: 1. NR4A1 expression in patients with acute myeloid leukemia was significantly higher than normal. 2. The expression of NR4A1 increased in a time- and concentration-dependent manner under the action of fenretinide. 3. After siNR4A1, the apoptosis of acute myeloid leukemia cells was significantly decreased, and the expression of apoptotic proteins was decreased. 4. The expression of apoptotic proteins was decreased after fenretinide combined with leptomycin treatment. After fenretinide treatment, the expression of NR4A1 was decreased in nuclear extracts, and the expression of mitochondrial extract was increased. After drug treatment, NR4A1 interacted with Bcl-2, and the bcl-2 BH3 domain in cytoplasm was exposed to play an anti-apoptotic effect. After successful modeling of AML mice, the survival rate of the fenretinide treatment group was significantly prolonged compared with the control group. The peripheral blood leukocytes, hemoglobin and platelet values were significantly different. The liver and spleen volume of the treatment group were significantly smaller than that of the control group. The mRNA and protein levels of NR4A1 in the spleen were significantly higher than those in the control group. Conclusion: The effect of fenretinide on acute myeloid leukemia cells induces NR4A1 expression and plays a pro-apoptotic effect. This apoptosis effect depends on the nuclear export of NR4A1. NR4A1 is located from the nucleus to the mitochondria, and binds to the Bcl-2 BH3 domain. Key words: NR4A1, fenretinide, acute myeloid leukemia, apoptosis, nuclear export Disclosures No relevant conflicts of interest to declare.

Purpose Smad ubiquitin regulatory factor 2(Smurf2) as a member of ubiquitinated E3 ligase was certificated to regulate the ubiquitination level of target gene. In this study, smurf2 as tumor suppressor was studied in acute B lymphoblastic leukemia, Histone deacetylases 3(HDAC3) which was connected with smurf2 was related with poor prognosis. It is further proved that smurf2 and HDAC3 were expected to be therapeutic targets for B-ALL cells and improve the prognosis of patients with B-ALL. Methods Realtime-PCR and westernblot were used to detect the expression of smurf2 and HDAC3 in healthy donors and B-ALL patients by us. Sup-B15 and CCRF-SB were treated with MG-132, small interfering RNA of smurf2 or HDAC3. Up-regulating smurf2 plasmid was also transfected to B-ALL cells by lipo3000. Flow cytometry and westernblot were used to validate the difference of apoptosis and cell cycle. Results Smurf2 mRNA in B-ALL patients was significantly lower than that in healthy donors. HDAC3 was higher in B-ALL patients than that in healthy donors. Protein level of smurf2 and HDAC3 was consistent with mRNA level. Up-regulating smurf2 by plasmid or MG-132 and silencing smurf2 could influence HDAC3, which further depressed JAK/STAT3 signal pathway inducing apoptosis of B-ALL cells. Sup-B15 and CCRF-SB were treated with siHDAC3 and MG-132 for 24h, we discovered that silencing HDAC3 enhanced the apoptosis level of B-ALL cells MG-132 inducing by depressing JAK/STAT3 pathway. It was expected to become a therapeutic target for improving the clinical prognosis of B-ALL patients. On the other hand, we found that MG-132 caused G2/M phase arrest in B-ALL cells and successfully inhibited the JAK/STAT3 pathway leading to apoptosis. Conclusions Silencing HDAC3 inhibited the JAK/STAT3 pathway and further enhanced apoptosis in B-ALL cells MG-132 inducing. HDAC3 and smurf2 were expected to become therapeutic targets for clinical treatment of acute B lymphocytic leukemia and improve survival rate of leukemia patients. Key words Smurf2; HDAC3; Apoptosis; Prognosis; Disclosures No relevant conflicts of interest to declare.