Activated natural killer cells predict poor clinical prognosis in high-risk B- and T-cell acute lymphoblastic leukemia.

B- and T-cell acute lymphoblastic leukemia (B/T-ALL) may be refractory or recur after therapy by suppressing host anticancer immune surveillance mediated specifically by natural killer (NK) cells. We delineated the phenotypic and functional defects in NK cells from high-risk patients with B/T-ALL using mass cytometry, flow cytometry, and in silico cytometry, with the goal of further elucidating the role of NK cells in sustaining acute lymphoblastic leukemia (ALL) regression. We found that, compared with their normal counterparts, NK cells from patients with B/T-ALL are less cytotoxic but exhibit an activated signature that is characterized by high CD56, high CD69, production of activated NK cell-origin cytokines, and calcium (Ca2+) signaling. We demonstrated that defective maturation of NK cells into cytotoxic effectors prevents NK cells from ALL from lysing NK cell-sensitive targets as efficiently as do normal NK cells. Additionally, we showed that NK cells in ALL are exhausted, which is likely caused by their chronic activation. We found that increased frequencies of activated cytokine-producing NK cells are associated with increased disease severity and independently predict poor clinical outcome in patients with ALL. Our studies highlight the benefits of developing NK cell profiling as a diagnostic tool to predict clinical outcome in patients with ALL and underscore the clinical potential of allogeneic NK cell infusions to prevent ALL recurrence.

Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-ß-catenin signaling.

Tyrosine kinase inhibitors (TKIs) are highly effective in treatment of chronic myeloid leukemia (CML) but do not eliminate leukemia stem cells (LSCs), which remain a potential source of relapse. TKI treatment effectively inhibits BCR-ABL kinase activity in CML LSCs, suggesting that additional kinase-independent mechanisms contribute to LSC preservation. We investigated whether signals from the bone marrow (BM) microenvironment protect CML LSCs from TKI treatment. Coculture with human BM mesenchymal stromal cells (MSCs) significantly inhibited apoptosis and preserved CML stem/progenitor cells following TKI exposure, maintaining colony-forming ability and engraftment potential in immunodeficient mice. We found that the N-cadherin receptor plays an important role in MSC-mediated protection of CML progenitors from TKI. N-cadherin-mediated adhesion to MSCs was associated with increased cytoplasmic N-cadherin-ß-catenin complex formation as well as enhanced ß-catenin nuclear translocation and transcriptional activity. Increased exogenous Wnt-mediated ß-catenin signaling played an important role in MSC-mediated protection of CML progenitors from TKI treatment. Our results reveal a close interplay between N-cadherin and the Wnt-ß-catenin pathway in protecting CML LSCs during TKI treatment. Importantly, these results reveal novel mechanisms of resistance of CML LSCs to TKI treatment and suggest new targets for treatment designed to eradicate residual LSCs in CML patients.

The Src and c-Kit kinase inhibitor dasatinib enhances p53-mediated targeting of human acute myeloid leukemia stem cells by chemotherapeutic agents.

The SRC family kinases (SFKs) and the receptor tyrosine kinase c-Kit are activated in human acute myeloid leukemia (AML) cells. We show here that the SFKs LYN, HCK, or FGR are overexpressed and activated in AML progenitor cells. Treatment with the SFK and c-KIT inhibitor dasatinib selectively inhibits human AML stem/progenitor cell growth in vitro. Importantly, dasatinib markedly increases the elimination of AML stem cells capable of engrafting immunodeficient mice by chemotherapeutic agents. In vivo dasatinib treatment enhances chemotherapy-induced targeting of primary murine AML stem cells capable of regenerating leukemia in secondary recipients. Our studies suggest that enhanced targeting of AML cells by the combination of dasatinib with daunorubicin may be related to inhibition of AKT-mediated human mouse double minute 2 homolog phosphorylation, resulting in enhanced p53 activity in AML cells. Combined treatment using dasatinib and chemotherapy provides a novel approach to increasing p53 activity and enhancing targeting of AML stem cells.

T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.

