Phenothiazines induce PP2A-mediated apoptosis in T cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that is frequently associated with activating mutations in NOTCH1 and dysregulation of MYC. Here, we performed 2 complementary screens to identify FDA-approved drugs and drug-like small molecules with activity against T-ALL. We developed a zebrafish system to screen small molecules for toxic activity toward MYC-overexpressing thymocytes and used a human T-ALL cell line to screen for small molecules that synergize with Notch inhibitors. We identified the antipsychotic drug perphenazine in both screens due to its ability to induce apoptosis in fish, mouse, and human T-ALL cells. Using ligand-affinity chromatography coupled with mass spectrometry, we identified protein phosphatase 2A (PP2A) as a perphenazine target. T-ALL cell lines treated with perphenazine exhibited rapid dephosphorylation of multiple PP2A substrates and subsequent apoptosis. Moreover, shRNA knockdown of specific PP2A subunits attenuated perphenazine activity, indicating that PP2A mediates the drug’s antileukemic activity. Finally, human T-ALLs treated with perphenazine exhibited suppressed cell growth and dephosphorylation of PP2A targets in vitro and in vivo. Our findings provide a mechanistic explanation for the recurring identification of phenothiazines as a class of drugs with anticancer effects. Furthermore, these data suggest that pharmacologic PP2A activation in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates has therapeutic potential.     

Activation of PI3K is indispensable for interleukin 7-mediated viability, proliferation, glucose use, and growth of T cell acute lymphoblastic leukemia cells

Interleukin (IL)-7 is essential for normal T cell development. Previously, we have shown that IL-7 increases viability and proliferation of T cell acute lymphoblastic leukemia (T-ALL) cells by up-regulating Bcl-2 and down-regulating the cyclin-dependent kinase inhibitor p27kip1. Here, we examined the signaling pathways via which IL-7 mediates these effects. We investigated mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase (Erk) and phosphatidylinositol-3-kinase (PI3K)-Akt (protein kinase B) pathways, which have active roles in T cell expansion and have been implicated in tumorigenesis. IL-7 induced activation of the MEK-Erk pathway in T-ALL cells; however, inhibition of the MEK-Erk pathway by the use of the cell-permeable inhibitor PD98059, did not affect IL-7-mediated viability or cell cycle progression of leukemic cells. IL-7 induced PI3K-dependent phosphorylation of Akt and its downstream targets GSK-3, FOXO1, and FOXO3a. PI3K activation was mandatory for IL-7-mediated Bcl-2 up-regulation, p27kip1 down-regulation, Rb hyperphosphorylation, and consequent viability and cell cycle progression of T-ALL cells. PI3K signaling was also required for cell size increase, up-regulation of CD71, expression of the glucose transporter Glut1, uptake of glucose, and maintenance of mitochondrial integrity. Our results implicate PI3K as a major effector of IL-7-induced viability, metabolic activation, growth and proliferation of T-ALL cells, and suggest that PI3K and its downstream effectors may represent molecular targets for therapeutic intervention in T-ALL.     

伴t(1;19)(q23;p13)和E2A-PBX1成人急性T淋巴细胞白血病一例报告附文献复习

目的 总结1例伴有t(1;19)和E2A-PBX1融合基因阳性的成人T细胞急性淋巴细胞白血病(ALL)患者的诊疗体会.方法 对1例成人T细胞ALL患者进行染色体核型分析,流式细胞术检测免疫表型,同时进行融合基因多重RT-PCR扩增.结果 患者染色体核型为47,XY,9p+,15p+,17q-,der(19),t(1;19)(q23;p13)[5]/46,XY[15].E2A-PBX1融合基因阳性表达.给予hyperCVAD(环磷酰胺+长春新碱+阿霉素+地塞米松)方案治疗后患者获血液学完全缓解,染色体核型复查为46,XY[10],E2A-PBX1融合基因检测阴性.结论 E2A-PBX1+ t(1;19)也可以发生于T细胞ALL,E2a-PBX1白血病细胞在不同的癌基因协同信号或微环境下转化方向可变.     

