Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34⁺) leukemic versus normal specimens. Our data indicate that CD34⁺ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34⁺ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34⁺ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34⁺ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34⁺ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.     

Ibrutinib is not an effective drug in primografts of TCF3-PBX1

AimThe Bruton's tyrosine kinase (BTK) inhibitor Ibrutinib (PCI-32765) is effective in patients with multiple myeloma, non-Hodgkin lymphoma and chronic lymphoblastic leukemia. We previously showed that primary cells of children with TCF3-PBX1 positive B-cell precursor acute lymphoblastic leukemia (BCP-ALL) express BTK and are sensitive to ibrutinib in vitro. However, preclinical studies in mice are lacking that justify clinical implementation.MethodsImmunocompromised NSG mice were engrafted with a luciferase-positive TCF3-PBX1 leukemic cell line or primary leukemic cells and treated with ibrutinib or placebo. Additionally, primary cells were exposed in vitro to 4 main induction drugs as monotherapy and in combination with ibrutinib.ResultsTreatment with ibrutinib of mice engrafted with a TCF3-PBX1 cell line, TCF3-PBX1 positive or TCF3-PBX1 negative primary leukemic cells did not result in prolonged life span compared to placebo treated mice. In vitro sensitivity to ibrutinib was unaltered in leukemic cells obtained from engrafted mice compared to the original material. However, ibrutinib treatment did not affect leukemic cell viability and tumor outgrowth, nor could lymphocytosis be detected. Ibrutinib was biologically active, since hCD19⁺ cells harvested from ibrutinib treated mice had no detectable levels of phospho-BTK at tyrosine 223 (pBTK Y223), whereas pBTK Y223 was still detectable in placebo treated cases. In combination tests, we noticed an antagonistic effect of ibrutinib on vincristine sensitivity, which was not observed for prednisolone, L-asparaginase and daunorubicin.ConclusionsWe conclude that ibrutinib is not the precision medicine of choice for TCF3-PBX1 positive BCP-ALL.     

The Functional Interplay Between the t(9;22)-Associated Fusion Proteins BCR/ABL and ABL/BCR in Philadelphia Chromosome-Positive Acute Lymphatic Leukemia

The hallmark of Philadelphia chromosome positive (Ph⁺) leukemia is the BCR/ABL kinase, which is successfully targeted by selective ATP competitors. However, inhibition of BCR/ABL alone is unable to eradicate Ph⁺ leukemia. The t(9;22) is a reciprocal translocation which encodes not only for the der22 (Philadelphia chromosome) related BCR/ABL, but also for der9 related ABL/BCR fusion proteins, which can be detected in 65% of patients with chronic myeloid leukemia (CML) and 100% of patients with Ph⁺ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96^ABL/BCR for the pathogenesis of Ph⁺ ALL. The co-expression of p96^ABL/BCR enhanced the kinase activity and as a consequence, the transformation potential of p185^BCR/ABL. Targeting p96^ABL/BCR by RNAi inhibited growth of Ph⁺ ALL cell lines and Ph⁺ ALL patient-derived long-term cultures (PD-LTCs). Our in vitro and in vivo stem cell studies further revealed a functional hierarchy of p96^ABL/BCR and p185^BCR/ABL in hematopoietic stem cells. Co-expression of p96^ABL/BCR abolished the capacity of p185^BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96^ABL/BCR for the pathogenesis of Ph⁺ ALL.     