Induction treatments for acute myeloid leukemia (AML) have remained largely unchanged for nearly 50 years, and AML remains a disease of poor prognosis. Allogeneic hematopoietic cell transplantation can achieve cures in select patients and highlights the susceptibility of AML to donor-derived immunotherapy. The interleukin-3 receptor α chain (CD123) has been identified as a potential immunotherapeutic target because it is overexpressed in AML compared with normal hematopoietic stem cells. Therefore, we developed 2 chimeric antigen receptors (CARs) containing a CD123-specific single-chain variable fragment, in combination with a CD28 costimulatory domain and CD3-ζ signaling domain, targeting different epitopes on CD123. CD123-CAR-redirected T cells mediated potent effector activity against CD123+ cell lines as well as primary AML patient samples. CD123 CAR T cells did not eliminate granulocyte/macrophage and erythroid colony formation in vitro. Additionally, T cells obtained from patients with active AML can be modified to express CD123 CARs and are able to lyse autologous AML blasts in vitro. Finally, CD123 CAR T cells exhibited antileukemic activity in vivo against a xenogeneic model of disseminated AML. These results suggest that CD123 CAR T cells are a promising immunotherapy for the treatment of high-risk AML.

A critical role for SHP2 in STAT5 activation and growth factor-mediated proliferation, survival, and differentiation of human CD34+ cells.

SHP2, a cytoplasmic protein-tyrosine phosphatase encoded by the PTPN11 gene, plays a critical role in developmental hematopoiesis in the mouse, and gain-of-function mutations of SHP2 are associated with hematopoietic malignancies. However, the role of SHP2 in adult hematopoiesis has not been addressed in previous studies. In addition, the role of SHP2 in human hematopoiesis has not been described. These questions are of considerable importance given the interest in development of SHP2 inhibitors for cancer treatment. We used shRNA-mediated inhibition of SHP2 expression to investigate the function of SHP2 in growth factor (GF) signaling in normal human CD34(+) cells. SHP2 knockdown resulted in markedly reduced proliferation and survival of cells cultured with GF, and reduced colony-forming cell growth. Cells expressing gain-of-function SHP2 mutations demonstrated increased dependency on SHP2 expression for survival compared with cells expressing wild-type SHP2. SHP2 knockdown was associated with significantly reduced myeloid and erythroid differentiation with retention of CD34(+) progenitors with enhanced proliferative capacity. Inhibition of SHP2 expression initially enhanced and later inhibited STAT5 phosphorylation and reduced expression of the antiapoptotic genes MCL1 and BCLXL. These results indicate an important role for SHP2 in STAT5 activation and GF-mediated proliferation, survival, and differentiation of human progenitor cells.

Persistence of leukemia stem cells in chronic myelogenous leukemia patients in prolonged remission with imatinib treatment.

Imatinib mesylate treatment markedly reduces the burden of leukemia cells in chronic myelogenous leukemia (CML) patients. However, patients remain at risk for relapse on discontinuing treatment. We have previously shown that residual BCR-ABL(+) progenitors can be detected in CML patients within the first 2 years of imatinib treatment. However, reduced rates of relapse and continued decline of BCR-ABL levels with prolonged treatment, together with the ability of selected patients to maintain remission after discontinuing treatment, led us to investigate whether prolonged imatinib exposure resulted in reduction or elimination of BCR-ABL(+) stem cells. We evaluated BCR-ABL expression in CD34(+)CD38(+) (38(+)) committed progenitors and CD34(+)CD38(-) (38(-)) stem/primitive progenitor cells in samples from CML patients on imatinib treatment for at least 4 years with cytogenetic and molecular response. High levels of BCR-ABL expression were maintained over time in the 38(-) stem cell fraction. The absolute frequency of BCR-ABL(+) cells as determined by limiting dilution analysis was consistently higher in 38(-) compared with 38(+) cells. Transplantation into NOD/SCID-IL2Rγ-chain knockout mice demonstrated that BCR-ABL(+) cells had long-term in vivo repopulating capacity. These results directly demonstrate that BCR-ABL(+) stem cells persist in CML patients despite prolonged treatment with imatinib, and support ongoing efforts to target this population.

Intrinsic suppression of type I interferon production underlies the therapeutic efficacy of IL-15-producing natural killer cells in B-cell acute lymphoblastic leukemia.