伴t(1;19)(q23;p13)和E2A-PBX1成人急性T淋巴细胞白血病一例报告附文献复习

目的 总结1例伴有t(1;19)和E2A-PBX1融合基因阳性的成人T细胞急性淋巴细胞白血病(ALL)患者的诊疗体会.方法 对1例成人T细胞ALL患者进行染色体核型分析,流式细胞术检测免疫表型,同时进行融合基因多重RT-PCR扩增.结果 患者染色体核型为47,XY,9p+,15p+,17q-,der(19),t(1;19)(q23;p13)[5]/46,XY[15].E2A-PBX1融合基因阳性表达.给予hyperCVAD(环磷酰胺+长春新碱+阿霉素+地塞米松)方案治疗后患者获血液学完全缓解,染色体核型复查为46,XY[10],E2A-PBX1融合基因检测阴性.结论 E2A-PBX1+ t(1;19)也可以发生于T细胞ALL,E2a-PBX1白血病细胞在不同的癌基因协同信号或微环境下转化方向可变.     

The SCFFBW7 ubiquitin ligase complex as a tumor suppressor in T cell leukemia

Recent studies have shown that activating mutations of NOTCH1 are responsible for the majority of T cell acute lymphoblastic leukemia (T-ALL) cases. Most of these mutations truncate its C-terminal domain, a region that is important for the NOTCH1 proteasome-mediated degradation. We report that the E3 ligase FBW7 targets NOTCH1 for ubiquitination and degradation. Our studies map in detail the amino acid degron sequence required for NOTCH1-FBW7 interaction. Furthermore, we identify inactivating FBW7 mutations in a large fraction of human T-ALL lines and primary leukemias. These mutations abrogate the binding of FBW7 not only to NOTCH1 but also to the two other characterized targets, c-Myc and cyclin E. The majority of the FBW7 mutations were present during relapse, and they were associated with NOTCH1 HD mutations. Interestingly, most of the T-ALL lines harboring FBW7 mutations were resistant to gamma-secretase inhibitor treatment and this resistance appeared to be related to the stabilization of the c-Myc protein. Our data suggest that FBW7 is a novel tumor suppressor in T cell leukemia, and implicate the loss of FBW7 function as a potential mechanism of drug resistance in T-ALL.     

Inhibition of IRAK1/4 sensitizes T cell acute lymphoblastic leukemia to chemotherapies

Signaling via the MyD88/IRAK pathway in T cells is indispensable for cell survival; however, it is not known whether this pathway functions in the progression of T acute lymphoblastic leukemia (T-ALL). Here, we determined that compared with thymic and peripheral T cells, T-ALL cells from patients have elevated levels of IRAK1 and IRAK4 mRNA as well as increased total and phosphorylated protein. Targeted inhibition of IRAK1 and IRAK4, either with shRNA or with a pharmacological IRAK1/4 inhibitor, dramatically impeded proliferation of T-ALL cells isolated from patients and T-ALL cells in a murine leukemia model; however, IRAK1/4 inhibition had little effect on cell death. We screened several hundred FDA-approved compounds and identified a set of drugs that had enhanced cytotoxic activity when combined with IRAK inhibition. Administration of an IRAK1/4 inhibitor or IRAK knockdown in combination with either ABT-737 or vincristine markedly reduced leukemia burden in mice and prolonged survival. IRAK1/4 signaling activated the E3 ubiquitin ligase TRAF6, increasing K63-linked ubiquitination and enhancing stability of the antiapoptotic protein MCL1; therefore, IRAK inhibition reduced MCL1 stability and sensitized T-ALL to combination therapy. These studies demonstrate that IRAK1/4 signaling promotes T-ALL progression through stabilization of MCL1 and suggest that impeding this pathway has potential as a therapeutic strategy to enhance chemotherapeutic efficacy.     

Heterogeneity of cultured leukemic lymphoid progenitor cells from B cell precursor acute lymphoblastic leukemia (ALL) patients.

Colony assays were performed for 50 patients with B cell precursor acute lymphoblastic leukemia (ALL). Blast colony formation was observed for 33 patients, and the plating efficiency (PE) showed a marked interpatient variation, which indicates a pronounced biological heterogeneity at the level of leukemic progenitor cells. Notably, the mean PE of leukemic B cell precursors from patients with a pseudodiploid or near-diploid karyotype with structural chromosomal abnormalities (SCA) was significantly higher than the mean PE of normal diploid or hyperdiploid cases. All patients who had SCA involving 7p13, 11q23-24, or 12p11-13, and patients with a Philadelphia chromosome had high PE values. The S phase percentage, expression of CD19 antigen, and relapse status were also correlated with PE. Significantly, colony blasts had slightly different surface marker profiles in each case and were common ALL antigen negative in 33% of cases, which indicates the existence of a marked immunological heterogeneity at the level of leukemic progenitor cells.     

