Primary Myeloid Leukemia Cells Research Articles

Curcumin induces apoptosis by inhibiting BCAT1 expression and mTOR signaling in cytarabine‑resistant myeloid leukemia cells.

Cytarabine is a key chemotherapy drug for treating leukemia; however, chemotherapy-induced multidrug resistance is a major cause of therapy failure or tumor recurrence. Current medical treatment strategies still cannot address the issue of multidrug resistance phenotypes in the treatment of leukemia. Curcumin counteracts tumor development by inducing apoptosis in cytarabine-resistant acute myeloid leukemia cells. Branched-chain amino acid transaminase 1 (BCAT1), an aminotransferase enzyme, acts on branched-chain amino acids. Moreover, the aberrant expression of BCAT1 has been observed in numerous cancer cells, and BCAT1 serves a critical role in the progression of myeloid leukemia. BCAT1 can interfere with cancer cell proliferation by regulating mTOR-mediated mitochondrial biogenesis and function. The present study aimed to investigate whether curcumin induces apoptosis by regulating BCAT1 expression and mTOR signaling in cytarabine-resistant myeloid leukemia cells. Four leukemia cell lines and three primary myeloid leukemia cells were treated with curcumin, and the expression and activity of BCAT1 and mTOR were investigated by reverse transcription-quantitative PCR, western blotting and α-KG quantification assay. The results demonstrated that curcumin inhibited BCAT1 expression in Kasumi-1, KG-1, HL60, cytarabine-resistant HL60, and cytarabine-resistant primary myeloid leukemia cells. Notably, tetrahydrocurcumin, a major metabolite of curcumin, and cytarabine had no inhibitory effect on BCAT1 expression. Furthermore, BCAT1 and mTOR signaling may modulate each other in cytarabine-resistant HL60 cells. The present results indicated that curcumin may induce apoptosis by inhibiting the BCAT1 and mTOR pathways. Thus, understanding the mechanism underlying curcumin-induced apoptosis in cytarabine-resistant cells can support the development of novel drugs for leukemia.

Jak-Stat Signaling Pathway Related Gene Expressions and Blood Biochemical indicators in Acute Promyelocytic Leukemia

Jak-Stat Signaling Pathway Related Gene Expressions and Blood Biochemical indicators in Acute Promyelocytic Leukemia

Pegylated liposomal doxorubicin for myeloid neoplasms

Pegylated liposomal doxorubicin (Peg-Dox) treatment resulted in a good outcome for patients with lymphoma and multiple myeloma, with reduced cardiotoxicity and an improved pharmacokinetic profile when compared to those of conventional doxorubicin. However, the use of Peg-Dox in myeloid neoplasms remains poorly studied. In this study, we first tested the role of Peg-Dox in the killing of myeloid cell lines and of primary myeloid leukemia cells. Then, a Peg-Dox-based protocol was used to treat patients with myeloid neoplasms. The results showed that the Peg-Dox and Peg-Dox-based protocols had a similar killing ability in myeloid cell lines and in primary myeloid leukemia cells compared to that of conventional doxorubicin. The complete remission rate was 87.5% and 100% for patients with refractory/relapsed acute myeloid leukemia and myelodysplastic syndrome with excess blasts, respectively, after treatment with Peg-Dox. All patients developed grade 3 or 4 hematological toxicity and recovered approximately 2 weeks after completing chemotherapy. No deaths or other severe complications were reported. Our results showed that Peg-Dox can be used in the treatment of myeloid neoplasms with high rates of complete remission and with mild complications.

Alginate foam-based three-dimensional culture to investigate drug sensitivity in primary leukaemia cells.

The development of assays for evaluating the sensitivity of leukaemia cells to anti-cancer agents is becoming an important aspect of personalized medicine. Conventional cell cultures lack the three-dimensional (3D) structure of the bone marrow (BM), the extracellular matrix and stromal components which are crucial for the growth and survival of leukaemia stem cells. To accurately predict the sensitivity of the leukaemia cells in an in vitro assay a culturing system containing the essential components of BM is required. In this study, we developed a porous calcium alginate foam-based scaffold to be used for 3D culture. The new 3D culture was shown to be cell compatible as it supported the proliferation of both normal haematopoietic and leukaemia cells. Our cell differential assay for myeloid markers showed that the porous foam-based 3D culture enhanced myeloid differentiation in both leukaemia and normal haematopoietic cells compared to two-dimensional culture. The foam-based scaffold reduced the sensitivity of the leukaemia cells to the tested antileukaemia agents in K562 and HL60 leukaemia cell line model and also primary myeloid leukaemia cells. This observation supports the application of calcium alginate foams as scaffold components of the 3D cultures for investigation of sensitivity to antileukaemia agents in primary myeloid cells.