BACKGROUND: Type I interferons (IFN-Is), secreted by hematopoietic cells, drive immune surveillance of solid tumors. However, the mechanisms of suppression of IFN-I-driven immune responses in hematopoietic malignancies including B-cell acute lymphoblastic leukemia (B-ALL) are unknown. METHODS: Using high-dimensional cytometry, we delineate the defects in IFN-I production and IFN-I-driven immune responses in high-grade primary human and mouse B-ALLs. We develop natural killer (NK) cells as therapies to counter the intrinsic suppression of IFN-I production in B-ALL. RESULTS: We find that high expression of IFN-I signaling genes predicts favorable clinical outcome in patients with B-ALL, underscoring the importance of the IFN-I pathway in this malignancy. We show that human and mouse B-ALL microenvironments harbor an intrinsic defect in paracrine (plasmacytoid dendritic cell) and/or autocrine (B-cell) IFN-I production and IFN-I-driven immune responses. Reduced IFN-I production is sufficient for suppressing the immune system and promoting leukemia development in mice prone to MYC-driven B-ALL. Among anti-leukemia immune subsets, suppression of IFN-I production most markedly lowers the transcription of IL-15 and reduces NK-cell number and effector maturation in B-ALL microenvironments. Adoptive transfer of healthy NK cells significantly prolongs survival of overt ALL-bearing transgenic mice. Administration of IFN-Is to B-ALL-prone mice reduces leukemia progression and increases the frequencies of total NK and NK-cell effectors in circulation. Ex vivo treatment of malignant and non-malignant immune cells in primary mouse B-ALL microenvironments with IFN-Is fully restores proximal IFN-I signaling and partially restores IL-15 production. In B-ALL patients, the suppression of IL-15 is the most severe in difficult-to-treat subtypes with MYC overexpression. MYC overexpression promotes sensitivity of B-ALL to NK cell-mediated killing. To counter the suppressed IFN-I-induced IL-15 production in MYChigh human B-ALL, we CRISPRa-engineered a novel human NK-cell line that secretes IL-15. CRISPRa IL-15-secreting human NK cells kill high-grade human B-ALL in vitro and block leukemia progression in vivo more effectively than NK cells that do not produce IL-15. CONCLUSION: We find that restoration of the intrinsically suppressed IFN-I production in B-ALL underlies the therapeutic efficacy of IL-15-producing NK cells and that such NK cells represent an attractive therapeutic solution for the problem of drugging MYC in high-grade B-ALL.

METTL3-Mediated m6A Modification Controls Splicing Factor Abundance and Contributes to Aggressive CLL.

RNA splicing dysregulation underlies the onset and progression of cancers. In chronic lymphocytic leukemia (CLL), spliceosome mutations leading to aberrant splicing occur in ∼20% of patients. However, the mechanism for splicing defects in spliceosome-unmutated CLL cases remains elusive. Through an integrative transcriptomic and proteomic analysis, we discover that proteins involved in RNA splicing are posttranscriptionally upregulated in CLL cells, resulting in splicing dysregulation. The abundance of splicing complexes is an independent risk factor for poor prognosis. Moreover, increased splicing factor expression is highly correlated with the abundance of METTL3, an RNA methyltransferase that deposits N6-methyladenosine (m6A) on mRNA. METTL3 is essential for cell growth in vitro and in vivo and controls splicing factor protein expression in a methyltransferase-dependent manner through m6A modification-mediated ribosome recycling and decoding. Our results uncover METTL3-mediated m6A modification as a novel regulatory axis in driving splicing dysregulation and contributing to aggressive CLL. SIGNIFICANCE: METTL3 controls widespread splicing factor abundance via translational control of m6A-modified mRNA, contributes to RNA splicing dysregulation and disease progression in CLL, and serves as a potential therapeutic target in aggressive CLL. See related commentary by Janin and Esteller, p. 176. This article is highlighted in the In This Issue feature, p. 171.

Targeting miR-126 disrupts maintenance of myelodysplastic syndrome stem and progenitor cells.

BACKGROUND: Myelodysplastic syndrome (MDS) arises from a rare population of aberrant hematopoietic stem and progenitor cells (HSPCs). These cells are relatively quiescent and therefore treatment resistant. Understanding mechanisms underlying their maintenance is critical for effective MDS treatment. METHODS: We evaluated microRNA-126 (miR-126) levels in MDS patients' sample and in a NUP98-HOXD13 (NHD13) murine MDS model along with their normal controls and defined its role in MDS HSPCs' maintenance by inhibiting miR-126 expression in vitro and in vivo. Identification of miR-126 effectors was conducted using biotinylated miR-126 pulldown coupled with transcriptome analysis. We also tested the therapeutic activity of our anti-miR-126 oligodeoxynucleotide (miRisten) in human MDS xenografts and murine MDS models. RESULTS: miR-126 levels were higher in bone marrow mononuclear cells from MDS patients and NHD13 mice relative to their respective normal controls (P < 0.001). Genetic deletion of miR-126 in NHD13 mice decreased quiescence and self-renewal capacity of MDS HSPCs, and alleviated MDS symptoms of NHD13 mice. Ex vivo exposure to miRisten increased cell cycling, reduced colony-forming capacity, and enhanced apoptosis in human MDS HSPCs, but spared normal human HSPCs. In vivo miRisten administration partially reversed pancytopenia in NHD13 mice and blocked the leukemic transformation (combination group vs DAC group, P < 0.0001). Mechanistically, we identified the non-coding RNA PTTG3P as a novel miR-126 target. Lower PTTG3P levels were associated with a shorter overall survival in MDS patients. CONCLUSIONS: MiR-126 plays crucial roles in MDS HSPC maintenance. Therapeutic targeting of miR-126 is a potentially novel approach in MDS.