Rearrangements of the tal-1 locus as clonal markers for T cell acute lymphoblastic leukemia.

Normal and aberrant immune receptor gene assembly each produce site-specific DNA rearrangements in leukemic lymphoblasts. In either case, these rearrangements provide useful clonal markers for the leukemias in question. In the t(1;14)(p34;q11) translocation associated with T cell acute lymphoblastic leukemia (T-ALL), the breakpoints on chromosome 1 interrupt the tal-1 gene. A site-specific deletion interrupts the same gene in an additional 26% of T-ALL. Thus, nearly one-third of these leukemias contain clustered rearrangements of the tal-1 locus. To test whether these rearrangements can serve as markers for residual disease, we monitored four patients with T-ALL; three of the leukemias contained a deleted (tald) and one a translocated (talt) tal-1 allele. These alleles were recognized by a sensitive amplification/hybridization assay. tald alleles were found in the blood of one patient during the 4th mo of treatment but not thereafter. Using a quantitative assay to measure the fraction of tald alleles in DNA extracts, we estimated that this month 4 sample contained 150 tald copies per 10(6) genome copies. The patient with t(1;14)(p34;q11) (talt) leukemia developed a positive assay during the 20th mo of treatment. By standard criteria, all four patients remain in complete remission 11-20 mo into treatment. We conclude that tal-1 rearrangements provide useful clonal markers for approximately 30% of T-ALLs.     

Clonal analysis of childhood acute lymphoblastic leukemia with "cytogenetically independent" cell populations

Acute lymphoblastic leukemia (ALL) is generally regarded as a clonal disease in which a single abnormal progenitor cell gives rise to neoplastic progeny. Five of 463 cases of childhood ALL with adequately banded leukemic cells were found to have two cytogenetically independent cell populations. In addition, two of the four cases tested had more than two rearranged immunoglobulin genes and (or) T cell receptor genes. To investigate the clonality of these unusual leukemias, we examined the neoplastic cells for X-linked markers extrinsic to the disease. Leukemic cells from each of the three patients heterozygous for an X-linked, restriction fragment length polymorphism showed a single active parental allele, suggesting that both apparently independent cell populations developed from a common progenitor. These cases provide evidence that leukemogenesis involves a multistep process of mutation and suggest that karyotypic abnormalities may be a late event of malignant transformation.     

Rat lymphoid cell lines with human T cell leukemia virus production. I. Biological and serological characterization

Cocultivation of spleen cells, lymph node cells, and thymocytes of female Wistar-King-Aptekman rats with short-term cultured male adult T cell leukemia (ATL) cells in the presence of 5-bromo-2'-deoxyuridine (BrdUrd) resulted in the establishment of rat lymphoid cell lines, TARS-1, TARL-2, and TART-1. Cytogenetic analysis of the three cell lines showed a female rat karyotype with 42 chromosomes. The surface phenotypes of TARS-1 and TART-1 were those of rat T cells. TARL-2 was only positive for rat Ia and leukocyte common antigens. The cell lines continuously produced a type C retrovirus, human T cell leukemia virus (HTLV) and expressed ATL-associated antigens. TARS-1 and TART-1, but not TARL-2 were transplantable into newborn syngeneic rats and nude mice. These results strongly indicate that HTLV not only immortalizes, but also transforms rat T cells in vitro. Adult rats immunized with either TARS-1 or TARL-2 produced antibodies specific for HTLV. The biochemical analysis of the antigens that reacted with rat sera revealed that they are the two HTLV-specific polypeptides, p24 and p28.     

Induction of human B cell antigens in non-T cell acute lymphoblastic leukemia.