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma (NHL). It is a group of adult lymphomas with a large clinical presentation and prognosis heterogeneity. In order to improve the cure rate of diffuse large B-cell lymphoma, it is necessary to find a new DLBCL treatment plan that can control the progression of the disease and explore its unknown potential therapeutic targets. Epigenetic modification has become a new treatment for leukemia in recent years, and histone deacetylase inhibitor (HDACi) is a novel drug for the treatment of DLBCL in recent years. However, in addition to effective clinical efficacy, Pan-HDACis has side effects. In contrast, LMK-235 is well tolerated as a novel inhibitor of HDAC4 and HDAC5. HACE1 is a potential tumor suppressor gene located on chromosome 6q21, which is involved in the regulation of tumor cell growth and apoptosis by mediating autophagy and ubiquitination of Rac1. The potential target of HACE1 as an HDAC inhibitor plays an important role in the occurrence, invasion and metastasis of human malignant tumors, but its underlying mechanism of HDACi-mediated apoptosis is still unclear. In this study, we investigated the potential mechanisms by which HACE1 regulates LMK-235-mediated apoptosis in DLBCL cells. In vitro, LMK-235 mediates apoptosis in DLBCL cells in a dose-dependent manner, and up-regulation of HACE1 by lentiviral transfection promotes LMK-235-induced apoptosis. At the same time, high levels of HACE1 can inhibit the expression of HO-1 at mRNA and protein levels. As a previous study in our laboratory, low expression of HO-1 enhanced the apoptotic rate of DLBCL cells. However, the correlation between HACE1 and HO-1 has not been reported. Therefore, we regulated HACE1 levels and found that HACE1 regulates HO-1, which affects the sensitivity of DLBCL cells to LMK-235. Our data show that HACE1 plays a key role in LMK-235-induced apoptosis in DLBCL cells. This provides new hope for the treatment of diffuse large B-cell lymphoma and is a potential new target for the treatment of diffuse large B-cell lymphoma. Disclosures No relevant conflicts of interest to declare.

The serine/threonine PIM protein kinases are critical regulators of turmorigenesis in mutiple hematologic malignancies and solid cancers. We used real-time PCR to detect the expression of PIM in B-cell acute lymphocytic leukemia (B-ALL) patients, and found the expression of PIM in B-ALL patients was significantly higher than that in normal controls. SMI-4a is a pan-PIM small molecule inhibitor, and this agent exhibits demonstrable preclinical antitumour activity in a wide range of hematologic malignant cell lines. To further explore the effect of SMI-4a on B-ALL cells, B-ALL cell lines CCRF-SB and Sup-B15 were treated with this small molecule inhibitor, and the results showed that SMI-4a inhibited B-ALL cell proliferation in a dose- and time-dependent manner. Moreover, SMI-4a significantly promoted B-ALL cell apoptosis and caused cell cycle arrest in the G0/G1 phase. The results of Western blot showed that SMI-4a increased the expression of Caspase-3, Caspase-9, Bax and P21, and decreased the expression of Bcl-2 and CDK4. Furthermore, we found that SMI-4a significantly inhibits the activation of the JAK2/STAT3 pathway and HO-1 interferes with the JAK2/STAT3 pathway to inhibit SMI-4a-induced ALL cell apoptosis. Finally, xenograft experiments in NOD/SCID mice were operated to investigate the potential role of SMI-4a in B-ALL tumorigenesis in vivo. To observe the effect of SMI-4a on tumor growth in vivo, NOD/SCID mice were transplanted with B-ALL devied cells, and the tumor-bearing mice were intraperitoneally injected with saline and SMI-4a, respectively. As a result, tretment with SMI-4a resulted in a significant inhibition on tumor growth. In addition, PIM inhibtor obviously reduced the volume and weight of B-ALL cell-derived tumors. TUNEL assay revealed the proportion of apoptotic cells was higher in the SMI-4a-treated group than in the control group. Taken together, our data showed PIM inhibitor (SMI-4a) significantly inhibits the growth of B-ALL cells in vitro and in vivo and promotes apoptosis and cell cycle arrest. This suppressive effect is mediated partly through inhibiting the JAK2/STAT3 pathway activation. Disclosures No relevant conflicts of interest to declare.