Daratumumab induces mechanisms of immune activation through CD38+ NK cell targeting.

Daratumumab (Dara), a multiple myeloma (MM) therapy, is an antibody against the surface receptor CD38, which is expressed not only on plasma cells but also on NK cells and monocytes. Correlative data have highlighted the immune-modulatory role of Dara, despite the paradoxical observation that Dara regimens decrease the frequency of total NK cells. Here we show that, despite this reduction, NK cells play a pivotal role in Dara anti-MM activity. CD38 on NK cells is essential for Dara-induced immune modulation, and its expression is restricted to NK cells with effector function. We also show that Dara induces rapid CD38 protein degradation associated with NK cell activation, leaving an activated CD38-negative NK cell population. CD38+ NK cell targeting by Dara also promotes monocyte activation, inducing an increase in T-cell costimulatory molecules (CD86/80) and enhancing anti-MM phagocytosis activity ex vivo and in vivo. In support of Dara's immunomodulating role, we show that MM patients that discontinued Dara therapy because of progression maintain targetable unmutated surface CD38 expression on their MM cells, but retain effector cells with impaired cellular immune function. In summary, we report that CD38+ NK cells may be an unexplored therapeutic target for priming the immune system of MM patients.

Targeting cell membrane HDM2: A novel therapeutic approach for acute myeloid leukemia.

The E3 ligase human double minute 2 (HDM2) regulates the activity of the tumor suppressor protein p53. A p53-independent HDM2 expression has been reported on the membrane of cancer cells but not on that of normal cells. Herein, we first showed that membrane HDM2 (mHDM2) is exclusively expressed on human and mouse AML blasts, including leukemia stem cell (LSC)-enriched subpopulations, but not on normal hematopoietic stem cells (HSCs). Higher mHDM2 levels in AML blasts were associated with leukemia-initiating capacity, quiescence, and chemoresistance. We also showed that a synthetic peptide PNC-27 binds to mHDM2 and enhances the interaction of mHDM2 and E-cadherin on the cell membrane; in turn, E-cadherin ubiquitination and degradation lead to membrane damage and cell death of AML blasts by necrobiosis. PNC-27 treatment in vivo resulted in a significant killing of both AML "bulk" blasts and LSCs, as demonstrated respectively in primary and secondary transplant experiments, using both human and murine AML models. Notably, PNC-27 spares normal HSC activity, as demonstrated in primary and secondary BM transplant experiments of wild-type mice. We concluded that mHDM2 represents a novel and unique therapeutic target, and targeting mHDM2 using PNC-27 selectively kills AML cells, including LSCs, with minimal off-target hematopoietic toxicity.

Daratumumab induces CD38 internalization and impairs myeloma cell adhesion.

Daratumumab (Dara), a human immunoglobulin G1 kappa (IgG1κ) monoclonal anti-CD38 antibody, has been approved by the U.S. Food and Drug Administration for the treatment of relapsed multiple myeloma (MM) as a single agent as well as in combination with immunomodulatory drugs (IMiDs) and proteasome inhibitors (PI). Although the scientific rationale behind the use of Dara in combination with IMiDs has been extensively explored, the molecular mechanisms underlying Dara-PI regimens have not yet been investigated. Here, we demonstrate that CD38 on the surface of MM cells is rapidly internalized after Dara treatment; we also show that Dara treatment impairs MM cell adhesion, an effect that can be rescued by using the endocytosis inhibitor Dynasore. Finally, we show that Dara potentiates bortezomib (BTZ) killing of MM cells in vitro and in vivo, independent of its function as an immune activator. In conclusion, our data show that Dara impairs MM cell adhesion, which results in an increased sensitivity of MM to proteasome inhibition.

