Leukemic Precursors Research Articles

C/EBPβ expressed By Bone Marrow Mesenchymal Stromal Cells Is Indispensable For Precursor B-Cell Lymphopoiesis

B-cell lymphopoiesis is critically dependent on the bone marrow microenvironment. Early B-cell lymphopoiesis is regulated by direct interaction with bone marrow stromal cells (BM-MSCs) and by soluble factors produced by BM-MSCs. The roles of transcription factors expressed by hematopoietic cells that involve early B-cell lymphopoiesis have been well investigated; those include PU.1, Ikaros, E2A, early B-cell factor, and paired box protein 5. By contrast, transcriptional factors expressed by BM-MSCs that are important for early B-cell lymphopoiesis remain unknown. We show that CCAAT-enhancer binding protein (C/EBP) β expressed by BM-MSCs contributes to the early B-cell lymphopoiesis. In addition, the proliferation of precursor B-cell acute lymphoblastic leukemia (B-ALL) cells is also associated with BM-MSCs, in which C/EBPβ is one of the regulatory transcription factors.When c-kit+ Sca-1+ lineage- (KSL) HSCs from Wild-type (WT) mice were co-cultured with C/EBPβ-deficient BM-MSCs in the presence of stem cell factor (SCF), Flt3-L, and interleukin (IL)-7 (Figure A), the generation of hematopoietic cells from KSL cells was significantly lower than when they were co-cultured with WT BM-MSCs. In addition, the generation of B220+ B-cells from KSL cells was also significantly lower when they were co-cultured with C/EBPβ-deficient BM-MSCs than when co-cultured with WT BM-MSCs. Detailed analysis of the generated B-cell subsets showed that differentiation of KSL cells into precursor B-cells was reduced and differentiation from pre-pro-B-cells to pro-B-cells/pre-BI cells was suppressed when cells were co-cultured with C/EBPβ-deficient BM-MSCs (Figure B). Therefore, C/EBPβ was an indispensable transcriptional factor expressed by BM-MSCs for supporting the differentiation of HSCs into precursor B-cells.We next examined the expression of B-cell lymphopoiesis-associated molecules in C/EBPβ-deficient BM-MSCs by quantitative real-time PCR analysis. Levels of IL-7 and SCF mRNA tended to be lower in C/EBPβ-deficient BM-MSCs than in WT BM-MSCs, although the difference was not statistically significant in our analysis. Levels of CXCL12/SDF-1 and Flt3-L mRNA were significantly lower in C/EBPβ-deficient BM-MSCs than in WT BM-MSCs. In addition, the protein concentration of CXCL12/SDF-1 was significantly lower in the culture supernatant of C/EBPβ-deficient BM-MSCs than that of WT BM-MSCs. The concentration of CXCL12/SDF-1 in the supernatant of BM-MSC co-cultures strongly correlated with the number of B220+ B-cells that differentiated from KSL cells. Thus, the impaired differentiation of HSCs into B-cells is associated, at least in part, with reduced production of CXCL12/SDF-1 by C/EBPβ-deficient BM-MSCs.Precursor B-ALL is a hematological disease characterized by malignant transformation of precursor B-cells. We examined whether C/EBPβ expressed by BM-MSCs has effects on the precursor B-ALL cells. When the precursor B-ALL cell line BaF3/Bcr-Abl cells was co-cultured with WT BM-MSCs, the number of BaF3/Bcr-Abl cells was significantly increased than when they were cultured alone. Interestingly, when the precursor B-ALL cell line BaF3/Bcr-Abl cells was co-cultured with C/EBPβ-deficient BM-MSCs, the number of BaF3/Bcr-Abl cells was reduced to the level similar to when they were cultured alone. This was true in co-cultures with BM-MSCs that were pharmacologically treated to down-regulate the C/EBPβ expression. As well as the correlation of SDF-1 concentration in co-cultures with precursor B-cell differentiation, the proliferation of BaF3/Bcr-Abl cells was associated with the correlation of SDF-1 concentration in co-cultures.In conclusion, this work demonstrates that C/EBPβ expressed by BM-MSCs is a critical transcriptional factor for both the differentiation of physiological precursor B-cells and pathological proliferation of leukemic precursor B-cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

PAX5 Fusion Genes Activate The STAT5 Signaling Pathway Through Lck Over-Expression