RNA editing signature during myeloid leukemia cell differentiation

Adenosine deaminases acting on RNA (ADARs) are key proteins for hematopoietic stem cell self-renewal and for survival of differentiating progenitor cells. However, their specific role in myeloid cell maturation has been poorly investigated. Here we show that ADAR1 is present at basal level in the primary myeloid leukemia cells obtained from patients at diagnosis as well as in myeloid U-937 and THP1 cell lines and its expression correlates with the editing levels. Upon phorbol-myristate acetate or Vitamin D3/granulocyte macrophage colony-stimulating factor (GM-CSF)-driven differentiation, both ADAR1 and ADAR2 enzymes are upregulated, with a concomitant global increase of A-to-I RNA editing. ADAR1 silencing caused an editing decrease at specific ADAR1 target genes, without, however, interfering with cell differentiation or with ADAR2 activity. Remarkably, ADAR2 is absent in the undifferentiated cell stage, due to its elimination through the ubiquitin–proteasome pathway, being strongly upregulated at the end of the differentiation process. Of note, peripheral blood monocytes display editing events at the selected targets similar to those found in differentiated cell lines. Taken together, the data indicate that ADAR enzymes play important and distinct roles in myeloid cells.

PUMILIO/FOXP1 signaling drives expansion of hematopoietic stem/progenitor and leukemia cells

AbstractRNA-binding proteins (RBPs) have emerged as important regulators of invertebrate adult stem cells, but their activities remain poorly appreciated in mammals. Using a short hairpin RNA strategy, we demonstrate here that the 2 mammalian RBPs, PUMILIO (PUM)1 and PUM2, members of the PUF family of posttranscriptional regulators, are essential for hematopoietic stem/progenitor cell (HSPC) proliferation and survival in vitro and in vivo upon reconstitution assays. Moreover, we found that PUM1/2 sustain myeloid leukemic cell growth. Through a proteomic approach, we identified the FOXP1 transcription factor as a new target of PUM1/2. Contrary to its canonical repressive activity, PUM1/2 rather promote FOXP1 expression by a direct binding to 2 canonical PUM responsive elements present in the FOXP1-3′ untranslated region (UTR). Expression of FOXP1 strongly correlates with PUM1 and PUM2 levels in primary HSPCs and myeloid leukemia cells. We demonstrate that FOXP1 by itself supports HSPC and leukemic cell growth, thus mimicking PUM activities. Mechanistically, FOXP1 represses the expression of the p21−CIP1 and p27−KIP1 cell cycle inhibitors. Enforced FOXP1 expression reverses shPUM antiproliferative and proapoptotic activities. Altogether, our results reveal a novel regulatory pathway, underscoring a previously unknown and interconnected key role of PUM1/2 and FOXP1 in regulating normal HSPC and leukemic cell growth.

In Vitro Apoptotic Effects of Farnesyltransferase blockade in Acute Myeloid Leukemia Cells.

Farnesyltransferase inhibitors (FTIs) are a class of oral anti-cancer drugs currently tested in phase I-II clinical trials for treatment of hematological malignancies. The in vitro effects of various FTIs (alpha-hydroxyfarnesylphosphonic acid, manumycin-A and SCH66336) were tested on CD34+ KG1a cell line and in primary acute myeloid leukemia (AML) cells from 64 patients. By cell viability and clonogeneic methylcellulose assays, FTIs showed a significant inhibitory activity in CD34+ KG1a and primary bone marrow (BM) leukemic cells from 56% of AML patients. FTIs also induced activation of caspase-3 and Fas-independent apoptosis, confirmed by the finding that inhibition of caspase-8 was not associated with the rescue of FTI-treated cells. We concluded that other cellular events induced by FTIs may trigger activation of caspase-3 and subsequent apoptosis, but the expression of proapoptotic molecules, as Bcl-2 and Bcl-XL, and antiapoptotic, as Bcl-X(s), were not modified by FTIs. By contrast, expression of inducible nitric oxide synthase (iNOS) was increased in FTI-treated AML cells. Our results suggest a very complex mechanism of action of FTIs that require more studies for a better clinical use of the drugs alone or in combination in the treatment of hematological malignancies.

Gradient Infiltration of Neutrophil Extracellular Traps in Colon Cancer and Evidence for Their Involvement in Tumour Growth.