SIRT1 Activation Disrupts Maintenance of Myelodysplastic Syndrome Stem and Progenitor Cells by Restoring TET2 Function.

Myelodysplastic syndrome (MDS), a largely incurable hematological malignancy, is derived from aberrant clonal hematopoietic stem/progenitor cells (HSPCs) that persist after conventional therapies. Defining the mechanisms underlying MDS HSPC maintenance is critical for developing MDS therapy. The deacetylase SIRT1 regulates stem cell proliferation, survival, and self-renewal by deacetylating downstream proteins. Here we show that SIRT1 protein levels were downregulated in MDS HSPCs. Genetic or pharmacological activation of SIRT1 inhibited MDS HSPC functions, whereas SIRT1 deficiency enhanced MDS HSPC self-renewal. Mechanistically, the inhibitory effects of SIRT1 were dependent on TET2, a safeguard against HSPC transformation. SIRT1 deacetylated TET2 at conserved lysine residues in its catalytic domain, enhancing TET2 activity. Our genome-wide analysis identified cancer-related genes regulated by the SIRT1/TET2 axis. SIRT1 activation also inhibited functions of MDS HSPCs from patients with TET2 heterozygous mutations. Altogether, our results indicate that restoring TET2 function through SIRT1 activation represents a promising means to target MDS HSPCs.

Bone marrow niche trafficking of miR-126 controls the self-renewal of leukemia stem cells in chronic myelogenous leukemia.

Leukemia stem cells (LSCs) in individuals with chronic myelogenous leukemia (CML) (hereafter referred to as CML LSCs) are responsible for initiating and maintaining clonal hematopoiesis. These cells persist in the bone marrow (BM) despite effective inhibition of BCR-ABL kinase activity by tyrosine kinase inhibitors (TKIs). Here we show that although the microRNA (miRNA) miR-126 supported the quiescence, self-renewal and engraftment capacity of CML LSCs, miR-126 levels were lower in CML LSCs than in long-term hematopoietic stem cells (LT-HSCs) from healthy individuals. Downregulation of miR-126 levels in CML LSCs was due to phosphorylation of Sprouty-related EVH1-domain-containing 1 (SPRED1) by BCR-ABL, which led to inhibition of the RAN-exportin-5-RCC1 complex that mediates miRNA maturation. Endothelial cells (ECs) in the BM supply miR-126 to CML LSCs to support quiescence and leukemia growth, as shown using mouse models of CML in which Mir126a (encoding miR-126) was conditionally knocked out in ECs and/or LSCs. Inhibition of BCR-ABL by TKI treatment caused an undesired increase in endogenous miR-126 levels, which enhanced LSC quiescence and persistence. Mir126a knockout in LSCs and/or ECs, or treatment with a miR-126 inhibitor that targets miR-126 expression in both LSCs and ECs, enhanced the in vivo anti-leukemic effects of TKI treatment and strongly diminished LSC leukemia-initiating capacity, providing a new strategy for the elimination of LSCs in individuals with CML.

HDAC8 Inhibition Specifically Targets Inv(16) Acute Myeloid Leukemic Stem Cells by Restoring p53 Acetylation.

Acute myeloid leukemia (AML) is driven and sustained by leukemia stem cells (LSCs) with unlimited self-renewal capacity and resistance to chemotherapy. Mutation in the TP53 tumor suppressor is relatively rare in de novo AML; however, p53 can be regulated through post-translational mechanisms. Here, we show that p53 activity is inhibited in inv(16)(+) AML LSCs via interactions with the CBFß-SMMHC (CM) fusion protein and histone deacetylase 8 (HDAC8). HDAC8 aberrantly deacetylates p53 and promotes LSC transformation and maintenance. HDAC8 deficiency or inhibition using HDAC8-selective inhibitors (HDAC8i) effectively restores p53 acetylation and activity. Importantly, HDAC8 inhibition induces apoptosis in inv(16)(+) AML CD34(+) cells, while sparing the normal hematopoietic stem cells. Furthermore, in vivo HDAC8i administration profoundly diminishes AML propagation and abrogates leukemia-initiating capacity of both murine and patient-derived LSCs. This study elucidates an HDAC8-mediated p53-inactivating mechanism promoting LSC activity and highlights HDAC8 inhibition as a promising approach to selectively target inv(16)(+) LSCs.