PAX5 is a transcription factor acting both as an activator and a repressor, known to be frequently altered (30% of the cases) in B Cell Precursor Leukemia (BCP-ALL). PAX5 aberrations include point mutations, deletions, amplifications and translocations with several partner genes. All the originated fusion genes retain the PAX5 DNA binding domain but acquire the regulatory regions of the partner gene, showing a dominant negative mechanism on endogenous PAX5. However, the function of PAX5 fusion proteins and their common mechanisms of leukemogenesis are poorly understood.We have established an in vitro model in which wild-type murine pre-BI cells were transduced with the most recurrent PAX5 fusion gene, PAX5/ETV6. Gene expression profiling of transduced PAX5/ETV6 pre-BI cells showed a significantly altered transcription profile compared to empty vector control. Interestingly, pathway analyses revealed that many of the genes implicated in the B-cell receptor (BCR) assembly and signaling were repressed by PAX5/ETV6. In addition, the downstream signaling of the BCR was non-functional through a block in IgM heavy chain rearrangement.Aim of the current study has been to elucidate the signaling pathway activated either in PAX5/ETV6 transduced pre-BI cells and in primary cells from patients carrying different PAX5 translocations, in order to overcome the absence of a functional BCR expression.Interestingly, among the top-ranked over-expressed genes in PAX5/ETV6 transduced pre-BI cells, the PAX5 repressed target gene LCK was up-regulated both at basal level (mean fold change by RQ-PCR in 3 different pre-BI lines: 1.79, p<0.001) and at all time points (0, 24, 48 and 72 hours) after o.n. IL-7 starvation (mean FC: 8.0, range 3.8-15.9, p<0.01). LCK is a src family kinase involved in STAT5 signaling pathway and known to play an important role in T cell survival.Indeed, for the first time we found LCK as significantly up-regulated in primary samples of patients carrying different PAX5 translocations, including PAX5/AUTS2, PAX5/CHFR, PAX5/SOX5 and PAX5/POM121C, when compared to BCP-ALL cases with wild-type PAX5, as shown by microarray analyses and confirmed by RT RQ-PCR (mean fold increase: 6.3, p=0.027).Strikingly, in addition to LCK overexpression, PAX5/ETV6 induction in pre-BI cells dephosphorylates the inhibitory domain of LCK (LCK Y505) (mean difference in LCKY505/total LCK ratio: 63%, ranging from 38% to 81% in 3 different pre-BI lines), thus hyper-activating the LCK signaling. By phosphoflow analysis we also demonstrated LCK-dependent STAT5 hyper-phosphorylation after IL7 stimulation at all time points in time course experiments (mean fold change: 2.0, range 1.77-2.18). Downstream in the pathway, as a confirmation, we found by RQ-PCR the up-regulation of cMYC, a STAT5 target (FC: 2.65, p<0.01, at 30 minutes and 3.85, p<0.01, at 24h after IL-7 stimulation). Also CCND2, a cMYC target, was upregulated (FC: 2.27, p<0.001, at 30 minutes and 1.83, p<0.001, at 24h after IL-7 stimulation). Moreover, PAX5/ETV6 induced pre-BI cells show an increase in the replicative S phase cells at all time points after o.n. IL7 withdrawal (+58%, +39% and 61%, p<0.001, for PAX5/ETV6 after 0, 24 and 48 hours, respectively) and a faster proliferation rate, likely due to a different STAT5 activation profile. This mechanism is specific and reversible, as the LCK inhibitor BIBF1120 reduces STAT5 phosphorylation in PAX5/ETV6 cells (-11% and -46% after 24 and 48 hours, respectively), thus decreasing the fraction of S phase cells (-20%, p<0.01 and -31%, p<0.001, after 24 and 48 hours, respectively) and leading to a proliferation rate comparable to the control cells.These results further sustain the role of PAX5 fusion proteins as aberrant transcriptional factors, leading to the over-expression and hyper-activation of LCK, with a role in the activation of signaling pathways alternative to BCR (i.e. STAT5 hyper-phosphorylation) to sustain survival of leukemic cells. The further comprehension of the signaling mechanisms activated by PAX5 fusion genes will be fundamental for the development of new strategies to arrest tumor proliferation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

MSH-TP15 Identified By Phage Display Technology Detects a Target Structure For Antibody-Mediated Killing In Multiple Myeloma