Apoptosis and neutrophils in the regulation of Ph-positive myeloid cell proliferation and differentiation ex vivo

Ex vivo proliferation and differentiation of Philadelphia chromosome-positive (Ph⁺) human myeloid cells (Ph+ cells) from chronic myeloid leukemia (CML) proceed via alternation stages of cell proliferation and neutrophil maturation. To regulate them, apoptosis is alternately blocked or induced with the help of neutrophils and expression of bcr/abl, bax, and bcl2. The regulation of apoptosis in main types of Ph⁺ cells depends on the alternation of (1) Ph⁺ cell proliferation and (2) neutrophil maturation and may follow two pathways. One consists in alternating blockages and inductions of apoptosis with initial maturation and subsequent proliferation under alternation stages as (2)-(1)-(2) and has not been described as yet. Neutrophil accumulation blocks apoptosis. As neutrophils are depleted, apoptosis is induced again. Its block accelerates proliferation with a new accumulation of neutrophils, which is followed by regular neutrophil death and a new induction of apoptosis. The way optimizes the proliferation efficiency (P/D index) with a regular alternation of maturation and proliferation, allowing the cycle of proliferation and differentiation to be completed. In another way, the alternation starts with proliferation as (1)-(2)-(1) at a lower neutrophil content) and leads to resistant decrease of the maximal apoptosis level by a factor of 3–8 as compared with (2)-(1)-(2) alternation. A stable block of apoptosis is observed in cells with prolonged stages of proliferation and maturation, leading to an accumulation of blasts and myelocytes with elevated bcr/abl expression and expression of bcl2 > bax. A stable block of apoptosis is associated with CML progression and in Ph⁺ cell lines. Cells follow the first pathway of the apoptotic regulation in chronic-phase CML. Ex vivo cultivation of Ph⁺ cells from individual CML patients was assumed to provide for a more exact diagnosis of the CML phase and optimizing the treatment.     

Proteinase-Activated Receptor 1 (PAR1) Regulates Leukemic Stem Cell Functions

External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients'' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34⁺ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1^−/− hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1^−/− leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance.     

Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome–positive acute lymphoblastic leukemia

Background aimsTo develop a treatment option for Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph⁺ALL) resistant to tyrosine kinase inhibitors (TKIs), we evaluated the anti-leukemic activity of T cells non-virally engineered to express a CD19-specific chimeric antigen receptor (CAR).MethodsA CD19.CAR gene was delivered into mononuclear cells from 10 mL of blood of healthy donors through the use of piggyBac-transposons and the 4-D Nucleofector System. Nucleofected cells were stimulated with CD3/CD28 antibodies, magnetically selected for the CD19.CAR, and cultured in interleukin-15–containing serum-free medium with autologous feeder cells for 21 days. To evaluate their cytotoxic potency, we co-cultured CAR T cells with seven Ph⁺ALL cell lines including three TKI-resistant (T315I-mutated) lines at an effector-to-target ratio of 1:5 or lower without cytokines.ResultsWe obtained ∼1.3 × 10⁸ CAR T cells (CD4⁺, 25.4%; CD8⁺, 71.3%), co-expressing CD45RA and CCR7 up to ∼80%. After 7-day co-culture, CAR T cells eradicated all tumor cells at the 1:5 and 1:10 ratios and substantially reduced tumor cell numbers at the 1:50 ratio. Kinetic analysis revealed up to 37-fold proliferation of CAR T cells during a 20-day culture period in the presence of tumor cells. On exposure to tumor cells, CAR T cells transiently and reproducibly upregulated the expression of transgene as well as tumor necrosis factor–related apoptosis-inducing ligand and interleukin-2.ConclusionsWe generated a clinically relevant number of CAR T cells from 10 mL of blood through the use of piggyBac-transposons, a 4D-Nulcleofector, and serum/xeno/tumor cell/virus-free culture system. CAR T cells exhibited marked cytotoxicity against Ph⁺ALL regardless of T315I mutation. PiggyBac-mediated CD19-specific T-cell therapy may provide an effective, inexpensive and safe option for drug-resistant Ph⁺ALL.     