The fetal liver (FL) serves as a predominant site for expansion of functional hematopoietic stem cells (HSCs) during mouse embryogenesis. However, the mechanisms for HSC development in FL remain poorly understood. In this study, we demonstrate that deletion of activating transcription factor 4 (ATF4) significantly impaired hematopoietic development and reduced HSC self-renewal in FL. In contrast, generation of the first HSC population in the aorta-gonad-mesonephros region was not affected. The migration activity of ATF4−/− HSCs was moderately reduced. Interestingly, the HSC-supporting ability of both endothelial and stromal cells in FL was significantly compromised in the absence of ATF4. Gene profiling using RNA-seq revealed downregulated expression of a panel of cytokines in ATF4−/− stromal cells, including angiopoietin-like protein 3 (Angptl3) and vascular endothelial growth factor A (VEGFA). Addition of Angptl3, but not VEGFA, partially rescued the repopulating defect of ATF4−/− HSCs in the culture. Furthermore, chromatin immunoprecipitation assay in conjunction with silencing RNA-mediated silencing and complementary DNA overexpression showed transcriptional control of Angptl3 by ATF4. To summarize, ATF4 plays a pivotal role in functional expansion and repopulating efficiency of HSCs in developing FL, and it acts through upregulating transcription of cytokines such as Angptl3 in the microenvironment.

Acute myeloid leukemia (AML) with MLL translocation is recognized as a group of aggressive hematopoietic malignancies with poor prognosis. An atypical homeodomain protein family (TALE family, three-amino-acid loop extension) has been reported to be involved in leukemogenesis and leukemia progression. For example, MEIS1 and PBX3 were abnormally expressed in examined AML patient samples when compared with their normal counterparts. While MEIS1 has been studied extensively, the precise role of PBX3 in leukemia development is still largely unknown. In this study, we explored the epigenetic regulatory network of PBX3 and the specific role of PBX3 in leukemia progression. By analyzing the clinical database, we found that the high expression of PBX3 iscorrelated with poor prognosis of AML patients. In MLL-fusion induced AML mouse models that were established by retrovirus-mediated ectopic expression of MLL-AF9 or MLL-NRIP3 genes in murine BM progenitor cells (c-Kit+). PBX3 also showed high expression level in leukemia stem cells (LSC) compared with normal HSPCs. Unlike other homeobox genes such as HOXA9 and MEIS1 that were both highly expressed in LSKs and LSCs, PBX3 only showed extremely high expression in LSCs in comparison with HSPCs or other normal hematopoietic cells. To explore the role of PBX3 in leukemia, we analyzed the epigenetic modification patterns along PBX3 gene. By analyzing the public ChIP-seq database, we found that increased activation mark H3K79me2 and decreased repressive mark H3K27me3 exhibited on PBX3 in LSCs. Additionally, transcription activating modifications H3K79 hypermethylation of PBX3 was only observed in LSCs but not LSKs or GMPs, consistent with increased PBX3 expression level in LSCs compared with HSPCs. To further confirm this, we assessed H3K79me2 level of PBX3 in purified c-Kit+ cells which are enriched with stem cells from leukemic or control mice by ChIP-qPCR. Consistently, ChIP-qPCR results revealed the similar trend with dataset analysis. Moreover, we also detected the transcription repressive mark H3K9me3 on PBX3 in c-Kit+ cells and found a dramatic decrease of H3K9me3 in LSCs in comparison with HSPCs. Thus, our results suggested that high expression level of PBX3 in LSCs might be caused by elevated activation mark H3K79me2 and depressed repressive mark H3K27me3 and H3K9me3. Considering that high expression level of PBX3 in LSCs may contribute to leukemic progression, we used the CRISPR/Cas9 system to delete PBX3 to examine its potential role in MLL-AF9 mediated AML development. We found that PBX3 inactivation significantly prolonged the survival of leukemic mice (median survival was 25 days of control, 31 days of SgPBX3-a, 30 days of SgPBX3-b, 10 mice per group). Morphological analysis revealed more leukemic blast cells with segment nuclei exhibited in PBX3 inactivation group, suggesting that PBX3 deficiency is associated with increased leukemic cell differentiation. Since LSC was considered to be sufficient to drive leukemogenesis and maintain leukemia progression, we investigated the impact of PBX3 inactivation on LSC capability. In vitro colony forming assay showed