Introduction Multiple Myeloma (MM) is a malignant plasma cell disorder with high incidence and mortality. Monoclonal antibodies represent promising new treatment options and are widely applied in cancer therapy, but none have yet been approved for the treatment of MM. Daratumumab and other antibodies have recently shown encouraging results in clinical trials, but there is still a need to identify plasma cell specific target structures suitable for this approach. Methods To identify novel plasma cell surface antigens or epitopes, synthetic human single-chain fragment variable (scFv) phage display libraries were screened by using a newly established subtractive, cellular screening approach. Pre-absorption steps with granulocytes and T lymphocytes were applied with subsequent positive selection for phages that show binding to plasma cell lines INA-6 and JK-6L. The binding patterns of the resulting phage antibodies on various cell types were evaluated by cellular ELISA and flow cytometry. Based on its favorable binding profile, MSH-TP15 was selected and the coding sequence was used to generate a fully human scFv-Fc fusion protein (mini-antibody) for functional assays. Results Screening of phage display libraries by using our novel strategy resulted in a number of phage clones with preferential binding to MM cells. MSH-TP15 was selected based upon its strong binding to five MM cell lines (INA-6, JK-6L, L363, RPMI-8226, U266), while not recognizing a B cell precursor leukemia cell line (SEM), an immature acute myeloid leukemia cell line (KG-1a), and T cell derived acute lymphoblastic leukemia cell lines (CEM, Jurkat). In addition to MM cells, two Burkitt lymphoma cell lines (Daudi, Ramos) were also positive. Importantly, patient-derived CD138-positive malignant plasma cells were recognized while no reactivity was seen on other mononuclear cells of the same patient. No significant binding was seen on peripheral blood T lymphocytes, NK cells, monocytes, and granulocytes from healthy donors. Additional screening on a panel of stably transfected cell lines that express antigens already considered for antibody-based MM therapy (CD38, CD40, CD56, CD70, CD138, CS1, FGFR3, IGF-1R, IL-6R) revealed no reactivity. To subsequently evaluate direct (e.g. growth inhibition) and indirect (e.g. antibody-dependent cell-mediated cytotoxicity; ADCC) antibody effector functions, the coding sequence was used to generate a scFv-Fc fusion protein. This mini-antibody successfully retained its prior established binding properties. The antibody by itself was not capable to induce significant growth inhibition of INA-6, L363, and RPMI-8226 plasma cell lines. In contrast, MSH-TP15 was able to trigger ADCC, an important killing mechanism of therapeutic antibodies, against these myeloma cells with peripheral blood mononuclear cells as effector cells. Conclusions Phage display technology together with a cell-based screening approach led to the generation of an antibody that preferentially binds to MM cells, and is effective in antibody-mediated killing mechanisms. The recognized antigen is distinct from any of the MM antigens currently explored as targets for antibody therapy, and therefore represents a promising target structure suitable for an antibody-based therapeutic approach in myeloma. Disclosures: No relevant conflicts of interest to declare.

Myeloid/NK cell acute leukemia with unique blast morphology: de novo or secondary leukemia?

Immature-type natural killer (NK) cell leukemia is rare and heterogeneous, and is variously classified as myeloid/NK cell precursor acute leukemia, myeloid/NK cell acute leukemia, or blastic NK cell lymphoma/leukemia [1–3]. Scott et al. [1] first described 20 cases of adult de novo acute myeloid leukemia showing a CD16/CD33/CD34/ CD56/HLA-DR phenotype. A few additional cases have since been reported, including that presented in this journal [4]. However, Suzuki et al. [2, 3] characterized myeloid/ NK cell precursor acute leukemia as a distinct entity, with a CD7/CD56/CD34/HLA-DR phenotype. Thus, myeloid/NK cell leukemia showing the CD7/CD56/ CD34/HLA-DR phenotype is considered to be more mature than myeloid/NK cell precursor leukemia [3]. Here, we report a case of myeloid/NK cell acute leukemia developing after chemoradiotherapy and with morphologically distinct characteristics. In March 2013, a 63-year-old man presented with poor appetite, lassitude, chest pain, and ophthalmic pain. He had undergone chemoradiotherapy (irradiation with 69.6 Gy/37 Fr and chemotherapy with cumulative doses of 280 mg/body carboplatin and 4,000 mg/body 5FU) for hypopharyngeal squamous cell carcinoma 5 months before referral. His complaints resolved gradually within 1 month; however, he developed anemia (Hb, 7.3 g/dL), thrombocytopenia (platelet count, 36 k/lL), and high lactate dehydrogenase (LDH, 836 IU/ L) in early April 2013. His condition rapidly deteriorated thereafter and he suffered respiratory distress and hepatic/ renal failure. There were no skin lesions, lymphadenopathy, or hepatosplenomegaly. No symptoms suspecting CNS disease were present. Repeat transfusions of red blood cells and platelets were performed. Blood analysis showed the following: white blood cell counts, 3,100/lL (5 % abnormal cells); Hb, 8.7 g/dL; platelet count, 63 K/lL; serum CRP, 2.89 mg/dL; aspartate aminotransferase (AST, 4,169 IU/L); alanine aminotransferase (ALT, 1,191 IU/L); LDH, 6,014 IU/L; total bilirubin, 6.2 mg/dL; direct bilirubin, 4.3 mg/dL; ferritin, 1,036 ng/mL; BUN, 48.2 mg/ dL; creatinine, 3.51 mg/dL; and uric acid, 19.6 mg/dL. The patient then developed coagulopathy, with a plasma D-dimer 2.7 lg/mL (normal \1.0 lg/mL), fibrinogen 140 mg/dL (normal 146–380 mg/dL) and antithrombin-III level of 33 % (normal 80–125 %). The bone marrow smear was cellular and showed 77 % blasts, which possessed round or oval nuclei with prominent nucleoli and a deeply stained cytoplasm with protrusions (mimicking plasmacytoid/megakaryoblastoid cells), some of which (6 %) showed weak staining for myeloperoxidase but stained negative for Periodic Acid-Schiff and iron. There were no azurophilic granules. In addition, we noted very large fishshaped blasts containing numerous nucleoli, large cells with mitotic figures, and unevenly divided binuclear blasts (Fig. 1a–d), Flow cytometry showed that bone marrow blasts were CD33 (80 %)/CD56 (96 %) but CD4 (\5 %)/CD7 (\5 %)/CD13 (\5 %)/CD34 (\5 %)/ CD41 (\5 %)/CD61 (\5 %) and HLA-DR (\5 %). Thus, the diagnosis of myeloid/NK cell leukemia was S. Imashuku (&) N. Kudo Division of Hematology, Takasago-seibu Hospital, 1-10-41 Nakasuji, Takasago 676-0812, Japan e-mail: shinim95@mbox.kyoto-inet.or.jp