BackgroundThe role of neutrophils in tumour biology is largely unresolved. Recently, independent studies indicated either neutrophil extracellular traps (NETs) or Tissue Factor (TF) involvement in cancer biology and associated thrombosis. However, their individual or combined role in colonic adenocarcinoma is still unexplored.MethodsColectomy tissue specimens and variable number of draining lymph nodes were obtained from ten patients with adenocarcinoma of the colon. NETs deposition and neutrophil presence as well as TF expression were examined by immunostaining. The effect of NETs on cancer cell growth was studied in in vitro co-cultures of Caco-2 cell line and acute myeloid leukemia primary cells. Proliferation and apoptosis/necrosis of cancer cells were analyzed by flow cytometry.ResultsTF-bearing NETs and neutrophil localization were prominent in tumour sections and the respective metastatic lymph nodes. Interestingly, neutrophil infiltration and NETs concentration were gradually reduced from the tumour mass to the distal margin. The in vitro-generated NETs impeded growth of cancer cell cultures by inducing apoptosis and/or inhibiting proliferation.ConclusionsThese data support further the role of neutrophils and NETs in cancer biology. We also suggest their involvement on cancer cell growth.

Efficient RNA Interference of Primary Leukemic Cells for Loss-of-Function Studies in Xenograft Mouse Models.

RNA interference (RNAi) is a powerful tool for efficient and highly specific gene silencing. Transduction with lentiviral vectors provides stable and long-term gene expression in slowly and non-dividing cells. This chapter describes how to couple these two technologies to efficiently silence specific genes in primary acute myeloid leukemia (AML) cells for subsequent xenotransplantation in immunocompromised mice. This approach could be used for loss-of-function studies aimed at identifying oncogenic targets in AML.

ANP32A Dysregulation Involves Histone Modifications and Contributes to Myeloid Leukemia

Genetic alterations as the initiator inducing leukemia also cause disorder of gene expression. Cooperation of these factors is known to be essential for leukemia development and remains to be further elucidated. In this study, we discovered that ANP32A dysregulation might contribute to myeloid leukemia. ANP32A expression was unanimously elevated in multiple online leukemia datasets and its upregulation was confirmed in human primary myeloid leukemia cells. Although ectopic expression of ANP32A did not promote leukemic cell proliferation, it did increase cell resistance to drug treatment such as TPA, Ara-C, VP16, and BCL2 inhibitor. Interestingly, ANP32A knockdown reduced cell proliferation and impaired colony formation in soft agar in various leukemic cells. ANP32A knockdown did so by inducing apoptosis and cell cycle arrest at G1 phase evidenced by upregulation of pro-apoptosis genes (BAK, BAD, cleaved caspase 3 and PARP) and downregulation of pro-survival or cell cycle progress genes (BCL2, CDK4, CCND1). The function of ANP32A knockdown to induce apoptosis and reduce colony formation was verified in human primary acute myeloid leukemia (AML) cells (Figure 1). However, re-introduction of BCL2, CDK4, or CCND1 failed to restore the impaired ability of colony formation by ANP32A knockdown, suggesting that apoptosis and cell cycle arrest may not be the direct effect of ANP32A knockdown.To probe how ANP32A would affect cell proliferation, we performed microarray analysis to identify potential ANP32A target genes including APOC1 and CCL26. Indeed, reintroduction of APOC1 or CCL26 significantly rescued colony formation ability while knockdown of APOC1 or CCL26 alone was sufficient to reduce colony formation. Further gene set enrichment analysis (GSEA) also revealed Notch signaling and histone modification signatures in ANP32A knockdown cells. To support this, ANP32A knockdown reduced intracellular Notch and NOTCH1 ovexpression significantly recovered the ability of colony formation. Furthermore, ANP32A knockdown also led to a global alteration of histone modifications including decrease of H3K9-acetylation (H3K9ac), H3K27-trimethylation (H3K27me3), and H3K4-trimethylation (H3K4me3) and increase of H3K9-trimethylation (H3K9me3). In fact, ChIP-PCR demonstrated that ANP32A bound to the promoter region of multiple target genes identified in microarray analysis and ANP32A downregulation caused decreased H3K27me3 and H3K4me3 at the same sites (Figure 2). These findings suggest that ANP32A may bind to the chromosome and involve histone modifications.To further test the potential function of ANP32A in leukemogenesis, we took advantage of MLL-AF9 fusion gene to immortalize bone marrow cells in vitro and compared wild-type (WT) cells to Anp32a knockout (Anp32a-/-) cells. We found that MLL-AF9-transduced Anp32a-/- bone marrow cells reproduced all phenotypes of leukemic cells with ANP32A knockdown: Anp32a-/- cells proliferated less, formed less colonies, exhibited more apoptosis, and showed cell cycle arrest at G1 phase compared to WT cells (Figure 3). These observations suggest a potential role of ANP32A in MLL-AF9-induced acute myeloid leukemia.Taken together, our studies have revealed ANP32A as a potential novel player to cooperate with other genetic alterations to induce leukemia. ANP32A upregulation may somehow involve histone modifications at genomic level that ultimately alter the expression of multiple genes and facilitate leukemic cell proliferation and survival. Thus ANP32A may serve as a potential target for developing novel therapy or biomarker for diagnosis and prognosis. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways.