The role of hERG1 K channels and a functional link between hERG1 K channels and SDF-1 in acute leukemic cell migration

Stromal cell-derived factor-1 (SDF-1) and its unique receptor, CXCR4, regulate stem/progenitor cell migration and retention in the bone marrow and are required for hematopoiesis. Recent studies found that hERG1 K⁺ channels were important regulators of tumor cell migration. In this study, we investigated whether SDF-1 induced acute leukemic cell migration associated with hERG1 K⁺ channels. Our results showed that E-4031, a specific hERG1 K⁺ channels inhibitor, significantly blocked SDF-1-induced migration of leukemic cell lines, primary acute leukemic cells, leukemic stem cells and HEK293T cells transfected with herg-pEGFP. The migration of phenotypically recognizable subsets gave the indication that lymphoblastic leukemic cells were inhibited more than myeloid cells while in the presence of E-4031 which maybe associated with herg expression. SDF-1 increased hERG1 K⁺ current expressed in oocytes and HEK293T cells transfected with herg-pEGFP. There were no significant changes of CXCR4 expression on both HL-60 cells and primary leukemic cells regardless if untreated or treated with E-4031 for 24 h (P > 0.05). The hERG1 K⁺ current increased by SDF-1 might contribute to the mechanism of SDF-1-induced leukemic cell migration. The data suggested that hERG1 K⁺ channels functionally linked to cell migration induced by SDF-1.     

Impaired immunomodulatory function of chronic myeloid leukemia cancer stem cells and the possible mechanism involved in it

Backgroud

Cancer stem cells (CSCs) are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Development of specific therapies targeted at CSCs holds hope for the improvement of survival and quality of life of cancer patients, especially for sufferers of metastatic disease. This is particularly true in chronic myeloid leukemia (CML).

Methods

In this study, we isolated fetal liver kinase-1-positive (Flk1⁺) cells carrying the BCR/ABL fusion gene from the bone marrow of Philadelphia chromosome-positive (Ph⁺) patients with stem cells property. We examined their biological characteristics as well as immunological function and further detected the possible molecular mechanism involved in the leukemia genesis.

Results

We showed that CML patient-derived Flk1⁺CD31^?CD34^? MSCs had normal morphology, phenotype and karyotype but appeared impaired immunomodulatory function. The capacity of Flk1⁺CD31^?CD34^? MSCs from CML patients to inhibit T lymphocyte activation and proliferation was impaired in vitro. CML patient-derived MSCs have dampening immunomodulatory functions, suggesting that the dysregulation of hematopoiesis and immune response might originate from MSCs rather than HSCs. These Ph⁺ putative CML hemangioblast upregulated TGF-β1 and resultantly activated matrix metalloproteinase-9 (MMP-9) to enhance s-KitL and s-ICAM-1 secretion, which activated c-kit⁺ HSCs from the quiescent state to proliferative state. Further studies showed that phosphatidylinositol-3 kinase (PI3K)/Akt/nuclear factor (NF)-κB signaling pathway was involved in CML pathogenesis.

Conclusions

Flk1⁺CD31^?CD34^? MSCs that express BCR/ABL leukemia oncogene are CSCs of CML and they play a critical role in the progression of CML through PI3K/Akt/NF-κB/MMP-9/s-ICAM-1/s-KitL signaling pathway beyond HSCs.     

Establishment of a Humanized APL Model via the Transplantation of PML-RARA-Transduced Human Common Myeloid Progenitors into Immunodeficient Mice

Recent advances in cancer biology have revealed that many malignancies possess a hierarchal system, and leukemic stem cells (LSC) or leukemia-initiating cells (LIC) appear to be obligatory for disease progression. Acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia characterized by the formation of a PML-RARα fusion protein, leads to the accumulation of abnormal promyelocytes. In order to understand the precise mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model involving retroviral transduction of PML-RARA into CD34⁺ hematopoietic cells from human cord blood and transplantation of these cells into immunodeficient mice. The leukemia well recapitulated human APL, consisting of leukemic cells with abundant azurophilic abnormal granules in the cytoplasm, which expressed CD13, CD33 and CD117, but not HLA-DR and CD34, were clustered in the same category as human APL samples in the gene expression analysis, and demonstrated sensitivity to ATRA. As seen in human APL, the induced APL cells showed a low transplantation efficiency in the secondary recipients, which was also exhibited in the transplantations that were carried out using the sorted CD34⁻ fraction. In order to analyze the mechanisms underlying APL initiation and development, fractionated human cord blood was transduced with PML-RARA. Common myeloid progenitors (CMP) from CD34⁺/CD38⁺ cells developed APL. These findings demonstrate that CMP are a target fraction for PML-RARA in APL, whereas the resultant CD34⁻ APL cells may share the ability to maintain the tumor.     