Acute Myeloid Leukemia: Potential for New Therapeutic Approaches Targeting mRNA Translation Pathways

Despite advances in molecular research related to acute myeloid leukemia (AML) and a better understanding of the mechanisms of leukemogenesis and pathophysiology of the disease, the pharmacological agents used in the treatment of AML have remained essentially unchanged for the last three decades. Advances in the clinical management of AML patients have been achieved by defining better molecular prognostic markers, but there remains a need for new targeted drugs that disrupt non-overlapping pathways in leukemia cells. The mTOR cellular cascade is critical for cell metabolism, growth, proliferation and survival. Extensive preclinical work suggests that targeting mTOR may provide a powerful approach to block AML precursor cells, while other findings suggest enhanced antileukemic effects by combining mTOR inhibitors with traditional chemotherapy. Such combinations may increase antileukemic responses further, offering unique ways to overcome leukemic cell resistance and to eliminate primitive leukemic precursors.

New heterocyclic chalcones. Part 6. Synthesis and cytotoxic activities of 5- or 6-(3-aryl- 2-propenoyl)-2(3H)-benzoxazolones

Abstract A number of chalcones bearing an oxazole cycle were synthesized by Claisen-Schmidt condensation of 5-acetyl-2(3H)-benzoxazolone or 6-acetyl-2(3H)-benzoxazolone and the appropriate aldehydes. The chalcones were evaluated for cytotoxic activity against several tumor cell lines – BV-173 (human B cell precursor leukemia), MCF-7 and MDA-MB-231 (human breast adenocarcinoma) using the MTT-dye reduction assay. The tested compounds exhibit concentration-dependent cytotoxic effects at micromolar concentrations. Exposure of the BV-173 tumor cell line to compound 3f results in strong mono- and oligonucleosomal fragmentation of genomic DNA, as evidenced by a ‘cell death detection’ ELISA kit, which unambiguously indicates that the induction of apoptosis is implicated in the cytotoxic mode of action of the tested compound.

Leukemic T-cell Precursors from T-lineage All Patients Are Characterized by Profound Ku80 Deficiency as well as Functional IK Deficiency with Very Low Ikaros Target Gene Expression Levels

We recently found that Ku regulates Ikaros (IK) function and Ku deficiency causes a lymphoproliferative disorder of thymic T-cell precursors with functional IK deficiency. We now examined the expression levels of validated IK target genes that harbor IK binding sites in primary leukemic cells from 1 104 pediatric ALL patients in relationship to Ku expression levels. Hierarchical cluster analysis of cellular gene expression profiles revealed a highly significant correlation between Ku expression level and expression levels of validated IK target genes. Notably, the expression levels of 12 IK-regulated lymphoid priming genes were significantly upregulated in human lymphocyte precursor cells from primary bone marrow specimens of pediatric patients with ALL expressing high levels of Ku. The observed striking Ku-dependency of the IK target gene expression levels taken together with the results of our earlier RNAi experiments indicate that IK function in human lymphocyte precursors is controlled by Ku expression levels. Leukemic T-cell precursors from children with T-lineage ALL were characterized by profoundly diminished Ku80 transcript levels as well as functional IK deficiency with very low IK target gene expression levels. These results extend our previous studies in mice and indicate that Ku-deficiency is also a contributor to the functional IK-deficiency in pediatric T-lineage ALL.

TLR Stimulation of Bone Marrow Lymphoid Precursors from Childhood Acute Leukemia Modifies Their Differentiation Potentials

Acute leukemias are the most frequent childhood malignancies worldwide and remain a leading cause of morbidity and mortality of relapsed patients. While remarkable progress has been made in characterizing genetic aberrations that may control these hematological disorders, it has also become clear that abnormalities in the bone marrow microenvironment might hit precursor cells and contribute to disease. However, responses of leukemic precursor cells to inflammatory conditions or microbial components upon infection are yet unexplored. Our previous work and increasing evidence indicate that Toll-like receptors (TLRs) in the earliest stages of lymphoid development in mice and humans provide an important mechanism for producing cells of the innate immune system. Using highly controlled co-culture systems, we now show that lymphoid precursors from leukemic bone marrow express TLRs and respond to their ligation by changing cell differentiation patterns. While no apparent contribution of TLR signals to tumor progression was recorded for any of the investigated diseases, the replenishment of innate cells was consistently promoted upon in vitro TLR exposure, suggesting that early recognition of pathogen-associated molecules might be implicated in the regulation of hematopoietic cell fate decisions in childhood acute leukemia.