that PBX3 inactivation group formed significantly fewer colonies of total numbers and type A clones. Flow cytometric analysis showed decreased LSCs frequency in murine leukemia cells of PBX3 inactivation group. Moreover, limiting dilution transplantation assays revealed 4-7 fold decreased of functional LSCs after PBX3 inactivation with the 1/102 of LSCs in control leukemic bone marrow in comparison with 1/442 in SgPBX3-a and 1/741 in SgPBX3-b respectively. Further analysis suggested that depletion of functional LSCs in PBX3 inactivation group mainly through accelerating apoptosis of LSCs, evidenced by both increased Annexin V positive cell frequency of whole BM blasts and LSCs in PBX3 inactivation group. In conclusion, we found PBX3 is epigenetically dis-regulated in LSCs of MLL-r AMLs and is essential for leukemia development. Inactivation of PBX3 could suppress leukemia progression by reducing LSCs frequency and impairing its function. More importantly, the differential expression of PBX3 in normal and malignant hematopoietic cells provides itself potentially as a prognostic marker and therapeutic target for MLL-rearranged leukemia. HD.G and YJ.C contributed equally to this work Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem cells (HSCs) exist as a rare population with two essential properties of self-renewal and differentiation. HSCs can give rise to all hematopoietic progenitor and mature cells. While critical for a full understanding of the hematopoietic process and HSC-related clinical applications, the mechanisms of self-renewal and differentiation of HSCs remain elusive. The PI3K-Akt signaling pathway plays essential roles in the regulation of hematopoiesis. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) activates multiple AGC kinases including Akt and is a pivotal regulator in this pathway. PDK1 phosphorylates Akt at its T308 residue and regulates the functional development of B and T cells during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined. In this study, we generated PDK1 conditional knockout mice Vav-Cre;PDK1fl/fl (PDK1Δ/Δ) to explore the roles of PDK1 in HSCs. While PDK1Δ/Δ mice have reduced B and T cell counts as previously described, their LT-HSCs and ST-HSCs were significantly increased in comparison with WT mice while MPPs and CMPs were decreased after PDK1 deletion, indicating that the loss of PDK1 perturbed the steady-state hematopoiesis. Furthermore, although deletion of PDK1 increased the frequency of HSCs, PDK1-deficient HSCs fail to reconstitute the hematopoietic system when PDK1-deficient HSCs were used in bone marrow transplantation and competitive transplantation experiments in comparison to the WT HSCs, indicating that PDK1 is vital for hematopoiesis. To explore the mechanisms by which PDK1 regulates HSC function, we examined the cell cycle status and found the percentage of PDK1Δ/Δ HSCs was decreased significantly in G0 stage while increased in G1 and S/G2/M phases. This suggests an increase in HSC exit from a quiescent state. Since MPPs were significantly decreased in bone marrow, we examined the percentage of Annexin V+ DAPI- PDK1Δ/Δ and WT MPPs and found that they are comparable. This indicates that apoptosis did not cause the decrease in MPPs. In addition, a total of 300 LT-HSCs from PDK1Δ/Δ or WT mice and competitor cells were transplanted into lethally irradiated recipient mice to examine whether the decrease in MPPs is due to a defect in HSC differentiation. We found that less than 1% of MPPs arose from PDK1Δ/Δ HSCs 12 weeks after transplantation, indicating that PDK1 is required for the differentiation from LT-HSCs to MPPs. Because the full activation of Akt requires cooperative phosphorylation at its S473 and T308 residues by mTORC2 and PDK1, respectively, we also investigated the function of HSCs in RictorΔ/Δ PDK1Δ/Δ (DKO) mice in conjunction with RictorΔ/Δ or PDK1Δ/Δ mice to explore how mTORC2 and/or PDK1 influence Akt function in HSCs. The flow cytometric analyses of peripheral blood and bone marrow samples revealed very similar parameters of RictorΔ/Δ PDK1Δ/Δ and PDK1Δ/Δ mice. Interestingly, Rictor seemed to exert a minimal impact on HSCs and MPPs. More importantly, in contrast to RictorΔ/Δ, RictorΔ/Δ PDK1Δ/Δ HSCs failed to reconstitute the hematopoietic system after transplantation as PDK1Δ/Δ HSCs, suggesting that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function. To explore the mechanism that leads to the defect in HSCs due to loss of PDK1, we assessed ROS levels in PDK1-deficient HSCs and found that PDK1-deficient LSKs and HSCs