Characteristics of preleukemia cells induced in mice.

A high proportion of irradiated C57BL/6 mice inoculated with the radiation leukemia virus D-RadLV develop overt T-cell leukemias originating in the thymus. In unirradiated hosts the incidence is much lower. As early as 10 days after injection of D-RadLV the bone marrow contains “preleukemia” cells which, although not frankly leukemic, will develop into leukemia cells if transferred into a specially pretreated recipient mouse. In the present report, certain properties of D-RadLV-induced leukemia and preleukemia cells are compared. In this model system, leukemia cells express the T-cell surface component Thy-1 (Thy-1⁺) whereas preleukemia cells do not (Thy-1^?). But preleukemia cells could be induced in vitro by thymopoietin or ubiquitin to become Thy-1⁺, suggesting that they are prothymocytes. Unlike leukemia cells, preleukemia cells injected into normal recipients immunized them against transplants of leukemias induced by the same D-RadLV virus. Evidently D-RadLV virus induces a critical change in prothymocytes which in a later (Thy-1⁺) phase of differentiation is manifest in overt leukemia transformation.     

Small Molecule ErbB Inhibitors Decrease Proliferative Signaling and Promote Apoptosis in Philadelphia Chromosome–Positive Acute Lymphoblastic Leukemia

The presence of the Philadelphia chromosome in patients with acute lymphoblastic leukemia (Ph⁺ALL) is a negative prognostic indicator. Tyrosine kinase inhibitors (TKI) that target BCR/ABL, such as imatinib, have improved treatment of Ph⁺ALL and are generally incorporated into induction regimens. This approach has improved clinical responses, but molecular remissions are seen in less than 50% of patients leaving few treatment options in the event of relapse. Thus, identification of additional targets for therapeutic intervention has potential to improve outcomes for Ph+ALL. The human epidermal growth factor receptor 2 (ErbB2) is expressed in ∼30% of B-ALLs, and numerous small molecule inhibitors are available to prevent its activation. We analyzed a cohort of 129 ALL patient samples using reverse phase protein array (RPPA) with ErbB2 and phospho-ErbB2 antibodies and found that activity of ErbB2 was elevated in 56% of Ph⁺ALL as compared to just 4.8% of Ph⁻ALL. In two human Ph+ALL cell lines, inhibition of ErbB kinase activity with canertinib resulted in a dose-dependent decrease in the phosphorylation of an ErbB kinase signaling target p70S6-kinase T389 (by 60% in Z119 and 39% in Z181 cells at 3 µM). Downstream, phosphorylation of S6-kinase was also diminished in both cell lines in a dose-dependent manner (by 91% in both cell lines at 3 µM). Canertinib treatment increased expression of the pro-apoptotic protein Bim by as much as 144% in Z119 cells and 49% in Z181 cells, and further produced caspase-3 activation and consequent apoptotic cell death. Both canertinib and the FDA-approved ErbB1/2-directed TKI lapatinib abrogated proliferation and increased sensitivity to BCR/ABL-directed TKIs at clinically relevant doses. Our results suggest that ErbB signaling is an additional molecular target in Ph⁺ALL and encourage the development of clinical strategies combining ErbB and BCR/ABL kinase inhibitors for this subset of ALL patients.     