Massive evolution of the immunoglobulin heavy chain locus in children with B precursor acute lymphoblastic leukemia

AbstractThe ability to distinguish clonal B-cell populations based on the sequence of their rearranged immunoglobulin heavy chain (IgH) locus is an important tool for diagnosing B-cell neoplasms and monitoring treatment response. Leukemic precursor B cells may continue to undergo recombination of the IgH gene after malignant transformation; however, the magnitude of evolution at the IgH locus is currently unknown. We used next-generation sequencing to characterize the repertoire of IgH sequences in diagnostic samples of 51 children with B precursor acute lymphoblastic leukemia (B-ALL). We identified clonal IgH rearrangements in 43 of 51 (84%) cases and found that the number of evolved IgH sequences per patient ranged dramatically from 0 to 4024. We demonstrate that the evolved IgH sequences are not the result of amplification artifacts and are unique to leukemic precursor B cells. In addition, the evolution often follows an allelic exclusion pattern, where only 1 of 2 rearranged IgH loci exhibit ongoing recombination. Thus, precursor B-cell leukemias maintain evolution at the IgH locus at levels that were previously underappreciated. This finding sheds light on the mechanisms associated with leukemic clonal evolution and may fundamentally change approaches for monitoring minimal residual disease burden.

Plerixafor (AMD3100) induces prolonged mobilization of acute lymphoblastic leukemia cells and increases the proportion of cycling cells in the blood in mice

The CXCR4 antagonist Plerixafor (AMD3100) induces the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. AMD3100 similarly induces the mobilization of human acute lymphoblastic leukemia (ALL) cells into the blood in mice. In this study, the temporal response of pre-B ALL cells to AMD3100 was compared with that of normal hematopoietic progenitor cells (HPC) using an NOD/SCID xenograft model of ALL and BALB/c mice, respectively. ALL cells remained in the circulation up to 6 hours after AMD3100 administration, by which time normal HPCs were no longer detectable. AMD3100 also increased the proportion of actively cycling ALL cells in the peripheral blood. Together, these data suggest that ALL cells are more sensitive to the effects of bone marrow disruption than normal progenitors. Using the NOD/SCID xenograft model, we demonstrated that AMD3100 increased the efficacy of the cell cycle specific drug vincristine, resulting in reduced disease levels in the blood and spleens of animals over 3 weeks and extended the survival of NOD/SCID mice with ALL. These data demonstrate that mobilizing agents can increase the therapeutic effect of cell cycle dependent chemotherapeutic agents.

Autophagic degradation of the BCR-ABL oncoprotein and generation of antileukemic responses by arsenic trioxide

AbstractWe provide evidence that arsenic trioxide (As2O3) targets the BCR-ABL oncoprotein via a novel mechanism involving p62/SQSTM1-mediated localization of the oncoprotein to the autolysosomes and subsequent degradation mediated by the protease cathepsin B. Our studies demonstrate that inhibitors of autophagy or cathepsin B activity and/or molecular targeting of p62/SQSTM1, Atg7, or cathepsin B result in partial reversal of the suppressive effects of AS2O3 on BCR-ABL expressing leukemic progenitors, including primitive leukemic precursors from chronic myelogenous leukemia (CML) patients. Altogether, these findings indicate that autophagic degradation of BCR-ABL is critical for the induction of the antileukemic effects of As2O3 and raise the potential for future therapeutic approaches to target BCR-ABL expressing cells by modulating elements of the autophagic machinery to promote BCR-ABL degradation.

Early T-cell precursor leukemia: a subtype of high risk childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia includes T-cell acute lymphoblastic leukemia (T-ALL) and B-cell acute lymphoblastic leukemia (B-ALL). In children, T-ALL usually has a worse prognosis than B-ALL, although childhood T-ALL prognoses have improved remarkably. The varying outcomes among T-ALL cases suggest that an unrecognized biological heterogeneity may contribute to chemo-resistance. Deep exploration of T-lymphocyte development in recent years has found a subgroup of patients with a phenotype that resembles early T-cell precursor, which confers a much poorer prognosis than any other form of T-ALL. This novel subtype of T-ALL was called early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). Flow cytometry data from T-ALL patients enrolled in Shanghai Children's Medical Center between July 2002 and October 2010 were assessed according to Dr. Campana's protocol. Among total 89 T-ALL cases, 74 cases had enough immunophenotype data available to differentiate between ETP (CD1a(-), CD8(-), CD5(dim), at least one marker of stem cell or myeloid lineage) and non-ETP. From these 74 subjects, 12 ETP-ALL cases (16.2%) were identified. The event-free survival (EFS) rate at 66.8 months was 11.1% ± 10.1% for ETP-ALL and 57.6% ± 5.6% for non-ETP-ALL (P = 0.003). The overall survival rates were 13.3% ± 11.0% for ETP-ALL and 64.7% ± 6.3% for non-ETP-ALL (P = 0.002). Our findings demonstrate that early T-cell precursor leukemia is a very high-risk subtype of acute lymphoblastic leukemia with poor prognosis.