SIRT1 activation by a c-MYC oncogenic network promotes the maintenance and drug resistance of human FLT3-ITD acute myeloid leukemia stem cells.

The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and is associated with poor prognosis. In such patients, FLT3 tyrosine kinase inhibitors (TKIs) are only partially effective and do not eliminate the leukemia stem cells (LSCs) that are assumed to be the source of treatment failure. Here, we show that the NAD-dependent SIRT1 deacetylase is selectively overexpressed in primary human FLT3-ITD AML LSCs. This SIRT1 overexpression is related to enhanced expression of the USP22 deubiquitinase induced by c-MYC, leading to reduced SIRT1 ubiquitination and enhanced stability. Inhibition of SIRT1 expression or activity reduced the growth of FLT3-ITD AML LSCs and significantly enhanced TKI-mediated killing of the cells. Therefore, these results identify a c-MYC-related network that enhances SIRT1 protein expression in human FLT3-ITD AML LSCs and contributes to their maintenance. Inhibition of this oncogenic network could be an attractive approach for targeting FLT3-ITD AML LSCs to improve treatment outcomes.

Activation of p53 by SIRT1 inhibition enhances elimination of CML leukemia stem cells in combination with imatinib.

BCR-ABL tyrosine kinase inhibitors (TKI) fail to eliminate quiescent leukemia stem cells (LSC) in chronic myelogenous leukemia (CML). Thus, strategies targeting LSC are required to achieve cure. We show that the NAD(+)-dependent deacetylase SIRT1 is overexpressed in human CML LSC. Pharmacological inhibition of SIRT1 or SIRT1 knockdown increased apoptosis in LSC of chronic phase and blast crisis CML and reduced their growth in vitro and in vivo. SIRT1 effects were enhanced in combination with the BCR-ABL TKI imatinib. SIRT1 inhibition increased p53 acetylation and transcriptional activity in CML progenitors, and the inhibitory effects of SIRT1 targeting on CML cells depended on p53 expression and acetylation. Activation of p53 via SIRT1 inhibition represents a potential approach to target CML LSC.

Altered hematopoietic cell gene expression precedes development of therapy-related myelodysplasia/acute myeloid leukemia and identifies patients at risk.

Therapy-related myelodysplasia or acute myeloid leukemia (t-MDS/AML) is a major complication of cancer treatment. We compared gene expression in CD34+ cells from patients who developed t-MDS/AML after autologous hematopoietic cell transplantation (aHCT) for lymphoma with controls who did not develop t-MDS/AML. We observed altered gene expression related to mitochondrial function, metabolism, and hematopoietic regulation in pre-aHCT samples from patients who subsequently developed t-MDS/AML. Progression to overt t-MDS/AML was associated with additional alterations in cell-cycle regulatory genes. An optimal 38-gene PBSC classifier accurately distinguished patients who did or did not develop t-MDS/AML in an independent group of patients. We conclude that genetic programs associated with t-MDS/AML are perturbed long before disease onset, and accurately identify patients at risk for this complication.

Role of BCR/ABL gene-expression levels in determining the phenotype and imatinib sensitivity of transformed human hematopoietic cells.

Increased levels of Bcr-Abl expression in chronic myelogenous leukemia (CML) cells are associated with disease progression and imatinib (IM) resistance. However, it is not clear if these associations are a direct result of elevated Bcr-Abl expression. We used a human transduction model of CML to directly investigate the role of varying Bcr-Abl expression levels in determining the phenotype and IM sensitivity of hematopoietic cells. CD34(+) cells were transduced with vectors coexpressing Bcr-Abl and GFP, and cells expressing low and high levels of GFP and Bcr-Abl (BA(lo) and BA(hi)) were selected. BA(hi) cells demonstrated enhanced activation of downstream proliferative and antiapoptotic signaling and enhanced proliferation and survival compared to BA(lo) cells. Freshly isolated BA(hi) CD34(+) cells and cell lines demonstrated increased IM-mediated growth inhibition likely reflecting Bcr-Abl dependence for growth and survival. CD34(+) cells expressing BCR/ABL kinase-mutant genes demonstrated resistance to IM-mediated inhibition of proliferation and viability, which was not enhanced by increased expression of BCR/ABL kinase-mutant genes. We conclude that Bcr-Abl overexpression results in increased proliferation and antiapoptotic signaling in CD34(+) cells, but may not play a direct role in IM resistance in progenitor cells expressing either wild-type or mutant BCR/ABL genes.