To investigate the effects of piperlongumine (PL), an anticancer alkaloid from long pepper plants, on the primary myeloid leukemia cells from patients and the mechanisms of action. Human BM samples were obtained from 9 patients with acute or chronic myeloid leukemias and 2 patients with myelodysplastic syndrome (MDS). Bone marrow mononuclear cells (BMMNCs) were isolated and cultured. Cell viability was determined using MTT assay, and apoptosis was examined with PI staining or flow cytometry. ROS levels in the cells were determined using DCFH-DA staining and flow cytometry. Expression of apoptotic and autophagic signaling proteins was analyzed using Western blotting. PL inhibited the viability of BMMNCs from the patients with myeloid leukemias (with IC50 less than 20 μmol/L), but not that of BMMNCs from a patient with MDS. Furthermore, PL (10 and 20 μmol/L) induced apoptosis of BMMNCs from the patients with myeloid leukemias in a dose-dependent manner. PL markedly increased ROS levels in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the antioxidant N-acetyl-L-cysteine abolished PL-induced ROS accumulation and effectively reduced PL-induced cytotoxicity. Moreover, PL markedly increased the expression of the apoptotic proteins (Bax, Bcl-2 and caspase-3) and autophagic proteins (Beclin-1 and LC3B), and phosphorylation of p38 and JNK in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the specific p38 inhibitor SB203580 or the specific JNK inhibitor SP600125 partially reversed PL-induced ROS production, apoptotic/autophagic signaling activation and cytotoxicity. Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways.

Medicarpin, a legume phytoalexin sensitizes myeloid leukemia cells to TRAIL-induced apoptosis through the induction of DR5 and activation of the ROS-JNK-CHOP pathway.

Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent with cancer cell-selective cell death inducing effect. However, the major limitation in the usage of TRAIL as a chemotherapeutic agent is the development of TRAIL resistance in many cancer types including myeloid leukemia. In this study, we report for the first time that Medicarpin (Med), a naturally occurring phytoalexin sensitizes myeloid leukemia cells to TRAIL-induced apoptosis. Combination of Med and TRAIL induced significantly higher apoptosis compared with that of the individual treatments of either agent alone through activation of both the extrinsic and the intrinsic cell death pathways characterized by the activation of caspases 8, 9, 3, and 7. Med treatment downregulated antiapoptotic proteins (Survivin, Bcl2, Bcl-xL, XIAP, and c-FLIP), upregulated pro-apoptotic proteins (Bax, Cytochrome C, Smac/Diablo, Bid, truncated Bid (tBid), p-eIF2α, Bip, and CHOP (CCAAT-enhancer binding protein homologous protein)), induced G2/M cell-cycle arrest, and increased the expression of the functional TRAIL receptor DR5 through activation of the ROS-JNK-CHOP pathway. Gain and loss of function studies clearly indicated that DR5 expression was critical for Med-induced TRAIL sensitization. The Med-induced TRAIL sensitization did not involve the NFkB signaling pathway or redistribution of DR5 in lipid rafts. The concomitant treatment with Med and TRAIL showed robust apoptotic effects in primary myeloid leukemia cells but had no toxic effects in primary human peripheral blood mononuclear cells (PBMCs). In conclusion, our results suggest that Med sensitizes myeloid leukemia cells to TRAIL-induced apoptosis through the upregulation of DR5 through activation of the ROS-JNK-CHOP pathway.

(6)-Gingerolinduced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression

The natural polyphenolic alkanone (6)-gingerol (6G) has established anti-inflammatory and antitumoral properties. However, its precise mechanism of action in myeloid leukemia cells is unclear. In this study, we investigated the effects of 6G on myeloid leukemia cells in vitro and in vivo. The results of this study showed that 6G inhibited proliferation of myeloid leukemia cell lines and primary myeloid leukemia cells while sparing the normal peripheral blood mononuclear cells, in a concentration- and time-dependent manner. Mechanistic studies using U937 and K562 cell lines revealed that 6G treatment induced reactive oxygen species (ROS) generation by inhibiting mitochondrial respiratory complex I (MRC I), which in turn increased the expression of the oxidative stress response-associated microRNA miR-27b and DNA damage. Elevated miR-27b expression inhibited PPARγ, with subsequent inhibition of the inflammatory cytokine gene expression associated with the oncogenic NF-κB pathway, whereas the increased DNA damage led to G2/M cell cycle arrest. The 6G induced effects were abolished in the presence of anti-miR-27b or the ROS scavenger N-acetylcysteine. In addition, the results of the in vivo xenograft experiments in mice indicated that 6G treatment inhibited tumor cell proliferation and induced apoptosis, in agreement with the in vitro studies. Our data provide new evidence that 6G-induced myeloid leukemia cell death is initiated by reactive oxygen species and mediated through an increase in miR-27b expression and DNA damage. The dual induction of increased miR-27b expression and DNA damage-associated cell cycle arrest by 6G may have implications for myeloid leukemia treatment.