Abstract LB-81: Autophagic degradation of BCR/ABL by arsenic trioxide and the role of cysteine cathepsins

Abstract Autophagy is increasingly an area of high potential therapeutic interest in hematopoietic malignancies, but its precise involvement in the control of leukemic cell growth and survival remain to be defined. We provide evidence that arsenic trioxide (As2O3) targets the BCR-ABL oncoprotein for degradation via autophagy, involving the p62/SQSTM1 and the protease cathepsin B. Utilizing either florescence probes or gold-conjugate antibodies we show co-localization of BCR-ABL and p62/SQSTM1 in autolysosomes. Also, by drug inhibition of either autophagy or cathepsin B, or by molecularly targeting p62/SQSMT1, Atg7 or cathepsin B, we documented reversal of the suppressive effect of As2O3 on BCR-ABL expressing cells, including primitive leukemic precursors from chronic myelogenous leukemia (CML) patients. Altogether, our data indicate that autophagy-induced degradation of BCR-ABL is critical for the generation of As2O3 antileukemic effects. These results raise the potential of targeting the autophagic machinery to enhance the antileukemic properties of arsenic trioxide on leukemia initiating stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-81. doi:1538-7445.AM2012-LB-81

Clinical and molecular characterization of early T-cell precursor leukemia: a high-risk subgroup in adult T-ALL with a high frequency of FLT3 mutations

A subgroup of pediatric acute T-lymphoblastic leukemia (T-ALL) was characterized by a gene expression profile comparable to that of early T-cell precursors (ETPs) with a highly unfavorable outcome. We have investigated clinical and molecular characteristics of the ETP-ALL subgroup in adult T-ALL. As ETP-ALL represents a subgroup of early T-ALL we particularly focused on this cohort and identified 178 adult patients enrolled in the German Acute Lymphoblastic Leukemia Multicenter studies (05/93–07/03). Of these, 32% (57/178) were classified as ETP-ALL based on their characteristic immunophenotype. The outcome of adults with ETP-ALL was poor with an overall survival of only 35% at 10 years, comparable to the inferior outcome of early T-ALL with 38%. The molecular characterization of adult ETP-ALL revealed distinct alterations with overexpression of stem cell-related genes (BAALC, IGFBP7, MN1, WT1). Interestingly, we found a low rate of NOTCH1 mutations and no FBXW7 mutations in adult ETP-ALL. In contrast, FLT3 mutations, rare in the overall cohort of T-ALL, were very frequent and nearly exclusively found in ETP-ALL characterized by a specific immunophenotype. These molecular characteristics provide biologic insights and implications with respect to innovative treatment strategies (for example, tyrosine kinase inhibitors) for this high-risk subgroup of adult ETP-ALL.

ANALYSIS OF THE NUMBER OF CELLS WITH CD34+CD38- AND CD34+CD38-CD19+ PHENOTYPES AS POTENTIAL LEUKEMIC STEM CELLS IN ACUTE LYMPHOBLASTIC LEUKEMIA IN CHILDREN

The number of the supposed leukemic stem cells in marrow samples of 54 patients with primary B-linear acute lymphoblastic leukemia was detected by the method of multiparametric flow cytofluorimetry in leukemia diagnosis (zero day). The level of minimal residual disease was estimated on zero and on the fifteenth days of induction therapy. In the course of the research it was found out that leukemic B-cell precursors with СD34+СD38-CD19+ phenotype prevailed among the cells with СD34+СD38-phenotype. The high percentage of both СD34+СD38-, СD34+СD38-, and СD34+СD38-CD19+ among the general population leukemic cells was associated with a worse response to the therapy. Therefore, the initial number of such cells can be considered as a prognostic marker in acute lymphoblastic leukemia in children.

Antileukemic effects of AMPK activators on BCR-ABL–expressing cells

AbstractThe mammalian target of rapamycin (mTOR) signaling pathway plays a critical role in growth and survival of BCR-ABL transformed cells. AMPK kinase is a metabolic sensor that exhibits suppressive effects on the mTOR pathway and negatively regulates mTOR activity. We report that AMPK activators, such as metformin and 5-aminoimidazole-4-carboxamide ribonucleotide, suppress activation of the mTOR pathway in BCR-ABL–expressing cells. Treatment with these inhibitors results in potent suppression of chronic myeloid leukemia leukemic precursors and Ph+ acute lymphoblastic leukemia cells, including cells expressing the T315I-BCR-ABL mutation. Altogether, our data suggest that AMPK is an attractive target for the treatment of BCR-ABL–expressing malignancies and raise the potential for use of AMPK activators in the treatment of refractory chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia.