Chromosome Studies in Leukemia

This review discusses the significance of chromosomal abnormalities found in leukemia with the bias of belief that these have a primary role or are the mechanism of action of leukemogenic agents. The Philadelphia chromosome (Ph¹) is present in marrow cells examined without culture at any stage of most patients with chronic granulocytic leukemia (CGL). the presence of this chromosome is of diagnostic and prognostic value.Varied chromosomal abnormalities have been found in acute leukemia. Each abnormality, which may be unique, is absent in remission, found again at relapse and is seldom changed by therapy. Abnormalities may be of number of chromosomes (aneuploid) or structural rearrangements resulting in “marker” chromosomes, Ranges of abnormal numbers of chromosomes, when present, usually have related patterns which suggest origin of several cell types from one initial cell. Cells from patients with increased risk of leukemia owing to genetic factors have a high incidence of chromosome breakage and structural rearrangements suggesting a mechanism for production of clones of abnormal, possibly leukemic, cells.     

Dramatic efficacy improvement of a DC-based vaccine against AML by CD25 T cell depletion allowing the induction of a long-lasting T cell response

Dendritic cell (DC)-based vaccination is a promising approach to enhance anti-tumor immunity that could be considered for acute myeloid leukemia (AML) patients with high-risk of relapse. Our purpose was to study the efficiency and to optimize the immunogenicity of a DC-based vaccine in a preclinical AML murine model. In this report, C57BL6 mice were vaccinated with DC pulsed with peptides eluted (EP) from the syngeneic C1498 myelomonocytic leukemic cell line in a prophylactic setting. In this model, a natural antileukemic immunity mediated by NK cells was observed in the control unloaded DC-vaccinated group. On the other hand, we showed that the cytotoxic antileukemic immune response induced by vaccination with eluted peptides pulsed-DC (DC/EP), in vitro and in vivo, was mainly mediated by CD4⁺ T cells. Treatment with anti-CD25 antibody to deplete CD4⁺ CD25⁺ regulatory T cells before DC-vaccination dramatically improved the antileukemic immune response induced by immunization, and allowed the development of long-lasting immune responses that were tumor protective after a re-challenge with leukemic cells. Our results suggest that this approach could be successful against weakly immunogenic tumors such as AML, and could be translated in human.     

CXCL12 is a costimulator for CD4+ T cell activation and proliferation in chronic lymphocytic leukemia patients

Activated T cells from patients with chronic lymphocytic leukemia (CLL) provide survival and proliferative signals to the leukemic clone within lymphoid tissues. Recruitment of both, CLL cells and T lymphocytes, to this supportive microenvironment greatly depends on CXCL12 production by stromal and myeloid cells. CXCL12 also supplies survival stimuli to leukemic B cells, but whether it exerts stimulatory effects on T lymphocytes from CLL patients is unknown. In order to evaluate the capacity of CXCL12 to increase CD4⁺ T cell activation and proliferation in CLL patients, peripheral blood mononuclear cells were cultured with or without recombinant human CXCL12 or autologous nurse-like cells, and then T cell activation was induced by anti-CD3 mAb. CXCL12 increases the proliferation and the expression of CD25, CD69, CD154, and IFNγ on CD3-stimulated CD4⁺ T cells from CLL patients, similarly in T cells from ZAP-70⁺ to ZAP-70^? patients. Autologous nurse-like cells establish a close contact with CD4⁺ T cells and increase their activation and proliferation partially through a CXCR4-dependent mechanism. In addition, we found that activated T cells in the presence of CXCL12 enhance the activation and proliferation of the leukemic clone. In conclusion, CXCL12 production by lymphoid tissue microenvironment in CLL patients might play a key dual role on T cell physiology, functioning not only as a chemoattractant but also as a costimulatory factor for activated T cells.     