Activation Of EphrinB2/EphB4 Influences Myeloid Leukemia Cell Migration and Invasion

Eph receptors and ephrin ligands are cell-surface molecules capable of bidirectional signaling that control cell-cell interactions, migration and invasion. However, their role and regulation in myeloid leukemia cells remain to be defined. To address the hypothesis that Ephrin/EphB is an important regulator of myeloid leukemia cell migration and invasion, we first screened the mRNA levels of 23 eph and ligand ephrin RTK family members in myeloid leukemia cells (K562, HL-60, THP-1) and mononuclear cells from healthy donors, then found that EphB4, EphA5, EfnA1 highly expressed in most myeloid leukemia cells compared to healthy donors(P<0.05). Both the mRNA and protein levels of EphB4 and EphA5 were detected in 13 primary myeloid leukemia cells (5 from patients with extramedullary leukemia among 13 cases) and 10 mononuclear cells from healthy donors by real-time RT-PCR and Immunoblot analysis. The results showed that both the mRNA and protein levels of EphB4 and EphA5 were higher in 13 primary myeloid leukemia cells relative to the 10 healthy donors (P=0.046). Moreover, the EphB4 were highly expressed in 5 patients with extramedullary leukemia compared with 8 patients without extramedullary leukemia. These findings indicated that EphB4 and EphA5 expression were correlated with the development of myeloid leukemia cells, moreover, EphB4 may be closely related with myeloid leukemia cell migration or invasion. To further clarified the question, migration were determined in leukemia cell lines (K562 cells) which were treated with clustered ephrinA1–Fc proteins, ephrinB2–Fc proteins and Fc proteins by transwell migration assay. Invasion were also determined by matrigel invasion assay. The results showed that, after ephrinB2–Fc stimulation, the numbers of K562 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (1.8 to 2.5-fold, P<0.05), meanwhile, the numbers of K562 cells invading the matrigel also enhanced (1.2 to 1.8-fold, P<0.05). However, after ephrinA1–Fc stimulation, the numbers of K562 cells migrating through transwell chamber didn't significantly increase compared to Fc proteins stimulation (P>0.05), and the numbers of K562 cells invading the matrigel also didn't enhanced (P>0.05). These findings indicated that ephrinB2–Fc could activate EphB4, leading to the change of myeloid leukemia cell migration and invasion. Further study may help to assess a promising potential of this protein to be used as a prognostic marker or as a target for a molecular therapy. Disclosures:No relevant conflicts of interest to declare.