exhibit greatly reduced ROS levels when compared with the control HSCs. Treating PDK1-deficient BM cells with BSO in vitro increased cellular ROS levels and the colony counts of PDK1-deficient BM cells significantly. Notably, the recovery effect was only observed with BSO concentrations lower than 0.03 mM. This suggests that ROS levels are precisely controlled in HSCs. Higher or lower ROS levels beyond the normal range are both harmful to normal HSC functions. Since increased SDFα expression is associated with cellular ROS levels in various cells including hematopoietic cells, we also treated PDK1Δ/Δ mice with SDFα and found that it couldpartially rescue the defective differentiation ability of PDK1-deficient HSCs. In addition, we found that PDK1 deletion could significantly prolong the life span and inhibit the leukemia development in murine T-ALL model via altering leukemic cell differentiation and proliferation. Taken together, PDK1 controls HSC differentiation via regulating cellular ROS levels and regulates malignant hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Abstract 1504 Leukemic cells are heterogeneous, so defining the differences of leukemic cell sub-populations in response to drug treatment may ultimately help design better strategies to maximize the efficacy of chemotherapeutic agents for patients. We have begun to study this issue with Notch1-induced T-cell acute lymphoblastic leukemia (T-ALL) in mice. The Notch1 signaling pathway plays an essential role in cell growth and differentiation, and activated mutations in the Notch1 gene are frequently observed in human T-ALL. Over-expression of the intracellular Notch1 domain (ICN1) in murine hematopoietic cells is able to induce robust T-ALL in mice. In our current study, we examined T-ALL cells, specifically the CD8 and CD4 double positive (CD8+CD4+) and CD8 single positive (CD8+CD4−) cell sub-populations, for their distinct sensitivities to drug treatment. We first treated T-ALL mice with a cell-cycle nonspecific chemical drug, cyclophosphamide (CTX), or a mTORC1 inhibitor, rapamycin, when the overall T-ALL cell population rose to more than 10% in mouse peripheral blood. We found that CTX treatment effectively reduced the T-ALL cells and led to an increase in the ratio of CD8+CD4+ versus CD8+CD4− T-ALL, indicating that CD8+CD4− T-ALL cells were more sensitive to CTX treatment. In contrast, rapamycin had the opposite effect. When compared with mice treated with rapamycin or CTX alone, the combination of the two drugs led to a significant improvement for T-ALL bearing mice with respect to leukemia initiation, progress and overall survival. We then set out to investigate the mechanism of different sensitivities of CD8+CD4+ and CD8+CD4− T-ALL cells upon CTX or rapamycin treatments. We found that CD8+CD4+ and CD8+CD4−T-ALL cell groups responded similarly to CTX treatment after 12 hours of treatment. However, CD8+CD4−T-ALL cells continued to undergo apoptosis 24 hours after CTX treatment. The expression of the anti-apoptosis gene Bcl-2 in CD8+CD4+ T-ALL cells was significantly higher than that in CD8+CD4− T-ALL cells, while the expression of the pro-apoptosis genes Bax, p53 and Noxa in CD8+CD4+ T-ALL cells was lower than that of CD8+CD4−T-ALL cells after 24 hour CTX treatment. This suggests that CD8+CD4+ and CD8+CD4− T-ALL cells activate apoptosis differently after CTX treatment. Interestingly, rapamycin treatment did not affect apoptosis in the same manner as CTX treatment, but instead, it arrested more T-ALL cells in the G0 phase. More importantly, only CD8+CD4+, but not CD8+CD4−T-ALL cells were sensitive to rapamycin treatment in arresting cell cycle at different time points. The expression levels of CDK2 and CDK4 were significantly lower in CD8+CD4+ T-ALL cells when compared with CD8+CD4− T-ALL cells, while p27 expression in CD8+CD4+ T-ALL cells was higher than that of CD8+CD4− T-ALL cells after rapamycin treatment for 3 or 10 days. These data provide a molecular basis for the distinct apoptotic and cell cycle arrest responses of CD8+CD4+ and CD8+CD4− T-ALL cell subsets to CTX or rapamycin treatment. Taken together, our study documents previously unappreciated, yet distinct properties of CD8+CD4+ and CD8+CD4−Notch1-induced T-ALL cells in the context of specific drug treatment and suggests a therapeutic potential of combining CTX and rapamycin treatment for T-ALL patients. This strategy may also help design better chemotherapeutical regimes for other types of leukemia and cancer. Disclosures: No relevant conflicts of interest to declare.