CXCR4 inhibitors selectively eliminate CXCR4-expressing human acute myeloid leukemia cells in NOG mouse model

The chemokine receptor CXCR4 favors the interaction of acute myeloid leukemia (AML) cells with their niche but the extent to which it participates in pathogenesis is unclear. Here, we show that CXCR4 expression at the surface of leukemic cells allowed distinguishing CXCR4^high from CXCR4^neg/low AML patients. When high levels of CXCR4 are expressed at the surface of AML cells, blocking the receptor function with small molecule inhibitors could promote leukemic cell death and reduce NOD/Shi-scid/IL-2Rγ^null (NOG) leukemia-initiating cells (LICs). Conversely, these drugs had no efficacy when AML cells do not express CXCR4 or when they do not respond to chemokine CXC motif ligand 12 (CXCL12). Functional analysis showed a greater mobilization of leukemic cells and LICs in response to drugs, suggesting that they target the interaction between leukemic cells and their supportive bone marrow microenvironment. In addition, increased apoptosis of leukemic cells in vitro and in vivo was observed. CXCR4 expression level on AML blast cells and their migratory response to CXCL12 are therefore predictive of the response to the inhibitors and could be used as biomarkers to select patients that could potentially benefit from the drugs.     

Inhibition of GATE-16 attenuates ATRA-induced neutrophil differentiation of APL cells and interferes with autophagosome formation

Autophagy is an intracellular bulk degradation process involved in cell survival upon stress induction, but also with a newly identified function in myeloid differentiation. The autophagy-related (ATG)8 protein family, including the GABARAP and LC3 subfamilies, is crucial for autophagosome biogenesis. In order to evaluate the significance of the GABARAPs in the pathogenesis of acute myeloid leukemia (AML), we compared their expression in primary AML patient samples, CD34⁺ progenitor cells and in granulocytes from healthy donors. GABARAPL1 and GABARAPL2/GATE-16, but not GABARAP, were significantly downregulated in particular AML subtypes compared to normal granulocytes. Moreover, the expression of GABARAPL1 and GATE-16 was significantly induced during ATRA-induced neutrophil differentiation of acute promyelocytic leukemia cells (APL). Lastly, knocking down GABARAPL2/GATE-16 in APL cells attenuatedneutrophil differentiation and decreased autophagic flux. In conclusion, low GABARAPL2/GATE-16 expression is associated with an immature myeloid leukemic phenotype and these proteins are necessary for neutrophil differentiation of APL cells.     

Anlotinib exerts potent antileukemic activities in Ph chromosome negative and positive B-cell acute lymphoblastic leukemia via perturbation of PI3K/AKT/mTOR pathway

ObjectivesDespite advances in the development of novel targeted therapies, the need for B-ALL alternative treatments has not been met. Anlotinib could blunt the proangiogenic activity of VEGFR, PDGFR, and FGFR, and has shown strong antitumor activities across multiple tumors. However, anlotinib cytotoxicity against B-ALL has not ever been evaluated, thus prompting us to initiate this study.MethodsExpression2Kinases program was used to identify potential treatment targets. Cell viability and apoptosis were determined by CCK-8 and Annexin V/PI staining kit, respectively. qRT-PCR and Western blotting were utilized to investigate the molecular mechanisms. In vivo antileukemia activity of Anlotinib was evaluated in a Ph⁺ B-ALL patient-Derived Xenograft (PDX) model.ResultsCompared with treatment-naive B-ALL cases, RR B-ALL patients had higher activities in the VEGF/VEGFR signaling and the PI3K/AKT/mTOR pathway. Exposure of Ph⁻ and Ph⁺ B-ALL cells to anlotinib resulted in significant cell viability reduction, apoptosis enhancement, and cell cycle arrest at G2/M phase. Importantly, anlotinib treatment led to remarkably decreased leukemia burdens and extended the survival period in a Ph⁺ B-ALL PDX model. Blockade of the role of the proangiogenic mediators, comprising VEGFR2, PDGFR-beta, and FGFR3, played a critical role in the cytotoxicity of anlotinib against Ph⁻ and Ph⁺ B-ALL. Moreover, anlotinib dampened the activity of PI3K/AKT/mTOR pathway that resides in the convergence of the three mentioned proangiogenic signals.ConclusionThis work provides impressive preclinical evidence of anlotinib against Ph⁻ and Ph⁺ B-ALL and raises a rationale for future clinical evaluation of this drug in the management of Ph⁻ and Ph⁺ B-ALL.