RAD52-Dependent Synthetic Lethality Eradicates Leukemia Stem Cells

Abstract 3▪▪This icon denotes a clinically relevant abstractWe showed that imatinib-naive and imatinib-treated BCR-ABL1 kinase-positive chronic myeloid leukemia in chronic phase (CML-CP) Lin−CD34+CD38− stem cells (LSCs) and Lin−CD34+CD38+ leukemia progenitor cells (LPCs) accumulate high numbers of the reactive oxygen species (ROS)-induced DNA double-strand breaks (DSBs) which, if not repaired, are lethal (Nieborowska-Skorska et al., Blood, 2012). Genome-wide microarray screen indicated that LSCs overexpress numerous genes responsible for homologous recombination repair (HRR) of DSBs. Direct targeting of RAD51, a key element in HRR, by small molecule inhibitor exerted anti-CML effect, but normal cells were also affected, indicating that RAD51 is not a preferable target. In normal cells HRR depends primarily on BRCA1/BRCA2-RAD51 pathway, while RAD52-RAD51 pathway may serve as a back-up. However BRCA1 protein is downregulated in CML, and RAD51(F259V) mutant (RAD52 binding-deficient) induced apoptosis and reduced cell growth in BCR-ABL1 –dependent manner, thus highlighting the potential role of alternative RAD52-driven HRR in CML. The absence of RAD52 (Rad52−/−) inhibited BCR-ABL1 –mediated cell cycle progression and clonogenic activity, induced apoptosis, reduced Lin−Kit+Sca1+CD34−Flt3− long-term LSCs and Lin−Kit+Sca1+CD34+Flt3− short-term LSCs, and abrogated leukemogenesis in murine model of CML. At the same time RAD52 was expendable in normal cells. In addition, RAD52 was essential to prevent accumulation of ROS-induced lethal DSBs in BCR-ABL1 –positive murine LSCs. Thus it appears that RAD52 is necessary to repair numerous ROS-induced DSBs in LSCs to promote leukemogenesis. BCR-ABL1 kinase interacts with and phosphorylates RAD52 on Y104, but phosphorylation-deficient RAD52(Y104F) mutant did not exert any negative effect on BCR-ABL1 kinase-driven leukemogenesis. This observation suggests that RAD52 activity is preserved in imatinib-treated cells. On the other hand, DNA binding-deficient RAD52(F79A) and RAD52(K102A) mutants (disrupt DNA binding domain I and II, respectively) inhibited clonogenic potential of Lin−CD34+ CML-CP cells and BCR-ABL1 –positive Lin−Kit+Sca1+ murine LSCs, but not normal counterparts. Therefore, RAD52-mediated DNA binding activity appears to be essential for BCR-ABL1 kinase-driven leukemogenesis, but not normal hematopoiesis. RAD52 DNA I binding groove containing F79 forms a pocket/niche. Peptide aptamer containing amino acid residues surrounding RAD52 F79, but not that with F79A substitution, inhibited RAD52 foci, RAD52-dependent RAD51 foci and HRR activity, resulting in elevation of the number of lethal DSBs and abrogation of clonogenic activity of BCR-ABL1-positive murine leukemia cells. Aptamer F79 reduced the growth of LSCs and LPCs from CML-CP and more aggressive CML-accelerated phase (CML-AP), depleted quiescent LSCs by attrition and eradicated BCR-ABL1 leukemia from SCID mice. Moreover, aptamer F79 enhanced the anti-CML effects of imatinib. The effect of aptamer F79 in BCR-ABL1 leukemia cells depended on downregulation of BRCA1 implicating “synthetic lethality”. At the same time the aptamer did not exert any measurable negative effects on normal cells and tissues. In conclusion, we postulate that CML-CP/AP cells are “addicted” to RAD52 and that targeting of DNA binding domain of RAD52 may induce “synthetic lethality” to eliminate proliferating LSCs/LPCs and to deplete quiescent LSCs. Moreover, anti-RAD52 F79 aptamer exerted anti-tumor activity in acute myeloid leukemia primary cells, and in cell lines derived from carcinomas of breast, ovary and pancreas displaying BRCA1 and/or BRCA2 deficiency. Thus, BRCA1/BRCA2ness-driven “addiction” to RAD52 may be selectively targeted to achieve “synthetic lethality” in wide-range of tumors while being harmless to normal cells. Disclosures:Copland:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria.

CS055 (Chidamide/HBI-8000), a novel histone deacetylase inhibitor, induces G1 arrest, ROS-dependent apoptosis and differentiation in human leukaemia cells

CS055 (Chidamide/HBI-8000) is a novel benzamide-type HDACi (histone deacetylase inhibitor), which has entered Phase I clinical trials in the U.S. and Phase II/III in China. In the present study, we investigated the effects of CS055 on proliferation, differentiation and apoptosis in human leukaemia cell lines and primary myeloid leukaemia cells. The results showed that at low concentrations (<1μM), CS055 induced G1 arrest. At moderate concentrations (0.5μM-2μM), CS055 induced differentiation, as determined by the increased expression of the myeloid differentiation marker CD11b. At relatively high concentrations (2μM-4μM), CS055 potently induced caspase-dependent apoptosis. Co-treatment with the ROS (reactive oxygen species) scavengers N-acetyl-L-cysteine or Tiron blocked CS055-induced cell differentiation and apoptosis, suggesting an essential role for ROS in these effects. Cytochrome c release and ROS-mediated mitochondrial dysfunction are involved in CS055-induced apoptosis of leukaemia. In addition to cell lines, CS055 also exhibits therapeutic effects in human primary leukaemia cells. Moreover, daily oral CS055 treatment of nude mice bearing HL60 cell xenografts suppressed tumour growth, induced tumour cell apoptosis and prolonged the survival of tumour-bearing mice. In conclusion, our findings demonstrate that CS055 is a novel HDACi with potential chemotherapeutic value in several haematological malignancies, especially leukaemia.