In vitro and in vivo chemosensitizing activity of LFM-A13, a dual-function inhibitor of Bruton's tyrosine kinase and polo-like kinases, against human leukemic B-cell precursors

The present study documents the chemosensitizing anti-leukemic activity of the leflunomide metabolite (LFM) analog, LFM-A13, a dual-function inhibitor of Bruton's tyrosine kinase (BTK) and Polo-like kinases (PLK), against human leukemic B-cell precursors. The results in 135 xenografted NOD/SCID mice regarding the anti-leukemic activity of GMP-grade LFM-A13, obtained with only 4-days of LFM-A13 therapy at nontoxic dose levels corresponding to 1-20% of its NOAEL (no observable advserse effect level), alone or in combination with the standard chemotherapy drug vincristine, demonstrate the potential of LFM-A13 as a new anti-leukemic drug candidate. All 82 LFM-A13-treated mice, including those receiving a combination of vincristine + LFM-A13 at the highest dose level of LFM-A13, tolerated their treatments well without weight loss, diarrhea, lethargy/ paralysis, other signs of morbidity, or mortality. The present study provides preclinical proof-of-principle for the development of LFM-A13 as a new chemosensitizing and apoptosis-promoting anti-leukemic agent and lends support to the hypothesis that the chemoresistance of relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) can be overcome by using LFM-A13 in combination with chemotherapy. Also presented are the results of a comprehensive meta-analysis of the overexpression of genes for LFM-A13 targeted kinases and their downstream effector molecules in B-lineage lymphoid malignancies utilizing the Oncomine database.

Morphological Atypia Represents Favorable Prognosis of Chronic Lymphocytic Leukemia in Japanese Patients

Abstract 4594 Background:B-chronic lymphocytic leukemia (B-CLL) is rare in Japan. Therefore, only a few data are available on the clinical characteristics of B-CLL for Japanese patients (pts). We reported that the rate of atypical CLL appeared to be higher in Japanese pts than that of Caucasians. Patients and Methods:This nation-wide study was performed from Oct. 2003 to March 2008, and was supported by Japanese Elderly Leukemia and Lymphoma Study Group. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Boards of the core members’ hospitals. The pts with CLL and CLL-like disease were registered. Precursor leukemia and adult T-cell leukemia/lymphoma were excluded. The smears of peripheral blood and bone marrow of all the registered pts were examined by the Diagnosis Committee by using the 4th edition of WHO classification and FAB classification. FAB classification defines typical CLL as prolymphocytes≤10% and atypical CLL including CLL/PLL as 11%≤prolymphocyte<55% and CLL mixed as prolymphocytes<10% and pleomorphic lymphocyte>15%. Mantle cell lymphoma was excluded by means of cytogenetics, fluorescent in situ hybridization of CCND1-IGH, and immunohistochemistry of CCND1. Results:One hundred and thirty-one evaluable pts were registered. There were 96 pts with B-CLL, 2 SLL, 1 B-PLL, 3 hairy cell leukemia, 16 leukemic phase of other B-cell lymphomas. Thirteen pts had NK or T cell leukemia/lymphoma with 7 T-LGL, 4 T-PLL, 1 NK-LGL, and 1 Sezary syndrome. Among B-CLL, there were 62 pts with typical morphology (typical CLL), 18 with atypical morphology (atypical CLL), 16 with intermediate morphology between typical and atypical CLL (intermediate CLL). The differences among 3 subgroups are shown in Table. No differences were found between the 3 morphological subgroups for age, sex, stage (modified Rai and Binet classification), hemoglobin level, platelet counts, the presence of hepatomegaly, splenomegaly and lympoadenopathy, ECOG-PS and the rate of treatment requirement. However, the incidence of CD5 and CD23 expression was higher in typical CLL than in atypical/intermediate morphology groups (P<0.001), i.e. 7% of the pts with atypical CLL showed co-expression of CD5 and CD23. The overall survival (OS) of typical CLL pts was lower than that of intermediate or atypical CLL pts (Figure). Conclusion:Morphological atypia was more frequent in Japanese B-CLL pts, partly because of the low incidence of typical CLL. The atypical/intermediate CLLs were associated with atypical immunophenotype (absence of CD5 and/or CD23) and favorable prognosis. Prospective study is ongoing to evaluate other prognositic factors including the implication of IgVH gene mutation status and ZAP-70 expression in Japan.TableTypicalIntermediateAtypicalPPatients No.621618Age, median6765670.50Sex (male/female)1.301.292.60.47CD20+98.4%100%88.9%0.16CD5+98.4%81.30%77.8%0.003CD5+23+86.5%50.0%7.0%<0.001No treatment45%50%61%0.49Outcome(died)29.8%0%12.5%0.018 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Distinct Metabolic Dependency of Normal and Leukemic Cells in a Mouse Model