Leukemic cells from 70% of patients with Ia+CALLA+ non-T cell acute lymphoblastic leukemia (ALL) express an antigen (B1) found on all normal B lymphocytes. In this study, ALL cells that do not express the B1 antigen were studied in an attempt to further elucidate the cellular lineage of these tumors. Non-T cell ALL lines and tumor cells isolated from patients with non-T cell ALL that are Ia + CALLA + B1- were studied in vitro with a variety of agents known to promote cellular differentiation. Phorbol diester (TPA) or phytohemagglutinin conditioned leukocyte culture media were capable of inducing the expression of B1 on all four non-T cell ALL lines tested. In contrast, B1 could not be induced under the identical conditions on a promyelocytic leukemia line or a T cell lymphoblastic leukemia line. With the induction of B1 on non-T cell ALL lines, cytoplasmic mu-heavy chain (c mu) became undetectable, whereas the expression of CALLA and Ia were unchanged. The expression of B1 was accompanied by a decrease of cellular proliferation and DNA synthesis, but not significant morphologic changes were noted. In addition, no other B or T cell antigens were detected. The cellular origin of non-T cell ALL was further investigated using tumor cells isolated from leukemic patients. Tumor cells from eight patients with Ia + CALLA + B1-c mu- ALL could be induced in vitro with TPA to express both B1 and c mu. In contrast, cells from five patients with Ia + CALLA-B1-c mu- non-T cell ALL could not be induced with TPA to express CALLA, B1 or c mu. These studies suggest that the non-T cell ALL are heterogeneous and represent a spectrum of early B cell differentiation including the pre- pre-B cell (Ia + CALLA + B1-c mu-), the intermediate pre-B cell (Ia + CALLA +B1 + c mu-), and finally the "true" pre-B cell (Ia + CALLA + B1 + c mu+). The cellular origin of the remaining Ia + CALLA-B1-c mu- form of non-T cell ALL (20%) is still unknown.     

Immunoglobulin mu-chain gene rearrangement in a patient with T cell acute lymphoblastic leukemia

A 6-yr-old girl with T cell acute lymphoblastic leukemia (ALL) is described. She had a mediastinal mass and her leukemic cells expressed T cell-associated antigens (Leu 1+, OKT3+, OKT9+, and OKT10+). When we examined genomic DNA from the leukemic cells, we detected Ig mu-chain gene rearrangement with germ-line configuration of light chain genes. As reported recently, detecting Ig gene rearrangement has become an important procedure for further classifying B cell precursor cells. This case, however, suggests that there is also heterogeneity among patients with T cell ALL, not only at the level of cell surface phenotypes, but also at the level of the Ig gene. These findings have major implications when we consider both the ontogenesis of these leukemic cells and the normal differentiation of human lymphocytes.     

Origin of human T-lymphotrophic virus I-positive T cell lines in adult T cell leukemia. Analysis of T cell receptor gene rearrangement

Using the clone-specific rearrangement of the T cell receptor gene as the genetic marker of the clonotype, we analyzed the clonal origin of the interleukin 2 (IL-2)-dependent human T-lymphotrophic virus I (HTLV-I)-positive T cell lines established from various adult T cell leukemia (ATL) patients. From a patient with chronic ATL, whose leukemic cells proliferated in vitro in response to IL-2, we repeatedly established leukemic T cell clones having the same rearrangement profile of the T beta chain gene as the leukemic cells. By contrast, established cell lines from acute ATL patients had different beta chain gene rearrangements from those of the leukemic cells. These HTLV-I+ T cell lines might not be the direct progeny of the leukemic cells, but that of T cells infected either in vivo or in vitro. These IL-2-reactive nonleukemic T cells might have been selected in vitro, because their leukemic cells failed to respond to IL-2, despite the expression of IL-2 receptor. The analysis of the T cell receptor gene rearrangement may give a new approach for the elucidation of the mechanism of leukemogenesis and the origin of the HTLV-I+ T cell lines in ATL.     

Common acute lymphoblastic leukemia antigen expressed on leukemia and melanoma cell lines has neutral endopeptidase activity.

We have previously reported that the amino acid sequence of the common acute lymphoblastic leukemia antigen (CALLA, CD10) translated from a normal human kidney cDNA clone is identical to that of neutral endopeptidase (NEP, EC 3.4.24.11). In this study, we show that by flow cytometry, a monoclonal antibody (135A3) produced against rabbit NEP reacted selectively with leukemia and melanoma cell lines expressing CALLA on their surface. A glycoprotein of apparent Mr 100,000 was immunoprecipitated from surface labeled NALM-1 leukemia or Mel-1477 melanoma cells with monoclonal antibodies to NEP (135A3) or CALLA (44C10). mRNAs hybridizing to a NEP-specific probe were present in CALLA+ leukemia and melanoma cell lines, but absent from CALLA- lines. NEP enzymatic activity was detected on intact cells from CALLA+ lines, but not CALLA- lines. The activity was blocked by two selective inhibitors of NEP, thiorphan and phosphoramidon. CALLA antigen purified from the NALM-6 leukemic cell line by affinity to 44C10-IgG Sepharose retained a peptidase activity that was completely blocked by thiorphan and phosphoramidon. Thus the CALLA antigen present at the surface of leukemia and melanoma cell lines is an enzymatically active neutral endopeptidase.