TMTP1, a novel tumor-homing peptide, specifically targets hematological malignancies and their metastases

TMTP1, a 5-amino acid peptide NVVRQ, obtained by using the flagella peptide library screening in our previous studies, can be used for the labeling of malignant in situ and metastatic lesions, and even micro-metastases. In this study, TMTP1 was assessed for its ability to specifically target the malignant hematopoietic cells and metastatic lesions of hematological malignancies. FITC-TMTP1 was chemically synthesized. Immunofluorescence assay and competitive test were carried out to determine the specific binding capacity of TMTPl to hematological malignant cell lines, including HL60, k562, SHI-1, Jurkat, Raji, El-4 and umbilical cord blood mononuclear cells. Mononuclear cells were isolated from the bone marrow of healthy subjects and patients with chronic myeloid leukemia. Then the cells were co-cultured with TMTP1 or scrambled peptides and the binding and affinity of TMTP1 peptide to the primary cells of hematological malignancies were flow cytometrically analyzed. The binding specificity of TMTP1 to target hematological malignancies was measured in vivo by intravenous injection of FITC-conjugated TMTP1 into El-4 lymphoma-bearing mice. The results showed that TMTP1 specifically bound to the cells of a series of hematological malignancies, including HL60, k562, Jurkat, Raji, El-4 and chronic myeloid leukemia primary cells but not to bone marrow mononuclear cells from healthy subjects. By contrast, TMTP1 could bind to the metastatic foci of lymphoma originating from the EL-4 cell line while the scrambled peptide failed to do so. Moreover, the occult metastases could be identified, with high specificity, by detecting FITC-TMTP1. We are led to conclude that TMTP1, as a novel tumor-homing peptide, can serve as a marker for primary malignant and metastatic lesions for the early diagnosis of hematological malignances and a carrier of anticancer drugs for cancer treatment.

Heat Shock Protein 90 Inhibition Depletes TrkA Levels and Signaling in Human Acute Leukemia Cells

Nerve growth factor (NGF) induces autophosphorylation and downstream progrowth and prosurvival signaling from the receptor tyrosine kinase TrkA. Overexpression or activating mutation of TrkA has been described in human acute myeloid leukemia cells. In the present study, we show the chaperone association of TrkA with heat shock protein 90 (hsp90) and the inhibitory effect of the hsp90 inhibitor, 17-DMAG, on TrkA levels and signaling in cultured and primary myeloid leukemia cells. Treatment with 17-DMAG disrupted the binding of TrkA with hsp90 and the cochaperone cdc37, resulting in polyubiquitylation, proteasomal degradation, and depletion of TrkA. Exposure to 17-DMAG inhibited NGF-induced p-TrkA, p-AKT, and p-ERK1/2 levels, as well as induced apoptosis of K562, 32D cells with ectopic expression of wild-type TrkA or the constitutively active mutant Delta TrkA, and of primary myeloid leukemia cells. Additionally, 17-DMAG treatment inhibited NGF-induced neurite formation in the rat pheochromocytoma PC-12 cells. Cotreatment with 17-DMAG and K-252a, an inhibitor of TrkA-mediated signaling, induced synergistic loss of viability of cultured and primary myeloid leukemia cells. These findings show that TrkA is an hsp90 client protein, and inhibition of hsp90 depletes TrkA and its progrowth and prosurvival signaling in myeloid leukemia cells. These findings also support further evaluation of the combined activity of an hsp90 inhibitor and TrkA antagonist against myeloid leukemia cells.

Abstract 1649: Pim kinase inhibitor, SGI-1776, induces apoptosis in acute myeloid leukemia primary cells

Abstract Small molecule SGI-1776 was found to be potent and selective inhibitor of Pim family serine-threonine kinases [IC50: Pim-1 (7 nM), Pim-2 (363 nM) and Pim-3 (69 nM)] without effects on other kinases except Flt-3 and haspin. In preclinical studies in chronic lymphocytic leukemia, SGI-1776 treatment resulted in apoptosis induction that was associated with a decrease in RNA synthesis, Mcl-1 protein level, and c-Myc phosphorylation (Chen, LS et al, Blood 2009, 114: 4150). Increased levels of Pim-2 are reported in acute myeloid leukemia (AML) as well as Flt-3 activating mutation, providing a rationale to test SGI-1776 in AML. We evaluated SGI-1776 using FLT3-ITD AML cell lines (MOLM-13, MV4-11), xenograft models, and in primary cells from AML patients, and our results indicate that SGI-1776 is effective in AML. In MOLM-13 and MV4-11 cell lines, the IC50 for viability was &amp;lt; 10 nM after treatment with SGI-1776 for 72 h, which was associated apoptosis induction and reduction in phosphoBad (Ser112) and Bad protein. In xenograft models of both cell lines, treatment with 135mg/kg SGI-1776 resulted in a consistent decline in tumor volume after 14 days. To further these investigations in primary cells, we obtained cells from AML patients including FLT-3 positive and mutated FLT3-ITD patients, and evaluated the potential for SGI-1776 to induce cell death. In vitro incubation of AML cells (n = 4) with 1, 3, and 10 μM SGI-1776 for 24 h resulted in an average increase in apoptosis of 5%,12% and 19% respectively, measured using Annexin-staining compared with untreated cells. Apoptosis induction was verified via PARP cleavage immunoblot, and apoptosis was observed in cells from FLT3 negative or mutated FLT3-ITD patients. In the samples evaluated, there was disparity in cytogenetic factors, Flt-3 status and prior chemotherapeutic treatment history. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. There was no observed change in phosphorylation of Pim targets histone H3 (Ser10) or p21 (Thr145), however protein levels of both total and phospho-Bad(Ser112) decreased following SGI-1776 treatment. In contrast, there was no change in anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1, and levels of pro-apoptotic proteins, Bak and Bax, were also unaffected. However, there was a decrease in phospho-c-Myc(Ser 62), which is a Pim target and associated with stabilization of c-Myc protein. Consequently, we evaluated the potential inhibition of c-Myc driven transcription by SGI-1776 by measuring total RNA synthesis. Following treatment with 1, 3 or 10 μM SGI-1776, there was a decrease in total RNA synthesis to 75%, 42% and 25% of control respectively, measured using a radioactive uridine incorporation assay (n = 4). Taken together, SGI-1776 was demonstrated to be an effective agent against AML in cell lines, xenograft models, and primary cells, and may be an effective new agent in the treatment of AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1649.