Abstract 759We hypothesized that metabolic differences between leukemia initiating cells and their normal counterparts represent a vulnerability in the leukemia initiating cell, which can be therapeutically exploited. To test this hypothesis, we used the MLL-AF9 acute myeloid leukemia (AML) model in mice. Actin-DsRed mouse bone marrow transduced with MLL-AF9 expressing retrovirus was used to produce serially transplantable leukemia. Leukemic granulocyte-monocyte precursors (L-GMPs), defined by others to be the leukemia initiating cells were flow sorted from secondary recipient mice and compared with normal GMPs (N-GMPs) from actin Ds-Red mice.Gene expression profiling, metabolomic profiling via liquid chromatography- mass spectrometry and an in vitro shRNA screen were used to identify metabolic pathways preferentially activated in leukemia initiating cells. Of 1574 defined metabolic enzymes, 44 were found to be differentially expressed between L-GMPs and their normal counterparts (N-GMPs). These together with 117 classic rate limiting enzymes were subjected to shRNA knockdown in vitro. Metabolomic profiling of both cell populations was used to corroborate findings from shRNA knockdowns.L-GMPs and N-GMPs were transduced with lentivirus expressing shRNAs of interest (5 shRNAs per gene) in a 384 well format, selected with puromycin and cultured for 72–96 hours in the presence of GFP-positive primary bone marrow stroma. The number of cells in each well at the end of this experiment was quantitated using an Image Xpress microscope. Genes, the knockdown of which by at least two independent shRNAs produced a two fold or more decrease in L-GMPs as compared to control wells and did not similarly decrease N-GMPs, were chosen for in vivo validation. Ten genes in the glycolysis pathway and TCA cycle, fatty acid metabolism and detoxification, and ketohexokinase were identified.Ketohexokinase, a rate-limiting enzyme in fructose metabolism was particularly potent and of interest given its potential to be exploited therapeutically. In vivo assessment of its relative ability to inhibit malignant versus normal hematopoietic cells is ongoing. These studies provide preliminary support for the hypothesis that specific metabolic circuits are differentially active in leukemia initiating cells in MLL-AF9 AML and may represent unique points of vulnerability that can be targeted therapeutically.Authors 1 and 2 contributed equally. Authors 3 and 4 contributed equally. Disclosures:No relevant conflicts of interest to declare.

Ca2+/NFAT Signaling Regulates the Expression CD38 and ZAP70 in Murine B Cells and Controls B1a Cell Homeostasis

Abstract 183NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT is inactivated and relocated to the cutoplasm by a network of several kinases including CK-1, GSK-3 and DYRK. Although identified originally as a major transcriptional regulator in T cells, it is now clear that NFAT transcription factors also possess important roles in other cells of the hematopoietic system including dendritic cells, mast cells, megakaryocytes and B cells.Here we have analyzed the role of NFAT2 in B cell development. Analysis of the role of this family member in hematopoiesis has been complicated by the fact that deletion of this gene is embryonic lethal around ED 13 because of defects in heart valve development. To circumvent this problem we generated mice with a conditional NFAT2 knock out allele (NFAT2fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2fl/fl mice to CD19-Cre mice, in which the Cre recombinase is expressed under the control of the B cell-specific cd19 promoter.B cells from these mice were isolated using CD19-labeled magnetic beads and subjected to analysis by flow cytometry. While CD19+ splenocytes from conditional NFAT2 knock-out mice occurred in normal numbers, these cells showed significantly reduced expression of CD38 and ZAP70 upon stimulation with anti-IgM antibody as compared to CD19+ splenocytes from wild-type controls. The reduction of these proteins could also be detected in B cells isolated from the peripheral blood and from bone marrow and was confirmed by western blotting and quantitative RT-PCR. CD38 and ZAP70 are well characterized prognostic factors in chronic lymphocytic leukemia (CLL) and their increased expression has been shown to correlate with poor patient survival. Our data indicate that the expression of these markers is at least in part regulated by Ca2+/NFAT signaling and that deregulation of this pathway can contribute to their overexpression in disease.Next we analyzed bone marrow and peritoneal lavages from conditional NFAT knock-out mice by flow cytometry. While we found no significant differences in the abundance of B cell subpopulations in bone marrow, we detected an almost complete absence of CD5+CD43+ B1a cells in the peritoneal cavity, clearly demonstrating the requirement of NFAT2 in the development of this subclass. B1a cells are a phenotypically and functionally distinct population of B cells which are long-lived and typically express CD5, CD43 and high levels of surface IgM together with low surface IgD and CD45 (B220). A human B cell equivalent of the murine B1a cell has been suggested as the leukemic precursor cell in chronic lymphocytic leukemia (CLL). To further delineate the role of NFAT2 in the development of B1a cells we determined the abundance of B1 progenitor cells (B1P) in bone marrow and spleen by FACS analysis. In NFAT2 knock-out mice we observed a significant reduction of the frequency of B220- CD19+ CD93+ B1P cells in bone marrow (0.8% vs. 4.7%) and spleen (0.16% vs. 0.82%) demonstrating that NFAT2 is essential for normal development of this precursor cell population.In summary, our data provide strong evidence that NFAT2 is critical for the expression of CD38 and ZAP70 in B cells and substantially controls B1a cell homeostasis implicating Ca2+/NFAT signaling as a potential target for the treatment of CLL. Disclosures:No relevant conflicts of interest to declare.