A Nonpeptidyl, Hydrazone Class, Thrombopoietin Receptor Agonist (TpoRA), SB559457, Selectively Kills Primary Human Myeloid Leukemia Cells

Treatment of thrombocytopenia, whether autoimmune, secondary to marrow failure, or iatrogenic, can become problematic. Stimulation of endogenous megakaryocyte (MEG) development might be an effective strategy and it was originally hoped that this could be accomplished with thrombopoietin (Tpo), the natural ligand of the thrombopoietin receptor (TpoR) and regulator of MEG development. When formulated Tpo proved immunogenic, development of small molecule TpoR agonists (TpoRA) was undertaken. In course of investigating the biological effects, and mechanism of action, of SB559457 (a nonpeptidyl, hydrazone class, TpoRA supplied by GlaxoSmithKline (GSK), Collegeville, PA) we found that the molecule robustly stimulated human megakaryocytopoiesis in vitro. In anticipation of TpoRA use in the treatment of patients with hematologic malignancies, we also evaluated SB559457's effect on human leukemia cell (HLC) growth in vitro since it is known that HLCs may express TpoR. Specifically, we examined the effects of SB559457 on samples obtained from 21 patients with AML, and 7 with ALL. Surprisingly, SB559457 not only failed to stimulate HLC growth, it proved toxic to primary acute myelogenous leukemia (AML). In 20 of 21 primary AML samples exposed to SB559457 (5μM) a large fraction (70–90%) of cells died between days 3 and 6 of culture, while untreated control cells survived. No significant effects on cell growth or viability were observed in the ALL patient samples. To investigate the mechanism(s) of AML cell killing, we examined signaling cascades initiated by SB559457 compared to recombinant human Tpo (rhTpo), which had no apparent effect on AML cell growth. Initially, we employed N2C-Tpo cells (a Tpo dependent megakaryoblastic cell line that express TpoR) and focused on kinases known to be phosphorylated after TpoR activation; STAT5, ERK, p70S6, and ribosomal kinase S6. When N2C-Tpo cells were stimulated with rhTpo (2.8μM, 30 min) all these kinases were highly phosphorylated. In contrast, none were phosphorylated in SB559457 (5μM, 30min) stimulated cells. Normal human CD34+ marrow cells were next examined using the same experimental conditions. In contrast to N2C-Tpo cells, ERK, and p70S6 kinases were both phosphorylated after exposure to SB559457 but STAT5 remained unphosphorylated. Since AML cells may require STAT5 activation for growth, we hypothesized that perturbation of STAT5 activation might be involved in the apoptotic mechanism. The molecular consequences of differential signaling were then pursued by Affymetrix GeneChip analysis. Remarkably, in 5 separate primary AML cell samples stimulated for 6 hours with either Tpo or SB559457 we found a statistically significant difference in expression in only 2 of 22000 genes represented on the chips: GAPDH and Redd1. Both of these genes are induced in stressed cells undergoing apoptosis. In addition, stimulation of primary AML cells with SB559457 resulted in increased phosphorylation of p70S6/S6 kinases, both downstream targets of mTOR kinase. Accordingly, we hypothesize that SB559457 mediated activation of mTOR pathway leads to a stress response in primary myeloid leukemia cells as reflected by the increased expression of Redd1 and GAPDH. Further investigation of this pathway, and the leukemic cell response to SB559457 exposure may lead to development of novel strategies for the treatment of myeloid leukemias.